Retrospective characterization of cell‒cell relationships in the tumor microenvironment provides significantly better predictive power than PD-L1 expression, tumor mutational burden (TMB), or gene expression profiles. In this small study assessing the safety and possible efficacy of gemcitabine and pembrolizumab in immunotherapy-naïve patients with NSCLC who have received prior treatment, we investigated both standard and novel immune parameters in 16 enrolled patients (NCT03083808). The combination of gemcitabine and pembrolizumab could be administered safely but did not demonstrate synergism compared with historical controls. Novel findings of this study are that elevated frequencies of regulatory T cells near CD3 T cells at baseline were associated with improved outcome to treatment (p < 0.05). Integrating this Treg/T cell relationship metric together with overall T-cell density yielded a tumor inflammation score that correlated (p < 0.002) with disease response. We postulate that this is indicative of an ongoing anticancer immune response. Additionally, while prior studies documented that IgG Ab responses to TAA can identify targets of a coordinated T and B cell response and evidence of immune surveillance, this study found that high autoantibody responses, while not statistically significant, trended toward a worse outcome (p = 0.06). This suggests to us that tumors may have developed mechanisms to escape the immune response to these TAAs.

Abstract Background: Despite advances in cancer genomics and immunotherapy, most cancer vaccine strategies target a limited set of canonical or mutated cancer antigens, capturing only a fraction of tumor antigenicity. Malignant cells generate a vast, largely unexplored repertoire of non-canonical “dark matter” antigens arising from the dark genome. Many of these antigens are frequently absent from normal tissues and enriched by and possibly responsible for oncogenic dysregulation. We have developed a vaccine strategy, DPV-001, that includes canonical and more than 400 dark matter cancer antigens and have evaluated DPV-001 in a combination immunotherapy trial for patients with HNSCC. This phase 1b trial tripled response rates over historical experience with anti-PD-1 and encouraged identification of the antigens targeted by the therapeutic anti-cancer immune response. Here we report the strategy we are employing and preliminary results on identifying targeted cancer antigens from patients on this trial. Methods: Eligible patients were randomized 1:1 to receive DPV-001 with or without a GITR agonist antibody (INCAGN-1876), with all patients receiving sequential PD-1 blockade (retifanlimab) starting day 15. Patient sera were analyzed by HuScan™ phage display immunoprecipitation (PhIP), covering 29,371 human proteins and splice isoforms. Antibody development was used as a surrogate for antigen-specific T cell responses. The canonical and dark matter cancer antigens in DPV-001, and an HNSCC HLA peptidome database, are being used to focus investigations of targets recognized by patients that received DPV-001. Results: PhIP detected Ab was assessed in 11 patients, including 7 patients from Arm 2 (received GITR). One-week post-infusion GITR serum levels were characterized as low (Avg 163 reads/million) in 3 patients and high (avg 585 reads/million) in 4 patients (p < 0.002). There were no significant differences between GITR Low and GITR High groups when comparing their smoking history, CPS score, or body weight. At baseline, patients in the GITR Low group had significantly fewer strong Ab signals (Avg 148) to the NCBI 35.1 proteome targets (>10x beads only maximum signal) compared to the GITR High group (avg 596, p=0.002) or Arm 1 (No-GITR, Avg 406, p=0.042). Characterization of B and T cell responses to HNSCC antigens is ongoing. Conclusions: This 18 pt trial of DPV-001 and sequenced PD-1 +/- GITR shows a promising RR. Identification of the antigens targeted in patients with objective clinical responses may enable development of a new generation of cancer vaccines. Citation Format: Traci Hilton, Ryan Meng, Guo Hui Gan, Tarsem Moudgil, Christopher Paustian, Noah Simons, Venkatesh Rajamanickam, Shawn M. Jensen, Elie Adwani, Sierra J. Smith, Matthew H. Taylor, Olivier Fesneau, Thomas M. Duhen, Irene Shih, Myungkyu Jang, Annie Long, Abigail Staeck, Tijana Jovanovic, Brady Bernard, Christie Tanisha, Noriko Iwamoto, Yuriko Minegishi, Koji Ueda, William L. Redmod, Lessli M. Rushforth, Takashi Shimada, Patrick Rethwisch, Carlo B. Bifulco, Hong-ming Hu, Walter J. Urba, Yoshinobu Koguchi, Marcus A. Couey, Richard B. Bell, Jianguo Huang, Eric Tran, Brian Piening, Rom Leidner, Bernard A. Fox. Search for targets of anti-cancer immunity in patients receiving an off-the-shelf cancer’s dark matter vaccine, DPV-001, combined with anti-PD-1 +/- anti-GITR in head and neck squamous cell cancer (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB425.

Improving responses to neoadjuvant therapy for patients with locally advanced rectal cancer has the potential to improve organ preservation and disease-free survival. Knowing which patients may need therapeutic escalation or de-escalation from standard-of-care treatment remains an area of investigation. We previously reported the primary and secondary endpoints of our single-arm study combining transforming growth factor beta receptor inhibitor, Galunisertib, with neoadjuvant chemoradiation in patients with locally advanced rectal cancer. Here we analyze RNA sequencing data obtained from tissue biopsies at baseline and after 2 weeks of galunisertib. Differences in expression of genes associated with MYC, inflammation, and epithelial-to-mesenchymal transition were observed between complete responders (CR) and <CR, with galunisertib upregulating MYC pathway expression in CR. Radiosensitivity and TGFβ response scores demonstrated limited ability to predict for response to galunisertib + chemoradiation. Typically treatment-resistant consensus molecular subtype 4 (CMS4), characterized by TGFβ expression, and metabolic subtype (CMS3) were associated with response to galunisertib + chemoradiation. Differences in correlations between RNA based measures of cell composition and immunohistologic quantification of infiltrates and extracted MRI parameters were observed for CIBERSORT, MCPcounter, and xCell methodologies. Based on these data, we hypothesize that the stromal radioresistant phenotype driven by TGFβ can be overcome by the addition of galunisertib to chemoradiation in rectal cancer.

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common adult soft-tissue sarcomas (STSs), yet therapeutic progress remains limited because of the absence of recurrent oncogenic driver mutations. To identify tumor suppressors contributing to UPS pathogenesis, we performed a customized in vivo CRISPR/Cas9 screen in mice. This approach identified BRCA1-associated protein 1 (BAP1) as a potent tumor suppressor in STS. Integrative analyses using RNA sequencing, multiplex immunohistochemistry, and flow cytometry revealed that Bap1-deficient sarcomas exhibited a markedly immunosuppressive tumor microenvironment. Consistent with these findings, BAP1 protein expression was reduced in human UPS, whereas polo-like kinase 1 (PLK1) expression was elevated. Functional studies demonstrated that PLK1 was required for the growth and survival of Bap1-deficient sarcomas. Pharmacologic inhibition of PLK1 with volasertib significantly suppressed tumor growth in both syngeneic and autochthonous mouse models. Moreover, combining PLK1 inhibition with anti–PD-1 therapy enhanced tumor control and improved survival compared with either treatment alone. Together, these results identify PLK1 as a potential therapeutic vulnerability in BAP1-deficient sarcomas and support further evaluation of combined PLK1 inhibition and immune checkpoint blockade as a treatment strategy for a subset of STSs.

Biliary tract cancers (BTC) pose clinical challenges due to poor chemotherapy response and aggressive disease course. We evaluated patient-derived tumor organoid-based drug sensitivity testing as a tool to guide therapy. In this multicenter study, 26 tumor organoids were successfully derived from 43 patients with BTC and tested with an average of 50 cancer-directed therapies using the Clinical Laboratory Improvement Amendments-certified PARIS assay. Despite most organoids being from late-stage disease, 24/26 (92.3%) exhibited strong sensitivity to one or more targeted agents. Active drugs included inhibitors of EGFR/HER2, MEK, ERK, BCR-ABL and SRC family, mTOR, PI3K, MDM2, BCL2, and BET. Drug sensitivities aligned with known genetic biomarkers but were also observed in cultures lacking them, indicating ex vivo testing can expand actionability beyond genomics. In five cases, results guided therapy; one patient with an FGFR-BICC1 fusion refractory to FGFR inhibitors responded to dasatinib, achieving symptomatic improvement, stable disease, and >8-month survival. SIGNIFICANCE:Ex vivo drug testing of tumor-derived organoids is clinically feasible and can be used to identify personalized treatment options for patients with BTC, to evaluate the functional relevance of genomic biomarkers, and to guide treatment in real time.

Undifferentiated pleomorphic sarcoma (UPS) is among the most common soft tissue sarcomas (STS) in adults. For decades, little therapeutic progress has been made for STSs, including UPSs. Targeted therapies for tumors driven by specific genetic mutations have proven to be more effective than standard chemotherapies. However, the molecular pathogenesis of UPSs remains unknown, hindering the development of targeted therapies for UPSs. Approximately 65% of UPSs harbor TP53 mutations, but somatic mutation of Trp53 alone is insufficient to induce sarcomas in vivo. The addition of Rb1 mutation alongside a Trp53 mutation induces sarcomas in vivo, though with low frequency of tumor onset and slow growth. The role of other genes in facilitating Trp53-driven sarcomas is largely unknown. Through a customized in vivo CRISPR/Cas9 screen of 35 genes commonly mutated in UPSs, followed by individual gene validation in vivo, we discovered that Bap1 knockout cooperates with Trp53 knockout to induce sarcomas in vivo. Furthermore, we demonstrated that Fat1 deletion increased the onset frequency and growth of Trp53 and Rb1-driven sarcomas in a genetically engineered mouse model. Through multiplex immunohistochemistry and flow cytometry, we found that mouse sarcomas induced by Trp53 and Rb1 mutations are significantly enriched with immune cells compared to other mouse sarcomas we generated in vivo. Finally, we show that PARP inhibition may be a potential targeted therapy for RB1-loss STSs and BRD4 inhibition may be a potential targeted therapy for FAT1-loss STSs. Jianguo Huang, Xingliang Liu, Warren Floyd, William Haugh, Andrea R. Daniel, Zhaoyu Sun, Nerissa T. Williams, Melissa J. Kasiewicz, Yaping Wu, Diana M. Cardona, Brian Piening, John T. Welle, Wesley K. Rosales, Venkatesh Rajamanickam, So Young Kim, Eric Xu, Lixia Luo, Yan Ma, Kristianne M. Oristian, Omar Lopez, Nicholas E. Sibinga, Rutulkumar Patel, Ziqiang Zhang, Alexander J. Lazar, Corinne M. Linardic, Brady Bernard, William L. Redmond, Walter J. Urba, David G. Kirsch. Direct in vivo CRISPR screen identifies BAP1 and FAT1 as potent tumor suppressors in sarcomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6707.

618 Background: Biliary tract cancers (BTC), including cholangiocarcinomas and gallbladder adenocarcinomas, present significant therapeutic challenges due to limited treatment options and poor prognoses. We report findings from the CLIA-certified PARIS assay, evaluating drug sensitivities of patient-derived tumor organoids (PDTOs) to a panel of oncology drugs. Methods: PDTOs were successfully cultured from 27 out of 46 live tumor samples obtained from 43 BTC patients. The majority of patients presented with advanced metastatic disease (60% stage IV, 13% stage III, 10% stage II, 4.4% stage I, 10% unknown) and had exhausted standard therapeutic options. Each PDTO culture underwent testing against an average of 50 drugs, encompassing chemotherapeutic agents and targeted therapies. Results: Comparative analysis of PDTO drug sensitivities with patients' prior treatment histories revealed non-responsiveness to >80% of drugs associated with clinical progression, including gemcitabine, cisplatin, and targeted therapies for FGFR translocations. In contrast, 100% (26/26) of the samples demonstrated significant sensitivity to one or more targeted agents, particularly inhibitors of EGFR, MEK, ERK, mTOR, PI3K, MDM2, BCL2, and BET families. Despite these shared sensitivities, each individual PDTO culture exhibited unique responses to targeted therapies, highlighting the genetic and phenotypic diversity between BTC patients. Comparison of ex vivo drug sensitivities with tumor genomic profiles validated oncogenic drivers such as HER2 amplification, KRAS and PIK3CA pathogenic mutations, correlating with sensitivities to EGFR/HER2 inhibitors, MEK inhibitors, and PI3K inhibitors, respectively. While mutations in KRAS , BRAF , BRCA1 , BRCA2 , ERBB2 , ERBB3 , or MET are present in less than 8% of cases, their role as biomarkers in BTCs requires further validation. PDTOs offer a promising tool to expedite this validation process. The PARIS assay results guided treatment decisions for five patients with metastatic disease, two of whom maintained treatment for over 4 weeks. Notably, two patients showed clinical benefit with everolimus (5 weeks) and dasatinib (11 weeks), experiencing symptom relief and reduced ascites. Intriguingly, 5 out of 7 PDTOs with FGFR alterations exhibited exceptional PARIS test responses to dasatinib, suggesting potential efficacy linked to FGFR activation. Conclusions: In conclusion, our study demonstrates that ex vivo drug testing of PDTO cultures can inform treatment selection and potentially accelerate drug approvals for BTC, leveraging therapies approved for other cancers. Early integration of such assays in patient management, possibly at diagnosis, holds promise for improving outcomes in this challenging disease.

Precision medicine relies on mutational profiling, but clinical actionability remains limited for many cancer patients. We evaluated the feasibility and actionability of ex-vivo drug sensitivity testing across solid tumors, focusing on rare cancers. This study uses the PARIS® CLIA-certified test, assessing patient-derived tumor cells (PDTCs) responses to a broad oncology drug panel. Tumor specimens (biopsies, fluids, or surgical samples) were shipped to our laboratory to arrive within 48h and cultured in either 3D or 2D with cancer type specific protocols to generate short term tumor cell cultures. PDTCs were tested with a panel of oncology drugs (up to 42 drugs /sample), customized for each patient based on cancer type, oncologist recommendations, and genomic features. Reports, ranking drug responses, were sent to oncologists within ∼3 weeks on average. 86 patients with diagnosis of rare cancers (occurring in fewer than 15 out of 100,000 people each year) were consented to the SEngine IRB protocol and had a successful PARIS® test. The overall actionability was 85%, defined by one or more drugs exhibiting exceptional/good responses based on absolute and relative metrics of drug responses in our database. The cohort included biliary tract cancers (n=26), sarcomas (n=15), low grade serous ovarian cancers (n=15), cancer of unknown primary (n=4), appendiceal cancers (n=4), esophageal and gastric cancers (n=6), brain and nervous system cancers (n=2). Notably, targeted therapies showed the strongest drug sensitivities, often revealing actionable insights beyond genomic predictions. In the LGSOC cohort in the recurrent setting the results demonstrated that the PARIS® test recapitulates the clinical resistance of LGSOC to most chemotherapies but can identify multiple targeted treatments for nearly all patients. Importantly, few of these agents wouldn't have been chosen based on standard molecular testing, and multiple patients have demonstrated months to years of disease stabilization, biomarker remission and good quality of life with PARIS® -informed therapies (Gray et al., NPJ 2023, and unpublished data). As an example, one LGSOC case, which did not present any actionable biomarkers and had progressed after standard chemotherapy, achieved disease stabilization with 29 months duration, no hospitalizations, following a PARIS®-informed therapy with regimens including Lapatinib, Everolimus, Trametinib and Letrozole. Currently, the patient has biomarker remission demonstrated by ctDNA and CA-125. Cases from other cancer types will be presented. The PARIS® test offers an unbiased ex-vivo measurements of oncology-drug responses and can identify responders to targeted therapies also in cases lacking biomarker-drug matches, particularly in rare tumors with complex genomics or gene fusions without targeted drugs and limited on-label treatment options. Rachele Rosati, Payel Chatterjee, Alex C. Rajewski, Marwah Al-Aloosi, Lauren R. Appleyard, Vaishnavi Pallem, Shalini Pereira, Robert L. Diaz, Christopher J. Kemp, Ryan Jones, Brady Bernard, Pashtoon M. Kasi, Kiran Turaga, Michael Castro, Tara Seery, Arun Singh, Sameek Roychowdhury, Melind Javle, Jill Alldredge, Soledad Jorge, Kathryn Pennington, Barbara A. Goff, Heidi J. Gray, Elizabeth M. Swisher, Carla Grandori. Functional precision medicine: Uncovering high actionability in rare cancers beyond genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4716.

Undifferentiated pleomorphic sarcoma (UPS) is among the most common soft tissue sarcomas (STS) in adults. For decades, little therapeutic progress has been made for STSs, including UPSs. Targeted therapies for tumors driven by specific genetic mutations have proven to be more effective than standard chemotherapies. However, the molecular pathogenesis of UPSs remains unknown, hindering the development of targeted therapies for UPSs. Approximately 65% of UPSs harbor TP53 mutations, but somatic mutation of Trp53 alone is insufficient to induce sarcomas in vivo. The addition of Rb1 mutation alongside a Trp53 mutation induces sarcomas in vivo, though with low frequency of tumor onset and slow growth. The role of other genes in facilitating Trp53-driven sarcomas is largely unknown. Through a customized in vivo CRISPR/Cas9 screen of 35 genes commonly mutated in UPSs, followed by individual gene validation in vivo, we discovered that Bap1 knockout cooperates with Trp53 knockout to induce sarcomas in vivo. Furthermore, we demonstrated that Fat1 deletion increased the onset frequency and growth of Trp53 and Rb1-driven sarcomas in a genetically engineered mouse model. Through multiplex immunohistochemistry and flow cytometry, we found that mouse sarcomas induced by Trp53 and Rb1 mutations are significantly enriched with immune cells compared to other mouse sarcomas we generated in vivo. Finally, we show that PARP inhibition may be a potential targeted therapy for RB1-loss STSs and BRD4 inhibition may be a potential targeted therapy for FAT1-loss STSs. Citation Format: Jianguo Huang, Xingliang Liu, Warren Floyd, William Haugh, Andrea R Daniel, Zhaoyu Sun, Nerissa T Williams, Melissa J Kasiewicz, Yaping Wu, Diana M Cardona, Brian Piening, John T. Welle, Wesley K Rosales, Venkatesh Rajamanickam, So Young Kim, Eric Xu, Lixia Luo, Yan Ma, Kristianne M Oristian, Omar Lopez, Nicholas E. S. Sibinga, Rutulkumar Patel, Ziqiang Zhang, Alexander J Lazar, Corinne M Linardic, Brady Bernard, William L Redmond, Walter J Urba, David G Kirsch. Direct in vivo CRISPR screen identifies BAP1 and FAT1 as potent tumor suppressors in sarcomagenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr A025.

Radiation therapy (RT) is a potent mediator of cancer cell death, but normal cells including T cells are killed in the treatment field. In this work we use the photoconvertible Kaede mouse model to identify cells that move from the tumor-draining lymph node (TdLN) to untreated (NT) and RT treated tumors in mice. We demonstrate selective recruitment of effector and memory CD8 T cells from the TdLN to tumors. By uniquely focusing on the recruited cells we see enrichment for progenitor exhausted cells in both NT and RT tumors, but over time their activation is distinct in RT tumors. RT results in upregulation of the activation/retention marker CD69 particularly in progenitor exhausted cells during the early phase of RT tumor refilling. Using irrelevant antigen vaccination, we demonstrate that CD69 upregulation is antigen non-specific. Tumors grown in IFNAR1-/- mice reveal that CD69 upregulation is IFN-mediated, resulting from RT-mediated adjuvant signaling in the irradiated tumors. scRNASeq analysis of photoconverted versus resident T cells demonstrates the distinct tumor environment of irradiated tumors redirects newly entered T cell differentiation. These data demonstrate an initial non-specific retention prior to antigen-specific engagement contributes to refilling of irradiated tumors with implications for therapy. This research was funded by NCI R01CA182311, NCI R01CA244142, and R01CA208644, and by the Providence Foundation. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Background NCPs derived from non-coding regions of the genome are a recently discovered source of non-mutated HLA-presented cancer antigens. Here we studied the immune response of a patient with NSCLC that received adjuvant treatment with DPV-001 immunotherapy that contained canonical and non-canonical antigens. The patient is disease free at 10 years. Materials and This patient received 7 doses of DPV-001 at 3-week intervals. PBMCs and serum were collected regularly to assess antibodies (PhIPseq), peripheral lymphocytes populations (flow cytometry), and TCR repertoires. A metastasis resected 5 months into treatment generated a tumor cell line and tumor-infiltrating lymphocyte (TIL) culture. The HLA-presented immunopeptidome of the patient’s NSCLC cell line was characterized by mass spectrometry and identified more than 300 NCP from 5’UTR. Antibody responses to the canonical protein downstream of the 5’UTR were used to prioritize peptides for detection of a T cell response in TIL. The TCR of a responding T cell was identified, expressed, and evaluated. Results The identified TCR recognized a NCP and autologous NSCLC cell line, it was not detected pretreatment. Current studies are evaluating whether this TCR recognizes other cancer cell lines. The detection of a T cell response to a NCP presented by the patient’s cancer expands the range of possible cancer antigens that can be targeted with the potential to improve outcomes for patients with cancer. Support: Murdock Charitable Trust, Providence Portland Medical Foundation, Nancy Lematta, Lynn Loacker, Cindy and Steve Harder, The Chiles Foundation, Robert W. Franz, Elsie Franz Finley and by NCI SBIR grant #R44 CA121612. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Renal cell carcinoma (RCC) is responsive to immunotherapy despite having a low tumor mutational burden. Interestingly, the degree of CD8+ T cell infiltration in RCC negatively correlates with outcomes, suggesting that a deeper understanding of the tumor microenvironment may provide critical insight to help improve clinical outcomes. We conducted a multi-omics approach including flow cytometry, multiplex immunohistochemistry, and single-cell RNA-seq to compare immune cell composition, gene expression, spatial distribution, and cell-cell interactions among a cohort of primary breast cancer, head and neck squamous cell carcinoma, non-small cell lung cancer, and RCC specimens. Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) revealed increased exhausted CD8 + (Tex) and CD4+CD8+ double positive (DP) T cells and decreased CD4+ T cells including regulatory T (Treg) cells in RCC samples compared to other tumor types. scRNA-seq analysis of TILs confirmed these results and showed Tex cells as a major population of CD8+ T cells. Using biomarkers for tumor-reactive cells including 4-1BB, CD39, and CD103 along with a gene signature to identify neo-antigen reactive T cells, we found that the Tex population contained putative tumor-reactive T cells and were not merely bystander cells. Exhausted CD8+ and DP T cells in RCC contain tumor-reactive T cells and may represent a novel cell population capable of targeting RCC. Funding: Funding was provided by a sponsored research grant from GlaxoSmithKline, the Providence Portland Medical Foundation, and the M. J. Murdock Charitable Trust. Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Single cell RNA-seq combined with CITE-seq and TCR-seq has allowed for a deep characterization of anticancer immunity. Here we report on a single patient with head and neck squamous cell cancer (HNSCC) who underwent initial resection in 2020. Twenty-one months later, when the patient recurred and went on a combination immunotherapy trial, they had three serial biopsies performed. Samples of blood and tumor from 2020 were sorted into 6 CD4 subsets: Blood Mem CD4, Blood Treg, PD1/ICOS(-/-) TIL, PD1/ICOS(+/+) TIL (putative tumor-specific CD4), PD1/ICOS(+/-) TIL, and TIL Treg. On progression the patient received a biologic containing >300 proteins for genes that are overexpressed by HNSCC followed by anti-PD-1 at wk 2, with biopsies taken at baseline, wk 2 and 8. Phenotypic and molecular studies were performed. Over 20% of the total peripheral T cell repertoire at the start of immunotherapy were CD4 clonotypes present in 2020; this shrank to less than 15% by week 8. Within the biopsy samples PD1+ TIL expanded while Treg contracted during immunotherapy. Blood Mem, Blood Treg and PD1/ICOS(-/-) TIL were all mainly found in the periphery, while PD-1 TIL were found in both blood and tumor. Interestingly, the majority of TIL Treg were later found in either the blood or tumor, not both. These results provide insights into how Immunotherapy may change the clonal and phenotypic distribution of CD4 T cells. Ethics Approval: PSJH IRB# 2020000480 Support: Incyte, Murdock Charitable Trust, Providence Portland Medical Foundation, Nancy Lematta, Lynn Loacker, Cindy and Steve Harder, The Chiles Foundation, Robert W. Franz and Elsie Franz Finley Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Treatment of head and neck squamous cell carcinoma (HNSCC) is complex, with immunotherapy demonstrating potential yet facing challenges due to the tumor’s unique immune microenvironment. Biomarker expression has been employed to predict immune responses, albeit with limited efficacy. We predicted that due to the complexity of the immune response, no singular biomarker could consistently forecast the efficacy of immunotherapy. Consequently, we implemented a multi-index strategy that encompassed a comprehensive study of the networks associated with HNSCC. Secretomes from 72 explants obtained from six HNSCC patients (comprising 72 secretome profiles: 18 untreated and 54 treated) were subjected to an information-theoretic analysis. The resultant phenotypes were corroborated in two external cohorts (TCGA, n = 518; GEO GSE159067, n = 102). This methodology revealed two reproducible co-expression phenotypes—Activation (Act) and Infiltration (Inf)—that were significantly correlated with T-cell functionality. Only tissues exhibiting both Act and Inf phenotypes demonstrated a favorable response to anti-PD-1 and anti-GITR ex vivo, and displayed an increased presence of CD8+ T-cells in proximity to cancer cells. External validation in two different RNA-seq cohorts reproduced the two phenotypes and verified that patients possessing both signatures had significantly prolonged overall survival following PD-1/PD-L1 therapy. This study emphasizes the importance of multiple-index characterization of HNSCC tissues in enhancing patient classification and predicting immunotherapy efficacy.

AbstractRetrospective characterization of cell-cell relationships in the tumor microenvironment provides significantly better predictive power than PD-L1 expression, tumor mutational burden (TMB), or gene expression profiles. In this small study assessing the safety and possible efficacy of gemcitabine and pembrolizumab in immunotherapy-naïve patients with NSCLC who have received prior treatment, we investigated both standard and novel immune parameters on 16 enrolled patients. The combination of gemcitabine and pembrolizumab could be administered safely but did not demonstrate synergism compared with historical controls. Novel findings of this study are that elevated frequencies of Tregulatory cells near CD3 T cells at baseline was associated with improved outcome to treatment (p<0.05). Integrating this Treg / T cell relationship metric together with overall T cell density yielded a tumor inflammation score which correlated (p<0.002) with disease response. We postulate this is indicative of ongoing anti-cancer immune response. Additionally, while prior studies documented that IgG Ab responses to TAA can identify targets of a coordinated T and B cell response and evidence of immune surveillance, this study found that high autoantibody responses, while not statistically significant, trended towards a worse outcome (p=0.06). This suggests to us that tumors may have developed mechanisms to escape the immune response to these TAAs.