Deficiency Signature Research Articles

Frequency and impact of a genome-wide homologous recombination deficiency signature (HRDsig+) on the genomic landscape of advanced colorectal cancer (CRC).

238 Background: There have been significant advancements in novel therapies that target the DNA damage response pathway, and utilizing scar-based measures of homologous recombination deficiency (HRD) beyond genomic alterations (GA) in BRCA1/2 may provide opportunities for additional biomarker-driven therapies for patients with advanced CRC. Methods: We identified 47,028 cases of advanced CRC for comprehensive genomic profiling (CGP) to examine all classes of GA and measure HRDsig+ status. HRDsig status was calculated using a broad set of genome-wide copy number features (PMID 37769224) and was correlated with microsatellite status, tumor mutation burden (TMB), genomic ancestry, trinucleotide mutational signatures, and PD-L1 IHC using the Dako 22C3 tumor proportional score (TPS) system. Categorical variables were compared using the Fisher exact test with the Benjamini-Hochberg adjustment to correct for false discovery rate. Results: A total of 796 (1.7%) advanced CRC tumors were HRDsig+ with a median age of 62 years for HRDsig+ and 63 years for HRDsig-. The HRDsig- cohort was more likely to be male compared to HRDsig+ (56.1% vs 50.4%; P=.004). Median GA per tumor was similar (range 5-6) in both groups. Genomic ancestry revealed significantly more African ancestry in the HRDsig- group (12.6% vs 8.8%; P=.002) and more European ancestry in the HRDsig+ group (75.8% vs 71.6%; P=.02). MSI-high status was more frequent in HRDsig- group as compared to the HRDsig+ group (5.5% vs 1.6%; P<.0001). Median TMB was higher in the HRDsig+ cohort (5.0 vs 3.6; P<.0001), as was frequency of TMB ≥ 10 mutations/Mb (16.2% vs 8.5%; P<.0001). Trinucleotide mutational signature distribution was similar in both cohorts with the exception that a tobacco signature was more frequent in the HRDsig+ group (1.5% vs 0.2%; P<.0001). Both PD-L1 low (1-49% TPS) expression (19.5% vs 13.1%; P=.005) and PD-L1 high (≥50% TPS) expression (4.4% vs 1.5%, P=.0021) were higher in HRDsig+ cases. GA in genes associated with HRD were BRCA1 (13.6% vs 1.1%; P<.0001), BRCA2 (8.4% vs 2.8%; P<.0001), ATM (8.0% vs 5.3%; P=.0003), and RAD21 (6.7% vs 3.2%; P<.0001), which were more frequent in the HRDsig+ group. In the HRDsig- group, 83.7%/88.8% of BRCA1 / BRCA2 mutated CRC were not bi-allelic, likely non-driver GA. ERBB2 GA were similar in both groups (5.3% vs 4.4%; P=0.31). KRAS GA were more frequent in the HRDsig+ group (49.1% vs 31.7%; P<.0001). Conclusions: This analysis is the largest assessment of HRD status in advanced CRC. HRDsig+ was 1.7% and is associated with co-occurring actionable GA compared to HRDsig- tumors and higher levels of PD-L1 TPS, which may provide opportunities to target these tumors with immunostimulatory therapies. Given the advances in novel DNA repair targeted therapies these results may provide insight for CRC clinical development for DNA repair combinations.

A study of the homologous recombination deficiency signature (HRDSig) status of advanced esophageal squamous cell carcinoma (AESCC) by utilizing comprehensive genomic profiling (CGP).

485 Background: The role of poly-ADP-ribose polymerase (PARP) inhibitors is currently under investigation as a potential therapeutic option for AESCC. Homologous recombination deficiency signature status (HRDSig) positivity has been shown to predict biallelic loss of BRCA1/2 which may simultaneously portend a worse prognosis and a sensitivity to poly-ADP-ribose polymerase (PARP) inhibitors. In this study, we attempt to characterize the genomic alterations present in AESCC based on HRDSig status utilizing comprehensive genomic profiling (CGP) techniques. Methods: 2,029 cases of AESCC underwent comprehensive genomic profiling with an examination of all genomic alterations (GA). MSI high status, tumor mutation burden (TMB) levels, genomic ancestry and genomic trinucleotide signatures were determined from the sequencing data. HRDSig status was calculated using a broad set of genome-wide copy number features (PMID 37769224). Results were compared using the Fisher exact system with the Benjamini-Hochberg adjustment to correct for false discovery. Results: 154 (7.6%) of the 2029 AESCC cases featured a positive HRDSig status (HRDSig+). The age (66-67) and gender (58%-63% male) distribution were similar in the HRDSig+ and HRDSig- AECSS cases as was the frequency of GA/tumor (9 for both). The median TMB was higher in the HRDSig+ (6.3 vs 3.8; P<.0001) as was frequency of TMB > 10 mutations/Mb (19.5% vs 10.0%; P=.004). An APOBEC trinucleotide signature was also more frequent in the HRDSig+ AESCC (11.0% vs 5.4%; P=.05). As anticipated, GA in genes associated with HRD including BRCA1 (10.4% vs 1.4%; P<.0001) and BRCA2 (14.3% vs 2.0%; P<.0001) was present. MTOR pathway activating mutations were also more frequent in the HRDSig+ AESCC group including PTEN (13.0% vs 7.3%; P=.06). Conclusions: With a 7.6% frequency, HRDSig+ status is a relatively rare event in AESCC. CGP with determination of HRDSig status in AESCC may prove useful in tailoring PARP inhibitor-based treatment regimens and may potentially uncover other genomic alterations that can aid in designing targeted therapy combinations in future. Characteristics of comprehensive genomic profiling of clinically advanced esophageal squamous cell carcinoma by HRDSig status. AESCC HRDSig- (n=1875) AESCC HRDSig+ (n=154) P-value Pathogenic genomic alterations BRCA1 1.4% (26) 10.4% (16) 0.00 BRCA2 2.0% (36) 14.3% (22) 0.00 PTEN 7.3% (137) 13.0% (20) 0.06 COSMIC trinucleotide signature APOBEC 5.4% (101) 11.0% (17) 0.05 Tumor mutational burden (TMB) Median TMB (range) (IQR) 3.8 (0-85) (2.5-6.3) 6.3 (0-61) (3.8-8.8) 0.00 TMB≥10 mut/Mb 10.0% (188) 19.5% (30) 0.00

Landscape of MET genomic alterations in colorectal cancer (CRC).

247 Background: MET signaling is implicated in the tumorigenesis of many solid tumors, including CRC. MET-targeted therapies are approved in non-small cell lung cancer with novel agents in clinical trials for tumors with MET exon 14 splice site mutations, MET amplifications, and recently MET expression. There is emerging therapeutic interest in targeting MET in CRC. Methods: Tumor samples from 50,500 cases of clinically advanced CRC were analyzed by hybrid capture-based comprehensive genomic profiling (CGP) that evaluated all classes of genomic alterations (GA). MSI-high status, tumor mutational burden (TMB), genomic ancestry, trinucleotide mutational signatures, and homologous recombination deficiency signature (HRDsig) were assessed for patients with activating MET GA. PD-L1 expression was determined by IHC (Dako 22C3 with TPS scoring system). Results were compared using the Fisher exact test with the Benjamini-Hochberg adjustment. Results: A total of 502 (1.0%) cases of activating GA of MET (METmut) were identified, including 418 cases (0.83%) with MET amplification (METamp), 10 (<0.1%) rearrangements, and 78 (0.1%) short variant (SV) mutations with 19 (<0.1%) cases of MET exon 14 splice site mutations. When compared with MET wild-type (METwt) CRC, the METmut CRC were of similar age (median 62 years), and numerically more likely to be male (61.4% vs 56.0%; P=.05). There were no differences in genomic ancestry (72% European ancestry for both groups). The METmut CRC featured more cases with a POLE signature (3.6% vs 0.4%; P=<0.0001) and more GA per tumor (7 vs 5; P<0.0001). MSI-high status was similar in both groups, with 4.2% in METmut and 6.0% in METwt (P=0.28). TMB levels were numerically higher in the METmut cases (TMB ≥ 10 mutations/Mb at 12.2% vs 9.0%; P=0.058). Low level PD-L1 expression (1-49% TPS) was similar in both groups (13.9% in METmut vs 13.3% in METwt), and while not statistically significant, high level PD-L1 expression (>50% TPS) was higher in the METmut group (4.3% vs 1.6%; P=0.056). The frequencies of HRDsig+ CRC were rare and similar in both groups with 1.5% in METmut and 1.7% in METwt. METmut CRC had lower frequencies of GA in KRAS (34.7% vs 48.8%; P<0.0001), NRAS (1.8% vs 4.2%; P=0.011), APC (72.5% vs 78.2%; P=0.009) and PIK3CA (12.2% vs 18.9%; P=0.0004), and higher frequencies of GA in CDK6 (10.2% vs 0.6%; P<0.0001), EGFR (5.2% vs 0.2%; P=0.0002), ERBB2 (9.6% vs 5.2%; P=0.0004), HGF (8.0% vs 0.8%; P<0.0001), POLE (4.2% vs 0.5%; P=0.005) and TP53 (84.1% vs 75.9%; P<0.0001). In METmut CRC, ERBB2 alterations were 4.1% amplifications, 2.9% sequence mutations, 0.9% with more than one mutation. BRAF V600E was seen in 5.9% in METmut CRC and 8.4% of METwt. Conclusions: CGP reveals significant differences in the genomic landscapes of METmut CRC and METwt CRC, which may guide selection of potential rational drug combinations with novel MET-targeted therapies.

Whole Genome Profiling of Primary and Metastatic Adrenocortical Carcinoma Unravels Significant Molecular Events

Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with limited treatment options and poor prognosis, with a 5-year survival rate of about 15 %. This study used whole genome sequencing to characterize the genomic landscape of five patients, one of them with both primary and metastatic samples. Key driver mutations were detected, including APC, JAK1, RFWD3 as well as other genes. Notably, a primary tumor harbored a RAD51 biallelic deleterious translocation, associated with homologous recombination deficiency signature. Large-scale copy neutral loss of heterozygosity (LOH) was identified in four tumors, three had TP53 mutations, with structural variants impacting genes as RB1, CDKN2A, and NF1. A genomic signature specific to mismatch repair was observed in a sample with MHS6 mutation. Two tumors presented novel fusions at TERT locus, including TERT::ZNF521. Comparative analysis between conventional and oncocytic ACC subtypes revealed no significant differences in mutation load, microsatellite instability, or specific gene enrichment. This comprehensive WGS analysis broadens the spectrum of genomic alterations in ACC, highlighting potential molecular targets and differences across subtypes that may inform future therapeutic strategies.

Single-Hit and Multi-hit PIK3CA Short Variant Genomic Alterations in Clinically Advanced Prostate Cancer: A Genomic Landscape Study.

Tumors harboring two or more PIK3CA short variant (SV) ("multi-hit") mutations have been linked to improved outcomes with anti-PIK3CA-targeted therapies in breast cancer. The landscape and clinical implications of multi-hit PIK3CA alterations in clinically advanced prostate cancer (CAPC) remains elusive. To evaluate the genomic landscape of single-hit and multi-hit PIK3CA genomic alterations in CAPC. The Foundation Medicine FoundationCore database was used to identify 19,978 CAPC tumors that underwent hybrid capture-based comprehensive genomic profiling to evaluate all classes of genomic alterations (GA) and determine tumor mutational burden (TMB), microsatellite instability (MSI), genomic ancestry, single-base substitution mutational signatures, and homologous recombination deficiency signature (HRDsig). Tumor cell PD-L1 expression was determined by IHC (Dako 22C3). 18,741 (93.8%) tumors were PIK3CA wild type (WT), 1155 (5.8%) featured single PIK3CA SV, and 82 (0.4%) featured multi-hit PIK3CA SVs. Single-hit (6.6 versus 3.8; p < 0.0001) and multi-hit (12.8 versus 3.8; p < 0.0001) featured more driver GA per tumor than PIK3CA WT CAPC, as well as higher prevalence of MMR mutational signature, MSI high status, and TMB levels versus PIK3CA WT (p < 0.0001). Other differences in GA included higher frequencies of GA in BRCA2 in multi-hit versus WT (18.3% versus 8.5%; p = 0.0191), ATM in multi-hit versus WT (13.4% versus 5.6%; p = 0.02) and PTEN in single-hit versus WT (40.2% versus 30.1%; p < 0.0001). Homologous recombination deficiency signatures were higher in PIK3CA WT versus single-hit (11.2% versus 7.6%; p = 0.0002). There were no significant differences in PD-L1 expression among the three groups. Identification of multi-hit PIK3CA GA in CAPC highlights a potentially unique phenotype that may be associated with response to anti-PIK3CA targeted therapy and checkpoint inhibition, supporting relevant clinical trial designs.

The coexistence of a BRCA2 germline and a DICER1 somatic variant in two first-degree cousins suggests their potential synergic effect

Cancer predisposition syndromes are recognized in about 10% of pediatric malignancies with several genes specifically involved in a subset of pediatric tumors such as DICER1, in pleuropulmonary blastoma, cystic nephroma, and brain sarcomas. By contrast, the role of BRCA1/2 in pediatric cancer predisposition is still under investigation. We present two cases of young first-degree cousins, both carrying a germline BRCA2 variant and developing tumors characterized by somatic DICER1 mutations. Patient 1 presented with a cystic nephroma harboring a somatic DICER1 variant (p.Asp1810Tyr), while patient 2 had a primary intracranial DICER1-mutated sarcoma showing a distinct somatic DICER1 variant (p.Asp1709Glu) as well as biallelic inactivation of TP53 (p.Val173Leu, VAF 91%) and APC (p.Ile1307Lys, VAF 95%) and a pathogenic variant in KRAS (p.Gln61His). Both patients carried the same germline BRCA2 variant (p.Arg2842Cys) of unknown significance. The same variant was found in the mother of patient 2 and in the father of patient 1, who are siblings. A homologous recombination deficiency signature was not identified in any of the two tumors, possibly suggesting a reduction of BRCA2 activity. The association of BRCA2 and DICER1 variants in our cases hints at a potential cooperative role in cancer pathogenesis. Further studies are warranted to elucidate the interplay between BRCA1/2 and DICER1 variants and their implications for cancer predisposition and treatment in pediatric patients.

Triple-Negative Breast Cancer: Molecular Particularities Still a Challenge.

Worldwide, breast cancer (BC) is one of the most common cancers in women and is responsible for the highest number of cancer-related deaths among women, with a special clinical behavior and therapy response. Triple-negative breast cancer (TNBC) is seen as a highly invasive BC, characterized by a short survival, higher mortality, recurrence, and metastasis when it is compared to the other BC subtypes. The molecular subtyping of TNBC based on mRNA expression levels does not accurately reflect protein expression levels, which impacts targeted therapy effectiveness and prognostic predictions. Most TNBC cases exhibit a high frequency of homologous recombination (HR) DNA repair deficiency (HRD) signatures and are associated with a complex genomic profile. Biomarker research in TNBC includes investigating genetic mutations, gene expression patterns, immune system-related markers, and other factors that can provide valuable information for diagnosis, treatment selection, and patient outcomes. Additionally, these biomarkers are often crucial in the development of personalized and precision medicine approaches, where treatments are customized to each patient's unique characteristics. This ongoing research is essential for improving the management and outcomes of TNBC, which is a challenging and heterogeneous form of breast cancer. The findings of this research have practical implications for refining treatment strategies, particularly in selecting appropriate systemic therapies and integrating traditional treatment modalities like surgery and radiotherapy into comprehensive care plans for TNBC patients.

Effectiveness of PARP Inhibitor Maintenance Therapy in Ovarian Cancer by BRCA1/2 and a Scar-Based HRD Signature in Real-World Practice.

The purpose of the study was to compare the effectiveness of PARP inhibitor maintenance therapy (mPARPi) in real-world practice by biomarker status [BRCA1/2 alterations (BRCAalt) and a homologous recombination deficiency signature (HRDsig)] in advanced ovarian cancer. Patients with ovarian cancer receiving first-line platinum-based chemotherapy and either mPARPi or no maintenance were included. Patient data were obtained by a US-based de-identified ovarian cancer Clinico-Genomic Database, from ∼280 US cancer clinics (01/2015-03/2023). Real-world progression-free survival (rwPFS) and overall survival (rwOS) were compared by biomarker status using Cox models, weighted by propensity scores. Of 673 patients, 160 received mPARPi [31.2% BRCAalt and 51.9% HRDsig(+)] and 513 no maintenance [15.6% BRCAalt and 34.1% HRDsig(+)]. BRCAalt patients receiving mPARPi versus no maintenance had favorable rwPFS [HR, 0.48; 95% confidence interval (CI), 0.26-0.87; P = 0.0154], as did BRCA wild-type (WT; HR, 0.76; 95% CI, 0.57-1.01; P = 0.0595). Favorable rwOS was not observed with mPARPi for BRCAalt or BRCA-WT. HRDsig(+) patients receiving mPARPi versus no maintenance had favorable rwPFS (HR, 0.36; 95% CI, 0.24-0.55; P < 0.001) and numerically favorable rwOS (HR, 0.46; 95% CI, 0.21-1.02; P = 0.0561). No differences were observed for HRDsig(-). mPARPi treatment interaction was observed for HRDsig(+) versus HRDsig(-) (rwPFS P < 0.001/rwOS P = 0.016) but not for BRCAalt versus BRCA-WT. Patients with BRCA-WT/HRDsig(+) receiving mPARPi had favorable rwPFS (HR, 0.40; 95% CI, 0.22-0.72; P = 0.003), whereas no difference was observed for BRCA-WT/HRDsig(-). HRDsig predicted benefit of mPARPi better than BRCAalt. Patients with HRDsig(+) status experienced favorable outcomes, even if they had BRCA-WT status. In contrast, patients with HRDsig(-) status did not show significant benefit from mPARPi treatment. HRDsig might predict benefit from mPARPi regardless of BRCAalt status.

A prognostic model based on selected cell state and cellular community scores in patients with advanced melanoma treated with immune checkpoint inhibitors (Ecotype-ICI score) as a predictor of ICI immunotherapeutic benefits.

9568 Background: We conducted an enhanced immune cell state atlas analysis of the data of patients with melanoma treated with ICI to investigate predictors of therapeutic benefits and mechanisms of resistance. Methods: Leveraging RNA-seq from melanoma patients (n=141) treated with ICI within the ORIEN Avatar project (NCT03977402), we constructed a prognostic model based on selected cell state and cellular community scores, also known as ecotype-ICI score ( Li. AACR 2023). We evaluated the model’s predictive value using transcriptomic data from high-risk melanoma patients treated with adjuvant ipilimumab (n=471) or interferon-α (n=248) as part of the E1609 phase 3 trial (1). RNA-seq data were initially deconvoluted for cellular community signatures using EcoTyper. The six prognostic carcinoma ecotype (CE) signatures identified (CE1, CE2, C6, CE7, CD9, CD10) were utilized for calculating a risk score based on a multivariable Cox model, which was trained using Avatar data. For additional external validation, we analyzed data from patients with metastatic melanoma treated with anti-CTLA4 ( Va. n=40), anti-PD1 ( Liu. n=121), and anti-PD1 alone or combined with anti-CTLA4 ( Gide. n=66). A series of ad hoc survival analyses, including Kaplan-Meier analysis, log-rank tests, and Cox regression were further applied. Results: The ecotype-ICI score showed strong prognostic significance in predicting overall survival (OS) in the entire group of E1609 patients (N=719; Cox P &lt; 0.0001, log-rank P &lt; 0.0001), the ipilimumab cohort (Cox P &lt; 0.000143, log-rank P =0.00011), but less so with interferon (Cox P= 0.06). CE9, characterized by its proinflammatory nature and IFN-gamma signaling, and CE10, noted for its canonical T cell state signatures, both demonstrated a leading predictive value according to multivariable Cox regression analysis. Interestingly, CE2, noted for its lymphocyte deficiency signature, also demonstrated complementary predictive value for outcomes following ipilimumab. Within the validation cohorts of public data sets noted above, the ecotype-ICI scores showed similarly strong prognostic significance in predicting OS (Gide. P = 0.045; Liu. P = 0.029; Va. P = 0.011). Ecotype-ICI also demonstrated a distinct distribution in the responder group (complete or partial response) compared to the progressive disease group, underscoring its potential as a discriminative biomarker for ICI response and survival outcomes. Conclusions: Our analysis has successfully established the utility of the immune cell state atlas in predicting therapeutic benefits with ICIs across diverse ICI treatment regimens in melanoma. We will update our results with data related to gene ontology, KEGG pathways and the most complementary pathway signatures at the meeting. 1. Tarhini, 2020.

Genome-wide somatic mutation analysis of sinonasal adenocarcinoma with and without wood dust exposure

BackgroundSinonasal adenocarcinoma is a rare cancer, encompassing two different entities, the intestinal-type sinonasal adenocarcinoma (ITAC) and the non-intestinal-type sinonasal adenocarcinoma (non-ITAC). Occurrence of ITAC is strongly associated with exposure to hardwood dusts. In countries with predominant exposure to softwood dust the occurrence of sinonasal adenocarcinomas is lower and the relative amount of non-ITACs to ITACs is higher. The molecular mechanisms behind the tumorigenic effects of wood dust remain largely unknown.MethodsWe carried out whole-genome sequencing of formalin-fixed paraffin-embedded (FFPE) samples of sinonasal adenocarcinomas from ten wood dust-exposed and six non-exposed individuals, with partial tobacco exposure data. Sequences were analyzed for the presence of mutational signatures matching COSMIC database signatures. Driver mutations and CN variant regions were characterized.ResultsMutation burden was higher in samples of wood dust-exposed patients (p = 0.016). Reactive oxygen species (ROS) damage-related mutational signatures were almost exclusively identified in ITAC subtype samples (p = 0.00055). Tobacco smoke mutational signatures were observed in samples of patients with tobacco exposure or missing information, but not in samples from non-exposed patients. A tetraploidy copy number (CN) signature was enriched in ITAC subtype (p = 0.042). CN variation included recurrent gains in COSMIC Cancer Gene Census genes TERT, SDHA, RAC1, ETV1, PCM1, and MYC. Pathogenic variants were observed most frequently in TP53, NF1, CHD2, BRAF, APC, and LRP1B. Driver mutations and copy number gains did not segregate by subtype.ConclusionsOur analysis identified distinct mutational characteristics in ITAC and non-ITAC. Mutational signature analysis may eventually become useful for documentation of occupation-related cancer, while the exact mechanisms behind wood dust-driven carcinogenesis remain elusive. The presence of homologous recombination deficiency signatures implies a novel opportunity for treatment, but further studies are needed.

Antileukemic potential of methylated indolequinone MAC681 through immunogenic necroptosis and PARP1 degradation

BackgroundDespite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence.MethodsTranscriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice.ResultsTranscriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients’ stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities.ConclusionsOverall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.

Abstract PO4-02-03: Distribution of MammaPrint, BluePrint, and Response Predictive Subtypes based on ImPrint and Reprint in ER+/HER2- Invasive Lobular Carcinoma – A FLEX sub study

Abstract Background: Invasive lobular carcinoma (ILC), the second most common histologic subtype of breast cancer, is increasingly recognized as a distinct tumor type compared to the more common invasive ductal carcinoma (IDC). While ILC is known to have lower rates of pathologic complete response to neoadjuvant chemotherapy, ILC tumors are biologically heterogenous. As such, genomic signatures might identify patients with ILC who might benefit from tailored treatment options. The gene expression signature MammaPrint (MP) classifies tumors as having a Low Risk or High Risk of distant recurrence. MP combined with BluePrint (BP), a molecular subtyping signature, categorizes tumors as Luminal A (MP Low Risk), Luminal B (MP High Risk), Basal or HER-2 type. ImPrint is a 53-gene signature that identifies HR+HER2- patients predicted to benefit from immune checkpoint inhibitors. RePrint is a 60-gene DNA repair deficiency (DRD) signature that identifies HR+HER2- patients who may benefit from PARP inhibitors with platinum agents. Response Predictive Subtypes (RPS), used in the I-SPY2 trial, combine clinical subtype and these genomic signatures to personalize treatment planning and improve outcomes. In this study, we determined the distribution of 3 RPS in HR+HER2-: (1) ImPrint-/RePrint-, (2) ImPrint-/RePrint+ (3) ImPrint+, in patients ILC compared to IDC enrolled in the FLEX trial. Methods: This study includes 1078 HR+HER2-women with ILC and 6078 with IDC enrolled in FLEX registry. FLEX (NCT03053193) is a prospective, observational trial that includes stage I-III breast cancer patients who undergo MammaPrint testing (with or without BluePrint) as standard of care, and consent to full transcriptome and clinical data collection. MP High Risk tumors were further stratified into High 1 and High 2. ImPrint and Reprint results were used to determine RPS. A two-tailed proportional z-test was used to assess differences between ILC and IDC as well as between RPS. Results: ILC patients had a significantly lower percentage of MP High Risk tumors in comparison to IDC. Among MP High Risk tumors, those with ILC had significantly more MP High 1 than patients with IDC. BP subtyping in ILC tumors showed significantly lower percentage of Basal, and Luminal B, and higher percentage of Luminal A compared to IDC tumors. For RPS, a higher percentage of ILC tumors were ImPrint-/RePrint- compared to IDC, whereas lower frequencies of ImPrint-/RePrint+ and ImPrint+ were found. Within the ILC ImPrint+ subgroup (n=14), 1 (7.1%) was classified as High 2 and 10 (71.4%) as High 1 and 3 (21.4%) as Low Risk. All 14 (100%) ImPrint+ patients were BP Luminal. In contrast, within the IDC MP High Risk ImPrint+ subgroup (n=328), 233 (71.0%) were High 2, 88 (26.8%) High 1 and 7 Low Risk (2.1%). Furthermore 193 (60.7%) were classified as BP Basal, 2 HER2 (0.6%) and 123 (38.7%) Luminal within this IDC RPS. Conclusions: This is the first study to investigate the distribution of Response Predictive Subtypes in ILC, which will be beneficial to optimize the treatment selection for patients with early-stage HR+/HER2- ILC. Though the percentage of ImPrint+ is lower in ILC, this study revealed a small subset of patients in ILC with potential response to Immunotherapy. Furthermore, these results underscore the heterogeneity of ILC tumors and generate further hypotheses to investigate the immune cell abundance in ILC compared to IDC and correlate immune cell abundance to ImPrint status. Table: Genomic and Tumor Characteristics of HR+HER2- ILC and IDC tumors Citation Format: Rita Mukhtar, Christina Yau, Denise Wolf, Adam Brufsky, Hannah Linden, Natasha Hunter, Reshma Mahtani, Abirami Sivapiragasam, Trevor Feinstein, Fengting Yan, Ian Grady, Priscilla McAuliffe, Michaela Tsai, Joyce O'Shaughnessy, Blanche Mavromatis, Sasha Davis, Josien Haan, William Audeh, Lavanya Samraj, FLEX Investigators' Group. Distribution of MammaPrint, BluePrint, and Response Predictive Subtypes based on ImPrint and Reprint in ER+/HER2- Invasive Lobular Carcinoma – A FLEX sub study [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 PO4-02-03.

Abstract P30: MUTATIONAL SIGNATURE ASSIGNMENT HETEROGENEITY IS WIDESPREAD AND CAN BE ADDRESSED BY ENSEMBLE APPROACHES

Abstract Single base substitution (SBS) mutational signatures have become standard practice in cancer genomics. In lieu of de novo signature extraction, reference signature assignment allows users to estimate the activities of pre-established SBS signatures within individual malignancies. Several tools have been developed for this purpose, each with differing methodologies. However, due to a lack of standardization, there may be inter-tool variability in signature assignment. We deeply characterized three assignment strategies and five SBS signature assignment tools. We observed that assignment strategy choice can significantly influence results and interpretations. Despite varying recommendations by tools, Refit performed best by reducing overfitting and maximizing reconstruction of the original mutational spectra. Even after uniform application of Refit, tools varied remarkably in signature assignments both qualitatively (Jaccard Index = 0.38 – 0.83) and quantitatively (Kendall tau-b = 0.18 – 0.76). This phenomenon was exacerbated for “flat” signatures such as the homologous-recombination repair deficiency (HRD) signature SBS3. An ensemble approach (EnsembleFit), which leverages output from all five tools, increased SBS3 assignment accuracy in BRCA1/2-deficient breast carcinomas. After generating synthetic mutational profiles for thousands of pan-cancer tumors, EnsembleFit reduced signature activity assignment error 15.9 – 24.7% on average using COSMIC and non-standard reference signature sets. We have also released the EnsembleFit web portal (https://www.ensemblefit.pittlabgenomics.com) for users to generate or download ensemble-based SBS signature assignments using any strategy and combination of tools. Overall, we show that signature assignment heterogeneity across tools and strategies is non-negligible and propose a viable, ensemble solution. Citation Format: Wu Jialun Andy, Akila Perera, Linganesan Kularatnarajah, Anna Korsakova, Jason James Pitt. MUTATIONAL SIGNATURE ASSIGNMENT HETEROGENEITY IS WIDESPREAD AND CAN BE ADDRESSED BY ENSEMBLE APPROACHES [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P30.

Abstract 1746: KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles

Abstract Background: Lung cancer is the 3rd most common cancer in the UK and leading cause of cancer mortality worldwide. Somatic RAS mutations are the most common oncogenes in human cancer, and KRAS mutations are the largest molecular subset of lung adenocarcinoma (LUAD). Each cancer genome accumulates a unique combination of somatic mutations, and these mutational signatures provide a genetic imprint of mutational processes that have occurred during tumorigenesis. Mutational signatures provide information on tumor aetiology/maintenance and could highlight potential therapeutic targets. This is the largest study to date to profile KRAS mutant LUAD mutational signatures using whole genome sequencing (WGS) data. Methods: Paired tumor and germline WGS data were obtained through the 100 000 Genomes Project (Genomics England). KRAS mutations were profiled using Ensembl Variant Effect Predictor (VEP) v109.0. Single base substitution (SBS), doublet base substitution (DBS), small insertion and deletions (ID) and copy number (CN) mutational signature extraction was performed using SigProfiler (COSMIC version 3.3). Statistical analysis and plotting were performed in R; a two-tailed p-value &amp;lt;0.05 was considered statistically significant. Results: 680 LUAD patients were included; 301 KRAS mutant (44%), 379 KRAS wild type (63%). Common KRAS variants were present, including G12C (38.9%), G12V (15.9%) and G12D (14.0%). 17 SBS, 9 DBS, 10 ID and 9 CN signatures were extracted and present in both KRAS mutant and wild type cohorts. KRAS mutant samples were enriched in smoking related signatures (SBS4, DBS2, ID3; odds (OR) ratio 5.54, 3.80, 4.97 respectively, p &amp;lt;0.001) and diploid CN signature (CN1, OR 1.41, p = 0.03). Conversely, KRAS mutant samples were less likely to have clocklike (ID5, OR 0.61, p = 0.012; ID8, OR 0.44, p &amp;lt;0.001), APOBEC (SBS2, OR 0.49, p &amp;lt;0.001; SBS13; 0.50, p &amp;lt;0.001), chromosomal instability (CN9, OR 0.63, p = 0.019), loss of heterozygosity (CN14, OR 0.47, p = 0.015), or homologous recombination deficiency (CN17, OR 0.58, p &amp;lt;0.001) signatures. Discussion: These results show distinct mutational signature profiles between KRAS mutant and wild type LUAD. As previously described, KRAS mutant LUAD is dominated by smoking related signatures; however, the majority of remaining signatures were more common in the KRAS wild type cohort where there was a statistically significant difference. This data suggests different mutational processes are observed in KRAS mutant compared to wild type LUAD. KRAS mutations are commonly clonal and thought to initiate tumorigenesis: timing signatures relative to KRAS mutations is necessary to further our understanding of KRAS cancer evolution and the subject of future work. Combined with clinical data integration, mutational signatures could provide prognostic information and inform predictive models. Citation Format: Laura Woodhouse, Aliah Hawari, Andreas Gruber, Colin Lindsay, David Wedge. KRAS mutant lung adenocarcinoma is associated with distinct mutational signature profiles [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 1746.

Abstract 3887: The origins and clinical impact of extrachromosomal DNA across 39 cancers

Abstract Extrachromosomal DNA (ecDNA) is emerging as a major contributor to cancer drug resistance and poor outcome in many cancer types. To understand the underlying genetic and mutational processes associated with ecDNA formation and presence, we conducted an analysis of whole-genome sequencing data from 14,778 cancer patients representing 39 tumor types from the Genomics England cohort. Our findings revealed focal amplifications driven by ecDNA in 17.1% of tumor samples, exhibiting varying frequencies across tissues. Notably, liposarcomas (55.4%) and HER2+ breast cancers (46.4%) displayed higher rates compared to lower rates found in pancreatic adenocarcinomas (2%). In addition to oncogenic amplifications such as HER2 derived from ecDNA during cancer evolution, ecDNAs that did not contain oncogenes were enriched for immunomodulatory genes (e.g., CXCL6, LRRC32, and HLA-G). Immunomodulatory ecDNAs exhibited reduced T cell infiltration. In the context of intrinsic and environmental mutational processes, deamination clock-like, tobacco smoking, and APOBEC cytidine deaminase signatures positively correlated with ecDNA presence. Conversely, mismatch repair deficiency (MMRd) and POLD1/POLE deficiency signatures exhibited a negative correlation. Mutational processes associated with tobacco exposure, UV light, and spontaneous deamination primarily occurred before ecDNA formation, while signatures of homologous recombination repair deficiency tended to manifest post-ecDNA formation.Notably, ecDNA was enriched at metastatic sites and significantly associated with shorter overall survival in multivariable analysis (HR 1.31, 95% CI 1.17-1.47), underscoring its clinical relevance. This study enhances our understanding of the mutational processes and tissue contexts that shape the ecDNA landscape in human cancer and highlights its implications for clinical outcomes. Citation Format: Chris Bailey, Oriol Pich, Kerstin Thol, Jens Luebeck, Georgia Stavrou, Thomas B. Watkins, Natasha E. Weiser, Wei-Ting Lu, Jeanette Kittel, Robert Bentham, Natasha Sharma, Roberto Salgado, Dave Moore, Maria Litovchenko, King L. Hung, Alex J. Cornish, Bhargavi Dameracharla, Serena Nik-Zainal, Vineet Bafna, Howard Chang, Nicholas McGranahan, Nnennaya Kanu, Genomics England Consortium, Paul S. Mischel, Mariam Jamal-Hanjani, Charles Swanton. The origins and clinical impact of extrachromosomal DNA across 39 cancers [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 3887.

Natural History of Germline BRCA1 Mutated and BRCA Wild-type Triple-negative Breast Cancer.

In germline BRCA1 mutated and BRCA wild-type patients, TNBC shows a branching evolutionary pattern of mutations with a single founding clone, are polyclonal throughout their disease course, and have widespread copy-number aberrations. This evolutionary pattern may be associated with treatment resistance or sensitivity and could be therapeutically exploited.

Real-world effectiveness of PARP inhibitors (PARPi) in metastatic castration-resistant prostate cancer (mCRPC) by genomic homologous recombination repair (HRR) alterations and homologous recombination deficiency signature (HRDsig).

186 Background: Multiple randomized controlled trials have demonstrated efficacy of poly ADP-ribose polymerase (PARP) inhibitors in patients with metastatic castration resistant prostate cancer (mCRPC) harboring deleterious BRCA1 or BRCA2 alterations (BRCAalt). Alterations in other DNA HRR gene pathways are common and the efficacy of PARPi in these populations is less clear. We sought to evaluate the real-world effectiveness of PARPi in commonly defined HRR groups and explore clinical validity of HRDsig to additionally predict outcomes on PARPi. Methods: This study used the nationwide (US-based) de-identified Flatiron Health-Foundation Medicine Prostate Cancer clinico-genomic database (FH-FMI CGDB), originating from approximately 280 US cancer clinics (~800 sites of care). Patients with mCRPC and tumor genomic profiling (tissue or liquid) who underwent single agent PARPi treatment were included, grouped by biomarkers with deleterious alterations detected: BRCA, ATM, other HRR ( ATR, ATRX, BAP1, BARD1, BRIP1, CHEK1/2, CDK12, FANCA, FANCL, MRE11, RAD51B/C/D, RAD54L, PALB2), or no HRR. Separately, patients with mCRPC and tumor tissue profiling were grouped by HRDsig status (+ vs -) using a machine learning- based algorithm built using a diverse set of copy number features. Kaplan Meier estimates and multivariable Cox PH models assessed time to next therapy (TTNT), time to treatment discontinuation (TTD), and real-world overall survival (rwOS). Results: 313 patients with mCRPC met inclusion criteria, of which 162 had tissue specimens for genomics evaluations and 151 had liquid profiling. In the tissue cohort, BRCAalt had more favorable TTNT (median 10.0 months, 95% CI 8.0 – 14.1) relative to ATM (median 3.8 months, 95% CI 2.7 – 6.9), other HRR (median 4.9 months, 95% CI 3.6 – 7.2) and no HRR (median 4.9, 95% CI 3.6 – 7.2 months). Other proxies for drug effectiveness (TTD, PSA kinetics) and rwOS had consistent results. More granular mutational analyses and liquid profiling data will be presented at the conference. Among evaluable patients without BRCAalt on tissue biopsy receiving PARPi, 5 of 75 (7%) were HRDsig(+) and had directionally more favorable (vs. HRDsig(-)) TTNT (HR: 0.39, 95%CI 0.14 – 1.12), TTD (0.46, 95%CI 0.16 – 1.31) and rwOS (HR: 0.27, 95%CI 0.07 – 1.14). Conclusions: We observe no significant outcomes difference between non- BRCAalt groups (defined as ATM, other HRR, and no HRR) in this cohort with respect to proxies of drug effectiveness. These results are largely consistent with biomarker-defined subgroup analyses from completed clinical trials. However, HRDsig may be able to identify a small non-BRCAalt subgroup with enhanced benefit. Associations of BRCAalt(-)/HRDsig(+) and PARPi performance deserve further attention in additional cohorts.

Abstract A021: Pre and post treatment transcriptomic analysis provide new insights on the mechanisms underlying the efficacy of PARPi and Androgen blockage in prostate cancer

Abstract Aim: Recent phase 3 trials (PROPEL, TALAPRO2) have demonstrated survival benefits when combining PARP inhibitors (PARPi) with androgen pathway inhibitors (API) in prostate cancer (PC) treatment. Importantly, this benefit is observed regardless of homologous repair deficiency (HRD) status, suggesting alternative underlying mechanisms beyond synthetic lethality. However, the complete identification of all the PC patients who can benefit from this combination therapy remains an unmet need. Therefore, further investigation into the distinct molecular pathways involved and identification of biomarkers of response is critical and is the aim of this study. Methods: We have performed an exploratory transcriptomic analysis including RNA-seq, differential expression analysis and gene set enrichment analyses (GSEA) of pre and post treatment biopsies of localized PC patients (pts) with higher risk of relapse and that were included in a window of opportunity (WoO) study. We have included pts recruited to the CANCAP-3 study that were exposed to either two weeks of olaparib alone or in combination with degarelix (1:1 randomization). A corresponding transcriptomic analysis of pre and post enzalutamide biopsies of patients recruited to the WoO study DARANA was also included. Results: GSEA results have shown p53 hallmark upregulation more pronounced in the combination cohort. In the combination cohort the androgen response genes were downregulated. Additionally, canonical cell cycle progression hallmarks (E2F and G2M checkpoint) were suppressed with a significant downregulation of the Prolaris signature. In the olaparib monotherapy cohort pts with the greatest PSA decline demonstrated large reductions in the Prolaris score. Furthermore, in the olaparib monotherapy cohort the two patients that relapsed within 4 years of follow up demonstrated greater Prolaris scores. Interestingly, a significant positive correlation between HR deficiency signatures and reduced prostate cancer proliferation index was observed across the study cohorts, although there was only minimal overlap in the genes constituting these two signatures. This correlation was maintained excluding HRD pts (n=2). Finally, this relationship was also observed following analysis of the DARANA trial. Conclusions: Tissue transcriptomic data analysis of PC pts can provide further insights on the possible biological mechanisms that underlie the effect of this drug combination. In this study the expression of HRD signature appeared to be in direct proportion to the reduction in cellular proliferation (Prolaris). This was seen in the combination treatment cohort, as well as in a separate API monotherapy study (DARANA). This suggests that androgen inhibition is the main driver for the reduction in HR signature. However, this phenomenon may represent a consequence of cell cycle arrest occurring more readily in cells with the greatest HR defect. Further studies are needed to further explain the unique upstream processes that contribute to an AR-supported DNA damage response, in the context of PARP inhibition. Citation Format: Ana Filipa Palma dos Reis, Toby Milne-Clark, Amit Dipak Amin, Liliya Nazlamova, Simon Pacey, Harveer Dev. Pre and post treatment transcriptomic analysis provide new insights on the mechanisms underlying the efficacy of PARPi and Androgen blockage in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A021.

Abstract C012: Integration of social variables resolves subclasses of tumor microenvironment

Abstract Intro: Recent work has identified subclasses of the tumor microenvironment, with most work emphasizing a robust adaptive immune response associated with ER-negative and/or basal-like tumors. Since adaptive immune responses may influence response to chemotherapy and may be targetable by immunotherapy, understanding population-level heterogeneity in immune response is important. Methods: We leveraged the Carolina Breast Cancer Study (CBCS), a large population-based study that oversampled young (≤50 years) and Black women with invasive breast cancer (BC). We performed NanoString gene expression profiling on tissue from 1,329 BC patients, including 704 (53%) Black and 625 (47%) non-Black women, and assayed genes representative of at least 10 individual immune cell types from both adaptive and innate arms of the immune system, homologous recombination deficiency signature and TP53 mutation status. Using a latent class approach that included variables for self-reported race, triple negative status, individual measures of socioeconomic status and community-level measures of advantage and disadvantage, we identified latent classes that integrated tumor microenvironment with tumor biology, individual and community level exposures under a Cells-to-Society conceptual model of breast cancer outcomes. Results: Latent Class Analysis identified 6 integrated classes of the tumor microenvironment, further subdividing a previously identified and validated adaptive immune class into two subgroups. The two adaptive classes were distinguished by different expression levels of B-cells, CD8+ T cells, cytotoxic cells and PD-L1, and differing frequency of basal-like subtype (Adaptive Class 1 RFD [95% CI]: 19.0 [11.9, 26.2]; Adaptive Class 2 RFD [95% CI]: 32.8 [23.3,42.3]) and presence of genomic instability gene expression subtypes (Adaptive Class-1 RFD [95% CI]: 22.1 [14.0, 30.1]; Adaptive Class-2 RFD [95% CI]: 37.5 [28.1,46.2]) relative to the immune-quiet tumors. Discussion: Social variables have complex effects on tumor biology and microenvironment, but latent class models and other model-based approaches may help to integrate biological data with other social factors to identify susceptible subgroups and to optimize precision medicine in diverse populations. Citation Format: Alina M. Hamilton, Kara McCormack, Marc A. Emerson, Melissa A. Troester, Terry Hyslop. Integration of social variables resolves subclasses of tumor microenvironment [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C012.

A BRCA2 germline mutation and high expression of immune checkpoints in a TNBC patient

Triple-negative breast cancer (TNBC) is the most aggressive subtype of mammary carcinoma. Here, we describe a case of an 81-year-old female diagnosed with ductal triple negative breast cancer with a germline pathogenic variant in BReast CAncer gene2 (BRCA2). Genetic testing also revealed the presence of four somatic mutations in the ephrin type-A receptor 3 (EphA3), TP53, BRCA1-associated protein (BAP1), and MYB genes. The BRCA2, TP53, and BAP1 gene mutations are highly predictive of a defective homologous recombination repair system and subsequent chromosomal instability in this patient. Coherently, the patient displayed a strong homologous recombination deficiency signature and high tumor mutational burden status, which are generally associated with increased probability of immune neoantigens formation and presentation, and with tumor immunogenicity. Analysis of immune checkpoint revealed high expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), suggesting that the patient might likely benefit from immunotherapies. Altogether, these findings support an unveiled link between BRCA2 inactivation, HR deficiency and increased expression of immune checkpoints in TNBC. This clinical case highlights the importance of screening TNBC patients for genetic mutations and TMB biomarkers in order to predict the potential efficacy of immunotherapy.