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Abstract Background We explored potential predictive biomarkers of immunotherapy response in patients with extensive-stage small-cell lung cancer (ES-SCLC) treated with durvalumab (D) + tremelimumab (T) + etoposide-platinum (EP), D + EP, or EP in the randomized phase 3 CASPIAN trial. Methods 805 treatment-naïve patients with ES-SCLC were randomized (1:1:1) to receive D + T + EP, D + EP, or EP. The primary endpoint was overall survival (OS). Patients were required to provide an archived tumor tissue block (or ≥ 15 newly cut unstained slides) at screening, if these samples existed. After assessment for programmed cell death ligand-1 expression and tissue tumor mutational burden, residual tissue was used for additional molecular profiling including by RNA sequencing and immunohistochemistry. Results In 182 patients with transcriptional molecular subtyping, OS with D ± T + EP was numerically highest in the SCLC-inflamed subtype (n = 10, median 24.0 months). Patients derived benefit from immunotherapy across subtypes; thus, additional biomarkers were investigated. OS benefit with D ± T + EP versus EP was greater with high versus low CD8A expression/CD8 cell density by immunohistochemistry, but with no additional benefit with D + T + EP versus D + EP. OS benefit with D + T + EP versus D + EP was associated with high expression of CD4 (median 25.9 vs. 11.4 months) and antigen-presenting and processing machinery (25.9 vs. 14.6 months) and MHC I and II (23.6 vs. 17.3 months) gene signatures, and with higher MHC I expression by immunohistochemistry. Conclusions These findings demonstrate the tumor microenvironment is important in mediating better outcomes with D ± T + EP in ES-SCLC, with canonical immune markers associated with hypothesized immunotherapy mechanisms of action defining patient subsets that respond to D ± T. Trial registration ClinicalTrials.gov, NCT03043872.

Summary: Background: Recent studies have reported the predictive and prognostic value of novel transcriptional factor-based molecular subtypes in small-cell lung cancer (SCLC). We conducted an in-depth analysis pairing multi-omics data with immunohistochemistry (IHC) to elucidate the underlying characteristics associated with differences in clinical outcomes between subtypes. Methods: IHC (n = 252), target exome sequencing (n = 422), and whole transcriptome sequencing (WTS, n = 189) data generated from 427 patients (86.4% males, 13.6% females) with SCLC were comprehensively analysed. The differences in the mutation profile, gene expression profile, and inflammed signatures were analysed according to the IHC-based molecular subtype. Findings: IHC-based molecular subtyping, comprised of 90 limited-disease (35.7%) and 162 extensive-disease (64.3%), revealed a high incidence of ASCL1 subtype (IHC-A, 56.3%) followed by ASCL1/NEUROD1 co-expressed (IHC-AN, 17.9%), NEUROD1 (IHC-N, 12.3%), POU2F3 (IHC-P, 9.1%), triple-negative (IHC-TN, 4.4%) subtypes. IHC-based subtype showing high concordance with WTS-based subtyping and non-negative matrix factorization (NMF) clusterization method. IHC-AN subtype resembled IHC-A (rather than IHC-N) in terms of both gene expression profiles and clinical outcomes. Favourable median overall survival was observed in IHC-A (15.2 months) compared to IHC-N (8.0 months, adjusted HR 2.3, 95% CI 1.4–3.9, p = 0.002) and IHC-P (8.3 months, adjusted HR 1.7, 95% CI 0.9–3.2, p = 0.076). Inflamed tumours made up 25% of cases (including 53% of IHC-P, 26% of IHC-A, 17% of IHC-AN, but only 11% of IHC-N). Consistent with recent findings, inflamed tumours were more likely to benefit from first-line immunotherapy treatment than non-inflamed phenotype (p = 0.002). Interpretation: This study provides fundamental data, including the incidence and basic demographics of molecular subtypes of SCLC using both IHC and WTS from a comparably large, real-world Asian/non-Western patient cohort, showing high concordance with the previous NMF-based SCLC model. In addition, we revealed underlying biological pathway activities, immunogenicity, and treatment outcomes based on molecular subtype, possibly related to the difference in clinical outcomes, including immunotherapy response. Funding: This work was supported by AstraZeneca, Future Medicine 2030 Project of the Samsung Medical Center [grant number SMX1240011], the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) [grant number 2020R1C1C1010626] and the 7th AstraZeneca-KHIDI (Korea Health Industry Development Institute) oncology research program.

Accurate diagnoses are crucial in determining the most effective treatment across different cancers. In challenging cases, morphology-based traditional pathology methods have important limitations, while molecular profiling can provide valuable information to guide clinical decisions. We present a 35-year female with lung cancer with choriocarcinoma features. Her disease involved the right lower lung, brain, and thoracic lymph nodes. The pathology from brain metastasis was reported as “metastatic choriocarcinoma” (a germ cell tumor) by local pathologists. She initiated carboplatin and etoposide, a regimen for choriocarcinoma. Subsequently, her case was assessed by pathologists from an academic cancer center, who gave the diagnosis of “adenocarcinoma with aberrant expression of β-hCG” and finally pathologists at our hospital, who gave the diagnosis of “poorly differentiated carcinoma with choriocarcinoma features”. Genomic profiling detected a KRAS G13R mutation and transcriptomics profiling was suggestive of lung origin. The patient was treated with carboplatin/paclitaxel/ipilimumab/nivolumab followed by consolidation radiation therapy. She had no evidence of progression to date, 16 months after the initial presentation. The molecular profiling could facilitate diagnosing of challenging cancer cases. In addition, chemoimmunotherapy and local consolidation radiation therapy may provide promising therapeutic options for patients with lung cancer exhibiting choriocarcinoma features.

Introduction: NSCLC transformation to SCLC has been best characterized with EGFR-mutant NSCLC, with emerging case reports seen in ALK, RET, and KRAS-altered NSCLC. Previous reports revealed transformed SCLC from EGFR-mutant NSCLC portends very poor prognosis and lack effective treatment. Genomic analyses revealed TP53 and RB1 loss of function increase the risk of SCLC transformation. Little has been reported on the detailed clinicogenomic characteristics and potential therapeutic targets for this patient population. Methods: In this study, we conducted a single-center retrospective analysis of clinical and genomic characteristics of patients with EGFR-mutant NSCLC transformed to SCLC. Demographic data, treatment course, and clinical molecular testing reports were extracted from electronic medical records. Kaplan-Meier analyses were used to estimate survival outcomes. Next generation sequencing-based assays was used to identify EGFR and co-occurring genetic alterations in tissue or plasma before and after SCLC transformation. Single-cell RNA sequencing (scRNA-seq) was performed on a patient-derived-xenograft model generated from a patient with EGFR-NSCLC transformed SCLC tumor. Results: A total of 34 patients were identified in our study. Median age at initial diagnosis was 58, and median time to SCLC transformation was 24.2 months. 68% were female and 82% were never smokers. 79% of patients were diagnosed as stage IV disease, and over half had brain metastases at baseline. Median overall survival of the entire cohort was 38.3 months from initial diagnoses and 12.4 months from time of SCLC transformation. Most patients harbored EGFR exon19 deletions as opposed to exon21 L858R alteration. Continuing EGFR tyrosine kinase inhibitor post-transformation did not improve overall survival compared with those patients where tyrosine kinase inhibitor was stopped in our cohort. In the 20 paired pretransformed and post-transformed patient samples, statistically significant enrichment was seen with PIK3CA alterations (p = 0.04) post-transformation. Profiling of longitudinal liquid biopsy samples suggest emergence of SCLC genetic alterations before biopsy-proven SCLC, as shown by increasing variant allele frequency of TP53, RB1, PIK3CA alterations. ScRNA-seq revealed potential therapeutic targets including DLL3, CD276 (B7-H3) and PTK7 were widely expressed in transformed SCLC. Conclusions: SCLC transformation is a potential treatment resistance mechanism in driver-mutant NSCLC. In our cohort of 34 EGFR-mutant NSCLC, poor prognosis was observed after SCLC transformation. Clinicogenomic analyses of paired and longitudinal samples identified genomic alterations emerging post-transformation and scRNA-seq reveal potential therapeutic targets in this population. Further studies are needed to rigorously validate biomarkers and therapeutic targets for this patient population.

The curative treatment of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC), utilizes radiation. The outcomes for HPV/p16-negative HNSCC are significantly worse than HPV/p16-positive tumors, with increased radiation resistance leading to worse locoregional recurrence (LRR) and ultimately death. This study analyzed the relationship between immune function and outcomes following radiation in HPV/p16-negative tumors to identify mechanisms of radiation resistance and prognostic immune biomarkers. A discovery cohort of 94 patients with HNSCC treated uniformly with surgery and adjuvant radiation and a validation cohort of 97 similarly treated patients were utilized. Tumor immune infiltrates were derived from RNAseq gene expression. The immune cell types significantly associated with outcomes in the discovery cohort were examined in the independent validation cohort. A positive association between high Th2 infiltration and LRR was identified in the discovery cohort and validated in the validation cohort. Tumor mutations in CREBBP/EP300 and CASP8 were significantly associated with Th2 infiltration. A pathway analysis of genes correlated with Th2 cells revealed the potential repression of the antitumor immune response and the activation of BRCA1-associated DNA damage repair in multiple cohorts. The Th2 infiltrates were enriched in the HPV/p16-negative HNSCC tumors and associated with LRR and mutations in CASP8, CREBBP/EP300, and pathways previously shown to impact the response to radiation.

Abstract Background Symptom expression in SARS-CoV-2 infection (COVID-19) may affect patients already symptomatic with cancer. Patient-reported outcomes (PROs) can describe symptom burden during the acute and postacute stages of COVID-19 and support risk stratification for levels of care. At the start of the COVID-19 pandemic, our purpose was to rapidly develop, launch through an electronic patient portal, and provide initial validation for a PRO measure of COVID-19 symptom burden in patients with cancer. Methods We conducted a CDC/WHO web-based scan for COVID-19 symptoms and a relevance review of symptoms by an expert panel of clinicians treating cancer patients with COVID-19 to create a provisional MD Anderson Symptom Inventory for COVID-19 (MDASI-COVID). English-speaking adults with cancer who tested positive for COVID-19 participated in the psychometric testing phase. Patients completed longitudinal assessments of the MDASI-COVID and the EuroQOL 5 Dimensions 5 Levels (EQ-5D-5L) utility index and visual analog scale, which were presented through an electronic health record patient portal. To test the validity of the MDASI-COVID to distinguish between known groups of patients, we hypothesized that patients hospitalized, including having a hospitalization extended, for COVID-19 versus those not hospitalized would experience higher symptom burden. Correlation of mean symptom severity and interference scores with relevant EQ-5D-5L scores tested concurrent validity. The reliability of the MDASI-COVID was evaluated by calculating Cronbach alpha coefficients and test-retest reliability was evaluated by calculating Pearson correlation coefficients between the initial assessment and a second assessment no more than 14 days later. Results The web-based scan found 31 COVID-19-related symptoms; rankings of a 14-clinician expert panel reduced this list to 11 COVID-specific items to be added to the core MDASI. Time from literature scan start in March 2020 to instrument launch in May 2020 was 2 months. Psychometric analysis established the MDASI-COVID’s reliability, known-group validity, and concurrent validity. Conclusions We were able to rapidly develop and electronically launch a PRO measure of COVID-19 symptom burden in patients with cancer. Additional research is needed to confirm the content domain and predictive validity of the MDASI-COVID and define the symptom burden trajectory of COVID-19.

The roles of preexisting auto-reactive antibodies in immune-related adverse events (irAEs) associated with immune checkpoint inhibitor therapy are not well defined. Here, we analyzed plasma samples longitudinally collected at predefined time points and at the time of irAEs from 58 patients with immunotherapy naïve metastatic non-small cell lung cancer treated on clinical protocol with ipilimumab and nivolumab. We used a proteomic microarray system capable of assaying antibody reactivity for IgG and IgM fractions against 120 antigens for systemically evaluating the correlations between auto-reactive antibodies and certain organ-specific irAEs. We found that distinct patterns of auto-reactive antibodies at baseline were associated with the subsequent development of organ-specific irAEs. Notably, ACHRG IgM was associated with pneumonitis, anti-cytokeratin 19 IgM with dermatitis, and anti-thyroglobulin IgG with hepatitis. These antibodies merit further investigation as potential biomarkers for identifying high-risk populations for irAEs and/or monitoring irAEs during immunotherapy treatment.Trial registrationClinicalTrials.gov identifier: NCT03391869.

Abstract Background Oral leukoplakia (OL) is associated with an increased risk for oral cancer (OC) development. Prediction of OL cancer progression may contribute to decreased OC morbidity and mortality by favoring early intervention. Current OL progression risk assessment approaches face large interobserver variability and is weakly prognostic. We hypothesized that convolutional neural networks (CNN)‐based histology image analyses could accelerate the discovery of better OC progression risk models. Methods Our CNN‐based oral mucosa risk stratification model (OMRS) was trained to classify a set of nondysplastic oral mucosa (OM) and a set of OC H&E slides. As a result, the OMRS model could identify abnormal morphological features of the oral epithelium. By applying this model to OL slides, we hypothesized that the extent of OC‐like features identified in the OL epithelium would correlate with its progression risk. The OMRS model scored and categorized the OL cohort (n = 62) into high‐ and low‐risk groups. Results OL patients classified as high‐risk (n = 31) were 3.98 (95% CI 1.36–11.7) times more likely to develop OC than low‐risk ones (n = 31). Time‐to‐progression significantly differed between high‐ and low‐risk groups (p = 0.003). The 5‐year OC development probability was 21.3% for low‐risk and 52.5% for high‐risk patients. The predictive power of the OMRS model was sustained even after adjustment for age, OL site, and OL dysplasia grading (HR = 4.52, 1.5–13.7). Conclusion The ORMS model successfully identified OL patients with a high risk of OC development and can potentially benefit OC early diagnosis and prevention policies.

Abstract Combined BRAF + MEK inhibition is FDA approved for BRAF V600E-mutant solid tumors except for colorectal cancer. However, beyond MAPK mediated resistance several other mechanisms of resistance such as activation of CRAF, ARAF, MET, P13K/AKT/mTOR pathway exist among other complex pathways. In the VEM-PLUS study, we performed a pooled analysis of four phase one studies evaluating the safety and efficacy of vemurafenib monotherapy and vemurafenib combined with targeted therapies (sorafenib, crizotinib, or everolimus) or carboplatin plus paclitaxel in advanced solid tumors harboring BRAF V600 mutations. When vemurafenib monotherapy was compared with the combination regimens, no significant differences in OS or PFS durations were noted, except for inferior OS in the vemurafenib and paclitaxel and carboplatin trial (P = 0.011; HR, 2.4; 95% CI, 1.22–4.7) and in crossover patients (P = 0.0025; HR, 2.089; 95% CI, 1.2–3.4). Patients naïve to prior BRAF inhibitors had statistically significantly improved OS at 12.6 months compared to 10.4 months in the BRAF therapy refractory group (P = 0.024; HR, 1.69; 95% CI 1.07–2.68). The median PFS was statistically significant between both groups, with 7 months in the BRAF therapy naïve group compared to 4.7 months in the BRAF therapy refractory group (P = 0.016; HR, 1.80; 95% CI 1.11–2.91). The confirmed ORR in the vemurafenib monotherapy trial (28%) was higher than that in the combination trials. Our findings suggest that, compared with vemurafenib monotherapy, combinations of vemurafenib with cytotoxic chemotherapy or with RAF- or mTOR-targeting agents do not significantly extend the OS or PFS of patients who have solid tumors with BRAF V600E mutations. Gaining a better understanding of the molecular mechanisms of BRAF inhibitor resistance, balancing toxicity and efficacy with novel trial designs are warranted.

Immune checkpoint inhibitors with or without chemotherapy are now standard of care for non-small cell lung cancer. However, the benefits of combination vs sequential therapy have not been fully explored. Here, the authors analysed 1,133 patient records and show combination therapy showed increased protection against early progression, but similar overall survival.

Abstract Antibody-drug conjugates (ADCs) are one of fastest growing classes of oncology drugs in modern drug development. By harnessing the powers of both cytotoxic chemotherapy and targeted therapy, ADCs are unique in offering the potential to deliver highly potent cytotoxic agents to cancer cells which express a pre-defined cell surface target. In lung cancer, the treatment paradigm has shifted dramatically in recent years, and now ADCs are now joining the list as potential options for lung cancer patients. Since 2020, the first ADC for NSCLC patients has been FDA-approved (trastuzumab deruxtecan) and two ADCs have been granted FDA Breakthrough Therapy Designation, currently under evaluation (patritumab deruxtecan, telisotuzumab vedotin). Furthermore, several early-phase trials are assessing various novel ADCs, either as monotherapy or in combinations with advanced lung cancer, and more selective and potent ADCs are expected to become therapeutic options in clinic soon. In this review, we discuss the structure and mechanism of action of ADCs, including insights from pre-clinical work; we summarize the ADCs’ recent progress in lung cancer, describe toxicity profiles of ADCs, and explore strategies designed to enhance ADC potency and overcome resistance. In addition, we discuss novel ADC strategies of interest in lung cancer, including non-cytotoxic payloads, such as immunomodulatory and anti-apoptotic agents.

Docetaxel +/− ramucirumab remains the standard-of-care therapy for patients with metastatic non-small-cell lung cancer (NSCLC) after progression on platinum doublets and immune checkpoint inhibitors (ICIs). The aim of our study was to investigate whether the cancer gene mutation status was associated with clinical benefits from docetaxel +/− ramucirumab. We also investigated whether platinum/taxane-based regimens offered a better clinical benefit in this patient population. A total of 454 patients were analyzed (docetaxel +/− ramucirumab n=381; platinum/taxane-based regimens n=73). Progression-free survival (PFS) and overall survival (OS) were compared among different subpopulations with different cancer gene mutations and between patients who received docetaxel +/− ramucirumab versus platinum/taxane-based regimens. Among patients who received docetaxel +/− ramucirumab, the top mutated cancer genes included TP53 (n=167), KRAS (n=127), EGFR (n=65), STK11 (n=32), ERBB2 (HER2) (n=26), etc. None of these cancer gene mutations or PD-L1 expression was associated with PFS or OS. Platinum/taxane-based regimens were associated with a significantly longer mQS (13.00 m, 95% Cl: 11.20–14.80 m versus 8.40 m, 95% Cl: 7.12–9.68 m, LogRank P=0.019) than docetaxel +/− ramcirumab. Key prognostic factors including age, histology, and performance status were not different between these two groups. In conclusion, in patients with metastatic NSCLC who have progressed on platinum doublets and ICIs, the clinical benefit from docetaxel +/− ramucirumab is not associated with the cancer gene mutation status. Platinum/taxane-based regimens may offer a superior clinical benefit over docetaxel +/− ramucirumab in this patient population.

Introduction: MET amplification is a known resistance mechanism to EGFR tyrosine kinase inhibitor (TKI) treatment in EGFR-mutant NSCLC. Dual EGFR-MET inhibition has been reported with success in overcoming such resistance and inducing clinical benefit. Resistance mechanisms to dual EGFR-MET inhibition require further investigation and characterization. Methods: Patients with NSCLC with both MET amplification and EGFR mutation who have received crizotinib, capmatinib, savolitinib, or tepotinib plus osimertinib (OSI) after progression on OSI at MD Anderson Cancer Center were included in this study. Molecular profiling was completed by means of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). Radiological response was assessed on the basis of Response Evaluation Criteria in Solid Tumors version 1.1. Results: From March 2016 to March 2022, 23 treatments with dual MET inhibitor and osi were identified with a total of 20 patients included. Three patients received capmatinib plus OSI after progression on crizotinib plus OSI. Median age was 64 (38–89) years old and 75% were female. MET amplification was detected by FISH in 14 patients in the tissue, NGS in 10 patients, and circulating tumor DNA in three patients. Median MET gene copy number was 13.6 (6.4–20). Overall response rate was 34.8% (eight of 23). In assessable patients, tumor shrinkage was observed in 82.4% (14 of 17). Median time on treatment was 27 months. Two of three patients responded to capmatinib plus OSI after progression on crizotinib plus OSI. Dual EGFR-MET inhibition was overall well tolerated. Two patients on crizotinib plus OSI and one pt on capmatinib plus OSI discontinued therapy due to pneumonitis. One pt discontinued crizotinib plus OSI due to gastrointestinal toxicity. Six patients were still on double TKI treatment. At disease progression to dual EGFR-MET inhibition, FISH and NGS on tumor and plasma were completed in six patients. Notable resistance mechanisms observed include acquired MET D1246H (n = 1), acquired EGFR C797S (n = 2), FGFR2 fusion (n = 1, concurrent with C797S), and EGFR G796S (n = 1, concurrent with C797S). Four patients lost MET amplification. Conclusions: Dual EGFR and MET inhibition yielded high clinical response rate after progression on OSI. Resistance mechanisms to EGFR-MET double TKI inhibition include MET secondary mutation, EGFR secondary mutation, or loss of MET amplification.

Histopathologic whole-slide images (WSI) are generally considered the gold standard for cancer diagnosis and prognosis. Survival prediction based on WSI has recently attracted substantial attention. Nevertheless, it remains a central challenge owing to the inherent difficulties of predicting patient prognosis and effectively extracting informative survival-specific representations from WSI with highly compounded gigapixels. In this study, we present a fully automated cellular-level dual global fusion pipeline for survival prediction. Specifically, the proposed method first describes the composition of different cell populations on WSI. Then, it generates dimension-reduced WSI-embedded maps, allowing for efficient investigation of the tumor microenvironment. In addition, we introduce a novel dual global fusion network to incorporate global and inter-patch features of cell distribution, which enables the sufficient fusion of different types and locations of cells. We further validate the proposed pipeline using The Cancer Genome Atlas lung adenocarcinoma dataset. Our model achieves a C-index of 0.675 (±0.05) in the five-fold cross-validation setting and surpasses comparable methods. Further, we extensively analyze embedded map features and survival probabilities. These experimental results manifest the potential of our proposed pipeline for applications using WSI in lung adenocarcinoma and other malignancies.

Abstract Background Lung cancer is the leading cause of cancer death, partially owing to its extensive heterogeneity. The analysis of intertumor heterogeneity has been limited by an inability to concurrently obtain tissue from synchronous metastases unaltered by multiple prior lines of therapy. Methods In order to study the relationship between genomic, epigenomic and T cell repertoire heterogeneity in a rare autopsy case from a 32-year-old female never-smoker with left lung primary late-stage lung adenocarcinoma (LUAD), we did whole-exome sequencing (WES), DNA methylation and T cell receptor (TCR) sequencing to characterize the immunogenomic landscape of one primary and 19 synchronous metastatic tumors. Results We observed heterogeneous mutation, methylation, and T cell patterns across distinct metastases. Only TP53 mutation was detected in all tumors suggesting an early event while other cancer gene mutations were later events which may have followed subclonal diversification. A set of prevalent T cell clonotypes were completely excluded from left-side thoracic tumors indicating distinct T cell repertoire profiles between left-side and non left-side thoracic tumors. Though a limited number of predicted neoantigens were shared, these were associated with homology of the T cell repertoire across metastases. Lastly, ratio of methylated neoantigen coding mutations was negatively associated with T-cell density, richness and clonality, suggesting neoantigen methylation may partially drive immunosuppression. Conclusions Our study demonstrates heterogeneous genomic and T cell profiles across synchronous metastases and how restriction of unique T cell clonotypes within an individual may differentially shape the genomic and epigenomic landscapes of synchronous lung metastases.

Introduction: Vidutolimod, a CpG-A TLR9 agonist, was investigated in a phase 1b study (CMP-001-003; ClinicalTrials.gov, NCT03438318) in combination with atezolizumab with and without radiation therapy (RT) in patients with advanced NSCLC. Methods: Patients with progressive disease after anti–programmed cell death protein 1 or programmed death-ligand 1 therapy received either vidutolimod and atezolizumab (part A) or vidutolimod, atezolizumab, and RT (part B). The primary objective was to evaluate the safety of vidutolimod and atezolizumab with and without RT. Key secondary end point was best objective response rate per Response Evaluation Criteria in Solid Tumors, version 1.1. Results: Between March 28, 2018, and July 25, 2019, a total of 29 patients were enrolled and received at least one dose of vidutolimod (part A, n = 13; part B, n = 16). Intratumoral injections of vidutolimod were administered successfully, including injection of visceral lesions. The most common treatment-related adverse events (≥30%) were flu-like symptoms and hypotension. No objective responses were observed; 23.1% and 50.0% of the patients in parts A and B, respectively, had stable disease as best response. In parts A and B, 15.4% and 25.0% of the patients, respectively, had tumor shrinkage (

The molecular determinants of lung cancer histologic subtypes are not well understood. Here the authors analyze lung cancers of mixed histology and find that histologic subtypes are associated with transcriptomic features rather than genomic profiles in most tumors.

Small-cell lung cancer (SCLC) is an aggressive disease with limited therapeutic options. Here the authors perform an immunogenomic analysis of limited-stage SCLC, revealing a homogeneous mutational landscape, but limited T-cell infiltration and a cold and heterogeneous T cell repertoire.

Introduction: Complete pathologic response (CPR) is an acceptable surrogate for survival in clinical trials but it occurs infrequently in patients with NSCLC receiving neoadjuvant chemotherapy (NCT). Therefore, we studied the impact of major pathologic response (MPR) for predicting survival of patients with NSCLC receiving NCT. We also tested a newly reported scoring system—the prognostic score (PRSC)—which combines T category, lymph node status, and MPR status. Methods: We analyzed CPR and MPR, defined as 0% and less than or equal to 10% viable tumor cells, respectively, in 339 patients with NSCLC with various histologic types who had been treated with NCT followed by complete surgical resection. We evaluated the relationships between CPR, MPR, or PRSC and overall survival using the Kaplan-Meier method and Cox regression multivariate models, accounting for known prognostic factors, such as age, gender, histologic subtype, and pathologic stage. Results: Among all 339 patients, the Kaplan-Meier method revealed that patients with CPR and MPR had better survival. MPR identified a favorable group of patients who experienced survival similar to patients with CPR. Nevertheless, patients with no MPR had a significantly reduced probability of survival. Furthermore, univariate and multivariate Cox proportional hazards regression analysis revealed that MPR and PRSC were significantly associated with overall survival. Conclusions: Our data suggest that MPR can be used as an end point for overall survival in different histologic types for evaluation of therapeutic agents in clinical trials exploring NCT. We also confirmed that PRSC had a prognostic impact, differentiating patients into three prognostic groups, but not superior compared with MPR alone or the TNM8 systems.

Granulomatous/sarcoid-like lesions have been reported in patients treated with immune checkpoint inhibitors (ICIs). Here the authors report the occurrence of “nodal immune flare”, an apparent radiological cancer progression in the nodes characterized by the absence of cancer and the presence of non-caseating granulomas, in patients with non-small cell lung cancer following neoadjuvant ICI treatment.

The evolution of immune landscape in lung adenocarcinoma (ADC) is largely unknown. Here the authors use a cohort of resected invasive lung ADC and its precursors and show a gradual increase of immunosuppression and decrease of anti-tumor response associated with specific genomic and epigenetic features.

K-ras–mutant lung adenocarcinoma (KM-LUAD) is associated with abysmal prognosis and is tightly linked to tumor-promoting inflammation. A human mAb, canakinumab, targeting the proinflammatory cytokine IL-1β, significantly decreased the risk of lung cancer in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study. Interestingly, we found high levels of IL-1β in the lungs of mice with K-rasG12D–mutant tumors (CC-LR mice). Here, we blocked IL-1β using an anti–IL-1β mAb in cohorts of 6- or 14-week-old CC-LR mice to explore its preventive and therapeutic effect, respectively. IL-1β blockade significantly reduced lung tumor burden, which was associated with reprogramming of the lung microenvironment toward an antitumor phenotype characterized by increased infiltration of cytotoxic CD8+ T cells (with high IFN-γ and granzyme B expression but low programmed cell death 1 [PD-1] expression) while suppressing neutrophils and polymorphonuclear (PMN) myeloid-derived suppressor cells. When querying the Cancer Genome Atlas data set, we found positive correlations between IL1B expression and infiltration of immunosuppressive PMNs and expression of their chemoattractant, CXCL1, and PDCD1 expressions in patients with KM-LUAD. Our data provide evidence that IL-1β blockade may be a preventive strategy for high-risk individuals and an alternative therapeutic approach in combination with currently available treatments for KM-LUAD.

It is known that invasive lung adenocarcinomas evolve from pre-cancerous dysplastic lesions. In this study, the authors show that evolution of pre-cancerous lesions is accompanied by DNA methylation alterations, and that global hypomethylation correlates with immune infiltration, mutational burden and copy number alterations.

Abstract Background Metastasis is the primary cause of cancer mortality accounting for 90% of cancer deaths. Our understanding of the molecular mechanisms driving metastasis is rudimentary. Results We perform whole exome sequencing (WES), RNA sequencing, methylation microarray, and immunohistochemistry (IHC) on 8 pairs of non-small cell lung cancer (NSCLC) primary tumors and matched distant metastases. Furthermore, we analyze published WES data from 35 primary NSCLC and metastasis pairs, and transcriptomic data from 4 autopsy cases with metastatic NSCLC and one metastatic lung cancer mouse model. The majority of somatic mutations are shared between primary tumors and paired distant metastases although mutational signatures suggest different mutagenesis processes in play before and after metastatic spread. Subclonal analysis reveals evidence of monoclonal seeding in 41 of 42 patients. Pathway analysis of transcriptomic data reveals that downregulated pathways in metastases are mainly immune-related. Further deconvolution analysis reveals significantly lower infiltration of various immune cell types in metastases with the exception of CD4+ T cells and M2 macrophages. These results are in line with lower densities of immune cells and higher CD4/CD8 ratios in metastases shown by IHC. Analysis of transcriptomic data from autopsy cases and animal models confirms that immunosuppression is also present in extracranial metastases. Significantly higher somatic copy number aberration and allelic imbalance burdens are identified in metastases. Conclusions Metastasis is a molecularly late event, and immunosuppression driven by different molecular events, including somatic copy number aberration, may be a common characteristic of tumors with metastatic plasticity.

Using a mini-library of 1062 lentiviral shRNAs targeting 40 nuclear hormone receptors and 70 of their co-regulators, we searched for potential therapeutic targets that would be important during in vivo tumor growth using a parallel in vitro and in vivo shRNA screening strategy in the non-small cell lung cancer (NSCLC) line NCI-H1819. We identified 21 genes essential for in vitro growth, and nine genes specifically required for tumor survival in vivo, but not in vitro: NCOR2, FOXA1, HDAC1, RXRA, RORB, RARB, MTA2, ETV4, and NR1H2. We focused on FOXA1, since it lies within the most frequently amplified genomic region in lung adenocarcinomas. We found that 14q-amplification in NSCLC cell lines was a biomarker for FOXA1 dependency for both in vivo xenograft growth and colony formation, but not mass culture growth in vitro. FOXA1 knockdown identified genes involved in electron transport among the most differentially regulated, indicating FOXA1 loss may lead to a decrease in cellular respiration. In support of this, FOXA1 amplification was correlated with increased sensitivity to the complex I inhibitor phenformin. Integrative ChipSeq analyses reveal that FOXA1 functions in this genetic context may be at least partially independent of NKX2-1. Our findings are consistent with a neomorphic function for amplified FOXA1, driving an oncogenic transcriptional program. These data provide new insight into the functional consequences of FOXA1 amplification in lung adenocarcinomas, and identify new transcriptional networks for exploration of therapeutic vulnerabilities in this patient population.

Relevant features of T cell repertoire in human cancer remain to be delineated. Here the authors show, by TCR sequencing in a large cohort of lung cancer patients, that while a majority of T cell clones are shared between tumor and adjacent lung tissue, less frequent tumor-unique T cell clones correlate with worse prognosis.

Abstract Introduction OX-40 co-stimulatory signaling plays a role in mounting anti-tumor immune responses and clinical trials targeting this pathway are ongoing. However, the association of with OX-40 protein expression with clinical outcomes and pathological features in non-small cell lung cancer (NSCLC) are largely unknown. Methods Surgically-resected stage I-III NSCLC specimens (N = 100) were stained by immunohistochemistry (IHC) for the following immune markers: OX-40, PD-L1, PD-1, CD3, CD4, CD8, CD45RO, CD57, CD68, FOXP3, granzyme B, and ICOS. Immune-related markers mRNA expression were also assessed. We evaluated the association of OX-40 levels with major clinicopathologic variables, including molecular driver mutations. Results OX-40 IHC expression was observed in all tested tumors, predominantly localized in the membrane of the tumor immune infiltrate, and was not associated with a specific clinicopathologic or molecular subtype. High OX-40 expression levels measured by IHC median score were associated with better overall survival (OS) (p = 0.002), independent of CD3/CD8, PD-L1, and ICOS expression. High OX-40 IHC score was associated with increased expression of immune-related genes such as CD3, IFN-gamma, ICOS, CD8, CXCL9, CXCL10, CCL5, granzyme K. Conclusions High OX-40 IHC expression in the tumor immune infiltrate is associated with favorable prognosis and increased levels of immune-related genes including IFN-gamma in patients with surgically resected stage I-III NSCLC. Its prognostic utility is independent of PD-L1 and other common markers of immune activation. High OX-40 expression potentially identifies a unique subgroup of NSCLC that may benefit from co-stimulation with OX-40 agonist antibodies and potentially enhance the efficacy of existing immune checkpoint therapies.

Introduction: The efficacy of targeted therapies in oncogene-driven lung adenocarcinomas (LUADs) has been well established; however, the benefit for EGFR-mutant or ALK-rearranged lung squamous cell carcinomas (LUSCs) is less known, partially owing to the rarity of the incidence. Methods: We reviewed the database of the MD Anderson Cancer Center and identified metastatic LUSC with classic EGFR or ALK alterations. Results: There were eight patients with EGFR-mutant LUSC (median age = 58 y) and six patients with EML4-ALK LUSC (median age = 50 y) who received tyrosine kinase inhibitors (TKIs) that were identified. Of the 14 patients, 11 (79%) were females and 12 (86%) were never smokers, similar to the demographics of EGFR or ALK LUAD. With TKI treatment, seven of eight cases of EGFR LUSC and four of six cases of ALK LUSC achieved partial response or stable disease, but the progression-free survival was 4.9 months and 2.9 months for EGFR-mutant and ALK-rearranged LUSC, respectively. In addition, we compared comutation profile of EGFR-mutant LUAD (The Cancer Genome Atlas, n = 46) versus LUSC (n = 19) and found that the comutation patterns are more consistent with squamous disease with a higher incidence of PIK3CA (p = 0.02) and KRAS or BRAF (p = 0.04) alterations. Conclusions: EGFR or ALK alterations occur in patients with LUSC, especially never-smoker females. TKI treatments render clinical benefit in disease control, but the duration was considerably truncated compared with those historically observed in LUAD.

Background: The benefit of chemotherapy combined with immunotherapy in EGFR-mutant lung adenocarcinoma (LUAD) patients whose tumor developed resistance to EGFR tyrosine kinase inhibitors (TKIs) is not thoroughly investigated. The goal of this retrospective cohort study is to assess the clinical efficiency of immunotherapy alone or in combination with chemotherapy in a real-world setting. Methods: This retrospective cohort study enrolled LUAD patients with EGFR sensitive mutations whose tumor had acquired resistance to EGFR TKIs and received systemic treatment with chemotherapy (chemo; n = 84), chemotherapy combined with immunotherapy (chemoIO; n = 30), chemotherapy plus bevacizumab with or without IO (withBev; n = 42), and IO monotherapy (IO-mono; n = 22). Clinical progression-free survival (PFS) and overall survival (OS) were evaluated. Associations of clinical characteristics with outcomes were assessed using univariable and multi-covariate Cox Proportional Hazards regression models. Results: A total of 178 patients (median age = 63.3; 57.9% females) with a median follow-up time of 42.0 (Interquartile range: 22.9–67.8) months were enrolled. There was no significant difference in PFS between chemoIO vs. chemo groups (5.3 vs. 4.8 months, p = 0.8). Compared to the chemo group, patients who received withBev therapy trended towards better PFS (6.1 months vs. 4.8; p = 0.3; HR 0.79; 95% CI: 0.52–1.20), while patients treated with IO-mono had inferior PFS (2.2 months; p = 0.001; HR 2.22; 95% CI: 1.37–3.59). Furthermore, PD-L1 level was not associated with PFS benefit in the chemoIO group. Patients with EGFR-mutant LUAD with high PD-L1 (≥50%) had shorter PFS (5.8 months) than non-EGFR/ALK LUAD patients who received chemoIO (12.8 months, p = 0.002; HR 0.22; 95% CI: 0.08–0.56) as first-line treatment. Chemotherapy-based therapy rendered similar benefit to patients with either EGFR exon19 deletion vs. L858R in the LUAD. Conclusions: This retrospective analysis revealed that immunotherapy provided limited additional benefit to chemotherapy in TKI-refractory EGFR-mutant LUAD. Chemotherapy alone or combined with bevacizumab remain good choices for patients with actionable EGFR mutations.

Abstract Background Preclinical evidence suggests that low-dose radiation may overcome the inhibitory effects of the tumor stroma and improve a tumor’s response to immunotherapy, when combined with high-dose radiation to another tumor. The aim of this study was to evaluate tumor responses to this combination in a clinical setting. Methods A post-hoc analysis of 3 ongoing immunoradiation trials was performed. Twenty-six (of 155) patients received low-dose radiation (1–20 Gy total), either as scatter from high-dose radiation or from intentional treatment of a second isocenter with low-dose radiation, were evaluated for response. The low-dose lesions were compared to lesions that received no radiation (

MicroRNAs (miRNAs) are short single-stranded RNAs, measuring 21 to 23 nucleotides in length and regulate gene expression at the post-transcriptional level through mRNA destabilization or repressing protein synthesis. Dysregulation of miRNAs can lead to tumorigenesis through changes in regulation of key cellular processes such as cell proliferation, cell survival, and apoptosis. miR-125a-5p has been implicated as a tumor suppressor miRNA in malignancies such as non-small cell lung cancer and colon cancer. However, the role of miR-125a-5p has not been fully investigated in head and neck squamous cell carcinoma (HNSCC). We performed microRNA microarray profiling of HNSCC tumor samples obtained from a prospective clinical trial evaluating the role of postoperative radiotherapy in head and neck cancer. We also mined through The Cancer Genome Atlas to evaluate expression and survival data. Biological experiments, including cell proliferation, flow cytometry, cell migration and invasion, clonogenic survival, and fluorescent microscopy, were conducted using HN5 and UM-SCC-22B cell lines. miR-125a-5p downregulation was associated with recurrent disease in a panel of high-risk HNSCC and then confirmed poor survival associated with low expression in HNSCC via the Cancer Genome Atlas, suggesting that miR-125a-5p acts as a tumor suppressor miRNA. We then demonstrated that miR-125a-5p regulates cell proliferation through cell cycle regulation at the G1/S transition. We also show that miR-125a-5p can alter cell migration and modulate sensitivity to ionizing radiation. Finally, we identified putative mRNA targets of miR-125a-5p, including ERBB2, EIF4EBP1, and TXNRD1, which support the tumor suppressive mechanism of miR-125a-5p. Functional validation of ERBB2 suggests that miR-125a-5p affects cell proliferation and sensitivity to ionizing radiation, in part, through ERBB2. Our data suggests that miR-125a-5p acts as a tumor suppressor miRNA, has potential as a diagnostic tool and may be a potential therapeutic target for the management and treatment of squamous cell carcinoma of the head and neck.

There has been a drastic increase in detection of lung nodules, many of which are precancers, preinvasive, minimally invasive or sometimes invasive lung cancers. Here, Hu et al. perform multi-region exome sequencing to discern the evolutional trajectory from precancers to invasive lung cancers.

Abstract Background Because patient-derived xenografts (PDXs) are grown in immunodeficient mouse strains, PDXs are regarded as lacking an immune microenvironment. However, whether patients’ immune cells co-exist in PDXs remains uncharacterized. Methods We cultured small pieces of lung PDX tissue in media containing human interleukin-2 and characterized the proliferated lymphocytes by flow cytometric assays with antibodies specific for human immune cell surface markers. Presence of immune cells in PDXs was also determined by immunohistochemical staining. Results Human tumor-infiltrating lymphocytes (TILs) were cultured from nine of 25 PDX samples (36%). The mean time of PDX growth in immunodeficient mice before obtaining TILs in culture was 113 days (range 63–292 days). The TILs detected in PDXs were predominantly human CD8+ T cells, CD4+ T cells, or CD19+ B cells, depending on cases. DNA fingerprint analysis showed that the TILs originated from the same patients as the PDXs. Further analysis of two PDX-derived CD8+ T cells showed that they were PD-1−, CD45RO+, and either CD62L+ or CD62L−, suggesting they were likely memory T cells. Immunohistochemical staining showed that human T cells (CD8+ or CD4+), B cells (CD19+), and macrophages (CD68+) were present in stroma or intraepithelial cancer structures and that human PD-L1 was expressed in stromal cells. Moreover, the patient-derived immune cells in PDX can be passaged to the F2 generation and may migrate to spleens of PDX-bearing mice. Conclusions Patient-derived immune cells co-exist in early passages of PDXs in some lung cancer PDX models. The CD8+ cells from PDXs were likely memory T cells. These results suggest that PDXs can be used for evaluating the functionality of immune components in tumor microenvironments.

It has been over three decades since the hepatocyte growth factor (HGF) ligand and its receptor MET proto-oncogene (MET) pathway was established as promoting cancer growth and metastasis. MET exon 14 skipping ( METex14 ) alterations occur in 3–4% of all non-small cell lung cancer (NSCLC) patients, typically in elderly patients (older than 70 years), and result in constitutive activation of the MET receptor by altering a region required for receptor degradation. Multi-kinase inhibitor of MET, such as crizotinib, and more recently selective MET inhibitors, such as capmatinib and tepotinib, have demonstrated clinical efficacy and safety in METex14 NSCLC patients in clinical trials. These results have led to the approval of MET inhibitors by regulatory agencies across the globe. The success also fueled the excitement of further development of therapeutic strategies to target METex14 in lung cancers. This article provides an overview of the clinical development program targeting METex14 in NSCLC, including small molecular tyrosine kinase inhibitors and anti-MET antibodies. Furthermore, combination therapy immune checkpoint inhibitors or other targeted therapies are also under development in various patient populations, with acquired resistance immune or targeted therapy. Clinical trials in different development stages are ongoing and more drugs targeted to c-MET will be available for NSCLC patients with METex14 skipping mutations in the future.

Small cell lung cancer (SCLC) may evolve under treatment. But tumor tissues are often not available to study evolution of SCLC. Here, the authors utilize circulating tumor DNA to investigate the genomic evolution and subclonal architecture of SCLC during therapy.

Abstract Background The clinical efficacy observed with inhibitors of programed cell death 1/programed cell death ligand 1 (PD-L1/PD-1) in cancer therapy has prompted studies to characterize the immune response in several tumor types, including lung cancer. However, the immunological profile of non–small cell lung carcinoma (NSCLC) treated with neoadjuvant chemotherapy (NCT) is not yet fully characterized, and it may be therapeutically important. The aim of this retrospective study was to characterize and quantify PD-L1/PD-1 expression and tumor-associated immune cells (TAICs) in surgically resected NSCLCs from patients who received NCT or did not receive NCT (non-NCT). Methods We analyzed immune markers in formalin-fixed, paraffin-embedded tumor tissues resected from 112 patients with stage II/III NSCLC, including 61 non-NCT (adenocarcinoma [ADC] = 33; squamous cell carcinoma [SCC] = 28) and 51 NCT (ADC = 31; SCC = 20). We used multiplex immunofluorescence to identify and quantify immune markers grouped into two 6-antibody panels: panel 1 included AE1/AE3, PD-L1, CD3, CD4, CD8, and CD68; panel 2 included AE1/AE3, PD1, granzyme B, FOXP3, CD45RO, and CD57. Results PD-L1 expression was higher (> overall median) in NCT cases (median, 19.53%) than in non-NCT cases (median, 1.55%; P = 0.022). Overall, density of TAICs was higher in NCT-NSCLCs than in non-NCT-NSCLCs. Densities of CD3+ cells in the tumor epithelial compartment were higher in NCT-ADCs and NCT-SCCs than in non-NCT-ADCs and non-NCT-SCCs (P = 0.043). Compared with non-NCT-SCCs, NCT-SCCs showed significantly higher densities of CD3 + CD4+ (P = 0.019) and PD-1+ (P

Although non-small cell lung cancer (NSCLC) patients benefit from standard taxane-platin chemotherapy, many relapse, developing drug resistance. We established preclinical taxane-platin-chemoresistance models and identified a 35-gene resistance signature, which was associated with poor recurrence-free survival in neoadjuvant-treated NSCLC patients and included upregulation of the JumonjiC lysine demethylase KDM3B. In fact, multi-drug-resistant cells progressively increased the expression of many JumonjiC demethylases, had altered histone methylation, and, importantly, showed hypersensitivity to JumonjiC inhibitors in vitro and in vivo. Increasing taxane-platin resistance in progressive cell line series was accompanied by progressive sensitization to JIB-04 and GSK-J4. These JumonjiC inhibitors partly reversed deregulated transcriptional programs, prevented the emergence of drug-tolerant colonies from chemo-naive cells, and synergized with standard chemotherapy in vitro and in vivo. Our findings reveal JumonjiC inhibitors as promising therapies for targeting taxane-platin-chemoresistant NSCLCs.

Some patients present with multiple lung tumours but it is unclear whether these are metastases or individual lesions. Here, the authors use genomics techniques to demonstrate in six patients that multiple tumours have individual genetic profiles and represent separate tumours.

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Severely symptomatic COVID-19 is associated with lung inflammation, pneumonia, and respiratory failure, thereby raising concerns of elevated risk of COVID-19-associated mortality among lung cancer patients. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for SARS-CoV-2 entry into lung cells. The single-cell expression landscape of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung cancer patients remains unknown. We sought to delineate single-cell expression profiles of ACE2 and other SARS-CoV-2-related genes in pulmonary tissues of lung adenocarcinoma (LUAD) patients. We examined the expression levels and cellular distribution of ACE2 and SARS-CoV-2-priming proteases TMPRSS2 and TMPRSS4 in 5 LUADs and 14 matched normal tissues by single-cell RNA-sequencing (scRNA-seq) analysis. scRNA-seq of 186,916 cells revealed epithelial-specific expression of ACE2, TMPRSS2, and TMPRSS4. Analysis of 70,030 LUAD- and normal-derived epithelial cells showed that ACE2 levels were highest in normal alveolar type 2 (AT2) cells and that TMPRSS2 was expressed in 65% of normal AT2 cells. Conversely, the expression of TMPRSS4 was highest and most frequently detected (75%) in lung cells with malignant features. ACE2-positive cells co-expressed genes implicated in lung pathobiology, including COPD-associated HHIP, and the scavengers CD36 and DMBT1. Notably, the viral scavenger DMBT1 was significantly positively correlated with ACE2 expression in AT2 cells. We describe normal and tumor lung epithelial populations that express SARS-CoV-2 receptor and proteases, as well as major host defense genes, thus comprising potential treatment targets for COVID-19 particularly among lung cancer patients.

The original version of this Article contained an error in Fig. 2, in which the left y-axis labels ‘tDNA’ and ‘ctDNA’ were inadvertently inverted. This has been corrected in the PDF and HTML versions of the Article.