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Elevated immunity to cancer-expressed antigens can be detected in people with no history of cancer and may contribute to cancer prevention. We have previously reported that MHC-restricted phosphopeptides are cancer-expressed antigens and targets of immune recognition. However, the extent to which this immunity reflects prior or ongoing phosphopeptide exposures was not investigated. In this study, we found that preexisting immune memory to cancer-expressed phosphopeptides was evident in most healthy donors, but the breadth among donors was highly variable. Although three phosphopeptides were recognized by most donors, suggesting exposures to common microbial/infectious agents, most of the 205 tested phosphopeptides were not recognized by peripheral blood mononuclear cells (PBMC) from any donor and the remainder were recognized by only 1 to 3 donors. In longitudinal analyses of 2 donors, effector immune response profiles suggested active reexposures to a subset of phosphopeptides. These findings suggest that the immunogens generating most phosphopeptide-specific immune memory are rare infectious agents or incipient cancer cells with distinct phosphoproteome dysregulations, and that repetitive immunogenic exposures occur in individual donors. Phosphopeptide-specific immunity in PBMCs and tumor-infiltrating lymphocytes from ovarian cancer patients was limited, regardless of whether the phosphopeptide was expressed on the tumor. However, 4 of 10 patients responded to 1 to 2 immunodominant phosphopeptides, and 1 showed an elevated effector response to a tumor-expressed phosphopeptide. As the tumors from these patients displayed many phosphopeptides, these data are consistent with lack of prior exposure or impaired ability to respond to some phosphopeptides and suggest that enhancing phosphopeptide-specific T-cell responses could be a useful approach to improve tumor immunotherapy.

Cholangiocarcinoma (CC) is an uncommon malignancy with increasing incidence and dismal prognosis. We conducted a comprehensive analysis of the CC tumor immune microenvironment (TIME) based on tumor location to identify therapeutic targets. We hypothesized that the TIME of CC would vary by primary tumor location and that high tumor infiltration by CD8+ T cells and low infiltration by M2 macrophages would be associated with improved survival. A retrospective analysis was conducted of 99 CC tumor samples surgically resected between 2000 and 2014. Tissue microarrays were constructed from each tumor and stained by immunohistochemistry for 24 markers of immune cells, immune activation or inhibition, programmed cell death-ligand 1, and mesothelin. Most tumors were amply infiltrated with by CD4+, CD8+, and FoxP3+ T cells, as well as by myeloid cells. Mesothelin expression ≥1+ by immunohistochemistry was found in 68% of tumors. We identified higher densities of M1 macrophages in primary distal extrahepatic CC, as well as metastatic lesions. Mesothelin expression was also significantly higher in distal extrahepatic CC. There was no association with survival of infiltration by CD4+, CD8+, or FoxP3+ T cells, mesothelin expression, or programmed cell death-ligand 1 percentage expression, however, high CD14+ myeloid cells and high CD163+ M2 macrophages were associated with worse survival. In conclusion, the CC TIME is a heterogenous milieu highly infiltrated by innate and adaptive immune cells, which differs based on primary tumor location and between primary tumors and metastatic lesions. The correlation of intratumoral M2 macrophages and myeloid cells with a worse prognosis may suggest promising immunotherapeutic targets in CC.

BACKGROUND Childhood cancer survivors (CCS) are at elevated risk of secondary malignancies (SM). Enhanced screening for SM is recommended, but compliance is poor. We hypothesized that CCS with adult-onset SM (colorectal cancer [CRC], melanoma, or breast cancer [BC]) would present with more advanced disease and have decreased overall survival (OS). METHODS The Surveillance, Epidemiology, and End Results Program was queried for patients diagnosed with cancer at age less than or equal to 18 also diagnosed with adult-onset CRC, melanoma, or BC. A cohort without a history of prior malignancy was likewise identified. Tumor features and clinical outcomes were compared. RESULTS CCS with a SM (n = 224) were compared with patients without a childhood cancer history (n = 1,392,670). CCS were diagnosed younger (BC = 37.6 vs. 61.3, p

The strength and durability of systemic anti-tumor immune responses induced by cancer vaccines depends on adjuvants to support an immunogenic vaccine site microenvironment (VSME). Adjuvants include water-in-oil emulsions with incomplete Freund's adjuvant (IFA) and combinations of toll-like receptor (TLR) agonists, including a preparation containing TLR4 and TLR9 agonists with QS-21 (AS15). IFA-containing vaccines can promote immune cell accumulation at the VSME, whereas effects of AS15 are largely unexplored. Therefore, we assessed innate and adaptive immune cell accumulation and gene expression at the VSME after vaccination with AS15 and compared to effects with IFA. We hypothesized that AS15 would promote less accumulation of innate and adaptive immune cells at the VSME than IFA vaccines. In two clinical trials, patients with resected high-risk melanoma received either a multipeptide vaccine with IFA or a recombinant MAGE-A3 protein vaccine with AS15. Vaccine site biopsies were obtained after one or multiple vaccines. T cells accumulated early after vaccines with AS15, but this was not durable or of the same magnitude as vaccination in IFA. Vaccines with AS15 increased durable expression of DC- and T cell-related genes, as well as PD-L1 and IDO1, suggesting complex activation and regulation of innate and adaptive immune function with AS15. These changes were generally greater with vaccines containing IFA, but IFA induced reduction in myeloid suppressor cells markers. Evidence of tertiary lymphoid structure (TLS) formation was observed with both adjuvants. Our findings highlight adjuvant-dependent changes in immune features at the VSME that may impact systemic immune responses.

Indoleamine 2-3 dioxygenase 1 (IDO1) expression may contribute to immunologic escape by melanoma metastases. However, a recent clinical trial failed to identify any clinical benefits of IDO1 inhibition in patients with unresectable metastatic melanoma, and prior characterizations of IDO1 expression have predominately studied primary lesions and local metastases, generating uncertainty regarding IDO1 expression in distant metastases. We hypothesized that IDO1 expression in such lesions would be low and correlated with decreased overall survival (OS). Metastases from patients (n=96) with stage IIIb to IV melanoma underwent tissue microarray construction and immunohistochemical staining for IDO1. Th1-related gene expression was determined quantitatively. Associations between OS and IDO1 expression were assessed with multivariate models. Of 96 metastatic lesions, 28% were IDOpos, and 85% exhibited IDO1 expression in

Background Immune checkpoint inhibition is part of standard systemic management for many advanced malignancies. Toxicities from this treatment approach are unpredictable, though usually reversible with management per established guidelines. Some patients suffer major morbidity and treatment-related mortality from these agents in an unpredictable manner. Cardiac and neurologic complications are rare, but can result in serious clinical consequences. Methods We describe the presentation, management, and outcomes of eight sequential cases of combined cardiac and neurologic toxicities resulting in severe illness and demonstrating lack of rapid response to immunosuppression. Results Our cohort consisted of six males and two females with an average age of 73.5 years (61–89 years). There were four patients with melanoma, and one patient each with urothelial carcinoma, renal cell carcinoma, breast cancer, and non-small cell lung cancer. Four patients received combination immunotherapy and four patients received monotherapy. The median time to presentation from treatment initiation was 27 days (11–132 days). All patients had a cardiovascular and neurologic toxicity, and most had hepatitis and myositis. The cardiac signs and symptoms were the prominent initial features of the clinical presentation. Each patient was managed by a multidisciplinary team and received a range of immunosuppressive agents. All patients died as a consequence of the immune related adverse events. Conclusions The evaluation of patients with cardiac adverse events from immunotherapy, should include assessment of overlapping toxicities such as myasthenia gravis and myositis. Providers should be aware of the potential for an extended duration of disability and slow improvement for certain toxicities as these expectations may factor prominently in goals of care decisions.

Vaccine therapies for treatment and prevention of cancer have seen modest degrees of efficacy with wide variation related to the tumor type, vaccine type, adjuvants and clinical setting for their study. Over the course of the last two decades, various peptide vaccines for breast cancer have been studied. The current leading peptide vaccine for human application is a HER2-based vaccine known as Nelipepimut-S, which has demonstrated immune activity and promising clinical activity in some settings. This review covers the development of this newer peptide vaccine for both HER2 amplified and non-amplified breast cancer.

BackgroundPancreatic cancer (PC) is a challenging target for immunotherapy due to its immune-suppressive microenvironment. Neoadjuvant chemoradiation (CRT) can increase the presence of tumor-infiltrating lymphocytes (TILs). We hypothesized that the combination of CRT and pembrolizumab can further expand and activate TILs.MethodsPatients with resectable or borderline resectable PC were randomized 2:1 to the investigational treatment (Arm A) of pembrolizumab 200mg IV every 3 weeks concurrently with CRT (capecitabine 825 mg/m2 orally twice daily and radiation 50.4 Gy in 28 fractions over 28 days) or CRT only (Arm B) prior to surgical resection. The primary endpoints were safety and difference in TILs density between Arm A and B assessed using multiplexed immunofluorescence on resected tumor specimens. As a correlate analysis, single cell RNA-sequencing (scRNA-seq) was performed to quantify gene expression in T cells from tumors and peripheral blood, and to track expanded T cell clonotypes in these compartments (n=4 patients Arm A; n=3 patients Arm B). The study was amended after enrollment of 37 patients to allow FOLFIRINOX prior to CRT, given changes in standard of care.Results37 patients were enrolled (24 Arm A, 13 Arm B). After neoadjuvant therapy, 13 patients had unresectable disease (9 on A, 4 on B), and 24 patients underwent surgery and were evaluable for the TILs primary endpoint (17 arm A, 7 arm B). The mean difference (A-B) in CD8+ T cell density was 36 cells/mm2 (95% CI -85 to 157, stdev 130) (p 0.48). Additional analysis did not show significant differences in activated cytotoxic T cells, regulatory T cells, M1- or M2-like polarized macrophages, or granulocytes. The median recurrence free survival (RFS) was 18.2 months on Arm A and 14.1 on Arm B (p 0.41). Overall survival was 27.8 months on Arm A and 24.3 on Arm B (p 0.68) with a median follow up of 2.2 years. The most common grade 3 treatment-related toxicities were lymphopenia reported in 29% on Arm A and 31% on Arm B, respectively followed by diarrhea in 8% on Arm A attributed to CRT. scRNA-seq revealed clonal expansion and expression of co-inhibitory markers among TIL subsets.ConclusionsThe combination of CRT and pembrolizumab is safe. Preliminary analysis shows that the addition of pembrolizumab to CRT has minimal effects on intratumoral densities of TILs and other immune cell populations. Single cell transcriptome analyses enable in-depth characterization of the functional responses of T cells to pembrolizumab in the setting of CRT.AcknowledgementsThis study was funded by MerckTrial RegistrationNCT02305186Ethics ApprovalThe study was conducted at 6 sites: University of Virginia, Dana Farber Cancer Institute, MD Anderson Cancer Center (MDACC), Mayo Clinic, Hartford Healthcare Cancer Center, and University of Miami. Written informed consent was provided by the study participants and the protocol was approved by the relevant local IRBs in each site.

BackgroundWe performed a clinical trial to evaluate safety and immunogenicity of a novel long peptide vaccine administered in combinations of incomplete Freund’s adjuvant (IFA) and agonists for TLR3 (polyICLC) and TLR7/8 (resiquimod). We hypothesized that T cell responses to minimal epitope peptides (MEPs) within the long peptides would be enhanced compared with prior vaccines with MEP themselves and that T cell responses would be enhanced with TLR agonists, compared with IFA alone.MethodsParticipants with resected stage IIB-IV melanoma were vaccinated with seven long melanoma peptides (LPV7) from tyrosinase, gp100, MAGE-A1, MAGE-A10, and NY-ESO-1, each containing a known MEP for CD8+ T cells, plus a tetanus helper peptide (Tet) restricted by Class II MHC. Enrollment was guided by an adaptive design to one of seven adjuvant combinations. Vaccines were administered at weeks 1, 2, 3, 6, 9, 12 at rotating injection sites. T cell and IgG antibody (Ab) responses were measured with IFN-gamma ELIspot assay ex vivo and ELISA, respectively.ResultsFifty eligible participants were assigned to seven study groups, with highest enrollment on arm E (LPV7+Tet+IFA+polyICLC). There was one dose-limiting toxicity (DLT) in Group E (grade 3 injection site reaction, 6% DLT rate). All other treatment-related adverse events were grades 1–2. The CD8+ T cell immune response rate (IRR) to MEPs was 18%, less than in prior studies using MEP vaccines in IFA. The CD8+ T cell IRR trended higher for IFA-containing adjuvants (24%) than adjuvants containing only TLR agonists (6%). Overall T cell IRR to full-length LPV7 was 30%; CD4+ T cell IRR to Tet was 40%, and serum Ab IRR to LPV7 was 84%. These IRRs also trended higher for IFA-containing adjuvants (36% vs 18%, 48% vs 24%, and 97% vs 60%, respectively).ConclusionsThe LPV7 vaccine is safe with each of seven adjuvant strategies and induced T cell responses to CD8 MEPs ex vivo in a subset of patients but did not enhance IRRs compared with prior vaccines using short peptides. Immunogenicity was supported more by IFA than by TLR agonists alone and may be enhanced by polyICLC plus IFA.Trial registration numberNCT02126579.

Checkpoint-blockade therapy (CBT) is approved for select colorectal cancer (CRC) patents, but additional immunotherapeutic options are needed. We hypothesized that vaccination with carcinoembryonic antigen (CEA) and Her2/neu (Her2) peptides would be immunogenic and well-tolerated by participants with advanced CRC. A pilot clinical trial (NCT00091286) was conducted in HLA-A2+ or -A3+ Stage IIIC-IV CRC patients. Participants were vaccinated weekly with CEA and Her2 peptides plus tetanus peptide and GM-CSF emulsified in Montanide ISA-51 adjuvant for 3 weeks. Adverse events (AE) were recorded per NIH Common Terminology Criteria for Adverse Events version 3. Immunogenicity was evaluated by interferon-gamma ELISpot assay of in-vitro sensitized peripheral blood mononuclear cells and lymphocytes from the sentinel immunized node (SIN). Eleven participants were enrolled and treated; one was retrospectively found to be ineligible due to HLA type. All eleven participants were included in AE and survival analyses, and the ten eligible participants were evaluated for immunogenicity. All participants reported AE: 82% were Grade 1-2, most commonly fatigue or injection site reactions. Two participants (18%) experienced treatment-related dose-limiting Grade 3 AE; both were self-limiting. Immune responses to Her2 or CEA peptides were detected in 70% of participants. Median overall survival was 16 months; among those enrolled with no evidence of disease (n=3), median overall survival was not reached after 10 years of follow-up. These data demonstrate that vaccination with CEA or Her2 peptides is well-tolerated and immunogenic. Further study is warranted to assess potential clinical benefits of vaccination in advanced CRC either alone or in combination with CBT.

Objective To understand role of barrier molecules in melanomas. Background We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. Methods FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. Results Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. Conclusions FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.

Background Obesity is a risk factor for colorectal cancer and possibly the formation of precancerous, colorectal polyps . Bariatric surgery is very effective for long-term weight loss; however, it is not known whether bariatric surgery decreases the risk of subsequent colonic neoplasia. We hypothesized that bariatric surgery would decrease the risk of developing colorectal lesions (new cancer and precancerous polyps). Methods We reviewed all patients (n = 3,676) who underwent bariatric surgery (gastric bypass, sleeve gastrectomy, or gastric banding) at the University of Virginia (Charlottesville, VA) 1985–2015. Obese, nonoperative patients (n = 46,873) from an institutional data repository were included as controls. Cases and controls were propensity score matched 1:1 by demographics, comorbidities, body mass index, and socioeconomic factors. The matched cohort was compared by univariate analysis and conditional logistic regression. Results A total of 4,462 patients (2,231 per group) with a median follow-up of 7.8 years were well-matched with no statistically significant baseline differences in initial body mass index (48 vs 49 kg/m2), sex, and age in addition to other comorbidities (all P > .05). The operative cohort had more weight loss (55.5% vs –1.4% decrease in excess body mass index, P .05). After risk adjustment, bariatric surgery was independently associated with a decrease in new colorectal lesions (OR 0.62, 95% CI 0.42–0.91, P = .016). Conclusion Bariatric surgery was associated with lesser, risk-adjusted incidence of new colorectal lesions in this large population of propensity matched patients undergoing bariatric surgery compared with a control group not undergoing bariatric surgery. These results suggest the benefits of bariatric surgery may extend beyond weight loss and mitigation of comorbidities.

The incidence of melanoma continues to increase even as advances in immunotherapy have led to survival benefits in advanced stages. Vaccines are capable of inducing strong, antitumor immune responses with limited toxicity. Some vaccines have demonstrated clinical benefit in clinical trials alone; however, others have not despite inducing strong immune responses. Recent advancements have improved vaccine design, and combining vaccines with other immunotherapies offers promise. This review highlights the underlying principles of vaccine development, common components of vaccines, and the remaining challenges and future directions of vaccine therapy in melanoma.

PURPOSE Current guidelines for postoperative management of patients with stage I-IIA cutaneous melanoma (CM) do not recommend routine cross-sectional imaging, yet many of these patients develop metastases. Methods that complement American Joint Committee on Cancer (AJCC) staging are needed to improve identification and treatment of these patients. A 31-gene expression profile (31-GEP) test predicts metastatic risk as low (class 1) or high (class 2). Prospective analysis of CM outcomes was performed to test the hypotheses that the 31-GEP provides prognostic value for patients with stage I-III CM, and that patients with stage I-IIA melanoma and class 2 31-GEP results have metastatic risk similar to patients for whom surveillance is recommended. MATERIALS AND METHODS Two multicenter registry studies, INTEGRATE (ClinicalTrials.gov identifier: NCT02355574 ) and EXPAND (ClinicalTrials.gov identifier: NCT02355587 ), were initiated under institutional review board approval, and 323 patients with stage I-III CM and median follow-up time of 3.2 years met inclusion criteria. Primary end points were 3-year recurrence-free survival (RFS), distant metastasis-free survival (DMFS), and overall survival (OS). RESULTS The 31-GEP was significant for RFS, DMFS, and OS in a univariate analysis and was a significant, independent predictor of RFS, DMFS, and OS in a multivariable analysis. GEP class 2 results were significantly associated with lower 3-year RFS, DMFS, and OS in all patients and those with stage I-IIA disease. Patients with stage I-IIA CM and a class 2 result had recurrence, distant metastasis, and death rates similar to patients with stage IIB-III CM. Combining 31-GEP results and AJCC staging enhanced sensitivity over each approach alone. CONCLUSION These data provide a rationale for using the 31-GEP along with AJCC staging, and suggest that patients with stage I-IIA CM and a class 2 31-GEP signature may be candidates for more intense follow-up.

The presence of tertiary lymphoid structures (TLS) is correlated with prolonged patient survival in a variety of solid cancers, including melanoma. However, few methods have been described that could enable a more comprehensive understanding of the organization and functionality of TLS in solid cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying TLS and intra-tumoral immune infiltrates in melanoma. The described methods are not limited to melanoma alone and could be used to evaluate tertiary lymphoid structures in a wide variety of human cancers.

BackgroundPeptide vaccines designed to stimulate melanoma-reactive CD4+ T cells can induce T cell and antibody (Ab) responses, associated with enhanced overall survival. We hypothesized that adding toll-like receptor 3 agonist polyICLC to an incomplete Freund’s adjuvant (IFA) would be safe and would support strong, durable CD4+ T cell and Ab responses. We also hypothesized that oral low-dose metronomic cyclophosphamide (mCy) would be safe, would reduce circulating regulatory T cells (T-regs) and would further enhance immunogenicity.Participants and methodsAn adaptive design based on toxicity and durable CD4+ T cell immune response (dRsp) was used to assign participants with resected stage IIA-IV melanoma to one of four study regimens. The regimens included a vaccine comprising six melanoma peptides restricted by Class II MHC (6MHP) in an emulsion with IFA alone (Arm A), with IFA plus systemic mCy (Arm B), with IFA+ local polyICLC (Arm C), or with IFA+ polyICLC+ mCy (Arm D). Toxicities were recorded (CTCAE V.4.03). T cell responses were measured by interferon γ ELIspot assay ex vivo. Serum Ab responses to 6MHP were measured by ELISA. Circulating T-regs were assessed by flow cytometry.ResultsForty-eight eligible participants were enrolled and treated. Early data on safety and dRsp favored enrollment on arm D. Total enrollment on Arms A-D were 3, 7, 6, and 32, respectively. Treatment-related dose-limiting toxicities (DLTs) were observed in 1/7 (14%) participants on arm B and 2/32 (6%) on arm D. None exceeded the 25% DLT threshold for early closure to enrollment for any arm. Strong durable T cell responses to 6MHP were detected ex vivo in 0%, 29%, 67%, and 47% of participants on arms A-D, respectively. IgG Ab responses were greatest for arms C and D. Circulating T-regs frequencies were not altered by mCy.Conclusions6MHP vaccines administered with IFA, polyICLC, and mCy were well tolerated. The dRsp rate for arm D of 47% (90% CI 32 to 63) exceeded the 18% (90% CI 11 to 26) rate previously observed with 6MHP in IFA alone. Vaccination with IFA+ polyICLC (arm C) also showed promise for enhancing T cell and Ab responses.

Ipilimumab (IPI) can enhance immunity to the cancer-testis antigen NY-ESO-1. A clinical trial was designed to assess safety, immunogenicity, and clinical responses with IPI + NY-ESO-1 vaccines and effects on the tumor microenvironment (TME). Patients with measurable NY-ESO-1+ tumors were enrolled among three arms: A) IPI + NY-ESO-1 protein + poly-ICLC (pICLC) + incomplete Freund’s adjuvant (IFA); B) IPI + NY-ESO-1 overlapping long peptides (OLP) + pICLC + IFA; and C) IPI + NY-ESO-1 OLP + pICLC. Clinical responses were assessed by irRC. T cell and Ab responses were assessed by ex vivo IFN-gamma ELIspot and ELISA. Tumor biopsies pre- and post-treatment were evaluated for immune infiltrates. Eight patients were enrolled: 5, 2, and 1 in Arms A-C, respectively. There were no DLTs. Best clinical responses were SD (4) and PD (4). T-cell and antibody (Ab) responses to NY-ESO-1 were detected in 6 (75%) and 7 (88%) patients, respectively, and were associated with SD. The breadth of Ab responses was greater for patients with SD than PD (p = .036). For five patients evaluable in the TME, treatment was associated with increases in proliferating (Ki67+) CD8+ T cells and decreases in RORγt+ CD4+ T cells. T cell densities increased for those with SD. Detection of T cell responses to NY-ESO-1 ex vivo in most patients suggests that IPI may have enhanced those responses. Proliferating intratumoral CD8+ T cells increased after vaccination plus IPI suggesting favorable impact of IPI plus NY-ESO-1 vaccines on the TME. List of Abbreviations: Ab = antibody; CTCAE = NCI Common Terminology Criteria for Adverse Events; DHFR/DHRP = dihydrofolate reductase; DLT = Dose-limiting toxicity; ELISA = enzyme-linked immunosorbent assay; IFA = incomplete Freund’s adjuvant (Montanide ISA-51); IFNγ = Interferon gamma; IPI = Ipilimumab; irRC = immune-related response criteria; mIFH = multispectral immunofluorescence histology; OLP = NY-ESO-1 overlapping long peptides; PBMC = peripheral blood mononuclear cells; PD = Progressive disease; pICLC = poly-ICLC (Hiltonol), a TLR3/MDA-5 agonist; RLT = Regimen-limiting Toxicity; ROI = regions of interest; RT = room temperature; SAE = serious adverse event; SD = stable disease; TEAE = treatment-emergent adverse events; TLR = toll-like receptor; TME = tumor microenvironment; TRAE = treatment-related adverse events.

The density of tumour-infiltrating lymphocytes (TILs) in melanoma is correlated with improved clinical prognosis; however, standardized TIL immunotyping and quantification protocols are lacking. Herein, we provide a review of the technologies being utilized for the immunotyping and quantification of melanoma TILs.

Sentinel lymph node biopsy (SLNB) is routinely recommended for clinically localized Merkel cell carcinoma (MCC); however, predictors of false negative (FN) SLNB are undefined.Patients from six centers undergoing wide excision and SLNB for stage I/II MCC (2005-2020) were identified and were classified as having either a true positive (TP), true negative (TN) or FN SLNB. Predictors of FN SLNB were identified and survival outcomes were estimated.Of 525 patients, 28 (5.4%), 329 (62.7%), and 168 (32%) were classified as FN, TN, and TP, respectively, giving an FN rate of 14.3% and negative predictive value of 92.2% for SLNB. Median follow-up for SLNB-negative patients was 27 months, and median time to nodal recurrence for FN patients was 7 months. Male sex (hazard ratio [HR] 3.15, p = 0.034) and lymphovascular invasion (LVI) (HR 2.22, p = 0.048) significantly correlated with FN, and increasing age trended toward significance (HR 1.04, p = 0.067). The 3-year regional nodal recurrence-free survival for males75 years with LVI was 78.5% versus 97.4% for females ≤75 years without LVI (p = 0.009). Five-year disease-specific survival (90.9% TN vs. 51.3% FN, p 0.001) and overall survival (69.9% TN vs. 48.1% FN, p = 0.035) were significantly worse for FN patients.Failure to detect regional nodal microscopic disease by SLNB is associated with worse survival in clinically localized MCC. Males, patients75 years, and those with LVI may be at increased risk for FN SLNB. Consideration of increased nodal surveillance following negative SLNB in these high-risk patients may aid in early identification of regional nodal recurrences.

Background Assessment of lymphatic status via sentinel lymph node (SLN) biopsy is an integral and crucial part of melanoma surgical oncology. The most common technique for sentinel node mapping is preoperative planar scintigraphy of an injected gamma-emitting lymphatic tracer followed by intraoperative node localization using a non-imaging gamma probe with auditory feedback. In recent years, intraoperative visualization of SLNs in 3D has become possible by coupling the probe to an external system capable of tracking its location and orientation as it is read out, thereby enabling computation of the 3D distribution of the tracer (freehand SPECT). In this project, the non-imaging probe of the fhSPECT system was replaced by a unique handheld gamma camera containing an array of sodium iodide crystals optically coupled to an array of silicon photomultipliers (SiPMs). A feasibility study was performed in which preoperative SLN mapping was performed using camera fhSPECT and the number of detected nodes was compared to that visualized by lymphoscintigraphy, probe fhSPECT, and to the number ultimately excised under non-imaging probe guidance. Results Among five subjects, SLNs were detected in nine lymphatic basins, with one to five SLNs detected per basin. A basin-by-basin comparison showed that the number of SLNs detected using camera fhSPECT exceeded that using lymphoscintigraphy and probe fhSPECT in seven of nine basins and five of five basins, respectively. (Probe fhSPECT scans were not performed for four basins.) It exceeded the number excised under non-imaging probe guidance for seven of nine basins and equaled the number excised for the other two basins. Conclusions Freehand SPECT using a prototype SiPM-based gamma camera demonstrates high sensitivity for detection of SLNs in a preoperative setting. Camera fhSPECT is a potential means for efficiently obtaining real-time 3D activity distribution maps in applications such as image-guided percutaneous biopsy, and surgical SLN biopsy or radioguided tumor excision.

Background Vaccines are a promising therapeutic for patients with advanced cancer, but achieving robust T-cell responses remains a challenge. Melanoma-associated antigen-A3 (MAGE-A3) in combination with adjuvant AS15 (a formulation of Toll-Like-Receptor (TLR)-4 and 9 agonists and a saponin), induced systemic CD4+ T-cell responses in 50% of patients when given subcutaneously/intradermally. Little is known about the transcriptional landscape of the vaccine-site microenvironment (VSME) of patients with systemic T-cell responses versus those without. We hypothesized that patients with systemic T-cell responses to vaccination would exhibit increased immune activation in the VSME, higher dendritic cell (DC) activation/maturation, TLR-pathway activation, and enhanced Th1 signatures. Methods Biopsies of the VSME were obtained from participants on the Mel55 clinical trial (NCT01425749) who were immunized with MAGE-A3/AS15. Biopsies were taken 8 days after immunization. T-cell response to MAGE-A3 was assessed in PBMC after in-vitro stimulation with recMAGE-A3, by IFNγ ELISPOT assay. Gene expression was assessed by RNAseq using DESeq2. Comparisons were made between immune-responders (IR), non-responders (NR), and normal skin controls. FDR p Results Four IR, four NR, and three controls were evaluated. The 500 most variable genes were used for principal component analysis (PCA). Two IR samples were identified as outliers on PCA and excluded from further analysis. There were 882 differentially expressed genes (DEGs) in the IR group vs the NR group (figure 1A). Unsupervised clustering of the top 500 DEGs revealed clustering according to the experimental groups (figure 1B). Of the 10 most highly upregulated DEGs, 9 were immune-related (figure 1C). Gene-set enrichment analysis revealed that immune-related pathways were highly enriched in IRs vs NRs (figure 1D). CD4 and CD8 expression did not differ between IR and NR (figure 2A), though both were higher in IR compared to control. Markers of DC activation/maturation were higher in IR vs NR (figure 2B), as were several Th1 associated genes (figure 2C). Interestingly, markers of exhaustion were higher in IR v NR (figure 2D). Expression of numerous TLR-pathway genes was higher in IR vs NR, including MYD88, but not TICAM1 (figure 2E). Conclusions These findings suggest a unique immune-transcriptional landscape in the VSME is associated with circulating T-cell responses to immunization, with differences in DC activation/maturation, Th1 response, and TLR signaling. Thus, immunologic changes in the VSME are useful predictors of systemic immune response, and host factors that modulate immune-related signaling at the vaccine site may have concordant systemic effects on promoting or limiting immune responses to vaccines. Trial Registration Samples for this work were collected from patients enrolled on the Mel55 clinical trial NCT01425749. Ethics Approval This work was completed after approval from the UVA institutional review board IRB-HSR# 15398.

Immunotherapy is now a cornerstone for cancer treatment, and much attention has been placed on the identification of prognostic and predictive biomarkers. The success of biomarker development is dependent on accurate and timely collection of biospecimens and high-quality processing, storage and shipping. Tumors are also increasingly used as source material for the generation of therapeutic T cells. There have been few guidelines or consensus statements on how to optimally collect and manage biospecimens and source material being used for immunotherapy and related research. The Society for Immunotherapy of Cancer Surgery Committee has brought together surgical experts from multiple subspecialty disciplines to identify best practices and to provide consensus on how best to access and manage specific tissues for immuno-oncology treatments and clinical investigation. In addition, the committee recommends early integration of surgeons and other interventional physicians with expertise in biospecimen collection, especially in clinical trials, to optimize the quality of tissue and the validity of correlative clinical studies in cancer immunotherapy.

Ex vivo expansion followed by reinfusion of tumor infiltrating leucocytes (TILs) has been utilized successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine interleukin-2 (IL-2) to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 post-reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic due to its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. Here we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high affinity IL-2 receptor for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident cytotoxic lymphocytes, such as CD8(+) T cells, NK cells, and γδT cells while wild-type IL-2 provides a growth advantage for CD4(+)Foxp3(+) T cells as well as myeloid cells. OMCPmutIL-2 expanded cytotoxic lymphocytes express higher levels of tumor homing receptors such as LFA-1, CD49a and CXCR3 which correlate with TIL localization to the tumor bed after intravenous injection. Consistent with this OMCPmutIL-2 expanded TILs provided superior tumor control compared to those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D expressing cytotoxic lymphocytes rather than indiscriminate expansion of all TILs.

BACKGROUND: Identifying factors that impact progression of surgery trainees into academic versus non-academic practices may permit tailoring residency experiences to promote academic careers in institutions charged with the training of future surgeon scientists. The aim of this study was to identify factors associated with progression of surgery trainees into academic versus non-academic practice. METHODS: A survey was distributed to 135 surgeons graduating from the University of Virginia residency program from 1964–2016, a single academic institution. Questions addressed practice type, research productivity, work/life balance, mentorship, and overall sentiment toward research and academic surgery. A 5-point Likert scale measured career satisfaction and influence of factors in practice setting choice. RESULTS: Of the 135 surveys that were electronically distributed, 69 participants responded (response rate: 51%). Of the 54 with known current practice types, 34 (63%) were academic and 20 (37%) non-academic. Academic surgeons reported more publications by the conclusion of surgery training (56% vs 25% with >10 publications, P = .02). More academic surgeons reported > $100,000 in student debt at graduation (44% vs 25%, P < .05). Factors encouraging an academic career were similar for both types of surgeons, including involvement in education of trainees and access to mentorship. Both groups were discouraged from an academic practice by requirements of grant-writing and funding responsibilities. Surgeons in academic practice were more likely to recommend surgery as a career to a current medical student (100% vs 70%, P = .001). CONCLUSION: This knowledge may help to tailor training experiences to promote academic careers. By supporting funding mechanisms and grant-writing programs, while encouraging mentorship and productive research experiences, current surgical trainees may be more enthusiastic about a career in academic practice.

Background Cancer immunotherapy has been firmly established as a standard of care for patients with advanced and metastatic melanoma. Therapeutic outcomes in clinical trials have resulted in the approval of 11 new drugs and/or combination regimens for patients with melanoma. However, prospective data to support evidence-based clinical decisions with respect to the optimal schedule and sequencing of immunotherapy and targeted agents, how best to manage emerging toxicities and when to stop treatment are not yet available. Methods To address this knowledge gap, the Society for Immunotherapy of Cancer (SITC) Melanoma Task Force developed a process for consensus recommendations for physicians treating patients with melanoma integrating evidence-based data, where available, with best expert consensus opinion. The initial consensus statement was published in 2013, and version 2.0 of this report is an update based on a recent meeting of the Task Force and extensive subsequent discussions on new agents, contemporary peer-reviewed literature and emerging clinical data. The Academy of Medicine (formerly Institute of Medicine) clinical practice guidelines were used as a basis for consensus development with an updated literature search for important studies published between 1992 and 2017 and supplemented, as appropriate, by recommendations from Task Force participants. Results The Task Force considered patients with stage II-IV melanoma and here provide consensus recommendations for how they would incorporate the many immunotherapy options into clinical pathways for patients with cutaneous melanoma. Conclusion These clinical guidleines provide physicians and healthcare providers with consensus recommendations for managing melanoma patients electing treatment with tumor immunotherapy. Electronic supplementary material The online version of this article (10.1186/s40425-018-0362-6) contains supplementary material, which is available to authorized users.

Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival. Experimental Design: A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD-scid-gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 (“don't eat me signal”) expression on tumor cells and characterize macrophages in the tumor microenvironment. In vitro engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival. Results: In vivo clodronate depletion experiments and NOD-scid-gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In in vitro engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival. Conclusions: These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. Clin Cancer Res; 24(6); 1415–25. ©2017 AACR.

Limited representation of intratumoral immune cells is a major barrier to tumor control. However, simply enhancing immune responses in tumor-draining lymph nodes or through adoptive transfer may not overcome the limited ability of tumor vasculature to support effector infiltration. An alternative is to promote a sustained immune response intratumorally. This idea has gained traction with the observation that many tumors are associated with tertiary lymphoid structures (TLS), which organizationally resemble lymph nodes. These peri- and intratumoral structures are usually, but not always, associated with positive prognoses in patients. Preclinical and clinical data support a role for TLS in modulating immunity in the tumor microenvironment. However, there appear to be varied functions of TLS, potentially based on their structure or location in relation to the tumor or the origin or location of the tumor itself. Understanding more about TLS development, composition, and function may offer new therapeutic opportunities to modulate antitumor immunity.

Background: tumor-infiltrating lymphocytes are prognostic in many human cancers. However, the prognostic value of lymphocytes infiltrating glioblastoma (GBM), and roles in tumor control or progression are unclear. We hypothesized that B and T cell density, and markers of their activity, proliferation, differentiation, or function, would have favorable prognostic significance for patients with GBM. Methods: initial resection specimens from 77 patients with IDH1/2 wild type GBM who received standard-of-care treatment were evaluated with multiplex immunofluorescence histology (mIFH), for the distribution, density, differentiation, and proliferation of T cells and B cells, as well as for the presence of tertiary lymphoid structures (TLS), and IFNγ expression. Immune infiltrates were evaluated for associations with overall survival (OS) by univariate and multivariate Cox proportional hazards modeling. Results: in univariate analyses, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.36, p = 0.001) and CD20+ cells (HR 0.51, p = 0.008), as well as CD8+Tbet+ cells (HR 0.46, p = 0.004), and RORγt+ cells (HR 0.56, p = 0.04). Conversely, IFNγ intensity was associated with diminished OS (HR 0.59, p = 0.036). In multivariable analyses, adjusting for clinical variables, including age, resection extent, Karnofsky Performance Status (KPS), and MGMT methylation status, improved OS was associated with high densities of proliferating (Ki67+) CD8+ cells (HR 0.15, p < 0.001), and higher ratios of CD8+ cells to CD4+ cells (HR 0.31, p = 0.005). Diminished OS was associated with increases in patient age (HR 1.21, p = 0.005) and higher mean intensities of IFNγ (HR 2.13, p = 0.027). Conclusions: intratumoral densities of proliferating CD8 T cells and higher CD8/CD4 ratios are independent predictors of OS in patients with GBM. Paradoxically, higher mean intensities of IFNγ in the tumors were associated with shorter OS. These findings suggest that survival may be enhanced by increasing proliferation of tumor-reactive CD8+ T cells and that approaches may be needed to promote CD8+ T cell dominance in GBM, and to interfere with the immunoregulatory effects of IFNγ in the tumor microenvironment.

BackgroundIntratumoral (IT) cancer therapies may enhance T cell activation and tumor infiltration when combined with systemic checkpoint blockade. This approach may improve treatment of advanced breast cancer, which is commonly resistant to immune therapy.MethodsA multicenter basket-style trial (NCT02643303) was performed in patients with advanced solid tumors, who received polyICLC IT 1mg x 6, then intramuscular (IM) x 3, combined with intravenous (IV) durvalumab 1500 mg q4W. Most were assigned to cohorts also receiving tremelimumab: 10 mg IT or 75 mg IV. Goals were to assess tolerability and clinical activity. Treated tumors were evaluated for immune infiltrates on days (d) 0, 15, and 29 by multiparameter immunofluorescence histology. A strong signal for clinical response was in breast cancer patients; thus, an expansion cohort was enrolled. We report analysis of that breast cancer subgroup.ResultsNineteen participants with treatment-refractory recurrent breast cancer with median 4 prior lines of therapy were enrolled and treated with IV durvalumab and IT/IM polyICLC. Seventeen also received tremelimumab (15 IT, 2 IV). Common treatment-related AEs were fatigue, injection site pain, and chills. There was one dose-limiting toxicity in a participant who received tremelimumab IV, and died with severe hyponatremia (DLT) and progressive disease. Objective clinical responses (1 complete; 4 partial (1 unconfirmed)) were observed in 5 (26%), including 2/9 patients with triple-negative breast cancer (TNBC) and 3/10 with non-TNBC. Median OS was longer for those with CR, PR, or SD (not reached) vs. those with PD or not evaluable (5 months): two responders remain alive at 34+ and 40+ months. In injected tumors, there were significant increases from d0 to d29 in numbers/mm2 of CD8+ T cells, CD20+ B cells, mature dendritic cells (DC), macrophages, and CD56+ NK cells, and in CD8+ cells with antigen-experience (CD45RO), cytotoxic function (granzyme B), activation (ICOS1), or proliferation (Ki67). CD8+ cells expressing LAG3 and TIM3 increased, as did PDL1+ tumor cells and stromal cells. There were no differences in cells expressing IDO, ARG1, CD39, or CD73. Among patients with objective response, vs. all others, proportions of intratumoral CD8+ cells expressing Ki67 increased (p < 0.04).ConclusionsIT tremelimumab and polyICLC plus systemic durvalumab is safe and has clinical activity in patients with advanced TNBC and non-TNBC. The therapy enhances intratumoral immune effectors and markers of T cell function in hypothesis-generating data that warrant confirmatory studies. Clinical response was associated with longer survival and increased CD8 T cell proliferation.Trial RegistrationNCT02643303Ethics ApprovalThe study has been performed with approval of the institutional review boards of each participating institution (Roswell Park Cancer Institute: STUDY 00000121/I291016; Mount Sinai School of Medicine: IRB-17-01692; University of Virginia: IRB # 19276; Cleveland Clinic: 18-694; Toledo: 300176; Dartmouth: STUDY00031630; Emory: IRB00099445). All participants give informed consent before enrolling and participating. The study was also performed with approval from the FDA

BackgroundA multipeptide vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe and immunogenic and has clinical activity. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus PD1 blockade.MethodsParticipants with measurable advanced melanoma, age ≥ 18 years, and ECOG performance status 0–1 were administered 6MHP vaccine intradermally and subcutaneously in an incomplete Freund’s adjuvant on days (D) 1, 8, 15, 43, 64, and 85. Pembrolizumab 200 mg was administered intravenously every 3 weeks for up to two years. Biopsies of accessible tumors at baseline and D22 were analyzed by multiparameter immunofluorescence histology. Primary endpoints were safety (CTCAE 4.03) and immunogenicity (ex vivo IFNγ ELIspot assay). Secondary and exploratory endpoints included changes in the tumor microenvironment (TME), and clinical outcomes.ResultsTwenty-two eligible participants were enrolled and treated, including 6 naïve to PD-1 Ab and 16 anti-PD-1 Ab-experienced. Median follow-up was 20 months. Treatment-related adverse events (any grade) experienced by >20% were injection site reactions, fatigue, anemia, nausea, fever, bruising, and rash. Treatment-related dose limiting toxicities (grade 3 elevated AST, skin ulcer, or uveitis) were observed in 3 (14%), which did not cross the study safety bound. Objective clinical responses were observed in 23% (1 CR, 4 PR), including 4/6 anti-PD-1 Ab-naive (67%) and one 1/16 anti-PD1 Ab-experienced (6%). Four participants (18%) had SD as best radiographic response (18%), all in the Ab-experienced cohort. T cell responses to 6MHP were detected in seven participants (32%) by week 13 and were associated with clinical response (CR/PR 80% vs. SD/PD 18%; p = 0.01). Overall survival was prolonged for anti-PD-1 Ab naïve vs experienced (p = 0.0048), for those with T cell response (p = 0.045, landmark analysis after week 13), and for those with objective response (p = 0.0148). TME evaluation in 12 participants revealed significant increases by D22 in the densities (per mm2) of CD8+ T cells (p = 0.0186), CD20+ B cells (P = 0.002), and Tbet+ cells (p = 0.034).ConclusionsIn patients with advanced melanoma, combined treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral T and B cells, as well as Th1 (Tbet+) cells, and induced T cell responses that were associated with objective response and with overall survival. The promising objective response rate and overall survival in patients naive to PD1 blockade supports consideration of a larger study to assess definitive benefit in that clinical setting.AcknowledgementsWe acknowledge the support of Merck for providing pembrolizumab at no charge, and the University of Virginia Cancer Center Support grant P30 CA044579 for support of shared resource facilities.Trial RegistrationThe clinical trial Mel64 (PATHVACS) is registered with Clinicaltrials.gov (NCT02515227).Ethics ApprovalThe clinical trial Mel64 (PATHVACS) was performed with IRB (#18174) and FDA approval (IND #10825) and is registered with Clinicaltrials.gov (NCT02515227). Written informed consent was obtained from each participant prior to participation in the study.

Background Breast cancer remains a leading cause of cancer death worldwide. There is evidence that immunotherapy may play a role in the eradication of residual disease. Peptide vaccines for immunotherapy are capable of durable immune memory, but vaccines alone have shown sparse clinical activity against breast cancer to date. Toll-like receptor (TLR) agonists and helper peptides are excellent adjuvants for vaccine immunotherapy and they are examined in this human clinical trial. Methods A vaccine consisting of 9 MHC class I-restricted breast cancer-associated peptides (from MAGE-A1, −A3, and -A10, CEA, NY-ESO-1, and HER2 proteins) was combined with a TLR3 agonist, poly-ICLC, along with a helper peptide derived from tetanus toxoid. The vaccine was administered on days 1, 8, 15, 36, 57, 78. CD8+ T cell responses to the vaccine were assessed by both direct and stimulated interferon gamma ELIspot assays. Results Twelve patients with breast cancer were treated: five had estrogen receptor positive disease and five were HER2 amplified. There were no dose-limiting toxicities. Toxicities were limited to Grade 1 and Grade 2 and included mild injection site reactions and flu-like symptoms, which occurred in most patients. The most common toxicities were injection site reaction/induration and fatigue, which were experienced by 100% and 92% of participants, respectively. In the stimulated ELIspot assays, peptide-specific CD8+ T cell responses were detected in 4 of 11 evaluable patients. Two patients had borderline immune responses to the vaccine. The two peptides derived from CEA were immunogenic. No difference in immune response was evident between patients receiving endocrine therapy and those not receiving endocrine therapy during the vaccine series. Conclusions Peptide vaccine administered in the adjuvant breast cancer setting was safe and feasible. The TLR3 adjuvant, poly-ICLC, plus helper peptide mixture provided modest immune stimulation. Further optimization is required for this multi-peptide vaccine/adjuvant combination. Trial registration ClinicalTrials.gov (posted 2/15/2012): NCT01532960. Registered 2/8/2012. https://clinicaltrials.gov/show/NCT01532960 Electronic supplementary material The online version of this article (10.1186/s40425-017-0295-5) contains supplementary material, which is available to authorized users.

BackgroundMost patients with advanced melanomas relapse after checkpoint blockade therapy. Thus, immunotherapies are needed that can be applied safely early, in the adjuvant setting. Seviprotimut-L is a vaccine containing human melanoma antigens, plus alum. To assess the efficacy of seviprotimut-L, the Melanoma Antigen Vaccine Immunotherapy Study (MAVIS) was initiated as a three-part multicenter, double-blind, placebo-controlled phase III trial. Results from part B1 are reported here.MethodsPatients with AJCC V.7 stage IIB-III cutaneous melanoma after resection were randomized 2:1, with stage stratification (IIB/C, IIIA, IIIB/C), to seviprotimut-L 40 mcg or placebo. Recurrence-free survival (RFS) was the primary endpoint. For an hypothesized HR of 0.625, one-sided alpha of 0.10, and power 80%, target enrollment was 325 patients.ResultsFor randomized patients (n=347), arms were well-balanced, and treatment-emergent adverse events were similar for seviprotimut-L and placebo. For the primary intent-to-treat endpoint of RFS, the estimated HR was 0.881 (95% CI: 0.629 to 1.233), with stratified logrank p=0.46. However, estimated HRs were not uniform over the stage randomized strata, with HRs (95% CIs) for stages IIB/IIC, IIIA, IIIB/IIIC of 0.67 (95% CI: 0.37 to 1.19), 0.72 (95% CI: 0.35 to 1.50), and 1.19 (95% CI: 0.72 to 1.97), respectively. In the stage IIB/IIC stratum, the effect on RFS was greatest for patients ConclusionsSeviprotimut-L is very well tolerated. Exploratory efficacy model estimation supports further study in stage IIB/IIC patients, especially younger patients and those with ulcerated melanomas.Trial registration numberNCT01546571.

9576 Background: While management of the nodal basin for melanoma has largely moved to observation for microscopic sentinel lymph node (SLN) metastasis, complete lymph node dissection (CLND) remains the current standard of care for melanoma patients with macroscopic, clinically detectable lymph node metastases (cLN). As CLND is associated with high surgical morbidity, we sought to study whether cLN may be safely managed by excision of only clinically abnormal nodes (precision lymph node dissection, PLND). Currently, a small subset of patients with cLN do not undergo CLND because of frailty or patient preference. We hypothesized that in these selected patients, PLND would provide acceptable regional control rates. Methods: Retrospective chart review was conducted at four academic tertiary care hospitals to identify melanoma patients who underwent PLND for cLN. cLN were defined as palpable or radiographically abnormal nodes. Recurrences were categorized as local/in-transit, same-basin lymph node, or distal lymph node/visceral. The primary outcome was isolated same-basin recurrence after PLND. Results: Twenty-one patients underwent PLND for cLN without synchronous distant metastases (characteristics of primary lesions summarized in Table). Reasons for forgoing CLND included patient preference (n=8), imaging indeterminate for distant metastases (n=2), comorbidities (n=4), loss to follow up (n=1), partial response to checkpoint blockade (n=1), or not reported (n=5). The inguinal node basin was the most common site (n=10), followed by the axillary (n=8) and cervical basins (n=3). A median of 2 nodes were resected at PLND, and 68% of resected nodes were positive for melanoma (median: 1, range: 1-3 nodes). Median follow-up was 23 months from PLND, and recurrence was observed in 28.6% of patients overall. Only 1 patient (4.8%) developed an isolated same-basin recurrence. The 3-year cumulative incidence of isolated same-basin recurrence was 5.3%, while risk of isolated local/in-transit recurrence or distant basin/visceral metastasis were 19.8% and 33.3%, respectively. Complications from PLND were reported in 1 patient (4.8%) and were limited to post-operative seroma and lymphedema. Conclusions: These pilot data suggest that PLND may offer acceptable regional disease control for cLN. Post-operative morbidity from PLND was also low, raising the possibility that PLND may provide adequate regional disease control without the morbidity associated with CLND. These data justify additional, prospective evaluation of PLND in selected patients.[Table: see text]

4128 Background: Pancreatic cancer (PC) is a challenging target for immunotherapy due to suppressive immune-microenvironment. Neoadjuvant chemoradiation (CRT) can increase the presence of tumor-infiltrating lymphocytes (TILs). We hypothesized that the combination of CRT and pembrolizumab can lead to further increase in TILs and their activation. Methods: Patients with resectable or borderline resectable PC were randomized 2:1 to the investigational treatment (Arm A) of pembrolizumab 200mg IV every 3 weeks concurrently with CRT (capecitabine 825 mg/m2 orally twice daily and radiation 50.4 Gy in 28 fractions over 28 days) or CRT only (Arm B) prior to surgical resection. The primary endpoints were treatment safety and density of TILs with the objective to estimate differences in TILs density between the investigational and the control arms. Immune cell densities were assessed using multiplexed immunofluorescence on resected tumor specimens. Densities of CD8+TILs were measured in 2-10 representative regions containing residual cancer per case and then averaged to obtain overall densities. The study was amended after enrollment of 37 patients to allow FOLFIRINOX prior to CRT, given changes in standard of care. Results: 37 patients were enrolled (24 Arm A and 13 Arm B). Post-neoadjuvant therapy, 13 patients had unresectable disease (9 on A and 4 on B), and 24 patients underwent surgery and were evaluable for the TILs primary endpoint (17 arm A and 7 arm B). The mean difference (A-B) in CD8+ cell density was 36 cells/mm2 (95% CI -85 to 157, stdev 130) (p 0.48). Additional analysis did not show significant differences in CD8+Ki67+ (activated cytotoxic T-cells), CD4+, and CD4+FOXP3+ (regulatory T cells), M1- or M2-like polarized macrophages, or granulocytes. The median recurrence free survival (RFS) was 18.2 months on Arm A and 14.1 on Arm B (p 0.41) and Overall Survival was 27.8 months on Arm A and 24.3 on Arm B (p 0.68) with a median follow up of 2.2 years. The most common grade 3 treatment-related toxicities were lymphopenia reported in 29% on Arm A and 31% on Arm B followed by diarrhea in 8% on Arm A attributed to CRT. There was only 1 DLT of increased ALT attributed to the combination on Arm A that resolved after holding the treatment and receiving steroids. There were no major surgical complications reported within 30 days post-surgery. Conclusions: The combination of CRT and pembrolizumab is safe. Preliminary analysis shows that the addition of pembrolizumab to CRT has minimal effects on several immune cell populations including CD8+TILs in the PC microenvironment. The study is currently enrolling 25 more patients who receive FOLFIRINOX prior to randomization to CRT+/- Pembrolizumab, which will help to dissect the immune modulatory effect of chemotherapy followed by CRT. Clinical trial information: NCT02305186.

BackgroundExperimental cancer vaccines are traditionally administered by injection in subcutaneous tissue or muscle, commonly with adjuvants that create chronic inflammatory depots. Injection of melanoma-derived peptides induces T cell responses; however, the depots that form following injection may inhibit optimization of the immune response. In skin, epidermal Langerhans cells (LC) are a dominant source of professional antigen presenting cells. We hypothesized that: (1) applying melanoma-derived peptides topically, in proximity to LC, could be immunogenic and safe, with low vaccine-site toxicity and (2) topical toll-like receptor 7 (TLR7) agonist would increase immunogenicity of the peptide vaccine.MethodsTwelve melanoma peptides plus a tetanus helper peptide were combined with granulocyte macrophage colony stimulating factor (GM-CSF) and were administered topically on days 1, 8, and 15, to 28 patients randomized to one of four adjuvant preparations: (1) incomplete Freund’s adjuvant (IFA); (2) IFA plus a TLR7 agonist (imiquimod) administered on days 0, 7, 14; (3) dimethyl sulfoxide (DMSO) or (4) DMSO+ imiquimod administered on day 0, 7, 14. Every 3 weeks thereafter (x 6), the peptides were combined with GM-CSF and were injected into the dermis and subcutis in an emulsion with IFA. Toxicities were recorded and immune responses assayed by ELIspot.ResultsCD8+ T cell responses to transdermal vaccination in DMSO occurred in 83% of participants in group 3 and 86% in group 4, and responses to vaccination in IFA were observed in 29% of participants in group 1 and 14% in group 2. Overall, 61% of participants had CD4+ T cell immune responses to the tetanus peptide, with large, durable responses in groups 3 and 4. Five of seven participants in group 4 had a severe rash, one that was dose limiting. Ten-year overall survival was 67% and disease-free survival was 44%.ConclusionsThese data provide proof of principle for immunogenicity in humans of transdermal immunization using peptides in DMSO. Further study is warranted into the pharmacokinetics and immunobiology of TLR agonists as vaccine adjuvants during transcutaneous application. Overall survival is high, supporting further investigation of this immunization approach.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and the second most common cause of cancer death worldwide. Current treatment options for patients with intermediate and advanced HCC are limited, and there is an unmet need for novel therapeutic approaches. HCC is an attractive target for immunomodulation therapy, since it arises in an inflammatory milieu due to hepatitis B and C infections and cirrhosis. However, a major barrier to the development and success of immunotherapy in patients with HCC is the liver's inherent immunosuppressive function. Recent advances in the field of cancer immunology allowed further characterization of immune cell subsets and function, and created new opportunities for therapeutic modulation of the immune system. In this review, we present the different immune cell subsets involved in potential immune modulation of HCC, discuss their function and clinical relevance, review the variety of immune therapeutic agents currently under investigation in clinical trials, and outline future research directions.

Background Funding toward surgical research through the National Institutes of Health has decreased relative to other medical specialties. This study was initiated to characterize features of academically successful surgeon-scientists and departments of surgery. We hypothesized that there may be decreases in young investigators obtaining independent National Institutes of Health awards and that successful academic departments of surgery may be depending increasingly on PhD faculty. Methods The National Institutes of Health RePORTER database was queried for grants awarded to departments of surgery during fiscal years 2003 and 2013. Grant summaries were categorized by research methodology. Training of the principal investigator and academic position were determined through the RePORTER database and publicly available academic biographies. Institutions were ranked by number of grants funded. Results Between 2003 and 2013, total surgery grants awarded decreased by 19%. The number of National Institutes of Health-funded, clinically active surgeons (MDs) decreased 11%, while funded PhDs increased 9%; however, clinically active junior faculty have comprised an increasing proportion of funded MDs (from 20–38%). Shifts in research topics include an increasing proportion of investigators engaged in outcomes research. Among institutions ranking in the top 20 for surgical research in both 2003 and 2013 ( N = 15), the ratio of MDs to PhDs was 2:1 in both fiscal years. Among institutions falling out of the top 20, this ratio was less than 1:1. Conclusion There has been an expansion of outcomes-based surgical research. The most consistently successful institutions are those that actively cultivate MD researchers. Encouragingly, the number of young, independently funded surgeon-scientists in America appears to be increasing.

Background In recent years, investigators have asserted that the 3 + 3 design lacks flexibility, making its use in modern early-phase trial settings, such as combinations and/or biological agents, inefficient. More innovative approaches are required to address contemporary research questions, such as those posed in trials involving immunotherapies. Design We describe the implementation of an adaptive design for identifying an optimal treatment regimen, defined by low toxicity and high immune response, in an early-phase trial of a melanoma helper peptide vaccine plus novel adjuvant combinations. Results Operating characteristics demonstrate the ability of the method to effectively recommend optimal regimens in a high percentage of trials with reasonable sample sizes. Conclusions The proposed design is a practical, early-phase, adaptive method for use with combined immunotherapy regimens. This design can be applied more broadly to early-phase combination studies, as it was used in an ongoing study of two small molecule inhibitors in relapsed/refractory mantle cell lymphoma.

Human telomerase reverse transcriptase (hTERT) is highly expressed in85% of human tumors and is thus considered as a good tumor-associated antigen candidate for vaccine development. We conducted a phase I study to investigate the safety, tolerability, clinical response, and immunogenicity of INVAC-1, a DNA plasmid encoding a modified hTERT protein in patients with relapsed or refractory solid tumors.INVAC-1 was either administered by intradermal route followed by electroporation or by Tropis, a needle-free injection system. Safety and tolerability were monitored by clinical and laboratory assessments. Progression-free survival and overall survival were reported using Kaplan-Meier survival analysis. Immunogenicity was studied by ELISpot, Luminex, and Flow Cytometry.Twenty-six patients were treated with INVAC-1 administered at three dose levels (100, 400, and 800 μg). Vaccination was well tolerated and no dose-limiting toxicity was reported. One treatment-related grade 3 SAE was reported. Fifty-eight percent of patients experienced disease stabilization. PFS was 2.7 months, median OS was 15 months, and 1-year survival was reached for 65% of patients. INVAC-1 vaccination stimulated specific anti-hTERT CD4 T-cell response as well as cytotoxic CD8 T-cell response. No evidence of peripheral vaccine-induced immunosuppression was observed.INVAC-1 vaccination was safe, well tolerated, and immunogenic when administered intradermally at the three tested doses in patients with relapsed or refractory cancers. Disease stabilization was observed for the majority of patients (58%) during the treatment period and beyond.

Background Cancer vaccines require adjuvants to induce effective immune responses; however, there is no consensus on optimal adjuvants. We hypothesized that toll-like receptor (TLR)3 agonist polyICLC or TLR4 agonist lipopolysaccharide (LPS), combined with CD4 T cell activation, would support strong and durable CD8+ T cell responses, whereas addition of an incomplete Freund’s adjuvant (IFA) would reduce magnitude and persistence of immune responses. Patients and methods Participants with resected stage IIB-IV melanoma received a vaccine comprised of 12 melanoma peptides restricted by Class I MHC (12MP), plus a tetanus helper peptide (Tet). Participants were randomly assigned 2:1 to cohort 1 (LPS dose-escalation) or cohort 2 (polyICLC). Each cohort included 3 subgroups (a-c), receiving 12MP + Tet + TLR agonist without IFA (0), or with IFA in vaccine one (V1), or all six vaccines (V6). Toxicities were recorded (CTCAE v4). T cell responses were measured with IFNγ ELIspot assay ex vivo or after one in vitro stimulation (IVS). Results Fifty-three eligible patients were enrolled, of which fifty-one were treated. Treatment-related dose-limiting toxicities (DLTs) were observed in 0/33 patients in cohort 1 and in 2/18 patients in cohort 2 (11%). CD8 T cell responses to 12MP were detected ex vivo in cohort 1 (42%) and in cohort 2 (56%) and in 18, 50, and 72% for subgroups V0, V1, and V6, respectively. T cell responses to melanoma peptides were more durable and of highest magnitude for IFA V6. Conclusions LPS and polyICLC are safe and effective vaccine adjuvants when combined with IFA. Contrary to the central hypothesis, IFA enhanced T cell responses to peptide vaccines when added to TLR agonists. Future studies will aim to understand mechanisms underlying the favorable effects with IFA. Trial registration The clinical trial Mel58 was performed with IRB (#15781) and FDA approval and is registered with Clinicaltrials.gov on April 25, 2012 (NCT01585350). Patients provided written informed consent to participate. Enrollment started on June 24, 2012. Electronic supplementary material The online version of this article (10.1186/s40425-019-0625-x) contains supplementary material, which is available to authorized users.

BACKGROUND: Nodal observation is safe for patients with microscopic melanoma metastasis in a sentinel lymph node (LN). Complete LN dissection (CLND) remains the standard of care for patients with clinically evident LN (cLN) metastases, even though about 40% have only one pathologic LN (pLN). We sought to identify clinical features associated with having one pLN among patients with cLNs. METHODS: Patients at three melanoma centers who underwent CLND for cLNs were identified. Clinicopathologic and imaging characteristics associated with one pLN were determined by multivariable logistic regression and classification tree analysis. RESULTS: Of 190 patients, 90 (47.4%) had one pLN and 100 (52.6%) had more than one pLN. By multivariable logistic regression, extremity versus truncal primary (odds ratio [OR] 2.15, p = 0.012), axillary versus superficial inguinal location (OR 3.89, p = 0.009), and preoperative cross-sectional imaging demonstrating more than one versus one cLN (OR 17.1, p < 0.001) were associated with more than one pLN. The negative predictive value for additional pathologic nodal disease of preoperative imaging was 70.9%, increasing to 74.4% for positron emission tomography/computed tomography. In the subgroup of patients with one cLN, the classification tree identified two groups with < 10% risk of additional pLNs: (1) Breslow thickness > 6.55 mm (n = 17); and (2) if unknown primary or Breslow thickness ≤ 6.55 mm, then LN diameter > 1.8 cm in the inguinal location (n = 22). CONCLUSION: The majority of patients with one cLN from melanoma by preoperative imaging will harbor no additional pathologic nodes on CLND. Safety of nodal observation after clinical nodal excision in these patients, particularly in an era of effective adjuvant therapies, deserves prospective evaluation.

INTRODUCTION: In contemporary oncology drug development, implementation of novel early-phase designs with the ability to address multiple research objectives are needed to better refine regimens. This paper describes an adaptive design strategy for identifying a range of optimal regimens based on two endpoints within multiple cohorts. The proposed design was developed to address objectives in an early-phase trial of cancer vaccines in combination with agonistic antibodies to CD40 and CD27. MATERIALS AND METHODS: We describe a model-based design strategy that was developed for a trial evaluating the safety and immunogenicity of vaccination with i) peptides plus CD40 antibody and TLR3 ligand, ii) systemic administration of an agonistic CD27 antibody, and c) to assess immune response from i) and ii) compared to optimal controls in participants with stage IIB-IV melanoma. RESULTS AND CONCLUSIONS: The proposed design is a practical adaptive method for use with combined immunotherapy regimens with multiple objectives within multiple cohorts of interest. Further advances in the effectiveness of cancer immunotherapies will require new approaches that include redefining optimal strategies to take multiple regimens forward into later phases, incorporating additional endpoints in the dose selection process and testing drug combination therapies to improve efficacy and reduce toxicity. Our goal is to facilitate acceptance and application of more novel designs in contemporary early development trials.

BACKGROUND: One of the purported reasons of ineffectiveness of immunotherapy for GBM is the meager immune response in the tumor microenvironment. We have observed that GBM can harbor dense B-cell infiltrate, but their prevalence and function is unknown. Pre-clinical studies have shown that B-cells can act as antigen-presenting cells which can stimulate T-cell proliferation; however, B-cells can also downregulate effector T-cells by secreting immunosuppressive cytokines including IL-10. Our study aims to determine the roles of B-cells in GBM and their association with patient outcomes. METHODS: Multiplexed immunohistochemistry, imaging and quantitation was performed on primary GBM samples obtained between 1/2008-8/2015. Intratumoral CD20(+)B-cells and CD8(+)T-cells infiltrate were enumerated from 3 serial GBM sections. Relevant prognostic clinical and molecular variables were reviewed. We also analyzed the association of MS4A1 gene encoding B-cell antigen CD20 with survival from the TCGA. RESULTS: We observed an average infiltrate of 5.5 CD20(+) cells/mm(2)and 36.7 CD8(+)cells/mm(2)tumor. B-cells were stratified into CD20(low)(12 patients) and CD20(high)groups (36 patients) with 0.24 B-cells/mm(2) cut off. Kaplan-Meier analyses indicated that the CD20(low)group had better overall survival (OS) than the CD20(high)cohort (31.7 versus 18.7 months; p=0.02), while a trend toward improved survival was observed with high CD8(+)T-cell infiltration (p=0.06). Cox proportional hazard demonstrated an association between decreased OS and CD20(high)cohort (HR=2.8, p=0.03), but no significance seen between CD8(+)infiltration and OS (p=0.16). A positive correlation was detected between CD8(+)and CD20(+)density (p CONCLUSIONS: Our results suggest that B-cell infiltrate carries an inverse correlation with survival. We hypothesize that CD20(+)B-cells may be regulatory B-cells, limiting immune activation in GBM. Further studies are underway to understand the role of B-cells in the GBM microenvironment.

SUMMARY: We analyze the transcriptome of baseline and on-therapy tumor biopsies from 101 patients with advanced melanoma treated with nivolumab (anti-PD-1) alone or combined with ipilimumab (anti-CTLA-4). We find that T cell infiltration and interferon-γ (IFNγ) signaling signatures correspond most highly with clinical response to therapy, with a reciprocal decrease in cell cycle and WNT signaling pathways in responding biopsies. We model the interaction in 58 human cell lines, where IFNγ in vitro exposure leads to a conserved transcriptome response unless cells have IFNγ receptor alterations. This conserved IFNγ transcriptome response in melanoma cells serves to amplify the antitumor immune response. Therefore, the magnitude of the antitumor T cell response and the corresponding downstream IFNγ signaling are the main drivers of clinical response or resistance to immune checkpoint blockade therapy. ETOC BLURB: Analyzing the transcriptome of biopsies of patients during immune checkpoint blockade therapy, Grasso et al. show that the increase of T cell infiltration and the downstream IFNγ signaling drive clinical responses.

Infiltration of non-small-cell lung cancer (NSCLC) by CD8+ T lymphocytes predicts improved patient survival; however, heterogeneity of intratumoral localization complicates this assessment. Strategies for tumor sampling may not accurately represent the whole tumor. We hypothesized that sampling strategies may alter the identification of tumors with high CD8 density and affect the prognostic significance. Twenty-three primary NSCLC tumors were immunohistochemically stained for CD8 and were assessed using automated software with eight different sampling strategies or the whole tumor. Results of all sampling strategies were compared to the whole tumor counts (paired t tests, Pearson’s r). Associations between CD8 densities and overall survival were assessed (log-rank test). Counts from all eight sampling strategies significantly correlated with whole tumor counts (p ≤ 0.001). However, the magnitude of CD8+ cell counts and categorization into high vs low infiltrate groups were affected by the sampling strategy. The most concordant values were derived from random sampling of 20 % of the tumor, a simulated core biopsy, or from sampling the tumor center. TIL infiltration was associated with survival when sampling the center (p = 0.038), but not the invasive margin (p > 0.2) or other strategies. Different tumor sampling strategies may yield discordant TIL density results and different stratification for risk assessment. Small biopsies may be particularly unrepresentative. Random sampling of larger tumor areas is recommended. Enumerating CD8+ T cells in the tumor center may have prognostic value.

Optimal approaches to induce T cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T cell recruitment and may be induced by IFN. This study tests the hypothesis that intratumoral administration of IFNγ will induce CXCL9-11 and will induce T cell recruitment and anti-tumor immune signatures in melanoma metastases.Nine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides and received IFNγ intratumorally. Effects on the tumor microenvironment were evaluated in sequential tumor biopsies. Adverse events (AEs) were recorded. T cell responses to vaccination were assessed in PBMC by IFNγ ELISPOT assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression, and gene expression.Vaccination and intratumoral administration of IFNγ were well tolerated. Circulating T cell responses to vaccine were detected in six of nine patients. IFNγ increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone, nor the addition of IFNγ promoted immune cell infiltration or induced anti-tumor immune gene signatures.The melanoma vaccine induced circulating T cell responses, but it failed to infiltrate metastases, thus highlighting the need for combination strategies to support T cell infiltration. A single intratumoral injection of IFNγ induced T cell-attracting chemokines; however, it also induced secondary immune regulation that may paradoxically limit immune infiltration and effector functions. Alternate dosing strategies or additional combinatorial treatments may be needed to promote trafficking and retention of tumor-reactive T cells in melanoma metastases.

Objective The purpose of this study was to assess regional recurrence rates of patients who underwent sentinel lymph node biopsy (SLNB) using radiocolloid guidance alone. Background Isosulfan blue dye is commonly used along with Tc99-labeled radiocolloid localization in SLNB for melanoma. Blue dye has, however, been associated with allergic reactions, long-term staining of skin, and increased cost. We hypothesized that the rate of regional recurrence when SLNB is performed with radiocolloid alone would be comparable to established reports using both radiocolloid and blue dye. Methods A prospectively collected database was retrospectively queried for patients who underwent SLNB for melanoma during the years 2005 through 2008. Data collected included patient demographics, primary lesion characteristics, operative details, and recurrence. The primary outcome was the rate of recurrence within the biopsied basin after negative SLNB's performed without isosulfan blue dye. Results In 215 patients, 279 nodal basins were identified. All patients underwent successful radiocolloid localization, and positive sentinel nodes were found in 40 patients (18.6%). Six of 175 patients with a negative SLNB developed a regional node recurrence as the first site of metastasis (3.4%). Among all 215 patients, 44 experienced recurrence of any kind (20.5%). Higher mitotic rate and Breslow depth were significantly associated with likelihood of recurrence. Conclusions Success rates, node positivity rates, and rates of regional recurrence after SLNB for melanoma using radiocolloid alone are acceptable and similar to those of prior reports using blue dye plus radiocolloid.