Biomarker-driven Clinical Trials Research Articles

IMMU-12. TRANSLATING COMBINED PD1 AND LAG3 INHIBITION FROM PRECLINICAL MODELS TO PATIENTS WITH REFRACTORY, DNA REPLICATION REPAIR DEFICIENT (RRD) GLIOBLASTOMA: AN IRRDC STUDY

Abstract BACKGROUND AND AIMS RRD-glioblastoma harbour high tumor mutation burden (TMB) and respond to anti-PD1 immune-checkpoint inhibition (ICI). However, not all respond, and the majority progress, highlighting the need for combinatorial therapies for sustained immune-surveillance. METHODS We performed transcriptomic analyses of human RRD-glioblastoma specimens for immune checkpoint expression, and accordingly, tested combined ICI in immunocompetent murine models. Based on these preclinical data, we treated refractory patients using a combination of anti-PD1+anti-LAG3 through single-patient trial/compassionate access. Complimentary immuno-genomic biomarker analyses including circulating tumor DNA (ctDNA) were performed to investigate mechanisms and track responses. RESULTS Human RRD-glioblastoma (n=80) demonstrated high LAG3 expression, providing a strong rationale for therapeutic targeting. We tested combined anti-PD1+anti-LAG3 inhibition in three immunocompetent RRD-glioblastoma murine models. In the anti-PD1-responsive (Nestin-CreMSH2LoxP/LoxP-POLES459F/+) model, anti-PD1+anti-LAG3 resulted in universal tumor response and superior survival to ICI-monotherapy. In the anti-PD1 resistant models (Mlh1-/-/NestinCre+/Trp53LoxP/LoxP and therapy-induced hypermutant ENU/Trp53-/- gliomas), anti-PD1+antiLAG3 improved survival, overcoming the lack of response to ICI-monotherapy. Biologically, high LAG3 expression and immune-exhaustion observed in CD8 T-cells after treatment with anti-PD1 was ablated following the addition of anti-LAG3. Serially transplanted, post-anti-PD1 treated tumors showed response, confirming, in-vivo, that resistance to anti-PD1 could be abrogated by anti-PD1+anti-LAG3. Seven children with refractory RRD-glioblastoma who had progressed after anti-PD1 treatment were treated using anti-PD1+anti-LAG3, resulting in objective radiological responses and prolonged ongoing survival. Tolerability was better than a previous study of combined CTLA4 and PD1 inhibition for similar patients. Paired immuno-genomic tumor analyses, serial blood flow-cytometry, T-cell receptor clonotype, and CSF ctDNA analyses provided novel insights into the mechanisms of immunological invigoration and first-in-human, radiological responses. CONCLUSIONS LAG3 is an effective target in refractory RRD-glioblastoma. Combined inhibition with anti-PD1 inhibition demonstrated radiological response, prolonged survival and manageable toxicities in patients, and unearthered mechanisms of immune-responses. The combination will now be tested in biomarker-driven clinical trials in RRD-glioblastoma and other immune-inflamed solid tumors.

Rapid label expansion based on public-private collaboration in IMPRESS-Norway.

e13529 Background: Access to new cancer drugs requires knowledge of biomarkers. As a consequence, there is an increasing access gap to new treatments between different cancer subgroups. Several precision medicine trials aim to address this gap through drug repurposing (1-3). IMPRESS-Norway is a national precision cancer medicine trial providing treatment to patients with advanced-stage disease after standard therapy. Patients with biomarkers are identified through the national infrastructure for precision diagnostics, InPreD, a part of the national healthcare services for stratification into biomarker-driven clinical trials. IMPRESS-Norway opened for inclusion on April 1st, 2021. Currently InPreD identifies biomarker in 31% of patients, and 50 % of patients included in treatment cohorts in IMPRESS-Norway are diagnosed with a rare cancer (4). The IMPRESS-Norway has, together with the public-private partnership CONNECT (Norwegian Cancer Precision Medicine Implementation Consortium), developed a roadmap for expanding access through ordinary reimbursement decisions. Methods: New drugs can efficiently be incorporated into the IMPRESS-Norway drug portfolio and tested across all relevant indications through a three-stage process. In stage 1 and 2, a total of 24 patients are recruited into a drug/diagnosis/biomarker-specific cohort, with drug provided by the company and a per-patient fee. If ≥ 5 patients exhibit clinical benefit (stable disease or better at week 16), the drug may undergo further testing in stage 3 (expansion cohorts). The company provides drugs during the first 16 weeks of treatment and with public reimbursement after week 16 for all patients experiencing clinical benefit. Results: Positive reimbursement decisions in Norway can be made based on knowledge of effect from single armed studies (5). Norwegian payers can therefore make reimbursement decisions based on results from the expansion cohort. Similar systems are set-up in the Netherlands (6). Several drugs are currently concluding stage 2 in IMPRESS-Norway. The first expansion cohort in IMPRESS-Norway (jointly with other DRUP-like trials in the EU Cancer Mission project PRIME-ROSE), exploring the effect of olaparib in patients with biallelic BRCA1/2 inactivation in non-approved diagnosis, is based on the DRUP-trial, exemplifying the European collaboration in these trials. Conclusions: Public-private collaboration facilitates rapid identification of eligible patients, irrespective of cancer subgroup, providing potential for new indications. IMPRESS-Norway presents a feasible strategy for rapid label expansion. 1. DLvan der Velden et al, Nature 2019; 574:127-31. 2. PM Mangat et al, JCO Prec Oncol 2018; 10. 3. Å Helland et al, J Trans Oncol 2022; 20. 4. Puco K et al., 2024 in press. 5. GL Fagereng et al, Front Pharmacol 2014; doi:10.3389. 6. SBW Doorn-Khosrovani et al. Ann Oncol 2019; 30: 663-5. Clinical trial information: NCT04817956 .

Improving ethnic and racial diversity in biomarker-driven clinical trials: A proof of concept with the BASECAMP-1 master prescreening study of patients with high-risk solid tumors with human leukocyte antigen-A*02 (HLA-A*02) loss of heterozygosity (LOH).

1604 Background: Diversity in clinical studies is essential to address health disparities, but racial and ethnic minorities are often underrepresented in clinical studies, especially in precision medicine studies using genetic data (1). BASECAMP-1 (NCT04981119) is an ongoing prescreening study to identify patients with unresectable advanced or metastatic solid tumors and tumor-associated HLA-A*02 LOH for the EVEREST-1 and -2 Tmod chimeric antigen receptor T-cell therapy trials. When enrollment began in 2021, eligibility for Part 1 was restricted to patients with germline HLA-A*02:01 based on preclinical data. Additional analysis revealed the blocker in the Tmod construct had activity across HLA-A*02 subtypes (Mock, et al. Mol Ther Oncolytics.2022). Eligibility for Part 1 was then expanded to all HLA-A*02 subtypes, allowing enrollment of more diverse patient populations. HLA-A*02 allele subtypes vary by ethnicity and race; the frequency of HLA-A*02:01 is 96% in non-Hispanic Whites, but ranges from 53% to 73% in other ethnicities and races. The frequency of all other HLA-A*02 allele subtypes (HLA-A*02:XX) is <5% in non-Hispanic Whites but up to 66% in other ethnicities and races (2,3). Methods: Patients eligible for BASECAMP-1 were identified at United States study sites. All patients screened before January 16, 2024, for Part 1 were included for demographic and central lab HLA-A typing. The % increase in eligibility and % of heterozygous HLA-A*02 patients with HLA-A*02:XX were calculated by ethnicity and race. Results: A total of 1124 patients were screened for germline HLA-A*02 typing; 431 patients were HLA-A*02:01 heterozygous, of whom 37 (9%) were Hispanic, 14 (3%) African American (AA), 2 (<1%) American Indian or Alaska Native, 17 (4%) Asian or Pacific Islander (API), and 324 (75%) non-Hispanic White (Table). The eligibility expansion identified 62 patients with HLA-A*02:XX heterozygosity, of whom 6 (10%) were Hispanic, 6 (10%) AA, 19 (31%) API, and 25 (40%) non-Hispanic White. Conclusions: By expanding enrollment to include HLA-A*02:XX, 16% more Hispanic, 43% more AA, and 112% more API patients were identified, improving the diversity in this trial compared with that of initial enrollment. 1. Aldrighetti, et al. JAMA Netw Open 2021. 2. Ellis, et al. Human Immunol. 2000. 3. Gragert, et al. Human Immunol.2013. Clinical trial information: NCT04981119 . [Table: see text]

Abstract PO4-05-07: Differential dynamics of fragmentomic and epigenetic circulating tumor DNA (ctDNA) features in first-line Palbociclib and Ribociclib treatment for Hormone Receptor (HR) positive, HER2 negative metastatic breast cancer (MBC)

Abstract Background: Cyclin-dependent kinase inhibitors (CDK4/6i) in combination with endocrine therapy (ET) represent the cornerstone for the management of HR positive, HER2 negative MBC. However, the lack of prospective cohorts comparing the distinctions among palbociclib (PAL), ribociclib (RIB) and abemaciclib (ABE) poses a significant clinical knowledge gap. The aim of this ancillary analysis was to explore the differential clinical impact of CDK4/6i by leveraging new ctDNA biomarkers in a prospective clinical study. Methods: A total of 114 patients (pts) with HR positive, HER2 negative MBC were prospectively enrolled in the multicenter MAGNETIC.1 study (CRO-2018-56) from January 2018 to January 2023. At baseline (BL) and every 3 months (mos.), ctDNA samples were collected concurrently with radiological restaging. The samples were then analyzed through methylation-specific (MS) droplet digital PCR (ddPCR), ddPCR fragmentomics, and next generation sequencing. Matched pairs of variations were tested through Wilcoxon signed rank test, and survival was analyzed by log-rank test. Results: Out of the 114 pts enrolled, 59 received PAL, 48 RIB, and 16 ABE. In the de novo subgroup, 51.4 % of pts were treated with PAL, 42.9% with RIB, and 5.7% with ABE. Among the pts who received PAL, 7 harbored an ESR1 mutation at BL (D538G: 2, H377R: 1, Y537N: 1, Y537S: 3). Additionally,12 pts had PIK3CA mutations, and 2 pts had AKT1 mutations. In the RIB group, 5 and 2 pts, respectively, had a PIK3CA- and AKT-mutated MBC, while no ESR1 mutations were detected. When comparing matched pairs of variations, it was found that at BL, at first radiological restaging (T1), and at 6 mos. after initiation of CDK4/6i (T2), only PAL showed significantly higher levels of promoter A and B methylation (promA and promB) at T1 (P = 0.0005 and P = 0.0056, respectively for promA and promB), A significant decrease in promA and promB levels was detected at T2 for PAL (P = 0.0005 and P < 0.0001 respectively for promA and promB) but not for RIB. Both PAL and RIB demonstrated a significant decrease in ctDNA short fragments at T1 (P = 0.0263 for PAL and P = 0.0001 for RIB), However, PAL showed a significant rebound at T2, with an increase in 80% of samples (P = 0.0014). The same was not seen with RIB. After a median follow-up of 34.8 mos., there were no significant differences between PAL and RIB in terms of PFS (P= 0.2573) and OS (P= 0.3783). The PFS rates at 12 and 24 mos. were 69% and 48% for PAL, and 76% and 64% for RIB. The OS rates at 12 and 24 mos. were 94% and 84% for PAL, compared to 97% and 82% of RIB, respectively. Conclusions This analysis of MAGNETIC.1 highlighted significant differences in ctDNA biomarkers between palbociclib and ribociclib, suggesting their potential impact on the development of endocrine resistance. Further biomarker-driven clinical trials leveraging this concept are needed to refine the sequencing algorithms for CDK4/6i. Citation Format: Claudia Noto, Linda Cucciniello, Elisabetta Molteni, Silvia Bolzonello, Alessandra Franzoni, Lucia Da Ros, Silvia Buriolla, Arianna Dri, Lorenzo Foffano, Simon Spazzapan, Stefania Russo, Brenno Pastò, Giulia Cudia, Giada Targato, Serena Della Rossa, Marta Bonotto, Alessandro Marco Minisini, Giuseppe Damante, Barbara Belletti, Lorenzo Gerratana, Fabio Puglisi. Differential dynamics of fragmentomic and epigenetic circulating tumor DNA (ctDNA) features in first-line Palbociclib and Ribociclib treatment for Hormone Receptor (HR) positive, HER2 negative metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-05-07.

Quantitative Assessment of Preanalytic Variables on Clinical Evaluation of PI3/AKT/mTOR Signaling Activity in Diffuse Glioma

Biomarker-driven therapeutic clinical trials require the implementation of standardized, evidence-based practices for sample collection. In diffuse glioma, phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR (PI3/AKT/mTOR) signaling is an attractive therapeutic target for which window-of-opportunity clinical trials could facilitate the identification of promising new agents. Yet, the relevant preanalytic variables and optimal tumor sampling methods necessary to measure pathway activity are unknown. To address this, we used a murine model for isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) and human tumor tissue, including IDH-wildtype GBM and IDH-mutant diffuse glioma. First, we determined the impact of delayed time-to-formalin fixation, or cold ischemia time (CIT), on the quantitative assessment of cellular expression of 6 phosphoproteins that are readouts of PI3K/AK/mTOR activity (phosphorylated-proline-rich Akt substrate of 40 kDa (p-PRAS40, T246), -mechanistic target of rapamycin (p-mTOR; S2448); -AKT (p-AKT, S473); -ribosomal protein S6 (p-RPS6, S240/244 and S235/236), and -eukaryotic initiation factor 4E–binding protein 1 (p-4EBP1, T37/46). With CITs ≥ 2 hours, typical of routine clinical handling, all had reduced or altered expression with p-RPS6 (S240/244) exhibiting relatively greater stability. A similar pattern was observed using patient tumor samples from the operating room with p-4EBP1 more sensitive to delayed fixation than p-RPS6 (S240/244). Many clinical trials utilize unstained slides for biomarker evaluation. Thus, we evaluated the impact of slide storage conditions on the detection of p-RPS6 (S240/244), p-4EBP1, and p-AKT. After 5 months, storage at −80°C was required to preserve the expression of p-4EBP1 and p-AKT, whereas p-RPS6 (240/244) expression was not stable regardless of storage temperature. Biomarker heterogeneity impacts optimal tumor sampling. Quantification of p-RPS6 (240/244) expression in multiple regionally distinct human tumor samples from 8 patients revealed significant intratumoral heterogeneity. Thus, the accurate assessment of PI3K/AKT/mTOR signaling in diffuse glioma must overcome intratumoral heterogeneity and multiple preanalytic factors, including time-to-formalin fixation, slide storage conditions, and phosphoprotein of interest.

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.

Comprehensive genomic profiling of squamous cell carcinoma of unknown primary presenting with liver metastases.

755 Background: Carcinomas of unknown primary (CUPs) are comprised of metastatic cancers for which a primary tumor of origin cannot be identified despite a full diagnostic workup. They frequently present with liver metastases, which portend a poor prognosis. Strategies employing comprehensive genomic profiling (CGP) to identify targeted treatments for CUPs have shown potential. Despite this, the molecular landscape of squamous cell carcinomas of unknown primary (SCCUPs) remains poorly defined. This study describes the results of CGP testing in patients with SCCUPs identified in the Foundation Medicine, Inc. database, with a focus on those with liver involvement. Methods: Cases of SCCUPs with FoundationOne CDx (F1CDx) assay results were identified based on a reported diagnosis of CUP and the presence of SCC histology. Cases then underwent central pathologist review and were excluded if a known primary site could be determined based on histology and disease location. Cases with liver involvement were selected based on the site of tissue biopsy. Genomic analyses of identified cases were assessed using the FoundationCORE database. Results: Sixty-nine cases of SCCUP presenting with liver involvement were identified. Alterations were observed in 102 of 324 (31.5%) genes evaluated by the F1CDx assay in at least 1 patient. The most frequently altered genes were TP53 (66.7%) , CDKN2A (33.3%) , CDKN2B (23.2%) , KRAS (21.7%; 0% G12C) , PIK3CA (18.8%), NFE2L2 (11.6%), SOX2 (11.6%), M2M2 (10.1%), and MTAP (10.1%). No patients had microsatellite instability-high disease, only 6 (8.7%) patients had a tumor mutational burden (TMB) ≥ 10, and 11 (15.9%) patients had evidence of HPV infection. Among 33 patients with available PDL-1 testing results, 7 (21.2%) had high-positive expression. An additional 374 cases of SCCUP were identified with involvement outside of the liver. Compared to these cases, patients presenting with liver involvement had less genomic alterations per tumor (5.10 vs 6.71, p=0.005), and were less likely to have TMB ≥ 10 (8.7% vs 38.0%; odds ratio [OR] 0.16, p<0.001). KRAS mutations were observed in 21.7% of those presenting with liver metastases compared to 8.3% of other SCCUP patients (OR 3.07, p=0.07). Conclusions: SCCUPs presenting with liver metastases had reduced numbers of genomic alterations and decreased TMB compared to other SCCUPs, suggesting these tumors may be less likely to respond to immunotherapy. Despite this, 36.2% of these patients had at least one alteration that could potentially guide targeted treatment decisions including entry into biomarker-driven clinical trials. Interestingly, there was a trend toward increased KRAS alterations in those with liver metastases. To our knowledge, this is the first report that describes the genomic landscape of SCCUPs presenting with liver metastases.

Challenges and opportunities in the immunotherapy era: balancing expectations with hope in small-cell lung cancer.

Small-cell lung cancer (SCLC) is a biologically aggressive subtype of lung cancer, a lethal disease characterized by rapid tumor growth, early relapse, a strong tendency for early widespread metastasis, and high genomic instability, making it a formidable foe in modern oncology practice. While the management of non-SCLC has been revolutionized in the era of immunotherapy, progress in SCLC has been more muted. Recent randomized phase III clinical trials have combined programmed death ligand-1 inhibitors to a chemotherapy backbone and demonstrated improved survival; however, the absolute benefit observed is short months. There is an undeniable urgent need for better responses, better agents, novel therapeutic approaches, and more rational, biomarker-driven clinical trials in SCLC. In this review, we discuss the rationale and current understanding of the biology of SCLC in the modern era of immunotherapy, discuss recent advances in front-line immunotherapeutic approaches that have changed clinical practice globally, provide an overview of some of the challenges and limitations that have staggered immune checkpoint blockade in SCLC, and explore some of the novel immunotherapeutic approaches currently being investigated.

Use of biomarkers in clinical trials and future developments that will help identify novel biomarkers.

Engineering new solutions for therapeutic benefit in Amyotrophic Lateral Sclerosis (ALS) has proved a difficult task to accomplish. This is largely the reflection of complexities at multiple levels, that require solutions to improve cost-effectiveness and outcomes. The main obstacle related to the condition's clinical heterogeneity, chiefly the broad difference in survival observed among ALS patients, imposes large populations studies and long follow-up to evaluate any efficacy. The emerging solution is composite clinical and biological parameters enabling prognostic stratification into homogeneous phenotypes for more affordable studies. From a therapeutic development perspective, the choice of a medicinal product requires the availability of treatment-specific biomarkers of target engagement to identify off-target effects based on the compound's putative modality of action. More importantly, there are no established biomarkers of treatment response that can complement clinical outcome measures and support futility and end of treatment analyses of efficacy. Ultimately the onus rests on the development of biomarkers encompassing the unmet needs of clinical trial design, from inclusion to efficacy. These readouts of the pathological process may be used in combination with clinical and paraclinical outcome measured, significantly reducing the time and financial burden of clinical studies. Progress towards a biomarker-driven clinical trial design in ALS has been possible thanks to the accurate detection of neurofilaments and of other immunological mediators in biological fluids with the disease progression, a step change enabling the testing of novel therapeutic agents in a new clinical trial setting. However, further progress remains to be made to find treatment specific target engagement biomarkers along with readouts of treatment response that can be reliably applied to all emerging therapies and clinical studies. Here we will cover the basic notions of biomarker development in ALS clinical trials, the most crucial unanswered questions and the unmet needs in the ALS biomarkers space.

Circulating Tumor Cells Prediction in Hormone Receptor Positive HER2-Negative Advanced Breast Cancer: A Retrospective Analysis of the MONARCH 2 Trial.

The MONARCH 2 trial (NCT02107703) showed the efficacy of abemaciclib, a cyclin-dependent kinase 4 & 6 inhibitor (CDK4/6i), in combination with fulvestrant for hormone receptor-positive, HER2-negative metastatic breast cancer (MBC). The aim of this analysis was to explore the prediction of circulating tumor cells (CTCs) stratification using machine learning for hypothesis generation of biomarker-driven clinical trials. Predicted CTCs were computed in the MONARCH 2 trial through a K nearest neighbor (KNN) classifier trained on a dataset comprising 2436 patients with MBC. Patients were categorized into predicted Stage IVaggressive (pStage IVaggressive, ≥5 predicted CTCs) or predicted Stage IVindolent (pStage IVindolent, <5 predicted CTCs). Prognosis was tested in terms of progression-free-survival (PFS) and overall survival (OS) through Cox regression. Patients classified as predicted pStage IVaggressive and predicted pStage Stage IVindolent were, respectively, 183 (28%) and 461 (72%). After multivariable Cox regression, predicted CTCs were confirmed as independently associated with prognosis in terms of OS, together with ECOG performance status, liver involvement, bone-only disease, and treatment arm. Patients in the pStage Stage IVindolent subgroup treated with abemaciclib experienced the best prognosis both in terms of PFS and OS. The treatment effect of abemaciclib on OS was then explored through subgroup analysis, showing a consistent benefit across all subgroups. This study is the first analysis of CTCs modeling for stage IV disease stratification. These results show the need to expand biomarker profiling in combination with CTCs stratification for improved biomarker-driven drug development.

Biomarker-driven prospective clinical trial in renal cell carcinoma: Developing machine learning models to allocate patients to treatment arms using RNA sequencing.

4525 Background: RNA sequencing has shown promise in defining the biology of individual renal cell carcinoma (RCC) tumors. However, these biomarkers have not yet been translated to the clinic for prospectively assigning optimal treatments to patients. Challenges for RNA-seq biomarker development include translating classifiers across different assays/platforms, normalization of data collected from single patients in the clinic and establishing robust thresholds for assigning prediction groups. We have designed the prospective phase II OPTIC RCC clinical trial (clinicaltrials.gov NCT05361720) to test the utility of an RNA-seq based biomarker in predicting treatment based on biologic drivers relevant to angiogenesis (anti-angiogenesis; TKI) and immune microenvironment (immunotherapy; IO). Here, we will describe the development and optimization of a machine learning model for assigning individual patients to biologically driven clusters in real time to facilitate RNA-seq based biomarker trials. Methods: We have utilized RNA-seq data from the IMmotion 151 trial (ref) to develop a machine learning model. Clusters were grouped into three based on their association to tumor biology and treatment: (1) Cluster 1+2 (angiogenic signature → TKI+IO therapy), (2) Cluster 4+5 (immune/proliferative signature → dual IO therapy), (3) Cluster 3+6 (neither signature → exclude). Random forest classifier was used to train a multi-class model to predict the three groups. The model is evaluated using bootstrapped cross-validation. Results: Our machine learning classifier was built using 188 genes and has a cross-validation accuracy of 85% and sensitivity of &gt;90% in predicting patients into one of the three biological clusters from our training data. Predictions of the classifier are significantly associated with progression-free survival across different treatments within each of the predicted groups. We also observed significant odds ratios when comparing responders (CR/PR/SD) to non-responders (PD) across the treatment groups. The model was then validated in two independent test sets treated with Angio and IO inhibitors: (1) 61 patient renal cell carcinoma cohort (2) 12 patient clear cell renal cell carcinoma cohort. We observed a significant enrichment of responders to Angio+IO treatment for the predicted Cluster_Angio patients compared to IO treatment (p=0.05), highlighting the importance of matching patients to their optimal treatment. Conclusions: We have developed an accurate machine learning model to assign individual patients to RNA-seq clusters in real time. This classifier will facilitate the prospective OPTIC RCC trial. If successful, our biomarker strategy will serve as a proof of concept for selecting optimal treatments for RCC patients.

The PLATON Network: An interactive platform for precision oncology.

TPS1613 Background: To increase the access of patients to precision oncology and personalized medicine, we need not only to undertake molecular profiling but rather we need to interlink patients, physicians and researchers using modern technological opportunities, increasing the likelihood of providing new therapies to patients with suitable targets. The PLATON Network (NCT05489250, IKF-T008) addresses these important issues. Methods: The PLATON Network (PLATON) is an interactive structure aiming at improving personalized medicine for all tumor entities (starting with upper GI tumors). The goal of the PLATON Network is to provide a platform for connecting medical practitioners, researchers and patients to personalized cancer treatments. The platform combines comprehensive molecular profiling, data on active clinical trials, remote data access with networking solutions, implemented substudies and associated educational and discussion forums. The main elements of PLATON are: a Platform-study design, an interactive Cloud-based WebApp, Networking, a Molecular Pathology Case Discussion Board, an educational forum, and a BioDataBank. The network will be composed of a global network of cancer centers and cancer specialists who can submit genomic data of their patients to PLATON. The WebApp allows members and associated scientists to view each other's current patients' clinical and molecular data and get into contact with the treating physicians or identify patients for ongoing biomarker-driven clinical trials using advanced algorithms. The PLATON Network also offers molecular testing via Foundation CDx and CDx Liquid for standardized NGS testing and functions as an umbrella for substudies within the platform design. PLATON enrolls patients during first line treatment and collects information on routine clinical data (baseline data, medical history, histopathology reports, treatment decisions, etc.). During follow up, all anti tumor therapy lines, tumor response and survival data are recorded. Importantly, quality of life data is captured monthly under therapy (with EORTC QLQ-30,-BIL21, -HCC18, -PAN26, -STO22 and EQ-5D-5L) and at further follow up (with EQ-5D-5L). So far, 110 cancer centers have joined the PLATON network. Over 200 patients with upper GI tumors received comprehensive molecular profiling and 162 cases were reviewed and discussed in the Molecular Pathology Case Discussion Board. Five sub-projects/-studies have been initiated under the umbrella of PLATON. The educational forum is in the planning stage. PLATON is conducted by the Institute of Clinical Cancer Research (IKF) in Frankfurt with the supervision of an independent scientific steering committee.

Abstract 244: Use of comprehensive molecular profiling to identify additional clinically-relevant alterations compared to targeted gene panels

Abstract 244: Use of comprehensive molecular profiling to identify additional clinically-relevant alterations compared to targeted gene panels

Biomarker-Driven Oncology Clinical Trials: Novel Designs in the Era of Precision Medicine

Oncology drug development historically has followed a path of sequential phase I, II, and III clinical trials using traditional trial designs, with the goal of achieving regulatory approval. These studies are often conducted with inclusion criteria that limit enrollment to a single tumor type or tumor site of origin, excluding other patients who might also respond. Increased use of precision medicine targeting biomarkers or specific oncogenic mutations has led to novel clinical trial designs that can evaluate these therapies in a less limited fashion. Master protocols such as basket trials, umbrella trials, and platform trials can, for example, evaluate histology-specific therapies targeting a common oncogenic mutation across multiple tumor types or screen for the presence of multiple different biomarkers rather than a single one. In other cases, they can lead to more rapid evaluation of a drug and evaluate targeted therapies in tumor types for which they are not yet currently indicated. As the use of complex biomarker-based master protocols increases, advanced practitioners must understand these novel trial designs, their advantages and disadvantages, and how their use may advance drug development and maximize the clinical benefits of molecular precision therapy.

EXTH-39. BIOMARKER DRIVEN TARGETING OF RECURRENT IDH MUTANT GLIOMA WITH ABEMACICLIB

Abstract Despite initial responsiveness to standard treatments like radiation and chemotherapy, IDH mutant gliomas inevitably recur, become more clinically aggressive, and lead to untimely death. The aggressive change in clinical outcome is driven by insensitivity to conventional methods of treatment, necessitating an improved therapeutic strategy at this stage of the disease. Greater than 20% of recurrent IDH mutant gliomas exhibit homozygous loss of CDKN2A, leading to the loss of the endogenous CDK4/6 inhibitor p16 and aberrant signaling through the CDK-RB pathway. We hypothesized that CDKN2A loss leads to enhanced sensitivity to CDK4/6 inhibitors which have been approved for use in a variety of other cancer types. We examined the sensitivity of a panel of patient-derived IDH mutant gliomas with either endogenous CDKN2A loss or CRISPR-mediated gene deletion to treatment with CDK4/6 inhibitors. We observed a correlation between CDKN2A loss and enhanced cytotoxicity following abemaciclib treatment in these glioma models. We have characterized the cell cycle profile following abemaciclib treatment by flow cytometry and western blotting in both sensitive and resistant lines. Further, abemaciclib treatment resulted in significant slowing of tumor growth rate in a subcutaneous patient-derived IDH mutant glioma model. Survival studies in an intracranial model are ongoing. These preclinical results provide foundational data to support a biomarker-driven clinical trial of CDK4/6 inhibitors in patients with recurrent IDH mutant glioma.

The Rocky Road from Preclinical Findings to Successful Targeted Therapy in Pleural Mesothelioma.

Pleural mesothelioma (PM) is a rare and aggressive disease that arises from the mesothelial cells lining the pleural cavity. Approximately 80% of PM patients have a history of asbestos exposure. The long latency period of 20-40 years from the time of asbestos exposure to diagnosis, suggests that multiple somatic genetic alterations are required for the tumorigenesis of PM. The genomic landscape of PM has been characterized by inter- and intratumor heterogeneity associated with the impairment of tumor suppressor genes such as CDKN2A, NF2, and BAP1. Current systemic therapies have shown only limited efficacy, and none is approved for patients with relapsed PM. Advances in understanding of the molecular landscape of PM has facilitated several biomarker-driven clinical trials but so far, no predictive biomarkers for targeted therapies are in clinical use. Recent advances in the PM genetics have provided optimism for successful molecular strategies in the future. Here, we summarize the molecular mechanism underlying PM pathogenesis and review potential therapeutic targets.

Comparison of PIK3CA Mutation Prevalence in Breast Cancer Across Predicted Ancestry Populations.

Understanding the differences in biomarker prevalence that may exist among diverse populations is invaluable to accurately forecast biomarker-driven clinical trial enrollment metrics and to advance inclusive research and health equity. This study evaluated the frequency and types of PIK3CA mutations (PIK3CAmut) detected in predicted genetic ancestry subgroups across breast cancer (BC) subtypes. Analyses were conducted using real-world genomic data from adult patients with BC treated in an academic or community setting in the United States and whose tumor tissue was submitted for comprehensive genomic profiling. Of 36,151 patients with BC (median age, 58 years; 99% female), the breakdown by predicted genetic ancestry was 75% European, 14% African, 6% Central/South American, 3% East Asian, and 1% South Asian. We demonstrated that patients of African ancestry are less likely to have tumors that harbor PIK3CAmut compared with patients of European ancestry with estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER+/HER2-) BC (37% [949/2,593] v 44% [7,706/17,637]; q = 4.39E-11) and triple-negative breast cancer (8% [179/2,199] v 14% [991/7,072]; q = 6.07E-13). Moreover, we found that PIK3CAmut were predominantly composed of hotspot mutations, of which mutations at H1047 were the most prevalent across BC subtypes (35%-41% ER+/HER2- BC; 43%-61% HER2+ BC; 40%-59% triple-negative breast cancer). This analysis established that tumor PIK3CAmut prevalence can differ among predicted genetic ancestries across BC subtypes on the basis of the largest comprehensive genomic profiling data set of patients with cancer treated in the United States. This study highlights the need for equitable representation in research studies, which is imperative to ensuring better health outcomes for all.

Increasing tissue requirements in lymphoma trials may exclude patients with high-risk disease or worse prognosis

AbstractAn enhanced understanding of the molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) has opened the door to clinical trials evaluating novel agents with subtype-specific activity. It is an emerging question whether core needle biopsies (CNB) can adequately meet the increasing tissue requirements of these clinical trials. This can potentially lead to selective enrollment of patients who can undergo excisional biopsy (EB). It is also important to know whether patients who can undergo extensive diagnostic work up differ in their disease characteristics and outcomes from those who cannot. In this observational study, we describe the characteristics, outcomes, and adequacy of diagnostic tissue in patients with newly diagnosed DLBCL and primary mediastinal large B-cell lymphoma who underwent EB vs CNB. Of the 1061 patients, 532 (49.8%) underwent EB and 529 (50.1%) underwent CNB. A significantly higher proportion of patients with CNB had advanced stage disease, an international prognostic index of ≥3, and inadequate tissue for molecular analyses. Patients with CNB had significantly worse 5-year event-free survival (67.6% vs 56.9%; hazard ratio [HR], 0.76; confidence interval [CI]95, 0.6-0.9, P < .001) and 5-year overall survival (76.4% vs 69.2%; HR, 0.8; CI95, 0.6-0.9, P < .001). Thus, patients who underwent CNB have poor-risk features and inferior outcomes on frontline chemoimmunotherapy, are more likely to have inadequate tissue for molecular analyses, and might not meet the tissue requirements of biomarker-driven clinical trials. Thus, the increasing tissue requirements of biomarker-driven clinical trials may result in the exclusion of patients with high-risk DLBCL who need novel agents.

Portuguese Consensus Recommendations for Next-Generation Sequencing of Lung Cancer, Rare Tumors, and Cancers of Unknown Primary Origin in Clinical Practice.

Next-generation sequencing (NGS) has been implemented in clinical oncology for diagnosis, prognosis, and therapeutic guidance. Among the various NGS applications in molecular oncology, we focused on the following topics: laboratory standards for targeted gene panels (somatic mutations) and therapeutic guidance based on NGS of lung cancer and rare cancers, namely sarcomas and cancers of unknown primary. Multiple quality control checkpoints should be addressed in the pre-analytical phase for good quality and interpretation of the NGS results. It includes tumor size and cellularity, tissue processing and decalcification, tumor fraction, tumor viability, fixatives, and staining. Communication between clinicians and laboratory support is also essential. In lung cancer, all patients with non-squamous non-small cell lung cancer should be tested with a NGS panel, and it should include not only genes with approved targeted therapies (ALK, BRAF, EGFR, MET, NTRK, RET, and ROS1) but also genes with potentially actionable genomic alterations (HER2 and KRAS). Since there is a lack of extensive knowledge regarding the use of NGS in rare tumors performing comprehensive genomic profiling, NGS panels to better manage the disease are recommended. Moreover, other patients with other incurable solid tumors may benefit from being included in biomarker-driven clinical trials. Multidisciplinary tumor boards with the participation of experts with the ability to integrate genomic profiling data are essential to tailor the best strategy for each patient. Considering that there are no national guidelines, this article aims to guide laboratory and clinical practice for the use of NGS in the context of lung cancer, rare tumors, and cancer of unknown primary in Portugal.

Abstract IA16: Trial enrichment and proof of concept studies

Abstract Identification of oncogene addiction loops has provided new therapeutic targets in oncology. Selective targeting of the ABL-BCR fusion in patients with chronic lymphocytic leukemia using imatinib was a major clinical breakthrough. Similar approaches in other solid tumors led to the approval of additional biomarker-driver molecular therapies (e.g., vemurafenib in BRAF mutated melanoma). Success of this approach was contingent on the identification of biomarkers that could help identify patients most likely to respond to these therapies. This spurred efforts to implement clinical trials able to detect efficacy in specific patient populations, specifically those enriched in the biomarker under evaluation. This has also been applied in hepatocellular carcinoma with mixed results. Approval of ramucirumab in patient with elevated AFP is the first example of successful trial enrichment, while tivantinib is paradigmatic of a negative biomarker enrichment trial. Lack of recurrent druggable mutations makes biomarker-driven clinical trials in HCC more difficult, which underscores the need for predictive biomarkers of immune therapies. Citation Format: Augusto Villanueva-Rodriguez. Trial enrichment and proof of concept studies [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr IA16.