Search

9559Background: Prior studies in pts with BRAFmM have shown increased density of tumor infiltrating lymphocytes (TIL) at 2 weeks (wks) with BRAF +/- MEK inhibition(i), but did not fully characteriz...

e15171Background: We designed a first-in-human clinical trial to evaluate safety and immunogenicity of LPV7, administered in vaccine adjuvant combinations of incomplete Freund’s adjuvant (IFA) and ...

Large centers have described triangular intermuscular space (TIS) sentinel nodes (SNs) for some melanomas of the back. However, their management remains controversial and poorly characterized, especially as related to the ipsilateral axillary node basin. The aim of this study was to summarize our experience with TIS SN, which may contribute to defining their appropriate surgical management.We performed a retrospective review on surgical patients from January 1993 to April 2009. Among 293 patients with upper back melanoma, data were collected on those with TIS SN.Fourteen patients (5%) with melanoma of the upper back had a TIS SN, 6 of whom (43%) were incorrectly identified at lymphoscintigraphy as axillary, and 11 of whom (79%) had a concurrent axillary SN. Micrometastatic disease was identified in TIS SN in two patients (14%) and in an axillary SN in one (9%). We found direct lymphatic drainage independently to the TIS and to the axilla, as well as a more typical pattern of drainage first to the TIS node and then to axillary nodes.We defined three patterns of lymphatic drainage to TIS and axillary nodes. The TIS and axilla are anatomically linked; patients with SN in both locations should undergo biopsies of both for optimal nodal staging. We recommend directed evaluation for TIS SN in patients with upper back melanomas and recommend clearing the TIS at the time of TIS SN biopsy. Melanoma can metastasize to TIS SN, and we discuss considerations for management of the axilla in patients with positive TIS nodes.

269 Background: Cholangiocarcinoma (CC) is a fatal malignancy with an unmet treatment need. With the approval of immunotherapy for solid tumors with mismatch repair (MMR) deficiency, there is a renewed interest in MMR testing. Little is known about the incidence of MMR deficiency in CC or its correlation to survival, immune cell infiltration, PD-L1 and other proteins expressed in CC such as mesothelin. Methods: CC tumors were identified from patients treated at the University of Virginia from 2000-2014. Tissue microarrays (TMAs) were constructed of 3-4 cores from each tumor and were stained by immunohistochemistry for MMR genes (MLH1, PMS2, MSH2, MSH6), mesothelin, PD-L1 and immune cells. TMAs were scanned using the Leica SCN400 and analyzed using the Digital Image Hub software. Stain intensity thresholds for defining positive cells were determined by two users and recorded as an average of all cores from each tumor. Mesothelin and PD-L1 expression were measured as a percentage of positive tumor cells. Correlation with overall survival was assessed using log-rank tests and classification and regression trees, with p values < 0.05 considered significant. Results: Ninety-one tumors were analyzed: 24 intrahepatic, 33 hilar, and 34 distal. MMR deficiency was found in 20 tumors (22%). None of the MMR deficient tumors co-expressed PD-L1 (>1%), which was found in 15% of the remaining tumors. T cell infiltration (CD4, CD8 and FoxP3) did not differ between MMR deficient or proficient tumors. Patients with MMR deficiency had a trend towards worse survival compared to those with proficiency (median OS: 19.2 vs. 28.1 months, p = 0.07). MMR deficient tumors showed a lower mesothelin expression compared to MMR proficient tumors, median 8 vs. 129 positive cells per TMA (p = 0.08). Patients with MMR deficiency and low mesothelin expression had a worse outcome compared to patients with MMR proficiency and high mesothelin expression (median OS: 14.5 vs. 30.0 months, p = 0.05). Conclusions: Given the high rate of MMR deficiency, all CC tumors should be tested and may benefit from anti-PD-1 therapy. The poor prognosis of MMR deficient CC may be independent of T-cell infiltration and additional studies are needed to better characterize the genetic and molecular landscape of this subset of tumors.

The mechanisms by which immune checkpoint blockade modulates tumor evolution during therapy are unclear. We assessed genomic changes in tumors from 68 patients with advanced melanoma, who progressed on ipilimumab or were ipilimumab-naive, before and after nivolumab initiation (CA209-038 study). Tumors were analyzed by whole-exome, transcriptome, and/or T cell receptor (TCR) sequencing. In responding patients, mutation and neoantigen load were reduced from baseline, and analysis of intratumoral heterogeneity during therapy demonstrated differential clonal evolution within tumors and putative selection against neoantigenic mutations on-therapy. Transcriptome analyses before and during nivolumab therapy revealed increases in distinct immune cell subsets, activation of specific transcriptional networks, and upregulation of immune checkpoint genes that were more pronounced in patients with response. Temporal changes in intratumoral TCR repertoire revealed expansion of T cell clones in the setting of neoantigen loss. Comprehensive genomic profiling data in this study provide insight into nivolumab's mechanism of action.

Purpose: Granulocyte/macrophage colony-stimulating factor (GM-CSF) administered locally together with vaccines can augment T-cell responses in animal models. Human experience has been limited to small and uncontrolled trials. Thus, a multicenter randomized phase II trial was done to determine whether local administration of GM-CSF augments immunogenicity of a multipeptide vaccine. It also assessed immunogenicity of administration in one versus two vaccine sites.Experimental Design: One hundred twenty-one eligible patients with resected stage IIB to IV melanoma were vaccinated with 12 MHC class Irestricted melanoma peptides to stimulate CD8+ T cells plus a HLA-DRrestricted tetanus helper peptide to stimulate CD4+ T cells, emulsified in incomplete Freund's adjuvant, with or without 110 g GM-CSF. Among 119 evaluable patients, T-cell responses were assessed by IFN- ELIspot assay and tetramer analysis. Clinical outcomes were recorded.Results: CD8+ T-cell response rates to the 12 MHC class Irestricted melanoma peptides (by day 50) with or without GM-CSF were 34 and 73, respectively (P < 0.001), by direct ELIspot assay. Tetramer analyses corroborated the functional data. CD4+ T-cell responses to tetanus helper peptide were higher without GM-CSF (95 versus 77; P = 0.005). There was no significant difference by number of vaccine sites. Three-year overall and disease-free survival estimates (95 confidence interval) were 76 (67-83) and 52 (43-61), respectively, with too few events to assess differences by study group.Conclusions: High immune response rates for this multipeptide vaccine were achieved, but CD8+ and CD4+ T-cell responses were lower when administered with GM-CSF. These data challenge the value of local GM-CSF as a vaccine adjuvant in humans. (Clin Cancer Res 2009;15(22):703644)

Background: There is disagreement about the behavior and optimal management of desmoplastic melanoma (DM), particularly regarding the incidence of lymph node (LN) involvement. Recently, investigators have noted the frequently heterogenous histologic composition of DM and have found significant differences between pure desmoplastic melanoma (PDM) (≥90% comprised of histologically typical DM) and mixed desmoplastic melanoma (MDM) [≥10% DM and >10% conventional melanoma (CM)]. Method: We reviewed 87 cases of DM comparing the histologic and clinical features of PDM (n = 44) to MDM (n = 43). Results: At surgical staging, there were LN metastases in 5 of 23 (22%) MDM patients, whereas all 17 PDM patients had negative LN biopsies (0%) (p = 0.04). PDM was less often clinically pigmented (36% vs. 67%) and had a lower mean mitotic index (1.3 vs. 3.0). Conclusions: There are differences between PDM and MDM, the most important of which is the incidence of LN involvement. Our findings support the clinical utility of classifying DM into pure and mixed subtypes because the negligible rate of nodal involvement in PDM does not support the routine performance of sentinel LN biopsy in this subgroup of melanoma patients. In contrast, the incidence of LN involvement in MDM is comparable to that of CM.

Vascular endothelial growth factor (VEGF) plays a vital role in tumor angiogenesis. VEGF is produced by human melanomas, and the VEGF receptor 2 (VEGFR-2) is expressed by most advanced stage melanomas, suggesting the possibility of an autocrine loop. Here, we show that bevacizumab, an anti-VEGF antibody, inhibits proliferation of VEGFR-2+ melanoma cell lines by an average of 41%; however, it failed to inhibit proliferation of VEGFR-2neg melanoma cell lines. The growth inhibitory effect of bevacizumab was eliminated by VEGFR-2 knockdown with small interfering RNA, showing that VEGF autocrine growth in melanoma is mediated through VEGFR-2. However, bevacizumab inhibition of autocrine signals did not completely inhibit cell proliferation nor cause cell death. Cell survival is mediated partially through mammalian target of rapamycin (mTOR), which is inhibited by rapamycin. Combination of bevacizumab with rapamycin caused loss of half of the VEGFR-2+ melanoma cells, but no reduction in the number of VEGFR-2neg melanoma cells. The results show (a) an autocrine growth loop active in VEGFR-2+ melanoma, (b) a nonangiogenic mechanism for inhibition of melanoma by blocking autocrine VEGFR-2 activation, and (c) a possible therapeutic role for combination of inhibitors of mTOR plus VEGF in selected melanomas. [Cancer Res 2008;68(11):4392–7]

Nine participants with epithelial ovarian, fallopian tube, or primary peritoneal carcinoma, who were human leukocyte antigen (HLA)-A1, HLA-A2, or HLA-A3, were eligible to enroll in a phase 1 study designed to assess the safety and immunogenicity of a peptide-based vaccine. Participants received 5 class I major histocompatibility complex-restricted synthetic peptides derived from multiple ovarian cancer-associated proteins plus a class II major histocompatibility complex-restricted synthetic helper peptide derived from tetanus toxoid protein. The vaccines were administered with granulocyte macrophage-colony stimulating factor in Montanide ISA-51 adjuvant over a 7-week period. All vaccine-related toxicities were grade 1 to 2, the most common being injection site reaction (grade 2, 100%), fatigue (grade 1, 78%), and headache (grade 1, 67%). Lymphocytes from the peripheral blood and a node draining a secondary vaccine site (sentinel immunized node) were harvested during the course of vaccination and T-cell responses to the peptides were evaluated using an enzyme-linked immunosorbent spot assay. CD8 T-cell responses were detected in 1 participant ex vivo and in 8 of 9 participants (89%) after in vitro stimulation. All 4 HLA-A2 and HLA-A3-restricted peptides were immunogenic. This includes 2 peptides, folate binding protein (FBP191-199) and Her-2/neu754-762, which had not previously been evaluated in vaccines in humans. Responding T cells required over 200 nM for half-maximal reactivity. These data support continued investigation of these peptides as immunogens for patients with ovarian cancer but, owing to low potency, also suggest a need for additional immunomodulation in combination with vaccines to increase the magnitude and to improve the quality of the T-cell responses.

Purpose: Human melanoma cells express shared antigens recognized by CD8+ T lymphocytes, the most common of which are melanocytic differentiation proteins and cancer-testis antigens. However, peptide vaccines for melanoma usually target only one or two MHC class I–associated peptide antigens. Because melanomas commonly evade immune recognition by selective antigen loss, optimization of melanoma vaccines may require development of more complex multipeptide vaccines.Experimental Design: In a prospective randomized clinical trial, we have evaluated the safety and immunogenicity of a vaccine containing a mixture of 12 peptides from melanocytic differentiation proteins and cancer-testis antigens, designed for human leukocyte antigen types that represent 80% of the melanoma patient population. This was compared with a four-peptide vaccine with only melanocytic differentiation peptides. Immune responses were assessed in peripheral blood and in vaccine-draining lymph nodes.Results: These data show that (a) the 12-peptide mixture is immunogenic in all treated patients; (b) immunogenicity of individual peptides is maintained despite competition with additional peptides for binding to MHC molecules; (c) a broader and more robust immune response is induced by vaccination with the more complex 12-peptide mixture; and (d) clinical outcome in this peptide vaccine trial correlates with immune responses measured in the peripheral blood lymphocytes.Conclusions: These data support continued investigation of complex multipeptide vaccines for melanoma.

Neurocristic hamartomas (NCH) are rare pigmented skin lesions based in the deep subcutaneous tissues that may be either congenital or acquired. The clinical importance of these lesions is the potential for misdiagnosis and the development of malignant melanomas over a poorly described time course. Histological pleomorphism precludes meaningful random biopsies as a means of cancer surveillance. We present the case of an extensive NCH in a 67-year-old man, with a reported duration of greater than 50 years and no current clinical or histological indication of malignancy. Incisional biopsies of nodular areas showed bland-appearing pigmented cells that extended into subcutaneous adipose tissue and skeletal muscle. The specimens contained numerous clusters of differing configurations and cell types. Positron emission tomography (PET) scanning was used as an adjunct to physical examination in follow up. A PET-avid mass was detected but proved to be a banal nodular melanocytic proliferation within the NCH. In conclusion, NCH may be characterized by extensive deep tissue involvement in the absence of overt malignancy. The possible development of malignant melanoma in such lesions warrants close surveillance.

In oncology, vaccine-based immunotherapy often investigates regimens that demonstrate minimal toxicity overall and higher doses may not correlate with greater immune response. Rather than determining the maximum tolerated dose, the goal of the study becomes locating the optimal biological dose, which is defined as a safe dose demonstrating the greatest immunogenicity, based on some predefined measure of immune response. Incorporation of adjuvants, new or optimized peptide vaccines, and combining vaccines with immune modulators may enhance immune response, with the aim of improving clinical response. Innovative dose escalation strategies are needed to establish the safety and immunogenicity of new immunologic combinations. We describe the implementation of an adaptive design for identifying the optimal treatment strategy in a multi-site, FDA-approved, phase I/II trial of a novel vaccination approach using long-peptides plus TLR agonists for resected stage IIB-IV melanoma. Operating characteristics of the design are demonstrated under various possible true scenarios via simulation studies. Overall performance indicates that the design is a practical Phase I/II adaptive method for use with combined immunotherapy agents. The simulation results demonstrate the method's ability to effectively recommend optimal regimens in a high percentage of trials with manageable sample sizes. The numerical results presented in this work include the type of simulation information that aid review boards in understanding design performance, such as average sample size and frequency of early trial termination, which we hope will augment early-phase trial design in cancer immunotherapy.

Purpose: A melanoma vaccine incorporating six peptides designed to induce helper T-cell responses to melanoma antigens has induced Th1-dominant CD4 + T-cell responses in most patients, and induced durable clinical responses or stable disease in 24% of evaluable patients. The present study tested whether this vaccine also induced antibody (Ab) responses to each peptide, and whether Ab responses were associated with T-cell responses and with clinical outcome. Experimental Design: Serum samples were studied from 35 patients with stage III-IV melanomas vaccinated with 6 melanoma helper peptides (6MHP). IgG Ab responses were measured by ELISA. Associations with immune response and overall survival were assessed by log-rank test and χ 2 analysis of Kaplan–Meier data. Results: Ab responses to 6MHP were detected by week 7 in 77% of patients, and increased to peak 6 weeks after the last vaccine and persisted to 6 months. Ab responses were induced most frequently to longer peptides. Of those with T-cell responses, 82% had early Ab responses. Survival was improved for patients with early Ab response ( P = 0.0011) or with early T-cell response ( P P = 0.0002). Conclusions: Vaccination with helper peptides induced both Ab responses and T-cell responses, associated with favorable clinical outcome. Such immune responses may predict favorable clinical outcome to guide combination immunotherapy. Further studies are warranted to understand mechanisms of interaction of these Abs, T-cell responses, and tumor control. Clin Cancer Res; 21(17); 3879–87. ©2015 AACR .

There has been a rush to convert discovery of new melanoma antigens into cancer vaccines for the therapy of melanoma. The result has been disappointing from a clinical standpoint. The premise behind rapid pursuit of peptide vaccines for melanoma therapy was that the spontaneous tumor-associated immune response was too weak to be effective. However, it is increasingly clear that the host-tumor relationship is a complex interplay of immune response, immune escape, and immune adaptation, with multiple layers of regulatory control and modulation of responses over time. The lesion in the immune response to cancer is much more complex than simply a weak immune response to defined antigens. Current results should serve as a call to take a closer look at immune regulatory processes and principles and to develop more comprehensive and multiagent approaches to modulate the host-tumor relationship. Development of effective immune therapy for cancer will require (a) more comprehensive and real-time immune monitoring in various tissue compartments and (b) patient-specific modulation of immune responses, informed by the real-time monitoring. Peptide antigens associated with MHC class I or class II molecules are the molecular targets for T-cell recognition of cancer. To characterize the host-tumor relationship and to optimize cancer vaccines, clinical studies using defined peptide antigens offer special opportunities to advance the field and thus have an important place in the ongoing development of effective immune therapy of melanoma.

Innovations and Challenges in Melanoma, chaired by Michael Atkins and cochaired by Ulrich Keilholz, John Kirkwood, and Jeffrey Sosman, was held July 15 to 16, 2005, in Cambridge, Massachusetts. The conference brought together leading experts in the fields of cancer research, medical oncology, surgical oncology, anatomic pathology, dermatology, and immunotherapy who wished to advance the field of melanoma treatment by exchanging information and perspectives regarding recent advances and recommendations for further study. The conference proceedings published in this educational supplement to Clinical Cancer Research are intended to provide timely information and recommendations on how genetics, biology, and data information can enhance our understanding of melanoma biology and help inform the use of therapies for this disease.

Meeting abstracts Cancer vaccines have promise as monotherapy or as part of combination immunotherapy regimens. Age and gender implications on immune response to cancer vaccinations have not previously been well described. There is uncertainty about including elderly patients as study participants

Meeting abstracts Intralesional therapies offer promise to modulate immune signatures within melanoma and other cancers, either as monotherapy or as a component of combination immune therapy. We have shown that the TLR2/6 agonists (MALP-2 and FSL-1) and IFNγ induce human melanoma cells to

Meeting abstracts The recMAGE-A3 protein has been administered intramuscularly (IM) with immunostimulant AS15 as an experimental immunotherapeutic. AS15 contains 3-O-desacyl-4'-monophosphoryl lipid A (MPL), QS-21, CpG 7909 and liposome. This MAGE-A3/AS15 immunotherapeutic has not been studied for

Immune-mediated control of tumors may occur, in part, through lysis of malignant cells by CD8+ T cells that recognize specific Ag-HLA class I complexes. However, tumor cell populations may escape T cell responses by immune editing, by preventing formation of those Ag-HLA complexes. It remains unclear whether the human immune system can respond to immune editing and recognize newly arising escape variants. We report an example of shifting immune responses to escape variants in a patient with sequential metastases of melanoma and long-term survival after surgery alone. Tumor cells in the first metastasis escaped immune recognition via selective loss of an HLA haplotype (HLA-A11, -B44, and -Cw17), but maintained expression of HLA-A2. In the second metastasis, immune escape from an immunodominant MART-1-specific T cell response was mediated by HLA class I down-regulation, resulting in a failure to present this epitope, but persistent presentation of a tyrosinase-derived epitope. Consequent to this modification in tumor Ag presentation, the dominant CTL response shifted principally toward a tyrosinase-targeted response, even though tyrosinase-specific CTL had been undetectable during the initial metastatic event. Thus, in response to immune editing of tumor cells, a patient’s spontaneous T cell response adapted, gaining the ability to recognize and to lyse “edited” tumor targets. The observation of both immune editing and immune adaptation in a patient with long-term survival after surgery alone demonstrates an example of immune system reactivity to counteract the escape mechanism(s) developed by tumor cells, which may contribute to the clinical outcome of malignant disease.

Purpose: To assess changes in serum cytokine levels in patients treated concomitantly with or without systemic low-dose IL-2. Vaccination targeted CTL responses to peptide antigens, and IL-2 was coadministered to expand activated CTL. Paradoxically, CTL responses were diminished in patients after 2 weeks of IL-2. We hypothesized that changes in the cytokine milieu may have contributed to this result. Experimental design: Serum samples were studied from 37 patients enrolled in two clinical trials of a melanoma peptide vaccine administered with or without low-dose IL-2 therapy. Twenty-two patients enrolled in the MEL36 trial received six weekly vaccinations with the four-peptide mixture and were randomized to receive subcutaneous IL-2 (3×106 IU/m2/day) daily for 6 weeks beginning either at week 1 (upfront group) or at week 4 (delayed group) of vaccine therapy. Fifteen patients on the MEL39 trial were treated with the same vaccine without concurrent IL-2 administration. Results: Circulating levels of IL-5 peaked 1 week after starting IL-2, followed 2 weeks later by a marked eosinophilia, correlating in magnitude with peak IL-5 serum levels. Levels of IFNγ, GM-CSF, IL-4, IL-10, and IL-12 had no observed relationship to IL-2 administration. At the time of the IL-5 serum peak, PBL responses to mitogen suggested a transient shift to Th2-dominance. Conclusions: Low-dose IL-2 appears to have induced a transient Th2-dominant secondary cytokine cascade at the time of vaccination, for which eosinophilia is a surrogate marker. For future vaccine therapies targeting cytotoxic T-cell responses, delaying IL-2 until after initiation of immune responses may be more effective.

In 2004, there were an estimated 147,000 new cases of colorectal carcinoma (CRC) and 57,000 deaths from this disease, ranking it third among causes of cancer-related death in the United States.1 Significant advances in systemic therapy for metastatic CRC, including targeted therapies, have improved survival, but even with combination therapy the median survival is only about 15 to 21 months.2,3 To continue to improve our therapies for metastatic CRC, we need a better understanding of the factors that lead to tumor progression and metastasis. In particular, the mechanisms regulating CRC cell invasion through the basement membrane of the colon and migration of the cells to form metastases need to be further investigated. Insulinlike growth factor-I (IGF-I) and its tyrosine kinase receptor (IGF-IR) have been implicated in the development and progression of a variety of human cancers,4–12 including CRC.13–15 IGF-I has been shown to be an important mediator of tumor cell migration and invasion,16–20 but the downstream pathways by which IGF-I induces these processes have not been fully elucidated. Hepatocyte growth factor/scatter factor (HGF) and its tyrosine kinase receptor c-Met have also been implicated in the pathogenesis of a wide variety of human malignancies,21–25 including CRC.26 Similar to IGF-I, HGF/c-Met signaling is known to induce tumor-cell migration and invasion.11,25,27–30 Recently, cooperation between receptors and their signaling pathways has been shown to be important in regulating cellular responses to various ligands. We theorized that IGF-IR and c-Met cooperate in mediating migration and invasion of human CRC cells, given the following findings. First, IGF-I and HGF lead to activation of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system in various malignancies.4,11,17,27,31,32 This is central to our hypothesis in that uPA has been shown to cleave pro-HGF to active HGF.33 Second, IGF-I signaling results in induction of hypoxia inducible factor-1α in pancreatic carcinoma cells,10 and hypoxia, likely acting via hypoxia inducible factor-1α, has been shown to increase c-Met levels in human lung, hepatocellular, and other carcinomas.34 Third, growth factor receptor-binding protein 2-associated binder-1 functions as the main substrate and docking protein regulating downstream signaling by c-Met and has been shown to function as a signaling intermediate for IGF-I as well.35 Fourth, IGF-I and HGF have been shown to function as comitogens in a rat hepatoma cell line.36 In the current study, we investigated the hypotheses that IGF-IR and c-Met cooperate to mediate migration and invasion of human CRC cells and that uPA/uPAR activation is required for IGF-I- and HGF-mediated migration and invasion. We used a c-Met ribozyme to inhibit c-Met function in KM12L4 human CRC cells and performed transwell migration and invasion assays. The c-Met ribozyme experiments demonstrated that c-Met function is critical for IGF-I-mediated cell migration and invasion and for constitutive invasion. In experiments inhibiting uPA or uPAR, we demonstrated that migration and invasion mediated by IGF-I and HGF are dependent on uPA/uPAR activation. This suggests that uPA and uPAR are downstream of IGF-I and IGF-IR and of HGF and c-Met. To our knowledge, this study is the first to identify tyrosine kinase receptor cooperation between IGF-IR and c-Met in human CRC and the role of uPA/uPAR in mediating IGF-I and HGF/c-Met effects.

The effectiveness of peptide-based cancer vaccines depends on the ability of peptides to bind to MHC molecules on the surface of antigen-presenting cells, where they reconstitute epitopes for cytotoxic T lymphocytes (CTLs). Multivalent vaccines have advantages over single-peptide vaccines; however, peptides may compete for binding to the same MHC molecules. In particular, it is possible that peptides with high affinity for MHC molecules prevent the binding of lower-affinity peptides. However, only small numbers of peptide/MHC complexes per cell are required for CTL recognition. Thus, the authors hypothesized that competition of peptides for MHC binding would not significantly reduce CTL recognition of individual peptides within a multiple-peptide mixture, and this hypothesis was tested by a series of experiments performed in vitro. In multiple experiments, two peptides with different affinities for HLA-A*0201 molecules were mixed at various concentrations and pulsed onto HLA-A2 cells, which were then evaluated for susceptibility to lysis by HLA-A*0201-restricted CTLs. CTL recognition of the melanoma peptides gp100(154-162) (KTWGQYWQV), gp100(280-288) (YLEPGPVTA), and tyrosinase(369-377D) (YMDGTMSQV) was maintained even when target cells were co-pulsed with equimolar concentrations of peptides with comparable or higher affinity for HLA-A2. In some cases, CTL recognition was maintained even when the higher-affinity peptide was present at concentrations several orders of magnitude higher than the target peptide. In addition, CTLs generated by in vitro stimulation with a peptide mixture developed reactivity to three different peptides, at a level comparable to that obtained by stimulation with each individual peptide separately. These data suggest that CTLs can respond to multiple peptides presented on the same antigen-presenting cells and justify further investigation, in clinical trials, of multiple-peptide cancer vaccines.

Melanoma progresses as a multistep process where the thickness of the lesion and depth of tumor invasion are the best prognostic indicators of clinical outcome. Degradation of the interstitial collagens in the extracellular matrix is an integral component of tumor invasion and metastasis, and much of this degradation is mediated by collagenase-1 (MMP-1), a member of the matrix metalloproteinase (MMP) family. MMP-1 levels increase during melanoma progression where they are associated with shorter disease-free survival. The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway is a major regulator of melanoma cell proliferation. Recently, BRAF has been identified as a common site of activating mutations, and, although many reports focus on its growth-promoting effects, this pathway has also been implicated in progression toward metastatic disease. In this study, we describe four melanoma cell lines that produce high levels of MMP-1 constitutively. In each cell line the Ras/Raf/MEK/ERK pathway is constitutively active and is the dominant pathway driving the production of MMP-1. Activation of this pathway arises due to either an activating mutation in BRAF (three cell lines) or autocrine fibroblast growth factor signaling (one cell line). Furthermore, blocking MEK/ERK activity inhibits melanoma cell proliferation and abrogates collagen degradation, thus decreasing their metastatic potential. Importantly, this inhibition of invasive behavior can occur in the absence of any detectable changes in cell proliferation and survival. Thus, constitutive activation of this MAPK pathway not only promotes the increased proliferation of melanoma cells but is also important for the acquisition of an invasive phenotype.

Purpose: Members of the SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome) family of cancer-testis antigens are promising targets for tumor immunotherapy because they are normally expressed exclusively during spermiogenesis on the adluminal side of the blood-testis barrier, an immune privileged compartment. Experimental Design and Results: This study analyzed the human SPANX genomic organization, as well as SPAN-X mRNA and protein expression in somatic and cancer cells. The SPANX family consists of five genes, one of which is duplicated, all located in a gene cluster at Xq27.1. From the centromere, the arrangement of the five SPANX genes mapped on one contiguous sequence is SPANXB, -C, -A1, -A2, and -D. Reverse transcription-PCR analyses demonstrated expression of SPAN-X mRNA in melanoma and ovarian cell lines, and virtual Northern analysis established SPANX gene expression in numerous cancer cell lines. Immunoblot analysis using polyclonal antisera raised against recombinant SPAN-X confirmed the translation of SPAN-X proteins in melanoma and ovarian tumor cell lines. The immunoreactive proteins migrated between Mr 15,000 and Mr 20,000 similar to those observed in spermatozoa. Immunoperoxidase labeling of melanoma cells and tissue sections demonstrated SPAN-X protein localization in the nucleus, cytoplasm, or both. Ultrastructurally, in melanoma cells with nuclear SPAN-X, the protein was associated with the nuclear envelope, a localization similar to that observed in human spermatids and spermatozoa. Significantly, the incidence of SPAN-X-positive immunostaining was greatest in the more aggressive skin tumors, particularly in distant, nonlymphatic metastatic melanomas. Conclusions: The data herein suggest that the SPAN-X protein may be a useful target in cancer immunotherapy.

Background: Patients have traditionally been considered candidates for sentinel node biopsy (SNBx) only at the time of wide local excision (WLE). We hypothesized that patients with prior WLE may also be staged accurately with SNBx. Methods: Seventy-six patients, including 18 patients from the University of Virginia and 58 from a multicenter study of SNBx led by investigators at the University of Vermont, who had previous WLE for clinically localized melanoma underwent lymphoscintigraphy with SNBx. Median follow-up time was 38 months. Results: Intraoperative identification of at least 1 sentinel node was accomplished in 75 patients (98.6%). The mean number of sentinel nodes removed per patient was 2.0. Eleven patients (15%) had positive sentinel nodes. Among the 64 patients with negative SNBx, 3 (4%) developed nodal recurrences in a sentinel node–negative basin simultaneous with systemic metastasis, and 1 (1%) developed an isolated first recurrence in a lymph node. Conclusions:This multicenter study more than doubles the published experience with SNBx after WLE and provides much-needed outcome data on recurrence after SNBx in these patients. These outcomes compare favorably with the reported literature for patients with SNBx at the time of WLE, suggesting that accurate staging of the regional lymph node bed is possible in patients after WLE.

Background Urogenital melanoma is a rare neoplasm with poor prognosis. Its management in the past involved radical vulvectomy and complete bilateral inguinofemoral lymphadenectomy. Sentinel lymph node biopsy is an accurate low-morbidity procedure when used in the context of cutaneous melanoma. However, prophylactic lymphadenectomy has not been shown to improve survival of melanoma patients. We wanted to determine the feasibility of sentinel lymph node biopsy in patients with female urogenital melanoma as a staging procedure. Methods Six patients with vulvar or vaginal melanomas underwent preoperative lymphatic mapping with (99m)Tc-labeled sulfur colloid followed by sentinel lymphadenectomy. In addition, we reviewed the literature on the application of sentinel lymph node biopsy in urogenital tract melanomas. Results One or more sentinel nodes were identified in all six patients by lymphoscintigraphy. All patients underwent sentinel lymphadenectomy, except for one patient with a deep vaginal melanoma that drained to pelvic nodes. The five successful cases had unilateral drainage patterns. None of the sentinel lymph nodes excised had tumor invasion. Combined with five other patients from the published literature, the success rate of localizing sentinel lymph nodes in the patients with urogenital melanoma approaches 100%. Conclusions This experience, plus reports of a small number of patients from three similar studies, supports the impression that sentinel lymph node biopsy is feasible for vulvar and vaginal melanoma.

Antigen-presenting cells (APCs) can induce tolerance or immunity. We describe a subset of human APCs that express indoleamine 2,3-dioxygenase (IDO) and inhibit T cell proliferation in vitro. IDO-positive APCs constituted a discrete subset identified by coexpression of the cell-surface markers CD123 and CCR6. In the dendritic cell (DC) lineage, IDO-mediated suppressor activity was present in fully mature as well as immature CD123+DCs. IDO+DCs could also be readily detected in vivo, which suggests that these cells may represent a regulatory subset of APCs in humans.

Background: The delineation of radial and vertical growth phases in primary cutaneous malignant melanomas has contributed to our understanding of melanoma progression and has enhanced the ability of pathologists to provide clinicians with meaningful prognostic information. Vertical growth phase (VGP) lesions have the potential to metastasize, but radial growth phase (RGP) melanomas are believed to lack competence for metastasis. Methods: We have identified three cases in which metastasis occurred in association with lesions initially interpreted as RGP melanomas. To determine whether these cases truly represented exceptional metastasizing RGP melanomas or VGP lesions incorrectly identified as RGP lesions, careful microscopic re-review of these cases was performed. Results: In one case, additional microscopic sectioning revealed a focus of vertical growth that was not evident on the original sections. In the other two cases, only radial growth was found. In one of these cases there was melanoma in situ with regressive changes, but no evidence for invasive melanoma. In the other, a RGP lesion was associated with an adjacent compound nevus with periadnexal involvement. Conclusions: These cases suggest that, while true RGP melanomas have an excellent prognosis, caution must be exercised in defining a lesion as having no metastatic potential when multiple sections of the primary lesion are unavailable, when the lesion is accompanied by regressive changes, or when there is an associated melanocytic nevus. It is possible that strictly defined RGP melanomas may metastasize in very rare cases. Our observations also suggest that metastatic potential is a function of numerous factors, and may not be evaluable on morphological grounds alone.

Not Available Study supported by Castle Biosciences.

Introduction: Nivolumab and ipilimumab (NIVO+IPI) in combination was more efficacious than nivolumab (NIVO) alone in advanced melanoma patients (pts) in the phase 3 CheckMate 067 study (Larkin et al, NEJM. 2015). To elucidate the differential mechanisms of action of NIVO+IPI vs NIVO, we assessed biomarkers in pts with advanced melanoma treated with these agents in the CheckMate 038 study. Methods: IPI-naive pts without brain metastases from parts 1-3 of the phase 1b CA209-038 study (NCT01621490) who received NIVO+IPI Q3W (1 mg/kg+3 mg/kg, n=53) or NIVO alone Q2W (3 mg/kg, n=52) were included in the analysis. Tumor and peripheral immune markers were assessed (Table), and compared across treatment groups and by best overall response (BOR) per RECIST v1.1. Results: Increases in tumor-infiltrating CD8 T cells were observed with both NIVO+IPI and NIVO treatment, with greatest increases in NIVO+IPI pts with CR/PR or SD vs other BOR groups (Table). Increases in tumor PD-L1 expression were observed with both treatments, with 10-fold greater increases in pts with CR/PR to NIVO+IPI vs pts with PD/NE to NIVO+IPI or CR/PR to NIVO alone. Reductions in circulating MDSCs were observed with NIVO+IPI treatment across BOR groups, but only in pts with CR/PR in the NIVO-treated group. Increases in CXCL9, CXCL10 and IFN-gamma were observed irrespective of BOR in pts treated with both NIVO+IPI and NIVO alone, with the greatest increases with NIVO+IPI treatment. Conclusion: NIVO+IPI vs NIVO alone was associated with consistent reductions in MDSCs and greater increases in IFN-gamma signaling. Pts with CR/PR to NIVO+IPI had the greatest increases in CD8 T cells and PD-L1 expression, suggesting enhanced T-cell effector function. This may partly explain the higher ORR observed for NIVO+IPI vs NIVO alone in CheckMate 067. Further analyses will elucidate the differential immunomodulatory effects of NIVO+IPI in combination vs NIVO monotherapy in pts with advanced melanoma. Table.Summary of results dataMedian change from baselinea (min, max)NIVO+IPI, n=53NIVO alone, n=52CR/PRSDPD/NECR/PRSDPD/NETumor immune markerbn%n%n%n%n%n%CD8 T cells1013.8 (1.3, 40.1)612.6 (0.3, 39.6)132.9 (-16.8, 33.0)34.3 (-17.3, 26.2)3-0.4 (-3.0, 7.8)40.5 (-1.3, 24.6)PD-L11041.0 (5.0, 93.0)51.0 (0.0, 55.0)94.0 (-1.0, 57.0)94.0 (-17.0, 30.0)54.0 (2.0, 70.0)142.0 (-53.0, 70.0)Peripheral immune markercn%n%n%n%n%n%MDSCs13-1.4 (-12.6, 3.7)7-5.8 (-9.9, 3.3)12-1.5 (-7.7, 4.5)3-4.0 (-5.2, 2.8)22.8 (-0.4, 5.9)55.6 (-7.8, 14.2)Immune serum cytokinednpg/mLnpg/mLnpg/mLnpg/mLnpg/mLnpg/mLCXCL9164294.5 (840, 53653)53670 (2153, 26270)152354 (402, 23188)14934 (85, 8970)9748 (190, 1765)17762 (-1250, 4490)CXCL1016410 (2, 4775)5345 (131, 2403)15369 (-20, 1706)14187 (-27, 768)995 (50, 932)17155 (-172, 1099)IFN-gamma160.40 (-0.02, 38.93)50.09 (-0.14, 0.48)150.11 (-0.41, 42.97)140.02 (-0.02, 0.64)90.02 (-0.16, 4.33)170.04 (-0.08, 0.19)CR/PR=complete response/partial response; SD=stable disease; PD/NE=progressive disease/not evaluable for BOR, but otherwise response evaluablea Baseline is defined as the last evaluable data prior to first dose treatment within 29 daysb Tumor biopsies collected day 8-36 on-treatment, assessed by immunohistochemistryc Whole-blood samples collected prior to third-dose NIVO+IPI or prior to fourth-dose NIVO alone, assessed by flow cytometryd Serum samples collected day 15+/-3 on-treatment, assessed by immunoassays Citation Format: Antoni Ribas, Salvador Martín-Algarra, Shailender Bhatia, Wen-Jen Hwu, Craig L. Slingluff, William H. Sharfman, F. Stephen Hodi, Walter J. Urba, Jason J. Luke, John B. Haanen, Margaret K. Callahan, Jedd D. Wolchok, Scott D. Chasalow, Petra Ross-Macdonald, Tina C. Young, Anila Qureshi, Christine E. Horak. Immunomodulatory effects of nivolumab and ipilimumab in combination or nivolumab monotherapy in advanced melanoma patients: CheckMate 038 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT073. doi:10.1158/1538-7445.AM2017-CT073

Background: Response to checkpoint blockade may be dependent on tumor mutational load and the presence of antigen-specific effector T cells in the tumor microenvironment; however, how blockade modulates these features during therapy is unclear. We assessed genomic changes in tumors from patients (pts) with advanced melanoma receiving nivolumab (nivo) who progressed on ipilimumab (ipi-P) or were ipi-naive (ipi-N). Methods: Tumor biopsies were collected pretreatment and 4 weeks post first nivo dose from ipi-N or ipi-P pts treated with nivo 3 mg/kg Q2W in the phase 1 open-label CA209-038 study (NCT01621490). Biopsies from 68 pts were analyzed by whole exome, transcriptome, and/or TCR sequencing (paired biopsies from 41, 42, and 34 pts, respectively). Results: Objective response rate (ORR) in the overall cohort (n=85) was 27% with similar ORR in ipi-N and ipi-P cohorts. In the genomic cohort (n=68), ORR was 23% with a similar number of complete or partial responses (CR/PR) in ipi-N and ipi-P pts (n=7 and n=8, respectively). Prior to treatment, mutational and neoantigen load were comparable, regardless of previous treatment. Following nivo treatment, both mutational and neoantigen load were reduced 5-fold in pts who responded (CR/PR; n=9) and 1.2-fold in pts with stable disease (SD, n=13) compared with a 1.1-fold increase in pts with progressive disease (PD, n=19). Intratumoral heterogeneity analysis before and after nivo demonstrated that CR/PR pts generally lost tumor mutation clones/subclones. Novel tumor mutation clones were observed in on-treatment samples from 2 CR/PR pts and all pts who progressed on nivo. Transcriptome analyses revealed significant increases in distinct tumor immune cell subsets (CD8+ T cells and NK cells) and immune checkpoint gene expression (LAG3, CTLA4, PCDC1, and CD274 [PD-L1]) following nivo, which were more pronounced in pts with CR/PR vs PD (log2 fold-changes of 1.24, 1.07, 1.71, and 0.74, respectively). Consistent with the transcriptome analyses, tumor-infiltrating lymphocytes, as assessed by immunohistochemistry, generally increased following nivo in pts who responded: 2.8 vs 1.9-fold change in CR/PR/SD vs PD in the ipi-P cohort; 4.8 vs 1.8-fold change in CR/PR/SD vs PD in the ipi-N cohort. Differences in treatment-related TCR repertoire diversity changes were apparent between pts who responded within the ipi-N and ipi-P cohorts: a decrease in the evenness of T-cell clonotype distribution was observed among pts with CR/PR/SD relative to pts with PD in the ipi-N cohort (P=0.036), but not in the ipi-P cohort. Conclusion: Nivo and ipi modulate T-cell repertoire and tumor mutational heterogeneity in pts with advanced melanoma, presenting potential mechanisms of action underlying successful nivo therapy. These data also show that prior ipi treatment may influence biological response to nivo, but further investigation is warranted. Citation Format: Timothy A. Chan, Nadeem Riaz, Jonathan J. Havel, Vladimir Makarov, Alexis Desrichard, Jennifer S. Sims, F. Stephen Hodi, Salvador Martín-Algarra, William H. Sharfman, Shailender Bhatia, Wen-Jen Hwu, Thomas F. Gajewski, Craig L. Slingluff, Sviatoslav M. Kendall, Han Chang, John-William Sidhom, Jonathan P. Schneck, Nils Weinhold, Christine E. Horak, Walter J. Urba. Immunogenomic analyses of tumor cells and microenvironment in patients with advanced melanoma before and after treatment with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2017-2988

9573 Background: DecisionDx-Melanoma has been validated as an accurate prognosticator of cutaneous melanoma (CM) metastasis risk. The GEP test classifies CM pts as Class 1 (low risk) or Class 2 (high risk). Interim survival analysis from two clinical registry studies (NCT02355574/NCT02355587) designed to prospectively evaluate outcomes in pts for whom the GEP test was performed is described. Methods: Eleven US dermatologic and surgical centers participated in the IRB-approved protocols. Physicians enrolled CM pts who were ≥16 years old and had successful GEP test results. Endpoints of recurrence-free (RFS), distant metastasis-free (DMFS) and melanoma-specific survival (MSS) were assessed using Kaplan-Meier and Cox regression analysis. As an interim analysis at year 3 of an expected 5-year study, the critical alpha level (p-value) was 0.01. Results: At the time of data extraction, 322 pts were accrued and completed at least one follow-up visit. Median age was 58 years (range 18-87), median Breslow thickness (BT) was 1.2mm, 55% were male, 20% (58/296) were ulcerated, and 15% (36/237 biopsied) had a positive sentinel lymph node (SLN). Median follow-up time was 1.5 years for pts without a recurrence. Of 25 recurrent cases, 80% (20/25) were Class 2 and 40% (10/25) were SLN-positive. Two percent of Class 1 pts had a recurrence compared to 6% (12/201 biopsied) of SLN-negative pts. Of the SLN-negative pts who recurred, 75% (9/12) were called Class 2. Combined GEP and SLN risk prediction identified 88% (21/24) of recurrences. Kaplan-Meier event rates for each class are shown in the table. In Cox multivariate analysis, BT and GEP Class 2 were significant predictors of recurrence (p

e15650 Background: Cholangiocarcinoma (CC) is a rapidly progressing malignancy with an unmet treatment need. Little is known about the CC tumor immune microenvironment or about relevant antigenic targets. We hypothesized that lack of T cell infiltration or PD-L1 expression may identify patients at high risk of death, and that mesothelin may be a relevant antigenic target. Methods: A retrospective analysis was conducted of CC tumors at the University of Virginia from 2000-2014. TMAs were constructed of 3-4 cores from each tumor and were stained by IHC for CD4 and CD8 tumor infiltrating lymphocytes (TILs), mesothelin and PD-L1. TMAs were scanned using the Leica SCN400 and analyzed using the Digital Image Hub software. Stain intensity thresholds for defining positive cells were determined by two users and recorded as an average of all cores from each tumor. Mesothelin and PD-L1 expression were measured as a percentage of positive tumor cells. TILs and protein expression were analyzed for association with overall survival, grouped as high or low expression based either on the median or the 33rdpercentile. Correlation with overall survival was assessed using a log rank test and a classification and regression tree with p-values < 0.05 being considered statistically significant. Results: Ninety-nine tumors were available for analysis: 26 intrahepatic, 37 hilar, and 36 distal. PD-L1 and mesothelin expression > 1% of tumor cells were found in 16% and 92% of tumors, respectively. CD4 and CD8 TILs were found in nearly all tumors (98% and 96%), with the majority showing intraepithelial CD4 and CD8 infiltration (73% and 68%). There were no significant associations between survival and PD-L1, mesothelin, or CD4 and CD8 infiltration. However when considered together, the group with low mesothelin/low CD8 (each below 33rdpercentile) had worse survival (9.1 months) compared to high mesothelin/high CD8 (25 months), high mesothelin/low CD8 (30.1 months) and low mesothelin/high CD8 (26.1 months), p = 0.015. Conclusions: CC tumors that lack CD8 infiltration and mesothelin expression have a poor prognosis. Mesothelin represents an attractive target in cholangiocarcinoma, opening the door for future immunotherapy for CC.

e21080 Background: A 31-GEP test is predictive of metastasis in cutaneous melanoma. We hypothesized that GEP testing would “upgrade” surveillance to routine imaging in at least 16% of stage IB/IIA patients, similar to the upgrade rate from sentinel node biopsy. Methods: A GEP score was obtained for consecutive patients with Stage IB/IIA melanoma treated between 6/2014-6/2016. A retrospective review of a prospectively collected database was performed. Results: 67 patients with Stage IB/IIA melanoma met inclusion criteria. In four cases, a GEP result was not available. Of the 63 tested cases, 68% were Stage IB (N = 43), and 32% were Stage IIA (N = 20). A high-risk result was seen in 12% of stage IB (5/43) and 42% of stage IIA (8/19) patients. Insurance denied coverage of scans in 1/13 patients with a high-risk GEP result. The remaining 12 Stage IB/IIA patients with high-risk scores were “upgraded” to high-intensity surveillance. With a median follow-up of 14 months, 1/13 patients with a high-risk GEP result developed distant metastases 21 months after diagnosis of a Stage IIA melanoma. Conclusions: Results from this retrospective single institution study show that GEP testing altered patient management in 19% of Stage IB/IIA patients. Early detection in 1/13 patients with high-risk scores in this series supports further investigation into stratifying traditionally low-risk patients by GEP testing.

4125 Background: Pancreatic cancer (PC) is a challenging target for immunotherapy.Tumor-infiltrating lymphocytes (TILs) do not reach the PC cells in significant numbers due to the presence of stroma and a suppressive microenvironment. Neoadjuvant chemoradiation (CRT) can increase the presence of TILs in the PC microenvironment. We hypothesized that combination of CRT and pembrolizumab can lead to further increase in TILs and their activation. Methods: Patients with resectable or borderline resectable PC have been randomized 2:1 to the investigational treatment (Arm A) to receive pembrolizumab 200mg IV every 3 weeks on days 1, 22, and 43 during concurrent CRT with capecitabine (825 mg/m2 orally twice daily, Monday-Friday, on days of radiation only) and radiation (50.4 Gy in 28 fractions over 28 days) or Arm B to receive only concurrent CRT with capecitabine. Restaging CT scan or MRI is performed at 4-6 weeks after completion of neoadjuvant treatment, and patients with resectable disease will undergo surgical resection. Here we report the preliminary safety data based on 22 enrolled patients. Results: As of February 3-2017,22 patients have been enrolled (14 Arm A and 8 Arm B). 50% of the patients had resectable disease (7 arm A; 4 arm B) and the other 50% had borderline resectable disease (7 Arm A; 4 arm B). Post-neoadjuvant therapy, 6 patients had unresectable disease (3 on each arm), and 14 patients underwent surgery (10 arm A and 4 arm B). There were 7 grade 3 treatment-related toxicities in Arm A (5 patients): 2 grade 3 diarrhea attributed to CRT; 4 grade 3 lymphopenias attributed to pembrolizumab, CRT or the combination; and one patient had elevated alkaline phosphatase probably related to the combination that met the definition of DLT and resolved after holding the treatment and receiving steroids. There was only one grade 3 toxicity on Arm B: lymphopenia attributed to CRT. No grade 4 toxicities have been reported on either arm. There were no major surgical complications reported within 30 days post-surgery. Conclusions: The combination of CRT and pembrolizuamb is safe based on the presented data. Clinical trial information: NCT02305186.

e21009 Background: The combination of Ipilimumab and Nivolumab is standard initial therapy in patients with advanced melanoma based on trials involving treatment-naïve patients. The benefit in those previously managed with checkpoint monotherapy is not well defined. Methods: We identified metastatic melanoma patients from our Immunotherapy database managed with combination Ipilimumab/Nivolumab after progression on prior checkpoint monotherapy. Baseline clinical factors, treatment history, combination therapy outcome by RECIST v1.1 and toxicity data were collected. Descriptive statistics were used to summarize the data. Given the small sample size and limited numbers of deaths, it is too early to look for preliminary associations between outcomes and clinicopathologic factors. Results: We identified 19 patients treated with combination Ipilimumab/Nivolumab after progression on prior checkpoint monotherapy. The cohort included 15 men and 4 women with an average age of 63 years. Thirteen patients had M1c disease, and 7 had a BRAF mutation. Patients had received up to four lines of prior immunotherapy including 9 treated with both prior anti-PD1 and anti-CTLA4 monotherapy. Seven patients completed all four doses of combination therapy with 6 proceeding onto maintenance nivolumab. Eight patients stopped treatment due to toxicity and 4 due to progressive disease. Thirteen patients had clinically significant toxicity, with rash, colitis, hepatitis, and hypophysitis reported most frequently. There were no treatment-related deaths. Overall, 2/19 patients (10.5%, 95% CI [1.3% to 33.1%]) had an objective response (CR+PR) and 9/19 patients (47.4%, 95% CI [24.5% to 71.1%]) had disease control (CR+PR+SD). Four of the patients had stable disease for over 6 months. Six of the 19 patients went on to receive subsequent treatment. Median follow-up for patients still alive was 7 months (range 1 to 20 months) and median survival was not reached. Six-month survival was 68.5% (95% CI [39.3% to 85.8%]) Conclusions: The combination of Ipilimumab and Nivolumab can result in melanoma control in patients with progression on prior checkpoint monotherapy with an expected toxicity profile.

TPS3106 Background: Immunotherapy has demonstrated promising antitumor activity in various advanced cancers. Combined tumor targeting from multiple drugs with unique mechanisms may provide further improved outcomes. Tremelimumab (TRE) is a CTLA-4 antibody and durvalumab (DUR) blocks PD-L1. Poly-ICLC is a toll-like receptor 3 agonist. Intratumoral (intra-T) injection of poly-ICLC directly alters the tumor microenvironment (TME), and by creating an in situ vaccination, may trigger a clinically effective systemic anti-tumor response when also combined with DUR and TRE. Methods: This is an ongoing Phase 1/2, open-label, multicenter study (NCT02643303). The study evaluates the use of intra-T administration of TRE and IV DUR + poly-ICLC (intra-T and intramuscular [IM]) to determine the safety, preliminary efficacy and immune activity of this regimen in patients with advanced, measurable, biopsy-accessible tumors: head and neck squamous cell carcinoma, breast cancer, sarcoma, merkel cell carcinoma, cutaneous T-cell lymphoma, melanoma, genitourinary cancer, and other solid tumors. Phase 1 determines the recommended combination dosing (RCD) for the regimen with dose de-escalation based on dose limiting toxicities (DLTs) and standard 3 + 3 rules. Starting doses are: DUR, 1500 mg IV; TRE, 75 mg IV; TRE, 10 mg intra-T; poly-ICLC, 1 mg intra-T/IM. Phase 1 starts with Cohort 1A (DUR + poly-ICLC). Upon demonstration of tolerability, enrollment proceeds with Cohort 1B (DUR + IV TRE + poly-ICLC) and Cohort 1C (DUR + intra-T TRE + poly-ICLC). The RCD is the highest dose at which < 2/6 patients have DLTs. In Phase 2, up to 66 evaluable patients are treated using the RCD regimen, with enrollment of 6 patients per tumor type initially, and enrollment of 6 additional patients per 3 tumor types contingent upon at least 1 response among the initial 6 patients. Study endpoints are RCD and safety, objective response rate, progression-free survival, and overall survival. Exploratory endpoints are biological activity, including effects on the TME and immunological responses. Enrollment opened on 28 Dec 2016. Clinical trial information: NCT02643303.

Background The purpose of this study is to compare the compositions of federally funded surgical research between 2003 and 2013, and to assess differences in funding trends between surgery and other medical specialties. Data Sources The National Institutes of Health (NIH) Research Portfolio Online Reporting Tool database was queried for grants within core surgical disciplines during 2003 and 2013. Funding was categorized by award type, methodology, and discipline. Application success rates for surgery and 5 nonsurgical departments were trended over time. Conclusions Inflation-adjusted NIH funding for surgical research decreased 19% from $270M in 2003 to $219M in 2013, with a shift from R-awards to U-awards. Proportional funding to outcomes research almost tripled, while translational research diminished. Nonsurgical departments have increased NIH application volume over the last 10 years; however, surgery’s application volume has been stagnant. To preserve surgery’s role in innovative research, new efforts are needed to incentivize an increase in application volume.

Cancer cells display novel phosphopeptides in association with MHC class I and II molecules. In this study, we evaluated two HLA-A2–restricted phosphopeptides derived from the insulin receptor substrate (IRS)-2 and the cell-cycle regulator CDC25b. These proteins are both broadly expressed in multiple malignancies and linked to cancer cell survival. Two phosphopeptides, termed pIRS-21097–1105 and pCDC25b38–46, served as targets of strong and specific CD8 T-cell memory responses in normal human donors. We cloned T-cell receptor (TCR) cDNAs from murine CD8 T-cell lines specific for either pIRS-21097–1105 or pCDC25b38–46. Expression of these TCRs in human CD8 T cells imparted high-avidity phosphopeptide-specific recognition and cytotoxic and cytokine-secreting effector activities. Using these cells, we found that endogenously processed pIRS-21097–1105 was presented on HLA-A2+ melanomas and breast, ovarian, and colorectal carcinomas. Presentation was correlated with the level of the Ser1100-phosphorylated IRS-2 protein in metastatic melanoma tissues. The highest expression of this protein was evident on dividing malignant cells. Presentation of endogenously processed pCDC25b38–46 was narrower, but still evident on HLA-A2+ melanoma, breast carcinoma, and lymphoblastoid cells. Notably, pIRS-21097–1105–specific and pCDC25b38–46–specific TCR-expressing human CD8 T cells markedly slowed tumor outgrowth in vivo. Our results define two new antigens that may be developed as immunotherapeutic agents for a broad range of HLA-A2+ cancers. Cancer Res; 74(23); 6784–95. ©2014 AACR.

In multiple forms of cancer, constitutive activation of type I IFN signaling is a critical consequence of immune surveillance against cancer; however, PBMCs isolated from cancer patients exhibit depressed STAT1 phosphorylation in response to IFN-α, suggesting IFN signaling dysfunction. Here, we demonstrated in a coculture system that melanoma cells differentially impairs the IFN-α response in PBMCs and that the inhibitory potential of a particular melanoma cell correlates with NOS1 expression. Comparison of gene transcription and array comparative genomic hybridization (aCGH) between melanoma cells from different patients indicated that suppression of IFN-α signaling correlates with an amplification of the NOS1 locus within segment 12q22-24. Evaluation of NOS1 levels in melanomas and IFN responsiveness of purified PBMCs from patients indicated a negative correlation between NOS1 expression in melanomas and the responsiveness of PBMCs to IFN-α. Furthermore, in an explorative study, NOS1 expression in melanoma metastases was negatively associated with patient response to adoptive T cell therapy. This study provides a link between cancer cell phenotype and IFN signal dysfunction in circulating immune cells.

Many peptide epitopes for cytotoxic T lymphocytes (CTLs) have been identified from melanocytic differentiation proteins. Vaccine trials with these peptides have been limited mostly to those associated with HLA-A2, and immune responses have been detected inconsistently. Cases of clinical regression have been observed after peptide vaccination in some trials, but melanoma regressions have not correlated well with T-cell responses measured in peripheral blood lymphocytes (PBLs). We vaccinated stage IV melanoma patients with a mixture of gp100 and tyrosinase peptides restricted by HLA-A1 (DAEKSDICTDEY), HLA-A2 (YLEPGPVTA and YMDGTMSQV) and HLA-A3 (ALLAVGATK) in an emulsion with GM-CSF and Montanide ISA-51 adjuvant. CTL responses were assessed in PBLs and in a lymph node draining a vaccine site (sentinel immunized node, SIN). We found CTL responses to vaccinating peptides in the SIN in 5/5 patients (100%). Equivalent assays detected peptide-reactive CTLs in PBLs of 2 of these 5 patients (40%). CTLs expanded from the SIN lysed melanoma cells naturally expressing tyrosinase or gp100. We demonstrated immunogenicity for peptides restricted by HLA-A1 and -A3 and for 1 HLA-A2 restricted peptide, YMDGTMSQV. Immune monitoring of clinical trials by evaluation of PBLs alone may under-estimate immunogenicity; evaluation of SIN provides a new and sensitive approach for defining responses to tumor vaccines and correlating these responses with clinical outcomes. This combination of an immunogenic vaccine strategy with a sensitive analysis of CTL responses demonstrates the potential for inducing and detecting anti-tumor immune responses in the majority of melanoma patients.

T-cell infiltration of melanoma is associated with enhanced clinical efficacy and is a desirable endpoint of immunotherapeutic vaccination. Infiltration is regulated, in part, by chemokine receptors and selectin ligands on the surface of tumor-specific lymphocytes. Therefore, we investigated the expression of two homing molecules, CXC chemokine receptor 3 (CXCR3) and cutaneous lymphocyte antigen (CLA), on vaccine-induced CD8 T cells, in the context of a clinical trial of a melanoma-specific peptide vaccine. Both CXCR3 and CLA have been associated with T-cell infiltration of melanoma. We show that a single subcutaneous/intradermal administration of peptide vaccine in Montanide adjuvant induces tumor-specific CD8 T cells that are predominantly positive for CXCR3, with a subpopulation of CXCR3+CLA+ cells. Addition of granulocyte macrophage colony—stimulating factor (GM-CSF) significantly enhances CXCR3 expression and increases the proportion of CLA-expressing cells. Concurrent with CXCR3 and CLA expression, vaccine-induced CD8 cells express high levels of T-bet, IFN-γ, and interleukin-12 receptor (IL-12Rβ1). Collectively, these studies show that peptide vaccination in adjuvant induces CD8 T cells with a phenotype that may support infiltration of melanoma. Cancer Immunol Res; 1(5); 332–9. ©2013 AACR.

Background Cancers produce soluble and cell-associated molecules that can suppress or alter antitumor immunity. Preclinical studies suggest the disease burden may alter the cytokine profile of helper T cell responses to cancer antigens. We studied cytokine production by helper T cells responding to vaccination with 6 melanoma helper peptides (6MHP) in blood and lymph nodes. Methods Twenty-three patients with stage IIIB-IV melanoma received a 6MHP vaccine. Antigen-reactive T cells from blood and draining lymph nodes were cultured, exposed to antigen, and then supernatants (days 2 and 5) were assayed for Th1 and Th2 cytokines. Results from 4 time points were compared to pre-vaccine levels. Results Cytokine responses to vaccinating peptides were observed in 83% of patients. Th1 favoring responses were most common (17 of 19 responders). The most abundant cytokines produced were IFN-γ and IL-5 in the PBMC’s. IL-2 responses predominated in cells obtained from draining lymph nodes in 2-day culture but not in 5-day cultures. Patients with clinically measurable disease produced similar levels of total cytokine and similar degree of Th1 polarization as patients with no evidence of disease (NED). Conclusions The MHC class II-associated peptides used in this study induced helper T cells with a Th1-biased cytokine response in both PBMC and sentinel immunized nodes. Most patients can mount a Th1 dominant response to these peptides. Future studies are needed to test newer vaccine adjuvants in combination with these peptides. Trial registration CDR0000378171, Clinicaltrials: NCT00089219.

Our knowledge of the immune system has grown tremendously in the 50 years since Coley used bacteria in an attempt to create a vaccine for cancer. The strategy for cancer vaccines has developed in that time as well. Both clinical and laboratory evidence suggests that melanoma is the more immunogenic of solid tumors. If treated early, melanoma can be controlled with surgery, but many patients continue to die from it. With our increased understanding of the immune system's interaction with melanoma, many clinical trials of melanoma vaccines are now underway. Vaccines designed to treat metastatic melanoma have shown some evidence of clinical effectiveness. This article outlines the current status of melanoma vaccination.

To investigate the ability of human dendritic cells (DC) to process and present multiple epitopes from the gp100 melanoma tumor-associated Ags (TAA), DC from melanoma patients expressing HLA-A2 and HLA-A3 were pulsed with gp100-derived peptides G9154, G9209, or G9280 or were infected with a vaccinia vector (Vac-Pmel/gp100) containing the gene for gp100 and used to elicit CTL from autologous PBL. CTL were also generated after stimulation of PBL with autologous tumor. CTL induced with autologous tumor stimulation demonstrated HLA-A2-restricted, gp100-specific lysis of autologous and allogeneic tumors and no lysis of HLA-A3-expressing, gp100+ target cells. CTL generated by G9154, G9209, or G9280 peptide-pulsed, DC-lysed, HLA-A2-matched EBV transformed B cells pulsed with the corresponding peptide. CTL generated by Vac-Pmel/gp100-infected DC (DC/Pmel) lysed HLA-A2- or HLA-A3-matched B cell lines pulsed with the HLA-A2-restricted G9154, G9209, or G9280 or with the HLA-A3-restricted G917 peptide derived from gp100. Furthermore, these DC/Pmel-induced CTL demonstrated potent cytotoxicity against allogeneic HLA-A2- or HLA-A3-matched gp100+ melanoma cells and autologous tumor. We conclude that DC-expressing TAA present multiple gp100 epitopes in the context of multiple HLA class I-restricting alleles and elicit CTL that recognize multiple gp100-derived peptides in the context of multiple HLA class I alleles. The data suggest that for tumor immunotherapy, genetically modified DC that express an entire TAA may present the full array of possible CTL epitopes in the context of all possible HLA alleles and may be superior to DC pulsed with limited numbers of defined peptides.

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8+cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4+T cells. The Melan-A/MART-151–73peptide was able to induce thein vitroexpansion of specific CD4+T cells derived from normal DR4+donors or from DR4+patients with melanoma when pulsed onto autologous dendritic cells. CD4+responder T cells specifically produced IFN-γ in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-151–73peptide and DR4+melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4+T cell immunoreactivity against the Melan-A/MART-151–73peptide typically coexisted with a high frequency of anti-Melan-A/MART-127–35reactive CD8+T cells in freshly isolated blood harvested from HLA-A2+/DR4+patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4+T cell responses against melanomain vivo.

Background Metastatic melanoma to abdominal and retroperitoneal viscera carries a poor prognosis possibly resulting in reluctance to offer surgical management. There is value in defining the morbidity of such surgery. Methods Review of a prospectively maintained database identified patients with metastatic melanoma to abdominal or retroperitoneal viscera who underwent surgery from 9/99 to 8/06. Results Nineteen patients underwent surgery for metastasis to abdominal or retroperitoneal viscera detected by clinical symptoms (80%), or imaging (20%). The median length of stay was 7 days. There was no perioperative mortality. Surgical complications occurred in four patients. At initial follow-up, 13 patients (68%) had returned to baseline function, 7 of which reported improvement. Four patients (21%) had minimal symptoms, and only two patients (11%) had significantly limited function. Median follow-up was 35 months, at which time 9 of the 19 patients (47%) were still alive, with 3- and 5-year Kaplan–Meier survival estimates of 53% (SE 12) and 45% (SE 12), respectively, and 2 of the 19 patients are alive at over 8 years since surgery. Conclusion In selected cases, surgery may have both palliative benefit and curative potential for patients with visceral metastases of melanoma. Surgical management of such patients should be encouraged in appropriate clinical settings. J. Surg. Oncol. 2009;100:481–483. © 2009 Wiley-Liss, Inc.

Peptide epitopes for tumor-reactive cytotoxic T-lymphocytes (CTL) have been identified on human cancers and are being used in tumor vaccine trials. However, the pharmacokinetics and pharmacodynamics of such peptides have been inadequately studied. It is predicted that immunogenic tumor peptides would have short half-lives in vivo. The goal of the present work was to evaluate the stability of the immunogenic peptide MART-1(27-35) in fresh normal human plasma (NHP) and to identify modifications that convey protection against enzymatic destruction without loss of immunogenicity. We evaluated the stability of the MART-1(27-35) peptide (AAGIGILTV) and modified forms of that peptide for stability and immune recognition in an in vitro model. The peptides were incubated in plasma for varied time intervals and evaluated for their ability to reconstitute the epitope for MART-1(27-35)-reactive CTL. Loss of CTL reactivity signaled loss of immunoreactive peptide. When 1 microM MART-1(27-35) peptide was incubated in plasma prior to pulsing on target cells, CTL reactivity was lost within 3 hr, and the calculated half-life of this peptide was 22 sec. This degradation was mediated by peptidases. The stability of MART-1(27-35) was markedly prolonged by C-terminal amidation and/or N-terminal acetylation (peptide capping), or by polyethylene-glycol modification (PEGylation) of the C-terminus. These modified peptides were recognized by CTL. The MART-1(27-35) peptide is very unstable in plasma. It is probable that it and other immunogenic peptides will be similarly unstable in vivo. Immunogenicity of these peptides might be enhanced by creating modifications that enhance stability.