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Concomitant inhibition of MEK and EGFR is synergistic in PC9KRAS-G12S and HCC827KRAS-G12S cells.

Supplementary figure legends

Activation of ERBB2 or MET leads to decreased sensitivity to AZD9291 and rociletinib.

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.

Purpose: To identify novel mechanisms of resistance to third-generation EGFR inhibitors in patients with lung adenocarcinoma that progressed under therapy with either AZD9291 or rociletinib (CO-1686).Experimental Design: We analyzed tumor biopsies from seven patients obtained before, during, and/or after treatment with AZD9291 or rociletinib (CO-1686). Targeted sequencing and FISH analyses were performed, and the relevance of candidate genes was functionally assessed in in vitro models.Results: We found recurrent amplification of either MET or ERBB2 in tumors that were resistant or developed resistance to third-generation EGFR inhibitors and show that ERBB2 and MET activation can confer resistance to these compounds. Furthermore, we identified a KRASG12S mutation in a patient with acquired resistance to AZD9291 as a potential driver of acquired resistance. Finally, we show that dual inhibition of EGFR/MEK might be a viable strategy to overcome resistance in EGFR-mutant cells expressing mutant KRAS.Conclusions: Our data suggest that heterogeneous mechanisms of resistance can drive primary and acquired resistance to third-generation EGFR inhibitors and provide a rationale for potential combination strategies. Clin Cancer Res; 22(19); 4837–47. ©2016 AACR.

Targeted and Sanger sequencing results at the EGFR locus for P6 and P7.

Expression of KRASG12S mutant results in decreased sensitivity to AZD9291 and rociletinib in EGFR mutated cells.

Supplemental Table 1-7 Supplemental Table 1: Diagnostic panels used for Next-generation sequencing (NGS) Supplemental Table 2: KEAP1 mutations Supplemental Table 3: NFE2L2 mutations Supplemental Table 4: Co-aberrations of KEAP1 Supplemental Table 5: Co-aberrations of NFE2L2 Supplemental Table 6: Distribution on FISH finding depending on histology Supplemental Table 7: Therapeutic outcome of patients receiving systemic therapy.

Main characteristics of the control group.

Effective targeted treatment strategies resulted from molecular profiling of lung cancer with distinct prevalent mutation profiles in smokers and non-smokers. Although Rn is the second most important risk factor, data for Rn-dependent driver events are limited. Therefore, a Rn-exposed cohort of lung cancer patients was screened for oncogenic drivers and their survival and genetic profiles were compared with data of the average regional population. Genetic alterations were analysed in 20 Rn-exposed and 22 histologically matched non-Rn exposed LC patients using targeted Next generation sequencing (NGS) and Fluorescence In Situ Hybridization (FISH). Sufficient material and sample quality could be obtained in 14/27 non-exposed versus 17/22 Rn-exposed LC samples. Survival was analysed in comparison to a histologically and stage-matched regional non-exposed lung cancer cohort (n = 51) for hypothesis generating. Median overall survivals were 83.02 months in the Rn-exposed and 38.7 months in the non-exposed lung cancer cohort (p = 0.22). Genetic alterations of both patient cohorts were in high concordance, except for an increase in MET alterations and a decrease in TP53 mutations in the Rn-exposed patients in this small hypothesis generating study.

Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r2 = 0.998) and phase 2 (r2 = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research.

We report on the incidental finding of a FOXL2 mutated adult granulosa cell tumour of the ovary with thecoma-like foci, a rare entity recently described by Jennifer N. Stall and Robert H. Young in a series of sixteen cases in 2019, displaying features differing from conventional adult granulosa cell tumour. Our aim is to specify the morphologic and molecular particularities of this presumably underrecognized finding, with a short presentation of the typical clinical context. Awareness of this rare and challenging neoplasm with indeterminate clinical course is crucial in routine diagnostics.

Resistance to MET inhibition occurs inevitably in MET-dependent non-small cell lung cancer and the underlying mechanisms are insufficiently understood. We describe resistance mechanisms in patients with MET exon 14 skipping mutation (METΔPre- and post-treatment biopsies were analysed by NGS (next generation sequencing), digital droplet PCR (polymerase chain reaction), and FISH (fluorescense in situ hybridization). A patient-derived xenograft model was generated in one case.Of 26 patients with MET tyrosine kinase inhibitor treatment, eight had paired pre- and post-treatment biopsies (Three with MET amplification, three with METΔResistance to MET inhibition is heterogeneous with on- and off-target mechanisms occurring regardless of the initial MET aberration. Switching therapy between different types of kinase inhibitors can lead to repeated responses in cases with second-site mutations. Controlled clinical trials in this setting with larger patient numbers are needed, as evidence to date is limited to preclinical data and case series.

MET tyrosine kinase receptor pathway activation has become an important actionable target in solid tumors. Aberrations in the MET proto-oncogene, including MET overexpression, the activation of MET mutations, MET mutations that lead to MET exon 14 skipping, MET gene amplifications, and MET fusions, are known to be primary and secondary oncogenic drivers in cancer; these aberrations have evolved as predictive biomarkers in clinical diagnostics. Thus, the detection of all known MET aberrations in daily clinical care is essential. In this review, current molecular technologies for the detection of the different MET aberrations are highlighted, including the benefits and drawbacks. In the future, another focus will be on the standardization of detection technologies for the delivery of reliable, quick, and affordable tests in clinical molecular diagnostics.

Predictive molecular testing has become an important part of the diagnosis of any patient with lung cancer. Using reliable methods to ensure timely and accurate results is inevitable for guiding treatment decisions. In the past few years, parallel sequencing has been established for mutation testing, and its use is currently broadened for the detection of other genetic alterations, such as gene fusion and copy number variations. In addition, conventional methods such as immunohistochemistry and in situ hybridization are still being used, either for formalin-fixed, paraffin-embedded tissue or for cytological specimens. For the development and broad implementation of such complex technologies, interdisciplinary and regional networks are needed. The Network Genomic Medicine (NGM) has served as a model of centralized testing and decentralized treatment of patients and incorporates all German comprehensive cancer centers. Internal quality control, laboratory accreditation, and participation in external quality assessment is mandatory for the delivery of reliable results. Here, we provide a summary of current technologies used to identify patients who have lung cancer with gene fusions, briefly describe the structures of NGM and the national NGM (nNGM), and provide recommendations for quality assurance.

BackgroundOver the past years, EGFR tyrosine kinase inhibitors (TKI) revolutionized treatment response. 1st-generation (reversible) EGFR TKI and later the 2nd –generation irreversible EGFR TKI Afatinib were aimed to improve treatment response. Nevertheless, diverse resistance mechanisms develop within the first year of therapy. Here, we evaluate the prevalence of acquired resistance mechanisms towards reversible and irreversible EGFR TKI.MethodsRebiopsies of patients after progression to EGFR TKI therapy (> 6 months) were targeted to histological and molecular analysis. Multiplexed targeted sequencing (NGS) was conducted to identify acquired resistance mutations (e.g. EGFR p.T790M). Further, Fluorescence in situ hybridisation (FISH) was applied to investigate the status of bypass mechanisms like, MET or HER2 amplification.ResultsOne hundred twenty-three rebiopsy samples of patients that underwent first-line EGFR TKI therapy (PFS ≥6 months) were histologically and molecularly profiled upon clinical progression. TheEGFRp.T790M mutation is the major mechanism of acquired resistance in patients treated with reversible as well as irreversible EGFR TKI. Nevertheless a statistically significant difference for the acquisition of T790M mutation has been identified: 45% of afatinib- vs 65% of reversible EGFR TKI treated patients developed a T790M mutation (p-value 0.02). Progression free survival (PFS) was comparable in patients treated with irreversibleEGFRirrespective of the sensitising primary mutation or the acquisition of p.T790M.ConclusionsTheEGFRp.T790M mutation is the most prominent mechanism of resistance to reversible and irreversible EGFR TKI therapy. Nevertheless there is a statistically significant difference of p.T790M acquisition between the two types of TKI, which might be of importance for clinical therapy decision.

PURPOSE Third-generation epidermal growth factor receptor ( EGFR) tyrosine kinase inhibitors (TKIs) are effective in acquired resistance (AR) to early-generation EGFR TKIs in EGFR-mutant lung cancer. However, efficacy is marked by interindividual heterogeneity. We present the molecular profiles of pretreatment and post-treatment samples from patients treated with third-generation EGFR TKIs and their impact on treatment outcomes. METHODS Using the databases of two lung cancer networks and two lung cancer centers, we molecularly characterized 124 patients with EGFR p.T790M-positive AR to early-generation EGFR TKIs. In 56 patients, correlative analyses of third-generation EGFR TKI treatment outcomes and molecular characteristics were feasible. In addition, matched post-treatment biopsy samples were collected for 29 patients with progression to third-generation EGFR TKIs. RESULTS Co-occurring genetic aberrations were found in 74.4% of EGFR p.T790-positive samples (n = 124). Mutations in TP53 were the most frequent aberrations detected (44.5%; n = 53) and had no significant impact on third-generation EGFR TKI treatment. Mesenchymal-epithelial transition factor ( MET) amplifications were found in 5% of samples (n = 6) and reduced efficacy of third-generation EGFR TKIs significantly (eg, median progression-free survival, 1.0 months; 95% CI, 0.37 to 1.72 v 8.2 months; 95% CI, 1.69 to 14.77 months; P ≤ .001). Genetic changes in the 29 samples with AR to third-generation EGFR TKIs were found in EGFR (eg, p.T790M loss, acquisition of p.C797S or p.G724S) or in other genes (eg, MET amplification, KRAS mutations). CONCLUSION Additional genetic aberrations are frequent in EGFR-mutant lung cancer and may mediate innate and AR to third-generation EGFR TKIs. MET amplification was strongly associated with primary treatment failure and was a common mechanism of AR to third-generation EGFR TKIs. Thus, combining EGFR inhibitors with TKIs targeting common mechanisms of resistance may delay AR.

Simple Summary Sinonasal carcinomas are rare tumors with an overall poor prognosis. Due to limitations in local therapeutic approaches, systemic neo-adjuvant or adjuvant therapies are becoming increasingly important in order to improve patient outcome. This study aimed to examine potentially therapeutic targetable molecular alterations in different sinonasal tumors, including deficiency in mismatch repair proteins and microsatellite instability as well as driver mutations. According to our results, immunohistochemical (IHC) analysis of mismatch repair (MMR) proteins and sequencing-based panel analysis should be integrated into the diagnostics of clinically aggressive inverted sinonasal papilloma (ISP) and sinonasal squamous cell carcinoma (SNSCC) in order to enable the therapeutic possibility of a targeted therapy. Abstract Due to limitations in local therapy approaches for sinonasal tumors, improvement in systemic therapies plays a pivotal role for prolongation of the patient’s survival. The aim of this study was to examine potential biomarkers, including deficiency in mismatch repair proteins (dMMR)/microsatellite instability (MSI-H) in sinonasal cancers and their precancerous lesions. A comprehensive analysis of 10 sinonasal cancer cell lines by whole exome sequencing, screening 174 sinonasal tumors by immunohistochemistry (IHC) for mismatch repair deficiency and next generation sequencing (NGS) of 136 tumor samples revealed a dMMR/MSI-H sinonasal squamous cell carcinoma (SNSCC) cell line based on a somatic missense mutation in MLH1 and an overall frequency of dMMR/MSI-H SNSCC of 3.2% (4/125). Targetable EGFR mutations were found in 89.3% (25/28) of inverted sinonasal papilloma (ISP) and in 60% (6/10) of ISP-associated carcinomas. While PIK3CA and EGFR mutations were not mutually exclusive, KRAS mutated tumors were an EGFR-wildtype. The effect of potential driver mutations in FGFR2, FGFR3, BRAF, HRAS, MAP2K1, PTEN, NOTCH1 and CARD11 need further investigation. Our results suggest that biomarker testing, including MMR-IHC and NGS panel analysis, should be integrated into the diagnostics of clinically aggressive ISPs and SNSCC to assess prognosis and facilitate therapeutic decisions.

Graphical abstract, In EGFR-treatment naive NSCLC patients, high-level MET amplification is detected in approximately 2–3% and is considered as adverse prognostic factor. Currently, clinical trials with two different inhibitors, capmatinib and tepotinib, are under way both defining different inclusion criteria regarding MET amplification from proven amplification only to defining an exact MET copy number. Here, 45 patient samples, including 10 samples without MET amplification, 5 samples showing a low-level MET amplification, 10 samples with an intermediate-level MET amplification, 10 samples having a high-level MET amplification by a MET/CEN7 ratio ≥2.0 and 10 samples showing a high-level MET amplification with GCN ≥6, were evaluated by MET FISH, MET IHC, a ddPCR copy number assay, a NanoString nCounter copy number assay and an amplicon-based parallel sequencing. The MET IHC had the best concordance with MET FISH followed by the NanoString copy number assay, the ddPCR copy number assay and the custom amplicon-based parallel sequencing assays. The concordance was higher in the high-level amplified cohorts than in the low- and intermediate-level amplified cohorts. In summary, currently extraction-based methods cannot replace the MET FISH for the detection of low-level, intermediate-level and high-level MET amplifications, as the number of false negative results is very high. Only for the detection of high-level amplified samples with a gene copy number ≥6 extraction-based methods are a reliable alternative.

Up to 40% of esophageal carcinomas have a biallelic intact TP53 gene. It is largely unclear how these carcinoma cells prevent apoptosis, what is the kind of pathway alterations, or whether therapeutically relevant alterations exist in this subgroup. We evaluated The Cancer Genome Atlas (TCGA) data to compare TP53-mutated with TP53–wild-type tumors regarding copy number variations, gene mutations, and expression patterns of protein-coding genes and miRNAs. Additionally, we analyzed up to 428 esophageal adenocarcinomas (EACs) in total using an ultra-deep parallel sequencing panel, immunohistochemistry, as well as fluorescence in situ hybridization. In the TCGA cohort, 17.3% has a biallelic intact TP53 gene. This group has a smaller average total size of somatic copy number variations. Some protein coding genes and miRNAs were differentially expressed between the TP53-wild-type and TP53-mutated group to emphasize mdm2, CCND2, TP73, or miRNA 150, 488, or 4662a. In addition, 50% of the TP53–wild-type tumors carry somatic mutations in at least one of the genes involved in the TP53 pathway. Our patient cohort revealed 41.3% TP53–wild-type tumors; 5.6% were MDM2 amplified. In accordance with the TCGA data, we did not find a prognostic relevance of TP53 in our tumor cohort as well. The mutation status of TP53 defines an important subtype in esophageal carcinoma. Our comprehensive molecular analysis revealed important and potentially therapeutically relevant genomic alterations in this subgroup.

Robust data on the outcome of MET-aberrant NSCLC with nontargeted therapies are limited, especially in consideration of the heterogeneity of MET-amplified tumors (METamp).A total of 337 tumor specimens of patients with MET-altered Union for International Cancer Control stage IIIB/IV NSCLC were analyzed using next-generation sequencing, fluorescence in situ hybridization, and immunohistochemistry. The evaluation focused on the type of MET aberration, co-occurring mutations, programmed death-ligand 1 expression, and overall survival (OS).METamp tumors (n = 278) had a high frequency of co-occurring mutations (80% for all amplification levels), whereas 57.6% of the 59 patients with MET gene and exon 14 (METex14) tumors had no additional mutations. In the METamp tumors, with increasing gene copy number (GCN), the frequency of inactivating TP53 mutations increased (GCN4: 58.2%; GCN ≥ 10: 76.5%), whereas the frequency of KRAS mutations decreased (GCN4: 43.2%; GCN ≥ 10: 11.8%). A total of 10.1% of all the METamp tumors with a GCN ≥ 10 had a significant worse OS (4.0 mo; 95% CI: 1.9-6.0) compared with the tumors with GCN10 (12.0 mo; 95% confidence interval [CI]: 9.4-14.6). In the METamp NSCLC, OS with immune checkpoint inhibitor (ICI) therapy was significantly better compared with chemotherapy with 19.0 months (95% CI: 15.8-22.2) versus 8.0 months (95% CI: 5.8-10.2, p0.0001). No significant difference in median OS was found between ICI therapy and chemotherapy in the patients with METex14 (p = 0.147).METex14, METamp GCN ≥ 10, and METamp GCN10 represent the subgroups of MET-dysregulated NSCLC with distinct molecular and clinical features. The patients with METex14 do not seem to benefit from immunotherapy in contrast to the patients with METamp, which is of particular relevance for the prognostically poor METamp GCN ≥ 10 subgroup.

Outcome of immune checkpoint inhibition in cancer can be predicted by measuring PDL1 expression of tumor cells. Search for additional biomarkers led to tumor mutational burden (TMB) as surrogate marker for neoantigens presented. While TMB was previously determined via whole exome sequencing (WES), there have been approaches with comprehensive gene panels as well. We sequenced samples derived from formalin-fixed tumors, a POLE mutated cell line and standard DNA by WES and five different panels. If available, normal tissue was also exome sequenced. Sequencing data was analyzed by commercial software solutions and an in-house pipeline. A robust Pearson correlation (R = 0.9801 ± 0.0167; mean ± sd; N = 7) was determined for the different panels in a tumor paired normal setting for WES. Expanded analysis on tumor only exome sequenced samples yielded similar correlation (R = 0.9439 ± 0.0632; mean ± sd; N = 14). Remaining germline variants increased TMB in WES by 5.761 ± 1.953 (mean ± sd.; N = 7) variants per megabase (v/mb) for samples including synonymous variants and 3.883 ± 1.38 v/mb for samples without synonymous variants compared to tumor-normal paired calling results. Due to limited sample numbers in this study, additional replication is suggested for a clinical setting. Remaining germline variants in a tumor-only setting and artifacts caused by different library chemistries construction might affect the results.

Aims The advent of specific ALK-targeting drugs has radically changed the outcome of patients with ALK translocated non-small-cell lung cancer (NSCLC). However, emerging resistance to treatment with ALK inhibitors in these patients remains a major concern. In previous studies, we analysed two ALK+ patient cohorts (TP53 wild-type/TP53 mutated) in terms of copy number alterations. All patients belonging to the TP53 wild-type group had mainly genetically stable genomes, with one exception showing chromosomal instability and amplifications of several gene loci, including TERT. Here, we aimed to determine the prevalence of TERT amplifications in these ALK+ lung cancer patients by analysing an independent cohort of 109 ALK translocated cases. We further analysed the copy numbers of numerous cancer-relevant genes and other genetic aberrations. Methods and results The prevalence of TERT amplifications was determined by means of FISH analyses. Copy numbers of 87 cancer-relevant genes were determined by NanoString nCounter® technology, FoundationOne® and lung-specific NGS panels in some of these TERT-amplified samples, and clinical data on patients with TERT-amplified tumours were collected. Our data revealed that five (4.6%) of all 109 analysed ALK+ patients harboured amplification of TERT and that these patients had genetically unstable genomes. Conclusions Our preliminary study shows that ALK+ adenocarcinomas should be evaluated in the context of their genomic background in order to more clearly understand and predict patients' individual course of disease.

Die personalisierte Therapie bietet Patienten mit fortgeschrittenem, nichtkleinzelligem Lungenkarzinom (NSCLC) die Chance zu einem langeren Uberleben. Fur ca. 15 % dieser Patienten ist bereits jetzt die treibermutationsgerichtete Therapie mit Kinaseinhibitoren der Standard. Fur Patienten ohne therapierbare Treibermutation ist die Immuntherapie bereits Standard, in Abhangigkeit von der PD-L1-Expression mit oder ohne Chemotherapie.

Although non-small-cell lung cancer is a leading cause of cancer-related deaths, the molecular characterization and classification of its genetic alterations has drastically changed treatment options and overall survival within the last few decades. In particular, tyrosine kinase inhibitors targeting specific molecular alterations, among other MET, have greatly improved the prognosis of non-small-cell lung cancer patients. Here, we compare the genomic background of a subset of non-small-cell lung cancer cases harboring either a MET high-level amplification (n = 24) or a MET exon 14 skipping mutation (n = 26), using next-generatison sequencing, fluorescence in situ hybridization, immunohistochemistry, and Nanostring nCounter® technology. We demonstrate that the MET-amplified cohort shows a higher genetic instability, compared with the mutant cohort (p

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non-small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co-occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53-mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC-overexpressing ALK+ TP53-mutated cells had a proliferative advantage compared to wild-type cells. ChIP-Seq data revealed MYC-binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53-mutated cells resulted in an upregulation of EML4-ALK, indicating a potential MYC-dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co-occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

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.

Der Vormarsch der personalisierten Therapie — getrieben durch die rasanten Fortschritte in der genomischen Analytik und die unerwartete Beschleunigung klinischer Entwicklungsprogramme — wandelt zunehmend das diagnostische und therapeutische Vorgehen beim fortgeschrittenen, nichtkleinzelligen Lungenkarzinom (NSCLC). Aktuell erhalten bereits circa 50 % der Patienten in der Erstlinie eine primar Biomarker-stratifizierte Behandlung durch eine zielgerichtete Therapie mit einem Tyrosinkinaseinhibitor oder eine Immuntherapie mit einem Immuncheckpointinhibitor. Der Einsatz einer biologisch-rationalen Therapie in einem praselektionierten Patientenkollektiv mit einem korrespondierenden, pradiktiven Biomarker („Schlussel-Schloss-Prinzip“ oder Prazisionsmedizin) ubersetzt sich — im Vergleich zur klassischen nicht selektiven, zytotoxischen Chemotherapie — in einen signifikanten Vorteil fur die Patienten mit verbesserten Remissionsraten und verlangertem Gesamtuberleben. Aktuelle Herausforderungen beinhalten die Uberwindung von Resistenzen, die Entwicklung pradiktiver Marker fur die Immuntherapie und die Implementierung personalisierter Therapien in der Breite der Versorgung.

// Carina Heydt 1, 2 , Sebastian Michels 2, 3 , Kenneth S. Thress 4 , Sven Bergner 5 , Jurgen Wolf 2, 3 and Reinhard Buettner 1, 2 1 Molecular Pathological Diagnostics, Institute of Pathology, University Hospital Cologne, Cologne, Germany 2 Center of Integrated Oncology Koln-Bonn, University Hospital Cologne, Cologne, Germany 3 Department I of Internal Medicine, Center for Integrated Oncology Koln-Bonn, University Hospital of Cologne, Cologne, Germany 4 Translational Science, Oncology iMED, AstraZeneca, Waltham, MA, USA 5 Medical Affairs, AstraZeneca Oncology, Wedel, Germany Correspondence to: Reinhard Buettner, email: reinhard.buettner@uk-koeln.de Keywords: epidermal growth factor receptor (EGFR); non-small-cell lung cancer (NSCLC); resistance; tyrosine kinase inhibitor (TKI); tumor heterogeneity Received: November 03, 2017 Accepted: February 21, 2018 Epub: March 08, 2018 Published: March 16, 2018 ABSTRACT Background. The identification and characterization of molecular biomarkers has helped to revolutionize non-small-cell lung cancer (NSCLC) management, as it transitions from target-focused to patient-based treatment, centered on the evolving genomic profile of the individual. Determination of epidermal growth factor receptor ( EGFR ) mutation status represents a critical step in the diagnostic process. The recent emergence of acquired resistance to “third-generation” EGFR tyrosine kinase inhibitors (TKIs) via multiple mechanisms serves to illustrate the important influence of tumor heterogeneity on prognostic outcomes in patients with NSCLC. Design. This literature review examines the emergence of TKI resistance and the course of disease progression and, consequently, the clinical decision-making process in NSCLC. Results. Molecular markers of acquired resistance, of which T790M and HER2 or MET amplifications are the most common, help to guide ongoing treatment past the point of progression. Although tissue biopsy techniques remain the gold standard, the emergence of liquid biopsies and advances in analytical techniques may eventually allow “real-time” monitoring of tumor evolution and, in this way, help to optimize targeted treatment approaches. Conclusions. The influence of inter- and intra-tumor heterogeneity on resistance mechanisms should be considered when treating patients using resistance-specific therapies. New tools are necessary to analyze changes in heterogeneity and clonal composition during drug treatment. The refinement and standardization of diagnostic procedures and increased accessibility to technology will ultimately help in personalizing the management of NSCLC.

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with poly ADP ribose polymerase (PARP) inhibitors. Tumor BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin-fixed paraffin embedded (FFPE), the tumor genome is complex, and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumors to compare different approaches to identify clinically important variants within FFPE tumor DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumor DNA samples ranging in genotype, quantity, quality, and variant allele frequency (VAF). Each laboratory performed their preferred next-generation sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification, or insufficient DNA. The use of hybridization capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardization for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies.

The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples. Venous blood from healthy donors was collected in tubes from Streck, Roche, and Qiagen. Samples were spiked with artificially fragmented EGFR T790M-mutated DNA and stored for different periods of time or spiked with different DNA amounts before plasma preparation. Extracted ctDNA was analyzed by amplification-refractory mutation system PCR. Mutant DNA, spiked into blood samples from healthy donors at quantities of 1 or 3 ng, was still reliably detectable in all samples after 7 days. EGFR T790M could be detected when spiking was performed with an amount of artificial ctDNA of 0.5 ng when tubes from Roche and Qiagen were used. Blood collection tubes from Roche and Qiagen are highly suitable for ctDNA stabilization and subsequent liquid biopsy testing. Even low ctDNA concentrations allow the detection of somatic mutations.

Primary cardiac tumours are rare and mostly benign lesions. Recent publications report that cardiac papillary fibroelastomas are the most common benign primary heart tumour, outnumbering myxomas. However, there is no consensus about their aetiology. We investigated the molecular profile of these tumours using next generation sequencing in a cohort of 16 cases. Eleven of 14 (79%) analysable tumours showed mutations of the KRAS oncogene. Our results provide unambiguous evidence that a significant proportion of these lesions are genuine neoplastic tumours caused by an oncogenic driver mutation.

Inflammatory myofibroblastic tumors are rare mesenchymal neoplasms frequently harboring oncogenic chromosomal rearrangements, most commonly, involving the ALK (anaplastic lymphoma kinase) gene. Treatment of this molecularly defined subgroup with the anaplastic lymphoma kinase inhibitor crizotinib has shown to be effective. However, comparable to lung adenocarcinoma, resistance inevitably develops. Second generation anaplastic lymphoma kinase inhibitors such as ceritinib are able to overcome acquired resistance to crizotinib. Here, we report the case of a patient with an inflammatory myofibroblastic tumors harboring a DCTN1-ALK fusion who developed resistance to crizotinib treatment. Next-generation sequencing of a rebiopsy sample revealed the acquisition of the ALK G1269A mutation as a mechanism of resistance. Therapy with ceritinib resulted in a short but profound clinical, metabolic and morphologic response. This case illustrates that (i) different tumor entities may share similar oncogenic driver mechanisms, rendering them vulnerable for the same therapeutic substances and (ii) likewise, the same mode of resistance may occur under targeted therapy among different tumor entities.