Humana Press Humana Press M E T H O D S I N M O L E C U L A R M E D I C I N E TM Lung Cancer Edited by Barbara Driscoll Volume I Molecular Pathology Methods and Reviews Lung Cancer Edited by Barbara Driscoll Volume I Molecular Pathology Methods and Reviews Genetic Alterations 3 3 From: Methods in Molecular Medicine, vol. 74: Lung Cancer, Vol. 1: Molecular Pathology Methods and Reviews Edited by: B. Driscoll © Humana Press Inc., Totowa, NJ 1 Characteristic Genetic Alterations in Lung Cancer Ignacio I. Wistuba and Adi F. Gazdar 1. Introduction Lung cancer is the most frequent cause of cancer deaths in both men and women in the U.S. (1). Although tobacco smoking is accepted as the number one cause of this devastating disease, our understanding of the acquired genetic changes leading to lung cancer is still rudimentary. Lung cancer is classifi ed into two major clinic-pathological groups, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) (2). Squamous cell carcinoma, adenocarcinoma, and large cell carcinoma are the major histologic types of NSCLC. As with other epithelial malignancies, lung cancers are believed to arise after a series of progressive pathological changes (preneoplastic lesions) (3). Many of these preneoplastic changes are frequently detected accompany- ing lung cancers and in the respiratory mucosa of smokers (3). Although many molecular abnormalities have been described in clinically evident lung cancers (4), relatively little is known about the molecular events preceding the development of lung carcinomas and the underlying genetic basis of tobacco- related lung carcinogenesis. To investigate the molecular abnormalities involved in the multistep patho- genesis of lung carcinomas, we have developed a fi ve-step analysis scheme that included the study of: 1) lung cancer cell lines; 2) microdissected primary lung tumors of the three major histologic types (SCLC, squamous cell carcinoma, and adenocarcinoma); and normal and abnormal respiratory epithelium from 3) lung cancer patients; 4) from smoker subjects without lung cancer; and from 5) never smoker subjects (see Fig. 1). Under this strategy we systematically search for mutations in tumor cell-lines specimens, and in archival tumor tissues, preneoplastic lesions, and normal epithelium, using paraffi n-embedded CH01,1-28,28pgs 07/22/02, 7:28 AM3 4 Wistuba and Gazdar materials. Recently, we have also analyzed genetic changes present in cytologic specimens bronchial brushes from smokers (5). In tissues samples, using a precise microdissection technique, under direct microscopic observation a variable number of cells from those areas are precisely isolated along with invasive primary tumor and stromal lymphocytes (as a source of normal constitutional DNA). Using polymerase chain reaction (PCR)-based techniques, these different specimens are examined for molecular abnormalities (mainly gene mutations and allele losses) at chromosomal regions frequently mutated or deleted in clinically evident lung carcinomas. The risk population for targeting lung cancer early detection efforts has been defi ned (current and heavy smokers, and patients who have survived one cancer of the upper aerodigestive tract). However, conventional morphologic methods for the identifi cation of premalignant cell populations in the airways have limitations. This has led to a search for other biological properties (including genetic changes) of respiratory mucosa that may provide new methods for assessing the risk of developing invasive lung cancer in smokers, for early detection, and for monitoring their response to chemopreventive regimens. Fig. 1. Schema showing the strategy developed to study the molecular abnormalities involved in the pathogenesis of lung cancer. CH01,1-28,28pgs 07/22/02, 7:28 AM4 Genetic Alterations 5 2. Tumor-Cell and Tissue-Specimens Methodologies Used in the Analysis of Lung Cancer Molecular Abnormalities To investigate the molecular abnormalities involved in the pathogenesis of lung cancers, we have utilized a panel of paired lung tumor cell lines and corresponding normal lymphoblastoid cells (6), as well as microdissection technique of archival paraffi n-embedded tumor and nonmalignant epithelial tissues (7–9) (see Fig. 2). Both methodologies have played a pivotal role in the study of the molecular abnormalities of the pathogenesis of lung cancer. 2.1. Paired Lung Tumor and Normal Lymphoblastoid Cell Lines Despite the pivotal role played by human lung cancer cell lines in biomedical research, there is a widespread belief in the scientifi c community that they are Fig. 2. Comparison of phenotypic properties between primary lung cancer tissues and their corresponding cancer cell lines. Upper panels, tumor tissue and corresponding cell line showing squamous cell differentiation with keratinization features. Lower panel, tumor tissue and corresponding cell line showing adenocarcinoma features with gland-like structures formation and p53 nuclear immunostaining. CH01,1-28,28pgs 07/22/02, 7:28 AM5 6 Wistuba and Gazdar not representative of the tumors from which they were derived. Lung cancer cell lines have demonstrated advanced molecular changes, including extensive chromosomal rearrangements, oncogene mutations, and multiple sites of allelic loss and gene amplifi cation (10,11). Thus, many investigators presume that loss of phenotypic properties and additional molecular changes develop during the prolonged time required for cell-culture establishment and subsequent passage. To investigate this phenomenon we compared the morphologic, phenotypic, and genetic changes in lung cancer cell lines and in their corresponding tumor tissues (12). We compared the properties of a series of 12 human NSCLC cell lines (cultured for a median period of 39 mo, range 12–69) and their corresponding archival tumor tissues. Other than differences in the degree of aneuploidy, the other properties studied demonstrated a remarkable degree of concordance between lung tumors and their corresponding cancer cell lines (see Table 1). These features included morphologic characteristics (see Fig. 2), presence of aneuploidy, immunohistochemical expression profi le for HER2/neu and p53 proteins, and a similar K-RAS and TP53 gene muta- tions allelic loss and MA pattern for multiple loci frequently deleted in lung carcinoma. The concordance between tumors and cell lines for all of the comparisons was independent of the time on culture, indicating that the properties of cell lines usually closely resemble those of their parental tumors for culture periods up to 69 mo. While p53 immunohistochemical protein expression was detected in all of the lung tumor cell lines and their corresponding tumor tissues (100% correlation). TP53 gene mutations in exons 5–8 were detected in 10 (83%) of 12 lung tumor cell lines, and six of those corresponding tumor tissues exhibited the identical TP53 gene mutation. K-RAS gene mutations at codon 12 were detected in two adenocarcinomas cell lines (17% of the NSCLCs and 33% of adenocarcinoma cases), and identical K-RAS mutations were identifi ed in their corresponding tumors. We also determined chromosomal deletions expressed by loss of heterozygosity (LOH) at 13 chromosomal regions frequently deleted in lung cancers. Nearly identical high LOH frequencies at all chromosomal regions analyzed were detected between tumors and theirs corresponding cell lines (see Table 1). For all of the individual markers there was an excellent correlation between tumors and cell lines (mean concordance of 89%). In all of the 115 (100%) comparisons, when allelic loss of a particular microsatellite was present in both the tumor and corresponding cell line, the identical parental allele was lost in both, confi rming that the allelic loss originated in the original tumor tissue. In addition, tumor cell did not develop greater frequency of genomic instability phenomenon in culture and they retain some of the unstable properties of their parental tumors after lengthy culture periods (up to 69 mo). CH01,1-28,28pgs 07/22/02, 7:28 AM6 Genetic Alterations 7 Our fi ndings also indicated that successfully cultured NSCLCs represent the general population of tumors and their cell lines are useful models for studying this important type of lung neoplasm. 2.2. Tissue Microdissection Technique The molecular examination of pathologically altered cells and tissues at the DNA, RNA, and protein level has revolutionized research and diagnostics in tumor pathogenesis. However, the inherent heterogeneity of primary tissues with an admixture of various reactive cell populations can affect the out- come and interpretation of molecular studies. Recently, microdissection of tissue sections and cytological preparations has been used increasingly for the Table 1 Comparison of Properties Between 12 Lung Cancer Tumor Tissues and Their Corresponding Cancer Cell Lines Frequency Feature Tumor tissue Cell lines Aneuploidy 100% 100% Protein immunohistochemical expression HER2/neu 25% 25% p53 protein 100% 100% Chromosomal region with LOH 3p25 38% 38% 3p22–24 55% 55% 3p21 58% 58% 3p14–21 22% 22% 3p14.2 (FHIT gene) 50% 50% 3p12 25% 25% Any 3p 67% 67% 5q22 (APC-MCC region) 44% 44% 8p23 91% 91% 8p22 91% 91% 8p21 58% 75% Any 8p 100% 100% 9p21 78% 89% 13q (RB gene) 33% 33% 17p (TP53 gene) 78% 89% Microsatellite Alteration (MA) 54% 58% Gene Mutations TP53 gene (Exons 5–8) 58% 83% K-RAS gene (Codons 12–13) 17% 17% CH01,1-28,28pgs 07/22/02, 7:28 AM7 8 Wistuba and Gazdar isolation of homogeneous, morphologically identifi ed cell populations, thus overcoming the obstacle of tissue complexity. In conjunction with sensitive analytical techniques, such as the PCR, microdissection allows precise in vitro examination of cell populations, such as normal epithelial or dysplastic cells, which are otherwise inaccessible for conventional molecular studies (see Fig. 3). However, most of manual microdissection techniques are time- consuming and require a high degree of manual dexterity, which limits their practical use. Microdissection under microscopic visualization using micromanipulator is very precise, but very time-consuming. Laser capture microdissection (LCM), a novel technique developed at the National Cancer Institute, is an important advance in terms of speed, ease of use, and versatility of microdissection (13). LCM is based on the adherence of visually selected cells to a thermoplastic membrane, which overlies the dehydrated tissue section and is focally melted by triggering of a low-energy infrared laser pulse. The melted membrane forms a composite with the selected tissue area, which Fig. 3. Representative example of precise tissue microdissection technique of bronchial epithelium (a and b) and adenocarcinoma of the lung (c and d) (before and after microdissection). Note that only tumor and epithelial cells were microdissected without contamination with stromal cells. CH01,1-28,28pgs 07/22/02, 7:28 AM8 Genetic Alterations 9 can be removed by simple lifting of the membrane. LCM can be applied to a wide range of cell and tissue preparations including paraffi n wax-embedded material. The use of immunohistochemical stains allows the selection of cells according to phenotypic and functional characteristics. Depending on the starting material, DNA, good-quality mRNA, and proteins can be extracted successfully from captured tissue fragments, down to the single-cell level. In combination with techniques like expression library construction, cDNA array hybridization, and differential display (14–16), the use of this microdissection technique has allowed to us to analyze minute amount of lung tissues and perform most of our studies on the genetic changes involved in lung cancer pathogenesis (7–9,12,17–25). 3. Overview of Molecular Abnormalities in Lung Cancer Several cytogenetic, allelotyping, and comparative genomic hybridization (CGH) studies have revealed that multiple genetic changes (estimated to be between 10 and 20) are found in clinically evident lung cancers, and involve known and putative recessive oncogenes as well as several dominant oncogenes (4). The major molecular changes detected in lung cancers are summarized in Table 1. 3.1. Growth Stimulation and Oncogenes Many growth factors or regulatory peptides and their receptors are expressed by cancer cells or adjacent normal cells in the lung, and thus provide a series of autocrine and paracrine growth stimulatory loops in this neoplasm (26). Several but not all components of these stimulatory pathways are proto-oncogene products. 3.2. Gastrin-Releasing Peptide (GRP)/Bombesin (BN) Autocrine Loop There is good evidence that the GRP/BN and GRP receptor autocrine loop is involved in the growth of lung cancer, particularly SCLC (26). Immuno- histochemical studies demonstrate that most SCLCs express the ligand por- tion of the autocrine loop GRP/BN, whereas NSCLC express GRP/BN less frequently (27). 3.3. Tyrosine Kinases Neuregulins and their receptors, the ERBB family of transmembrane recep- tor tyrosine kinases (ERBB1 and ERBB2), constitute a potential growth stimulatory loops in lung cancer (27). However, NSCLCs but not SCLCs often demonstrate abnormalities of ERBB gene family. The KIT proto-oncogene, which encodes yet another tyrosine kinase receptor, CD117, and its ligand, stem cell factor (SCF), are co-expressed in many SCLC (28). Other putative CH01,1-28,28pgs 07/22/02, 7:28 AM9 10 Wistuba and Gazdar loops involve insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), and the type I insulin-like growth factor receptor (IGF-1R), which are frequently co-expressed in SCLC, as well as platelet-derived growth factor (PDGF) and its receptor (4). 3.4. MYC Family MYC family genes are frequently altered in SCLC and include MYC, MYCN, and MYCL, all of which can be involved in SCLC pathogenesis. Of the well- characterized MYC genes, MYC is most frequently activated in both SCLC and NSCLC, whereas abnormalities of MYCN and MYCL usually only affect SCLC. In nearly all cases, only one MYC family member is activated in each individual tumor. Activation of the MYC genes may occur via gene amplifi cation (20–115 copies per cell) or via transcriptional dysregulation, both of which lead to protein overexpression (29). Amplifi cation or overexpression of MYC family members has been reported more frequently in SCLCs (18–31%) than NSCLCs (8–20%) (27). 3.5. BCL-2 , BAX, and Apoptosis There is accumulating evidence that tumor cells acquire the ability to escape pathways leading cells to undergo programmed cell death (apoptosis) when exposed to conditions such as growth factor deprivation or DNA damage. Key members of the normal apoptotic pathways are the BCL-2 proto-oncogene product and the TP53 TSG product. BCL-2 protects cells from the apoptotic process and thus probably plays a role in determining the chemotherapy response of cancer cells. Whereas BCL-2 protein immunohistochemical expression (and thus upregulation) is present in most SCLCs (75–95%), BCL-2 immunostaining is far less frequent in NSCLC (10–35%) (4). BAX, which is a BCL-2 related protein that promotes apoptosis, is a downstream transcriptional target of p53. BAX and BCL-2 immunostaining are inversely related in neuroendocrine lung cancers, with most SCLCs having high BCL-2 and low BAX expression (30). Recent CGH studies have shown that lung cancer cell lines and tumor tissues demonstrate increased copy number consistent with amplifi cation of underlying dominant oncogenes at several chromosomal regions, including 1p, 1q, 2p, 3q, 5q, 11q, 16p, 17q, 19q, and Xq (31). Some of these regions, such as 1p32 (L-MYC), 2p25 (N-MYC), and 8q24 (C-MYC) contain known dominant oncogenes, while in others the genes need to be identifi ed. 3.6. Recessive Oncogenes The list of recessive oncogenes that are involved in lung cancer is likely to include as many 10–15 known and putative genes (4). These include changes CH01,1-28,28pgs 07/22/02, 7:28 AM10 Genetic Alterations 11 in TP53 (17p13), RB (13q14), p16 6ink4 (9p21), and new candidate recessive oncogenes in the short arms of chromosome 3 (3p) at 3p12 (DUTT1 gene), 3p14.2 (FHIT gene), 3p21 (RASFF1 gene), 3p22-24 (BAP-1 gene), and 3p25 regions (4). Recessive oncogenes are believed to be inactivated via a two-step process involving both alleles. Knudson has proposed that the fi rst “hit” frequently is a point mutation, while the second allele is subsequently inactivated via a chromosomal deletion, translocation, or other event such as methylation (32). 3.6.1. TP53 Gene Loss of p53 function allows inappropriate survival of genetically damaged cells, setting the stage for the accumulation of multiple mutations and the subsequent evolution of a cancer cell (33). Missense TP53 mutations prolong the protein’s half-life, leading to accumulation of high levels of mutant p53 protein readily detected by immunohistochemistry. Multiple studies have shown abnormal p53 protein expression by immunohistochemistry in 40–70% of lung cancer (4). TP53 abnormalities play a critical role in lung cancer pathogenesis (4). Chromosome 17p13 sequences, the site of the TP53 locus, are frequently hemizygously lost in SCLC (90%) and NSCLC (65%) (9), and mutational inactivation of the remaining allele occurs in 50–75% of these neoplasms (34). TP53 mutations in lung tumors correlate with cigarette smoking and are mostly the G-T transversions expected of tobacco-smoke car- cinogens (33). Furthermore, in lung cancers a relationship has been described between mutational hot-spots at the TP53 gene and adduct hot-spots caused by benzo[α]pyrene metabolites of cigarette smoke (35). 3.6.2. The p16-Cyclin D1-CDK4-Retinoblastoma Pathway In SCLC, this pathway is usually disrupted by retinoblastoma gene (RB) inactivation, while cyclin D1, CDK4, and p16 abnormalities are rare in SCLC but common (particularly p16) in NSCLC (4). The major growth-suppressing function of RB protein is to block G1-S progression. Inactivation of both RB alleles at chromosomal region 13q14 is common in SCLC (36), with protein abnormalities reported at frequencies of over 90% (37). There is frequent loss of one the RB 13q14 alleles. Functional loss of the remaining RB allele can include deletion, nonsense mutations, or splicing abnormalities, leading to a truncated RB protein encoded by the remaining allele. 3.6.3. PTEN/MMAC1 A new TSG, PTEN (Phosphatase and Tensin homolog deleted on chromo- some 10), also called MMAC1 (Mutated in Multiple Advanced Cancers), has been identifi ed and localized to chromosome region 10q23.3 (38). Allelo- CH01,1-28,28pgs 07/22/02, 7:28 AM11 [...]... lung specimens resected for lung cancer (63) They also occur in CH 01, 1-28,28pgs 16 07/22/02, 7:28 AM Genetic Alterations 17 Table 2 Major Differences in the Pathogenesis of SCLC and NSCLC SCLC 20%–25% Yes GRP/GRP receptor NDF/ERBB 80% ~20% 13 0% 80% 10 0% 13 q 11, Xq22 .1 22% >90% 17 2% Not studied ~40% 14 1% 10 –40% various genes* Unknown 90% 68% Frequency Neuroendocrine cells Putative autocrine loop SCF/KIT RAS mutations MYC amplification BCL-2 IHC TP53 abnormalities LOH Mutation p53 IHQ RB abnormalities LOH rb abnormalities (IHC) p166ink4 abnormalities LOH Mutation p16 IHC PTEN/MMAC1... overexpression TP53 LOH and mutation High 10 % 9p 21, 17 p/TP5 31 High 13 % TP53 LOH and mutation K-RAS mutation Intermediate 54% 8p 21- 23, 9p 21, 17 p/TP53 Intermediate 10 % Genetic Alterations CH 01, 1-28,28pgs Table 3 Summary of the Histopathological and Molecular Abnormalities in the Major Three Types of Lung Cancer Low 90% 5q 21, 8p 21 23, 9p 21, 17 p/TP53 Low 68% 21 07/22/02, 7:29 AM 22 Wistuba and Gazdar that... PTEN/MMAC1 mutations were detected in only 11 % of lung cancers, including both SCLC and NSCLC tumors (40) 3.6.4 Other Candidate TSGs TSG1 01 is a recently discovered candidate TSG that maps to 11 p15 ( 41) It has been reported that the mutant TSG1 01 transcript was expressed simultaneously with wild-type TSG1 01 transcript in almost all SCLC cell lines In contrast, normal lung tissue, as well primary NSCLC specimens,... al (19 98) Mutation analysis of the PTEN/MMAC1 gene in lung cancer Oncogene 17 , 15 57 15 65 41 Ponting, C P., Cai, Y D., and Bork, P (19 97) The breast cancer gene product TSG1 01: a regulator of ubiquitination? J Mol Med 75, 467–469 42 Mollenhauer, J., Wiemann, S., Scheurlen, W., Korn, B., Hayashi, Y., Wilgenbus, K K., et al (19 97) DMBT1, a new member of the SRCR superfamily, on chromosome 10 q25.3-26 .1 is... TSGs of lung cancer on chromosome 3p The Von Hippel-Lindau (VHL) TSG at 3p25 and the BRCA1-associated protein, BAP -1, at 3p 21 However, these genes have been infrequently mutated in lung cancer, including SCLC (4) Recently, a new candidate TSG, DUTT1, has been cloned residing in the U2020 3p12 deletion region and crossing a small ( >10 0 KB) lung cancer homozygous deletion at 3p12 (49) However, DUTT1 tumor-suppressing... have found different patterns of allelic loss involving the two major types of NSCLC (squamous cell and adenocarcinoma), with higher CH 01, 1-28,28pgs 15 07/22/02, 7:28 AM 16 Wistuba and Gazdar incidences of deletions at 17 p13 (TP53), 13 q14 (RB), 9p 21 (p166ink4), 8p 21- 23, and several 3p regions in squamous cell carcinomas These results suggest that more genetic changes accumulate during tumorigenesis... microdissection Am J Pathol 15 6, 445–452 17 Sugio, K., Kishimoto, Y., Virmani, A., Hung, J Y., and Gazdar, A F (19 94) K-ras mutations are a relatively late event in the pathogenesis of lung carcinomas Cancer Res 54, 5 811 –5 815 CH 01, 1-28,28pgs 24 07/22/02, 7:29 AM Genetic Alterations 25 18 Hung, J., Kishimoto, Y., Sugio, K., Virmani, A., McIntire, D D., Minna, J D., and Gazdar, A F (19 95) Allele-specific chromosome... PTEN/MMAC1 loci LOH TSG1 01 abnormal transcripts DMBT1 abnormal expression 3p LOH various regions 4p LOH various regions 4q LOH various regions 8p 21- 23 LOH Other specific LOH regions Microsatellite alterations Promoter hypermethylation RASSF1 gene RARβ gene Other genes Preneoplastic changes Histopathology LOH multiple loci MA frequency NSCLC Relatively known 31% 11 % 15 %–20% 18 %–20% 10 %–35% GRP, Gastrin-releasing... protein from the lung tumour suppressor locus 3p 21. 3 Nat Genet 25, 315 – 319 CH 01, 1-28,28pgs 26 07/22/02, 7:29 AM Genetic Alterations 27 47 Sozzi, G., Veronese, M L., Negrini, M., Baffa, R., Corticelli, M G., Inoue, H., et al (19 96) The FHIT gene at 3p14.2 is abnormal in lung cancer Cell 85, 17 –26 48 Fong, K M., Biesterveld, E J., Virmani, A., Wistuba, I., Sekido, Y., Bader, S A., et al (19 97) FHIT and . abnormalities (IHC) 90% 15 %–30% p16 6ink4 abnormalities LOH 53% 66% Mutation < ;1% 10 %–40% p16 IHC 0% 10 % 30%–70% PTEN/MMAC1 loci LOH 11 91% 14 1% TSG1 01 abnormal transcripts ~10 0% 11 0% DMBT1 abnormal. Frequency 10 % 54% 90% Chromosomal regions 9p 21, 17 p/TP5 31 8p 21- 23, 9p 21, 17 p/TP53 5q 21, 8p 21 23, 9p 21, 17 p/TP53 Genetic instability High Intermediate Low Frequency 13 % 10 % 68% CH 01, 1-28,28pgs. 11 00% 14 3% 3p LOH various regions 1& gt;90% >80% 4p LOH various regions 11 50% ~20% 4q LOH various regions 11 80% 13 0% 8p 21- 23 LOH 80%–90% 80% 10 0% Other specifi c LOH regions 1q23, 9q22-32, 10 p15,