Carmen Behrens

Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma

To understand the molecular pathways involved in the pathogenesis of squamous cell lung carcinoma, we obtained DNA from 94 microdissected foci from 12 archival surgically resected tumors including histologically normal epithelium (n=13), preneoplastic lesions (n=54), carcinoma is situ (CIS) (n=15) and invasive tumors (n=12). We determined loss of heterozygosity (LOH) at 10 chromosomal regions (3p12, 3p14.2, 3p14.1-21.3, 3p21, 3p22-24, 3p25, 5q22, 9p21, 13q14 RB, and 17p13 TP53) frequently deleted in lung cancer, using 31 polymorphic microsatellite markers, including 24 that spanned the entire 3p arm. Our major findings are as follows: (1) Thirty one percent of histologically normal epithelium and 42% of mildly abnormal (hyperplasia/metaplasia) specimens had clones of cells with allelic loss at one or more regions; (2) There was a progressive increase of the overall LOH frequency within clones with increasing severity of histopathological changes; (3) The earliest and most frequent regions of allelic loss occurred at 3p21, 3p22-24, 3p25 and 9p21; (4) The size of the 3p deletions increased with progressive histologic changes; (5) TP53 allelic loss was present in many histologically advanced lesions (dysplasia and CIS); (6) Analyses of 58 normal and non-invasive foci having any molecular abnormality, indicated that 30 probably arose as independent clonal events, while 28 were potentially of the same clonal origin as the corresponding tumor; (7) Nevertheless, when the allelic losses in the 30 clonally independent lesions and their clonally unrelated tumors were compared the same parental allele was lost in 113 of 125 (90%) of comparisons. The mechanism by which this phenomenon (known as allele specific mutations) occurs is unknown; (8) Four patterns of allelic loss in clones were found. Histologically normal or mildly abnormal foci had a negative pattern (no allelic loss) or early pattern of loss while all foci of CIS and invasive tumor had an advanced pattern. However dysplasias demonstrated the entire spectrum of allelic loss patterns, and were the only histologic category having the intermediate pattern. Our findings indicate that multiple, sequentially occurring allele specific molecular changes commence in widely dispersed, apparently clonally independent foci, early in the multistage pathogenesis of squamous cell carcinomas of the lung.

Aldehyde Dehydrogenase Activity Selects for Lung Adenocarcinoma Stem Cells Dependent on Notch Signaling

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component, implicating Notch signaling in lung cancer stem cell maintenance.

Co-occurring genomic alterations define major subsets of KRAS - mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities

UNLABELLED The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. SIGNIFICANCE Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B-the latter coupled with low TTF1 expression-define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities.

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.

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.

A 12-gene set predicts survival benefits from adjuvant chemotherapy in non-small cell lung cancer patients

Purpose: Prospectively identifying who will benefit from adjuvant chemotherapy (ACT) would improve clinical decisions for non–small cell lung cancer (NSCLC) patients. In this study, we aim to develop and validate a functional gene set that predicts the clinical benefits of ACT in NSCLC. Experimental Design: An 18-hub-gene prognosis signature was developed through a systems biology approach, and its prognostic value was evaluated in six independent cohorts. The 18-hub-gene set was then integrated with genome-wide functional (RNAi) data and genetic aberration data to derive a 12-gene predictive signature for ACT benefits in NSCLC. Results: Using a cohort of 442 stage I to III NSCLC patients who underwent surgical resection, we identified an 18-hub-gene set that robustly predicted the prognosis of patients with adenocarcinoma in all validation datasets across four microarray platforms. The hub genes, identified through a purely data-driven approach, have significant biological implications in tumor pathogenesis, including NKX2-1, Aurora Kinase A, PRC1, CDKN3, MBIP, and RRM2. The 12-gene predictive signature was successfully validated in two independent datasets (n = 90 and 176). The predicted benefit group showed significant improvement in survival after ACT (UT Lung SPORE data: HR = 0.34, P = 0.017; JBR.10 clinical trial data: HR = 0.36, P = 0.038), whereas the predicted nonbenefit group showed no survival benefit for 2 datasets (HR = 0.80, P = 0.70; HR = 0.91, P = 0.82). Conclusions: This is the first study to integrate genetic aberration, genome-wide RNAi data, and mRNA expression data to identify a functional gene set that predicts which resectable patients with non–small cell lung cancer will have a survival benefit with ACT. Clin Cancer Res; 19(6); 1577–86. ©2013 AACR.

Immunohistochemical Expression of Basic Fibroblast Growth Factor and Fibroblast Growth Factor Receptors 1 and 2 in the Pathogenesis of Lung Cancer

Purpose: To identify the patterns of protein expression of basic fibroblast growth factor (bFGF) and FGF receptors 1 and 2 in non-small cell lung carcinoma (NSCLC) and their role in the early pathogenesis of squamous cell carcinoma (SCC) of the lung. Experimental Design: Archived tissue from NSCLC (adenocarcinoma and SCC; n = 321) and adjacent bronchial epithelial specimens (n = 426) were analyzed for the immunohistochemical expression of bFGF, FGFR1, and FGFR2, and the findings were correlated with clinicopathologic features of the patients. Results: High expression of bFGF, FGFR1, and FGFR2 was shown in most NSCLC tumors. The pattern of expression for all markers varied according to tumor histologic type and cellular localization. Cytoplasmic expression scores were significantly higher in tumors than in normal epithelia. Nuclear bFGF (P = 0.03) and FGFR1 (P = 0.02) levels were significantly higher in women than in men. Although cytoplasmic FGFR1 expression was significantly higher (P = 0.002) in ever smokers than in never smokers, nuclear FGFR1 (P = 0.0001) and FGFR2 (P = 0.003) expression was significantly higher in never smokers. Different prognostic patterns for the expression of these markers were detected for both NSCLC histologic types. Dysplastic changes showed significantly higher expression of all markers compared with squamous metaplasia. Conclusions: bFGF, FGFR1, and FGFR2 are frequently overexpressed in SCC and adenocarcinoma of the lung. bFGF signaling pathway activation may be an early phenomenon in the pathogenesis of SCC and thus an attractive novel target for lung cancer chemopreventive and therapeutic strategies.

Epithelial-Mesenchymal Transition Is Associated with a Distinct Tumor Microenvironment Including Elevation of Inflammatory Signals and Multiple Immune Checkpoints in Lung Adenocarcinoma.

Purpose: Promising results in the treatment of non–small cell lung cancer (NSCLC) have been seen with agents targeting immune checkpoints, such as programmed cell death 1 (PD-1) or programmed death ligand-1 (PD-L1). However, only a select group of patients respond to these interventions. The identification of biomarkers that predict clinical benefit to immune checkpoint blockade is critical to successful clinical translation of these agents. Methods: We conducted an integrated analysis of three independent large datasets, including The Cancer Genome Atlas of lung adenocarcinoma and two datasets from MD Anderson Cancer Center (Houston, TX), Profiling of Resistance Patterns and Oncogenic Signaling Pathways in Evaluation of Cancers of the Thorax (named PROSPECT) and Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (named BATTLE-1). Comprehensive analysis of mRNA gene expression, reverse-phase protein array, IHC, and correlation with clinical data were performed. Results: Epithelial–mesenchymal transition (EMT) is highly associated with an inflammatory tumor microenvironment in lung adenocarcinoma, independent of tumor mutational burden. We found immune activation coexistent with elevation of multiple targetable immune checkpoint molecules, including PD-L1, PD-L2, PD-1, TIM-3, B7-H3, BTLA, and CTLA-4, along with increases in tumor infiltration by CD4+Foxp3+ regulatory T cells in lung adenocarcinomas that displayed an EMT phenotype. Furthermore, we identify B7-H3 as a prognostic marker for NSCLC. Conclusions: The strong association between EMT status and an inflammatory tumor microenvironment with elevation of multiple targetable immune checkpoint molecules warrants further investigation of using EMT as a predictive biomarker for immune checkpoint blockade agents and other immunotherapies in NSCLC and possibly a broad range of other cancers. Clin Cancer Res; 22(14); 3630–42. ©2016 AACR. See related commentary by Datar and Schalper, p. 3422