Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

MET Fusions in NSCLC: Clinicopathologic Features and Response to MET Inhibition.

INTRODUCTION: MET fusions have been described only rarely in NSCLC. Thus, data on patient characteristics and treatment response are limited. We here report histopathologic data, patient demographics, and treatment outcome including response to MET tyrosine kinase inhibitor (TKI) therapy in MET fusion-positive NSCLC. METHODS: Patients with NSCLC and MET fusions were identified mostly by RNA sequencing within the routine molecular screening program of the national Network Genomic Medicine, Germany. RESULTS: We describe a cohort of nine patients harboring MET fusions. Among these nine patients, two patients had been reported earlier. The overall frequency was 0.29% (95% confidence interval: 0.15-0.55). The tumors were exclusively adenocarcinoma. The cohort was heterogeneous in terms of age, sex, or smoking status. We saw five different fusion partner genes (KIF5B, TRIM4, ST7, PRKAR2B, and CAPZA2) and several different breakpoints. Four patients were treated with a MET TKI leading to two partial responses, one stable disease, and one progressive disease. One patient had a BRAF V600E mutation as acquired resistance mechanism. CONCLUSIONS: MET fusions are very rare oncogenic driver events in NSCLC and predominantly seem in adenocarcinomas. They are heterogeneous in terms of fusion partners and breakpoints. Patients with MET fusion can benefit from MET TKI therapy.

Tumour area infiltration and cell count in endoscopic biopsies of therapy-naive upper GI tract carcinomas by QuPath analysis: implications for predictive biomarker testing.

Guidelines regulate how many (tumour-bearing) tissue particles should be sampled during gastric cancer biopsy to obtain representative results in predictive biomarker testing. Little is known about how well these guidelines are applied, how the number of tissue particles correlates with the actual tumour-infiltrated area and how many absolute tumour cells are captured. The study included endoscopic biopsies of untreated carcinomas of the upper gastrointestinal (GI)-tract during the 2016-2020 review period. Archival (H&E)-stained histological sections were digitised and the tumour areas were manually annotated. The tumour-bearing tissue area and absolute carcinoma cell count per case were determined by image analysis and compared with a reference primary surgical specimen. Biopsies from 253 patients were analysed. The following mean values were determined: (a) tumour tissue particle number: 6.5 (range: 1-25, standard deviation (SD) = 3.33), (b) number of tumour-bearing tissue particles: 4.7 (range: 1-20, SD = 2.80), (c) tumour-infiltrated area: 7.5 mm2 (range: 0.18-59.46 mm2, SD = 6.67 mm2), (d) absolute tumour cell count: 13,492 (range: 193-92,834, SD = 14,185) and (e) tumour cell count in a primary surgical specimen (tumour size: 6.7 cm): 105,200,176. The guideline-recommended tissue particle count of 10 was not achieved in 208 patients (82.2%) and the required tumour-bearing tissue particle count of 5 was not achieved in 133 patients (52.6%). Tissue particle count, tumour-infiltrated area and tumour cell count were only weakly correlated. Most cases featured an infiltrated area ≥ 4.5 mm2 (156, 61.7%). Cases with more tissue particles showed only a moderate increase in infiltrated area and tumour cells compared to cases with fewer particles. Biopsies are often used to determine predictive biomarkers, particularly Her2/neu and PD-L1. Diagnostic standards to ensure representative material have been suggested in guidelines to reduce false-negative predictions. However, the real-world practice seems to substantially deviate from recommended standards. To the best of our knowledge, this is the first systematic study describing the relationships between endoscopic tissue fragment number, actual infiltrated tumour area and carcinoma cell number. The data question the tissue particle number as a quality assessment parameter. We advocate histopathological reports indicating on which basis statements on therapy-relevant biomarkers were made. Digital pathology has the potential to objectively quantify the tissue for documentation, quality assessment and future clinical studies.

Somatic rearrangements causing oncogenic ectodomain deletions of FGFR1 in squamous cell lung cancer.

The discovery of frequent 8p11-p12 amplifications in squamous cell lung cancer (SQLC) has fueled hopes that FGFR1, located inside this amplicon, might be a therapeutic target. In a clinical trial, only 11% of patients with 8p11 amplification (detected by FISH) responded to FGFR kinase inhibitor treatment. To understand the mechanism of FGFR1 dependency, we performed deep genomic characterization of 52 SQLCs with 8p11-p12 amplification, including 10 tumors obtained from patients who had been treated with FGFR inhibitors. We discovered somatically altered variants of FGFR1 with deletion of exons 1-8 that resulted from intragenic tail-to-tail rearrangements. These ectodomain-deficient FGFR1 variants (ΔEC-FGFR1) were expressed in the affected tumors and were tumorigenic in both in vitro and in vivo models of lung cancer. Mechanistically, breakage-fusion-bridges were the source of 8p11-p12 amplification, resulting from frequent head-to-head and tail-to-tail rearrangements. Generally, tail-to-tail rearrangements within or in close proximity upstream of FGFR1 were associated with FGFR1 dependency. Thus, the genomic events shaping the architecture of the 8p11-p12 amplicon provide a mechanistic explanation for the emergence of FGFR1-driven SQLC. Specifically, we believe that FGFR1 ectodomain-deficient and FGFR1-centered amplifications caused by tail-to-tail rearrangements are a novel somatic genomic event that might be predictive of therapeutically relevant FGFR1 dependency.

KEAP1/NFE2L2 Pathway Signature Outperforms KEAP1/NFE2L2 Mutation Status and Reveals Alternative Pathway-Activating Mutations in NSCLC.

INTRODUCTION: Activation of the antioxidant KEAP1/NFE2L2 (NRF2) pathway leads to increased glutamine dependence and an aggressive phenotype in NSCLC. Because this pathway has been explored as a clinical target, we developed a transcriptomic signature for identifying KEAP1/NFE2L2-activated tumors. METHODS: A total of 971 NSCLC samples were used to train an expression signature (K1N2-score) to predict KEAP1/NFE2L2 mutations. There were 348 in-house NSCLCs that were analyzed using a NanoString expression panel for validation. RESULTS: The 46-gene K1N2 score robustly predicted KEAP1/NFE2L2 mutations in the validation set irrespective of histology and mutation (area under the curve: 89.5, sensitivity: 90.2%), suggesting that approximately 90% of KEAP1/NFE2L2 mutations are pathway-activating. The K1N2-score outperformed KEAP1/NFE2L2 mutational status when predicting patient survival (score p = 0.047; mutation p = 0.215). In K1N2 score-positive but KEAP1/NFE2L2 wild-type samples, enrichment testing identified SMARCA4/BRG1 and CUL3 mutations as mimics of KEAP1/NFE2L2 mutations. CONCLUSIONS: The K1N2-score identified KEAP1/NFE2L2-activated NSCLC by robustly detecting KEAP1/NFE2L2mut cases and discovering alternative genomic activators. It is a potential means for selecting patients with a constitutively active KEAP1/NFE2L2 pathway.

Resistance to MET inhibition in MET-dependent NSCLC and therapeutic activity after switching from type I to type II MET inhibitors.

OBJECTIVES: 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 (METΔex14), MET amplification, and MET fusion and report treatment outcomes after switching therapy from type I to type II MET inhibitors. MATERIALS AND METHODS: Pre- 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. RESULTS: Of 26 patients with MET tyrosine kinase inhibitor treatment, eight had paired pre- and post-treatment biopsies (Three with MET amplification, three with METΔex14, two with MET fusions (KIF5B-MET and PRKAR2B-MET).) In six patients, mechanisms of resistance were detected, whereas in two cases, the cause of resistance remained unclear. We found off-target resistance mechanisms in four cases with KRAS mutations and HER2 amplifications appearing. Two patients exhibited second-site MET mutations (p.D1246N and p. Y1248H). Three patients received type I and type II MET tyrosine kinase inhibitors sequentially. In two cases, further progressive disease was seen hereafter. The patient with KIF5B-MET fusion received three different MET inhibitors and showed long-lasting stable disease and a repeated response after switching therapy, respectively. CONCLUSION: Resistance 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.

High-Throughput Profiling of Colorectal Cancer Liver Metastases Reveals Intra- and Inter-Patient Heterogeneity in the EGFR and WNT Pathways Associated with Clinical Outcome.

Seventy percent of patients with colorectal cancer develop liver metastases (CRLM), which are a decisive factor in cancer progression. Therapy outcome is largely influenced by tumor heterogeneity, but the intra- and inter-patient heterogeneity of CRLM has been poorly studied. In particular, the contribution of the WNT and EGFR pathways, which are both frequently deregulated in colorectal cancer, has not yet been addressed in this context. To this end, we comprehensively characterized normal liver tissue and eight CRLM from two patients by standardized histopathological, molecular, and proteomic subtyping. Suitable fresh-frozen tissue samples were profiled by transcriptome sequencing (RNA-Seq) and proteomic profiling with reverse phase protein arrays (RPPA) combined with bioinformatic analyses to assess tumor heterogeneity and identify WNT- and EGFR-related master regulators and metastatic effectors. A standardized data analysis pipeline for integrating RNA-Seq with clinical, proteomic, and genetic data was established. Dimensionality reduction of the transcriptome data revealed a distinct signature for CRLM differing from normal liver tissue and indicated a high degree of tumor heterogeneity. WNT and EGFR signaling were highly active in CRLM and the genes of both pathways were heterogeneously expressed between the two patients as well as between the synchronous metastases of a single patient. An analysis of the master regulators and metastatic effectors implicated in the regulation of these genes revealed a set of four genes (SFN, IGF2BP1, STAT1, PIK3CG) that were differentially expressed in CRLM and were associated with clinical outcome in a large cohort of colorectal cancer patients as well as CRLM samples. In conclusion, high-throughput profiling enabled us to define a CRLM-specific signature and revealed the genes of the WNT and EGFR pathways associated with inter- and intra-patient heterogeneity, which were validated as prognostic biomarkers in CRC primary tumors as well as liver metastases.

Rebiopsy in advanced non-small cell lung cancer, clinical relevance and prognostic implications.

INTRODUCTION: Rebiopsies of non-small cell lung cancers (NSCLC) are mainly performed to (i) cover the evolution of potentially amenable resistance mechanisms against a targeted therapy, and (ii) to identify new therapeutic targets which were not detected in the initial diagnostic biopsy. Comprehensive systematic analyses evaluating the value of rebiopsies are missing. METHODS: Clinical databases from two large comprehensive cancer center networks were queried following prespecified criteria to identify prospectively entered NSCLC cases with at least one rebiopsy at disease progression. Clinicopathological and biomarker findings including multigene sequencing were correlated with clinical outcomes. RESULTS: From a total of 17,477 stage IV NSCLC patients, a cohort of 403 evaluable patients undergoing at least one rebiopsy of a primary tumor or metastasis was retrieved. Changes in biomarker profiles as compared to baseline were observed in 48.9%. In 31.3% of cases, findings of potential therapeutic relevance were revealed, including 18 patients (4.4%) with a targetable marker only detected at rebiopsy. New findings were more frequent (greater than50%) in NSCLC with EGFR/ALK/ROS1 alterations, including mutations of the dominant oncogene, TP53 mutations, and MET or ERBB2 amplifications. Patients undergoing rebiopsy exhibited superior overall survival compared to a control group, irrespective of presence (HR 0.28) or absence (HR 0.20, both p < 0.001) of a therapeutically targetable aberration. CONCLUSIONS: Rebiopsies at progression of advanced NSCLC are strongly supported by a high rate of clinically relevant findings. Current clinical practice selects a patient population with exceptional outcomes, which merits further characterization.

Lymphangiosis carcinomatosa independently affects long-term survival of Non-Small Cell Lung Cancer patients.

OBJECTIVE: The significance of postoperatively diagnosed Lymphangiosis Carcinomatosa (L1) as an independent risk factor for long-term survival in Non-Small Cell Lung Cancer (NSCLC) remains controversial. We analyzed the effect of L1 on postoperative survival in stage I, II and III NSCLC-patients. METHODS: We investigated all consecutive patients with NSCLC between January 2012 and December 2019 who underwent an anatomical resection and radical lymphadenectomy at our institute. L1-were compared to L0-patients. All patients received adjuvant chemotherapy in accordance with European guidelines. 3- and 5- year survival rates and median-survival were assessed. To investigate whether L1 is an independent risk factor, we carried out a multivariate cox regression and a pair-match analysis looking at different properties such as TNM. RESULTS: A total of 641 patients (L0: 74%; L1: 26%) were analyzed. Baseline characteristics were comparable between groups. The mean age was 65.3 ± 10.2 years and 64.9 ± 9.4 years in the L0 and L1-groups respectively (p-value = 0.703). 58.5% of L0-patients were male (L1: 62.7%; p-value = 0.351). Overall survival in the L1-group was significantly shorter compared to the L0-group (L1: 42.3 ± 2.8; L0: 67.6 ± 2.1 months; p-value<0.0001). We confirmed this finding in a pair-matched analysis (L0: 73.9 ± 4.7 months; L1: 42.2 ± 4.2; p-value = 0.009). 3- and 5-year survival were significantly shorter for L1-patients (3-year: L0: 65.9%; L1: 35.9%; p-value<0.0001) (5-year: L0: 34.9%; L1: 7.5%; p-value<0.0001). CONCLUSION: L1 is an independent risk factor for long-term survival of patients with NSCLC. This cohort supports that the L0/L1 status should be included in pathological reports. We suggest to further include L0/L1-status in guideline recommendations for NSCLC patients.

Optimized PD-L1 scoring of gastric cancer.

BACKGROUND: PD-1/PD-L1-Immunotherapy has been approved for gastric carcinoma. PD-L1 assessment by immunohistochemistry is the principle biomarker. Are biopsies able to map the actual PD-L1 status of the entire tumor? METHODS: Whole tumor slides of 56 gastric carcinoma were analyzed to determine the distribution of PD-L1 positive cells in the entire tumor areas. Tissue micro arrays with four cores of the tumor surface, which represents the endoscopically accessible biopsy zone, were built from the same tumors. The PD-L1 CPS value was determined separately for each core. Preoperative diagnostic biopsies were available for 22 of the tumors. PD-L1 prevalence, sensitivity and specificity were analyzed using the whole tumor slides as reference scores. Molecular subtyping was performed and related to the PD-L1 status. RESULTS: 27.3% of cases were PD-L1 negative (CPS < 1), 43.6% showed low PD-L1 expression (CPS ≥ 1 to < 5), 12.7% moderate (CPS ≥ 5 to < 10) and 16.4% strong expression (CPS ≥ 10). The biopsies showed best test characteristics if four surface biopsies were analyzed combined, i.e., the CPS was calculated across all four biopsies. The prevalence showed a distribution similar to the resection specimens, sensitivity was 0.73 and specificity 1.0. Using fewer surface biopsies decreased sensitivity and specificity and caused false-negative classifications. Compared to the TMAs, the preoperative biopsies showed reduced sensitivity (0.412). CONCLUSIONS: This is the first comprehensive study to optimize PD-L1 assessment in gastric cancer using endoscopically available tissue. The obtained PD-L1 prevalence is consistent with data of current clinical studies. Calculation of the test characteristics shows that surface biopsies can be indicative of the true PD-L1 status based on the resection specimen. However, an adequate number of biopsies is required. In this study, n = 4 biopsies yielded best results.

"Interchangeability" of PD-L1 immunohistochemistry assays: a meta-analysis of diagnostic accuracy.

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.

Tumor Vessel Normalization, Immunostimulatory Reprogramming, and Improved Survival in Glioblastoma with Combined Inhibition of PD-1, Angiopoietin-2, and VEGF.

Glioblastoma (GBM) is a non-T-cell-inflamed cancer characterized by an immunosuppressive microenvironment that impedes dendritic cell maturation and T-cell cytotoxicity. Proangiogenic cytokines such as VEGF and angiopoietin-2 (Ang-2) have high expression in glioblastoma in a cell-specific manner and not only drive tumor angiogenesis and vascular permeability but also negatively regulate T-lymphocyte and innate immune cell responses. Consequently, the alleviation of immunosuppression might be a prerequisite for successful immune checkpoint therapy in GBM. We here combined antiangiogenic and immune checkpoint therapy and demonstrated improved therapeutic efficacy in syngeneic, orthotopic GBM models. We observed that blockade of VEGF, Ang-2, and programmed cell death protein-1 (PD-1) significantly extended survival compared with vascular targeting alone. In the GBM microenvironment, triple therapy increased the numbers of CTLs, which inversely correlated with myeloid-derived suppressor cells and regulatory T cells. Transcriptome analysis of GBM microvessels indicated a global vascular normalization that was highest after triple therapy. Our results propose a rationale to overcome tumor immunosuppression and the current limitations of VEGF monotherapy by integrating the synergistic effects of VEGF/Ang-2 and PD-1 blockade to reinforce antitumor immunity through a normalized vasculature.

Safety and Efficacy of Crizotinib in Patients With Advanced or Metastatic ROS1-Rearranged Lung Cancer (EUCROSS): A European Phase II Clinical Trial.

INTRODUCTION: ROS1 rearrangements are found in 1% of lung cancer patients. Therapeutic efficacy of crizotinib in this subset has been shown in early phase trials in the United States and East Asia. Here we present data on efficacy and safety of a prospective phase II trial evaluating crizotinib in European ROS1-positive patients (EUCROSS). PATIENTS AND METHODS: The trial was a multicenter, single-arm phase II trial (Clinicaltrial.gov identifier: NCT02183870). Key eligibility criteria included patients who were 18 years of age or older with advanced/metastatic lung cancer and centrally confirmed ROS1-rearranged lung cancer (fluorescence-in situ hybridization). Treatment included 250 mg crizotinib twice daily. The primary endpoint was investigator-assessed objective response rate (ORR) (Response Evaluation Criteria in Solid Tumors, version 1.1). Key secondary endpoints were progression-free survival (PFS), overall survival, efficacy by independent radiologic review, safety, health-related quality of life, and molecular characterization of tumor tissue. RESULTS: Thirty-four patients received treatment. Four patients were excluded from efficacy analysis. Investigator ORR was 70% (95% confidence interval [CI]: 51-85; 21 of 30 patients) and median PFS was 20.0 months (95% CI: 10.1-not reached). Two patients with ROS1 wild-type sequences assessed by DNA sequencing had progression as best response. CD74-ROS1-positive patients had a trend towards a higher ORR and longer median PFS. TP53-co-mutant patients had a significantly shorter median PFS than wild-type patients (7.0 months, 95% CI: 1.7-20.0 versus 24.1 months, 95% CI: 10.1-not reached; p = 0.022). Treatment-related adverse events were documented in 33 of 34 patients (97%). CONCLUSIONS: Crizotinib is highly effective and safe in patients with ROS1-rearranged lung cancer. ROS1-/TP53-co-aberrant patients had a significantly worse outcome compared to TP53 wild-type patients.

Clinical impact of PD-L1 and PD-1 expression in squamous cell cancer of the vulva.

PURPOSE: Squamous cell carcinoma of the vulva (SQCV) is the fifth most common cancer in women and accounts for about 5% of all genital cancers in women. The PD-L1 signaling pathway is activated in many malignant neoplasms and its blockade enhances anti-cancer immunity. The aim of our study was to examine the protein expression of PD-L1 and PD-1 in squamous cell cancer of the vulva, its correlations with clinicopathologic features and prognostic value. METHODS: Patients with SQCV treated in one institution were used for the analyses. PD-L1 immunohistochemistry was performed on 4 µm-thick section of the respective FFPE tissue blocks using the 28-8 antibody. PD-L1 scoring was performed separately for tumour cells (TC) and tumour associated immune cells. DNA was extracted to determine HPV status. Kaplan-Meier estimates for disease-free-survival and overall-survival were calculated and compared by log-rank test. RESULTS: PD-L1 expression in tumour cells could be observed in 32.9% of the patients. The expression of PD-L1 in peritumoural immune cells was confirmed in 91.4% of the patients. A significant correlation between PD-L1 expression in tumour cells and tumour stage was detected (p = 0.007). PD-L1 expression was independent from HPV status. Using the log-rank test we could not prove any significant differences in disease-free survival (p = 0.434) and overall survival (p = 0.858). Regression analysis showed that nodal status is a predictive factor of survival (p < 0.001). CONCLUSION: The present study showed that a relevant amount of patients with squamous cell cancer of the vulva express PD-L1 in both, tumour cells and tumour-associated immune cells. Furthermore, the significant correlation of PD-L1 expression in TCs with tumour stage indicated the clinical impact of PD-L1 expression during tumour development. These data indicate that SQCV might be amenable to immune checkpoint-inhibition and constitute a rational for the future clinical trials.

K-ras Mutation Subtypes in NSCLC and Associated Co-occuring Mutations in Other Oncogenic Pathways.

INTRODUCTION: Although KRAS mutations in NSCLC have been considered mutually exclusive driver mutations for a long time, there is now growing evidence that KRAS-mutated NSCLC represents a genetically heterogeneous subgroup. We sought to determine genetic heterogeneity with respect to cancer-related co-mutations and their correlation with different KRAS mutation subtypes. METHODS: Diagnostic samples from 4507 patients with NSCLC were analyzed by next-generation sequencing by using a panel of 14 genes and, in a subset of patients, fluorescence in situ hybridization. Next-generation sequencing with an extended panel of 14 additional genes was performed in 101 patients. Molecular data were correlated with clinical data. Whole-exome sequencing was performed in two patients. RESULTS: We identified 1078 patients with KRAS mutations, of whom 53.5% had at least one additional mutation. Different KRAS mutation subtypes showed different patterns of co-occurring mutations. Besides mutations in tumor protein p53 gene (TP53) (39.4%), serine/threonine kinase 11 gene (STK11) (19.8%), kelch like ECH associated protein 1 gene (KEAP1) (12.9%), and ATM serine/threonine kinase gene (ATM) (11.9%), as well as MNNG HOS Transforming gene (MET) amplifications (15.4%) and erb-b2 receptor tyrosine kinase 2 gene (ERBB2) amplifications (13.8%, exclusively in G12C), we found rare co-occurrence of targetable mutations in EGFR (1.2%) and BRAF (1.2%). Whole-exome sequencing of two patients with co-occurring phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA) mutation revealed clonality of mutated KRAS in one patient and subclonality in the second, suggesting different evolutionary backgrounds. CONCLUSION: KRAS-mutated NSCLC represents a genetically heterogeneous subgroup with a high frequency of co-occurring mutations in cancer-associated pathways, partly associated with distinct KRAS mutation subtypes. This diversity might have implications for understanding the variability of treatment outcome in KRAS-mutated NSCLC and for future trial design.

Genomic Profiling Identifies Outcome-Relevant Mechanisms of Innate and Acquired Resistance to Third-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Therapy in Lung Cancer.

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.

Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors.

The emergence of acquired resistance against targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. In a subgroup of these patients we identified an association between selection of EGFRT790M-negative but EGFRG724S-positive subclones and osimertinib resistance. We demonstrate that EGFRG724S limits the activity of third-generation EGFR inhibitors both in vitro and in vivo. Structural analyses and computational modeling indicate that EGFRG724S mutations may induce a conformation of the glycine-rich loop, which is incompatible with the binding of third-generation TKIs. Systematic inhibitor screening and in-depth kinetic profiling validate these findings and show that second-generation EGFR inhibitors retain kinase affinity and overcome EGFRG724S-mediated resistance. In the case of afatinib this profile translates into a robust reduction of colony formation and tumor growth of EGFRG724S-driven cells. Our data provide a mechanistic basis for the osimertinib-induced selection of EGFRG724S-mutant clones and a rationale to treat these patients with clinically approved second-generation EGFR inhibitors.

Genetic instability and recurrent MYC amplification in ALK-translocated NSCLC: a central role of TP53 mutations.

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.

Physical basis of the 'magnification rule' for standardized Immunohistochemical scoring of HER2 in breast and gastric cancer.

BACKGROUND: Detection of HER2/neu receptor overexpression and/or amplification is a prerequisite for efficient anti-HER2 treatment of breast and gastric carcinomas. Immunohistochemistry (IHC) of the HER2 protein is the most common screening test, thus precise and reproducible IHC-scoring is of utmost importance. Interobserver variance still is a problem; in particular in gastric carcinomas the reliable differentiation of IHC scores 2+ and 1+ is challenging. Herein we describe the physical basis of what we called the 'magnification rule': Different microscope objectives are employed to reproducibly subdivide the continuous spectrum of IHC staining intensities into distinct categories (1+, 2+, 3+). METHODS: HER2-IHC was performed on 120 breast cancer biopsy specimens (n = 40 per category). Width and color-intensity of membranous DAB chromogen precipitates were measured by whole-slide scanning and digital morphometry. Image-analysis data were related to semi-quantitative manual scoring according to the magnification rule and to the optical properties of the employed microscope objectives. RESULTS: The semi-quantitative manual HER2-IHC scores are correlated to color-intensity measured by image-analysis and to the width of DAB-precipitates. The mean widths ±standard deviations of precipitates were: IHC-score 1+, 0.64 ± 0.1 µm; score 2+, 1.0 ± 0.23 µm; score 3+, 2.14 ± 0.4 µm. The width of precipitates per category matched the optical resolution of the employed microscope objective lenses: Approximately 0.4 µm (40×), 1.0 µm (10×) and 2.0 µm (5×). CONCLUSIONS: Perceived intensity, width of the DAB chromogen precipitate, and absolute color-intensity determined by image-analysis are linked. These interrelations form the physical basis of the 'magnification rule': 2+ precipitates are too narrow to be observed with 5× microscope objectives, 1+ precipitates are too narrow for 10× objectives. Thus, the rule uses the optical resolution windows of standard diagnostic microscope objectives to derive the width of the DAB-precipitates. The width is in turn correlated with color-intensity. Hereby, the more or less subjective estimation of IHC scores based only on the staining-intensity is replaced by a quasi-morphometric measurement. The principle seems universally applicable to immunohistochemical stainings of membrane-bound biomarkers that require an intensity-dependent scoring.