Optimal Neoantigen Cancer Vaccines Target CD8+ and CD4+ T Cells with Multiple Antigens.

Cancer vaccines targeting mutated neoantigens offer promise for prevention of cancer recurrence and for treatment of established cancers. Questions remain about whether vaccines need to target multiple neoantigens and whether they need to target both CD8+ and CD4+ T cells. In this issue, Garzia and colleagues demonstrate the importance of including multiple antigens to stimulate both CD4+ T cells and CD8+ T cells for treatment of established cancer. See related article by Garzia et al., p. 440 (4).

Multipeptide vaccines for melanoma in the adjuvant setting: long-term survival outcomes and post-hoc analysis of a randomized phase II trial.

The critical roles of CD4+ T cells have been understudied for cancer vaccines. Here we report long-term clinical outcomes of a randomized multicenter phase II clinical trial (NCT00118274), where patients with high-risk melanoma received a multipeptide vaccine targeting CD8+ T cells (12MP) and were randomized to receive either of two vaccines for CD4+ (helper) T cells: 6MHP (6 melanoma-specific helper peptides), or tet (a nonspecific helper peptide from tetanus toxoid). Cyclophosphamide (Cy) pre-treatment was also assessed. Primary outcomes for T cell responses to 12MP, 6MHP, and tet were previously reported, suggesting immunogenicity of both vaccines but that CD8 T cell responses to 12MP were lower when tet was replaced with 6MHP. Here, in post-hoc analyses, we report durable prolongation of overall survival by adding 6MHP instead of tet. That benefit was experienced only by male patients. A favorable interaction of 6MHP and Cy is also suggested. Multivariable Cox regression analysis of the intent-to-treat population identify vaccine arm (12MP + 6MHP+Cy) and patient sex (male) as the two significant predictors of enhanced survival. These findings support the value of adding cognate T cell help to cancer vaccines and also suggest a need to assess the impact of patient sex on immune therapy outcomes.

Applications of the partial-order continual reassessment method in the early development of treatment combinations.

Combination therapy is increasingly being explored as a promising approach for improving cancer treatment outcomes. However, identifying effective dose combinations in early oncology drug development is challenging due to limited sample sizes in early-phase clinical trials. This task becomes even more complex when multiple agents are being escalated simultaneously, potentially leading to a loss of monotonic toxicity order with respect to the dose. Traditional single-agent trial designs are insufficient for this multi-dimensional problem, necessitating the development and implementation of dose-finding methods specifically designed for drug combinations. While, in practice, approaches to this problem have focused on preselecting combinations with a known toxicity order and applying single-agent designs, this limits the number of combinations considered and may miss promising dose combinations. In recent years, several novel designs have been proposed for exploring partially ordered drug combination spaces with the goal of identifying a maximum tolerated dose combination, based on safety, or an optimal dose combination, based on toxicity and efficacy. However, their implementation in clinical practice remains limited. In this article, we describe the application of the partial order continual reassessment method and its extensions for combination therapies in early-phase clinical trials. We present completed trials that use safety endpoints to identify maximum tolerated dose combinations and adaptively use both safety and efficacy endpoints to determine optimal treatment strategies. We discuss the effectiveness of the partial-order continual reassessment method and its extensions in identifying optimal treatment strategies and provide our experience with executing these novel adaptive designs in practice. By utilizing innovative dose-finding methods, researchers and clinicians can more effectively navigate the challenges of combination therapy development, ultimately improving patient outcomes in the treatment of cancer.

Cancer-oocyte SAS1B protein is expressed at the cell surface of multiple solid tumors and targeted with antibody-drug conjugates.

BACKGROUND: Sperm acrosomal SLLP1 binding (SAS1B) protein is found in oocytes, which is necessary for sperm-oocyte interaction, and also in uterine and pancreatic cancers. Anti-SAS1B antibody-drug conjugates (ADCs) arrested growth in these cancers. However, SAS1B expression in cancers and normal tissues has not been characterized. We hypothesized that SAS1B is expressed on the surface of other common solid cancer cells, but not on normal tissue cells, and might be selectively targeted therapeutically. METHODS: SAS1B expression in human normal and cancer tissues was determined by immunohistochemistry, and complementary DNA (cDNA) libraries were employed to PCR amplify human SAS1B and its transcripts. Monoclonal antibodies (mAbs) to human SAS1B were generated using mouse hybridomas. SAS1B deletion constructs were developed to map SAS1B's epitope, enabling the creation of a blocking peptide. Indirect immunofluorescence (IIF) of human transfected normal and cancer cells was performed to assess SAS1B expression. SAS1B intracellular versus surface expression in normal and tumor tissues was evaluated by flow cytometry after staining with anti-SAS1B mAb, with specificity confirmed with the blocking peptide. Human cancer lines were treated with increasing mAb and ADC concentrations. ATP was quantitated as a measure of cell viability. RESULTS: SAS1B expression was identified in a subset of human cancers and the cytoplasm of pancreatic islet cells. Two new SAS1B splice variants were deduced. Monoclonal antibodies were generated to SAS1B splice variant A. The epitope for mAbs SB2 and SB5 is between SAS1B amino acids 32-39. IIF demonstrated intracellular SAS1B expression in transfected kidney cells and on the cell surface of squamous cell lung carcinoma. Flow cytometry demonstrated intracellular SAS1B expression in all tumors and some normal cells. However, surface expression of SAS1B was identified only on cancer cells. SB2 ADC mediated dose-dependent cytotoxic killing of multiple human cancer lines. CONCLUSION: SAS1B is a novel cancer-oocyte antigen with cell surface expression restricted to cancer cells. In vitro, it is an effective target for antibody-mediated cancer cell lysis. These findings support further exploration of SAS1B as a potential therapeutic cancer target in multiple human cancers, either with ADC or as a chimeric antigen receptor-T (CAR-T) cell target.

PD-1 Blockade Induces Reactivation of Nonproductive T-Cell Responses Characterized by NF-κB Signaling in Patients with Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) trials have evaluated CTLA-4 and/or PD-(L)1 blockade in patients with advanced disease in which bulky tumor burden and limited time to develop antitumor T cells may have contributed to poor clinical efficacy. Here, we evaluated peripheral blood and tumor T cells from patients with PDAC receiving neoadjuvant chemoradiation plus anti-PD-1 (pembrolizumab) versus chemoradiation alone. We analyzed whether PD-1 blockade successfully reactivated T cells in the blood and/or tumor to determine whether lack of clinical benefit could be explained by lack of reactivated T cells versus other factors. EXPERIMENTAL DESIGN: We used single-cell transcriptional profiling and TCR clonotype tracking to identify TCR clonotypes from blood that match clonotypes in the tumor. RESULTS: PD-1 blockade increases the flux of TCR clonotypes entering cell cycle and induces an IFNγ signature like that seen in patients with other GI malignancies who respond to PD-1 blockade. However, these reactivated T cells have a robust signature of NF-κB signaling not seen in cases of PD-1 antibody response. Among paired samples between blood and tumor, several of the newly cycling clonotypes matched activated T-cell clonotypes observed in the tumor. CONCLUSIONS: Cytotoxic T cells in the blood of patients with PDAC remain sensitive to reinvigoration by PD-1 blockade, and some have tumor-recognizing potential. Although these T cells proliferate and have a signature of IFN exposure, they also upregulate NF-κB signaling, which potentially counteracts the beneficial effects of anti-PD-1 reinvigoration and marks these T cells as non-productive contributors to antitumor immunity. See related commentary by Lander and DeNardo, p. 474.

Multicenter randomized controlled trial of neoadjuvant chemoradiotherapy alone or in combination with pembrolizumab in patients with resectable or borderline resectable pancreatic adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a challenging target for immunotherapy because it has an immunosuppressive tumor microenvironment. Neoadjuvant chemoradiotherapy can increase tumor-infiltrating lymphocyte (TIL) density, which may predict overall survival (OS). We hypothesized that adding programmed cell death protein 1 (PD-1) blockade to chemoradiotherapy would be well tolerated and increase TILs among patients with localized PDAC. METHODS: Patients were randomized 2:1 to Arm A (receiving pembrolizumab plus chemoradiotherapy (capecitabine and external beam radiation)) or Arm B (receiving chemoradiotherapy alone) before anticipated pancreatectomy. Primary endpoints were (1) incidence and severity of adverse events during neoadjuvant therapy and (2) density of TILs in resected tumor specimens. TIL density was assessed using multiplexed immunofluorescence histologic examination. RESULTS: Thirty-seven patients were randomized to Arms A (n=24) and B (n=13). Grade ≥3 adverse events related to neoadjuvant treatment were experienced by 9 (38%) and 4 (31%) patients in Arms A and B, respectively, with one patient experiencing dose-limiting toxicity in Arm A. Seventeen (71%) and 7 (54%) patients in Arms A and B, respectively, underwent pancreatectomy. Median CD8+ T-cell densities in Arms A and B were 67.4 (IQR: 39.2-141.8) and 37.9 (IQR: 22.9-173.4) cells/mm2, respectively. Arms showed no noticeable differences in density of CD8+Ki67+, CD4+, or CD4+FOXP3+ regulatory T cells; M1-like and M2-like macrophages; or granulocytes. Median OS durations were 27.8 (95% CI: 17.1 to NR) and 24.3 (95% CI: 12.6 to NR) months for Arms A and B, respectively. CONCLUSIONS: Adding pembrolizumab to neoadjuvant chemoradiotherapy was safe. However, no convincing effect on CD8+ TILs was observed.

Barriers to immune cell infiltration in tumors.

Increased immune cell infiltration into tumors is associated with improved patient survival and predicts response to immune therapies. Thus, identification of factors that determine the extent of immune infiltration is crucial, so that methods to intervene on these targets can be developed. T cells enter tumor tissues through the vasculature, and under control of interactions between homing receptors on the T cells and homing receptor ligands (HRLs) expressed by tumor vascular endothelium and tumor cell nests. HRLs are often deficient in tumors, and there also may be active barriers to infiltration. These remain understudied but may be crucial for enhancing immune-mediated cancer control. Multiple intratumoral and systemic therapeutic approaches show promise to enhance T cell infiltration, including both approved therapies and experimental therapies. This review highlights the intracellular and extracellular determinants of immune cell infiltration into tumors, barriers to infiltration, and approaches for intervention to enhance infiltration and response to immune therapies.

Intratumoral IFN-γ or topical TLR7 agonist promotes infiltration of melanoma metastases by T lymphocytes expanded in the blood after cancer vaccine.

BACKGROUND: Immune-mediated melanoma regression relies on melanoma-reactive T cells infiltrating tumor. Cancer vaccines increase circulating melanoma-reactive T cells, but little is known about vaccine-induced circulating lymphocytes (viCLs) homing to tumor or whether interventions are needed to enhance infiltration. We hypothesized that viCLs infiltrate melanoma metastases, and intratumoral interferon (IFN)-γ or Toll-like receptor 7 (TLR7) agonism enhances infiltration. METHODS: Patients on two clinical trials (Mel51 (NCT00977145), Mel53 (NCT01264731)) received vaccines containing 12 class I major histocompatibility complex-restricted melanoma peptides (12MP). In Mel51, tumor was injected with IFN-γ on day 22, and biopsied on days 1, 22, and 24. In Mel53, dermal metastases were treated with topical imiquimod, a TLR7 agonist, for 12 weeks, and biopsied on days 1, 22, and 43. For patients with circulating T-cell responses to 12MP by IFN-γ ELISpot assays, DNA was extracted from peripheral blood mononuclear cells (PBMCs) pre-vaccination and at peak T-cell response, and from tumor biopsies, which underwent T-cell receptor sequencing. This enabled identification of clonotypes induced in PBMCs post-vaccination (viCLs) and present in tumor post-vaccination, but not pre-vaccination. RESULTS: Six patients with T-cell responses post-vaccination (Mel51 n = 4, Mel53 n = 2) were evaluated for viCLs and vaccine-induced tumor infiltrating lymphocytes (viTILs). All six patients had viCLs, five of whom were evaluable for viTILs in tumor post-vaccination alone. Mel51 patients had viTILs identified in day 22 tumors, post-vaccination and before IFN-γ (median = 2, range = 0-24). This increased in day 24 tumors after IFN-γ (median = 30, range = 4-74). Mel53 patients had viTILs identified in day 22 tumors, post-vaccination plus imiquimod (median = 33, range = 2-64). Three of five evaluable patients across both trials had viTILs with vaccination alone. All five had enhancement of viTILs with tumor-directed therapy. viTILs represented 0.0-2.9% of total T cells after vaccination alone, which increased to 0.6-8.7% after tumor-directed therapy. CONCLUSION: Cancer vaccines induce expansion of new viCLs, which infiltrate melanoma metastases in some patients. Our findings identify opportunities to combine vaccines with tumor-directed therapies to enhance T-cell infiltration and T cell-mediated tumor control. These combinations hold promise in improving the therapeutic efficacy of antigen-specific therapies for solid malignancies.

Evaluation of Preoperative and Intraoperative Mobile Gamma Camera Imaging in Sentinel Lymph Node Biopsy for Melanoma Independent of Preoperative Lymphoscintigraphy.

INTRODUCTION: Sentinel lymph node biopsy (SLNB) is a standard practice for staging cutaneous melanoma. High false-negative rates have an increased interest in adjunctive techniques for localizing SLNs. Mobile gamma cameras (MGCs) represent potential tools to enhance SLNB performance. METHODS: An institutional review board approval was obtained for this study (ClinicalTrials.gov ID NCT01531608). After obtaining informed consent, 20 eligible melanoma patients underwent 99mTc sulfur colloid injection and standard lymphoscintigraphy with a fixed gamma camera (FGC). A survey using a 20 cm square MGC, performed immediately preoperatively by the study surgeon, was used to establish an operative plan while blinded to the FGC results. Subsequently, SLNB was performed using a gamma probe and a novel 6 cm diameter handheld MGC. RESULTS: A total of 24 SLN basins were detected by FGC. Prior to unblinding, all 24 basins were identified with the preoperative MGC and the operative plan established by preoperative MGC imaging was confirmed accurate by review of the FGC images. All individual sentinel lymph nodes were identified during intraoperative MGC imaging, and in 5/24 (21%) cases, surgeon-reported additional clinically useful information was obtained from the MGC. CONCLUSIONS: Preoperative MGC images provide information consistent with FGC images for planning SLNB and in some cases provide additional information that aided in surgical decision-making.

Tertiary lymphoid structures in desmoplastic melanoma have increased lymphocyte density, lymphocyte proliferation, and immune cross talk with tumor when compared to non-desmoplastic melanomas.

Tertiary lymphoid structures (TLS) are ectopic lymphoid structures that can arise in human cancers and are associated with improved overall survival (OS) and response to immune checkpoint blockade (ICB) in several cancers, including non-desmoplastic metastatic melanoma (NDMM). Desmoplastic melanoma (DM) has one of the highest response rates to ICB, and we previously identified that primary DM (PDM) contains TLS. Despite the association of TLS with survival and ICB response, it is unknown whether TLS or associated markers of immune activity can differ between PDM and NDMM. We hypothesized that PDM would contain higher frequencies of TLS than NDMM, that T and B-cell densities and proliferation would be greater in TLS of PDM than TLS of NDMM, and that proliferation rates of T and B-cells in PDM TLS would be concordant with those of intratumoral lymphocytes. We found that four features of TLS in PDM distinguish them from TLS in NDMM. TLS were peritumoral in NDMM but intratumoral in PDM. CD8+ T-cell and CD20+ B-cell densities and proliferative fractions were higher in PDM TLS than NDMM TLS. Additionally, the proliferative fractions of T- and B-cells were concordant between the TLS and tumor site in PDM and discordant in NDMM. Collectively, these data suggest that TLS and associated immune markers can differ across melanoma subsets and suggest that PDM TLS may be more immunologically active and have enhanced immune cell trafficking between tumor and TLS compared to NDMM.

Multicenter Experience with Neoadjuvant Therapy in Melanoma Highlights Heterogeneity in Contemporary Practice.

OBJECTIVE: To determine the feasibility and impact of neoadjuvant therapy (NT) in patients who present with advanced melanoma amenable to surgical resection. SUMMARY BACKGROUND DATA: Given current effective systemic therapy for melanoma, the use of NT is being explored in patients with advanced melanoma with disease amenable to surgical resection. METHODS: Prospective data from 3 institutions was obtained in patients with clinically evident Stage III/IV melanoma who underwent NT. The primary objective was to compare recurrence-free survival between patients who had pathologic complete response (pCR) to those with persistent disease. RESULTS: NT was offered to 45 patients, with 43 patients initiating various NT regimens including PD-1 antagonist (PD-1) therapy (N = 16), PD-1 plus ipilimumab (N = 10), BRAF/MEK inhibitor therapy (N = 14), a combination of those three (N = 1), and talimogene laherparepvec (TVEC) (N = 2). Thirty-two (74.1%) patients underwent surgery whereas 11 patients did not undergo surgery for these reasons: clinical CR (N = 7), progressive disease not amenable to resection (N = 3), and ongoing therapy (N = 1). 12 of 32 patients (37.5%) had pCR with these therapies: PD-1 (N = 4), PD-1 plus ipilimumab (N = 2), BRAF/MEK (N = 4), combination (N = 1), and TVEC (N = 1). At median follow-up of 16.4 months there was only 1 recurrence in the pCR group and patients with a pCR had significantly improved recurrence-free survival compared to patients without pCR (p = 0.004). CONCLUSIONS: Despite variability in NT regimens across institutions, NT for melanoma is feasible and associated with improved prognosis in patients who achieve a pCR. Maximizing rates of pCR could improve prognosis for patients with advanced melanoma.

Regional control after precision lymph node dissection for clinically evident melanoma metastasis.

INTRODUCTION: Completion lymph node dissection (CLND) for microscopic lymph node metastases has been replaced by observation; however, CLND is standard for clinically detectable nodal metastases (cLN). CLND has high morbidity, which may be reduced by excision of only the cLN (precision lymph node dissection [PLND]). We hypothesized that same-basin recurrence risk would be low after PLND. METHODS: Retrospective review at four tertiary care hospitals identified patients who underwent PLND. The primary outcome was 3-year cumulative incidence of isolated same-basin recurrence. RESULTS: Twenty-one patients underwent PLND for cLN without synchronous distant metastases. Reasons for forgoing CLND included patient preference (n = 11), comorbidities (n = 5), imaging indeterminate for distant metastases (n = 2), partial response to checkpoint blockade (n = 1), or not reported (n = 2). A median of 2 nodes (range: 1-6) were resected at PLND, and 68% contained melanoma. Recurrence was observed in 33% overall. Only 1 patient (5%) developed an isolated same-basin recurrence. Cumulative incidences at 3 years were 5.0%, 17.3%, and 49.7% for isolated same-basin recurrence, any same-basin recurrence, and any recurrence, respectively. Complications from PLND were reported in 1 patient (5%). CONCLUSIONS: These pilot data suggest that PLND may provide adequate regional disease control with less morbidity than CLND. These data justify prospective evaluation of PLND in select patients.

A Step Toward Personalized Surgical Decision Making: Machine Learning Predicts 1 Versus Numerous Melanoma Lymph Node Metastases Using RNA-sequencing.

OBJECTIVE: Develop a predictive model to identify patients with 1 pathologic lymph node (pLN) versus >1 pLN using machine learning applied to gene expression profiles and clinical data as input variables. BACKGROUND: Standard management for clinically detected melanoma lymph node metastases is complete therapeutic LN dissection (TLND). However, >40% of patients with a clinically detected melanoma lymph node will only have 1 pLN on final review. Recent data suggest that targeted excision of just the single enlarged LN may provide excellent regional control, with less morbidity than TLND. The selection of patients for less morbid surgery requires accurate identification of those with only 1 pLN. METHODS: The Cancer Genome Atlas database was used to identify patients who underwent TLND for melanoma. Pathology reports in The Cancer Genome Atlas were reviewed to identify the number of pLNs. Patients were included for machine learning analyses if RNA sequencing data were available from a pLN. After feature selection, the top 20 gene expression and clinical input features were used to train a ridge logistic regression model to predict patients with 1 pLN versus >1 pLN using 10-fold cross-validation on 80% of samples. The model was then tested on the remaining holdout samples. RESULTS: A total of 153 patients met inclusion criteria: 64 with one pLN (42%) and 89 with >1 pLNs (58%). Feature selection identified 1 clinical (extranodal extension) and 19 gene expression variables used to predict patients with 1 pLN versus >1 pLN. The ridge logistic regression model identified patient groups with an accuracy of 90% and an area under the receiver operating characteristic curve of 0.97. CONCLUSIONS: Gene expression profiles together with clinical variables can distinguish melanoma metastasis patients with 1 pLN versus >1 pLN. Future models trained using positron emission tomography/computed tomography imaging, gene expression, and relevant clinical variables may further improve accuracy and may predict patients who can be managed with a targeted LN excision rather than a complete TLND.

A nano-enhanced vaccine for metastatic melanoma immunotherapy.

Aim: Despite the huge advancements in cancer therapies and treatments over the past decade, most patients with metastasized melanoma still die from the disease. This poor prognosis largely results from resistance to conventional chemotherapies and other cytotoxic drugs. We have previously identified 6 antigenic peptides derived from melanomas that have proven efficacious for activating CD4+ T cells in clinical trials for melanoma. Our aim was to improve pharmacodynamics, pharmacokinetic and toxicological parameters by individually encapsulating each of the 6 melanoma helper peptides within their own immunogenic nanoliposomes. Methods: We modified these liposomes as necessary to account for differences in the peptides' chemical properties, resulting in 3 distinct formulations. To further enhance immunogenicity, we also incorporated KDO2, a TLR4 agonist, into the lipid bilayer of all nanoliposome formulations. We then conducted in vivo imaging studies in mice and ex vivo cell studies from 2 patient samples who both strongly expressed one of the identified peptides. Results: We demonstrate that these liposomes, loaded with the different melanoma helper peptides, can be readily mixed together and simultaneously delivered without toxicity in vivo. These liposomes are capable of being diffused to the secondary lymphoid organs very quickly and for at least 6 days. In addition, we show that these immunogenic liposomes enhance immune responses to specific peptides ex vivo. Conclusion: Lipid-based delivery systems, including nanoliposomes and lipid nanoparticles, have now been validated for pharmacological (small molecules, bioactive lipids) and molecular (mRNA, siRNA) therapeutic approaches. However, the utility of these formulations as cancer vaccines, delivering antigenic peptides, has not yet achieved the same degree of commercial success. Here, we describe the novel and successful development of a nanoliposome-based cancer vaccine for melanoma. These vaccines help to circumvent drug resistance by increasing a patient's T cell response, making them more susceptible to checkpoint blockade therapy.

Phase I/II clinical trial of a helper peptide vaccine plus PD-1 blockade in PD-1 antibody-naïve and PD-1 antibody-experienced patients with melanoma (MEL64).

BACKGROUND: A vaccine containing 6 melanoma-associated peptides to stimulate helper T cells (6MHP) is safe, immunogenic, and clinically active. A phase I/II trial was designed to evaluate safety and immunogenicity of 6MHP vaccines plus programmed death 1 (PD-1) blockade. PARTICIPANTS AND METHODS: Participants with advanced melanoma received 6MHP vaccines in an incomplete Freund's adjuvant (6 vaccines over 12 weeks). Pembrolizumab was administered intravenously every 3 weeks. Tumor biopsies at baseline and day 22 were analyzed by multiplex immunohistochemistry. Primary end points were safety (Common Terminology Criteria for Adverse Events V.4.03) and immunogenicity (ex vivo interferon-γ ELISpot assay). Additional end points included changes in the tumor microenvironment (TME) and clinical outcomes. RESULTS: Twenty-two eligible participants were treated: 6 naïve to PD-1 antibody (Ab) and 16 PD-1 Ab-experienced. Median follow-up was 24.4 months. Most common treatment-related adverse events (any grade) included injection site reactions, fatigue, anemia, lymphopenia, fever, elevated aspartate aminotransferase, pruritus, and rash. Treatment-related dose-limiting toxicities were observed in 3 (14%) participants, which did not cross the study safety bound. A high durable T cell response (Rsp) to 6MHP was detected in only one participant, but twofold T cell Rsps to 6MHP were detected in 7/22 (32%; 90% CI (16% to 52%)) by week 13. Objective clinical responses were observed in 23% (1 complete response, 4 partial responses), including 4/6 PD-1 Ab-naïve (67%) and 1/16 PD-1 Ab-experienced (6%). Overall survival (OS) was longer for PD-1 Ab-naïve than Ab-experienced participants (HR 6.3 (90% CI (2.1 to 28.7)). In landmark analyses at 13 weeks, OS was also longer for those with T cell Rsps (HR 6.5 (90% CI (2.1 to 29.2)) and for those with objective clinical responses. TME evaluation revealed increased densities of CD8+ T cells, CD20+ B cells, and Tbet+ cells by day 22. CONCLUSIONS: Treatment with the 6MHP vaccine plus pembrolizumab was safe, increased intratumoral lymphocytes, and induced T cell Rsps associated with prolonged OS. The low T cell Rsp rate in PD-1 Ab-experienced participants corroborates prior murine studies that caution against delaying cancer vaccines until after PD-1 blockade. The promising objective response rate and OS in PD-1 Ab-naïve participants support consideration of a larger study in that setting.

CD103 and periplakin are potential biomarkers for response of metastatic melanoma to pembrolizumab.

This study was designed to screen for preliminary evidence of predictive markers of melanoma response to PD-1 blockade. We hypothesized that the following immune markers would be positive predictors of response: increased densities of CD103 + CD8 + T cells or Th1 lineage T-bet + T cells, high expression of CXCL9-11 and presence of tertiary lymphoid structures. Conversely, we hypothesized that the high expression of barrier molecules would be a negative predictor of response. Patients with advanced melanoma treated with pembrolizumab were identified, and clinical response as well as overall survival data were collected. Tumor samples were evaluated by multiplex immunofluorescence histology. All statistical analyses were performed in R Studio and Microsoft Excel using the Mann-Whitney U test, chi-square test, Spearman's rank correlation and Kaplan-Meier survival curves. Sixty-five advanced melanoma patients were identified, of whom 46 met inclusion criteria and were included in this study. Increased densities ( P = 0.04) and proportions ( P = 0.02) of CD8 + T cells expressing CD103 + were associated with complete response (CR) to pembrolizumab. Improved survival was associated with increased proportions of CD8 + cells expressing CD103 ( P = 0.0085) as well as decreased density of periplakin + cells ( P = 0.012) and periplakin + SOX10 + cells ( P = 0.0012). The density and proportion of CD8 + T cells expressing CD103 + positively correlated with PD-L1 expression, though PD-L1 expression was not significantly correlated with outcomes. This screening study found that increased density and proportion of CD8 + T cells expressing CD103 and decreased density of periplakin were associated with positive outcomes in patients with melanoma metastases treated with pembrolizumab and may warrant further study.