(1) Workshop on “Molecular Pathology”
Our purpose was to consider the role and impact of studies of the molecular pathogenesis of human cancers. The workshop was organized by Dr. Robert W. Miller and Dr. Haruo Sugano, with the assistance of Dr. William G. Nelson and Dr. Yusuke Nakamura, and held at the Hawaii Prince Hotel in Honolulu, Hawaii, on February 12-13. 1996. The scope of the workshop presentations covered many common cancers, including cancers of the lung, liver, breast, prostate, colon, and head and neck, and chronic myclogenous leukemia, as well as considering rate cancers, such as those arising in patients afflicted with Werner syndrome. Emphasis was placed on new molecular techniques and strategies and the application of these tools to the discovery of new somaticallly altered cancer genes, to the analysis of altered cancer gene product function(s), and to improving cancer diagnosis and staging. Dr. Nelson prepared the summaries of presentations by the US participants and Dr. Miller did so for the Japanese.
Topographic Analysis of Multistep Tumor Progression.
Dr. Darryl Shibata opened the workshop with a presentation that addressed the mechanism by which individual carcinoma cells comprising human cancers exhibit marked genetic heterogencity. He described an experimental alpproach involving careful microdissection of normal and neoplastic colonic tissue, located in different topographic regions within single tumor resection specimens. DNAs extracted from different regions of a carcinoma tended to reveal concordant mutations in p53, K-ras, and APC genes suggesting that such mutations arose early during carcinogenesis, at least before the prolification of the majority of the carcinoma cells. Of interest, an examination of regional variations in microsatellite repeat lengths in colonic carcinoma lesions exhibiting microsatellite repeat length instability (likely attributable to early loss of mismatch repair activity) permitted an assessment of cancer cell genetic legacy analogous to approaches undertaken to elucidate the genetic legacy of populations of organisms. In vitro analysis of the acquisition of microsatellite repeat length alterations supported the proposition that the extent of diversity increased with the number of cell replications. In vivo, assessment of the extent of microsatellite repeat length heterogeneity in colonic neoplasms indicated a greater repeat length diversity in adenomas than in nearby carcinomas, supporting the hypothesis that the adenomas antedated, and likely originated, the carcinomas.
Application of Fish to Diagnosis of Cancer in Breast Tumors.
Fluorescence in situ hybridization (FISH) on metaphase or interphase chromosomes, coupled with molecular and cytogenetic information, is a powerful tool for gene mapping and cancer genetics. Dr. Joji Inozawa investigated FISH as a tooi for diagnosis of primary breast tumors by seeking numerical aberrations of chromosomes 1, 11, and 17 by multicolor FISH of selected probes to cell nuclei derived from fine-needle aspiration biopsies of 106 breast tumors. The tumors consists of 78 primary breast tumors, 2 phyllode tumors and 26 benign breast tumors. Sufficient materials for FISH analysis could be obtained by aspiration biopsy in 98 cases of l06 breast tumors (93.4%). To define aneusomy of chromosomes l, Il, and 17, multicolor FISH was carried out on normal breast tissues obtained by fine-needle biopsy with centromere-specific probes for each chromosome, and hybridization signals were examined in more than 20O interphase nuclei. None of the benign tumors and phyllode tumors showed evidence of aneusomy for any of these chromosomes. However, 71 of the 74 breast cancers (95.9%) for which sufficient material was available showed aneusomy for at least one of the three chromosomes tested. Further, FISH analysis suggested a possible correlation between aneusomy of chromosome 17 and metastasis to regional lymph nodes (chi-square test, p<0.05). In this series, twelve cancers had been diagnosed as “class II or class III” preoperatively by conventional cytology of aspiration biopsy material. Ten of these tumors showed aneusomy for at least one chromosome and were classified as malignant by the FISH analysis. On the other hand, two of three intraductal papillomas, representing one of the most difficult types of tumor to diagnose histologically, had been diagnosed as “class IV” preoperatively. In the three intraductal papiilomas, however, we could not detected aneusomy for tested chromosomes by FISH and so we classified them as a benign group. These results suggest that FISH analysis of fine-needle aspiration biopsies can be a practical and useful method for diagnosis of carcinoma in breast tumors.
Identification and Detection of Mutant Genes Relevant to Cancer Diagnosis.
Dr. Jeffrey Sklar described an innovative strategy for the discovery of somatically altered genes in human cancers located at particular chromosomal loci. To develop this new strategy, initial studies were directed at the chromosome region encompassing 7q22, a region suspected to harbor a tumor suppressor (or tumor metastasis suppressor) gene for prostate cancer. The approach presented involved annealing of prostate cell cDNAs, prepared from prostate cell mRNAs Via reverse transcription-PCR using oligonucleotide primers that were highly degenerate at their 3 ends and sequence tagged at their 5’ ends to permit ready reamplification, to normal metaphase chromosomes in situ. Chromosome region-specific cDNAs were then isolated by microdissection of the chromosomal region of interest (e.g. 7q22). Prostate cell cDNAs annealing to this chromosomal area were recovered by reamplification via PCR using primers complementary to the sequence tags comprising the 5’ end of the original degenerate RT-PCR cDNA primers. By this strategy, genes expressed in specific tissue specimens (such as in normal or neoplastic prostate tissues) and located at specific chromosomal loci could be selectively recovered for further study. Dr. Sklar also presented a novel strategy for mutation detection that his laboratory has applied to screening chromosomal locus-specific tissue-specific cDNAs generated using the approach described above. This mutation detection strategy employed bacteriophage resolvases T4 endonuclease VII and T7 endonuclease I to nick hetcroduplex DNAs prepared from annealing mismatched normal/tumor cDNA mixtures. Dr. Sklar claimed that this technique has been found to detect as many as 95% of mutations. Thus far, most of the genes known to be somatically altered during human cancer pathogenesis have been discovered via laborious positional cloning efforts directed at chromosomal regions known to be frequently lost in cancer cells. The application of new gene discovery strategies, such as those discussed by Dr. Sklar, may greatly accelerate the identification of new critical genes for human cancer development and for malignant cancer progression.
Genetic and Epigenetic Mechanisms of Tumor Progression: Invasion and Metastasis of Liver and Other Digestive Organ Cancers.
Most human liver cancer is associated with hepatitis B or C infection. Long after infection with these viruses, hyperplastic foci and small nodular lesions (adenomatous hyperplasia), which do not destroy substantially the underlying liver structure, develop in liver showing chronic hepatitis or cirrhosis. Some of these nodules show structural abnormalities and are diagnosed as well differentiated HCC (early HCC). Increase in cell proliferation occurs at an early stage, and the presence of polyploid cells in early HCC is important for progression. Late stage events may include p53 mutations, loss of heterozygosity of chromosomes 4, 16, 17 and others, and hypervascularization. Also observed are nodule-in-noduie lesions composed of a peripheral area of early HCC and a central area of less well-differentiated HCC. These findings clearly indicate the multistage nature of human hepatocarcinogenesis consisting of clonal expansion and successive subclonal progression.
Invasion and metastasis are critically important biological manifestations of tumor progression. In this area, our studies have been focused on the cadherin system, which mediates Ca ++ -dependent homophilic cell-cell adhesion. Studies carried out by our group and others have indicated that the cadherin-mediated cell adhesion system acts as an “invasion suppressor.” This system in inactivated by multiple mechanisms in carcinoma cells that are poorly differentiated and highly invasive. As in the mechanism for inactivating tumor suppressor genes in tumor progression, mutations have been found in the genes for E-cadherin, a major cadherin in epithelial cells, and its undercoat proteins,!!
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! catenins, which connect E-cadherin to actin filaments and establish firm cell-cell adhesion. Transcriptional inactivation of E-cadherin expression was also shown to play a significant role, and we recently clarified that CpG-methylation of the E-cadherin promoter is the underlying mechanism. In addition, it was found that tyrosine-phophorylation of the E-cadherin-catenin complex inactivated cell adhesion, and that the tyrosine kinase oncogene product c-erbB-2 is associated with!!! catenin. From these findings, we propose that the mechanisms for inactivation of a particular function are multiple, and include genetic and epigenetic alterations. Tumor development and progression may represent the sum of these multiple alterations in key systems regulating cell growth and differentiation. Functional Analysis of the von Hippel-Lindau Gene.
Dr. William Kaelin discussed how functional studies of the products of genes thought to be critical targets for mutation or other alteration in specific cancers can lead to new insights about into cancer pathogenesis. He presented the results of an analysis of the function of the VHL-1 gene product (pVHL). VHL-1 is the gene targeted for inactivation in the inherited von Hippel-Lindau syndrome (VHL), characterized by the development of renal carcinomas. cerebellar hemangiomas, and pheochromocytomas, In addition. VHL-1 also appeared to be inactivated in the majority of sporadic renal carcinomas and cerebellar hemangiomas. Using a strategy involving immunoprecipitation of protein complexes recovered from cell lysates, using!!
! -pVHI_ antibodies, the transcriptional elongation proteins elongin A and elongin B were found to bind wild-type pVHL, but not mutant pVHL species, with great specificity in such a way as to inhibit their transcriptional elongation activities. In another series of studies. introduction of cDNA encoding wild-type pVHL polypeptides, but not cDNA encoding mutant pVHL polypeptides into VHL-1 -/-renal carcinoma cells resulted in an abrogation of tumorgenesis in nude mice. In addition, restoration of wild-type pVHL function in renal carcinoma cells also resulted in restoration of the normal oxygen-dependent regulation of a number of “hypoxia-inducible” genes. Dr. Kaelin suggested that abnormalities in the regulation of such “hypoxia-inducible” genes might partly account for a number of features characteristically exhibited by renal cell carcinomas, including abundant neovascularization, exuberant erythropoietin production, etc. He offered to participate in several collaborative studies with Japanese investigator to better characterize the regulation of “hypoxia-inducible” genes in other human cancers. Clearly, to fully elucidate the molecular pathogenesis of various human cancers. functional studies of cancer gene products, such as those accomplished by Dr. Kaelin, will be more and more needed to complement ongoing cancer gene discovery efforts. von Hippel-Lindau disease is a hereditary cancer syndrome characterized by the development of multiple tumors including renal carcinomas, pheochromocytomas, and vascular tumors of the central nervous system. Tumor development in this setting is due to dual inactivation of a tumor suppressor gene (VHL-1) located at chromosome 3p25. In keeping with Knudson’s Hypothesis, this gene is mutated or silenced in the vast majority of sporadic renal cell carcinomas and cerebellar hemangioblastomas as well. The protein encoded by VHI-1 is an ÅH30 kD serine phosphoprotein (pVHL) whose predicted primary sequence bears little resemblance to other proteins in currently available databases. Reintroduction of a wild-type, but not mutant. VHL cDNA into VHL-/-renal carcinoma cells has been shown to inhibit their ability to form tumors in nude mice. Recently, we and others have shown that al frequently mutated region of p VHL binds VHL binds to elongin B and C in vitro and in vivo. Elongin B and C, when bound to a third protein, elongin A, generate an active transcriptional elongation complex called elongin or SIII. pVHL competes with elongin A for binding to elongin B and C, thereby inhibiting elongin/SIII activity. In the simplest view, tumor suppression by pVHL is due, at least in part, to this biochemical activity. According to this model, certain genes which are important for renal cell growth and development would be regulated by the availability of elongin/SIII. Experiments are currently in progress to test whether inhibition of elongin/SIII function, per se, will inhibit renal carcinoma cell growth and to identify genes which might be regulated by elongin/SIII.
Molecular Pathology of Tuberous Sclerosis (TSC2) Gene.
The Eker rat hereditary renal carcinoma (RC) is an excellent example of a mendelian dominant predisposition to a specific cancer in an experimental animal. Dr. Okio Hino’s group recently reported that a germline insertion in the rat homologue of the human tuberous sclerosis (TSC2) gene gives rise to the dominantly inherited cancer in the Eker rat model (Nature Genetics, 9: 7074, 1995) and a tumor suppressor nature for Tsc2 gene function (Proc. Natl. Acad. Sci. USA, 90: 327-33 1, 1993, Cancer Res., 54: 2633-2635, 1994., Cancer Res., 55: 989-990, 1995., Biochem. Biophys. Res. Commun. in press). Investigation of extra-renal primary tumors co-occurring In Eker rats late in life (at 2 years) additionally revealed enhanced development of probable hemangiosarcomas of the spleen, uterine leiomyosarcomas, and pituitary adenomas, although the demonstrated predilection for these extra-renal tumors was not as complete as with RCs (Cancer Lett. 83: 117-121, 1994. Transplantation Proceedings 27: l_529-1531, 1995). We further dctected LOH in all but the splenic tumors, suggesting that inactivation of the Tsc2 gene is also a critical event in the pathogenesis of these extra-renal lesions (Jpn. J. Cancer Res. 86: 828-832. 1995). Microsatellite instability was not observed in Eker rat tumors (Molecular Carcinogenesis, 14: 2327. 1995). Mutation of the von Hippel-Lindau (VHL) tumor suppressor gene is not an event in rat renal carcinogenesis, at least in our systems (Jpn. J. Cancer Res., 86: 905-909, 1995).
The phenotype of tuberous sclerosis in humans differs from that in the Eker rat, except for the occurrence of RCs (in humans, angiomyolipomas are more common). Since nothing is known about the molecular mechanism in human tuberous sclerosis, the Eker rat has special potential value for elucidating TSC2 gene role in renal carcinogenesis, as well as studying species-specific differences in tumorgenesis, phenotype-specific mutations and/or cell-type specific carcinogenesis. We determined the entire cDNA including several alternative splicing variants and the exon-intron organization of the rat Tsc2 gene (Nucleic Acids Res., 23: 2608-2613, 1995). The rat Tsc2 product shows a 92% amino acid identity to the human counterpart. Surprisingly, the rat Tsc2 gene consists of 41 exons, although there is non-coding exon(s) in the 5’ upstream region. We have already found intragenic mutations in the Tsc2 gene by PCR-SSCP analysis and transcriptional activation domains in the C-terminus of the Tsc2 product. While a C-terminal truncated Tsc2 protein was localized in the nucleus, the full length protein is predominantly found in the perinuclear region of cytoplasm (Cancer Res. in press).
Signaling by Normal and Leukemogenic Abl Proteins.
Dr. Charles Sawyers, who has focused his investigative efforts on the Bcr-Abl fusion protein stereotypically generated as a consequence of the Philadelphia chromosome translocation in human leukemias, presented data concerning the function of c-Abl and Bcr-Abl tyrosine kinases in cellular growth-stimulating and growth-inhibiting signal transduction pathways, In studies of c-Abl overexpression in model fibroblasts in vitro, a critical interaction of c-Abl and p53 was identified. The consequence of this interaction appeared to be enhancement of p53 transcriptional transactivation activity and growth arrest, suggesting that the wild-type c-Abl polypeptide might function as a growth-inhibitory tyrosine kinase. In another series of studies, the leukemogenic polypeptides Bcr-Abl and v-Abl were found to activate Ras-dependent signal transduction pathways culminating in cellular proliferation and transformation. Of interest, Bcr-Abl appeared to activate the Jun kinase (JNK) pathway, increasing transcription from Jun-responsive gene promoters, but not to activate the extracellular signal regulated kinase (ERK) pathway. Curiously, v-Abl appeared to activate both JNK and ERK signal transduction pathways, Thus, the progressive elucidation of the functions of normal and abnormal Abl polypeptides has resulted in plausible hypotheses for how somatic alterations of the c-Abl gene fuel leukemogenesis. In addition, the mechanistic dissection of signal transduction pathways involving c-Abl and Bcr-Abl undertaken by Dr. Sawyers has yielded a substantial number of leads for candidate anti-leukemic drugs and drug targets.
The Abl gene encodes a nuclear tyrosine kinase protein whose normal function remains to be defined. In Philadelphia chromosome positive leukemias, a Abl fusion protein called Bcr-Abl is generated which is the primary pathogenic event in these leukemias. Dr. Sawyers’ laboratory is investigating the function of normal Abl and Bcr-Abl by examining signal transduction pathways affected by each protein.
Growth suppression by the Rb and p53 tumor suppressor proteins is mediated through effects on cell cycle regulatory proteins at the G1/S transition. Because overexpression of c-Abl induces G l arrest in fibroblasts (Sawyers et al, Cell 77: 121, 1994), it was reasoned that c-Abl may also affcct cell cycle proteins which regulate G1. We used fibroblasts containing disruptions of the Rb or p53 genes to genetically test the role of these proteins in c-Abl growth suppression. We find that c-Abl requires p53 but not Rb to suppress growth. c-Abl binds p53 in vitro and enhances p53 dependent transcription from a promoter containing p53 DNA binding sites. An Abl mutant which no longer binds p53 does not enhance p53 transcriptional activity and fails to suppress growth. These findings provide a novel link between a growth inhibitory tyrosine kinase and the p53 tumor suppressor protein.
The leukemogenic tyrosine kinase fusion proteins Bcr-Abl and v-Abl activate a Ras-dependent pathway required for transformation (Sawyers et al, J. Exp. Med. 181:307, 1995). We measured effects of Bcr-Abl and v-Abl on two mitogen activated protein kinase (MAPK) cascades downstream from Ras-the extracellular signal regulated kinase (ERK) pathway and the Jun kinase (JNK) pathway. We find that Bcr-Abl primarily activatcs thc JNK pathway and not the ERK pathway, whereas v-Abl activates both. Bcr-Abl enhances transcription from Jun responsive promoters in a Ras, MEKK (MEK kinase) and JNK-dependent fashion, and dominant negative mutants of c-Jun impair transformation by Bcr-Abl. These findings implicate the JNK pathway in mediating transformation by a human leukemia oncogene.
Genetic Alterations in Human Breast Cancer.
Human breast cancers frequently show loss of heterozygosity (LOH) and amplification at specific chromosomal regions. To understand the role of these genetic alterations in tumor development and progression, Dr. Mitsuru Emi’s group examined 457 cases of primary breast cancers for LOH at chromosomal regions 16q24, 17pl3.3, 17q21, and amplification of the erbB2 locus at 17ql 1.2 and the c-myc locus at 8q24. They looked for a relationship to lymph node metastasis, histological type and tumor stage. LOH at 17q21 was more frequently observed in tumors of solid-tubular type (41/75, 55%) than the other types (48/187, 26 %) (p<0.0001). LOH at 17p13.3 was more frequent in tumors of scirrhous and solid-tubular types (77/141, 55%, and 48/88, 55%) than the other types (29/89, 33%) (p=0.0004). The association of allelic loss at 17pl3.3 and 17q21 with specific types of tumor indicates that loss or inactivation of tumor suppressor genes at these location may exert an effect in a histological type-specific manner. In this series, we found 22 tumors with three or more genetic alterations in 187 tumors that are free of lymph node metastasis. Among them, 12 of 122 tumors at a stage; 4 of 90 tumors of well-differentiated histological type are included. Although these tumors are considered to have a relatively good prognosis when a conventional classification of diagnosis is used, we suspect that, though small in proportion, they may actually have a poorer prognoses than those predicted from the conventional clinical classification We propose that they be treated as a new high-risk group in the operative and/or postoperative management of these patients.
To investigate possible relationships between genetic alterations and hormonal deregulation during breast cancer development and/or progression, we examined 616 primary breast cancers for loss of heterozygosity (LOH) at chromosomal regions 16q24, 17pl3.3, and 17q21, and for amplifications of the ERBB2 and c-MYC loci. A comparison of estrogen receptor (ER) and progesterone receptor (PgR) status in tumor cells with data concerning these genetic alterations revealed that LOH at 17q21 was significantly correlated with absence of ER (p<0.0003) or PgR (p<0.0001), and with the absence both (p<0.0001). Similarly, a significant association was observed between amplification of ERBB2 and the absence of either ER or PgR. LOH at 17pl3.3 was associated with the absence of PgR (p<0.01). These data suggest a possible relationship of specific genetic changes on chromosome 17 with hormonal deregulation in the progression of breast cancer.
Predisposing mutations in a BRCA1 gene have been recently identified in germline 17q-linked breast and ovarian cancer families. To understand the frequency, distribution, and nature of BRCA1 mutations in Japanese breast cancer patients, we screened 1,000 unselected primary cancers for mutations in exon 11, which accounts for 61% of the entire BRCA1 coding sequence. Using a method based on multiplex single strand conformational polymorphism (SSCP), analysis of multiple restriction fragments generated by restriction-enzyme digestion of amplified DNA, we identified eight mutations including four that we had previously found in a subset of these cases. All eight were germline mutations; four of them were nonsense mutations or small deletions resulting in premature stop codons, and the other four were missense mutations. The Japanese carriers of these mutant BRCA1 alleles had developed breast cancers at ages ranging from 45 to 62, five of them bilaterally. Taking into account the effect of various factors such as life-lime risk of breast cancer, screening efficiency, and the region examined, we roughly estimate that 2-3% of breast cancer in Japan is attributable to BRCA1 mutation and that I in 1,500-2,000 Japanese women carry a germline mutation in the BRCA1 gene.
Molecular Analysis of Colorectal Cancer.
Dr. Yusuke Nakamura’s group have investigated somatic and germline mutations of the genes associated with hereditary colorectal cancer syndromes, familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC). Mutations of the APC (adenomatous polyposis coli) gene was found in 70% of 150 FAP patients that were screened over the entire coding region by an RNase protection assay. Most of the mutations found would cause truncation of the gene product; two-thirds of them were frameshift mutations and the remaining one-third were nonsense mutations. Only 5% of the mutations so far found were considered to induce substitution of an amino acid. These results imply that detection of truncated APC protein by antibody may be an easy and simple method for presymptomatic diagnosis of individuals carrying APC mutations. Somatic mutations of the APC gene were also found frequently in colorectal carcinomas as well as adenomas; the type of the somatic mutations detected were almost similar to that of the germline mutations. When adenomas in FAP patients were divided into three groups based on their size (<3mm, 3-10mm, or >10mm) or their pathohistological grade (mild, moderate, or severe dysplasia), somatic APC mutations were detected even in adenomas of <3mm with moderate dysplasia while K-ras mutations were rare in these tumors. Hence, inactivation of both alleles of the APC gene is considered to play a significant role in development of colorectal adenoma.
In addition to molecular analysis of tumors, we attempted molecular diagnosis of micrometastasis of colorectal cancer to lymph nodes. Despite apparently radical removal of their primary tumors, almost half of patients with colorectal cancer die from local or distant tumor relapse. The important determinants of recurrence and survival are the extent of the primary tumor, its cellular differentiation, and the involvement of regional lymph nodes. In these prognostic factors, the presence of metastatic tumors in regional lymph nodes of a patient who has undergone resection of a colorectal cancer is of major prognostic significance. On the basis of conventional histology, even among patients without lymph node metastasis, 20-30% will die as a result of distant metastasis or local recurrences within 5 years. We previously established the mutant allele-specific amplification (MASA) method that is capable of detecting one cancer cell among 2,000 normal cells in lymph nodes. To examine the prognostic significance of genetically detectable tumor cells in regional lymph nodes of colorectal cancer patients, we screened 120 colorectal cancers without histologically-detectable lymph node metastasis, for K-ras (codon 12, 13, and 61) or p53 mutations (exons 5-8) and examined corresponding regional lymph nodes at the genetic level. Patients with genetically “positive” lymph nodes had higher risk of recurrences within 5 years after surgery (p<0.0001) and none of the patients who had no genetically-positive lymph node metastasis had recurrences for at least 5 years after operation. Genetic diagnosis of lymph node metastasis may become a useful prognostic factor and be a selective marker for postoperative intensive adjuvant chemotherapy.
Prostate Cancer.
Hypermethylation of regulatory sequences at the locus of the &Mac185;-class glutathione S-transferalse gene GSTP1 was detected in 20 of 20 human prostatic carcinoma tissue studied, but not in normal tissues or prostatic tissues exhibiting benign hyperplasia. In addition, a striking decrease in GSIP1 expression was found to accompany human prostatic carcinogenesis. Immunohistochemical staining with anti-GSTP1 antibodies failed to detect the enzyme in 88 of 91 prostatic carcinomas. In vitro, GSTP1 was limited to human prostatic cancer cell lines containing GSTP1 alleles with hypomethylated promoter sequences; a human prostatic cancer cell line containing only hypermethylated GSTP1 promoter sequences did not express GSTP1 mRNA or polypeptides. Methylation of cytidine nucleotides in GSTP1 regulatory sequences constitute the most common genomic alteration yet described for human prostate cancer.
Dr. William Nelson attempted to show how knowledge of an early somatic genome alteration accompanying human prostatic carcinogenesis, the inactivation of gene encoding the caircinogen-defense enzyme GSTP1, might provide new opportunities for rational prostate cancer prevention. The data presented revealed that prostate cancers contained transcriptionally-silenced GSTP1 genes as a consequence of extensive somatic “CpG island” methylation changes, involving the promoter region of the gene. GSTP1 inactivation appeared to occur early during the process of prostate cancer pathogenesis and to be present in all, or nearly all. prostate cancer cell. Dr. Nelson proposed that the resultant crippling of carcinogen defenses might render normal prostatic epithelial cells vulnerable to neoplastic transformation and neoplastic prostatic cells vulnerable to malignant progression. This proposal formed the basis of a translationall research hypothesis for prostate cancer prevention: if inadequate defenses against electrophilic carcinogens promotes the development of life-threatening prostate cancers, then therapeutic intervention targeted at augumentation of electrophile detoxification capacities might abrogate or attenuate prostatic carcinogenesis. To test this hypothesis, Dr. Nelson’s laboratory has initiated a screening program for carcinogen-defense enzyme inducer compounds as candidate drugs or dietary supplements for prostate cancer chemoprevention. Dr. Nelson also proposed a collaborative study with Dr. Hirohashi to ascertain whether GSTP1 inactivation might also commonly occur during hepntic carcinogenesis.
Head and Neck Cancer: Molecular Diagnosis and Staging.
Dr. Li Mao demonstrated the utility of cancer-associated molecular markers as adjuncts to cancer diagnosis, staging, and treatment monitoring. For example, analysis of p53 tumor suppressor gene mutations may help discern the behavior of head and neck squamous carcinomas following surgical resection. Studies presented demonstrated that detection of tumor-specific mutant p53 sequences at the surgical margins of resection of head and neck squamous carcinomas portended a poorer prognosis than when such mutant sequences were not detected. DNA microsatellite markers were shown to be potentially useful as molecular diagnostic and screening tools. Using a panel of microsatellite markers derived from different chromosomal loci. a number of cancer DNA specimens derived from a variety of different human cancer types were found to contain somatic alterations affecting at least one microsatellite marker. By employing such a microsatellite marker panel as a screen for clonal somatic genome lesions, cancer cells were successfully detected in urine, sputum, and at the margins of surgical resections. Dr. Mao suggested that clonal microsatellite alteration detection might complement conventional cytopathology in cancer diagnosis and screening. In all, data presented by Dr. Mao, as well as by Dr. Nakamura, illustrated the potential clinical applications of markers derived from molecular pathogenesis research studies.
In head and neck cancers, current techniques may not detect small numbers of cancer cells at the margins of resection or in cervical lymph nodes. They identified 25 patients with primary squamous-cell carcinoma of the head and neck containing a p53 mutation who appeared to have had complete tumor resection on the basis of a negative histopathological assessment. In 13 of these 25 patients, molecular analysis was positive for a p53 mutation in at least one tumor margin. In 5 of 13 patients with positive margins by the method (38%), the carcinoma has recurred locally, as compared with none of the 12 with negative margins. Furthermore, molecular analysis identified neoplastic cells in 6 of 28 lymph nodes (21%) that were initially negative by histopathological assessment.
Genomic Imprinting and Epigenetic Changes in Human Lung Cancer.
Genomic imprinting at 11p15 may play a role in certain pediatric tumors such as Wilms’ tumor, based on the findings of selective maternal loss of this chromosomal region. Recently, epigenetic inactivation of a candidate tumor suppressor, H19, which is known to be paternally imprinted and expressed exclusively from the maternal allele, was reported by two independent groups, suggesting that it may play a role in pediatric oncogenesis. In contrast, we found that loss of imprinting (LOI) of H19 is a frequent event in lung cancer but not in another common adult cancer, liver cancer, and that LOI of H19 is often associated with its overexpression in lung cancers retaining both parental alleles with hypomethylation of the promoter region. Although frequent and tissue-specific occurrence of LOI of H19 warrants further investigation, these data are in direct contradiction to a putative tumor suppressor gene role of H19 and do not provide sufficient explanation for frequent occurrence of allelic loss at 11p15 in lung cancer. In this regard, we recently found that the p57Kip2 gene, a newly described member of the p21 cyclin-dependent kinase (CDK) inhibitor family which is thought to negatively regulate the cell cycle at the G1 checkpoint, is paternally imprinted with maternal expression. The active maternal alleles had been selectively lost in 11 of 13 (85%,) lung cancer cases carrying 11p15 deletions, this being a significant bias (P =0.02 for a two-tailed test). These data suggest that genomic imprinting may play a role in the oncogenesis of not only rare pediatric tumors but also this common cancer of adults, The data also suggest that the imprinted p57Kip2 CDK inhibitor gene is a potential target for maternally biased 11p15 deletions.
To study possible involvement of altered DNA methylation in the oncogenesis of lung cancer, Dr. Takashi Takahashi’s group investigated the methylation status of the Rb locus at 13ql4 as well as that of the bcl-2 locus at 18q21 in 134 lung cancer specimens, representing all major histological subtypes. As a result, they identified the occurrence of tumor-specific aberrant hypermethylation at the bcl-2 locus at 18q21 specifically in non-small cell lung cancers (NSCLCs) and among NSCLCs such epigenetic aberrations were observed most frequently (about 40%) in adenocarcinomas. Interestingly, allelic loss at the bcl-2 locus was also seen in 40% of adenocarcinomas. This frequency was also the highest among values for the various histological subtypes of lung cancers. They found that the presence of aberrant hypermethylation does not correlate with the relative levels of bcl-2 expression. The bcl-2 gene itself might consequentiy be an innocent bystander of epigenetic changes, since it has been suggested that DNA methylation can form a methylation domain starting from certain scquence elements, termed “centers of methylation.” These results suggest that this chromosomal region may harbor a putative tumor suppressor gene especially for adenocarcinomas, and that aberrant hypermethyiation might be involved in inactivation processes, perhaps by silencing gene expression and/or causing a high rate of mutation of methylated cytosine residues. In contrast, altered methylation of the Rb locus, which is often hypermethylated in retinoblastomas, appears to be quite rare in lung cancers, if it is at all present, suggesting tumor-type and locus specific nature of aberrant DNA methylation in cancers.
Excess of Rare Cancers in Werner’s Syndrome (Adult Progeria): Another Key to Understanding Oncogenesis?
The association between genetic disorders and diverse cancers has provided clues for laboratory research into carcinogenesis, as exemplified by Li-Fraumeni syndrome. Soft-tissue sarcoma and benign meningioma have been associated with Werner’s syndrome (WS), an autosomal recessive disorder characterized by premature aging, more commonly reported in Japan than elsewhere in part because of inbreeding. In the literature were found 124 cases-reports of neoplasia and WS from Japan and 34 from outside Japan, 1939 through August 1995. They reveal a greater diversity of neoplasia in WS than was previously known. In Japanese there were 130 cancers, 15 benign meningioma, and 5 myeloid disorders, as compared with 31, 7 and 2 respectively in non-Japanese. The ratio of epithelial to non-epithelial cancers was about 1:1 for Japanese and for non-Japanese instead of the usual 10:1. Both series had excesses of soft-tissue sarcoma, osteosarcoma. myeloid disorders and benign meningioma. In addition, the Japanese had an excess of thyroid cancer (26 vs 2 cases in non-Japanese) and melanoma (21 vs 3), including -5 intranasal and 13 of the feet. These malignancies accounted for 57% of all cancer in WS as compared with 2010 expected, based on the Osaka population at 25-64 years of age. Multiple tumors were reported in 19 Japanese and 5 non-Japanese. First-degree relatives were affected in 9 Japanese families; 6 were concordant as to site and/or cell type. The high frequency of thyroid cancer and melanoma in the Japanese, not found outside Japan may be related to racial differences, in accord with laboratory evidence of alleles closely linked to the WRN gene in Japanese but not in Caucasians. Li-Fraumeni syndrome shares with WS a propensity to osteosarcoma and soft-tissue sarcoma, but, in addition, has an excess of breast carcinoma, brain tumors, leukemia, and adrenocortical carcinoma. The role of the p53 gene in carcinogenesis and normal human biology has been clarified by molecular studies of this gene.
Dr. Miller discussed the important inferences that can be gleaned from the study of genetic disorders associated with susceptibility to cancer development. Werner’s syndrome, an autosomal recessive disorder affecting Japanese and non-Japanese and characterized by features of premature aging, appeared to be associated with increased risk of development of a wide variety of cancers in both Japanese and in non-Japanese. Of interest, the ratio of epithelial cancers to non-epithelial cancers associated with Werner’s syndrome appeared far less than the ratio observed for general population. Also, in Japan, excess numbers of thyroid cancers and melanomas, especially intranasal melanomas and melanomas of the feet, have been reported. Werner’s syndrome thus provides an opportunity for consideration of a specific inherited genetic lesion (recently discovered and reported) in the context of both racial factors (other inherited modifying genes) and environmental factors in cancer development.
PARTICIPANTS
UNITED STATES
Dr. Darryl K. Shibata
Department of Pathology
University of Southern California School of Medicine
1200 North State Street
Number 736
Los Angeles, CA 90033
Tel: (213) 226-7067 Fax: (213) 226-2686
Dr. Charles S. Sawyers
University of California at Los Angeles
11-934 Factor Bldg. Hematology/Oncology
l0833 LeConte Avenue
Los Angeles, CA 90095
Tel: (31O) 825-4321 Fax: (31O) 825-6192
Dr. William G. Nelson
Marburg 411 James Buchanan Institute of Urology
Johns Hopkins Oncology Center
600 North Wolfe Street
Baltimore, MD 21287-8936
Tel: (410) 614-1661 Fax: (41O) 9_55-O12_5
Dr. William Kaelin
Dana Farber Cancer Center
44 Binney Street
Boston, MA 02115
Tel: (617) 632-3975 Fax: (617) 632-4381
Dr. Jeffrey Sklar
Department of Pathology
Brigham and Women’s Hospital
Harvard Medical School
75 Francis Street
Boston, MA 02115
Tel: (617) 732-7446 Fax: (617) 732-7449
Dr. Li Mao
University of Texas/M.D. Anderson Cancer Center
1515 Holcombe Boulevard
Box #80 Houston, TX 77030
Tel: (713) 792-6363 Fax: (713) 796-8655
Dr. Robert W. Miller
Scientist Emeritus Genetic Epidemiology Branch
National Cancer Institute, EPN-400
Bethesda, MD 20892-7360
Tel: (301) 496-5785 Fax: (301) 496-1854
JAPAN
Dr. Mitsuru Emi
Department of Molecular Biology
Institute of Gerontology Nippon Medical School
Kosugi, Nakahara-ku
Kawasaki 211, and
Cancer Institute Kami-Ikebukuro, Toshima-ku
Tokyo 170
Dr. Okio Hino
Department of Experimental Pathology
Cancer Institute 1-37-1
Kami-Ikebukero, Toshima-ku
Tokyo 170
Dr. Setsuo Hirohashi
National Cancer Center
Research Institute
1-1, Tsukiji 5-chome, Chuo-ku
Tokyo 104
Dr. Johji Inazawa
Department of Hygiene
Kyoto Prefectural University of Madicine
Kamigyo-ku
Kyoto 602
Dr. Yusuke Nakamura
Laboratory of Molecular Medicine
Institute of Medical Science
The University of Tokyo
Minato, Tokyo 108
Dr. Takashi Takahashi
Laboratory of Ultrastructure Research
Aichi Cancer Center Research Institute
1-1 Kanokoden, Chikusa-ku
Nagoya 464
Dr. Haruo Sugano
Director Emeritus
The Cancer Institute
1-37-1, Kami-Ikebukuro, Toshima-ku
Tokyo 170
(2) Workshop on “Cancer in Werner’s Syndrome”
Introduction:
This seminar on Werner’s syndrome (WS) represents the extraordinary benefit that can come from the exchange of ideas through a series of meetings sponsored by the US-Japan Cooperative Cancer Research Program. The advantage of having two seminars a year allows ideas developed at one to be further developed through additional seminars. Thus, in March 1994 a workshop was held on cancer clusters. Dr. Makoto Goto, a rheumatologist at the Tokyo Metropolitan Hospital, spoke on clustering of cancer in Werner’s syndrome, a subject not previously given any attention by oncologists. Because of cousin marriages in Japan, homozygotes for the recessive WRN gene occur more often than usual and produce children with the disorder. As a result, there are many more WS cases in Japan than elsewhere and the link to cancer can be explored. The neoplasia appears in rare forms, such as melanoma of the feet and intranasal passages.
Dr. Goto was working alone without funds. Over the next two years, two Japanese and one American, who attended the seminar, assisted him in bringing the paper to publication-on the same day that cloning of a WRN gene was reported. The function of the gene should reveal the reason for the occurrence of unusual cancers in WS, which, as tumor suppressor genes have done before, may shed light on a broad group of cancers in the general population, beyond those found in this rare syndrome.
At a seminar the following year on genetic syndromes with high risk of cancer, the patterns of neoplasia in various syndromes were compared, which proved helpful in the discussion of the WS paper. Discovery of its gene, a helicase, adds to the information on the Bloom syndrome gene, also a helicase, a discovery made earlier in the year. Persons with Bloom syndrome also are especially prone to cancer, but of the usual sort: non-lymphocytic leukemia, non-Hodgkin’s lymphoma, and aerodigestive tract cancer. The 1996 seminar brings together scientists long interested in WS but not in cancer, and oncologists who know the cancers but not the syndrome.
The report on an excess of rare cancers in Werner’s syndrome was published: Goto M, Miller RW, Ishikawa Y, Sugano H: Cancer Epidemiol. Biomarkers and Prev. 5:239-246, 1996. The report on cloning a WRN gene was by a Seattle group (Yu C-E et al: Science 272:258-262, 1966).
Hierarchical Deterioration of Body Systems in Werner’s Syndrome.
Werner’s syndrome (WS), a genetically-determined (autosomal recessive) premature aging syndrome, has been viewed as a disorder that may reveal mechanisms of natural aging. WS patients manifest relatively uniform signs of various elderly phenomena, plus malignancy (sarcoma and thyroid carcinoma). The average life-span is 46 years and the major causes of death are malignancy and atherosclerotic disorders. Numerous in vitro experiments of WS cells have suggested their striking similarity to cell changes with natural aging. Cancer rates in the normal population rises rapidly in the aged, although the exact role of aging per se in the development of cancer remains an open question.
Despite the fact that much attention has been paid to this unique syndrome, only small numbers can be studied because of its rarity. Until now, 1100 patients with WS have been reported worldwide, about 80% of them from Japan. The majority of Japanese patients (70% are offspring of first-cousin marriages) are the descendants of Heike Family (patient clusters in and around Heike villages), which was defeated 800 years ago. Based on genotyping data of the WRN gene locus (D8S339), patients can be classified into 3 groups. The locations of their hometowns correspond to their genotypes.
Extensive clinical investigation into 81O Japanese patients with WS suggests that their signs and symptoms, which appear in a sequential manner, can be grouped into 4 major clinical categories: skeletal, ondocrine, immune, and neural. For example, among major clinical signs and symptoms observed in this syndrome, skin changes (scleroderma-like), gray hair and cataracts can be classified as skeletal-based (connective tissue) clinical manifestations (affecting 100% of WS patients); hypogonadism, thyroid dysfunction, and diabetes mellitus as endocrine-based (70% affected); and depressed NK activity, autoantibody production, and autoimmune diseases as immune-based (5Oc’/(, affected); and brain atrophy and dementia as nervous system-based (3Oolo affected) .
After failures of the adolescent growth spurt, hierarchical deterioration of the 4 body systems becomes evident at mean ages of 32 for the skeletal system, 36 for the endocrine system and 40 for the immune and nervous systems. Thereafter, increased deaths from atherosclerotic disorders and malignancy occur.
Unfortunately, the data on natural aging of the 4 body-systems among the general population are not sufficient to compare with those observed in WS. The mechanisms by which the deterioration of these systems in WS occur in a hierarchical fashion should be clarified after the WRN gene is cloned and is function determined.
Clinical Aspects of Werner’s Syndrome in Europe.
Twenty-four European patients suspected of being affected with Werner’s syndrome (WS) were collected on the basis of the major criteria; bilateral cataracts, short stature, characteristic facies and dermatologic alterations, premature graying and/or thinning of scalp hair, evidence consistent with autosomal recessive inheritance, associated or not with other signs and symptoms as reviewed at the Hague meeting in 1989. Six of them were temporarily excluded because of lack of clinical evidence, too early onset, presence of unusual symptoms or possibly dominant inheritance. The mean age of the selected patients was 25±7 years at the onset and 44±9 years at the time of the study. In two families consanguinity was present and in two others 3 and 2 siblings were affected, respectively. Three sporadic patients had relatives affected with premature graying or aging, ulcerations, coronary heart disease or cancers.
First symptoms were generally graying or sparse hair, slight atrophic changes of the skin and bilateral cataracts (operated very early in many patients). The overall appearance was similar to the classical WS phenotype with bird-like facies, pinched nose, irregular teeth, changing of the voice, stocky trunk, thin limbs, and abnormal fat distribution. Stature was not as small as expected: 1m57cm in our series (70% were females) vs. 1m57cm in males and 1m46cm in females in previous European studies. Nevertheless, 57% of the patients were generally underweight female BMI<19, male BMI<20 (Body-mass index: W/H2. The most prominent symptoms were cataracts; hyperkeratosis; ulcerations of toes, Achilles tendons, ankles, knees and elbows; scleroderma-like changes with muscle wasting and osteoporosis. Changes of voice, hypermelanosis and osteoarthritis were not rare while hyaluronic aciduria hypcrcholesterolemia (2.05±0.42mg%) and confirmed NIDD were not as prevalent as in other reports. The age of puberty was generally normal or slightly delayed (14±7 years). Female patients gave birth to 19 children whereas males fathered 3 children. Secondary sexual underdevelopment occurred in two-thirds of patients. These subjects had sparse, irregular menses, abortions (4) and stillbirth followed by infertility (1). Menopause was at 37±7 years in females. There were 3 abortions in relatives. Gynecomastia, small testicles, and low testosterone levels were seen in males. Chromosome analysis was usually normal in blood leukocytes but fibroblasts revealed breakage and rearrangements consistent with the concept of “variegated translocation mosalcrsm” In half of the patients.
In many patients, the combination of skin atrophy, ulcerations, ankylosis, and bone lesions resulted in painful and severely handicapped walking. Malignant and benign tumors were seen in WS patients (cerebral meningioma, soft tissue spinocellular carcinoma, thyroid adenomas, and two metastasized breast cancers). In relatives we observed colon cancer, breast cancers, and two prostate cancers. Atherosclerosis was found in at least half of the patients, mainly involving the arteries of the lower limbs. Severe vascular insufficiency was frequent despite sympathectomy in two patients. The insufficiency sometimes resulted in partial amputations. Degenerative changes involved aortic or pulmonary valves as well as coronary or cerebral arteries, leading to congestive heart failure, myocardial infarction or stroke. In our series, low or absent pulses, reduced Doppler flow and arterial calcifications seemed to be better markers than hypertension or lipid abnormalities.
Most patients were Caucasian (89%), including three Sicilians and one Belgian. Two others were Turkish (but of Armenian origin). Their phenotypes were compared to previous publications in Europe and other regions of the world in order to determine if some traits were presumably more specific to European populations.
Clinicopathological Characteristics of Cancer Arising in Werner’s Syndrome, Particularly Thyroid Cancer and Osteosarcoma.
Dr. Yuichi Ishikawa investigated pathological characteristics of thyroid carcinoma and osteosarcoma arising in Werner’s syndrome (WS). By using a Japanese medical index, IgakuChuo-Zasshi (Japan Centra Revuo Medicina), and collected information of 26 thyroid carcinomas and 9 osteosarcomas among 810 WS cases reported from 1964-1994. For osteosarcoma, the pathology slides of 8 cases were available. No slides have been examined yet for thyroid carcinoma. As controls, incidence registries of each tumor were employed, which were published as a Gann Monograph on Cancer Research vol. 43 by CRC Press in the United States and Japan in 1995. Additionally, osteosarcoma cases of the Cancer Institute Hospital were used for more detailed comparisons.
Among 26 thyroid carcinomas, 24 were clinical cancer and 2 were discovered incidentally at autopsy. Mean ages were 39 years for WS cases and 49 for controls. The ratio of papillary to follicular carcinoma is 0.75 versus I for WS patients and 5.6:1 for controls. Female: male ratios were 2:1 for papillary and 3:1 for follicular carcinoma, approximately half of those of controls.
Osteosarcoma in WS had the following characteristics: (1) Mean ages were 40 for WS cases and 21 for controls. (2) Female to male ratios were 8:1 for WS cases and 2:3 for controls. (3) Most of WS osteosarcomas developed at unusual sites such as distal tibia and patella while usual osteosarcoma preferentially occurs at distal femur or proximal tibia. (4) In terms of histological subtypes, chondroblastic and fibroblastic osteosarcomas were common in WS cases whereas in the general population most are osteoblastic. For further characterization of WS osteosarcoma, we compared them with adult type osteosarcoma (30 years of age or older) seen in the Canccr Institute Hospital. As a result, clinicopathological aspects such as’ age distribution, F:M ratio, skeletal sites, sites of each bone and histological subtypes were quite similar between the two.
In WS thyroid carcinoma, we found low papillary/follicular ratio whereas it is well-known that high papillary/follicular ratio is related to the iodine rich area. Therefore, WS thyroid carcinomas may represent the effect of an iodine-deficient area. For ostcosarcoma, WS tumors are similar to adult-type osteosarcoma in several ways.
Werner’s Syndrome and Cancer in Japanese vs Non-Japanese, as Compared with Cancer in Other Chromosomal Instability Syndromes.
It has long been known that outside Japan WS is associated with an excess of soft-tissue sarcoma, osteosarcoma, and benign meningioma. Because of inbreeding in Japan, the syndrome, a recessive trait, is more common there. In 1994, at a US-Japan workshop on cancer clusters, M. Goto presented a case-series of WS and cancer in Japan. The number of cases was large, and had not been exhaustively reviewed. Dr. Miller suggested that al complete inventory of cases be made and analyzed using the approach his group has used for over 30 years in studying case series for links between cancer and congenital malformations. Also present were Yuichi Ishikawa, a pathologist especially interested in thorotrast-induced osteosarcoma, and Haruo Sugano, President Emeritus of the Cancer Institute, whose alert screening of the local medical literature led him to the rheumatologist, Dr. Goto, who was then invited to the US-Japan workshop. These four authors collaborated for the next two years to bring the report to publication (Cancer Epidemiology, Biomarkers and Prevention 5:239-246. 1996).
Review of the world literature revealed 124 cases-reports of neoplasia and WS from Japan and 34 from outside Japan, 1939 through August 1995. The diversity of neoplasia in WS was greater than previously known. In Japanese there were 127 cancers, 14 benign meningioma, and 5 myeloid disorders, as compared with 30, 7 and 2 respectively in non-Japanese. The ratio of epithelial to non-epithelial cancers was about 1:1 for Japanese and for non-Japanese instead of the usual 10:1. Both series had excesses of STS, osteosarcoma, myeloid disorders, and benign meningioma. In addition, the Japanese had an excess of thyroid cancer (20 vs 2 case in non-Japanese) and melanoma (21 vs 3), including 5 intranasal and 13 of the feet. STS, osteosarcoma, melanoma, and thyroid carcinoma accounted for 57% of all cancer in WS as compared with 2% expected, based on the Osaka population at 25-64 years of age. Multiple tumors were reported In 19 Japanese and 5 non-Japanese. In Japan 9 first-degree relatives had WS and cancer 6 of whom were concordant as to site and/or cell type. The WRN gene has been mapped to chromosome 8p. The high frequency of thyroid cancer and melanoma in the Japanese, not found in Caucasians, may be related to a report of linkage disequilibrium with the WRN gene in Japanese, but not in Caucasians; and haplotype differences within and between the two races, suggesting multiple independent mutations.
Thus the WRN gene affects aging and tumorigenesis. The tumors in Japan include thyroid carcinoma and acral lentiginous melanoma, which were not excessive in WS patients outside Japan. Study of the association between a genetic disorder and diverse cancers has in the past provided laboratory research with new approaches to understanding carcinogenic mechanisms.
The cytogenetic abnormalities in WS have been called variegated translocation mosaicism, because the abnormalities range from a partial deletion of a single chromosome to multiple translocations of several chromosomes, some occurring as clones (discussed by Drs. Nichols and Monnat). Cytogenetic instability characterizes 3 syndromes with high risk of cancer: Bloom syndrome, Fanconi syndrome, and ataxia-telangiectasia. Each differs from the others in the cancers to which it predisposes. Bloom syndrome is prone to leukemia, especially nonlymphocytic in childhood; to carcinoma, especially enteric in early adulthood; and to non-Hodgkin’s lymphoma throughout the life-span.1 Fanconi anemia predisposes to acute non-lymphocytic leukemia in particular; to liver neoplasia as a complication of treatment with androgens; and to carcinoma — oropharyngeal, gastrointestinal or gynecologic.2 Ataxia-telangiectasia predisposes mostly to non-Hodgkin’s lymphoma, to a moderate excess of acute lymphocytic leukemia, a small excess of Hodgkin’s lymphoma, and to a scattering of gastric carcinoma.3 None of the three chromosome instability syndromes has an excess of sarcoma. as observed in WS. Li-Fraumeni syndrome is associated with such an excess, plus other cancers unlike those in WS: breast carcinoma, brain tumors, leukemia, and adrenocortical carcinoma.4 The role of the p53 gene in carcinogenesis and normal human biology has been clarified by molecular studies of Li-Fraumeni syndrome.
Notes:
1. German J. Bloom syndrome: a mendelian prototype of somatic mutational disease. Medicine 1993;72:393-406.
2. Young NS, Alter BP. Aplastic Anemia: Acquired and Inherited. Philadelphia: WB Saunders, 1994;294-300.
3. Spector BD, Filipovich AH, Perry GS, Kersey JH. Epidemiology of cancer in ataxia-telangiectasia. In: Bridges BA, Harnden DG (eds). Ataxia-telangiectasia-a cellular and molecular link between cancer, neuropathology, and immune deficiency. New York, John Wiley, 1982:103-38.
4. Li FP, Fraumeni JF Jr, Mulvihill JJ, et al. A cancer family syndrome in twenty-four kindreds. Cancer Res 1988;48:5358-62.
Melanoma in Werner’s Syndrome.
The three main types of melanoma are superficial spreading and nodular (related to sun exposure) and acral lentiginous melanoma (ALM), which is not rclated to sun exposure. ALM occurs on the palms’, soles or mucosa, with an incidence of 1.6 per million per year in whites, blacks and Asians. Sun-related types of melanoma are rare in blacks and Japanese. It is remarkable that 13 cases occurred on the feet and 5 in the nasal passages over 20-30 years among perhaps 10,000 Japanese with Werner’s syndrome. This predisposition provides an opportunity to determine the genesis of this form of melanoma as distinguished from sun-related types. Dr. Wallace Clark, an expert in melanoma pathology, may be interested in examining histologic specimens to see if these neoplasms differ from ALM in the general population.
Altered expression of multiple connective tissue components in Werner’s syndrome fibroblasts.
Werner’s syndrome (WS) is a rare inherited disorder, thought to be a “premature aging disease.” WS is also assumed to be a disorder of systemic connective tissue metabolism based on many characteristic clinical findings such as short stature, calcification of soft tissues, numerous mesenchymal tumors, excessive urinary hyaluronic acid, and scleroderma-like skin. There have been many reports of connective tissue abnormalities in the skin of patients with WS. Also noted is the existence of homogenized and hyalinized thick collagen bundles and the increased absolute amount of collagen and glycosaminoglycan in the affected skin of patients with WS. With this in mind, Dr, Atsushi Hatamochi’s group carried out several studies of the connective tissue components using cultured dermal fibroblasts from patients with Werner’s syndrome.
We investigated gene expression of collagens I, III and VI. the main components of collagenous protein in the dermis, in WS by measuring mRNA and protein production levels in six fibroblast strains from patients with WS and comparing them in same population for doubling levels of fibroblasts from age-matched healthy subjects. A coordinate increase of the!!
!1(I) and!!!1(III) collagen mRNA levels was observed in WS fibroblast strains. The levels of type VI collagen mRNA were decreased in Werner’s syndrome fibroblast strains, and these were in parallel in all three chains (!!!1,!!!2 and!!!3) of type VI collagen. However, no qualitative abnormality of these mRNA transcripts was found by Northern blot analysis. Changes in type I and type VI collagen mRNA correlated well with the production levels of corresponding proteins, as determined by immunologic assays.Results demonstrate that gene expression of type I and type III collagen in Werner’s syndrome fibroblasts is increased, whereas that of type VI collagen is decreased. It has been reported that total glycosaminoglycan synthesis and collagen synthesis (type I and type 111) are increased in cultured Werner’s syndrome fibroblasts. Recently, increased expression of fibronectin in WS fibroblasts cultured in serum rich medium has been reported. Findings and the results of previous studies suggest that changes in the expression of multiple connective tissue constituents are characteristic of fibroblasts from WS, and these changes may be related to the abnormality in connective tissue in this syndrome.
The question raised in this meeting is whether the pattern of aberrations of connective tissue components synthesis in WS fibroblasts is similar to that in aged fibroblasts. It is known that expression levels of type I and type III collagen and glycosaminoglycan are decreased and fibronectin is over-expressed in normal fibroblasts undergoing replicative senescence. In preliminary studies, it was observed that the expression level of type VI collagen is also decreased in in vitro aged fibroblasts. Therefore, it was concluded that expression levels of type I and type III collagen and glycosaminoglycan in WS fibroblasts differ from those in aged fibroblasts, whereas the pattern of fibronectin and type VI collagen expression levels in WS syndrome fibroblasts are similar to those in aged fibroblasts.
Cytogenetic Instability of Werner’s Syndrome Cells.
The first indication of genetic instability or a mutator phenotype in Werner’s syndrome was found in chromosome studies in fibroblast cultures derived from these patients. These chromosomal changes were primarily in the form of clonal stable structural abnormalities in which translocations predominated. This type of change has been termed variegated translocation mosaicism (Hoehn et al., 1975). While clonal translocations are not unique to Werner’s syndrome and indeed can be found in both embryo and adult normal fibroblast cultures, the frequency of cells with translocations is usually very low under most circumstances, while in Werner’s the number of cells exhibiting one or more clonal translocations can achieve 900% or 100%. It was also observed in the first several studies that fibroblasts lacked acute unstable chromosomal rearrangements and breaks usually seem to accompany stable translocations. It was also found that these were not seen in peripheral lymphocytes and, indeed, in the first several studies variegated translocation mosaicism was not seen in peripheral lymphocytes. Since that time there are reports that these can be seen. These differences in observation may be due to different media used to grow cells or different mutational sites within the Werner’s syndrome gene as well as other possibilities. In addition, reports by Gebhart et al. would indicate that peripheral blood chromosomes do not have an increased sensitivity to clastogen-induced chromosome breakage or SCEs. Both of these observations present differences between the Werner’s cells and the usual chromosome fragility syndromes. It also invites the possibility that the mechanism of breakage may be different than the usual breakage-reunion sequence believed to occur. With the recent demonstrations that changes in telomere length and telomerase activity have been correlated both in vivo and in vitro with senescence and tumorigenesis and the reports that indicated both the presence of interstitial telomere sequences and telomere sequences at the break points of some rearrangements invites speculation that a telomere abnormality could be participating in the mechanism of this somewhat unusual chromosome breakage syndrome
References:
Hoehn, H. et al. Cytogenet. Cell Genet. 15,282 (1975).
Gebhart, E. et al. Human Genet. 70, 324 (1985).
Gebhart, E. et al. Human Genet. 80, 135 (1988)
Genetic Instability in Werner’s Syndrome Disease Pathogenesis.
Werner’s syndrome (WS; McKusick catalog #27770) is an uncommon autosomal recessive disease whose phenotype caricatures premature aging: WS patients develop, as young adults, many of the phenotypic changes that are associated with normal aging and are at increased risk for cancer. atherosclerotic cardiovascular disease and diabetes mellitus. Several different types of karyotypic and molecular genetic instability have been identified in cells and cell lines from WS patients. A summary of current information on instability in different cell lines or lincages is given in the table below. Much of the information used to assemble this summary was recently reviewed (see Monnat, Exper. Gerontol. 27:447-453 (1992)). chromosomal instability — 1° L + F lines*
forward HPRT mutation rate — F lines
deletion mutator phenotype — F lines
!!
!TG-resistant blood T-tymphs-1° T-cells!!
!glycophorin-A (GPA) variants-RBC’s !!
!mitotic recombination-RBC’s, F lines? *key:1° = primary cells/strains; lines = immortalized cell lines; L = lymphocytcs; F = fibroblasts; RBC’s = red blood cells/erythroid lineage; HPRT = X-linked hypoxanthine phosphoribosyltransferase gene; TG = 6-thioguanine.
The WRN locus has been mapped to chromosome region 8p11.1-21.1, near markers D8S339 and GSR. Once we know the nature of the WRN gene product and the spectrum of mutations at WRN that are responsible for WS, we should be able to understand several unanswered questions about WS: First, is there a unitary biochemical explanation for the several different types of genetic instability displayed by WS cells and cell lines? Second, is the WS clinical phenotype generated indirectly, by virtue of the mutator phenotype conferred by mutations at WRN. or is there a more direct pathogenetic link between mutant forms of the WRN gene product and the WS clinical phenotype? Third, what, if any, phenotype is conferred by the presence of singkmutant WRN alleles in different human cell lineages? Finally, what, if any, role do somatic mutations at the WRN locus play in age-associated disease processes such as cancer and atherosclerotic vascular disease?
The post-cloning era of WS research is almost upon us. It promises to be richly rewarding in that we will at last have good ways to begin to study the pathogenesis of this fascinating disease and roles of the WRN gene product in human cellular and organismal biology.
Kinetics and Replicative Declines in Cultivated Lymphocytes and Fibroblasts in Werner’s Syndrome.
The decreased life span of Werner’s fibroblasts in vitro compared to wild-type cells is due to an increased rate of irreversible exit from the cell cycle. In general, Werner’s fibroblasts show an increased frequency to become non-cycling five to six times greater than wild-type cells. T-lymphocytes from Werner’s patients do not show any difference in growth properties in vitro compared to T-lymphocytes from normal people. These observations add further support to the notion that Werner’s is a mesodermal disease. However, the discrepancy between our T lymphocyte results and the clear evidence for increased mutability in Werner’s syndrome cells raises serious questions about the relationship of the in vitro data to the phenotype of the patients. The question raised by this discrepancy of whether the growth properties or the hypermutability is of greater importance in the phenotype of Werner’s patients. It appears that the syndrome gene has relevance either or both to DNA integrity and DNA damage or to the cell cycle.
Fine Structure Physical and Genetic Mapping at the WRN Locus.
Dr. Dennis Drayna’s group have adopted a linkage disequilibrium mapping strategy to define more precisely the location of the Werner’s syndrome gene. They derived a group of P1/PAC contigs across the WRN region, including a central contig consisting of 25 unique clones. This contig contains the markers D8S339 and GSR, and spans a distance of greater than 1 Mb. These clones were used to derive 15 novel CA-repeat markers which were usefully polymorphic in the Japanese population. Marker order and intermarker distances were estimated by STS content mapping within our P1 contig. The resultant genotypes confirmed the existence of two ancestral haplotypes within this population. A number of analyses were applied to the genotypic data. including examination of Pexcess at individual markers, multipoint disequilibrium measurements and examination of individual chromosomes containing partial ancestral haplotypes. These analyses all suggest the most likely location for the WRN is in the immediate region of D8S339) and GSR.
Positional Cloning of the Werner’s Syndrome Locus.
Positional cloning methods are underway to identify the Werner’s syndrome (WRN) gene. Approximately 70 subjects with Werner’s syndrome (WS) have been identified and are under study for genetic mapping. Many are from consanguineous marriages, which has permitted the use of homozygosity mapping methods. Using this panel of families and traditional meiotic mapping methods, recombinants were identified which place the WRN locus as follows: Otelomere 8p—D8S131/D8S137—WRN/D8S339/GSR—D8S278—D8S283/D8S259/D8S71—D8S87—centromere. Using WS subjects of Japanese descent, the D8S339/GSR region was shown to be in linkage disequilibrium with WS. This D8S339/CJSR region has been the focus of our positional cloning efforts.
A yeast artificial chromosome (YAC) contig was assembled using polymorphic dinucleotide repeat markers D8S339 and GSR as sequenced-tagged sites (STS’s) to screen the Genethon Mega YAC library. Additional STS’s were identified by randomly cloning fragments from these YAC’s and these STS’s and used to identify flanking YAC’s. The final contig spans more than 3 Mb. Because YAC’s can be chimeric and unstable, STS’s from the YAC’s were used to screen arrayed Pl and cosmid libraries to replicate the contig as a combined P1 and cosmid contig. P1 and cosmid clones are now being used to identify additional dinuclcotide repeat polymorphic markers for disequilibrium mapping, and for candidate gene identification. Three methods have been used to identify genes in the region. First, cDNA selection has been performed using both YAC’s and Pl clones. Second, exon trapping was performed using P1 and cosmid clones. Third Pl clones across the region have been sequenced, and the resulting genomic sequence used to search data bases for known genes and expressed sequence tagged (EST) sequences. The genomic sequence has also been analyzed using the gene-finding algorithm GRAIL. Gene fragments identified through the use of these methods are then used to screen cDNA libraries to obtain full-length clones. Approximately 15 genes have been identified by a combination of these methods. These genes are now being screened as candidates for WS mutations.
Recent Advances in Werner’s Syndrome Gene Research.
To identify the WRN gene that causes Werner’s syndrome (WS), we have been conducting positional cloning. The initial efforts have been in generating a high resolution physical map, a detailed restriction and a cleavage map, followed by a transcription map, so the molecular genetic approaches to define the gene mutation will proceed on a solid basis.
The Werner’s syndrome gene (WRN) located at 8p11.2-12.1 is thought to be between D8S 131 and D8S87 that includes the closest marker D8S339. The order of major markers in this region was determined and the physical distances between them were estimated by dual-color fluorescent in situ hybridization (FISH) using P1, PAC and cosmid clone DNAs as probes. The final overall order of telomere D8S 131-D8S339-GSR-PP2A -D8S87-centromere was determined. The distance from D8S131 to D8S339 was found to be 815 kb and that between D8S339 and D8S87 was found to be 838 kb. Thus, the distance between D8S131 and D8S87 was estimated to be 1598 kb. Similar results were obtained from four normal adults; a Japanese male and female and a Caucasian male tlnd female. To our surprise, the distance between D8S131 and D8S87 was much shorter than previously estimated by recombination analysis, i.e. 8.3 cM equivalent to 8.3 Mb in physical distance. This experiment provided the basis for constructing a complete P1 contig of the WRN locus for the end of the positional cloning of the gene.
P1/PAC DNAs were used to produce a contig map that contains a WS locus (1.3 Mbp) representing the highest linkage disequilibriums obtained from the analysis with over 60 Japanese WS patients. Earlier, we tested YAC (yeast artificial chromosome) clones for this purpose, but it often gave rise to puzzling data stemming from the chimeric DNA rearrangement.
A contig map that covers the region from D8S131 to DS283 was completed, and each P1/PAC DNA was subjected to restriction enzyme cleavage mapping analysis, isolation of new STS (sequence tagged site) markers used for further narrowing the WS area, and/or exon trapping experiments.
Each overlapping P1/PAC DNA was purified and used for isolating the encoded exons by a method which used the splicing vector pSPL3 (Church et al. Nature Genetics, 6, 98-105, 1994). From 17 P1/PAC DNA, a total of 246 distinct exons (or exon-like DNA fragments) were obtained, and they were subjected to further analyses including the determination of the sequence and an accession to various data bases.
An exon-joining was carried out to link two adjacent exons by PCR, and the elongated exons (exon di-or tri-mers) were used as probes to screen for cDNAs from appropriate cDNAs libraries. TAIL-PCR (thermal asymmetric interlaced PCR); (Liu and Whittier, Genomics 25, in press 1995) has also been carried out with these exons in an attempt to detect new mRNA species. mRNAs derived from the WS region are cloned and investigated extensively with respect to differences in the nucleotide sequence between normals and patients.
More than 30 new patients have so far been identified in 18 months, from whom blood specimens were obtained. The fresh specimens (usually l0 ml) were split into three; one third for DNA extraction, one third for B-cell proliferation after EBV infection, and the other third for tlctiviltion of T-cells by OKT-3 and IL-2. These cells were kept frozen at -115°C in 10% DMSO/ 90% fetal calf serum. The EBV transformed B-cells are being used to compare the life-span of cells with cells of normal or patient origin.
Haplotype Analysis of the WRN Region.
Werner’s syndrome (WS) is a rare, autosomal recessive disorder, which has been mapped to the 8p11.1-21.1 region. Because WS is so rare and because many patients are from consanguineous marriages, fine localization of the gene by traditional meiotic mapping methods is unlikely to succeed. Identification of disease-specific haplotypes provides an alternative strategy for localizing the disease locus, and is the strategy which we have been using to narrow the critical region containing WRN. This strategy involves three stages: (1) identifying markers which are in linkage disequilibrium with WRN, as determined by pairwise tests, (2) use of a subject of these markers to construct haplotypes in cases and controls, and (3) use of the haplotypes to identify probable recombinants which have occurred during the evolution of the region, thus narrowing the probable location of WRN.
Dr. Ellen Wijsman’s group have performed pairwise tests to investigate the presence or absence of linkage disequilibrium for about three dozen markers spanning the region between D8S133 and D8S164. Because of the large number of alleles at most of these predominantly microsatellite markers, statistical tests typically used to evaluate evidence of linkage disequilibrium may give erroneous results. Thus we have compared the results obtained with a number of possible tests, and show that a Monte Carlo Markov chain method of approximating the Fisher exact test (the ideal test) gives the best results, while the more common approach of pooling observations in order to use a chi-square test gives the worst results. The linkage disequilibrium tests suggest that WRN is between D8S278 on the centromeric side, and C41C3S3 on the telomeric side.
A subset of 21 markers between D8S137 and D8S278, which contains the interval from C41C3S3 through D8S278, was used to construct haplotypes in Japanese and Caucasian cases and controls, using an EM-algorithm to estimate haplotype frequencies in situations where phase was ambiguous. We find evidence for three predominant disease-associated haplotypes in the Japanese population, accounting for approximately 70% of the Japanese haplotypes. One of these accounts for more than half of the Japanese haplotypes. Individuals with this haplotype originate from all over Japan. In the Caucasian population there are also three most common haplotypes, but these only account for about 1/3 of the Caucasian haplotypes. These more common caucasian haplotypes are all found in individuals with Sardinian or German ancestry. Ancestral recombinants defined by these haplotypes narrow the critical region containing WRN to the interval between Y807T2 and Y896R9. Issues which complicate interpretation of the results include (1) a high mutation rate among alleles at these microsatellite loci, (2) phase ambiguities in the control population, (3) variability in heterozygosity among marker loci, and (4) ethnic heterogeneity in the Caucasian population.
Werner’s Syndrome, Cellular Senescence, Telomeres and Telomerase.
The aim of this project is to see possible differences, between EBV-transformed B lymphocytes from Werner’s syndrome (WS) patients and from unaffected donors, in l) the maximal PDL, 2) establishing the frequency of immortal cell lines (proliferating over 100 PDL), 3) telomerase activity, and 4) telomere repeat length. Experiments are still ongoing and, therefore, conclusions should be awaited. Tentative results are as follows: 1) 11 of 28 cultures from WS and 5 of 20 cultures from unaffected donors came to crisis. No apparent differences were observed in PDLs at crisis between WS and unaffected donors. Maximal PDLs at crisis varied from 33 (an unaffected case) through 96 (a WS case). 2) One of 20 cultures from unaffected donors appeared to be immortalized (rapidly growing over 130 PDL). No culture from WS patients became immortalized so far. 3) Immortalized cultures had high telomerase activity before and after 100 PDLs. Another culture from an unaffected donor recently turned to high telomerase activity and continued prolifering (71 PDL at present), though not yet determined to be immortalized. All other cultures had weak or no telomerase activity. 4) No apparent difference was observed in telomere repeat length between EBV-lymphocytes from WS and unaffected donors. Crisis observed in EBV-lymphocytes is likely to be equivalent to M2 stage, not M1 stage, of SV40-transformed fibroblasts. WS phenotype (premature senescence) may not be expressed in M2 stage of EBV-transformed B lymphocytes. Dr. D. Drayna argued that EBV-lymphocytes became immortalized without exception in his experience of over 1000 cases. Culture conditions and/or criteria of immortalization may be different. Dr. G. M. Martin argued that telomere repeat length was not shorter in fibroblasts from WS patients than those from age-matched control donors, but that the shortening rate of telomere DNA with increasing PDLs was higher in the former than in the latter. However, WS fibroblasts reached the M1 stage (senescence) when telomere length was still longer than that of normal senescent cells, indicating premature senescence (M1 stage) of WS fibroblasts in terms of telomere length.
Genetic Regulation of Telomerase in Multiple Pathways to Cellular Senescence.
Telomerase activity has been detected in immortalized and tumor cells in vitro and in primary tumor tissues, and represents an important difference between normal cells and cancer cells. It has been proposed that telomere shortening causes cellular senescence. Genes involved in the senescent program have been mapped to over 10 different loci by the introduction of human chromosomes via microcell fusion and restoration of the senescence program. One or more of the senescence genes may suppress telomerase activity in tumor cells, resulting in telomere shortening and cellular senescence.
Reintroduction of a normal chromosome 3 into a RCC immortal cell line restored the program of cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells with a normal chromosome 3. Thus, restoration of the cellular senescence program by chromosome 3 is associated with repression of telomerase function in RCC cells. Radiation hybrid experiments showed the region to be 3pl4-21. In addition, several other findings that the telomerase function is one of the pathways involved in immortalization and each pathway may involve multiple genes. According to the hypothesis, the WRN gene could be a regulatory gene to enhance the expression of one of the genes involved in the cellular senescence program.
The Werner’s Syndrome in the Context of Evolutionary Biological Theories of Aging.
Mammalian gerontologists generally use the terms aging and senescing interchangeably, referring to the insidious and gradual development, after the attainment of the adult, sexually mature phenotype, of alterations in structure and function at many levels of organization. A few of these changes are adaptive, representing attempts by the organism to maintain homeostasis, but most are nonadaptive; an example would be the loss of proliferative homeostasis that leads to the emergence of a variety of neoplasms about half way through the usual life spans of all mammals so far examined for age-related pathologies. At the population level, these increasing structural and functional disabilities are associated with exponential increases in age-specific death rates. Given the paucity of evidence for group selection, essentially all evolutionary biologists regard aging as non-adaptive to the species, simply representing phenotypes that have escaped the force of natural selection. Three classes of gene action appear to underlie aging: l) Constitutional mutations or allelic variations at loci selected because of enhanced reproductive fitness, but which exhibit deleterious effects in late phases of the life history, when the force of natural selection is attenuated (“antagonistic pleiotropy”). 2) Constitutional mutations or polymorphisms that have neutral effects during early life but that reach some threshold of phenotypic expression in late life. 3) Constitutional mutations or polymorphisms whose phenotypic effects have been postponed as a result of suppressor mutations at other loci.
Given the findings that reproductive fitness is clearly impaired in the Werner’s syndrome, some authors (e.g., Rose, 1991) argue that the disorder cannot be regarded as a suitable model for aging research. There are at least four counter-arguments, however: l) The heterozygous state does not appear to impact upon reproductive fitness, but may impact upon common aging phenotypes, perhaps including neoplasia. 2) There may exist allelic variants at that locus representing different degrees of “leakiness,” even when homozygous, with relatively delayed onsets and with unimpaired reproductive fitness. 3) Suppressor mutations may be evolving over many generations to delay the times of onset. 4) The Werner’s syndrome mutation may define a locus, allelic variation at which could potentially lead to enhanced structure and function over the life course (“antigeroid” genotypes).
Werner’s syndrome is of interest to evolutionary biologists on another level. Somatic cells from homozygotes exhibit dramatically accelerated rates of clonal attenuation. It has therefore been widely viewed as a model for the study of accelerated aging at the cellular level. The process of clonal attenuation, however, appears to occur with cells obtained from the embryo, as shown in the original classic studies of Hayflick and Moorhead. The process could not possibly have escaped the force of natural selection and should not, strictly speaking, be regarded as a process of aging, Nevertheless, it has important consequences for aging, in that it could contribute to the development of aberrations in proliferative homeostasis in aging tissues (regional multi-focal atrophies and, via altered cell-cell regulation, focal hyperplasias). Some workers believe that the process of clonal attenuation evolved to protect the organism from neoplasia, but I have argued that there were likely to have been stronger selective forces involved in the regulation of organ growth and development (Martin, 1993).
References:
Rose, Michael R. Evolutionary Biology of Aging. Oxford University Press, New York, 1991.
Martin, George M. Clonal attenuation: causes and consequences. J. Gerontol. 48:B171-172, 1993.
PARTICIPANTS
UNITED STATES
Dr. Dennis Drayna
Mercator Genetics Inc.
Director of Research and Genetics
4040 Campbell Avenue
Menlo Park CA 94025
Tel: (415) 617-0881 Fax: (415) 617-0883
E-mail: drayna@jmercator.com
Dr. George M. Martin
Department of Pathology Box 357470
University of Washington
Seattle WA 68195
Tel: (206) 543-5088 Fax: (206) 685-8356
E-mail: gmmartin@u.washington.edu
Dr. Raymond J. Monnat, Jr.
Department of Pathology Box 357470
University of Washington
Seattle WA 68195
Tel: (206) 543-6585 Fax: (206) 685-8540
E-mail: monnat@u.washington.edu
Dr. Robert W. Miller
Scientist Emeritus
Genetic Epidemiology Branch
National Cancer Institute, EPN-400
Bethesda, MD 20892-7360
Tel: (301) 496-5785 Fax: (301) 496-1854
E-mail: millerr@epndce.nci.nih.gov
Dr. Warren W. Nichols
Merck, Sharp & Dohme Labs
Bldg 44-1
West Point PA 19486
Tel: (215) 652-7980 Fax: (215) 652-3888
Dr. Charles Puissan
Unite de Genetique Clinique
Centre Hospitalier Universitaire D’Amiens
Hopital Nord 60054 Amiens Cedex
Pediatrie I (Dr Puissan)
Tel: (33) 22.66.82.60 Fax: (33) 22.66.82.94
Dr. Gerard Schellenberg
GRECC (182 B)
VA Medicai Center
1660 South Columbia Way
Seattle WA 98108
Tel: (206) 764-2701 Fax: (206) 764-2569
E-mail: zachdad@u.washington.cdu
Dr. Sydney Shall
University of Sussex at Brighton
Cell and Molecular Biology Laboratory
Falmer, Brighton BNI 90G England
Tel: 441 273 606 755 Fax: 441 273 678 433
Dr. Margaret A. Tucker
Genetic Epidemiology Branch
National Cancer Institute. EPN 439
Bethesda MD 20892-7372
Tel: (301 ) 496-4375 Fax: (301) 402-4489
Dr. Ellen Wijsman
Department of Medical Genetics
Box 357720
University of Washington
Seattle WA 98195
Tel: (206) 543-8987 Fax: (206) 616-1973
E-mail: wijsman@u.washington.edu
JAPAN
Dr. Makoto Goto
Director, Division of Rheumatic Diseases
Tokyo Metropolitan Otsuka Hospital
2-8-1, Minami-Otsuka, Toshima-ku, Tokyo 170
Tel: 03-3941-3211 Fax: 03-3941-9557
Dr. Yuichi Ishikawa
Department of Pathology Associate
The Cancer Institute
1-37-1, Kami-Ikebukuro, Toshima-ku, Tokyo 170
Tel: 03-3918-0111 (4324) Fax: 03-5394-3923
Dr. Atsushi Hatamochi
Department of Dermatology
Chiba University School of Medicine
1-8-1 Inohana, Chuo-ku, Chiba 260
Tel: 043-222-7171 Fax: 043-226-2128
Dr. Toshinori Ide
Professor, Hiroshima University
1-2-3, Kasumi, Minami-ku, Hiroshima City,
Hiroshima 734
Tel: 082-257-5290 Fax: 082-257-5294
Dr. Mitsuo Oshimura
Professor
Faculty of Medicine, Tottori University
Nishimachi 86, Yonago, Tottori 683
Tel: 0859-34-8260 Fax: 0859-34-8134
Dr. Yasuhiro Furuichi
Director
AGENE Research Institute
Kajiwara 200, Kamakura, Kanagawa 247
Tel: 0467-46-4910 Fax: 0467-48-6595
Dr. Haruo Sugano
Director Emeritus, Cancer Institute
1-37-1. Kami-Ikebukuro. Toshima-ku Tokyo 170
Tel: 03-3917-5764 Fax: 03-3917-7564