Large-scale State Transitions Research Articles

Genomic instability in non-breast/ovarian malignancies of individuals with germline pathogenic variants in BRCA1/2.

Individuals with germline pathogenic variants (gPVs) in BRCA1 or BRCA2 (BRCA1/2) are at a high risk of breast- and ovarian carcinomas (BOCs) with BRCA1/2-deficiency and homologous recombination deficiency (HRD) that can be detected by analysis of genome-wide genomic instability features such as large-scale state transitions, telomeric allelic imbalances and genomic loss-of-heterozygosity. Malignancies with HRD are more sensitive to platinum-based therapies and PARP inhibitors. Here, we aim to investigate the fraction of non-BOC malignancies that have BRCA1/2-deficiency and genomic instability features. The full tumor history of a large historical clinic-based consecutive cohort of 2,965 individuals with gPVs in BRCA1/2 was retrieved via the Dutch nationwide pathology databank (Palga). In total, 169 non-BOC malignancies were collected and analyzed with targeted next-generation sequencing and shallow whole-genome sequencing to determine somatic second hit alterations and genomic instabilities indicative of HRD, respectively. BRCA1/2-deficiency was detected in 27% (21/79) and 23% (21/90) of 20 different types of non-BOC malignancies of individuals with gPVs in BRCA1 and BRCA2, respectively. These malignancies had a higher genomic instability score than BRCA1- or BRCA2-proficient malignancies (P < .001 and P < .001, respectively). BRCA1/2-deficiency and genomic instability features were found in 27% and 23% of a broad spectrum of non-BOC malignancies in individuals with gPVs in BRCA1 and BRCA2, respectively. Evaluation of the effectivity of PARP-inhibitors in these individuals should be focused on tumors with confirmed absence of a wild type allele.

Increased Synthetic Cytotoxicity of Combinatorial Chemoradiation Therapy in Homologous Recombination Deficient Tumors

BackgroundHomologous recombination deficient (HRD) tumors are exquisitely sensitive to platinum-based chemotherapy and when combined with RT, leads to improved overall survival in multiple cancer types. Whether a subset of tumors with distinct molecular characteristics demonstrate increased benefit from cisplatin and RT (c-RT) is unclear. We hypothesized that HRD tumors, whether associated with BRCA mutations or genomic scars of HRD, exhibit exquisite sensitivity to c-RT, and that HRD may be a significant driver of c-RT benefit. MethodsSensitivity to c-RT was examined using isogenic and sporadic breast cancer cell lines. HRD was assessed using four assays: RT-induced Rad51 foci, a DR-GFP reporter assay, a genomic scar (large scale state transitions, LST), and clonogenic survival assays (CSA). Whole genome sequencing of 4 breast tumors from a phase 2 clinical trial of neoadjuvant c-RT in triple negative breast cancer (TNBC) was performed and defined HRD utilizing HRDetect. ResultsBRCA1/2 deficient cell lines displayed functional HRD based on the Rad51 functional assay, with c-RT to RT or cisplatin interaction ratios (IR) of 1.11 and 26.84 for the BRCA1 isogenic pair at 2uM Cisplatin and 6Gy, respectively. The highest LST lines demonstrated HRD and synthetic cytotoxicity to c-RT with IR at 2Gy and Cisplatin 20uM of 7.50, and the lowest LST line with IR of 0.65. Of 4 evaluable patients on the Phase 2 trial, one achieved pathologic complete response (pCR) with corresponding HRD based on multiple genomic scar scores including HRDetect and LST scores, compared with patients without pCR. ConclusionsHRD breast cancers, whether identified by BRCA1/2 mutation status, functional tests or mutational signatures, appear to be significantly more sensitive to c-RT compared to isogenic controls or tumors without HRD mutational signature. HRD tumors may be exquisitely sensitive to c-RT and warrants further clinical investigation to guide a precision oncology approach.

Genomic Insights Into High-Grade Infarct-Associated Bone Sarcomas

Sarcomas rarely develop in bones previously compromised by infarcts. These infarct-associated sarcomas often present as undifferentiated pleomorphic sarcomas (UPS), and their genetic characteristics are poorly understood. High-grade UPS of bone are typically treated with a combination of surgery and chemotherapy, similar to osteosarcoma. We conducted a detailed clinicopathologic and genomic analysis of 6 cases of intraosseous sarcomas arising from histologically and radiographically confirmed bone infarcts. We analyzed 523 genes for sequence-level mutations using next-generation sequencing with the TruSight Oncology 500 panel and utilized whole-genome single nucleotide polymorphism Microarray (OncoScan CNV) to detect copy number alterations and loss of heterozygosity (LOH). Genomic instability was assessed through homologous recombination deficiency (HRD) metrics, incorporating LOH, telomeric allelic imbalance, and large-scale state transitions. Fluorescence in situ hybridization and immunohistochemistry validated the findings. The cohort included 3 men and 3 women, with a median age of 70 years, and tumors located in the femur and tibia. Five of the 6 patients developed distant metastases. Treatment involved surgery and chemotherapy or immune checkpoint inhibitors. Genomic analysis revealed significant complexity and high HRD scores, ranging from 32 to 57 (with a cutoff of 32). Chromosome 12 alterations, including segmental amplification or chromothripsis, were observed in 4 cases. Notably, MDM2 amplification, confirmed by fluorescence in situ hybridization, was detected in 2 cases. Homozygous deletion of CDKN2A/B was observed in all six cases. Tumor mutational burden levels ranged from 2.4 to 7.9 mutations per megabase. Notable pathogenic mutations included H3-3A mutations (p.G35R and p.G35W), and mutations in HRAS, DNMT3A, NF2, PIK3CA, POLE, and TP53, each in one case. These results suggest that high-grade infarct-associated sarcomas of bone, whereas sharing high levels of structural variations with osteosarcoma, may exhibit potentially less frequent TP53 mutations and more common CDKN2A/B deletions. This points to the possibility that the mutation spectrum and disrupted pathways could be distinct from conventional osteosarcoma.

Dependency-aware action planning for smart home.

How can a smart home system control a connected device to be in a desired state? Recent developments in the Internet of Things (IoT) technology enable people to control various devices with the smart home system rather than physical contact. Furthermore, smart home systems cooperate with voice assistants such as Bixby or Alexa allowing users to control their devices through voice. In this process, a user's query clarifies the target state of the device rather than the actions to perform. Thus, the smart home system needs to plan a sequence of actions to fulfill the user's needs. However, it is challenging to perform action planning because it needs to handle a large-scale state transition graph of a real-world device, and the complex dependence relationships between capabilities. In this work, we propose SmartAid (Smart Home Action Planning in awareness of Dependency), an action planning method for smart home systems. To represent the state transition graph, SmartAid learns models that represent the prerequisite conditions and operations of actions. Then, SmartAid generates an action plan considering the dependencies between capabilities and actions. Extensive experiments demonstrate that SmartAid successfully represents a real-world device based on a state transition log and generates an accurate action sequence for a given query.

Homologous recombination deficiency score for predicting efficacy of platinum-based chemotherapy and PARPi maintenance therapy in ovarian cancer.

e17543 Background: Currently, homologous recombination deficiency (HRD) has been confirmed to be a predictive biomarker for poly-ADP-ribose-polymerase inhibitors (PARPi) in ovarian cancer, while its effects on the clinical outcomes of platinum-based chemotherapy and PARPi maintenance therapy are not assessed rigorously in ovarian cancer. We developed an HRD assay to evaluate the effect of HRD scores on the platinum-based chemotherapy and PARPi maintenance therapy in Chinese patients with ovarian cancer. Methods: HRD testing was performed on the samples obtained at primary cytoreductive surgery. The progression-free survival (PFS) was observed among the BRCA-mutated/HRD-positive and BRCA wild-type/HRD-negative patients. Univariate and multivariate COX analyses were performed to test the association of PFS with different variables, and hazards ratios (HR) and 95% confidence interval (CI) were calculated. The two-tailed p value less than 0.05 was considered statistically significant. Results: Totally 74 patients who received platinum-based chemotherapy were eligible for the study, among whom 81.1% responded to the initial platinum-based chemotherapy, 33.8% harbored BRCA1/2 mutations and 74.3% were HRD-positive. The median HRD score of platinum-sensitive patients (n=60) was 48.5, significantly higher than the 31 of platinum-resistant patients (n=14, p=0.0035). Analysis of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI) and large-scale state transitions (LST) between platinum-sensitive and platinum-resistant patients showed significant differences in TAI ( p=0.0012) and LST ( p=0.004). Compared with HRD-negative patients, HRD-positive patients had a significantly longer median PFS (10 vs. 15 months, p=0.017), and multivariate analysis further indicated that HRD-positive status (HR, 0.423, 95%CI: 0.191-0.939, p=0.035) was a significant predictor for the improvement of PFS. Conclusions: The HRD assay developed in our study can accurately predict the response to platinum-based chemotherapy and PARPi efficacy in patients with ovarian cancer, providing powerful evidence for implementation of genetic testing and guidance of clinical treatment in ovarian cancer.

Improved detection of homologous recombination deficiency using WES sequencing: Insights into a Chinese pan-cancer cohort.

e17503 Background: Homologous recombination (HR) deficiency has emerged as a novel biomarker for the clinical use of PARP inhibitors in advanced ovarian cancer, and may also be of value in guiding the clinical use of PARP inhibitors and platinum-based drugs in breast cancer, prostate cancer, and other tumors. This significant finding assists in precision medication and prognosis judgments for patients. Methods: This research incorporated a Chinese pan-cancer cohort comprising 1033 patients diagnosed across thirteen cancer types. All matched tumor tissues and peripheral blood samples were processed for the whole exome sequencing. The HRD scoring was conducted with ScarHRD R package with defined parameters for the heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST) status. HR deficiency were defined as an HRD-score ≥50 or BRCA1/2 deficient, while HR proficiency were defined as an HRD-score &lt;50 and BRCA1/2 intact. Results: Ovarian cancer demonstrates the highest HRD prevalence at 51.85%, surpassing the BRCA1/2 mutation rate of 20.37%. Breast cancer follows with an HRD rate of 45.45%, yet no BRCA1/2 mutations are detected. Other cancers like pancreatic, gastric, and sarcomas also present notable HRD rates (29.41%, 36.84%, and 29.79%, respectively) without corresponding BRCA1/2 mutations. In contrast, cancers such as urological, lung, intestinal, and neurological tumors exhibit a mismatch between HRD rates and BRCA1/2 mutation presence, few patients with notable BRCA1/2 mutations did not have consisted HR-deficient rate, and vice versa. Conclusions: Analysis HRD-score can help to screen a larger population of PARP inhibitor beneficiaries and provide hope to more patients. In the future we will do more dimensional data analysis statistics on this cohort. [Table: see text]

Abstract PO3-14-08: Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets

Abstract Background: Metaplastic breast cancer (MpBC) is a rare histologic subtype of triple negative breast cancer (TNBC) with an aggressive behavior and poor outcome. Previous studies by whole-exome and targeted sequencing showed that MpBCs display a dominant mutational signature 3, associated to homologous recombination (HR) deficiency (HRD). Tumors displaying HRD are associated with response to therapies causing double strand breaks. Nonetheless, MpBCs consistently display significantly lower rates of response to neoadjuvant chemotherapy than non-metaplastic TNBCs and effective therapeutic alternatives are an unmet clinical need. Here we sought to assess the HR features of MpBCs using whole-genome sequencing (WGS), and to evaluate the expression in MpBCs of antigens that are targets of novel antibody drug conjugates (ADCs) in common forms of breast cancer. In addition, we sought to assess the efficacy of ADCs and drugs targeting the DNA damage response (DDR) using preclinical MpBC models. Materials and Methods: We subjected 25 primary MpBC to WGS. HRDetect, the ‘gold standard’ tool for assessment of HRD, was utilized. Mutational signatures were inferred using Signal. 31 MpBCs were subjected to RNA-sequencing to assess the gene expression levels ADC targets compared to non-MpBCs from The Cancer Genome Atlas (TCGA). Efficiency of ADCs and DDR drugs was evaluated using the MpBC cell models HCC1806, BT549 and Hs578T in vitroand/or using HCC1806 xenografts in athymic mice in vivo. Results: Although the majority of MpBCs (13/25) displayed a dominant SBS mutational signature 3, only a small subset of cases (3/25) were classified as HRD by HRDetect. These three bona fide HRD cases were found to harbor bi-allelic inactivation of BRCA1 (n=2) or RAD51C (n=1), and displayed various genomic features of HRD, including deletions with microhomology, enrichment in tandem duplications, rearrangement signatures 3 and 5, and high large scale state transitions (LST) and telomeric allelic imbalance (NtAI) scores. Most MpBCs (22/25), however, were not classified as HRD by HRDetect displaying an incomplete HRD phenotype, with only partial genomic features of HRD. 52% of MpBCs (13/25) displayed APOBEC mutagenesis exposure, which was present in the three bona fide HRD MpBCs. RNA-sequencing analyses revealed detectable ERBB2 and TROP2 expression in all MpBCs interrogated, although at lower levels than in non-MpBCs from TCGA. The HCC1806, BT549 and Hs578T MpBC cell models were found to be sensitive to Sacituzumab Govitecan and Trastuzumab Deruxtecan (T-DXd) in cell viability and colony formation assays, and to T-DXd in vivo. These MpBC cell models were also found to be sensitive to DDR compounds, such as ATR, WEE1 and PKMYT1 inhibitors in vitro, alone and in combination with T-DXd or Sacituzumab Govitecan. Conclusions: Most MpBCs display an incomplete HRD genomic scar, except for those cases harboring bi-allelic inactivation of HRD genes. APOBEC mutagenesis is operative in MpBCs and co-exists with HRD. MpBCs express ERBB2 and TROP2, and MpBC cell models are sensitive to T-DXd and Sacituzumab govitecan, as well as DDR compounds, targeting the incomplete HRD phenotype of MpBCs, alone and in combination. Taken together, these findings warrant further studies in patients with MpBCs testing the therapeutic efficacy of agents targeting the incomplete HRD phenotype, as well as the expression of ADC targets in these aggressive rare forms of TNBCs. Citation Format: Fresia Pareja, Andrea Gazzo, Higinio Dopeso, David Brown, Pier Selenica, Yingjie Zhu, Juan Blanco-Heredia, Laxmi Gusain, Xin Pei, Giacomo Montagna, Nour Abuhadra, Hanna Y Wen, Edi Brogi, Nadeem Riaz, Sarat Chandarlapaty, Maurizio Scaltriti, Larry Norton, Simon Powell, Jorge Reis-Filho, Britta Weigelt. Metaplastic Breast Cancer: Whole Genome Sequencing Analysis and Emerging Therapeutic Targets [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 PO3-14-08.

Abstract PO5-04-04: Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer

Abstract Introduction Blood-based liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional tissue biopsy is not feasible. Recently, the novel anti-HER2 antibody-drug conjugate, Trastuzumab Deruxtecan (T-DXd), has shown effectiveness against MBC with low HER2 expression detected by tissue biopsy characterized as HER2-low (immunohistochemically 1+ or 2+ and ERBB2 amplification-negative via in situ hybridization) and is being explored in an ultra-low population. Here we report on the application of a liquid biopsy platform designed to sensitively detect low levels of HER2 protein expression in CTCs from MBC patients. Methods Blood samples from 704 metastatic breast cancer patients as well as control cells lines were collected for cell-based and cell-free DNA analysis. After plasma isolation, nucleated cells deposited on glass slides went through immunofluorescent staining and imaging. CTCs were identified using Epic Sciences’ digital imaging and machine learning algorithms, and a subset of CTCs were isolated and sequenced for genomic quantification of large scale-state transitions (LSTs) as well as copy number variants (CNVs). The HER2 status was based upon on ASCO-CAP guidelines and the previous tissue biopsy results from the ordering healthcare provider, as documented on the DefineMBC™ test requisition form. Results Within a DefineMBC early-access program (EAP), 93% of patients with MBC had detectable CTCs. Among ERBB2-non-amp patients tested with the DefineMBC assay, 25% of patients with MBC had findings that were reported as consistent with HER2-low in the CAP-CLIA setting. To explore the lower boundaries of HER2 protein expression, we created and validated an MCF-7 HER2-negative ERBB2 knock-out cell line and utilized its 95% confidence level threshold (271 MFI) as a negative fluorescence cutoff for very low HER2 expression. Based upon publicly available CCLE datasets, MDA-MB-453, MCF7, and MCF7 ERBB2 KO breast cancer cell lines were selected for their high and low HER2 RNA expression levels and were corroborated via observed HER2 immunofluorescence median levels of 15740, 381, and 188 MFI respectively. In an exploratory analysis, among ERBB2-non-amp patients, we retrospectively applied the 271 MFI HER2 cutoff to the EAP cohort and identified 38% of patients with very low HER2 expression. We examined the impact of the HER2 ultra-low cutoff in patients not eligible for T-DXd treatment. Among patients reported as IHC=0 or with unknown IHC status from the most recent tissue biopsy, 44% and 42%, respectively, may qualify for treatment with T-DXd. Conclusion Using our higher sensitivity assay that combines CTC immunofluorescence and single-cell genomic analysis, we examined the lower boundary of HER2 protein expression. Feasibility data support further characterization of a very low HER2 expression phenotype, for the eventual inclusion into our comprehensive cancer profiling solution. To date, studies with biomarker expression have been limited to tissue biopsy, which may not always yield contemporaneous sampling in the metastatic setting. These results offer a liquid biopsy test that might identify patients potentially responsive to HER2-directed therapies. Citation Format: Giuseppe Di Caro, David Bourdon, Andrew Kunihiro, Alessandra Cunsolo, Ernest Lam, Megan Slade, Martin Blankfard, Lee Schwartzberg. Development of a method to detect very low levels of HER2 expression in Circulating Tumor Cells (CTCs) by liquid biopsy in patients with metastatic breast cancer [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 PO5-04-04.

Abstract PO4-06-12: Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test

Abstract Introduction: Breast cancer is a highly heterogeneous disease and is a leading cause of death for women worldwide. Triple-negative breast cancer (TNBC), the most aggressive form of the disease, is clinically defined by the lack of expression of estrogen receptor (ER) and progesterone receptor (PR), as well as lack of over-expression of human epidermal growth factor receptor-2 (HER2). Approximately 70% of all TNBC cases exhibit basal-like cells, characterized by high basal expression of keratins as well as extensive genomic instability with copy number gains and losses across most chromosomes. In this study we show that novel single cell genomic evaluation coupled with immunofluorescence (IF) protein analysis of CTCs can detect TNBC with basal-like genomic features as well as basal-like tumors with expression of ER and HER2. Materials and Methods: Epic Sciences’ DefineMBC™ liquid biopsy test characterizes CTC proteomics and genomics as well as ctDNA for the treatment of metastatic breast cancer. Blood samples from 813 metastatic breast cancer patients, with either newly diagnosed or progressing disease, with historical tissue subtype [HR(+)HER2(-), HR(+/-)HER2(+), and TNBC] supplied by the referring physician were collected and tested. Up to 5 CTCs were selected and isolated from each sample based on morphology and IF signal. DNA was extracted from individual cells, and whole genome amplified and sequenced at low coverage. Copy number analysis was performed to determine copy number gains and losses across chromosomes, as well as large scale-state transitions (LSTs) and ERBB2 amplification. CTCs characterized with high chromosomal instability (e.g., exhibiting &amp;gt;30 LSTs; 1Q firestorm and 8Q stair step) and high levels of cytokeratin expression (mean fluorescence intensity signal &amp;gt;10K) were categorized as basal-like. Results: By previous metastatic tissue biopsy, 77% were HR(+), 8% were HER2(+), and 12% were TNBC. CTCs categorized in DefineMBC as basal-like subtype (9.8 % of all samples tested) showed to be highly present in patients called as TNBC (79% basal-like). Additionally, 54% of HR(+) and 23% of HER2(+) had basal-like CTCS. Patients characterized with TNBC by metastatic tissue biopsy and by DefineMBC exhibited basal-like cells in 88% of the cases. Conclusion: The results show a novel clinical assay which utilizes CTC protein detection as well as single-cell genomic analysis to identify tumors driven by basal-like cells. Generally, TNBC is classified with negative biomarker expression. We show that CTCs in TNBC display genomically basal-like cells. CTCs from HR(+) and HER2(+) tumors may express basal-like features with preserved ER and HER2 expression. The comprehensive nature of DefineMBC™ utilizing genomic analysis of detected CTCs has provided a new tool to diagnose both basal-like TNBC and basal-like hormone receptor positive and HER2 over-expressing tumors. Citation Format: Alessandra Cunsolo, David Bourdon, Ernest Lam, Giuseppe Di Caro, Nilesh Dharajiya, Timothy Pluard, Lee Schwartzberg. Distinction of basal-like and triple-negative basal-like breast cancers utilizing a novel comprehensive single-cell liquid biopsy-based test [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 PO4-06-12.

Establishing the homologous recombination score threshold in metastatic prostate cancer patients to predict the efficacy of PARP inhibitors

BackgroundThe homologous recombination deficiency (HRD) score serves as a promising biomarker to identify patients who are eligible for treatment with PARP inhibitors (PARPi). Previous studies have suggested a 3-biomarker Genomic Instability Score (GIS) threshold of ≥ 42 as a valid biomarker to predict response to PARPi in patients with ovarian cancer and breast cancer. However, the GIS threshold for prostate cancer (PCa) is still lacking. Here, we conducted an exploratory analysis to investigate an appropriate HRD score threshold and to evaluate its ability to predict response to PARPi in PCa patients. MethodsA total of 181 patients with metastatic castration-resistant PCa were included in this study. Tumor tissue specimens were collected for targeted next-generation sequencing for homologous recombination repair (HRR) genes and copy number variation (CNV) analysis. The HRD score was calculated based on over 50,000 single-nucleotide polymorphisms (SNP) distributed across the human genome, incorporating three SNP-based assays: loss of heterozygosity, telomeric allelic imbalance, and large-scale state transition. The HRD score threshold was set at the last 5th percentile of the HRD scores in our cohort of known HRR-deficient tumors. The relationship between the HRD score and the efficacy in 16 patients of our cohort who received PARPi treatment were retrospectively analyzed. ResultsGenomic testing was succeeded in 162 patients. In our cohort, 61 patients (37.7%) had HRR mutations (HRRm). BRCA mutations occurred in 15 patients (9.3%). The median HRD score was 4 (ranged from 0 to 57) in the total cohort, which is much lower than that in breast and ovarian cancers. Patients who harbored HRRm and BRCA or TP53 mutations had higher HRD scores. CNV occured more frequently in patients with HRRm. The last 5th percentile of HRD scores was 43 in the HRR-mutant cohort and consequently HRD high was defined as HRD scores ≥43. In the 16 patients who received PARPi in our cohort, 4 patients with a high HRD score achieved an objective response rate (ORR) of 100% while 12 patients with a low HRD score achieved an ORR of 8.3%. Progression-free survival (PFS) in HRD high patients was longer compared to HRD low patients, regardless of HRRm. ConclusionsA HRD score threshold of 43 was established and preliminarily validated to predict the efficacy of PARPi in this study. Future studies are needed to further verify this threshold.

Prediction of homologous recombination deficiency from Oncomine Comprehensive Assay Plus correlating with SOPHiA DDM HRD Solution.

Poly(ADP-ribose) polymerase (PARP) inhibitors are used for targeted therapy for ovarian cancer with homologous recombination deficiency (HRD). In this study, we aimed to develop a homologous recombination deficiency prediction model to predict the genomic integrity (GI) index of the SOPHiA DDM HRD Solution from the Oncomine Comprehensive Assay (OCA) Plus. We also tried to a find cut-off value of the genomic instability metric (GIM) of the OCA Plus that correlates with the GI index of the SOPHiA DDM HRD Solution. We included 87 cases with high-grade ovarian serous carcinoma from five tertiary referral hospitals in Republic of Korea. We developed an HRD prediction model to predict the GI index of the SOPHiA DDM HRD Solution. As predictor variables in the model, we used the HRD score, which included percent loss of heterozygosity (%LOH), percent telomeric allelic imbalance (%TAI), percent large-scale state transitions (%LST), and the genomic instability metric (GIM). To build the model, we employed a penalized logistic regression technique. The final model equation is -21.77 + 0.200 × GIM + 0.102 × %LOH + 0.037 × %TAI + 0.261 × %LST. To improve the performance of the prediction model, we added a borderline result category to the GI results. The accuracy of our HRD status prediction model was 0.958 for the test set. The accuracy of HRD status using GIM with a cut-off value of 16 was 0.911. The Oncomine Comprehensive Assay Plus provides a reliable biomarker for homologous recombination deficiency.

Leveraging Off-Target Reads in Panel Sequencing for Homologous Recombination Repair Deficiency Screening in Tumor

Targeted tumor only sequencing has become a standard practice in cancer diagnostics. This study aims to develop an approach for robust copy number variant (CNV) calling in tumor samples using only off-target region (OTR) reads. We also established a clinical use case for homologous recombination deficiency (HRD) score estimation (HRDest) using the sum of telomeric allelic imbalance (TAI) and large-scale state transitions (LST) scores without the need for loss of heterozygosity (LOH) information. We demonstrated a strong correlation between HRD score and the sum of TAI + LST in the TCGA cohort (rho = 0.99, p < 2.2x 10-16) and in a clinical in-house cohort of 34 tumors (rho = 0.9, p = 5.1 x 10-13) comparing WES and targeted sequencing data. We compared HRDest scores from 1086 clinical cases to the TCGA data set and showed that there were no significant differences in HRD score distribution within the analyzed tumor types. As a control, commercially available HRD standards were also sequenced and the HRDest scores obtained from the OTR reads were well within the HRD reference range provided by the manufacturer. In conclusion, we showed that OTR reads of tumor-only panel sequencing can be used to determine genome-wide CNV profiles and to approximate HRD scores.

Abstract 2328: Next-generation sequencing-based regression algorithm to determine homologous recombination deficiency scores in a pan-cancer cohort

Abstract Background: Homologous recombination deficiency (HRD) inhibits double strand breaks from being repaired in DNA, leading to cancer cells failing to recover themselves and cell death. Previous studies found that HRD-positive ovarian cancer patients had more significant clinical benefits from poly ADP-ribose polymerase inhibitors (PARPi) treatment. For accurate detection of HRD, next-generation sequencing (whole exome, whole genome) was used to identify large-scale genomic aberrations including telomeric allelic imbalance (HRD-TAI score), loss of heterozygosity profiles (HRD-LOH score), and large-scale state transitions (HRD-LST score). So far, many studies (HRDetect, SigMA, CHORD and shallowHRD) have been conducted to accurately determine HRD in a pan-cancer cohort. As the study progressed, it has been revealed that PARPi is effective when applied to patients with HRD in not only ovarian cancer but also breast cancer, prostate cancer, and pancreatic cancer. Purpose: To develop an algorithm that accurately predict HRD score in patients with ovarian cancer, breast cancer, prostate cancer, and pancreatic cancer utilizing a machine learning algorithm. Methods: We used whole-genome sequencing (WGS) data of 710 samples from 309 patients, whole-exome sequencing (WES) data of 4,650 samples from 2,193 patients from the pan-cancer cohort of the TCGA (TCGA-OV, TCGA-BRCA, TCGA-PRAD, and TCGA-PAAD). The HRD-TAI/HRD-LST/HRD-LOH scores were calculated through structural variation analyses. After data cleaning processes, machine-learning models were trained and tested on 228 out of 2,502 samples, and validation was performed on 1,248 out of 2,502 samples from the TCGA dataset. Results: To assess the performance of the machine-learning regressor, the concordance between predictions and annotations was quantified by calculating the R squared (𝑅;;;^2). As a result of training using machine learning algorithm, we achieved a high 𝑅;;;^2 (0.904) with a RMSE (root mean squared error) score (7.649) for a pan-cancer cohort. Conclusions: Our regressor was robust and accurate when applied to 4 cancer types. Using our systematic pan-cancer analysis, we found novel insights into the mechanisms of HRD across cancer types with potential contribution to clinical practice. Citation Format: Boram Choi, Youn Jin Choi. Next-generation sequencing-based regression algorithm to determine homologous recombination deficiency scores in a pan-cancer cohort [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 2328.

Pathogenic germline variants in non-BRCA1/2 homologous recombination genes in ovarian cancer: Analysis of tumor phenotype and survival

ObjectiveTo define molecular features of ovarian cancer (OC) with germline pathogenic variants (PVs) in non-BRCA homologous recombination (HR) genes and analyze survival compared to BRCA1/2 and wildtype (WT) OC. MethodsWe included patients with OC undergoing tumor-normal sequencing (MSK-IMPACT) from 07/01/2015–12/31/2020, including germline assessment of BRCA1/2 and other HR genes ATM, BARD1, BRIP1, FANCA, FANCC, NBN, PALB2, RAD50, RAD51B, RAD51C, and RAD51D. Biallelic inactivation was assessed within tumors. Progression-free (PFS) and overall survival (OS) were calculated from pathologic diagnosis using the Kaplan-Meier method with left truncation. Whole-exome sequencing (WES) was performed in a subset. ResultsOf 882 patients with OC, 56 (6.3%) had germline PVs in non-BRCA HR genes; 95 (11%) had BRCA1-associated OC (58 germline, 37 somatic); and 59 (6.7%) had BRCA2-associated OC (40 germline, 19 somatic). High rates of biallelic alterations were observed among germline PVs in BRIP1 (11/13), PALB2 (3/4), RAD51B (3/4), RAD51C (3/4), and RAD51D (8/10). In cases with WES (27/35), there was higher tumor mutational burden (TMB; median 2.5 [1.1–6.0] vs. 1.2 mut/Mb [0.6–2.6]) and enrichment of HR-deficient (HRD) mutational signatures in tumors associated with germline PALB2 and RAD51B/C/D compared with BRIP1 PVs (p < 0.01). Other features of HRD, including telomeric-allelic imbalance (TAI) and large-scale state transitions (LSTs), were similar. Although there was heterogeneity in PFS/OS by gene group, only BRCA1/2-associated OC had improved survival compared to WT OC (p < 0.01). ConclusionsOCs associated with germline PVs in non-BRCA HR genes represent a heterogenous group, with PALB2 and RAD51B/C/D associated with an HRD phenotype.

Combination of genomic instability score and TP53 status for prognosis prediction in lung adenocarcinoma

The genomic instability (GI) /homologous recombination deficiency (HRD) score, calculated as the sum of the events of loss of heterozygosity (LOH), large-scale state transition (LST) and telomere allele imbalance (TAI), is used to guide the choice of treatment in several cancers, but its relationship with genomic features, clinicopathological characteristics and prognosis in lung cancer is poorly understood, which could lead to population bias in prospective studies. We retrospectively analyzed 1011 lung cancer patients whose tumor samples were successfully profiled by high-throughput sequencing panel including GI/HRD score. Alterations of many cancer suppressor genes were associated with higher GI/HRD scores, biallelic inactivation of TP53 was correlated with a high GI/HRD score. A combination of two gene alterations exhibited a higher GI/HRD scores than single gene alterations. The GI/HRD score was associated with advanced stages in lung adenocarcinoma but not in lung squamous cell carcinoma. Furthermore, patients with higher GI/HRD scores had significantly shorter overall survival and progression-free survival than patients with lower GI/HRD scores. Finally, patients with a combination of a higher GI/HRD scores and TP53 alteration exhibited an extremely poor prognosis compared with patients with a lower GI/HRD scores and wild-type TP53 (overall survival, training cohort, hazard ratio (HR) = 8.56, P < 0.001; validation cohort, HR = 6.47, P < 0.001; progression-free survival, HR = 4.76, P < 0.001). Our study revealed the prognostic value of the GI/HRD score in lung adenocarcinoma, but not for all lung cancer. Moreover, the combination of the GI/HRD score and TP53 status could be a promising strategy to predict the prognosis of patients with lung adenocarcinoma.

Normalized LST Is an Efficient Biomarker for Homologous Recombination Deficiency and Olaparib Response in Ovarian Carcinoma.

The efficiency of the Myriad Homologous Recombination Deficiency (HRD) test to guide the use of poly (ADP-ribose) polymerase (PARP) inhibitors has been demonstrated in several phase III trials. However, a need exists for alternative clinically validated tests. A novel biomarker for HRD was developed using The Cancer Genome Atlas database and, as part of the ENGOT HRD European Initiative, applied to 469 samples from the PAOLA-1/ENGOT-ov25 trial. Results were compared with the Myriad myChoice Genomic Instability Score (GIS) with respect to the progression-free survival in the olaparib + bevacizumab and placebo + bevacizumab arms. Analysis of the TCGA cohort revealed that a normalization of the number of large-scale state transitions by the number of whole-genome doubling events allows a better separation and classification of HRD samples than the GIS. Analysis of the PAOLA-1 samples, using the Geneva test (OncoScan + nLST), yielded a lower failure rate (27 of 469 v 59 of 469) and a hazard ratio of 0.40 (95% CI, 0.28 to 0.57) compared with 0.37 for Myriad myChoice (BRCAm or GIS+) in the nLST-positive samples. In patients with BRCAwt, the Geneva test identified a novel subpopulation of patients, with a favorable 1-year PFS (85%) but a poor 2-year PFS (30%) on olaparib + bevacizumab treatment. The proposed test efficiently separates HRD-positive from HRD-negative patients, predicts response to PARP inhibition, and can be easily deployed in a clinical laboratory for routine practice. The performance is similar to the available commercial test, but its lower failure rate allows an increase in the number of patients who will receive a conclusive laboratory result.

An in-house testing for detection of homologous recombination repair deficiency (HRD) in patients with solid tumors.

e15025 Background: HRD is associated with sensitivity towards PARP inhibitors (PARPi) in various cancer types, such as ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer, and its determination is used as a biomarker for therapy decision making. HRD leads to the larger genome alterations, called “genomic scars“, which comprise loss of heterozygosity (LOH), telomeric allelic imbalance (TAI) and large scale state transitions(LST). The detection of SNPs is capable of measuring chromosomal abnormalities including genomic scars as mentioned above. Methods: Using a panel targeting more than 50,000 SNPs that evenly spaced across the entire human genome and the next generation sequencing technique, we developed an in-house testing for HRD detection, namely HRD score, which was computed by combining the measurements of large genomic defects including LOH, TAI, and LST. Then, we detected 44 ovarian cancer patients without BRCA1/2 mutations and 14 ovarian cancer patients harboring BRCA1/2 mutations to explore the application possibility of HRD score 42 as the threshold for HRD patients. Furthermore, we studied the HRD score distribution of 494 solid tumor patients. Results: For the 44 ovarian cancer patients without BRCA mutation, 50% (22/44) of them had the HRD score greater than 42. For the 14 ovarian cancer patients harboring BRCA1/2 mutations, 100%(14/14) of them had the HRD score greater than 42. For the 494 solid tumor patients, 38.66% (191/494) of them had the HRD score greater than 42. Conclusions: For this in-house HRD testing, the percentage of ovarian cancer patients without BRCA mutation who had HRD score greater than 42 was consistent with former report. Besides, the HRD score of all the 14 ovarian cancer patients harboring BRCA1/2 mutations were greater than 42, which was consistent with the facts. Therefore, it seems applicable to define the patients with HRD score greater than 42 as HRD positive. In addition, there is a wide range of demands for comprehensive implementation of HRD testing for other tumor entities.

Liquid biopsy identification of ERBB2 amplified and HER2 expressing metastatic breast cancer: Comparison and combination of cell and cell-free platforms.

3061 Background: Liquid biopsies are a non-invasive diagnostic approach for detecting circulating tumor cells (CTCs) or circulating tumor DNA (ctDNA) that may provide clinically actionable information for treatment decisions for metastatic breast cancer (MBC) patients when a conventional biopsy is otherwise infeasible. Here we report a comprehensive liquid biopsy platform including: 1) quantitative immunofluorescent HER2 and ER protein expression in CTCs (ctcIF), 2) determination of ERBB2 amplification and the number of Large-scale State transitions (LST) by single-cells CTC genomics (ctcDNA) and 3) alterations in plasma ctDNA. Methods: Blood samples from 457 progressing metastatic breast cancer patients and 25 healthy donors (HDs) as controls were collected for cell &amp; cell-free DNA analysis. After plasma isolation, nucleated cells were plated, &amp; slides were bio-banked for Immunofluorescent staining &amp; subsequent imaging. CTCs were identified using Epic Sciences digital imaging &amp; machine learning algorithms, and individual CTCs were sequenced for genomic quantification of LSTs and Copy Number Variants (CNV). Bio-banked plasma was analyzed to detect ctDNA alterations with high clinical relevance (Class IA). Results: Within this cohort of 457 MBC patients, the presence of CTCs, ctcDNA (LST+) and ctDNA alterations were detected in 98%, 58%, and 42%, respectively, while no CTCs and no ctDNA alterations were detected in the HD cohort, suggesting high specificity. ctcDNA genomics was more sensitive than ctDNA in detecting ERBB2amp in MBC patients (16%, and 2%, respectively). A mean variant allele frequency (VAF) of &lt; 25%, which is the threshold for detecting a two-fold ERBB2 amplification by ctDNA, was present in 20% and 80% of ERBB2amp MBC detected by the ctDNA and ctcDNA platform, respectively, suggesting that the ctcDNA platform can identify the majority of ERBB2amp among patients with a low ctDNA fraction. HER2exp or ERexp expression by ctcIF were detected in 67% and 67% of MBC patients, respectively. A liquid biopsy classification of HER2 status by combining the three platforms (ctcIF, ctcDNA, and ctDNA) identified that in the tested population, 16% were ERBB2amp, 52% were HER2 expressing (HER2exp and ERBB2nonamp), and 27% were HER2neg (HER2- and ERBB2nonamp). To identify MBC patients that may potentially change treatment as a result of the test we identified a small cohort of MBC patients (n=46) with HER2- disease and tissue biopsy IHC=0, 74% of these patients had HER2exp CTC consistent with HER2 low expression which may aid therapeutic decisions in later line MBC. Conclusions: Here we report a comprehensive liquid biopsy profile combining ctcIF, ctcDNA, and ctDNA platforms with high sensitivity and specificity in determining clinically actionable HER2 and ER biomarker status that may impact therapeutic decision-making in MBC patients.

The clinical value of homologous recombination deficiency score in prostate cancer.

e17011 Background: Aberrations in homologous recombination repair (HRR) genes serve as biomarkers for guiding the treatment decision of PARP inhibitors in prostate cancer (PCa). Homologous recombination deficiency (HRD) score which directly shows the consequence of the loss of HRR function (genomic scars) may be a more reliable biomarker for identifying patients who are sensitive to PARP inhibitors. Methods: This study involved 50 patients with PCa, including mHSPC (n = 37), mCRPC (n = 11), and locally advanced PCa (n = 2). Tumor tissue specimens were collected for targeted next-generation sequencing for 406 cancer-related genes and HRD detection. HRD score was calculated based on over 50,000 single-nucleotide polymorphisms (SNP) distributed across the human genome, incorporating three SNP-based assays: loss of heterozygosity, telomeric allelic imbalance, and large-scale state transition. All the experiments were performed in OrigiMed, a College of American Pathologists accredited and Clinical Laboratory Improvement Amendments certified laboratory. Results: The median HRD score was 34.5 (ranged from 14 to 64). With the median value as cutoff, the mutation rates of HRR were 32% (8/25) and 12% (3/25) in the high and low HRD score groups, respectively. BRCA2 mutations (P = 0.011), AR mutations (P = 0.032), RB1 mutations (P = 0.035) and altered HRR pathway (P = 0.017) were associated with high HRD scores. The mutation frequencies of AR and TP53 were significantly higher in mCRPC compared to mHSPC ( AR: 45.5% [5/11] vs 2.7% [1/37], P &lt; 0.001; TP53: 63.6% [7/11] vs 24.3% [9/37], P = 0.027). A total of 9 patients carried HRR germline mutations (5 BRCA2, 1 BRIP1, 1 RAD50, 1 RAD51D, and 1 ATM). It was found that patients with BRCA2 germline mutations had a higher HRD score (median = 37) than those with other germline mutations ( BRIP1: 25, RAD50: 19, RAD51D: 35, ATM: 36). In the 6 patients who received HRD-related treatments (including cisplatin based chemotherapy or olaparib), 1 of the 2 patients with low HRD score had PSA progression after 251 days (the other one was lost to follow up), while no PSA progression was observed in the 4 patients with high HRD score with a median follow-up of 107 days (range: 40-274 days). A mCRPC case with high HRD score (37) and HRR wild-type who received second line docetaxel+cisplatin even achieved undetectable PSA. Another mHSPC patient with a high HRD score (37) and a BRCA2 germline mutation also had no detectable PSA after first-line docetaxel+cisplatin therapy. Conclusions: To our knowledge, this is the first study in China reporting the association of HRD score and platinum-based treatments in PCa. Our results supported the possibility that HRD score could be predictive for the efficacy of platinum-based chemotherapy.

Correlation of homologous recombination deficiency (HRD) score with response to the first-line treatment of immune checkpoint inhibitors plus chemotherapy in non-small cell lung cancer.

e21121 Background: Homologous recombination (HR) gene mutations were reported to be associated with efficiency of immune checkpoint inhibitors (ICIs). However, up to date, no recognized standard has been set up for definition of HR mutation (+), which limited its clinical application. Homologous recombination deficiency (HRD) score reflects genomic scar induced by HR genes alteration and disfunction. Thus, it may be worth to explore whether HRD score could predict the efficacy of ICIs-based therapy. Methods: We collected tumor tissues from metastatic non-small cell lung cancer (NSCLC) patients who received first-line treatment of ICIs combined with platinum-based chemotherapy. HRD score testing were carried by BGI genomic scar analysis kit, which calculates the sum of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), large-scale state transitions (LST) scores, and corrected by ploidy of tumor samples, through next generation sequencing(NGS) data. The association of HRD score and response to ICIs plus chemotherapy were analyzed. Results: 22 EGFR/ALK wild-type metastatic NSCLC patients were included. The average HRD score was 24.57, varying from -26.37 to 92.34. Threshold traversal was performed based on analysis of the progression free survival (PFS) data from the included NSCLC patients. HRD score 31 or more were define as HRD (+). Kaplan-Meier PFS Survival analysis showed prolonged median PFS (mPFS) in HRD (+) versus HRD (-) NSCLC patients (N/A vs. 7.0 ms, Log-rank P = 0.029; HR 0.20, 95%CI 0.04-0.96, likelihood-ratio P = 0.03). The objective response rate (ORR) was 45.5% (5/11) in HRD(+) versus 27.2% (3/11) in HRD(-) patients. In patients with PD-L1 TPS≥1% (22C3), the mPFS was N/A vs. N/A, and the ORR was 75.0% (3/4) vs. 37.5% (3/8), in HRD (+) vs. HRD(-) patients. In patients with PD-L1 TPS &lt; 1% (22C3), the mPFS was 8.5 ms vs.2.0 ms (Log-rank P = 0.0085; HR 0.08, 95%CI 0.01-0.82, likelihood-ratio P = 0.02), and the ORR were 28.6% (2/7) vs. 0%(0/3), in HRD (+) vs. HRD(-) patients. Conclusions: In addition to PD-L1 expression level, HRD score would be a potential promising biomarker for predicting the efficiency of ICIs plus platinum-based chemotherapy in NSCLC patients.