Hisayuki Shigematsu

Somatic Mutations of the HER2 Kinase Domain in Lung Adenocarcinomas

Mutations in the epidermal growth factor receptor gene (EGFR) in lung cancers predict for sensitivity to EGFR kinase inhibitors. HER2 (also known as NEU, EGFR2, or ERBB2) is a member of the EGFR family of receptor tyrosine kinases and plays important roles in the pathogenesis of certain human cancers, and mutations have recently been reported in lung cancers. We sequenced the tyrosine kinase domain of HER2 in 671 primary non-small cell lung cancers (NSCLC), 80 NSCLC cell lines, and 55 SCLCs and other neuroendocrine lung tumors as well as 85 other epithelial cancers (breast, bladder, prostate, and colorectal cancers) and compared the mutational status with clinicopathologic features and the presence of EGFR or KRAS mutations. HER2 mutations were present in 1.6% (11 of 671) of NSCLC and were absent in other types of cancers. Only one adenocarcinoma cell line (NCI-H1781) had a mutation. All HER2 mutations were in-frame insertions in exon 20 and target the identical corresponding region as did EGFR insertions. HER2 mutations were significantly more frequent in never smokers (3.2%, 8 of 248; P=0.02) and adenocarcinoma histology (2.8%, 11 of 394; P=0.003). In 394 adenocarcinoma cases, HER2 mutations preferentially targeted Oriental ethnicity (3.9%) compared with other ethnicities (0.7%), female gender (3.6%) compared with male gender (1.9%) and never smokers (4.1%) compared with smokers (1.4%). Mutations in EGFR, HER2, and KRAS genes were never present together in individual tumors and cell lines. The remarkable similarities of mutations in EGFR and HER2 genes involving tumor type and subtype, mutation type, gene location, and specific patient subpopulations targeted are unprecedented and suggest similar etiologic factors. EGFR, HER2, and KRAS mutations are mutually exclusive, suggesting different pathways to lung cancer in smokers and never smokers.

Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers

Somatic mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) gene in lung cancers have generated enormous interest, because they predict for sensitivity to TK inhibitors (TKIs). While mutational status is of great importance in determining response to TKIs, it is not the sole factor, and evidence is accumulating that EGFR gene amplification, other members of the EGFR family (HER2, HER3) and genes downstream of EGFR signaling (KRAS, BRAF), may be involved in cancer pathogenesis and the response of TKIs. EGFR mutations occur in highly selected subpopulations of lung cancer patients: adenocarcinoma histology, never‐smoker status, East Asian ethnicity and female gender. The recent finding of “a resistance associated” mutation for TKIs also provides new insights into this complicated mechanism. Thus, molecular‐based studies to analyze the biological functions and to assess TKI sensitivity depending on the type of mutations are required. Epidemiological studies to identify possible carcinogenic factor(s) affecting different subpopulations are also of interest. In addition, for optimal therapeutic approach a comprehensive understanding of the genes related to EGFR signaling pathway, including RAS/RAF/MAPK and PI3K‐AKT pathways, are required.

Increased HER2 Gene Copy Number Is Associated With Response to Gefitinib Therapy in Epidermal Growth Factor Receptor–Positive Non–Small-Cell Lung Cancer Patients

PURPOSE In non-small-cell lung cancer (NSCLC), response to tyrosine kinase inhibitors (TKIs) is significantly associated with the presence of increased copy number and/or activating mutations of the epidermal growth factor receptor gene (EGFR). Preclinical data indicate that HER2, a member of the EGFR family, could enhance TKI sensitivity. PATIENTS AND METHODS HER2 gene copy numbers per cell were evaluated by fluorescent in situ hybridization (FISH) in 102 NSCLC patients treated with gefitinib, and previously evaluated for EGFR status by FISH, immunohistochemistry, and presence of mutations. RESULTS Patients with HER2 high copy number (high polysomy and gene amplification [HER2 FISH positive]) represented 22.8% of patients, and compared with patients with no or low gain (HER2 FISH negative), had significantly better objective response (OR, 34.8% v 6.4%; P = .001), disease control rate (DCR, 56.5% v 33.3%; P = .04), time to progression (TTP, 9.05 v 2.7 months; P = .02), and a trend toward longer overall survival (OS, 20.8 v 8.4 months; P = .056). HER2 protein expression investigated by immunohistochemistry was positive in only five of 72 (7%) patients analyzed and all 89 patients tested by DNA sequencing were negative for mutations in HER2 exon 20. Patients with HER2 FISH-positive tumors displaying increased expression of EGFR protein, gene gain, or mutations (EGFR positive) had a significantly better OR, DCR, TTP, and OS than patients negative for both receptors. CONCLUSION Increased copy number of the HER2 gene is associated with gefitinib sensitivity in EGFR-positive patients, supporting use of HER2 FISH analysis for selection of patients for TKI therapy.

PIK3CA Mutations and Copy Number Gains in Human Lung Cancers

We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non-small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference-mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.

EGFR Tyrosine Kinase Domain Mutations Are Detected in Histologically Normal Respiratory Epithelium in Lung Cancer Patients

To determine whether EGFR tyrosine kinase domain mutations are early events in the pathogenesis of lung adenocarcinomas, we tested for the presence of EGFR mutations in histologically normal bronchial and bronchiolar epithelia from lung adenocarcinomas bearing the common EGFR mutations. DNA was extracted from microdissected tissue obtained from 21 tumors with known EGFR mutations, 16 tumors without mutation, and 90 sites of normal bronchial and bronchiolar epithelium from the same surgical specimens. With the use of PCR and direct DNA sequencing, EGFR mutations identical to the tumors were detected in the normal respiratory epithelium in 9 of 21 (43%) patients with EGFR mutant adenocarcinomas but none in patients without mutation in the tumors. The finding of mutations being more frequent in normal epithelium within tumor (43%) than in adjacent sites (24%) suggests a localized field effect phenomenon. Our findings indicate that mutation of the tyrosine kinase domain of EGFR is an early event in the pathogenesis of lung adenocarcinomas, and suggest EGFR mutations as an early detection marker and chemoprevention target.

Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells

Background Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes. Methodology/Principal Findings We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival. Conclusions MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.

Alterations in Genes of the EGFR Signaling Pathway and Their Relationship to EGFR Tyrosine Kinase Inhibitor Sensitivity in Lung Cancer Cell Lines

Background Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs. Methodology/Principal Findings We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3 HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results:  1. mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance.  2. mKRAS was associated with increased in vitro resistance to gefitinib. Conclusions/Significance Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.

Aberrant methylation of trail decoy receptor genes is frequent in multiple tumor types

TNF‐related apoptosis‐inducing ligand (TRAIL) selectively induces programmed cell death (apoptosis) in various cancer cells but not in normal cells. TRAIL is known to bind to 4 different receptors, 2 proapoptotic (DR4 and DR5), and 2 potentially antiapoptotic receptors lacking death domains (DcR1 and DcR2). Aberrant promoter methylation and resultant silencing of tumor suppressor genes play an important role in the pathogenesis of many tumor types. Recently aberrant methylation of TRAIL decoy receptors was reported in pediatric tumor cell lines and neuroblastomas. We examined the methylation and expression status of TRAIL receptor genes in cancers of breast, lung, mesothelioma, prostate, bladder, cervix, ovary, brain and in hematopoietic malignancies. Aberrant methylation of DcR1 or DcR2 was present in 70% of primary breast cancers, 31% of primary lung cancers, in 63% of primary malignant mesothelioma (MM), in 60% of prostate cancer, in 42% of bladder cancer, in 100% of cervical cancer, in 43% of ovarian cancer, in 41% of lymphoma, in 26% of leukemia and in 56% of multiple myeloma. Methylation of DR4 and DR5 was rare in all the tumor types examined. Methylation of all the 4 receptors was rare in non malignant tissues. In cell lines, aberrant methylation of DcR1 was present in 11 of 23 (48%) breast, 10 of 27 (37%) lung and 3 of 7 (43%) MM, whereas aberrant methylation of DcR2 was present in 17 of 23 (74%) breast, 13 of 27 (48%) lung and 5 of 7 (71%) MM. The concordance between loss of gene expression and aberrant methylation ranged from 70–100%. Treatment with 5‐aza‐2′‐deoxycytidine restored DcR1 and DcR2 expression in 9 methylated cell lines confirming that aberrant methylation was the cause for silencing of DcR1 and DcR2 expression. Our results demonstrate that DcR1 and DcR2 genes are frequently methylated in various tumor types, and that the role of decoy receptors in tumor pathogenesis needs to be re‐evaluated.

Mutational and Epigenetic Evidence for Independent Pathways for Lung Adenocarcinomas Arising in Smokers and Never Smokers

Genetic and epigenetic alterations are considered to play important roles in lung cancer. Recent studies showed that EGFR and K-RAS mutations exhibited a mutually exclusive pattern in adenocarcinoma of the lung, suggesting the presence of two independent oncogenic pathways. However, it is unknown how epigenetic alterations were involved in lung carcinogenesis mediated by EGFR or K-RAS mutation. In this study, we examined the relationship between genetic and epigenetic alterations in 164 cases of lung adenocarcinoma. Somatic mutations were determined by direct sequence of EGFR exons 18 to 21 and K-RAS codons 12 and 13. Methylation status of p16(INK4a), RASSF1A, APC, RARbeta, and CDH13, frequently methylated in lung cancer, was determined by methylation-specific PCR and the degree of methylation was defined as the methylation index. Multivariate analysis adjusted for age, sex, and smoking dose showed that the probability of having EGFR mutation was significantly lower among those with p16(INK4a) and CDH13 methylation than in those without [p16(INK4a): odds ratio (OR), 0.07; 95% confidence interval (95% CI), 0.02-0.33; CDH13: OR, 0.34; 95% CI, 0.15-0.77] and the methylation index was significantly lower in EGFR mutant cases than in wild type (OR, 0.70; 95% CI, 0.52-0.95). By contrast, K-RAS mutation was significantly higher in p16(INK4a) methylated cases than in unmethylated cases (OR, 4.93; 95% CI, 1.54-15.7) and the methylation index was higher in K-RAS mutant cases than in wild type with marginal significance (OR, 1.46; 95% CI, 0.95-2.25). Our results indicate the differences in the evolvement of epigenetic alterations between the EGFR- and K-RAS-mediated tumorigenesis and suggest the specific interaction of genetic and epigenetic changes in tumorigenesis of lung cancer.

Polymorphisms, mutations, and amplification of the EGFR gene in non-small cell lung cancers

Background The epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, −216 (G/T or T/T) and −191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers. Methods and Findings We examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African–Americans, and Mexican–Americans. We sequenced the four exons (18–21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP −216 (G/T or T/T) and SNP −191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP −216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%–54.7%) of mutant tumors compared with 25.9% (20.6%–31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%–88.4%) than in wild-type tumors (43.5%, 31.8%–55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles. Conclusions The three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs −216 and −191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.