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.

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.

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.

B-cell-specific conditional expression of Myd88p.L252P leads to the development of diffuse large B-cell lymphoma in mice.

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

Interlaboratory concordance of PD-L1 immunohistochemistry for non-small-cell lung cancer.

AIMS: Programmed death ligand 1 (PD-L1) immunohistochemistry has become a mandatory diagnostic test in the treatment of lung cancer. Several research initiatives have started to harmonise the five PD-L1 immunohistochemistry assays that have been used in clinical trials. Here, we report data on interlaboratory and interassay concordance for commercial assays ('assays') and laboratory-developed tests (LDTs) at 10 German testing sites. METHODS AND RESULTS: To assess interlaboratory concordance, a tissue microarray containing 21 pulmonary carcinoma specimens was centrally prepared. Pre-cut sections were stained at 10 sites by the use of assays 28-8, 22C3, SP263, and SP142, as well as 11 LDTs. Assay performance was evaluated with a second tissue microarray containing 11 cell lines with defined PD-L1 expression. Quality control was centrally performed by manual and digital analyses. The assays yielded reproducible IHC staining patterns at all sites. In agreement with previous studies, 22C3, 28-8 and SP263 showed similar staining patterns, whereas SP142 was distinct. Among the LDTs, six of 11 protocols showed staining patterns similar to those of assays 22C3 and 28-8. Interlaboratory concordance of tumour cell scoring by use of a six-step system was moderate (Light's κ = 0.43-0.69), whereas the clinically approved cut-offs of ≥1% and ≥50% showed substantial concordance (κ = 0.73-0.89). Immune cell scoring by the use of SP142 yielded moderate concordance (κ = 0.42). CONCLUSIONS: The data confirm the previously described staining patterns of the assays, and show that they can be reproducibly employed at different sites. LDTs with staining results similar to those of the assays are implementable, but have to be carefully validated.

Optimized expression-based microdissection of formalin-fixed lung cancer tissue.

Analysis of specific DNA alterations in precision medicine of tumors is crucially important for molecular targeted treatments. Lung cancer is a prototypic example and one of the leading causes of cancer-related deaths worldwide. One major technical problem of detecting DNA alterations in tissue samples is cellular heterogeneity, that is, mixture of tumor and normal cells. Microdissection is an important tool to enrich tumor cells from heterogeneous tissue samples. However, conventional laser capture microdissection has several disadvantages like user-dependent selection of regions of interest (ROI), high costs for dissection systems and long processing times. ROI selection in expression-based microdissection (xMD) directly relies on cancer cell-specific immunostaining. Whole-slide irradiation leads to localized energy absorption at the sites of most intensive staining and melting of a membrane covering the slide, so that tumor cells can be isolated by removing the complete membrane. In this study, we optimized xMD of lung cancer tissue by enhancing staining intensity of tumor cell-specific immunostaining and processing of the stained samples. This optimized procedure did not alter DNA quality and resulted in enrichment of mutated EGFR DNA from lung adenocarcinoma specimens after xMD. We here also introduce a quality control protocol based on digital whole-slide scanning and image analysis before and after xMD to quantify selectivity and efficiency of the procedure. In summary, this study provides a workflow for xMD, adapted and tested for lung cancer tissue that can be used for lung tumor cell dissection before diagnostic or investigatory analyses.

Harmonized PD-L1 immunohistochemistry for pulmonary squamous-cell and adenocarcinomas.

Immunohistochemistry of the PD-L1 protein may be predictive for anti-PD-1 and anti-PD-L1 immunotherapy in pulmonary adenocarcinoma and in clinically unselected cohorts of so-called non-small-cell lung cancer. Several PD-L1 immunohistochemistry assays with custom reagents and scoring-criteria are developed in parallel. Biomarker testing and clinical decision making would profit from harmonized PD-L1 diagnostics. To assess interobserver concordance and PD-L1 immunohistochemistry staining patterns, 15 pulmonary carcinoma resection specimens (adenocarcinoma: n=11, squamous-cell carcinoma: n=4) were centrally stained with the assays 28-8, 22C3, SP142, and SP263 according to clinical trial protocols. The slides were evaluated independently by nine pathologists. Proportions of PD-L1-positive carcinoma cells and immune cells were scored according to a 6-step system that integrates the criteria employed by the four PD-L1 immunohistochemistry assays. Proportion scoring of PD-L1-positive carcinoma cells showed moderate interobserver concordance coefficients for the 6-step scoring system (Light's kappa=0.47-0.50). The integrated dichotomous proportion cut-offs (≥1, ≥5, ≥10, ≥50%) showed good concordance coefficients (κ=0.6-0.8). Proportion scoring of PD-L1-positive immune cells yielded low interobserver concordance coefficients both for the 6-step-score (κ<0.2) and the dichotomous cut-offs (κ=0.12-0.25). The assays 28-8 and 22C3 stained similar proportions of carcinoma cells in 12 of 15 cases. SP142 stained fewer carcinoma cells compared to 28-8, 22C3, and SP263 in four cases, whereas SP263 stained more carcinoma cells in nine cases. SP142 and SP263 stained immune cells more intensely. The data indicate that carcinoma cells can be reproducibly scored in PD-L1 immunohistochemistry for pulmonary adenocarcinoma and squamous-cell carcinoma. No differences in interobserver concordance were noticed among the tested assays. The scoring of immune cells yielded low concordance rates and might require specific standardization. The four tested PD-L1 assays did not show comparable staining patterns in all cases. Thus, studies that correlate staining patterns and response to immunotherapy are required to test the significance of the observed differences.

Impact of epidermal growth factor receptor (EGFR) cell surface expression levels on effector mechanisms of EGFR antibodies.

The epidermal growth factor receptor (EGFR) is a widely expressed Ag that is successfully targeted in tumor patients by mAbs or tyrosine kinase inhibitors. A clinical study in non-small cell lung cancer patients demonstrated a positive correlation between EGFR expression levels and the therapeutic efficacy of the EGFR mAb cetuximab. However, the impact of EGFR expression on the different mechanisms of action (MoAs) triggered by the EGFR mAb has not been defined. In this study, BHK-21 cells were stably transfected to express different EGFR levels, which were quantified by immunofluorescence and immunohistochemistry and compared with EGFR levels of clinical non-small cell lung cancer samples. These cells were used to systematically investigate the impact of target Ag expression levels on Fab- or Fc-mediated MoAs of EGFR mAb. A negative correlation between EGFR levels and potency of Fab-mediated MoA was observed. Interestingly, Ab-dependent cell-mediated cytotoxicity (ADCC) by NK cells, monocytes, or polymorphonuclear cells as well as complement-dependent cytotoxicity positively correlated with the number of EGFR molecules. In comparison with ADCC by mononuclear cells, polymorphonuclear cell-mediated ADCC and complement-dependent cytotoxicity required higher EGFR expression levels and higher mAb concentrations to trigger significant tumor cell killing. This correlation between EGFR expression levels and Fc-mediated MoA was confirmed in an independent panel of human tumor cell lines carrying diverse genetic alterations. Furthermore, RNA interference-induced knockdown experiments reinforced the impact of EGFR expression on tumor cell killing by EGFR mAb. In conclusion, these results suggest that EGFR expression levels may determine distinct patterns of MoAs that contribute to the therapeutic efficacy of EGFR mAb.

Challenging dedifferentiated liposarcoma identified by MDM2-amplification, a report of two cases.

BACKGROUND: Liposarcoma is the most frequent soft tissue sarcoma. Well differentiated liposarcoma may progress into dedifferentiated liposarcoma with pleomorphic histology. A minority additionally features myogenic, osteo- or chondrosarcomatous heterologous differentiation. Genomic amplification of the Mouse double minute 2 homolog (MDM2) locus is characteristic for well differentiated and dedifferentiated liposarcomas. Detection of MDM2 amplification may supplement histopathology and aid to distinguish liposarcoma from other soft tissue neoplasia. CASE PRESENTATION: Here we present two cases of dedifferentiated liposarcoma with challenging presentation. Case 1 features a myogenic component. As the tumour infiltrated the abdominal muscles and showed immunohistochemical expression of myogenic proteins, rhabdomyosarcoma had to be ruled out. Case 2 has an osteosarcomatous component resembling extraosseous osteosarcoma. The MDM2 status was determined in both cases and helped making the correct diagnosis. Overexpression of MDM2 and co-overexpression of Cyclin-dependent kinase 4 is demonstrated by immunohistochemistry. The underlying MDM2 amplification is shown by fluorescence in situ hybridisation. Since low grade osteosarcoma may also harbour MDM2 amplification it is emphasised that the amplification has to be present in the lipomatous parts of the tumour to distinguish liposarcoma from extraosseous osteosarcoma. CONCLUSIONS: The two cases exemplify challenges in the diagnoses of dedifferentiated liposarcoma. Liposarcoma often has pleomorphic histology and additionally may feature heterologous components that mimic other soft tissue neoplasms. Amplification of MDM2 is characteristic for well differentiated and dedifferentiated liposarcomas. Determination of the MDM2 status by in situ hybridisation may assist histopathology and help to rule out differential diagnoses.

Upregulation and epigenetic modification of the creatine transporter SLC6A8 in non-small cell lung cancer.

INTRODUCTION: Lung cancer is a major cause of cancer-related death worldwide and effective therapies, besides surgery, are available only for a small proportion of patients. Since cellular respiration is known to be broadly altered in malignant tumors, the cellular processes of respiration can be a potential therapeutic target. One important element of cellular respiration is creatine and its transport by the creatine transporter SLC6A8. Here we describe the expression of SLC6A8 at the RNA and protein level, epigenetic modifications as well as survival analysis in NSCLC tissues and matched controls. MATERIALS AND METHODS: We analyzed epigenetic modifications of the SLC68A gene in 32 patients, of which 18 were additionally analyzed by transcriptome analysis. The expression of SLC6A8 at the protein level was assessed by immunohistochemistry using an independent cohort and correlated with clinicopathological data including survival. Kaplan-Meier analysis was performed to analyze the possible effects of the transcriptional levels of SLC6A8 in another separate cohort (n=1925). RESULTS: SLC6A8 loci are epigenetically modified in NSCLC compared with tumor-free controls. SLC6A8 is upregulated in NSCLC at the RNA and protein level. High mRNA expression of SLC6A8 was associated with an overall poor prognosis in lung adenocarcinoma patients and displayed the strongest adverse prognostic effect in male smokers with adenocarcinomas. Results of transcriptome analysis were partially confirmed at the protein level. CONCLUSIONS: Our results suggest an important role of creatine and its transport via SLC6A8 in NSCLC.

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.

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.

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.

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.

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.

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.

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.