Marcel Wiesweg

High Prevalence of Concomitant Oncogene Mutations in Prospectively Identified Patients with ROS1-Positive Metastatic Lung Cancer

Objectives: Chromosomal rearrangements involving ROS1 define a rare entity of lung adenocarcinomas with exquisite sensitivity to molecularly targeted therapy. We report clinical outcomes and genomic findings of patients with ROS1‐positive lung cancer who were prospectively identified within a multiplex biomarker profiling program at the West German Cancer Center. Methods: Standardized immunohistochemical (IHC) analysis, fluorescence in situ hybridization (FISH), and hotspot mutation analyses were performed in 1345 patients with advanced cancer, including 805 patients with metastatic lung adenocarcinoma. Clinical and epidemiological data were retrieved from the institutional database. Results: ROS1 positivity by IHC analysis was detected in 25 patients with lung cancer (4.8% of lung adenocarcinomas), including 13 patients (2.5%) with ROS1 FISH positivity with a cutoff of at least 15% of events. Of the ROS1 IHC analysis–positive cases, 36% presented with concomitant oncogenic driver mutations involving EGFR (six cases, five of which were clinically validated by response to EGFR‐targeting agents), KRAS (two cases), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha gene (PIK3CA), and BRAF. Three cases initially classified as ROS1 FISH–negative passed the threshold of 15% positive events when repeat biopsies were analyzed at progression. The median overall survival of the ROS1‐positive patients (104 months) was significantly superior to that of the 261 patients with EGFR/anaplastic lymphoma kinase/ROS1–negative lung adenocarcinoma (24.4 months, p = 0.044). Interestingly, the overall survival of the 13 ROS1‐positive patients with lung cancer from initiation of pemetrexed‐based chemotherapy was significantly prolonged when compared with that of 169 pemetrexed‐treated patients with EGFR/anaplastic lymphoma kinase/ROS1–negative adenocarcinoma (p = 0.01). Conclusions: ROS1‐positive metastatic lung adenocarcinomas frequently harbor concomitant oncogenic driver mutations. Levels of ROS1 FISH–positive events are variable over time. This heterogeneity provides additional therapeutic options if discovered by multiplex biomarker testing and repeat biopsies.

HER2 expression and markers of phosphoinositide-3-kinase pathway activation define a favorable subgroup of metastatic pulmonary adenocarcinomas

OBJECTIVES Pulmonary adenocarcinomas (ADC) can be sub-grouped based on dominant oncogenic drivers. EGFR mutations define an entity of metastatic ADC with favorable prognosis and high susceptibility to EGFR tyrosine kinase inhibition. In contrast, the clinical impact of additional ERBB family members in ADC is less defined. To this end we prospectively studied HER2 expression, gene amplification, and mutation in relation to outcome of patients with advanced or metastatic ADC. MATERIALS AND METHODS Diagnostic tumor biopsies from 193 sequential patients with stage III/IV ADC were prospectively studied for HER2 expression by immunohistochemistry (IHC). Cases with IHC scores 2+ or 3+ were analyzed by HER2 chromogenic in situ hybridization (CISH), and sequencing of HER2 exons 20 and 23. Additional prospectively determined biomarkers included PTEN, cMET, pAKT, and pERK expression, KRAS, EGFR, BRAF and PIK3CA mutations, and ALK fluorescence ISH (FISH). RESULTS AND CONCLUSION HER2-IHC was feasible in 176 (91.2%) cases. Of 53 (30%) cases with IHC scores 2+/3+, 45 (85%) could be studied by CISH and 34 (64%) by sequencing. The lower number of HER2-mutational analyses resulted from exhaustion of tumor tissue and DNA following mutational analysis of KRAS, EGFR, BRAF and PIK3CA. HER2 amplification was detected in 4 cases (2.3%), while no mutation was found. HER2 expression correlated with expression of pAKT and cMET. Expression of HER2 and pAKT was associated with favorable overall survival in stage IV disease. HER2-expressing ADC more frequently harbored KRAS mutations, while HER2 expression was absent in all 4 cases with BRAF mutation. HER2-IHC was not predictive of HER2 gene amplification or mutation, which both were rare events in prospectively studied patients with advanced or metastatic ADC. Expression of HER2 and pAKT define a population of patients with stage IV ADC with a distinct disease course, who could benefit from specifically tailored pharmacotherapies.

Molecular dissection of effector mechanisms of RAS -mediated resistance to anti-EGFR antibody therapy

Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR), cetuximab and panitumumab, are a mainstay of metastatic colorectal cancer (mCRC) treatment. However, a significant number of patients suffer from primary or acquired resistance. RAS mutations are negative predictors of clinical efficacy of anti-EGFR antibodies in patients with mCRC. Oncogenic RAS activates the MAPK and PI3K/AKT pathways, which are considered the main effectors of resistance. However, the relative impact of these pathways in RAS-mutant CRC is less defined. A better mechanistic understanding of RAS-mediated resistance may guide development of rational intervention strategies. To this end we developed cancer models for functional dissection of resistance to anti-EGFR therapy in vitro and in vivo. To selectively activate MAPK- or AKT-signaling we expressed conditionally activatable RAF-1 and AKT in cancer cells. We found that either pathway independently protected sensitive cancer models against anti-EGFR antibody treatment in vitro and in vivo. RAF-1- and AKT-mediated resistance was associated with increased expression of anti-apoptotic BCL-2 proteins. Biomarkers of MAPK and PI3K/AKT pathway activation correlated with inferior outcome in a cohort of mCRC patients receiving cetuximab-based therapy. Dual pharmacologic inhibition of PI3K and MEK successfully sensitized primary resistant CRC models to anti-EGFR therapy. In conclusion, combined targeting of MAPK and PI3K/AKT signaling, but not single pathways, may be required to enhance the efficacy of anti-EGFR antibody therapy in patients with RAS-mutated CRC as well as in RAS wild type tumors with clinical resistance.

Pathogenic and targetable genetic alterations in 70 urachal adenocarcinomas: Genetic alterations in 70 urachal adenocarcinomas

Urachal cancer (UrC) is a rare but aggressive malignancy often diagnosed in advanced stages requiring systemic treatment. Although cytotoxic chemotherapy is of limited effectiveness, prospective clinical studies can hardly be conducted. Targeted therapeutic treatment approaches and potentially immunotherapy based on a biological rationale may provide an alternative strategy. We therefore subjected 70 urachal adenocarcinomas to targeted next‐generation sequencing, conducted in situ and immunohistochemical analyses (including PD‐L1 and DNA mismatch repair proteins [MMR]) and evaluated the microsatellite instability (MSI) status. The analytical findings were correlated with clinicopathological and outcome data and Kaplan‐Meier and univariable/multivariable Cox regression analyses were performed. The patients had a mean age of 50 years, 66% were male and a 5‐year overall survival (OS) of 58% and recurrence‐free survival (RFS) of 45% was detected. Sequence variations were observed in TP53 (66%), KRAS (21%), BRAF (4%), PIK3CA (4%), FGFR1 (1%), MET (1%), NRAS (1%), and PDGFRA (1%). Gene amplifications were found in EGFR (5%), ERBB2 (2%), and MET (2%). We detected no evidence of MMR‐deficiency (MMR‐d)/MSI‐high (MSI‐h), whereas 10 of 63 cases (16%) expressed PD‐L1. Therefore, anti‐PD‐1/PD‐L1 immunotherapy approaches might be tested in UrC. Importantly, we found aberrations in intracellular signal transduction pathways (RAS/RAF/PI3K) in 31% of UrCs with potential implications for anti‐EGFR therapy. Less frequent potentially actionable genetic alterations were additionally detected in ERBB2 (HER2), MET, FGFR1, and PDGFRA. The molecular profile strengthens the notion that UrC is a distinct entity on the genomic level with closer resemblance to colorectal than to bladder cancer.

MET Expression in Advanced Non–Small-Cell Lung Cancer: Effect on Clinical Outcomes of Chemotherapy, Targeted Therapy, and Immunotherapy

&NA; We studied the effect of MET expression in a prospectively recruited cohort of patients (n = 384) with advanced non–small‐cell lung cancer (NSCLC). High MET expression was associated with inferior outcome of epidermal growth factor receptor‐targeting therapy, but with a more favorable outcome with programmed death 1/programmed death ligand 1‐directed therapy. MET mutations were detected at low prevalence (0.78%) and MET‐mutated NSCLC appeared—on an anecdotal basis—to respond less to immunotherapy. Background: The receptor tyrosine kinase MET is implicated in malignant transformation, tumor progression, metastasis, and acquired treatment resistance. We conducted an analysis of the effect of MET expression and MET genomic aberrations on the outcome of patients with advanced or metastatic pulmonary adenocarcinomas prospectively enrolled in an institutional precision oncology program. Patients and Methods: Standardized immunohistochemistry (IHC) analyses of MET and markers of pathway activation were available in 384 patients, and next‐generation sequencing‐based MET hotspot mutation analyses were available from 892 patients. Clinical data were retrieved with a median follow‐up from initial diagnosis of 37 months. Results: High MET expression, defined as MET IHC 3+ or MET H‐Score in the upper quartile, was observed in 102 of 384 patients (26.6%). MET exon 14 mutations were only detected in 7 of 892 patients (0.78%). High MET expression correlated with activation markers of the mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) pathways only in cases without Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR), v‐Raf murine sarcoma viral oncogene homolog B (BRAF), anaplastic lymphoma kinase (ALK) and proto‐oncogene tyrosine‐protein kinase ROS (ROS1) aberrations. There was no association of MET expression with outcome during chemotherapy. High MET expression negatively affected the outcome during EGFR‐targeting therapy but was associated with more favorable results with programmed death 1/programmed death ligand 1 (PD‐L1)‐directed therapy, independent of smoking history, PD‐L1 expression or KRAS mutation. Two patients with MET exon 14 mutation and high PD‐L1 expression failed to respond to pembrolizumab. Conclusion: MET expression affects the outcomes of targeted therapies in non–small‐cell lung cancer, thus supporting the development of biomarker‐informed combination strategies. The interaction of MET expression and MET mutation with immune checkpoint inhibitor therapy is novel and merits further investigation.

Rapid and Highly Sensitive Detection of Therapeutically Relevant Oncogenic Driver Mutations in EBUS-TBNA Specimens From Patients With Lung Adenocarcinoma

&NA; We compared light cycler reverse transcription polymerase chain reaction (LCRT‐PCR) and next generation sequencing (NGS) to detect exon 19 deletion of epidermal growth factor receptor (EGFRdelEx19) and Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) exon 2 mutations in endobronchial ultrasound‐guided transbronchial needle aspirations (EBUS‐TBNAs). LCRT‐PCR additionally detected 2 KRAS exon 2 mutations and 3 EGFRdelEx19 mutations that were not detected using NGS. LCRT‐PCR is a highly sensitive method to rapidly detect mutations of therapeutic relevance. Background: First‐line afatinib treatment prolongs overall survival in patients with metastatic non–small‐cell lung cancer (NSCLC) harboring exon 19 deletion of epidermal growth factor receptor (EGFRdelEx19) mutations. In contrast, Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) mutations are negative predictors for benefit from EGFR‐targeting agents. Endobronchial ultrasound‐guided transbronchial needle aspiration (EBUS‐TBNA) is well‐established for lung cancer diagnosis and staging. Next generation sequencing (NGS) allows for simultaneous interrogation for multiple mutations but has limitations (required tumor tissue amount, assay times). Reverse transcription polymerase chain reaction (RT‐PCR) using light‐Cycler technology (LCRT‐PCR) can rapidly and sensitively detect somatic mutations from NSCLC patients. In the present study, we analyzed the feasibility of LCRT‐PCR for rapid EGFRdelEx19 and KRAS exon 2 mutation detection in EBUS‐TBNA samples and compared the LCRT‐PCR and NGS results. Materials and Methods: A total of 48 EBUS‐TBNA samples from 47 patients with a confirmed diagnosis of pulmonary adenocarcinoma were analyzed using LCRT‐PCR (as previously described) and NGS (MiSeq; Illumina) using targeted resequencing and a customized multiplex PCR panel. The processing time was ˜1 week for the NGS and < 24 hours for the LCRT‐PCR analyses. Results: All (100%) EGFRdelEx19 and KRAS exon 2 mutations detected by NGS were detected by LCRT‐PCR. In addition, LCRT‐PCR detected 2 KRAS exon 2 mutations and 3 EGFRdelEx19 mutations that were not detected by NGS. Conclusion: LCRT‐PCR is a highly sensitive method to rapidly detect mutations of therapeutic relevance (eg, EGFRdelEx19 and KRAS exon 2) in EBUS‐TBNAs from NSCLC patients. It is of value as an initial assay for first‐line treatment decisions.

Phosphorylation of p70 Ribosomal Protein S6 Kinase β-1 is an Independent Prognostic Parameter in Metastatic Colorectal Cancer

Background Deregulation of signal transduction pathways plays a critical role in oncogenesis of colorectal cancer (CRC) and directly affects sensitivity to targeted therapies. Against this background we developed a comprehensive biomarker profiling program including markers of downstream signaling to study their association with clinical outcomes. Patients and Methods A prospectively studied cohort of 160 patients with metastatic CRC was included. Standard diagnostic workup included mutational analyses of Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS), and v‐Raf murine sarcoma viral oncogene homolog B (BRAF). In addition, markers of mitogen‐activated protein kinase (MAPK), phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) and mammalian target of rapamycin pathway activation (phosphorylation of extracellular signal‐regulated kinase [ERK], AKT, and p70 ribosomal protein S6 kinase &bgr;‐1 [p70S6K]) were studied using standardized immunohistochemistry. Results There was a significant correlation between markers of ERK and AKT activation in the full cohort. In addition, phosphorylation of p70S6K correlated strongly with ERK and AKT phosphorylation and primary tumor localization in the right colon. Subgroup analyses specified these correlations to patients with all‐RAS wild type tumors. In contrast, tumors harboring RAS mutations predominantly exhibited ERK phosphorylation. Interestingly, patients with CRC showing high p70S6K phosphorylation (highest quartile) had a significantly inferior overall survival (hazard ratio [HR], 2.4; P = .002) irrespective of RAS mutational status. This effect remained significant in multivariate analysis (P = .002). A patient subgroup characterized by high p70S6K phosphorylation and right‐sided primary tumors had a particularly poor prognosis with a dramatically inferior overall survival (HR, 5.2; P < .001). Patients with right‐sided primary tumor and low p70S6K phosphorylation had responses to anti‐epidermal growth factor receptor antibody‐based therapies and overall survival similar to patients with left‐sided primary tumors. Conclusion High phosphorylation of p70S6K is a novel, independent biomarker for poor prognosis, in particular in patients with right‐sided primary tumors. Micro‐Abstract We studied a cohort of patients (n = 160) with advanced colorectal cancer prospectively recruited in a biomarker profiling program. Among markers of pathway activation, high phosphorylation of p70 ribosomal protein S6 kinase &bgr;‐1 (p70S6K) was identified as a novel, independent biomarker for poor prognosis. The combination of a right‐sided primary tumor and high p70S6K phosphorylation formed a subgroup with dramatically inferior outcome.