Arvind K. Virmani

Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer.

Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender‐related changes in non‐small cell lung cancer (NSCLC). We investigated smoking‐related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs. We examined 514 cases of NSCLC and 84 corresponding nonmalignant lung tissues from 4 countries (USA, Australia, Japan and Taiwan) for the methylation status of 7 genes known to be frequently methylated in lung cancers [p16, RASSF1A (RAS association domain family 1), APC, RARβ, CDH13, MGMT and GSTP1]. Multivariate analyses were used for data analysis. Adenocarcinoma was the major histologic type in women and never smokers; analyses that involved smoke exposure and gender were limited to this histology. Our major findings are a) methylation status of any single gene was largely independent of methylation status of other genes; b) the rates of methylation of p16 and APC and the mean Methylation Index (MI), a reflection of the overall methylation status, were significantly higher in ever smokers than in never smokers; c) the mean MI of tumors arising in former smokers was significantly lower than the mean of current smokers; d) the methylation rates of APC, CDH13 and RARβ were significantly higher in adenocarcinomas than in squamous cell carcinomas; e) methylation rates of MGMT and GSTP1 were significantly higher in the USA and Australian cases than in those from Japan and Taiwan; and (f) no significant gender‐related differences in methylation patterns were noted. Our findings demonstrate important smoke exposure, histologic type and geography‐related differences in the methylation profiles of NSCLC tumors.

Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer.

The goal of our study was to develop a panel of tumor cell lines along with paired non‐malignant cell lines or strains collected from breast cancers, predominantly primary tumors. From a total of 189 breast tumor samples consisting of 177 primary tumors and 12 metastatic tissues, we established 21 human breast tumor cell lines that included 18 cell lines derived from primary tumors and 3 derived from metastatic lesions. Cell lines included those from patients with germline BRCA1 and FHIT gene mutations and others with possible genetic predisposition. For 19 tumor cell lines, we also established one or more corresponding non‐malignant cell strains or B lymphoblastoid (BL) lines, which included 16 BL lines and 7 breast epithelial (2) or stromal (5) cell strains. The present report describes clinical, pathological and molecular information regarding the normal and tumor tissue sources along with relevant personal information and familial medical history. Analysis of the breast tumor cell lines indicated that most of the cell lines had the following features: they were derived from large tumors with or without axillary node metastases; were aneuploid and exhibited a moderate to poorly differentiated phenotype; were estrogen receptor (ER)‐ and progesterone receptor (PR)‐negative; and overexpressed p53 and HER2/neu proteins. Of 13 patients with primary breast cancers receiving curative intent mastectomies, 7 were dead after a mean period of 10 months. Our panel of paired tumor and non‐malignant cell lines should provide important new reagents for breast cancer research. Int. J. Cancer 78:766–774, 1998.

Mutation analysis of the PTEN/MMAC1 gene in lung cancer

We studied PTEN/MMAC1, a newly discovered candidate tumor suppressor gene at 10q23.3, for mutations in lung cancer. One hundred and thirty-six lung cancer cell line DNAs (66 small cell lung cancers, SCLC, 61 non-small cell lung cancers, NSCLC, four mesotheliomas, five extrapulmonary small cell cancers) were analysed for PTEN/MMAC1 homozygous deletions and five (8%) SCLC lines showed homozygous deletions interrupting the PTEN/MMAC1 gene. Using single stranded conformation polymorphism (SSCP) analysis, we screened the PTEN/MMAC1 open reading frame of 53 lung cancer cell line cDNAs for point mutations and found that 3/35 SCLCs and 3/18 NSCLCs contained homozygous amino acid sequence altering mutations. Northern blot analysis revealed that expression of the PTEN/MMAC1 gene was considerably lower in all the tumor cell lines with point mutations while no expression was detected for cell lines with PTEN/MMAC1 homozygous deletions. Mutation analysis of 22 uncultured, microdissected, primary SCLC tumors and metastases showed two silent mutations, and two apparent homozygous deletions. We also discovered a processed pseudogene (PTEN2) which has 98.5% nt identity to PTEN/MMAC1, that needs to be accounted for in cDNA mutation analysis. Our findings suggest that genetic abnormalities of the PTEN/MMAC1 gene are only involved in a relatively small subset of lung cancers.

Allelotyping demonstrates common and distinct patterns of chromosomal loss in human lung cancer types

Allelic loss is a hallmark of tumor suppressor gene (TSG) inactivation. We have allelotyped 29 paired lymphoblastoid and lung cancer cell lines derived from 11 patients with small cell (SCLC) and 18 patients with non‐small cell lung carcinomas (NSCLC). Statistical analysis indicated that a threshold of 30% separated non‐random allelic loss from the random genetic deletions of malignancy. We have identified non‐random allelic loss at 42 of 54 (78%) specific chromosomal regions examined, with 22 regions (52%) common between the two major lung cancer histologic types. There were 3 regions (7%) with allelic loss specific for SCLC and 17 regions (41%) specific for NSCLC. Furthermore, there were significant differences in loss of heterozygosity (LOH) frequencies between NSCLC and SCLC at 13 regions on eight chromosome arms (3p, 5q, 6q, 9p, 10q, 11p, 13q, and 19p). Eight homozygous deletions were present in seven cell lines at four regions, 3p12, 3p14.2, 9p21, and 10q23–25. We have also identified novel sites of chromosomal deletions. In particular, there was frequent loss at 11p13 in SCLC and loss at 6p21.3 and 13q12.3 in NSCLC. In this study, we demonstrate that a) non‐random allelic losses in lung cancer involve multiple regions; b) some losses are common to both NSCLC and SCLC subtypes, whereas others are subtype specific; c) there are genetic deletions at novel chromosomal regions; and d) several homozygous deletions have been noted. Our studies demonstrate the usefulness of continuous cell lines for detailed allelotyping, for comparing genetic abnormalities between SCLC and NSCLC, and for identifying homozygous deletions. Genes Chromosomes Cancer 21:308–319, 1998.

Aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers

An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor β‐2 (RARβ‐2), CDH13 (H‐cadherin), p16INK4a (p16), RASSF1A (RAS association domain family I) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens. In addition, we also analyzed sputum samples from 30 never smokers for methylation of these genes. Our major findings are: (i) At least one gene was methylated in one or more specimens from 48% of the smokers. However, methylation was statistically significant less frequently in never smokers compared to smokers. (ii) In general, methylation occurred more frequently in samples from the central airways (sputum, bronchial brushes) compared to the peripheral airways (BAL) and only occasionally in the oropharynx. (iii) RARβ‐2 was the most frequently methylated gene, whereas the frequency of methylation for the other genes was lower. (iv) Data from sputum samples and bronchial brushes were comparable. Our findings suggest that detection of methylation should be investigated as an intermediate marker for lung cancer risk assessment and response to chemopreventive regimens.

High-resolution chromosome 3p allelotyping of breast carcinomas and precursor lesions demonstrates frequent loss of heterozygosity and a discontinuous pattern of allele loss

We performed high-resolution allelotyping for loss of heterozygosity (LOH) analysis on microdissected samples from 45 primary breast cancers, 47 mammary preneoplastic epithelial foci, and 18 breast cancer cell lines, using a panel of 27 polymorphic chromosome 3p markers. Allele loss in some regions of chromosome 3p was detected in 39 of 45 (87%) primary breast tumors. The 3p21.3 region had the highest frequency of LOH (69%), followed by 3p22-24 (61%), 3p21.2-21.3 (58%), 3p25 (48%), 3p14.2 (45%), 3p14.3 (41%), and 3p12 (35%). Analysis of all of the data revealed at least nine discrete intervals showing frequent allele loss: D3S1511-D3S1284 (U2020/DUTT1 region centered on D3S1274 with a homozygous deletion), D3S1300-D3S1234 [fragile histidine triad (FHIT)/FRA3B region centered on D3S1300 with a homozygous deletion], D3S1076-D3S1573, D3S4624/Luca2.1-D3S4597/P1.5, D3S1478-D3S1029, D3S1029 (with a homozygous deletion), D3S1612-D3S1537, D3S1293-D3S1597, and D3S1597-telomere; it is more than likely that additional localized regions of LOH not examined in this study also exist on chromosome 3p. In multiple cases, there was discontinuous allele loss at several 3p sites in the same tumor. Twenty-one of 47 (45%) preneoplastic lesions demonstrated 3p LOH, including 12 of 13 (92%) ductal carcinoma in situ, 2 of 7 (29%) apocrine metaplasia, and 7 of 25 (28%) usual epithelial hyperplasia. The 3p21.3 region had the highest frequency of LOH in preneoplastic breast epithelium (36%), followed by 3p21.2-21.3 (20%), 3p14.2/FHIT region (11%), 3p25 (10%), and 3p22-24 (5%). In 39 3p loci showing LOH in both the tumor and accompanying preneoplasia, 34 (87%) showed loss of the same parental allele (P = 1.2 x 10(-6), cumulative binomial test). In addition, when 21 preneoplastic samples showing LOH were compared to their accompanying cancers, 67% were clonally related, 20% were potentially clonally related but were divergent, and 13% were clonally unrelated. Overall this demonstrated the high likelihood of clonal relatedness of the preneoplastic foci to the tumors. We conclude that: chromosome 3p allele loss is a common event in breast carcinoma pathogenesis; involves multiple, localized sites that often show discontinuous LOH with intervening markers retaining heterozygosity; and is seen in early preneoplastic stages, which demonstrate clonal relatedness to the invasive cancer.

Steroid-hormone receptors in cell lines and tumor biopsies of human lung cancer

Female gender is a significant independent favorable prognostic factor in lung cancer. To study the possible role of sex hormones in lung cancer, the expression of sex‐steroid receptors and the glucocorticoid receptor was investigated in 29 lung‐cancer cell lines stemming from small‐cell lung cancer (SCLC) and non‐small‐cell lung cancer (NSCLC) by means of immunocytochemistry, ligand‐binding assays and RNA expression via polymerase chain reaction. In at least 2 methods of investigation, NSCLC cell lines showed a low expression of estrogen receptor in 6, progesterone receptor in 13 and androgen receptor in 12 out of 17 cases examined; sex‐steroid‐receptor expression was virtually absent in SCLC cell lines. The glucocorticoid receptor was expressed in all 29 cell lines studied. Additionally, 52 tumor samples from primary lung cancer were investigated for their receptor expression by means of immunohistochemistry. Among patients with primary lung‐cancer sex‐steroid‐receptor expression in tumor biopsies was detected most frequently in female patients (in 69% of 16 cases, vs. 42% of 36 tumors from men) and in patients with adenocarcinoma. Further research will focus on these subgroups. Immunohistology is a feasible method of studying steroid‐receptor expression in lung cancer.

Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes.

AIMS/BACKGROUND: Telomerase, an enzyme associated with cellular immortality, is expressed by most malignant tumours, but is inactive in normal somatic cells except for male germ cells and proliferating stem cells. Thus, the measurement of telomerase activity in tissue samples may provide useful diagnostic and prognostic information. The aim of this study was to determine whether telomerase expression is useful for the detection of occult malignant cells in lymph nodes. METHODS: Telomerase activity was compared with histological findings in 123 surgically removed lymph nodes submitted for routine or frozen section diagnosis. Telomerase activity was measured using a modified, semi-quantitative PCR-based telomeric repeat amplification protocol (TRAP). The assay was adapted for single 5 microns OCT embedded cryostat sections. In either fresh tissues or cryostat sections, normalised activity was linear when compared with protein concentration. Furthermore, using an in situ hybridisation method, the human telomerase RNA (hTR) component was measured in a subset of negative and positive nodes. RESULTS: Most (96%) of the 97 histologically negative nodes expressed low levels of activity (mean value of positive samples = 3.0 units/microgram protein) which may be derived from activated lymphocytes that express telomerase activity. All 26 malignant nodes (17 metastases, nine lymphomas) expressed telomerase (mean value = 17.8 units/microgram protein). The rank order levels between the two groups differed significantly (p = 0.0002). In situ results showed clearly that the hTR was expressed relatively highly in metastatic cancer cells and at lower levels in germinal centres of secondary follicles. CONCLUSIONS: Although expression of telomerase by activated lymphocytes may limit its usefulness, measurement of enzyme activity combined with detection of hTR using in situ hybridisation may assist in the histopathological diagnosis of lymph nodes.

Comparative Genomic Hybridization Reveals Complex Genetic Changes in Primary Breast Cancer Tumors and Their Cell Lines

DNA copy number changes were characterized by comparative genomic hybridization (CGH) in 18 breast cancer cell lines. In 5 of these, the results were comparable with those from the primary tumors of which the cell lines were established. All of the cell lines showed extensive DNA copy number changes, with a mean of 16.3 +/- 1.1 aberrations per sample (range 7-26). All of the cell lines had a gain at 8q22-qter. Other common gains of DNA sequences occurred at 1q31-32 (89%), 20q12-q13.2 (83%), 8q13 (72%), 3q26.1-qter (67%), 17q21-qter (67%) 5p14 (61%), 6p22 (56%), and 22pter-qter (50%). High-level amplifications were observed in all cell lines; the most frequent minimal common regions were 8q24.1 (89%), 20q12 (61%), 1q41 (39%), and 20p11.2 (28%). Losses were observed less frequently than gains and the minimal common regions of the most frequent losses were Xq11-q12 (56%), Xp11.2-pter (50%), 13q21 (50%), 8p12-pter (44%), 4p13-p14 (39%), 6q15-q22 (39%), and 18q11.2-qter (33%). Although the cell lines showed more DNA copy number changes than the primary tumors, all aberrations, except one found in a primary tumor, were always present in the corresponding cell line. High-level amplifications found both in primary tumors and cell lines were at 1q, 8q, 17q, and 20q. The DNA copy number changes detected in these cell lines can be valuable in investigation of tumor progression in vitro and for a more detailed mapping and isolation of genes implicated in breast cancer.

Aberrant methylation of TMS1 in small cell, non small cell lung cancer and breast cancer.

TMS1 (target of methylation‐induced silencing) is a CpG island‐associated gene that functions in the regulation of apoptosis and encodes a caspase recruitment domain, a recently described motif found in apoptotic signaling molecules. Recent evidence suggests that silencing of genes in the apoptotic pathway contribute to human carcinogenesis. We examined the DNA methylation status of the TMS1 promoter in lung and breast tumor tissues, tumor cell lines and nonmalignant tissues by methylation‐specific polymerase chain reaction (MSP) and its mRNA expression by reverse transcription PCR. Aberrant methylation of TMS1 was present in 70% (40 of 57) of small cell lung cancer (SCLC) cell lines and 41% (13 of 32) of SCLC tumor tissues, 48% (29 of 61) of non small cell lung cancer (NSCLC) cell lines and 40% (28 of 70) of NSCLC tumor tissues and 46% (12 of 26) of breast cancer cell lines and 32% (20 of 63) of breast tumor tissues. Methylation was absent in the peripheral blood lymphocytes and buccal epithelium from healthy volunteers, as well as in nonmalignant lung tissues and was rare in nonmalignant breast tissues 7% (2 of 30). DNA methylation was confirmed by sequence analysis and the methylation status correlated inversely with TMS1 RNA expression in 18 cell lines tested. RNA expression was restored by treatment with the demethylating agent 5‐aza‐2′‐deoxycytidine, in 4 of 4 methylated cell lines that lacked the TMS1 transcript. Our results suggest that methylation of TMS1 may play a role in the pathogenesis of small cell and non small lung and breast cancers.