Pre-treatment Samples Research Articles

Abstract 2427: The development of a tissue-agnostic genome-wide methylome enrichment MRD assay for applications across the cancer care continuum for head and neck malignancies

Abstract Background: Plasma-based tests to quantify circulating cell-free DNA cancer signal have emerged as viable applications across the cancer continuum, from early detection to optimal disease management. Here we demonstrate the feasibility of a tissue-agnostic, genome-wide methylome enrichment platform based on cell-free methylated DNA immunoprecipitation and high throughput sequencing (cfMEDIP-seq) for cancer detection, cancer signal quantification, and prognostication in head and neck cancer (HNC). Methods: Pre-treatment plasma samples from individuals with newly diagnosed stage I-IV HPV+ or HPV- HNC were analyzed with a bisulfite-free, non-degradative, genome-wide methylome enrichment platform using 5-10 ng of cell-free DNA. For cancer detection, a machine learning classifier used differentially methylated regions to distinguish cancers from non-cancer controls. The area under the receiver operating characteristic curve (AUC) and 95% confidence intervals were calculated. Cancer signals were quantified from average normalized counts across informative methylated regions and a 95% specificity threshold. For prognostication, events were defined as recurrence, progression, or death due to HNC, whichever occurred earliest. Time to event was compared for samples with cancer signal quantities above versus below the threshold. Post-treatment and longitudinal plasma samples from individuals with Stage I-IVB HNC (HPV+ and HPV- included) will be analyzed for recurrence prediction and detection of relapse. More than 100 patients and 300 samples will be analyzed. Results: For cancer detection, 92 pre-treatment plasma samples from HNC cases were distinguished from 674 controls with an AUC of 0.96 (0.94, 0.98). The AUC was 0.93 (0.86, 1.0) for Stage I, 0.93 (0.83, 1.0) for Stage II, 0.96 (0.94, 0.99) for Stage III, and 0.97 (0.96, 0.99) for Stage IV. For prognostication, 91 pre-treatment samples were included (7 stage I, 16 stage II, 23 stage III, 45 stage IV). Median follow-up time was 50.6 months with 27 events. Likelihood of recurrence or progression was significantly higher in samples with cancer signal above the threshold [hazard ratio 5.4 (95% CI 2.25, 12.95), log-rank P<0.001]. In the upcoming analysis, data will be reported on the ability to predict recurrence and relapse in post-treatment samples. Conclusions: The cfMeDIP-seq approach demonstrated robust detection of HNC, across all stages and subtypes, and the ability to predict recurrence and progression from pre-treatment samples. We will report training data with cross validation to predict recurrence and relapse using post-treatment and longitudinal sampling. Collectively, data from these studies indicate that genome wide methylome enrichment has multiple use cases across the care continuum for patients with HNC. Citation Format: Geoffrey Liu, Jun Min, Yarong Wang, Justin Burgener, Ben Brown, Karen Budhraja, Junjun Zhang, Owen Hall, Shu Yi Shen, Martha Pienkowski, Shao Hui Huang, Laurie Ailles, Katrina Rey-McIntyre, Jeremy B. Provance, Eduardo Sosa, Cynthia Frye, Scott Bratman, Brian Allen, Joshua T. Jones, Abel Licon, Jing Zhang, Anne-Renee Hartman, Daniel D. De Carvalho. The development of a tissue-agnostic genome-wide methylome enrichment MRD assay for applications across the cancer care continuum for head and neck malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2427.

Abstract 5186: Spatial single-cell deconvolution of the bone marrow microenvironment to define predictive biomarker for response to immunotherapy in patients treated with a check point inhibitor in smoldering multiple myeloma, phase II trial of nivolumab in combination with lenalidomide and low dose dexamethasone

Abstract Background:The immune system is critical for response and resistance in all cancer types and therapies. Patients with Smoldering Multiple Myeloma (SMM) have altered immune cell composition in the bone marrow (BM). However, little is known about the role of BM spatial immune profiling in patients treated with checkpoint inhibitors in MM. Methods: We performed spatial profiling with imaging mass cytometry of trephine biopsies of the BM (n=15; pre-treatment n=7 and post-treatment n=8) from patients with high-risk SMM enrolled in a phase II trial of nivolumab in combination with lenalidomide and low dose dexamethasone (NCT02903381). Patients received 6 cycles of induction therapy of nivolumab (240mg) at days 1 and 15, in combination with lenalidomide (25mg) at days 1-21 and dexamethasone at days 1, 8, and 15. The induction phase was followed by nivolumab (240mg) and lenalidomide (25mg) maintenance for another 6 cycles. A treatment cycle was defined as 28 consecutive days for a total of 12 months period. Event monitoring was performed up to 3 years. The primary efficacy outcome was progression-free survival 2 years after treatment. Results: Eight patients were enrolled on this study from January 2017 to June 2017. The study was put on hold due to the toxicity of checkpoint inhibitors observed in other trials of MM. The median age for all patients at enrolment was 60 years (range 49 to 81), with 5 males (62%) and 3 females (38%). All patients met criteria for high risk SMM disease. Median follow-up for all 8 patients was 38.7 months. Median overall survival was not reached; median progression-free survival was 33.8 months. Seven total patients had a response of Minimal Response (MR) or better, and 4 patients who initially responded eventually progressed. The total number of patients who were followed for at least two years and remained progression-free was 4 of 8 evaluable patients (50%; 90% CI: 19 - 81%).Spatial profiling with a panel of 37 markers on the pre-treatment samples allowed us to assess the states and abundances of multiple immune subpopulations, including rare regulatory NK cells. To assess the accuracy of our cell type annotation, we correlated the proportion of malignant plasma cells identified per patient with the value determined by pathologists for clinical diagnosis. These measures were significantly positively correlated (R=0.92, p<0.01). Spatial neighborhood analysis revealed statistically significant interactions enriched in the responders, even with our small sample size (t-test; p < 0.05). Conclusions and future directions: This pilot study suggests that BM spatial immune profiling before receiving immune check point inhibitors can be a biomarker for response in patients with SMM. Citation Format: Yoshinobu Konishi, Giuseppe Tarantino, Yiwen He, Romanos Sklavenitis-Pistofidis, Sujal I. Shah, Katherine Towle, Christian J. Cea-Curry, Oksana Zavidij, Ruben D. Carrasco, Scott J. Rodig, Jon C. Aster, David Liu, Gad Getz, Irene M. Ghobrial. Spatial single-cell deconvolution of the bone marrow microenvironment to define predictive biomarker for response to immunotherapy in patients treated with a check point inhibitor in smoldering multiple myeloma, phase II trial of nivolumab in combination with lenalidomide and low dose dexamethasone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5186.

Abstract 7634: HER2 extracellular domain in serum samples from HER2+ metastatic breast cancer patients as a potential biomarker of progression-free survival

Abstract Introduction: The detection of serum biomarkers holds potential for non-invasive methods for early cancer detection, monitoring of disease progression, and assessment of treatment responses leading to earlier interventions that may significantly improve patient outcomes. Human epidermal growth factor receptor 2 (HER2) overexpression plays a key role in the prognosis of HER2+ breast cancer (BC). While HER2 is well-established as a cell surface marker, its soluble extracellular domain (ECD) in serum has gained attention as a potential prognostic biomarker. Elevated levels of HER2 ECD (>15ng/ml) have been associated with a poor outcome. However only a small percentage of primary BC patients have levels of HER2 ECD >15ng/ml. Research has suggested that HER2 targeted therapies can influence the rate of HER2 ECD shedding. Therefore, this study aimed to explore the association between circulating HER2 ECD levels with progression-free survival (PFS) and overall survival (OS) in patients with metastatic HER2+ BC. Methods: Matched Pre and post treatment serum samples (n=25) from BC patients enrolled in TH V THL (ICORG (CTRIAL-IE) 11-10/NCT01526369) clinical trial were assessed. Human ErbB2 (HER2) ELISA Kit (Invitrogen) was used to detect levels of soluble HER2 ECD in the serum. Serum levels were stratified as Low (<300pg/ml n=5), Intermediate (300-700pg/ml n=10) or High (>700pg/ml n=10). Patient data correlation was used to assess PFS and OS, Kaplan-Meier survival curves were constructed based on the serum levels of HER2 ECD. Significance p values were determined using log-rank (Mantel-Cox) test. Results: Levels of HER2 ECD detected (94-1730pg/ml) did not exceed 15ng/ml for any sample. There was no significant difference in HER2 ECD levels between treatment arms (TH vs. THL) for PFS (p=0.4685) or OS (p=0.1376). There was no change between the pre and post treatment HER2 ECD serum levels in the samples tested (p=0.344). However, when pre-treatment samples were stratified based on Low, Intermediate or High HER2 ECD levels, there was a significant increase in PFS for patients in the High cohort. Median PFS increased from 8.31 months in the Low cohort to 13.57 months in the Intermediate cohort. The median PFS increased further to 22.24 months in the High cohort (Log rank p=0.0197). The same parameters were applied for the analysis of OS, which also improved as the level of HER2 ECD increased. Median OS increased from 35.19 months (Low) to 68.7 months (Intermediate), with a Median OS not reached for the High cohort (Log rank p=0.077). Conclusions: Patients with HER2 ECD levels above 700pg/ml display a survival advantage in this limited study. Further validation and prospective studies are warranted to fully establish the clinical utility of HER2 ECD as a biomarker in metastatic HER2+ BC. Citation Format: Debbie O'Reilly, Marc Nolan, Ausra Teiserskiene, Bryan T. Hennessy, John Crown, Denis M. Collins. HER2 extracellular domain in serum samples from HER2+ metastatic breast cancer patients as a potential biomarker of progression-free survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7634.

Abstract 5151: High serum kidney injury marker-1 and high baseline tumor PD-L1 protein expression levels are independently associated with treatment effect in adjuvant nivolumab plus ipilimumab vs placebo in localized clear cell renal cell carcinoma

Abstract Introduction: CheckMate 914 (CM-914) Part A is a double-blind, phase III randomized trial of the Nivolumab (NIVO) plus Ipilimumab (IPI) vs placebo (PBO) in localized ccRCC. Our prior report from this study suggested a disease-free survival (DFS) benefit for NIVO+IPI among patients with Fuhrman grade 4, TNM stages PT2a and PT4, or sarcomatoid features, although the sample size was limited. It is known that high circulating KIM-1 is associated with worse DFS after nephrectomy. In this exploratory post hoc analysis, we investigated whether high KIM-1 may help identify a subset of patients who benefit from adjuvant NIVO+IPI. Methods: Patients (n=816) with RCC after nephrectomy were randomized in CM-914 Part A to receive NIVO+IPI or PBO as previously described. Assessment of KIM-1 levels was performed using enzyme linked immunoassay (ELISA) on pre-treatment (n=584) and matched on-treatment blood samples (n=584). We used pre-treatment tumor samples to assess PD-L1% tumor cell expression (%TC) in an PD-L1 IHC 28-8 pharm Dx assay. The association between biomarkers and DFS outcomes was investigated by Kaplan-Meier (KM) and Cox proportional hazards analysis. Results: Median baseline serum KIM-1 level was 102 (9.9 - 1055.7) pg/mL. Serum KIM-1 levels were higher in males vs females, ≥65 yrs vs <65 yrs, Asian vs white patients, and patients with partial vs radical nephrectomy. In the PBO arm, subjects with highest quartile of pre-treatment KIM-1 had significantly worse DFS than those from the three lower quartiles. In contrast, this DFS risk among the subjects within the highest KIM-1 quartile was mitigated with NIVO+IPI treatment. Among patients within the highest quartile of pre-treatment KIM-1, there was trend for better DFS for NIVO+IPI versus PBO, HR=0.6 (0.34-1.04). Increase in KIM-1 during study therapy was positively associated with higher DFS rate in both arms. Multivariable analysis showed that PD-L1 %TC was predictive at predefined PD-L1 cutoffs (>=1%, >=5%, and >=10%), associating with improved DFS compared to placebo. Subjects with high PD-L1 expression had a DFS benefit from NIVO+IPI independent of KIM-1. Conclusion: Circulating KIM-1 and tumor PD-L1 expression may enrich for benefit from IO therapy in adjuvant ccRCC and hence holds promise for informing risk stratification and patient inclusion in neoadjuvant or adjuvant clinical trials. Citation Format: Sai Vikram Vemula, Wenxin Xu, Yu Wang, Xiaowen Liu, Jorge Ruiz de Somocurcio, David McDermott, Jun Li, Rupal Bhatt, Chung-Wei Lee, Burcin Simsek, Saurabh Gupta, Robert Motzer. High serum kidney injury marker-1 and high baseline tumor PD-L1 protein expression levels are independently associated with treatment effect in adjuvant nivolumab plus ipilimumab vs placebo in localized clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5151.

Abstract 6804: High IL12family levels are associated with pathologic complete response amongst HER2- patients in the neoadjuvant I-SPY2 TRIAL

Abstract Background: Immune contexture of the tumor microenvironment (TME) is associated with response to therapy. We hypothesize that expression of interleukin (IL) family of cytokines which plays essential roles in the activation and differentiation of immune cells, shapes the landscape of therapeutic response in breast cancers. We leverage published gene expression, molecular and clinical data from the I-SPY2 Trial (PMID35623341) - a neoadjuvant trial for locally advanced breast cancers. We explored the contribution of expression-based signatures of IL signaling at pre-treatment in relation to pathological complete response (pCR). Methods: We curated 32 IL gene sets from literature. Signatures scores characterizing IL signaling were calculated from gene expression profiles of pre-treatment tumor samples from 987 I-SPY2 patients in the first 10 treatment arms of the trial. Association of IL signatures with pCR in HR/HER2 receptor subtypes, and the five I-SPY response-predictive subtypes (RPS) were evaluated using non-parametric tests. Significance levels were defined at adj. p < 0.05. Correlations of IL signatures with multiplex immunofluorescence staining of immune infiltrates in the PD1-inhibitor (PD1i) arm were also assessed. Results: High expression of IL signatures in HER2- patients, regardless of HR status, was associated with pCR especially in the Trebananib, Veliparib/Carboplatin (VC) and PD1i arms. In the PD1i arm, expression of almost all IL signatures was highly correlated with density of T, CD8+ cytotoxic T, CD8- T, and B cells and their colocalization with tumor cells; IL-6 signatures correlated with density of proliferating tumor cells.Within RPS subtypes, enrichment of IL signatures in HER2-/Immune+ such as pro-inflammatory IL-1 and IL-12 signaling was associated with pCR in the VC, PD1i, Ganetespib, and control arms. Furthermore, we identified an IL-12 signature in the HER2-/Immune-/DRD- subtype associated with pCR, with responders characterized by high IL-12 family signaling (Fisher adj. p < 0.05). IL-12 family signaling was positively associated with CD8+ cytotoxic T, Th1 and B cell enrichment, IFN-γ signaling, and inflammatory response overall and in HER2- breast cancers. Conclusion: High pre-treatment expression of IL signatures was associated with HER2- receptor and RPS HER2-/Immune+ subtype-specific response to I-SPY2 agents, and the colocalization of immune and tumor cells in the tumor bed. In HER2- breast cancers, high levels of IL-12 family signaling were associated with response to therapy and enrichment of known IL-12-related pathways that stimulate effective anti-cancer response including CD8+ T cell expansion, Th1 cell differentiation, B cell activation, IFN-γ secretion, and inflammatory response. These findings suggest that IL signaling may serve as a valuable predictive biomarker in this patient population. Citation Format: Kingsley V. Chow, Silver Al Khafaji, Denise Wolf, Christina Yau, Kailey Dubinsky, Elene Tsopurashvili, Kirthiga Ramalingam, Fluorentine van Nouhuijs, Zheyun Xun, Anna van Montfort, Diane Heditsian, Douglas Yee, Laura Esserman, Michael Campbell, Laura van't Veer, Rosalyn W. Sayaman. High IL12family levels are associated with pathologic complete response amongst HER2- patients in the neoadjuvant I-SPY2 TRIAL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6804.

Abstract 1211: Biomarker analysis of pembrolizumab and ramucirumab neoadjuvant therapy for PD-L1-positive stage IB-IIIA lung cancer: EAST ENERGY trial

Abstract Background: Neoadjuvant treatments with novel combination immunotherapies for resectable non-small cell lung cancer (NSCLC) are being developed. Combination therapy with antiangiogenic agents and anti-PD-1/PD-L1 antibody has shown enhanced antitumor effects. EAST ENERGY is a single-arm multicenter phase 2 trial of neoadjuvant pembrolizumab and ramucirumab in patients with PD-L1-positive stage IB-IIIA NSCLC (NCT04040361). In this study, we aimed to identify biomarkers that predicts the efficacy of the neoadjuvant therapy. Furthermore, we aimed to elucidate the effect of the neoadjuvant therapy on the tumor microenvironment (TME). Methods: Patients enrolled in the EAST ENERGY trial (n=24) for whom pre- and post-treatment specimens were available (n=20) were included in this study. RNA-sequencing and multiplex immunohistochemistry (mIHC) were used to examine the association between the immunological phenotype of the TME and major pathological response (MPR), and changes in the TME before and after treatment. Results: MPR was achieved in 50.0% (12/24) of patients. Biomarker analyses with RNA-sequencing revealed that high expression of cancer-associated genes, and upregulation of interferon-gamma and interferon-alpha response in pre-treatment tumor were associated with MPR (q value<0.001). mIHC in pre-treatment samples revealed that abundance of CD8+ T cells in the pre-treatment TME was associated with MPR (p=0.039). On the other hand, infiltration of immunosuppressive cells such as Treg or M2-macrophages were not associated with resistance for the treatment (p=0.836, 0.639, respectively). Next, to clarify the effect of the neoadjuvant therapy on the TME, comparative analyses of pre- and post-treatment samples was performed. In gene set enrichment analysis, the neoadjuvant therapy enhanced gene set associated with interferon-gamma response (q value=0.051). Furthermore, angiogenesis-related gene set were downregulated after the treatment (q value=0.090). mIHC analyses revealed that the number of CD8+ T cells in the TME was significantly increased after the neoadjuvant therapy (p<0.001). Notably, Tregs/CD8+ T cells ratio and the number of M2-macrophage in the TME were significantly decreased after the neoadjuvant therapy (p=0.032, 0.003, respectively). Conclusion: In the neoadjuvant therapy with pembrolizumab and ramucirumab, immune-activated status before treatment serves as a potential biomarker for predicting treatment response. Moreover, combining ramucirumab with pembrolizumab reduced immunosuppressive cells in the TME and inhibited tumor angiogenesis. These results suggest that this combination therapy modifies the TME and augments antitumor immunity. Larger clinical trial is warranted to assess the efficacy of neoadjuvant therapy with ramucirumab and pembrolizumab in patients with PD-L1-positive NSCLC. Citation Format: Kotaro Nomura, Shogo Kumagai, Shohei Koyama, Keiju Aokage, Yoshihisa Shimada, Kiyotaka Yoh, Masashi Wakabayashi, Miki Fukutani, Hideki Furuya, Tomohiro Miyoshi, Kenta Tane, Joji Samejima, Tetsuro Taki, Takuo Hayashi, Jun Matsubayashi, Genichiro Ishii, Norihiko Ikeda, Hiroyoshi Nishikawa, Masahiro Tsuboi. Biomarker analysis of pembrolizumab and ramucirumab neoadjuvant therapy for PD-L1-positive stage IB-IIIA lung cancer: EAST ENERGY trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1211.

Abstract 2955: HPV E6* and senescence-related gene expression in cervical cancer

Abstract Previous work in our lab found that increased expression of the E6* variant of the HPV E6 viral protein was significantly correlated with worse response to standard-of-care chemoradiation (CRT) and progression-free survival, independently of HPV genotype. In vitro validation showed that overexpression of E6* induces senescence in an HPV 16-positive cervical cancer cell line. Given these findings, we hypothesized that increased E6* expression mediates resistance to CRT by inducing a reversible state of tumor cell senescence. We created a senescence score using ten genes that have been consistently reported in the literature as associated with senescence. Using an institutional cohort of 68 patients with cervical cancer with prospectively collected clinical outcome data, we performed bulk RNA sequencing to assess senescence scores before CRT and determine whether there is an association between senescence scores and E6* expression. Using a second institutional cohort of 35 patients with cervical cancer treated with CRT with pre and posttreatment tumor biopsies, we performed bulk RNA sequencing to evaluate the same relationship and determine whether expression of senescence-related genes was affected by CRT. Nine cervical cancer cell lines stratified into low- and high-E6* expression groups will be assessed regarding their senescence score, beta-galactosidase staining, and SASP expression before and after CRT. In our first cohort, there was no correlation between E6*:E6 expression ratio and senescence score, regardless of HPV genotype. In our second cohort, CRT increased senescence score in CRT-treated samples (p < 0.01), but p16INK4a expression significantly decreased compared to pre-treatment samples (p < 0.01). Increased senescence score was not associated with cancer recurrence. There was no significant difference in senescence scores between different HPV genotypes before therapy. Tumors that were positive for HPV 16 or HPV 18 had significantly lower senescence scores compared to HPV negative tumors (p = 0.037 and p = 0.038, respectively) after CRT. Among HPV 16-positive tumors (n = 12), there was no correlation between E6*:E6 expression ratio and senescence score before or after CRT. We found no association between senescence score and E6* expression, and between senescence score and cancer recurrence after CRT in our dataset. Additionally, our results suggest that p16INK4a might not be an appropriate senescence marker in tumors that have intrinsically high p16INK4a expression, such as in cervical cancer, reinforcing the need for a cervical cancer-specific senescence signature. We are currently testing beta-galactosidase and SASP expression in vitro and in vivo before and after CRT. Citation Format: Louise Medina Bengtsson, Matthew Inkman, Naoshad Muhammad, Leahan Castillo, Naomi Msengi, Tyler McKinnish, Nishanth Gabriel, Jin Zhang, Julie Schwarz. HPV E6* and senescence-related gene expression in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2955.

Abstract 3660: Plasma cell free DNA hydroxymethylation profiling reveals anti-PD1 treatment response and resistance biology in non-small cell lung cancer

Abstract Background: Treatment with immune checkpoint inhibitors (ICIs) targeting programmed death-1 (PD-1) can yield durable anti-tumor responses, yet not all patients respond to ICIs. Current approaches to select patients who may benefit from anti-PD-1 treatment are insufficient. 5-hydroxymethylation (5hmC) analysis of plasma-derived cell free DNA (cfDNA) presents a novel non-invasive approach for identification of therapy response biomarkers which can tackle challenges associated with tumor biopsies such as tumor heterogeneity and serial sample collection. Methods: 151 blood samples were collected from 31 non-small cell lung cancer (NSCLC) patients before therapy start and at multiple timepoints whilst on therapy. Blood samples were processed to obtain plasma-derived cfDNA, followed by enrichment of 5hmC-containing cfDNA fragments through biotinylation via a two-step chemistry and binding to streptavidin coated beads. 5hmC-enriched cfDNA and whole genome libraries were prepared in parallel and sequenced to obtain whole hydroxymethylome and whole genome plasma profiles, respectively. Results: Comparison of on-treatment timepoint to matched pre-treatment samples from same patients revealed that anti-PD-1 treatment induced distinct changes in plasma cfDNA 5hmC profiles of responders, as judged by RECIST, relative to non-responders. In responders, 5hmC accumulated over genes involved in immune activation such as IFNγ and IFNα response, inflammatory response, and TNFα signaling, whereas in non-responders 5hmC increased over epithelial to mesenchymal transition genes. The Molecular Response to anti-PD-1 treatment, as measured by 5hmC changes in plasma cfDNA profiles were observed early, starting with the first cycle of treatment. Comparison of pre-treatment plasma samples revealed that anti-PD-1 treatment response- and resistance-associated genes can be captured by 5hmC profiling of plasma-derived cfDNA. Conclusions: These results demonstrate that 5hmC profiling can identify response and resistance associated biological pathways in plasma samples, offering a novel method for non-invasive prediction and monitoring of immunotherapy response in NSCLC. Citation Format: Gulfem D. Guler, Yuhong Ning, Ceyda Coruh, Tierney Phillips, Maryam Nabiyouni, Kyle Hazen, Aaron Scott, Wayne Volkmuth, Samuel Levy. Plasma cell free DNA hydroxymethylation profiling reveals anti-PD1 treatment response and resistance biology in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3660.

Abstract 5532: Differences in immune contexture across metastatic sites and tissue compartments in ovarian cancer

Abstract Introduction: Characterizing the immune contexture within the tumor microenvironment (TME) can provide valuable biological insights that may impact treatment regimens and improve clinical outcomes. The advent of neoadjuvant chemotherapy has provided a unique opportunity to study matched, pre- and post- treatment sample pairs, but these samples are often obtained from different metastatic sites. Innate differences in the immune profiles across metastatic sites could therefore be a confounding factor when attempting to determine treatment-related effects. Furthermore, immune contexture can also vary by region within the TME, so results could vary depending on the regions of interest (ROIs) selected for analysis. Methods: 36 matched, advanced primary ovarian cancer samples were obtained pre- and post- treatment with neoadjuvant chemotherapy aimed at reducing tumor burden. The first biopsy was taken at the time of initial diagnosis, whereas the second biopsy was collected after 3-4 rounds of carboplatin paclitaxel chemotherapy. Each slide was processed using a 26-plex multiplex immunohistochemistry (mIHC) assay, which interrogates immune cell densities and functional states for a broad range of immune cell populations, including CD8+ T cells, Tregs, TH1s, B cells, dendritic cells (DCs), granulocytes, macrophages, and monocytes. Tumor and stroma ROIs were selected by a board-certified pathologist using H&E slides immediately adjacent to the mIHC samples, and cell densities were calculated separately for each of the two tissue compartments. Results: When comparing pre-treatment samples from the omentum (n = 8) with post-treatment samples from the omentum (n = 14), all of the statistically significant changes were found within the tumor compartment, with higher densities in the post-treatment samples for CD8+ T cells (p = 0.009), Tregs (p = 0.012), DCs (p = 0.04), and monocytes (p = 0.027). In contrast, when comparing post-treatment samples from the omentum (n = 14) and the ovary (n = 14), all of the significant differences were found within the stroma compartment, with higher densities in the omentum for CD8+ T cells (p = 0.002), Tregs (p = 0.043), TH1s (p = 0.003), and macrophages (p = 0.048). Conclusions: These results demonstrate that the site of metastasis can be a significant confounding factor when making statistical comparisons between variables such as timepoint and that differences in immune cell density before and after treatment may be more apparent in the tumor, while differences between tissue types may be more apparent in the stroma. Further research into the immune profiles of different metastatic sites is warranted, but our findings demonstrate the importance of considering both tissue type and ROI selection strategy when designing a study with the intent to detect differences between longitudinal patient samples. Citation Format: Benjamin Tate, Elias Pavlatos, Teresa Kim, Kiaya Wilson, Erik Cid, Jian Rao, Gottfried Konecny, Joanna Pucilowska. Differences in immune contexture across metastatic sites and tissue compartments in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5532.

Abstract 6425: Harnessing big data from pan-cancer concerning more than 1300 patients with immune checkpoint inhibitors identifies novel predictive biomarkers

Abstract Immune-checkpoint inhibitors (ICIs) have revolutionized cancer therapy, yet many patients face the challenges of either not benefiting or developing resistance to ICIs. Advances in genome sequencing allow the exploitation of high-dimension oncological data in the research and development of precision immuno-oncology. We conducted a comprehensive literature review involving cancer patients treated with ICIs [anti-programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1), anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4), or their combination] and collected available transcriptomic data from pretreatment biopsies along with corresponding clinical outcomes. Biomarkers for ICI response were independently investigated within each study using the logistic regression method and combined with the odds ratio (OR) for meta-analysis. A total of 210 formalin-fixed paraffin-embedded pan-cancer samples with pretreatment biopsies from the National Cancer Center retrospective cohort were collected for validation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) were employed to validate the stable and clinically applicable utility of the genomic biomarker identified in our analysis. Across 18 studies, we identified 1106 cancer patients who received immunotherapy with pre-treatment transcriptomic data, including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), stomach adenocarcinoma (STAD), glioblastoma (GBM), and bladder urothelial carcinoma (BLCA), of which 8421 genes were available in all datasets. Meta-analysis based on a common effect model revealed that 332 genes were positively correlated with a favorable response to ICIs, and 404 genes were negatively associated with the therapeutic outcome, with all pooled OR being statistically significant (P <0.05). We identified a novel biomarker, ZBED2 (Zinc Finger BED-Type Containing 2), which demonstrated superior predictive value over other biomarkers for ICI efficacy (pooled OR, 0.596; 95% CI, 0.450-0.788, P =0.002). Similarly, RT-qPCR (AUC: 0.821) and IHC (AUC: 0.789) results further validated the predictive utility for ICI response in the real-world cohort of 210 cancer patients with pretreatment samples receiving ICIs. Additionally, we observed that the expression of ZBED2 was favorably associated with the overall survival of patients receiving immunotherapy (HR: 0.72). In summary, our study provides a in-depth analysis of potentially predictable biomarkers for ICIs in cancer and highlights the potential of large-scale meta-analyses in identifying stable biomarkers. ZBED2 emerges as a novel predictive biomarker for ICIs and may guide the implementation of risk-related therapeutic strategies. Citation Format: Peng Wu, Chaoqi Zhang, Ao Shen, Xuanyu Gu, Nan Sun, Jie He. Harnessing big data from pan-cancer concerning more than 1300 patients with immune checkpoint inhibitors identifies novel predictive biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6425.

Molecular and Clinical Determinants of Acquired Resistance and Treatment Duration for Targeted Therapies in Colorectal Cancer.

Targeted therapies have improved outcomes for patients with metastatic colorectal cancer, but their impact is limited by rapid emergence of resistance. We hypothesized that an understanding of the underlying genetic mechanisms and intrinsic tumor features that mediate resistance to therapy will guide new therapeutic strategies and ultimately allow the prevention of resistance. We assembled a series of 52 patients with paired pretreatment and progression samples who received therapy targeting EGFR (n = 17), BRAF V600E (n = 17), KRAS G12C (n = 15), or amplified HER2 (n = 3) to identify molecular and clinical factors associated with time on treatment (TOT). All patients stopped treatment for progression and TOT did not vary by oncogenic driver (P = 0.5). Baseline disease burden (≥3 vs. <3 sites, P = 0.02), the presence of hepatic metastases (P = 0.02), and gene amplification on baseline tissue (P = 0.03) were each associated with shorter TOT. We found evidence of chromosomal instability (CIN) at progression in patients with baseline MAPK pathway amplifications and those with acquired gene amplifications. At resistance, copy-number changes (P = 0.008) and high number (≥5) of acquired alterations (P = 0.04) were associated with shorter TOT. Patients with hepatic metastases demonstrated both higher number of emergent alterations at resistance and enrichment of mutations involving receptor tyrosine kinases. Our genomic analysis suggests that high baseline CIN or effective induction of enhanced mutagenesis on targeted therapy underlies rapid progression. Longer response appears to result from a progressive acquisition of genomic or chromosomal instability in the underlying cancer or from the chance event of a new resistance alteration.

Untranslated regions (UTRs) are a potential novel source of neoantigens for personalised immunotherapy.

Neoantigens, mutated tumour-specific antigens, are key targets of anti-tumour immunity during checkpoint inhibitor (CPI) treatment. Their identification is fundamental to designing neoantigen-directed therapy. Non-canonical neoantigens arising from the untranslated regions (UTR) of the genome are an overlooked source of immunogenic neoantigens. Here, we describe the landscape of UTR-derived neoantigens and release a computational tool, PrimeCUTR, to predict UTR neoantigens generated by start-gain and stop-loss mutations. We applied PrimeCUTR to a whole genome sequencing dataset of pre-treatment tumour samples from CPI-treated patients (n = 341). Cancer immunopeptidomic datasets were interrogated to identify MHC class I presentation of UTR neoantigens. Start-gain neoantigens were predicted in 72.7% of patients, while stop-loss mutations were found in 19.3% of patients. While UTR neoantigens only accounted 2.6% of total predicted neoantigen burden, they contributed 12.4% of neoantigens with high dissimilarity to self-proteome. More start-gain neoantigens were found in CPI responders, but this relationship was not significant when correcting for tumour mutational burden. While most UTR neoantigens are private, we identified two recurrent start-gain mutations in melanoma. Using immunopeptidomic datasets, we identify two distinct MHC class I-presented UTR neoantigens: one from a recurrent start-gain mutation in melanoma, and one private to Jurkat cells. PrimeCUTR is a novel tool which complements existing neoantigen discovery approaches and has potential to increase the detection yield of neoantigens in personalised therapeutics, particularly for neoantigens with high dissimilarity to self. Further studies are warranted to confirm the expression and immunogenicity of UTR neoantigens.

Clinical and genomic characterization of chemoradiation-resistant HPV-positive oropharyngeal squamous cell carcinoma.

Most patients with HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) have an excellent response to chemoradiation, and trials are now investigating de-escalated treatment. However, up to 25% of patients with HPV-positive OPSCC will experience recurrence, and up to 5% will even progress through primary treatment. Currently, there are no molecular markers to identify patients with poor prognosis who would be harmed by de-escalation. Herein we report the clinical and genomic characteristics of persistent HPV-positive OPSCC after definitive platinum-based chemoradiation therapy. Patients with HPV-positive OPSCC treated with curative intent platinum-based chemoradiation between 2007 and 2017 at two institutions and with a persistent locoregional disease were included. We evaluated clinical characteristics, including smoking status, age, stage, treatment, and overall survival. A subset of five patients had tissue available for targeted exome DNA sequencing and RNA sequencing. Genomic analysis was compared to a previously published cohort of 47 treatment-responsive HPV+ OPSCC tumors after batch correction. Mutational landscape, pathway activation, and OncoGPS tumor states were employed to characterize these tumors. Ten patients met the inclusion criteria. The tumor and nodal stages ranged from T1 to T4 and N1 to N2 by AJCC 8th edition staging. All patients were p16-positive by immunohistochemistry, and eight with available in situ hybridization were confirmed to be HPV-positive. The 1-year overall survival from the time of diagnosis was 57%, and the 2-year overall survival was 17%. TP53 mutations were present in three of five (60%) persistent tumors compared to 2% (one of 47) of treatment-responsive HPV-positive tumors (p = 0.008). Other genes with recurrent mutations in persistent HPV-positive OPSCC tumors were NF1, KMT2D, PIK3C2B, and TFGBR2. Compared to treatment-responsive HPV-positive tumors, persistent tumors demonstrated activation of DNA Repair and p53, EMT, MYC, SRC, and TGF-beta signaling pathways, with post-treatment samples demonstrating significant activation of the PI3K-EMT-Stem pathways compared to pretreatment samples. Chemoradiation-resistant HPV-positive OPSCC occurs infrequently but portends a poor prognosis. These tumors demonstrate higher rates of p53 mutation and activation of MYC, SRC, and TGF-beta pathways. A comparison of tumors before and after treatment demonstrates PI3K-EMT-Stem pathways post-treatment in HPV-positive tumors with persistent disease after platinum-based chemoradiation.

Biological insights from plasma proteomics of non-small cell lung cancer patients treated with immunotherapy.

Immune checkpoint inhibitors have made a paradigm shift in the treatment of non-small cell lung cancer (NSCLC). However, clinical response varies widely and robust predictive biomarkers for patient stratification are lacking. Here, we characterize early on-treatment proteomic changes in blood plasma to gain a better understanding of treatment response and resistance. Pre-treatment (T0) and on-treatment (T1) plasma samples were collected from 225 NSCLC patients receiving PD-1/PD-L1 inhibitor-based regimens. Plasma was profiled using aptamer-based technology to quantify approximately 7000 plasma proteins per sample. Proteins displaying significant fold changes (T1:T0) were analyzed further to identify associations with clinical outcomes using clinical benefit and overall survival as endpoints. Bioinformatic analyses of upregulated proteins were performed to determine potential cell origins and enriched biological processes. The levels of 142 proteins were significantly increased in the plasma of NSCLC patients following ICI-based treatments. Soluble PD-1 exhibited the highest increase, with a positive correlation to tumor PD-L1 status, and, in the ICI monotherapy dataset, an association with improved overall survival. Bioinformatic analysis of the ICI monotherapy dataset revealed a set of 30 upregulated proteins that formed a single, highly interconnected network, including CD8A connected to ten other proteins, suggestive of T cell activation during ICI treatment. Notably, the T cell-related network was detected regardless of clinical benefit. Lastly, circulating proteins of alveolar origin were identified as potential biomarkers of limited clinical benefit, possibly due to a link with cellular stress and lung damage. Our study provides insights into the biological processes activated during ICI-based therapy, highlighting the potential of plasma proteomics to identify mechanisms of therapy resistance and biomarkers for outcome.

Impact of metronomic trabectedin combined with low-dose cyclophosphamide on sarcoma microenvironment and correlation with clinical outcome: results from the TARMIC study.

Soft tissue sarcomas (STS) are diverse mesenchymal tumors with few therapeutic options in advanced stages. Trabectedin has global approval for treating STS patients resistant to anthracycline-based regimens. Recent pre-clinical data suggest that trabectedin's antitumor activity extends beyond tumor cells to influencing the tumor microenvironment (TME), especially affecting tumor-associated macrophages and their pro-tumoral functions. We present the phase I/II results evaluating a combination of metronomic trabectedin and low-dose cyclophosphamide on the TME in patients with advanced sarcomas. 50 patients participated: 20 in phase I and 30 in phase II. Changes in the TME were assessed in 28 patients using sequential tumor samples at baseline and day two of the cycle. Treatment notably decreased CD68 + CD163 + macrophages in biopsies from tumor lesions compared to pre-treatment samples in 9 of the 28 patients after 4 weeks. Baseline CD8 + T cell presence increased in 11 of these patients. In summary, up to 57% of patients exhibited a positive immunological response marked by reduced M2 macrophages or increased CD8 + T cells post-treatment. This positive shift in the TME correlated with improved clinical benefit and progression-free survival. This study offers the first prospective evidence of trabectedin's immunological effect in advanced STS patients, highlighting a relationship between TME modulation and patient outcomes.This study was registered with ClinicalTrial.gov, number NCT02406781.

Physiologic responses of Cownose Rays following freshwater dips for treatment of capsalid monogeneans.

Two Cownose Rays Rhinoptera bonasus were presented for management of a severe capsalid monogenean infection Benedeniella posterocolpa in a mixed species habitat at an aquarium. A series of freshwater (FW) dips were elected to mitigate parasite resistance to praziquantel due to the endemic monogenean population in the system. A pretreatment blood sample was opportunistically obtained, and subsequent samples were processed due to development of clinical signs. While the first FW dip was tolerated well and did not induce abnormal behavior, the second led to one individual presenting with tachypnea, pallor, and lethargy. Marked hematological abnormalities requiring medical intervention occurred in both individuals after the second dip. After treatment with elasmobranch Ringer's fluid therapy, sodium bicarbonate, prednisolone acetate, and vitamin E/selenium in the more severely affected ray, and elasmobranch Ringer's alone in the second ray, hematologic derangements and symptoms resolved. Hemoconcentration, hyperproteinemia, hyperlactatemia, and hyperglycemia were attributed to a stress response. A selective loss of urea after exposure to FW resulted in decreased plasma osmolality. Given the severity of the stress response and associated complications observed, hyposalinity treatments should be utilized with caution in this species. If this therapy is employed, the clinician is advised to be prepared to administer supportive care coinciding with the FW dip if necessary.

Clonal hematopoiesis of indeterminate potential (CHIP) and cardiovascular toxicity from androgen deprivation therapy in men with prostate cancer.

312 Background: Androgen deprivation therapy (ADT) with agonists of the gonadotropin releasing hormone (GnRH) receptor (e.g. leuprolide) is associated with coronary artery disease and major adverse cardiovascular (CV) events. CHIP, an age-related expanded somatic blood cell clone without other hematologic abnormalities, is associated with inflammation and accelerated atherosclerosis. Whether CHIP is associated with adverse CV outcomes in patients receiving ADT is unknown. Methods: Twenty-five men with localized prostate cancer pursuing radiotherapy plus ≥ 6 months of leuprolide were enrolled on a prospective biorepository. Using pre-treatment blood samples, whole exome sequencing libraries were generated; identified variants were filtered to retain &lt;1% MAF variants found amongst the three most common CHIP genes ( DNMT3A, TET2, ASXL1). Pre-treatment and 6-month blood samples from each patient subsequently were analyzed using a commercially available, validated 4-protein risk score that is predictive of 4-year major adverse CV events (Prevencio Inc HART CVE, Seattle WA). Multivariable logistic regression measured the association of CHIP with increased CV risk after initiation of leuprolide, defined as a clinically significant rise in 4-protein risk score at six months compared with baseline. Results: Median age was 70 years (IQR 65-76), 10 men (40%) were Black, and 4 men (16%) had a history of myocardial infarction. CHIP gene variants were found in 7 men (28%). Amongst the men with CHIP variants, 6 (85.7%) were Black. Amongst the entire cohort, 11/25 (44%) men had a clinically significant rise in CV risk score after six months of ADT. These men, compared with those who did not experience a rise in CV risk, were more often Black (72.7% versus 14.3%), on anti-hypertensive medication (72.7% versus 14.3%), and had higher baseline LDL cholesterol level (median 98 versus 81 mg/dL). On univariate analysis, harboring ≥1 CHIP variant was associated with a significantly increased odds of having an elevated CV risk score after 6 months of ADT. On multivariable analysis, after adjusting for race, anti-hypertensive medication use, and baseline LDL cholesterol, harboring ≥1 CHIP variant was associated with increased odds of having an elevated CV risk score after 6 months of ADT (adjusted OR 1.44, 95% CI 0.88-1.83), which did not meet statistical significance but limited by small sample size. Conclusions: Six months of GnRH-agonist leuprolide resulted in a rise in CV risk score in 44% of this cohort. CHIP variants, identified in 28% of this cohort, was associated with increased odds of experiencing a rise in CV risk score. Notably, Black men were more likely to harbor CHIP and experience increased CV risk after 6 months of leuprolide, respectively. Larger studies are underway to determine if CHIP variants may serve as a biomarker of ADT-induced CV risk, especially in older Black men.

Nucleosome profiling at AR binding sites (ARBS) to provide a prognostic tool for metastatic castration-resistant prostate cancer (mCRPC).

42 Background: Prostate cancer is the second leading cause of cancer death for men in the U.S., and metastatic castration-resistant prostate cancer (mCRPC) is the most advanced form. In previous work, we have reported prognostic significance of PTEN-PI3K-AKT pathway and DNA damage response aberrations detected in cell-free DNA (cfDNA) of mCRPC. Here we utilized nucleosome profiling pattern at androgen receptor (AR) binding sites to further investigate AR signaling pathway dysregulation of mCRPC. Methods: Using the next-generation sequencing PredicineScore liquid biopsy assay (low-pass whole genome sequencing, LP-WGS), we profiled cfDNA fragment coverage centered around AR binding sites (ARBS). For 118 mCRPC patient samples, we quantified sample-level ARBS nucleosome profiling abnormality score (ARBS score) by comparing its centric fragment coverage with normal plasma background using standard Z-score. ARBS score was correlated with clinical outcomes including PSA response rate (PSA RR), progression-free survival and overall survival (OS). Results: By comparing ARBS nucleosome profiling and normal plasma background, we found that mCRPC plasma samples had significant ARBS nucleosome depletion, reflective of transcription factor binding and persistent AR signaling. Notably, ARBS nucleosome depletion was prostate cancer specific, and did not occur in other examined cancer types. ARBS score was highly correlated with tumor fraction (R=0.82), indicating that high tumor fraction mCRPC sample had higher AR binding. Importantly, we found that high ARBS score (&gt;2) in pre-treatment samples (n=86) was associated with significantly shorter OS and lower PSA RR compared with low ARBS (≤2) group for patients receiving AR pathway inhibitor (ARPI) or taxane chemotherapy (13.4 vs. 19.4 months, p=7.3×10-3, 45.7% vs. 77.5%, p=5.2×10-3). By integrating genome-wide DNA methylation profiling, we further found that ARBS was hypo-methylated in mCRPC patients, and identified a strong negative correlation between DNA methylation beta value and ARBS score (R=-0.83). Conclusions: ARBS nucleosome profiling abnormality inferred from LP-WGS can serve as a key prognostic tool for mCRPC patients. Patients with high ARBS score had worse clinical outcomes, highlighting the potential clinical utility of cfDNA fragmentomics analysis for mCRPC patient stratification. Integration of mutation, copy number variation and fragmentomics profiling of ARBS may further enhance our understanding of molecular aberrations and treatment outcomes in mCRPC.

Abstract A032: Low non-immune stromal cell levels and high hypoxic tumor microenvironments are associated with pathologic complete response in the neoadjuvant I-SPY2 TRIAL

Abstract Background: Non-immune components of the tumor microenvironment (TME) play important roles in cancer. We explore two important components of the TME: non-immune stromal cell populations including fibroblasts, adipocytes, and endothelial cells; and hypoxic conditions. We leverage published gene expression-based biomarkers of stromal cell types and hypoxia, and molecular and clinical data from the I-SPY2 TRIAL (PMID: 35623341) – an adaptive neoadjuvant trial for locally advanced breast cancers. We assess the contribution of stromal cell populations and oxygen-poor environments at pre-treatment in shaping the landscape of immune enrichment and therapeutic response in breast cancers. Methods: We curated 14 stromal and 11 hypoxia gene sets, in addition to 35 immune gene sets from literature. Signatures scores of stromal, hypoxic, and immune TME were calculated from gene expression profiles of pre-treatment tumor samples from I-SPY2 patients (n=987) in the first 9 treatment arms of the trial plus control arms. Correlation of stromal and hypoxia signatures with immune signatures were assessed, and association of TME features with HER2/HR receptor status, I-SPY response-predictive subtype (RPS), and pathological complete response (pCR) were evaluated using non-parametric tests. All p-values were adjusted for multiple testing. Significance levels were defined at adj. p&amp;lt;0.05. Results: Both stromal and hypoxia signatures showed significant differential enrichment across HR/HER2 and RPS subtypes, and association with pCR. Several stromal signatures including adipocytes, endothelial cells, mesenchymal stem cells, and overall stromal score had the lowest enrichment in HR-/HER2- breast cancers, and in the RPS HER2-/immune+ subtype. Moreover, most stromal signatures showed anti-correlation with CD8+ and CD4+ T cell enrichment. Stromal signatures including endothelial cells, smooth muscle cells, and overall score were significantly lower in patients who achieved pCR. Two hypoxia gene sets demonstrated highly significant enrichment in HR- versus HR+ samples regardless of HER2 status. All hypoxia signatures varied significantly across RPS subtypes, with the highest scores in HER2-/Immune-/DRD+, especially relative to HER2-/Immune-/DRD- and HER2+/BP-Luminal subtypes. Hypoxia signatures strongly linked to hypoxia inducible factor (HIF) and angiogenesis via VEGF were positively associated with pCR. These hypoxia signatures showed treatment-specific associations with pCR, specifically in agents targeting mechanisms with known interactions with hypoxia-induced biology: trebananib (angiogenesis inhibitor), ganetespib (HSP90 inhibitor) and veliparib/carboplatin (DNA damage repair inhibitor). Conclusions: Hypoxia and stromal cell gene signatures show breast cancer subtype-specific biology. Relative absence of non-immune stromal cell types is associated with higher immune infiltration and higher pCR rates, while specific hypoxia pathways were associated with response to therapeutic agents targeting hypoxia-related pathways. Citation Format: Kailey Dubinsky, Elene Tsopurashvili, Denise M. Wolf, Zheyun Xu, Silver Al Khafaji Alkhafaji, Anna van Montfort, Christina Yau, Diane Heditsian, Allison Fiscalini, Laura J. Esserman, Laura van ‘t Veer, Rosalyn W. Sayaman. Low non-immune stromal cell levels and high hypoxic tumor microenvironments are associated with pathologic complete response in the neoadjuvant I-SPY2 TRIAL [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A032.

Predicting disease recurrence in limited disease small cell lung cancer using cell-free DNA-based mutation and fragmentome analyses.

Limited disease (LD) small cell lung cancer (SCLC) treated with definitive concurrent chemoradiotherapy (CCRT) potentially experience disease recurrence. We investigated the feasibility of circulating-tumor DNA (ctDNA)-based genomic and fragmentome analyses to assess the risk of recurrence. Targeted sequencing was conducted using pre-treatment and on-treatment blood samples from definitive CCRT-treated patients with LD-SCLC (n=50). Based on 12-month recurrence-free survival (RFS), patients were categorized into persistent-response (PeR, n=29) and non-PeR (n=21) groups. Fragmentome analysis was conducted using ctDNA fragments of different lengths: P1 (100-155 bp) and P2 (160-180 bp). Patients with TP53 (n=15) and RB1 (n=11) mutation in on-treatment samples demonstrated significantly shorter RFS than patients with wild-type (WT) (P=0.05, P=0.0014, respectively). Fragmentome analysis of all available on-treatment samples (n=26) revealed that the non-PeR group (n=10) had a significantly higher P1 range (P=0.003) and lower P2 range (P=0.002). The areas under the curves for P1, P2, and the fragmentation ratio (P1/P2) in distinguishing the PeR and non-PeR were 0.850, 0.725, and 0.900, respectively. Using optimal cut-off, longer RFSs were found with the low-fragmentation-ratio group than with the high-fragmentation-ratio group (not reached vs. 7.6 months, P=0.002). Patients with both WT RB1 and a low-fragmentation-ratio (n=10) showed better outcomes than patients with both mutated RB1 and a high-fragmentation-ratio (n=10; hazard ratio, 7.55; 95% confidence interval: 2.14-26.6; P=0.002). RB1 mutations and high fragmentation ratios correlated with early disease recurrence. Analyzing ctDNA could help in predicting early treatment failure and making clinical decisions for high-risk patients.