Ulrich Gerigk

Definition of a fluorescence in-situ hybridization score identifies high- and low-level FGFR1 amplification types in squamous cell lung cancer

We recently reported fibroblast growth factor receptor-type 1 (FGFR1) amplification to be associated with therapeutically tractable FGFR1 dependency in squamous cell lung cancer. This makes FGFR1 a novel target for directed therapy in these tumors. To reproducibly identify patients for clinical studies, we developed a standardized reading and evaluation strategy for FGFR1 fluorescence in-situ hybridization (FISH) and propose evaluation criteria, describe different patterns of low- and high-level amplifications and report on the prevalence of FGFR1 amplifications in pulmonary carcinomas. A total of 420 lung cancer patients including 307 squamous carcinomas, 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for FGFR1 amplification using a dual color FISH. We found heterogeneous and different patterns of gene copy numbers. FGFR1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas. High-level amplification (as defined by an FGFR1/centromer 8 (CEN8) ratio ≥2.0, or average number of FGFR1 signals per tumor cell nucleus ≥6, or the percentage of tumor cells containing ≥15 FGFR1 signals or large clusters ≥10%) was detected at a frequency of 16% and low-level amplification (as defined by ≥5 FGFR1 signals in ≥50% of tumor cells) at a frequency of 4%. We conclude that FGFR1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas. Standardized reporting of FGFR1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with FGFR1 inhibitors in clinical studies. Thus, our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials.

Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients

Introduction: The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons. Methods: In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed. Results: We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas. Conclusion: Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.

Rationale for Treatment of Metastatic Squamous Cell Carcinoma of the Lung Using Fibroblast Growth Factor Receptor Inhibitors

BACKGROUND We previously identified amplification of the fibroblast growth factor receptor 1 gene (FGFR1) as a potential therapeutic target for small-molecule inhibitor therapy in squamous cell lung cancer (L-SCC). Currently, clinical phase I trials are underway to examine whether patients with FGFR1-amplified L-SCC benefit from a targeted therapy approach using small-molecule inhibitors. Because most patients with lung cancer present with metastatic disease, we investigated whether lymph node metastases in L-SCC share the FGFR1 amplification status of their corresponding primary tumor. METHODS The study cohort consisted of 72 patients with L-SCC, 39 with regional lymph node metastases. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded tissue of the primary tumors and, where present, of the corresponding lymph node metastasis. A biotin-labeled target probe spanning the FGFR1 locus (8p11.22-23) was used to determine the FGFR1 amplification status by fluorescence in situ hybridization. RESULTS FGFR1 amplification was detected in 16% (12 of 72) of all primary L-SCCs. In metastatic tumors, 18% (seven of 39) of the lymph node metastases displayed FGFR1 amplification with an exact correlation of FGFR1 amplification status between tumor and metastatic tissue. CONCLUSIONS FGFR1 amplification is a common genetic event occurring at a frequency of 16% in L-SCCs. Moreover, lymph node metastases derived from FGFR1-amplified L-SCCs also exhibit FGFR1 amplification. Therefore, we suggest that the FGFR1 amplification is a clonal event in tumor progression. Beyond this biologically relevant observation, the findings carry potential therapeutic implications in that small-molecule inhibitors may be applicable to the treatment of a subset of patients with metastatic L-SCC.

Clinicopathological Characteristics of RET Rearranged Lung Cancer in European Patients

Introduction Rearrangements of RET are rare oncogenic events in patients with non–small cell lung cancer (NSCLC). While the characterization of Asian patients suggests a predominance of nonsmokers of young age in this genetically defined lung cancer subgroup, little is known about the characteristics of non‐Asian patients. We present the results of an analysis of a European cohort of patients with RET rearranged NSCLC. Methods Nine hundred ninety‐seven patients with KRAS/EGFR/ALK wildtype lung adenocarcinomas were analyzed using fluorescence in situ hybridization for RET fusions. Tumor specimens were molecularly profiled and clinicopathological characteristics of the patients were collected. Results Rearrangements of RET were identified in 22 patients, with a prevalence of 2.2% in the KRAS/EGFR/ALK wildtype subgroup. Co‐occurring genetic aberrations were detected in 10 patients, and the majority had mutations in TP53. The median age at diagnosis was 62 years (range, 39–80 years; mean ± SD, 61 ± 11.7 years) with a higher proportion of men (59% versus 41%). There was only a slight predominance of nonsmokers (54.5%) compared to current or former smokers (45.5%). Conclusions Patients with RET rearranged adenocarcinomas represent a rare and heterogeneous NSCLC subgroup. In some contrast to published data, we see a high prevalence of current and former smokers in our white RET cohort. The significance of co‐occurring aberrations, so far, is unclear.

Regional screening network for characterization of the molecular epidemiology of non-small cell lung cancer (NSCLC) and implementation of personalized treatment.

CRA10529 Background: Personalized treatment of genetically stratified subgroups has the potential to substantially improve outcome in NSCLC. A major challenge now is to implement high-quality molecular diagnostics and personalized treatment strategies in routine clinical practice also outside of highly specialized academic centers. METHODS We have established a molecular screening network in the catchment area of our comprehensive cancer center encompassing about 2.5 million inhabitants in March 2010 after review of the local ethics committee (10-242). Lung adenocarcinoma (AD) was screened centrally for ALK translocations, mutations in KRAS, EGFR, BRAF and PIK3CA and for amplification of ERBB2. Squamous cell carcinoma (SQ) was analyzed for FGFR1 amplifications. RESULTS 2032 NSCLC samples were acquired of which 1782 in the Cologne-Bonn area indicating a capture rate of 60-70% of all NSCLC samples in the area. Material was suitable for molecular analysis in 77%. Distribution of histological subtypes was as expected (AD 63.4%, SQ 26.7, large cell carcinoma 1.4%, adenosquamous cell carcinoma 1.8%, carcinoid 0.1%, NSCLC NOS 6.7%. In AD the following frequencies of genetic lesions were detected: KRAS (32%), EGFR (13%), ALK (3%), BRAF (2%), PIK3CA (2%), ERBB2 (2%). EGFR mutations were highly enriched in the lepidic and micropapillary subtype of AD (30-32%), whereas the solid subtype only harboured a very small amount of the tested oncogenic lesions. In SQ FGFR1 amplification was detected in 78/500. Overall 40% of all NSCLC samples harboured potentially tractable oncogenic lesions. All patients with ALK translocations received crizotinib when clinically indicated. 75% of the stage IIIB/IV patients with activating EGFR mutations received EGFR-TKI treatment. In addition, clinical trials have been initiated to provide personalized treatment options to all patients with tractable genetic lesions. CONCLUSIONS High-quality molecular diagnostics and identification of patients for personalized treatment approaches is feasible in daily clinical routine for the majority of diagnostic samples also in a non-academic setting.

Clinical and Pathological Characteristics of KEAP1- and NFE2L2-Mutated Non–Small Cell Lung Carcinoma (NSCLC)

Purpose: KEAP1 and NFE2L2 mutations are associated with impaired prognosis in a variety of cancers and with squamous cell carcinoma formation in non–small cell lung cancer (NSCLC). However, little is known about frequency, histology dependence, molecular and clinical presentation as well as response to systemic treatment in NSCLC. Experimental Design: Tumor tissue of 1,391 patients with NSCLC was analyzed using next-generation sequencing (NGS). Clinical and pathologic characteristics, survival, and treatment outcome of patients with KEAP1 or NFE2L2 mutations were assessed. Results: KEAP1 mutations occurred with a frequency of 11.3% (n = 157) and NFE2L2 mutations with a frequency of 3.5% (n = 49) in NSCLC patients. In the vast majority of patients, both mutations did not occur simultaneously. KEAP1 mutations were found mainly in adenocarcinoma (AD; 72%), while NFE2L2 mutations were more common in squamous cell carcinoma (LSCC; 59%). KEAP1 mutations were spread over the whole protein, whereas NFE2L2 mutations were clustered in specific hotspot regions. In over 80% of the patients both mutations co-occurred with other cancer-related mutations, among them also targetable aberrations like activating EGFR mutations or MET amplification. Both patient groups showed different patterns of metastases, stage distribution and performance state. No patient with KEAP1 mutation had a response on systemic treatment in first-, second-, or third-line setting. Of NFE2L2-mutated patients, none responded to second- or third-line therapy. Conclusions: KEAP1- and NFE2L2-mutated NSCLC patients represent a highly heterogeneous patient cohort. Both are associated with different histologies and usually are found together with other cancer-related, partly targetable, genetic aberrations. In addition, both markers seem to be predictive for chemotherapy resistance. Clin Cancer Res; 24(13); 3087–96. ©2018 AACR.