Variant Allele Frequency Research Articles

Limitations in next-generation sequencing-based genotyping of breast cancer polygenic risk score loci

Considering polygenic risk scores (PRSs) in individual risk prediction is increasingly implemented in genetic testing for hereditary breast cancer (BC) based on next-generation sequencing (NGS). To calculate individual BC risks, the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) with the inclusion of the BCAC 313 or the BRIDGES 306 BC PRS is commonly used. The PRS calculation depends on accurately reproducing the variant allele frequencies (AFs) and, consequently, the distribution of PRS values anticipated by the algorithm. Here, the 324 loci of the BCAC 313 and the BRIDGES 306 BC PRS were examined in population-specific database gnomAD and in real-world data sets of five centers of the German Consortium for Hereditary Breast and Ovarian Cancer (GC-HBOC), to determine whether these expected AFs can be reproduced by NGS-based genotyping. Four PRS loci were non-existent in gnomAD v3.1.2 non-Finnish Europeans, further 24 loci showed noticeably deviating AFs. In real-world data, between 11 and 23 loci were reported with noticeably deviating AFs, and were shown to have effects on final risk prediction. Deviations depended on the sequencing approach, variant caller and calling mode (forced versus unforced) employed. Therefore, this study demonstrates the necessity to apply quality assurance not only in terms of sequencing coverage but also observed AFs in a sufficiently large cohort, when implementing PRSs in a routine diagnostic setting. Furthermore, future PRS design should be guided by the technical reproducibility of expected AFs across commonly used genotyping methods, especially NGS, in addition to the observed effect sizes.

Misclassification of a frequent variant from PMS2CL pseudogene as a PMS2 loss of function variant in Brazilian patients.

PMS2, a Lynch Syndrome gene, presents challenges in genetic testing due to the existence of multiple pseudogenes. This study aims to describe a series of cases harboring a variant in the PMS2CL pseudogene that has been incorrectly assigned to PMS2 with different nomenclatures. We reviewed data from 647 Brazilian patients who underwent multigene genetic testing at a single center to identify those harboring the PMS2 V1:c.2186_2187delTC or V2:c.2182_2184delACTinsG variants, allegedly located at PMS2 exon 13. Gene-specific PCR and transcript sequencing was performed. Among the 647 individuals, 1.8% (12) carried the investigated variants, with variant allele frequencies ranging from 15 to 34%. By visually inspecting the alignments, we confirmed that both V1 and V2 represented the same variant and through gene-specific PCR and PMS2 transcript analysis, we demonstrated that V1/V2 is actually located in the PMS2CL pseudogene. Genomic databases (ExAC and gnomAD) report an incidence of 2.5 - 5.3% of this variant in the African population. Currently, V1 is classified as "uncertain significance" and V2 as "conflicting" in ClinVar, with several laboratories classifying them as "pathogenic". We identified a frequent African PMS2CL variant in the Brazilian population that is misclassified as a PMS2 variant. It is likely that V1/V2 have been erroneously assigned to PMS2 in several manuscripts and by clinical laboratories, underscoring a disparity-induced matter. Considering the limitations of short-read NGS differentiating between certain regions of PMS2 and PMS2CL, using complementary methodologies is imperative to provide an accurate diagnosis.

Similarities and differences between brain and skin GNAQ p.R183Q driven capillary malformations.

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~90% of syndromic and non-syndromic CM specimens and is present in CD31pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimen from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1, and were surrounded by MRC1pos/LYVE1pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM.

Mutational variant allele frequency profile as a biomarker of response to immune checkpoint blockade in non-small cell lung Cancer

IntroductionIdentifying new biomarkers for predicting immune checkpoint inhibitors (ICIs) response in non-small cell lung cancer (NSCLC) is crucial. We aimed to assess the variant allele frequency (VAF)-related profile as a novel biomarker for NSCLC personalized therapy.MethodsWe utilized genomic data of 915 NSCLC patients via cBioPortal and a local cohort of 23 patients for model construction and mutational analysis. Genomic, transcriptomic data from 952 TCGA NSCLC patients, and immunofluorescence (IF) assessment with the local cohort supported mechanism analysis.ResultsUtilizing the random forest algorithm, a 15-gene VAF-related model was established, differentiating patients with durable clinical benefit (DCB) from no durable benefit (NDB). The model demonstrated robust performance, with ROC-AUC values of 0.905, 0.737, and 0.711 across training (n = 313), internal validation (n = 133), and external validation (n = 157) cohorts. Stratification by the model into high- and low-score groups correlated significantly with both progression-free survival (PFS) (training: P < 0.0001, internal validation: P < 0.0001, external validation: P = 0.0066) and overall survival (OS) (n = 341) (P < 0.0001). Notably, the stratification system was independent of PD-L1 (P < 0.0001) and TMB (P < 0.0001). High-score patients exhibited an increased DCB ratio and longer PFS across both PD-L1 and TMB subgroups. Additionally, the high-score group appeared influenced by tobacco exposure, with activated DNA damage response pathways. Whereas, immune/inflammation-related pathways were enriched in the low-score group. Tumor immune microenvironment analyses revealed higher proportions of exhausted/effector memory CD8 + T cells in the high-score group.ConclusionsThe mutational VAF profile is a promising biomarker for ICI therapy in NSCLC, with enhanced therapeutic stratification and management as a supplement to PD-L1 or TMB.

BIOM-12. LIQUID BIOPSIES IN CNS TUMORS: A COMBINED APPROACH TO PROFILING CSF

Abstract In precision medicine, molecular profiling of pediatric tumors is crucial for diagnosis, prognosis, and therapeutic decision-making. Traditional biopsy methods, often invasive, have inherent risks and may not capture a tumor’s heterogeneity. Liquid biopsy, however, offers a minimally invasive and comprehensive alternative. We demonstrate its utility in diagnosing and monitoring brain tumors using cell-free DNA (cfDNA) from cerebrospinal fluid (CSF), where conventional cfDNA plasma analysis has shown low sensitivity. Through digital droplet PCR, we analyzed 23 plasma samples from patients with BRAFV600E or H3K27M gliomas, detecting H3K27M in a single case. Conversely, CSF samples revealed 8 positives out of 9 for these mutations, underscoring CSF’s diagnostic value. We advanced our methodology by developing a 21-gene hybrid-capture panel paired with low-pass whole-genome sequencing to pinpoint copy number variations. In our reference sample set, the panel achieved a detection limit with variant allele frequencies as low as 0.5%. With 10ng of cfDNA, we reached 83% sensitivity and 100% specificity, which increased to 100% sensitivity with a 30ng input. Using this assay, we profiled 141 CSF samples from 119 patients, achieving 70% positivity rate in active disease cases in a validation cohort—ranging from 50% in low-grade gliomas to 84% in high-grade gliomas and 75% in medulloblastomas. Intriguingly, of 14 queried tumor samples lacking biopsy confirmation, half showed positive ctDNA results using the panel and/or low-pass whole-genome sequencing. This approach allowed for differential diagnoses in suspected medulloblastoma relapses, tailored therapeutic interventions based on CSF profiles, and effective treatment response monitoring. In summary, our integrated liquid biopsy strategy showcases a powerful, clinically applicable tool that significantly enhances patient care in pediatric neuro-oncology.

Plasma circulating tumor DNA unveils the efficacy of PD-1 inhibitors and chemotherapy in advanced gastric cancer

Programmed Death Receptor 1 (PD-1) inhibitors, when combined with chemotherapy, have exhibited notable effectiveness in enhancing the survival outcomes of patients afflicted with advanced gastric cancer. However, it is important to acknowledge that not all patients derive substantial benefits from this therapeutic approach, highlighting the crucial necessity of identifying efficacious biomarkers to inform immunotherapy interventions. In this study, we sought to investigate the predictive utility of circulating tumor DNA (ctDNA) as a biomarker in a cohort of 30 patients diagnosed with advanced gastric cancer, all of whom underwent first-line treatment involving PD-1 inhibitor administration alongside chemotherapy. We procured peripheral blood samples both at baseline and following the completion of two treatment cycles. Additionally, baseline tissue specimens were collected for the purpose of genomic alteration assessment, employing both 47-gene and 737-gene next-generation sequencing panels for plasma and tumor tissue, respectively. We delineated a ctDNA response as the eradication of maximum variant allele frequencies relative to baseline levels. Notably, the objective response rate among individuals exhibiting a ctDNA response proved significantly superior in comparison to non-responders (P = 0.0073). Furthermore, patients who manifested a ctDNA response experienced markedly prolonged progression-free survival (PFS) and overall survival (OS) when juxtaposed with those devoid of a ctDNA response (median PFS: 15.6 vs. 6.0 months, P = 0.003; median OS: not reached [NR] vs. 9.0 months, P = 0.011). In summation, patients with advanced gastric cancer receiving first-line treatment with PD-1 inhibitors and chemotherapy, dynamic changes in ctDNA can serve as a potential biomarker for predicting treatment efficacy and long-term outcomes.

A diagnostic challenge of KIT p.V559D and BRAF p.G469A mutations in a paragastric mass.

A patient with gastrointestinal stroma tumor (GIST) and KIT p.V559D and BRAF p.G469A alterations was referred to our institutional molecular tumor board (MTB) to discuss therapeutic implications. The patient had been diagnosed with B-cell chronic lymphocytic leukemia (CLL) years prior to the MTB presentation. GIST had been diagnosed 1 month earlier. After structured clinical annotation of the molecular alterations and interdisciplinary discussion, we considered BRAF/KIT co-mutation unlikely in a treatment-naïve GIST. Discordant variant allele frequencies furthermore suggested a second malignancy. NGS of a CLL sample revealed the identical class 2 BRAF alteration, thus supporting admixture of CLL cells in the paragastric mass, leading to the detection of 2 alterations. Following the MTB recommendation, the patient received imatinib and had a radiographic response. Structured annotation and interdisciplinary discussion in specialized tumor boards facilitate the clinical management of complex molecular findings. Coexisting malignancies and clonal hematopoiesis warrant consideration in case of complex and uncommon molecular findings.

Bronchial Basaloid Papillary Tumor of Uncertain Malignant Potential: A Case Report.

Bronchial papillomas are benign tumors, which can be divided into different subtypes based on the cellular features. So far, no bronchial papilloma with basaloid cell features has been reported. We report a bronchial basaloid papillary tumor in a 67-year-old woman. Tumor recurrence and malignant transformation were observed after a long-term follow-up. The clinical, histological, immunohistochemical, and genetic features were reappraised. The primary tumor was characterized by papillary growth pattern and basaloid neoplastic cells, only a small amount of neoplastic cells showed mature characteristics. The tumor originated from respiratory epithelium and had a low proliferation index by Ki-67. Keratin (KRT) 5/6 and KRT7 showed patchy or partial positivity. Myoepithelial markers were negative. P63 was diffusely positive, but it was negative in the small amount of tumor cells with mature characteristics. The common genetic alterations (EGFR, KRAS, BRAF V600E, HER2, and ALK) of lung cancers were not detected. However, tumor recurrence was observed in the mediastinum and esophagus 12 years after surgery. The recurrent tumor had a morphology overlapping with that of the primary tumor; however, it displayed significantly malignant characteristics. The recurrent tumor was not related to high-risk HPV. A high variant allele frequency was observed in tumor suppressor gene BRCA1, TP53, oncogene GNA11, and KIT, which were all missense mutations. Considering the bland features of the primary tumor and the fact of tumor recurrence and undisputed malignant transformation, the basaloid papillary tumor was considered a tumor with uncertain malignant potential.

A randomized study evaluating tailoring of advanced/metastatic colorectal cancer (mCRC) therapy using circulating tumor DNA (ctDNA): TACT-D.

LBA3557 Background: Identifying non-responders to expensive salvage therapies with modest benefits and substantial treatment related adverse events (TRAEs) (e.g. regorafenib [Reg] or TAS102 [Tas] in mCRC) is key to precision care. Retrospective studies suggest that ctDNA changes at timepoints (4-10 weeks [wks] into therapy) before radiographic assessment may predict treatment outcomes. However, prospective studies assessing early ctDNA changes are lacking and clinical utility remains uncertain. Methods: TACT-D is a randomized study to validate early dynamic changes in ctDNA (ΔctDNA: change in either maximum variant allele frequency (maxVAF) or mean VAF in predicting treatment response/resistance. Patients (pts) with mCRC clinically eligible for Reg/Tas were randomly assigned 2:1 to either standard of care (SOC) or ctDNA arm. All pts had ctDNA sequencing by Guardant360 CDx assay on cycle 1 day 1 (C1D1) and C1D15. On SOC arm, pts were restaged at 8 wks. On ctDNA arm, ΔctDNA (C1D15 - C1D1) was run in real-time and increase in ctDNA (ΔctDNA &gt; 0) triggered early radiographic restaging. Therapy was continued for responders (RECISTv1.1 stable disease/response) and stopped for progression (non-responders). Co-primary endpoints were: 1) comparison of TRAEs among study arms and 2) association of ΔctDNA and objective response rate (RR). Key secondary endpoints were progression free (PFS) and overall (OS) survival. Study was powered (82%; 2-sided α = .05) to detect 30% decrease in toxicity. Results: Between 4/2019 and 8/2023, 100 pts were randomized; 80 evaluable had median age of 56 years, 46% were females, 44% and 56% received Tas and Reg, respectively. Baseline ctDNA levels (ρ 0.90) and ΔctDNA (ρ 0.68) using maxVAF (reported below) and mean VAF showed strong correlation (P &lt; .001). Median ΔctDNA for entire cohort was -47% with no significant difference by treatment arms (SOC -45% v Exp -58%, P = .79) and therapy (Tas -71 v Reg -44, P = .19). ΔctDNA increased in 18% pts. Grade 3/4 TRAEs (32% v 40%, P = .62) did not differ significantly between arms. No significant association was seen between ΔctDNA and RR (OR .88, P = 1.0), PFS (HR .99, P = .88) and OS (HR 1.00, P = .64). Notably, higher baseline maxVAF was strongly associated with response (median maxVAF: 21.8% in non-responders v 3.4% in responders), PFS (HR 1.02) and OS (HR 1.03) (all P &lt; .001). After adjusting for baseline maxVAF, ΔctDNA was found to be associated with both PFS (HR 1.87, P = .038) and OS (HR 3.55, P = .001). Conclusions: In the first prospective study of clinical utility of monitoring ctDNA in mCRC, baseline ctDNA was strongly prognostic for clinical benefit from salvage therapies in mCRC. Adjusted for this prognostic impact, ΔctDNA between C1D1 and C1D15 was predictive of clinical outcomes. Efforts are needed to establish novel signatures, optimal cutoffs/intervals for assessing ctDNA response in mCRC, tailored to pts and their therapy. Clinical trial information: NCT03844620 .

Multi-regional sequencing reveals the genetic and immune heterogeneity of non-cancerous tissues in gastric cancer.

Gastric cancer (GC) is one of the most heterogeneous tumors. However, research on normal tissue adjacent to the tumor (NAT) is very limited. We performed multi-regional omics sequencing on 150 samples to assess the genetic basis and immune microenvironment in NAT and matched primary tumor or lymph node metastases. NATs demonstrated different mutated genes compared with GC, and NAT genomes underwent independent evolution with low variant allele frequency. Mutation profiles were predominated by aging and smoking-associated signatures in NAT instead of signatures associated with genetic instability. Although the immune microenvironment within NATs shows substantial intra-patient heterogeneity, the proportion of shared TCR clones among NATs is five times higher than that of tumor regions. These findings support the notion that subclonal expansion is not pronounced in NATs. We also demonstrated remarkable intra-patient heterogeneity of GCs and revealed heterogeneity of focal amplification of CD274 (encoding PD-L1) that leads to differential expression. Finally, we identified that monoclonal seeding is predominant in GC, which is followed by metastasis-to-metastasis dissemination in individual lymph nodes. These results provide novel insights into GC carcinogenesis. © 2024 The Pathological Society of Great Britain and Ireland.

Allele Frequencies of Genetic Variants Associated with Varroa Drone Brood Resistance (DBR) in Apis mellifera Subspecies across the European Continent.

Implementation of marker-assisted selection (MAS) in modern beekeeping would improve sustainability, especially in breeding programs aiming for resilience against the parasitic mite Varroa destructor. Selecting honey bee colonies for natural resistance traits, such as brood-intrinsic suppression of varroa mite reproduction, reduces the use of chemical acaricides while respecting local adaptation. In 2019, eight genomic variants associated with varroa non-reproduction in drone brood were discovered in a single colony from the Amsterdam Water Dune population in the Netherlands. Recently, a new study tested the applicability of these eight genetic variants for the same phenotype on a population-wide scale in Flanders, Belgium. As the properties of some variants varied between the two studies, one hypothesized that the difference in genetic ancestry of the sampled colonies may underly these contribution shifts. In order to frame this, we determined the allele frequencies of the eight genetic variants in more than 360 Apis mellifera colonies across the European continent and found that variant type allele frequencies of these variants are primarily related to the A. mellifera subspecies or phylogenetic honey bee lineage. Our results confirm that population-specific genetic markers should always be evaluated in a new population prior to using them in MAS programs.

Observed germline BRCA1/2 and TP53 tumor genomic profiling allele frequencies.

e22533 Background: Tumor genomic profiling (TGP) is increasingly used to assist with cancer treatment planning. While these tests are not intended to find germline variants, they are a possible incidental outcome. Currently there are no clear guidelines on when to suspect if a somatic variant identified on TGP may need confirmatory germline genetic testing. Variant allele frequency (VAF) is one tool commonly used to assess if a variant may be germline. While some studies have demonstrated germline VAF ranges on TGP, the data is limited. The purpose of this study was to review VAF ranges of BRCA1/2 and TP53 variants at a large reference laboratory to better understand VAF ranges of confirmed germline variants. Methods: A retrospective review of test results from 184 patients who underwent TGP and hereditary cancer germline testing at a large reference laboratory was performed. Patients were included in the study if they had both TGP and germline testing performed, regardless of the order of testing. Results were analyzed to determine variants identified and VAF. Analysis of BRCA1/2 and TP53 variants included patients who had variants identified on TGP and had germline testing that would have confirmed the variants if present in the germline. The VAFs were obtained, and simple statistics were used to describe the findings. Results: Twenty-one patients were found to have 23 BRCA1/2 variants identified on TGP (2 patients had 2 BRCA1/2 TGP variants). Of these TGP variants, 16 were confirmed on germline testing (69.6%). The TGP VAF for these patients ranged from 38.2% to 85.1%. The mean was 61.0% and the median was 61.8%. The remaining 7 TGP variants that were not confirmed on germline testing ranged from 7.2% to 63.4%. The mean was 31.1% and the median was 28%. Thirty-eight patients were found to have 41 TP53 variants identified on TGP (3 patients had 2 TP53 TGP variants). Of these patients, 1 had a TP53 variant confirmed on germline testing with a VAF of 44.7%. The 40 remaining variants in this dataset were not confirmed on germline testing. The VAF for these 40 variants ranged from 1.3% to 95.3%. The mean was 38.4% and the median was 34.5%. Conclusions: In this study, 69.6% of BRCA1/2 TGP variants were confirmed on germline testing compared to 2.4% of TP53 TGP variants. Confirmed germline variants identified on TGP had VAFs that were &gt; 38%. It’s important to note that TP53 variants may have very high VAFs (up to 95.3% in this study) and not be.

Tumor purity as confounder for the determination of tumor mutational burden (TMB).

e14656 Background: TMB determination is a prerequisite to stratify advanced cancer patients for pembrolizumab based on the histology-agnostic FDA approval. The accuracy of TMB measurement is challenged by low tumor purity resulting in a lower number of detected somatic variants for a fixed threshold of variant allele frequency (VAF) and a lower measured TMB level as a consequence. Methods: We derived a mathematical formula describing the influence of tumor purity on the VAF of somatic variants, in which the DNA copy number of the corresponding region of the tumor genome is a parameter. We simulated the effect of tumor purity in a cohort of 4,900 tumors from the TCGA project with tumor purity of at least 80%. TMB was calculated as the number of missense mutations with VAF greater or equal than 5%. Tumors were classified as TMB-high when harboring at least 199 missense mutations. This cutpoint corresponds to 10 mut/Mb as derived in the TMB bridging study. We compared tumors with a simulated tumor purity of 70%, 60%, ..., and 10% with tumors with a tumor purity of 80%, which served as reference. Results: The median of TMB in the pan-cancer cohort was reduced by 2%, 10%, and 32% for tumor purity of 60%, 40%, and 20% compared to the reference of 80%. For the 10% tumors with the worst estimation, TMB was reduced even by 12%, 35%, and 77% or more, respectively. In the pan-cancer cohort, the sensitivity to detect high TMB was 98%, 90%, and 62% for tumor purity of 60%, 40%, and 20%, respectively. For head and neck cancer, lung adenocarcinoma, lung squamous cell carcinoma, and cutaneous melanoma, the sensitivity to detect high TMB was 80%, 89%, 92%, and 91%, respectively, for tumors of tumor purity 40%. Conclusions: High tumor purity is critical for accurate determination of the TMB and to reach a high sensitivity concerning the selection of patients for immunotherapy. As shown by the simulations, the sensitivity to detect high TMB is close to 100% for tumor purity of at least 60%, while a substantial proportion of patients that could receive immunotherapy is missed for tumors with purity of 40% or less. Potential ways out of the TMB underestimation include increasing the sequencing coverage and lowering the VAF threshold for mutation calling or using clonal TMB with an adapted cutpoint instead of total TMB as biomarker for immunotherapy guidance.

Association between CH mutation in TET2and solid cancer in elderly Asian women.

10585 Background: Clonal hematopoiesis (CH), defined by the expansion of mutated hematopoietic cells due to somatic mutations acquired during aging, is associated with increased age and inflammatory conditions. CH serves as a predictor for the risks of cancer and cardiovascular events, highlighting the importance of early detection of genetic alterations for proactive intervention. As Asian societies are experiencing rapid aging alongside genetic and environmental variations distinct from Western cohorts, we aimed to evaluate the predictive significance of CH in solid cancer in an Asian cohort. Methods: We conducted a nested case-control study within Singapore Longitudinal Ageing Study 1 (SLAS1; NCT03405675). Chinese females (aged 55.0-83.9), comprising 31 patients with solid cancers (breast, lung, colon) who developed cancer during the follow-up, and 29 controls, were randomly selected from the SLAS1. SLAS1 is a community-based longitudinal aging cohort study. Subjects aged 55 years or older, capable of self-ambulation, were recruited between 2003 and 2005 (n=2804) and were followed up in 2005-2007, 2008-2009, and 2018-2020. We linked SLAS1 with records from the Singapore National Death Registry to identify subjects with cancers as cause of death during this period (median follow-up period 17 years). Buffy coat samples taken at baseline were sequenced using the 42-gene target HemeMark panel (Lucence, Singapore) with a reported 0.1% limit of detection. Chi-square test and t-test were performed to compare the frequency of CH variants and the mean number of mutations per person in the case and control group. We evaluated multiple variant allele frequency (VAF) thresholds on an exploratory basis (0.1% to 2%). The relationship between CH mutations and the likelihood of developing cancer was investigated through a logistic regression model, considering CH mutation defined at various cutoff points. Results: CH was found in 29.0% (9/31) of cases and 37.9% (11/29) of controls, with common mutated genes being TET2 (8/31, 25.8%), ASXL1 (4/31, 12.9%), and DNMT3A (2/31, 6.5%) in case group (VAF threshold=0.5%). Multi-gene CH did not predict cancer at any threshold (with the lowest P value of 0.5858 corresponding to VAF 0.5), Genes correlating with increased cancer risk included TET2 with adjusting for age, which was notably significant at thresholds of 0.3% and above (VAF 0.3%, adjusted OR=2.25, 95% CI: 1.14-4.47, p=0.0201; 0.5%, adjusted OR=4.12, 95% CI: 2.00-8.88, p=0.0002). Conclusions: CH determined through multi-genes was not associated with solid cancers, but TET2 mutations at 0.3-0.5% VAF thresholds were more prevalent in cancer cases, as compared to the current 2% threshold used for calling CHIP. The finding in this nested case-control study, conducted within a cohort with a 17-year follow-up, implies a potential association between TET2 mutation and solid cancer. Further study is warranted on a larger cohort.

An analysis of likely germline events by tumor tissue testing on large somatic panels.

e22524 Background: The increase in large panel testing has facilitated the detection of numerous events, including those in genes associated with inherited cancer. Further, large panel sequencing may uncover variants in these genes in non-inherited cancers. Hence the frequency of pathogenic/likely pathogenic (P/LP) events in some common germline genes was studied across cancers in the Indian population. Methods: A statistical model was used to derive a method to label variants as likely germline. Variants in hereditary breast and ovarian cancer (HBOC) and mismatch repair (MMR) genes from 1553 cases sequenced at our referral laboratory on the TruSight Oncology 500 panel (TSO500) were analyzed for their variant allele frequency (VAF) distribution. A 3 component Gaussian mixture model (intended to separate somatic, germline heterozygous, homozygous variants) was fit to this VAF distribution and a cutoff was determined such that the area under the curve of the somatic component above the cutoff was below 0.01. This gave a threshold value of 40% VAF for determining likely germline variants (LGVs). Data from 1713 TSO500 cases was then analyzed to identify LGVs. Results: The dataset of 1713 cases comprised 20.67% NSCLC, 12.61% breast, 12.49% ovarian and 10.5% colorectal cancer and &lt; 10% of other cancer types including 2.39% pancreatic and 2.04% prostate cancer. 23.8% of ovarian, 17.14% of prostate, 12.5% of breast and 12.2% of pancreatic cancer cases had variants in BRCA1/BRCA2. Among these, 18.69% of ovarian, 17.14% of prostate, 8.33% of breast and 7.32% of pancreatic cancers had LGVs. LGVs in BRCA1/2 were also found in 3.33% of colorectal and 1.13% of NSCLC. The BRCA1/BRCA2 LGV ratio was found to be 3.0 in ovarian, 1.0 in breast and 2.0 in pancreatic but 0.2 in prostate and colorectal and 0.33 in NSCLC cancer. Adding other HBOC genes (ATM, BRCA1/2, BRIP1, CHEK2, PALB2, RAD51C, RAD51D) increased the LGV frequency to 22.43% of ovarian, 22.86% of prostate, 11.11% of breast, 9.76% of pancreatic, 4.44% of colorectal and 2.26% of NSCLC. MMR gene LGVs were seen in 1.23% of all cancers mainly in 10.71% of endometrial and 4.44% of colorectal cancers. Unlike the HBOC genes, MMR gene variants were not observed across multiple cancers and did not show a predominantly germline distribution (somatic/LGV 1:1). In contrast, the background P/LP frequency calculated from gnomAD v4 South Asian data (excluding those with conflicting labels) was 0.74% for BRCA1/2, 1.82% for the extended HBOC list and 0.5% for the MMR genes. Conclusions: Somatic testing was used to identify LGVs and determine the frequencies of HBOC and MMR genes in cancers in the Indian population. Somatic testing also identified LGVs in multiple tissues at frequencies above the background P/LP frequency for this population, suggesting a possible role for these genes in other cancers. This information underscores the need for further study and may be useful to define a clinically relevant subset of patients.

Clonal hematopoiesis of indeterminate potential (CHIP): Outcomes and adverse events in patients with solid tumors.

3521 Background: CHIP is the clonal expansion of hematopoietic stem cells following acquisition of somatic mutations. It is a common incidental finding in cell-free DNA (cfDNA). We investigated the incidence of CHIP in cfDNA from patients with solid tumors and explored its association with outcomes and adverse events (AEs). Methods: We reviewed cfDNA results from a local prospective solid tumor cohort (PREDICT-L) and two randomized trials: CCTG CO.26 (durvalumab + tremelimumab [D+T] or best supportive care [BSC] in metastatic colorectal cancer [mCRC]) and CCTG PA.7 (gemcitabine and nab-paclitaxel [GN] +/- D+T in metastatic pancreatic adenocarcinoma [mPDAC]). CHIP+ was defined as any mutation in DNMT3A, TET2, ASXL1or ATM with a variant allele frequency (VAF) 2% or higher. Variants were considered germline if the VAF was &gt;40%, or were annotated as germline by respective assays. The first line of treatment after cfDNA was reviewed for grade 3 or higher and dose-limiting AEs. Results: The prevalence of CHIP was 10%-21% and was more common in older patients (p=0.003). There was no association between CHIP and sex (p=0.80) or ECOG status (p=0.31). The VAF of CHIP variants was similar across all panels, and DNMT3A was the gene most frequently mutated in all cohorts. Patients with CHIP in PA.7 treated with immunotherapy (IO) had improved progression-free survival (PFS) versus patients without CHIP (p=0.036, p-interaction=0.061 [multivariable]). However, patients with CHIP treated with chemotherapy (Chemo) showed a trend towards worse overall survival (OS) in the PREDICT-L cohort (p=0.057). There was no significant difference in the rates of AEs between the CHIP+ versus CHIP- groups for those treated with Chemo or IO. Conclusions: CHIP is common in patients with solid tumors. Although not appearing to impact rates of AEs, CHIP may impact outcomes from Chemo or IO. [Table: see text]

Circulating tumor DNA as a predictive biomarker for pathologic response after treatment with neoadjuvant immunotherapy for localized dMMR/MSI-H colorectal cancer.

3612 Background: Neoadjuvant immunotherapy (IO) has shown promise as a potential non-surgical, curative therapy in pts with localized dMMR/MSI-H CRC. However, response assessment to IO is challenging due to difficulty measuring luminal tumors, the invasive nature of endoscopy, and known discordance between these assessment modalities. ctDNA may serve as an important, novel marker of response in this population. We evaluated pre-treatment ctDNA and ctDNA clearance and their association with pathologic complete response (pCR) in pts with localized dMMR/MSI-H CRC treated with IO. Methods: We performed a 70-gene, tumor-agnostic, NGS-based ctDNA assay (LB-70) to detect somatic mutations in a CLIA setting on 49 plasma samples collected serially from 13 pts with localized dMMR/MSI-H CRC treated with neoadjuvant pembrolizumab (mean and median cycles per pt was 8; range 1 - 16) prior to definitive surgery as part of a prospective phase 2 trial (NCT04082572). NGS was also performed on each pt’s tumor tissue to confirm mutations identified in ctDNA were tumor-specific. We defined “ctDNA clearance” as a reduction in mutational variant allele frequency (VAF) to a threshold below LB-70’s limit of detection (&lt;0.3% VAF) after neoadjuvant IO in mutations that were present in both plasma and tumor tissue. We conducted a descriptive analysis to evaluate the association between the presence of pre-treatment (“baseline”) ctDNA, ctDNA clearance, and pCR. Results: On a median number of 3 samples per pt, mutations in baseline ctDNA were detected in 7/13 pts and were confirmed orthogonally in all pts with matched tumor tissue. The mean and median VAF was 8.5% and 0.7% (range 0.3% - 26.9%). Among 10/13 pts with a pCR, none recurred clinically after median follow-up 29.7 mo. Three pts did not experience a pCR, 1 of whom had disease recurrence after surgery. pCR was observed in 6/6 pts without detectable ctDNA at baseline, while only 4/7 pts with detectable ctDNA at baseline had a pCR (100% vs 57%). Of the 7 pts with detectable ctDNA at baseline, 5 cleared their ctDNA in the neoadjuvant period, among whom, 4 had a pCR (80% likelihood of pCR with ctDNA clearance). Notably, the 1 pt who did not have a pCR despite clearing ctDNA had a near-pCR (carcinoma identified in 1/91 lymph nodes, none in cecum or colon). The mean and median number of cycles to ctDNA clearance was 2.8 and 2 (range 1 - 8). For the 2 pts who did not clear their ctDNA, neither (0%) had a pCR. Conclusions: Clearance of ctDNA following neoadjuvant IO appears to predict pCR in this pilot analysis of pts with localized dMMR/MSI-H CRC, even after a median of 2 cycles. The absence of detectable ctDNA at baseline, perhaps a surrogate for lower tumor burden at initial diagnosis, may also predict a higher likelihood for pCR. Our work suggests utility of ctDNA as a biomarker for improving risk-stratification and response assessment sensitivity.

Analytical and clinical validation of a personalized, tumor-informed circulating tumor DNA assay for minimal residual disease detection.

e14545 Background: The detection of minimal residual disease (MRD) through circulating tumor DNA (ctDNA) analysis provides an advanced molecular method for early identification of surgical patients at high risk of recurrence. In this study, we present the development of mClear, a personalized, tumor-informed next-generation sequencing (NGS) assay for MRD detection. Furthermore, we report the analytical and clinical validation processes conducted to evaluate the performance of the MRD assay. Methods: mClear is designed to assess MRD in patients with solid tumors. Whole exome sequencing (WES) was performed on tumor tissues and matched peripheral blood samples to detect somatic variants comprehensively. Personalized MRD assays were carefully designed by selecting up to 40 prioritized variants, including single nucleotide variants (SNVs), small insertions and deletions (InDels), and fusions while ensuring coverage of 21 additional key driver genes. For analytical validation, we utilized a pan-cancer MRD ctDNA quality control product (LDT-999, LDT Bioscience), characterized by known variant allele frequency (VAF) gradients of 0%, 0.005%, 0.05%, and 0.5%, confirmed through droplet digital PCR (ddPCR). We conducted 18 replicate tests at VAF gradients of 0.5% and 0.05%, 23 replicates at the 0.005% VAF gradient, and 16 replicates at the 0% VAF gradient. For clinical validation, preoperative and postoperative blood samples from a cohort of 264 pan-cancer patients were analyzed. ctDNA collected for MRD detection underwent ultra-deep next-generation sequencing (100,000× average depth of coverage). The library preparation process commenced with an input of 30ng DNA. Results: At VAF gradients of 0.5% and 0.05%, the assay achieved an excellent sensitivity of 100%, and at VAF gradients of 0.005% and 0%, it demonstrated a sensitivity of 95.7% and a specificity of 100%. Preoperative ctDNA was detected in 86.7% (72/83) of the pan-cancer patients with 71.4% (5/7), 90.9% (10/11), 85.5% (47/55), and 100.0% (10/10) in pathological stage (pStage)I, pStage II, pStage III, and pStage IV, respectively. In the pan-cancer cohort, the positive rate of longitudinal MRD was 35.7% (75/210) with 17.8% (21/118), 50.0% (13/26), 55.1% (27/49), and 82.4% (14/17) in pStage I, II, III, and IV, respectively. In four patients who relapsed, MRD detection during surveillance preceded radiological recurrence by a lead time of 1-8 months. Conclusions: Our personalized MRD assay demonstrated excellent analytical sensitivity and specificity with a limit of detection (LoD) of 0.005% VAF at the sample level. Furthermore, it displayed high clinical sensitivity for the detection of ctDNA in the pan-cancer cohort, highlighting its potential for monitoring of recurrence.

TP53 mutation screening for patients at risk of myeloid malignancy.

6546 Background: Cellular therapy is a promising approach for many diseases but is associated with a risk of subsequent myelodysplastic syndrome and/or acute myeloid leukemia, also known as therapy-related myeloid neoplasms (tMN). This phenomenon has recently been described with allogeneic hematopoietic cell transplantation (HCT) and gene therapy for sickle cell disease (SCD). Mutation(s) in functional domains of TP53 are often associated with the development of tMN but currently no prospective screening or monitoring technology is available. Methods: A next-generation sequencing (NGS) assay with unique molecular identifiers (UMI) and bioinformatic noise-suppression was designed to discover somatic mutations at a variant allele frequency (VAF) 1% throughout the TP53gene. The background error profile at each base position was calculated using blood genomic DNA from healthy donors. The minimal detectable allele fraction with 95% confidence (MDAF) was calculated for all reported known AML/tMN TP53 hotspots. Orthogonal validation was performed using droplet digital PCR (ddPCR). Results: The median error corrected sequencing depth at 62 known TP53 hotspot regions was 14,512 (95% CI: 14,769 – 15,430) with a theoretical median de novo NGS variant discovery limit of 0.058% (range: 0.037% - 0.13%). Experimental evidence of assay performance characteristics was produced using serial dilutions of 4 missense, 2 splicing, and 1 frameshift TP53 variants with 100% discovery specificity observed (lowest detected VAF range: 0.05 – 0.12%). High correlation was observed in quantification using NGS discovery and ddPCR validation ( r = 0.958, p&lt;0.0001). Pre- and post-HCT clinical samples from 19 patients with SCD, 11 of which had graft failure including 4 who developed tMN, were screened for TP53 mutations. In all tMN cases, the AML-associated TP53 variant was detectable at least 6 months and up to 2.5 years prior to clinical diagnosis of myeloid malignancy. In contrast, no pathogenic TP53 variants were detected in 15 SCD patients who underwent HCT but did not develop tMN. Conclusions: This study provides generalizable evidence that ultra-sensitive discovery of TP53 mutations prior to cellular therapy is possible and could be a potentially valuable clinical tool to screen for tMN risk.

Association of germline HSD3B1 (c.1100) genotype with tumoral and clinical outcomes in breast and endometrial cancers.

10587 Background: A germline single-nucleotide polymorphism in HSD3B1 results in adrenal-permissive (c.1100 C) and adrenal-restrictive (c.1100 A) phenotypes with respect to androgen synthesis. In pre- and post-menopausal patients with breast cancer (n=3548 and 7871) and endometrial cancer (n=1588 and 8439), we annotated samples as adrenal permissive (CC/AC) or restrictive (AA) based on HSD3B1 variant status and then examined tumoral characteristics and clinical outcomes. Methods: We utilized tumors from the Caris Life Sciences Precision Oncology Alliance database and inferred germline HSD3B1 c.1100 genotype using variant allele frequencies (VAFs): c.1100 C VAF of 0% defined the AA genotype, VAF of 40-60% defined the AC genotype, and VAF of 100% defined the CC genotype. We explored associations of each HSD3B1genotypes with other genomic (WES) and transcriptomic (RNA-seq) features, as well as with survival outcomes. To performed consider menopausal status we used the patient’s age at the time of biopsy (&gt;55 indicating post-menopausal). Results: In breast cancer (BC) and endometrial cancer (EC), the permissive HSD3B1genotype had greater mRNA expression of the estrogen receptor ( ESR1) and co-alterations of the TP53 (p&lt;0.0001). In post-menopausal ECs, the permissive genotype was associated with increased alterations in the PI-3-kinase pathway ( PTEN, PIK3CA, PIK3R1, p&lt;0.0001). In pre-menopausal HER2+ BCs, the restrictive genotype was associated with worse survival (HR=1.40, 95% CI = 1.05-1.85, p=0.027) and exhibited increases in hallmark GSEA signatures including epithelial-to-mesenchymal transition. Post-menopausal basal BCs with the permissive genotype had numerically higher PD-L1 protein expression (50% vs 13%) and less PI-3-kinase alterations ( PTEN, PIK3R1, p&lt;0.01). Post-menopausal clear cell ECs with the restrictive genotype exhibited worse survival (HR=1.60, 95% CI=1.07-2.38, p=0.03) and were 3-fold more likely to be TMB-high or MSI-high (p&lt;0.05). Distinctly, post-menopausal endometrioid ECs of the restrictive genotype were less likely to be TMB- or MSI-high (p&lt;0.05). Conclusions: Breast and endometrial cancers can be classified as adrenal-permissive or -restrictive based on HSD3B1 c.1100 genotype. This classification distinguishes clinical outcomes and tumoral features, particularly when stratifying by menopausal status. Importantly, these genotypes are differentially associated with druggable pathways (PI-3-kinase) and markers of immune sensitivity (PD-L1, TMB, MSI). [Table: see text]