Ximing Tang

KRAS Mutation Is an Important Predictor of Resistance to Therapy with Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non–Small-Cell Lung Cancer

Purpose:EGFR gene mutations and increased EGFR copy number have been associated with favorable response to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) in patients with non–small-cell lung cancer (NSCLC). In contrast, KRAS mutation has been shown to predict poor response to such therapy. We tested the utility of combinations of these three markers in predicting response and survival in patients with NSCLC treated with EGFR-TKIs. Experimental Design: Patients with advanced NSCLC treated with EGFR-TKI with available archival tissue specimens were included. EGFR and KRAS mutations were analyzed using PCR-based sequencing. EGFR copy number was analyzed using fluorescence in situ hybridization. Results: The study included 73 patients, 59 of whom had all three potential markers successfully analyzed. EGFR mutation was detected in 7 of 71 patients (9.8%), increased EGFR copy number in 32 of 59 (54.2%), and KRAS mutation in 16 of 70 (22.8%). EGFR mutation (P < 0.0001) but not increased EGFR copy number (P = 0.48) correlated with favorable response. No survival benefit was detected in patients with either of these features. KRAS mutation correlated with progressive disease (P = 0.04) and shorter median time to progression (P = 0.0025) but not with survival. Patients with both EGFR mutation and increased EGFR copy number had a >99.7% chance of objective response, whereas patients with KRAS mutation with or without increased EGFR copy number had a >96.5% chance of disease progression. Conclusion:KRAS mutation should be included as indicator of resistance in the panel of markers used to predict response to EGFR-TKIs in NSCLC.

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.

Effect of KRAS Oncogene Substitutions on Protein Behavior: Implications for Signaling and Clinical Outcome

BACKGROUND Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non-small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting. METHODS We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan-Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided. RESULTS Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor-dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers. CONCLUSIONS Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.

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.

Nuclear factor-κB (nf-κB) is frequently expressed in lung cancer and preneoplastic lesions

Nuclear factor‐κB (NF‐κB), a key transcription factor thought to play a major role in carcinogenesis, regulates many important signaling pathways involved in tumor promotion. Although NF‐κB can be activated in lung cancer cell lines by tobacco exposure, there have been no studies of the expression of NF‐κB in lung cancer pathogenesis.

High expression of ErbB family members and their ligands in lung adenocarcinomas that are sensitive to inhibition of epidermal growth factor receptor

Recent findings in tumor biopsies from lung adenocarcinoma patients suggest that somatic mutations in the genes encoding epidermal growth factor receptor (EGFR) and Kirsten ras (KRAS) confer sensitivity and resistance, respectively, to EGFR inhibition. Here, we provide evidence that these genetic mutations are not sufficient to modulate the biological response of lung adenocarcinoma cells to EGFR inhibition. We found high expression of ErbB family members, ErbB ligands, or both in three models that were sensitive to EGFR inhibition, including alveolar epithelial neoplastic lesions in mice that develop lung adenocarcinoma by oncogenic KRAS, human lung adenocarcinoma cell lines, and tumor biopsies from lung adenocarcinoma patients. Thus, lung adenocarcinoma cells that depend on EGFR for survival constitutively activate the receptor through a combination of genetic mutations and overexpression of EGFR dimeric partners and their ligands.

Prognostic Factors in Resected Stage I Non–Small-Cell Lung Cancer: A Multivariate Analysis of Six Molecular Markers

PURPOSE To analyze the prognostic significance of six molecular biomarkers (death-associated protein kinase [DAPK] promoter methylation, interleukin-10 [IL-10] protein expression, cyclooxygenase-2 [COX-2] mRNA expression, human telomerase reverse transcriptase catalytic subunit [hTERT] mRNA expression, retinoic acid receptor-beta [RAR-beta] mRNA expression, and K-ras mutational status) in stage I non-small-cell lung cancer (NSCLC) patients. PATIENTS AND METHODS Biomarker analyses were performed on tumors from 94 patients with stage I NSCLC who underwent surgical resection at our institution. A minimum follow-up period of 5 years was required. DAPK methylation was assessed by methylation-specific polymerase chain reaction (PCR). RAR-beta, COX-2, and hTERT mRNA levels were determined by in situ hybridization with digoxigenin-labeled antisense riboprobes. K-ras mutation status was determined by the PCR-primer introduced restriction with enrichment for mutant alleles method. IL-10 protein expression was analyzed by immunohistochemistry using a polyclonal antihuman IL-10 antibody. Cancer-specific survival was analyzed with a Cox proportional hazards model. To identify independent prognostic factors, a stepwise selection method was used. RESULTS DAPK methylation, IL-10 lack of expression, COX-2 expression, hTERT expression, RAR-beta expression, and K-ras mutations were observed in 46.8%, 29.8%, 59.6%, 34.0%, 23.4%, and 34.0% of patients, respectively. In the final model, DAPK methylation and IL-10 lack of expression were significant negative prognostic factors for cancer-specific survival, whereas COX-2 expression was of borderline significance. CONCLUSION In this cohort of resected stage I NSCLC patients, molecular markers that independently predict cancer-specific survival have been identified. The prognostic roles of DAPK methylation, IL-10, and other biomarkers in NSCLC merit further investigation.

HER Family Receptor Abnormalities in Lung Cancer Brain Metastases and Corresponding Primary Tumors

Purpose: To compare the characteristics of deregulation of HER receptors and their ligands between primary tumor and corresponding brain metastases of non–small cell lung carcinoma (NSCLC). Experimental Design: Fifty-five NSCLC primary tumors and corresponding brain metastases specimens were examined for the immunohistochemical expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR, Her2, Her3, and phosphorylated Her3, and their ligands EGF, transforming growth factor-α, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, neuregulin (NRG) 1, and NRG2. Analysis of EGFR copy number using fluorescence in situ hybridization and mutation by PCR-based sequencing was also done. Results: Metastases showed significantly higher immunohistochemical expression of EGF (membrane: brain metastases 66.0 versus primary tumors 48.5; P = 0.027; nucleus: brain metastases 92.2 versus 67.4; P = 0.008), amphiregulin (nucleus: brain metastases 53.7 versus primary tumors 33.7; P = 0.019), phosphorylated EGFR (membrane: brain metastases 161.5 versus primary tumors 76.0; P < 0.0001; cytoplasm: brain metastases 101.5 versus primary tumors 55.9; P = 0.014), and phosphorylated Her3 (membrane: brain metastases 25.0 versus primary tumors 3.7; P = 0.001) than primary tumors did. Primary tumors showed significantly higher expression of cytoplasmic transforming growth factor-α(primary tumors 149.8 versus brain metastases 111.3; P = 0.008) and NRG1 (primary tumors 158.5 versus brain metastases 122.8; P = 0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time point. Conclusions: Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptor–related abnormalities from primary lung tumors.

Sex determining region Y-box 2 (SOX2) is a potential cell-lineage gene highly expressed in the pathogenesis of squamous cell carcinomas of the lung

Background Non-small cell lung cancer (NSCLC) represents the majority (85%) of lung cancers and is comprised mainly of adenocarcinomas and squamous cell carcinomas (SCCs). The sequential pathogenesis of lung adenocarcinomas and SCCs occurs through dissimilar phases as the former tumors typically arise in the lung periphery whereas the latter normally arise near the central airway. Methodology/Principal Findings We assessed the expression of SOX2, an embryonic stem cell transcriptional factor that also plays important roles in the proliferation of basal tracheal cells and whose expression is restricted to the main and central airways and bronchioles of the developing and adult mouse lung, in NSCLC by various methodologies. Here, we found that SOX2 mRNA levels, from various published datasets, were significantly elevated in lung SCCs compared to adenocarcinomas (all p<0.001). Moreover, a previously characterized OCT4/SOX2/NANOG signature effectively separated lung SCCs from adenocarcinomas in two independent publicly available datasets which correlated with increased SOX2 mRNA in SCCs. Immunohistochemical analysis of various histological lung tissue specimens demonstrated marked nuclear SOX2 protein expression in all normal bronchial epithelia, alveolar bronchiolization structures and premalignant lesions in SCC development (hyperplasia, dysplasia and carcinoma in situ) and absence of expression in all normal alveoli and atypical adenomatous hyperplasias. Moreover, SOX2 protein expression was greatly higher in lung SCCs compared to adenocarcinomas following analyses in two independent large TMA sets (TMA set I, n = 287; TMA set II, n = 511 both p<0.001). Furthermore, amplification of SOX2 DNA was detected in 20% of lung SCCs tested (n = 40) and in none of the adenocarcinomas (n = 17). Conclusions/Significance Our findings highlight a cell-lineage gene expression pattern for the stem cell transcriptional factor SOX2 in the pathogenesis of lung SCCs and suggest a differential activation of stem cell-related pathways between squamous cell carcinomas and adenocarcinomas of the lung.

Epithelial-to-mesenchymal transition in the development and progression of adenocarcinoma and squamous cell carcinoma of the lung

Epithelial-to-mesenchymal transition is a process in which cells undergo a developmental switch from an epithelial to a mesenchymal phenotype. We investigated the role of this phenomenon in the pathogenesis and progression of adenocarcinoma and squamous cell carcinoma of the lung. Archived tissue from primary tumors (n=325), brain metastases (n=48) and adjacent bronchial epithelial specimens (n=192) were analyzed for immunohistochemical expression by image analysis of E-cadherin, N-cadherin, integrin-αvβ6, vimentin, and matrix metalloproteinase-9. The findings were compared with the patients’ clinicopathologic features. High expression of the epithelial-to-mesenchymal transition phenotype (low E-cadherin and high N-cadherin, integrin-αvβ6, vimentin, and matrix metalloproteinase-9) was found in most lung tumors examined, and the expression pattern varied according to the tumor histologic type. Low E-cadherin membrane and high N-cadherin cytoplasmic expression were significantly more common in squamous cell carcinoma than in adenocarcinoma (P=0.002 and 0.005, respectively). Dysplastic lesions had significantly lower expression of the epithelial-to-mesenchymal transition phenotype than the squamous cell carcinomas, and integrin-αvβ6 membrane expression increased stepwise according to the histopathologic severity. Brain metastases had decreased epithelial-to-mesenchymal transition expression compared with primary tumors. Brain metastases had significantly lower integrin-αvβ6 membrane (P=0.04), N-cadherin membrane, and cytoplasm (P<0.0002) expression than the primary tumors. The epithelial-to-mesenchymal transition phenotype is commonly expressed in primary squamous cell carcinoma and adenocarcinoma of the lung; this expression occurs early in the pathogenesis of squamous cell carcinoma. Brain metastases showed characteristics of reversed mesenchymal-to-epithelial transition. Our findings suggest that epithelial-to-mesenchymal transition is a potential target for lung cancer chemoprevention and therapy.