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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.

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.

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.

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.

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.

Infiltration of cancers by T cells is associated with improved patient survival and response to immune therapies; however, optimal approaches to induce T cell infiltration of tumors are not known. This study was designed to assess whether topical treatment of melanoma metastases with the TLR7 agonist imiquimod plus administration of a multipeptide cancer vaccine will improve immune cell infiltration of melanoma metastases.Eligible patients were immunized with a vaccine comprised of 12 melanoma peptides and a tetanus toxoid-derived helper peptide, and imiquimod was applied topically to metastatic tumors daily. Adverse events were recorded, and effects on the tumor microenvironment were evaluated from sequential tumor biopsies. T cell responses were assessed by IFNγ ELIspot assay and T cell tetramer staining. Patient tumors were evaluated for immune cell infiltration, cytokine and chemokine production, and gene expression.Four eligible patients were enrolled, and administration of imiquimod and vaccination were well tolerated. Circulating T cell responses to the vaccine was detected by ex vivo ELIspot assay in 3 of 4 patients. Treatment of metastases with imiquimod induced immune cell infiltration and favorable gene signatures in the patients with circulating T cell responses. This study supports further study of topical imiquimod combined with vaccines or other immune therapies for the treatment of melanoma.

3033Background: Melanoma metastases limit infiltration and function of antitumor T cells in part by immunosuppression with IDO1. INCB024360 (epacadostat) is an IDO1 inhibitor that normalizes serum ...

Methods to induce T cell responses to protein vaccines have not been optimized. The immunostimulant AS15 has been administered with the recombinant MAGE-A3 protein (recMAGE-A3) i.m. but not i.d. or s.c. This study tests hypotheses that the i.d./s.c. route is safe and will increase CD4(+) and CD8(+) T cell responses to MAGE-A3.Twenty-five patients with resected stage IIB-IV MAGE-A3(+) melanoma were randomized to immunization with recMAGE-A3 combined with AS15 immunostimulant (MAGE-A3 immunotherapeutic) either i.m. (group A, n = 13) or i.d./s.c. (group B, n = 12). Adverse events were recorded. Ab responses to MAGE-A3 were measured by ELISA. T cell responses to overlapping MAGE-A3 peptides were assessed in PBMC and a sentinel immunized node (SIN) after 1 in vitro stimulation with recMAGE-A3, by IFN-γ ELISPOT assay and by flow cytometry for multifunctional (TNF-α/IFN-γ) responses.Both routes of immunization were well tolerated without treatment-related grade 3 adverse events. All patients had durable Ab responses. For all 25 patients, the T cell response rate by ELISPOT assay was 30 % in SIN (7/23) but only 4 % (1/25) in PBMC. By flow cytometry, multifunctional CD8(+) T cell responses were identified in one patient in each group; multifunctional CD4(+) T cell response rates for groups A and B, respectively, were 31 and 64 % in SIN and 31 and 50 % in PBMC.The MAGE-A3 immunotherapeutic was well tolerated after i.d./s.c. administration, with trends to higher CD4(+) T cell response rates than with i.m. administration. This study supports further study of AS15 by i.d./s.c. administration.

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

A major obstacle to cancer rejection is the failure of T cells to infiltrate and function within the tumor microenvironment (TME). Direct modulation of the TME to promote T cell homing and activation may enhance tumor control. Two pilot clinical trials were conducted to test the hypotheses that either intratumoral interferon-gamma (IFN-γ, Trial A, NCT00977145), or application of a TLR7 agonist, imiquimod (Trial B, NCT01264731) would induce favorable immune signatures and infiltration of CD8+ T cells into the TME.