Immunogenic Chemotherapy Sensitizes Tumors to Checkpoint Blockade Therapy.

Checkpoint blockade immunotherapies can be extraordinarily effective, but might benefit only the minority of patients whose tumors are pre-infiltrated by T cells. Here, using lung adenocarcinoma mouse models, including genetic models, we show that autochthonous tumors that lacked T cell infiltration and resisted current treatment options could be successfully sensitized to host antitumor T cell immunity when appropriately selected immunogenic drugs (e.g., oxaliplatin combined with cyclophosphamide for treatment against tumors expressing oncogenic Kras and lacking Trp53) were used. The antitumor response was triggered by direct drug actions on tumor cells, relied on innate immune sensing through toll-like receptor 4 signaling, and ultimately depended on CD8(+) T cell antitumor immunity. Furthermore, instigating tumor infiltration by T cells sensitized tumors to checkpoint inhibition and controlled cancer durably. These findings indicate that the proportion of cancers responding to checkpoint therapy can be feasibly and substantially expanded by combining checkpoint blockade with immunogenic drugs.

Thoracic nuclear protein in testis (NUT) carcinoma: expanded pathological spectrum with expression of thyroid transcription factor-1 and neuroendocrine markers.

AIMS: Nuclear protein in testis (NUT) carcinoma, an aggressive tumour driven by NUTM1 rearrangements, often involves the lung/mediastinum and shows squamous differentiation. We encountered an index patient with a thoracic NUT carcinoma diagnosed by molecular testing, showing extensive pleural involvement and diffuse thyroid transcription factor-1 (TTF-1) expression, initially suggestive of lung adenocarcinoma with pseudomesotheliomatous growth. We thus gathered an institutional series of thoracic NUT carcinomas to examine their pathological spectrum. METHODS AND RESULTS: We searched for thoracic NUT carcinomas in our surgical pathology files and in 2289 consecutive patients with primary thoracic tumours investigated with RNA-based assays. We performed NUT immunohistochemistry on 425 additional lung adenocarcinomas. Collectively, we identified six patients (five men and one woman; age 31-80 years; four never-smokers) with thoracic NUT carcinomas confirmed by molecular testing (including five with positive NUT immunohistochemistry). They died at 2.3-12.9 months (median, 2.8 months) after presentation. Two patients were diagnosed by histopathological assessment, and the remaining four (including the index patient) were diagnosed by molecular testing. Analysis of the index case revealed expression of multiple neuroendocrine markers and TTF-1; no ultrastructural evidence of neuroendocrine differentiation was noted. No additional NUT-positive cases were found by immunohistochemical screening. CONCLUSIONS: Although NUT carcinoma classically shows squamous differentiation, it can rarely express TTF-1 (even diffusely) and/or multiple neuroendocrine markers. This immunophenotypic spectrum may lead to diagnostic confusion with pulmonary adenocarcinoma, neuroendocrine tumour, and others. To circumvent this pitfall, NUT immunohistochemistry and/or NUTM1 molecular testing should be considered in primitive-appearing tumours, regardless of their immunophenotypic features.

Differential expression of PD-L1 and IDO1 in association with the immune microenvironment in resected lung adenocarcinomas.

Like programmed cell death ligand 1 (PD-L1), indoleamine 2,3-dioxygenase 1 (IDO1) is known to exert immunosuppressive effects and be variably expressed in human lung cancer. However, IDO1 expression has not been well studied in lung adenocarcinoma. PD-L1 and IDO1 expression was evaluated in 261 resected lung adenocarcinomas using tissue microarrays and H-scores (cutoff: 5). We compared IDO1 and PD-L1 expression with clinical features, tumor-infiltrating lymphocytes, HLA class I molecule expression, molecular alterations, and patient outcomes. There was expression of PD-L1 in 89 (34%) and IDO1 in 74 (29%) cases, with co-expression in 49 (19%). Both PD-L1 and IDO1 were significantly associated with smoking, aggressive pathologic features, and abundant CD8+ and T-bet+ (Th1 marker) tumor-infiltrating lymphocytes. PD-L1 expression was also associated with preserved HLA class I molecule expression (p = 0.002). Compared to PD-L1+/IDO1+ and PD-L1+ only cases, significantly fewer IDO1+ only cases had abundant CD8+ and T-bet+ tumor-infiltrating lymphocytes (p < 0.001, respectively). PD-L1 expression was significantly associated with EGFR wild-type (p < 0.001) and KRAS mutants (p = 0.021), whereas isolated IDO1 expression was significantly associated with EGFR mutations (p = 0.007). As for survival, PD-L1 was a significant predictor of decreased progression-free and overall survival by univariate but not multivariate analysis, while IDO1 was not associated with progression-free or overall survival. Interestingly, there was a significant difference in the 5-year progression-free and overall survival (p = 0.004 and 0.038, respectively), where cases without PD-L1 or IDO1 expression had the longest survival, and those with PD-L1 alone had the shortest survival. While PD-L1+/-IDO1 expression is observed in association with HLA class I expression, cytotoxic T lymphocyte/Th1 microenvironments, EGFR wild-type, and KRAS mutations, isolated IDO1 expression does not demonstrate these associations, suggesting that IDO1 may serve a distinct immunosuppressive role in lung adenocarcinomas. Thus, further investigation of IDO1 may demonstrate its role as a potential biomarker for patients who undergo anti-PD-1/PD-L1 therapy.

PD-L1 expression in colorectal cancer is associated with microsatellite instability, BRAF mutation, medullary morphology and cytotoxic tumor-infiltrating lymphocytes.

Programmed cell death 1 (PD-1) and its ligand (PD-L1) are key suppressors of the cytotoxic immune response. PD-L1 expression on tumor cells may be induced by the immune microenvironment, resulting in immune escape (adaptive immune resistance), and an adverse prognosis in many malignancies. In colorectal carcinoma the response to PD-1/PD-L1 inhibition is correlated with microsatellite instability. However, little is known about the clinicopathologic, molecular, and prognostic characteristics of colorectal carcinoma with PD-L1 expression. We performed immunohistochemistry for PD-L1 on 181 cases of colorectal carcinoma with known microsatellite instability and mutational status, and correlated PD-L1 expression with clinicopathologic features including tumor-infiltrating lymphocyte burden/immunophenotype, tumor mutational profile, and disease-specific survival. PD-L1 was expressed in tumors from 16 patients (9%) who were more often older (P=0.006) and female (P=0.035), with tumors exhibiting a larger size (P=0.013), but lower stage (P<0.001). PD-L1 expression was associated with increased CD8 and TBET-positive tumor-infiltrating lymphocytes, medullary phenotype, poor differentiation, microsatellite instability, BRAF mutation (P<0.001 for each), and a lower frequency of KRAS mutation (P=0.012). On multivariate analysis, PD-L1 expression was associated with medullary morphology and frequent CD8-positive tumor-infiltrating lymphocytes, suggesting adaptive immune resistance. PD-L1 positivity was not predictive of survival in the entire cohort, but it was associated with a lower disease-specific survival within the microsatellite-instability high cohort. PD-L1 expression in colorectal carcinoma is associated with clinicopathologic and molecular features of the serrated pathway of colorectal carcinogenesis, and is associated with a worse outcome within microsatellite-unstable tumors. These findings support the role of PD-L1 expression in providing normally immunogenic colorectal carcinoma a means of immune evasion and a more aggressive biology, provide a potential mechanistic explanation for the favorable response of microsatellite-unstable colorectal carcinoma to PD-1/PD-L1 pathway blockade, and suggest potential predictive and prognostic roles of PD-L1 immunohistochemistry in colorectal carcinoma.

Successful Treatment of an Aggressive Tracheal Malignancy With Immunotherapy.

Immune checkpoint inhibitors are emerging as therapeutic options for oncology patients in whom conventional treatment regimens have failed. These immunotherapies counteract tumor-induced tolerance and have been shown to be effective in thoracic malignancies, including non-small cell lung cancer (NSCLC). This report highlights the successful use of nivolumab-an immunotherapeutic agent that binds to proteins involved in T-cell proliferation-for the management of recurrent tracheal squamous cell cancer after exhaustion of conventional surgical, chemotherapeutic, and radiation therapy options. Observations provide a strong indication of the potential value of checkpoint inhibitors for managing a wide array of thoracic malignancies.

Programmed Cell Death Ligand (PD-L1) Expression in Stage II and III Lung Adenocarcinomas and Nodal Metastases.

INTRODUCTION: Programmed death ligand 1 (PD-L1) expression determined by immunohistochemistry (IHC) may serve as a predictive biomarker for anti-PD-1/PD-L1 therapies; however, little is known about intertumoral heterogeneity of PD-L1 expression determined by IHC in lung adenocarcinomas (ADCs), and there have been conflicting results on the prognostic role of PD-L1 expression in ADCs. METHODS: PD-L1 expression was evaluated in resected stage II and III ADCs by using various cutoffs and correlated with clinicopathologic parameters and survival. PD-L1 expression was also compared between the primary tumor and lymph node metastases. RESULTS: There were 109 study cases. PD-L1 expression was seen in 56 (51%), 43 (39%), and 19 (17%) when cutoffs of at least 1%, at least 5%, and at least 50%, respectively, were used. Abundant intratumoral CD8-positive T cells were a significant predictor of the expression in the primary tumor, with cutoffs of 1% and 5% (p < 0.001 for both) by multivariate analysis, whereas they were a nonsignificant predictor of the expression with a 50% cutoff (p = 0.076). PD-L1 expression was concordant between the primary tumor and nodal metastasis in most of the cases, but it was discrepant in up to 38%. The discrepancy was attributed in part to different predominant histologic patterns between the primary and metastatic tumors. In the entire cohort, PD-L1 expression with all cutoffs had no bearing on 5-year recurrence-free survival. CONCLUSIONS: PD-L1 expression is associated with abundant intratumoral CD8-positive T cells in resected ADCs, suggesting a predictive role of PD-L1 expression in anti-PD-1/PD-L1 therapies; however, the possible intertumoral heterogeneity of PD-L1 expression raises a concern about selecting the most appropriate sample for PD-L1 IHC.

Napsin A/p40 antibody cocktail for subtyping non-small cell lung carcinoma on cytology and small biopsy specimens.

BACKGROUND: Subtyping non-small cell lung carcinomas (NSCLC) into adenocarcinoma (ACA) or squamous cell carcinoma (SQCC) is important for treatment and specimen triage for molecular studies. To preserve tissue for molecular studies in cytology/small biopsy specimens, a 2-antibody cocktail for NSCLC subtyping was developed. METHODS: Markers for lung ACA (thyroid transcription factor 1 and napsin A) and SQCC (cytokeratin 5/6 and p40) were evaluated on tissue microarrays (TMAs) with 143 ACA and 98 SQCC specimens. The napsin A/p40 combination was selected for NSCLC subtyping and validated on the TMA as well as on a cohort of cell block/small biopsy specimens from 80 poorly differentiated NSCLCs. RESULTS: Using TMA analysis, the napsin A-positive (+)/p40± immunophenotype identified ACA with 94% sensitivity and 100% specificity, whereas the napsin A (negative)-/p40+ immunophenotype identified SQCC with 100% sensitivity and specificity. On the validation cohort of 80 cell block and small biopsy specimens, the napsin A/p40 cocktail accurately subtyped 63 of 70 NSCLC (90%) as ACA or SQCC using the subsequent surgical resection as reference histology. Of the remaining 17 cases, 15 were classified as NSCLC-not otherwise specified based on a napsin A-/p40- immunophenotype; their corresponding resections were diagnosed as ACA (7 cases), large cell carcinoma (7 cases), or pleomorphic carcinoma (1 case). Two additional large cell carcinoma cases showed a napsin A-/p40+ or napsin A+/p40+ profile in the preoperative cell block/small biopsy sample. CONCLUSIONS: A napsin A/p40 cocktail can accurately subtype NSCLC into ACA and SQCC in most cell block/small biopsy specimens of poorly differentiated NSCLC. In the minority of cases in which the napsin A/p40 immunophenotype is indeterminate, additional stains may be necessary for precise classification. Cancer Cytopathol 2016;124:472-84. © 2016 American Cancer Society.

Programmed Cell Death Ligand 1 Expression in Resected Lung Adenocarcinomas: Association with Immune Microenvironment.

INTRODUCTION: Programmed cell death ligand 1 (PD-L1) expression on tumor cells can be upregulated via activation of CD8+ cytotoxic T lymphocytes (CTLs) or the T helper cell (Th1) pathway, counterbalancing the CTL/Th1 microenvironment. However, PD-L1 expression in association with subtypes of tumor-associated lymphocytes and molecular alterations has not been well characterized in lung adenocarcinomas. METHODS: PD-L1 expression was evaluated in 261 resected lung adenocarcinomas using tissue microarrays and various scoring systems, and was correlated with clinicopathologic/molecular features, including the extent/subtype of tumor-associated lymphocytes (i.e., CD8, T-bet [Th1 transcription factor], and GATA3 [Th2 transcription factor]), and patient outcomes. RESULTS: PD-L1 expression was present in 129 (49%), 95 (36.5%), and 62 (24%) cases using cutoffs of ≥1%, ≥5%, and ≥50%, respectively, 98 (38%) by H score and 72 (28%) by immune score. PD-L1 expression was associated with abundant CD8+ and/or T-bet+ tumor-infiltrating lymphocytes and EGFR wild-type, significant smoking history, and aggressive pathologic features. In addition, concurrent PD-L1 expression and abundant CD8+ tumor-associated lymphocytes were seen in 25% of KRAS mutants or cases with no alterations by clinical molecular testing as opposed to only 7.4% of EGFR mutants. PD-L1 expression was significantly associated with decreased progression-free and overall survival rates by univariate analysis, but not by multivariate analysis. CONCLUSION: PD-L1 expression in resected lung adenocarcinomas is frequently observed in the presence of CTL/Th1 microenvironment, in particular in those with KRAS mutations or no common molecular alterations, suggesting that blockade of the PD-1/PD-L1 axis may be a promising treatment strategy to reinstitute active immune response for at least a subset of such patient populations.

EGFR Mutations and ALK Rearrangements Are Associated with Low Response Rates to PD-1 Pathway Blockade in Non-Small Cell Lung Cancer: A Retrospective Analysis.

PURPOSE: PD-1 inhibitors are established agents in the management of non-small cell lung cancer (NSCLC); however, only a subset of patients derives clinical benefit. To determine the activity of PD-1/PD-L1 inhibitors within clinically relevant molecular subgroups, we retrospectively evaluated response patterns among EGFR-mutant, anaplastic lymphoma kinase (ALK)-positive, and EGFR wild-type/ALK-negative patients. EXPERIMENTAL DESIGN: We identified 58 patients treated with PD-1/PD-L1 inhibitors. Objective response rates (ORR) were assessed using RECIST v1.1. PD-L1 expression and CD8(+) tumor-infiltrating lymphocytes (TIL) were evaluated by IHC. RESULTS: Objective responses were observed in 1 of 28 (3.6%) EGFR-mutant or ALK-positive patients versus 7 of 30 (23.3%) EGFR wild-type and ALK-negative/unknown patients (P = 0.053). The ORR among never- or light- (≤10 pack years) smokers was 4.2% versus 20.6% among heavy smokers (P = 0.123). In an independent cohort of advanced EGFR-mutant (N = 68) and ALK-positive (N = 27) patients, PD-L1 expression was observed in 24%/16%/11% and 63%/47%/26% of pre-tyrosine kinase inhibitor (TKI) biopsies using cutoffs of ≥1%, ≥5%, and ≥50% tumor cell staining, respectively. Among EGFR-mutant patients with paired, pre- and post-TKI-resistant biopsies (N = 57), PD-L1 expression levels changed after resistance in 16 (28%) patients. Concurrent PD-L1 expression (≥5%) and high levels of CD8(+) TILs (grade ≥2) were observed in only 1 pretreatment (2.1%) and 5 resistant (11.6%) EGFR-mutant specimens and was not observed in any ALK-positive, pre- or post-TKI specimens. CONCLUSIONS: NSCLCs harboring EGFR mutations or ALK rearrangements are associated with low ORRs to PD-1/PD-L1 inhibitors. Low rates of concurrent PD-L1 expression and CD8(+) TILs within the tumor microenvironment may underlie these clinical observations. Clin Cancer Res; 22(18); 4585-93. ©2016 AACRSee related commentary by Gettinger and Politi, p. 4539.

Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations.

UNLABELLED: BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance. SIGNIFICANCE: RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.