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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.

Purpose:IL12 promotes adaptive type I immunity and has demonstrated antitumor efficacy, but systemic administration leads to severe adverse events (AE), including death. This pilot trial investigated safety, efficacy, and immunologic activity of intratumoral delivery of IL12 plasmid DNA (tavo) via in vivo electroporation (i.t.-tavo-EP) in patients with Merkel cell carcinoma (MCC), an aggressive virus-associated skin cancer.Patients and Methods:Fifteen patients with MCC with superficial injectable tumor(s) received i.t.-tavo-EP on days 1, 5, and 8 of each cycle. Patients with locoregional MCC (cohort A, N = 3) received one cycle before definitive surgery in week 4. Patients with metastatic MCC (cohort B, N = 12) received up to four cycles total, administered at least 6 weeks apart. Serial tumor and blood samples were collected.Results:All patients successfully completed at least one cycle with transient, mild (grades 1 and 2) AEs and without significant systemic toxicity. Sustained (day 22) intratumoral expression of IL12 protein was observed along with local inflammation and increased tumor-specific CD8+ T-cell infiltration, which led to systemic immunologic and clinical responses. The overall response rate was 25% (3/12) in cohort B, with 2 patients experiencing durable clinical benefit (16 and 55+ months, respectively). Two cohort A patients (1 with pathologic complete remission) were recurrence-free at 44+ and 75+ months, respectively.Conclusions:I.t.-tavo-EP was safe and feasible without systemic toxicity. Sustained local expression of IL12 protein and local inflammation led to systemic immune responses and clinically meaningful benefit in some patients. Gene electrotransfer, specifically i.t.-tavo-EP, warrants further investigation for immunotherapy of cancer.

Patient demographic and tumor pathology

Image cytometry infiltration results by layer

Combination immunotherapy tumor slice culture experiment replicates

The impact of liver metastases on immune checkpoint-inhibitor effectiveness in patients with solid-tumor malignancies has been the focus of several recent clinical and translational studies. We review the literature describing the immune functions of the liver and particularly the mechanistic observations in these studies. The initial clinical observation was that pembrolizumab appeared to be much less effective in melanoma and non–small cell lung cancer (NSCLC) patients with liver metastasis. Subsequently other clinical studies have extended and reported similar findings with programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) inhibitors in many cancers. Two recent translational studies in animal models have dissected the mechanism of this systemic immune suppression. In both studies CD11b+ suppressive macrophages generated by liver metastasis in a two-site MC38 model appear to delete CD8+ T cells in a FasL-dependent manner. In addition, regulatory T-cell (Treg) activation was observed and contributed to the distal immunosuppression. Finally, we discuss some of the interventions reported to address liver immune suppression, such as radiation therapy, combination checkpoint blockade, and Treg depletion.

BackgroundMerkel cell carcinoma (MCC) often responds to PD-1 pathway blockade, regardless of tumor-viral status (~80% of cases driven by the Merkel cell polyomavirus (MCPyV)). Prior studies have characterized tumor-specific T cell responses to MCPyV, which have typically been CD8, but little is known about the T cell response to UV-induced neoantigens.MethodsA patient in her mid-50s with virus-negative (VN) MCC developed large liver metastases after a brief initial response to chemotherapy. She received anti-PD-L1 (avelumab) and had a partial response within 4 weeks. Whole exome sequencing (WES) was performed to determine potential neoantigen peptides. Characterization of peripheral blood neoantigen T cell responses was evaluated via interferon-gamma (IFNγ) ELISpot, flow cytometry and single-cell RNA sequencing. Tumor-resident T cells were characterized by multiplexed immunohistochemistry.ResultsWES identified 1027 tumor-specific somatic mutations, similar to the published average of 1121 for VN-MCCs. Peptide prediction with a binding cut-off of ≤100 nM resulted in 77 peptides that were synthesized for T cell assays. Although peptides were predicted based on class I HLAs, we identified circulating CD4 T cells targeting 5 of 77 neoantigens. In contrast, no neoantigen-specific CD8 T cell responses were detected. Neoantigen-specific CD4 T cells were undetectable in blood before anti-PD-L1 therapy but became readily detectible shortly after starting therapy. T cells produced robust IFNγ when stimulated by neoantigen (mutant) peptides but not by the normal (wild-type) peptides. Single cell RNAseq showed neoantigen-reactive T cells expressed the Th1-associated transcription factor (T-bet) and associated cytokines. These CD4 T cells did not significantly exhibit cytotoxicity or non-Th1 markers. Within the pretreatment tumor, resident CD4 T cells were also Th1-skewed and expressed T-bet.ConclusionsWe identified and characterized tumor-specific Th1-skewed CD4 T cells targeting multiple neoantigens in a patient who experienced a profound and durable partial response to anti-PD-L1 therapy. To our knowledge, this is the first report of neoantigen-specific T cell responses in MCC. Although CD4 and CD8 T cells recognizing viral tumor antigens are often detectible in virus-positive MCC, only CD4 T cells recognizing neoantigens were detected in this patient. These findings suggest that CD4 T cells can play an important role in the response to anti-PD-(L)1 therapy.

Background CD39 expression is upregulated in the tumor microenvironment (TME), which is characterized by high levels of extracellular ATP (eATP) and low pH. CD39 catalyzes the rate-limiting degradation step of this immunostimulatory ATP, leading to a rise in immunosuppressive adenosine (ADO). CD39 displays broad expression on endothelial cells and macrophages, which represents a significant peripheral sink for CD39 targeting antibodies. Our strategy aims to circumvent this problem through delivery of pH-selective anti-CD39 blocking antibodies that will achieve a high target occupancy in the tumor, maintaining eATP and inhibiting ADO generation to enhance anti-tumor immunity. Methods Anti-CD39 antibodies were generated through a yeast-based screening platform and characterized for efficacy of blocking CD39 activity. Cell lines (HEK293 stably overexpressing CD39, parental HEK293, CD39+ SK-MEL28, and CD39high ARH77) or soluble CD39 protein were incubated with candidate antibodies. CD39 enzymatic activity was determined using CellTiter Glo 2.0 assay (Promega) and EnzCheck Phosphate Assay (Thermo Fisher). Results Monovalent affinity of 83 candidate antibodies was measured; KDs ranged from 1 × 10−9 - 5 × 10−7M under both physiological (pH 7.4) and acidic (pH 6.0) conditions. All 83 antibodies bound CD39-overexpressing HEK293 cells at pH 6.0; 76 bound CD39-overexpressing HEK293 cells at pH 7.4. We identified 30 antibodies (100 nM) that block CD39 activity on HEK293-CD39 cells (threshold: > 5% inhibition) under either pH 7.4 or pH 6.0 conditions. 26 antibodies inhibit CD39 activity at pH 7.4, whereas 22 antibodies block CD39 at pH 6.0. Among these 30 antibodies, ten (at 200 nM) inhibit CD39 activity on SK-MEL28 cells (moderate CD39 expression) at both pH 7.4 and pH 6.0; 5 of these preferentially inhibit CD39 at pH 6.0 (threshold: > 7.5% inhibition). The 30 antibodies selected above were tested on ARH77 cells (high CD39 expression). Among them, 16 antibodies inhibited CD39 enzymatic activity beyond 10% at pH 6.0 (maximal inhibition: 43%) and > 6.5% at pH 7.4 (maximal inhibition: 35%). All 16 suppressed soluble CD39 activity under both pH conditions. A set of 8 antibodies demonstrating higher efficacy of CD39 inhibition on both cell lines at pH 6.0 versus pH 7.4 were prioritized for lead optimization. Summary and conclusions A panel of 83 antibodies were identified and characterized for inhibition of CD39 enzymatic activity at neutral and acidic pH. The 8 most promising antibodies in terms of pH-selective CD39 binding and inhibition are currently undergoing lead optimization. This pH-dependent TME targeting strategy may alleviate undesirable properties of on-target/off-tumor binding such as target-mediated drug disposition (that results in suboptimal pharmacokinetic properties) and effectively bolster the specific blocking of CD39 in the local tumor environs. Citation Format: Aiping Bai, Elise Renahan, Kanam Malhotra, Thomas Thisted, F. Donelson Smith, Robert H Pierce, Edward H van der Horst. Identification of conditionally active antibodies that selectively block CD39 activity in the acidic tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4645.

Purpose: To characterize changes in the soft-tissue sarcoma (STS) tumor immune microenvironment induced by standard neoadjuvant therapy with the goal of informing neoadjuvant immunotherapy trial design. Experimental Design: Paired pre- and postneoadjuvant therapy specimens were retrospectively identified for 32 patients with STSs and analyzed by three modalities: multiplexed IHC, NanoString, and RNA sequencing with ImmunoPrism analysis. Results: All 32 patients, representing a variety of STS histologic subtypes, received neoadjuvant radiotherapy and 21 (66%) received chemotherapy prior to radiotherapy. The most prevalent immune cells in the tumor before neoadjuvant therapy were myeloid cells (45% of all immune cells) and B cells (37%), with T (13%) and natural killer (NK) cells (5%) also present. Neoadjuvant therapy significantly increased the total immune cells infiltrating the tumors across all histologic subtypes for patients receiving neoadjuvant radiotherapy with or without chemotherapy. An increase in the percentage of monocytes and macrophages, particularly M2 macrophages, B cells, and CD4+ T cells was observed postneoadjuvant therapy. Upregulation of genes and cytokines associated with antigen presentation was also observed, and a favorable pathologic response (≥90% necrosis postneoadjuvant therapy) was associated with an increase in monocytic infiltrate. Upregulation of the T-cell checkpoint TIM3 and downregulation of OX40 were observed posttreatment. Conclusions: Standard neoadjuvant therapy induces both immunostimulatory and immunosuppressive effects within a complex sarcoma microenvironment dominated by myeloid and B cells. This work informs ongoing efforts to incorporate immune checkpoint inhibitors and novel immunotherapies into the neoadjuvant setting for STSs.

As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a mullti-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or “cuboids” with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

Purpose: IL12 promotes adaptive type I immunity and has demonstrated antitumor efficacy, but systemic administration leads to severe adverse events (AE), including death. This pilot trial investigated safety, efficacy, and immunologic activity of intratumoral delivery of IL12 plasmid DNA (tavo) via in vivo electroporation (i.t.-tavo-EP) in patients with Merkel cell carcinoma (MCC), an aggressive virus-associated skin cancer. Patients and Methods: Fifteen patients with MCC with superficial injectable tumor(s) received i.t.-tavo-EP on days 1, 5, and 8 of each cycle. Patients with locoregional MCC (cohort A, N = 3) received one cycle before definitive surgery in week 4. Patients with metastatic MCC (cohort B, N = 12) received up to four cycles total, administered at least 6 weeks apart. Serial tumor and blood samples were collected. Results: All patients successfully completed at least one cycle with transient, mild (grades 1 and 2) AEs and without significant systemic toxicity. Sustained (day 22) intratumoral expression of IL12 protein was observed along with local inflammation and increased tumor-specific CD8+ T-cell infiltration, which led to systemic immunologic and clinical responses. The overall response rate was 25% (3/12) in cohort B, with 2 patients experiencing durable clinical benefit (16 and 55+ months, respectively). Two cohort A patients (1 with pathologic complete remission) were recurrence-free at 44+ and 75+ months, respectively. Conclusions: I.t.-tavo-EP was safe and feasible without systemic toxicity. Sustained local expression of IL12 protein and local inflammation led to systemic immune responses and clinically meaningful benefit in some patients. Gene electrotransfer, specifically i.t.-tavo-EP, warrants further investigation for immunotherapy of cancer.

PURPOSE To assess the efficacy of pembrolizumab in patients with advanced relapsed or refractory mycosis fungoides (MF) or Sézary syndrome (SS). PATIENTS AND METHODS CITN-10 is a single-arm, multicenter phase II trial of 24 patients with advanced MF or SS. Patients were treated with pembrolizumab 2 mg/kg every 3 weeks for up to 24 months. The primary end point was overall response rate by consensus global response criteria. RESULTS Patients had advanced-stage disease (23 of 24 with stage IIB to IV MF/SS) and were heavily pretreated with a median of four prior systemic therapies. The overall response rate was 38% with two complete responses and seven partial responses. Of the nine responding patients, six had 90% or more improvement in skin disease by modified Severity Weighted Assessment Tool, and eight had ongoing responses at last follow-up. The median duration of response was not reached, with a median response follow-up time of 58 weeks. Immune-related adverse events led to treatment discontinuation in four patients. A transient worsening of erythroderma and pruritus occurred in 53% of patients with SS. This cutaneous flare reaction did not result in treatment discontinuation for any patient. The flare reaction correlated with high PD-1 expression on Sézary cells but did not associate with subsequent clinical responses or lack of response. Treatment responses did not correlate with expression of PD-L1, total mutation burden, or an interferon-γ gene expression signature. CONCLUSION Pembrolizumab demonstrated significant antitumor activity with durable responses and a favorable safety profile in patients with advanced MF/SS.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome, and high-dimensional analyses of patient samples reveal the heterogeneity of the immune TME. Macrophages infiltrate TMEs and generally associate with tumor progression, but the underlying mechanisms are incompletely understood. Because experimental systems are needed to elucidate the functional properties of these cells, we developed a humanized mouse model reconstituted with human immune cells and human melanoma. We used two strains of recipient mice, supporting or not supporting the development of human myeloid cells. We found that human myeloid cells favored metastatic spread of the primary tumor, thereby recapitulating the cancer-supportive role of macrophages. We next analyzed the transcriptome of human immune cells infiltrating tumors versus other tissues. This analysis identified a cluster of myeloid cells present in the TME, but not in other tissues, which do not correspond to canonical M2 cells. The transcriptome of these cells is characterized by high expression of glycolytic enzymes and multiple chemokines and by low expression of gene sets associated with inflammation and adaptive immunity. Compared with humanized mouse results, we found transcriptionally similar myeloid cells in patient-derived samples of melanoma and other cancer types. The humanized mouse model described here thus complements patient sample analyses, enabling further elucidation of fundamental principles in melanoma biology beyond M1/M2 macrophage polarization. The model can also support the development and evaluation of candidate antitumor therapies.

Cutaneous T cell lymphomas (CTCL) are rare but aggressive cancers without effective treatments. While a subset of patients derive benefit from PD-1 blockade, there is a critically unmet need for predictive biomarkers of response. Herein, we perform CODEX multiplexed tissue imaging and RNA sequencing on 70 tumor regions from 14 advanced CTCL patients enrolled in a pembrolizumab clinical trial (NCT02243579). We find no differences in the frequencies of immune or tumor cells between responders and non-responders. Instead, we identify topographical differences between effector PD-1+ CD4+ T cells, tumor cells, and immunosuppressive Tregs, from which we derive a spatial biomarker, termed the SpatialScore, that correlates strongly with pembrolizumab response in CTCL. The SpatialScore coincides with differences in the functional immune state of the tumor microenvironment, T cell function, and tumor cell-specific chemokine recruitment and is validated using a simplified, clinically accessible tissue imaging platform. Collectively, these results provide a paradigm for investigating the spatial balance of effector and suppressive T cell activity and broadly leveraging this biomarker approach to inform the clinical use of immunotherapies., PD-1 blockade is effective for only a subset of patients with cutaneous T cell lymphomas. Here, the authors report a spatial biomarker that uses immune and cancer cell topography to predict response to PD-1 blockade in this disease.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome. High dimensional analyses of patient samples can reveal the composition and heterogeneity of the immune TME. In particular, macrophages are known for their cancer-supportive role, but the underlying mechanisms are incompletely understood, and experimental in vivo systems are needed to test the functional properties of these cells. We characterized a humanized mouse model, reconstituted with a human immune system and a human melanoma, in which: (1) human macrophages support metastatic spread of the tumor; and (2) tumor-infiltrating macrophages have a specific transcriptional signature that faithfully represents the transcriptome of macrophages from patient melanoma samples and is associated with shorter survival. This model complements patient sample analyses, enabling the elucidation of fundamental principles in melanoma biology, and the development and evaluation of candidate therapies.

BackgroundDedifferentiated liposarcoma (DDLPS) is one of the most common soft tissue sarcoma subtypes and is devastating in the advanced/metastatic stage. Despite the observation of clinical responses to PD-1 inhibitors, little is known about the immune microenvironment in relation to patient prognosis.MethodsWe performed a retrospective study of 61 patients with DDLPS. We completed deep sequencing of the T-cell receptor (TCR) β-chain and RNA sequencing for predictive modeling, evaluating both immune markers and tumor escape genes. Hierarchical clustering and recursive partitioning were employed to elucidate relationships of cellular infiltrates within the tumor microenvironment, while an immune score for single markers was created as a predictive tool.ResultsAlthough many DDLPS samples had low TCR clonality, high TCR clonality combined with low T-cell fraction predicted lower 3-year overall survival (p=0.05). Higher levels of CD14+ monocytes (p=0.02) inversely correlated with 3-year recurrence-free survival (RFS), while CD4+ T-cell infiltration (p=0.05) was associated with a higher RFS. Genes associated with longer RFS included PD-1 (p=0.003), ICOS (p=0.006), BTLA (p=0.033), and CTLA4 (p=0.02). In a composite immune score, CD4+ T cells had the strongest positive predictive value, while CD14+ monocytes and M2 macrophages had the strongest negative predictive values.ConclusionsImmune cell infiltration predicts clinical outcome in DDLPS, with CD4+ cells associated with better outcomes; CD14+ cells and M2 macrophages are associated with worse outcomes. Future checkpoint inhibitor studies in DDLPS should incorporate immunosequencing and gene expression profiling techniques that can generate immune landscape profiles.

The main objective of our study was to understand the impact of immune cell composition and the tumor-reactivity of tumor infiltrating lymphocytes (TIL) in HPV-positive (HPV

Interferon-γ (IFNγ) has been studied as a cancer treatment with limited evidence of clinical benefit. However, it could play a role in cancer immunotherapy combination treatments. Despite high expression of immunogenic cancer–testis antigens, synovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) have a cold tumor microenvironment (TME), with few infiltrating T cells and low expression of major histocompatibility complex class I (MHC-I). We hypothesized that IFNγ treatment could drive inflammation in a cold TME, facilitating further immunotherapy. We conducted a phase 0 clinical trial treating 8 SS or MRCL patients with weekly systemic IFNγ. We performed pre- and posttreatment biopsies. IFNγ changed the SS and MRCL TME, inducing tumor-surface MHC-I expression and significant T-cell infiltration (P < 0.05). Gene-expression analysis suggested increased tumor antigen presentation and less exhausted phenotypes of the tumor-infiltrating T cells. Newly emergent antigen-specific humoral and/or T-cell responses were found in 3 of 7 evaluable patients. However, increased expression of PD-L1 was observed on tumor-infiltrating myeloid cells and in some cases tumor cells. These findings suggest that systemic IFNγ used to convert SS and MRCL into “hot” tumors will work in concert with anti–PD-1 therapy to provide patient benefit.

Therapeutic cancer vaccines have met limited clinical success. In the setting of cancer, the immune system is either tolerized and/or has a limited tumor-specific T cell repertoire. In this study, we explore whether intratumoral (IT) vaccination with an HPV vaccine can elicit quantitative and qualitative differences in immune response as compared to intramuscular (IM) vaccination to overcome immune resistance in established tumors. We report that IT administration of an HPV-16 E7 peptide vaccine formulated with polyinosinic–polycytidylic acid (Poly (I:C)) generated an enhanced antitumor effect relative to IM delivery. The elicited anti-tumor effect with IT vaccination was consistent among the vaccinated groups and across various C57BL/6 substrains. IT vaccination resulted in an increased frequency of PD-1(hi) TILs, which represented both vaccine-targeted and non-vaccine-targeted tumor specific CD8(+) T cells. Overall, the CD8(+)/Treg ratio was increased within the tumor microenvironment using IT vaccination. We also assessed transcriptional changes in several immune-related genes in the tumor microenvironment of the various treated groups, and our data suggest that IT vaccination leads to upregulation of a broad complement of immunomodulatory genes, including upregulation of interferon gamma (IFNγ) and antigen presentation and processing machine (APM) components. IT vaccine delivery is superior to traditional IM vaccination routes with the potential to improve tumor immunogenicity, which has potential clinical application in the setting of accessible lesions such as head and neck squamous cell carcinomas (HNSCCs).

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapies, including immune-checkpoint blockade. We investigated two distinct strategies to modulate tumor-associated macrophages (TAM) to enhance cellular therapy targeting mesothelin in an autochthonous PDA mouse model. Administration of an antibody to colony-stimulating factor (anti-Csf1R) depleted Ly6Clow protumorigenic TAMs and significantly enhanced endogenous T-cell intratumoral accumulation. Despite increasing the number of endogenous T cells at the tumor site, as previously reported, TAM depletion had only minimal impact on intratumoral accumulation and persistence of T cells engineered to express a murine mesothelin-specific T-cell receptor (TCR). TAM depletion interfered with the antitumor activity of the infused T cells in PDA, evidenced by reduced tumor cell apoptosis. In contrast, TAM programming with agonistic anti-CD40 increased both Ly6Chigh TAMs and the intratumoral accumulation and longevity of TCR-engineered T cells. Anti-CD40 significantly increased the frequency and number of proliferating and granzyme B+ engineered T cells, and increased tumor cell apoptosis. However, anti-CD40 failed to rescue intratumoral engineered T-cell IFNγ production. Thus, although functional modulation, rather than TAM depletion, enhanced the longevity of engineered T cells and increased tumor cell apoptosis, ultimately, anti-CD40 modulation was insufficient to rescue key effector defects in tumor-reactive T cells. This study highlights critical distinctions between how endogenous T cells that evolve in vivo, and engineered T cells with previously acquired effector activity, respond to modifications of the tumor microenvironment.

BACKGROUND: ROR1 is a type 1 transmembrane tyrosine kinase receptor that plays a critical role in embryonic and fetal development. ROR1 has been described as a possible oncogene and is expressed in numerous malignancies including TNBC and non-small cell lung cancer (NSCLC). We are conducting a first-in-human trial targeting ROR1 with CAR-T cells in patients with advanced TNBC and NSCLC. The cellular construct employed targets the Ig/Fz portion of the extracellular domain of ROR1 and contains 4-1BB/CD3ζ intracellular signaling domain. The manufacturing process utilizes autologous peripheral blood lymphocytes, separated into CD4 and CD8 subsets, which are independently cultured with anti-CD3/anti-CD28 beads and IL-2, then transduced with a lentiviral vector encoding the ROR1 CAR. The CAR-T cell product is formulated in a 1:1 ratio of CD4+ and CD8+ CAR-T cells. METHODS: This ongoing phase I trial (NCT02706392) is evaluating the safety of administering ROR1 CAR-T cells in escalating doses (3.3x105, 1x106, 3.3x106 and 1x107 cells/kg) following lymphodepletion with cyclophosphamide-containing regimens using a continual reassessment method (CRM) for dose escalation. TNBC patients with adequate organ function and performance status, measurable disease, and tumors expressing ROR1 (>20% by IHC) are eligible for enrollment. Persistence of CAR-T cells in blood, cytokine levels, measures of immunogenicity and multi-parametric flow cytometry are being evaluated at multiple time points. Imaging assessments by RECIST 1.1 are performed day 28 - 90, then at 6 and 12 months, and every 6 months as clinically indicated to estimate efficacy. RESULTS: To date, 4 TNBC patients (age range 38-67) have been enrolled, treated and are evaluable for response. Patients had received prior therapies for metastatic disease (range 3-11). 3 of 4 had visceral metastases. No dose-limiting toxicities, severe neurotoxicity or severe cytokine release syndrome (sCRS) were observed at dose levels 1 and 2. Two patients experienced grade 1 CRS. 2 of 4 patients had evidence of CAR-T cell expansion between days 14 and 20, with peak CD8+ CAR-T up to 232.1 cells/uL. Analysis of surface phenotype revealed upregulation of inhibitory receptors on CAR-T cells at the peak of expansion, confirmed by RNA seq. Post-treatment tumor biopsy in patient with partial response revealed an influx of CD3+ T cells and macrophages suggesting ROR1 CAR-T cell trafficking. Two patients received 2 CAR-T cell infusions. Two patients had confirmed stable disease at 15 weeks and 19 weeks, respectively. One patient has stable disease after 1st CAR-T cell infusion, then confirmed partial response after 2nd CAR-T cell infusion which has persisted for 14 weeks. Results will be updated. CONCLUSIONS: ROR1+ CAR-T cells can be safely transferred, expand in vivo in patients with TNBC. Current efforts are directed at understanding and overcoming the mechanisms that limit homing, persistence, and/or function at tumor sites. The trial is continuing with dose-escalation. Funding provided by 8RO1 CA114536-11 and Juno Therapeutics. Citation Format: Specht JM, Lee SM, Turtle C, Berger C, Balakrishnan A, Srivastava S, Viollet V, Veatch J, Gooley T, Mullane E, Chaney CN, Rader C, Pierce RH, Gottardo R, Maloney DG, Riddell SR. A phase I study of adoptive immunotherapy for ROR1+ advanced triple negative breast cancer (TNBC) with defined subsets of autologous T cells expressing a ROR1-specific chimeric antigen receptor (ROR1-CAR) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-13.

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.

Hp infection was found to alter metaplasia and accelerate dysplasia in mice. Thus, Hp can promote gastric preneoplastic progression, in addition to its known role in initiating chronic inflammation., More than 80% of gastric cancer is attributable to stomach infection with Helicobacter pylori (Hp). Gastric preneoplastic progression involves sequential tissue changes, including loss of parietal cells, metaplasia and dysplasia. In transgenic mice, active KRAS expression recapitulates these tissue changes in the absence of Hp infection. This model provides an experimental system to investigate additional roles of Hp in preneoplastic progression, beyond its known role in initiating inflammation. Tissue histology, gene expression, the immune cell repertoire, and metaplasia and dysplasia marker expression were assessed in KRAS+ mice +/−Hp infection. Hp+/KRAS+ mice had severe T-cell infiltration and altered macrophage polarization; a different trajectory of metaplasia; more dysplastic glands; and greater proliferation of metaplastic and dysplastic glands. Eradication of Hp with antibiotics, even after onset of metaplasia, prevented or reversed these tissue phenotypes. These results suggest that gastric preneoplastic progression differs between Hp+ and Hp− cases, and that sustained Hp infection can promote the later stages of gastric preneoplastic progression.

Background Efficacy of anti-PD-1 therapy is attributed to the presence of infiltrating antigen-specific CD8+ T-cells. Despite the success of anti-PD-1 therapy, many patients with SCCHN present with immune desert or immune excluded tumors and only 13–18% of patients achieve tumor reductions. Given this low response rate, it is imperative to combine agents that generate or expand anti-tumor T cells, such as vaccines, with anti-PD-1 therapies. SNS-301 is a first-in-class, bacteriophage-based immune activating agent targeting human aspartate β-hydroxylase (ASPH), a tumor associated antigen overexpressed in multiple tumor types. SNS-301 is a self-adjuvanted vaccine consisting of λ-bacteriophage engineered to express an immunogenic fragment of ASPH fused to the phage gpD coat protein, previously shown to be well tolerated and generate an immune response (Phase 1, NCT03120832). The objectives of this trial are to evaluate safety, immunogenicity and preliminary efficacy of SNS-301 in combination with pembrolizumab in patients that did not achieve tumor reductions on anti-PD-1/PD-L1 therapy alone. Methods The study consists of an initial safety-run-in followed by a two-stage design. SNS-301 is delivered intradermally in addition to pembrolizumab in up to 30 patients with locally advanced unresectable or metastatic/recurrent SCCHN. Patients must have actively received anti-PD-1 therapy for ≥12 weeks, with a best response of stable disease (SD) or unconfirmed progressive disease (PD) per iRECIST. Patients provide pre, on-treatment and biopsies at PD (optional) to characterize the tumor microenvironment using NanostringTM, multiplex immunohistochemistry, and correlate with clinical outcomes. Blood samples are collected to evaluate T cell responses using flow cytometry, ELISA, ELISPOT. Results As of July 23, 2020, 9 patients were enrolled. Median duration of ongoing anti-PD therapy was 37 weeks (range 20–101). The combination was well-tolerated with no DLTs and mostly Grade 1–2 unrelated adverse events. Two Grade 3 events were reported: hypertension (not related) and dehydration (related), the later reported as serious adverse event. Of seven patients eligible for efficacy analysis, one patient with PD-L1 negative disease had a partial response with a reduction of 29% at week 6 with deepening of the response to 43% at week 12 and one patient with progressive disease at study entry had stabilization of disease at week 6 and 12. Another two patients had stable disease for 30+ weeks and three patients had PD. Additional efficacy and immunological analyses are ongoing. Conclusions Early data show that the combination of SNS-301 and pembrolizumab has manageable toxicity and capacity to achieve long-term disease stability and objective tumor responses. Trial Registration NCT04034225 Ethics Approval This study has been approved by WIRB (20190628) as well as several institutional IRBs.

More than 80% of gastric cancer is attributable to stomach infection with Helicobacter pylori (Hp), even though the bacterium is not always present at time of diagnosis. Infection is thought to lead to cancer by promoting the accumulation of oncogenic mutations downstream of inflammation; once oncogenic pathways become activated, infection may become dispensable for cancer development. Gastric preneoplastic progression involves sequential changes to the tissue, including loss of parietal cells, spasmolytic polypeptide-expressing metaplasia (SPEM), intestinal metaplasia (IM) and dysplasia. In mice, active KRAS expression recapitulates these tissue changes in the absence of Hp infection. This model provides an experimental system to investigate whether Hp infection has additional roles in preneoplastic progression, beyond initiating inflammation. Mice were assessed by evaluating tissue histology, gene expression changes, the immune cell repertoire, and expression of metaplasia and dysplasia markers. Compared to Hp-/KRAS+ mice, Hp+/KRAS+ mice had i) severe T cell infiltration and altered macrophage polarization; ii) altered expression of metaplasia markers, including increased expression of CD44v9 (SPEM) and decreased expression of TFF3 (IM); iii) more dysplastic (TROP2+) glands; and iv) greater proliferation of metaplastic and dysplastic glands. Hp was able to persistently colonize the stomach during the onset of these tissue changes, and eradication of Hp with antibiotics prevented metaplastic, dysplastic and proliferation marker changes. Collectively, these results suggest that gastric preneoplastic progression differs between Hp+ and Hp-cases, and that sustained Hp infection can promote the later stages of gastric preneoplastic progression, in addition to its established role in initiating chronic inflammation.

BackgroundThough currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies.MethodsUsing lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies.ResultsHere, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins.ConclusionsOur data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Patients with metastatic soft tissue sarcoma (STS) have a poor prognosis and few available systemic treatment options. Trabectedin is currently being investigated as a potential adjunct to immunotherapy as it has been previously shown to kill tumor-associated macrophages. In this retrospective study, we sought to identify biomarkers that would be relevant to trials combining trabectedin with immunotherapy. We performed a single-center retrospective study of sarcoma patients treated with trabectedin with long-term follow-up. Multiplex gene expression analysis using the NanoString platform was assessed, and an exploratory analysis using the lasso-penalized Cox regression and kernel association test for survival (MiRKAT-S) methods investigated tumor-associated immune cells and correlated their gene signatures to patient survival. In total, 147 sarcoma patients treated with trabectedin were analyzed, with a mean follow-up time of 5 years. Patients with fewer prior chemotherapy regimens were more likely to stay on trabectedin longer (pairwise correlation = −0.17, p = 0.04). At 5 years, increased PD-L1 expression corresponded to worse outcomes (HR = 1.87, p = 0.04, q = 0.199). Additionally, six immunologic gene signatures were associated with up to 7-year survival by MiRKAT-S, notably myeloid-derived suppressor cells (p = 0.023, q = 0.058) and M2 macrophages (p = 0.03, q = 0.058). We found that the number of chemotherapy regimens prior to trabectedin negatively correlated with the number of trabectedin cycles received, suggesting that patients may benefit from receiving trabectedin earlier in their therapy course. The correlation of trabectedin outcomes with immune cell infiltrates supports the hypothesis that trabectedin may function as an immune modulator and supports ongoing efforts to study trabectedin in combination with immunotherapy. Furthermore, tumors with an immunosuppressive microenvironment characterized by macrophage infiltration and high PD-L1 expression were less likely to benefit from trabectedin, which could guide clinicians in future treatment decisions.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Summary Adoptive therapy using chimeric antigen receptor-modified T cells (CAR-T cells) is effective in hematologic but not epithelial malignancies, which cause the greatest mortality. In breast and lung cancer patients, CAR-T cells targeting the tumor-associated antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) infiltrate tumors poorly and become dysfunctional. To test strategies for enhancing efficacy, we adapted the KrasLSL-G12D/+;p53f/f autochthonous model of lung adenocarcinoma to express the CAR target ROR1. Murine ROR1 CAR-T cells transferred after lymphodepletion with cyclophosphamide (Cy) transiently control tumor growth but infiltrate tumors poorly and lose function, similar to what is seen in patients. Adding oxaliplatin (Ox) to the lymphodepletion regimen activates tumor macrophages to express T-cell-recruiting chemokines, resulting in improved CAR-T cell infiltration, remodeling of the tumor microenvironment, and increased tumor sensitivity to anti-PD-L1. Combination therapy with Ox/Cy and anti-PD-L1 synergistically improves CAR-T cell-mediated tumor control and survival, providing a strategy to improve CAR-T cell efficacy in the clinic.

Whereas systemic IL12 is associated with potentially life-threatening toxicity, intratumoral delivery of IL12 through tavokinogene telseplasmid electroporation (tavo) is safe and can induce tumor regression at distant sites. The mechanism by which these responses are mediated is unknown but is presumed to result from a cellular immune response. In a phase II clinical trial of tavo (NCT01502293), samples from 29 patients with cutaneous melanoma with in-transit disease were assessed for immune responses induced with this treatment. Within the blood circulating immune cell population, we found that the frequencies of circulating PD-1+ CD4+ and CD8+ T cells declined with treatment. Circulating immune responses to gp100 were also detected following treatment as measured by IFNγ ELISpot. Patients with a greater antigen-specific circulating immune response also had higher numbers of CD8+ T cells within the tumor. Clinical response was also associated with increased intratumoral CD3+ T cells. Finally, intratumoral T-cell clonality and convergence were increased after treatment, indicating a focusing of the T-cell receptor repertoire. These results indicated that local treatment with tavo can induce a systemic T-cell response and recruit T cells to the tumor microenvironment.

Gastrointestinal stromal tumor (GIST) is a devastating disease, especially in the setting of metastasis. The natural progression of GIST has been significantly altered by the development of small molecule tyrosine kinase inhibitors (TKIs), including imatinib, sunitinib, and regorafenib, all of which are FDA approved. However, TKIs are not always well-tolerated, and the refractory disease continues to be a problem. For these reasons, alternative treatments are needed. In this report, we discuss a patient with metastatic wild-type (WT) GIST refractory to multiple TKIs, but with a durable clinical response to the anti-programmed cell death protein 1 (PD-1) antibody, nivolumab. This report suggests that continued research evaluating checkpoint inhibitors in GIST is warranted.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor–immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.

Background Most glioblastomas recur near prior radiation treatment sites. Future clinical success will require achieving and optimizing an “abscopal effect,” whereby unirradiated neoplastic cells outside treatment sites are recognized and attacked by the immune system. Radiation combined with anti–programmed cell death ligand 1 (PD-L1) demonstrated modest efficacy in phase II human glioblastoma clinical trials, but the mechanism and relevance of the abscopal effect during this response remain unknown. Methods We modified an immune-competent, genetically driven mouse glioma model (forced platelet derived growth factor [PDGF] expression + phosphatase and tensin homolog loss) where a portion of the tumor burden is irradiated (PDGF) and another unirradiated luciferase-expressing tumor (PDGF + luciferase) is used as a readout of the abscopal effect following systemic anti–PD-L1 immunotherapy. We assessed relevance of tumor neoepitope during the abscopal response by inducing expression of epidermal growth factor receptor variant III (EGFRvIII) (PDGF + EGFRvIII). Statistical tests were two-sided. Results Following radiation of one lesion, anti–PD-L1 immunotherapy enhanced the abscopal response to the unirradiated lesion. In PDGF-driven gliomas without tumor neoepitope (PDGF + luciferase, n = 8), the abscopal response occurred via anti–PD-L1 driven, extracellular signal-regulated kinase–mediated, bone marrow–derived macrophage phagocytosis of adjacent unirradiated tumor cells, with modest survival implications (median survival 41 days vs radiation alone 37.5 days, P = 0.03). In PDGF-driven gliomas with tumor neoepitope (PDGF + EGFRvIII, n = 8), anti–PD-L1 enhanced abscopal response was associated with macrophage and T-cell infiltration and increased survival benefit (median survival 36 days vs radiation alone 28 days, P = 0.001). Conclusion Our results indicate that anti–PD-L1 immunotherapy enhances a radiation- induced abscopal response via canonical T-cell activation and direct macrophage activation in glioblastoma.

Chimeric antigen receptors (CAR) are synthetic molecules that provide new specificities to T cells. Although successful in treatment of hematologic malignancies, CAR T cells are ineffective for solid tumors to date. We found that the cell-surface molecule c-Met was expressed in ∼50% of breast tumors, prompting the construction of a CAR T cell specific for c-Met, which halted tumor growth in immune-incompetent mice with tumor xenografts. We then evaluated the safety and feasibility of treating metastatic breast cancer with intratumoral administration of mRNA-transfected c-Met-CAR T cells in a phase 0 clinical trial (NCT01837602). Introducing the CAR construct via mRNA ensured safety by limiting the nontumor cell effects (on-target/off-tumor) of targeting c-Met. Patients with metastatic breast cancer with accessible cutaneous or lymph node metastases received a single intratumoral injection of 3 × 107 or 3 × 108 cells. CAR T mRNA was detectable in peripheral blood and in the injected tumor tissues after intratumoral injection in 2 and 4 patients, respectively. mRNA c-Met-CAR T cell injections were well tolerated, as none of the patients had study drug–related adverse effects greater than grade 1. Tumors treated with intratumoral injected mRNA c-Met-CAR T cells were excised and analyzed by immunohistochemistry, revealing extensive tumor necrosis at the injection site, cellular debris, loss of c-Met immunoreactivity, all surrounded by macrophages at the leading edges and within necrotic zones. We conclude that intratumoral injections of mRNA c-Met-CAR T cells are well tolerated and evoke an inflammatory response within tumors. Cancer Immunol Res; 5(12); 1152–61. ©2017 AACR.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity tumor. In this study, we examined the basis for the activity of programmed cell death protein (PD-1)-based immune checkpoint therapy that is being explored widely in head and neck cancers. Using multispectral imaging, we systematically investigated the OTSCC tumor microenvironment (TME) by evaluating the frequency of PD-1 expression in CD8+, CD4+, and FoxP3+ tumor-infiltrating lymphocytes (TIL). We also defined the cellular sources of PD-1 ligand (PD-L1) to evaluate the utility of PD-1:PD-L1 blocking antibody therapy in this patient population. PD-L1 was expressed in 79% of the OTSCC specimens examined within the TME. Expression of PD-L1 was associated with moderate to high levels of CD4+ and CD8+ TILs. We found that CD4+ TILs were present in equal or greater frequencies than CD8+ TILs in 94% of OTSCC and that CD4+FOXP3neg TILs were colocalized with PD-1/PD-L1/CD68 more frequently than CD8+ TILs. Both CD4+PD1+ and CD8+PD1+ TILs were anergic in the setting of PD-L1 expression. Overall, our results highlight the importance of CD4+ TILs as pivotal regulators of PD-L1 levels and in determining the responsiveness of OTSCC to PD1-based immune checkpoint therapy. Cancer Res; 77(22); 6365–74. ©2017 AACR.

6029 Background: The absence of infiltrating antigen-specific CD8+ T-cells at baseline is associated with low response rates to PD-1 blockade. SCCHN tumors often exclude effector T cells, and 2nd line response rates are low (13-18%). Highly immunogenic, antigen specific antitumor vaccines may expand intratumoral CD8+ T cells, potentially increasing durable response rates to PD-1 blockade. SNS-301 is a first-in-class, bacteriophage-based immune activating agent targeting human aspartate β-hydroxylase (ASPH), a tumor associated antigen overexpressed in multiple tumor types. SNS-301 is a self-adjuvanted vaccine consisting of λ-bacteriophage engineered to express an immunogenic fragment of ASPH fused to the phage gpD coat protein. The study objectives are to evaluate safety, immunogenicity and preliminary efficacy of SNS-301 added to pembrolizumab in patients (pts) not achieving tumor reductions on PD-1 blockade alone. Methods: Intradermal SNS-301 was combined with pembrolizumab in pts with locally advanced unresectable (LA) or metastatic/recurrent (met) SCCHN with a best response of stable disease (SD) or unconfirmed progressive disease (uPD) on ongoing PD-1 blockade > 12 weeks. Pts provided pre and on-treatment biopsies to characterize the tumor microenvironment using Nanostring and multiplex immunohistochemistry (mIHC). Blood samples were collected to evaluate B and T cell responses using ELISA/ELISPOT assays. Results: As of February 4, 2021, 13 pts were enrolled. Median duration of PD-1 blockade was 48 weeks (range 14-114) at study entry. There were no DLTs & mostly Grade 1-2 unrelated adverse events. Only two related Grade 3 events were reported: rash & dehydration (also a serious adverse event). Ten pts were evaluable for efficacy: 1 pt with PD-L1 negative (neg) disease & SD on pembrolizumab monotherapy achieved a partial response (PR; -52% at 8 months), 4 pts achieved SD & 5 pts had progressive disease. Two of the pts with SD had long-lasting duration (8 & 10 months) of which the latter had PD-L1 neg disease. One pt with uPD at enrollment achieved SD for 4 months. Analyses of pre- & on-treatment biopsies from the PR pt demonstrated an increase in infiltrating CD8+ T cells, PD-L1 expression & PD-1/PD-L1 proximity measures. Nanostring analysis demonstrated increased gene expression signatures for immune cells in the PR pt that was concordant with the mIHC & clinical outcome. Conclusions: The combination of SNS-301 and pembrolizumab was well-tolerated and resulted in encouraging clinical efficacy in pts not expected to respond to PD-1 blockade alone. Translational data suggest cellular response to SNS-301 and transformation of a poorly inflamed tumor to an immunologically active tumor in a responding pt (PR). Based on these data, an additional cohort will start enrolling PD-1 blockade naïve pts with LA/met SCHNN in the front-line setting. Clinical trial information: NCT04034225.

Author(s): Kohli, Karan; Yao, Lu; Nowicki, Theodore Scott; Zhang, Shihong; Black, Ralph Graeme; Schroeder, Brett A; Farrar, Erik A; Cao, Jianhong; Sloan, Heather; Stief, Dawn; Cranmer, Lee D; Wagner, Michael J; Hawkins, Douglas S; Pillarisetty, Venu G; Ribas, Antoni; Campbell, Jean; Pierce, Robert H; Kim, Edward Y; Jones, Robin L; Riddell, Stanley R; Yee, Cassian; Pollack, Seth M | Abstract: BackgroundSynovial sarcoma (SS) and myxoid/round cell liposarcoma (MRCL) are ideal solid tumors for the development of adoptive cellular therapy (ACT) targeting NY-ESO-1, as a high frequency of tumors homogeneously express this cancer-testes antigen. Data from early phase clinical trials have shown antitumor activity after the adoptive transfer of NY-ESO-1-specific T cells. In these studies, persistence of NY-ESO-1 specific T cells is highly correlated with response to ACT, but patients often continue to have detectable transferred cells in their peripheral blood following progression.MethodWe performed a phase I clinical trial evaluating the safety of NY-ESO-1-specific endogenous T cells (ETC) following cyclophosphamide conditioning. Peripheral blood mononuclear cells (PBMCs) from treated patients were evaluated by flow cytometry and gene expression analysis as well as through ex vivo culture assays with and without IL-15.ResultsFour patients were treated in a cohort using ETC targeting NY-ESO-1 following cyclophosphamide conditioning. Treatment was well tolerated without significant toxicity, but all patients ultimately had disease progression. In two of four patients, we obtained post-treatment tumor tissue and in both, NY-ESO-1 antigen was retained despite clear detectable persisting NY-ESO-1-specific T cells in the peripheral blood. Despite a memory phenotype, these persisting cells lacked markers of proliferation or activation. However, in ex vivo culture assays, they could be induced to proliferate and kill tumor using IL-15. These results were also seen in PBMCs from two patients who received gene-engineered T-cell receptor-based products at other centers.ConclusionsETC targeting NY-ESO-1 with single-agent cyclophosphamide alone conditioning was well tolerated in patients with SS and those with MRCL. IL-15 can induce proliferation and activity in persisting NY-ESO-1-specific T cells even in patients with disease progression following ACT. These results support future work evaluating whether IL-15 could be incorporated into ACT trials post-infusion or at the time of progression.

We explored the association between liver metastases, tumor CD8+ T-cell count, and response in patients with melanoma or lung cancer treated with the anti-PD-1 antibody, pembrolizumab. The melanoma discovery cohort was drawn from the phase I Keynote 001 trial, whereas the melanoma validation cohort was drawn from Keynote 002, 006, and EAP trials and the non–small cell lung cancer (NSCLC) cohort from Keynote 001. Liver metastasis was associated with reduced response and shortened progression-free survival [PFS; objective response rate (ORR), 30.6%; median PFS, 5.1 months] compared with patients without liver metastasis (ORR, 56.3%; median PFS, 20.1 months) P ≤ 0.0001, and confirmed in the validation cohort (P = 0.0006). The presence of liver metastasis significantly increased the likelihood of progression (OR, 1.852; P < 0.0001). In a subset of biopsied patients (n = 62), liver metastasis was associated with reduced CD8+ T-cell density at the invasive tumor margin (liver metastasis+ group, n = 547 ± 164.8; liver metastasis− group, n = 1,441 ± 250.7; P < 0.016). A reduced response rate and shortened PFS was also observed in NSCLC patients with liver metastasis [median PFS, 1.8 months; 95% confidence interval (CI), 1.4–2.0], compared with those without liver metastasis (n = 119, median PFS, 4.0 months; 95% CI, 2.1–5.1), P = 0.0094. Thus, liver metastatic patients with melanoma or NSCLC that had been treated with pembrolizumab were associated with reduced responses and PFS, and liver metastases were associated with reduced marginal CD8+ T-cell infiltration, providing a potential mechanism for this outcome. Cancer Immunol Res; 5(5); 417–24. ©2017 AACR.

Background Interleukin 12 (IL-12) is a pivotal regulator of innate and adaptive immunity. We conducted a prospective open-label, phase II clinical trial of electroporated plasmid IL-12 in advanced melanoma patients (NCT 01502293). Patients and methods Patients with stage III/IV melanoma were treated intratumorally with plasmid encoding IL-12 (tavokinogene telseplasmid; tavo), 0.5 mg/ml followed by electroporation (six pulses, 1500 V/cm) on days 1, 5, and 8 every 90 days in the main study and additional patients were treated in two alternative schedule exploration cohorts. Correlative analyses for programmed death-ligand 1 (PD-L1), flow cytometry to assess changes in immune cell subsets, and analysis of immune-related gene expression were carried out on pre- and post-treatment samples from study patients, as well as from additional patients treated during exploration of additional dosing schedules beyond the pre-specified protocol dosing schedule. Response was measured by study-specific criteria to maximize detection of latent and potentially transient immune responses in patients with multiple skin lesions and toxicities were graded by the Common Terminology Criteria for Adverse Events version 4.0 (CTCAE v4.0). Results The objective overall response rate was 35.7% in the main study (29.8% in all cohorts), with a complete response rate of 17.9% (10.6% in all cohorts). The median progression-free survival in the main study was 3.7 months while the median overall survival was not reached at a median follow up of 29.7 months. A total of 46% of patients in all cohorts with uninjected lesions experienced regression of at least one of these lesions and 25% had a net regression of all untreated lesions. Transcriptomic and immunohistochemistry analysis showed that immune activation and co-stimulatory transcripts were up-regulated but there was also increased adaptive immune resistance. Conclusions Intratumoral Tavo was well tolerated and led to systemic immune responses in advanced melanoma patients. While tumor regression and increased immune infiltration were observed in treated as well as untreated/distal lesions, adaptive immune resistance limited the response.

Oncolytic viruses induce local tumor destruction and inflammation. Whether virotherapy can also overcome immunosuppression in noninfected tumor areas is under debate. To address this question, we have explored immunologic effects of oncolytic herpes simplex viruses (oHSVs) in a genetically engineered mouse model of isocitrate dehydrogenase (IDH) wild-type glioblastoma, the most common and most malignant primary brain tumor in adults. Our model recapitulates the genomics, the diffuse infiltrative growth pattern, and the extensive macrophage-dominant immunosuppression of human glioblastoma. Infection with an oHSV that was armed with a UL16-binding protein 3 (ULBP3) expression cassette inhibited distant tumor growth in the absence of viral spreading (abscopal effect) and yielded accumulation of activated macrophages and T cells. There was also abscopal synergism of oHSV(ULBP3) with anti–programmed cell death 1 (anti–PD-1) against distant, uninfected tumor areas; albeit consistent with clinical trials in patients with glioblastoma, monotherapy with anti–PD-1 was ineffective in our model. Arming oHSV with ULBP3 led to upregulation of antigen processing and presentation gene sets in myeloid cells. The cognate ULBP3 receptor NKG2D, however, is not present on myeloid cells, suggesting a noncanonical mechanism of action of ULBP3. Overall, the myeloid-dominant, anti–PD-1–sensitive abscopal effect of oHSV(ULBP3) warrants further investigation in patients with IDH wild-type glioblastoma.

Immune checkpoint inhibitor (ICI) treatment has recently become a first-line therapy for many non–small cell lung cancer (NSCLC) patients. Unfortunately, most NSCLC patients are refractory to ICI monotherapy, and initial attempts to address this issue with secondary therapeutics have proven unsuccessful. To identify entities precluding CD8(+) T cell accumulation in this process, we performed unbiased analyses on flow cytometry, gene expression, and multiplexed immunohistochemical data from a NSCLC patient cohort. The results revealed the presence of a myeloid-rich subgroup, which was devoid of CD4(+) and CD8(+) T cells. Of all myeloid cell types assessed, neutrophils were the most highly associated with the myeloid phenotype. Additionally, the ratio of CD8(+) T cells to neutrophils (CD8/PMN) within the tumor mass optimally distinguished between active and myeloid cases. This ratio was also capable of showing the separation of patients responsive to ICI therapy from those with stable or progressive disease in 2 independent cohorts. Tumor-bearing mice treated with a combination of anti-PD1 and SX-682 (CXCR1/2 inhibitor) displayed relocation of lymphocytes from the tumor periphery into a malignant tumor, which was associated with induction of IFN-γ–responsive genes. These results suggest that neutrophil antagonism may represent a viable secondary therapeutic strategy to enhance ICI treatment outcomes.

Purpose: Pancreatic ductal adenocarcinoma (PDA) is rarely cured, and single-agent immune checkpoint inhibition has not demonstrated clinical benefit despite the presence of large numbers of CD8+ T cells. We hypothesized that tumor-infiltrating CD8+ T cells harbor latent antitumor activity that can be reactivated using combination immunotherapy. Experimental Design: Preserved human PDA specimens were analyzed using multiplex IHC (mIHC) and T-cell receptor (TCR) sequencing. Fresh tumor was treated in organotypic slice culture to test the effects of combination PD-1 and CXCR4 blockade. Slices were analyzed using IHC, flow cytometry, and live fluorescent microscopy to assess tumor kill, in addition to T-cell expansion and mobilization. Results: mIHC demonstrated fewer CD8+ T cells in juxtatumoral stroma containing carcinoma cells than in stroma devoid of them. Using TCR sequencing, we found clonal expansion in each tumor; high-frequency clones had multiple DNA rearrangements coding for the same amino acid binding sequence, which suggests response to common tumor antigens. Treatment of fresh human PDA slices with combination PD-1 and CXCR4 blockade led to increased tumor cell death concomitant with lymphocyte expansion. Live microscopy after combination therapy demonstrated CD8+ T-cell migration into the juxtatumoral compartment and rapid increase in tumor cell apoptosis. Conclusions: Endogenous tumor-reactive T cells are present within the human PDA tumor microenvironment and can be reactivated by combined blockade of PD-1 and CXCR4. This provides a new basis for the rational selection of combination immunotherapy for PDA. See related commentary by Medina and Miller, p. 3747

Non-response to anti-PD-1 antibodies is characterized by a “cold” tumor microenvironment lacking “exhausted” CD8+ T cells. We report on the first prospective Phase 2 trial of intratumoral plasmid IL-12 and pembrolizumab in advanced melanoma with “exhausted” T-cell (peCTL) poor tumors that had a low pre-test probability of response to anti-PD-1 Ab monotherapy. Patients had a 43% objective response rate (n=23, RECIST 1.1) with 38% complete responses. Interrogation of samples demonstrated that, while intratumoral IL-12 triggered a rapid increase in immune signaling and licensing of the tumor microenvironment, the compensatory adaptive resistance blunted clinical responses. However, by disrupting the PD-1/PD-L1 axis with pembrolizumab, the IL-12/IFN-γ feed-forward cycle was sustained, driving intratumoral cross-presenting DC subsets with increased TIL and TCR clonality and, ultimately, systemic cellular immune responses. Thus, combination of intratumoral plasmid IL-12 with pembrolizumab transformed poorly immunogenic lesions into effective in situ vaccines, attaining clinical activity in low peCTL tumors.

Anti-PD-1 immune checkpoint blockers can induce sustained clinical responses in cancer but how they function invivo remains incompletely understood. Here, we combined intravital real-time imaging with single-cell RNA sequencing analysis and mouse models to uncover anti-PD-1 pharmacodynamics directly within tumors. We showed that effective antitumor responses required a subset of tumor-infiltrating dendritic cells (DCs), which produced interleukin 12 (IL-12). These DCs did not bind anti-PD-1 but produced IL-12 upon sensing interferon γ (IFN-γ) that was released from neighboring Tcells. In turn, DC-derived IL-12 stimulated antitumor Tcell immunity. These findings suggest that full-fledged activation of antitumor Tcells by anti-PD-1 is not direct, but rather involves Tcell:DC crosstalk and is licensed by IFN-γ and IL-12. Furthermore, we found that activating the non-canonical NF-κB transcription factor pathway amplified IL-12-producing DCs and sensitized tumors to anti-PD-1 treatment, suggesting a therapeutic strategy to improve responses to checkpoint blockade.

Background: Chimeric antigen receptor (CAR) T cell therapy targeting CD19 results in marked tumor regression for patients with B-cell malignancies. It would be ideal to extend the success of CAR T cell therapy to common epithelial cancers. MUC1* is a post-translationally modified/cleaved form of mucin 1 (MUC1) that is frequently expressed on breast tumors, functions as a growth factor receptor, and is a promising antigen for CAR T cell therapy. Minerva Biotechnologies developed a CAR T (huMNC2-CAR44) that specifically recognizes the cleaved form of MUC1* and does not bind to full-length or MUC1* negative cells. huMNC2-CAR44 product consists of autologous T cells transduced with a lentiviral vector encoding humanized MNC2-scFv (MUC1* targeting head), sequences from CD8 α;; leader, hinge and transmembrane domains, 4-1BB and CD3ζ domains. Trial Design: NCT04020575 is a phase I study evaluating the safety and anti-tumor activity of adoptively transferred autologous T cells genetically modified to express huMNC2-CAR44 in patients with metastatic MUC1* breast cancer. After screening, leukapheresis is performed, CD8+ and CD4+ T cells are selected, transduced with huMNC2-CAR44, expanded, and antigen stimulated in vitro. Lymphodepletion with cyclophosphamide and fludarabine is followed 36-96 hours later by infusion of huMNC2-CAR44 CAR T cells in escalating doses (3.3 x 105 CAR+ T cells/kg - 1 x 107 CAR+ T cells/kg). Eligibility: Key inclusion criteria include metastatic breast cancer of known ER, PR and HER2 status which has MUC1* membrane expression > or = 30% by immunohistochemistry, measurable or evaluable disease, receipt of standard systemic therapies known to confer benefit, age >18, informed consent, adequate organ function, and KPS > or = 60%. Patients with active autoimmune disease or uncontrolled infection, contraindication to cyclophosphamide, anticipated survival < 3 months, and/or untreated CNS metastases are not eligible. Specific Aims: The primary objective is to identify the maximum tolerated dose of huMNC2-CAR44 T cells by CTCAE v5 and Lee criteria. Secondary objectives include persistence and phenotype of adoptively transferred huMNC2-CAR44 T cells and preliminary antitumor activity in all patients with measurable disease by RECIST 1.1. Exploratory objectives include trafficking of huMNC2-CAR44 T cells to tumor sites, effector function of huMNC2-CAR44 T cells in vivo, association between tumor MUC1* expression and huMNC2-CAR44 T cell persistence and response, change in tumor immune microenvironment by multiplex immunohistochemistry in pre and post-treatment tumor biopsies. Statistical Design: Dose escalation or de-escalation is tested in cohorts of 3 patients each using standard "3+3" dose-finding targeting a T cell dose that is associated with a true DLT rate < 33% and > than 17%. DLT period is between day 0 and 28. Once the MTD has been determined, up to 15 more patients will be enrolled in each of 3 expansion cohorts (Luminal, HER2 positive, and TNBC) to estimate the anti-tumor activity in these patient populations and to inform future huMNC2-CAR44 T cell trials. Present accrual: Study is open to screening and enrollment in dose escalation. Up to 69 patients may be enrolled in dose escalation and expansion phases. Contact information: To refer patients or obtain more information, please contact immunotherapy@seattlecca.org. Citation Format: Jennifer Marie Specht, David Maloney, Cecilia Yeung, Qian (Vicky) Wu, Cynthia Bamdad. Phase I study of adoptive immunotherapy for advanced MUC1* positive breast cancer with autologous T cells engineered to express a chimeric antigen receptor, huMNC2-CAR44 specific for a cleaved form of MUC1 (MUC1*) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-13-07.

More than 80% of gastric cancer is attributable to stomach infection with Helicobacter pylori (Hp). Gastric preneoplastic progression involves sequential tissue changes, including loss of parietal cells, metaplasia and dysplasia. In transgenic mice, active KRAS expression recapitulates these tissue changes in the absence of Hp infection. This model provides an experimental system to investigate additional roles of Hp in preneoplastic progression, beyond its known role in initiating inflammation. Tissue histology, gene expression, the immune cell repertoire, and metaplasia and dysplasia marker expression were assessed in KRAS+ mice +/−Hp infection. Hp+/KRAS+ mice had severe T-cell infiltration and altered macrophage polarization; a different trajectory of metaplasia; more dysplastic glands; and greater proliferation of metaplastic and dysplastic glands. Eradication of Hp with antibiotics, even after onset of metaplasia, prevented or reversed these tissue phenotypes. These results suggest that gastric preneoplastic progression differs between Hp+ and Hp− cases, and that sustained Hp infection can promote the later stages of gastric preneoplastic progression.

Cutaneous T-cell lymphoma (CTCL) is a rare, incurable CD4+ T cell malignancy of the skin with a 5-year survival rate of less than 30% in advanced stages. Immune checkpoint inhibitors, such as anti-PD-1 antibodies, have shown dramatic clinical efficacy in multiple advanced cancers, but the majority of cancer patients do not respond to these treatments. The clinical use of immunotherapies will increase considerably in the near future; therefore, predictive biomarkers of response to stratify patients for treatment are needed to limit potentially devastating adverse effects and reduce costs for healthcare systems. A clinical trial of the anti-PD-1 antibody pembrolizumab in CTCL showed that 38% of patients have a durable clinical response. However, standard tests, including comprehensive immunohistochemistry and single-cell quantification of PD-1 expression, have so far failed to identify a predictive biomarker for pembrolizumab response in this cohort. We reasoned that deep profiling of the CTCL tumor microenvironment (TME) using CODEX–a novel technology that allows for highly multiplexed tissue microscopy with >50 simultaneous parameters–could provide insight into the mechanisms of pembrolizumab response and enable prediction. We analyzed the CTCL TME using a tissue microarray of matched biopsies taken before and after pembrolizumab therapy in 7 responders and 7 non-responders. Imaging of 55 markers allowed discriminating malignant CD4+ tumor cells from reactive CD4+ T cells based on nuclear size and differential expression of CD7, CD25 and Ki-67. Unsupervised machine learning followed by supervised curation identified 21 different cell type clusters with spatial information. Integrating these data using advanced computational analysis revealed 10 distinct, conserved cellular neighborhoods (CNs) in the CTCL TME that changed in frequency and distribution during pembrolizumab therapy. In responders, effector-type CNs, including a tumor/dendritic cell CN and a tumor/CD4+ T cell CN, were significantly increased after treatment. In contrast, in non-responders, an immunosuppressive-type CN enriched in regulatory T cells was significantly increased before and after therapy. Importantly, the global frequencies in the tissues of the cell types defining these CNs were not different between patient groups. In addition, RNA sequencing of matched tissue sections revealed higher expression of effector-type cytokines and chemokines in responders. In sum, highly multiplexed analysis of the CTCL TME architecture in combination with RNA sequencing allows discovering novel, predictive spatial biomarkers of immunotherapy response and will pave the way for future studies that functionally address the identified cell types and cellular interactions. Citation Format: Christian M. Schürch, Darci J. Phillips, Belén Rivero Gutierrez, Magdalena Matusiak, Salil S. Bhate, Graham L. Barlow, Steven P. Fling, Nirasha Ramchurren, Robert H. Pierce, Martin A. Cheever, Michael S. Khodadoust, Robert West, Youn H. Kim, Garry P. Nolan. Cellular neighborhoods predict pembrolizumab response in cutaneous T cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6669.

e18510 Background: SNS-301 is a first-in-class immune activating agent targeting human aspartyl (asparaginyl) β-hydroxylase (ASPH). ASPH is overexpressed in +20 tumor types. SNS-301 is an inactivated λ-bacteriophage, engineered to express a highly immunogenic fragment of ASPH fused to the phage gpD coat protein, previously shown to safely and effectively generate CD4, CD8 and B cell responses in a Phase I study (NCT04034225). Anti-PD-1 antibodies such as pembrolizumab and nivolumab can induce durable clinical responses in 13-16% SCCHN patients. While ASPH is frequently expressed in SCCHN, effective de novo responses to this tumor-specific antigen may be limited due to immune tolerance. Our hypothesis is that SNS-301 will break tolerance and trigger a significant anti-ASPH T-cell immune response, which in combination with anti-PD-1 blockade, will result in improved anti-tumor responses in this difficult-to-treat patient population. Methods: This is a Phase I/II study of SNS-301 delivered intradermally in combination with pembrolizumab. The population consists of 30 patients with ASPH+ locally advanced unresectable or metastatic/recurrent SCCHN who are actively receiving pembrolizumab or nivolumab for ≥12 weeks and have a best response of stable disease (SD) or first evidence of progressive disease (PD). A safety run-in will be performed for safety and any dose limiting toxicities (DLT) using a modified rolling six design prior to proceeding to full enrollment. Patients will provide a pre-SNS-301 tumor sample, an on-treatment tumor biopsy and an optional biopsy at PD to characterize the tumor microenvironment and correlate with clinical outcomes. SNS-301 in combination with pembrolizumab is given until confirmed PD or unacceptable toxicity. Study Objectives: Safety, tolerability and anti-tumor activity such as objective response rate, duration of response, progression-free and overall survival, immune responses, tumor/immune biomarkers and their associated treatment outcome. Results: Three patients have been enrolled. There have been no DLTs. Emerging safety data includes G1 adverse events with one G1 injection site pain. Patient status includes 1 PD and 2 patients with SD of at least six weeks duration. Immunological analyses will be presented. Conclusions: Early safety/efficacy warrant further evaluation of this combination therapy. Trial will be proceeding to full enrollment after the safety run-in is completed. Clinical trial information: NCT04034225.

e19009 Background: Breast cancer, the most common cancer in sub-Saharan Africa (SSA), is characterized by poor survival. An accurate assessment of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor 2 receptor (HER2) status, typically via immunohistochemistry (IHC), is considered essential to provide prognostic data and guide therapeutic decision-making. However, due to inaccessible IHC services, these data are often unavailable in many parts of SSA; alternate methods need to be explored. Given the lab infrastructure developed in response to the HIV pandemic, RT-PCR testing is more readily accessible and feasible in SSA. Here we assess the potential of RT-PCR in evaluating the receptor status of women with breast cancer in Uganda. Methods: We enrolled women with a new diagnosis of invasive breast cancer at the Uganda Cancer Institute. Demographic and clinical data were obtained. A formalin-fixed paraffin embedded (FFPE) specimen was utilized for quantitative RT-PCR, using a validated assay for the detection of the ER, PR and HER2. Receptor expression levels were expressed as relative quantity (RQ) compared to housekeeping genes (CALM2). HER2 IHC results were categorized as negative (score 0 or 1+) or positive (3+); 2+ results (n=6) were excluded as FISH testing was not performed. Unstained slides were sent to the Fred Hutchinson Cancer Research Center for IHC. Receiver operating characteristic (ROC) analysis was applied to compare RT-PCR to IHC (gold standard). Results: We analyzed interim data (anticipated N=100 of an ongoing study) from 32 women aged 35 to 56 years. The majority of women (25, 78%) presented with advanced stage disease. Of the 32 cancers, 18 were ER+ (56%), 10 were PR+ (31%), 9 were HER2+ (28%) and 8 were triple negative (25%) by IHC. From ROC analysis, the AUC were 0.94, 0.95, and 0.81 for ER, PR, and HER2 respectively with high sensitivity and specificity (Table). Conclusions: Despite the tremendous need, the ability to detect the ER, PR, and HER2 via IHC in SSA is limited. Here we demonstrate the favorable test characteristics of RT-PCR when compared to IHC. Given the relatively wide accessibility of RT-PCR and endocrine therapy for breast cancer, as well as the recent inclusion of trastuzumab in the WHO’s Essential Medicines List, the results of this study have both direct diagnostic and therapeutic implications. Clinical trial information: NCT03518242 . [Table: see text]