Boston Foundation

Gene copy number (CN) amplifications and protein overexpression are common drug targets and detection relies on various methodologies, including NGS-based CN, IHC, and ISH. We investigated the pan-tumor landscape of amplifications and developed AmpRatio, a novel method of CN quantitation. Pan-tumor tissue (N=486,340) and liquid (N=85,635) samples underwent hybrid capture-based comprehensive genomic profiling. A genome-wide CN model for each sample was generated to estimate the purity, ploidy, and segment-level CN. AmpRatio was calculated by dividing gene CN/sample ploidy. A US-based de-identified clinico-genomic database was utilized to assess the relationship between ERBB2 AmpRatio and HER2 IHC/FISH and outcomes on anti-HER2 therapies. Amplifications with varying degrees of gain were reported in 38.6% of pan-tumor tissue samples, most frequently MYC (5.6%), 11q13 (5.2%), ERBB2 (5.2%), and CCNE1 (3.2%). ERBB2 AmpRatio was associated with HER2 positivity by IHC/FISH in gastroesophageal (overall percent agreement [OPA] 90%) and breast (OPA 95%) cancers. Among patients treated with anti-HER2 therapies, ERBB2 AmpRatio significantly stratified outcomes within the ERBB2-amplified and IHC-defined HER2+ and HER2-low/ultralow populations. High concordance (sensitivity 88%) of amplification detection in liquid biopsy vs tissue was associated with higher AmpRatio and ctDNA tumor fraction ≥20%. CN amplifications are prevalent and diverse biomarkers and AmpRatio is variable across genes and tumor types. ERBB2 AmpRatio is associated with outcomes to HER2-directed therapies and may have utility alongside IHC for clinical decision making. With the increasing number of therapies targeting amplifications/overexpression, it will be important to define harmonized methods for CN quantification for optimal patient selection.

Background/Objectives: Emerging evidence suggests that multiple micronutrient supplements (MMS) provide additional benefits for maternal and neonatal health compared with iron and folic acid (IFA) supplements. To achieve effective coverage, acceptability, and adherence—and to inform a nationwide rollout of MMS—it is essential to understand the context-specific factors that shape implementation. This study evaluated the pilot implementation of MMS in Rwanda to identify key enablers, areas for improvement, and challenges related to antenatal care (ANC) attendance and MMS use. Methods: Data were collected through a survey of 3257 women who attended ANC services, seven focus group discussions with 35 ANC attendees, and key informant interviews with 20 ANC nurses and 21 community health workers. Results: Pregnant women reported high ANC attendance (74%) and MMS consumption (79%), largely driven by strong motivation and awareness of MMS benefits. Strategies to remember daily intake and to manage side effects supported adherence, as did reminders, motivation, and information from family members and healthcare providers. Limited patient-centered counselling, financial constraints, barriers to accessing ANC services, and product stock-outs were key areas for strengthening service delivery in Rwanda. Conclusions: Sustaining high ANC attendance and MMS adherence as the program transitions from the pilot phase to national scale-up is essential. Improving counseling quality and strengthening supply chains may reinforce ANC services and support sustained MMS adherence, with benefits for maternal and child health.

The BRAF inhibitor encorafenib (Enco) plus the anti-EGFR antibody cetuximab (Cetux) improved overall survival, objective response rate, and progression-free survival in previously treated BRAFV600E-mutant metastatic colorectal cancer in BEACON, a phase III randomized trial, leading to regulatory approval for this indication. To support rapid, plasma-based testing for BRAFV600E identification, clinical validity of a ctDNA-based assay, FoundationOneLiquid CDx (F1LCDx), was assessed against the reference tumor-based clinical trial assay (CTA) in liquid biopsy-evaluable samples from BEACON and commercially obtained tissue-matched plasma samples. Pretreatment tissue samples were collected in BEACON to confirm BRAF mutational status using the central single gene PCR assay. Concordance between the CTA and liquid biopsy tests was assessed, and clinical validity of liquid biopsy testing was examined using clinical outcomes from BEACON. Of the 523 evaluable patients, 433 with matched tissue and plasma samples had CTA and F1LCDx results available (BEACON, n = 328; commercial, n = 105). A strong concordance in detecting BRAFV600E was found between F1LCDx and CTA, with a positive percent agreement of 87.2% and negative percent agreement of 97.1%. Among 42 F1LCDx-/CTA+ samples, 41 (97.6%) had ctDNA tumor fraction <1%. Among samples with ctDNA tumor fraction >1%, the positive percent agreement was 99.4% and negative percent agreement was 86.7%. Clinical outcomes with Enco plus Cetux were similar between those identified as F1LCDx+/CTA+ and CTA+ overall. This study supports using liquid biopsies as a clinically valid assay for identifying BRAFV600E alterations in patients with metastatic colorectal cancer, particularly when ctDNA tumor fraction was >1%. In the phase III BEACON trial, which established Enco plus Cetux as a standard of care for previously treated BRAFV600E-mutant metastatic colorectal cancer, mutational status was confirmed through testing of tumor tissue. To support rapid, less invasive testing for BRAFV600E in plasma, this retrospective study assessed a ctDNA-based assay and found strong concordance between the liquid biopsy test and the tumor-based assay in detecting BRAFV600E.

Purpose:Many preclinical hypotheses, including reciprocal feedback activation between the androgen receptor (AR)–PI3K pathway in PTEN loss and AR signaling inhibitor–induced “BRCAness” regardless of BRCA1/2 status, have struggled to translate into clinical benefit for patients with metastatic prostate cancer. This gap in translatability, particularly in metastatic castration-resistant prostate cancer (mCRPC), may stem from a limited understanding of prostate cancer evolution. A key challenge is the correlation between early-stage tumor genetics and mCRPC. By examining clinical, genomic, and molecular changes over time, we aimed to refine clinical trial design.Experimental Design:Using a comprehensive dataset from electronic health records with genomic profiling, a shift in the prognostic value of biomarkers from metastatic hormone-sensitive prostate cancer (mHSPC) to mCRPC was observed. Additionally, genomic and transcriptomic analyses of primary tumors from the IPATential150 trial and mCRPC samples from the Stand Up To Cancer cohort examined the changing AR–PI3K signaling correlation.Results:PI3K–AKT pathway alterations lost their prognostic significance in later stages. Although AR and PI3K–AKT signaling were inversely correlated in primary tumors, this relationship was disrupted in mCRPC, independent of PIK3CA/AKT1/PTEN status. We identified broad transcriptional rewiring associated with AR signaling, increasing tumor heterogeneity. Improving the understanding of early-stage disease, we identified a high-risk mHSPC subset enriched for AR alterations. Additionally, in a subset of patients, AR ligand-binding domain mutations preceded amplification, potentially leading to preferential amplification of the mutant AR form.Conclusions:Our findings underscore the dynamic nature of prostate tumor biology and emphasize the need for translational research to validate preclinical hypotheses in clinically relevant settings, ultimately improving trial design and therapeutic strategies.

221 Background: CH results from fitness-enhancing mutations in hematopoietic stem cells that accumulate with age. Deep sequencing of LBx, a standard of care for guiding therapy selection in advanced PCa, can be confounded by sensitive detection of variants (vars) arising from both ctDNA and CH-derived DNA. Here we report the prevalence of CH vars in PCa and other cancers and describe an algorithmic method for accurately distinguishing CH from tumor signal, even at low variant allele frequencies (VAF). Methods: Plasma cell-free DNA from 1813 patients with cancer (n = 270 PCa) was sequenced using FoundationOneLiquid CDx (F1LCDx) in a CLIA lab setting. In parallel, DNA from buffy coats was extracted, sheared, and sequenced on F1LCDx to identical depth, to establish ground truth for CH var identity. This cohort was used to train a machine learning variant origin prediction (VOP) model incorporating fragmentomics and other sequencing features to assign probabilities of origin (germline, tumor, or CH) to all short variants across the 324 genes baited on the assay. Only pathogenic vars were considered. Results: 1247/1813 (69%) of LBx detected ≥1 CH var, including 209/270 (77%) of PCa LBx (mean 1.8 vars/sample). CH contributed confounding vars in clinically relevant genes including CHEK2, ATM, BRAF, TP53, and BRCA2 in 90/270 (33%) of PCa LBx (Table). Median VAF of CH vars in these genes was 0.3% in LBx and 0.7% in WBC. CH vars enriched in PCa vs other cancers were: TET2 (23.0% of PCa LBx vs 13.6% of non-PCa), ASXL1 (14.4% vs 7.5%), SF3B1 (7.0 vs 2.3%), and TERT promoter (5.2% vs 1.3%), FDR &lt;0.05 for all. In the pan-cancer cohort, the VOP algorithm identified CH vars with 95.2% (2613/2745) sensitivity, 94.1% (3741/3975) specificity, and 91.8% (2613/2847) positive predictive value (PPV). For CH vars with VAF&lt;1%, VOP had 95.0% (1622/1707) sensitivity, 1021/1169 (87.3%) specificity, and 91.6% (1622/1770) PPV. Conclusions: Equal-depth DNA sequencing of plasma and matched WBC reveals higher CH prevalence in LBx than previously reported: 77% of PCa LBx with a CH var, including 33% with a CH var in clinically relevant genes . Many potentially actionable vars have low VAF in LBx, and filtering CH via shallower depth WBC sequencing than that of LBx risks leaving low VAF CH vars to be mistaken for tumor vars. Equal-depth WBC sequencing or an algorithmic method validated using equal-depth WBC sequencing is required for accurate identification of low VAF CH confounders in LBx. Percentages of LBx with only CH vars/total LBx with vars detected in genes clinically relevant in PCa. Gene PCa Pan-cancer CHEK2 19/25 (76%) 143/168 (85%) ATM 18/29 (62%) 156/227 (69%) BRAF 2/5 (40%) 10/48 (21%) TP53 46/120 (38%) 235/996 (24%) BRCA2 2/12 (17%) 3/37 (8%) PALB2 0/3 (0%) 4/17 (24%) BRCA1 0/2 (0%) 5/28 (18%) CDK12 0/7 (0%) 3/17 (18%)