Deciphering the immunosuppressive tumor microenvironment in ALK- and EGFR-positive lung adenocarcinoma.

INTRODUCTION: The advent of immune checkpoint blockade (ICB) has led to significantly improved disease outcome in lung adenocarcinoma (ADC), but response of ALK/EGFR-positive tumors to immune therapy is limited. The underlying immune biology is incompletely understood. METHODS: We performed comparative mRNA expression profiling of 31 ALK-positive, 40 EGFR-positive and 43 ALK/EGFR-negative lung ADC focused on immune gene expression. The presence and levels of tumor infiltration lymphocytes (TILs) as well as fourteen specific immune cell populations were estimated from the gene expression profiles. RESULTS: While total TILs were not lower in ALK-positive and EGFR-positive tumors compared to ALK/EGFR-negative tumors, specific immunosuppressive characteristics were detected in both subgroups: In ALK-positive tumors, regulatory T cells were significantly higher compared to EGFR-positive (fold change: FC = 1.9, p = 0.0013) and ALK/EGFR-negative tumors (FC = 2.1, p = 0.00047). In EGFR-positive tumors, cytotoxic cells were significantly lower compared to ALK-positive (FC = - 1.7, p = 0.016) and to ALK/EGFR-negative tumors (FC = - 2.1, p = 2.0E-05). A total number of 289 genes, 40 part of cytokine-cytokine receptor signaling, were differentially expressed between the three subgroups. Among the latter, five genes were differently expressed in both ALK-positive and EGFR-positive tumors, while twelve genes showed differential expression solely in ALK-positive tumors and eleven genes solely in EGFR-positive tumors. CONCLUSION: Targeted gene expression profiling is a promising tool to read out tumor microenvironment characteristics from routine diagnostic lung cancer biopsies. Significant immune reactivity including specific immunosuppressive characteristics in ALK- and EGFR-positive lung ADC, but not a total absence of immune infiltration supports further clinical evaluation of immune-modulators as partners of ICB in such tumors.

NTRK testing: First results of the QuiP-EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA-based NGS assays.

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA-/DNA-based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH-based and 18 in the NGS-based round robin test, the latter additionally subdivided into low-input and high-input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin-fixed and paraffin-embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS-based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH- and tNGS-based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA-based tNGS panels.

Testing NTRK testing: Wet-lab and in silico comparison of RNA-based targeted sequencing assays.

NTRK fusions involving three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, and NTRK3 and a variety of fusion partners were identified as oncogenic drivers across many cancer types. Drugs that target the chimeric protein product require the identification of the underlying gene fusion. This advocates the diagnostic use of molecular assays ranging from fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR)/Sanger approaches to targeted next-generation sequencing (NGS). Immunohistochemistry may be used as a screening tool and adjunct diagnostic assay in this context. Although FISH and RT-PCR/Sanger approaches are widely adopted in routine diagnostics, current experience with targeted RNA-based NGS is limited. Here, we report on the analysis of major assays (TruSight TST170 and TruSight RNA Fusion [Illumina]; Archer FusionPlex Solid Tumor, Archer FusionPlex Lung, and Archer FusionPlex Oncology [Archer]; Oncomine Comprehensive Assay v3 RNA and Oncomine Focus RNA [Thermo Fisher Scientific]) that are commercially available. The data set includes performance results of a multicentric comparative wet-lab study as well as an in silico analysis on the ability to detect the broad range of NTRK fusions reported until now. A test algorithm that reflects assay methodology is provided. This data will support implementation of targeted RNA sequencing in routine diagnostics and inform screening and testing strategies that have been brought forward.

Current Management and Future Perspective in Pleural Mesothelioma.

Pleural mesothelioma is an aggressive malignancy arising from pleural mesothelial cell lining, predominantly associated with prior exposure to asbestos. The ban on asbestos use has led to its lower incidence in many countries, but globally the disease burden is expected to rise. Therefore, well-planned research is needed to develop more effective, tolerable and affordable drugs. The development of novel treatment has been too slow, with only two regimens of systemic therapy with robust phase 3 data approved formally to date. The treatment scenario for resectable disease remains controversial. However, recent developments in the understanding of disease and clinical trials have been encouraging, and may add better treatment options in the coming years. In this review, we discuss the current treatment options for pleural mesothelioma and shed light on some recent studies and ongoing trials.

A gene expression signature associated with B cells predicts benefit from immune checkpoint blockade in lung adenocarcinoma.

Immune checkpoint blockade (ICB) expands the therapeutic options for metastatic lung cancer nowadays representing a standard frontline strategy as monotherapy or combination therapy, as well as an option in oncogene-addicted NSCLC after exhaustion of targeted therapies. Predictive markers are urgently needed, since only a minority of patients benefits from ICB, while serious adverse effects of immunotoxicity may occur. The study cohort included 43 ICB-treated metastatic lung adenocarcinoma showing long-term response (n = 16), rapid progression (n = 21) or intermediate patterns of response (n = 6). Lung biopsies acquired before initiation of ICB were analyzed by targeted mRNA expression profiling of 770 genes. Level and proportions of 14 immune cell types were estimated using characteristic gene expression signatures. Abundance of B cells (HR = 0.66, p = .00074), CD45+ cells (HR = 0.61, p = .01) and total TILs (HR = 0.62, p = .025) was associated with prolonged progression-free survival after ICB treatment. In a ROC analysis, B cells (AUC = 0.77, p = .0055) and CD45+ cells (AUC = 0.73, p = .019) predicted benefit of ICB, which was not the case for PD-L1 mRNA (AUC = 0.54, p = .72) and PD-L1 protein expression (AUC = 0.68, p = .082). Clustering of 79 candidate predictive markers identified among 770 investigated genes revealed two distinct predictive clusters which included cytotoxic cell or macrophage markers, respectively. In summary, targeted gene expression profiling was feasible using routine diagnostics biopsies. This study proposes B cells and total TILs as complementary predictors of ICB benefit in NSCLC. While further preferably prospective validation is required, gene expression profiling could be integrated in the routine diagnostic work-up complementing existing NGS protocols.

Targeting rare and non-canonical driver variants in NSCLC - An uncharted clinical field.

OBJECTIVES: Implementation of tyrosine kinase inhibitors (TKI) and other targeted therapies was a main advance in thoracic oncology with survival gains ranging from several months to years for non-small-cell lung cancer (NSCLC) patients. High-throughput comprehensive molecular profiling is of key importance to identify patients that can potentially benefit from these novel treatments. MATERIAL AND METHODS: Next-generation sequencing (NGS) was performed on 4500 consecutive formalin-fixed, paraffin-embedded specimens of advanced NSCLC (n = 4172 patients) after automated extraction of DNA and RNA for parallel detection of mutations and gene fusions, respectively. RESULTS AND CONCLUSION: Besides the 24.9 % (n = 1040) of cases eligible for approved targeted therapies based on the presence of canonical alterations in EGFR exons 18-21, BRAF, ROS1, ALK, NTRK, and RET, an additional n = 1260 patients (30.2 %) displayed rare or non-canonical mutations in EGFR (n = 748), BRAF (n = 135), ERBB2 (n = 30), KIT (n = 32), PIK3CA (n = 221), and CTNNB1 (n = 94), for which targeted therapies could also be potentially effective. A systematic literature search in conjunction with in silico evaluation identified n = 232 (5.5 %) patients, for which a trial of targeted treatment would be warranted according to available evidence (NCT level 1, i.e. published data showing efficacy in the same tumor entity). In conclusion, a sizeable fraction of NSCLC patients harbors rare or non-canonical alterations that may be associated with clinical benefit from currently available targeted drugs. Systematic identification and individualized management of these cases can expand applicability of precision oncology in NSCLC and extend clinical gain from established molecular targets. These results can also inform clinical trials.