ESHG Posters 2

Germline Mutations in BRCA1 and BRCA2 genes in the Czech Hereditary Forms of Breast / Ovarian Cancer

E. Machackova, M. Navratilova, H. Pavlu, D. Valik, M. Hruba, L. Foretova;
Masaryk Memorial Cancer Institute, Brno, CZECH REPUBLIC.

Background: It is estimated that 5-10% of all breast cancers can be of hereditary origin. Germline mutations in highly penetrant cancer susceptibility genes BRCA1 and BRCA2 could cause genetic predisposition to breast and ovarian cancers.
Material and Methods: Molecular genetic analysis of BRCA1 and BRCA2 genes was performed in 150 high-risk breast and breast/ovarian cancer families and in 25 women diagnosed with early-onset sporadic breast cancers below 40 years. Protein truncation test and heteroduplex analysis followed by sequencing was carried out on genomic DNA isolated from blood samples. The genetic counseling and preventive clinical follow-up of gene carriers is part of the genetic program.
Results: A germline disease causing mutation was found in 63 screened high-risk unrelated families (42%), 41 mutations (12 different) in BRCA1 gene and 22 mutations (15 different) in BRCA2 gene. The most frequently detected mutations in BRCA1 gene were 5385-5386insC (14 families) and 3819-3823del5 (7 families). Two novel frame shift mutations were detected in BRCA1gene: 2616-2617ins10; 3761-3762del2. Three novel frame shift mutations were detected in BRCA2 gene: 5073-5074del2; 6677-6678del2; 6866delC. Within sporadic early-onset breast cancer cases no disease-causing mutation was found.
Conclusion: Germline mutations in BRCA1 and BRCA2 genes are responsible for a significant fraction of familial breast and ovarian cancer cases in the Czech Republic.
Contract grant sponsor: Ministry of Health of the Czech Republic: MZ00020980501;
and Internal Grant Agency of the Ministry of Health of the Czech Republic: NC5561-3 and NC6396-3.

Y. Engwall¹, M. Nordling¹, J. Lundberg¹, A. Bergman¹, T. Martinsson¹, Z. Einbeigi², P. Karlsson², A. Wallgren², J. Wahlström¹;
¹Dept. of Clinical Genetics, Sahlgrenska Univ. Hospital/East, Göteborg, SWEDEN, ²Dept. of Oncology, Sahlgrenska Univ. Hospital/Sahlgrenska, Göteborg, SWEDEN.

Germline mutations in the hereditary breast/ovarian cancer causative genes BRCA1 and BRCA2 are considered to constitute approximately 6-10% of these cancers. The frequency of female mutation carriers with breast/ovarian cancer depends on the population studied, and display considerable variation in coincidence with ethnic and geographical diversity. Mutations are mainly found as small insertions, deletions or substitutions, but also as exon-wide deletions. We performed mutation analyses in 116 patients, selected under informed consent, from the Sahlgrenska University Hospital, Gothenburg, Sweden. The genes were initially screened using the Protein Truncation Test (PTT) on genomic DNA (BRCA1 exon 11, BRCA2 exon 10, 11) and cDNA from RT-PCR (all other exons) for truncating mutations. All mutations but one was detected with PTT; the remaining one with dHPLC. Automated DNA sequencing of the detected mutations revealed seven different frameshift mutations, two nonsense mutations and one large deletion. Four of these have not been reported earlier: BRCA1 409-410delCA; 2229-2230delAA; 3029delA; 1912 T>G. BRCA mutations were found in 34% of the screened.families; this is comparable to frequencies reported in other European studies. Notably, a western Swedish founder mutation (BRCA1 3171ins5) accounted for 27 of the 37 mutations detected in the 116 families. Our results are furthermore in accordance with the observation that frameshift mutations in the first two-thirds of BRCA1 are associated with a higher risk of ovarian relative to breast cancer than are truncating mutations in the last one-third of the gene.

An investigation into the methylation status of oestrogen receptor beta and its subsequent expression in human breast cancer

F. J. Cooke¹, P. Balfe¹, A. H. McCann², P. Dervan³, M. Kennedy³, M. Kerin¹;
¹Conway Institute of Biomolecular and Biomedical Research, University College Dublin / Mater Hospital, Dublin, IRELAND, ²Conway Institute for Biomolecular and Biomedical Research, University College Dublin, IRELAND, ³Department of Pathology, Mater Misericordiae Hospital, Eccles St, Dublin 7, IRELAND.

Since the discovery of the novel oestrogen receptor beta (ERb) much investigation has centered on the role of this new marker in the prognosis of breast cancer and response to adjuvant tamoxifen. The aim of this study is to assess the methylation status of the ERb promoter with respect to ERb expression by immunohistochemistry in a preliminary cohort of 25 primary human breast tumors. In addition we sought to correlate ERa, ERb and c-erbB2 status with tumor staging and prognosis.
Fresh tissue was prospectively collected from screen detected and symptomatic palpable breast tumors managed in the Mater Misericordiae and Mater Private Hospitals. DNA was extracted from the frozen tissue using the standard phenol-chloroform approach. The DNA was subsequently quantified by spectrophotometry and its integrity verified on a 1% agarose gel. To allow Methylation Specific PCR, the DNA was modified by a sodium bisulphate procedure and amplified using methylation specific primers. Resultant products were analysed on an 8% PAGE and confirmed with sequencing.
Table of Contents
Table 1. Histological diagnosis and immunohistochemical status


Diagnosis	Total	ERa status (% positive)	ERb status (% positive)
Invasive Ductal	22	20/22 (91%)	16/22(73%)
Invasive Lobular	3	3/3 (100%)	2/3(66%)

With the establishment of this novel MSP approach to analyzing the methylation status of the ERb promoter in human breast cancer, we intend to correlate the findings with the immunohistochemical results obtained with the 14C8 monoclonal antibody. These studies are of special importance in the context of epigenetic reversibility as a potential therapeutic option.

M. Carvallo¹, L. Palma¹, M. Gallardo¹, C. Rousseau², M. King²;
¹Universidad Catolica de Chile, Santiago, CHILE, ²University of Washington, Seattle, WA.

Two genes have been described until today as responsible for familiar breast cancer, BRCA1 and BRCA2. Several studies have demonstrated that the frequency of mutations in either gene is variable depending on the population analysed. Since this effect may be due to the ethnic origin of the families selected, we were interested in knowing the incidence of BRCA1 and BRCA2 mutations in Chilean families. We previously reported a very low frequency of mutations in the BRCA1 gene (10%). This study shows the analysis of BRCA2 mutations in the same group of families. The families were selected by standard criteria. All exons encoding the BRCA2 gene were PCR amplified and analysed through SSCA, heteroduplex and DNA sequencing. We found in one family the 6174delT mutation in exon 11, which has been extensively reported in families with Ashkenazi-Jewish ancestors. The patient carrying the mutation informed about Ashkenazi-Jewish ancestors in her family. The second mutation found is 6857delAA, also present in exon 11. This is a very interesting mutation for our study, since it has been described previously in three families from Spanish origin. Due to high percentage of admixture of the Chilean population with Spanish colonisers during the XVI and XVII centuries, it is highly probable that the mutation has a common ancestor. Also it is interesting to note that the incidence of BRCA1 and BRCA2 mutations in the Spanish population is below 20% each. (Financed by FONDECYT 1011076)

Telomerase enzyme activity and chromosome abnormalities detected by combined G-banding and comparative genomic hybridization in primary breast cancer

A. Papadopoulou¹, T. Trangas¹, H. Tsarouha¹, M. Texeira², E. Dimitriadis¹, P. Ioannidis¹, S. Heim³, N. Pandis¹;
¹"G. Papanikolaou" Research Center, "Saint Savvas" Oncological Hospital of Athens, Athens, GREECE, ²Portugese Oncology Institute, Porto, PORTUGAL, ³The Norwegian Radium Hospital, Oslo, NORWAY.

Several structural and numerical chromosomal abnormalities have been recorded in breast cancer. It has been suggested that some chromosomal aberrations may be the result of telomere dysfunction in rapidly proliferating cells. The de novo activation of telomerase in cancer possibly provides a survival mechanism curtailing further chromosomal aberrations. In order to investigate the relation of telomerase level of expression and the extent of chromosomal aberrations, 62 primary breast carcinomas were studied. Telomerase activity was measured using a PCR based TRAP assay and 92% of the tumors were found to express Telomerase with relative activity ranging from 0 – 3839.6. Genetic alterations were determined by G banding and Comparative Genomic Hybridization analysis. 97% of the tumors exhibited chromosomal aberrations ranging from 0-45. The average number of genetic alteration recorded by G-banding and CGH was 10.98. No correlation was observed between Telomerase activity levels and the number of genetic alteration in the overall sample population. However when tumor samples with below average genetic alteration numbers were considered as a separate group, a statistically significant positive correlation was recorded between Telomerase activity levels and number of genetic alterations (R=0.339, p=0.032). This relationship was inversed in the sample group with above average genetic alterations but it was not statistically significant (R=-0.127; p=0.574). These results suggest that Telomerase may be activated in the initial steps of carcinogenesis perhaps to maintain chromosomal integrity of the rapidly proliferating tumor cells while at the later stages alternative survival mechanisms have evolved.

Selective genetic screening in 250 Belgian breast and ovarian cancer families identifies BRCA1 or BRCA2 mutations in 20% of cases

G. Michils, K. Minner, B. Vankeirsbilck, E. Legius, G. Matthijs;
Center for Human Genetics, Leuven, BELGIUM.

Germline mutations of the BRCA genes are associated with familial breast and/or ovarium cancer, or with early onset of breast cancer (<35y) in the absence of a familial history. In families attending a cancer genetic clinic mutations are typically identified in 15-20% of the analysed families. Most mutations are truncating and spread all over the coding regions, but at a higher frequency in the large exons (exon 11 of BRCA1 and exons 10 and 11 of BRCA2). We chose to analyse these large exons together with exons with Belgian founder mutations, Ashkenazi mutations, hot spots and exonic deletions.
DNA samples of a cohort of 250 unrelated affected patients were included in this study: 140 samples were analysed by Enzymatic Mutation Detection (EMD), while 110 samples were screened by Denaturing High Performance Liquid Chromatography (DHPLC). Exon deletions were analysed by amplification across breakpoint junctions.
By screening selected exons of BRCA1 and BRCA2, 21 germline truncating mutations and one exonic deletion were found in 48 of 250 families. In addition, one pathogenic missense mutation has been identified in BRCA1: M1V. DHPLC has advantages in comparison to EMD, mainly because it is semi-automatable. This study shows that a limited screening of BRCA1 and BRCA2 results is a high yield of mutations in our clinical sample (20%). This screening could be offered to a large group of females while an exhaustive screening could be performed in a more selected group. Analysis of additional exons by DHPLC is going on in such a selected group.

A. Lyakhovich¹^,2, M. Shekhar¹;
¹Wayne State University, Detroit, MI, ²Institute of Molecular Biology and Biophysics, Novosibirsk, RUSSIAN FEDERATION.

Treatment with DNA damaging drugs is commonly used to localize breast cancer. It causes DNA damage and leads to genomic instability and/or apoptosis as a result of mutation or altered expression of genes associated with DNA repair, in particular RAD6. RAD6 (Ubch2) protein is present at low amounts in cytoplasm of normal human breast cells, while in metastatic breast cancer cells it is up-regulated and localized in the nucleus. We have demonstrated, that overexpression of exogenous RAD6 cDNA in MCF10A human breast epithelial cells induced cell-cell fusion generating multinucleated cells, centrosome amplification, abnormal mitosis and aneuploidy. We found that exposure of MCF10A cells with cisplatin or adriamycin resulted in enhancement of RAD6 mRNA/protein level which was post-transcriptionally regulated and post-translationally stabilized. RAD6 protein is predominantly expressed during late S/G2 phases. Its localization in cells at specific stages of mitosis reveals the lack of association of RAD6 with condensed chromatin. Co-localization of RAD6 protein with g-tubulin on centrosomes is maintained throughout the interphase and mitotic stages of the cell cycle. We were able to show for the first time that in drug-treated cells RAD6 is associated with p53-p14ARF-MDM2 in the nucleus. The expression of RAD6 correlates with human breast cancer stage and can be used as a marker to predict response to chemotherapy. Our findings suggest that RAD6 at low levels may play a significant role in the maintenance of genomic integrity of mammalian cells, high levels of RAD6 probably over a certain threshold may cause genomic instability.

Elevated CA-125 serum level as an example of correlation between cell biology, carcinogenesis, positive family story. A case of woman from the breast/ovarian cancer family, with mutation in BRCA1 gene.

Characteristic feature of the neoplasm evolution is long-lasting process. The priority of oncology is to diagnose cancer in its pre-clinical stage of development. It is extremely important in families, which have positive family story. The point is searching for substances, presence of which in the blood would give evidence to the presence of cancer. In case of CA-125, its production in tumor is significantly higher than in a normal cell. Level depends on mass of the living tumor cells.
Here we report a case of patient, whose family was affected by three breast and one ovarian cancer. Because strong aggregation breast and ovarian cancer- the woman performed genetic test. It showed mutation in BRCA1 gene, exon 20-5382insC. The next two mutations were proven in her sister daughters.
In all performed clinical examinations (mammography, ultrasonographic examination of the breasts, transvaginal ultrasonography) there were no pathology. But the serum level CA-125 was highly increased, accordingly:108 and 125 IU/L ( normal: 30 IU/L). She underwent prophylactic oophrectomy, and at the time of surgery tumor was suspected. Histopathology gave the final solution: poorely diffrentiated adenocarcinoma, only in one site of the left ovary. As a consequence, the woman started few courses of chemotheraphy.
We want to conclude, that we should perform BRCA1 and BRCA2 tests in families with strong aggregation of breast and ovarian cancer. We should have a high suspicion of cancer, when serum level of Ca-125 is highly increased. We want to undreline the strategy of prevention.

Androgen Receptor CAG Repeat Length in Jewish Israeli Women who are BRCA1/2 Mutation Carriers: Association with Breast/Ovarian Cancer Phenotype

E. Dagan¹, E. Friedman², T. Paperna¹, N. Carmi², R. Gershoni-Baruch¹;
¹Rambam Medical Center, Haifa, ISRAEL, ²Sheba Medical Center, Tel-Aviv, ISRAEL.

BRCA1/2 mutation carriers are at increased lifetime risk for developing breast and/or ovarian cancer. Yet, the genetic or environmental factors that govern the phenotypic expression of mutant BRCA1/2 alleles remain elusive. The CAG repeat, within exon 1 of the Androgen Receptor (AR) gene is reportedly associated with breast cancer phenotype in BRCA1 mutation carriers. To extend this observation, we genotyped 227 BRCA1/2 mutation carriers for the polymorphic AR CAG repeat, and correlated allele size with breast/ovarian cancer morbidity parameters. Of 227 BRCA1/2 carriers, 169 were BRCA1 mutation carriers and 58 carried a BRCA2 mutation. Seventy-nine women had unilateral breast cancer, 15 - bilateral breast cancer, 41 - ovarian cancer, 14 - breast and ovarian cancer and 78 were asymptomatic mutation carriers. Mean age at diagnosis in women with either or both neoplasms was 46.7±11.2 years, and that of the asymptomatic group - 45.8±9.4 years, a statistically insignificant difference. The AR CAG repeat ranged from 8-28 in all tested women. Mean number of AR CAG repeat was not statistically different between affected (18.3±2.4) and asymptomatic mutation carriers (18.6±2.1). AR CAG repeat among patients with early onset (<42 years) breast cancer was significantly shorter (17.5±2.3) compared with asymptomatic individuals (18.6±2.1) (p<0.01), and the shorter allele - the younger the age at diagnosis. This study does not provide conclusive evidence of association between AR CAG repeat size and breast or ovarian cancer risk. However, a small effect of a short AR CAG allele size on breast cancer penetrance at early age was noted.

Association of 5382insC Mutation with SNPs of BRCA1 Gene and the Mutation Frequency in Russia

A. N. Loginova¹, N. I. Pospekhova¹, L. N. Lubchenko², E. V. Khomich¹, I. V. Kuzmina¹, A. V. Budilov³, V. M. Zakharyev³, E. K. Ginter¹, R. F. Garkavtseva², A. V. Karpukhin¹;
¹Research Centre For Medical Genetics, Moscow, RUSSIAN FEDERATION, ²Cancer Research Centre, Moscow, RUSSIAN FEDERATION, ³Engelgardt Institute of Molecular Biology, Moscow, RUSSIAN FEDERATION.

A high predominance of 5382insC in BRCA1 gene mutation spectrum (80% of all mutations) of patients with familial breast/ovarian cancer and a set of 11 SNPs on an extent of the gene were found. This set of SNPs in strong linkage disequilibrium and consensus sequence defined two most frequent haplotypes (named B and A). The haplotype frequencies in cancer patients and in control individuals were not different significantly. However, the haplotype A to the haplotype B ratio in genomes with the 5382insC mutation was approximately three times higher than these haplotypes ratio in the population (P < 0.04). The same difference between patients under 5382insC mutation and control group was observed for genotype frequencies (P < 0.04) with odds ratio equal 3.3 in favor of AA among patients. The reason for observed frequency difference may be the mutation - haplotype A linkage. But it is interesting that a ratio of genotype AA and AB frequencies under the mutation is different in patients and control individuals. This may suggest on operation of other factors in addition to linkage. It should be noted that the haplotypes A and B frequencies in Russian and West-European patients with 5382insC were the same (P = 0.40).
The frequency of 5382insC mutation revealed in Russia is highest among investigated populations. The proportion of this mutation is next highest in East-European countries and common in West-Europe. These data jointly with the same SNP haplotypes found in Russia and West-Europe are suggestive on East-European origin of 5382insC mutation.

BRCA2 mutations and polymorphisms in Russian patients with familial breast/ovarian cancer

E. V. Khomich¹, N. I. Pospekhova¹, L. N. Lubchenko², A. N. Loginova¹, I. V. Kuzmina¹, A. V. Budilov³, V. M. Zakharyev³, E. K. Ginter¹, R. F. Garkavtseva², A. V. Karpukhin¹;
¹Research Centre for Medical Genetics, Moscow, RUSSIAN FEDERATION, ²Cancer Research Centre, Moscow, RUSSIAN FEDERATION, ³Engelgardt Institute of Molecular Biology, Moscow, RUSSIAN FEDERATION.

BRCA1 mutations were found in 40% of a sample of breast/ovarian cancer families in Russia. In present study BRCA2 gene sequence variations among cancer families of the rest part of the sample were investigated.
There were only 11% of the probands with deleterious BRCA2 mutations. Two deleterious mutations - 2001del4 and 4816insG - are new. Missence mutations of unclear significance were revealed in 19% of the cases. One of that (N1808K) and two single nucleotide polymorphisms (SNPs) are revealed for the first time. The gene variant S384F that was thought to be unclear significance mutation is evidently polymorphism because was found in common with deleterious mutation of BRCA2 gene. SNPs of 12 types on an extent of BRCA2 gene were found. Six of those were in coding regions of the gene. A variant N372H that known as confers an increased breast cancer risk under HH homozygote, in 12% of the cases was homozygosis on HH with allele frequency equals 0.31. At the same time, a variant IVS11+80del4 with the similar allele frequency was not found as homozygote. It is interesting that we found a frequency of 203G/A polymorphism significantly higher in comparison with results in BIC data base, although the frequencies of other frequent SNPs were not so different. BRCA2 SNPs of control group are under investigation at present.

Prevalence of BRCA1 gene 5382insC mutation in St.Petersburg patients with familial breast cancer.

N. A. Grudinina¹, E. P. Lamber²;
¹Insitute for Experimental Medicine, Saint Petersburg, RUSSIAN FEDERATION, ²Institute for Experimental Medicine, Saint Petersburg, RUSSIAN FEDERATION.

The BRCA1 gene mutation are the common cause of familial breast cancer. The risk of breast cancer development in women with germline BRCA1 gene mutations approaches 90% during life span. We have created the DNA collection from St. Petersburg familial breast cancer patients and demonstrated nearly the same frequency of BRCA1 gene 5382insC mutation in both Slavic and Ashkenazi Jewish patients with familial breast cancer. 5382insC mutation of the BRCA1 gene was found in 1 Ashkenazi Jewish family with familial breast cancer out of 9 studied and in 3 Slavic families with familial breast cancer out of 20 studied. 5382insC mutation was found neither in Ashkenazi and Slavic patients with sporadic breast cancer, nor in control group, that consists of 50 Slavic and 50 healthy Ashkenazi patients unselected in respect of familial breast cancer. Previously 5382insC mutation of the BRCA1 gene was reported in number of familial ovary cancer patients from Moscow and thus 5382insC mutation of the BRCA1 gene may be the common cause of familial breast and ovary cancer in whole Russia. However, the mutation spectra specificity from other countries in BRCA1 gene is expected in Russian population and some new BRCA1 gene mutations are in process of characterization now. The elucidation of BRCA1 gene mutation spectra in St. Petersburg familial breast cancer will help to provide genetic counseling in breast cancer families and improve treatment patients with high risk of breast cancer in the future. The current research was supported in part by RFBR grant 01-04-49627.

Altered expression of the candidate tumor suppressor gene, WWOX, in human breast tumors

K. Driouch¹, H. Prydz², R. Lidereau¹, E. Frengen²;
¹INSERM E0017/Oncogénétique, Centre René Huguenin, F-92211 St-Cloud, FRANCE, ²Biotechnology Centre of Oslo, University of Oslo, N-0316 Oslo, NORWAY.

The presence of putative tumor-suppressor genes on chromosome 16q23.2-24.1 has been suggested by LOH analysis in breast cancer as well as other cancer types. This region overlaps with the fragile site FRA16D and the region of homozygous deletions found in various cancers. We have previously constructed a 1.2 Mb contig map and used this resource to assign transcripts to the LOH region. This resulted in the identification of the WWOX/FOR gene.
The mouse homologue of the WWOX protein has been defined as an apoptogenic protein and an essential partner of p53 in cell death. Thus WWOX is a strong candidate tumor-suppressor gene. We have performed an expression study of the WWOX gene in a series of human breast tumors and breast cancer cell lines, and detected altered WWOX expression at high frequency in cancer cells. Furthermore, identification of two distinct alternative WWOX transcripts expressed at high levels in human tumors suggests an involvement of the WWOX gene in cancer progression. We have initiated functional studies of WWOX in human cells in order to characterize the role of the WWOX protein in normal as well as cancerous cells.
This work was supported by the Research Council of Norway, the Norwegian Cancer Society, the Ligue Nationale de Lutte Contre le Cancer (LNCC), and the Association pour la Recherche sur le Cancer (ARC).

Investigation of APC mutations of a patient with FAP and her family members by heterodublex analyses

B. Tunca¹, M. Menigatti², P. Benatti², G. Cecener¹, M. Pedroni², A. Scarselli², F. Borghi², E. Sala², T. Yýlmazlar³, A. Zorluoglu³, U. Egeli¹, O. Yerci⁴, M. Ponz de Leon²;
¹University of Uludag, Medical Faculty, Department of Medical Biology and Genetics, Bursa, TURKEY, ²Dipartimento di Medicina Interna, Universita di Modena, Modena, ITALY, ³University of Uludag, Medical Faculty, Department of General Surgery, Bursa, TURKEY, ⁴University of Uludag, Medical Faculty, Department of Pathology, Bursa, TURKEY.

Familial adenomatous polyposis coli (FAP) is an autosomal dominant disease characterised by the presence of 100 or more colorectal adenomatous polyps. Mutations in the adenomatous polyposis gene (APC) gene primarily responsible for the development of this disease.
In this study, we examined one patient with FAP and 21 family members including one effected person from FAP and 20 nonsemptomatic persons. Our proband case who have a retinal lesions (congenital hypertrophy of the retinal pigment epithelium, called CHRPE) and hundreds adenomatous polyps on all colon and rectum is a 36 years old woman. We isolated DNA from pheripheral blood samples of proband and her family members by proteinaz K incubation and phenol-chloroform extraction. We studied E,D, F, and G segments of exon 15 of APC gene by heterodublex analyses (HDA). For staining, we used non-radioactive silver staining method. We determined mutation in 5 person from this family in segment F of exon 15 of APC. Two of them were patients with FAP (one is ourproband case) and another three persons were non semptomatic family members. Result of sequencing analysis of these cases, we determined T deletion at position 3554 causing a frameshift mutation in APC gene.

Involvement of APC/beta-catenin signalling and E-cadherin in sporadic colon cancer

T. Cacev¹, R. Spaventi², K. Pavelic¹, S. Kapitanovic¹;
¹Rudjer Boskovic Institute, Zagreb, CROATIA, ²Pliva d.d., Zagreb, CROATIA.

Activation of APC/beta-catenin signalling pathway by mutation in the APC or beta-catenin gene contributes to colorectal carcinogenesis. E-cadherin is involved in control of intercellular adhesion and acts as an invasion supressor. We examined 60 cases of human sporadic colon cancer and corresponding normal tissue samples to evaluate the loss of heterozygosity (LOH) and presence of mutations at the APC, beta-catenin and E-cadherin gene loci.
DNAs were used for PCR, RFLP, VNTR and LOH analysis. To analyze LOH at the APC gene loci we used three RFLP intragenic markers (exon 11 RsaI, exon 15 MspI, and exon 15 AspHI). The presence of the mutations in the amplicon 15H of the APC gene, and APC gene mutation in codon 1309 were analyzed as well. To analyze mutations in the beta-catenin gene we amplified exon 3 and the intronic sequences flanking it from tumor DNAs. For the LOH analysis of E-cadherin gene locus we used D16S752 polymorphic marker.
The informativity for all three APC intragenic markers was 53.3 % (32 of 60 assayed), and 25 % of tumors (8 of 32 informative) demonstrated LOH. We found two APC gene mutations in our tumor samples: a deletion in codon 1309, and an insertion in the amplicon 15H of the APC gene. In 3.3 % of tumor samples (2 of 60 tested) the mutation of the beta-catenin gene was found. The informativity of D16S752 E-cadherin gene polymorphic marker was 75% (45 of 60 tested) and 28.8 % of tumors (13 of 45 informative) demonstrated LOH.

Genetic analysis of APC gene and the diagnostics of familial adenomatous polyposis in pediatrics

H. Kapitanovic Vidak, T. Cacev, K. Pavelic, S. Kapitanovic;
Rudjer Boskovic Institute, Zagreb, CROATIA.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease. Patients with FAP develop hundreds to thousands of adenomatous polyps in the colon and rectum during their second or third decades and one or more of them can progress to cancer. Children of affected individuals are at 50% risk of inheriting the disease. Because FAP patients have a very high risk of colorectal cancer, identification of the individual risk in family members is important to prevent cancer deaths. For these at risk members of the family, annual endoscopy is recommended. The method of providing such accurate presymptomatic diagnosis is to determine whether a family member has inherited the particular germ-line mutation of the adenomatous polyposis coli (APC) gene carried by the affected parent.
Genomic DNAs were isolated from peripheral blood of patients and their relatives. Polymerase chain reaction (PCR) was performed using specific pairs of primers. PCR products were analyzed by electrophoresis on a Spreadex EL 300 gels.
The genetic analysis confirmed the APC gene codon 1309 germ-line mutation in two children not yet having colorectal adenomas, but having inherited APC gene mutation from their mother who died from colon carcinoma. APC gene mutation analysis also confirmed the diagnosis of FAP in one child having colorectal adenomas as a first case of FAP in that family.
We use APC gene mutations analysis in presymptomatic diagnostics but also to confirm the diagnosis of FAP. Children confirmed as a gene mutation carriers can be early included in surveillance program and treatment.

M. Kohoutová¹, J. Stekrová¹, V. Jirásek², J. Kotlas¹, V. Kebrdlová¹;
¹Institute of Biology and Medical Genetics of the First Faculty of Medicine and General Teaching Hospital, Charles University, Prague, CZECH REPUBLIC, ²1st Medical Department of the First Faculty of Medicine and General Teaching Hospital, Charles University, Prague, CZECH REPUBLIC.

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disease characterised by the development of hundreds to thousands of colorectal adenomatous polyps and by the progression to carcinomas. Causative germline mutations have been described in the adenomatous polyposis coli (APC) gene. In the present study the entire APC coding region has been screened for germline mutations in 45 unrelated Czech FAP families. Using PCR, DGGE analysis and DNA sequencing we found 30 mutations, twelve of which were found to be novel: seven frameshift mutations (exon 9 and 15), two nonsense mutations (exon 9 and 11) and three splicing mutations (intron 11 and 14). In previously reported mutations we identified ten frameshift mutations in exon 15 and six nonsense mutations (exon 5, 9 and 15). The common 5 bp deletions at codons 1309 and 1061 were identified in five families (16,6%) and substitution 2805C>A was found in three cases (10%). Identified mutations result in the classical form of FAP except the 1 bp deletion at codon 409 (exon 9), which caused the attenuated form of FAP. In addition, one missense mutation was revealed in exon 3 although missense mutations are rarely observed in FAP. It remains to be determined whether this substitution represents the disease-causing mutation. Two samesense variations were detected in exon 15. The presence of one of them correlates with disease occurrence in the affected family. Thus the clinical significance of this mutation cannot be excluded.
Supported by the grant project MS CR:CEZ:J13/98:111100004.

Denaturing High-Performance Liquid Chromatographie (DHPLC) in Screening for Mutations in Exon 15 of the APC (Adenomatous Polyposis Coli) Gene

W. Heinritz, A. Kujat, S. Strenge, K. Peisker, U. G. Froster;
University of Leipzig, Department of Human Genetics, Leipzig, GERMANY.

FAP (OMIM: *175100, McKusick 1986) is a rare form of hereditary colorectal cancer. Germline mutations of the APC gene were reported in patients with Familial Adenomatous Polyposis (FAP). Inactivation of the APC gene plays a significant role in the development of early onset colon cancer based on polyposis of the colorectum. The location of germline mutations in the APC gene appears to correlate with the clinical phenotype (number of colorectal adenomas, concomitants like occurence of further adenomas in other digestive organs, desmoid tumors and Congenital Hypertrophy of the Retinal Pigmental Epithel [CHRPE]). To provide a fast mutation screening we analyzed the region of the APC gene where more than 40% of the mutations in FAP are described (exon 15-4 to 15-8). We established DHPLC (Denaturing High Performance Liquid Chromatography) mutation analysis followed by automated sequencing of suspicious fragments. We investigated 9 patients with a clinical diagnosis of FAP. Three sequence variations could be identified: 1 polymorphism and 2 mutations (3597del2A at codon 1199 with termination of protein translation at amino acid position 1206; 3949GàC at codon 1317, E1317Q). We describe the optimized conditions for DHPLC for this gene. According to our results DHPLC is an efficient and fast screening method to identify mutations in the APC gene which can be applied to the other exons of the APC gene for a fast and cost reducing mutation screening. The rapid mutation screening will optimize further diagnostic and therapeutic strategies in families with hereditary colon cancer.

S. Kapitanovic¹, T. Cacev¹, K. Pavelic¹, R. Spaventi¹^,2;
¹Rudjer Boskovic Institute, Zagreb, CROATIA, ²PLIVA d.d., Zagreb, CROATIA.

Colorectal carcinomas are characterized by multiple genetic alterations that occur during tumorigenesis. Several tumor suppressor genes associated with colorectal carcinoma have been identified: MCC and APC on chromosome 5q, p53 on chromosome 17p, nm23-H1 on chromosome 17q, and DCC and DPC4 on chromosome 18q. We examined 60 cases of human sporadic colon cancer and corresponding normal tissue samples to evaluate the loss of heterozygosity (LOH) at the NF1 gene loci. The purpose of this study was also to evaluate whether the LOH at the NF1 gene is associated with clinicopathological characteristics in sporadic colon cancer.
DNAs were used for PCR, RFLP, VNTR, and LOH analysis. PCR was performed using specific pairs of primers. PCR products were analyzed by RFLP analysis, and VNTR analysis. To analyze LOH at the NF1 gene loci we used three polymorphic markers: one RFLP marker (exon 5 RsaI) and two VNTR markers (IVS27AAAT2.1 and IVS38GT53.0).
Using these three polymorphic markers 50 (83.3%) patients were found heterozygous and informative for LOH analysis. DNA from 9 (18%) tumors exhibited LOH at the NF1 locus. The majority NF1 gene LOH was observed in Dukes' A (56%), in the well differentiated tumors (43%), and in the tumors that were smaller than 5cm (67%).
Conclusion: Our results support the view that malignant progression is a consequence of more than one genetic change and suggest that inactivation of NF1 gene plays a role in a multistep process of colon tumor progression as an early event.

Complete characterization of the colon cancer cell line HT29 clone 19A by multicolor banding (MCB)

A. Kuechler¹^,2, A. Weise¹, S. Michel¹, B. Pool-Zobel³, A. Schaeferhenrich³, A. Heller¹, H. Starke¹, T. G. Wendt², U. Claussen¹, T. Liehr¹;
¹Institute of Human Genetics and Anthropology, Jena, GERMANY, ²Department of Radiotherapy, Jena, GERMANY, ³Department of Nutritional Toxicology, Institute of Nutrition, Jena, GERMANY.

The human colorectal adenocarcinoma cell line HT29 subclone 19A was recently characterized by M-FISH (Eur J Hum Genet 2001, Vol 9/S1, p138, P0193) and the following composite karyotype was established:
64~69,XX,+del(Xp),+1,+der(1)t(1;11;16),+2,+der(2)t(1;2),+der(3)ins(3;12),+der(4)t(2;4),+5,+del(5q),+7,+7,-8,+dup(8),+del(9),+der(9)t(6;9;X;9),+10,+11,+11,+del(11p),+del(11q),+12,-13,-13,+i(13q),+i(13q),+der(13)t(5;i(13q)),+15,+16,+17,+del(18),-19,+del(19),+der(19)t(5;19),+der(19)t(17;19),+20,+20,+22,+22[cp10]. Using M-FISH, it was possible to identify the chromosomes involved in aberrations, but not to define their exact breakpoints. In order to further clarify the translocation breakpoints and to characterize possible iso-chromosomes, multicolor banding (MCB) was applied at the 400 band level according to Mrasek et al. (Cytogenet Cell Genet 93:242-248). MCB-analyses were performed on all aberrant chromosomes of this composite karyotype, i.e. on the following eighteen chromosomes: #1, #2, #3, #4, #5, #6, #8, #9, #11, #12, #13, #16, #17, #18, #19, #20, #22 and X. Where necessary for exact definition of rearrangements, MCB-probes were combined with centromere specific and whole chromosome painting probes. The resulting karyotype is as follows: 64~69,XX,+del(X)(p11.2-->qter),+del(1)(p35-->qter),+2,+der(2)t(1;2)(1q32-->1qter;2pter-->2q11),-3,+i(3)(q10),+der(3)ins(3;12)(3pter-->3p12::12p12::3p12-->3qter),+der(4)t(2;4)(2q35-->2qter;4pter-->4q11),+5,+del(5)(pter-->q11.2),+dic(6;9)t(6;9;X;9)(6pter-->6q10;9q10-->9q21;Xp21.1-->Xp11.3;9q21-->9qter),+7,+7,-8,+dup(8)(qter-->q10::q10-->q24::hsr::q24-->qter),+10,+11,+del(11)(p13-->qter),+der(11)t(11;?)(11pter-->11q24;?),+12,-13,-13,+i(13q),+i(13q),+der(13)t(5;13)(13qter-->13q10::13q10-->13q21::5q31-->5qter),+15,+del(16)(pter-->q13),+der(17)t(19;17)(19pter-->19p11;17p11-->17qter),+i(18p),-19,+der(19)t(5;19)(5pter-->5p11;19q10-->19qter),+20,+20,+22,+der(22)t(17;22;17) [cp10].
In summary, the MCB-technique was suitable to define all translocation breakpoints apart from one (i.e. der(22)t(17;22;17) which consists of only very little chromosomal material). Thus, MCB is a very useful tool for detailed analyses of chromosomal rearrangements.
Supported by DFG (PO284/6-1), Wilhelm Sander-Stiftung (99.105.1) and EU (ICA2-CT-2000-10012 and QLRT-1999-31590).

Optimization of DHPLC experimental conditions for mutation analysis of the hereditary non polyposis colon cancer (HNPCC) genes hMLH1 and hMSH2

M. Schmitt¹, M. Gribba², J. M. Limacher³, S. Olschwang⁴, J. L. Mandel²;
¹Laboratoire de diagnostic génétique, Strasbourg, FRANCE, ²IGBMC, Illkirch, FRANCE, ³Service d'Oncologie, Hopital Civil, Strasbourg, FRANCE, ⁴CEPH Diagnostics - Laboratoire de Génétique Médicale, Paris, FRANCE.

Denaturating high-performance liquid chromatography (DHPLC) is an efficient method for the detection of point mutations in disease-related genes.
Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary non polyposis colon cancer (HNPCC), hMLH1 and hMSH2 represent the major cause of hNPCC.
We have recently applied the DHPLC mutation detection to the 16 exons of hMSH2 and 19 exons of hMLH1 genes. To test sensitivity and reproducibility of DHPLC, we have first determined the best DHPLC conditions on the wild type sequences, followed by the study of 35 sequence variants previously found by sequencing DNA samples of HNPCC patients. All of the 35 mutations were detected using DHPLC (sensitivity 100%).
We then used DHPLC to analyse 18 patients with colorectal cancer not fulfilling all the Amsterdam criteria. We have found two mutations : Y43C in exon 1 of hMSH2 (unpublished yet) affecting a highly conserved residue and a 790+1G to A in intron 9 of hMLH1 (previously described), one of them did not fulfill the Amsterdam criteria. This low mutation yield could be due to the patients inclusion criteria. We have also found many polymorphisms, with a much higher frequency than previously published.
In conclusion, DHPLC is a rather rapid and inexpensive technology that may be used to screen for mutations colorectal cancer patients where HNPCC may be suspected but who do not fulfill stricter criteria.

Unusual Findings Of APC Gene Analyses In suspected FAP Cases From The Republic Of Macedonia

A. J. Dimovski¹, A. M. Stefanovska¹, T. Josifovski², M. Panovski², D. Jashar³, G. Zografski³, G. D. Efremov¹;
¹Macedonian Academy of Sciences and Arts, RCGEB, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, ²Clinic for Abdominal Surgery, Faculty of Medicine, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, ³Institute of Oncology, Faculty of Medicine, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA.

AIM: Characterization of the molecular basis of FAP in Macedonia.
SUBJECTS: Patients with multiple adenomatous polyposis of the large intestine confirmed by histopathological evaluation.
METHODS: DGGE of exons 1-14, PTT and heteroduplex analysis of exon 15, RT-PCR of exons 1-15, sequencing of the 5' end, and Southern blot analysis of the APC gene.
RESULTS: Six unrelated cases (one female and five males) with multiple polyposis of the colon were enrolled in this study. Of the six patients, only one had a positive familial history. In the female patient the number of polyps was much lower (<100) than the number observed in the male subjects (>1,000). Detailed DNA analyses of the APC gene revealed the presence of rare (unusual) defects in two patients. A large deletion, removing the entire APC gene, was found in the patient with a positive familial history. A somatic mosaicism for a deletion removing exons 2-14 was detected in the female patient. No abnormalities at the DNA level and no allelic imbalance in the expression profile of the APC gene were detected in the other four patients. Abnormal APC gene transcripts were found in the peripheral blood of two of these (deletion of exons 9-13 and exon 14, respectively) that could not be explained by any defect at the DNA level.
CONCLUSION: The unusual findings of our study indicate that there are genes other than the APC which might influence the development of multiple colorectal adenomas, either through an APC related mechanism or through other pathways.

Linkage mapping of FAP disease modifier locus in a large family with a known APC mutation

M. Plasilova, Z. Dobbie, K. Heinimann, H. Müller;
Human Genetics, Division of Medical Genetics, University Clinics Basel, SWITZERLAND.

Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal cancer predisposition syndrome caused by germline mutations within the adenomatous polyposis coli (APC) gene. Mouse model studies and broad phenotypic variability observed both within and among affected families indicate, that in FAP disease expression also other genetic and environmental factors must play an important role. Their identification would substantially improve possibilities of genetic counseling of FAP patients by enabling the precise prediction of the disease severity. A large FAP kindred which has been previously reported by our group and harbours an adenine deletion at codon 1982 of the APC gene represents an ideal model for studying FAP modifiers. Though carrying the same mutation, the affected subjects (45) present with variable colonic and extracolonic manifestations which are in several branches transmitted through the generations. Performed simulation studies revealed a high potential of this pedigree to detect a modifier locus. Here, the results of linkage analysis of 20 candidate regions and eventually results on the genome wide screening for a modifier gene in FAP condition will be presented.

Monozygotic twins showing variable expression of Muir-Torre syndrome due to MSH2 mutation.

S. R. Forrester¹, C. Baker², V. Kimonis³, M. Schneider¹;
¹Southern Illinois University School of Medicine, Department of Pediatrics, Division of Genetics and Metabolism, Springfield, IL, ²Southern Illinois University School of Medicine, Department of Internal Medicine, Division of Dermatology, Springfield, IL, ³3 Harvard Medical School, Division of Genetics and Metabolism, Boston, MA.

Muir-Torre syndrome (MTS) is an autosomal dominant genodermatosis characterized by skin tumors associated with visceral malignancies. MTS shares many clinical similarities with Hereditary Nonpolyposis Colorectal Cancer (HNPCC), and germ-line mutations in DNA mismatch repair (MMR) genes have also been found in MTS families. We present monozygotic sisters with MTS caused by a point mutation (IVS5+3A>T) in the 3’ splice site of exon 5 of MSH2 resulting in the deletion of this exon from the mRNA, thus encoding a truncated protein. One sister developed her first squamos cell carcinoma at 50 years, and at age 53 had two sebaceous carcinomas and one adenoma removed. The second sister had uterine cancer at age 40, metastatic colon cancer at 43, thyroid cancer at 45, and sebaceous adenoma at 49 years. Their mother is deceased at the age of 41 from uterine and liver cancer, and the maternal grandfather was diagnosed with colon cancer at 50 years. Kindreds with this same MMR mutation are described in the literature, and the types of cancers represented in these families were quite varied and included colon, uterine, rectal, endometrial, brain, ovarian, ureter, gastric, breast, duodenal, sebaceous, bone, thyroid, and lung cancer. Therefore, genetic counseling must emphasize the inability to establish any correlation between the site of the individual mutation and spectrum of tumor types and the use of appropriate surveillance methods. This family demonstrates the intrafamilial variability of carcinogenesis even among monozygotic twins, and suggests that non-genetic factors are important in modifying the expression of this syndrome.

Mutations of N- and K-Ras, p53 and FMS genes in myelodysplastic syndromes in children

B. Jekic¹, V. Bunjevacki¹, M. Kuzmanovic², I. Novakovic¹, L. Lukovic¹;
¹Faculty of Medicine, Belgrade, YUGOSLAVIA, ²Institute for Mother and Child Health, Belgrade, YUGOSLAVIA.

Myelodysplastic syndromes arise from molecular-genetic disorder of myeloid stem cell, characterized by dysfunction of myeloid, monocytic, erythroid and megakaryocytic lineages and high risk of evolution to ALL. Hence, MDS are considered to be preleukemic states and are a model for studying mechanisms of leukemic transformation. Ras, FMS and p53 were found to be the most frequently mutated genes in adults with MDS. We have used PCR-SSCP method and sequencing to examine mutations in these genes in 35 archival bone marrow samples of children with MDS collected in last ten years in Institute for Mother and Child Health. 22 DNA samples were successfully amplified with primers for N-Ras (exons 1 and 2), K-Ras (exons 1 and 2) and FMS (region including codon 969) genes and 11 with primers for p53 gene (exons 5, 6, 7, 8 and 9). One of analyzed samples harbored mutation in first exon, and two in second exon of N-Ras gene. In two samples were detected mutations in second exon of K-Ras gene. We have not detected mutations in analyzed regions of neither p53 nor FMS genes. These findings may suggest that mutations of Ras genes play important role in the development of MDS in children.

The relationship of chromosomes 7,9,10,17 aneuploidies and p53 gene alterations between the low and high grade astrocytomas, using interphase FISH technique

U. Egeli¹, T. Yakut¹, A. Bekar², M. Doygun², E. Ogul³;
¹Department of Medical Biology and Genetics, Faculty of Medicine, University of Uludag, Bursa, TURKEY, ²Department of Neurosurgery, Faculty of Medicine, University of Uludag, Bursa, TURKEY, ³Department of Neurology, Faculty of Medicine, University of Uludag, Bursa, TURKEY.

In the present study, we examined chromosomes 7, 9, 10 and 17 aneuploidies and p53 gene alteration on surgical fresh tissue samples of 29 different grades of astrocytomas by using flourescence in situ hybridization (FISH). Eleven of these astrocytomas were low grade (5 pilocytic and 6 grade II) and eighteen astrocytomas were high grade (6 anaplastic and 12 gioblastoma multiforme). All samples were classified according to the WHO classification of tumours of the central nervous system and none of the patients received preoperative chemotherapy or radiotherapy. The results showed that 2 of 11 low-grade and in 6 of 18 high-grade tumours had trisomy 7. One of 11 low-grade and one of 18 high-grades had monosomy 9. Three of 11 low-grade and in 5 of 18 high-grade tumours had monosomy 10. One of 11 low-grade and 2 of 18 high-grade tumours had monosomy 17. Three of 11 low-grade and 6 of 18 high-grade astrocytomas had deletion of p53 gene, although there were not monosomy 17. Based on these findings, chromosomes 9 and 17 aneuploidies are not exclusive to low and high grade astrocytomas. However we identified monosomy 10 and p53 deletions similary rates between low and high grade astrocytomas, gain of chromosome 7 was identified in high-grade astrocytomas nearly two times more than low grade ones. Thus, we suggest that loss of chromosome 10 and p53 gene abnormalities to be earlier event than gain of chromosome 7 for carcinogenesis of astrocytomas.

J. Mares¹, M. Babjuk², M. Trkova¹, J. Duskova³, V. Soukup², P. Goetz¹, Z. Sedlacek¹;
¹Institute of Biology and Clinical Genetics, 2nd Medical School, Charles University, Prague, CZECH REPUBLIC, ²Urology Clinic, 1st Medical School, Charles University, Prague, CZECH REPUBLIC, ³Institute of Pathological Anatomy, 1st Medical School, Charles University, Prague, CZECH REPUBLIC.

Transitional cell carcinoma belongs to a very heterogenous group of neoplasms. Prognosis of a patient at the time of diagnosis is a basic problem of the bladder cancer therapy and has encouraged the search for prognostic markers. The role and possible oncogenic activity of the PAX genes have been discussed recently. A candidate gene PAX5 is situated on the 9p21-23, the region of the most frequent genetic changes in bladder cancer, as well as another signal transduction gene SHB. Concerning the prognostic potential of p53 mutations, recent research yielded contradictory results. The aim of the study was to define new combinations of prognostic markers reflecting biological behaviour of individual tumours in order to identify patients at risk for tumour progression. We investigated 44 patients with superficial bladder cancer and 20 controls for p53 mutations in exons 5-9 and adjacent intronic sequences by the SSCP and direct genomic sequencing. One mutation (del 128 Pro) was detected among the 44 tumours (2.3 %). 36 patients overexpressed at least one of the PAX5 or SHB genes, 26 of them overexpressed both genes. The staging and grading of these pacients were generally higher then of those without increased expression. The correlation of PAX5 and SHB expression and clinical and histopathological data was evaluated. In conclusion, our results indicate that combination of expression data might be used as a diagnostic tool in superficial bladder cancer. Supported by the grant IGA MZ NC/5961-3.

Loss of heterozygosity of p53 gene in gastic carcinoma in the region of easten Turkey

I. Pirim¹, A. Karaman², M. Ikbal¹;
¹Ataturk Universty, Erzurum, TURKEY, ²State Hospital, Erzurum, TURKEY.

Loss of heterozygosity effecting various chromosomes has been characterized on tumor of many human cancers. Tumor suppressor gene p53 was found to be primer target for that losses. In our study, we examined 41 patients with gastric neoplasm for loss of heterozygozity effecting the p53 gene by using PCR/RFLP technique. The samples were run on to agarose gel and visualized on UV. Cancerous lesion of wet tissues from 25 of 41 patients was taken together with their peripheral blood samples. 6 patients was inoperable, so only blood was taken and paraffin tissue of 10 patients were examined for allelic losses. The PCR was carried out by using two sets of primers, both amplified 72. codon of exon 4 of p53 gene. The primer called G-H gave amplified fragment of 66 bp and the other I-J gave 247bp fragment. These fragment were subjected to restriction enzyme BstUI for detecting LOH. 18 out of 41 patients exhibited heterozygosity loss 43,6 % and many of these LOH positive cases had lenf metastasis. We could not determined any relation between p53 LOH positivity and sex or age. Finally, it has been shown that LOH in p53 gene are common in gastric cancer and play important role for cancer progression.

P. Goetz¹, M. Trkova¹, L. Foretova², V. Krutilkova³, R. Kodet¹, J. Mares¹, Z. Sedlacek¹;
¹Charles University, Prague, CZECH REPUBLIC, ²Masaryk Memorial Cancer Institute, Brno, CZECH REPUBLIC, ³University Hospital Motol, Prague, CZECH REPUBLIC.

A strong genetic determination is observed in about 5% of all cancer cases. These patients belong to families with high cancer incidence and/or suffer from early onset tumours or multiple or multifocal malignancies. Many cases of hereditary predisposition to cancer are due to a germline mutation in one of the tumour suppressor genes. Some familial cancer syndromes show predisposition to a particular type of cancer (e.g. breast or colon cancer). The much rarer Li-Fraumeni syndrome (LFS) is distinct because members of LFS families suffer from a wide spectrum of different malignancies including sarcomas, brain tumours, breast cancer, adrenocortical carcinomas and other tumours. The cancer predisposition in most of these families is due to a germline mutation in the TP53 gene. It is generally expected that the tumours in most of such predisposed persons arise after the wild type TP53 allele is lost in a particular cell clone in a carrier individual. We show on our material that many tumours from germline TP53 mutation carriers retain the wild type TP53 allele. The development of these tumours in LFS individuals must therefore be based on another mechanism of the TP53 gene silencing than simple DNA loss. Alternatively, one functional TP53 allele may still be present in these tumours. We also compare these observations with records in our web database of published germline TP53 mutations, which is a very useful tool for different analyses of this intriguing syndrome. Supported by grant IGA MZ CR NC/6513-3.

Gene expression following tet-regulated reexpression of wt p53 in lung cancer cells

I. Wieland¹, A. Brüning², H. Burtscher³, U. H. Weidle³;
¹Otto-von-Guericke University, Magdeburg, GERMANY, ²Institute for Cell Biology (Cancer Research), University Essen Medical School, Essen, GERMANY, ³Roche Diagnostics GmbH, Penzberg, GERMANY.

The tumor suppressor p53 is inactivated in a wide range of human tumors. In non-small cell lung carcinoma (NSCLC) cell line NCI-H358 p53 and p16INK4a/p15INK4b are deficient while Rb is expressed. This condition occurs frequently in native human NSCLC and, therefore, appears to be particularly suited for studying the effects of reexpression of wild-type (wt) p53 in lung cancer cells. We generated the wt p53 inducible NSCLC line H358B22 using a tetracycline/doxycline-regulated (tet-on) expression system. High-level reexpression of wt p53 suppressed proliferation of H358B22 cells completely. Most growth arrested cells stayed viable over a period of 1 week. Therefore, p53 appears to function mainly as an inducer of cell cycle arrest rather than as an inducer of apoptosis in these cells. This growth inhibitory effect of wt p53 is reversible after 24 h of p53 induction, but it becomes irreversible after 48 h of wt p53 induction followed by resilencing of the exogenous wt p53. Therefore, genes regulated in growth arrested H358B22 cells were investigated by microarrays (Affymetrix), RT-PCR and Western blotting. Most differences in gene expression were reversible upon resilencing of exogenous wt p53. However, in irreversibly arrested H358B22 cells a subset of genes including Bax, Fas, p27KIP1, p21WAF1, B-myb, cyclin A and IGF-BP3 escaped reversibility.

GSTM1 null, GSTT1 null, GSTP1 (Ile105Val) and CYPA1 (T6235C) Genotypes in Childhood Acute Leukemia

G. Balta¹, E. Ozyurek¹, U. Ertem², G. Hicsonmez¹, C. Altay¹, A. Gurgey¹;
¹Hacettepe University, Pediatric Hematology Unit, Ankara, TURKEY, ²Sami Ulus Children's Hospital, Ankara, TURKEY.

The purpose of the present study is to elucidate the role of GSTM1 null, GSTT1 null, GSTP1 (Ile105Val) and CYPA1 (T6235C) polymorphisms in the etiology of childhood acute leukemia. The study showed that: A) Frequencies of the genotypes were almost identical in 145 ALL patients and 186 healthy controls. Differences in the frequencies were not statistically significant in all genotypes (>p 0.2). The frequency of GSTM1 and GSTT1 double null genotype was lower in ALL (9.9%) than controls (13%). In ALL patients: 1- No statistically significant differences were found in the frequencies of genotypes between patients belonging to B cell (73) and non B cell lineage (41), yet the frequency of GSTT1 genotype was lower in the group non-B cell (17%) than B cell and control (23%). 2- No differences were found in frequencies of the genotypes between male and female patients. 3- There was no differences in distribution of the genotypes among age groups, except frequency of the GSTT1 genotype was lower in patients 10-17 years (17%) than 0-2, 2-9 years (23%) and control. 4- The frequency of CYPA1 polymorphism was statistically significant in group of patients with WBC count 10.000-50.000 at presentation (58%) than <10.000 (20%), >50.000 (21%) (p 0.01) and control (29%). Frequency of GSTT1 genotype was lower in patients with >50.000 (10%) than others and control (23%). B) Statistically significant association was found in the frequencies of GSTT1 genotype between AML patients (3.4%) and control (23%) (p 0.016) while no association was observed for other genotypes.

Increased accuracy of leukemia diagnosis by combined analysis of morphology and FISH using the Duet automatic cell scanning system.

C. Kaplinski¹, I. Hardan¹, M. Daniely², A. Toren¹, A. Shimoni¹, A. Avigdor¹, M. Reichart², A. Nagler¹, T. Kaplan², F. Brok-Simoni¹, G. Rechavi¹, N. Amariglio¹, L. Trakhtenbrot¹;
¹Dept. of Pediatric Hemato-Oncology and Inst. of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer, ISRAEL, ²BioView Ltd., Rehovot, ISRAEL.

Fluorescence in situ hybridization (FISH) is a valuable tool in clinical practice of leukemia. However, high false positive and false negative rates complicate the interpretation of results. These limitations are especially important in follow-up examinations, and in detection of minimal residual disease (MRD). Recently, the Duet scanning system (BioView Ltd, Rehovot, Israel) was introduced. The system provides two important features: Automatic scanning of large number of cells, and combined analysis of morphology and FISH on the same cells. Prior to scanning, blood samples are processed according to a unique protocol, which allows removal of RBC and 2 consecutive staining of WBC (giemsa or immunocytochemistry and FISH). This approach was applied to 80 samples of various hematological malignancies in order to: a) Determine the lineage of cells carrying specific chromosomal rearrangement. b) Enhance FISH analysis accuracy in leukemic cells. c) Determine clonality and maturity of residual recipient/donor cells in bone marrow transplantation. d) Determine the maturity of cells carrying chromosomal rearrangements in MRD. The results were compared to the diagnosis given by routine methods. We found that the Duet system enabled increased specificity and sensitivity of leukemic cells detection. Scanning automatically large numbers of cells provided rapid and efficient identification of rare cells in MRD cases (up to one leukemic cells in 15,000 WBC). The combined morphologic and FISH information of suspected cells enhanced the specificity of leukemic cells detection and reduced FP drawbacks. These preliminary results indicate the advantage of using such approach in diagnosis of leukemic diseases.

M. D. Tischkowitz¹, N. V. Morgan¹, C. Eddy¹, S. Ball², S. E. Langabeer³, I. Vorechovsky⁴, R. Stoeger¹, D. Grimwade¹^,3, C. G. Mathew¹, S. V. Hodgson¹;
¹GKT School of Medicine, London, UNITED KINGDOM, ²Department of Haematology, St George's Hospital, London, UNITED KINGDOM, ³Department of Haematology, University College, London, UNITED KINGDOM, ⁴Department of Bioscience, Karolinska Institute, Stockholm, SWEDEN.

Fanconi Anaemia (FA) is an autosomal recessive disorder characterised by congenital abnormalities, defective haemopoesis and a greatly increased risk of Acute Myeloid Leukaemia (AML). We are investigating whether mutations in the FA genes might predispose to the development of sporadic AML. Quantitative fluorescent PCR was used to screen archival DNA from peripheral blood or bone marrow from AML cases for deletions in the cloned FA genes, FANCA, C, D2, E, F, G. Of the 103 samples successfully screened for the FANCA gene, four heterozygous deletions were found (see table). Sequence analysis of the other allele in these four cases did not locate a second mutation. A sodium bisulphite conversion assay was developed to detect methylation of the FANCA CpG island. There was no evidence of allele inactivation by hypermethylation in these 4 samples, nor in a further 28 non-deleted samples. No deletions were found on screening the FANCC, D2, E, F and G genes in 64, 68, 31, 42 and 51 samples respectively. FANCA is a large gene (43 exons, 80kb) with a high incidence of deletion mutations in affected FA patients. These results show that such deletions may also be found in sporadic AML and may have contributed to leukaemogenesis.

Characteristics of FANCA deletion samples (*= deletions endpoints undefined)
Sample	Type	Cytogenetics	FANCA Deletion
1	Male 65 yrs FAB M2	43,XY,del(5)(q15q3?3),-6,-7, r(7),i(8)q),add(16)(q?24),-17,-22, +der(?)t(?;6)(?;?p11)	heterozygous ex5-43*
2	Female 45 yrs FAB M1	44.XX,add(2)(q2),add(5)(q?),-7, +?10, -12, add(16)(q2), -18, -20, +mar[4]	heterozygous ex19-21
3	Male 56 yrs FAB M6	44,XY,del(1)(q21q25),add(4)(q? 25),-5,-7,-11,-12,del(12)(q21q24),add(13)(q13), add(16)	heterozygous 11-21
4	Female 69 yrs	N/A	heterozygous ex 5-43*

H. Bruchova, R. Brdicka;
Institute of Hematology and Blood Transfusion, Department of Molecular Genetics, Prague 2, CZECH REPUBLIC.

Array hybridization technique represents a useful method for the expression profiling of large gene sets during disease processes. Using this technology we studied gene expression in childhood acute lymphoblastic leukemia (ALL) patients. For detection of transcription activity we used Human Cancer cDNA Nylon Arrays (Clontech, USA) with 588 genes that can be involved in transformation. Total RNA was isolated from bone marrow leukocytes of patients at the time of diagnosis (previously untreated). The standard sample was prepared by RNA mixing of control individuals (bone marrow donors). Our objectives were to identify genes that were differentially expressed in ALL and might contribute to the disease development (and characterization).
Obtained gene expression profiles of patients were compared with the standard profile. The majority of patient genes showed the similar gene activity as in the control sample (e.g. gluthatione-S-transferase homolog, vimentin, rho-GAP hematopoietic protein C1, rho GDP dissociation inhibitor 2, fau etc.). Many genes displayed significant expression changes only in some patients. In a few genes it was possible to observe similar significant changes of gene expression in most patients ( eg. PCNA, MMP8). These changes might be associated with common stream of the disease process and they can be studied in more detail. Supported by the grant IGA MZ CR no. NM/5901-3.

The relationship between the chromosomal rearrangement complexity and disease agresivity in some cases of leukemia

A. G. Lungeanu¹, A. Arghir², A. Lupu³, D. Mut-Popescu⁴, N. Berbec⁴, L. Popescu⁵;
¹National Institute, Bucharest, ROMANIA, ²"Victor Babes" Institute, Bucharest, ROMANIA, ³"Carol Davila"University, Bucharest, ROMANIA, ⁴"Carol Davila" University, Bucharest, ROMANIA, ⁵"Carol Davila" University, "Victor Babes" Institute, Bucharest, ROMANIA.

Among over 100 patients with myeloid and lymphoid leukemias investigated cytogeneticaly during the last 15 months, in four cases, disease evolution was determined by the complexity and nature of chromosomal abnormalities identified at the first presentation. First case, a 22 years old man with L3type ALL, exhibited: del 3q26;del 5p13; t(8;14)(q24;q13);del 9p11q11 and inv 15p12qter in all cells from bone marrow. He died after four months. The second case, a woman of 62 years old with acute leukemia weak-differentiated, refractory to treatment, showed 48-54 chromosomes and 3-4 markers derived from chromosomes 5 and 12. She died in the next three weekes. The third case, a young man of 27 years old, with acute myeloid leukemia, apart of Ph chromosome presented del11q21 and del16q22. The rapid death of the three cases was a powerful prove of positive correlation between the complexity of chromosomal changes and disease agresivity. In change, a constitutional translocation t(3;5)(q26;q21) identified in a 72 years old woman with ET, conferred favourable evolution of the disease after a succesfull treatment with HU. So, we appreciate that, if in the first three cases of myeloid and lymphoid leukemias could be a direct relationship between the complexity of genomic rearrangements identified at the onset and agresive development of the disease, in the fourth case of ET, constitutional translocation t(3;5), seems to be not involved in the etiology of the disease.
Acknowledgements: VIASAN project 089, ANSTI 5195/1999-2001, and Schering-Plough Central East Ag.

D. Szczesniak¹, J. Kocki¹, M. Cioch², A. Dmoszynska², B. Marzec¹, J. Wojcierowski¹;
¹Department of Medical Genetics, Medical Academy, Lublin, POLAND, ²Department of Hematology, Medical Academy, Lublin, POLAND.

The negative regulators of cell cycle like cyclin dependent kinases inhibitors genes, Rb family genes and p53 gene play important role as inhibitors of cell proliferation. Incorrect expression of these genes may cause disturbances in cell machinery, uncontrolled cell division and consequently malignant transformation.In our research we examined the level of cell cycle negative regulators genes expression on mRNA level in bone marrow samples in patients with acute leukemia before treatment.For detection of mRNA we used the Multi Probe RNase protection Assay System (RiboQuant). We analyzed the expression of cyclin dependent kinases genes (p16 and p21 family), Rb family genes and p53 gene.Obtained results show significantly high level of p53, p27, p19 and p18 mRNA, while the level of Rb and p16 mRNA is very low in the examined cells.The correlation of the results with the level of other cell cycle regulators expressions and clinical data may give us important information about new prognostic factors in hematological malignancies.

Submicroscopic deletion at the breakpoint in chromosome der(9) in Ph+ acute lymphoblastic leukemia (ALL)

I. F. Loncarevic-Barcena¹, B. Shell¹, H. J. Fricke², M. Prechtel¹, M. Ziegler¹, U. Claussen¹;
¹Humangenetik und Anthropologie, Jena, GERMANY, ²Klinik für Innere Medizin II, Jena, GERMANY.

Submicroscopic deletions in the breakpoint region of chromosome der(9)t(9;22) are found in ~25% of patients with chronic myeloid leukemia (CML). Notably, these deletions are strongly associated with a shorter life expectancy when non transplanted CML patients are compared. We present molecular and clinical data of a 22 year old male patient with a t(9;22) positive B-cell specific acute lymphoblastic leukemia (B-ALL) that exhibit a deletion in chromosome der(9). In ALL this deletion was first and uniquely detected so far in one of 48 ALL patients investigated by Reid et al (abstract: 1334, ASH meeting 2001). The rare occurrence of this deletion in ALL makes it difficult to evaluate the clinical impact. The deletion we found is located proximal to the breakpoint in der(9)t(9;22). RT-PCR detected a b3a2 BCR-ABL and failed to detect an ABL-BCR transcript. No response to therapy was achieved with a high dose protocol (Hölzer Studie 5/93). The patient was subjected to salvage therapy with Idarubicin/AraC and reached a partial remission with 20-25% BCR-ABL positive cells at day 125 after initial therapy. At present, STI571 is administered and a stem cell donor is searched. The data confirm that deletions in der(9) can also be found in Ph+ ALL. The clinical significance of this rare deletion in ALL is unknown and has to be evaluated by increasing the study cohort.

Acute monocytic leukemia and multiple abnormalities in a child with duplication of 1q detected by GTG-banding and SKY.

M. R. Baruffi¹, C. A. Scrideli², J. A. Squire³, J. Karaskowa³, E. S. Ramos⁴, B. Heck⁴, L. G. Tone⁴;
¹Ribeirao Preto Medice School, University of Sao Paulo, Ribeirao Preto, SP, BRAZIL, ²Ribeirao Preto Medicine School, University of Sao Paulo, Ribeirão Preto, SP, BRAZIL, ³University of Toronto, Toronto, ON, CANADA, ⁴Ribeirao Preto Medicine School, University of Sao Paulo, Ribeirao Preto, SP, BRAZIL.

Patients with 1q duplication have demonstrated a wide range of multiple congenital abnormalities, but a clinical delineation of a trisomy 1q “syndrome” was proposed. Alterations involving this same chromosomal region have also being described in various hematopoietic malignant disorders and a series of candidate genes that may be associated with neoplasia have been described in this region. We describe a female girl with low birth weight, microcephaly, mid facial hipoplasia, synophris, short palpebral fissures, epicanthic fold, beak-like nose, narrow palate, teeth abnormalities, cardiac defect, syndactily, and motor delay, that presented, at 18 months of age, an acute monocytic leukemia (FAB-M5) according to cytological, histochemical and immunophenotyping features. The patient failed to achieve remission, and died 2 months after diagnosis. Cytogenetic study of the bone marrow cells by GTG-banding showed: 44~48,XX,-X[9],dup(1)(q23q44)[35],+2[27],-6[13],+7[4],-8[3],-9[7],+9[2],+10[3],+11[13], +12[5],-14[5],-15[4],-17[7],-18[13],-19[3],+21[5],-22[5],+22[2],+mar[5]cp[35].Spectral karyotyping (SKY) was also performed to identify the aberrations 46,XX,der(1)dup(1)(q23q44)t(1;1)(p36;q32),der(6)t(6;8)(p25;q13),+11,der(11)t(11;18)(q10;q10).Peripheral blood cytogenetic analysis was not performed due to repeated blood products transfusions and the precocious patient death. The dismorphological features with the dup(1q) founded in all bone marrow cells analyzed suggest that this is probably a constitutional chromosome alteration and the first, in our knowledge, association of a trisomy 1q"syndrome" with AML.
Supported by: FAEPA, FAPESP, CCS, NCIC

A. Carrió¹, D. Costa¹, A. Arias¹, R. Queralt¹, F. Cervantes², J. Aguilar³, D. Colomer³, E. Campo³;
¹Servei de Genètica. CDB. Hospital Clínic, Barcelona, SPAIN, ²Servei d'Hematologia. Hospital Clínic, Barcelona, SPAIN, ³Unitat d'Hematopatologia. CDB. Hospital Clínic, Barcelona, SPAIN.

A 90-95% of patients diagnosed with Chronic Myeloid Leukemia (CML) show the Philadelphia chromosome (Ph) as a result of the t(9;22)(q34;q11). About 5-10% of CML show the variant forms: simple (22q11qter is translocated into a chromosome other than 9) and complex (three or more chromosomes are involved).
We present cytogenetic, fluorescence in situ hybridization (FISH), and molecular analyses in three cases of the complex variant.
The chromosome bands involved were 11q13 (two cases) and 1p36.1. The FISH analyses (locus specific, centromeric and whole chromosome painting) showed that the bcr/abl fusion gene was in chromosome 22 in all three cases, suggesting a complex variant rather than a clonal evolution.

Characterisation of Acute Myeloid Leukemias (AML) with complex aberrant karyotype using gene expression analysis and mutation screening of candidate genes

S. Mergenthaler, C. Schoch, S. Schnittger, A. Kohlmann, W. Kern, M. Dugas, M. Klaus, J. Christodoulou, W. Hiddemann, T. Haferlach;
Labor für Leukämiediagnostik, Klinikum Grosshadern, Munich, GERMANY.

AML represent a pathogenetically and prognostically heterogeneous group. For differentiation of AML-subgroups, cytogenetics offers the most evaluated and established criteria today. 10-15% of AML-patients show complex aberrant karyotypes in leukemic blasts, associated with a most adverse progression of the disease. So far, no crucial candidate genes relevant for the pathogenesis were identified.
Data obtained by 24color-FISH and CGH in 50 AML-cases with complex aberrant karyotype demonstrated a much larger percentage of loss than gain of genetic material. Frequent observations included deletions of the entire chromosomes 5 and 7, as well as interstitial deletions within their long arms.
These deletions may represent the first of two required mutation events to deplete a tumorsuppressor gene’s function. Alternatively, haploinsufficiency with only one mutation event might already be sufficient to reduce the physiologically necessary amount of gene product.
To evaluate both models, we currently investigate 25 of these AML-cases with complex aberrant karyotype more precisely on molecular genetic level: utilizing gene expression analysis (GeneChip U133) and mutation screening (Single Strand Conformation Polymorphism Analysis, SSCPA) we focus on 20 functionally relevant candidate genes on chromosomes 5 and 7, involved in apoptosis, cell cycle/transcription regulation and DNA repair mechanisms.
So far, SSCPA in 25 AML-patients and 10 healthy controls using different conditions excluded the General Transciption Factor GTF2H2 (5q12.2-q13.3), involved in transcription/transcription-mediated DNA-repair, as a major candidate gene in complex AML-pathogenesis.
Our future investigations aim at providing a deeper insight into basic pathogenetic mechanisms of complex AML with subsequent implementation in prognosis and therapy strategy.

M. Alkan¹, C. Ulger¹, G. A. Toruner¹, M. Muhammed², S. Damani², P. Tolias¹^,3, M. Schwalb¹, J. Dermody¹;
¹Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, ²Spectral Genomics, Houston, TX, ³Center for Applied Genomics, Public Health Research Institute, Newark, NJ.

Comparative genomic hybridization (CGH) is a method, used to detect, and map DNA sequence copy number differences between two genomes in a single experiment. Although it is a valuable technique, the lower resolution of CGH compared to molecular genetic techniques have limited its application. The utilization of microarray technology in CGH analysis has the potential to meet these challenges. For this purpose, a well-characterized promyelocytic cell line HL-60 was analyzed by using Human BAC Array- 3MB system, which was developed by Spectral Genomics^TM. The glass array is composed of 1003 non-overlapping BAC library clones which encompass the human genome with a resolution of 3 Megabases. In HL-60, amplification of the region of 8q24, an extra copy of chromosome 18, and deletions in loci of 5q11.2-q31, 6q12, 8p23, 9p21.3-p22, 10p12-15, 14q22-q31, 16q21, and 17p12-17p13.3 were detected. These results are largely concordant with the previously reported aberrations, and indicate that this system might be an alternative to conventional CGH analysis.

High-throughput tissue microarray analysis of 11q13 genes amplification (CCND1, FGF3/FGF4, FGF3, EMS1) in urinary bladder cancer

B. Zaharieva¹, R. Simon², T. Gasser³, G. Sauter², D. Toncheva¹;
¹Department of Medical Genetics, Medical Faculty Sofia, Sofia, BULGARIA, ²Institute of Pathology, University of Basel, Basel, SWITZERLAND, ³Urologic Clinics, Cantonal Hospital Liestal, Liestal, SWITZERLAND.

Gene amplification is a common mechanism for oncogene overexpression. High-level amplifications at 11q13 had been repeatedly found in bladder cancer by comparative genomic hybridization and by other techniques. Putitative candidate oncogenes located in this region are CCND1, EMS1, FGF3 and FGF4. To evaluate the involvement of these genes in bladder cancer, we screened a tissue microarray (TMA) containing 2317 samples by FISH. Among all tumors with 11q13 amplifications (13.3%) 68.3% had all 4 genes amplified, 19.5% had amplified CCND1, FGF4 and FGF3 together, 0.8% had FGF4, FGF3 and EMS1 coamplified. Single amplification of CCND1 was found in 9% of the tumors, while 1,6% had single amplification of EMS1 and 0.8% - of FGF4 suggesting that CCND1 is the major target gene in the 11q13 amplicon in bladder cancer. The frequency of both gains and amplifications of all genes increased significantly from stage pTa to pT1-4 and from low to high grade tumors. Increased copy number changes of all 4 genes were associated with survival of patients with tumors from all stages and progression of pT1 tumors and were not associated with recurrence of pTa tumors and survival of patients with pT2-4 tumors. Tumors with gains of FGF3, FGF3/FGF4 and EMS1 were shown to behave like tumors with amplifications rather than like normal tumors contrary to CCND1 where gained tumors behaved like CCND1 normal rather than like CCND1 amplified tumors.

Dystrophic Scoliosis And Genetic Polymorphisms In Patients With Neurofibromatosis

S. Funke¹, E. Morava², M. Czakó³, B. Cser⁴, G. Kosztolányi⁴, T. Illés⁵;
¹Dep. Medical Genetics and Dep. of Obstetrics and Gynecology, University of Pécs, Pécs, HUNGARY, ²University of Pécs, Pécs, HUNGARY, ³MTA-PTE Clinical Genetic Research Group, University of Pécs, Pécs, HUNGARY, ⁴Department of Medical Genetics and Child Development, University of Pécs, Pécs, HUNGARY, ⁵Department of Orthopedics, University of Pécs, Pécs, HUNGARY.

The dystrophic form of scoliosis in neurofibromatosis 1 (NF1) is often associated with severe decrease in bone mineral density, significantly hindering reconstructive bone surgery. Osteoporosis has been found to be associated with distinct polymorphisms of the vitamin D receptor gene (VDR), oestrogen receptor gene (OER) and the collagen 1a1 gene (COL1A1) in the general population. The purpose of the present case-control study was to evaluate the hypothesis, whether the genotypes at these three polymorphic loci are associated with decreased bone mineral density in scoliotic patients with neurofibromatosis. Genotype of 21 selected NF1 patients with scoliosis and decreased bone mineral density was compared to 21 patients with non-scoliotic NF1 with normal bone density. Patients with idiopathic scoliosis with normal bone density measurements (21) have been also assessed for the same genetic polymorphisms. In this pilot study of altogether 63 individuals no association was found between VDR and OER polymorphisms, and the phenotype. The genetic marker distribution in the idiopathic scoliosis (IS) group did not significantly differ from that of scoliotic NF1 patients. However, we observed a threefold prevalence of the homozygous polymorphism (CC) over the heterozygous form (Cc) of the COL1A1 gene in non-scoliotic NF1 patients compared to patients with scoliosis presenting with an almost equal distribution in this genotype. This difference was not statistically significant. The sample size of this pilot study is not large enough to draw a final conclusion. A possible protective role of CC genotype in non-scoliotic NF patients deserves reevaluation in a larger group of patients.

E. Leisti;
Oulu University Hospital, Department of Radiology, Oulu, FINLAND.

In a population-based study on neuroradiological imaging of individuals with NF1, 10 of 124 studied patients (8%) presented with intracranial tumours other than optic gliomas or T2 hyperintense lesions. Six patients aged 6 to 53 years had an astrocytoma, one patient had a suspected astrocytoma which proved histologically to be normal brain tissue, one patient had a small lipoma in the interpeduncular cistern, one patient had a hypophyseal adenoma and one patient presented with an enhancing lesion of the cavernous sinus. - The astrocytomas showed wide variation in their behaviour and MR imaging. Four of the tumours were progressive, one histologically confirmed astrocytoma disappeared spontaneously and one astrocytoma appeared within a previously detected T2 hyperintense lesion, which was partly located in the region of the optic radiation and had remained stable for several years. The results indicate that the fate of an astrocytoma in NF1 cannot be predicted on the basis of one imaging only, but that the patients need close follow-up.

Do some additional chromosome rearrangements mean a favourable T-cell prolymphocytic leukemia prognosis with preserved alkylator based treatment sensitivity?

N. Kokalj Vokac¹, S. Zver², A. Erjavec¹, B. Zagradisnik¹, A. Zagorac¹, D. Zontar², P. Cernelc²;
¹Maribor Teaching Hospital, Maribor, SLOVENIA, ²Univ.Clinical Center Ljubljana, Ljubljana, SLOVENIA.

A 77-year-old woman came to haematological department because of leucocytosis, (WBC 276x109/L; Hgb 111 g/L, Plt 239x109/L), sweating and weight loss. Bone marrow biopsy revealed 80% lymphoid-cell infiltration. Morphologically cells appeared as prolymphocytes and immunohistochemically they were CD3, CD4, CD5 positive, while being CD8, CD20, CD30, CD43, CD56, TIA-1 and Granzyme B negative. Flow-cytometrically performed T-lymphoid immunological markers CD2, CD3, CD4, CD5 and CD7 were highly, more than 90% positive. Diagnosis of T-cell prolymphocytic leukemia (T-PLL) was made and the later was confirmed also by cytogenetic analysis. T-PLL specific chromosome rearrangements were observed: inv(14)(q11q32), i(8)(q10) and del(11)(q22q23) involving ATM gene. Complex translocation of X chromosome, probably involving MTCP1 gene, was present on der(X)t(X;3)(q28;p25)t(X;16)(p14;q12). There were several other chromosome rearrangements observed, including chromosomes 5, 6, 13, 14, 17, 20 and 22. Classical cytogenetic analysis was confirmed by FISH, using Cytocell Octochrome Multiprobe System, and some locus specific DNA probes.
T-PL leukemia is aggressive, and refractory to alkylator-based therapy, with a median survival of 7 months. Treatment options are highly immunosupressive 2-CDA, Pentostatin or Campath 1-H. Because of the patient's advanced age, we have started with chlorambucil 10 mg/m2 for 5 consecutive days, repeatingly every 4 weeks. After 4 cycles of chlorambucil the patient was without any clinical symptoms, with WBC 23,4x109/L, Hgb 128g/L, Plt 256x109/L. To our knowledge, the additional chromosomal rearrangements: der(6)t(X;6)(p14;q25), der(13)t(13;14)(q22;q11), t(5;13)(q34;p11), r(17)(p13q21), t(17;20)(q21;q13), 22p+ , were not yet described in the literature. Some of them may means favourable T-PL prognosis because of preserved alkylator agent treatment sensitivity.

Prognostic Significance Of Small Cell Clones With Hyperdiploidy In Childhood Acute Lymphoblastic Leukemia (all).

Z. Zemanova¹, K. Michalová¹^,2, L. Sindelarova¹, J. Brezinova², S. Kurkova², P. Smisek³, O. Hrusak⁴, J. Stary³;
¹Center of Oncocytogenetics, General Faculty Hospital and 1st Medical Faculty of Charles University, Prague, CZECH REPUBLIC, ²Institute of Haematology and Blood Transfusion, Prague, CZECH REPUBLIC, ³2nd Department of Pediatrics, Faculty Hospital Motol, Prague, CZECH REPUBLIC, ⁴Institute of Immunology, 2nd Medical Faculty of Charles University, Prague, CZECH REPUBLIC.

Children with ALL and chromosome hyperdiploidy in bone marrow cells have a better prognosis in contrast to those with other cytogenetic abnormalities. Therefore early detection of hyperdiploid cell clones is important and can lead to appropriate less aggressive therapy protocol with the lower risk of the late side effect. For the assessment of hyperdiploidy we use consecutive double target interphase FISH (I-FISH) with combination of alpha-satellite and/or locus-specific probes for 10 chromosomes most frequently overrepresented in hyperdiploid clones (200 interphase nuclei analysed per slide and probe-mix, cut-off level 2.5% tested on controls, standard deviation not exceed 0.5%). I-FISH is quick and sensitive screening method which enables to find even "hidden" small pathological clones. Structural and/or complex chromosomal aberrations in hyperdiploid cells were analysed by multi-color FISH (mFISH).
During the last four years we examined prospectively or retrospectively 88 children with ALL (56 boys, 32 girls; mean age 8 years). Various level of hyperdiploidy was found in 60 children (68%). The extent of pathological clones being 2.5 - 100%. Small clones under 10% were detected in 18 patients (20,5%). Structural or complex chromosomal rearrangements together with hyperdiploidy were found in 23 patients (26%). We compare FISH and cytogenetic findings, results of DNA analysis by flow cytometry and clinical course of the disease in all patients with a particular respect to the prognostic significance of small pathological clones and complex chromosomal rearrangements.
This work was supported by grants IGA MZ CR NE 6472-3, GACR 301/01/0200 and IGA MZ CR 6406-3.

Cytogenetic studies in T-cell acute lymphoblastic leukemia: a report of 35 cases.

N. Douet-Guilbert¹, M. J. Le Bris², A. Herry², F. Morel², G. Le Calvez³, V. Marion³, J. F. Abgrall³, C. Berthou¹, M. De Braekeleer²;
¹Service d'Hématologie clinique, CHU, Brest, FRANCE, ²Service de Cytogénétique, Cytologie et Biologie de la Reproduction, UBO & CHU, Brest, FRANCE, ³Service d'Hématologie biologique, CHU, Brest, FRANCE.

A total of 198 patients with acute lymphoblastic leukemia (ALL), including 189 at diagnosis and 9 at relapse, had a cytogenetic analysis on a bone marrow sample between 1981 and 2001. Thirty-five ALL (17.7%) were of the T cell lineage. The immunophenotyping performed on 32 cases showed that 30 were true T-cell ALL, one was mixed T-cell/B-cell and one mixed T-cell/Myeloid. The 35 patients were distributed in 14 children and 21 adults. The quality of the chromosomal preparations was too poor to allow a feasible identification in 2 cases and one culture did not yield metaphases. Karyotyping was sucessfully performed in 32 patients. A normal karyotype was observed in 5 of the 13 pediatric cases (38.5%) and in 5 of the 19 adult cases (26.3%). These values are within the range observed in other series of T cell-ALL. Numerical chromosome abnormalities were rare, 77.3% of the abnormal karyotypes (17/22) being pseudodiploid. Translocations involving band 14q11 were observed in 7 patients whereas band 12p13 was deleted in 2 cases and translocated in a further 3 cases. The short arm of chromosome 11 was involved in 4 translocations, band 11p13 in 2 and band 11p15 in another 2 cases [t(4;11) and t(1;4;11)]. Other recurring structural rearrangements include del(6q) in 3 cases and del(5q) in 2. Most of these recurrent abnormalities are different from those of B-lineage ALL. Some are known to involve T cell receptor genes whereas others can lead to the discovery of new genes that are important to T-lineage leukemogenesis.

Detection of Philadelphia Chromosome in Chronic Myelogenous and Acute Lymphoblastic Leukemia in two locations in Ecuador.

J. C. Ruiz-Cabezas;
Hospital Dr. Juan Tanca Marengo SOLCA, Guayaquil, ECUADOR.

A previous study sustained that there may be a difference in the presence in Philadephia (Ph) Chromosome t(9;22) in the studied ecuadorian series due to the ethnical content and geographical location (Quito, 2800m) of the studied human group.
We present here data that supports that there is no influence of these aspect in the presence of this chromosomal marker in cases of Chronic Myelogenous and Acute Lymphoblastic Leukemias (CML and ALL, respectively) in Ecuador.
The study was performed in two major laboratories in Guayaquil (at the sea level) and Quito (2800 m over the sea level) and included a population with varied ethnical content. The described cases correspond to ALL and CML diagnosed by bone marrow smears and Immunocytochemistry, this are 199 cases of ALL and 295 cases of CML studied in both cities.
The CML presented and average frequency of Ph+ of 85%, and the ALL cases had a frequency of 14%.
No statistical difference in the presence of Ph Chromosome in neither CML or ALL was found in the studied cases at the coast area in relation to the frequencies published for Quito and to the world statistics, although in a previous publication of the molecular analysis of Quito’s cases it was shown the presence of a particular pattern of the abl-bcr rearrangements.

Molecular and cytogenetic changes in STI571 (Gleevec) treated Acute Lymphoblastic Ph + Leukemia

Targeting the tyrosine kines activity of Bcr-Abloncoprotein is effective therapeuitc option of Ph-chromosome positive CML and ALL.
However, accumulating clinical experience describes emerging tumour resistance to STI571 tyrosine kinase inhibitor in the treatment course.
A 36-year old woman with relapsing Philadelphia positive Acute Lymphoblastic leukemia (Ph+ALL) was treated with STI571 (600 mg/d). After 3 months haematological response in terms of correcting leukopenia, reducing the number of immature cells in the bone marrow by 50% and decreasing the need for platenet and haemoglobin transfusion was seen. However, in the sixth month of treatment patient stopped responding to the drug with rapidly incresing number of blasts. At that point standard G-banding of leukaemic cells identifed additional chromosomal changes:del(6q) and t(11;14)(q13;q32). Molecurarly, we were able to detect Major and Minor-breakpoint Bcr gene rearrangements in the fusion with the Abl gene while molecular cytogenetics showed amplification of the Bcr-Abl gene detected as the emergence of an extra Bcr-Abl gene copy in 17%of cells.PCR amplification of the BCL1-IgH fusion gene was negative and there was no BCL1 expression by the blasts (immunocytochemistry). Biological mechanisms of these genetic events are unknown and multiplication of Philadelphia chromosome can be related to the acquired.

A. Amiel, N. Bouaron, R. Sharony, D. Kidron, M. Fejgin;
Meir Hospital, Kfar-Saba, ISRAEL.

Confined placental mosaicism (CPM) in term placental tissues is usually diagnosed by conventional cytogenetic analysis and more recently by fluorescence in situ hybridization (FISH) of the trophoblast. In this study, we describe the use of comparative genomic hybridization (CGH) for detection of chromosomal aneuploidy in 26 fresh and 14 paraffin embedded placentas and evaluate the sensitivity of this novel approach for CPM diagnosis in multiple placental samples.
We applied CGH technique to samples taken from various sites of placentas originating from abnormal pregnancies (23 IUGRs, one with fetal malformation, one with toxemia, one with hydrocephalus and 2 undetectable MSAFP). In the control cases (7 normal and 5 with known aneuploidy) CGH concurred with the known karyotype.
The most common aberration in the IUGR cases was the addition of a whole or part of X chromosome. Other aberrations such as addition of Y chromosome , addition of 13(q22) and loss of chromosome 17 where found in other cases. There was also one IUGR case of trisomy 8 (in one site) and 47,XXY found in all sites. In the two cases with the MSAFP=O monosomy 16 was detected (in one case on both sites searched). Some of the results were confirmed by the FISH technique.
Our results demonstrate the usefulness of CGH technique in the genetic evaluation of fresh and paraffin embedded placentas in problematic pregnancies even when its morphology is normal.

Detection of chromosomal aneuploidy in spontaneous abortions using comparative genomic hybridization (CGH)

N. V. Ostroverkhova, S. A. Nazarenko, I. N. Lebedev, A. D. Cheremnykh;
Institute of Medical Genetics, Tomsk, RUSSIAN FEDERATION.

Chromosomal aneuploidy is a common cause of abnormal prenatal development. Comparative genomic hybridization (CGH) provides a rapid and comprehensive detection chromosomal gains and losses in the test genome and maps the aneuploidies onto normal metaphase chromosomes. Among the 52 tissue culture of spontaneous abortions, 10 cases showed failure of fetal cell growth in culture and could not be identified reliably by conventional cytogenetics. CGH analysis was successfully performed for detection of chromosomal aneuploidy in spontaneously aborted specimens with tissue culture failure. Balanced karyotype profiles were obtained for 5 samples. All of them were analysed by fluorescence in situ hybridization (FISH) with centromere-specific DNA probes to exclude polyploidy. Nothing cells with abnormal level of ploidy were found. Five spontaneous abortions (50%) have monosomy 22 and trisomy 10, 14, 18 and 21. Monosomy 22 identified by CGH is one of the most rare aneuploidies in spontaneous abortions. In all cases with an indication of chromosomal imbalance by CGH, FISH with chromosome-specific DNA probe was performed to confirm the presence of aneuploidy. As determined by FISH analysis two cases with trisomy 10 and monosomy 22 were mosaics with frequency of abnormal cell line 68% and 33% respectively. Advantages and limitations of CGH for a detection of complete and mosaic forms of aneuploidy are discussed.

Marker chromosome identification with chromosome microdissection and reverse FISH and CGH

Y. H. Cho, J. Y. Lee, J. H. Kyhm, H. K. Seo, C. H. Lee;
Department of Medical Genetics, College of Medicine, Hanyang University, Seoul, REPUBLIC OF KOREA.

Reverse painting fluorescent in situ hybridization (FISH) on the normal metaphase with probes generated by chromosome microdissection and comparative genomic hybridization (CGH) are powerful methods to identify the origin of marker chromosomes. Three cases having unidentified marker chromosomes were studied by reverse painting FISH and CGH. Reverse FISH probes were generated from five copies of each marker chromosomes dissected with micromanipulator, amplified with DOP-PCR, and labeled with fluorochromes. The probes were hybridized to normal metaphases and the origin of marker chromosomes could be determined. Three marker chromosomes were identified as derivative chromosome 15 inducing partial trisomy of 15q, duplication of the short arm of chromosome 17 and duplication of the short arm and deletion of the part of the long arm of chromosome X. CGH showed concordant results with reverse FISH.

Reexamination of chromosome 2 rearrangements characterized by multicolor banding (MCB) by region-specific FISH probes

A. Weise, H. Starke, A. Heller, U. Claussen, T. Liehr;
Institute of Human Genetics and Anthropology, Jena, GERMANY.

Conventional banding techniques often fail to characterize the exact nature of chromosomal rearrangements. The MCB technique has demonstrated to improve the definition of chromosomal breakpoints (e.g. Starke et al., 2001, PrenatDiag, 21, 1049-1052, Dufke et al., 2001, Europ J Hum Genet 9, 572-576). Here MCB was applied to identify human chromosome 2 breakpoints in two clinical cases. To show how precise the correlation between the MCB pseudocolors and the GTG banding works the results of MCB were reexamined using region-specific YAC or BAC probes. The chosen band resolution of chromosome 2 was 400 bands per haploid karyotype. Case 1 presented with primary mental retardation and severe delayed speech development. The boy had a der(9)t(2;9)(2q24.2;9p11.2) according to GTG banding. MCB showed, however, that the translocation was balanced although it seemed to be not according to GTG banding; new karyptype: t(2;9)(q24.2;p24.3). Case 2 showed primary mental retardation, tendency to seizures, craniofacial dysmorphisms and adipositas. The type of aberration in this male patient could not be defined by GTG-banding (inv(2)(p11q23)+dup?or.inv(2)(p21q24.1)+del?). MCB could characterize the rearrangement as inv(2)(p15q24.3). In both cases the results of MCB were confirmed with a panel of region specific YAC/BAC probes. In all 20 MCB metaphases analyzed per case the breakpoints appeared within the same pseudo-colored bands. Thus, the highly reproducible MCB pattern, can be used to characterize abnormalities that remain cryptic or unresolvable in G-banding analysis. Supported by DFG (436 RUS 17/40/00; PO284/6-1), Wilhelm Sander-Stiftung (99.105.1), the EU (ICA2-CT-2000-10012 and QLRT-1999-31590). Dr. Rocchi (Bari, Italy) is acknowledged for YAC/BACs.

Identification of satellite sequences in metaphase and interphase with peptide nucleic acid (PNA) probes using multicolor fluorescence in situ hybridization.

K. L. Taneja¹, B. Williams¹, R. H. Singer²;
¹Applied Biosystems, Bedford, MA, ²Albert Einstein College of Medicine, Bronx, NY.

Multiplex fluorescence in situ hybridization (M-FISH) can be used to detect marker chromosomes, chromosomal rearrangements in cancer, prenatal diagnosis etc. Regular M-FISH requires a large amount of labeled DNA, the hybridization time is longer and is less informative in interphase nuclei compared to standard FISH. We have designed and developed directly labeled PNA probes to distinguish up to 2ⁿ-1 chromosomes (where n is the number of different fluorochromes) using an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images. Peptide nucleic acids (PNA) are synthetic mimics of DNA in which the phosphodiester backbone has been replaced with 2-aminoethyl glycine linkages, but maintaining the four natural nucleobases. PNA probes bind to the complementary DNA sequence obeying Watson-Crick base pairing, however the neutral backbone of the PNA molecule allows for the PNA/DNA binding to occur more rapidly and more tightly than DNA/DNA binding. Chromosome specific composite PNA probe sets were generated from the human satellite sequences, in which the different fluorochromes were incorporated to address specific issues, like identification of marker chromosomes and anueploidies. With four fluorophores, we were able to enumerate up to 15 chromosomes in both metaphase spreads and interphase nuclei in a single hybridization experiment. Our data suggests that multiplex fluorescence in situ hybridization (M-FISH) using PNA probes could have wide clinical utility, particularly in detection and enumeration of chromosomes in a given sample. Multi-parameter hybridization analysis should facilitate the study in molecular cytogenetics and probe-based diagnosis of pathogens.

Multicolor fluorescent in situ hybridization in neuronal cells as an approach for identification of low level chromosomal aneuploidy in the brain

Y. B. Yurov¹, V. M. Vostrikov¹, S. G. Vorsanova², V. V. Monachov¹, I. Y. Iourov¹;
¹National Center of Mental Health, Moscow, RUSSIAN FEDERATION, ²Intitute of Pediatrics and Children Surgery, Moscow, RUSSIAN FEDERATION.

Fluorescence in situ hybridization (FISH) of DNA-DNA or DNA-RNA using post-mortem brain samples is an approach to study a low-level chromosomal aneuploidy and selective expression of specific genes in brain of patients with neuropsychiatric diseases. We have performed a pilot molecular-cytogenetic analysis of post-mortem brain of schizophrenic patients. Multicolor FISH on two post-mortem brain samples of normal and six schizophrenic individuals (area 10 of cortex) was applied. A set of DNA probes for FISH included (i) centromeric alphoid DNA probes for chromosomes 7, 8, 13 and 21, 18, X, Y;(ii) classical satellite DNA probes for chromosomes 1 and 16 and (iii) region-specific DNA probes for chromosomes 13, 21 and 22. Statistically significant level of aneuploidy (up to 3-4% of neurons) involving chromosome X and 18 was detected in two post-mortem brains of patients with schizophrenia. The multicolor FISH assay could be applied to study low level of chromosomal aneuploidy, intranuclear distribution and conformation of heterochromatin, abnormal patterns of chromosomal organization and functional gene expression in situ in post-mortem brain at many neurogenetic diseases. Schizophrenia and Rett syndrome are the diseases of special interest for extended molecular-cytogenetic analysis as both of them could suspect alterations in chromatin conformation and differential gene expression in brain cells. Supported by Copernicus 2 grant.

J. M. Lapierre, G. Joly, M. Prieur, O. Raoul, M. C. de Blois, N. Morichon-Delvallez, P. Gosset, M. Vekemans, S. P. Romana, C. Turleau;
Hop.Necker-Enfants Malades, Paris, FRANCE.

Comparative Genomic Hybridization (CGH) is able to identify the origin of extra or missing chromosome material when either the small size of the segment or a non-discriminatory banding pattern does not allow a cytogenetic diagnosis. It has also the potential to detect both terminal and interstitial rearrangements. We illustrate here the contribution of CGH in the delineation of 13 different cases studied in our laboratory. In most cases, CGH was performed to characterize a rearrangement detected using classical cytogenetic methods i.e identification of extra or missing chromosome material, confirmation of an imbalance or accurate definition of the chromosome breakpoints. In all these cases CGH was decisive. In some other cases, classical cytogenetics (550 to 850 bands) did not detect any chromosome imbalance when CGH detected a chromosome imbalance in several cases. All abnormal results were confirmed using whole chromosome painting and/or FISH with subtelomeric probes. In conclusion, CGH is a very powerful method to analyze an unbalanced rearrangement already identified using classical cytogenetics and requiring further characterization . When no chromosomal rearrangement is observed, CGH could be an alternative to multiprobe FISH study of all subtelomeric regions. However its use as a screening tool in unexplained mental retardation remains limited due to the difficulty of obtaining chromosomal preparations allowing high quality hybridizations on a regular basis.

E. Dagan, S. Gil;
University of Haifa - Department of Nursing, Haifa, ISRAEL.

Three predominant mutations in BRCA1/2 genes have been found in 3% of the Jewish Ashkenazi population. Such mutations significantly increase lifetime risk for developing breast and/or ovarian cancer. The present study focuses on the impact of being BRCA1/2 mutation carrier on women’s mental health. A retrospective study was conducted in the oncogenetic clinic at Rambam medical center, Israel. One hundred and thirty eight women were recruited and evaluated regarding their medical history and mental health state using the BSI (The Brief Symptom Inventory; Derogatise 1982). All women were genotyped for BRCA1/2 founder mutations. Of the 138 women, 39 (28%) were mutation carriers. Breast cancer was diagnosed in 69 (50%) women. The mean age at diagnosis was 45.7±10.7 years and at the interview was 50±10.5 years. Univariate analysis of Variance (ANOVA) [Morbidity (with/without breast cancer) X Mutation (carrier/non-carrier)] revealed significant effect for morbidity, mutation, and the interaction on four sub-scales of the BSI and on its total score (GSI). Apparently, asymptomatic mutation carriers expressed the highest levels of somatization (F=30.0; p<.001), depression (F=9.1; p<.01), interpersonal sensitivity (F=4.5; p<.05) hostility (F=14.4; p<.001), and GSI (F=8.9; p<.01). It may be that healthy women who carry a mutation are more stressed regarding their health status than breast cancer mutation carriers.

Familial or sporadic? Unexpected results in the diagnosis of hereditary breast cancers

C. Schiffer, T. Voigtländer, R. Klaes;
Institute of Human Genetics, Heidelberg, GERMANY.

Genetic counseling and risk assessment in families with breast/ovarian cancer is regularly based on pedigree analysis. However, familial and sporadic cases may occur in the same family. We report on three families with unexpected segregation of BRCA1/2 mutations in affected and unaffected family members.
Family No 1: The female proband, who presented with breast cancer at 28 years of age, carried the common T300G mutation. Surprisingly her mother, diagnosed with breast cancer at 33 years of age, was tested negative for this mutation. T300G was identified in the proband´s healthy father.
Family No 2: Three siblings (one man, two women) and their deceased father had been diagnosed with breast cancer. The brother and one sister, diagnosed at 40 years of age, carried the common 2041insA mutation in the BRCA2 gene. The other sister, diagnosed at 60 years of age, tested negative for this mutation.
Family No 3: The female proband presented with breast cancer at 37 years of age. She carried a novel BRCA1-splice mutation(4304+2insAdel21bp). Her mother, diagnosed with breast cancer at 55 years, was tested negative, although she had two affected sisters. The proband`s healthy father however with an unremarkable family history carried the splice mutation. br />We conclude that for exact risk assessment and genetic counseling in the hereditary breast/ovarian cancer syndrome, each affected and unaffected family member at risk should be tested.

J. C. Coyne¹, J. Stopfer², K. Calzone¹;
¹University of Pennsylvania Health System, Philadelphia, PA, ²University of Pennsylvania, Philadelphia, PA.

Study Design: Retrospective follow up study of individuals who notified that they are carriers of a BRCA1/BRCA2 mutation. Participants received and returned by mail an assessment of the pattern of their disclosure of the results of their genetic testing in their family.
Instrumentation: Self-report questionnaire follow up assessment of mutation carriers who have received results.
Most probands (60%) were the first in their family to receive results, and almost half (47.3%) had agreements with family members prior to testing to disclose results. Probands with such agreements were more likely to have family members present during genetic counseling and results disclosure, C2 (1) = 4.0, p < .05. Individuals with such an agreement reported that a sense of obligation, encouragement from their physician and family members, and being asked by family members were stronger determinants of their decision to share results than did probands without a prior agreement (all ps < .001). Probands with such an agreement were more likely to endorse the following factors as facilitating disclosure: support from close family relationships, their physicians’ support, concern that family members be able to use information to make healthcare decisions for themselves and their children, and being asked directly by family members (all p < .05). These data suggest that the family context is a crucial determinant of how genetic testing information is disseminated, and that interventions aimed at improving dissemination of genetic testing information need to focus on agreements to disseminate test results made prior to the receipt of results.

Genetic polymorphisms of biotransformation enzymes and susceptibility to breast cancer

J. Sarmanova¹, S. Susova¹, I. Gut¹, J. Adamek², K. Kubackova², P. Soucek¹;
¹National Institute of Public Health, Prague, CZECH REPUBLIC, ²Faculty Hospital in Motol, Prague, CZECH REPUBLIC.

Breast cancer is the most common malignancy in women and second leading cause of death from cancer. The genetically variable biotransformation enzymes: epoxide hydrolase (EPHX), NADPH-quinone oxidoreductase (NQO1), and glutathione S-transferases (GST's) metabolize drugs, carcinogens, and natural products. In addition, it is generally accepted that majority of human cancers results from exposure to environmental carcinogens. Considering the role in the metabolism of chemicals played by biotransformation enzymes, we aimed at determining whether any association exists between genetic polymorphisms of biotransformation enzymes and individual susceptibility to breast cancer in Czech women.
Genotyping analyses were performed by PCR-RFLP to determine the frequency of polymorphisms in EPHX (exons 3 and 4), GSTM1 (deletion), GSTP1 (exon 5), GSTT1 (deletion) and NQO1 (exon 6). The study population consisted of 169 breast cancer cases and 231 healthy controls.
No association between polymorphisms in EPHX, GSTM1, GSTP1, and GSTT1 and breast cancer was found. On the contrary, a significantly different distribution of genotypes in NQO1 between controls and breast cancer group was confirmed by chi-square test (P=0.003, chi-square=11.83, DF=2). We have observed significantly higher frequency of mutated genotype S/S in patients in comparison with controls (8.1% vs. 1.3%). Homozygous mutant genotype S/S leads to complete lack of activity NQO1. Moreover, the involvement of NQO1 in colorectal cancer and tumor resistance to anticancer drugs was implicated.
Our results suggest that NQO1 may be an important factor in susceptibility to breast cancer and its role should be further investigated.
This study was supported by grant GACR No.: 310/01/1537.

N. D. Rendtorff¹, B. Mogensen², K. Brondum-Nielsen¹, M. Schwartz²;
¹Department of Medical Genetics, The John F. Kennedy Institute, Glostrup, DENMARK, ²Molecular Genetics Laboratory, Department of Clinical Genetics, Rigshospitalet, Copenhagen, DENMARK.

Tuberous Sclerosis Complex (TSC) is an autosomal dominantly inherited disorder characterized by development of benign tumours (hamartomas) in many organs. Hamartoma formation in the central nervous system is associated with some of the most problematic clinical manifestations of TSC, and can lead to intellectual handicap, epilepsy and autism. Inactivating mutations in either of two tumour supressor genes, TSC1 or TSC2, is the cause of this syndrome.
Here we have established a mutational analysis for TSC1 and TSC2. For the 21 coding exons of TSC1, we have developed a mutation identification assay that combines long-range PCR with automated sequencing. For mutation screening of the 41 coding exons of TSC2, we have developed a rapid and efficient denaturing gradient gel electrophoresis (DGGE) assay.
We are currently collecting DNA samples from Danish tuberous sclerosis patients. Presently, we are carrying out mutational analysis on DNA from peripheral blood from 25 Danish TSC patients (15 sporadic and 10 familial cases). Furthermore, Southern blot analyses using TSC1 and TSC2 cDNA probes are also carried out. Sofar, we have identified a total of 11 mutations, 4 of which have been identified previously in another lab and 9 of which are novel mutations. In one patient mosaicism was detected. A number of polymorphisms were also detected.

M. Somer¹, L. M. Messiaen², K. Aittomäki¹;
¹Clinical Genetics Unit, Helsinki University Central Hospital, Helsinki, FINLAND, ²Center Medical Genetics, University Central Hospital, Gent, BELGIUM.

An 11-year-old girl of Albanian origin was diagnosed to have neurofibromatosis 1, but she also had features of the Noonan syndrome including short stature (-4.2 SD), pulmonary valvular stenosis, shield chest, posteriorly rotated earlobes with thick helices, and high palate. The diagnosis of neurofibromatosis type 1 was made on the basis of several cafe-au-lait spots, axillary freckling, and bilateral Lisch noduli of the iris. The MRI studies showed both bright signals in left globus pallidus and hippocampus and a thick medulla oblongata. She had mild to moderate developmental delay.
In molecular genetic studies, a nonsense mutation (R816X) was identified in the NF1 gene. The parents and the 5 siblings had no signs of either neurofibromatosis 1 or the Noonan syndrome, and neither one of the parents carried the mutation.
Previously, Bahuau et al.(1998) found the same mutation in a family segregating both NF1 and Noonan syndrome. They suggested a coincidental occurrence of the two conditions, having evidence from their pedigree that both phenotypes co-localize and that another locus for Noonan syndrome resides on 17q in close vicinity of the NF1 gene. They also identified this mutation in 3/184 (1.6%) individuals with only classical NF1. However, the independent finding of the same R816X mutation in an unrelated patient displaying both phenotypes suggests that this particular mutation may bring about the expression of the Noonan phenotype in individuals with NF1.

Application of denaturing high-performance liquid chromatography-based analysis to neurofibromatosis type 1

A. De Luca¹, A. Buccino¹, D. Gianni¹, S. Giustini², A. Richetta², L. Divona², S. Calvieri², R. Mingarelli¹, B. Dallapiccola¹;
¹CSS-Mendel Institute, Rome, ITALY, ²Institute of Clinical Dermatology, University of Rome "La Sapienza", Rome, ITALY.

Neurofibromatosis type 1 (NF1; MIM# 162200) is a common autosomal dominant disorder, characterised by café-au-lait spots, peripheral neurofibromas, Lisch nodules and freckling. The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of NF1 mutations are private and rare, generating high allelic diversity with a restricted number of recurrent mutations. Denaturing high-performance liquid chromatography (DHPLC) has been recently introduced as a rapid and highly sensitive method for detecting sequence alterations, well suited to mutation detection. We have scanned 17 exons of the NF1 gene using DHPLC method in a series of 39 NF1 patients. Five novel mutations (496delGTTT, E725X, G848E, 1148insG, 4481delAG), plus two mutated alleles previously reported (499delTGTT, L847P) have been identified. In addition we detected one silent mutation (G846A), three rare intron changes (730-6)A®C, (1063-28)C®G, (1063-24)delT, and one apparent polymorphism (4368-46)G®C. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.
This work was supported by the Italian Ministry of Health and Associazione Romana Ricerca Dermatologica (ARRD)

Mutation characterization in patients with type I Neurofibromatosis: towards a routine diagnostic test

A. Honsberger, C. DeLozier-Blanchet, M. Morris;
Divison Medical Genetics, Geneva University Medical Faculty, Geneva, SWITZERLAND.

Neurofibromatosis type I, an autosomal dominant condition with an incidence of about 1/3000, results from mutations in the NF1 gene. The variability in clinical expression is striking, with symptoms varying from "cosmetic" to lethal. This variability is apparently not due to locus heterogeneity, since mutations in the NF1 gene are responsible for the quasi-totality of cases. Allelic heterogeneity may explain a proportion, as over 400 mutations have been reported. However, major intrafamilial variation in disease expression also occurs, making the establishment of genotype-phenotype correlations difficult. A reliable molecular diagnostic test is needed to allow earlier diagnosis, carrier detection and clinical follow-up. However, the size of the gene and the diversity of mutations currently makes testing difficult outside of a research context. We have done molecular analysis on 38 NF1 patients using a multi-step DNA and RNA-based protocol, using SSCA of selected exons, RT-PCR and PTT followed by sequencing of variants. This approach has allowed us to define mutations in nearly half (17 of 38) of our patients, with work still ongoing. The mutations defined, 11 of which have not been previously described, include 7 nonsense, 4 splice-site, 4 insertion-deletions and 2 missense mutations.
Attempts will be made to correlate clinical symptoms with specific mutations, as a standard set of clinical information has been collected.
On the basis of these and published results we now propose a routine diagnostic test which compromises acceptable cost and reasonable sensitivity, thus responding to a frequent demand of patients and physicians.

Characterization of 2p aberrations in classical Hodgkin lymphoma by means of FICTION reveals recurrent involvement of the REL and BCL11A loci

J. I. Martin-Subero¹, S. Gesk¹, L. Harder¹, T. Sonoki², P. W. Tucker³, B. Schlegelberger¹, W. Grote¹, F. J. Novo⁴, M. J. Calasanz⁴, M. L. Hansmann⁵, M. J. S. Dyer⁶, R. Siebert¹;
¹Institute of Human Genetics, Kiel, GERMANY, ²Internal Medicine II, Kumamoto University, Kumamoto, JAPAN, ³Institute of Cellular and Molecular Biology, Austin, TX, ⁴University of Navarra, Pamplona, SPAIN, ⁵University of Frankfurt, Frankfurt, GERMANY, ⁶University of Leicester, Leicester, UNITED KINGDOM.

The genetic background of classical Hodgkin lymphoma (cHL) is widely unknown. A common feature of the tumor cells in HL is the constitutive activation of the NF-kB transcription factor. In a subset of cases this might be due to the presence of the LMP1 of the EBV or mutations in the NF-kB inhibitors IkB-alpha or IkB-epsilon. Recent comparative genomic hybridization studies have shown gains in chromosome arm 2p as the most common imbalance in cHL. The minimal region of gain contained two candidate oncogenes, REL and BCL11A. Overexpression of REL due to genetic amplification might contribute to NF-kB activation. The transcriptional repressor BCL11A has been shown to be overexpressed in cHL cell lines and may play a role in B-cell transformation through the BCL6 pathway. Here, we investigated the involvement of REL and BCL11A in 44 primary cases of cHL by combined immunophenotyping and interphase cytogenetics (FICTION technique). A median 2p13 copy number above the tetraploid range was detected in 24 (55%) cases. Adjustment for centromere 2 copy number indicated gains of 2p13 in 11 of 31 cHL (35%) with 8 (26%) high-level amplifications. One case displayed selective amplification of the REL locus not affecting BCL11A, and another case showed signal patterns suggesting a breakpoint in the region spanned by the REL probe. According to these data, REL rather than BCL11A may be the target of the 2p13 alterations in cHL, although a role for BCL11A in cHL cannot be ruled out due to its frequent coamplification.

Gene mutation in the SDHB gene in sporadic pheochromocytoma induces the same functional consequences as SDHD gene mutation in hereditary paraganglioma

A. Gimenez-Roqueplo¹, J. Favier², P. Rustin³, P. Plouin⁴, X. Jeunemaitre¹;
¹Département de Génétique Moléculaire, Paris, FRANCE, ²INSERM U36, Paris, FRANCE, ³INSERM U393, Paris, FRANCE, ⁴Département d'Hypertension Artérielle, HEGP, Paris, FRANCE.

The genetics of neural crest-derived tumors was recently transformed by the discovery of mutations in SDHD, SDHC and SDHB genes. The goal of this study was to better understand the functional consequences of SDHD and SDHB gene mutations.The first patient had a mediastinal pheochromocytoma and was a member of a family with inherited paraganglioma.The second patient had a malignant nonfamilial pheochromocytoma. The search for mutations in the SDHD and SDHB genes was performed by direct sequencing in germ-line and tumoral DNA. LOH was tested with several fluorescent microsatellites of 11q and 1p chromosome regions. The activity of respiratory-chain enzymes was tested by measuring the succinate cytochrome c reductase activities on the tumor homogenates. The influence of the mutations on the hypoxic pathway was tested by in situ hybridization, immunohistochemistry and real-time quantitative RT-PCR.A nonsense mutation of the SDHD gene was detected in the first patient and a missense mutation of the SDHB gene in the second. These two mutations were associated with a LOH in tumors on 11q and 1p chromosomes, respectively. Enzymatic experiments showed a complete and selective loss of complex II electron transfer activity in both the SDHD- and SDHB-inherited pheochromocytomas. Immunohistochemistry, in situ hybridization and quantitative RT-PCR revealed a high level of expression of angiogenic factors EPAS1, VEGF and its receptors in both tumors.Mutation in the SDHB gene induced a dramatic disturbance of mitochondrial and hypoxia pathways similar to those induced by SDHD gene mutation, which might be important to trigger tumorigenesis of pheochromocytomas.

Investigation of a potential role of the putative tumor suppressor gene EXTL1 in neuroblastoma development

D. Mathysen¹, W. Van Hul¹, N. Van Roy², F. Speleman², W. Wuyts¹;
¹Department of Medical Genetics, University of Antwerp, Antwerp, BELGIUM, ²Department of Medical Genetics, University of Ghent, Ghent, BELGIUM.

Neuroblastoma, a frequent pediatric tumor of the sympathetic nervous system, is characterized by a wide variety in outcome, ranging from rapid progression of the disease associated with poor prognosis to a quite unusual and rather high rate of spontaneous tumor regression that correlates with excellent prognosis. It has been shown that molecular abnormalities often recognized in neuroblastomas, such as MYCN-amplification or deletion of the distal part of chromosome 1p are correlated with the outcome of the disease. Two neuroblastoma suppressor loci are thought to be located on chromosome 1p36. Recently, the EXTL1 gene was suggested to be a candidate neuroblastoma suppressor gene because of its chromosomal localization in the 1p36.1 region between the translocation breakpoints observed in the UHG-NP and GI-ME-N neuroblastoma-derived cell lines, and its presumed tumor suppressor capacity. To evaluate this hypothesis, we performed 1p-deletion analysis and mutation screening of the EXTL1 coding region on tumor genomic DNA originating from 25 neuroblastoma-derived cell lines and 30 neuroblastomas. Deletion of a fragment of chromosome 1p, including the EXTL1 locus, has been observed in at least 23 of the 55 investigated neuroblastoma samples. Only one mutation (C83G; Ser28Cys) has been observed in the DNA of neuroblastoma cell line STA-NB3. This combination of deletion and mutation analysis allows us to conclude that the EXTL1 gene does not play a leading role in neuroblastoma etiology, despite the fact that it is often deleted.

Plasma DNA Microsatellite Analysis for Bladder Cancer Follow-up may give False Positive Results.

D. Fornari¹, H. Vibits², J. V. Jepsen², J. D. Olesen¹, M. Schwartz³, K. Steven², T. Horn¹, S. Larsen¹;
¹Department of Pathology, Herlev University Hospital of Copenhagen, Herlev, DENMARK, ²Department of Urology, Herlev University Hospital of Copenhagen, Herlev, DENMARK, ³Department of Clinical Genetics, Rigshospitalet University Hospital of Copenhagen, Copenhagen, DENMARK.

Purpose: Patients with non-invasive bladder cancer develop frequent recurrences, a part of which progresses into invasive disease. Intensive follow-up is therefore required.
We studied the possibility of following-up bladder cancer patients by microsatellite investigation of plasma DNA for LOH (Loss of Heterozygosity) and MSI (Microsatellite Instability).
Materials and methods: Sixteen microsatellite markers were amplified in plasma, leukocyte and, when available, tissue DNA of 40 patients and 20 healthy controls by use of fluorescent primers. The plasma was filtered prior to DNA extraction in order to exclude the presence of cells.
Results:


Classification	Tissue LOH/cases analyzed	Plasma LOH/cases analyzed
Controls	0 % (0/10)	5 % (1/20)
Patients (Ta + T1-4)	68 % (25/37)	25 % (10/40)

Only two patients displayed LOH in both tissue and plasma, however the markers involved were not the same.
The tissue MSI frequency was 0 % in the controls and 5 % in the cancer patients. As for the plasma MSI, in most cases it was enough to reduce the number of PCR cycles in order for it to disappear.
Conclusions: Plasma microsatellite analysis was found to be of little use for bladder cancer follow-up. Attention should be paid to allelic drop-out and overamplification as they may give, respectively, artifact LOH and MSI.

M. T. Akbari, F. Behjati;
Akbari Medical Genetics Laboratory, 98 Taleghani Avenue, Tehran, IRAN (ISLAMIC REPUBLIC OF).

The investigation was carried out on bone marrow and peripheral blood samples of 224 Iranian patients presented or followed up for various types of leukaemia in years 1999 and 2000. The samples were mainly referred from three major haematology-oncology centres at Tehran. The Cell cultureing and bandings were carried out according to standard protocols. Chromosome analysis was performed following ISCN guidelines. The patients were from different leukaemic groups, the major ones being: CML, AML, ALL and MDS. There were more males than females: 133 and 91 respectively with approximately 1.5:1 ratio. In terms of sample types, 210 had BM aspiration, whreas peripheral blood was used only in 14 cases. The common typical chromosome abnormalities as well as rare and combined forms were observed. The overall chromosome abnormality rate obtained was about 50%. The breakdown figures for different categories were as follows: 73% in CML, 41% in AML, 26% in ALL, 30% in MDS and 61% in other types.
Compared to the published data, the observed rate in the present study is considered low to average. The main reason being the patients selection criteria at the initial diagnosis stage.

The role of microsatellite instability in patients with gastric cancer: A case-control study in high-frequent area.

M. Yaghoobi, L. Gholamrezaei, S. Mamaghani, M. Sohrabi, A. Sayyari, F. Imanzadeh, A. Farsar;
Research Unit, Department of Gastroenterology, Mofid Medical Center, Shahid Beheshti Uni. Med. Sci., Tehran, IRAN (ISLAMIC REPUBLIC OF).

Introduction: Microsatellite instability (MSI) in gastrointestinal cancers is a process in developing of gastric cancer. We studied MSI in 8 MS region in gastric cancer tissue of the patients with a history of gastric cancer in their first-degree-relative and healthy family memebers.
Methods: Patients expired of gastric cancer with a positive family history of gastric cancer in the first-degree relative and their healthy first-degree relatives were included in the study. Paraffin-blocked tissues were obtained from pathology department. Biopsies from the intact gastric tissue were also taken from healthy members. DNA was extracted and reserved in 4oC. MSI was checked based on 8 MS markers including BAT25, BAT26, BAT40, D2S123, D5S346, D13S170, D17S250 and TP53.
Results: 20 patients were enrolled in the study. Low-level MSI was detected in 9 (45%) and high-level MSI in 3(15%). 8 patients (40%) was MSS. 20 healthy members were randomly selected and included in the study. Low-level MSI was detected in 4 (20%) and the other cases (85%) were MSS. Intestinal metaplasia was detected in 2 patents with low-level MSI.
Conclusion: This study showed that MSI could be one of the most important screening tests in detecting first-degree relatives of patients with gastric cancer.

U. Chalapathi Rao¹, M. Ravindrababu², U. Ratnamala², B. Solanki³, J. Solanki⁴, U. Radhakrishna²;
¹Green Cross Blood Bank & Genetic Centre, Ahmedabad, INDIA, ²Green Cross blood bank & Genetic Centre, Ahmedabad, INDIA, ³Department of Gynaecology, Civil Hospital, Kheda, INDIA, ⁴Department of Animal Genetics & Breeding, Veterinary college, Gujarat Agriculture University, Anand, INDIA.

Hydatidiform mole (HYDM) (OMIM 231090) is the product of malformed human pregnancy with the incidence ranging from 1/250 to 1/1500 pregnancies depending on ethnic groups. It was classified into two different types. Complete hydatidiform mole (CHM) and partial hydatidiform mole (PHM). CHM is characterized by gross hydropic swelling of almost all of the chorionic villi with loss of intravillous vascularity and resembling bunches of grapes, usually with an absence of a fetus or fetal tissue such as blood or amniotic membrane. PHM is generally accompanied by a fetus, or shows evidence of a previous existence of a fetus by the presence of erythroblasts or fetal membrane. The gene responsible for HYDM have been mapped to chromosome 19q13.3-q13.4 (Hum. Molec. Genet. 8:667-671, 1999; Europ. J. Hum. Genet. 8: 641-644, 2000). We have studied three large Indian pedigrees with an autosomal recessive HYDM. Pedigrees consist of 65 individuals, including 18 affecteds. The Severity of the disease was quite variable among the families. The desease occurred in two or more pregnancies in two or more sisters of the same pedigree. Detailed clinical, ultrasonographic, morphological and histological studies were carried out on 14 affecteds from the three pedigrees. The affected status of remaining individuals was collected from the family records. Karyotype analysis of six affecteds representing the three pedigrees showed no chromosomal anomaly. Linkage studies with markers closely linked to HYDM will either confirm allelism to this locus or provide evidence for genetic heterogeneity.

Polymorphisms C825T in GNB3 gene and Pro/Leu at codon 10 in TGFbeta1 gene and the risk of prostate cancer

T. Hajdinjak, B. Zagradisnik, N. Kokalj-Vokac, K. Kisner;
Maribor Teaching Hospital, Maribor, SLOVENIA.

Essential role for G proteins and TGFbeta in prostate cells signaling, growth and differentiation was recently recognized. Beta 3 subunit is functional part of most G proteins signaling cascades with its polymorphism C825T being functionally significant. TGFbeta1 level was found to be predictor for progression of prostate cancer and polymorphism in codon 10 is associated with variations in tissue TGFbeta1 level.
A group of 86 patients with histologically proven prostate cancer was compared to 200 apparently healthy controls.
C825T polymorphism distribution was 14% TT, 44% CT, 42% CC among patients and 8% TT, 46% CT and 46% CC among controls. The difference for the frequency of homozygosity TT was not significant between the groups (p=0.12, OR 1.87, 95%CI 0.84-4.1).
Proline (C) and leucine (T) distribution among cancer patients was 11% CC, 55% CT, 34% TT and among controls 17% CC, 49% CT, 34% TT. The difference for the frequency of homoygosity CC was not significant between the groups (p=0.25, OR 0.64, 95%CI 0.30-1.37).
The two studied polymorphisms were not found to be significantly correlated with the risk of prostate cancer in Slovenian/Caucasian population, although trend toward increased risk for TT in C825T GNB3 was noted. These findings do not exclude influence of these polymorphisms on progression and prognosis of prostate cancer, which warrants further studies.

Genetic changes of chromosomal region 10q24 in malignant lymphomas: Detection of aberrations affecting the NFKB2/LYT10 gene locus by FISH

S. Gesk¹, C. Kahl², L. Harder², L. French³, M. Earthrowl³, J. I. Martin-Subero², B. Schlegelberger², D. G. Oscier⁴, J. A. Martinez-Climent⁵, E. Callet-Bauchu⁶, F. Sole⁷, P. Deloukas³, R. Siebert²;
¹supported by Deutsche Krebshilfe,Institute of Human Genetics, University Hospital Kiel, Kiel, GERMANY, ²Institute of Human Genetics, University Hospital Kiel, Kiel, GERMANY, ³Sanger Centre, Hinxton, UNITED KINGDOM, ⁴Royal Bournemouth Hospital, Bournemouth, UNITED KINGDOM, ⁵Dept. of Hematology and Oncology, University of Valencia, Valencia, SPAIN, ⁶Lab. d`Hematology, Centre Hospitalier, Lyon, FRANCE, ⁷Dept. of Pathology, Hospital del Mar IMAS IMIM, Barcelona, SPAIN.

Various genetic changes affecting chromosomal region 10q24 have been shown to be recurrent in malignant lymphomas. Among those, the translocation t(10;14)(q24;q32) is supposed to activate the NFKB2/LYT10 gene in 10q24 via its juxtaposition nexto the IGH gene in 14q32. In addition, the NFKB2/LYT10-locus has been reported to be targeted by deletions particularly in cutaneous lymphomas. In the present study, we established an interphase FISH assay for detecting alterations of NFKB2/LYT10-locus. As probes, differentially labelled BAC-clones from a contig of chromosomal region 10q24 were applied which cover at least 400kb on each side of the NFKB2/LYT10-locus. The diagnostic cut-off levels of the FISH probe set in interphase cells were determined in normal controls. A cell line known to carry breaks in both NFKB2 alleles served as positive control. In all five studied B-cell lymphomas with cytogenetically proven t(10;14)(q24;q32) a break within the IGH locus but not in the NFKB2 locus was detected. From 14 lymphomas with chromosomal aberrations in 10q, only a single case displayed a signal constellation indicating a breakpoint within the NFKB2/LYT10-locus. Four cases showed signal patterns indicating deletions of the NFKB2/LYT10-locus. Similarly, a loss of the NFKB2/LYT10-locus was detected in 8/18 cutaneous T-cell lymphomas. These findings indicate that the NFKB2 gene very likely is not the sole target of the t(10;14)(q24;q32) and that deletions affecting the NFKB2/LYT10-locus are recurrent particularly in cutaneous T-cell lymphomas. Finally, our results show the established double-color FISH assay to provide a new routinely applicable tool for diagnosing recurrent breakpoints and imbalances in NFKB2/LYT10-locus.

p16INK4a, p15INK4b, p14ARF, Rb1 and ECAD Genes Aberrant Methylation in Various Cancers.

V. Zemlyakova¹, L. Lubchenko², I. Zborovskaya², V. Strelnikov³, V. Artamonov⁴, M. Nemtsova¹;
¹Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, RUSSIAN FEDERATION, ²Research Oncological Center, Moscow, RUSSIAN FEDERATION, ³Institute for Molecular Medicine, Moscow Medical Academy, Moscow, RUSSIAN FEDERATION, ⁴Russian State Medical University, Moscow, RUSSIAN FEDERATION.

Alterations of DNA methylation pattern have been recognized as common changes in human cancers.
We investigated the frequency of p16INK4a, p15INK4b, p14ARF, retinoblastoma (Rb1) and E-cadherin (ECAD) genes aberrant methylation in different cancers: breast cancer (60 samples), non-small lung cancer - NSLC - (35 samples), nephroblastoma (10 samples), retinoblastoma (50 samples), acute lymphoblastic leukemia - ALL - (30 samples), chronic lymphoblastic leukemia - CLL - (15 samples).
Methylation in the breast cancer samples was detected in 14% for Rb1 promoter, 27% for p16INK4a promoter, 41% for p16INK4a exon1, 40% for ECAD promoter; for nephroblastoma - 70% for Rb1 promoter, 50% for p16INK4a promoter, 50% for p16INK4a exon1, 50% for ECAD promoter; for NSLC - 12% for Rb1 promoter, 18% for p16INK4a promoter, 66% for p16INK4a exon1, 72% for ECAD promoter; for ALL - 16% for Rb1 promoter, 26% for p16INK4a promoter, 26% for p16INK4a exon1, 20% forECAD promoter; for CLL - 13% for Rb1 promoter, 26% for p16INK4a promoter, 26% for p16INK4a exon1, 6% for ECAD promoter; for retinoblastoma - 28% for Rb1 promoter, 16% for p16INK4a promoter, 57% for p16INK4a exon1, 59% for ECAD promoter. No methylation of p15INK4b and p14ARF was detected in our samples. In a number of samples we have shown joint methylation of several genes.
Studies of joint genes methylation and determination of methylation profile in tumors will allow to define a functional role of genes in carcinogenesis, as well as to develop practical approaches to the early diagnostics of cancer.

Joint methylation of p16INK4a, Rb1, ECAD genes in cancer samples
	p16 Rb1	p16 ECAD	Rb1 ECAD	p16 Rb1 ECAD
ALL	9%	9%	6%	6%
CLL	12%	6%	6%	6%
Breast cancer	14%	51%	8%	8%
NSLC	11%	53%	11%	11%
Retinoblastoma	16%	42%	18%	12%
Nephroblastoma	40%	50%	40%	40%

Frequency of the C282Y and H63D mutations of HFE gene in patients with malignant glioblastomas from Ukraine

V. N. Pampuha¹, A. P. Cherchenko², L. A. Livshits¹;
¹Institute of Molecular Biology and Genetics, Kiev, UKRAINE, ²Institute of Neurosurgery, Kiev, UKRAINE.

The discovery of the HFE gene allows us to study the molecular basis of iron overload disorders. In hereditary haemochromatosis high frequency of the C282Y and H63D mutations of HFE gene established, but their role in neoplastic transformation are still under investigation. For elucidation the association beetween HFE gene mutations and risk malignant gliomas development we investigated the frequency of the HFE mutations on DNA of 38 patients with malignant glioblastomas and 97 normal healthy subjects from Ukraine. The C282Y and H63D mutations were detected after PCR amplification of exons 2 and 4 HFE gene followed by restriction endonuclease digestion with RsaI for C282Y and BclI for H63D. Statistical data analysis was performed by Fishers exect test.
The allele frequency of the C282Y mutation in the normal population and malignant glioblastomas patients were 0.021 and 0.026, respectivelly ( p=0.32 ). The allele frequency of the H63D mutation in the normal subjects and malignant gliomas patients were 0.17 and 0.13, respectivelly( p=0.11 ).
These findings did not provide evidence of association between higher level of H63D mutation and malignant glioblastomas in contrast to the date obtained by F. Martines di Montemuros et al. (2001).The obtained tendency of association malignant glioblastomas with lower frequency of H63D mutation in genotype will be check for major patients group with malignant glioblastomas.

A. Cirakoglu¹, S. Hacihanefioglu¹, Y. Tarkan-Argüden¹, A. Deviren¹, S. Berrak², C. Canpolat²;
¹Dept. of Genetics, Div. of Biomedical Sciences, Cerrahpasa Medical School, Istanbul University, Istanbul, TURKEY, ²Dept.of Pediatric Hematology Oncology, Marmara University Hospital, Istanbul, TURKEY.

It is known that clonal chromosomal changes in childhood ALL are nonrandom. They are important markers for diagnosis, prognosis and relaps. The median survival time following relaps and clonal complex chromosomal changes are related. Our case was four years old boy. He was diagnosed with ALL-L1 a year ago. He had weakness, fever, massive lymphadenopathy (LAP) and hepatosplenomegaly. He was considered poor risk patient and treated with chemotherapy and radiotherapy because of central nervous system involvement. Relaps was occured a year later. Bone marrow sample was analysed cytogenetically after relaps. The karyotype was 46,XY,t(3;17)(q23?;p13),t(5;12)(q31;p13),inv(11)(p15q12)[11]/46,XY[8]. The patient died from ARDS after convulsions likely due to toxicity of drugs.
t(5;12)(q31-33;p12-13) was reported in childhood and adult myelodysplastic syndromes. Extensive research of literature failed to demonstrate any published on t(3;17)(q23?;p13) in leukemias. Whereas the breakpoint of 17p13 had been involved in a t(11;17)(q23;p13) in ANLL. inv(11)(p15q12) has not been reportedin literature, but the breakpoint of 11p15 had been reported in inv(11)(p15q23) in ANLL and MDS and in t(7;11)(p15;p15) in ANLL and CML in blast crisis.
To our knowledge there is no reported case with such karyotypic abnormalities. Since the patient had not been referred us for cytogenetic examination at the time of diagnosis,it remains unknown whether this anomaly was present in earlier stages of the disease or occured after relaps.

B. Popovic¹, J. Milasin¹, B. Jekic², I. Novakovic²;
¹Faculty of Stomatology, Belgrade, YUGOSLAVIA, ²Faculty of Medicine, Belgrade, YUGOSLAVIA.

The aim of the present study is to establish the relationship between mutations in exons 1 and 2 of the H-Ras gene and clinicopathological features of oral squamous cell carcinoma (OSCC). It is well known that H-Ras gene is one of a family of Ras genes, which encode p21 plasma membrane protein involved in signal transduction. Point mutations within codons 12 and 13(exon 1) or codon 61(exon 2) are frequently present in various tumours including OSCC. In our study, in order to detect these mutations, we isolated DNA from 20 paraffin embedded OSCC specimens. Using PCR technique, it was possible to analyse 10 of 20 (50%) samples, probably, because of DNA degradation, which occurred during paraffin embedding. According to the TNM staging, 5 of 10 successful amplified samples were stage III and 5 stage II. These histopathological parameters suggest that our analyzed samples, might have a higher incidence of H-Ras mutations, but to confirm this correlation, PCR products will be screened by method for detection mutations–SSCP. Also, the additional cases will be tested and their prognostic significance will be discussed.

H. Sennana Sendi¹, H. Elghezal¹, M. Gribaa¹, B. Meddeb², H. Ben Abid², A. Hafsia², A. Saad¹;
¹Service de Cytogénétique, CHU Farhat Hached, Sousse, TUNISIA, ²Service d'Hématologie, CHU Aziza Othmana, Tunis, TUNISIA.

We report herein a 9-year old boy undergoing a cytogenetic investigation for pancytopenia. A bone marrow aspirate revealed aplastic anemia. A diagnosis of fanconi anemia was confirmed by an increased number of chromosomal breaks and rearrangements in peripheral blood lymphocytes cultured in the presence of mitomycin C. The patient had no phenotypic manifestations. Bone marrow RHG-banded karyotype showed deletion del(21)(q22). Fluorescence in situ hybridization (FISH) analysis revealed a cryptic translocation t(1;21)(p36;q22).This translocation have been reported previously in two old patients who developed acute myeloid leukemia secondary to toxic exposure (after radiation exposure in one case, and after treatment with antitopoisomerase II in the second case), but this is the first report of t(1;21)(p36;q22) in a child with fanconi anemia.

B. Ravcukova, J. Kadlecova, I. Valaskova, J. Sterba, R. Gaillyova;
University of Children´s hospital, Brno, CZECH REPUBLIC.

Bone marrow metastasis often occurs in patients with neuroblastoma; therefore a sensitive assay to detect circulating neuroblastoma cells in bone marrow (BM) and peripheral blood (PB) is needed. The feasibility and clinical value of using the reverse transcriptase (RT) polymerase chain reaction (PCR) to amplify mRNA for tyrosinase hydroxylase (TH), the first enzyme of catecholamine synthesis, was evaluated to detect neroblastoma cells in patient samples.
A total of 32 patients with neuroblastoma were studied. After preparation of complementary DNA, the PCR was performed to amplify the TH gene. Amplified products were analyzed in polyacrylamide gel or by using the GeneScan Analysis on ABI PRISM 310.The specificity of the amplified products was checked by sequencing analysis and neuroblastoma cells were detected at a level of 1 per 10⁵ normal PB mononuclear cells by this method.TH mRNA was in 19 of the 63 BM samples, in 6 of the 53 PB samples and in 4 of the 8 tumor samples.
RT-PCR of TH mRNA is a sensitive and specific method for detection of circulating neuroblastoma cells in BM and PB samples. The clinical significance of these very low levels of neuroblastoma cells detected by RT-PCR requires further investigation. We conclude that, by combining multiple molecular markers and independent screening techniques, we may be able to overcome tumor heterogeneity and expedite the detection of microscopic disease in the clinical management of NB. This work is supported from the Ministry of the Health in the Czech Republic ( MZ 0006526 97 05)

T. K. Petrova¹, M. Tzancheva², A. Gegova², G. Dineva¹, M. Marinov², H. Kadyan², P. Pentchev², D. Nedin², A. Alexandrova², J. Ilieva², D. Damyanov², V. Mitev³, I. Kremensky¹;
¹Laboratory of Molecular Pathology, University Hospital of Obstetrics and Gynecology, Sofia, BULGARIA, ²Queen Jovanna Hospital, Medical University, Sofia, BULGARIA, ³Department of Chemistry and Biochemistry, Medical University, Sofia, BULGARIA.

Colorectal cancer (CRC) is the most common gastro- intestinal neoplasia. Hereditary nonpolyposis colorectal cancer is the most common type of familial CRC. Germline mutations in one of DNA mismatch repair genes are known to be responsible for the HNPCC phenotype. Their mutations induce microsatelite instability (MSI). Approximately 12-17% of the unselected tumors also show MSI.
Highly efficient set of five markers- D2S123, BAT26, D5S346, D18S35 and FGA have been selected for detecting the MSI.
A total of 108 patients with colorectal cancer have been included in the current study. The analysis was performed using two detection methods- denaturing polyacrylamide gels followed by silver staining and/or automated fluorescence detection (ALFExpress Biotech, Pharmacia).
We detected 16 tumours with MSI. These cases were reliably detected by the two methods. 64,3% of them were family cases. In 65% of the MSI cases the tumors were right localized, compared with 27% in the whole group patients. We found strong correlation between the right tumor localization and MSI-H (MSI-high) phenotypes.
The histopathological evaluation showed the presence of mucin production in 64,3% of the MSI cases, compared with 15,9% in the microsatelite stable tumors. Most of the cases had not cancer cell metastasis in the regional lymph nodes (71,4%). All the right localized tumors were included in this group. No long distance metastases in MSI positive tumors were found. These data were corelated with the better prognosis of the MSI positive colorectal cancers especially the tumors with right colon origin.

PCR detection and typing of HPV in Bulgarian patients with invasive cervical cancer

D. Georgiev¹, L. N. Georgieva², T. Arsov², B. Slavchev¹, I. Karagyozov¹, A. Galabov³, D. Toncheva²;
¹University Hospital of Obstetrics and Gynaecology "Maichin dom", First Clinics of Gynaecology, Sofia, BULGARIA, ²Department of Medical Genetics, Medical University Sofia, Sofia, BULGARIA, ³Institute of Microbiology, Department of Virology, Bulgarianan Academy of Sciencies, Sofia, BULGARIA.

Human Papillomavirus (HPV) infection is considered to be an important risk factor for cervical cancer development. The polymerase chain reaction (PCR) method enables detection and typing of large number of HPV types and is widely used in HPV diagnostics. Here we present results from PCR HPV typing in cervical cancer patients obtained for the first time in Bulgaria.
HPV diagnostics was carried out on DNA samples obtained from 70 paraffin-embedded biopsies from Bulgarian women diagnosed with invasive cervical cancers during the last 2 years. The samples were initially tested with MY09/MY11 consensus primers located within the L1 region of HPV genome, which covers a broad spectrum of HPV types. Gamma globin gene amplification was carried out as an internal positive control. The positive samples were subsequently tested with specific primers for the most common HPV types (6, 11, 16, 18, 31 and 33).
We detected HPV in 45 cases (64.28%). Mixed infection was observed in more than 40% of the positive cases. HPV 16 was detected in 25 cases (31.6%), HPV 18 in 19 (24.1%), HPV 6 in 15 (19.0%), HPV 11 in 9 (11.4%), HPV 31 in 7 (8.9%) and HPV 33 in 4 cases(5.0%).
Our initial results indicate the highest prevalence of HPV types 16 and 18. This confirms the association of high-risk types HPV16 and 18 with invasive cervical cancers.

Amplification of the androgen receptor gene in primary prostate cancer due to X chromosome aneuploidy

A. Röpke¹, K. John¹, A. Erbersdobler², P. Hammerer³, M. Stumm¹, P. F. Wieacker¹;
¹Institute of Human Genetics, Otto-von-Guericke-University, Magdeburg, GERMANY, ²Institute of Pathology, Universitätskrankenhaus Hamburg-Eppendorf, Hamburg, GERMANY, ³Department of Urology, Universitätskrankenhaus Hamburg-Eppendorf, Hamburg, GERMANY.

In the majority of patients with prostate cancers (PC), the growth of the tumor is androgen-dependent. In advanced PC, when radical treatment of prostate cancer is not curative, androgen deprivation therapy has proved to be an effective palliative therapy. In this study we analysed isolated nuclei from 31 prostatectomy specimens from PC patients without preoperative therapy for amplification of the androgen receptor (AR) gene by fluorescence in situ hybridization (FISH). For this purpose, an AR gene probe (Vysis) was used in combination with an X centromere control probe. In 11 out of 31 PC, additional X chromosomes (disomy to tetrasomy) with the corresponding AR gene could be detected in more than 20% of the analysed nuclei. In two of these patients, over 40% of the nuclei of the PC tissue showed two or more fluorescence signals. Control analyses with centromere probes of chromosomes X, Y and 18 confirmed these high level of X aneuploidies. Both patients have advanced stage PC (pT4,pN1 and pT3b,pN0). FISH analysis on normal prostate tissue detected in both patients in less than 10% of the nuclei additional X chromosomes. Furthermore, the analysis of 11 normal prostate tissues showed that an average of 8% of the nuclei had two or more signals for the AR and X centromere probe.
We conclude that additional AR gene copies are present in a subgroup of primary PC prior to anti-androgen therapy. This may be an important factor for initial anti-androgen resistance.
This study was founded by Dr. Mildred Scheel Stiftung.

M. Papezova¹, A. Krepelova¹, V. Kebrdlova¹, T. Dolezal¹, L. Foretova², P. Plevova³;
¹1st Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC, ²Masaryk Memorial Cancer Institute, Brno, CZECH REPUBLIC, ³Faculty Hospital, Ostrava, CZECH REPUBLIC.

Microsatellite instability (MSI) is characteristic feature of colorectal cancer with loss of mismatch repair (MMR) in tumor cells. MSI is found in tumors of patients with HNPCC and in 15-20% of sporadic colorectal tumors. In connection with mutation analysis of MSH2 and MLH1 genes in patients with HNPCC, we studied MSI in 47 unrelated patients with colorectal cancer. Of these, 8 patients fulfilled Amsterdam criteria (AMS+), 27 patients were familial (AMS-) and 12 were sporadic cases (Spor). Two mononucleotide (BATRII, BAT26) and five dinucleotide (D2S123, D3S1029, D5S346, D17S250, D18S58) loci were analysed. Initially, MSI was determined in denaturing polyacrylamide gel stained with ethidium bromide, then fragmentation analysis with fluorescent primers on ABI Prism 310 Genetic Analyzer was introduced and both methods were compared. Tumors were classified as MSI-H (high degree of MSI, 2 or more loci with MSI), MSI-L (low degree of MSI, 1 unstable locus), and MSS (stable, no MSI detected). Results of MSI analysis in tumors are shown in table. Number of patients with germ-line mutation in MSH2 or MLH1 gene is shown in parentheses. In patients with MSI-H tumor and negative result of mutation detection, analysis of methylation status of MLH1 gene promoter is in progress. This work was supported by Grant Agency of Charles University (Grant No. 17/2001).


Group of patients	MSI-H tumors	MSI-L tumors	MSS tumors	Total
AMS+	5 (5)	1 (0)	2 (0)	8 (5)
AMS-	5 (2)	1 (0)	21 (1)	27 (3)
Spor	2 (0)	0	10 (2)	12 (2)
All	12 (7)	2 (0)	33 (3)	47 (10)

M. Grzmil¹, D. Mury¹, W. Engel¹, P. Thelen², R. Ringert², B. Hemmerlein³, H. Radzun³, P. Burfeind¹;
¹Institute of Human Genetics, University of Göttingen, Göttingen, GERMANY, ²Department of Urology, University of Göttingen, Göttingen, GERMANY, ³Department of Pathology, University of Göttingen, Göttingen, GERMANY.

Prostate cancer is the most frequently diagnosed solid tumor in men, and the second leading cause of cancer death in males from western countries. One of the key issues in prostate cancer research is to develop molecular markers that can effectively detect and distinguish the progression and malignancy of prostate tumors. In order to analyze differential gene expression of putative tumor markers labeled cDNA probes were generated from capsule-invasive prostate tumor (stage pT3a) and normal prostate tissue. The cDNA probes were hybridized with an Atlas Select Human Tumor cDNA Expression Array (Clontech) with immobilized cDNAs of differentially expressed genes from five different human tumors, e.g. bladder, breast, liver, lung and prostate carcinoma. In total, 46 known and unknown genes were identified to be up-or down-regulated in prostate carcinoma. The known genes showing a differential expression pattern in prostate tumor samples included transcription factors, protooncogenes and other proteins, e.g. Krox 24, c-jun, spermidyne acetyltransferase, ribosomal proteins, clusterin and prostate secretory protein 94. In addition, by using both Northern blot analyses on whole tumor RNA and real time RT-PCR on RNA from tumor cryosections enhanced expression of seven unknown genes was verified in prostate tumors as compared to normal prostate tissue. To further circumvent the problem of tissue heterogeneity, RNA from microdissected prostate tumor tissue samples was isolated and analyzed for differential gene expression. The identification of these tumor-specific expression patterns could lead to the establishment of genetic fingerprints of prostate tumors as a versatile tool for diagnostic and prognostic purposes.

S. Braun¹^,2, K. Riemann¹^,2, M. Pfister², K. Sotlar³, H. Zenner⁴, N. Blin¹, C. Pusch¹, S. Kupka¹^,2;
¹Department of Anthropology and Human Genetics, University of Tübingen, GERMANY, ²Department of Otolaryngology, University of Tübingen, GERMANY, ³Department of Pathology, University of Tübingen, GERMANY, ⁴Department of Otolaryngology,, University of Tübingen, GERMANY.

Paragangliomas of the head and neck region are usually benign tumors developing from chemoreceptors of paraganglionic origin in the majority of patients. These receptors play an important role in sensing and regulation of the blood CO2-level. Genetic alterations in the mitochondrial enzyme complex II (SDH), which is involved in respiratory chain and citric acid cycle reactions, have been shown to lead to sporadic as well as familiar cases of these tumors. Therefore we analyzed our collective containing sporadic cases of patients with paragangliomas for genetic changes in the SDH-genes SDHD, SDHC and SDHB. We detected several new DNA mutations in samples derived from tumor patients. Furthermore we demonstrated loss of heterozygosity (LOH) usually connected with oncogenesis of various tumors. Elucidation of the genetic regions involved in tumor development is a basis for understanding their contribution to normal and pathogenic cell physiology.

A fixed cascade of genomic changes in a murine tumor progression model for pancreatic adenocarcinoma

B. Schreiner¹, U. Zechner², D. Baur², F. Greten²^,3, M. Wagner², H. Hameister¹, R. Schmid²;
¹Department of Human Genetics, University of Ulm, GERMANY, ²Department of Internal Medicine I, University of Ulm, GERMANY, ³Department of Pharmacology, University of California, San Diego, CA.

P53+/- knockout mice overexpressing TGFa in a pancreas specific manner develop adenocarcinoma of the pancreatic ducts. This established animal model reflects human pancreatic cancer disease.
To investigate secondary genomic changes, more than 40 tumors were analyzed by CGH. In about 40% of the tumors gain of proximal chromosome 11 and loss of its distal part including p53 was detected. Due to this loss no wildtype p53-allel is left.
In addition to this recurrent aberration pattern further cancer progression follows two alternate routes: Overrepresentation of chromosome 15 including the Myc locus (A), or more rarely, loss of the distal part of chromosome 14 including the Rb-locus (B). It seems that these aberrations occur exclusively because in none of the aforementioned tumors a combination of these events was detected. On the average mice bearing tumors of subtype B develop tumors about 75 days earlier than mice with tumors of subtype A. These data indicate that there are at least two different pathways in pancreatic tumor formation: Proliferation through Rb-deletion or c-myc overexpression and activation.
To analyze the extension of the amplified regions of chromosome 11 and 15 cell lines were investigated by FISH and real-time PCR. On proximal chromosome 11 the amplification-unit extends about 18 cM. Together with Egfr, the c-Rel gene gets amplified which provides antiapoptotic activity. In contrast, the Myc amplification-unit of chromosome 15 is much smaller.
In the meanwhile a second series of pancreatic adenocarcinoma with TGFa / p19ARF+/- was analyzed by CGH. These tumors show less chromosomal aberrations.

Breakpoint Analysis of a Novel Recurrent Chromosomal Translocation (14;20)(q32;q12) in a Human Multiple Myeloma Cell Line.

G. R. Boersma-Vreugdenhil, T. Peeters, J. Kuipers, B. Bast;
Dept. of Immunology, UMC, Utrecht, NETHERLANDS.

Recurrent chromosomal translocations are regularly involved in hematological malignancies. Translocations involving the Immunoglobulin Heavy chain (IgH) region at chromosome 14q32 are a hallmark in human B cell malignancies including multiple myeloma (MM). Using Fluorescent In Situ Hybridization (FISH) we already demonstrated the ubiquitous presence of translocations in this region in MM cell lines, with a diverse array of translocation partners. Recently we have found a high percentage (92%) of der14t(14;?)(q32;?) in fresh MM samples as well.
Apart from the four major 14q32 translocations (involving 4p16, 6p25, 11q13 and 16q23), we reported another recurrent translocation, i.e. t(14;20)(q32;q12). Here, we describe the cloning and characterization of the breakpoint of this UM3 cell line.
Using FiberFISH techniques the breakpoint was detected in the switch gamma-1 region of chromosome14. Next, a genomic phage library of the UM3 cell line was screened with a gamma-1 probe resulting in a clone that spans the breakpoint. Sequence analysis pinpointed the breakpoint of chromosome 14 in the mu enhancer, adjacent to the recombined switch mu/switch gamma-1 genes.
Screening a P1 library picked up a larger genomic fragment covering the breakpoint at chromosome 20. Screening of a UM3 derived cDNA library with this P1 clone resulted in a cDNA clone of 3.5kb. The first known gene located downstream of the breakpoint at 20q12 is the oncoprotein MAF-B, pointing to MAF-B as one of the candidate target genes involved in t(14;20) in multiple myeloma. Functional studies to such an effect are underway.

Increased noise as an effect of haploinsufficiency of the tumor suppressor gene Neurofibromatosis type 1 in vitro

D. Kaufmann¹, S. Schrank², W. Vogel¹, H. Gruler², R. Kemkemer²;
¹Department of Human Genetics, University of Ulm, GERMANY, ²Department of Biophysics, University of Ulm, GERMANY.

In human diseases related to tumor suppressor genes, it is suggested that only the complete loss of the protein results in specific symptoms such as tumor formation, whereas simple reduction of protein quantity to 50%, called haploinsufficiency, essentially does not affect cellular behavior. Using a model of gene expression it was presumed that haploinsufficiency is related to an increased noise in gene expression also in vivo [Cook, D. L., Gerber, A. N. & Tapscott, S. J. (1998) Proc. Natl. Acad. Sci. USA 95, 15641-15646]. Here, we demonstrate that haploinsufficiency of the tumor suppressor gene Neurofibromatosis type 1 (NF1) results in an increased variation of dendrite formation in cultured NF1 melanocytes. These morphological differences between NF1 and control melanocytes can be described by a mathematical model where the cell is considered a self-organized automaton. The model describes the adjustment of the cells to a set point and includes a noise term which allows for stochastic processes. It describes the experimental data of control and NF1 melanocytes. In the cells haploinsufficient for NF1 we found an altered signal-to-noise ratio detectable as increased variation in dendrite formation in two out of three investigated morphological parameters. We also suggest that in vivo NF1 haploinsufficiency results in an increased noise in a cellular regulation and that this effect of haploinsufficiency might be found also in other tumor suppressors.

RB1 structural and functional pathology, methylation pattern of p16, p15, p14 and ECAD genes in retinoblastoma patients.

O. Babenko¹, V. Zemlyakova¹, S. Saakyan², V. Kozlova³;
¹Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, RUSSIAN FEDERATION, ²Moscow Institute for Eye Diseases, Moscow, RUSSIAN FEDERATION, ³National Scientific Oncology Centre, Moscow, RUSSIAN FEDERATION.

The aim of our investigation was to research molecular anomalies causing retinoblastoma. Retinoblastoma is an embryonic malignant tumor of retina with an incidence of 1:13000 which is found together with structural abnormalities in RB1 tumor-suppressor gene. Some cases are obviously caused by methylation of RB1 gene promoter region. SSCP and heteroduplex analysis were used for mutation screening, microsatellite analysis (intron 2, 20, D13S262, D13S284) - for loss of heterozygosity (LOH), methylation pattern analysis of RB1 promoter region - for functional mutation.
We have studied 60 families with different forms of retinoblastoma. Analysis of PCR products mobility by using of SSCP and heteroduplex analyses revealed 47 mutations in different RB1 gene exons and introns. All familial and sporadic bilateral cases of retinoblastoma had germinal mutations. Complete deletion of RB1 was revealed in two sporadic cases. Loss of heterozygosity of at least one of intragenic markers was found in 71% of analyzed tumors. Methylation pattern anomalies of the RB1 gene promoter region were found in 27% retinoblastomas.
Having not found any abnormalities in same tumors we undertook a study of methylation status of p16 gene (INK4a) promoter region as it functions as an upstream regulator of pRB activity and others important tumor-suppressor gene: p15 (INK4b), p14 (ARF) and ECAD in retinoblastoma tumors. We found abnormal methylation p16 promoter region - 11% cases, in exon 1 of p16 - 46% tumors and methylation of ECAD promoter - in 59% retinoblastomas. These data confirm necessity of methylation profile studies in different tumor types.

Telomerase activity as a predictive marker for in vitro chemo-reponsiveness of bilharzial bladder cancer

A. M. Metwally¹, H. M. Khaled¹, I. Abdel Salam¹, M. S. Aly²;
¹National Cancer Institute, Cairo University, Cairo, EGYPT, ²Faculty of Science, Cairo University (Beni Suef branch), Cairo, EGYPT.

The DNA component of telomeres is synthesized by a specialized reverse transcriptase enzyme,the telomerase.More than 90% of human cancers are telomerase positive where most normal tissues or benign tumors contained low or undetectable levels.We wanted to know if there would be any correlation between the in vitro sensitivity of bilharzial bladder cancer to different chemotherapeutic agents and the telomerase activity.The present study included 33 samples taken from bladder cancer patients treated at the National Cancer Institute,Cairo University.Three anticancer drugs (gemcitabine,taxotere,and navelbine) were applied as single agents or in combination.Telomerase activity was positive in 28 out of the 33(85%)cases studied.The drug gemcitabine was tested on all 33 samples included in the study,while taxotere and navelbine were tested on 26 and 28 samples respectively.Simultaneous application of the 3 drugs was tested on 7 samples. No sensitivity could be detected to any of the 33 samples tested for gemcitabine (0%).Among the 26 samples tested for taxotere,3 samples showed in vitro sensitivity (9%).Navelbine has demonstrated in vitro activity in 1/27 (9%)samples.Among 7 samples tested for simultaneous application of the 3 drugs,the activity of the combination was demonstrated in one sample(14%). There was highly significant correlation between the telomerase activity and the in vitro sensitivity to both the drugs taxotere and navelbine (p.00016 and .0005 for taxotere and .012 and .0421 for navelbine).Thus it may be concluded that telomerase activity may be used as a predictive marker for responsiveness to chemotherapy in bilharzial bladder cancer patients.However,clinical correlations are clearly needed before reaching such a conclusion.

Conversion of ALL-L2 with a double 12;21 translocation to JMML with a 4;11 translocation: A cytogenetic, morphological and immunophenotypic study.

Abstract
The phenotypic conversion of acute lymphocytic leukemia (ALL) into juvenile myelomonocytic leukemia (JMML) is a rare event, especially in the pediatric population. We describe a comprehensive cytogenetic and flowcytometric study performed from bone marrow and peripheral cells from such a child enabling us to determine the origin of his JMML. A four-year-old boy diagnosed with double t(12;21), CALLA+, pre-B ALL was treated as-per BFM protocols on the low risk arm. A routine cytogenetic analysis performed at 17 months into treatment failed to detect t(12;21) in bone marrow (BM) cells. However, a novel translocation, namely, t(4;11), involving the MLL gene at 11q23 was detected in monocytes, mature granulocytes and in immature myeloid/monocytic cells. No cytogenetic abnormalities were found either in EBV-transformed B or in PHA-stimulated T lymphoid cells. Flowcytometric analysis demonstrated an asynchronous expression of the antigenic determinants in populations of granulocyte and monocytoid cells: Low levels of CD14, an unusually high level of CD15, and no CD13 or HLA-DR antigens were observed in 60% of monocytes, while 74% of myeloid cells expressed no CD13. Debate exists as to whether JMML is a disorder of the pluripotent hemopoietic stem cells or of the committed myeloid/erythroid/megakaryocytic (GEMM) stem cell. Our results indicate that the transformation from B-cell ALL to JMML in this case, occurred, most probably in the GEMM stem cells without involving the lymphoid cell line. The role of the initial double t(12;21) aberration in the evolution of the disease is not clear.

Z. S. Klestova¹, V. N. Balatsky², R. A. Golubets¹;
¹The Institute of Veterinary Medicine, Kyiv, UKRAINE, ²The Institute of Pig breeding, Poltava, UKRAINE.

Many items of human and animals leucosis pathogenesis are not clear. We studied leucosis virus influence on animals' cells genome and change of exchange processes in an organism in case of long term infection. As a model we used Bovine Leukemia virus (BLV) and cattle of black-mottle species (in fifties experimental and check animals). Peculiarity of cattle pathogenic properties is its tropism to lymphocytes. After have studied locus BM-315 of chromosome #5 cattle's lymphocytes of infected cattle within 4 months we had defined 1 allele sized 152 pairs of nucleotides. In 4 months two alleles sized 152 and 166 pairs of nucleotides were found in the same animals. One animal in the studied locus on first analyses were found two alleles sized 160 and 156 pairs of nucleotides. In 4 months in the same animal were found 2 alleles sized 158 and 152 pairs of nucleotides. In locus MAF-50 of cattle’s cells chromosome #4 was also registered change of allele’s number and sizes in comparison with check animals. Thus was found the change in parts of animals’ cells chromosomes #4 and #5 in BLV infected animals. Comparative RAPD-PCR analyses revealed individual changes on strips quantity in RAPD-spectrum of experimental animals in ontogenesis in comparison with check animals after a time. This testifies to changes in genome of studied cells. RAPD-PCR analyses is more informative for small sampling of animals evaluation. It is also preferable for comparing with analyses of single high-polymer locuses.

A. Humeny¹, T. Bonk¹, J. Gebert², C. Sutter², M. von Knebel-Döberitz², C. M. Becker¹;
¹Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Erlangen, GERMANY, ²Sektion für Molekulare Diagnostik und Therapie, Chirurgische Klinik, Universität Heidelberg, Heidelberg, GERMANY.

Insertion as well as deletion of repetitive units in microsatellites arise as a consequence of DNA polymerase slippage during DNA replication. The DNA mismatch repair (MMR) system eliminates the initial insertion-deletion loops to reduce the error rates in DNA replication. Defects in the MMR system result in an increased loss or gain of repeat units in microsatellites, commonly known as microsatellite instability (MSI). These processes play an important role in cancer development and therefore serve as markers for tumorigenesis. Due to this reason, efficient methodical approaches for the reliable detection of MSI with prospects for high throughput screening are needed. Matrix assisted laser desorption ionization - time of flight - mass spectrometry (MALDI-TOF-MS) possesses these general features as shown for genotyping of single nucleotide polymorphisms (SNPs). Here, we present a MALDI-TOF-MS based genotyping approach for MSI affecting mono nucleotide repeats in somatic tissues important in hereditary nonpolyposis colorectal cancer (HNPCC). Following PCR amplification of genomic regions including the microsatellites and primer extension reactions over the microsatellites to produce informative molecular masses, MSI was detected by MALDI-TOF-MS. The analysis of peak integral ratios in a single spectrum of the peaks representing insertions or deletions in comparison to the full length microsatellites lead to a relative quantification of MSI. MALDI-TOF-MS based genotyping results were confirmed completely by conventional DNA sequencing and electrophoresis. Due to its accuracy, short runtimes and low costs, this procedure possesses the potential for high throughput screening of MSI to replace gel based methods.

Chromosome mechanisms involved in the inactivation of hSNF5/INI1 leading to rhabdoid tumors.

M. F. Rousseau-Merck¹, I. Legrand¹, I. Versteege¹, N. Sévenet¹, P. Heiman², O. Delattre¹, A. Aurias¹;
¹INSERM U509, Paris, FRANCE, ²Hopital Erasme, Bruxelles, BELGIUM.

Rhabdoid tumors are highly malignant pediatric cancers. Observations of biallelic alterations or deletions of hSNF5/INI1 in these tumors as well as in murine models provide evidence of a tumor suppressor gene acting in this pathology. A precedent work demonstrated that the major mechanisms associated to the inactivation of hSNF5/INI1 are mitotic recombinations or putative non disjunction/duplication leading to partial or total isodisomy.
In an attempt to further characterize the main chromosomal mechanisms involved in the hSNF5/INI1 inactivation in rhabdoid tumors, we report here the molecular cytogenetic data obtained with 18 rhabdoid cell lines harboring hSNF5/INI1 mutation or deletion using 10 different markers located all along the chromosome 22q11.2-q12 region. The FISH results show that several deletions may occured in the cases carrying chromosome 22 abnormalities. The translocations including the hSNF5/INI1 consensus region are always associated with an homozygous deletion of variable size. By contrast, no deletion could be detected in any of the cases exhibiting an apparently normal karyotype.
Besides common mechanisms of mitotic recombination or non disjunction/ duplication occuring in 60% of either retinoblastoma or rhabdoid tumors, other specific chromosome mechanisms seem to be involved in each category of tumors for inactivating the respective tumor suppressor genes. Translocations associated with homozygous deletions and monosomy cases associated with mutations are mainly described in the rhabdoid tumors. Inactivation by two different mutations is mainly found in retinoblastoma. These differences may be related to the presence of low copy repeat families in the proximal 22q region leading to an increased chromosome instability.

Application of FISH and microsatellite markers for monitoring chimerism in Bulgarian patients after bone marrow transplantation

D. Koynova¹, B. Zaharieva¹, S. Atanasova¹, G. Mihailov², B. Avramova², M. Jordanova², L. Garcheva², D. Bobev², D. Toncheva¹;
¹Department of Medical Genetics, Medical Faculty Sofia, Sofia, BULGARIA, ²Children Hospital for oncohematological disorders, Sofia, BULGARIA.

Development of effective molecular-diagnostic methods for monitoring chimerism is an important tool for determining the risk of relapse in patients after allogenic bone marrow transplantation. Evaluation of the chimerism status in these patients provides substantial information about the replacement of host cells with donor cells during the posttransplantation period at the level of extremely small number of cells. Two male patients were studied by FISH with alternatively labeled X and Y probes after transplantation from sister and mother donors respectively. In the first case the percentage of donor cells increased in cultured lymphocytes from periferal blood from 6 to 70% in about 10 days period starting one month after the transplantation and by now the patient has achieved full remission. The second patient showed 97% chimerism on a bone marrow smear at the 51^st day of transplantation and is still under observation. The three male-to-male donor-recipient pairs were studied by genotyping of ACPP, D3S1282, D3S1509, SST, RHO, D3S1212, SLC, SERT and DAT1 microsattelite loci by PCR. In the case of brother donation SERT proved to be informative and showed chimerism 3 months after the transplantation in blood sample followed by recurrence of the illness. In the case of unrelated donor ACPP was found to be informative but unfortunately the patient died before the first monitoring. In the third case (father donor) none of the markers was informative. Polymorphic microsattelite markers and FISH can be used as powerfull tools in identification of donor-recipient differences.

FISH analysis of 11q13 gene amplifications on tissue microchips of transitional cell carcinomas from Bulgarian patients

D. Toncheva¹, T. Arsov², B. Zaharieva¹, C. Georgiev³, T. Todorov³, G. Sauter⁴;
¹Department of Medical Genetics, Medical Faculty Sofia, Sofia, BULGARIA, ²Institute of Immunology and Human Genetics, Faculty of Medicine, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, ³Department of pathological anatomy, Medical Faculty Sofia, Sofia, BULGARIA, ⁴Institute of Pathology, University of Basel, Basel, SWITZERLAND.

Gene amplification results in an increased dosage of the affected gene. It represents one of the major molecular pathways through which the oncogenes are activated during tumorogenesis. The most frequently amplified proto-oncogenes in human tumors mainly belong to one of the three erbB, ras and myc families, or to the 11q13 locus. Candidate oncogenes located at 11q13 are CCND1 (PRAD1, bcl-1), EMS1, FGF3 (Int-2) and FGF4 (hst1, hstf1). We report results from a screening for gains and amplifications of CCND1, FGF3/FGF4, FGF3 and EMS1 by fluorescence in situ hybridization (FISH) using tissue microarray (TMA) containing urinary tract tumor samples from 207 Bulgarian patientsAll 4 genes were successfully analysed in 106 tumors. Three of these tumors (2,83%) had the 4 genes amplified, three tumors (2,83%) had amplifications of CCND1, FGF3/FGF4 and FGF3 together, two of which had gain for EMS1 and one was normal for EMS1. No tumor had amplification of a single gene. 8 of the tumors had gain for all 4 genes (7,5%), 3 tumors (2,83%) had gain only for CCND1, 2 tumors (1,9%) had gain only for CCND1 and FGF4 and 1 tumor had gain for all 3 genes without CCND1. Three of the patients had Balkan Endemic Nephropathy (BEN). Interestingly, two of the three BEN tumors studied (stage pTa and pT3 respectively) had genetic gain at 11q13, the third did not react. The BEN associated pTa tumor was the only pTa tumor with 11q13 copy number change of all studied tumors.

Association of the G289S polymorphism in the HSD17B3 Gene with Prostate Cancer Risk in Italian Men

K. Margiotti¹^,2, E. Kim², L. Pearce², E. Spera¹, G. Novelli¹, J. Reichardt²;
¹University of Tor Vergata, Rome, ITALY, ²USC Keck School of Medicine, Los Angeles, CA.

Prostate cancer is a significant public health problem. Substantial data support an important role for androgens in the etiology of this disease. The human HSD17B3 gene encodes the testicular (or type III) 17b-hydroxysteroid dehydrogenase enzyme which is involved in testosterone biosynthesis in men. We have investigated the G289S (glycine at codon 289 replaced by serine) polymorphism at the HSD17B3 locus as a potential candidate SNP (single nucleotide polymorphism) for prostate cancer risk in constitutional (“germline”) DNA from 103 Italian prostate cancer cases and 109 Italian “centenarian” controls to assess the role of this SNP in susceptibility to prostate cancer. The G289S polymorphism appears to confer a statistically significant increased risk for prostate cancer (OR= 2.5; p= 0.04) in this pilot study.
Our preliminary data are consistent with an important role of the G289S SNP in prostate cancer susceptibility. Thus, the HSD17B3 gene may be an important candidate gene for prostate cancer risk.

Supported by the NCI, USA (JR) the Ministero della Sanita’, ITALY (GN)

The G289S polymorphism at the HSD17B3 Locus and Prostate Cancer
Genotype	Controls	Cases
GG(wt)	101	86
GS+SS	8 (8+0)	17(15+2)	OR=2.5
			p=0.04
The heterozygote (GS) and homozygote (SS) genotypes were combined in the analysis. GG (wt) is the normal genotype. OR is the odds ratio and p is the p-value

A Single DNA chemical modification by cisplatin may cause long-range changes in protein binding to DNA. Results of computer modeling.

V. B. Teif¹^,2, V. I. Vorob'ev³, D. Y. Lando⁴;
¹Institute of Bioorganic Chemistry, Minsk, BELARUS, ²Institute of Molecular and Atomic Physics, Belarus National Academy of Sciences, Minsk, BELARUS, ³Institute of Cytology, Russian Academy of Sciences, St.-Petersburg, RUSSIAN FEDERATION, ⁴Institute of Bioorganic Chemistry, Belarus National Academy of Sciences, Minsk, BELARUS.

Anticancer drug cisplatin covalently binds to DNA in vivo. The sites of DNA platination are recognized by several nuclear proteins. In particular, histone H1 and some of HMG proteins bind to platinated DNA with binding constants hundred times greater in comparison with unmodified DNA. Using computer modeling, we investigated binding of proteins to DNA treated by cisplatin. Binding of proteins to DNA is characterized by contact cooperativity as well as long-range interaction. Each platinated site is characterized by 100 times higher statistical weight for protein binding in comparison with non-modified ones. As a result the map of binding, i.e. the probability of each DNA base pair to be bound to a protein was calculated. It was found that chemical modification of one or several DNA base pairs by cisplatin strongly changes the character of protein binding to DNA. This effect is strongly dependent on the position of modified site. It was shown that a single chemical modification of DNA gives rise to a dramatic rearrangement in protein positions. If one protein covers 15 base pairs then the area of rearrangement involves up to 200 base pairs. Cisplatin changes the region(s) of preferable binding of proteins. In the case of chromatin, platination might cause primary nucleosome repositioning and strong secondary stabilization of their locations.
This work was supported by BRFFI (X99R-099)

Mixed Polyposis Syndrome in patients referred to genetic counselling clinic as Familial Adenomatous Polyposis patients.

C. Marchese¹, M. Montera², M. Torrini², E. Picco³, L. Locatelli⁴, M. Bossotti⁵, C. Mareni²;
¹Ospedale Mauriziano, Torino, ITALY, ²Department of Internal Medicine, University of Genova, Genova, ITALY, ³Ospedale Valdese, Pomaretto, ITALY, ⁴Ospedale Valdese, Torino, ITALY, ⁵Ospedale Gradenigo, Torino, ITALY.

The polyposis syndromes are a heterogeneous group of disease characterized by multiple lesions in the intestinal tract. The classification of polyposis syndromes is based on clinical observation of affected pedigree, on hystopathological diagnosis, on extracolonic manifestations. The better characterized autosomal dominant syndromes predisposing to colorectal cancer are associated to APC gene known to cause familial adenomatous polyposis (FAP) and to mismatch repair genes hMLH1 and hMSH2 associated with hereditary nonpolyposis colorectal cancer (HNPCC). Peutz Jegher syndrome (PJS) and juvenile polyposis (JP) are characterized by amartomatous or hyperplastic polyps readily distinguishable from adenomatous polyps of FAP. Recently the mixed hereditary polyposis syndrome (HMPS) has been described as a new rare syndrome as only two small families and a large kindred were reported. Genetic linkage suggested that the HMPS locus lies on the proximal part of the long arm of chromosome 6. We report our experience in genetic counselling with 53 patients referred as FAP patients. In 32 out of 46 patients with adenomatous polyps APC gene mutation was detected. 7 presented an hystological feature of mixed adenomatous and hyperplastic polyps. No APC gene mutation or microsatellite instability (MSI) were detected. Three presented familiarity, four were apparently sporadic. Mean age of polyps detection was 40.5 (13-57).
Increasing cases of mixed polyposis syndrome are seen in genetic counselling clinic but many questions raises about its classification and about surveillance of patients and at risk individuals. The syndrome is rare but an effort should be done, through collaborative studies, to identify the responsible

Germline mutations in the ccm1 gene, encoding krit1, in patients with cerebral cavernous malformations.

V. Marini¹, L. Ferrera¹, F. Pigatto², F. Alberti³, E. Sanzaro³, G. Viale⁴, P. Origone¹, C. Mareni⁵, C. Garrè¹;
¹Department of Oncology, Biology and Genetics, University of Genova, Genova, ITALY, ²Department of Neurological Science and Vision, University of Genova, Genova, ITALY, ³Division of Neurology, Sanremo Hospital, Imperia, ITALY, ⁴Department of Specialistic Surgery Science, Anaesthesiology and Organ Transplantation, University of Genova, Genova, ITALY, ⁵Department of Internal Medicine, University of Genova, Genova, ITALY.

Cerebral cavernous malformations (CCM) are congenital vascular anomalies of the brain, constituting approximately 10 to 20% of cerebral vascular lesions and with a frequency of 0.5% in the general population. The most common symptoms in affected patients are seizures, focal neurological deficits, migraine and/or intracranial haemorrhage, although some patients are asymptomatic.
Both sporadic and familial forms have been identified. Familial forms exhibit autosomal dominant inheritance with variable expression. In familial patients multiple lesions occur and the frequency of haemorrhages is higher. About 50% of familial forms are linked to mutations of the CCM1 gene (Chr 7q21-22) encoding Krit1 protein (736 aa). To date 27 mutations have been described all leading to non-sense stop codons and to a truncated protein.
We analysed 18 unrelated patients/families (11 sporadic cases, 2 familial cases and 5 unclassified cases) by the SSCP technique and sequencing analysis.
In the 2 familial cases, we found 2 different mutations leading to a truncated protein:
- 1302delGAAT, previously described as giving rise to a protein of 435 aa
- IVS8 -13 C->G, a new intronic substitution leading to a protein of 385 aa.
Moreover, a GTA->GTG (V660V) polymorphism was also observed in control subjects.
Using 6 extragenic polymorphic markers, we are evaluating the loss of heterozigosity in tumour tissue from CCM patients.
The aim is to identify CCM1 gene mutations and to determine the incidence of familial cases in the Italian population. Moreover, we propose to ascertain whether the "Knudson's double-loss mechanism" is involved in CCM disease.

The 825C allele of the gene GNB3 encoding the G-protein -3 subunit is associated with an increased risk for developing colorectal cancer

D. Öfner¹, M. Zitt¹, H. Menzel², K. W. Schmid³, U. Frey⁴, W. Siffert⁴;
¹University Clinic for Surgery, Innsbruck, AUSTRIA, ²Institute of medical Biology and human Genetics, Innsbruck, AUSTRIA, ³Institute of Pathology, Essen, GERMANY, ⁴Institute of Pharmacology, Essen, GERMANY.

Colorectal cancer is one of the major malign disease in all Western countries. Little is known about the pathogenesis of this disease. Like many other disorders, the development of colorectal cancer is multifactorial with a certain genetic contribution. We screened patients from Tirol with colorectal cancer for the C825T polymorphism in the gene encoding the G-protein beta-3 subunit. The frequency of the genotypes was 3% TT, 35% TC and 63% CC (n=157) which is significantly (p<0.0013) different from a local control population (randomly recruited healthy blood donors). The frequency of genotypes in the control population was 11% TT, 43% TC and 46% CC (n=188). Carriers of the 825C allele have a 1.89 fold increased risk (95% CI, 1.32-2.72) to develop colorectal cancer compared to carriers of the 825T allele. We observed also a gene-dose effect since homozygotes for 825C-allele have a higher risk to develop colorectal cancer than heterozygotes, both compared to homozygotes with the T-allele. CC versus TT gives an odds ratio of 4.83 (95% CI, 1.67-17.01) and CC versus CT gives an odds ratio of 1.70 (95% CI, 1.06-2.72). The protein of the GNB3 gene is part of a G-protein heterotrimer involved in signal transduction. The messenger RNA with the 825T allele is spliced differently but the resulting protein is still active. There is evidence that the 825T allele is associated with enhanced G-protein reactivity and hypertension, obesity and lately, that it is a genetic marker for enhanced T cell response.

Chromosome Insitability and Sister Chromatid Exchange Studies in Patients with Esophageal Carcinoma

O. Sahin, M. Ikbal, T. Tos, I. Pirim, C. Gundogdu, A. Yilmaz;
Ataturk Universitiy, Erzurum, TURKEY.

Cytogenetic studies have been carried out using convansional technique in peripheral blood lymphocytes in patiens with cancers. Sister chromatid exchanges (SCE) are resiprocal exchanges between sister chromatids. It has been reported that the ferquency of SCE in peripheral blood lymphocytes is significantly higher in patiens with variety type cancer than in normal individuals. This study assesed the frequencies of SCE and chromosome abnormality in peripheral lymphocytes of 28 patients with esophageal carcinoma and 20 controls. Peripheral lymphocyt cells were cultured with conventional culture methods. The blood samples were obtained from the patiens after histopatologic confirmation of the malignancy but before the initiation of chemotherapy or radiotherapy. The frequncy of aberrant metaphases appear to be significant in patients with esophageal carcinoma. The mean SCE ferquencies were 10.46 ± 0.48 and 6.82 ± 0.38 per methaphase in patients and controls, respectively. The increase of SCE frequency in cancer patients was stastically significant ( p< 0.001), but no seen in controls. Our results suggest that patients with esophageal carcinoma show a degree of chromosomal insitability that might be related to a predisposition to neoplasia.

Cytogenetic and molecular genetic characterization of patients with neuroblastoma in Yugoslavia

M. Djurisic¹, M. Guc-Scekic², D. Radivojevic², T. Lalic², S. Djuricic¹, D. Djokic²;
¹Mother and Child Health Institute of Serbia, Belgrade, YUGOSLAVIA, ²Mother and Child Health Institute of Serbia "Dr Vukan Cupic", Belgrade, YUGOSLAVIA.

Diagnostic of neuroblastoma (NB) is complex process involving multiple analyses from different samples (blood, urine, bone marrow, tumor tissue). Determination of genetic parameters involved in NB is one of the most significant analysis with great prognostic value.
In past ten years, in Laboratory of Medical Genetics, Mother and Child Health Institute “Dr. Vukan Cupic”, Belgrade, 80 patients with NB were analysed. Samples from bone marrow were cytogenetically analysed in 78 cases using standard preparation of chromosomes and G banding. In 3 patients beside cytogenetic we used FISH technique for detection of LOH for 1p36 and in one patient we combined these two techniques with PCR to detect deletion of 1p36. Two patients were analysed using just one molecular technique, FISH for 1p36 deletion in first case and PCR for 1p36 deletion in second case.
Results were following: 44 of 78 (56,4%) cytogenetically examined patients had normal karyotype while 34 (43,2%) had aberrant karyotype. 1p36 deletion was detected with FISH technique in one case while other analysed patients had no 1p 36 deletion. Cytogenetic findings in two of these trhee patient were normal but the one with deletion had also hiperdiplod clone in bone marrow. Analysis with PCR in two patients for 1p36 region showed intact chromosome 1.

R. A. Caruso¹, L. Rigoli², C. Di Bella², E. Cavallaro³, D. C. Salpietro²;
¹Dpt of Human Pathology, Messina, ITALY, ²Unità Operativa di Genetica ed Immunologia Pediatrica Policlinico Universitario, Messina, ITALY, ³Unità Operativa di Terapia Subintensiva e Dialisi Policlinico Universitario, Messina, ITALY.

Mitochondrial abnormalities have been observed in many human cancers,including changes in structure, number,respiratory enzyme components and transport systems.Mitochondrial DNA (mtDNA) can be modified by many carcinogens because its repair is less efficient compared with nuclear DNA.The mtDNA non-coding region, which contain hypervariable regions HV1 and HV2, origin of replication, the D-loop region and both origins of transcriptions,exibits a high degree of sequence polymorphisms. In this study, we examined in some gastric adenocarcinomas the mitochondrial D-loop region.The 15 primary gastric cancers were obtained from gastrectomies.MtDNA was extracted from paraffin-embedded tissues.A 445-bp portion of the human mitochondrial D-loop (bases 75 to 520) was amplified with four sets of overlapping PCR primers.For each tumor and normal fraction was performed the PCR-SSCP gel analysis.In four cancers, similar but altered bands distinctly different from the patterns obtained from adjacent normal tissue were observed. These altered bands were cloned and sequenced to reveal identical 50-bp deletions that involved flanking 9-bp direct repeats.This deletion eliminates a functional region.Antimitochondrial immunoreactivity was revealed in the supranuclear portion of adenocarcinoma cells.The deletion was not observed in normal mucosa.Mitochondrial mutations in human solid tumors have been reported in isolated case reports.One recent study sequenced a portion of the mitochondrial D-loop in colorectal cancers and failed to find mutations.Instead, in our study the 50 bp deletion was found in four out of 15 adenocarcinomas. These findings document the presence of somatic mitochondrial alterations in gastric cancer, which may reflect the environmental and genetic influence operative during tumor progression.

Blast Crisis in Philadelphia Negative CML: Clinical Findings and Molecular Cytogenetic Characterization of a 47,XX,+i(11)(q10) Cell Line.

W. Emberger¹, W. Olipitz², S. Sodia¹, E. Petek¹, H. Zierler¹, P. M. Kroisel¹, K. Wagner¹;
¹Institute of Medical Biology and Human Genetics, Karl-Franzens University, Graz, AUSTRIA, ²Departement of Medicine, Division of Hematology, Karl-Franzens University, Graz, AUSTRIA.

The presence of an abnormal karyotype is well known in Philadelphia negative CML. Cases without detectable cytogenetic aberrations are rare. The correlation between cytogenetic, morphologic, and clinical data is widely unclear. We report on a female patient who was first diagnosed with CML at an age of 69 years. At this time cytogenetic evaluation revealed a normal karyotype and no BCR/ABL transcripts could be detected using polymerase chain reaction (PCR). Litalir therapy was performed and after a chronic phase of three years she developed a CML blast crisis. The blasts where characterized as FAB M4 and cytogenetic evaluation was performed. A clone with a 47,XX,+i(11)(q10) karyotype resulting in a partial tetrasomy for 11q could be found. Cytogenetic data was confirmed by molecular cytogenetic analysis. Palliative Aloxan therapy was applied. The patient died in the 9th month of the blast crisis.
To our knowledge the presence of an isochromosome 11q in a CML blast crisis has never been reported before. The possibility that the 47,XX,+i(11)(q10) clone developed from the CML cell line or the occurrence of a secondary leukaemia will be discussed.

Rapid quantitative monitoring of mixed chimerism using amplification a highly discriminative PCR-STR system after bone marrow transplant

M. Shahrooei, A. Aleyasin, K. Darvishi, K. Alimoghaddam, A. Ghavamzadeh;
National Research Center for Genetic Engineering and Biotechnology, Tehran, IRAN (ISLAMIC REPUBLIC OF).

Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) is an important way for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are shown to be sensitive and rapid method. The intent of the present study was to test this approach for the quantification of mixed chimerism using six to twelve STR assay after boon marrow transplantation. The feasibility of this assay and the accuracy of quantitative results were tested using serial cell mixtures of unrelated individuals. Sequential analysis of individual chimerism status was performed in 63 patients who underwent BMT in Shariati Hospital, Tehran, Iran. Mixed chimerism (MC) was found in 12 of the patients from four weeks till nine months. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 53) except for one patient who was homologous even for his HLA genotypes with donor genotype. This procedure allows rapid and sensitive quantification of mixed chimerism after boon marrow transplantaion.

Chromosome imbalances in oligodendroglial tumors detected by Comparative Genomic Hybridization

V. Bourdon¹, G. Plessis², F. Chapon³, J. Derlon⁴, P. Jonveaux⁵;
¹Laboratoire de Génétique - EA 3441, Vandoeuvre les Nancy, FRANCE, ²Service de Génétique, CHU, Caen, FRANCE, ³Laboratoire de Neuropathologie, CHU, CAEN, FRANCE, ⁴Service de Neurochirurgie, CHU, Caen, FRANCE, ⁵Laboratoire de Génétique - EA 3441, CHU, Nancy, FRANCE.

Morphologic criteria for diagnosing and classifying oligodendroglial tumors remain the gold standard. Nevertheless, the addition of molecular approaches such as LOH, and Comparative Genomic Hybridization (CGH) can provide a means of arbitrating difficult or borderline cases, and establishing objective, reproducible standards, which will be tested prospectively for their ability to predict prognosis and responsiveness to therapy. Here we report a study on 25 oligodendroglial tumors (7 well-differentiated oligodendrogliomas, 16 anaplastic oligodendrogliomas and 2 oligoastrocytomas) analysed by CGH. Losses of 1p and 19q, known as common markers of oligodendroglial tumors, were observed in 36% (9/25) and 68% (17/25) of cases respectiveley and 32 % (8/25) of the tumors displayed both losses. These 8 tumors with 1p/19q losses have other abnormalities, except one case. The most prevalent deviations associated with this 1p/19q losses were deletions of chromosome 22 (3/8) and gains of the region 13(q21qter) (4/8). Loss of 9p was essentially restricted to anaplastic ooligodendrogliomas (4/16) and occured in tumors with intact 1p or 19q. Interestingly, six tumors (24%) showed gain of chromosome 7 associated in 3 cases with an amplification of the EGFR region (7p11), and loss of chromosome 10, alterations known to be preferentially involved in the progression of astrocytic tumors. This study confirms previous reports and shows that oligodendroglial tumors carry heterogeneous genetic alterations . In addition, these findings suggest that genes localized to these common chromosomal regions play a role in the tumorogenesis of oligodendrogliomas.

Detection of Illegitimate Rearrangements within the Immunoglobulin Light Chain Loci in B-cell Malignancies.

A. N. Silahtaroglu¹, T. S. Poulsen², C. G. Gisselø², N. Tommerup¹, H. E. Johnsen²;
¹Wilhelm Johansen Research Centre for Functional Genomics, IMBG, Panum Institute, University of Copenhagen, Copenhagen, DENMARK, ²The Research Laboratory, Department of Haematology L, Herlev Hospital, University of Copenhagen, Copenhagen, DENMARK.

Translocations involving the immunoglobulin loci are recurring events of B-cell oncogenesis. However, only a minority of translocations involves the immunoglobulin light chain loci; the kappa light chain (IGK) located at 2p11.2 and the lambda light chain (IGL) located at 22q11.2. We characterized clones from bacterial artificial chromosomes (BAC) libraries, spanning the IGK and IGL loci, for detection of illegitimate rearrangements within the loci by fluorescence in situ hybridization (FISH). Within the IGL region we have identified six end sequenced probes (22M5, 1152K19, 2036J16, 3188M21, 3115E23, and 274M7) covering the IGL variable (IGLV) cluster and two probes (165G5 and 31L9) covering the IGL constant (IGLC) cluster however within the IGK region five probes (479P12, 969D7, 316G9, 122B6, and 2575M7) have been identified covering the IGK variable (IGKV) cluster, and one probe (1021F21) covering the IGK constant (IGKC) cluster. A series of 25 cell lines of different origin have been analyzed for the presence of a translocation involving the immunoglobulin light chain by dual color FISH where the split of the variable cluster and the constant cluster indicated a translocation.
This study is supported by The Danish Cancer Society.
¤ The two authors have contributed to the work equally.

Cytogenetic Profiling Could be an Adjunct to Differential Diagnosis and Prognosis of Unknown Primary Tumors

H. Tsarouha¹, D. Pantou², A. Papadopoulou³, L. Mahaira³, I. Kyriazoglou⁴, N. Apostoloikas⁵, S. Markidou⁶, T. Trangas³, N. Pandis³, G. Bardi³;
¹Department of Genetics, " Saint Savas" Oncological Hospital, Athens, GREECE, ²Department of Genetics, "Saint Savas" Oncological Hospital, Athens, GREECE, ³Department of Genetics "Saint Savas" Oncological Hospital, Athens, GREECE, ⁴Orthopaedics, "SAINT Savas" Oncological Hospital, Athens, GREECE, ⁵Histopathology, "Saint Savas" Oncological Hospital, Athens, GREECE, ⁶Cytology, "SaintSavas" Oncological Hospital, Athens, GREECE.

Unknown primary tumors (UPT) constitute an entity, with great clinical and biological interest. The patients’ clinical features are heterogeneous and their prognosis is very difficult to predict. The present study aims to 1.identify UPT-associated cytogenetic aberrations, 2.evaluate the efficacy of cytogenetic analysis in differentiating metastatic carcinomas from lymphomas and sarcomas, and 3.assess the potential of genetic characteristics in UPT prognosis.
G-banding analysis was performed in surgical biopsies from 20 UPT at diagnosis. In cases with tumor material available after G-banding, CGH and interphase FISH were performed in 10 and 5 cases respectively.
The cytogenetic investigation revealed clonal chromosome aberrations in all, but one, of 18 successfully analyzed samples. In the total series of tumors, the breakpoints 1q21, 6q15, 7q22, 11p12-13, 11q21-22, 12cen, and 17p11-12 were most frequently involved in structural rearrangements, whereas the most common imbalances were losses of 1p, 6q, 8p, 9p, 11p, 11q, and 13q. Which of the above chromosomal sites are important to UPT pathogenesis and harbor genes that might determine the highly aggressive phenotype of this diagnostic entity needs to be further investigated. CGH analysis enhanced the classical cytogenetic findings by revealing imbalances in 7 out of 10 cases, including 2 cytogenetically not informative. Interphase FISH analysis, using locus-specific, break-apart probes for IgH(14q32) and ALK(2p23) revealed a lymphoma diagnosis in 3 UPT, histologically classified as malignant neoplasms.
A preliminary correlation analysis between the cytogenetic profile of the tumors and the patients’ survival showed that the increase of karyotypic complexity was associated with decrease of patient’ survival.

Methylation-Associated Transcriptional Silencing of E-Cadherin in Association with beta-Catenin Expression in Sporadic Colorectal Carcinomas.

G. A. Garinis¹, P. Menounos¹, N. Spanakis², G. P. Patrinos¹, G. Papadopoulos³, G. Karavitis³, G. Peros³;
¹Nursing Military Academy, Athens, GREECE, ²Medicanalysis Research Institute, Athens, GREECE, ³Nikaia Hospital, Department of Surgery, Athens, GREECE.

We investigated the possibility of an epigenetically associated loss-of-E-cadherin function in SCRCs by examining the methylation status of the e-cadherin promoter by means of the methylation-specific PCR (MSP), in tumour and adjacent normal tissues derived from 63 SCRC patients and correlated it with gene transcriptional silencing, at both the RNA and protein level and beta-catenin mRNA expression. Data were associated with patients' clinicopathological features. A more than two-fold decreased expression of e-cadherin gene was observed in 29/63 carcinomas (46%) versus 11/63 (17.5%) and 23/63 (34.3%) that was found to be increased or unaltered respectively. ICH examination revealed reduced E-cadherin protein expression in 21/63 (33,3%) of carcinomas versus 42/63 (66.7%) with increased E-cadherin expression. Decreased e-cadherin gene expression was significantly associated with E-cadherin ICH detection (P=0.0002) and was paralleled with a decreased beta-catenin expression in 70% of the carcinomas examined (P=0.001). Thirty-four out of 61 cases (50,7%) were reported as hypermethylated in e-cadherin promoter locus versus 27/61 (40.3%) that were found to be unmethylated. The methylation status of the e-cadherin gene promoter was significantly associated with E-cadherin expression at both the RNA and the protein level (P=0.002, P=0.004 respectively). A significant association was observed between E-cadherin ICH detection, lymph node metastasis and/or tumour stage (P=0.007, P=0.01 respectively). In agreement with prior work demonstrating that somatic mutations and loss of heterozygosity (LOH) of e-cadherin gene are rare or absent in the vast majority of SCRCs studied, we have found consistent aberrant methylation-associated decrease of E-cadherin expression suggesting an epigenetically mediated loss-of-E-cadherin function.

A new frameshift AML1/ETO fusion transcript in a patient with t(8;21) positive acute myeloid leukaemia

A. Lasa¹, M. J. Carnicer¹, J. F. Nomdedeu¹, J. Llorente², A. Aventín¹, S. Brunet¹, M. Baiget¹, J. Sierra¹;
¹Hospital Sant Pau, Barcelona, SPAIN, ²Hospital Joan XXIII, Tarragona, SPAIN.

Acute myeloid leukaemia (AML) is a heterogeneous disease, with individual cases showing variability in clinical presentation, blast cell morphology, therapeutic response and long-term prognosis. One of the most frequent cytogenetic abnormalities in AML is t(8;21)(q22;q22), found in approximately 10-15% of the cases. The t(8;21) fuses the AML gene to the ETO gene also identified as MTG8 (myeloid translocation gene on chromosome 8), generating predominant PCR products of a constant size (260bp), corresponding to an in-frame fusion of AML1 exon 5 to ETO exon 2.
We present one AML patient in wich an abnormal AML1-ETO fusion transcript was observed by RT-PCR. The sequence of this purified product revealed a 50 bp frameshift deletion in exon 2 of the ETO gene. The loss of 50 bp originates a disruption of the reading frame of this transcript creating a stop codon 48 aa downstream. As a consecuence of this deletion, the expected protein will be a truncated form. Due to the fact that: a) AML breakpoints are clustered between exon 5 and exon 6, and ETO breakpoints are located upstream of exon 2 and, b) the deletion is located in exon 2 of the ETO gene, the structure of this truncated fusion-protein will included only 31 aa of the ETO gene.
AML/ETO has been shown to fuction as a transcriptional activator that is critical for the tissue-specific expression of a number of haemopoietic specific genes. This case shows that most ETO sequence may be dispensable in AML/ETO+ leukemias.

G. G. Neskovic¹, E. Veljkovic¹, R. Dzodic², B. Dimitrijevic¹;
¹INS-Vinca, Belgrade, YUGOSLAVIA, ²Institute of Oncology and Radiology of Serbia, Belgrade, YUGOSLAVIA.

Medullary thyroid carcinoma (MTC) is rare malignant disease with poor prognosis. Point mutations in the RET oncogene are the hallmark in the molecular pathogenesis of this disease. This malignancy presents with sporadic and inherited etiology. In the view of the fact that genetic penetrance is close to 100%, early detection of inherited mutations in the RET oncogene offers the basis for prevention of MTC. This report describes initial efforts to establish genotype/phenotype relations in Serbia. We analyzed DNA from 35 tissue specimens of 21 patients histopathologically diagnosed as MTC and from 6 blood specimens of their relatives. Namely, two patients provided informed consent for the gene analysis of family members where heritable nature of mutated RET was confirmed. RET gene (exons: 10, 11, 13, 15 and 16) was scanned for mutations by single strand conformation polymorphism (SSCP) and heteroduplex analysis (HD). Mutations characterized by direct cycle sequencing and restriction fragment length polymorphism (RFLP).
Germline mutations were detected in 4 patients from distinct families. These mutations were: TGC618AGC, TGC629CGC, ATG918ACG and TGC634CGC. Four patients were characterized by somatically acquired mutations in the RET oncogene. One of sporadic cases presented with mutation in exon 10, TTG610TCG, (Leu->Trp) not previously described in literature. This initial research effort is currently being extended to population wide scope.

S. Kiuru-Kuhlefelt¹^,2, M. Allinen³, P. Huusko³, P. Vahteristo², H. Eerola², R. Winqvist³, H. Nevanlinna², K. Aittomäki¹^,2;
¹University of Helsinki, Helsinki, FINLAND, ²Helsinki University Central Hospital, Helsinki, FINLAND, ³University of Oulu and Oulu University Hospital, Oulu, FINLAND.

Li-Fraumeni syndrome (LFS) is a dominantly inherited cancer predisposition caused by mutations in the p53 gene. The cancer spectrum in LFS is broad complicating the identification of families. Recently Chompret et al. (2001) introduced new LFS-criteria based on the occurrence of narrow spectrum tumors including breast, sarcoma, brain, and adrenocortical cancer. The criteria include: 1. A proband with a narrow spectrum tumor <36 years with a 1° relative with a narrow spectrum tumor <46 years (other than breast cancer, if the proband had this) or with multiple tumors. 2. A proband with two narrow spectrum tumors, the first of which diagnosed <36 years. 3. A proband with adrenocortical carcinoma regardless of the age or family history. The more stringent classical criteria include: a proband with sarcoma <46 years, a 1° relative with cancer <46 years, and another 1° or 2° relative with cancer <45 years or sarcoma at any age. We used both criteria to categorize 14 families with a clinical LFS-suspicion. p53 mutation screening had also been performed. Two mutation positive families fulfilled both criteria. Ten families, of which five were mutation positive, fulfilled only the new criteria. Two mutation negative families fulfilled neither of the criteria, although one proband had both breast cancer and sarcoma, and the other had sarcoma and a sister with breast cancer. None of these cancers were, however, diagnosed <36 years. In our series, the specificity and sensitivity for the old criteria were 100% and 29%, and for the new 58% and 100%, respectively.

Molecular cytogenetic profile of invasive transitional cell urinary bladder cancer determined by comparative genomic hybridisation

T. Arsov¹, B. Zaharieva², N. Kalchishkova², C. Damianov³, V. Tabakov³, B. Tzingilev³, C. Georgiev⁴, T. Todorov⁴, D. Toncheva²;
¹Institute for immunobiology and human genetics, Faculty of Medicine, University in Skopje, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, ²Department of Medical Genetics, Medical Faculty Sofia, Sofia, BULGARIA, ³Division of Urology, National Center of Oncology, Bulgaria, Sofia, BULGARIA, ⁴Department of Pathology, Medical Faculty Sofia, Sofia, BULGARIA.

We analyzed chromosomal abnormalities (CA) in 25 tumor specimens originating from 25 Bulgarian patients with invasive transitional cell cancer of the urinary bladder by comparative genome hybridization (CHG). A total of 168 CA were detected in 21 cases (6,7 aberrations/case), the remaining 4 being normal. The distribution of the CA (gains-CG, loses-CL, whole chromosome gains-WCG) for the whole group and pT1/pT2-4, and G2/G3 subgroups are given in the table.


	PT1	pT2-4	G2	G3	Total
CG	56 (3,7/case)	64 (6,4/case)	83 (4,6/case)	37 (5,3/case)	120 (4,8/case)
CL	5 (0,3/case)	21 (2,1/case)	22 (1,2/case)	4 (0,6/case)	26 (1/case)
WCG	12 (0,8/case)	10 (1/case)	12 (0.7/case)	10 (1,4/case)	22 (0,9/case)
Number CA	73 (4,8/case)	95 (9,5/case)	117 (6,5/case)	51 (7,3/case)	168 (6,7/case)
Number cases	15	10	18	7	25

Individual case analysis of CG demonstrated that the most frequently affected regions were 1p, 1q, 2q, 3p, 7q, 16p, 17q, 19p and 19q (representing 51,4% of all chromosome gains). CL on 9, 6 and 10 represented 46% of all chromosome losses. WCG were found most frequently for chromosome 19 (5 cases).
Multiple case analysis included combined bar analysis and pool chromosome analysis. Combined bar analysis demonstrated that the minimal overlapping regions of CG were 1p36, 1q12.11-12.12, 3q12-13, 3q27, 12q12.2-12.4, 19p12 and 19q12. The 3q12-13 was characteristic for the pT1 whereas 1p36 for the pT2-4 group.
Multiple case CGH analysis (pool chromosome analysis) demonstrated significant CG at regions 1q12.11-12.13, 16q11-12 and 19p for the whole group, 1q12.11-12.13, 6q14.5-16.3 and 19p for the pT1, 19p for the pT2-4 and G2 group, and 2q12.3-14.1, 8q21.31-ter, 11q13.1-14.1, 11q14.31-ter, 12q14.2-ter, 13q32-33, 15q21.11-ter, 16q, 18q21-23.1, 19q, 20q11.41-45, 21q22.21-ter for the G3 subgroup.

Frequency of frameshift alterations in polynucleotide repeat-containing genes in HNPCC tumors

A. Paoloni-Giacobino¹, C. Rey-Berthoed², A. Couturier², S. E. Antonarakis¹, P. Hutter²;
¹Division of Medical Genetics, Geneva University Hospital, SWITZERLAND, ²Unit of Genetics, Institut Central des Hôpitaux Valaisans, Sion, SWITZERLAND.

DNA sequences made of mono-, di- and trinucleotide sequences are prone to replication errors and thus constitute mutational hot spots. This is well illustrated by the occurrence of DNA microsatellite instability in tumors from patients affected with hereditary non-polyposis colorectal cancer (HNPCC), resulting from a defect in a gene that controls post-replicative DNA mismatch repair (MMR). We analysed 10 tumors (9 colorectal and 1 ovary carcinomas) from HNPCC patients carrying a germline mutation in the MMR gene MLH1. For each tumor, error rates were measured by sequencing 20 to 50 cloned amplicons from 4 genes involved in either cell proliferation or apoptosis. The polynucleotide tracts selected consisted in a 10 A coding repeat in the TGFbRII gene, an 8 G coding repeat in the BAX gene, a 7 A coding repeat in the CASP1 gene, and a 7 CCA repeat in the 3’-UTR of the APP gene. Substantial inter-tumors variations were observed in the pattern of alterations, with error rates varying between 12 and 80% for TGFbRII, 2 and 84% for BAX, 0 and 30% for CASP1 and 0 to 18% for APP. In contrast with previous results obtained not from single molecule analysis, the BAX error rate did not exceed 20 % in 9 tumors. High error rates in more than one gene in a same tumor suggested additive selective effects from different alterations. These data on somatic frameshifts in specific genes may contribute to better tumor classification and outcome prediction.

VHL gene mutations in Czech and Slovak patients with VHL disease and sporadic hemangioblastoma

A. Krepelova¹, J. Plas², E. Kantorova³, F. Cisarik⁴, M. Puschauerova⁵, V. Kovacova⁶;
¹Institute of Biology and Medical Genetics, 1st Medical Faculty, Charles University, Prague, CZECH REPUBLIC, ²Clinics of Neurosurgery, 1st Medical Faculty, Charles University, Prague, CZECH REPUBLIC, ³Department of Medical Genetics, Municipal Hospital, Ceske Budejovice, CZECH REPUBLIC, ⁴Department of Medical Genetics, Municipal Hospital, Zilina, SLOVAKIA, ⁵Department of Medical Genetics, Municipal Hospital, Presov, SLOVAKIA, ⁶Department of Medical Genetics, Faculty Hospital, Bratislava, SLOVAKIA.

The Von Hippel-Lindau syndrome is a dominantly inherited familial cancer syndrome with an incidence of 1 in 53000 to 85000 persons, predisposing to retinal, cerebellar, and spinal hemangioblastoma, renal cell carcinoma, pheochromocytoma and pancreatic tumors. The disease is caused by germ-line mutations in the VHL tumor suppressor gene. Between 1996 and 2001, sixteen unrelated probands fulfilling clinical criteria of VHL disease and seven patients with sporadic hemangioblastoma (2 retinal, 5 cerebellar) were included to the study. Detection of mutations in the VHL gene was performed by Southern blot analysis, DGGE of exons 2 and 3, and DNA sequencing of exon 1 in all samples and of exon 2 or 3 in the DGGE-positive samples. Germ-line mutations of the VHL gene were identified in 13/16 (81%) patients with VHL disease and in 2/7 (29%) patients with sporadic hemangioblastoma. Among the identified mutations one mutation was novel (Y112S) and 14 mutations have been already described by others (P25L, S65L, S65W, F76del, S80N, L101R, G132X, R161X, 2x R167W, 3x R167Q, 10kb genomic deletion of exon 3). Except the P25L substitution, which could represent a polymorphism, the mutations are pathogenic. Moreover, one rare variant, IVS2+8c>t, was also identified in one healthy individual. In seven families, the identification of mutation in proband was followed by presymptomatic DNA testing in 29 relatives at risk. Negative results of mutation analysis in 3 patients with familial VHL disease require further study. This work was supported by the Grant Agency of Charles University (Grant No. 36/1996).

Cyclin D1 Cd242 G-A Polymorphism is not a Risk Factor for Colorectal Cancer in Patients from The Republic Of Macedonia

A. M. Stefanovska¹, T. Josifovski², M. Panovski², D. Jasar³, G. Zografski³, K. Stefanovski¹, G. D. Efremov¹, A. J. Dimovski¹;
¹Macedonian Academy of Sciences and Arts, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, ²Clinic for Abdominal Surgery, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA, ³Institute of Oncology, Skopje, THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA.

Cyclin D1 is involved in the regulation of the transition from G1 to S phase of the cell cycle and is often over-expressed in tumors. A G->A polymorphism at CD242 of the CCND1 gene was implicated as an important risk factor for development of colorectal cancer at a younger age, both among HNPCC and sporadic cases. We evaluated the prevalence of the CCND1 polymorphism using RFLP-PCR among 136 colorectal cancer patients and 170 normal controls from Macedonia. The allele frequencies and the distribution of different genotypes of the case subjects was similar to those of the controls (A/G allele 0.55/0.45 and 0.51/0.49, respectively; AA/AG/GG genotypes 33.1%/44.1%/22.8% and 25.3%/51.2%/ 23.5%, respectively). No differences of allele frequencies and genotype distribution were observed when cases were grouped by age, Dukes stage, localization, histological type, MSI, p53 and 18q status (p>0.05). The observed differences in the CCND1 genotypes between colorectal cancer patients from Macedonia and the USA are similar to the differences that were observed for the transforming growth factor b-type I receptor polymorphism (Stefanovska et al., Cancer Res., 61:8351, 2001), and strongly suggests that certain environmental factors influence colorectal cancerogenesis through multiple mechanisms.

The role of the H-ras oncogene in neurometabolism: Do depression and cancer stem from a common etiology?

An association between depression and cancer has long-been recognized. Essentially, the prevailing hypothesis is that neurometabolic disturbances inherent in depression effect a change in endocrine and immunological systems making one at greater risk for cancer. More recently, molecular epidemiologists have postulated that the linkage between depression and later cancer onset might be genetic in nature. Specifically, the H-ras oncogene has recently been implicated as a source of dysfunctional neurometabolism, presenting as an alternative hypothesis to the prevailing view that cancer results from a weakened immune system. Essentially, it has been theorized that dysregulation of this cancer gene results in impaired serotonin and dopamine synthesis secondarily. However, whatever the cause of depression/cancer comorbidity, studies have failed to confirm a definite link between depression and later onset of cancer. I expect that two confounding variables exist within the most recent depression/cancer correlational studies. First, these studies have failed to incorporate the growing body of literature delineating the role of the H-ras oncogene in neurometabolism. It is plausible that a dysfunctional H-ras gene may be the causal link between depression and cancer. If so, longitudinal studies seeking to correlate depression and cancer should parse out cancers known to be associated with the H-ras gene. Second, existing studies fail to employ an accurate classification and diagnosis of depression. My hypothesis is that a depression/cancer correlation is caused by a dysfunctional H-ras oncogene which acts primarily to disrupt neurometabolism, the serotonin and dopamine biosynthetic pathways specifically, and secondarily causes cancer over time.

Identification of Deoxyribonucleic Acid Copy Number Changes in Larynx Carcinoma by Comparative Genomic Hybridization

G. Luleci¹, I. Keser¹, A. Toraman¹, G. Ozbilim², K. Guney³;
¹Akdeniz University, Faculty of Medicine, Department of Medical Biology and Genetics, Antalya, TURKEY, ²Akdeniz University, Faculty of Medicine, Department of Pathology, Antalya, TURKEY, ³Akdeniz University, Faculty of Medicine, Department of Otolaryngology, Head and Neck Surgery, Antalya, TURKEY.

Carcinomas of the head and neck represent 5% of all human cancers, squamous cell carcinoma being the most important group. Head and neck carcinomas including laryngeal carcinoma have been investigated recently by various molecular, cytogenetic, and molecular cytogenetic techniques.
In this study, comparative genomic hybridization(CGH) technique was used to identify DNA copy number changes in 15 paraffin-embedded tissue samples of the larynx carcinoma. Of these patients, 13 were male, and 2 were female. DNA copy number changes were detected in 10 of the 15 patients(66.6%). While 3 of the 10 patients had polyploidy, other 7 patients were found to have gains and losses on different chromosomes. 5 cases had normal CGH profiles. The results of the study were compared with literature reported previously and similar findings were detected in chromosomes 5p, 7q, and 18p in larynx carcinoma. Also, loss of chromosome 22q13-qter was found as a novel site in a case with larynx carcinoma.
Although the number of tumor samples investigated is rather low, our results suggest that the chromosomal loci which affect the differentation and progression of the larynx carcinomas can be detected by CGH technique.

Cytogenetic and molecular response in CML patients on Interferon-a 2b (IFN-a 2b) therapy using conventional cytogenetics and FISH analysis

R. Talwar¹, K. Kucheria², V. P. Chaudhry²;
¹All India Institute of Medical Sciences, New Delhi, India, New Delhi, INDIA, ²All India Institute of Medical Sciences, New Delhi, INDIA.

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterised by the presence of Philadelphia (Ph¹) chromosome resulting from balanced reciprocal translocation, t(9;22)(q34;q11) leading to the formation of bcr/abl fusion gene. Studies have shown that IFN-a therapy induces both cytogenetic response (reduction in Ph+ cells) and molecular response (reduction in the bcr/abl+ cells) in a significant proportion of CML patients thereby improving their prognosis and survival. To the best of our knowledge, no published reports are available from India using molecular methods for evaluation of minimal residual disease. The present study was conducted to evaluate the cytogenetic and molecular response in CML patients on Interferon-a 2b (IFN-a 2b) therapy. Sequential cytogenetic analysis was done using standard methods in 45 CML patients on IFN-a 2b therapy up to a variable period of 3 years. Further dual colour Fluorescence In Situ Hybridisation (FISH) analysis using specific probes for bcr and abl genes was done to assess the molecular response. Complete cytogenetic response (CCR) was observed in 8 patients. Of these 8 patients in CCR, 4 were negative for the bcr/abl fusion gene implying a complete cytogenetic and molecular response while the remaining 4 showed bcr/abl fusion signals representing residual disease. Thus the present study stresses on the need for sequential cytogenetic and molecular analysis in CML patients on therapy. The importance of using FISH on interphase nuclei and poorly spread metaphases that cannot be analysed using conventional cytogenetics is also highlighted.

High frequencies of primary multiple melanomas in families with CDKN2A mutations.

S. Majore¹, C. Catricalà², F. Binni¹, P. De Simone², C. De Bernardo³, L. Eibenschutz², P. Grammatico¹;
¹Medical Genetics, University "La Sapienza", Rome, ITALY, ²San Gallicano Dermatological Institute, Rome, ITALY, ³Medical Genetics, San Camillo-Forlanini Hospital, Rome, ITALY.

About 10 % of melanoma is inherited in an autosomal dominant fashion with variable penetrance and 50-80% of the families are linked with 9p21. CDKN2A, located in 9p21, consists of three coding exons and encodes the cell cycle inhibitor, p16. This protein plays a role as a negative regulator of the cyclin D1/CDK4/p16/pRb signaling pathway, the major growth control pathway in the cell cycle. In our study, on a wide sample of melanoma-prone families, we found 8 pedigrees in which a CDKN2A mutation was evidenced, some already described in literature and some as a new mutation (Pro48Thr, ivs1+2(T-C), 201delC). Analyzing clinical data of these 8 families we observed that in 6 (75%) it was present at least one multiple primary melanoma (MPM). The presence of a MPM in a so large amount of the mutated cases brings us to consider CDKN2A mutations as responsible to a high constitutional risk for melanocytes transformation. For this reason, we think relevant to include CDKN2A mutational screening in all the families in which there is one patient with MPM, even if not familial.
In addition in 2 out of our 8 families a larynx carcinoma was also present, supporting the hypothesis of a related risk for this tumor in CDKN2A mutation carriers.
Al last, we also observed, in 5 out of these 8 pedigrees, the presence of only two melanoma patients suggesting the opportunity to extend CDKN2A mutations analysis also to families with less then three affected relatives, at least in Italian population.

Mutational and expression analysis of the NF1 gene argues against a role as tumor suppressor in sporadic pilocytic astrocytomas

K. Wimmer¹, M. Eckart¹, B. Meyer-Puttlitz², C. Fonatsch¹, T. Pietsch²;
¹Universität Wien, Institut für Medizinische Biologie, Wien, AUSTRIA, ²Universität Bonn, Institut für Neuropathologie, Bonn, GERMANY.

Children with Neurofibromatosis type I (NF1) have an increased risk for developing pilocytic astrocytomas (PAs). LOH studies demonstrate frequent loss of the NF1 gene in NF1-associated PAs. Further, it has been demonstrated that loss of neurofibromin in a NF1-associated PA is associated with elevated Ras-GTP levels. However, conflicting results on the role of the NF1 gene in the development of sporadic PAs have been reported. Therefore, we investigated 14 sporadic PAs for NF1 mutation and for LOH within the NF1 locus. The protein truncation test, which identifies approximally 80% of the mutations found in NF1 patients, failed to detect NF1 mutations in 10 analyzed tumors. LOH analysis was unable to reveal evidence for disruption of the NF1 gene in 11 informative cases. The GTPase-activating domain of the NF1 gene is expressed in two isoforms. Using real-time PCR we investigated the ratio of the isoforms in 14 sporadic PAs and compared it to the ratios in normal adult tissues, glioblastomas and neuroblastomas. In accordance with previous reports we found marked predominance of the type II transcripts in PAs as well as in two glioblastomas and in all analyzed adult tissues including brain. In contrast, marked predominance of the type I transcripts were observed in neuroblastomas. Our results argue against a role of the NF1 gene as tumor suppressor in sporadic PAs and suggest that the predominant expression of the type II NF1 transcript in PAs reflects the differentiation stage of the cells rather than being a response to elevated cell proliferation.

Characteriziation of differentially expressed candidate genes associated with gynaecological tumors

A. Sadr-Nabavi¹, M. Du², B. Betz², D. Schmidt¹, E. Dahl³, S. Gelling³, B. Hinzmann³, P. R. Kreutzfeld⁴, R. Schmutzler⁴, A. Meindl¹, D. Niederacher², G. C. C. Coordinator: A. Rosenthal³;
¹Abt. Päd. Genetik der Kinderpoliklinik der LMU, München, GERMANY, ²Molekulargenetisches Labor der Universitätsfrauenklinik, Düsseldorf, GERMANY, ³metaGen Pharmaceuticals GmbH, Berlin, GERMANY, ⁴Labor für Molekulare Onkologie, Universitätsfrauenklinik, Bonn, GERMANY.

More than 600 candidate genes were identified as differentially expressed in gynaecological tumors by "in-silico" approaches and fifty of them, considered as either putative tumor suppressor genes or oncogenes, were selected for further analysis in sporadic gynaecological tumors by the GCC (Gynaecological Cancer Consortium).
To confirm the electronic Northern data, seven putative TSGs were investigated by hybridization of cancer arrays (Clontech cancer profiling arrays) against gene-specific probes. Two genes (designated bn39, bn40) were found to be expressed lower in up to 80% of breast or ovarian cancer samples compared to matched corresponding normal tissues, respectively. These data could be supported by real time PCR (TaqMan) performed in matched tissue samples collected at our hospitals.
For each of the two determined TSG candidates, 20 LOH positive tumor samples were screened for mutations by the DHPLC technique. No functional mutations were found in one downregulated candidate gene, only one frame-shift mutation and one missense mutation were detected in the other assumed TSG (bn40). To characterize three putative oncogenes, expression was analysed by hybridization of cancer arrays and assumed amplification was determined by real-time PCR or quantitative differential PCR. One candidate gene (bt11) was shown to be frequently over-expressed especially in ovarian cancer while gene amplification could not be shown.
Conclusions: About 30% of the nominated candidate genes identified by database screening were shown to be differentially expressed in-vivo. Down-regulation of gene expression in cancer tissues might be explained by epigenetic inactivation/activation mechanisms rather than mutations or gene amplification.

HNPCC - two different entities in regard of mutation analysis and clinical phenotype

Y. Müller-Koch¹, H. Vogelsang², G. Keller³, R. Kopp⁴, P. Lohse⁵, M. Gross⁶, U. Schiemann⁶, G. Baretton⁶, D. Aust⁶, B. Kerker⁶, G. Henke⁶, J. Daum⁶, E. Holinski-Feder⁷;
¹University, Dept. Medical Genetics, GERMANY, ²University, Dept. Surgery, Munich, GERMANY, ³University, Pathology, Munich, GERMANY, ⁴Department of Surgery, Munich, GERMANY, ⁵Department clinical chemistry, Munich, GERMANY, ⁶University, Munich, GERMANY, ⁷University, Dept. Medical Gentics, Munich, GERMANY.

HNPCC is caused by heritable mutations in the DNA mismatch repair genes.
For 254 patients that fullfilled one of the Bethesda Kriteria 1-7, mutation analysis for hMLH1, hMSH2 and hMSH6, microsatellite analysis and immunohistochemistry for hMLH1, hMSH2 and hMSH6 was performed. 25 pathogenic mutations and 24 missense variations were found. Data concerning the sensitivity of microsatellite analysis and immunohistochemistry will be presented.
The cohort included 51 families fullfilling the Amsterdam Criteria. First, 11 patients with MSI-H tumors and truncating mutations. Second 5 patients with MSI-H tumors without mutation in hMLH1, hMSH2 and hMSH6. Third 7 patients with MSI-H tumors and missense mutations in hMLH1 or hMSH2 or truncating mutations in hMSH6. Fourth, 6 patients with MSS or MSI-L tumors with suspected missense mutations in hMLH1 or hMSH2 or truncation mutatins in hMSH6 and fith, 22 patients with MSS tumors without mutations in hMLH1, hMSH2 or hMSH6.
Clinical and molecular data of the first and the fourth group revealed differences concerning age of onset, tumor spectrum within the families, tumor localisation and pathohistological features. There is an earlier age of onset and a broader spectrum of tumors in the families of group 1. Tumors are more right sided and more frequently show the typical HNPCC-associated histopathological features. In hMSH2 endometrial cancer seems to cluster with mutations in exon13. Life expectancy of female mutation carriers is increased compared to male mutation carriers.These data point towards the existence of at least two entities of hereditary colon cancer other than FAP.

Frequent epigenetic silencing of the CpG island promoter of RASSF1A in thyroid carcinoma

U. Schagdarsurengin¹, O. Gimm², C. Hoang-Vu², H. Dralle², G. Pfeifer³, R. Dammann¹;
¹AG Tumorgenetik, Med. Fakultaet, University Halle, Halle, GERMANY, ²Klinik fuer Allgemein-, Viszeral- und Gefaesschirurgie, University Halle, Halle, GERMANY, ³Dep. of Biology, Beckmann Research Institute, City of Hope Cancer Center, Duarte, CA.

LOH of chromosome 3p21 is one of most frequent alterations in solid tumors. RASSF1A-isoform is epigenetically inactivated in a variety of human primary tumors. We investigated expression and methylation status of RASSF1 gene in 38 primary thyroid tumors (1 PDTC, 5 MTC, 10 FTC, 9 UTC, 13 PTC) and 9 thyroid cancer cell lines. In all cell lines the RASSF1A promoter CpG-island was completely methylated and expression was absent. Treatment of these cell lines with DNA methylation inhibitor 5-aza-2-deoxycytidine reactivated the transcription of RASSF1A. In 71% of primary thyroid carcinomas the RASSF1A-promoter was hypermethylated. Methylation frequency was higher in aggressive forms of thyroid carcinoma ( 80% of MTC, 78% of UTC and 70% of FTC) compared to 62% in more benign PTC. RASSF1A-inactivation was detected in all stages of thyroid carcinoma scored by pTNM-classification. Additionally, we analyzed the methylation frequency of CpG-island of cell cycle inhibitor p16INK4a in the same thyroid tumors. The p16-gene was inactivated in 56% and 25% of cell lines and primary tumors, respectively. p16 methylation was detected in 56% of UTC, in 10% of FTC and in 25% of PTC, but not in MTC. In UTC, which belongs to the most aggressive carcinomas in humans, the most common combined inactivation of RASSF1A and p16 was detected. In general, 90% of tumors with p16 inactivation were also silenced for RASSF1A expression. However, RASSF1A hypermethylation was detected three times more frequently in thyroid cancers. Thus, RASSF1A inactivation may play a crucial role in the malignancy of thyroid carcinoma.

Analysis of genomic copy number and expression of genes in the chromosomal band 8q11 in hepatoblastoma

A. Zatkova¹, J. Rouillard², B. J. Lamb², R. Kuick², M. Eckart¹, D. von Schweinitz³, C. Fonatsch¹, T. Pietsch⁴, S. M. Hanash², K. Wimmer¹;
¹Institut für Medizinische Biologie,Universität Wien, Wien, AUSTRIA, ²Department of Pediatrics, University of Michigan, Ann Arbor, MI, ³Abteilung für Kinderchirurgie,Universtitäts-Kinderspital Beider Basel, Basel, SWITZERLAND, ⁴Institut für Neuropathologie, Universität Bonn, Bonn, GERMANY.

Recently, the correlation of comparative genomic hybridization (CGH) results with clinical data in a large series of hepatoblastomas, has uncovered that gain or amplification of chromosomal 8q material is associated with poor prognosis of this highly malignant childhood tumor. The minimal amplified region was defined to chromosomal bands 8q11.2-q13. In an attempt to identify hepatoblastoma-related genes in this region we implemented a strategy that combined restriction landmark genomic scanning (RLGS) and a genomic copy number assay based on real-time PCR. RLGS analysis uncovered six chromosome 8 derived fragments amplified in one hepatoblastoma. Virtual genomic scann, a novel informatic tool for sequence prediction of RLGS fragments, identified the sequence of five of these fragments. Thus, the critical region was defined to chromosomal band 8q11 extending at least between the two genes, SOX17 and Lyn. Four microsatellite markers and genes located between or adjacent to these genes as well as four control markers were selected for genomic copy number analysis. Seven of 19 tumors investigated (37%) showed gain or amplification in this region, including three tumors in which a gain was undetectable by CGH analysis. The expression of five genes and ESTs, located within the newly defined minimal amplified region was assayed by real-time RT-PCR in 10 hepatoblastomas. The gene encoding the transcription factor PLAG1 showed increased RNA expression in all but one hepatoblastoma when compared to normal liver. The possible role of PLAG1 as an activator of fetal growth factor IGF2 in hepatoblastoma is currently investigated.

M. Stefanovic¹, A. Begonja¹, E. Topic¹, I. Curcic², A. Simundic¹;
¹University Hospital "Sestre milosrdnice", Zagreb, CROATIA, ²Dubrava University Hospital, ENT Department, Zagreb, CROATIA.

We investigated the possible association of drug metabolizing enzyme system CYP P450 CYP2D6 and its null alleles (CYP2D6*3, *4, *5, *6, *7, and *8) with incidence of tumors in patients having head and neck cancer (HNC). It is known that persons bearing two null alleles poorly metabolize some common drugs (Poor Metabolizer Phenotype – PM) as well as other foreign and carcinogenic substances. Persons with only one disrupted CYP2D6 gene (bearing one normal and one null allele) are considered to be Intermediate metabolizer phenotype (IM). We genotyped 145 controls, and 42 HNC patients by Multiplex Allele Specific PCR on whole blood DNA. Study results showed allelic frequencies for *3, *4 and *6 alleles (only alleles observed) in controls to be 1.4%, 11.0% and 1.0%, respectively; among them we found 2.1% PMs and 22.8% IMs. In cancer patient’s group allelic frequencies for *3, *4 and *6 were 1.2%, 19.0% and 3,6% respectively, and no other alleles were found; among them we found 2.4% PMs and 42.9% IMs. Results of our study showed statistically significant difference for genotype frequencies (Chi-square; p=0.025) and predicted phenotype (Chi-square; p=0.034). IM phenotype showed to be responsible for increased risk to HNC (Odds ratio 2.6; 95%CI= 1.248 - 5.193). To confirm our preliminary findings, further study on a larger group is planned.
E-mail: mario.stefanovic@zg.hinet.hr

Glutathione S-transferase polymorphisms influence plasma antioxidants level and oxidative DNA damage.

A. Ficek¹, M. Dusinská¹, A. Horská¹, K. Raslová¹, H. Petrovská¹, B. Vallová¹, M. Drlicková¹, S. G. Wood², A. Stupáková¹, J. Gasparovic¹, P. Bobek¹, A. Nagyová¹, Z. Kováciková¹, P. Blazicek³, U. Liegebel⁴, A. R. Collins²;
¹Department of Molecular and Genetic Toxicology, Institute of Preventive and Clinical Medicine, Bratislava, SLOVAKIA, ²Rowet Research Institute, Aberdeen, UNITED KINGDOM, ³Hospital of Ministry of Defence, Bratislava, SLOVAKIA, ⁴Friedrich-Schiller-Universitat, Jena, GERMANY.

Glutathione S-transferases (GST), xenobiotic detoxifying enzymes, involve in defences against oxidative stress and metabolism of plenty carcinogens. Hence the GST polymorphisms are supposed to be significant determinants of individual cancer risk.
We screened 155- middle aged men (51 smokers and 104 non-smokers) for GSTT1null, GSTM1null and GSTP1b polymorphisms and compared them with parameter of oxidative stress at the level of oxidative DNA damage measured in lymphocytes, and with plasma antioxidants level.
Smokers had on average significantly lower levels of plasma antioxidants and higher amounts of oxidised purines and pyrimidines measured in lymphocyte DNA. The observed genotype frequencies were represented as follows: 48% GSTM1null, 21% GSTT1null and 11% GSTP1b/b. The GSTT1 null genotype was associated with decreased Vitamin C concentration compared to GSTT1+ genotype, while Vitamin C was higher in GSTM1 null compared with GSTM1+. The homozygous GSTP1 a/a genotype was associated with significantly higher levels of GST activity measured in lymphocytes, in comparison with the b/b genotype. Using multifactorial statistical analysis significant interactions were found between smoking, GSTP1 genotype, plasma Vitamin C, and purine base damage in lymphocyte DNA. Vitamin C concentrations were substantially higher in b/b non-smokers compare with b/b smokers, whereas this phenomenon was not observed neither in a/a nor a/b groups. The b/b smokers had on average about twice as much oxidised purine base damage as the non-smokers with that genotype, and higher levels than the other smokers. In contrast, the link between smoking and oxidised pyrimidines in DNA was seen only in the GSTT1 null group.

V. van Scherpenzeel Thim, C. Verellen-Dumoulin, C. Sibille;
Center for Human Genetics,UCL,St-Luc, Brussels, BELGIUM.

The Carney triad is a rare association of gastric epithelioid leiomyosarcoma, functioning extra-adrenal paraganglioma and pulmonary chondroma, affecting specifically young individuals. It probably has an autosomal dominant pattern of inheritance. However, the molecular basis of this unusual syndrome has not yet been elucidated. We report a new case of incomplete Carney triad in a female who presented first with a pulmonary chondroma at age 21. At age 27, she developed a primary gastric leiomyoblastoma. Seven years later, a total gastrectomy was performed following the discovery of two additional gastric tumors. Intriguingly, karyotypic analysis of PHA-stimulated peripheral blood lymphocytes revealed an apparently balanced translocation t(1;19) (p11-13;p11-12). This chromosomal abnormality seemed to be inherited from her phenotypically normal father. Possible mechanisms whereby such a familial translocation could have a pathogenic effect in our patient include a chimeric fusion gene generated by a complex rearrangement, a de novo duplication of a protooncogene or a de novo microdeletion in the translocated chromosomes disrupting a putative tumor suppressor gene at the breakpoints. This is the first report of a cytogenetic abnormality associated with the Carney triad. The translocation breakpoints in this patient may become candidate regions for susceptibility genes causing this uncommon disorder. Further genetic investigations are carried out by Fluorescent in situ hybridization (FISH) to map potential genes in the 1p11-13 breakpoint region, interestingly containing the N-RAS and notch2 genes.

Hypermethylation of the 5´ promoter region represses Caveolin-1 gene expression in a human prostate cancer cell line

J. Häusler, P. Geyer, J. Häussler, W. Vogel;
University of Ulm, Ulm, GERMANY.

LOH of chromosomal region 7q31.1 has been implicated in the pathogenesis of many human cancers, including prostate cancer. The genes encoding Caveolin-1 and -2 are localized at 7q31.1. The Cav-1 promoter contains several CpG dinucleotides of which four are methylated in two human breast cancer cell lines. They fail to express Cav-1 mRNA, suggesting an epigenetic mechanism of Cav-1 gene regulation in these cell lines. We´re investigating the role of Cav-1 in prostate cancer by investigating the expression of Cav-1 in human cell lines derived from normal prostate and prostate cancer. Our findings show that Cav-1 expression is absent from the cell line LNCaP on RNA and protein level. To test the hypothesis that DNA methylation in the promoter correlates with down-regulation of Cav-1 gene expression, we determined the methylation status of additional prostate cell lines. A minimal promoter of Cav-1 contains seven CpG sites and we found a very heterogeneous methylation profile at four of these. Only the promoter region of LNCaP showed almost complete methylation. The functional importance of these CpG sites was demonstrated by an in vitro reporter gene assay which revealed that the cav-1 promoter activity in vitro is regulated by methylation of four CpG sites within its minimal promoter region. We conclude therefore that repression of the Cav-1 gene in LNCaP cells is due to DNA methylation. Furthermore, results from bandshift assays demonstrated that a yet unknown methyl-CpG-binding protein interacts with the methylated Cav-1 promoter region examined and that this protein is different from MeCP2.

A. D'Angelo¹, A. Andre'¹, V. Aglio¹, S. Olivieri², L. Garzia¹, G. Arrigoni², R. Lanzi², A. Ballabio¹, M. Zollo¹;
¹Telethon Institute of Genetics and Medicine (TIGEM), Naples, ITALY, ²HSR San Raffaele, Milan, ITALY.

We report here a functional characterization of prune protein and its possible correlation with Neuroblastoma cancer. A newly identified phosphodiesterase
(PDE) PDE11A was found to contain a catalytic site motif equally present in the human prune protein and corresponding to the third DHH motif of prune.
A scintillation proximity assay was performed to investigate prune phosphodiesterase activity both on transiently transfected COS-7 crude extracts and on the purified histidine-tagged prune protein produced by the Baculovirus expression system. We demonstrate that prune is able to act as a phosphodiesterase preferentially on cAMP substrate. Furthermore, prune is able to interact with NM23-H1, an antimetastatic protein but its interaction is impaired with NM23-H1S120G, a mutation associated with advanced stages of Neuroblastoma. By in vivo co-immunoprecipitations and interaction mating assays we demonstrate the interaction between nm23-H2 and a series of described nm23-H2 protein mutants.
PRUNE protein is predominantly a cytoplasmic protein. By immunofluorescence experiments we investigated prune and nm23 localization in SK-N-SH, SK-N-BE, SH-5YSY and IMR-32 Neuroblastoma-derived cell-lines. A prune predominant nuclear localization was observed for the first time. Expression of prune protein was examined by immunohistochemistry analysis on paraffin embedded tissues from 5 Neuroblastoma affected patients revealing high protein expression
in the nucleus and its association with poor outcome. We are preparing stable clones of SH-5YSY cells by transfection (pBABE retroviral vector) of prune cDNA in order to study the growth properties of the stabilized cells and isolate other putative prune nuclear interactors by mono-dimensional SDS-page analysis and Mass Spectrometry.