Jaime Rodriguez Canales

Co-occurring genomic alterations define major subsets of KRAS - mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities

UNLABELLED The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. SIGNIFICANCE Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B-the latter coupled with low TTF1 expression-define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities.

Phase II trial of everolimus and erlotinib in patients with platinum-resistant recurrent and/or metastatic head and neck squamous cell carcinoma

BACKGROUND Enhanced phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the key adaptive changes accounting for epidermal growth factor receptor (EGFR) inhibitor-resistant growth in head and neck squamous cell carcinoma (HNSCC). We designed a phase II clinical trial of EGFR tyrosine kinase inhibitor (TKI), erlotinib, in association with the mTOR inhibitor, everolimus, based on the hypothesis that the downstream effects of Akt through inhibition of mTOR may enhance the effectiveness of the EGFR-TKI in patients with recurrent/metastatic HNSCC. PATIENTS AND METHODS Patients with histologically or cytologically confirmed platinum-resistant HNSCC received everolimus 5 mg and erlotinib 150 mg daily orally until disease progression, intolerable toxicity, investigator or patient decision. Cytokines and angiogenic factors profile, limited mutation analysis and p16 immunohistochemistry status were included in the biomarker analysis. RESULTS Of the 35 assessable patients, 3 (8%) achieved partial response at 4 weeks, 1 confirmed at 12 weeks; overall response rate at 12 weeks was 2.8%. Twenty-seven (77%) patients achieved disease stabilization at 4 weeks, 11 (31%) confirmed at 12 weeks. Twelve-week progression-free survival (PFS) was 49%, median PFS 11.9 weeks and median overall survival (OS) 10.25 months. High neutrophil gelatinase lipocalin (P = 0.01) and vascular endothelial growth factor (VEGF) (P = 0.04) plasma levels were significantly associated with worse OS. CONCLUSIONS The combination of erlotinib and everolimus did not show significant benefit in unselected patients with platinum-resistant metastatic HNSCC despite a manageable toxicity profile. Markers of tumor invasion and hypoxia identify a group of patients with particularly poor prognosis. CLINICAL TRIAL NUMBER NCT00942734.

ZEB1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis

Lung cancer is the leading cause of cancer-related deaths, primarily due to distant metastatic disease. Metastatic lung cancer cells can undergo an epithelial-to-mesenchymal transition (EMT) regulated by various transcription factors, including a double-negative feedback loop between the microRNA-200 (miR-200) family and ZEB1, but the precise mechanisms by which ZEB1-dependent EMT promotes malignancy remain largely undefined. Although the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. Investigating the collaborative effect of EMT and ECM in the metastatic process reveals increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in ZEB1-activated mesenchymal lung cancer cells. In addition, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and ZEB1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principal isoform that crosslinks and stabilizes insoluble collagen deposition in tumor tissues. In turn, focal adhesion formation and FAK/SRC signaling is activated in mesenchymal tumor cells by crosslinked collagen in the ECM. Our study is the first to validate direct regulation of LOX and LOXL2 by the miR-200/ZEB1 axis, defines a novel mechanism driving tumor metastasis, delineates collagen as a prognostic marker, and identifies LOXL2 as a potential therapeutic target against tumor progression.

Sarcomatoid renal cell carcinoma has a distinct molecular pathogenesis, driver mutation profile and transcriptional landscape.

Purpose: Sarcomatoid renal cell carcinoma (SRCC) ranks among the most aggressive clinicopathologic phenotypes of RCC. However, the paucity of high-quality, genome-wide molecular examinations of SRCC has hindered our understanding of this entity. Experimental Design: We interrogated the mutational, copy number, and transcriptional characteristics of SRCC and compared these data with those of nonsarcomatoid RCC (RCC). We evaluated whole-exome sequencing, single-nucleotide polymorphism, and RNA sequencing data from patients with SRCC (n = 65) and RCC (n = 598) across different parent RCC subtypes, including clear-cell RCC, papillary RCC, and chromophobe RCC subtypes. Results: SRCC was molecularly discrete from RCC and clustered according to its parent RCC subtype, though with upregulation of TGFβ signaling across all subtypes. The epithelioid (E-) and spindled (S-) histologic components of SRCC did not show differences in mutational load among cancer-related genes despite a higher mutational burden in S-. Notably, sarcomatoid clear-cell RCC (SccRCC) showed significantly fewer deletions at 3p21-25, a lower rate of two-hit loss for VHL and PBRM1, and more mutations in PTEN, TP53, and RELN compared with ccRCC. A two-hit loss involving VHL predicted for ccRCC and a better prognosis, whereas mutations in PTEN, TP53, or RELN predicted for SccRCC and worse prognosis. Conclusions: SRCC segregates by parent subtype, and SccRCC has a fundamentally different early molecular pathogenesis, usually lacking the classic 3p21-25 deletion and showing distinctive mutational and transcriptional profiles. These features prompt a more precise molecular classification of RCC, with diagnostic, prognostic, and therapeutic implications. Clin Cancer Res; 23(21); 6686–96. ©2017 AACR. See related commentary by Bergerot et al., p. 6381

Development of an Immune-Pathology Informed Radiomics Model for Non-Small Cell Lung Cancer

With increasing use of immunotherapy agents, pretreatment strategies for identifying responders and non-responders is useful for appropriate treatment assignment. We hypothesize that the local immune micro-environment of NSCLC is associated with patient outcomes and that these local immune features exhibit distinct radiologic characteristics discernible by quantitative imaging metrics. We assembled two cohorts of NSCLC patients treated with definitive surgical resection and extracted quantitative parameters from pretreatment CT imaging. The excised primary tumors were then quantified for percent tumor PDL1 expression and density of tumor-infiltrating lymphocyte (via CD3 count) utilizing immunohistochemistry and automated cell counting. Associating these pretreatment radiomics parameters with tumor immune parameters, we developed an immune pathology-informed model (IPIM) that separated patients into 4 clusters (designated A-D) utilizing 4 radiomics features. The IPIM designation was significantly associated with overall survival in both training (5 year OS: 61%, 41%, 50%, and 91%, for clusters A-D, respectively, P = 0.04) and validation (5 year OS: 55%, 72%, 75%, and 86%, for clusters A-D, respectively, P = 0.002) cohorts and immune pathology (all P < 0.05). Specifically, we identified a favorable outcome group characterized by low CT intensity and high heterogeneity that exhibited low PDL1 and high CD3 infiltration, suggestive of a favorable immune activated state. We have developed a NSCLC radiomics signature based on the immune micro-environment and patient outcomes. This manuscript demonstrates model creation and validation in independent cohorts.

Spindle cell and pleomorphic (“sarcomatoid”) carcinomas of the lung: an immunohistochemical analysis of 86 cases

Spindle cell and pleomorphic carcinomas are currently grouped among sarcomatoid carcinomas of the lung. Because of their unusual occurrence, these tumors have not been properly assessed by immunohistochemistry. We performed a comprehensive immunohistochemical analysis of 86 of these tumors. Seventy-four pleomorphic carcinomas (57 with differentiated elements) and 12 spindle cell carcinomas were subjected to immunohistochemistry with CAM5.2, cytokeratin (CK) 7, thyroid transcription factor 1, napsin A, CK5/6, p40, desmocollin 3, Sox2, calretinin, and D2-40. The percentage of positive tumor cells as well as the staining intensity were evaluated and scored. The spindle/giant elements were positive for CAM5.2 (93%), CK7 (79%), thyroid transcription factor 1 (41%), napsin A (20%), calretinin (20%), Sox2 (13%), CK5/6 (9%), p40 (8%), D2-40 (6%), and desmocollin 3 (3%). Of 29 cases in which immunohistochemistry was performed on spindle/giant cell and corresponding differentiated elements, 21 (72%) showed a consistent staining pattern in both components, whereas in 8 cases (28%), the immunophenotype in the spindle/giant cells was less lineage-specific than in the differentiated component. Therefore, we consider that 42% of neoplasms otherwise classified as sarcomatoid carcinoma can be reclassified as adenocarcinoma and 14% as squamous cell carcinoma, while the remaining 44% failed to show a more specific immunophenotype. The use of a comprehensive immunohistochemical panel allows reclassification of the majority of sarcomatoid carcinomas as poorly differentiated variants of adenocarcinoma or squamous cell carcinoma. Such reclassification will facilitate clinical management and allow molecular testing and pursuit of targeted treatment strategies. Application of immunohistochemistry should become the standard in the workup of pulmonary sarcomatoid carcinomas.

Programmed Death Cell Ligand 1 (PD-L1) Is Associated With Survival in Stage I Non–Small Cell Lung Cancer

Programmed cell death ligand (PD-L1) has been studied as a predictive immunotherapy biomarker. We investigated PD-L1 expression in the whole tumor and in tumor-infiltrating macrophages (TIMs) as a prognostic biomarker in surgically resected pathologic stage I non-small cell lung cancer. Pathologic specimen from 113 patients with stage I lung cancer (pT1-2a, N0, M0, tumor size 1-5 cm, 79 adenocarcinoma, 34 squamous cell carcinoma) were analyzed for PD-L1 expression in the tumor and in the TIMs using immunohistochemistry and image analysis. Statistics included recursive partitioning, univariable, multivariable, and Kaplan-Meier analyses. Patients whose tumors expressed <4.7% PD-L1 (N = 87) experienced significantly better overall survival (OS) (P = 0.001) than patients with PD-L1 >4.7% (N = 26). Patients with PD-L1 expression in macrophages <6.3% (N = 24) also experienced significantly better (P = 0.005) OS than patients with >6.3% (N = 89). The best outcomes were observed in patients with low PD-L1 expression in both tumor and macrophages with 5-year OS of 94% (N = 17). Contrarily, patients with high PD-L1 expression in both tumor and macrophages experienced 5-year OS of 20% (N = 19). Low PD-L1 expression in the tumor and in the TIMs was independently associated with survival in multivariable analysis (P = 0.000 and P = 0.030, respectively). Lower PD-L1 % expression in the tumor and in the TIMs seems to be associated with significantly better OS in surgically resected stage I lung cancer. Additional studies are needed to validate PD-L1 as a prognostic biomarker in lung cancer and to study the mechanisms of intratumoral immune response.

Development and Validation of a Predictive Radiomics Model for Clinical Outcomes in Stage I Non-Small Cell Lung Cancer

PURPOSE To develop and validate a radiomics signature that can predict the clinical outcomes for patients with stage I non-small cell lung cancer (NSCLC). METHODS AND MATERIALS We retrospectively analyzed contrast-enhanced computed tomography images of patients from a training cohort (n = 147) treated with surgery and an independent validation cohort (n = 295) treated with stereotactic ablative radiation therapy. Twelve radiomics features with established strategies for filtering and preprocessing were extracted. The random survival forests (RSF) method was used to build models from subsets of the 12 candidate features based on their survival relevance and generate a mortality risk index for each observation in the training set. An optimal model was selected, and its ability to predict clinical outcomes was evaluated in the validation set using predicted mortality risk indexes. RESULTS The optimal RSF model, consisting of 2 predictive features, kurtosis and the gray level co-occurrence matrix feature homogeneity2, allowed for significant risk stratification (log-rank P < .0001) and remained an independent predictor of overall survival after adjusting for age, tumor volume and histologic type, and Karnofsky performance status (hazard ratio [HR] 1.27; P < 2e-16) in the training set. The resultant mortality risk indexes were significantly associated with overall survival in the validation set (log-rank P = .0173; HR 1.02, P = .0438). They were also significant for distant metastasis (log-rank P < .05; HR 1.04, P = .0407) and were borderline significant for regional recurrence on univariate analysis (log-rank P < .05; HR 1.04, P = .0617). CONCLUSIONS Our radiomics model accurately predicted several clinical outcomes and allowed pretreatment risk stratification in stage I NSCLC, allowing the choice of treatment to be tailored to each patients individual risk profile.

Combining Immune Checkpoint Blockade and Tumor-Specific Vaccine for Patients With Incurable Human Papillomavirus 16–Related Cancer: A Phase 2 Clinical Trial

Importance In recurrent human papilloma virus (HPV)–driven cancer, immune checkpoint blockade with anti–programmed cell death 1 (PD-1) antibodies produces tumor regression in only a minority of patients. Therapeutic HPV vaccines have produced strong immune responses to HPV-16, but vaccination alone has been ineffective for invasive cancer. Objective To determine whether the efficacy of nivolumab, an anti–PD-1 immune checkpoint antibody, is amplified through treatment with ISA 101, a synthetic long-peptide HPV-16 vaccine inducing HPV-specific T cells, in patients with incurable HPV-16–positive cancer. Design, Setting, and Participants In this single-arm, single-center phase 2 clinical trial, 24 patients with incurable HPV-16–positive cancer were enrolled from December 23, 2015, to December 12, 2016. Duration of follow-up for censored patients was 12.2 months through August 31, 2017. Interventions The vaccine ISA101, 100 &mgr;g/peptide, was given subcutaneously on days 1, 22, and 50. Nivolumab, 3 mg/kg, was given intravenously every 2 weeks beginning day 8 for up to 1 year. Main Outcomes and Measures Assessment of efficacy reflected in the overall response rate (per Response Evaluation Criteria in Solid Tumors, version 1.1). Results Of the 24 patients (4 women and 20 men; 22 with oropharyngeal cancer; median age, 60 years [range, 36-73 years]), the overall response rate was 33% (8 patients; 90% CI, 19%-50%). Median duration of response was 10.3 months (95% CI, 10.3 months to inestimable). Five of 8 patients remain in response. Median progression-free survival was 2.7 months (95% CI, 2.5-9.4 months). Median overall survival was 17.5 months (95% CI, 17.5 months to inestimable). Grades 3 to 4 toxicity occurred in 2 patients (asymptomatic grade 3 transaminase level elevation in 1 patient and grade 4 lipase elevation in 1 patient), requiring discontinuation of nivolumab therapy. Conclusions and Relevance The overall response rate of 33% and median overall survival of 17.5 months is promising compared with PD-1 inhibition alone in similar patients. A randomized clinical trial to confirm the contribution of HPV-16 vaccination to tumoricidal effects of PD-1 inhibition is warranted for further study. Trial Registration ClinicalTrials.gov identifier: NCT02426892

Abstract 627:NLRC5co-mutations are associated with impaired antigen presentation and immune exclusion inKRAS-mutant lung adenocarcinoma

Background: We recently reported that co-occurring genetic events constitute major determinants of the molecular diversity of KRAS-mutant lung adenocarcinoma (LUAC) (Skoulidis et al., Cancer Discovery, 2015). However, comprehensive evaluation of the functional impact of KRAS co-mutations on key cancer hallmarks is thus far lacking. Here, we find that inactivating mutations in NLRC5, a major transactivator of MHC class I molecules, are significantly enriched in KRAS-mutant LUAC and examine the impact of NLRC5 loss on the composition of the tumor immune microenvironment. Methods: Our cohorts consist of 513 LUACs from the TCGA (145 KRAS-mutant), 152 chemotherapy-naive surgically resected LUAC from the PROSPECT cohort, 20 platinum-refractory KRAS-mutant LUAC from the BATTLE-2 clinical trial, as well as a panel of 31 KRAS-mutant NSCLC cell lines. Analysis of immune cell sub-population was performed using automated IF-based enumeration. Antigen presentation score was defined as the geometric mean mRNA expression of HLA-A, HLA-B, HLA-C and β2M. Results: In an unbiased analysis for genes significantly co-mutated with KRAS in LUAC (TCGA cohort) we identified NLRC5 (NLR family, CARD domain containing 5), encoding a recently discovered major transactivator of MHC class I genes (~11% of KRAS-mutant LUAC, odds ratio 2.99, P=0.0197).The spectrum of NLRC5 somatic mutations includes several nonsense and frameshift mutations, as well as missense mutations, many of which are predicted to abrogate normal NLRC5 function. In the TCGA cohort, KRAS/NLRC5 co-mutated tumors exhibited lower antigen presentation score compared to KRAS-mutant NLRC5 wild-type tumors (P=0.0369, t-test). Among KRAS-mutant LUAC from the TCGA, PROSPECT, BATTLE-2 cohorts expression of NLRC5 mRNA correlated tightly with the expression of core antigen presentation pathway components including HLA-A, HLA-B, HLA-C, β2M, TAP1, TAP2, PSMB8 and PSMB9 [in BATTLE-2: HLA-A r=0.7616, P=9.572e-05, HLA-B r=0.834, P=4.884e-06, HLA-C r=0.8029, P=2.036e-05, TAP1 r=0.8189, P=1.009e-05 ]. Similar results were obtained in a panel of 31 KRAS-mutant NSCLC cell line. Thus, both mutational and non-mutational mechanisms can account for NLRC5 inactivation. Finally, in a tumor microarray encompassing surgically resected, chemotherapy naive LUAC (PROSPECT), NLRC5-low tumors (lower tertile for NLRC5 mRNA expression, N=34), exhibited reduced density of infiltrating CD3+ (P Conclusions: Co-mutations in NLRC5, are enriched in KRAS-mutant LUAC and are associated with immune exclusion. KRAS co-mutations can shape the tumor immune micro-environment and may therefore predict for response - or lack thereof- to immunotherapy. Citation Format: Ferdinandos Skoulidis, Taghreed Hirz, Xiang Dong Lee, Jaime Rodriguez Canales, Edwin R. Parra, Pan Tong, Carmen Behrens, Vassiliki A. Papadimitrakopoulou, Jing Wang, Ignacio Wistuba, John V. Heymach. NLRC5 co-mutations are associated with impaired antigen presentation and immune exclusion in KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 627. doi:10.1158/1538-7445.AM2017-627