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Adjuvant therapy; Breast cancer; Olaparib Terapia adyuvante; Cáncer de mama; Olaparib Teràpia adjuvant; Càncer de mama; Olaparib Background The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. Patients and methods One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. Results With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI −0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. Conclusion With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals. Funding for this work, which was conducted as a collaborative partnership among the Breast International Group, NRG Oncology, Frontier Science, AstraZeneca, and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, U.S.A. (MSD), was provided by the National Institutes of Health (grant numbers: U10CA 180868, UG1CA 189867, and U10CA 180822) and by AstraZeneca as part of an alliance between AstraZeneca and MSD. Provision of olaparib and placebo was from AstraZeneca.

Purpose: To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)–resistant triple-negative breast cancer (TNBC) and ER+HER2– breast cancer disease. Experimental Design: Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2–n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2– cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC. Results: RCB II/III TNBC and ER+HER2– breast cancer were immunologically “cold” at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16–) within both the local TIME and peripheral white cell populations. Conclusions: This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

Supplementary Table S1: CDK12 expression in relation to clinicopathological parameters for the unselected TMA series; Supplementary Table S2: CDK12 expression in relation to clinicopathological parameters for the HER2-positive Herceptin treated series; Supplementary Table S3: CDK12 expression in relation to clinicopathological parameters for the METABRIC TMA series; Supplementary Table S4: Univariate and multivariate analysis of CDK12 in the TMA cohorts; Supplementary Table S5: CDK12 mutations in breast cancer. Taken from cBioportal (42,43); Supplementary Table S6: Correlations of CDK12 mutations, methylation, gene expression and ERBB2 copy number in primary breast cancers from TCGA; Supplementary Table S7: Correlations of CDK12 mutations and gene expression of DNA repair genes in primary tumors from METABRIC. P values from heteroscedastic 2-tailed, t-test; Supplementary Table S8: Correlations of CDK12 protein expression, and miRNA expression in primary tumors from METABRIC. Wilcoxon rank P values are corrected for multiple testing; Supplementary Table S9: Correlations of CDK12 protein expression and gene expression of DNA repair genes in primary tumors from METABRIC. Limma analysis corrected for multiple testing; Supplementary Table S10: Association of CDK12 absent and intermediate (0, 2-6) versus high (7-8) expression with DNA repair proteins in unselected and TNBC. P values from Fishers exact test.

Disruption of Cyclin-Dependent Kinase 12 (CDK12) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However, CDK12 has also been proposed as an oncogene in breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer–specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers. Mol Cancer Ther; 17(1); 306–15. ©2017 AACR.

Supplementary table descriptions

S1. LDL-cholesterol does not affect the viability of human γÎ' T cells. S2. Effects of LDL-cholesterol on Vï�¤2+ T cells after 36 or 48 hours of culture. S3. Effects of LDL-cholesterol on additional immune functions of γÎ' T cells. S4. Effect of LDL-cholesterol on expanded Vγ9VÎ'2 T cells cultured in the presence of human plasma. S5. Effects of receptor blockade on breast cancer cell targeting by γÎ' T lymphocytes. S6: Cholesterol-rich diet reduces multifunctional anti-tumour γÎ' infiltrating lymphocytes.

Vγ9Vδ2 T cells, the main subset of γδ T lymphocytes in human peripheral blood, are endowed with antitumor functions such as cytotoxicity and IFNγ production. These functions are triggered upon T-cell receptor–dependent activation by non-peptidic prenyl pyrophosphates (“phosphoantigens”) that are selective agonists of Vγ9Vδ2 T cells, and which have been evaluated in clinical studies. Because phosphoantigens have shown interindividual variation in Vγ9Vδ2 T-cell activities, we asked whether metabolic resources, namely lipids such as cholesterol, could affect phosphoantigen-mediated Vγ9Vδ2 T-cell activation and function. We show here that Vγ9Vδ2 T cells express the LDL receptor upon activation and take up LDL cholesterol. Resulting changes, such as decreased mitochondrial mass and reduced ATP production, correlate with downregulation of Vγ9Vδ2 T-cell activation and functionality. In particular, the expression of IFNγ, NKG2D, and DNAM-1 were reduced upon LDL cholesterol treatment of phosphoantigen-expanded Vγ9Vδ2 T cells. As a result, their capacity to target breast cancer cells was compromised both in vitro and in an in vivo xenograft mouse model. Thus, this study describes the role of LDL cholesterol as an inhibitor of the antitumor functions of phosphoantigen-activated Vγ9Vδ2 T cells. Our observations have implications for therapeutic applications dependent on Vγ9Vδ2 T cells. Cancer Immunol Res; 6(4); 448–57. ©2018 AACR.

Supplementary Data from Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy–Resistant Early Breast Cancers

Background The efficacy and safety of olaparib compared with placebo in the subset of patients from Japan in the phase 3 OlympiA trial (NCT02032823) are reported here and contextualized with reference to the global OlympiA population. Methods Patients with germline BRCA1 and/or BRCA2 pathogenic variants and HER2-negative, high-risk early breast cancer who had received neoadjuvant or adjuvant chemotherapy and completed local treatment were eligible. Patients were randomized 1:1 to receive olaparib or placebo for 1 year. Primary endpoint: invasive disease-free survival (IDFS). Secondary endpoints: distant disease-free survival (DDFS), overall survival (OS), and safety. Data are reported from the first pre-specified interim analysis (data cut-off [DCO] March 27, 2020) and the second, event driven, pre-specified interim analysis of OS (DCO July 12, 2021) in patients from Japan. Results 140 patients were randomized in Japan (olaparib, n = 64; placebo, n = 76). At the first pre-specified interim analysis (median follow-up: 2.9 years), hazard ratios (HRs) for adjuvant olaparib compared with placebo were 0.5 for IDFS (95% confidence interval [CI] 0.18–1.24) and 0.41 for DDFS (95% CI 0.11–1.16). At the second pre-specified interim analysis of OS, three deaths occurred in the olaparib group versus six deaths in the placebo group (HR, 0.62 [95% CI 0.13–2.36]). Findings were consistent with those for the global population. No new safety signals were observed. Conclusions While this analysis in a Japanese subset of patients was not powered to detect population-related treatment differences, efficacy and safety analysis results were consistent with the global OlympiA population, suggesting the findings from the global study are generalizable to clinical practice in Japan.

Supplementary Figure from Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy–Resistant Early Breast Cancers

Purpose:To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)–resistant triple-negative breast cancer (TNBC) and ER+HER2– breast cancer disease.Experimental Design:Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2–n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2– cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC.Results:RCB II/III TNBC and ER+HER2– breast cancer were immunologically “cold” at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16–) within both the local TIME and peripheral white cell populations.Conclusions:This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

Supplementary Data from Tiling Path Genomic Profiling of Grade 3 Invasive Ductal Breast Cancers

Patient demographic of low and high quartiles of HMOX1 expressing tumors from the METABRIC dataset

Purpose: Unprecedented clinical outcomes have been achieved in a variety of cancers by targeting immune checkpoint molecules. This preclinical study investigates heme oxygenase-1 (HO-1), an immunosuppressive enzyme that is expressed in a wide variety of cancers, as a potential immune checkpoint target in the context of a chemotherapy-elicited antitumor immune response. We evaluate repurposing tin mesoporphyrin (SnMP), which has demonstrated safety and efficacy targeting hepatic HO in the clinic for the treatment of hyperbilirubinemia, as an immune checkpoint blockade therapy for the treatment of cancer.Experimental Design: SnMP and genetic inactivation of myeloid HO-1 were evaluated alongside 5-fluorouracil in an aggressive spontaneous murine model of breast cancer (MMTV-PyMT). Single-cell RNA sequencing analysis, tumor microarray, and clinical survival data from breast cancer patients were used to support the clinical relevance of our observations.Results: We demonstrate that SnMP inhibits immune suppression of chemotherapy-elicited CD8+ T cells by targeting myeloid HO-1 activity in the tumor microenvironment. Microarray and survival data from breast cancer patients reveal that HO-1 is a poor prognostic factor in patients receiving chemotherapy. Single-cell RNA-sequencing analysis suggests that the myeloid lineage is a significant source of HO-1 expression, and is co-expressed with the immune checkpoints PD-L1/2 in human breast tumors. In vivo, we therapeutically compare the efficacy of targeting these two pathways alongside immune-stimulating chemotherapy, and demonstrate that the efficacy of SnMP compares favorably with PD-1 blockade in preclinical models.Conclusions: SnMP could represent a novel immune checkpoint therapy, which may improve the immunological response to chemotherapy. Clin Cancer Res; 24(7); 1617–28. ©2018 AACR.

Purpose: To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes.Experimental Design: Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs.Results: We show that basal-like and HER-2 tumors are characterized by “sawtooth” and “firestorm” genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification.Conclusions: Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

Supplementary Figure S1â",HO-1 expression in MMTV-PyMT tumors; Supplementary Figure S2â",Effects of SnMP and chemotherapy combination therapy; Supplementary Figure S3â",HO-1+ TAMs in murine models of breast cancer; Supplementary Figure S4â",Genetic inactivation and therapeutic inhibition of myeloid HO-1 in the MMTV-PyMT model; Supplementary Figure S5â",HO-1 in human cancer; Supplementary Figure S6â",tSNE clustering identifies stromal and epithelial cell types from breast cancer patient single-cell RNA sequencing data; Supplementary Figure 7â", Single cell RNA-Seq gene expression for checkpoint molecules grouped by inferred cell type; Supplementary Figure S8â",HO-1 protein in myeloid cells in human cancer; Supplementary Figure S9â", Comparative efficacies of therapeutically targeting HO-1 and PD-1 alongside paclitaxel treatment in 4T1 tumors; Supplementary Figure S10â",Improving checkpoint blockade therapy using SnMP.

Supplementary Figures 1-7 from FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer

The glycome acts as an essential interface between cells and the surrounding microenvironment. However, changes in glycosylation occur in nearly all breast cancers, which can alter this interaction. Here, we report that profiles of glycosylation vary between ER-positive and ER-negative breast cancers. We found that genes involved in the synthesis of sialyl-Lewis x (sLex; FUT3, FUT4, and ST3GAL6) are significantly increased in estrogen receptor alpha-negative (ER-negative) tumors compared with ER-positive ones. SLex expression had no influence on the survival of patients whether they had ER-negative or ER-positive tumors. However, high expression of sLex in ER-positive tumors was correlated with metastasis to the bone where sLex receptor E-selectin is constitutively expressed. The ER-positive ZR-75-1 and the ER-negative BT20 cell lines both express sLex but only ZR-75-1 cells could adhere to activated endothelial cells under dynamic flow conditions in a sLex and E-selectin–dependent manner. Moreover, L/P-selectins bound strongly to ER-negative MDA-MB-231 and BT-20 cell lines in a heparan sulfate (HS)–dependent manner that was independent of sLex expression. Expression of glycosylation genes involved in heparan biosynthesis (EXT1 and HS3ST1) was increased in ER-negative tumors. Taken together, our results suggest that the context of sLex expression is important in determining its functional significance and that selectins may promote metastasis in breast cancer through protein-associated sLex and HS glycosaminoglycans. Cancer Res; 71(24); 7683–93. ©2011 AACR.

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Supplementary Table S1. List of genes with an absolute copy number of h >=5 for >=25% of TNBCs.

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PDF file - 1575K, Supplementary Figure 1. Set-up of the two bone marrow metastatic niche models - Inhibitory Niche (Bone Marrow Cell lines, BMCL) and Supportive Niche (Human Bone Marrow Stromal Cells, BMSC). Supplementary Figure 2. Set-up of bone marrow metastatic niche models, including coculture with mouse BMSC and the cell lines 293t, fetal kidney, U2OS and MG63, osteosarcoma and organ-specific metastatic sub-lines of MDA-MB-231, 1833 BoM and 4175 LM2. Supplementary Figure 3. Control experiments for set-up of bone marrow metastatic niche models. Supplementary Figure 4 Breast cancer cells in IN co-culture did not undergo apoptosis were viable and capable to proliferate. Supplementary Figure 5. Conditioned medium from IN culture induced cell cycle arrest in breast cancer cells. Supplementary Figure 6. Effects of small molecule inhibitors SB431542 (Alk5), SB203580 (p38) and S1042 (RTK) in 3D co-cultures of breast cancer cell lines. Supplementary Figure 7. Treatment with small molecule inhibitors induced growth of BCC in the subcutaneous implantation model. Supplementary Figure 8 Breast cancer cell lines proliferation in orthotopic and metastasis mouse models (intratibial and intracardiac injection).

Supplementary Methods, Figure Legends 1-7 from FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer

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The targeting of Homologous Recombination deficient (HRD) malignancy using a synthetic lethal strategy based on the inhibition and trapping of PARP1 on DNA in manner that leads to tumour selective effects via dependency on the function of HR gene products such as BRCA1, BRCA2 and PALB2 for DNA replication fitness is now well established. However, there is a need to improve both the frequency and duration of response in the licensed indications and to explore combination PARP inhibitor (PARPi) strategies that may be effective a broader range of breast cancers with functional deficiencies in HR and the wider DNA damage response. ATR inhibitors exacerbate replication stress that is toxic to HR deficient cells. Combinations of an ATR inhibition (ATRi) and PARPi have been shown to be synergistic and to be active in PARPi resistant pre-clinical model contexts. The development of combination strategies of these agents and of platinums and ATRi have been limited by combinatorial toxicity but have recently reported results in breast cancer. PARP1 is part of a family of PARP enzymes and currently licensed PARPi inhibit several family members that underpin some of their toxicity. New PARP1 selective agents have recently reported results in early phase trials. I will review some of the mechanistic rationale, preclinical data and relevant clinical trial data in my lecture. Citation Format: Andrew Tutt. ATR inhibitors and PARP1 selective PARP inhibitors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr ED12-3.

Since the proof of concept of synthetic lethality between poly(ADP-ribose) polymerase inhibition and loss of BRCA1/2 homologous recombination (HR) function in preclinical models and early phase clinical trials, poly(ADP-ribose) polymerase inhibitors (PARPi) are increasing part of standard-of-care treatment for advanced breast cancers with BRCA gene mutations. The field has also recently seen benefits for PARPi in early breast cancer in those with germline BRCA1 and BRCA2 pathogenic mutations, and signals that synthetic lethal affects may occur in tumors with deficiencies in HR caused by germline, somatic, or epigenetic dysregulation of a number of HR genes. Despite the evidence of the synthetic lethal effects of PARPi, they are not always effective in HR defective cancers, and as they become part of standard of care in breast cancer, the study of prevalence of distinct mechanisms of resistance to PARPi and cross-resistance with other DNA-damaging agents such as platinum in breast cancer will be important and may inform therapy choices.

BackgroundPoly(adenosine diphosphate-ribose) polymerase inhibitors target cancers with defects in homologous recombination repair by synthetic lethality. New therapies are needed to reduce recurrence in patients with BRCA1 or BRCA2 germline mutation-associated early breast cancer.MethodsWe conducted a phase 3, double-blind, randomized trial involving patients with human epidermal growth factor receptor 2 (HER2)-negative early breast cancer with BRCA1 or BRCA2 germline pathogenic or likely pathogenic variants and high-risk clinicopathological factors who had received local treatment and neoadjuvant or adjuvant chemotherapy. Patients were randomly assigned (in a 1:1 ratio) to 1 year of oral olaparib or placebo. The primary end point was invasive disease-free survival.ResultsA total of 1836 patients underwent randomization. At a prespecified event-driven interim analysis with a median follow-up of 2.5 years, the 3-year invasive disease-free survival was 85.9% in the olaparib group and 77.1% in the placebo group (difference, 8.8 percentage points; 95% confidence interval [CI], 4.5 to 13.0; hazard ratio for invasive disease or death, 0.58; 99.5% CI, 0.41 to 0.82; P

Background: BRCA1 promoter methylation (PM) is an early initiating event in cancer, occurring in 3 to 65.2% of all breast tumors, and 30 to 65% of triple negative tumors. BRCA1 PM has been associated with defective homologous recombination repair (HRR), early onset of breast and ovarian cancer, and improved clinical response to adjuvant chemotherapy. Historically, there has been no diagnostic assay that comprehensively evaluates both BRCA1 PM and genomic alterations in cell-free circulating tumor DNA (ctDNA). Here, we describe the novel detection of BRCA1 PM and genomic alterations in a cohort of patients with breast cancer using GuardantINFINITY, a liquid biopsy assay interrogating 800+ genes and genome-wide methylation detection. Method: We assessed for BRCA1 PM in ctDNA from 274 patients with late-stage breast cancer. Genomic sequencing of 800+ genes and PM profiling of 398 genes was performed by GuardantINFINITY. The positive calling threshold for PM was established by comparing cell-free DNA derived from patients with cancer and cancer-free donors. The limit of detection (LoD) was determined through in silico and experimental titrations of ctDNA from clinical samples and cell lines with known gene PM into the plasma of cancer-free donors. Results: Among the 274 patients with advanced breast cancer, 8 (2.9%) had germline pathogenic mutations in BRCA1, BRCA2, or ATM. BRCA1 PM was detected in 11/274 (4.0%) patients at the predefined threshold of >99% specificity. BRCA1 PM detection in this cohort was 8.9% (8/90) when excluding samples with low tumor shedding ( Conclusion: GuardantINFINITY, a plasma-based diagnostic assay, detected both BRCA1 PM and genomic alterations in this unspecified advanced breast cancer cohort. The BRCA1 PM detection rates of 4.0-8.9% are consistent with values previously reported in the literature. As BRCA1 PM has important prognostic and therapeutic implications for the management of breast (as well as ovarian) cancers, additional studies are warranted to further describe the PM patterns across breast cancer subtypes and how these patterns both influence and are influenced by disease evolution and therapeutic response. Liquid biopsy thus serves as a suitable method to noninvasively identify and monitor changes in both genomics and epigenomics. Citation Format: Jennifer Yen, Sai Chen, Colby Jenkins, Brooke Overstreet, Yu Fu, Jun Zhao, Tingting Jiang, Leylah Drusbosky, Stephen Pettitt, Michael Dorschner, Lauren Lawrence, Han-Yu Chuang, Andrew Tutt. BRCA1 promoter methylation in sporadic breast cancer patients detected by liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6603.

Background The TNT trial (NCT00532727) showed no evidence of carboplatin (C) superiority over docetaxel (D) overall in metastatic triple negative breast cancers (TNBC), but a C benefit was observed in the pre-specified sub-group analysis in patients with a gBRCA1/2 mutation (Tutt et al, Nat Med 2018). Given only ~30% of patients have a gBRCA1/2 mutation, broader predictive biomarkers of response are needed. In this cohort we previously found that DNA Damage Response (DDR) signatures were associated with improved C response in chemotherapy (CT) naïve patients only (Tovey et al, ASCO 2020). Since DDR activities influence tumour immune-microenvironment, we explored the predictive ability of immune cell markers and performed integrative analyses on multi-omics features to identify novel TNBC subgroups. Patients and Methods Tumour infiltrating lymphocytes (TILs) were evaluated on haematoxylin and eosin stained primary tumour (PT) slides for 222/376 TNT patients. Formalin-fixed paraffin-embedded PT tissues from 186/376 TNT patients were successfully profiled using total RNA-sequencing. Matched recurrence (REC) was also sequenced for 13 patients. Twenty-five immune signatures were assessed. Logistic regression and restricted mean progression free survival (PFS) were applied to delineate the relationship of these features with treatment outcomes. Random forest clustering of multi-omics DDR and immune biology markers, including gene expression signatures and mutation/methylation status, was applied to identify subgroups. We further molecularly characterised these clusters through supervised clustering of 693 gene expression “modules” (sets of co-expressed genes), immune cell deconvolution and genomic scars. Results Immune gene expression signatures and TILs were highly correlated. Average immune infiltration based on ConsensusTME was lower in mutated/methylated tumours compared with BRCA1 wildtype tumours (p=0.04). Immune signature score markers decreased from PT to REC, demonstrating a dynamic immune microenvironment. In the overall population and when restricting to prior CT treated patients, high immune infiltration (gene expression based & TILs) was associated with response to D while C response rates were not associated with immune scores (interaction p-values< 0.05). This did not translate to a PFS benefit. Multi-omics clustering identified 6 biological subgroups including immune enriched, immune depleted, DDR deficient and proficient clusters as well as 2 small clusters with no obvious distinguishing features. Immune enriched TNBC were predominantly basal-like immune activated with high B-cell/T-cell diversity. Immune depleted TNBC showed higher activity of proliferation and DDR pathway modules. DDR proficient tumours had low expression of immune markers and enrichment for ESR1/PGR expression, markers of extra cellular formation, cell structure, lipid metabolism and proliferation. The DDR deficient cluster was enriched for proliferation and demonstrated high number of TILs despite no apparent enrichment for gene expression-based immune modules. In the prior CT treated cohort, the immune enriched cluster had preferential response to D (62.5% (D) vs. 29.4% (C); p=0.02). The immune depleted cluster had preferential response to C (8.0% (D) vs. 40.0% (C); p=0.01). Numbers were too small to assess differential response within the other clusters or in the CT naïve cohort. Conclusions Tumours with high immune features have high response to D while those with low immune features have preferential response to C in advanced TNBC. Combining multi-omics markers of DDR deficiency and immune biology can identify clusters of patients with distinct biological profiles and differential treatment specific response rates. Citation Format: Holly Tovey, Orsolya Sipos, Katherine A Hoadley, Joel S Parker, Jelmar Quist, Sarah Kernaghan, Lucy Kilburn, Roberto Salgado, Sherene Loi, Richard D Kennedy, Ioannis Roxanis, Patrycja Gazinska, Sarah E. Pinder, Judith Bliss, Charles M. Perou, Syed Haider, Andrew Tutt, Anita Grigoriadis, Maggie Chon U Cheang. Histopathological and molecular immune landscape and DNA damage response signatures to predict response to carboplatin and docetaxel in TNT trial TNBC cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-06.