BIOLOGY AND DIAGNOSIS PROGRAM AREA

REPORTS ON SEMINARS

(1) Genetic Diagnosis of Cancer

ABSTRACTS OF US PARTICIPANTS:
Marx SJ, Agarwal SK, Koster M09 Kim YS, Hoppner C, Goldsmith P, Spiegel AM, Burns AL (NIDDK), Emmert-Buck MR, Debelonko LV, Zhuang Z, Lubonsky IA, Liotta LA (MCI), Guru SC, Manickam P, Crabtree JS, Olufomi SE, Collins FS, Chandrasekharappa SA (NNORI)

MENI: EARLY FINDINGS WITH A NOVEL AND RECENTLY CLONED TUMOR SUPPRESSOR GENE.
Multiple endocrine neoplasia type I (MENI) is expressed as tumors of the parathyroid, the gastrointestinal endocrine tissues, and the anterior pituitary. Additional tumors include foregut carcinoids, thyroid tumors, and angiofibromas or lipomas of skin. Most of the associated tumors cause morbidity by hormone hypersecrstion, but the gastrinomas and carcinoid tumors have high malignant potential. A mainly intramural NIH consortium recently identified the MENI gone by a rigorous positional cloning approach. The MENI gene was novel with no important homologous proteins or even with any recognizable signature domains. It predicted an encoded protein (enin of 610 amino acids that was widely expressed. Analysis for germline mutation revealed mutations in 47 of 50 families with MENI. There was no correlation of mutations with selected phenotypes. We tested sporadic tumors of the types previously recognized to show frequent loss of heterozygosity (LOH) at 11q13. Overall we found approximately 20% mutation of MENI in sporadic tumors of parathyroid, gastrinoma, insulinoma, pituitary tumor, and carcinoid. Compilation of the germline and somatic MENI mutations indicated that two thirds predicted protein truncation and one third were missense. The missense mutations were spread randomly and did not point to regulatory hot spots. Current efforts are directed at understanding the normal and abnormal roles of menin.

MICRODISSECTION TECHNOLOGY: APPLICATIONS TO GENE DISCOVERY AND MOLECULAR DIAGNOSIS.
Lance A. Liotta, Rodrigo Chuaqui, David Krizman, Zhongping Zhuang Kristina Cole, Michael Emmert Buck, and Robert Bonner*, National Cancer Institute, and *National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD. USA
Understanding the molecular forces driving premalignant progression will offer new strategies for early detection and intervention. Microhybridization array technology Is becoming ever more sophisticated (see abstract of Dr. Trent). Imagine a prostate or breast specific set of thousands of genes on a slide. Further imagine that we can sample each pathologic stage of progression, Including the metastatic lesion, before and after treatment, and obtain a fingerprint of the fluctuating sets of genes in actual human tissue biopsies. Under direct microscopic visualization, Laser Capture Microdissection (LCM)* permits rapid one step procurement of selected call populations directly from a section of complex heterogeneous tissue. A transparent ethylene vinyl acetate (e.v.a.) thermoplastic film Is applied to the surface of the tissue section on a glass slide hold in a standard microscope stage (see abstract of Dr. Bonner). The operator moves the slide under a fixed pulsed laser which irradiates the a.v.a. film above the chosen calls. The laser pulse energy is absorbed (>99%) by the film which adheres tightly to the underlying selected calls, which are selectively procured when the film Is removed directly into PCR amplification buffer. LCM can be applied to fixed stained tissue sections with preservation of protein, DNA, and RNA, and has potential utility for chip based genetic panel testing. From a single patient’s tissue section RNA or DNA can be procured from normal epithelium, hyperplasia, in situ carcinoma, and invasive carcinoma. Under direct microscopic visualization of stained fixed tissue, individual areas corresponding to cellular regions ranging in size from less than 30 microns to 300 microns are procured and then analyzed. The timing and persistence during progression of somatic genetic alterations, or gene expression fluctuations, can now be analyzed directly in human tissue microscopic lesions. Microdissection based LOH analysis has revealed alterations in many genetic loci which are first detected in cancer precursor stages. Microdissection has now permitted the development of cDNA libraries representing normal epithelium, atypical hyperplasia, in situ, invasive lesions, and metastatic foci, all from the same patient. Microdissection derived cDNA libraries are being developed for human solid tumors under the NCI CGAP program (see abstracts of Krizman, Cole and Emmert-Buck). Snap shot cDNA libraries of microdissected human tumor premalignant stages can impact on the search for specific patterns of gene expression which correlate with progression, invasion, or metastasis to specific sites such as bone.
* Science, Vol 2749 pp. 998-1001, 1996
* Science, Vol 278, pp.1481-30 1997

Application of Molecular Detection to the Integrated Management of Early Lung Cancer.
James L. Mulshins, M.D., Head, Intervention Section, CCBD, MB, DCS, NCI, NIH, Rockville, MD 20850
Lung cancer is the most commonly fatal cancer worldwide. The current mortality rate for this cancer approaches 90% with the overwhelming majority of this disease caused by exposure to tobacco combustion products. Unfortunately, with the current burden of tobacco consumption in the world today and the long incubation time of lung cancer, the high death rate from this cancer will not change for decades despite success with smoking cessation measures. Using a recently reported approach to population-based lung cancer screening with sputum-based immunocytology, we were able to detect the majority of lung cancer cases in the pro-invasive state in the preliminary analysis of two ongoing trials (Clin Cancer Ros 3:2237, 1997). Controlling the progression of early genetic changes of lung cancer provides an opportunity to exploit local chemoprovention-drug delivery strategies. The field of cancer injury comprises the airway tissue In the direct path of the tobacco-combustion products. Through the use of aerosolized-delivery technologies and other local delivery approaches, we postulate that high drug levels can be administered with minimal systemic toxicity by exclusively targeting the cancer field. Using now molecular diagnostic technologies, the success of these delivery approaches can be monitored by serial bronchoscopic analysis of the airway epithelium to evaluated for regression of clonal populations of genetical injured cells. If the integration of these now approaches can be coordinated, this may allow for success in reducing the occurrence of the final lethal phase of aerodigestive cancers.

Identification of motility and metastasis genes in a Drosophila melanogaster model
Elisa Woodhouse and Lance Liotta, Laboratory of Pathology, MCI, NIN, 9000 Rockville Pike, Bethesda, MD 20892

We have developed a Drosophila melanogaster tumor model characterized by the loss of function in the lethal giant larvae (lgl) gene, which produces tissues that grow, invade extensively, and metastasize widely upon transplantation. The goal of this project is to identify genes which are important in motility and metastasis. The first approach we have taken is the identification of now motility and metastasis genes by the generation of second site mutations in an lgl hoterozygous mutant. The metastatic properties of tissues from these mutants can then be compared to lgl mutants. Second site mutations were generated by P element mediated mutagenesis. Mobilization of genetically engineered P elements is a method for generating mutations In random genes which can then be cloned with relative case. When the P element is mobilized, it inserts into a new chromosomal location. Often the P element will insert into a gene and will disrupt the expression of the gene. A collection of second and third chromosome mutation in an lgl background has been generated. Presently, approximately 32,000 lgl haterozygotes for P element transposition have been screened and with a frequency of P element mobilization of 1.1%. Tissues from the Drosophila lines which were generated for comparison to lgl tissues are being analyzed by in vivo transplantation methods. The genes in which mutations suppress or enhance metastasis will be cloned and sequenced and the functions of the proteins analyzed. The second approach we are using is to analyze the effects of mutations in genes which are known to encode signaling proteins on the metastasis of lgl tissues. Specifically, we have made Drosophila lines which are lgl mutants and contain one of the following: loss of function of rolled (the Drosophila MAP kinase gone), constitutively activated Drosophila Ras, constitutively activated Drosophila Raf. constitutively activated human Raf, loss of function of the Drosophila ras gene, and a hypomorphic allele of the Drosophila ras gene. The affects of these genetic changes on the motility and invasion assays of lgl tissues are being tested. We have adapted in vitro motility and invasion assays for lgl calls.

Evaluation of Tissue Fixation on Histology and Integrity of RNA/DNA and Protein
Kristina Cole, Paul Duray, Rodrigo Chuaqui, John Gillespie, Tracy Hatcher@, Stanley Hamiltonffi and Jonathan Epsteing, Lance Liotta and Michael Emmert-Buck, and S. Steven Bova#. Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, 10/2A33, 9000 Rockville Pike, Bethesda, MD 20892 # Department of Pathology, Pathology Building, Johns Hopkins University, Baltimore, MD 21287

Background: In routine pathology paraffin embedding of patient tissues has several advantages over tissue freezing. These include convenient archiving and improved histologic detail. The latter becomes particularly important when diagnosing premalignant lesions. Prior to paraffin embedding the tissue must be fixed to preserve the tissue morphology and prevent autolysis. The most routinely used fixative is 10% neutral buffered formalin. Unfortunately, formalin cross links nucleic acids to protein limiting the molecular analysis from these tissues. Recently is has been demonstrated that alcohol based fixation prior to embedding renders the nucleic acids amenable to PCR and random hexamer primed RT-PCR.
Specific Aim In this study we sought to identify a fixative that would provide adequate histology for routine pathologic diagnosis while maintains RNA, DNA and protein integrity better than 10% neutral buffered formalin. In the hope of applying this technique to gene discovery and gene profiling of laser capture microdissected specimens, expression patterns were compared between RNA derived from paraffin embedded and frozen tissues. Methods Kidney and prostate tissue was obtained directly from the operating room, cut into equal sized pieces and placed into one of 13 fixatives or frozen immediately. The tissue was fixed for either 15 hrs or 5 days, cleared and paraffin embedded In a conventional tissue processor and blocked in paraffin. Five micrometer sections were cut onto glass slides. One set of coded slides were H&E stained and reviewed by pathologists at Johns Hopkins and MCI. The pathologists were asked to rate the sections on nuclear and cytoplasmic morphology, tissue architecture and overall usability , The remaining slides were dedicated for assessment of molecular preservation of RNA, DNA and protein (immunohistochemistry). RNA extracted from the paraffin embedded tissue was oligodT reverse transcribed, flanked with oligonucleotides of known sequence and amplified by PCR. The amplification smears were visualized on an agarose gel treated with ethidium bromide. To compare expression patterns, radiolabelled cDNAs obtained from laer capture microdissected prostate epithelium from frozen of paraffin embedded tissue were hybridized to a nylon 588 gene expression array. Results By averaging the histology rankings based on fixative alone 70% Ethanol + 100% Methanol (3:1) was rated the best followed by 70% Ethanol fixation. 10% neutral buffered formalin was rated fourth based on histology. Interobserver and intraobserver variability was observed. Integrity of RNA extracted from the paraffin embedded tissue was extremely sensitive to type of fixation. The aldehyde based fixatives 10% neutral buffered formalin and Safefix resulted in little to no recovery of RNA. The alcohol based fixatives preserved the integrity of the RNA. In particular, 70% Ethanol resulted in the strongest intensity and largest products. Preliminary experiments suggest that expression patterns between RNA derived from 70% ethanol fixed paraffin embedded and frozen microdissected tissues are very similar as assessed by hybridization to the cDNA array.

cDNA Sequencing and Analysis of PB39: A Novel Gone Up-regulated in Prostate Cancer
Kristina A. Cole, Rodrigo F. Chuaqui, Kenneth KatzQ, Svetiana Pack, Zongping Zhuang, Catherine E. Cole, John C. Lynag, Marston Linchanti, Lance A. Liotta, and Michael R. Emmert Buck, Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, 1012A33, 9000 Rockville Pike, Bethesda, MD, 20892 # Urologic Oncology Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, 10/2B47, 9000 Rockville Pike, Bethesda, MD 20892 @National Center for Biotechnology Information
We recently identified a novel gene (PB39) whose expression is up-regulated in human prostate cancer using tissue microdissection-based differential display analysis. In the present study we report the full length sequencing of PB39 cDNA, genomic localization of the PB39 gene, and genomic sequence of the mouse homologue. The full length human cDNA is 2317 nucleotides in length and contains an open reading frame of 559 amino acids which does not show homology with any reported human genes. The N-terninus contains charged amino acids and a helical loop pattern suggestive of an srp leader sequence for a secreted protein. Fluorescent in-situ hybridization using PB39 cDNA as probe mapped the gene to chromosome 11p1.1-1.2. Comparison of PB39 cDNA sequence with murine sequence available in the public database identified a region of previously sequenced mouse genomic DNA showing high sequence homology with human PB39. Based on alignment and comparison to the human cDNA the mouse genomic sequence suggests there are eight exons in the mouse gene spread over approximately 100 kb of genomic sequence. Further analysis of PB39 expression in human tissues shows the presence of a unique splice variant mRNA which appears to be primarily associated with fetal tissues and tumors. Interestingly, the unique splice variant appears in prostatic intraepithelial neoplasia (PIN), a microscopic precursor lesion of prostate cancer. The current data supports the hypothesis that PB39 plays a role in the development of human prostate cancer, and will be useful in the analysis of the gene product in further human and murine studies.

ABSTRACTS OF JAPANESE PARTICIPANTS
Under the U.S.-Japan Cooperative Cancer Research Program, seven speakers from Japan and 12 from the United States) met to discuss the latest research advances in molecular diagnosis of cancer. A wide variety of topics including development of novel technologies, identification of novel molecular targets and direct clinical applications in cancer patients were discussed by the participants.

Dr. Yoshinori Murakami described their ongoing efforts of genetic complementation study through the transfer of chromosomal fragments or YAC clones into human cancer cells. By conducting microcell-mediated chromosome transfer of various portions of human 10p fragments, his group has narrowed down to a 6 cM fragment on 10p14-p15 as a potential subchromosomal location of a putative prostate tumor suppressor gene. Dr. Murakami also reported on the use of YAC clones to identify a putative tumor suppressor gene at 11q23. Each YACs spanning a 5cM region on chromosome 11q23 was tagged with the neomycin resistance gene by homologous recombination in yeast and introduced into a human NSCLC cell line, A549, by spheroplast fusion. The resulting clones of one of the YACs suppressed the tumorigenicity when 1x10⁵ cells were injected into nude mice, suggesting that the YAC clone contains a tumor suppressor gene. Further refinement of the localization is now being undertaken using subportions of the parental YAC clone.
Dr. Yoshio Miki described ethnic differences in terms of the germline BRCA1 and BRCA2 mutations. To examine the nature and frequency of BRCA1 and BRCA2 mutations in Japanese families in the category of autosomal-dominantly inherited breast cancer, they screened 78 unrelated such families. By examining the entire coding sequences as well as exon-intron boundaries of both genes by PCR-SSCP and multiplex-SSCP analyses, they identified possible disease-causing BRCA1 alterations in the affected members of 15 families and BRCA2 mutations in another 14 families. In 15 of the 29 families, the affected individuals carried missense mutations in contrast to the previous reports that most of the germline mutations identified in America and Europe were deletions or nonsense mutations. He noted that their results showing clear disticntions between American and Japanese breast cancer families will be of vital importance for future genetic testing.
Dr. Takashi Takahashi spoke on the genes involved in the pathogenesis of lung cancers. The chromosome regions including 3p, 8p, 9p, 11p, 13q, 17p, 18q and 22q have been shown to be frequently deleted in lung cancer, suggesting the possible existence of tumor suppressor genes at these chromosomal regions. He began his talk by presenting some data of their earlier work on p53 mutations regarding its importance in the oncogenic process and usefulness in both molecular epidemiological studies and prediction of patients’ prognosis. He also showed that Smad2 and Smad4/DPC4 at 18q21 are inactivated in a fraction of lung cancers and that these in vivo mutants are indeed defective in transcriptional activation. Another possible target he also dealt with was p57^KIP2 at 11p15.5. He presented evidence for maternal imprinting of this cyclin-dependent kinase inhibitor gene in normal lung and showed the presence of significantly selective loss of the active maternal allele of p57^KIP2, suggesting that p57^KIP2 may play a role in lung cancer development. He concluded that better understanding of the molecular pathogenesis should allow us to establish a basis for designing novel strategies for better diagnosis, treatment and prevention of this fatal disease.
Dr. Yasuhiko Kaneko described metaphase cytogenetic and/or interphase 2-color FISH analyses using 1p telomeric and 1q near-centromeric probes on 246 patients with neuroblastomas, which included 186 patients found by mass screening at 6 months of age, and 60 diagnosed clinically after the negative results by the screening. He noted significant relationship between clinical characteristics and the constitution of chromosome 1. Ninety % of the mass screening-positive tumors were those with trisomy 1, pentasomy 1, or a mixed population of cells with trisomy 1 and cells with tetrasomy 1 as well as with 1p deletion. In contrast, 55% of the mass screening-negative tumors had 1p deletion with either disomy 1 or tetrasomy 1. Multivariate analysis revealed that the constitution of chromosome 1 had the largest contribution on the event free survival, followed by the outcome of mass screening, and the stage of the disease. MYCN amplification or the primary site provided no significant prognostic information, although univariate analysis showed significant influences of these factors on the event free survival. He concluded that the 2-color FISH analysis may be the most useful tool to predict the clinical outcome of neuroblastoma patients.
Dr. Masafumi Taniwaki described potential application of fluorescence in situ hybridization (FISH) analysis to molecular diagnosis of cancers. He noted that FISH technique has the advantage to demonstrate the genetic defects involving DNA segment ranging from 10 kb to 1~2 Mb in size in interphase nuclei. Accordingly, interphase FISH is particularly useful to detect the specific genomic rearrangements in cells of most differentiated malignancies and solid tumors, in which no metaphase was available for precise analysis of the karyotype. To simultaneously analyze multiple genetic rearrangements, they used yeast artificial chromosome (YAC) and P1-derived artificial chromosome (PAC) clones as FISH probes and have established a comprehensive diagnostic protocol to detect gene rearrangement specific to subtype of non-Hodgkin’s lymphoma (NHL). The IgH translocation was detected in 44% of 120 patients with NHL. The specific oncogenes were identified as partners of the IgH translocation as follows; BCL2 in 26% of patients with the IgH translocation, BCL6 in 23%, BCLI in 15%, c-MYC in 13%, and PAX-5 in 4%. Using two YAC probes specific to 11q24, t(11;22) was shown to be present in interphase nuclei of Ewing’s sarcoma. Split signals resulting from rearrangement of FLI-1 gene were detected in 70% of Ewing’s sarcoma and peripheral neuroepithelioma, demonstrating that the method is useful for the diagnosis of Ewing sarcoma family of tumors. He also added that interphase FISH with oncogene probes may complement the pathologic diagnosis of cancer.
Dr. Masao Seto described their experience in molecular diagnosis of lymphoma especially on mantle cell lymphoma (MCL) with BCL1 translocation at 11q13. While Dr. Seto group and others had suggested that the PRAD 1/cyclin D1 gene is a candidate BCL1 gene, their analysis of a variant translocation t(11;22)(q13;q11) nailed down that the PRAD1 gene is the BCL1 gene. Dr. Seto and his colleagues produced a highly specific monoclonal antibody, 5D4, against PRAD1/cyclin D1 gene product, which could stain formalin-fixed paraffin-embedded tissue of MCL. Immunostaining study on a larger number of various lymphomas revealed that positive staining occurred in 80 % of MCL, but not in other types of lymphoma, indicating that the antibody is useful for MCL diagnosis. Based on their preliminary results showing that 5D4 staining-negative MCLs had a better prognosis than those with positive staining, they have initiated a nationwide study. Although the study is still on going (128 cases of 5D4 positive MCL and 23 cases of 5D4 negative MCL), the 5D4 positive MCLs appear to constitute a distinct molecular pathologic entity with very poor prognosis. He also discussed about the relation between CD5+ diffuse large cell lymphoma and CD5+ MCLs.
Dr. Haruo Sugiyama reported on monitoring of minimal residual disease of leukemias and staging of myelodysplastic syndrome (MDS) by quantitation of Wilms tumor suppressor WT1 gene transcript. They found that WT1 is aberrantly overexpressed in almost all leukemic cells regardless of types of leukemias (AML, ALL, or CML) and thus that WT1 is a new tumor marker for leukemic blast cells. By the quantitation of WT1 transcript (WT1 assay), one leukemic cell in 10³-10⁴ normal bone marrow (BM) cells and in 10⁵ normal peripheral blood (PB) cells was detectable. He stated that they could detect relapse by the WTI assay 2-24 months prior to apparent clinical relapse. The WT1 levels that predicted with certainty patients relapse were 10^-2 in BM and 10^-3 in PB, where WT1 expression level of leukemic cell line K562 was defined as 1.0. He also showed possible clinical application of the WT1 assay to staging of MDS. High WT1 expression levels in PB predicted progression to overt leukemia within one year. As to biological consequence of overexpression of WTI in leukemia cells, he showed that WT1 antisense oligomers inhibited growth of lenkemic cells and that WTI transfected 32D cl3 (IL-3-dependent myeloid progenitor cell line) failed to differentiate and instead proliferated by the substitution of G-CSF for IL-3.

Japanese participants:

Dr. Yasuhiko Kaneko
Department of Medical Oncology, Saitama Cancer Center Hospital, Ina-cho, Kita-adachi-gun, Saitama 362, Japan
Phone: +81 (487) 22-1111
Fax: +81 (48) 722-1129
E-mail : kaneko@saitama-cc.go.jp

Dr. Yoshio Miki
Department of Molecular Diagnosis, the Cancer Institute, Japanese Foundation for Cancer Research, 1-37-1 Kamiikebukuro, Toshima-ku, Tokyo 170, Japan
Phone / Fax: +81 (3) 5394-3926
E-mail: Error! Bookmark not defined.

Dr. Yoshinori Murakami,
Oncogene Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104, Japan, National Cancer Center Research Institute
Phone: (03) 3542-2511 ext. 4806
Fax: (03) 5565-9535
E-mail: Error! Bookmark not defined.

Dr. Masao Seto
Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464, Japan
Phone & Fax: +81 (52) 764-2982
E-mail: Error! Bookmark not defined.

Dr. Haruo Sugiyama
Department of Clinical Laboratory Science, Osaka University Medical School, 1-7 Yamadaoka, Suita 565, Japan
Phone & Fax: +81 (6) 879-2593
E-mail: Error! Bookmark not defined.

Dr. Takashi Takahashi (organizer)
Laboratory of Ultrastructure Research, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464, Japan
Phone: +81 (52) 764-2983
Fax: +81 (52) 763-5233
E-mail: Error! Bookmark not defined.

Dr. Masafumi Taniwaki
Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kawaramachi, Kamikyo-ku, Kyoto 602, Japan
Phone: +81 (75) 251 -5519
Fax: +81 (75) 251-0710
E-mail: Error! Bookmark not defined.

American participants:
Dr. Diane Arthur, National Cancer Institute
Dr. Ilan Kirsch, National Cancer Institute
Dr. David Krizman, National Cancer Institute
Drs. Lance Liotta (organizer) & Kristina Cole, National Cancer Institute
Dr. Steve Marx, National Institute of Diabetes and Digestive and Kidney Diseases
Dr. James Mulshine, National Cancer Institute
Dr. Lou Staudt, National Cancer Institute
Dr. Patricia Steeg, National Cancer Institute & Dr. Allen Shearn, Johns Hopkins University
Dr. Jeffrey Trent, National Center for Human Genome Research
Dr. Elisa Woodhouse, National Cancer Institute