Single-cell spatial landscapes of the lung tumour immune microenvironment.

Single-cell technologies have revealed the complexity of the tumour immune microenvironment with unparalleled resolution1-9. Most clinical strategies rely on histopathological stratification of tumour subtypes, yet the spatial context of single-cell phenotypes within these stratified subgroups is poorly understood. Here we apply imaging mass cytometry to characterize the tumour and immunological landscape of samples from 416 patients with lung adenocarcinoma across five histological patterns. We resolve more than 1.6 million cells, enabling spatial analysis of immune lineages and activation states with distinct clinical correlates, including survival. Using deep learning, we can predict with high accuracy those patients who will progress after surgery using a single 1-mm2 tumour core, which could be informative for clinical management following surgical resection. Our dataset represents a valuable resource for the non-small cell lung cancer research community and exemplifies the utility of spatial resolution within single-cell analyses. This study also highlights how artificial intelligence can improve our understanding of microenvironmental features that underlie cancer progression and may influence future clinical practice.

Single-cell and single-nucleus RNA-sequencing from paired normal-adenocarcinoma lung samples provide both common and discordant biological insights.

Whether single-cell RNA-sequencing (scRNA-seq) captures the same biological information as single-nucleus RNA-sequencing (snRNA-seq) remains uncertain and likely to be context-dependent. Herein, a head-to-head comparison was performed in matched normal-adenocarcinoma human lung samples to assess biological insights derived from scRNA-seq versus snRNA-seq and better understand the cellular transition that occurs from normal to tumoral tissue. Here, the transcriptome of 160,621 cells/nuclei was obtained. In non-tumor lung, cell type proportions varied widely between scRNA-seq and snRNA-seq with a predominance of immune cells in the former (81.5%) and epithelial cells (69.9%) in the later. Similar results were observed in adenocarcinomas, in addition to an overall increase in cell type heterogeneity and a greater prevalence of copy number variants in cells of epithelial origin, which suggests malignant assignment. The cell type transition that occurs from normal lung tissue to adenocarcinoma was not always concordant whether cells or nuclei were examined. As expected, large differential expression of the whole-cell and nuclear transcriptome was observed, but cell-type specific changes of paired normal and tumor lung samples revealed a set of common genes in the cells and nuclei involved in cancer-related pathways. In addition, we showed that the ligand-receptor interactome landscape of lung adenocarcinoma was largely different whether cells or nuclei were evaluated. Immune cell depletion in fresh specimens partly mitigated the difference in cell type composition observed between cells and nuclei. However, the extra manipulations affected cell viability and amplified the transcriptional signatures associated with stress responses. In conclusion, research applications focussing on mapping the immune landscape of lung adenocarcinoma benefit from scRNA-seq in fresh samples, whereas snRNA-seq of frozen samples provide a low-cost alternative to profile more epithelial and cancer cells, and yield cell type proportions that more closely match tissue content.

Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

Transcriptomic data helps refining classification of pulmonary carcinoid tumors with increased mitotic counts.

Pulmonary neuroendocrine neoplasms are classified by WHO as either typical or atypical carcinoids, large cell (LCNEC) or small cell (SCLC) neuroendocrine carcinoma based on mitotic count, morphology, and necrosis assessment. LCNEC with low mitotic count and sharing morphologic features with carcinoids are in a gray zone for classification and their rare prevalence and the paucity of studies precludes proper validation of the current grading system. In this study, we aim to investigate their clinicopathological and transcriptomic profiles. Lung resection specimens obtained from 18 patients diagnosed with carcinoids or LCNEC were selected. Four of them were characterized as borderline tumors based on a mitotic rate ranging between 10 and 30 mitoses per 2 mm2. Comprehensive morphological and immunohistochemical (IHC) evaluation was performed and tumor-based transcriptomic profiles were analyzed through unsupervised clustering. Clustering analysis revealed two distinct molecular groups characterized by low (C1) and high (C2) proliferation. C1 was comprised of seven carcinoids and three borderline tumors, while C2 was comprised of seven LCNEC and one borderline tumor. Furthermore, patients in cluster C1 had a better recurrence-free survival compared with patients in cluster C2 (20% vs 75%). Histological features, IHC profile, and molecular analysis showed that three out of four borderline tumors showed features consistent with carcinoids. Therefore, our findings convey that the current diagnostic guidelines are suboptimal for classification of pulmonary neuroendocrine tumors with increased proliferative index and carcinoid-like morphology. These results support the emerging concept that neuroendocrine tumors with carcinoid-like features and mitotic count of <20 mitoses per 2 mm2 should be regarded as pulmonary carcinoids instead of LCNEC.

V-domain Ig-containing suppressor of T-cell activation (VISTA), a potentially targetable immune checkpoint molecule, is highly expressed in epithelioid malignant pleural mesothelioma.

V-domain Ig-containing suppressor of T-cell activation (VISTA) is an immune checkpoint gene that inhibits anti-tumor immune responses. Since most malignant pleural mesotheliomas do not respond to anti-programmed cell death(-ligand)1 (PD-(L)1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA4) therapy and given the recent finding of The Cancer Genome Atlas Study that pleural mesothelioma displays the highest expression of VISTA among all cancers studied, we examined VISTA expression in a large pleural mesothelioma cohort. VISTA and PD-L1 immunohistochemistry were performed on tissue microarray of immunotherapy-naive pleural mesotheliomas (254 epithelioid, 24 biphasic and 41 sarcomatoid) and ten whole-tissue sections of benign pleura (VISTA only). Percentages of tumor and inflammatory cells with positive staining were assessed. Optimal prognostic cutoff percentages were determined using maximally selected rank statistics. Overall survival was evaluated using Kaplan-Meier methods and Cox proportional hazard analysis. All benign mesothelium expressed VISTA. Eighty-five percent of 319 and 38% of 304 mesotheliomas expressed VISTA and PD-L1 (88% and 33% of epithelioid, 90% and 43% of biphasic, and 42% and 75% of sarcomatoid), respectively. Median VISTA score was significantly higher in epithelioid (50%) (vs. biphasic [20%] and sarcomatoid [0]) (p < 0.001), while median PD-L1 score was significantly higher in sarcomatoid tumors (20%) (vs. biphasic and epithelioid [both 0%]) (p < 0.001). VISTA and PD-L1 were expressed in inflammatory cells in 94% (n = 317) and 24% (n = 303) of mesothelioma, respectively. Optimal prognostic cutoffs for VISTA and PD-L1 were 40% and 30%, respectively. On multivariable analysis, VISTA and PD-L1 expression in mesothelioma were associated with better and worse overall survival (p = 0.001 and p = 0.002), respectively, independent of histology. In a large cohort of mesothelioma, we report frequent expression of VISTA and infrequent expression of PD-L1 with favorable and unfavorable survival correlations, respectively. These findings may explain poor responses to anti-PD-(L)1 immunotherapy and suggest VISTA as a potential novel target in pleural mesothelioma.

Stage IV lung carcinoids: spectrum and evolution of proliferation rate, focusing on variants with elevated proliferation indices.

The spectrum and evolution of proliferation rates in stage IV lung carcinoids is poorly defined. In particular, there are limited data on the prevalence and characteristics of tumors exceeding the standard upper proliferative criteria-as defined largely based on early-stage carcinoids-in metastatic setting. Sixty-six patients with stage IV lung carcinoids were identified, and all evaluable samples (n = 132; mean 2 samples per patient) were analyzed for mitotic counts and Ki-67 rate. Clinicopathologic and genomic features associated with elevated proliferation rates (>10 mitoses per 2 mm2 and/or >20% hot-spot Ki-67), and evolution of proliferation rates in serial specimens were analyzed. We found that mitoses and/or Ki-67 exceeded the standard criteria in 35 of 132 (27%) samples, primarily (31/35 cases) at  metastatic sites. Although neuroendocrine neoplasms with >10 mitoses per 2 mm2 are currently regarded as de facto neuroendocrine carcinomas, the notion that these cases are part of the spectrum of carcinoids was supported by (1) well-differentiated morphology, (2) conventional proliferation rates in other samples from same patient, (3) genetic characteristics, including the lack of RB1/TP53 alterations in all tested samples (n = 19), and (4) median overall survival of 2.7 years, compared to <1 year survival of stage IV neuroendocrine carcinomas in the historic cohorts. In patients with matched primary/metastatic specimens (48 pairs), escalation of mitoses or Ki-67 by ≥10 points was observed in 35% of metastatic samples; clonal relationship in one pair with marked proliferative progression was confirmed by next-generation sequencing. Notably, escalation of proliferation rate was documented in a subset of metastases arising from resected typical carcinoids, emphasizing that the diagnosis of typical carcinoid in primary tumor does not assure low proliferation rate at metastatic sites. In conclusion, stage IV lung carcinoids frequently exceed the standard proliferative criteria established for primary tumors, and commonly exhibit proliferative escalation at metastatic sites. Despite the overlap of proliferation rates, these tumors show fundamental morphologic, genomic and clinical differences from neuroendocrine carcinomas, and should be classified separately from those tumors. Awareness of the increased proliferative spectrum in metastatic carcinoids is critical for their accurate diagnosis. Further studies are warranted to explore the impact of proliferation indices on prognosis and therapeutic responses of patients with metastatic carcinoids.

Therapeutic Potential of Afatinib for Cancers with ERBB2 (HER2) Transmembrane Domain Mutations G660D and V659E.

We previously reported on a family with hereditary lung cancer, in which a germline mutation in the transmembrane domain (G660D) of avian erythroblastic leukemia viral oncogene homolog 2 (erb-b2 receptor tyrosine kinase 2) (ERBB2; human epidermal growth factor receptor 2 [HER2]) seemed to be responsible for the cancer predisposition. Although few data are available on treatment, anti-ERBB2 therapeutic agents may be effective for ERBB2-mutant cancers. The familial lung cancer patient in one of the authors' institutes developed bone metastasis with enlarging lung tumors and was treated with the ERBB2 inhibitor afatinib. We also encountered a patient with ampullary adenocarcinoma with ERBB2 G660D and S310F comutations in another institute of the authors', revealed by comprehensive genomic profiling. This patient was then treated with afatinib and also achieved transitory response. We also searched for ERBB2 transmembrane mutations in various types of cancers in PubMed, The Cancer Genome Atlas (TCGA), and the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) database. Besides our two cases, two patients with V659E mutations were found via PubMed. Three potential patients were found in TCGA. In addition, MSK-IMPACT allowed identification of three additional urothelial carcinomas with G660D mutations and two lung adenocarcinomas with V659E mutations. Our experience suggests that establishing a database of integrated information regarding the clinical genome and therapeutic outcome of patients with recurrent but less common mutations is essential to implement precision oncology. KEY POINTS: Rare but targetable mutations such as avian erythroblastic leukemia viral oncogene homolog 2 (erb-b2 receptor tyrosine kinase 2) (ERBB2; human epidermal growth factor receptor 2 [HER2]) transmembrane domain (TMD) mutations can be detected by comprehensive genomic profiling.Afatinib may be effective for patients with cancer with ERBB2 (HER2) TMD mutations.In order to implement precision oncology, it is important to establish a database of integrated information regarding the clinical genomes and therapeutic outcomes of patients with recurrent but less common mutations.

Visual and automated assessment of matrix metalloproteinase-14 tissue expression for the evaluation of ovarian cancer prognosis.

The purpose of this study was to evaluate whether the membrane type 1 matrix metalloproteinase-14 (or MT1-MMP) tissue expression, as assessed visually on digital slides and by digital image analysis, could predict outcomes in women with ovarian carcinoma. Tissue microarrays from a cohort of 211 ovarian carcinoma women who underwent a debulking surgery between 1993 and 2006 at the CHU de Québec (Canada) were immunostained for matrix metalloproteinase-14. The percentage of MMP-14 staining was assessed visually and with the Calopix software. Progression was evaluated using the CA-125 and/or the RECIST criteria according to the GCIG criteria. Dates of death were obtained by record linkage with the Québec mortality files. Adjusted hazard ratios of death and progression with their 95% confidence intervals were estimated using the Cox model. Comparisons between the two modalities of MMP-14 assessment were done using the box plots and the Kruskal-Wallis test. The highest levels of MMP-14 immunostaining were associated with nonserous histology, early FIGO stage, and low preoperative CA-125 levels (P<0.05). In bivariate analyses, the higher level of MMP-14 expression (>40% of MMP-14-positive cells) was inversely associated with progression using visual assessment (hazard ratio=0.39; 95% confidence interval: 0.18-0.82). A similar association was observed with the highest quartile of MMP-14-positive area assessed by digital image analysis (hazard ratio=0.48; 95% confidence interval: 0.28-0.82). After adjustment for standard prognostic factors, these associations were no longer significant in the ovarian carcinoma cohort. However, in women with serous carcinoma, the highest quartile of MMP-14-positive area was associated with progression (adjusted hazard ratio=0.48; 95% confidence interval: 0.24-0.99). There was no association with overall survival. The digital image analysis of MMP-14-positive area matched the visual assessment using three categories (>40% vs 21-40 vs <20%). Higher levels of MMP-14 immunostaining were associated with standard factors of better ovarian carcinoma prognosis. In women with serous carcinoma, high expression of MMP-14 was associated with lower progression.

Polygenic inheritance and its interplay with smoking history in predicting lung cancer diagnosis: a French-Canadian case-control cohort.

BACKGROUND: The most near-term clinical application of genome-wide association studies in lung cancer is a polygenic risk score (PRS). METHODS: A case-control dataset was generated consisting of 4002 lung cancer cases from the LORD project and 20,010 ethnically matched controls from CARTaGENE. A genome-wide PRS including >1.1 million genetic variants was derived and validated in UK Biobank (n = 5419 lung cancer cases). The predictive ability and diagnostic discrimination performance of the PRS was tested in LORD/CARTaGENE and benchmarked against previous PRSs from the literature. Stratified analyses were performed by smoking status and genetic risk groups defined as low (<20th percentile), intermediate (20-80th percentile) and high (>80th percentile) PRS. FINDINGS: The phenotypic variance explained and the effect size of the genome-wide PRS numerically outperformed previous PRSs. Individuals with high genetic risk had a 2-fold odds of lung cancer compared to low genetic risk. The PRS was an independent predictor of lung cancer beyond conventional clinical risk factors, but its diagnostic discrimination performance was incremental in an integrated risk model. Smoking increased the odds of lung cancer by 7.7-fold in low genetic risk and by 11.3-fold in high genetic risk. Smoking with high genetic risk was associated with a 17-fold increase in the odds of lung cancer compared to individuals who never smoked and with low genetic risk. INTERPRETATION: Individuals at low genetic risk are not protected against the smoking-related risk of lung cancer. The joint multiplicative effect of PRS and smoking increases the odds of lung cancer by nearly 20-fold. FUNDING: This work was supported by the CQDM and the IUCPQ Foundation owing to a generous donation from Mr. Normand Lord.

Retrospective Assessment of Complementary Liquid Biopsy on Tissue Single-Gene Testing for Tumor Genotyping in Advanced NSCLC.

Biomarker testing is key for non-small cell lung cancer (NSCLC) management and plasma based next-generation sequencing (NGS) is increasingly characterized as a non-invasive alternative. This study aimed to evaluate the value of complementary circulating tumor DNA (ctDNA) NGS on tissue single-gene testing (SGT). Ninety-one advanced stage NSCLC patients with tumor genotyping by tissue SGT (3 genes) followed by ctDNA (38 genes amplicon panel) were included. ctDNA was positive in 47% (n = 43) and identified a targetable biomarker in 19 patients (21%). The likelihood of positivity on ctDNA was higher if patients had extra-thoracic disease (59%) or were not under active treatment (59%). When compared to SGT, ctDNA provided additional information in 41% but missed a known alteration in 8%. Therapeutic change for targeted therapy based on ctDNA occurred in five patients (5%), while seven patients with missed alterations on ctDNA had EGFR mutations or ALK fusions. The median turnaround time of ctDNA was 10 days (range 6-25), shorter (p = 0.002) than the cumulative delays for the tissue testing trajectory until biomarker availability (13 d; range 7-1737). Overall, the results from this study recapitulate the potential and limitations of ctDNA when used complementarily to tissue testing with limited biomarker coverage.

A Simplified Version of the IASLC Grading System for Invasive Pulmonary Adenocarcinomas With Improved Prognosis Discrimination.

Tumor grading enables better management of patients and treatment options. The International Association for the Study of Lung Cancer (IASLC) Pathology Committee has recently released a 3-tier grading system for invasive pulmonary adenocarcinoma consisting of predominant histologic patterns plus a cutoff of 20% of high-grade components including solid, micropapillary, and complex glandular patterns. The goal of this study was to validate the prognostic value of the new IASLC grading system and to compare its discriminatory performance to the predominant pattern-based grading system and a simplified version of the IASLC grading system without complex glandular patterns. This was a single-site retrospective study based on a 20-year data collection of patients that underwent lung cancer surgery. All invasive pulmonary adenocarcinomas confirmed by the histologic review were evaluated in a discovery cohort (n=676) and a validation cohort (n=717). The median duration of follow-up in the combined dataset (n=1393) was 7.5 years. The primary outcome was overall survival after surgery. The 3 grading systems had strong and relatively similar predictive performance, but the best parsimonious model was the simplified IASLC grading system (log-rank P =1.39E-13). The latter was strongly associated with survival in the validation set ( P =1.1E-18) and the combined set ( P =5.01E-35). We observed a large proportion of patients upgraded to the poor prognosis group using the IASLC grading system, which was attenuated when using the simplified IASLC grading system. In conclusion, we identified a histologic simpler classification for invasive pulmonary adenocarcinomas that outperformed the recently proposed IASLC grading system. A simplified grading system is clinically convenient and will facilitate widespread implementation.

Consensus Recommendations to Optimize the Detection and Reporting of NTRK Gene Fusions by RNA-Based Next-Generation Sequencing.

The detection of gene fusions by RNA-based next-generation sequencing (NGS) is an emerging method in clinical genetic laboratories for oncology biomarker testing to direct targeted therapy selections. A recent Canadian study (CANTRK study) comparing the detection of NTRK gene fusions on different NGS assays to determine subjects' eligibility for tyrosine kinase TRK inhibitor therapy identified the need for recommendations for best practices for laboratory testing to optimize RNA-based NGS gene fusion detection. To develop consensus recommendations, representatives from 17 Canadian genetic laboratories participated in working group discussions and the completion of survey questions about RNA-based NGS. Consensus recommendations are presented for pre-analytic, analytic and reporting aspects of gene fusion detection by RNA-based NGS.

CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

Prognostic value of complex glandular patterns in invasive pulmonary adenocarcinomas.

Prognostic stratification of patients surgically resected with invasive pulmonary adenocarcinoma must be improved. Previous studies reported that complex glandular patterns (CGPs), cribriform and fused gland growth patterns, are associated with unfavorable prognosis. The goal of this study is to evaluate the prognostic value of CGPs in patients with resected stage I-IV lung adenocarcinoma. The presence of CGPs as a minor to predominant component was tested for association with overall survival (OS, n = 676) and relapse-free survival (RFS, n = 463) after surgery. CGPs were observed in 284 tumors (42.0%). Cribriform and fused gland were the predominant patterns in 35 and 37 cases, respectively. The presence of cribriform pattern was associated with worse RFS, but not OS. The fused gland pattern alone or grouped into CGPs with the cribriform pattern was not associated with OS and RFS. As a predominant pattern, cribriform was associated with the worse survival compared to the 5 recognized histologic patterns. Patients with fused gland-predominant tumors had 5-year survival that ranged between papillary- and micropapillary-predominant tumors. We conclude that cribriform-predominant, but not fused gland-predominant, is a subtype with poor prognosis similar to the solid and micropapillary subtypes. In contrast, the presence of a minor component of fused gland or CGPs (cribriform + fused gland) is not associated with survival. The cribriform pattern alone offers prognosis stratification improvement, but this effect is attenuated when combined into CGPs to define a subset of acinar-predominant tumors with poor prognosis. This argues against combining cribriform and fused gland into CGPs to summarize high-grade patterns.

Performance of an RNA-Based Next-Generation Sequencing Assay for Combined Detection of Clinically Actionable Fusions and Hotspot Mutations in NSCLC.

INTRODUCTION: With its expanding list of approved and emerging therapeutic indications, NSCLC is the exemplar tumor type requiring upfront assessment of several biomarkers to guide clinical management. Next-generation sequencing allows identification of different types of molecular alterations, each with specific analytical challenges. Library preparation using parallel DNA and RNA workflows can overcome most of them, but it increases complexity of laboratory operations, turnaround time, and costs. We describe the performance characteristics of a 15-gene RNA panel on the basis of anchored multiplex polymerase chain reaction for combined detection of clinically relevant oncogenic fusion transcripts and hotspot small variants. METHODS: Formalin-fixed, paraffin-embedded NSCLC clinical samples (N = 58) were used along cell lines and commercial controls to validate the assay's analytical performance, followed by an exploratory prospective cohort (N = 87). RESULTS: The raw assay sensitivity for hotspot mutations and fusions was 83% and 93%, respectively, reaching 100% after filtering for key assay metrics. Those include quantity and quality of input of nucleic acid and sequencing metric from primers on housekeeping genes included in the assay. In the prospective cohort, driver alterations were identified in most cases (≥58%). CONCLUSIONS: This ultrafocused RNA-next-generation sequencing assay offers an advantageous option with single unified workflow for simultaneous detection of clinically relevant hotspot mutations and fusions in NSCLC, focusing on actionable gene targets.

Tumor Mutational Burden by Whole-Genome Sequencing in Resected NSCLC of Never Smokers.

BACKGROUND: Data are scarce about tumor mutational burden (TMB) as a biomarker in never smokers with non-small cell lung cancer (NSCLC). METHODS: TMB was assessed by whole-genome sequencing (WGS) and compared with in silico reduced whole-exome sequencing (WES) and targeted commercial next-generation sequencing (NGS) gene panels in 92 paired tumor-normal samples from never smokers who underwent NSCLC resection with curative intent. Analyses were performed to test for association with survival after surgery and to identify the optimal prognostic TMB cutoff. RESULTS: Tumors of never smokers with NSCLC had low TMB scores (median 1.57 mutations/Mb; range, 0.13-17.94). A TMB cutoff of 1.70 mutations/Mb was associated with a 5-year overall survival of 58% in the high-TMB (42% of cases) compared with 86% in low-TMB patients (Wald P = 0.0029). TMB scores from WGS and WES were highly correlated (Spearman ρ = 0.93, P < 2.2e-16). TMB scores from NGS panels demonstrated high intraindividual fluctuations and identified high-TMB patients with 65% concordance in average compared with WGS. CONCLUSIONS: In resected NSCLC of never smokers, high TMB was associated with worse prognosis. WES provided a good estimate of TMB while targeted NGS panels seem to lack adequate depth and resolution in the setting of low mutation burden. IMPACT: TMB is a prognostic indicator of survival in resected NSCLC from individuals who never smoked. In this setting of low mutation counts, TMB can be accurately measured by WGS or WES, but not NGS panels.

BCG vaccination provides protection against IAV but not SARS-CoV-2.

Since the vast majority of species solely rely on innate immunity for host defense, it stands to reason that a critical evolutionary trait like immunological memory evolved in this primitive branch of our immune system. There is ample evidence that vaccines such as bacillus Calmette-Guérin (BCG) induce protective innate immune memory responses (trained immunity) against heterologous pathogens. Here we show that while BCG vaccination significantly reduces morbidity and mortality against influenza A virus (IAV), it fails to provide protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In contrast to IAV, SARS-CoV-2 infection leads to unique pulmonary vasculature damage facilitating viral dissemination to other organs, including the bone marrow (BM), a central site for BCG-mediated trained immunity. Finally, monocytes from BCG-vaccinated individuals mount an efficient cytokine response to IAV infection, while this response is minimal following SARS-CoV-2. Collectively, our data suggest that the protective capacity of BCG vaccination is contingent on viral pathogenesis and tissue tropism.

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next-generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods.

Detection of DNA damage in oocytes of small ovarian follicles following phosphoramide mustard exposures of cultured rodent ovaries in vitro.

Healthy oocytes are critical for producing healthy children, but little is known about whether or not oocytes have the capacity to identify and recover from injury. Using a model ovotoxic alkylating drug, cyclophosphamide (CPA), and its active metabolite, phosphoramide mustard (PM), we previously showed that PM (≥3µM) caused significant follicle loss in postnatal day 4 (PND4) mouse ovaries in vitro. We now investigate whether PM induces DNA damage in oocytes, examining histone H2AX phosphorylation (γH2AX), a marker of DNA double-strand breaks (DSBs). Exposure of cultured PND4 mouse ovaries to 3 and 0.1µM PM induced significant losses of primordial and small primary follicles, respectively. PM-induced γH2AX was observed predominantly in oocytes, in which foci of γH2AX staining increased in a concentration-dependent manner and peaked 18-24h after exposure to 3-10µMPM. Numbers of oocytes with ≥5 γH2AX foci were significantly increased both 1 and 8days after exposure to ≥1µMPM compared to controls. Inhibiting the kinases that phosphorylate H2AX significantly increased follicle loss relative to PM alone. In adult mice, CPA also induced follicle loss in vivo. PM also significantly decreased primordial follicle numbers (≥30µM) and increased γH2AX foci (≥3µM) in cultured PND4 Sprague-Dawley rat ovaries. Results suggest oocytes can detect PM-induced damage at or below concentrations which cause significant follicle loss, and there are quantitative species-specific differences in sensitivity. Surviving oocytes with DNA damage may represent an increased risk for fertility problems or unhealthy offspring.

Canadian Consensus Recommendations on the Management of MET-Altered NSCLC.

In Canada, the therapeutic management of patients with advanced non-small cell lung cancer (NSCLC) with rare actionable mutations differs between provinces, territories, and individual centres based on access to molecular testing and funded treatments. These variations, together with the emergence of several novel mesenchymal-epithelial transition (MET) factor-targeted therapies for the treatment of NSCLC, warrant the development of evidence-based consensus recommendations for the use of these agents. A Canadian expert panel was convened to define key clinical questions, review evidence, discuss practice recommendations and reach consensus on the treatment of advanced MET-altered NSCLC. Questions addressed by the panel include: 1. How should the patients most likely to benefit from MET-targeted therapies be identified? 2. What are the preferred first-line and subsequent therapies for patients with MET exon 14 skipping mutations? 3. What are the preferred first-line and subsequent therapies for advanced NSCLC patients with de novo MET amplification? 4. What is the preferred therapy for patients with advanced epidermal growth factor receptor (EGFR)-mutated NSCLC with acquired MET amplification progressing on EGFR inhibitors? 5. What are the potential strategies for overcoming resistance to MET inhibitors? Answers to these questions, along with the consensus recommendations herein, will help streamline the management of MET-altered NSCLC in routine practice, assist clinicians in therapeutic decision-making, and help ensure optimal outcomes for NSCLC patients with MET alterations.