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Approximately 10% to 15% of human cancers lack detectable telomerase activity, and a subset of these maintain telomere lengths by the telomerase-independent telomere maintenance mechanism termed alternative lengthening of telomeres (ALT). The ALT phenotype, relatively common in subtypes of sarcomas and astrocytomas, has rarely been reported in epithelial malignancies. However, the prevalence of ALT has not been thoroughly assessed across all cancer types. We therefore comprehensively surveyed the ALT phenotype in a broad range of human cancers. In total, two independent sets comprising 6110 primary tumors from 94 different cancer subtypes, 541 benign neoplasms, and 264 normal tissue samples were assessed by combined telomere-specific fluorescence in situ hybridization and immunofluorescence labeling for PML protein. Overall, ALT was observed in 3.73% (228/6110) of all tumor specimens, but was not observed in benign neoplasms or normal tissues. This is the first report of ALT in carcinomas arising from the bladder, cervix, endometrium, esophagus, gallbladder, kidney, liver, and lung. Additionally, this is the first report of ALT in medulloblastomas, oligodendrogliomas, meningiomas, schwannomas, and pediatric glioblastoma multiformes. Previous studies have shown associations between ALT status and prognosis in some tumor types; thus, further studies are warranted to assess the potential prognostic significance and unique biology of ALT-positive tumors. These findings may have therapeutic consequences, because ALT-positive cancers are predicted to be resistant to anti-telomerase therapies.

The authors address a group of loosely associated, characteristically benign soft tissue neoplasms that exhibit partial myoid differentiation. The entities share similarities in morphology and in nomenclature that have historically created confusion. The authors attempt to clarify the distinct architectural patterns and the corresponding immunophenotypic and ultrastructural features that distinguish myofibroma, myopericytoma, myoepithelioma, and myofibroblastoma.

Differentiated (simplex) vulvar intraepithelial neoplasia (VIN) is an uncommon variant of VIN characterized by highly differentiated morphology, making it a potential diagnostic pitfall. It may arise in the background of lichen sclerosus, and unlike most VIN, is not causally associated with human papilloma virus infection. It occurs in an older demographic and is thought to be the precursor of aggressive, invasive vulvar squamous cell carcinoma. For this reason, the timely and accurate diagnosis of this unusual lesion is crucial. The clinical and histologic features of a case of a 70-year-old woman with newly diagnosed differentiated (simplex) VIN arising in a background of long-standing lichen sclerosus is reported, and the historic aspects, current terminology, and diagnostic criteria of differentiated (simplex) VIN are reviewed.

The cooperation of tumor-infiltrating lymphocytes and tertiary lymphoid tissue in early-stage colorectal carcinoma further corroborates the strong immune influences on tumor progression and patient outcome. Immune factors in the tumor microenvironment may warrant inclusion in pathology reports and staging systems for prognostication and prediction of therapeutic response. Clin Cancer Res; 20(8); 2023–5. ©2014 AACR.

Nasopharyngeal carcinoma (NPC) is an EBV-driven tumor that shows variable expression of PD-L1 and ~20% objective response rate to anti-PD-1 monotherapy. As novel immune checkpoint inhibitors are being developed, combination therapies may allow for more effective treatment of both newly diagnosed and relapsed NPC. We characterized the TME in 13 cases of EBV+ NPC from the Johns Hopkins Pathology archives (7 primary tumors, 6 metastases). EBV status was confirmed with EBER ISH. Immunohistochemistry (IHC) was conducted on all cases for CD3, CD4, CD8, CD20, FoxP3, PD-1, PD-L1, LAG-3, TIM-3, GITR, IDO, COX2, and pSTAT3. Gene expression profiling (GEP) was performed on 7 cases with sufficient material (4 primary lesions, 3 metastases), using multiplex qRT-PCR for a panel of 61 candidate immune-related genes (Duffield, Blood Advances 2017). The immunosuppressive ligand PD-L1 was expressed on tumor cells in 11/13 cases (mean 22% tumor cells+, range 0-57%), as well as on infiltrating macrophages. The NPC inflammatory infiltrate was diverse, including CD4+, CD8+, CD20+ and CD68+ cells, and showed variable expression of immune-regulatory molecules. In all 13 cases, lymphocytes expressing PD-1 (mean 36% positive, range 8-70%), LAG-3 (7%; 1-30%) and GITR (12%; 2-27%) were found. FoxP3+ and TIM-3+ lymphocytes were infrequent. IDO+ macrophages were also infrequent; however, 7/13 NPCs showed expression of the immunosuppressive metabolic enzyme IDO by a proportion of tumor cells. Compared to 12 EBV+ Hodgkin lymphomas (Duffield, Blood Advances 2017), EBV+ NPCs demonstrated a Th17 cytokine profile with overexpression of IL1A, IL17RC, IL23A, and IL23R. The generation of pathogenic Th17 responses requires phosphorylation of the STAT3 transcription factor, and IHC confirmed that a subset of inflammatory cells in all NPC cases expressed pSTAT3 (mean 10%; range 1-40%). Additionally, upregulated gene expression characterizing activated macrophages was found (IDO, IL1A, IL12A, LYZ, TLR3). Of note, several molecules upregulated in the NPC TME are capable of inducing PD-L1 expression on human monocytes in vitro, including IL-1A and IL-32-gamma (Taube, Clin Cancer Res 2015; Duffield, Blood Advances 2017). Importantly PTGS2 (COX2), with known proinflammatory and immunosuppressive properties, was over-expressed in NPCs along with the downstream modulator CXCL8 (IL-8); IHC revealed COX2 expression in tumor cells but not infiltrating immune cells. In summary, NPC is characterized by markers of an immunosuppressive TME, including immune checkpoints and metabolic modulators. While these findings should be explored in a larger cohort, they have potential implications for designing combination NPC treatment regimens with anti-PD-1, which might include inhibitors of LAG-3, IDO, IL-17/-23, COX2, and/or IL-8. Funded by the Bristol-Myers Squibb International Immuno-Oncology Network and NCI R01 CA142779 Citation Format: Amy S. Duffield, Maria Libera Ascierto, Robert A. Anders, Janis M. Taube, Tracee L. McMiller, Elizabeth L. Engle, Alan K. Meeker, Alan E. Berger, Drew M. Pardoll, Richard F. Ambinder, Suzanne L. Topalian. The immunosuppressive tumor microenvironment (TME) in nasopharyngeal carcinoma: implications for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4750.

Approximately 20% of advanced SCCHNs respond to anti-PD-(L)1. We aimed to inform rational combination therapy development, which might have greater efficacy, by understanding the potential immune effects of the oncogenic human papillomavirus (HPV) in SCCHN, and by characterizing potentially targetable immune checkpoints in the tumor microenvironment (TME). Immunohistochemistry (IHC) was performed on archival SCCHN specimens from 27 treatment-naive patients. HPV testing with p16 IHC was confirmed by HPV genotyping. IHC for PD-L1 assessed percentage of positive tumor cells, and whether PD-L1 was expressed in an adaptive or constitutive pattern (i.e., PD-L1+ tumor cells juxtaposed to TILs or in the absence of TILs, respectively). PD-L1 on infiltrating immune cells was scored separately. IHC for CD3, CD4, CD8, CD20, CD68, FoxP3, PD-1, PD-L2, LAG-3, TIM-3, GITR and IDO was quantified using Halo image analysis for density of positive cells. Among 27 SCCHN specimens, 14 were HPV(-) and 13 were HPV+ (one HPV58, 12 HPV16). In 19 of 27 specimens (70%), ≥5% of tumor cells had PD-L1 cell surface expression by IHC, including 9 HPV+ and 10 HPV(-) cases. Among 23 tumors expressing any level of PD-L1 (≥1% tumor cells+), 12 displayed an adaptive PD-L1 expression pattern, 5 a constitutive pattern, and 6 were mixed. All tumors were infiltrated by immune cells (ICs). Notably, 26 of 27 specimens (96%) contained PD-L1+ immune cells (range 5-80% of ICs expressing PD-L1). The proportion of PD-L1+ ICs exceeded PD-L1+ tumor cells in 21/26 (81%) cases. Neither the proportion of PD-L1+ tumor cells nor infiltrating immune cells correlated with tumor viral status. When compared to HPV(-) tumors, HPV+ tumors contained significantly higher densities of CD3+, CD4+, CD8+, CD20+, and PD-1+ cells (p500 IDO+ cells/mm2 in 17/27 specimens), IDO was expressed by tumor cells as well as ICs in 12/27 (44%) cases (range 5-95% tumor cells+). In summary, we found that both HPV+ and (-) SCCHNs abundantly express PD-L1 on tumor and/or stromal cells, although HPV+ tumors are more heavily infiltrated by ICs. Multiple other immune checkpoints are expressed in these tumors, providing options for therapeutic co-targeting. Tumor DNA sequencing is in progress to explore the genetic basis for constitutive vs. adaptive PD-L1 expression. These studies are expected to provide a comprehensive portrait of the SCCHN TME, with implications for future immunotherapy development. Funded by the Bristol-Myers Squibb International Immuno-Oncology Network and NCI R01 CA142779 Citation Format: Farah Succaria, Pia Kvistborg, Elizabeth L. Engle, Tracee L. McMiller, Elizabeth Thompson, Alan E. Berger, John Haanen, Suzanne L. Topalian, Janis M. Taube. Characterization of the tumor immune microenvironment in head and neck squamous cell carcinoma (SCCHN) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4693.

Purpose Programmed death-1 (PD-1), an inhibitory receptor expressed on activated T cells, may suppress antitumor immunity. This phase I study sought to determine the safety and tolerability of anti–PD-1 blockade in patients with treatment-refractory solid tumors and to preliminarily assess antitumor activity, pharmacodynamics, and immunologic correlates. Patients and Methods Thirty-nine patients with advanced metastatic melanoma, colorectal cancer (CRC), castrate-resistant prostate cancer, non–small-cell lung cancer (NSCLC), or renal cell carcinoma (RCC) received a single intravenous infusion of anti–PD-1 (MDX-1106) in dose-escalating six-patient cohorts at 0.3, 1, 3, or 10 mg/kg, followed by a 15-patient expansion cohort at 10 mg/kg. Patients with evidence of clinical benefit at 3 months were eligible for repeated therapy. Results Anti–PD-1 was well tolerated: one serious adverse event, inflammatory colitis, was observed in a patient with melanoma who received five doses at 1 mg/kg. One durable complete response (CRC) and two partial responses (PRs; melanoma, RCC) were seen. Two additional patients (melanoma, NSCLC) had significant lesional tumor regressions not meeting PR criteria. The serum half-life of anti–PD-1 was 12 to 20 days. However, pharmacodynamics indicated a sustained mean occupancy of > 70% of PD-1 molecules on circulating T cells ≥ 2 months following infusion, regardless of dose. In nine patients examined, tumor cell surface B7-H1 expression appeared to correlate with the likelihood of response to treatment. Conclusion Blocking the PD-1 immune checkpoint with intermittent antibody dosing is well tolerated and associated with evidence of antitumor activity. Exploration of alternative dosing regimens and combinatorial therapies with vaccines, targeted therapies, and/or other checkpoint inhibitors is warranted.

9505Background: MCC is a rare, aggressive skin cancer commonly associated with the oncogenic Merkel cell polyomavirus (MCPyV). The PD-1/PD-L1 immunosuppressive pathway is often upregulated in MCC, ...

The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility.

The production of engineered cartilage from mesenchymal stem cells is a rapidly developing field. Potential applications include the treatment of degenerative joint disease as well as the treatment of traumatic and surgical bone injury. Prior to clinical application, however, further characterization of the morphology, ultrastructure, biocompatibility, and performance of the engineered tissue is warranted. To achieve this, human mesenchymal stem cells (hMSCs) were grown in vitro in pellet culture for 3 weeks in chondrogenic medium conditions. The resultant engineered cartilage was compared to native adult and fetal tissue. Routine histology, special stains, and ultrastructural and quantitative histomorphometric analyses were performed. The engineered tissue demonstrated a similar chondrocyte phenotype, collagen fibril appearance, and matrix distribution when compared to native cartilage. By histomorphometric analysis, the cell density of the engineered cartilage was between that of native fetal and adult cartilage. The cell-to-matrix ratio and cellular area fraction of engineered cartilage samples was significantly greater than in adult samples, but indistinguishable from fetal cartilage samples, supporting the hypothesis that hMSC-engineered cartilage regeneration may mimic fetal cartilage development.

Mutational activation of the BRAF oncogene is the most common genetic alteration in cutaneous melanoma. Potentially, BRAF mutation analysis of sentinel lymph node (SLN) biopsies could enhance the detection of micrometastases and improve the accuracy of nodal staging for patients with melanoma. Nodal nevi are small aggregates of benign nevus cells that are commonly encountered in the SLNs of patients with melanoma. The status of the BRAF gene in nodal nevi is not known, but this unresolved issue is of critical importance to any future detection strategies that use genetic alterations as biomarkers of metastatic spread. Twenty-six nodal nevi from 26 patients were evaluated for the thymine (T)-->adenine (A) missense mutation at nucleotide 1796 of the BRAF gene using the LigAmp assay, which can detect 1 mutant allele among 10,000 wild-type alleles. For each case, a matching volume of adjacent lymphoid tissue was used as a negative control. BRAF mutations were detected in 13 of the 26 nodal nevi, but in just 1 of the 26 adjacent controls (50% vs. 4%, P

This Case Study describes a 60-year-old woman who presented with an enlarging subcutaneous lesion on her right flank, 6 months after having undergone treatment for transitional cell carcinoma (TCC) of the bladder. The article highlights that rare cutaneous TCC metastases can mimic common benign and inflammatory processes, and the authors suggest treatment options for such patients. Background A 60-year-old female with a 6-month history of muscle-invasive transitional cell carcinoma of the bladder presented with an enlarging subcutaneous lesion involving her right flank. She had previously undergone radical cystectomy, bilateral pelvic lymphadenectomy and ileal orthotopic neobladder reconstruction. Investigations Axial fused fluorodeoxyglucose PET–CT of the chest, abdomen and pelvis, fine needle aspiration with direct ultrasound guidance, excisional biopsy and immunohistochemistry. Diagnosis Subcutaneous and liver metastases of transitional cell carcinoma. Management Wide local excision of the subcutaneous lesion followed by combination gemcitabine–cisplatin chemotherapy. Gemcitabine was administered at a dose of 1,000 mg/m2 on days 1, 8, and 15, and cisplatin was administered at a dose of 75 mg/m2 on day 1; the schedule was repeated every 28 days for three cycles.

Background: Decreased numbers of Langerhans cells (LCs) in the cervix of human immunodeficiency virus (HIV)-infected women are believed to contribute to the progression of human papilloma virus (HPV)-related squamous intraepithelial lesions. However, this impairment of local immunity has not been well studied in the vulva. The objective of this study was to compare the S100+ LC density in high-grade vulvar intraepithelial neoplasia (VIN) in HIV-positive and HIV-negative women. Methods: HIV-positive and HIV-negative patients with high-grade VIN, 48 (55%) and 40 (45%), respectively, were identified by retrospective chart review. Smoking status of patients was noted. The mean LC count per high-power field (HPF) was determined using S100 immunohistochemical staining. In situ hybridization was performed to detect HPV DNA types 16 and 18. Results: Mean S100+ LC counts for HIV-positive and HIV-negative patients were 5.82 and 9.86 per HPF, respectively (p = 0.0026). LC counts in HIV-positive and HIV-negative patients were compared between smoking and nonsmoking groups (HIV-positive p = 0.4812, HIV-negative p = 0.2821). Conclusions: HIV-positive patients with high-grade VIN had significantly lower LC counts compared with HIV-negative patients. This suggests that local vulvar immunity as evaluated by S100+ LCs is impaired in HIV-positive women, possibly contributing to the progression of HPV-related vulvar lesions.

Background Limited adjuvant treatment options exist for patients with high-risk surgically resected melanoma. This first-in-human study investigated the safety, tolerability and immunologic correlates of Melanoma GVAX, a lethally irradiated granulocyte–macrophage colony stimulating factor (GM-CSF)-secreting allogeneic whole-cell melanoma vaccine, administered in the adjuvant setting. Methods Patients with stage IIB-IV melanoma were enrolled following complete surgical resection. Melanoma GVAX was administered intradermally once every 28 days for four cycles, at 5E7 cells/cycle (n = 3), 2E8 cells/cycle (n = 9), or 2E8 cells/cycle preceded by cyclophosphamide 200 mg/m2 to deplete T regulatory cells (Tregs; n = 8). Blood was collected before each vaccination and at 4 and 6 months after treatment initiation for immunologic studies. Vaccine injection site biopsies and additional blood samples were obtained 2 days after the 1st and 4th vaccines. Results Among 20 treated patients, 18 completed 4 vaccinations. Minimal treatment-related toxicity was observed. One patient developed vitiligo and patches of white hair during the treatment and follow-up period. Vaccine site biopsies demonstrated complex inflammatory infiltrates, including significant increases in eosinophils and PD-1+ lymphocytes from cycle 1 to cycle 4 (P

Despite similar components, the heterogeneity of skin characteristics across the human body is enormous. It is classically believed that site-specific fibroblasts in the dermis control postnatal skin identity by modulating the behavior of the surface-overlying keratinocytes in the epidermis. To begin testing this hypothesis, we characterized the gene expression differences between volar (ventral; palmoplantar) and nonvolar (dorsal) human skin. We show that KERATIN 9 (KRT9) is the most uniquely enriched transcript in volar skin, consistent with its etiology in genetic diseases of the palms and soles. In addition, ectopic KRT9 expression is selectively activated by volar fibroblasts. However, KRT9 expression occurs in the absence of all fibroblasts, although not to the maximal levels induced by fibroblasts. Through gain-of-function and loss-of-function experiments, we demonstrate that the mechanism is through overlapping paracrine or autocrine canonical WNT–β-catenin signaling in each respective context. Finally, as an in vivo example of ectopic expression of KRT9 independent of volar fibroblasts, we demonstrate that in the human skin disease lichen simplex chronicus, WNT5a and KRT9 are robustly activated outside of volar sites. These results highlight the complexities of site-specific gene expression and its disruption in skin disease.

Purpose: Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1+ versus PD-L1(−) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1+ melanoma microenvironment coordinately contributing to immunosuppression. Experimental Design: Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1+ versus PD-L1(−) melanomas. Results: Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1+ melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1+ melanomas, involved in CD8+ T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells. Conclusions: These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti–PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies. Clin Cancer Res; 21(17); 3969–76. ©2015 AACR.

Focal tumor cell PD-L1 expression adjacent to TIL can be used as a surrogate marker of an ongoing antitumor host response, which may be unleashed by PD-1 blockade. Tumor cell PD-L1 expression is superior to TIL PD-1 expression and the presence of TIL alone, when predicting response to anti-PD-1 therapy.

Type 2 diabetes is characterized by increased plasma triglyceride levels and a fourfold increase in ischemic heart disease, but the mechanism is unclear. CD36 is a receptor/transporter that binds fatty acids of lipoproteins. CD36 deficiency has been linked with insulin resistance. There is strong evidence of in vivo interaction between platelets and atherogenic lipoproteins suggesting that atherogenic triglyceride-rich lipoproteins, such as VLDL, that are increased in diabetic dyslipidemia are important in this process. This study demonstrates that VLDL binds to the platelet receptor CD36, enhances platelet thromboxane A2 production, and causes increased collagen-mediated platelet aggregation. VLDL enhanced collagen-induced platelet aggregation by 1) shortening the time taken for aggregation to begin (lag time) to 70% of control (P = 0.001); 2) increasing maximum aggregation to 170% of control (P = 0.008); and 3) increasing thromboxane production to 3,318% of control (P = 0.004), where control represents platelets stimulated with collagen (100%). A monoclonal antibody against CD36 attenuated VLDL-enhanced collagen-induced platelet aggregation by 1) inhibiting binding of VLDL to platelets by 75% (P = 0.041); 2) lengthening lag time to 190% (P < 0.001); and 3) decreasing thromboxane production to 8% of control (P < 0.001). In support of this finding, platelets from Cd36-deficient rats showed no increase in aggregation, thromboxane production, and VLDL binding in contrast to platelets from rats expressing CD36. These data suggest that platelet Cd36 has a key role in VLDL-induced collagen-mediated platelet aggregation, possibly contributing to atherothrombosis associated with increased VLDL levels.

8508 Background: Anti-PD-1 therapy produces objective and often durable responses in ~20% of unselected patients (pts) with metastatic non-small cell lung cancer (NSCLC). However, the role of PD-1 blockade in treating resectable NSCLC is unknown. This is the first study to test nivolumab in the neoadjuvant setting. This trial design provides an opportunity to examine anti-PD-1 mechanism of action and immunologic correlates of outcomes. Methods: Patients with Stage IB - IIIA NSCLC received 2 doses of nivolumab 3mg/kg over 4 weeks before surgery. The primary endpoint was safety in 20 patients with resected NSCLC. Efficacy was explored using objective pathologic response criteria. Correlative studies of the tumor immune microenvironment, tumor mutation and predicted neoantigen loads, and changes in T cell receptor (TCR) clonality in tumor and blood pre and post treatment were conducted. Results: 22 pts were treated. Nivolumab was well-tolerated and no surgeries were delayed. 1 pt withdrew from study preop without progression or toxicity. Among the 21 attempted resections, 1 tumor was unresectable. 9/21 (43%, 95% CI 24-63%) had a major pathologic response ( < 10% viable tumor cells in resection specimen). With a median postop follow-up of 9 months, 18 pts (86%) remain alive and recurrence free. Pre-treatment tumor exome sequencing showed a correlation between both tumor mutation and predicted neoantigen loads with pathologic response. Multiplex immunohistochemistry of pre- and post-treatment tumors showed an influx of PD-1+CD8+ T cells into responding tumors. TCR sequencing demonstrated that expanded peripheral T cell clones after treatment match clones found in the tumor. Conclusions: Neoadjuvant nivolumab in resectable NSCLC did not delay surgery. Major pathologic response rate was encouraging and compares favorably to outcomes with cisplatin-based neoadjuvant chemotherapy. Genomic analyses suggest that higher mutational and neoantigen burden could result in deeper pathologic response. Immunologic analyses support the detection of intra-tumoral T cell clones in the blood after treatment with nivolumab and may provide further insight into the molecular and immunologic features of response and non-response to PD-1 blockade. Clinical trial information: NCT02259621.

PD-L1 expression in melanoma correlates with response to PD-1 pathway–blocking antibodies. Aberrant tumor-cell PD-L1 expression may be oncogene driven and/or induced by IFNγ. Melanomas express PD-L1 in association with tumor-infiltrating lymphocytes (TIL), but the potential contribution of the BRAF V600E mutation (BRAFmut) to induced PD-L1 expression has not been determined. Fifty-two archival melanocytic lesions were assessed for PD-L1 expression, TIL infiltration, and BRAFmut simultaneously. IFNγ-induced PD-L1 expression in cultured melanomas was assessed in parallel according to BRAF status. Melanocyte PD-L1 expression was observed in 40% of specimens, and BRAFmut was observed in 42% of specimens, but no significant concordance was found between these variables. Almost all melanocytes displaying PD-L1 expression were observed to be adjacent to TILs, irrespective of BRAF status. TIL− lesions were not more likely to be associated with BRAFmut, when compared with TIL+ lesions. Baseline expression of PD-L1 by melanoma cell lines was virtually nil, regardless of BRAFmut status, and the intensity of IFN-induced PD-L1 expression in melanoma cell lines likewise did not correlate with BRAF mutational status. PD-L1 expression in melanocytic lesions does not correlate with the BRAFmut. Thus, distinct populations of melanoma patients will likely benefit from BRAF inhibitors versus PD-1 pathway blockade. Cancer Immunol Res; 3(2); 110–5. ©2014 AACR.

We examined the immune microenvironment of primary colorectal cancer using immunohistochemistry, laser capture microdissection/qRT-PCR, flow cytometry, and functional analysis of tumor-infiltrating lymphocytes. A subset of colorectal cancer displayed high infiltration with activated CD8+ cytotoxic T lymphocyte (CTL) as well as activated Th1 cells characterized by IFNγ production and the Th1 transcription factor TBET. Parallel analysis of tumor genotypes revealed that virtually all of the tumors with this active Th1/CTL microenvironment had defects in mismatch repair, as evidenced by microsatellite instability (MSI). Counterbalancing this active Th1/CTL microenvironment, MSI tumors selectively demonstrated highly upregulated expression of multiple immune checkpoints, including five—PD-1, PD-L1, CTLA-4, LAG-3, and IDO—currently being targeted clinically with inhibitors. These findings link tumor genotype with the immune microenvironment, and explain why MSI tumors are not naturally eliminated despite a hostile Th1/CTL microenvironment. They further suggest that blockade of specific checkpoints may be selectively efficacious in the MSI subset of colorectal cancer. Significance: The findings reported in this article are the first to demonstrate a link between a genetically defined subtype of cancer and its corresponding expression of immune checkpoints in the tumor microenvironment. The mismatch repair–defective subset of colorectal cancer selectively upregulates at least five checkpoint molecules that are targets of inhibitors currently being clinically tested. Cancer Discov; 5(1); 43–51. ©2014 AACR. See related commentary by Xiao and Freeman, p. 16 This article is highlighted in the In This Issue feature, p. 1

Chordomas are rare malignant tumors that are postulated to arise from remnants of the notochord. Currently, the interaction between chordomas and the host immune system is poorly understood. The checkpoint protein, PD-1 is expressed by circulating lymphocytes and is a marker of activation and exhaustion. Its ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), are expressed on a variety of human cancers; however this pathway has not been previously reported in chordomas. We used flow cytometric and RT-PCR analysis in three established primary and recurrent chordoma cell lines (U-CH1, U-CH2, and JHC7) as well as immunohistochemical analysis of chordoma tissues from 10 patients to identify and localize expression of PD-1 pathway proteins. PD-1 ligands are not constitutively expressed by chordoma cells, but their expression is induced in the setting of pro-inflammatory cytokines in all cell lines examined. In paraffin embedded tissues, we found that tumor infiltrating lymphocytes expressed PD-1 in 3/6 cases. We also found that, although chordoma cells did not express significant levels of PD-L1, PD-L1 expression was observed on tumor-infiltrating macrophages and tumor infiltrating lymphocytes. Our study suggests that PD-1, PD-L1, and PD-L2 are present in the microenvironment of a subset of chordomas analyzed. Future studies are needed to evaluate the contribution of the PD-1 pathway to the immunosuppressive microenvironment of chordomas.

Numerous complications can be observed in the post-transplant period among recipients of hematopoietic stem cells including graft-versus-host disease (GVHD), which is associated with significant morbidity and mortality. On the other hand, graft versus tumor (GVT) effect is a well-described phenomenon in patients with hematologic malignancies and has also been reported in renal cell cancer, ovarian cancer, breast carcinoma, and melanoma. We describe spontaneous regression of a cutaneous invasive squamous cell carcinoma and multifocal atypical intraepidermal proliferations in a patient with chronic graft-versus-host disease following initiation of extracorporeal photopheresis (ECP). This observation raises questions regarding the GVT in cutaneous neoplasms and potential immunomodulatory effects of ECP.

Purpose: Immunomodulatory drugs differ in mechanism-of-action from directly cytotoxic cancer therapies. Identifying factors predicting clinical response could guide patient selection and therapeutic optimization. Experimental Design: Patients (N = 41) with melanoma, non–small cell lung carcinoma (NSCLC), renal cell carcinoma (RCC), colorectal carcinoma, or castration-resistant prostate cancer were treated on an early-phase trial of anti–PD-1 (nivolumab) at one institution and had evaluable pretreatment tumor specimens. Immunoarchitectural features, including PD-1, PD-L1, and PD-L2 expression, patterns of immune cell infiltration, and lymphocyte subpopulations, were assessed for interrelationships and potential correlations with clinical outcomes. Results: Membranous (cell surface) PD-L1 expression by tumor cells and immune infiltrates varied significantly by tumor type and was most abundant in melanoma, NSCLC, and RCC. In the overall cohort, PD-L1 expression was geographically associated with infiltrating immune cells (P < 0.001), although lymphocyte-rich regions were not always associated with PD-L1 expression. Expression of PD-L1 by tumor cells and immune infiltrates was significantly associated with expression of PD-1 on lymphocytes. PD-L2, the second ligand for PD-1, was associated with PD-L1 expression. Tumor cell PD-L1 expression correlated with objective response to anti–PD-1 therapy, when analyzing either the specimen obtained closest to therapy or the highest scoring sample among multiple biopsies from individual patients. These correlations were stronger than borderline associations of PD-1 expression or the presence of intratumoral immune cell infiltrates with response. Conclusions: Tumor PD-L1 expression reflects an immune-active microenvironment and, while associated other immunosuppressive molecules, including PD-1 and PD-L2, is the single factor most closely correlated with response to anti–PD-1 blockade. Clin Cancer Res; 20(19); 5064–74. ©2014 AACR.

Basal cell carcinoma (BCC) of the vulva is rare and may be confused with the much more commonly encountered high-risk human papillomavirus (HPV)-related basaloid squamous cell carcinoma (SCC). The HPV status of BCCs is not well established. This study assesses the utility of p16 and BerEP4 expression patterns and high-risk HPV detection for distinction of these tumors. Thirteen cases of vulvar BCC were analyzed by immunohistochemistry for p16 and BerEP4 expression. HPV status was assessed by in situ hybridization (ISH) with a high-risk HPV wide-spectrum probe and HPV 16 and 18 type-specific probes. All tumors (13/13) demonstrated patchy p16 positivity, with

Purpose Programmed cell death 1 (PD-1) is an inhibitory receptor expressed by activated T cells that downmodulates effector functions and limits the generation of immune memory. PD-1 blockade can mediate tumor regression in a substantial proportion of patients with melanoma, but it is not known whether this is associated with extended survival or maintenance of response after treatment is discontinued. Patients and Methods Patients with advanced melanoma (N = 107) enrolled between 2008 and 2012 received intravenous nivolumab in an outpatient setting every 2 weeks for up to 96 weeks and were observed for overall survival, long-term safety, and response duration after treatment discontinuation. Results Median overall survival in nivolumab-treated patients (62% with two to five prior systemic therapies) was 16.8 months, and 1- and 2-year survival rates were 62% and 43%, respectively. Among 33 patients with objective tumor regressions (31%), the Kaplan-Meier estimated median response duration was 2 years. Seventeen patients discontinued therapy for reasons other than disease progression, and 12 (71%) of 17 maintained responses off-therapy for at least 16 weeks (range, 16 to 56+ weeks). Objective response and toxicity rates were similar to those reported previously; in an extended analysis of all 306 patients treated on this trial (including those with other cancer types), exposure-adjusted toxicity rates were not cumulative. Conclusion Overall survival following nivolumab treatment in patients with advanced treatment–refractory melanoma compares favorably with that in literature studies of similar patient populations. Responses were durable and persisted after drug discontinuation. Long-term safety was acceptable. Ongoing randomized clinical trials will further assess the impact of nivolumab therapy on overall survival in patients with metastatic melanoma.

Primary effusion lymphoma (PEL) is a rare and aggressive lymphoma that arises in the context of immunosuppression and is characterized by co-infection with Epstein–Barr virus (EBV) and human herpesvirus-8/Kaposi sarcoma-associated herpesvirus (HHV-8/KSHV). It was originally described as arising in body cavity effusions, but presentation as a mass lesion (extracavitary PEL) is now recognized. Here, we describe a case of PEL with an initial presentation as an intravascular lymphoma with associated skin lesions. The patient was a 53-year-old man with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) who presented with fevers, weight loss and skin lesions concerning for Kaposi sarcoma (KS). A skin biopsy revealed no evidence of KS; however, dermal vessels contained large atypical cells that expressed CD31 and plasma cell markers but lacked most B- and T-cell antigens. The atypical cells expressed EBV and HHV-8. The patient subsequently developed a malignant pleural effusion containing the same neoplastic cell population. The findings in this case highlight the potential for unusual intravascular presentations of PEL in the skin as well as the importance of pursuing microscopic diagnosis of skin lesions in immunosuppressed patients.

Numerous complications can be observed in the post-transplant period among recipients of hematopoietic stem cells including graft-versus-host disease (GVHD), which is associated with significant morbidity and mortality. On the other hand, graft versus tumor (GVT) effect is a well-described phenomenon in patients with hematologic malignancies and has also been reported in renal cell cancer, ovarian cancer, breast carcinoma, and melanoma. We describe spontaneous regression of a cutaneous invasive squamous cell carcinoma and multifocal atypical intraepidermal proliferations in a patient with chronic graft-versus-host disease following initiation of extracorporeal photopheresis (ECP). This observation raises questions regarding the GVT in cutaneous neoplasms and potential immunomodulatory effects of ECP.

Cytochrome P-450 2C9 is the principle enzyme that terminates the anticoagulant effect of warfarin. Genetic polymorphisms inCYP2C9 producing variants with altered catalytic properties have been identified. Patients (n = 561) with a target international normalized ratio (INR) of 2.5 who had been treated with warfarin for more than 2 months were anonymously genotyped for the wild-typeCYP2C9*1 allele and the 2C9*2 and2C9*3 variants. The mean maintenance dose of warfarin in patients who were wild-type for both alleles was 5.01 mg. The maintenance dose of warfarin was significantly related to genotype (Kruskall-Wallis, χ2 = 17.985, P = .001) with mean maintenance doses in patients with variant alleles between 61% and 86% of that in wild-type patients. The odds ratio for the2C9*2 allele in patients with a maintenance dose of 1.5 mg or less was 5.42 (95% CI 1.68-17.4). The odds ratio for one or more variant alleles in patients developing an INR of 8.0 or greater was 1.52 (95% CI 0.64-3.58). The SD of the mean INR, percentage of high INRs, and person-time spent in range were determined as parameters of stability. There was no difference between patients grouped according to genotype for any parameter of stability. This study confirmed an association between CYP2C9 genotype and warfarin sensitivity. However, the possession of a variant allele does not increase the likelihood of severe over-anticoagulation or stability of anticoagulation during long-term therapy.

Purulent pericarditis is a cardiac emergency that can be difficult to diagnose and can be rapidly fatal. We report the case of a child with Down syndrome and recent atrial and ventricular septal defect repair who died from Haemophilus influenzae serotype f pericarditis.

We present a 57-year-old man with erosive lichen sclerosus isolated to the infraorbital area.

Dermal-based combined squamous and melanocytic neoplasms are emerging clinicopathologic entities that tend to appear on sun-exposed areas of elderly patients. The biologic behavior of such cutaneous neoplasms remains uncertain because of their rarity. Histopathologic differential includes the following diagnostic entities: (1) dermal squamomelanocytic tumor, (2) melanocytic matricoma, and (3) rare histologic variant of pilomatrical carcinoma, the so-called pilomatrical carcinoma with intralesional melanocytes. Herein, we present a novel case of locally invasive dermal squamomelanocytic tumor. A 72-year-old man presented with a pigmented papule on nasal ala that was clinically concerning for basal cell carcinoma. Histopathologic evaluation demonstrated atypical melanocytic cells architecturally and intimately intermixed with single units and clusters of atypical squamous cells. Most notable feature of this case is focal matrical differentiation and locally invasive tumor growth, characterized by multifocal perineural invasion.

The B7/CD28 family has profound modulatory effects in immune responses and constitutes an important target for the development of novel therapeutic drugs against human diseases. Here we describe a new CD28 homologue (CD28H) that has unique functions in the regulation of the human immune response and is absent in mice. CD28H is constitutively expressed on all naive T cells. Repetitive antigenic exposure, however, induces a complete loss of CD28H on many T cells, and CD28H negative T cells have a phenotype of terminal differentiation and senescence. After extensive screening in a receptor array, a B7-like molecule, B7 homologue 5 (B7-H5), was identified as a specific ligand for CD28H. B7-H5 is constitutively found in macrophages and could be induced on dendritic cells. The B7-H5/CD28H interaction selectively costimulates human T-cell growth and cytokine production via an AKT-dependent signalling cascade. Our study identifies a novel costimulatory pathway regulating human T-cell responses.

Human papillomavirus-associated head and neck squamous cell carcinomas (HPV-HNSCC) originate in the tonsils, the major lymphoid organ that orchestrates immunity to oral infections. Despite its location, the virus escapes immune elimination during malignant transformation and progression. Here, we provide evidence for the role of the PD-1:PD-L1 pathway in HPV-HNSCC immune resistance. We show membranous expression of PD-L1 in the tonsillar crypts, the site of initial HPV infection. In HPV-HNSCCs that are highly infiltrated with lymphocytes, PD-L1 expression on both tumor cells and CD68+ tumor-associated macrophages is geographically localized to sites of lymphocyte fronts, whereas the majority of CD8+ tumor-infiltrating lymphocytes express high levels of PD-1, the inhibitory PD-L1 receptor. Significant levels of mRNA for IFN-γ, a major cytokine inducer of PD-L1 expression, were found in HPV+ PD-L1(+) tumors. Our findings support the role of the PD-1:PD-L1 interaction in creating an “immune-privileged” site for initial viral infection and subsequent adaptive immune resistance once tumors are established and suggest a rationale for therapeutic blockade of this pathway in patients with HPV-HNSCC. Cancer Res; 73(6); 1733–41. ©2012 AACR.

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1). Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity. Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas. In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues, but staining on paraffin-embedded slides had been a challenge until recently. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

We present a 57-year-old man with erosive lichen sclerosus isolated to the infraorbital area.

Cathepsin K is consistently and diffusely expressed in alveolar soft part sarcoma (ASPS) and a subset of translocation renal cell carcinomas (RCCs). However, cathepsin K expression in human neoplasms has not been systematically analyzed. We constructed tissue microarrays (TMA) from a wide variety of human neoplasms, and performed cathepsin K immunohistochemistry (IHC). Only 2.7% of 1,140 carcinomas from various sites exhibited cathepsin K labeling, thus suggesting that among carcinomas, cathepsin K labeling is highly specific for translocation RCC. In contrast to carcinomas, cathepsin K labeling was relatively common (54.6%) in the 414 mesenchymal lesions studied, including granular cell tumor, melanoma, and histiocytic lesions, but not paraganglioma, all of which are in the morphologic differential diagnosis of ASPS. Cathepsin K IHC can be helpful in distinguishing ASPS and translocation RCC from some but not all of the lesions in their differential diagnosis.

As immuno-oncology takes center stage in the battle against cancer, the need for biomarkers has become even more acute, with response rates continuing in the 20%–30% range and the menu of options, including combination therapies, growing at an accelerating pace. The specific immunoarchitecture characteristics of the microenvironment in which a particular tumor grows may be both prognostic and predictive of response to these new immunotherapies. Multiplex immunofluorescence is the most effective, efficient way to identify specific immune cell types, their location, and their state of activation, as well as the presence of immunoactive molecular expression, all at the same time. This method is highly beneficial for exploring immune evasion mechanisms and finding potential biomarkers that allow researchers to assess the mechanism of action and predict and track response. This live, online seminar will take the viewer through validation of this multiplexing technique, including a comparison with singleplex immunofluorescence and standard (chromogenic) immunohistochemistry, as well as an assessment of intersite reproducibility and the influence of staining order on quantitation. View the Webinar

Tumors may evade immune attack by constitutive (oncogene-driven) and/or adaptive (IFN-g inducible) expression of PD-L1. PD-1/PD-L1 blockade can mediate tumor regression in immunocompetent patients. In HIV-infected patients, developing tumors may face little immune selection pressure and therefore may not evolve to evade immune attack. Expression of PD-L1 has not been systematically assessed in cancers from HIV-infected people. In the current study, immunohistochemistry for PD-L1, and CD3 and CD68 (immune cells, ICs) was performed on biopsies from 46 anal SCCs, including 27 from HIV-infected and 19 from uninfected patients. The proportion of cases with tumor cell PD-L1 expression was similar in patients with and without HIV (52% vs. 47%, respectively, p = 0.76), as was the presence of moderate/severely dense ICs (33% vs. 37%, p = 0.81) (Table). Among HIV-infected patients, 19% of anal SCC tumors (5/27) both expressed PD-L1 and had moderate/severe IC infiltration. Tumors from HIV-infected and uninfected patients had similar densities of CD68+ macrophages (mean 517 vs. 404 cells/mm2, p = 0.33) and CD3+ T- lymphocytes (mean 501 vs. 428 cells/mm2, p = 0.57). A component of adaptive PD-L1 expression (juxtaposed to tumor infiltrating ICs) was also observed in both groups (56% vs. 47%, p = 0.58), consistent with comparable T-cell functionality. Further studies will explore the expression of other immune checkpoint proteins and lymphocyte subsets. Despite expectations that cancers from HIV-infected patients would show reduced inflammation, our preliminary findings demonstrate an immune-reactive microenvironment in both HIV+ and HIV- anal SCCs and suggest that anti-PD-1/PD-L1 therapies should be evaluated in anal SCC patients. Tumor infiltrating ICTotalHIV- a (n = 19)HIV+ a (n = 27)P-value cNone-mildTotal3012 (40%)18 (60%)0.66PD-L1(+) b145 (36%)9 (64%)PD-L1(-)167 (44%)9 (56%)Moderate-severeTotal167 (44%)9 (56%)0.95PD-L1(+) b94 (44%)5 (56%)PD-L1(-)73 (43%)4 (57%)a Anal SCC tumors in HIV-infected patients include 19 tumors identified through the AIDS-Cancer Specimen Resource and 8 tumors identified at Johns Hopkins Hospital. Anal SCC tumors in HIV-uninfected patients include 1 tumor identified through the AIDS-Cancer Specimen Resource and 18 tumors identified at Johns Hopkins Hospital.b Defined as ≥5% of tumor cells expressing cell surface PD-L1 by immunohistochemistry with the 5H1 monoclonal antibody.c P-values are for the comparison of PD-L1 expression between HIV+ and HIV- anal SCCs within strata of tumor infiltrating ICIC = immune cells Citation Format: Elizabeth L. Yanik, Suzanne L. Topalian, Genevieve Kaunitz, Jessica Esandrio, Tricia Cottrell, Janis M. Taube. The tumor immune microenvironment is similar in anal squamous cell carcinomas (SCCs) from HIV-infected and uninfected patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1464.

Background: In previously treated MEL patients (pts), the results of an early phase I trial with NIVO monotherapy (CA209-003) demonstrated tumor responses that were durable even after treatment discontinuation (Topalian et al. J Clin Oncol 2014;32:1020). We report extended follow-up with 5-year overall survival (OS) data from this study. Methods: IPI-naïve pts (N = 107) who had received 1-5 prior systemic therapies for MEL were treated with NIVO (0.1, 0.3, 1, 3, or 10 mg/kg) every 2 weeks for ?96 weeks. Pts were followed for OS, progression-free survival (PFS), long-term safety, and response duration after discontinuing NIVO treatment. Pts began treatment in October 2008, and current data were analyzed in October 2015 with a minimum follow-up of 45 months (time from when the last pt received his or her first dose of NIVO). Results: Median age of the pts was 61 years, 67% were male, 97% had an ECOG performance status of 0 or 1, 62% had received ?2 prior systemic therapies, and 36% had elevated lactate dehydrogenase levels at baseline. In all 107 pts, the 60-month OS rate was 34% (95% confidence interval [CI]: 25-43) and median OS was 17.3 months (95% CI: 12.5-37.8) (Table). OS rates appeared to plateau at ∼48 months, although further follow-up is needed. Similar results were observed with NIVO at 3 mg/kg, the currently approved monotherapy dose (Table). At the last timepoint for tumor assessment, PFS rates at 30 months were 18.6% and 25.7% for all pts and those who received NIVO at 3 mg/kg, respectively. Conclusions: This analysis represents the longest survival follow-up of pts who received anti-PD-1 therapy in a clinical study. In this heavily pretreated population of MEL pts, these results suggest durable, long-term survival following NIVO monotherapy, with 34% of pts alive at 5 years. Characteristics of long-term survivors and updated safety data will also be presented. OS ratesNIVO 3 mg/kg (n=17)All Patients (N=107)OS rate, % (95% CI)*12-month64.7 (37.7-82.3)62.7 (52.6-71.2)24-month47.1 (23.0-68.0)48.0 (38.1-57.2)36-month41.2 (18.6-62.6)42.1 (32.4-51.4)48-month35.3 (14.5-57.0)34.8 (25.7-44.1)60-month35.3 (14.5-57.0)33.6 (24.6-42.9)Median OS, months (95% CI)20.3 (7.2-NR)17.3 (12.5-37.8)*Based on Kaplan-Meier estimates.NR, not reached. Citation Format: F. Stephen Hodi, Harriet Kluger, Mario Sznol, Richard Carvajal, Donald Lawrence, Michael Atkins, John Powderly, William Sharfman, Igor Puzanov, David Smith, Philip Leming, Evan Lipson, Janis Taube, Robert Anders, Christine Horak, Joel Jiang, David McDermott, Jeffrey Sosman, Julie Brahmer, Drew Pardoll, Suzanne Topalian. Durable, long-term survival in previously treated patients with advanced melanoma (MEL) who received nivolumab (NIVO) monotherapy in a phase I trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT001.

Introduction: Randomized controlled trials of platinum-based neoadjuvant chemotherapy (NAC) for bladder cancer have shown that patients who achieve a pathologic response to NAC exhibit 5 y survival rates of approximately 80–90% while NAC resistant (NR) cases exhibit 5 y survival rates of approximately 30–40%. These findings highlight the need to predict who will benefit from conventional NAC and the need for plausible alternatives. Methods: The pre-treatment biopsy tissues from a cohort of 41 patients with muscle invasive bladder who were treated with NAC were incorporated in tissue microarray and immunohistochemistry for PD-L1, CD8, and FOXP3 was performed. Percentage of PD-L1 positive tumor cells was measured. Tumor-infiltrating lymphocytes (TIL) densities, along with CD8 and Treg-specific TILs, were measured. Results: TIL density was strongly correlated with tumor PD-L1 expression, consistent with the mechanism of adaptive immune resistance in bladder cancer. Tumor cell PD-L1 expression was not a significant predictor of response. Neither was the CD8 nor Treg TIL density associated with response. Intriguingly though, the ratio of CD8 to Treg TIL densities was strongly associated with response (p = 0.0003), supporting the hypothesis that the immune system plays a role in the response of bladder cancer to chemotherapy. Discussion: To our knowledge, this is the first report in bladder cancer showing that the CD8 to Treg TIL density in the pre-treatment tissues is predictive for conventional NAC response. These findings warrant further investigations to both better characterize this association in larger cohorts and begin to elucidate the underlying mechanism(s) of this phenomenon.

Purpose: Results from the first-in-human phase I trial of the anti–programmed death-1 (PD-1) antibody BMS-936558 in patients with treatment-refractory solid tumors, including safety, tolerability, pharmacodynamics, and immunologic correlates, have been previously reported. Here, we provide long-term follow-up on three patients from that trial who sustained objective tumor regressions off therapy, and test the hypothesis that reinduction therapy for late tumor recurrence can be effective. Experimental Design: Three patients with colorectal cancer, renal cell cancer, and melanoma achieved objective responses on an intermittent dosing regimen of BMS-936558. Following cessation of therapy, patients were followed for more than 3 years. A patient with melanoma who experienced a prolonged partial regression followed by tumor recurrence received reinduction therapy. Results: A patient with colorectal cancer experienced a complete response, which is ongoing after 3 years. A patient with renal cell cancer experienced a partial response lasting 3 years off therapy, which converted to a complete response, which is ongoing at 12 months. A patient with melanoma achieved a partial response that was stable for 16 months off therapy; recurrent disease was successfully treated with reinduction anti-PD-1 therapy. Conclusion: These data represent the most prolonged observation to date of patients with solid tumors responding to anti-PD-1 immunotherapy and the first report of successful reinduction therapy following delayed tumor progression. They underscore the potential for immune checkpoint blockade with anti-PD-1 to reset the equilibrium between tumor and the host immune system. Clin Cancer Res; 19(2); 462–8. ©2012 AACR.

Background Blockade of programmed death 1 (PD-1), an inhibitory receptor expressed by T cells, can overcome immune resistance. We assessed the antitumor activity and safety of BMS-936558, an antibody that specifically blocks PD-1. Methods We enrolled patients with advanced melanoma, non–small-cell lung cancer, castrationresistant prostate cancer, or renal-cell or colorectal cancer to receive anti–PD-1 antibody at a dose of 0.1 to 10.0 mg per kilogram of body weight every 2 weeks. Response was assessed after each 8-week treatment cycle. Patients received up to 12 cycles until disease progression or a complete response occurred. Results A total of 296 patients received treatment through February 24, 2012. Grade 3 or 4 drugrelated adverse events occurred in 14% of patients; there were three deaths from pulmonary toxicity. No maximum tolerated dose was defined. Adverse events consistent with immune-related causes were observed. Among 236 patients in whom response could be evaluated, objective responses (complete or partial responses) were observed in those with non–small-cell lung cancer, melanoma, or renal-cell cancer. Cumulative response rates (all doses) were 18% among patients with non–small-cell lung cancer (14 of 76 patients), 28% among patients with melanoma (26 of 94 patients), and 27% among patients with renal-cell cancer (9 of 33 patients). Responses were durable; 20 of 31 responses lasted 1 year or more in patients with 1 year or more of follow-up. To assess the role of intratumoral PD-1 ligand (PD-L1) expression in the modulation of the PD-1–PD-L1 pathway, immunohistochemical analysis was performed on pretreatment tumor specimens obtained from 42 patients. Of 17 patients with PD-L1–negative tumors, none had an objective response; 9 of 25 patients (36%) with PD-L1–positive tumors had an objective response (P = 0.006). Conclusions Anti–PD-1 antibody produced objective responses in approximately one in four to one in five patients with non–small-cell lung cancer, melanoma, or renal-cell cancer; the adverse-event profile does not appear to preclude its use. Preliminary data suggest a relationship between PD-L1 expression on tumor cells and objective response. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT00730639.)