Tumor Molecular Profiling Research Articles

Natural history study of patients with pancreatic acinar cell carcinoma: A 3-year prospective longitudinal analysis.

764 Background: Pancreatic acinar cell carcinoma (PACC) is a rare exocrine tumor of the pancreas that comprises 0.2-2% of all pancreatic malignancies. There are no prospective randomized studies in this disease and most data about PACC comes from retrospective database analyses and case reports. Methods: The Cancer Moonshot-funded My Pediatric and Adult Rare Tumor (MyPART) network’s Rare Solid Tumor Natural History study (NCT03739827) is a prospective, single-institution observational study that enrolls patients of all ages with rare solid tumors (<15 cases per 100,000 people per year), including PACC. Patients can participate remotely (field cohort) or visit the NIH for annual evaluations (in-person cohort). All participants complete medical and family histories, patient reported outcomes (PROs) measures, and provide saliva for DNA analysis. Relevant data such as clinical and family histories, surgical and molecular pathology, and imaging results are extracted from patient medical records and entered in the central study database. Archival tumor samples (when available) are analyzed using a 500+ gene panel (TruSight500, Illumina) and discussed in a molecular tumor board. Patients participating in the in-person cohort undergo additional evaluations including clinical examination, imaging studies, genetic counseling, and blood sample collection for clinical and research purposes (standard clinical labs, germline DNA/RNA, immune phenotypes, cytokines) as indicated. Results: From 09/2021 until 09/2024, 17 PACC patients (n=13 for the field cohort, n=4 for the in-person cohort) were accrued. The mean age of diagnosis is 64.7 (48-78) years, and the male to female ratio is 14:3. At the time of diagnosis, 6 patients had Stage I or II, 1 had Stage III, and 10 had Stage IV disease. The median follow-up time since accrual is 7.6 months, and 4 patients have died. No patients have been lost to follow-up. Of the 6 patients diagnosed with early-stage disease, 5 had surgery and all received adjuvant chemotherapy, while 1 patient is receiving neoadjuvant chemotherapy. Among the 9 patients diagnosed with advanced disease, 8 received platinum-based regimens as a first-line therapy. Tumor molecular profiling was available for 16 patients. The most common tumor mutations observed were in CDKN2A (29.4%), BRCA2 (29.4%), and SMAD4 (23.5%). Six patients received targeted therapies matched to their tumor molecular profile. Conclusions: The demographic profile of our cohort aligns with previous studies, with a mean age of diagnosis in the early-to-mid 60s and a high male to female ratio. Our cohort highlights a shift in first-line treatment to platinum-based therapies over the past decade. Many PACC tumors have targetable mutations found in molecular profiling, and the efficacy of matched therapy warrants further exploration. Clinical trial information: NCT03739827 .

A global comparative study on real-world clinical and genomic differences in advanced biliary tract cancer.

628 Background: Biliary tract cancers (BTC) are aggressive malignancies with poor survival outcomes and limited treatment options. This study compared the epidemiological and molecular differences of advanced BTC patients through a global collaboration involving the Mayo Clinic and National Cancer Center Hospital East. Methods: We included patients with advanced BTC who underwent comprehensive tumor molecular profiling who received at least 30 days of systemic therapy. Patients were grouped based on specific alterations. Overall survival (OS) was calculated from the start of first-line therapy until death and compared among subgroups using Cox regression models. Results: 332 (36%) out of 932 patients had at least one actionable mutation : somatic BRCA1/2 mutations N=106, FGFR2 fusions N=61, HER2 amplifications N=65, KRAS G12C/D mutations N=46, IDH1 mutations N=38, and TMB >10 mut/Mb N=16. BRCA1/2 -mutated BTC were more commonly found in older Asian males with intrahepatic tumors and often presented with peritoneal and liver metastases upon initial diagnosis. FGFR2 fusions were more prevalent in younger White females with intrahepatic tumors, while HER2 amplifications were mainly seen in Asian females with gallbladder tumors. KRAS mutations were primarily observed in Asian males, and IDH1 mutations were common in White males with intrahepatic tumors, frequently presenting with liver metastasis. Survival outcomes varied significantly based on the alteration type: patients with FGFR2 fusions had the longest median OS (mOS) at 23.3 months (mo) (95%CI 18.4-34.9), followed by those with IDH1 mutations (19.3 mo; 95% CI 17.4-NE) and TMB >10 mut/Mb (17.8 mo; 95% CI 13.6-NE). BRCA mutations had a mOS of 16 mo (95% CI 14.6-19.8), while HER2 amplifications and KRAS mutations had mOS of 15.5 (95% CI 11.9-20.2) and 11.6 mo (95% CI 10.0-20.9), respectively. Conclusions: These findings underscore the heterogeneity in clinical and genomic characteristics among BTC patients, highlighting the importance of tailored therapeutic approaches. Baseline patient characteristics stratified by mutation status. KRAS G12C/D (N=46) BRCA 1/2 (N=106) IDH1 (N=38) HER2 amplification(N=65) FGFR2 fusion(N=61) TMB>10 mut/Mb(N=16) P-value Median Age, year 67 68 64 66 52 73 <0.001 Female, n (%) 19 (41) 34 (32) 18(47) 34 (52) 45(74) 6 (38) <0.001 Race, n (%) <0.001 Asian 31 (67) 97 (92) 13 (34) 51 (79) 10 (16) 6 (38) White 14 (30) 8 (8) 24 (63) 13 (20) 48 (79) 10 (63) Primary tumor location, n (%) <0.001 Intrahepatic 28 (68) 45 (45) 33 (100) 19 (31) 54(95) 10 (67) Extrahepatic 9 (22) 27(27) 0 (0) 10 (16) 2 (4) 4 (27) Gallbladder 4 (10) 28 (28) 0 (0) 32 (53) 1 (2) 1 (7) Sites of Metastasis at diagnosis, n (%) Abdominal Wall / Peritoneal 9 (24) 15 (19) 4(13) 7 (12) 6 (10) 4 (27) 0.309 Bone 33 (92) 70 (90) 26 (87) 2 (3) 9(15) 0 (0) 0.212 Liver 20 (49) 52 (61) 28 (82) 45 (73) 45 (76) 6 (38) 0.001 Lung 26 (70) 65 (82) 22 (69) 36 (58) 27(45) 11 (69) 0.006

Real-world impact of matched targeted therapy on survival in advanced biliary tract cancer: An international collaborative study.

536 Background: The prognosis of advanced BTC remains poor. Although targeted agents have expanded late-line options, many patients (pts) become ineligible for these therapies after progressing on first-line treatment. Herein, we examined the real-world impact of targeted therapy on survival in advanced BTC. Methods: In this international collaborative study between the Mayo Clinic (US) and the National Cancer Center East (Japan), we included pts with advanced BTC with comprehensive tumor molecular profiling who had received at least 30 days of systemic therapy. Pts were categorized into 3 groups: 1) non-actionable, 2) pts with alterations who received matched targeted therapy at any point during their treatment (matched group), and 3) those with mutations who did not receive matched therapy (unmatched group). OS was calculated from the start date of first-line therapy until death and compared among three subgroups using Cox regression models. Results: Of 932 pts, 366 (39.3%) had an actionable alteration, and 135 of these received matched therapy. 566 (61.7%) did not have an actionable alteration (Table). The most common alterations were somatic BRCA1/2 (N=106, 11.4%), HER2 amplification (N=65, 7%), FGFR2 fusion (N=61, 6.5%), KRAS G12C/D (N=46, 4.9%), and IDH1 (N=38, 4.1%). 78.7% of FGFR2 fusions were found in Caucasian pts, whereas BRCA1/2 (97.5%), HER2 (78.5%), and KRAS G12C/D (67.4%) alterations were more prevalent in Asian pts. The median OS was significantly longer in the matched group (21.4 months; 95% CI 18.4-27.9) compared to the unmatched group (14.6 months; 95% CI 12.8-17.6) and the non-actionable group (17.2 months; 95% CI 15.5-19.0). The 36-month OS rate was 33% in the matched group vs. 7% in the unmatched group (p < 0.0001). After adjusting for age, gender, prior surgical resection, primary tumor location, and country of origin, matched targeted therapy remained a strong independent predictor for improved OS (HR 0.61; 95% CI 0.48-0.79). Conclusions: These real-world findings suggest that matched targeted therapies significantly improve survival in advanced BTC, underscoring the need to incorporate them earlier in the treatment course and address barriers to treatment access. The regional prevalence of actionable alterations should also be considered when developing new targeted therapies. Patient baseline characteristics stratified by actionable alterations and targeted therapy. Non-actionable(N=566) Unmatched group(N=231) Matched group(N=135) Site, n (%) Japan 409 (72%) 166 (72%) 50 (37%) US 157 (28%) 65 (28%) 85 (63%) Age at Dx > 65, n (%) 324 (57%) 122 (53%) 54 (40%) Female, n (%) 235 (42%) 92 (40%) 77 (57%) Primary tumor location (category), n (%) Intrahepatic 217 (43%) 115 (55%) 99 (76%) Extrahepatic 150 (30%) 39 (19%) 17 (13%) Gallbladder 142 (28%) 54 (26%) 14 (11%) Prior surgical resection, n (%) 220 (42%) 74 (34%) 48 (37%)

Personalized profiling in the field of senology

The concept of personalized medicine is becoming increasingly important. The possibilities of diagnostics include not only genetic and molecular tumor profiles, but also the use of precise and individual imaging techniques. The development and implementation of suitable diagnostic procedures with high sensitivity and specificity, which are at the same time tailored to the individual risk factors and biological characteristics of the patient, remain achallenge. To enable personalized profiling, comprehensive diagnostics must be established that take into account all parameters such as imaging, molecular and genetic markers as well as real-world data and the use of artificial intelligence. This article sheds light on different approaches to personalized diagnostics in breast cancer and highlights the current clinical standard, innovative areas of research and the resulting challenges. The highest hurdles for newer imaging techniques are the standardization of image analysis and the validation of these techniques in large clinical trials. The use of artificial intelligence requires not only appropriate technical and medical expertise, but also asensitive approach to issues such as data protection and patient privacy. Real-world registries offer insights into real world treatment situations and are therefore of great importance.

Molecular profile of salivary gland tumors: discovering new targets for therapy

Introduction. Due to variety of morphological subtypes and poor clinical response, comprehensive molecular profiling for salivary gland cancers become a part of treatment strategy for chemotherapy-resistant cases. Tumors of the salivary glands include more than 20 histological types distinguished by their biological characteristics, response to therapy, and prognosis. The search for specific therapeutically significant markers and mutations for various histological types of salivary gland tumors may allow the development of a diagnostic algorithm and improve patient treatment results.Aim. To search for therapeutically significant molecular and genetic targets and immunohistochemical markers in various histological types of malignant salivary gland tumors.Materials and methods. We analyzed data on 280 patients with unresectable recurrent and metastatic salivary gland cancer to identify the main characteristic therapeutically significant immunohistochemical (androgen receptor (AR), epidermal growth factor receptor type 2 (HER2neu), epidermal growth factor receptor (EgfR), programmed cell death receptor 1 (pD-L1) (CpS), сD117, estrogen receptor (ER), progesterone receptor (pR), pan-TRk) and molecular genetic targets (ALK, BRAF, ERBB2, KIT, MET, RET, ROS1, SMO, AR, HRAS, PIK3CA, PDGFRA, NTRK1-3) depending on the histological subtype of the tumor.Results. Based on the results obtained, in comparison with previous studies, a greater diversity of therapeutically significant mutations, fusions and immunohistochemical markers was revealed for various histological types of tumors. Interestingly, increased expression of AR was detected not only in salivary duct carcinoma, but also in carcinoma ex pleomorphic adenoma, adenocarcinoma not otherwise specified, myoepithelial, mucoepidermoid, adenoid cystic, acinic cell, polymorphous adenocarcinomas. ETV6-NTRK3 gene fusions in addition to secretory carcinoma were detected in mucoepidermoid and salivary duct carcinomas. mutations and fusions in the RET gene have been identified in ductal and mucoepidermoid carcinomas, as well as adenocarcinoma not otherwise specified.Conclusion. The results of the study allow to expand the group of patients for molecular genetic and IHC testing in order to individualize therapy.

Pan-cancer secreted proteome and skeletal muscle regulation: insight from a proteogenomic data-driven knowledge base.

Large-scale, pan-cancer analysis is enabled by data driven knowledge bases that link tumor molecular profiles with phenotypes. A debilitating cancer-related phenotype is skeletal muscle loss, or cachexia, which occurs partly from tumor products secreted into circulation. Using the LinkedOmicsKB knowledge base assembled from the Clinical Proteomics Tumor Analysis Consortium proteogenomic analysis, along with catalogs of human secretome proteins, ligand-receptor pairs and molecular signatures, we sought to identify candidate pan-cancer proteins secreted to blood that could regulate skeletal muscle phenotypes in multiple solid cancers. Tumor proteins having significant pan-cancer associations with muscle were referenced against secretome proteins secreted to blood from the Human Protein Atlas, then verified as increased in paired tumor vs. normal tissues in pan-cancer manner. This workflow revealed seven secreted proteins from cancers afflicting kidneys, head and neck, lungs and pancreas that classified as protein-binding activity modulator, extracellular matrix protein or intercellular signaling molecule. Concordance of these biomarkers with validated molecular signatures of cachexia and senescence supported relevance to muscle and cachexia disease biology, and high tumor expression of the biomarker set associated with lower overall survival. In this article, we discuss avenues by which skeletal muscle and cachexia may be regulated by these candidate pan-cancer proteins secreted to blood, and conceptualize a strategy that considers them collectively as a biomarker signature with potential for refinement by data analytics and radiogenomics for predictive testing of future risk in a non-invasive, blood-based panel amenable to broad uptake and early management.

An Open-Label Phase II Trial of Pembrolizumab, an Immune Checkpoint Inhibitor Alone or in Combination With Oral Azacitidine as Second-Line Therapy for Advanced Head and Neck Squamous Cell Cancers.

Sensitivity to immune checkpoint inhibitor (ICI) therapy depends in part on the genetic and epigenetic makeup of cancer cells, and CD8 T-lymphocytes that mediate immune responses. Epigenetics are heritable reversible changes in gene expression that occur without any changes in the nuclear DNA sequence or DNA copy number. i. To determine if non-cytotoxic oral azacytidine when combined with pembrolizumab can improve ORRs of ICI treatment in patients with recurrent/metastatic tumors of head and neck region. i. To evaluate the clinical effectiveness endpoints and toxicity of oral azacytidine when combined with pembrolizumab. ii. To assess the induction of a T-cell response among the study subjects. iii. To examine the hypotheses on the predictive biomarkers of response to pembrolizumab, and the mechanisms of resistance. Our trial is a Phase 2 randomized study of immunotherapy drug pembrolizumab given in combination with azacitidine (HMA). The intervention model includes "Parallel assignment," with the primary purpose of the trial being treatment. The primary effectiveness endpoint is overall RECIST-defined response. To accomplish this goal, 232 patients will be randomized 1:1 (116 in each arm), respectively, to azacitidine plus pembrolizumab or pembrolizumab only groups. In this trial, molecular profiling of tumor and peripheral blood samples will be conducted which will enable in gaining biological insights for survival benefit. The expected primary outcome assessed at a time frame of 2 years includes the objective response rate of patients measured as per RECIST 1.1 criteria. The secondary outcomes assessed at 2 years include progression-free survival, time to progression, overall survival, and incidence of treatment-emergent adverse events. The findings of this trial will have translational implications, in terms of immune reprogramming induced by epigenetic therapy among a subset of advanced H & N cancer patients in a clinical setting.

Everolimus Through Plasmatic Concentrations in Cancer Patients: Prospective Longitudinal Observational Multicentric Study (DIANA-1 Project).

Background: Everolimus, an oral inhibitor of the mammalian target of rapamycin (mTOR), is actually used to prevent organ transplant rejection and treat metastatic breast, renal, and neuroendocrine cancers. Despite significant pharmacokinetic variability among patients, routine therapeutic drug monitoring (TDM) is not commonly used in oncology. Methods: The aim of this multicenter, prospective observational cohort study is to assess the prevalence of everolimus minimum concentration at a steady state (Cminss) falling outside the therapeutic range (10-26.3 ng/mL) during a routine TDM programme. Sixty patients with metastatic breast, neuroendocrine, or renal cancers, either starting or continuing everolimus treatment according to hospital protocols, are to be included between 1st of January 2024 and 31st of December 2025 (patients undergoing clinical trials are excluded). We hypothesize that 30-50% of our patients and their blood samples will not achieve the target optimal plasma concentrations. Blood samples are collected every 4-6 weeks to monitor drug levels. The secondary goal is to explore correlation between out-of-range everolimus levels and factors such as demographic and anthropometric data, treatment specifics, lab results, genetic polymorphisms, and the presence of toxicity. Conclusions: This study could offer valuable insights into optimizing dosing strategies and may contribute to future research on personalizing everolimus and other anticancer treatments. This personalized approach seeks to tailor therapy not only to the tumour's molecular profile but also to the individual characteristics of each patient, improving both drug selection and dosing precision.

Employing the Oncolytic Vesicular Stomatitis Virus in Cancer Virotherapy: Resistance and Clinical Considerations.

Vesicular Stomatitis Virus (VSV) has emerged as a promising candidate for various clinical applications, including vaccine development, virus pseudotyping, and gene delivery. Its broad host range, ease of propagation, and lack of pre-existing immunity in humans make it ideal for therapeutic use. VSV's potential as an oncolytic virus has garnered attention; however, resistance to VSV-mediated oncolysis has been observed in some cell lines and tumor types, limiting its effectiveness. This review provides a detailed analysis of recent advances in VSV-based oncolysis, focusing on resistance mechanisms such as sustained type-I IFN signaling, upregulation of ISGs, immune cell activation, the tumor microenvironment (TME), and tumor-intrinsic factors. Strategies to overcome resistance include enhancing viral oncoselectivity, inhibiting IFN responses, modulating the TME, and combining VSV with chemotherapies, radiation, and immune checkpoint inhibitors. Several VSV-based phase I/II clinical trials show promise; however, addressing resistance and developing novel strategies to enhance therapeutic efficacy are essential for realizing the full potential of VSV oncolytic virotherapy. Future research should focus on patient-specific approaches, as tumor heterogeneity implies varying resistance mechanisms. Personalized treatments tailored to tumor molecular profiles, along with identifying biomarkers predictive of resistance to VSV oncolysis, will enhance patient selection and enable more effective, individualized VSV-based therapies.

Cuproplasia-Related Gene Signature: Prognostic Insights for Glioma Therapy

Abstract Background Adult-type diffuse gliomas encompass nearly a quarter of all primary tumors found in the CNS, including astrocytoma, oligodendroglioma and glioblastoma. Histopathological tumor grade and molecular profile distinctly impact patient survival. Despite treatment advancements, patients with recurrent glioma have a very poor clinical outcome, warranting improved risk stratification to determine therapeutic interventions. Various studies have shown that copper is a notable trace element that is crucial for biological processes and has been shown to display pro-tumorigenic functions in cancer, particularly gliomas. Methods Differential gene expression, Cox regression, and Least Absolute Shrinkage and Selection Operator (LASSO) regression were used to identify a 19 copper-homeostasis-related gene (CHRGs) signature using TCGA lower-grade glioma (LGG) and glioblastoma (GBM) cohorts. The GLASS Consortium dataset was used as an independent validation cohort. Enrichment analysis revealed involvement of the signature in various cancer-related pathways and biological processes. Using this CHRG signature, a risk score model and a nomogram was developed to predict survival in glioma patients. Results Our prognostic CHRG signature stratified patients into high- and low-risk groups, demonstrating robust predictive performance. High-risk groups showed poorer survival outcomes. The nomogram model integrating CHRG signature and clinical features accurately predicted 1-, 3-, and 5-year survival rates in both training and test sets. Conclusion The identified 19-gene CHRG signature holds promise as a prognostic tool, enabling accurate risk stratification and survival prediction in glioma patients. Integrating this signature with clinical characteristics enhances prognostic accuracy, underscoring its potential clinical utility in optimizing therapeutic strategies and patient care in glioma management.

Precision radiotherapy with molecular-profiling of CNS tumours.

Diagnoses of CNS malignancies in the primary and metastatic setting have significantly advanced in the last decade with the advent of molecular pathology. Using a combination of immunohistochemistry, next-generation sequencing, and methylation profiling integrated with traditional histopathology, patient prognosis and disease characteristics can be understood to a much greater extent. This has recently manifested in predicting response to targeted drug therapies that are redefining management practices of CNS tumours. Radiotherapy, along with surgery, still remains an integral part of treating the majority of CNS tumours. However, the rapid advances in CNS molecular diagnostics have not yet been effectively translated into improving CNS radiotherapy. We explore several promising strategies under development to integrate molecular oncology into radiotherapy, and explore future directions that can serve to use molecular diagnostics to personalize radiotherapy. Evolving the management of CNS tumours with molecular profiling will be integral to supporting the future of precision radiotherapy.

Non-invasive multi-cancer detection using DNA hypomethylation of LINE-1 retrotransposons.

The detection of circulating tumor DNA, which allows non-invasive tumor molecular profiling and disease follow-up, promises optimal and individualized management of patients with cancer. However, detecting small fractions of tumor DNA released when the tumor burden is reduced remains a challenge. We implemented a new highly sensitive strategy to detect base-pair resolution methylation patterns from plasma DNA and assessed the potential of hypomethylation of LINE-1 retrotransposons as a non-invasive multi-cancer detection biomarker. The DIAMOND (Detection of Long Interspersed Nuclear Element Altered Methylation ON plasma DNA) method targets 30-40,000 young L1 scattered throughout the genome, covering about 100,000 CpG sites and is based on a reference-free analysis pipeline. Resulting machine learning-based classifiers showed powerful correct classification rates discriminating healthy and tumor plasmas from 6 types of cancers (colorectal, breast, lung, ovarian, gastric cancers and uveal melanoma including localized stages) in two independent cohorts (AUC = 88% to 100%, N = 747). DIAMOND can also be used to perform copy number alterations (CNA) analysis which improves cancer detection. This should lead to the development of more efficient non-invasive diagnostic tests adapted to all cancer patients, based on the universality of these factors.

Molecular heterogeneity in urothelial carcinoma and determinants of clinical benefit to PD-L1 blockade

Checkpoint inhibitors targeting programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) have revolutionized cancer therapy across many indications including urothelial carcinoma (UC). Because many patients do not benefit, a better understanding of the molecular mechanisms underlying response and resistance is needed to improve outcomes. We profiled tumors from 2,803UC patients from four late-stage randomized clinical trials evaluating the PD-L1 inhibitor atezolizumab by RNA sequencing (RNA-seq), a targeted DNA panel, immunohistochemistry, and digital pathology. Machine learning identifies four transcriptional subtypes, representing luminal desert, stromal, immune, and basal tumors. Overall survival benefit from atezolizumab over standard-of-care is observed in immune and basal tumors, through different response mechanisms. A self-supervised digital pathology approach can classify molecular subtypes from H&E slides with high accuracy, which could accelerate tumor molecular profiling. This study represents a large integration of UC molecular and clinical data in randomized trials, paving the way for clinical studies tailoring treatment to specific molecular subtypes in UC and other indications.

Abstract IA007: Modulating cfDNA biology to enhance liquid biopsies

Abstract Liquid biopsies could transform cancer care but require higher sensitivity to detect early-stage tumors and minimal residual disease (MRD) and to enable tumor molecular profiling and therapy response monitoring for most patients. In this talk, I will focus on the significant biological bottlenecks upstream of cell-free DNA (cfDNA) testing. I will posit that in many circumstances, insufficient circulating tumor DNA (ctDNA) drawn in a blood tube cannot be overcome by assay technology alone, and that in vivo modulation of cfDNA biology may also be required to enhance liquid biopsies. Inspired by widespread use of diagnostic drugs in other areas of medicine, we sought to create the first “priming agents” for liquid biopsies that could be administered before a blood draw to recover more ctDNA. I will describe two such approaches—a monoclonal antibody against double-stranded DNA and a liposome nanoparticle—that briefly attenuate cfDNA clearance by shielding it from nuclease digestion and cellular uptake. These enabled >10x more ctDNA to be collected when administered within 1-2 hours of a blood draw in mice. This improved the analytical limit of detection for ctDNA testing by >10-fold, provided more complete representation of the tumor genome in each blood sample, and increased the sensitivity for detection of small tumors from <10% to >75% in mice. Envisioning a future where liquid biopsies are optionally enhanced with priming, I will also explore a unique potential pairing with our tumor-informed, whole-genome, mutation enrichment sequencing MRD test called MAESTRO, and how it stands to enable routine ctDNA detection below 1 part per million. Citation Format: Viktor Adalsteinsson. Modulating cfDNA biology to enhance liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr IA007.

CTNI-73. ASSESSMENT OF MOLECULAR ALTERATIONS IN NEWLY DIAGNOSED GBM MGMT-UNMETHYLATED PATIENTS TREATED WITH VAL-083

Abstract Glioblastoma (GBM) commonly features molecular alterations in the TERT, EGFR, PTEN, and TP53 genes, which contributes to uncontrolled cell growth, evasion of apoptosis, and enhanced tumor cell survival. VAL-083 is a bi-functional DNA-targeting agent which rapidly induces inter-strand DNA cross-links at N7 and O6-guanine inducing double-strand breaks causing cell death independently of MGMT DNA repair and DNA mismatch repair deficiency. VAL-083 was evaluated in an open-label, biomarker-driven, phase 2 trial in MGMT-unmethylated, bevacizumab-naïve GBM patients. The molecular profile of tumors from a newly diagnosed GBM patient subgroup who received VAL-083 alone after chemo-radiation with concurrent TMZ, was determined in order to correlate with clinical outcomes. Thirty-four (34/36, 94.4%) patients who received 30 mg/m2 VAL-083 (days 1-3 of 21-day cycle) had available next-generation sequencing data on their primary tissue. Patient median age was 54.8 years (5-95pth: 29.8-66.0), median KPS was 90 (5-95pth: 80-100) and 58.8% were males. The median number of cycles of VAL-083 was 7.5 (5-95pth: 1-12). There were 5 dose reductions due to toxicity. The median time to progression (PFS) and overall survival (OS) were 9.2 mo (95%CI: 7.6-10.2) and 16.5 mo (95%CI: 13.3-19.0), respectively. The mean number of molecular alterations was 3.9 (5-95pth 1-8). Six (17.6%) patients had gene fusions. The most common molecular alterations were TERT promoter (91.2%), EGFR amplification (amp) (52.9%), PTEN (50.0%), EGFR (20.6%), and TP53 (20.6%). Additional alterations (in ≥2 patients; 6%) included EGFR-EGFR (EGFRvIII), BRCA2, CDKN2A/B, CDK4/6 amp, MDM2/4 amp, NF1, NOTCH1, PIK3R1 and RB1. While TP53 mutations showed a trend as predictors for time to progression (P 0.027), this was not observed with OS. None of the molecular alterations were predictors of number of treatment cycles or dose reductions. No specific molecular alterations were identified that predicted either improved or poorer patient outcome, during treatment with VAL-083. Clinicaltrials.gov identifier: NCT02717962.

NCOG-35. IDENTIFYING PROGNOSTIC FACTORS IN PEDIATRIC DIFFUSE MIDLINE GLIOMAS, H3 K27-ALTERED

Abstract INTRODUCTION Diffuse midline gliomas, H3 K27-altered (DMG) are rare, grade 4 tumors localized to the diencephalon, brainstem, and cervical spine. DMGs, characterized by a K27M missense mutation in histone H3, often cannot be safely resected and predominantly present with poor outcomes in children. Few prognostic factors for DMGs have been identified and/or need to be further elucidated, including adolescent age of onset, re-irradiation status, and tumor molecular profile (i.e., PIK3CA mutation). Herein, we describe the clinical outcomes of DMG patients at our institution. METHODS Pediatric patients (<18 y/o) between 2015 and 2023 with biopsy-proven DMG were reviewed. Demographic, treatment, and tumor characteristics (histologic and molecular) were recorded. Adolescent-onset DMGs were defined as patients ≥10 y/o at diagnosis and early-onset DMGs were defined as patients <10 y/o. Progression free survival (PFS) and overall survival (OS) were calculated according to the Kaplan Meier-method. Multivariate regression analyses were performed. RESULTS Twenty-one patients were included (median age=9 y/o). Early-onset DMGs (n=13) progressed significantly earlier and experienced significantly greater mortality than adolescent-onset DMGs (n=8); PFS and OS at 12 months was 7.7% and 23% for early-onset DMGs and 50% and 87.5% for adolescent-onset DMGs (PFS: p<0.01, OS: p<0.02). Patients that received re-irradiation for progressive disease (n=9, mean=84Gy) had an OS of 75% at 12 months, which was significantly greater than an OS of 23% at 12 months (p<0.05) for patients that received no re-irradiation (n=12, mean=53Gy). PIK3CA mutation status was identified as an independent predictor of survival, with PIK3CA-mutant DMGs (n=7) having a median survival of 4.1 months versus 9.7 months for PIK3CA-WT DMGs. CONCLUSION We describe a pediatric cohort that presents several possible prognostic factors of survival in DMGs. Our findings on adolescent age of onset, if further corroborated, has the potential to inform the design and enrollment of DMG clinical trials.

STEM-15. THE IMPACT OF TUMOR TREATING FIELDS (TTFIELDS) ON CANCER STEM-LIKE CELLS ISOLATED FROM THE SUB-VENTRICULAR ZONE OF GLIOBLASTOMA PATIENTS

Abstract Treatment of glioblastoma (GBM) is challenging due to its heterogeneous nature, invasive potential, and poor response to standard-of-care treatments. Areas of residual disease represent the source of the recurrent tumor that is fatal for GBM patients. However, targeting the residual disease is not offered as part of the standard of care because these areas are difficult to identify. Our laboratory was the first to identify and characterize residual disease in the sub-ventricular zone (SVZ) of the lateral ventricles of GBM patients. In this study, we are examining the impact of Tumor Treating Fields (TTFields) on treatment-resistant cancer stem-like cells (CSCs) isolated from the SVZ of 12 GBM patients using single-cell transcriptomics and functional phenotyping analysis. These cells are maintained in conditions that preserve the molecular profile of the original patient tumor, thus representing bona fide models to study the impact of TTFields on GBM residual disease. Our results show that: CSCs isolated from the SVZ from different individuals show different sensitivity to TTFields, the anti-proliferative effects of TTFields are maintained at different plating cell densities, and when the frequency of TTFields increase from 50 kHz to 200 kHz, CSCs undergo morphological changes (e.g., fewer spheres and fewer connections between cells). We used 100 μM Temozolomide and ionizing radiation (10 Gy) mimicking the standard of care of GBM patients, and the growth of the treatment-resistant CSCs was inhibited by TTFields (200 kHz). To elucidate how TTFields impact the cellular and molecular characteristics of CSCs in their microenvironment, ongoing work focuses on single-cell transcriptomics and functional phenotyping analysis. These results extend our understanding of the mechanisms of action of TTFields, specifically on how TTFields have the potential to alter the cytokine-mediated cross-talk between CSCs of the SVZ and their microenvironment.

BIOM-51. MOLECULAR SUBTYPES, CLINICAL CHARACTERISTICS, AND THERAPEUTIC IMPLICATIONS FOR CNS TUMORS WITHBCOR/BCORL1 FUSION/INTERNAL TANDEM DUPLICATION

Abstract Central nervous system (CNS) tumors with either BCOR internal tandem duplication (BCOR ITD) or gene fusions involving BCOR/BCORL1 (BCOR FUS) are recognized as tumor types with characteristic molecular and clinical features but lacking clear therapeutic guidance. In an extensive international effort, we compiled a retrospective cohort of 229 (148 BCOR ITD, 81 BCOR FUS) unique cases via molecular profiling of CNS tumors. By analyzing DNA methylation patterns of 220 tumors we confirmed distinct epigenetic profiles of BCOR ITD versus BCOR FUS. The majority (91 percent) of BCOR FUS primary tumors was localized in the supratentorial region whereas BCOR ITD were localized across all anatomical CNS compartments. Median patient age at diagnosis was 20 years for BCOR FUS and 4 years for BCOR ITD. Metastatic disease was rare in BCOR FUS patients but more common in BCOR ITD tumors where 10 percent presented with metastatic disease and one third showed CNS or (rarely) extra-CNS metastasis in the course of disease. This was also reflected in clinical outcome as 5-year overall survival (OS) was 89 percent in BCOR FUS but 61 percent in BCOR ITD. In contrast, 5-year progression-free survival (PFS) was comparable reaching 27 percent in BCOR FUS and 30 percent in BCOR ITD. Integrating treatment details we found a trend towards benefit of gross-total resection on OS (BCOR FUS, n=18, p<0.08; BCOR ITD, n=67, p<0.12) but not for PFS (BCOR FUS, n=16, p<0.33; BCOR ITD, n=64, p<0.59). Radiotherapy was associated with improved PFS (BCOR FUS, n=18, p<0.01; BCOR ITD, n=70, p<0.001) and OS (BCOR FUS, n=20, p<0.01; BCOR ITD, n=73, p<0.001). Further in-depth analyses of clinical outcome parameters (primary metastatic disease, radiotherapy approach, chemotherapy type, patient age) are currently ongoing. In summary, we provide first robust annotation of CNS BCOR tumor types, clinical outcomes and therapy.

BIOM-04. SINGLE-CELL RNA SEQUENCING PROTOCOL OF THE HUMAN METASTATIC PROSTATE SPINE TUMOR MICROENVIRONMENT

Abstract INTRODUCTION 90% of spine tumor patients can develop metastases with debilitating complications, such as sensorimotor dysfunction, paralysis, and spinal instability. As spine tumor patients have limited eligibility to participate in clinical trials and have heterogenous tumor pathology, there is a distinct lack of therapeutic targets and prognostication markers of disease. Thus, there is a distinct need for molecular profiling of spine tumors. OBJECTIVE We describe an OR-to-bench-top processing of spine tumors for single-cell omic studies, with samples from a metastatic prostate cancer patient to demonstrate the ability to compare molecular markers and microenvironments of difficult-to-process spine tumors and adjacent normal tissue. METHODS Under IRB protocol #Pro00101198, patients undergoing spine surgery with the Department of Neurosurgery at Duke University were consented for tissue collection. Vertebral osseous metastases and adjacent normal tissue were confirmed histologically (H&E, immunohistochemistry) and radiologically (MRI, PET, CT, and x-ray). Tissues were manually and enzymatically homogenized in serum-free media with collagenase A and DNAse I to obtain a single cell suspension. Cells were then filter sterilized, erythrolyzed, counted to determine live-death ratio, resuspended to 10-20 million cells per mL, then cryopreserved for downstream standard library preparation for single-cell sequencing using the 10X genomics platform. Downstream analysis was performed using Seurat pipeline, InferCNV analysis, and SingleR. RESULTS P404S and PSA expression confirmed tumor was prostate in origin. Radiological imaging and H&E staining confirmed the presence of T7 osseous metastasis. Uniform manifold approximation and project (UMAP) plots identified 12 unique clusters of immune cell subpopulations between normal and tumorous vertebrae, with their own subsets of differentially expressed genes. CONCLUSION We have developed a protocol to generate granular single cell molecular profiling for spine tumors and metastases. Applications of this protocol can aid in identifying therapeutic targets, molecular predictors of disease, and potentiate increased eligibility of patients for clinical trials.

TMIC-46. TRANSCRIPTIONAL HETEROGENEITY AND MECHANISTIC PATHWAYS OF RECURRENT GLIOBLASTOMA: INSIGHTS FROM A PRE-CLINICAL RECURRENT TUMOR MODEL

Abstract Despite aggressive therapy combining surgical resection, radiation and chemotherapy, glioblastoma (GBM) almost always recurs. Currently, there is no standard treatment for recurrent GBM, and patient inclusion in clinical trials is based on the genetic and molecular profile of the primary tumor even though the effects of chemoradiation on the transcriptional subtype in matched primary and recurrent GBM remain largely understudied. Paralleling the GLASS consortium, we sought to perform a longitudinal assessment of GBM recurrence after therapy based on molecular subtype with the hypothesis that recurrent tumors exhibit a distinct transcriptional landscape different from treatment-naive tumors. To address this, we utilized primary tumor-derived glioma stem cells (GSCs) representing classical and proneural subtypes. We then undertook in vivo intracranial implantation studies and performed standard concomitant treatment that mimics the Stupp protocol. Upon bulk RNAseq data analyses of untreated (n=21) and recurrent tumors (n=17) we identified that while the molecular subtype of recurrent tumors does not change post therapy, their transcriptional profile is more heterogenous compared to untreated tumors for both subtypes. Hierarchical cluster analyses revealed distinct sub-clusters of recurrent tumors. Common mechanisms of recurrence include upregulation of angiogenesis pathways and PI3k/Akt/mTOR signaling pathways. Interestingly, recurrent tumors exhibit upregulation of genes involved in cellular metabolism and membrane transport/ potential. The source of heterogeneity in recurrent tumors in both subtypes appears to be due to the diversity in expression of gene sets involved in hallmark of cancer pathways such as cell cycle, Notch and Wnt signaling pathways and heterogeneity in oncogenic signatures and cell states resembling fetal or neural cells. Further research into the recurrent tumor microenvironment by spatial multi-omics approaches using this pre-clinical recurrent GBM model will elucidate the linkage between cellular as well as molecular heterogeneity and enable identification of targets that can help drive therapeutic decisions for recurrent GBM patients.