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Cutaneous T cell lymphomas (CTCLs) are skin cancers with poor survival rates and limited treatments. While immunotherapies have shown some efficacy, the immunological consequences of administering immune-activating agents to CTCL patients have not been systematically characterized. We apply a suite of high-dimensional technologies to investigate the local, cellular, and systemic responses in CTCL patients receiving either mono- or combination anti-PD-1 plus interferon-gamma (IFN-γ) therapy. Neoplastic T cells display no evidence of activation after immunotherapy. IFN-γ induces muted endogenous immunological responses, while anti-PD-1 elicits broader changes, including increased abundance of CLA + CD39 + T cells. We develop an unbiased multi-omic profiling approach enabling discovery of immune modules stratifying patients. We identify an enrichment of activated regulatory CLA + CD39 + T cells in non-responders and activated cytotoxic CLA + CD39 + T cells in leukemic patients. Our results provide insights into the effects of immunotherapy in CTCL patients and a generalizable framework for multi-omic analysis of clinical trials., Competing Interests: Declaration of interests E.W.N. is a co-founder, advisor, and shareholder of ImmunoScape and is an advisor for Neogene Therapuetics and NanoString Technologies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Merkel cell carcinoma is a skin cancer often driven by Merkel cell polyomavirus (MCPyV) with high rates of response to anti-PD-1 therapy despite low mutational burden. MCPyV-specific CD8 T cells are implicated in anti-PD-1-associated immune responses and provide a means to directly study tumor-specific T cell responses to treatment. Using mass cytometry and combinatorial tetramer staining, we find that baseline frequencies of blood MCPyV-specific cells correlated with response and survival. Frequencies of these cells decrease markedly during response to therapy. Phenotypes of MCPyV-specific CD8 T cells have distinct expression patterns of CD39, cutaneous lymphocyte-associated antigen (CLA), and CD103. Correspondingly, overall bulk CD39 + CLA + CD8 T cell frequencies in blood correlate with MCPyV-specific cell frequencies and similarly predicted favorable clinical outcomes. Conversely, frequencies of CD39 + CD103 + CD8 T cells are associated with tumor burden and worse outcomes. These cell subsets can be useful as biomarkers and to isolate blood-derived tumor-specific T cells., Competing Interests: Declaration of interests E.W.N. is a co-founder, advisor, and shareholder for ImmunoScape Pte. Ltd., a scientific advisory board member and shareholder for Neogene Therapeutics, and a scientific advisory board member for Nanostring Biotechnologies and Trojan Biotechnologies. D.M.K. and P.N. are co-inventors on an institutionally owned patent concerning MCPyV-specific TCRs., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

Introduction: Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens., Methods: Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins., Results and Discussion: The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community., Competing Interests: JW is a consultant to CellCarta. S.D.S. receives research funding from ADC Therapeutics, AstraZeneca, Bayer, BeiGene, De Novo Biopharma, Enterome, Genentech, Incyte Corporation, Kymera Therapeutics, Merck Sharp & Dohme Corp., MorphoSys, Nanjing Pharmaceuticals Co., Ltd., and Viracta Therapeutics and provides consultancy to ADC Therapeutics, AstraZeneca, BeiGene, Epizyme, Karyopharm, Kite Pharma, Incyte, Numab Therapeutics, and AbbVie. AG receives funding from Merck, I-Mab Bio, IgM Bio, Takeda, Gilead, AstraZeneca, Agios, Janssen, BMS, SeaGen, Teva, and Genmab and provides consultancy to Incyte, Kite, MorphoSys/Incyte, ADCT, Acrotech, Merck, Karyopharm, Servier, BeiGene, Cellectar, Janssen, SeaGen, Epizyme, I-Mab Bio, Gilead, Genentech, Lilly, Caribou, and Fresenius-Kabi. AP is the Founder of Precision Assays and a consultant to CellCarta. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Whiteaker, Zhao, Schoenherr, Huang, Lundeen, Voytovich, Kennedy, Ivey, Lin, Murillo, Lorentzen, Colantonio, Caceres, Roberts, Knotts, Reading, Perry, Richardson, Garcia-Buntley, Bocik, Hewitt, Chowdhury, Vandermeer, Smith, Gopal, Ramchurren, Fling, Wang and Paulovich.)

The PD-1 inhibitor pembrolizumab is effective in treating Sézary syndrome, a leukemic variant of cutaneous T-cell lymphoma. Our purpose was to investigate the effects of pembrolizumab on healthy and malignant T cells in Sézary syndrome and to discover characteristics that predict pembrolizumab response. Samples were analyzed before and after 3 weeks of pembrolizumab treatment using single-cell RNA-sequencing of 118,961 peripheral blood T cells isolated from six Sézary syndrome patients. T-cell receptor clonotyping, bulk RNA-seq signatures, and whole-exome data were integrated to classify malignant T-cells and their underlying subclonal heterogeneity. We found that responses to pembrolizumab were associated with lower KIR3DL2 expression within Sézary T cells. Pembrolizumab modulated Sézary cell gene expression of T-cell activation associated genes. The CD8 effector populations included clonally expanded populations with a strong cytotoxic profile. Expansions of CD8 terminal effector and CD8 effector memory T-cell populations were observed in responding patients after treatment. We observed intrapatient Sézary cell heterogeneity including subclonal segregation of a coding mutation and copy number variation. Our study reveals differential effects of pembrolizumab in both malignant and healthy T cells. These data support further study of KIR3DL2 expression and CD8 immune populations as predictive biomarkers of pembrolizumab response in Sézary syndrome., Competing Interests: The authors report there are no competing interests to declare., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

To uncover underlying mechanisms associated with failure of indoleamine 2,3-dioxygenase 1 (IDO1) blockade in clinical trials, we conducted a pilot, window-of-opportunity clinical study in 17 patients with newly diagnosed advanced high-grade serous ovarian cancer before their standard tumor debulking surgery. Patients were treated with the IDO1 inhibitor epacadostat, and immunologic, transcriptomic, and metabolomic characterization of the tumor microenvironment was undertaken in baseline and posttreatment tumor biopsies. IDO1 inhibition resulted in efficient blockade of the kynurenine pathway of tryptophan degradation and was accompanied by a metabolic adaptation that shunted tryptophan catabolism toward the serotonin pathway. This resulted in elevated nicotinamide adenine dinucleotide (NAD + ), which reduced T cell proliferation and function. Because NAD + metabolites could be ligands for purinergic receptors, we investigated the impact of blocking purinergic receptors in the presence or absence of NAD + on T cell proliferation and function in our mouse model. We demonstrated that A2a and A2b purinergic receptor antagonists, SCH58261 or PSB1115, respectively, rescued NAD + -mediated suppression of T cell proliferation and function. Combining IDO1 inhibition and A2a/A2b receptor blockade improved survival and boosted the antitumor immune signature in mice with IDO1 overexpressing ovarian cancer. These findings elucidate the downstream adaptive metabolic consequences of IDO1 blockade in ovarian cancers that may undermine antitumor T cell responses in the tumor microenvironment.

Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1 + CD4 + T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies., (© 2021. The Author(s).)

We introduce a new method for single-cell cytometry studies, FAUST, which performs unbiased cell population discovery and annotation. FAUST processes experimental data on a per-sample basis and returns biologically interpretable cell phenotypes, making it well suited for the analysis of complex datasets. We provide simulation studies that compare FAUST with existing methodology, exemplifying its strength. We apply FAUST to data from a Merkel cell carcinoma anti-PD-1 trial and discover pre-treatment effector memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. Using FAUST, we then validate these correlates in cryopreserved peripheral blood mononuclear cell samples from the same study, as well as an independent CyTOF dataset from a published metastatic melanoma trial. Finally, we show how FAUST's phenotypes can be used to perform cross-study data integration in the presence of diverse staining panels. Together, these results establish FAUST as a powerful new approach for unbiased discovery in single-cell cytometry., Competing Interests: A patent for the application of the FAUST algorithm to cytometry datasets has been applied for on behalf of the Fred Hutchinson Cancer Research Center. The research described in this paper was completed while E.G. was conducting research and working at the Fred Hutchinson Cancer Research Center. E.G. declares ownership interest in Ozette Technologies and was an employee of Ozette Technologies when the manuscript was revised to respond to peer review. G.F. has received consulting income from Takeda and research support from Janssen Pharmaceuticals and declares ownership interest in Ozette Technologies. R.G. has received consulting income from Juno Therapeutics, Takeda, Infotech Soft, and Celgene, has received research support from Janssen Pharmaceuticals and Juno Therapeutics, and declares ownership in Ozette Technologies and Modulus Therapeutics. Trial funds for CITN-07 were in part provided by Celldex. Trial funds for CITN-09 were in part provided by Merck., (© 2021 The Authors.)

Background: Sipuleucel-T (sip-T) is a Food and Drug Administration (FDA)-approved autologous cellular immunotherapy for metastatic castration-resistant prostate cancer (mCRPC). We hypothesized that combining sip-T with interleukin (IL)-7, a homeostatic cytokine that enhances both B and T cell development and proliferation, would augment and prolong antigen-specific immune responses against both PA2024 (the immunogen for sip-T) and prostatic acid phosphatase (PAP)., Methods: Fifty-four patients with mCRPC treated with sip-T were subsequently enrolled and randomized 1:1 into observation (n=26) or IL-7 (n=28) arms of a phase II clinical trial (NCT01881867). Recombinant human (rh) IL-7 (CYT107) was given weekly×4. Immune responses were evaluated using flow cytometry, mass cytometry (CyTOF), interferon (IFN)-γ ELISpot, 3 H-thymidine incorporation, and ELISA., Results: Treatment with rhIL-7 was well tolerated. For the rhIL-7-treated, but not observation group, statistically significant lymphocyte subset expansion was found, with 2.3-2.6-fold increases in CD4+T, CD8+T, and CD56 bright NK cells at week 6 compared with baseline. No significant differences in PA2024 or PAP-specific T cell responses measured by IFN-γ ELISpot assay were found between rhIL-7 and observation groups. However, antigen-specific T cell proliferative responses and humoral IgG and IgG/IgM responses significantly increased over time in the rhIL-7-treated group only. CyTOF analyses revealed pleiotropic effects of rhIL-7 on lymphocyte subsets, including increases in CD137 and intracellular IL-2 and IFN-γ expression. While not powered to detect clinical outcomes, we found that 31% of patients in the rhIL-7 group had prostate specific antigen (PSA) doubling times of >6 months, compared with 14% in the observation group., Conclusions: Treatment with rhIL-7 led to a significant expansion of CD4+ and CD8+ T cells, and CD56 bright natural killer (NK) cells compared with observation after treatment with sip-T. The rhIL-7 treatment also led to improved antigen-specific humoral and T cell proliferative responses over time as well as to increased expression of activation markers and beneficial cytokines. This is the first study to evaluate the use of rhIL-7 after sip-T in patients with mCRPC and demonstrates encouraging results for combination approaches to augment beneficial immune responses., Competing Interests: Competing interests: MM and AG are employees of RevImmune who provided CYT107 for this trial. LF, RKP and PM served in advisory/consulting roles for Dendreon. EYY has received grant funding from Dendreon., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer associated with poor survival. Programmed cell death-1 (PD-1) pathway inhibitors have shown high rates of durable tumor regression compared with chemotherapy for MCC. The current study was undertaken to assess baseline and on-treatment factors associated with MCC regression and 3-year survival, and to explore the effects of salvage therapies in patients experiencing initial non-response or tumor progression after response or stable disease following first-line pembrolizumab therapy on Cancer Immunotherapy Trials Network-09/KEYNOTE-017., Methods: In this multicenter phase II trial, 50 patients with advanced unresectable MCC received pembrolizumab 2 mg/kg every 3 weeks for ≤2 years. Patients were followed for a median of 31.8 months., Results: Overall response rate to pembrolizumab was 58% (complete response 30%+partial response 28%; 95% CI 43.2 to 71.8). Among 29 responders, the median response duration was not reached (NR) at 3 years (range 1.0+ to 51.8+ months). Median progression-free survival (PFS) was 16.8 months (95% CI 4.6 to 43.4) and the 3-year PFS was 39.1%. Median OS was NR; the 3-year OS was 59.4% for all patients and 89.5% for responders. Baseline Eastern Cooperative Oncology Group performance status of 0, greater per cent tumor reduction, completion of 2 years of treatment and low neutrophil-to-lymphocyte ratio were associated with response and longer survival. Among patients with initial disease progression or those who developed progression after response or stable disease, some had extended survival with subsequent treatments including chemotherapies and immunotherapies., Conclusions: This study represents the longest available follow-up from any first-line anti-programmed death-(ligand) 1 (anti-PD-(L)1) therapy in MCC, confirming durable PFS and OS in a proportion of patients. After initial tumor progression or relapse following response, some patients receiving salvage therapies survived. Improving the management of anti-PD-(L)1-refractory MCC remains a challenge and a high priority., Trial Registration Number: NCT02267603., Competing Interests: Competing interests: PN reports grants from Bristol-Myers Squibb and EMD-Serono; advisory fees from EMD-Serono, Pfizer and Merck & Co.; travel expenses from Sanofi/Regeneron and Merck & Co. and has a pending patent related to high-affinity T-cell receptors that target the Merkel polyomavirus. SB reports personal fees from Bristol-Myers Squibb, EMD-Serono and personal fees and other from Sanofi-Genzyme; grants from Bristol-Myers Squibb, EMD-Serono, Merck & Co., NantKwest, Novartis, Immune Design, Oncosec, Exicure, Nektar; and personal fees from Castle Biosciences. EJ reports grants from Merck & Co., Bristol-Myers Squibb and Sanofi/Regeneron; personal fees from Bristol-Myers Squibb, Novartis, Array BioPharma, Macrogenics, Sanofi/Regeneron and Genentech. RK reports grants from Merck & Co., Bristol-Myers Squibb and Regeneron; advisory fees from Merck & Co., Bristol-Myers Squibb, Regeneron, Novartis and Array. ASB reports personal fees from Bayer, Deciphera and EMD Serono. BAH reports grants from Merck & Co., Tempest Therapeutics, Olatec Therapeutics, A*STAR Singapore, Sanofi, Leap Therapeutics, GSK and AstraZeneca; personal fees from Merck & Co., Novartis, G1 Therapeutics and CE Concepts; travel fees from ASCO and ASCI and patents related to dendritic cell vaccines, immunotherapy biomarkers and methods for augmenting anti-PD-1 therapy. CC reports a pending patent related to high-affinity T-cell receptors that target the Merkel polyomavirus. JT reports consulting/advisory fees from Merck & Co, Bristol-Myers Squibb, AstraZeneca and Compugen; and a grant from Bristol-Myers Squibb. EJ, MK and BHM are employees of Merck & Co. SPF reports research funding from Merck. MAC reports research funding from Merck & Co. SLT reports that she or an immediate family member has stock and other ownership interests in Aduro Biotech, DNAtrix, Dracen Pharmaceuticals, Dragonfly Therapeutics, Ervaxx, Five Prime Therapeutics, Potenza Therapeutics, RAPT, Tizona Therapeutics, Trieza Therapeutics and WindMIL; a consulting or advisory role in Amgen, DNAtrix, Dragonfly Therapeutics, Dynavax, Ervaxx, Five Prime Therapeutics, Immunocore, Immunomic Therapeutics, Janssen Pharmaceuticals, MedImmune/AstraZeneca, Merck & Co., RAPT and WindMIL; research grants from Bristol-Myers Squibb and Compugen; patents, royalties and/or other intellectual property with Aduro Biotech, Arbor Pharmaceuticals, Bristol-Myers Squibb, Immunomic Therapeutics, NexImmune and WindMIL and travel, accommodations, and expenses from Bristol-Myers Squibb, Dragonfly and Five Prime Therapeutics., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Generating responses to tumor antigens poses a challenge for immunotherapy. This phase II trial (NCT02129075) tested fms-like tyrosine kinase 3 (Flt3) ligand pre-treatment enhancement of responses to dendritic cell (DC)-targeting vaccines. We evaluated a regimen of Flt3L (CDX-301) to increase DCs and other antigen-presenting cells, poly-ICLC (TLR3 agonist that activates DCs) and a vaccine comprising anti-DEC-205-NY-ESO-1, a fusion antibody targeting CD205, linked to NY-ESO-1. High-risk melanoma patients were randomized to vaccine, with and without CDX-301. The end point was immune response to NY-ESO-1. Flt3L increased peripheral monocytes and conventional DCs (cDCs), including cross-presenting cDC1 and cDC2 and plasmacytoid DCs. Significant increases in humoral and T-cell responses and activation of DCs, natural killer cells and T cells were elicited. Transcriptional analyses revealed gene signatures associated with CDX-301 induction of an early, durable immune response. This study reveals in vivo effects of Flt3L on innate immune cells in the setting of vaccination, leading to an immunogenic vaccine regimen., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Clinical benefit from programmed cell death 1 receptor (PD-1) inhibitors relies on reinvigoration of endogenous antitumor immunity. Nonetheless, robust immunological markers, based on circulating immune cell subsets associated with therapeutic efficacy are yet to be validated., Methods: We isolated peripheral blood mononuclear cell from three independent cohorts of melanoma and Merkel cell carcinoma patients treated with PD-1 inhibitor, at baseline and longitudinally after therapy. Using multiparameter flow cytometry and cell sorting, we isolated four subsets of CD8 + T cells, based on PD-1 and TIGIT expression profiles. We performed phenotypic characterization, T cell receptor sequencing, targeted transcriptomic analysis and antitumor reactivity assays to thoroughly characterize each of these subsets., Results: We documented that the frequency of circulating PD-1 + TIGIT + (DPOS) CD8 + T-cells after 1 month of anti-PD-1 therapy was associated with clinical response and overall survival. This DPOS T-cell population was enriched in highly activated T-cells, tumor-specific and emerging T-cell clonotypes and T lymphocytes overexpressing CXCR5, a key marker of the CD8 cytotoxic follicular T cell population. Additionally, transcriptomic profiling defined a specific gene signature for this population as well as the overexpression of specific pathways associated with the therapeutic response., Conclusions: Our results provide a convincing rationale for monitoring this PD-1 + TIGIT + circulating population as an early cellular-based marker of therapeutic response to anti-PD-1 therapy., Competing Interests: Competing interests: SRR has served as an advisor and has patents licensed to Juno Therapeutics, a Celgene/Bristol-Myers Squibb company; is a founder and employee of Lyell Immunopharma; and has served on advisory boards for Adaptive Biotechnologies and Nohla. PN serves as a paid consultant for EMD Serono. Bristol Myers Squibb has provided research support to PN’s institution. RG has received consulting income from Juno Therapeutics, Takeda, Infotech Soft, Celgene, has received research support from Janssen Pharmaceuticals and Juno Therapeutics, and declares ownership in Cellspace Biosciences. SR and ZW are employed by QIAGEN, however, the studies were conducted in the absence of any potential conflict of interest. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To assess the efficacy of pembrolizumab in patients with advanced relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS)., Patients and Methods: CITN-10 is a single-arm, multicenter phase II trial of 24 patients with advanced MF or SS. Patients were treated with pembrolizumab 2 mg/kg every 3 weeks for up to 24 months. The primary end point was overall response rate by consensus global response criteria., Results: Patients had advanced-stage disease (23 of 24 with stage IIB to IV MF/SS) and were heavily pretreated with a median of four prior systemic therapies. The overall response rate was 38% with two complete responses and seven partial responses. Of the nine responding patients, six had 90% or more improvement in skin disease by modified Severity Weighted Assessment Tool, and eight had ongoing responses at last follow-up. The median duration of response was not reached, with a median response follow-up time of 58 weeks. Immune-related adverse events led to treatment discontinuation in four patients. A transient worsening of erythroderma and pruritus occurred in 53% of patients with SS. This cutaneous flare reaction did not result in treatment discontinuation for any patient. The flare reaction correlated with high PD-1 expression on Sézary cells but did not associate with subsequent clinical responses or lack of response. Treatment responses did not correlate with expression of PD-L1, total mutation burden, or an interferon-γ gene expression signature., Conclusion: Pembrolizumab demonstrated significant antitumor activity with durable responses and a favorable safety profile in patients with advanced MF/SS.

Purpose: Merkel cell carcinoma (MCC) is an aggressive skin cancer often caused by the Merkel cell polyomavirus. Clinical trials of programmed cell death-1 pathway inhibitors for advanced MCC (aMCC) demonstrate increased progression-free survival (PFS) compared with historical chemotherapy data. However, response durability and overall survival (OS) data are limited., Patients and Methods: In this multicenter phase II trial (Cancer Immunotherapy Trials Network-09/Keynote-017), 50 adults naïve to systemic therapy for aMCC received pembrolizumab (2 mg/kg every 3 weeks) for up to 2 years. Radiographic responses were assessed centrally per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1., Results: Among 50 patients, the median age was 70.5 years, and 64% had Merkel cell polyomavirus-positive tumors. The objective response rate (ORR) to pembrolizumab was 56% (complete response [24%] plus partial response [32%]; 95% CI, 41.3% to 70.0%), with ORRs of 59% in virus-positive and 53% in virus-negative tumors. Median follow-up time was 14.9 months (range, 0.4 to 36.4+ months). Among 28 responders, median response duration was not reached (range, 5.9 to 34.5+ months). The 24-month PFS rate was 48.3%, and median PFS time was 16.8 months (95% CI, 4.6 months to not estimable). The 24-month OS rate was 68.7%, and median OS time was not reached. Although tumor viral status did not correlate with ORR, PFS, or OS, there was a trend toward improved PFS and OS in patients with programmed death ligand-1-positive tumors. Grade 3 or greater treatment-related adverse events occurred in 14 (28%) of 50 patients and led to treatment discontinuation in seven (14%) of 50 patients, including one treatment-related death., Conclusion: Here, we present the longest observation to date of patients with aMCC receiving first-line anti-programmed cell death-1 therapy. Pembrolizumab demonstrated durable tumor control, a generally manageable safety profile, and favorable OS compared with historical data from patients treated with first-line chemotherapy.

Background: Merkel cell carcinoma (MCC) is an aggressive skin cancer that frequently responds to anti-PD-1 therapy. MCC is associated with sun exposure and, in 80% of cases, Merkel cell polyomavirus (MCPyV). MCPyV-specific T and B cell responses provide a unique opportunity to study cancer-specific immunity throughout PD-1 blockade therapy., Methods: Immune responses were assessed in patients (n = 26) with advanced MCC receiving pembrolizumab. Peripheral blood mononuclear cells (PBMC) were collected at baseline and throughout treatment. MCPyV-oncoprotein antibodies were quantified and T cells were assessed for MCPyV-specificity via tetramer staining and/or cytokine secretion. Pre-treatment tumor biopsies were analyzed for T cell receptor clonality., Results: MCPyV oncoprotein antibodies were detectable in 15 of 17 (88%) of virus-positive MCC (VP-MCC) patients. Antibodies decreased in 10 of 11 (91%) patients with responding tumors. Virus-specific T cells decreased over time in patients who had a complete response, and increased in patients who had persistent disease. Tumors that were MCPyV(+) had a strikingly more clonal (less diverse) intratumoral TCR repertoire than virus-negative tumors (p = 0.0001)., Conclusions: Cancer-specific T and B cell responses generally track with disease burden during PD-1 blockade, in proportion to presence of antigen. Intratumoral TCR clonality was significantly greater in VP-MCC than VN-MCC tumors, suggesting expansion of a limited number of dominant clones in response to fewer immunogenic MCPyV antigens. In contrast, VN-MCC tumors had lower clonality, suggesting a diverse T cell response to numerous neoantigens. These findings reveal differences in tumor-specific immunity for VP-MCC and VN-MCC, both of which often respond to anti-PD-1 therapy.

Background: We recently reported a 56% objective response rate in patients with advanced Merkel cell carcinoma (MCC) receiving pembrolizumab. However, a biomarker predicting clinical response was not identified., Methods: Pretreatment FFPE tumor specimens (n = 26) were stained for CD8, PD-L1, and PD-1 by immunohistochemistry/immunofluorescence (IHC/IF), and the density and distribution of positive cells was quantified to determine the associations with anti-PD-1 response. Multiplex IF was used to test a separate cohort of MCC archival specimens (n = 16), to identify cell types expressing PD-1., Results: Tumors from patients who responded to anti-PD-1 showed higher densities of PD-1+ and PD-L1+ cells when compared to non-responders (median cells/mm 2 , 70.7 vs. 6.7, p = 0.03; and 855.4 vs. 245.0, p = 0.02, respectively). There was no significant association of CD8+ cell density with clinical response. Quantification of PD-1+ cells located within 20 μm of a PD-L1+ cell showed that PD-1/PD-L1 proximity was associated with clinical response (p = 0.03), but CD8/PD-L1 proximity was not. CD4+ and CD8+ cells in the TME expressed similar amounts of PD-1., Conclusions: While the binomial presence or absence of PD-L1 expression in the TME was not sufficient to predict response to anti-PD-1 in patients with MCC, we show that quantitative assessments of PD-1+ and PD-L1+ cell densities as well as the geographic interactions between these two cell populations correlate with clinical response. Cell types expressing PD-1 in the TME include CD8+ T-cells, CD4+ T-cells, T regs , and CD20+ B-cells, supporting the notion that multiple cell types may potentiate tumor regression following PD-1 blockade.

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

Within a panel of 15 colon carcinoma cell lines we have characterized the p53 gene status using immunocytochemistry (ICC), SSCP and direct sequence analysis. Extension of this analysis to the use of ICC on 104 colonic lesions, representative of different stages of colonic neoplastic progression, showed an absence of detectable p53 nuclear staining in preneoplastic polyp lesions (20 cases) with staining of 52% (25/48) of primary colon carcinomas and 81% (29/36) of hepatic metastases, suggestive of an increased incidence of p53 mutations in late stage lesions of colonic cancer. To address this issue more directly, we analysed 18 primary colon carcinomas and hepatic metastases excised coincidentally from the same patients. In ICC, p53 nuclear staining was recorded in matching lesions from eight individuals where direct sequencing revealed identical mutations in each case. In four individuals no ICC staining was detected in either lesion and molecular analysis revealed wild type sequence in exons 4-9. In six individuals p53 nuclear staining was observed in the hepatic metastases of patients but not the primary lesion. Molecular analysis revealed point mutation events in hepatic metastases from these patients which were not detected in the primary tumor. The point mutations identified in colon carcinomas were predominantly transition events (83%) located in previously characterized colon hotspot regions. These results demonstrate an increased incidence of p53 mutations associated with secondary lesions of colorectal tumors suggestive of a role for p53 in the establishment of colorectal hepatic metastases.

Twenty-one invasive squamous-cell carcinomas (SCC) of the bladder from Schistosoma-hematobium-infected patients were examined immunohistochemically for the expression of p53, Rb, EGFR and c-erbB-2 proteins; and screened by single-strand conformation polymorphism and sequencing for mutations in the ras (H, N, K) codon hotspots (12, 13, 61) and p53 (exons 4-9) genes. Positive staining for p53, EGFR and c-erbB-2 was reported in 38, 67 and 28% of tumors respectively. Only one of the tumors, the only one that was poorly differentiated, displayed an absence of nuclear Rb staining. Ras alterations were detected in the H-ras gene in 3 tumors, 2 of which harbored a codon-13 (Gly-->Arg) and one a codon-12 (Gly-->Ser) point mutation. p53 mutations were recorded in 12 tumors (57%), 6 of which stained positively for p53. Four tumors had exon-7 mutations (codons 235, 241 and 249; one tumor had 2 exon-7 mutations). Eight tumors were mutated in exon 8 (codons 264, 271, 273, 285, 286, 288 and 294), 5 of which harbored multiple mutations. One tumor had an insertion/deletion event in exon 9. The frequency of detection of over-expression of EGFR and c-erbB-2 in bilharzial-bladder lesions is comparable to that reported in TCC, contrasting with the infrequent loss of Rb expression found in invasive lesions associated with schistosomiasis infection. However, the detection of multiple p53 mutations in these lesions is suggestive of the involvement of a carcinogenic agent with maintenance of preferential activation of the H-ras gene.

Background: Urinary cytology has long been used as a noninvasive screen for the detection of urinary tract cancer but is limited by the generation of false positive and false negative results. More recently, molecular changes associated with urothelial neoplastic progression have been identified in DNA from urine sediments, demonstrating an alternative approach for identifying neoplastic change in the bladder., Purpose: The purpose of this prospective study was to determine the value of detection of H-ras (also known as HRAS) mutations in urine sediment DNA as a clinical indicator of tumor presence, recurrence, and/or progression., Methods: Urine sediments were collected from 100 patients presenting with bladder tumors, with follow-up samples collected from 19 patients. DNA extracted from urine sediments was analyzed for changes in exon 1 of the H-ras gene, using single-strand conformation polymorphism (SSCP) analysis. A representative number of aberrant H-ras/SSCP migrating bands were excised and sequenced to confirm the presence of a mutation. Human bladder specimens were obtained from patients (93 of the 100 patients initially and 18 of the 19 patients studied by follow-up) and histologically evaluated for tumor content and grade., Results: Mutations in exon 1 of the H-ras gene were detected in urine sediments from 44% (44 of 100) of the patients; concordant results were obtained by cytologic analysis, where 33% (31 of 93) of the patients displayed positive cytology. Analysis of the distribution of abnormalities with tumor grade revealed greater detection of low-grade (1-2) lesions using ras analysis (47%) compared with cytology (16%). In contrast, cytology was more effective in identifying the presence of carcinoma in situ. Combined results from these two approaches substantially increased the sensitivity of tumor detection, resulting in the identification of tumors in 60% of patients., Conclusions: Identification of H-ras mutations in DNA from urine sediments facilitates the detection of low-grade bladder tumors and, in combination with cytology, increases the overall tumor detection from 33% to 60%. Preliminary results in patient follow-up suggest that detection of H-ras mutations may have some clinical utility in detecting the presence of abnormal cells in the absence of an overt lesion following cystoscopy or positive cytology.

Objective: To establish whether specific K-ras alterations are predictive of less aggressive tumor behavior and subsequently those patients who are most likely to benefit from resection of hepatic metastases from colorectal carcinoma., Design: Evaluation of long-term survivors of hepatic resection for metastases of colorectal carcinoma (median survival, 85 months)., Results: DNA, extracted from 26 paraffin-embedded hepatic metastases from 19 patients, was analyzed using single-strand conformation polymorphism and direct sequence analysis of codons 12 and 13 of the K-ras gene. Seven of 19 patients were found to harbor K-ras mutations. A similar frequency and spectrum of K-ras mutational events was detected in 14 patients with short-term survival following pathologic diagnosis of hepatic metastasis., Conclusions: Neither the presence of a K-ras mutational event nor the precise nucleotide change are predictive of less aggressive tumor behavior, and genetic alterations at this locus alone cannot be used to select patients undergoing resection of hepatic metastases from colorectal carcinoma.

A series of 111 human bladder tumors were screened using oligonucleotide mutant specific probes, restriction enzyme analysis and single-stranded confirmation polymorphism (SSCP) for the presence of H-ras activation events. Thirty-three tumors were found to harbor H-ras mutations where a glycine to valine (G-->T) change in codon 12 was the most common point mutation recorded (26 tumors). Additional mutations involved glycine to cysteine at codon 13 (2 tumors) and glutamine to arginine/lysine/leucine at codon 61 (3/1/1 tumors, respectively). Ambiguous signals recorded with oligonucleotide probes were further analyzed using SSCP analysis revealing the presence of H-ras mutations in restricted regions of some tumors. The apparent sensitivity of SSCP enabled us to extend this study to DNA isolated from urine sediments where 4 of the 9 patients studied showed representation of mutant H-ras. Our study demonstrates a sensitive, non-invasive assay for the screening of urine-borne cells, with no requirement for prior knowledge of the mutational change at the H-ras locus.

Recent identification of genetic alterations in colon polyps and tumors has allowed construction of a hypothesis for the molecular basis of colon carcinogenesis. The consistency of observed genetic changes has inspired enthusiastic anticipation of new diagnostic tools and interventions for colon cancer. Appropriate animal models are crucial to the testing of molecular postulates as well as the development of markers and therapies for colon carcinogenesis. We discuss herein the various animal models that are currently used for the study of colon cancer as well as those that hold promise for the future. The contributions, drawbacks, and potential uses for the chemical carcinogen model, the multiple intestinal neoplasia model, transgenic animals, and the reconstruction model in the study of colon carcinoma are presented.

Prostaglandin production by two continuous human esophageal carcinoma cell lines HCU 18 and HCU 39 derived from poorly and moderately differentiated source tumors, respectively, was investigated. Behavior of both lines in vitro and upon sc inoculation into athymic randombred BALB/c nude mice was also assessed. Approximately half the xenografts induced by HCU 18 cells were invasive, whereas those initiated by HCU 39 cells were all well encapsulated. Although metastases were not detected in mice given injections of HCU 39 cells, metastatic tumors developed in 2 mice inoculated with HCU 18 cells. In addition, HCU 18 cells produced significantly more prostaglandin E (PGE) and prostaglandin F (PGF) than HCU 39 cells. These findings suggest a relationship between PGE and PGF production by human esophageal carcinoma cells and their invasive and metastatic potential in athymic mice.

The effects of gamma-linolenic acid (GLA) on growth of cells of the continuous murine sarcoma line M52B were investigated in vitro. Prostaglandin (PG) production by these cells after GLA treatment was also measured. GLA inhibited the growth of M52B cells and became overtly toxic at high doses or after long periods of exposure to lower doses. The inhibitory effects of GLA were accompanied by an increase in PGE production by M52B cells. However, the rise in PGE was not statistically significant. Accordingly the extent to which PGE may contribute to the inhibition observed with GLA remains unclear. In order to establish whether these in vitro effects could be reproduced in vivo, athymic nude mice bearing murine sarcoma allografts were fed either standard laboratory diets or diets supplemented with 35% evening primrose oil, which contains 10% GLA. As there was no significant difference in tumour volumes between the two groups at the end of the treatment period, the oil-enriched diet was concluded to be without effect on tumour growth in this in vivo model.