Biomarkers For Patient Selection Research Articles

Immunotherapy in Colorectal Cancer - Finding the Achilles' Heel.

AbstractColorectal cancer treatment has evolved considerably in the last decade with the development of immunotherapies. Immune checkpoint inhibitor therapies have brisk and durable responses in patients with advanced microsatellite instability-high colorectal cancer, both surgically resectable and unresectable; however, patients with microsatellite stable colorectal cancer in general do not respond to the same therapy. Emerging evidence shows that immune checkpoint inhibitors may elicit responses in subsets of patients with microsatellite stable colorectal cancer, especially when combined with other anticancer agents that can modulate the tumor microenvironment. Therefore, rationally designed therapeutic combinations involving immune checkpoint inhibitors, as well as the development of predictive biomarkers for optimal patient selection, have emerged as two key areas of active research. In addition, other immunotherapeutic agents such as cell-based therapies and bispecific T-cell engagers are beginning to be studied in preclinical and early-phase settings. Although by no means a universal treatment strategy, immunotherapy can elicit responses in microsatellite stable colorectal cancer and further research is needed to extend their benefit to patients with microsatellite stable colorectal cancer. Here, we review the current state of immunotherapeutic regimens for microsatellite stable colorectal cancer.

Pan-cancer analysis of super-enhancer-induced LINC00862 and validation as a SIRT1-promoting factor in cervical cancer and gastric cancer

Immune checkpoints inhibitors are effective but it needs more precise biomarkers for patient selection. We explored the biological significance of LINC00862 in pan-cancer by bioinformatics. And we studied its regulatory mechanisms using chromatin immunoprecipitation and RNA immunoprecipitation assays etc. TCGA and single-cell sequencing data analysis indicated that LINC00862 was overexpressed in the majority of tumor and stromal cells, which was related with poor prognosis. LINC00862 expression was related with immune cell infiltration and immune checkpoints expression, and had a high predictive value for immunotherapy efficacy. Mechanistically, LINC00862 competitively bound to miR-29c-3p to unleash SIRT1’s tumor-promoting function. SIRT1 inhibitor-EX527 were screened by virtual screening and verified by in vitro and vivo assays. Notably, acetyltransferase P300-mediated super-enhancer activity stimulated LINC00862 transcription. Collectively, LINC00862 could be a diagnostic and prognostic biomarker. LINC00862 could also be a predictive biomarker for immunotherapy efficacy. Super-enhancer activity is the driver for LINC00862 overexpression in cervical cancer and gastric cancer.

Abstract PO3-04-12: SUMIT-BC: Phase 2 randomized study of fulvestrant with or without the cyclin-dependent kinase 7 inhibitor (CDK7i) samuraciclib in advanced hormone receptor positive (HR+) breast cancer after CDK4/6i

Abstract Background: CDK7 has 3 critical roles in cancer: enhanced transcription of oncogenes and upregulation of anti-apoptotic genes, acceleration of the cell cycle through phosphorylation of other CDKs and driving estrogen receptor (ER) resistance to hormonal therapy. Inhibition of CDK7 is therefore a potential novel anti-cancer therapeutic strategy. In an initial single-arm evaluation samuraciclib (CT7001), a once daily (QD) oral CDK7 inhibitor has demonstrated a favourable safety profile and clinical activity in combination with fulvestrant, a selective estrogen receptor degrader (SERD), in patients with HR+/HER2- advanced breast cancer who have previously been treated with a CDK4/6 inhibitor. There was evidence of enhanced benefit in patients with no detectable TP53 mutation from baseline circulating tumor DNA (ctDNA) analysis and in patients without baseline liver metastases [1]. Samuraciclib is associated with low grade gastrointestinal (GI) adverse events such as nausea, vomiting and diarrhea managed with simple prophylaxis and patient counselling. An instant release capsule formulation was used in the initial clinical evaluation of samuraciclib requiring patients to take multiple capsules designed to rapidly release a large amount of material high in the GI tract. In this study a novel single tablet formulation will be administered which may enhance GI tolerability (2). This Phase 2 open-label randomised study will evaluate the efficacy, safety, pharmacokinetics and Quality of Life (QoL) impact of samuraciclib in combination with fulvestrant in comparison to a fulvestrant monotherapy control arm. Consistent with the principles of the current Project OPTIMUS initiative, 2 dose levels of samuraciclib will be evaluated [3]. All patients will undergo baseline ctDNA evaluation of TP53 mutation status to permit a prospective evaluation of its patient selection biomarker potential. Trial Design: Eligible patients will provide written informed consent, be aged 18 years or older, have histologically or cytologically confirmed ER+/HER2- advanced or metastatic breast cancer not amenable to resection or radiotherapy of curative intent, have received an aromatase inhibitor in combination with a CDK4/6 inhibitor in either the adjuvant or advanced setting, be receiving a luteinizing hormone-releasing hormone agonist if pre/perimenopausal and have RECIST v1.1 evaluable disease. Prior SERD, mammalian target of rapamycin inhibitor (mTORi) or chemotherapy for advanced breast cancer are not permitted. All patients will undergo baseline Guardant360 ctDNA analysis to establish their TP53 mutation status among others. 60 patients will be enrolled and will all receive fulvestrant 500mg IM administered on days 1, 15 and 29 and then monthly thereafter. Patients will be randomized 1:1:1 to fulvestrant alone, fulvestrant and samuraciclib 240mg QD or fulvestrant and samuraciclib 360mg QD. Patients will undergo RECIST v1.1 evaluation at baseline every 8-weeks until week 48, followed by every 12-weeks thereafter. The pharmacokinetics of samuraciclib and fulvestrant will be studied though the first 6 months of the study, with highest intensity during month 1. To evaluate Quality of Life (QoL) The Functional Assessment of Cancer Therapy -Breast questionnaire (FACT-B) will be completed. The primary endpoint is clinical benefit response at 24 weeks. Secondary endpoints are tolerability, progression free survival, overall response rate, duration of response, and the pharmacokinetics of fulvestrant and samuraciclib. QoL and correlations between ctDNA detectable TP53 mutations and efficacy/safety finding in this patient population will be reported. The study opened for recruitment in June 2023.

Addressing the Challenges of PD-1 Targeted Immunotherapy in Cancer Treatment

Background: Immune checkpoint inhibitors targeting the programmed cell death protein 1 (PD-1)/PD-ligand 1 (PD-L1) axis have revolutionized cancer treatment, demonstrating remarkable clinical efficacy across multiple malignancies. However, their broader applicability and therapeutic potential remain constrained by several challenges. Objective: This review aims to provide a comprehensive overview of the current challenges associated with PD-1 targeted immunotherapy, encompassing resistance mechanisms, patient selection, toxicity management, and the development of novel combination strategies. Methods: A comprehensive literature search was conducted across PubMed, Embase, and Web of Science databases to identify and critically analyze studies investigating the challenges and limitations of PD-1 targeted immunotherapy in cancer treatment. Results: The primary challenges identified include: (1) Intrinsic and acquired resistance mechanisms, such as alterations in antigen presentation, tumor microenvironment factors, and immune cell dysfunction; (2) Lack of reliable predictive biomarkers for patient selection and response monitoring; (3) Immune-related adverse events and their effective management; (4) Optimization of combination regimens with other immunotherapies, targeted therapies, or conventional treatments; (5) Financial and accessibility barriers limiting the broader implementation of immunotherapy. Conclusion: PD-1 targeted immunotherapy has transformed cancer treatment, addressing the current challenges is imperative to maximize its therapeutic potential and broaden its applicability. Ongoing research efforts focused on elucidating resistance mechanisms, developing predictive biomarkers, managing toxicities, and exploring novel combination strategies hold promise for overcoming these limitations. Interdisciplinary collaborations and continued investment in immunotherapy research are essential to ensure that the benefits of these groundbreaking therapies are accessible to a wider patient population.

Prognostic value of hemogram parameters in osteosarcoma: The French OS2006 experience.

Previous studies have shown that neutrophil-to-lymphocyte (NLR) ratio at diagnosis and early lymphocytes recovery on doxorubicin-based chemotherapy, may impact the outcome in patients with osteosarcoma (OST). This study aimed to evaluate the prognostic value of hemogram parameters in patients with OST treated with high-dose methotrexate and etoposide/ifosfamide (M-EI) chemotherapy. We retrospectively analyzed the prognostic value of various hemogram parameters at diagnosis and during therapy in a large consecutive cohort of patients with OST included in the French OS2006 trial and treated with M-EI chemotherapy. A total of 164 patients were analyzed. The median age was 14.7years (interquartile range [IQR]: 11.7-17). Median follow-up was 5.6years (IQR: 3.3-7.7years). Three-year event-free survival (EFS) and overall survival (OS) were 71.5% (95% confidence interval [CI]: 64%-78%) and 86.4% (95% CI: 80%-91%), respectively. In univariate analysis, blood count parameters at diagnosis and early lymphocyte recovery at Day 14 were not found prognostic of survival outcomes. By contrast, an increase of NLR ratio at Day 1 of the first EI chemotherapy (NLR-W4) was associated with reduced OS in univariate (p=.0044) and multivariate analysis (hazards ratio [HR]=1.3, 95% CI: 1.1-1.5; p=.002), although not with EFS. After adjustment on histological response and metastatic status, an increase of the ratio NLR-W4 of 1 was associated with an increased risk of death of 30%. We identified NLR-W4 as a potential early biomarker for survival in patients with OST treated with M-EI chemotherapy. Further studies are required to confirm the prognostic value of NLR and better identify immune mechanisms involved in disease surveillance.

Circulating Tumor DNA in Diffuse Large B-Cell Lymphoma: from Bench to Bedside?

Diffuse large B-cell lymphoma (DLBCL) is a curable disease with variable outcomes due to underlying heterogeneous clinical and molecular features-features that are insufficiently characterized with our current tools. Due to these limitations, treatment largely remains a "one-size-fits-all" approach. Circulating tumor DNA (ctDNA) is a novel biomarker in cancers that is increasingly utilized for risk stratification and response assessment. ctDNA is readily detectable from the plasma of patients with DLBCL but has not yet been incorporated into clinical care to guide treatment. Here, we describe how ctDNA sequencing represents a promising technology in development to personalize the care of patients with DLBCL. We will review the different types of ctDNA assays being studied and the rapidly growing body of evidence supporting the utility of ctDNA in different treatment settings in DLBCL. Risk stratification by estimation of tumor burden and liquid genotyping, molecular response assessment during treatment, and monitoring for measurable residual disease (MRD) to identify therapy resistance and predict clinical relapse are all potential applications of ctDNA. It is time for clinical trials in DLBCL to utilize ctDNA as an integral biomarker for patient selection, response-adapted designs, and surrogate endpoints. As more ctDNA assays become commercially available for routine use, clinicians should consider liquid biopsy when treatment response is equivocal on imaging. Incorporating MRD may also guide decision-making if patients experience severe treatment toxicities. Though important barriers remain, we believe that ctDNA will soon be ready to transition from bench to bedside to individualize treatment for our patients with DLBCL.

There and Back Again: A Perspective on 20 Years of CYP4Z1.

CYP4Z1, a highly expressed CYP gene in breast cancer, was one of the last CYPs to be identified in the human genome, some twenty years ago. CYP4 enzymes typically catalyze w-hydroxylation and metabolize w3 and w6 polyunsaturated fatty acids (PUFAs) to bioactive lipid metabolites that can influence tumor growth and metastasis. These attributes of CYP4Z1 make it an attractive target for new chemotherapeutic drug design, as a potential biomarker for selection of patients that might respond favorably to drugs and for developing enzyme inhibitors as potential therapeutic agents. This review summarizes the current state of knowledge regarding the advancing biochemistry of CYP4Z1, its role in breast cancer and the recent synthesis of selective chemical inhibitors of the enzyme. We identify gaps that need to be filled to further advance this field and present new experimental data on recombinant CYP4Z1 expression and purification of the active catalytic form. Significance Statement In breast cancer, an unmet need is the availability of highly effective therapeutic agents, especially for triple negative breast cancer. The relevance of the work summarized in this mini-review is that it identifies a new potential drug target, CYP4Z1, and discusses ways in which the gene product's catalytic activity might be modulated in order to combat this malignancy and limit its spread.

Abstract CT033: Avelumab combined with regorafenib in solid tumors with tertiary lymphoid structures: A phase 2 REGOMUNE trial cohort

Abstract Background: Mature tertiary lymphoid structures (mTLS) predict improved outcome in patients treated with immune checkpoint inhibitors (ICIs). However, a significant proportion of patients with TLS-positive tumors do not respond to ICI when used as a single agent and may therefore benefit from combination therapy. The multikinase inhibitor, regorafenib, deplete regulatory T cells, an immune population associated with resistance to ICI in TLS-positive tumors. We report the results of the PD-L1 inhibitor avelumab combined with regorafenib in patients with mTLS-positive advanced solid tumors. Methods: ‘Regomune’ is an open-label, multicenter phase II study assessing the combination of avelumab (10mg/kg IV every 14 days) with regorafenib (160 mg daily for 3 weeks in a 4-week cycle) in patients with advanced mTLS-positive solid tumors. The mTLS status was centrally assessed as previously described (Vanhersecke et al Nature Cancer 2021). All patients had confirmed progressive disease at inclusion, based on a central review of imaging. The primary efficacy endpoint was a 6-month non-progression rate using RECIST v1.1 based on blinded central review. 29 assessable patients were deemed necessary, and to meet the primary endpoint, at least 8 patients needed to be progression-free at 6 months. Results: Between January 2021 and July 2022, 132 patients (5 centers) underwent mTLS screening. Of these, 55 (41.7%) were identified as mTLS+, and 38 were included in the study. The top five histological subtypes were MSS colorectal cancer (15.8%), sarcoma (13.1%), oesogastric (10.5%), biliary tract (7.9%), and pancreatic cancer (7.9%). The most frequent grade 3/4 adverse events were palmar-plantar erythrodysesthesia syndrome (23.7%), maculo-papular rash (18.4%), fatigue, and oral mucositis (7.9% each). No treatment-related deaths were reported. Of the 34 patients assessed for efficacy, 16 (47%) showed tumor shrinkage, with 9 achieving a partial response (26.7%) and 7 having stable disease (20.6%). The median duration of response was 6 months. Twelve patients (comprising various tumor types including MSS colorectal cancer, small bowel carcinomas, biliary tract cancer, sarcomas, thyroid and gynecological tumors.) were progression-free at 6 months, marking the study’s primary endpoint achievement. The median progression-free survival and overall survival were 3.6 months and 8.6 months, respectively. Spatial transcriptomics, multiplex immunofluorescence (IF) of tumor tissues, and comprehensive plasma proteomics analysis have collectively uncovered critical biomarkers that significantly determine both sensitivity and resistance to treatment. Conclusions: This is the first histology-agnostic clinical trial employing mTLS as a biomarker for patient selection for treatment with an immune checkpoint inhibitor-based regimen. Durable responses were recorded, even in cases typically resistant to immunotherapy, confirming the predictive value of mTLS. Trial Registration NCT03475953 Citation Format: Antoine Italiano, Sophie Cousin, Carine Bellera, Jean-Philippe Guegan, Jean-Philippe Metges, Antoine Adenis, Rastilav Bahleda, Philippe Cassier, Coralie Cantarel, Michele Kind, Jean Palussiere, Lucile Vanhersecke, Alban Bessede. Avelumab combined with regorafenib in solid tumors with tertiary lymphoid structures: A phase 2 REGOMUNE trial cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT033.

Identification of Novel Molecular and Clinical Biomarkers of Survival in Glioblastoma Multiforme Patients: A Study Based on The Cancer Genome Atlas Data.

Glioblastoma multiforme (GBM) is the most prevalent type of brain tumour; although advancements in treatment have been made, the median survival time for GBM patients has persisted at 15 months. This study is aimed at investigating the genetic alterations and clinical features of GBM patients to find predictors of survival. GBM patients' methylation and gene expression data along with clinical information from TCGA were retrieved. The most overrepresented pathways were identified independently for each omics dataset. From the genes found in at least 30% of these pathways, one gene that was identified in both sets was further examined using the Kaplan-Meier method for survival analysis. Additionally, three groups of patients who started radio and chemotherapy at different times were identified, and the influence of these variations in treatment modality on patient survival was evaluated. Four pathways that seemed to negatively impact survival and two with the opposite effect were identified. The methylation status of PRKCB was highlighted as a potential novel biomarker for patient survival. The study also found that treatment with chemotherapy prior to radiotherapy can have a significant impact on patient survival, which could lead to improvements in clinical management and therapeutic approaches for GBM patients.

Targeting the tumor microenvironment, a new therapeutic approach for prostate cancer.

A growing number of studies have shown that in addition to adaptive immune cells such as CD8 + T cells and CD4 + T cells, various other cellular components within prostate cancer (PCa) tumor microenvironment (TME), mainly tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (MDSCs), have been increasingly recognized as important modulators of tumor progression and promising therapeutic targets. In this review, we aim to delineate the mechanisms by which TAMs, CAFs and MDSCs interact with PCa cells in the TME, summarize the therapeutic advancements targeting these cells and discuss potential new therapeutic avenues. We searched PubMed for relevant studies published through December 10 2023 on TAMs, CAFs and MDSCs in PCa. TAMs, CAFs and MDSCs play a critical role in the tumorigenesis, progression, and metastasis of PCa. Moreover, they substantially mediate therapeutic resistance against conventional treatments including anti-androgen therapy, chemotherapy, and immunotherapy. Therapeutic interventions targeting these cellular components have demonstrated promising effects in preclinical models and several clinical trials for PCa, when administrated alone, or combined with other anti-cancer therapies. However, the lack of reliable biomarkers for patient selection and incomplete understanding of the mechanisms underlying the interactions between these cellular components and PCa cells hinder their clinical translation and utility. New therapeutic strategies targeting TAMs, CAFs, and MDSCs in PCa hold promising prospects. Future research endeavors should focus on a more comprehensive exploration of the specific mechanisms by which these cells contribute to PCa, aiming to identify additional drug targets and conduct more clinical trials to validate the safety and efficacy of these treatment strategies.

Abstract 2560: Genomic biomarkers of survival in patients (pts) with metastatic castrate-sensitive prostate cancer (mCSPC) undergoing androgen deprivation therapy (ADT) intensification (ADTi)

Abstract Background: ADTi with androgen receptor pathway inhibitors (ARPI) with or without docetaxel improves survival outcomes in pts with mCSPC. Even with ADTi, 20% of these pts progress within a year and experience poor overall survival (OS). Identifying baseline genomic biomarkers predicting poor survival outcomes in pts receiving ADTi remains an unmet need. Methods: In this IRB-approved retrospective study, pts with a new diagnosis of mCSPC who underwent comprehensive genomic profiling of primary prostate tissue or metastatic site or cell-free DNA and treated with ADTi (ADT with ARPI or docetaxel) were included. Genomic alterations with an incidence > 5% were included in the analysis. Study endpoints: progression-free survival (PFS) was defined from the start of therapy for mCSPC to progression (per PCWG-2) or death from any cause. OS was defined from the start of therapy for mCSPC to the date of death from any cause or censored at the last follow-up. A multivariable analysis using the Cox proportional hazards model was used, adjusting for age at mCSPC diagnosis, disease volume (high vs. low), Gleason score (≥ 8 vs. < 8), Log2PSA at baseline, and initial timing of metastasis (de novo vs. non de novo). A subgroup analysis by intensification treatment received (ARPI vs. docetaxel) was also performed. Missing baseline characteristics such as volume of disease, Gleason score, and Log2PSA were multiply imputed on 50 chained equations using “mice” package in R. All the analyses were done using R version 4.3.1. Results: A total of 289 pts were included in the study. The baseline characteristics of the cohort will be presented in the meeting. The alterations included in the analysis were TP53 (31.8%), TMPRSS2 (30.4%), PTEN (17%), APC (10.4%), SPOP (9%), RB1 (6.2%), BRCA2 (5.2%), and CDK12 (5.2%). In the multivariable analysis, TP53 (HR 1.87, 95% CI 1.29-2.72, p = 0.001), RB1 (HR 2.56, 95% CI 1.45-4.53, p = 0.001), PTEN (HR 1.77, 95% CI 1.15-2.72, p = 0.009), and BRCA2 (HR 2.68, 95% CI 1.47-4.89, p = 0.002) were associated with significantly shorter PFS while TP53 (HR 1.78, 95% CI 1.1-2.88, p = 0.019), RB1 (HR 4.78, 95% CI 2.53-9.02, p < 0.001), and PTEN (HR 2.37, 95% CI 1.37-4.1, p = 0.003) were associated with significantly worse OS. Results of the subgroup analysis by intensification treatment (ARPI vs. docetaxel) will also be presented in the meeting. Conclusions: In this real-world study, we identified genomic biomarkers that could be associated with shorter PFS and OS in pts with mCSPC treated with ADTi. Upon external validation, these results may aid in developing a clinical-genomic risk stratification model, patient counseling, and treatment selection. Citation Format: Arshit Narang, Georges Gebrael, Chadi Hage Chehade, Nicolas Sayegh, Yeonjung Jo, Beverly Chigarira, Nishita Tripathi, Ayana Srivastava, Clara Tandar, Benjamin L. Maughan, Umang Swami, Neeraj Agarwal. Genomic biomarkers of survival in patients (pts) with metastatic castrate-sensitive prostate cancer (mCSPC) undergoing androgen deprivation therapy (ADT) intensification (ADTi) [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 2560.

Abstract 2485: A combined mregDC and Treg signature associates with antitumor efficacy of CCR4 antagonist tivumecirnon FLX475

Abstract Cancers evade immune surveillance through multiple mechanisms including the recruitment of immunosuppressive regulatory T cells (Treg). Tivumecirnon (formerly known as FLX475), a small molecule oral CCR4 antagonist under clinical evaluation, blocks the binding of CCR4 to its ligands CCL17 and CCL22 and thereby reduces Treg infiltration into the tumor microenvironment (TME). This relieves Treg-mediated immune suppression resulting in increased antitumor immunity. Our clinical study (NCT03674567) demonstrated that tivumecirnon can block the local accumulation of Treg in the TME and has clinical activity as monotherapy as well as in combination with pembrolizumab. In both mouse and human tumors, we identified a subset of tumor infiltrating dendritic cells (DC) producing high levels of CCL22, previously identified as mregDC, suggesting a potential role in the CCR4-dependent recruitment of Treg. To study the role of mregDC in the recruitment of Treg, non-clinical studies were performed. CD45+ immune cells from the TME of MB49 tumor bearing mice treated with tivumecirnon or vehicle and were analyzed by flow cytometry, scRNA-seq and CITE-seq. We confirmed that mregDC cells are the primary producer of CCL22 in the TME and that tivumecirnon treatment reduced the number of Treg cells in the TME and increased the percentage of IFN gamma and granzyme B expressing CD4+ T cells. Based on these findings we interrogated tumor biopsies from patients from our clinical study prior to (n = 33), and after approximately 6 weeks of treatment (n = 22, pre/post-treatment paired samples) with tivumecirnon +/- pembrolizumab. We performed RNAseq and compared to published biopsy data from anti-PD-1 treated patients. We found that higher mregDC + Treg signature levels at baseline significantly (p < 0.001) associated with response to tivumecirnon and pembrolizumab therapy. Importantly, analysis of published datasets revealed that higher baseline mregDC and Treg levels are not associated with response to anti-PD-1 monotherapy. Analysis of TCGA gene expression data demonstrated a positive correlation with mregDC and Treg cells across human tumor types, indicating a previously unknown relationship between these cell types. Further application of this mregDC and Treg signature to the TCGA dataset identified a subset of cancer types resistant to prior treatments that could potentially benefit from combinatorial treatment with tivumecirnon. Overall, this study demonstrated that inhibition of the CCR4 pathway using tivumecirnon can disrupt the immunosuppressive mregDC – Treg axis leading to enhanced antitumor activity. Furthermore, the levels of mregDC and Treg may be used as a biomarker for selection of patients more likely to respond to tivumecirnon in combination with anti-PD-1. Additional studies are required to confirm these results Citation Format: Adam Grant, Juraj Adamik, Jingtao Qiu, Jacob Wert, Molly Grandcolas, Scott Jacobson, Rakesh K. Goyal, William Ho, Shoji Ikeda, Marvin Au, Damian Trujillo, Michael Chisamore, Denise de Almeida Nagata, Mohsen S. Ghomi, Dirk G. Brockstedt, Paul D. Kassner, George Katibah. A combined mregDC and Treg signature associates with antitumor efficacy of CCR4 antagonist tivumecirnon FLX475 [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 2485.

Abstract 2112: Identification of biomarkers associated with sensitivity to a novel PCNA inhibitor (AOH1996) by CRISPRi screening

Abstract Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is essential for cancer cell growth and survival. We discovered a cancer-associated isoform of PCNA (caPCNA), which is ubiquitously and highly expressed in a broad range of cancer cells and tumor tissues but is not significantly expressed in non-malignant cells. Further studies of this novel caPCNA isoform as an anti-cancer drug target led to the identification of a cancer distinct region of PCNA which is partly delineated by the L126-Y133 peptide sequence and the discovery of a small molecule (AOH1996), which binds to this cancer distinct region as shown by crystallization studies. By targeting caPCNA, AOH1996 selectively kills cancer cells by interfering with the resolution of transcription-replication conflicts (TRC). Importantly, it causes no discernable toxicity at 6 times of its effective dose in mice. AOH1996 is currently in a phase 1 clinical trial for adult solid tumors at City of Hope (IND 138273). To identify genes that may be used as biomarkers for patient selection or potential targets for combination therapies, we screened a CRISPR interference (CRISPRi) library, which targets genes that are involved in cell signaling and cell cycle regulation. After culturing transduced cells in the presence of DMSO or 250 nM AOH1996 (corresponding to IC20) in triplicates for 14 generations, we measured the relative abundance of each guide RNA (gRNA) construct. Significant changes in the abundance of the gRNA constructs in AOH1996-treated cells relative to that in DMSO-treated cells were determined by the MAGeCK method. The target genes of the depleted or enriched gRNAs are predominantly involved in DNA repair, cell cycle regulation, and transcription regulation, which is consistent with the action mechanism of AOH1996. We validated the effect of these genes on sensitivity to AOH1996 and identified a panel of target proteins whose up or down-regulation is associated with sensitivity to AOH1996. Some of these proteins are targets of known chemotherapeutic drugs, which may be used in combination with AOH1996 in the clinic. Citation Format: Long Gu, Ming li, Chun-Wei Chen, Robert J. Hickey, Linda H. Malkas. Identification of biomarkers associated with sensitivity to a novel PCNA inhibitor (AOH1996) by CRISPRi screening [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 2112.

Abstract 2866: NAPRTsilencing in rhabdomyosarcoma confers therapeutic vulnerabilities to NAD+depletion

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma among children, and new treatments are needed to improve survival. Cells primarily synthesize nicotinamide adenine dinucleotide (NAD) through the Preiss-Handler and Salvage pathways driven by nicotinic acid phosphoribosyltransferase (NAPRT) and nicotinamide phosphoribosyltransferase (NAMPT), respectively. NAMPT inhibitors (NAMPTi) have been tested in clinical trials, but their efficacy has been limited by (a) lack of appropriate biomarkers for patient selection, and (b) significant dose-limiting toxicities (DLTs), including myelosuppression and retinal toxicity, although the latter has been described only in preclinical studies. Previous work from our group indicates that loss of NAPRT expression by tumor-specific promoter CpG island methylation may provide a novel biomarker for sensitivity to NAMPTi in a variety of tumors. Here, we sought to determine if RMS harbors NAPRT silencing that confers a synthetic lethal interaction with NAMPTi. We probed methylation data within the Cancer Dependency Map (DepMap) and found a high frequency of NAPRT gene promoter hypermethylation in RMS models. Predicted NAPRT silencing was confirmed at the protein level by western blot in a panel of RMS cell lines. In vitro growth delay assays demonstrated that NAPRT-silenced cells are exquisitely sensitive to NAMPTi even in the presence of nicotinic acid (NA) supplementation, while NAPRT-expressing cells were completely rescued. This sensitivity to NAMPTi was further mirrored by changes in NAD levels. In vivo, NAMPTi induced significant tumor regression in a NAPRT-silenced RMS xenograft model. To further establish the translational relevance, NAPRT protein expression in a TMA of 51 human RMS tumors was assessed via IHC staining using a newly validated NAPRT monoclonal antibody (4A5D7). Based on a semi-quantitative scoring system accounting for the percentage of positive tumor cells and staining intensity, 28% of RMS samples demonstrated loss of NAPRT protein expression. Overall, these data suggest that NAPRT loss may serve as a therapeutic target in RMS by inducing a synthetic lethal interaction with NAD+ depleting agents. Future studies in patient-derived xenograft (PDX) models will determine the optimal cut-off of NAPRT expression that portends NAMPTi sensitivity, paving the way for the development of biomarker-driven clinical trials in RMS. Ongoing studies will also evaluate the efficacy of combining NAMPTi with existing cytotoxic and DNA damaging chemotherapy in these difficult-to-treat tumors. Citation Format: Sophia J. Zhao, Katelyn J. Noronha, Prateek Bhardwaj, Karlie N. Lucas, Ranjini K. Sundaram, Collin D. Heer, Raffaella Morotti, Josh Spurrier, Mitch Raponi, Ranjit S. Bindra, Juan C. Vasquez. NAPRTsilencing in rhabdomyosarcoma confers therapeutic vulnerabilities to NAD+depletion [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 2866.

Abstract 5181: Early activity and biomarker evaluation of NT219 in combination with cetuximab in a Phase 1/2 study of recurrent/metastatic squamous cell carcinoma of the head and neck (R/M SCCHN)

Abstract Background. NT219 is a novel small molecule, dual inhibitor, that uniquely triggers the degradation of Insulin Receptor Substrates 1/2 (IRS) and the dephosphorylation and suppression of STAT3. These two major oncogenic targets in the tumor and tumor microenvironment (TME) play a key role in drug resistance, particularly to EGFR inhibitors. Previously, we demonstrated synergistic effect of NT219 and cetuximab in multiple SCCHN patient-derived xenograft (PDX) models. HPV-negative R/M SCCHN patients have poor survival and demonstrate IGF1R/IRS and STAT3 upregulation. We report biomarker results from dose escalation of NT219 in combination with cetuximab in patients (pts) with immunotherapy-refractory R/M SCCHN (NCT04474470). Method. Biopsies from 11 enrolled SCCHN patients treated with different dose levels of NT219 (6, 12, 24 and 50 mg/kg) were obtained and evaluated using Immunohistochemistry and pathological analysis. Evaluation of potential biomarkers included measurements of IRS1/2 levels, as well as IGF1R and STAT3 phosphorylation (pIGF1R, pSTAT3) in tissue biopsies, and H-score was defined for tumor cells, infiltrating immune cells, and fibroblasts in the TME. Membrane completeness (mc) parameter was included in pIGF1R scoring. Results. NT219 Cmax and AUC demonstrated a dose dependent increase, with 50mg/kg dose mean human AUC reaching Human Equivalent Dose efficacy of animal models. Indeed, at this dose, anti-tumor activity in humans was also demonstrated with 2 confirmed partial responses (PR) reported. At the 50 mg/kg NT219 dose, out of 4 SCCHN pts, for whom pre-treatment biopsies were available, 3 were HPV negative and 2 of them achieved PR. Pretreatment biopsies of these 2 pts demonstrated high activated IGF1R membranal staining (Avg. mcH-score: 285, p-value:) and high activated STAT3 nuclear staining (Avg. H-score: 285), in contrast to the non-responding SCCHN pts (30 and 33, with p-value 0.017, 0.004 respectively). Inhibition of intratumoral IGF1R/IRS and STAT3 was demonstrated in on-treatment biopsies. Analysis will be presented at the conference. Conclusion. NT219 in combination with cetuximab demonstrated safety and early activity with 2 confirmed PRs at 50mg/kg dose level in R/M SCCHN pts. This dose level was the first dose at which the observed NT219 exposure was within the range of the efficacious dose levels in pre-clinical models. Detection of activated IGF1R and STAT3 in responding pts, compared to the non-responding pts, suggests a potential use of these targets as biomarkers for future patient selection. This notion is further substantiated by the demonstration of NT219 on-target effects in on-treatment tumor biopsies. Upregulation of NT219 targets, IGF1R and STAT3, in HPV-negative SCCHN pts and their response to the NT219+cetuximab treatment, support further evaluation of this patient population. Citation Format: Ari J. Rosenberg, Shumei Kato, Daniel Johnson, Aron Popovtzer, Vi K. Chiu, Ravit Geva, Hava Ben-David, Tomer Meirson, Michael Schickler, Hadas Reuveni. Early activity and biomarker evaluation of NT219 in combination with cetuximab in a Phase 1/2 study of recurrent/metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) [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 5181.

Abstract 903: AURIGIN: A comprehensive single-cell OMICs atlas of human development and an AI/ML framework to classify and identify the drivers of tumor plasticity and altered cellular state

Abstract Altered cellular state and plasticity have been increasingly identified as drivers of poor prognosis and treatment-resistance in cancer. Identifying therapeutics that correct altered cellular state and plasticity meets a broad need in the field of oncology, particularly in tumors resistant to standard-of-care therapy. Tumor plasticity and altered cellular states encompass many potential configurations, including dedifferentiated and transdifferentiated tumors, so classifying tumors based on their specific cell state profile is critical to identifying the gene drivers that are specific to that altered state. For that reason, we have developed AURIGIN - a platform that combines a comprehensive single-cell OMICs atlas of human development with an AI/ML framework that can classify cell state plasticity and identify the genes and pathways that drive those specific altered cellular states. AURIGIN is more comprehensive than previously disclosed single cell atlases in that it focuses and enriches the diverse stem, progenitor, and developmental states commonly involved in plastic cell states of cancer. The platform standardizes and integrates these states with multiple atlases of differentiated adult tissues to create a unified atlas of human development. In addition, the AI/ML paradigm of AURIGIN is embedded with developmental biology models that enable it to deconvolute state heterogeneity within tumor indications for precise identification of the most relevant gene targets for novel therapies. AURIGIN also unifies classified plasticity across tissue types by identifying altered cellular states that span multiple indications. Furthermore, we present the implementation of AURIGINDRIVE ML models that integrate pathway information, physical interaction datasets, and regulatory annotations to accurately predict the gene targets at the top of hierarchy of control of tumor plasticity. AURIGIN also enables and accelerates target validation by identifying models that map to the relevant classified plastic states. Similarly, AURIGIN defines clinically relevant patient selection and treatment efficacy biomarkers that are specifically defined from the classified plastic cellular state. We demonstrate the success of AURIGIN for therapeutic discovery. In sum, AURIGIN amplifies the value of multiOMIC tumor data in cellular state and plasticity target discovery by mapping the data into a developmental biology-informed ML framework. Citation Format: Joseph DeBartolo, Henry Wilson, Kimberly S. Straley, Maulasri Bhatta, Sambad Sharma, Sara Sinicropi-Yao, James Neef, Betty Chan, Andrew McRiner, Mark Bittinger, Laura Antipov, Katharine E. Yen, Thomas G. Graeber, David S. Millan. AURIGIN: A comprehensive single-cell OMICs atlas of human development and an AI/ML framework to classify and identify the drivers of tumor plasticity and altered cellular state [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 903.

Abstract 7176: Optimization of T cell co-stimulatory agonists: A semi-mechanistic PKPD model integrating drug properties and tumor-immune interactions

Abstract Background: Despite the success of some immune checkpoint inhibitors in the treatment of cancer, T-cell co-stimulation has faced limited clinical success due to the intricacies around the optimal engagement of agonistic antibodies to co-stimulatory receptors, and the absence of biomarkers for patient selection. Understanding agonist-binding characteristics, pharmacokinetics, T cell subpopulation activation and differentiation kinetics, T cell life span in the tumor microenvironment, and the impact of T cell/tumor interactions on tumor growth kinetics is crucial for the positive clinical outcomes of T cell agonists. Currently there is not a suitable model to describe these complex interactions. Methods: We investigated the pharmacokinetic (PK), pharmacodynamics (PD), and anti-tumor efficacy of an anti-TNFR2 agonist, HFB3-1, and an anti-OX40 agonist, HFB10-1, in syngeneic tumor models. Flow cytometry was employed to profile post-treatment tumor infiltrating lymphocytes (TILs) in dissociated tumor tissues. In vitro binding affinity was determined using Biolayer Interferometry. A semi-mechanistic model, integrating PK, tumor growth, and immune interaction networks (interactions among Teff, Treg, and tumor cells), was developed in Berkeley Madonna (version 10.5.1). This was subsequently adapted to a human tumor growth model with T cell interaction networks to assess the impact of doses and dosing regimens on tumor growth inhibition. Results: A semi-mechanistic PKPD model was successfully developed incorporating drug properties including binding affinity, agonistic effect, ADCC activity, and pharmacokinetics, and tumor growth characteristics including tumor growth kinetics, TIL composition, interaction, proliferation, and turnover. This model was applied to investigate the effect of FcγR, dose, and dosing frequency on in vivo efficacy. Following incorporation of human PK and human tumor microenvironment properties into the model, tumor growth characteristics were simulated under varying human dosing regimens. Simulation results indicated that lower and less frequent doses optimize T cell co-stimulation for optimal anti-tumor activity. Conclusion: Our semi-mechanistic PKPD model elucidates the intricate interactions between T cell co-stimulatory agonists and the tumor-immune cells in the tumor microenvironment. The model advocates for pulsatile agonism as the optimal anti-tumor activity for T cell co-stimulation agents. This model serves as a valuable tool for guiding dose and dose regimen optimization for T cell co-stimulatory agonists in clinical development. Citation Format: Jinping Gan, William Hedrich, Yun-Yueh Lu, Wenhua Xu, Robert H. Andtbacka, Francisco Adrian, Liang Schweizer. Optimization of T cell co-stimulatory agonists: A semi-mechanistic PKPD model integrating drug properties and tumor-immune interactions [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 7176.

Abstract 5139: PD1-PDL1 interaction as a superior predictor for response to immune checkpoint therapy in NSCLC patients

Abstract Background: Immune checkpoint therapies (ICT) are now widely introduced in clinical oncology and most often guided by PDL1 expression in diagnostic biopsies. However, the predictive value of this testing is only modest, leading to over- and undertreatment. Conceptually, the prevention of binding of ligand PDL1 to PD1 receptor is decisive for the efficacy of ICT. Therefore, directly detecting the spatial PD1-PDL1 interaction would provide more predictive information. Methods: A second-generation in situ proximity ligation assay (isPLA; Navinci Diagnostics) was established to detect PD1-PD-L1 interactions in diagnostic patient samples and linked to an automated analysis pipeline (QuPath). For basic validation, we used pan-cancer tissue microarrays (TMA) including 16 solid tumor types, and a non-small cell lung cancer (NSCLC) TMA (359 ICT-naïve surgically treated patients). In addition, we analyzed diagnostic tissue biopsies from 75 advanced NSCLC patients treated with anti-PD1-PDL1 regiments. Results: The pan-cancer analysis revealed varying levels of PD1-PDL1 interaction among the solid cancer types, with the lowest levels detected in liver cancer and the highest in testicular cancer. A general positive association was observed between the literature-reported objective ICT response rate (ORR) per tumor type and its corresponding PD1-PDL1 interaction status. Conventional immunohistochemical analysis of the ICT-naïve, surgically treated NSCLC cohort revealed variable protein expression level for both, PDL1 and PD1, for 200 cases, but only 108 (54%) of them demonstrated a detectable PD1-PDL1 interaction with our isPLA assay. EGFR mutated cases showed generally lower PD1-PDL1 interaction scores (p=0.01). PD1-PDL1 interaction was not associated with survival (p=0.46). A subsequent analysis of the ICT-NSCLC cohort revealed a significant positive association between PD1-PDL1 interaction status and prolonged overall survival upon ICT (median survival 14.8 vs 32.3 months; p=0.022). The standard tumor proportional score (TPS) of membranous PDL1 expression did not correlate with ICT-specific survival (p=0.40). Conclusions: The second generation isPLA successfully distinguishes a previously difficult-to-define patient subset with positive PD1-PDL1 interaction status. Our data suggest that the PD1-PDL1 interaction is a superior clinical biomarker for patient selection for ICT in NSCLC. The interaction analysis is applicable to sections from minute diagnostics biopsies and accessible by light microscopy either semi-quantitatively by a pathologist or by an automated image analysis program. Thus, the assay holds great potential to be integrated into routine clinical pathological workflow to stratify cancer patients for ICT. Citation Format: Patrick Micke, Amanda Lindberg, Rebecca Artursson, Louise Hellberg, Hui Yu, Max Backman, Johan Botling, Hans Brunnström, Artur Mezheyeuski, Johan Isaksson, Carina Strell. PD1-PDL1 interaction as a superior predictor for response to immune checkpoint therapy in NSCLC patients [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 5139.

Abstract 6404: GRB2 stabilizes RAD51 at reversed replication forks restricting genomic instability and associated innate immune response

Abstract Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adaptor for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination. Citation Format: Zamal Ahmed, Zu Ye, Shengfeng Xu, Yin Shi, Xueqian Cheng, Yuan Zhang, Sunetra Roy, Sarita Namjoshi, Michael A. Longo, Todd Link, Katharina Schlacher, Guang Peng, Dihua Yu, Bin Wang, John Tainer. GRB2 stabilizes RAD51 at reversed replication forks restricting genomic instability and associated innate immune response [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 6404.

Abstract 5203: Identification of prognostic and predictive biomarkers for palbociclib add-on therapy using cancer panel sequencing of cell-free DNA in patients with HR-positive HER2-negative advanced and metastatic breast cancer with fulvestrant resistance

Abstract Purpose: The combination of cyclin-dependent kinase 4/6 inhibitors with endocrine therapies has shown promise for improving the survival of patients with hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative advanced and metastatic breast cancer (ABC/MBC). The FUTURE trial (UMIN000029294) demonstrated that adding palbociclib after acquiring fulvestrant resistance is potentially safe and effective. To further identify biomarkers for patient selection, this preplanned translational sub-study aims to use cancer panel sequencing of cell-free DNA (cfDNA) to discover prognostic and predictive biomarkers for additional palbociclib treatment after acquiring fulvestrant resistance in patents with HR-positive/HER2-negative ABC/MBC. Methods: Herein, we utilized 149 cfDNA samples from 61 patients with disease progression after fulvestrant monotherapy and who subsequently received fulvestrant and palbociclib. Circulating cfDNA was analyzed at the time of palbociclib addition after fulvestrant resistance, on day 15 of cycle one (D15), and at the end of treatment (EOT) using the ThruPLEX ctDNA Assay, which can identify single or multi nucleotide variants, DNA insertions, and deletions in up to 34 cancer-associated genes. Results: The genetic landscape of patients with BC undergoing palbociclib treatment was dynamic and changed over the course of treatment. Regarding progression-free survival, alterations in NRAS (Hazard Ratio (HR): 11.97, P < 0.005)), MAP3K1 (HR: 6.86, P < 0.005), and TP53 (HR: 3.6, P = 0.02) were identified in cfDNA on D15 and were linked to poor prognoses. Alterations in CBFB at baseline (HR: 5.15, P = 0.01), in BRCA2 (HR: 7.17, P < 0.005) and NRAS (HR: 5.32, P < 0.005) at D15, and ESR1 (HR: 4.37, P = 0.01) at EOT were also linked to poor prognosis, whereas alterations in WEE1 (HR: -5.84, P = 0.02) at EOT were associated with a favorable prognosis. Alterations in ESR1, WEE1, CBFB, and BRCA2 were prognostic factors at peak frequencies during treatment. Moreover, our research uncovered a novel observation that as treatment progressed, the relationship between prognostic factors focused on alterations in ESR1 and TP53 tended to weaken. Conclusion: Cancer panel testing for cfDNA identified prognostic and predictive biomarkers for palbociclib add-on therapy after acquiring fulvestrant resistance in patients with HR-positive/HER2-negative ABC/MBC. These findings have important implications for patient selection and personalized treatment strategies in such patients. Citation Format: Takashi Takeshita, Takayuki Iwamoto, Naoki Niikura, Kenichi Watanabe, Yuichiro Kikawa, Kokoro Kobayashi, Nobutaka Iwakuma, Takuho Okamura, Hiroshi Tada, Shinji Ozaki, Toshitaka Okuno, Uhi Toh, Yutaka Yamamoto, Michiko Tsuneizumi, Hiroshi Ishiguro, Norikazu Masuda, Shigehira Saji. Identification of prognostic and predictive biomarkers for palbociclib add-on therapy using cancer panel sequencing of cell-free DNA in patients with HR-positive HER2-negative advanced and metastatic breast cancer with fulvestrant resistance [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 5203.