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Purpose: Clinical implications of neoadjuvant immunotherapy in patients with locally advanced but resectable head and neck squamous cell carcinoma (HNSCC) remain largely unexplored., Patients and Methods: Patients with resectable HNSCC were randomized to receive a single dose of preoperative durvalumab (D) with or without tremelimumab (T) before resection, followed by postoperative (chemo)radiotherapy based on multidisciplinary discretion and 1-year D treatment. Artificial intelligence (AI)-powered spatial distribution analysis of tumor-infiltrating lymphocytes and high-dimensional profiling of circulating immune cells tracked dynamic intratumoral and systemic immune responses., Results: Of the 48 patients enrolled (D, 24 patients; D+T, 24 patients), 45 underwent surgical resection per protocol (D, 21 patients; D+T, 24 patients). D±T had a favorable safety profile and did not delay surgery. Distant recurrence-free survival (DRFS) was significantly better in patients treated with D+T than in those treated with D monotherapy. AI-powered whole-slide image analysis demonstrated that D+T significantly reshaped the tumor microenvironment toward immune-inflamed phenotypes, in contrast with the D monotherapy or cytotoxic chemotherapy. High-dimensional profiling of circulating immune cells revealed a significant expansion of T-cell subsets characterized by proliferation and activation in response to D+T therapy, which was rare following D monotherapy. Importantly, expansion of specific clusters in CD8+ T cells and non-regulatory CD4+ T cells with activation and exhaustion programs was associated with prolonged DRFS in patients treated with D+T., Conclusions: Preoperative D±T is feasible and may benefit patients with resectable HNSCC. Distinct changes in the tumor microenvironment and circulating immune cells were induced by each treatment regimen, warranting further investigation., (©2024 American Association for Cancer Research.)

Purpose: Impaired MHCI-presentation and insensitivity to immune effector molecules are common features of immune checkpoint blockade (ICB)-resistant tumors and can be, respectively, associated with loss of β2 microglobulin (B2M) or impaired IFNγ signaling. Patients with ICB-resistant tumors can respond to alternative immunotherapies, such as infusion of autologous tumor-infiltrating lymphocytes (TIL). CD4+ T cells can exert cytotoxic functions against tumor cells; however, it is unclear whether CD4+ T-cell responses can be exploited to improve the clinical outcomes of patients affected by ICB-resistant tumors., Experimental Design: Here, we exploited CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing to reproduce immune-resistant tumor phenotypes via gene knockout (KO). To determine the role of cytotoxic CD4+ TILs in ICB-resistant tumors, we investigated CD4+ TIL-mediated cytotoxicity in matched pairs of TILs and autologous melanoma cell lines, used as a model of patient-specific immune-tumor interaction. Around 40% of melanomas constitutively express MHC Class II molecules; hence, melanomas with or without natural constitutive MHC Class II expression (MHCIIconst+ or MHCIIconst-) were used., Results: CD4+ TIL-mediated cytotoxicity was not affected by B2M loss but was dependent on the expression of CIITA. MHCIIconst+ melanomas were killed by tumor-specific CD4+ TILs even in the absence of IFNγ-mediated MHCII upregulation, whereas IFNγ was necessary for CD4+ TIL-mediated cytotoxicity against MHCIIconst- melanomas. Notably, although tumor-specific CD4+ TILs did not kill JAK1KO MHCIIconst- melanomas even after IFNγ stimulation, sensitivity to CD4+ TIL-mediated cytotoxicity was maintained by JAK1KO MHCIIconst+ melanomas., Conclusions: In conclusion, our data indicate that exploiting tumor-specific cytotoxic CD4+ TILs could help overcome resistance to ICB mediated by IFNγ-signaling loss in MHCIIconst+ melanomas. See related commentary by Betof Warner and Luke, p. 3829., (©2023 American Association for Cancer Research.)

In this CCR Translations, we discuss the potential for tumor-infiltrating lymphocyte therapy to overcome immune checkpoint inhibitor resistance through CD4+-mediated and MHC-II-dependent killing. Validating these results from human tumors has potential to improve the clinical application of adoptive cellular transfer in advanced cancers. See related article by Draghi et al., p. 3937., (©2023 American Association for Cancer Research.)

Purpose: Immune checkpoint blockade (ICB) agents and adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) are prominent immunotherapies used for the treatment of advanced melanoma. Both therapies rely on activation of lymphocytes that target shared tumor antigens or neoantigens. Recent analysis of patients with metastatic melanoma who underwent treatment with TIL ACT at the NCI demonstrated decreased responses in patients previously treated with anti-PD-1 agents. We aimed to find a basis for the difference in response rates between anti-PD-1 naïve and experienced patients., Patients and Methods: We examined the tumor mutational burden (TMB) of resected tumors and the repertoire of neoantigens targeted by autologous TIL in a cohort of 112 anti-PD-1 naïve and 69 anti-PD-1 experienced patients., Results: Anti-PD-1 naïve patients were found to possess tumors with higher TMBs (352.0 vs. 213.5, P = 0.005) and received TIL reactive with more neoantigens (2 vs. 1, P = 0.003) compared with anti-PD-1 experienced patients. Among patients treated with TIL ACT, TMB and number of neoantigens identified were higher in ACT responders than ACT nonresponders in both anti-PD-1 naïve and experienced patients. Among patients with comparable TMBs and predicted neoantigen loads, treatment products administered to anti-PD-1 naïve patients were more likely to contain T cells reactive against neoantigens than treatment products for anti-PD-1 experienced patients (2.5 vs. 1, P = 0.02)., Conclusions: These results indicate that decreases in TMB and targeted neoantigens partially account for the difference in response to ACT and that additional factors likely influence responses in these patients. See related commentary by Blass and Ott, p. 2980., (©2022 American Association for Cancer Research.)

Purpose: Immunotherapies targeting immune checkpoint molecules have shown promising treatment for a subset of cancers; however, many "cold" tumors, such as prostate cancer, remain unresponsive. We aimed to identify a potential targetable marker relevant to prostate cancer and develop novel immunotherapy., Experimental Design: Analysis of transcriptomic profiles at single-cell resolution was performed in clinical patients' samples, along with integrated analysis of multiple RNA-sequencing datasets. The antitumor activity of YY001, a novel EP4 antagonist, combined with anti-programmed cell death protein 1 (PD-1) antibody was evaluated both in vitro and in vivo ., Results: We identified EP4 (PTGER4) as expressed in epithelial cells and various immune cells and involved in modulating the prostate cancer immune microenvironment. YY001, a novel EP4 antagonist, inhibited the differentiation, maturation, and immunosuppressive function of myeloid-derived suppressor cells (MDSC) while enhancing the proliferation and anticancer functions of T cells. Furthermore, it reversed the infiltration levels of MDSCs and T cells in the tumor microenvironment by overturning the chemokine profile of tumor cells in vitro and in vivo . The combined immunotherapy demonstrated a robust antitumor immune response as indicated by the robust accumulation and activation of CD8 + cytotoxic T cells, with a significantly decreased MDSC ratio and reduced MDSC immunosuppression function., Conclusions: Our study identified EP4 as a specific target for prostate cancer immunotherapy and demonstrated that YY001 inhibited the growth of prostate tumors by regulating the immune microenvironment and strongly synergized with anti-PD-1 antibodies to convert completely unresponsive prostate cancers into responsive cancers, resulting in marked tumor regression, long-term survival, and lasting immunologic memory., (©2021 American Association for Cancer Research.)

Purpose: While evidence indicates that Fusobacterium nucleatum ( F. nucleatum ) may promote colorectal carcinogenesis through its suppressive effect on T-cell-mediated antitumor immunity, the specific T-cell subsets involved remain uncertain., Experimental Design: We measured F. nucleatum DNA within tumor tissue by quantitative PCR on 933 cases (including 128 F. nucleatum -positive cases) among 4,465 incident colorectal carcinoma cases in two prospective cohorts. Multiplex immunofluorescence combined with digital image analysis and machine learning algorithms for CD3, CD4, CD8, CD45RO (PTPRC isoform), and FOXP3 measured various T-cell subsets. We leveraged data on Bifidobacterium , microsatellite instability (MSI), tumor whole-exome sequencing, and M1/M2-type tumor-associated macrophages [TAM; by CD68, CD86, IRF5, MAF, and MRC1 (CD206) multimarker assay]. Using the 4,465 cancer cases and inverse probability weighting method to control for selection bias due to tissue availability, multivariable-adjusted logistic regression analysis assessed the association between F. nucleatum and T-cell subsets., Results: The amount of F. nucleatum was inversely associated with tumor stromal CD3 + lymphocytes [multivariable OR, 0.47; 95% confidence interval (CI), 0.28-0.79, for F. nucleatum -high vs. -negative category; P trend = 0.0004] and specifically stromal CD3 + CD4 + CD45RO + cells (corresponding multivariable OR, 0.52; 95% CI, 0.32-0.85; P trend = 0.003). These relationships did not substantially differ by MSI status, neoantigen load, or exome-wide tumor mutational burden. F. nucleatum was not significantly associated with tumor intraepithelial T cells or with M1 or M2 TAMs., Conclusions: The amount of tissue F. nucleatum is associated with lower density of stromal memory helper T cells. Our findings provide evidence for the interactive pathogenic roles of microbiota and specific immune cells., (©2021 American Association for Cancer Research.)

Purpose: Triple-negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Antibodies targeting programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1) have entered the therapeutic landscape in TNBC, but only a minority of patients benefit. A way to reliably enhance immunogenicity, T-cell infiltration, and predict responsiveness is critically needed., Patients and Methods: Using mouse models of TNBC, we evaluate immune activation and tumor targeting of intratumoral IL12 plasmid followed by electroporation (tavokinogene telseplasmid; Tavo). We further present a single-arm, prospective clinical trial of Tavo monotherapy in patients with treatment refractory, advanced TNBC (OMS-I140). Finally, we expand these findings using publicly available breast cancer and melanoma datasets., Results: Single-cell RNA sequencing of murine tumors identified a CXCR3 gene signature (CXCR3-GS) following Tavo treatment associated with enhanced antigen presentation, T-cell infiltration and expansion, and PD-1/PD-L1 expression. Assessment of pretreatment and posttreatment tissue from patients confirms enrichment of this CXCR3-GS in tumors from patients that exhibited an enhancement of CD8 + T-cell infiltration following treatment. One patient, previously unresponsive to anti-PD-L1 therapy, but who exhibited an increased CXCR3-GS after Tavo treatment, went on to receive additional anti-PD-1 therapy as their immediate next treatment after OMS-I140, and demonstrated a significant clinical response., Conclusions: These data show a safe, effective intratumoral therapy that can enhance antigen presentation and recruit CD8 T cells, which are required for the antitumor efficacy. We identify a Tavo treatment-related gene signature associated with improved outcomes and conversion of nonresponsive tumors, potentially even beyond TNBC., (©2021 American Association for Cancer Research.)

Purpose: Regorafenib is synergistic with immune checkpoint inhibition in colorectal cancer preclinical models., Patients and Methods: This was a single-arm, multicentric phase II trial. Regorafenib was given 3 weeks on/1 week off, 160 mg every day; avelumab 10 mg/kg i.v. was given every 2 weeks, beginning at cycle 1, day 15 until progression or unacceptable toxicity. The primary endpoint was the confirmed objective response rate under treatment, as per RECIST 1.1. The secondary endpoints included a 1-year nonprogression rate, progression-free survival (PFS), and overall survival (OS), safety and biomarkers studies performed on sequential tumor samples obtained at baseline and at cycle 2 day 1., Results: Forty-eight patients were enrolled in four centers. Forty-three were assessable for efficacy after central radiological review. Best response was stable disease for 23 patients (53.5%) and progressive disease for 17 patients (39.5%). The median PFS and OS were 3.6 months [95% confidence interval (CI), 1.8-5.4] and 10.8 months (95% CI, 5.9-NA), respectively. The most common grade 3 or 4 adverse events were palmar-plantar erythrodysesthesia syndrome ( n = 14, 30%), hypertension ( n = 11, 23%), and diarrhea ( n = 6, 13%). High baseline infiltration by tumor-associated macrophages was significantly associated with adverse PFS (1.8 vs. 3.7 months; P = 0.002) and OS (3.7 months vs. not reached; P = 0.002). Increased tumor infiltration by CD8 + T cells at cycle 2, day 1 as compared with baseline was significantly associated with better outcome., Conclusions: The combination of regorafenib + avelumab mobilizes antitumor immunity in a subset of patients with microsatellite stable colorectal cancer. Computational pathology through quantification of immune cell infiltration may improve patient selection for further studies investigating this approach., (©2021 American Association for Cancer Research.)

Purpose: The use of high-dose per fraction radiotherapy delivered as stereotactic body radiotherapy is a standard of care for prostate cancer. It is hypothesized that high-dose radiotherapy may enhance or suppress tumor-reactive immunity. The objective of this study was to assess both antitumor and immunosuppressive effects induced by high-dose radiotherapy in prostate cancer coclinical models, and ultimately, to test whether a combination of radiotherapy with targeted immunotherapy can enhance antitumor immunity., Experimental Design: We studied the effects of high-dose per fraction radiotherapy with and without anti-Gr-1 using syngeneic murine allograft prostate cancer models. The dynamic change of immune populations, including tumor-infiltrating lymphocytes (TIL), T regulatory cells (Treg), and myeloid-derived suppressive cells (MDSC), was evaluated using flow cytometry and IHC., Results: Coclinical prostate cancer models demonstrated that high-dose per fraction radiotherapy induced a rapid increase of tumor-infiltrating MDSCs and a subsequent rise of CD8 TILs and circulating CD8 T effector memory cells. These radiation-induced CD8 TILs were more functionally potent than those from nonirradiated controls. While systemic depletion of MDSCs by anti-Gr-1 effectively prevented MDSC tumor infiltration, it did not enhance radiotherapy-induced antitumor immunity due to a compensatory expansion of Treg-mediated immune suppression., Conclusions: In allograft prostate cancer models, high-dose radiotherapy induced an early rise of MDSCs, followed by a transient increase of functionally active CD8 TILs. However, systemic depletion of MDSC did not augment the antitumor efficacy of high-dose radiotherapy due to a compensatory Treg response, indicating blocking both MDSCs and Tregs might be necessary to enhance radiotherapy-induced antitumor immunity., (©2020 American Association for Cancer Research.)

Purpose: Tumor-infiltrating immune cells play a key role in tumor progression. The purpose of this study was to analyze whether the immune infiltrate predicts benefit from postoperative radiotherapy in a large randomized breast cancer radiotherapy trial., Experimental Design: In the SweBCG91RT trial, patients with stage I and II breast cancer were randomized to breast-conserving surgery (BCS) and postoperative radiotherapy or to BCS only and followed for a median time of 15.2 years. The primary tumor immune infiltrate was quantified through two independent methods: IHC and gene expression profiling. For IHC analyses, the absolute stromal area occupied by CD8 + T cells and FOXP3 + T cells, respectively, was used to define the immune infiltrate. For gene expression analyses, immune cells found to be prognostic in independent datasets were pooled into two groups consisting of antitumoral and protumoral immune cells, respectively., Results: An antitumoral immune response in the primary tumor was associated with a reduced risk of breast cancer recurrence and predicted less benefit from adjuvant radiotherapy. The interaction between radiotherapy and immune phenotype was significant for any recurrence in both the IHC and gene expression analyses ( P = 0.039 and P = 0.035) and was also significant for ipsilateral breast tumor recurrence in the gene expression analyses ( P = 0.025)., Conclusions: Patients with an antitumoral immune infiltrate in the primary tumor have a reduced risk of any recurrence and may derive less benefit from adjuvant radiotherapy. These results may impact decisions regarding postoperative radiotherapy in early breast cancer., (©2020 American Association for Cancer Research.)

Purpose: Recurrent tumors (RT) of head and neck squamous cell carcinoma (HNSCC) occur in up to 60%, with poor therapeutic response and detrimental prognosis. We hypothesized that HNSCC RTs successfully evade antitumor immune response and aimed to reveal tumor immune microenvironment (TIME) changes of primary tumors (PT) and corresponding RTs., Experimental Design: Tumor-infiltrating leukocytes (TIL) of 300 PTs and 108 RTs from two large independent and clinically well-characterized HNSCC cohorts [discovery cohort (DC), validation cohort (VD)] were compared by IHC. mRNA expression analysis of 730 immune-related genes was performed for 18 PTs and RTs after adjuvant chemoradiotherapy (CRT). The effect of chemotherapy and radiation resistance was assessed with an in vitro spheroid/immunocyte coculture model., Results: TIME analysis revealed overall decrease of TILs with significant loss of CD8 + T cells (DC P = 0.045/VC P < 0.0001) and B lymphocytes (DC P = 0.036/VC P < 0.0001) in RTs compared with PTs in both cohorts. Decrease predominantly occurred in RTs after CRT. Gene expression analysis confirmed loss of TILs ( P = 0.0004) and B lymphocytes ( P < 0.0001) and showed relative increase of neutrophils ( P = 0.018), macrophages ( P < 0.0001), dendritic cells ( P = 0.0002), and mast cells ( P = 0.0057) as well as lower overall expression of immune-related genes ( P = 0.018) in RTs after CRT. Genes involved in B-lymphocyte functions and number of tertiary lymphoid structures showed the strongest decrease. SPP1 and MAPK1 were upregulated in vivo and in vitro , indicating their potential suitability as therapeutic targets in CRT resistance., Conclusions: HNSCC RTs have an immunosuppressive TIME, which is particularly apparent after adjuvant CRT and might substantially contribute to poor therapeutic response and prognosis., (©2020 American Association for Cancer Research.)

Purpose: The clinical implications of tumor-infiltrating T cell subsets and their spatial distribution in biliary tract cancer (BTC) patients treated with gemcitabine plus cisplatin were investigated., Materials and Methods: A total of 52 BTC patients treated with palliative gemcitabine plus cisplatin were included. Multiplexed immunohistochemistry was performed on tumor tissues, and immune infiltrates were separately analyzed for the stroma, tumor margin, and tumor core., Results: The density of CD8+ T cells, FoxP3- CD4+ helper T cells, and FoxP3+ CD4+ regulatory T cells was significantly higher in the tumor margin than in the stroma and tumor core. The density of LAG3- or TIM3-expressing CD8+ T cell and FoxP3- CD4+ helper T cell infiltrates was also higher in the tumor margin. In extrahepatic cholangiocarcinoma, there was a higher density of T cell subsets in the tumor core and regulatory T cells in all regions. A high density of FoxP3- CD4+ helper T cells in the tumor margin showed a trend toward better progression-free survival (PFS) (p=0.092) and significantly better overall survival (OS) (p=0.012). In multivariate analyses, a high density of FoxP3- CD4+ helper T cells in the tumor margin was independently associated with favorable PFS and OS., Conclusion: The tumor margin is the major site for the active infiltration of T cell subsets with higher levels of LAG3 and TIM3 expression in BTC. The density of tumor margin-infiltrating FoxP3- CD4+ helper T cells may be associated with clinical outcomes in BTC patients treated with gemcitabine plus cisplatin.

Purpose: Patients with estrogen receptor- and/or progesterone receptor-positive, early breast cancer benefit from hormonal treatment, yet high global death burdens due to high prevalence and long-term recurrence risk call for biomarkers to guide additional treatment approaches., Experimental Design: From a prospective, observational study of postmenopausal early breast cancer patients treated with tamoxifen or aromatase inhibitors, gene expression analyses of 612 tumors was performed using the NanoString Breast Cancer 360 panel to interrogate 23 breast cancer pathways. Candidate signatures associated with disease subtype and event-free survival (EFS) were obtained by cluster analysis, Cox modeling, and conditional inference trees, and were independently tested in 613 patients from BreastMark. Tumor-infiltrating lymphocytes (TIL) were assessed on tissue sections, and mutational burden was assessed in 36 tumors by whole-exome sequencing., Results: PAM50-derived classification distinguished lower-risk (Luminal A) from higher-risk subtypes (Luminal B, P = 0.04; HER2, P = 0.006; Basal, P = 0.008). In higher-risk patients, shorter EFS was associated with low androgen receptor [HR = 3.61; 95% confidence interval (CI), 1.72-7.56; P = 0.001] or high BRCAness signature expression (HR = 3.58; 95% CI, 1.19-10.7; P = 0.023). BRCAness was independently confirmed as a predictor of shorter EFS (HR = 2.64; 95% CI, 1.31-5.34; P = 0.007). About 13%-15% of patients, enriched for high-grade, higher-risk subtypes ( P ≤ 0.0001), had strong expression of the Tumor Inflammation Signature (TIS) suggestive of an inhibited antitumor immune response. TIS scores were strongly associated with TIL numbers ( P < 1e-30) but not with tumor mutation status., Conclusions: BRCA-related DNA repair deficiency and suppressed tumor immune responses may be clinically relevant predictors of endocrine therapy complementing treatment options in subgroups of hormone-sensitive early breast cancer., (©2020 American Association for Cancer Research.)

Purpose: Invasive lobular carcinoma (ILC) represents the second most common histologic breast cancer subtype after invasive ductal carcinoma (IDC). While primary ILC has been extensively studied, metastatic ILC has been poorly characterized at the genomic and immune level., Experimental Design: We retrospectively assembled the multicentric EuroILC series of matched primary and metastatic samples from 94 patients with estrogen receptor (ER)-positive ILC. Stromal tumor-infiltrating lymphocytes (sTILs) were assessed by experienced pathologists. Targeted sequencing and low pass whole-genome sequencing were conducted to detect mutations and copy-number aberrations (CNAs). We compared the frequencies of the alterations in EuroILC with those from patients with ER-positive metastatic ILC ( n = 135) and IDC ( n = 563) from MSK-IMPACT., Results: Low sTIL levels were observed in ILC metastases, with higher levels in the mixed nonclassic histology. Considering ILC metastases from EuroILC and MSK-IMPACT, we observed that >50% of tumors harbor genomic alterations that have previously been associated with endocrine resistance. A matched primary/metastasis comparison in EuroILC revealed mutations ( AKT1, ARID1A, ESR1, ERBB2 , or NF1 ) and CNAs ( PTEN or NF1 deletion, CYP19A1 amplification) associated with endocrine resistance that were private to the metastasis in 22% (7/32) and 19% (4/21) of patients, respectively. An increase in CDH1, ERBB2, FOXA1 , and TBX3 mutations, in CDH1 deletions and a decrease in TP53 mutations was observed in ILC as compared with IDC metastases., Conclusions: ILC metastases harbor genomic alterations that may potentially explain endocrine resistance in a large proportion of patients, and present genomic differences as compared with IDC metastases., (©2020 American Association for Cancer Research.)

Purpose: While immune checkpoint inhibitors such as anti-PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long-term responses. IL12 promotes antitumor immunity in mouse models; however, systemic recombinant IL12 had significant toxicity and limited efficacy in early clinical trials., Experimental Design: We therefore designed a novel intratumoral IL12 mRNA therapy to promote local IL12 tumor production while mitigating systemic effects., Results: A single intratumoral dose of mouse (m)IL12 mRNA induced IFNγ and CD8 + T-cell-dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to rechallenge. Antitumor activity of mIL12 mRNA did not require NK and NKT cells. mIL12 mRNA antitumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy-resistant model, antitumor immunity induced by mIL12 mRNA was enhanced by anti-PD-L1. mIL12 mRNA also drove regression of uninjected distal lesions, and anti-PD-L1 potentiated this response. Importantly, intratumoral delivery of mRNA encoding membrane-tethered mIL12 also drove rejection of uninjected lesions with very limited circulating IL12p70, supporting the hypothesis that local IL12 could induce a systemic antitumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL12 mRNA (MEDI1191) induced dose-dependent IL12 production, downstream IFNγ expression and TH1 gene expression., Conclusions: These data demonstrate the potential for intratumorally delivered IL12 mRNA to promote TH1 TME transformation and robust antitumor immunity. See related commentary by Cirella et al., p. 6080 ., (©2020 American Association for Cancer Research.)

Purpose: High-grade gliomas (HGGs) are central nervous system tumors with poor prognoses and limited treatment options. Vocimagene amiretrorepvec (Toca 511) is a retroviral replicating vector encoding cytosine deaminase, which converts extended release 5-fluorocytosine (Toca FC) into the anticancer agent, 5-fluorouracil. According to preclinical studies, this therapy kills cancer cells and immunosuppressive myeloid cells in the tumor microenvironment, leading to T-cell-mediated antitumor immune activity. Therefore, we sought to elucidate this immune-related mechanism of action in humans, and to investigate potential molecular and immunologic indicators of clinical benefit from therapy., Patients and Methods: In a phase I clinical trial (NCT01470794), patients with recurrent HGG treated with Toca 511 and Toca FC showed improved survival relative to historical controls, and some had durable complete responses to therapy. As a part of this trial, we performed whole-exome DNA sequencing, RNA-sequencing, and multiplex digital ELISA measurements on tumor and blood samples., Results: Genetic analyses suggest mutations, copy-number variations, and neoantigens are linked to survival. Quantities of tumor immune infiltrates estimated by transcript abundance may potentially predict clinical outcomes. Peak values of cytokines in peripheral blood samples collected during and after therapy could indicate response., Conclusions: These results support an immune-related mechanism of action for Toca 511 and Toca FC, and suggest that molecular and immunologic signatures are related to clinical benefit from treatment., (©2020 American Association for Cancer Research.)

Purpose: Pembrolizumab improved survival in patients with recurrent or metastatic head and neck squamous-cell carcinoma (HNSCC). The aims of this study were to determine if pembrolizumab would be safe, result in pathologic tumor response (pTR), and lower the relapse rate in patients with resectable human papillomavirus (HPV)-unrelated HNSCC., Patients and Methods: Neoadjuvant pembrolizumab (200 mg) was administered and followed 2 to 3 weeks later by surgical tumor ablation. Postoperative (chemo)radiation was planned. Patients with high-risk pathology (positive margins and/or extranodal extension) received adjuvant pembrolizumab. pTR was quantified as the proportion of the resection bed with tumor necrosis, keratinous debris, and giant cells/histiocytes: pTR-0 (<10%), pTR-1 (10%-49%), and pTR-2 (≥50%). Coprimary endpoints were pTR-2 among all patients and 1-year relapse rate in patients with high-risk pathology (historical: 35%). Correlations of baseline PD-L1 and T-cell infiltration with pTR were assessed. Tumor clonal dynamics were evaluated (ClinicalTrials.gov NCT02296684)., Results: Thirty-six patients enrolled. After neoadjuvant pembrolizumab, serious (grades 3-4) adverse events and unexpected surgical delays/complications did not occur. pTR-2 occurred in eight patients (22%), and pTR-1 in eight other patients (22%). One-year relapse rate among 18 patients with high-risk pathology was 16.7% (95% confidence interval, 3.6%-41.4%). pTR ≥10% correlated with baseline tumor PD-L1, immune infiltrate, and IFNγ activity. Matched samples showed upregulation of inhibitory checkpoints in patients with pTR-0 and confirmed clonal loss in some patients., Conclusions: Among patients with locally advanced, HPV-unrelated HNSCC, pembrolizumab was safe, and any pathologic response was observed in 44% of patients with 0% pathologic complete responses. The 1-year relapse rate in patients with high-risk pathology was lower than historical., (©2020 American Association for Cancer Research.)

Purpose: Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by highly chemotherapy-resistant recurrence in the majority of patients. To effectively treat SCLC, we have developed a unique and novel IgG-like T-cell engaging bispecific antibody (ITE) that potently redirects T-cells to specifically lyse SCLC cells expressing Delta-like ligand 3 (DLL3), an antigen that is frequently expressed on the cell surface of SCLC cells, with no to very little detectable expression in normal tissues., Experimental Design: The antitumor activity and mode of action of DLL3/CD3 ITE was evaluated in vitro using SCLC cell lines and primary human effector cells and in vivo in an SCLC xenograft model reconstituted with human CD3 + T-cells., Results: Selective binding of DLL3/CD3 ITE to DLL3-positive tumor cells and T-cells induces formation of an immunological synapse resulting in tumor cell lysis and activation of T-cells. In a human T-cell engrafted xenograft model, the DLL3/CD3 ITE leads to an increase in infiltration of T-cells into the tumor tissue resulting in apoptosis of the tumor cells and tumor regression. Consistent with the mode of action, the DLL3/CD3 ITE treatment led to upregulation of PD-1, PD-L1, and LAG-3., Conclusions: This study highlights the ability of the DLL3/CD3 ITE to induce strictly DLL3-dependent T-cell redirected lysis of tumor cells and recruitment of T-cells into noninflamed tumor tissues leading to tumor regression in a preclinical in vivo model. These data support clinical testing of the DLL3/CD3 ITE in patients with SCLC., (©2020 American Association for Cancer Research.)

Purpose: Immune checkpoint inhibitor therapy is emerging as the promising option for patients with advanced hepatocellular carcinoma. We aimed to investigate the heterogeneity of different tumor nodules of the same patient with multifocal hepatocellular carcinomas in response to immunotherapy and its molecular mechanisms., Experimental Design: We attained 45 surgical tumor samples including 33 small and 12 large nodules from 12 patients with multifocal hepatocellular carcinoma and evaluated genomic and immune heterogeneity among tumors through whole-genome sequencing and RNA sequencing. IHC was performed to validate the expression of immune markers. The responses to anti-programmed cell death protein-1 (PD-1) therapy in patients with multifocal hepatocellular carcinoma were evaluated., Results: The small and large tumors within the same patient presented with similar genomic characteristics, indicating their same genomic origin. We further found the small tumors had higher immune cell infiltration including more CD8 + T cells, M1 macrophages, and monocytes as compared with large tumors. Besides, the expression of interferon signature predictive of response to anti-PD-1 therapy was significantly upregulated in the small tumors. Moreover, the immune pathways were more vigorous along with less active proliferation pathways in the small tumors. In keeping with this, we found that small nodules were more sensitive to anti-PD-1 therapy than large nodules in patients with multifocal hepatocellular carcinoma., Conclusions: The small tumors in patients with multifocal hepatocellular carcinoma had higher immune cell infiltration and upregulation of immune pathways as compared with the large tumors, which can partially explain the different responses of small and large tumors in the same case to anti-PD-1 therapy., (©2020 American Association for Cancer Research.)

Purpose: Despite promising advances in breast cancer immunotherapy, augmenting T-cell infiltration has remained a significant challenge. Although neither individual vaccines nor immune checkpoint blockade (ICB) have had broad success as monotherapies, we hypothesized that targeted vaccination against an oncogenic driver in combination with ICB could direct and enable antitumor immunity in advanced cancers., Experimental Design: Our models of HER2 + breast cancer exhibit molecular signatures that are reflective of advanced human HER2 + breast cancer, with a small numbers of neoepitopes and elevated immunosuppressive markers. Using these, we vaccinated against the oncogenic HER2Δ16 isoform, a nondriver tumor-associated gene (GFP), and specific neoepitopes. We further tested the effect of vaccination or anti-PD-1, alone and in combination., Results: We found that only vaccination targeting HER2Δ16, a driver of oncogenicity and HER2-therapeutic resistance, could elicit significant antitumor responses, while vaccines targeting a nondriver tumor-specific antigen or tumor neoepitopes did not. Vaccine-induced HER2-specific CD8 + T cells were essential for responses, which were more effective early in tumor development. Long-term tumor control of advanced cancers occurred only when HER2Δ16 vaccination was combined with αPD-1. Single-cell RNA sequencing of tumor-infiltrating T cells revealed that while vaccination expanded CD8 T cells, only the combination of vaccine with αPD-1 induced functional gene expression signatures in those CD8 T cells. Furthermore, we show that expanded clones are HER2-reactive, conclusively demonstrating the efficacy of this vaccination strategy in targeting HER2., Conclusions: Combining oncogenic driver targeted vaccines with selective ICB offers a rational paradigm for precision immunotherapy, which we are clinically evaluating in a phase II trial (NCT03632941)., (©2020 American Association for Cancer Research.)

The development of the most successful cancer immunotherapies in solid tumors, immune-checkpoint blockade, has focused on factors regulating T-cell activation. Until recently, the field has maintained a predominately T-cell centric view of immunotherapy, leaving aside the impact of innate immunity and especially myeloid cells. Dendritic cells (DC) are dominant partners of T cells, necessary for initiation of adaptive immune responses. Emerging evidence supports a broader role for DCs in tumors including the maintenance and support of effector functions during T-cell responses. This relationship is evidenced by the association of activated DCs with immune-checkpoint blockade responses and transcriptional analysis of responding tumors demonstrating the presence of type I IFN transcripts and DC relevant chemokines. T-cell-inflamed tumors preferentially respond to immunotherapies compared with non-T-cell-inflamed tumors and this model suggests a potentially modifiable spectrum of tumor microenvironmental immunity. Although host and commensal factors may limit the T-cell-inflamed phenotype, tumor cell intrinsic factors are gaining prominence as therapeutic targets. For example, tumor WNT/β-catenin signaling inhibits production of chemokine gradients and blocking DC recruitment to tumors. Conversely, mechanisms of innate immune nucleic acid sensing, normally operative during pathogen response, may enhance DC accumulation and make tumors more susceptible to cancer immunotherapy. Elucidating mechanisms whereby DCs infiltrate and become activated within tumors may provide new opportunities for therapeutic intervention. Conceptually, this would facilitate conversion of non-T-cell-inflamed to T-cell-inflamed states or overcome secondary resistance mechanisms in T-cell-inflamed tumors, expanding the proportion of patients who benefit from cancer immunotherapy., (©2020 American Association for Cancer Research.)

Purpose: Clinical trials with immune checkpoint inhibition in sarcomas have demonstrated minimal response. Here, we interrogated the tumor microenvironment (TME) of two contrasting soft-tissue sarcomas (STS), rhabdomyosarcomas and undifferentiated pleomorphic sarcomas (UPS), with differing genetic underpinnings and responses to immune checkpoint inhibition to understand the mechanisms that lead to response., Experimental Design: Utilizing fresh and formalin-fixed, paraffin-embedded tissue from patients diagnosed with UPS and rhabdomyosarcomas, we dissected the TME by using IHC, flow cytometry, and comparative transcriptomic studies., Results: Our results demonstrated both STS subtypes to be dominated by tumor-associated macrophages and infiltrated with immune cells that localized near the tumor vasculature. Both subtypes had similar T-cell densities, however, their in situ distribution diverged. UPS specimens demonstrated diffuse intratumoral infiltration of T cells, while rhabdomyosarcomas samples revealed intratumoral T cells that clustered with B cells near perivascular beds, forming tertiary lymphoid structures (TLS). T cells in UPS specimens were comprised of abundant CD8 + T cells exhibiting high PD-1 expression, which might represent the tumor reactive repertoire. In rhabdomyosarcomas, T cells were limited to TLS, but expressed immune checkpoints and immunomodulatory molecules which, if appropriately targeted, could help unleash T cells into the rest of the tumor tissue., Conclusions: Our work in STS revealed an immunosuppressive TME dominated by myeloid cells, which may be overcome with activation of T cells that traffic into the tumor. In rhabdomyosarcomas, targeting T cells found within TLS may be key to achieve antitumor response., (©2020 American Association for Cancer Research.)

Purpose: Adenocarcinoma of the uterine cervix is the second most common type of cervical cancer after squamous cell carcinoma (SCC). Although both subtypes are treated similarly, patients with adenocarcinoma have a worse prognosis. In this study, immunologic features of the tumor microenvironment in these two subsets were pursued with potential therapeutic implications., Experimental Design: The immune microenvironment of primary tumors and nonmetastatic tumor-draining lymph nodes (TDLN) was compared between patients with cervical adenocarcinoma ( n = 16) and SCC ( n = 20) by polychromatic flow cytometry and by transcriptional profiling of the primary tumors ( n = 299) using publicly available data from The Cancer Genome Atlas (TCGA)., Results: Flow cytometric analyses revealed intact T-cell differentiation in TDLNs, but hampered effector T-cell trafficking to the primary tumors in adenocarcinoma, as compared with SCC. TCGA analysis demonstrated higher expression of chemokines involved in effector T-cell homing (CXCL9/10/11) in SCC primary tumors as compared with adenocarcinoma primary tumors, which was highly correlated to a transcriptional signature for type I conventional dendritic cells (cDC1). This was consistent with elevated frequencies of CD141/BDCA3 + cDC1 in primary tumor SCC samples relative to adenocarcinoma and correspondingly elevated levels of CXCL9 and CXCL10 in 24-hour ex vivo cultures. Hampered cDC1 recruitment in adenocarcinoma was in turn related to lower transcript levels of cDC1-recruiting chemokines and an elevated β-catenin activation score and was associated with poor overall survival., Conclusions: Our data have identified an opportunity for the investigation of potentially novel therapeutic interventions in adenocarcinoma of the cervix, that is, β-catenin inhibition and cDC1 mobilization., (©2020 American Association for Cancer Research.)

Purpose: Resistance to anti-PD1-based immune checkpoint blockade (ICB) remains a problem for the treatment of metastatic melanoma. Tumor cells as well as host myeloid cells can express the immune checkpoint ligand CD155 to regulate immune cell function. However, the effect of tumor CD155 on the immune context of human melanoma has not been well described. This observational study characterizes tumor CD155 ligand expression by metastatic melanoma tumors and correlates results with differences in immune cell features and response to ICB., Experimental Design: Pretreatment tumor specimens, from 155 patients with metastatic melanoma treated with ICB and from 50 patients treated with BRAF/MEK-directed targeted therapy, were assessed for CD155 expression by IHC. Intratumor T-cell features were analyzed using multiplex-immunohistofluorescence for CD8, PD1, and SOX10. Correlations were made between CD155 tumor level and bulk tumor RNA sequencing results, as well as clinical RECIST response and progression-free survival., Results: High pretreatment CD155 tumor levels correlated with high parenchymal PD1 + CD8 + /CD8 + T-cell ratios (PD1 tR ) and poor response to anti-PD1 therapy. In PDL1 negative tumors, high CD155 tumor expression was associated with patients who had poor response to combination anti-PD1/CTLA4 therapy., Conclusions: Our findings are the first to suggest that tumor CD155 supports an increase in the fraction of PD1 + CD8 + T cells in anti-PD1 refractory melanoma tumors and, further, that targeting the CD155 pathway might improve response to anti-PD1 therapy for patients with metastatic melanoma., (©2020 American Association for Cancer Research.)

Purpose: Previous studies suggest that androgen deprivation therapy (ADT) promotes antitumor immunity in prostate cancer. Whether a vaccine-based approach can augment this effect remains unknown., Patients and Methods: We conducted a neoadjuvant, randomized study to quantify the immunologic effects of a GM-CSF-secreting allogeneic cellular vaccine in combination with low-dose cyclophosphamide (Cy/GVAX) followed by degarelix versus degarelix alone in patients with high-risk localized prostate adenocarcinoma who were planned for radical prostatectomy., Results: Both Cy/GVAX plus degarelix and degarelix alone led to significant increases in intratumoral CD8 + T-cell infiltration and PD-L1 expression as compared with a cohort of untreated, matched controls. However, the CD8 + T-cell infiltrate was accompanied by a proportional increase in regulatory T cells (Treg), suggesting that adaptive Treg resistance may dampen the immunogenicity of ADT. Although Cy/GVAX followed by degarelix was associated with a modest improvement in time-to-PSA progression and time-to-next treatment, as well as an increase in PD-L1, there was no difference in the CD8 + T-cell infiltrate as compared with degarelix alone. Gene expression profiling demonstrated that CHIT1 , a macrophage marker, was differentially upregulated with Cy/GVAX plus degarelix compared with degarelix alone., Conclusions: Our results highlight that ADT with or without Cy/GVAX induces a complex immune response within the prostate tumor microenvironment. These data have important implications for combining ADT with immunotherapy. In particular, our finding that ADT increases both CD8 + T cells and Tregs supports the development of regimens combining ADT with Treg-depleting agents in the treatment of prostate cancer., (©2020 American Association for Cancer Research.)

Purpose: Immune dysregulation is described in multiple myeloma. While preclinical models suggest a role for altered T-cell immunity in disease progression, the contribution of immune dysfunction to clinical outcomes remains unclear. We aimed to characterize marrow-infiltrating T cells in newly diagnosed patients and explore associations with outcomes of first-line therapy., Experimental Design: We undertook detailed characterization of T cells from bone marrow (BM) samples, focusing on immune checkpoints and features of immune dysfunction, correlating with clinical features and progression-free survival., Results: We found that patients with multiple myeloma had greater abundance of BM regulatory T cells (Tregs) which, in turn, expressed higher levels of the activation marker CD25 compared with healthy donors. Patients with higher frequencies of Tregs had shorter PFS and a distinct Treg immune checkpoint profile (increased PD-1, LAG-3) compared with patients with lower frequencies of Tregs. Analysis of CD4 and CD8 effectors revealed that low CD4effector (CD4 eff ):Treg ratio and increased frequency of PD-1-expressing CD4 eff cells were independent predictors of early relapse over and above conventional risk factors, such as genetic risk and depth of response. Ex vivo functional analysis and RNA sequencing revealed that CD4 and CD8 cells from patients with greater abundance of CD4 eff PD-1 + cells displayed transcriptional and secretory features of dysfunction., Conclusions: BM-infiltrating T-cell subsets, specifically Tregs and PD-1-expressing CD4 effectors, negatively influence clinical outcomes in newly diagnosed patients. Pending confirmation in larger cohorts and further mechanistic work, these immune parameters may inform new risk models, and present potential targets for immunotherapeutic strategies., (©2020 American Association for Cancer Research.)

Purpose: Salivary gland carcinomas (SGC) are rare, aggressive cancers with high rates of recurrence and distant metastasis. These factors, and a lack of active systemic therapies, contribute to poor clinical outcome. Response rates with immune checkpoint blockade have been low, although clinical data remain sparse. To improve the efficacy of therapies, a more comprehensive understanding of relevant molecular alterations and immunologic processes is needed., Experimental Design: To characterize the immune microenvironment and neoantigen landscape of SGCs, we performed RNA sequencing (RNA-seq) in 76 tumors representing the three most lethal histologies: adenoid cystic carcinoma (ACC), myoepithelial carcinoma (MECA), and salivary duct carcinoma (SDC). We analyzed transcriptomic profiles, tumor-infiltrating immune cell populations, and measures of T-cell activation/dysfunction. In 37 cases also undergoing exome sequencing, we analyzed somatic mutations and neoantigens., Results: SDCs exhibited high levels of immune infiltration, with corresponding higher levels of T-cell dysfunction, and higher mutational load. In contrast, ACCs were characterized by an immune-excluded microenvironment, the presence of M2-polarized macrophages and myeloid-derived suppressor cells, and very low mutational load. MECAs were more heterogeneous, with both immune-low and immune-high phenotypes represented. Across all SGCs, levels of immune infiltration were associated with mutation- and fusion-derived neoantigens, and with aggressive clinical behavior., Conclusions: These findings provide new insights into the immune microenvironment and neoantigen landscape of SGCs, showing that mechanisms of immune escape appear to differ by histology. These data nominate potential immunologic vulnerabilities and may help guide the next steps of investigation in precision immunotherapy for these difficult-to-treat cancers., (©2020 American Association for Cancer Research.)

Purpose: Patients with triple-negative breast cancer (TNBC) with homologous recombination deficient tumors achieve significantly higher pathologic complete response (pCR) rates when treated with neoadjuvant platinum-based therapy. Tumor-infiltrating lymphocytes (TIL) are prognostic and predictive of chemotherapy benefit in early stage TNBC. The relationship between TILs, BRCA1/2 mutation status, and homologous recombination deficiency (HRD) status in TNBC remains unclear., Experimental Design: We performed a pooled analysis of five phase II studies that included patients with TNBC treated with neoadjuvant platinum-based chemotherapy to evaluate the association of TILs with HRD status (Myriad Genetics) and tumor BRCA1/2 mutation status. Furthermore, the relationship between pathologic response assessed using the residual cancer burden (RCB) index and HRD status with adjustment for TILs was evaluated., Results: Among 161 patients, stromal TIL (sTIL) density was not significantly associated with HRD status ( P = 0.107) or tumor BRCA1/2 mutation status ( P = 0.391). In multivariate analyses, sTIL density [OR, 1.23; 95% confidence interval (CI), 0.94-1.61; P = 0.139] was not associated with pCR, but was associated with RCB 0/I status (OR 1.62; 95% CI, 1.20-2.28; P = 0.001). HRD was significantly associated with both pCR (OR 12.09; 95% CI, 4.11-44.29; P = 7.82 × 10 -7 ) and RCB 0/I (OR 10.22; 95% CI, 4.11-28.75; P = 1.09 × 10 -7 ) in these models., Conclusions: In patients with TNBC treated with neoadjuvant platinum-based therapy, TIL density was not significantly associated with either tumor BRCA1/2 mutation status or HRD status. In this pooled analysis, HRD and sTIL density were independently associated with treatment response, with HRD status being the strongest predictor., (©2019 American Association for Cancer Research.)

Purpose: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer, which can be effectively controlled by immunotherapy with PD-1/PD-L1 checkpoint inhibitors. However, a significant proportion of patients are characterized by primary therapy resistance. Predictive biomarkers for response to immunotherapy are lacking., Experimental Design: We applied Bayesian inference analyses on 41 patients with MCC testing various clinical and biomolecular characteristics to predict treatment response. Further, we performed a comprehensive analysis of tumor tissue-based immunologic parameters including multiplexed immunofluorescence for T-cell activation and differentiation markers, expression of immune-related genes and T-cell receptor (TCR) repertoire analyses in 18 patients, seven objective responders, and 11 nonresponders., Results: Bayesian inference analyses demonstrated that among currently discussed biomarkers only unimpaired overall performance status and absence of immunosuppression were associated with response to therapy. However, in responders, a predominance of central memory T cells and expression of genes associated with lymphocyte attraction and activation was evident. In addition, TCR repertoire usage of tumor-infiltrating lymphocytes (TILs) demonstrated low T-cell clonality, but high TCR diversity in responding patients. In nonresponders, terminally differentiated effector T cells with a constrained TCR repertoire prevailed. Sequential analyses of tumor tissue obtained during immunotherapy revealed a more pronounced and diverse clonal expansion of TILs in responders indicating an impaired proliferative capacity among TILs of nonresponders upon checkpoint blockade., Conclusions: Our explorative study identified new tumor tissue-based molecular characteristics associated with response to anti-PD-1/PD-L1 therapy in MCC. These observations warrant further investigations in larger patient cohorts to confirm their potential value as predictive markers., (©2020 American Association for Cancer Research.)

Purpose: To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease., Experimental Design: Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data ( n = 230) and single-cell RNA-sequencing (scRNA-seq) data ( n = 52,698)., Results: TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes ( SFTPA1, SFTPB , and NAPSA ) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment., Conclusions: The expression of surfactant metabolism-related genes ( SFTPA1, SFTPB , and NAPSA ) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches., (©2020 American Association for Cancer Research.)

Purpose: The impact of EGFR tyrosine kinase inhibitors (TKI) on the tumor immune microenvironment (TME) in non-small cell lung cancer (NSCLC) is unclear., Experimental Design: We retrospectively identified 138 patients with EGFR -mutated NSCLC who underwent rebiopsy after progression during EGFR-TKI treatment. PD-L1 and CD73 expression in tumor cells and tumor-infiltrating lymphocyte (TIL) density at baseline and after progression were determined by IHC. Tumor mutation burden (TMB) was determined by next-generation sequencing., Results: The proportion of patients with a PD-L1 expression level of ≥50% (high) increased from 14% before to 28% after EGFR-TKI ( P = 0.0010). Whereas CD8 + and FOXP3 + TIL densities were significantly lower after EGFR-TKI treatment than before, CD8 + TIL density was maintained in tumors with a high PD-L1 expression level. Expression of CD73 in tumor cells after EGFR-TKI treatment was higher than that before in patients with a high PD-L1 expression level. TMB tended to be higher after EGFR-TKI treatment than before (3.3→4.1 mutations/Mbp, P = 0.0508). Median progression-free survival for subsequent treatment with antibodies to PD-1 was longer for patients with a high than for those with a low PD-L1 expression after EGFR-TKI (7.1 vs. 1.7 months, P = 0.0033), and two of five patients whose PD-L1 expression level changed from low to high after EGFR-TKI treatment achieved a PFS of >6 months., Conclusions: EGFR-TKI treatment was associated with changes in the TME of EGFR -mutated NSCLC, and such changes may provide clues for optimization of subsequent PD-1 inhibitor treatment., (©2020 American Association for Cancer Research.)

Purpose: In breast cancer, bevacizumab increased pCR rate but not long-term survival and no predictive markers are available to identify patients with long-term benefit from the drug., Experimental Design: We profiled 289 pretherapeutic formalin-fixed, paraffin-embedded (FFPE) biopsies of HER2-negative patients from the GeparQuinto trial of neoadjuvant chemotherapy ± bevacizumab by exome-capture RNA-sequencing (RNA-seq). In a prospectively planned study, we tested molecular signatures for response prediction. IHC validation was performed using tissue microarrays., Results: We found strong agreement of molecular and pathologic parameters as hormone receptors, grading, and lymphocyte infiltration in 221 high-quality samples. Response rates (49.3% pCR overall) were higher in basal-like (68.9%) and HER2-enriched (45.5%) than in luminal B (35.7%), luminal A (17.9%), and normal-like (20.0%) subtypes. T-cell (OR = 1.60; 95% confidence interval, 1.21-2.12; P = 0.001), proliferation (OR = 2.88; 95% CI, 2.00-4.15; P < 0.001), and hypoxia signatures (OR = 1.92; 95% CI, 1.41-2.60; P < 0.001) significantly predicted pCR in univariate analysis. In a prespecified multivariate logistic regression, a small hypoxia signature predicted pCR (OR = 2.40; 95% CI, 1.28-4.51; P = 0.006) with a significant interaction with bevacizumab treatment ( P = 0.020). IHC validation using NDRG1 as marker revealed highly heterogenous expression within tissue leading to profound loss of sensitivity in TMA analysis, still a significant predictive value for pCR was detected ( P = 0.025)., Conclusions: Exome-capture RNA-seq characterizes small FFPE core biopsies by reliably detecting factors as for example ER status, grade, and tumor-infiltrating lymphocytes levels. Beside molecular subtypes and immune signatures, a small hypoxia signature predicted pCR to bevacizumab, which could be validated by IHC. The signature can have important applications for bevacizumab treatment in different cancer types and might also have a role for novel combination therapies of bevacizumab with immune checkpoint inhibition., (©2020 American Association for Cancer Research.)

Purpose: To evaluate the safety and feasibility of preoperative locoregional cytokine therapy (IRX-2 regimen) in early-stage breast cancer, and to evaluate for intratumoral and peripheral immunomodulatory activity., Patients and Methods: Sixteen patients with stage I-III early-stage breast cancer (any histology type) indicated for surgical lumpectomy or mastectomy were enrolled to receive preoperative locoregional immunotherapy with the IRX-2 cytokine biological (2 mL subcutaneous × 10 days to periareolar skin). The regimen also included single-dose cyclophosphamide (300 mg/m 2 ) on day 1 to deplete T-regulatory cells and oral indomethacin to modulate suppressive myeloid subpopulations. The primary objective was to evaluate feasibility (i.e., receipt of therapy without surgical delays or grade 3/4 treatment-related adverse events). The secondary objective was to evaluate changes in stromal tumor-infiltrating lymphocyte score. The exploratory objective was to identify candidate pharmacodynamic changes for future study using a variety of assays, including flow cytometry, RNA and T-cell receptor DNA sequencing, and multispectral immunofluorescence., Results: Preoperative locoregional cytokine administration was feasible in 100% ( n = 16/16) of subjects and associated with increases in stromal tumor-infiltrating lymphocytes ( P < 0.001). Programmed death ligand 1 (CD274) was upregulated at the RNA ( P < 0.01) and protein level [by Ventana PD-L1 (SP142) and immunofluorescence]. Other immunomodulatory effects included upregulation of RNA signatures of T-cell activation and recruitment and cyclophosphamide-related peripheral T-regulatory cell depletion., Conclusions: IRX-2 is safe in early-stage breast cancer. Potentially favorable immunomodulatory changes were observed, supporting further study of IRX-2 in early-stage breast cancer and other malignancies., (©2019 American Association for Cancer Research.)

Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) who actively smoke during treatment have worse survival compared with never-smokers and former-smokers. We hypothesize the poor prognosis in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response. We characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers., Experimental Design: Tumor specimens were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pancytokeratin by multiplex immunofluorescence, whole-exome sequencing, and RNA sequencing. Immune markers were measured in tumor core, tumor margin, and stroma., Results: Our data indicate that current smokers have significantly lower numbers of CD8 + cytotoxic T cells and PD-L1 + cells in the TIM compared with never- and former-smokers. While tumor mutation burden and mutant allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFNα and IFNγ response pathways in current smokers. Gene expression of canonical IFN response chemokines, CXCL9 , CXCL10 , and CXCL11 , are lower in current smokers than in former smokers, suggesting a mechanism for the decreased immune cell migration to tumor sites., Conclusions: These results suggest active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T cells, likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management., (©2019 American Association for Cancer Research.)

Purpose: We recently reported a 17.5% objective RECIST 1.1 response rate in a phase II study of pembrolizumab in patients with advanced sarcoma (SARC028). The majority of responses occurred in undifferentiated pleomorphic sarcoma (UPS) and dedifferentiated liposarcoma (DDLPS). We sought to determine whether we can identify immune features that correlate with clinical outcomes from tumor tissues obtained pre- and on-treatment., Patients and Methods: Pretreatment ( n = 78) and 8-week on-treatment ( n = 68) tumor biopsies were stained for PD-L1 and multiplex immunofluorescence panels. The density of positive cells was quantified to determine associations with anti-PD-1 response., Results: Patients that responded to pembrolizumab were more likely to have higher densities of activated T cells (CD8 + CD3 + PD-1 + ) and increased percentage of tumor-associated macrophages (TAM) expressing PD-L1 pre-treatment compared with non-responders. Pre-treatment tumors from responders also exhibited higher densities of effector memory cytotoxic T cells and regulatory T cells compared with non-responders. In addition, higher density of cytotoxic tumor-infiltrating T cells at baseline correlated with a better progression-free survival (PFS)., Conclusions: We show that quantitative assessments of CD8 + CD3 + PD-1 + T cells, percentage of TAMs expressing PD-L1, and other T-cell densities correlate with sarcoma response to pembrolizumab and improved PFS. Our findings support that multiple cell types present at the start of treatment may enhance tumor regression following anti-PD-1 therapy in specific advanced sarcomas. Efforts to confirm the activity of pembrolizumab in an expansion cohort of patients with UPS/DDLPS are underway., (©2020 American Association for Cancer Research.)

Purpose: The success of checkpoint blockade against glioblastoma (GBM) has been disappointing. Anti-PD-1 strategies may be hampered by severe T-cell exhaustion. We sought to develop a strategy that might license new efficacy for checkpoint blockade in GBM., Experimental Design: We characterized 4-1BB expression in tumor-infiltrating lymphocytes (TIL) from human GBM. We implanted murine tumor models including glioma (CT2A), melanoma (B16), breast (E0771), and lung carcinomas intracranially and subcutaneously, characterized 4-1BB expression, and tested checkpoint blockade strategies in vivo ., Results: Our data reveal that 4-1BB is frequently present on nonexhausted CD8 + TILs in human and murine GBM. In murine gliomas, 4-1BB agonism and PD-1 blockade demonstrate a synergistic survival benefit in a CD8 + T-cell-dependent manner. The combination decreases TIL exhaustion and improves TIL functionality. This strategy proves most successful against intracranial CT2A gliomas. Efficacy in all instances correlates with the levels of 4-1BB expression on CD8 + TILs, rather than with histology or with intracranial versus subcutaneous tumor location. Proffering 4-1BB expression to T cells licenses combination 4-1BB agonism and PD-1 blockade in models where TIL 4-1BB levels had previously been low and the treatment ineffective., Conclusions: Although poor T-cell activation and severe T-cell exhaustion appear to be limiting factors for checkpoint blockade in GBM, 4-1BB agonism obviates these limitations and produces long-term survival when combined with anti-PD-1 therapy. Furthermore, this combination therapy is limited by TIL 4-1BB expression, but not by the intracranial compartment, and therefore may be particularly well-suited to GBM., (©2019 American Association for Cancer Research.)

Purpose: Radiotherapy (RT) is frequently used for local control of solid tumors using equal dose per fraction. Recently, single high-dose radiation has been used for ablation of solid tumors. In this report, we provide a novel immunological basis for radiation dose fractionation consisting of a single high-dose radiotherapy, followed by postablation modulation (PAM) with four daily low-dose fractions (22 Gy + 0.5 Gy × 4) to reprogram the tumor microenvironment by diminishing immune suppression, enabling infiltration of effector cells and increasing efficacy of tumor control., Experimental Design: Palpable 3LL and 4T1 tumors in C57Bl/6 and Balb/c mice were irradiated with the Small-Animal Radiation Research Platform irradiator, and tumor growth and survival were monitored. Immunomodulation of tumor and immune cells in vitro and in vivo characterization of tumor-infiltrating immune effector cells were performed by FACS. For systemic application of PAM-RT, whole-lung irradiation was administered in 4T1-bearing Balb/c mice., Results: We report significant tumor growth delays and increased survival in 3LL tumor-bearing mice with PAM. Primary tumor PAM-RT increased infiltration of immune effector cells and decreased Treg in irradiated tumors and secondary lymphoid organs. In a model of murine metastatic breast cancer (4T1), we demonstrated that systemic PAM-RT to the whole lung, 12 days after primary tumor ablative radiotherapy, increased survival with suppression of pulmonary metastases., Conclusions: We provide a novel immunologic basis for radiation dose fractionation consisting of a single high dose of radiotherapy followed by daily low-dose PAM-RT fractionation to improve the immunogenic potential of ablative radiotherapy., (©2019 American Association for Cancer Research.)

Purpose: Despite the well-known prognostic value of the tumor-immune microenvironment (TIME) in colorectal cancers, objective and readily applicable methods for quantifying tumor-infiltrating lymphocytes (TIL) and the tumor-stroma ratio (TSR) are not yet available., Experimental Design: We established an open-source software-based analytic pipeline for quantifying TILs and the TSR from whole-slide images obtained after CD3 and CD8 IHC staining. Using a random forest classifier, the method separately quantified intraepithelial TILs (iTIL) and stromal TILs (sTIL). We applied this method to discovery and validation cohorts of 578 and 283 stage III or high-risk stage II colorectal cancers patients, respectively, who were subjected to curative surgical resection and oxlaliplatin-based adjuvant chemotherapy., Results: Automatic quantification of iTILs and sTILs showed a moderate concordance with that obtained after visual inspection by a pathologist. The K-means-based consensus clustering of 197 TIME parameters that showed robustness against interobserver variations caused colorectal cancers to be grouped into five distinctive subgroups, reminiscent of those for consensus molecular subtypes (CMS1-4 and mixed/intermediate group). In accordance with the original CMS report, the CMS4-like subgroup (cluster 4) was significantly associated with a worse 5-year relapse-free survival and proved to be an independent prognostic factor. The clinicopathologic and prognostic features of the TIME subgroups have been validated in an independent validation cohort., Conclusions: Machine-learning-based image analysis can be useful for extracting quantitative information about the TIME, using whole-slide histopathologic images. This information can classify colorectal cancers into clinicopathologically relevant subgroups without performing a molecular analysis of the tumors., (©2019 American Association for Cancer Research.)

Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with benefit to trastuzumab and chemotherapy in patients with early-stage HER2 + breast cancer. The predictive value of TILs, TIL subsets, and other immune cells in patients receiving chemotherapy-sparing lapatinib plus trastuzumab treatment is unclear. Experimental Design: Hematoxylin and eosin-stained slides ( n = 59) were used to score stromal (s-)TILs from pretreatment biopsies of patients enrolled in the neoadjuvant TBCRC006 trial of 12-week lapatinib plus trastuzumab therapy (plus endocrine therapy for ER + tumors). A 60% threshold was used to define lymphocyte-predominant breast cancer (LPBC). Multiplexed immunofluorescence (m-IF) staining (CD4, CD8, CD20, CD68, and FoxP3) and multispectral imaging were performed to characterize immune infiltrates in single formalin-fixed paraffin-embedded slides ( n = 33)., Results: The pathologic complete response (pCR) rate was numerically higher in patients with LPBC compared with patients with non-LPBC (50% vs. 19%, P = 0.057). Unsupervised hierarchical clustering of the five immune markers identified two patient clusters with different responses to lapatinib plus trastuzumab treatment (pCR = 7% vs. 50%, for cluster 1 vs. 2 respectively; P = 0.01). In multivariable analysis, cluster 2, characterized by high CD4 + , CD8 + , CD20 + s-TILs, and high CD20 + intratumoral TILs, was independently associated with a higher pCR rate ( P = 0.03). Analysis of single immune subpopulations revealed a significant association of pCR with higher baseline infiltration by s-CD4, intratumoral (i-) CD4, and i-CD20 + TILs., Conclusions: LPBC was marginally associated with higher pCR rate than non-LPBC in patients with lapatinib plus trastuzumab treated HER2 + breast cancer. Quantitative assessment of the immune infiltrate by m-IF is feasible and may help correlate individual immune cell subpopulations and immune cell profiles with treatment response., (©2019 American Association for Cancer Research.)

The RAS family of proteins is at the apex of several pathways implicated in a multitude of epithelial cancers but has remained stubbornly resistant to the wave of targeted small molecules and antibodies that have revolutionized clinical oncology. KRAS, the most commonly mutated of the isoforms, represents an attractive target for treatment, given its ubiquity, central role as a driver mutation, and association with poor prognosis. This review is a comprehensive summary of the existing approaches to targeting KRAS spanning small-molecule inhibitors, cancer vaccines, and with a focus on trials in adoptive cell therapy. Here we explain how the limitations of existing drugs and nonspecific immune-based therapies are circumvented with techniques in modern immunotherapy. The successes outlined represent the most promising path to finally targeting the prototypical "undruggable" RAS oncogene family., (©2019 American Association for Cancer Research.)

Purpose: Pancreatic cancer remains one of the most lethal cancers, and late detection renders most tumors refractory to conventional therapies. Development of cancer prophylaxis may be the most realistic option for improving mortality associated with this disease. Here, we develop a novel individualized prophylactic and therapeutic vaccination regimen using induced pluripotent stem cells (iPSC), gene editing, and tumor-targeted replicating oncolytic viruses., Experimental Design: We created a Virus-Infected, Reprogrammed Somatic cell-derived Tumor cell (VIReST) regime. iPSCs from healthy cells were induced to pancreatic tumor cells using in situ gene editing via stable provision of KRas G12D and p53 R172H tumor driver mutations. These cells were preinfected with oncolytic Adenovirus (AdV) as prime or Vaccinia virus (VV) as boost, to improve vaccine immunogenicity, prior to delivery of vaccines in a sequential regime to young KPC transgenic mice, genetically programmed to develop pancreatic cancer, to prevent and delay disease development., Results: Tumor cells preinfected with oncolytic AdV as prime or VV as boost were the best regime to induce tumor-specific immunity. iPSC-derived tumor cells were highly related in antigen repertoire to pancreatic cancer cells of KPC transgenic mice, suggesting that an individual's stem cells can provide an antigenically matched whole tumor cell vaccine. The VIReST vaccination primed tumor-specific T-cell responses, resulting in delayed disease emergence and progression and significantly prolonged survival of KPC transgenic mice. Importantly, this regime was well-tolerated and nontoxic., Conclusions: These results provide both proof of concept and a robust technology platform for the development of personalized prophylactic cancer vaccines to prevent pancreatic malignancies in at-risk individuals., (©2019 American Association for Cancer Research.)

Purpose: In breast cancer, response rates to immune therapies are generally low and differ significantly across molecular subtypes, urging a better understanding of immunogenicity and immune evasion., Experimental Design: We interrogated large gene-expression data sets including 867 node-negative, treatment-naïve breast cancer patients (microarray data) and 347 breast cancer patients (whole-genome sequencing and transcriptome data) according to parameters of T cells as well as immune microenvironment in relation to patient survival., Results: We developed a 109-immune gene signature that captures abundance of CD8 tumor-infiltrating lymphocytes (TIL) and is prognostic in basal-like, her2, and luminal B breast cancer, but not in luminal A or normal-like breast cancer. Basal-like and her2 are characterized by highest CD8 TIL abundance, highest T-cell clonality, highest frequencies of memory T cells, and highest antigenicity, yet only the former shows highest expression level of immune and metabolic checkpoints and highest frequency of myeloid suppressor cells. Also, luminal B shows a high antigenicity and T-cell clonality, yet a low abundance of CD8 TILs. In contrast, luminal A and normal-like both show a low antigenicity, and notably, a low and high abundance of CD8 TILs, respectively, which associates with T-cell influx parameters, such as expression of adhesion molecules., Conclusions: Collectively, our data argue that not only CD8 T-cell presence itself, but rather T-cell clonality, T-cell subset distribution, coinhibition, and antigen presentation reflect occurrence of a CD8 T-cell response in breast cancer subtypes, which have been aborted by distinct T-cell-suppressive mechanisms, providing a rationale for subtype-specific combination immune therapies., (©2019 American Association for Cancer Research.)

Purpose: Regulatory T cells (Tregs) expressing CC chemokine receptor 4 (CCR4) can suppress antitumor immune responses and are associated with poor prognoses in several cancers. We assessed the safety and efficacy of combined mogamulizumab (anti-CCR4 antibody) and nivolumab [anti-programmed death-1 (PD-1) antibody] in immunotherapy-naïve patients with advanced/metastatic solid tumors., Patients and Methods: This study (NCT02476123) comprised dose-escalation (3+3 design) and expansion parts. Patients received nivolumab (3.0 mg/kg) every 2 weeks, with mogamulizumab (0.3 or 1.0 mg/kg in dose escalation, 1.0 mg/kg in expansion) once weekly for 4 weeks, then every 2 weeks, until progression or unacceptable toxicity. Primary objective was safety; secondary objectives were antitumor effects, pharmacokinetics, and immunogenicity. Exploratory biomarker analyses were conducted., Results: Ninety-six patients were enrolled (July 2015-November 2016): six patients in the dose-escalation part and 90 in the expansion part. No dose-limiting toxicities were observed in the dose-escalation part. Grade 3/4 treatment-related adverse events (TRAEs) occurred in 29% of patients in the expansion part (no grade 5 TRAEs). The most frequent TRAEs were rash (39%), rash maculopapular (20%), diarrhea (13%), stomatitis (12%), and pruritus (11%). There were four (27%) confirmed tumor responses among 15 patients with hepatocellular carcinoma, and one confirmed and two unconfirmed responses among 15 patients with pancreatic adenocarcinoma. During treatment, populations of effector Tregs (CD4 + CD45RA - FoxP3 high ) decreased and CD8 + T cells in tumor-infiltrating lymphocytes increased., Conclusions: Combining an anti-PD-1 antibody, nivolumab, with a Treg-depleting anti-CCR4 antibody, mogamulizumab, provides an acceptable safety profile, antitumor activity, and a potentially effective option in cancer immunotherapy., (©2019 American Association for Cancer Research.)

Purpose: High levels of tumor-infiltrating lymphocytes (TIL) before neoadjuvant chemotherapy (NAC) are associated with higher pathologic complete response (pCR) rates and better survival in triple-negative breast cancer (TNBC) and HER2 -positive breast cancer. We investigated the value of TIL levels by evaluating lymphocyte infiltration before and after NAC., Experimental Design: We assessed stromal TIL levels in 716 pre- and posttreatment matched paired specimens, according to the guidelines of the International TIL Working Group., Results: Pre-NAC TIL levels were higher in tumors for which pCR was achieved than in cases with residual disease (33.9% vs. 20.3%, P = 0.001). This was observed in luminal tumors and TNBCs, but not in HER2 -positive breast cancers ( P = 0.001). The association between pre-NAC TIL levels and pCR was nonlinear in TNBCs ( Interaction = 0.001). The association between pre-NAC TIL levels and pCR was nonlinear in TNBCs ( P = 0.005). Mean TIL levels decreased after chemotherapy completion (pre-NAC TILs: 24.1% vs. post-NAC TILs: 13.0%, P < 0.001). This decrease was strongly associated with high pCR rates, and the variation of TIL levels was strongly inversely correlated with pre-NAC TIL levels ( r = -0.80, P < 0.001). Pre-NAC TILs and disease-free survival (DFS) were associated in a nonlinear manner ( P < 0.001). High post-NAC TIL levels were associated with aggressive tumor characteristics and with impaired DFS in HER2 -positive breast cancers (HR, 1.04; confidence interval, 1.02-1.06; P = 0.001), but not in luminal tumors or TNBCs ( P Interaction = 0.04)., Conclusions: The associations of pre- and post-NAC TIL levels with response to treatment and DFS differ between breast cancer subtypes. The characterization of immune subpopulations may improve our understanding of the complex interactions between pre- or post-NAC setting, breast cancer subtype, response to treatment, and prognosis., (©2019 American Association for Cancer Research.)

Purpose: OX40 agonist-based combinations are emerging as a novel avenue to improve the effectiveness of cancer immunotherapy. To better guide its clinical development, we characterized the role of the OX40 pathway in tumor-reactive immune cells. We also evaluated combining OX40 agonists with targeted therapy to combat resistance to cancer immunotherapy. Experimental Design: We utilized patient-derived tumor-infiltrating lymphocytes (TILs) and multiple preclinical models to determine the direct effect of anti-OX40 agonistic antibodies on tumor-reactive CD8 + T cells. We also evaluated the antitumor activity of an anti-OX40 antibody plus PI3Kβ inhibition in a transgenic murine melanoma model ( Braf mutant, PTEN null), which spontaneously develops immunotherapy-resistant melanomas., Results: We observed elevated expression of OX40 in tumor-reactive CD8 + TILs upon encountering tumors; activation of OX40 signaling enhanced their cytotoxic function. OX40 agonist antibody improved the antitumor activity of CD8 + T cells and the generation of tumor-specific T-cell memory in vivo . Furthermore, combining anti-OX40 with GSK2636771, a PI3Kβ-selective inhibitor, delayed tumor growth and extended the survival of mice with PTEN-null melanomas. This combination treatment did not increase the number of TILs, but it instead significantly enhanced proliferation of CD8 + TILs and elevated the serum levels of CCL4, CXCL10, and IFNγ, which are mainly produced by memory and/or effector T cells., Conclusions: These results highlight a critical role of OX40 activation in potentiating the effector function of tumor-reactive CD8 + T cells and suggest further evaluation of OX40 agonist-based combinations in patients with immune-resistant tumors., (©2019 American Association for Cancer Research.)

Purpose: Multiple negative regulators restrict the ability of T cells to attack tumors. This work demonstrates the role of PI3K-interacting protein 1 (Pik3ip1) in restraining T-cell responses and antitumor immunity., Experimental Design: An anti-Pik3ip1 mAb was generated to identify the Pik3ip1 expression pattern of hematopoietic cells. Pik3ip1 -/- mice and a Pik3ip1 fusion protein were generated to investigate the effect of Pik3ip1 on T-cell-mediated antitumor immunity in MC38 and B16-F10 tumor models. Immunoblotting and confocal microscopy were used to identify inhibitory effects of Pik3ip1 on T-cell receptor (TCR) signaling. Pik3ip1 expression was quantified, and its impact on T-cell function in human tumors was measured., Results: We demonstrated that Pik3ip1 was predominantly expressed on T cells and served as an essential rheostat for T-cell-mediated immunity. A Pik3ip1 genetic deficiency led to enhanced T-cell responsiveness upon immunization with a neoantigen. Pik3ip1 -/- mice exhibited a marked increase in antitumor immunity and were resistant to tumor growth. Furthermore, Pik3ip1 extracellular domain fusion protein enhanced MC38 tumor growth was observed. Mechanistically, we found that Pik3ip1 inhibited TCR signaling by mediating the degradation of SLP76 through Pik3ip1 oligomerization via its extracellular region. Consistent with the results from the mouse models, PIK3IP1 expression correlated with T-cell dysfunction in human tumors., Conclusions: Our data reveal a critical role for Pik3ip1 as a novel inhibitory immune regulator of T-cell responses and provide a potential molecular target for cancer immunotherapy., (©2019 American Association for Cancer Research.)

Purpose: Protein expression in formalin-fixed, paraffin-embedded tissue is routinely measured by IHC or quantitative fluorescence (QIF) on a handful of markers on a single section. Digital spatial profiling (DSP) allows spatially informed simultaneous assessment of multiple biomarkers. Here we demonstrate the DSP technology using a 44-plex antibody cocktail to find protein expression that could potentially be used to predict response to immune therapy in melanoma. Experimental Design: The NanoString GeoMx DSP technology is compared with automated QIF (AQUA) for immune marker compartment-specific measurement and prognostic value in non-small cell lung cancer (NSCLC). Then we use this tool to search for novel predictive markers in a cohort of 60 patients with immunotherapy-treated melanoma on a tissue microarray using a 44-plex immune marker panel measured in three compartments (macrophage, leukocyte, and melanocyte) generating 132 quantitative variables., Results: The spatially informed variable assessment by DSP validates by both regression and variable prognostication compared with QIF for stromal CD3, CD4, CD8, CD20, and PD-L1 in NSCLC. From the 132 variables, 11 and 15 immune markers were associated with prolonged progression-free survival (PFS) and overall survival (OS). Notably, we find PD-L1 expression in CD68-positive cells (macrophages) and not in tumor cells was a predictive marker for PFS, OS, and response., Conclusions: DSP technology shows high concordance with QIF and validates based on both regression and outcome assessment. Using the high-plex capacity, we found a series of expression patterns associated with outcome, including that the expression of PD-L1 in macrophages is associated with response., (©2019 American Association for Cancer Research.)

Purpose: Clinically aggressive meningiomas (MGMs) are rare but treatment-resistant tumors in need for more effective therapies. Because tumor-infiltrating T lymphocytes (TILs) are essential for successful immunotherapy, we assessed TIL numbers and their activation status in primary (p-) and recurrent (r-) meningiomas and their impact on survival., Experimental Design: Presence of TILs was analyzed in 202 clinically well-annotated cases ( n = 123 pMGMs and n = 79 rMGMs) focusing on higher-grade meningiomas [ n = 97 World Health Organization (WHO) °II, n = 62 WHO°III]. TILs were quantified by a semiautomated analysis on whole-tissue sections stained by multicolor immunofluorescence for CD3, CD8, FOXP3, and programmed cell death protein 1 (PD-1)., Results: Median T-cell infiltration accounted for 0.59% TILs per total cell count. Although there were no significant WHO°-dependent changes regarding helper (CD3 + CD8 - FOXP3 - ) and cytotoxic (CD3 + CD8 + FOXP3 - ) TILs in pMGMs, higher number of cytotoxic TILs were associated with an improved progression-free survival (PFS) independent of prognostic confounders. rMGMs were characterized by lower numbers of TILs in general, helper, and cytotoxic TILs. The additional analysis of their activation status revealed that a proportion of PD-1 + CD8 + TILs within the TIL population was significantly decreased with higher WHO grade and in rMGMs. Furthermore, lower proportions of PD-1 + CD8 + TILs were associated with inferior PFS in multivariate analyses, arguing for PD-1 as activation rather than exhaustion marker., Conclusions: We identified higher numbers of CD3 + CD8 + FOXP3 - TILs and proportions of PD-1-expressing CD3 + CD8 + FOXP3 - TILs as novel biomarkers for better survival. These findings might facilitate the selection of patients who may benefit from immunotherapy and argue in favor of an intervention in primary rather than recurrent tumors., (©2019 American Association for Cancer Research.)