Purpose The survival benefit with adjuvant chemotherapy for patients with resected stage II-III non-small-cell lung cancer (NSCLC) is modest. Efforts to develop prognostic or predictive biomarkers in these patients have not yielded clinically useful tests. We report findings from the Lung Adjuvant Cisplatin Evaluation (LACE)-Bio-II study, in which we analyzed next-generation sequencing and long-term outcomes data from > 900 patients with early-stage NSCLC treated prospectively in adjuvant landmark clinical trials. We used a targeted gene panel to assess the prognostic and predictive effect of mutations in individual genes, DNA repair pathways, and tumor mutation burden (TMB). Methods A total of 908 unmatched, formalin-fixed, paraffin-embedded, resected lung cancer tumor specimens were sequenced using a targeted panel of 1,538 genes. Stringent filtering criteria were applied to exclude germline variants and artifacts related to formalin fixation. Disease-free survival, overall survival, and lung cancer-specific survival (LCSS) were assessed in Cox models stratified by trial and adjusted for treatment, age, sex, performance score, histology, type of surgery, and stage. Results Nonsynonymous mutations were identified in 1,515 genes in 908 tumor samples. High nonsynonymous TMB (> 8 mutations/Mb) was prognostic for favorable outcomes (ie, overall survival, disease-free survival, and LCSS) in patients with resected NSCLC. LCSS benefit with adjuvant chemotherapy was more pronounced in patients with low nonsynonymous TMBs (≤ 4 mutations/Mb). Presence of mutations in DNA repair pathways, tumor-infiltrating lymphocytes, TP53 alteration subtype, and intratumor heterogeneity was neither prognostic nor predictive. Statistically significant effect of mutations in individual genes was difficult to determine due to high false-discovery rates. Conclusion High nonsynonymous TMB was associated with a better prognosis in patients with resected NSCLC. In addition, the benefit of adjuvant chemotherapy on LCSS was more pronounced in patients with low nonsynonymous TMBs. Studies are warranted to confirm these findings.

Purpose : Anti-PD1/PD-L1 immunotherapy has demonstrated success in the treatment of advanced non-small cell lung cancer (NSCLC). Recently, PD1/PD-L1 blockade also has demonstrated interesting results in small trials of neo-adjuvant treatment in Stage IB-IIIA NSCLC. In addition, several clinical trials using anti-PD1/PD-L1 as an adjuvant or neo-adjuvant treatment in resectable stage NSCLC patients are ongoing. However, few analyses of anti- PD1/PD-L1 immunotherapy related biomarkers in early stage squamous cell lung carcinoma (SqCLC) have been reported. In this study, we evaluated PD-L1 protein expression, tumor mutation burden, and expression of an immune gene signature in early stage SqCLC, providing data for identifying the potential role for anti-PD1/PD-L1 treatment in early stage SqCLC patients. Experimental Design and Results : A total of 255 early stage SqCLC patient specimens were identified within the Strategic Partnering to Evaluate Cancer Signatures (SPECS) program participating centers. PD-L1 protein expression by IHC was evaluated using the Dako PD-L1 22C3 pharmDx kit on the Dako Link 48 auto-stainer. Tumor Mutation Burden (TMB) was calculated based on data from targeted genome sequencing. The T-effector and IFN-γ gene signature was determined from Affymetrix gene chip data from frozen specimens. The prevalence of PD-L1 expression was 9.8% at a tumor proportion score (TPS) cutoff of ≥ 50%. PD- L1 mRNA and PD-L2 mRNA positively correlated with PD-L1 protein expression on tumor cells (TCs) and tumor-infiltrating immune cells (TIICs). PD-L1 protein expression on TIICs was correlated with the T-effector and IFN-y gene signature (P<0.001), but not with TMB. For tumor cells, all of these biomarkers were independent of each other. And neither PD-L1 protein expression, TMB, or T-effector and IFN-γ gene signatures were independently prognostic for patient outcomes. Conclusions : Evaluation of PD-L1 expression, TMB, and T-effector and IFN-γ gene signatures in the early-stage SqCLC cohort were found to be independent of each other and none were associated with overall survival. Results also support the hypothesis that PD-L1 expression is regulated by an intrinsic mechanism on tumor cells and an adaptive mechanism on immune cells.

Multiple myeloma (MM) is a disease of copy number variants (CNVs), chromosomal translocations, and single-nucleotide variants (SNVs). To enable integrative studies across these diverse mutation types, we developed a capture-based sequencing platform to detect their occurrence in 465 genes altered in MM and used it to sequence 95 primary tumor-normal pairs to a mean depth of 104×. We detected cases of hyperdiploidy (23%), deletions of 1p (8%), 6q (21%), 8p (17%), 14q (16%), 16q (22%), and 17p (4%), and amplification of 1q (19%). We also detected IGH and MYC translocations near expected frequencies and non-silent SNVs in NRAS (24%), KRAS (21%), FAM46C (17%), TP53 (9%), DIS3 (9%), and BRAF (3%). We discovered frequent mutations in IGLL5 (18%) that were mutually exclusive of RAS mutations and associated with increased risk of disease progression (p = 0.03), suggesting that IGLL5 may be a stratifying biomarker. We identified novel IGLL5/IGH translocations in two samples. We subjected 15 of the pairs to ultra-deep sequencing (1259×) and found that although depth correlated with number of mutations detected (p = 0.001), depth past ~300× added little. The platform provides cost-effective genomic analysis for research and may be useful in individualizing treatment decisions in clinical settings.