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PURPOSE Triple-negative breast cancer (TNBC) is a heterogeneous disease with variable response to neoadjuvant therapy (NAT). Pathologic complete response (pCR) has become a prognostic marker for overall and disease-free survival. The aim of this study was to determine if dynamic contrast-enhanced (DCE)-MRI after 2 and/or 4 cycles of NAT can identify patients with a high likelihood of achieving pCR, triaging them to standard of care (SOC), or, when appropriate, to de-escalation trials. Conversely, we aimed to identify chemoresistant tumors that are unlikely to achieve pCR and may benefit from escalated targeted trials. METHOD AND MATERIALS 309 patients with stage I-III TNBC underwent DCE-MRI (temporal resolution: 9-12 sec) at baseline (BL), 2 cycles (C2), and 4 cycles (C4) of SOC doxorubicin/cyclophosphamide (AC) NAT as part of a prospective IRB-approved study (NCT02276443). Tumor volumes of the index lesion were calculated using 3 axis measurements during the early phase of the DCE-MRI (60s). Percent tumor volume reduction (TVR) between BL, C2, and C4 was calculated. Patients were randomly assigned to a training or a validation cohort in a 1:1 ratio. pCR was assessed at surgery after completion of SOC NAT. Correlation between pCR and TVR was evaluated using ROC analysis. RESULTS Of 309 TNBC patients, 136 (44%) achieved pCR. Following 2 cycles of NAT, TVR >80% was predictive of pCR (chemosensitivity), while TVR ≤ 55% was predictive of non-pCR (chemoresistance) with PPV 80%, NPV 89%, AUC 0.811 (0.73~0.893, p< 0.0001) in the training cohort, and PPV 82%, NPV 85%, AUC 0.815 (CI:0.736~0.894, p< 0.0001) in the validation cohort. Following 4 cycles of NAT, TVR >90% was predictive of pCR, while TVR ≤80% was predictive of non-pCR with PPV 80%, NPV 84%, AUC 0.827 (0.756~0.898, p< 0.0001) in the training cohort and with PPV 73%, NPV 82%, AUC 0.785 (CI:0.709~0.862, p< 0.001) in the validation cohort. Using this model, the pCR status was correctly classified in 50% of TNBC patients using C2 DCE-MRI in the training cohort, and 54% in the validation cohort. Only 8% were misclassified in the training cohort, and 10% in the validation cohort. Using C4 DCE-MRI, the pCR status of 61% and 57% of TNBC was correctly classified in the validation and the testing cohorts, respectively. 12% were misclassified in the validation cohort, and 21% in the testing cohort. CONCLUSION DCE-MRI after 2 and 4 cycles of AC-based NAT correctly predicted the pCR status of 54% and 57% of TNBC patients, respectively, as either excellent responders or nonresponders with high AUC 0.811 and 0.827. This may allow patients to be triaged to SOC NAT with option of de-escalation or early targeted therapies for non-responders. Citation Format: Mary S. Guirguis, Beatriz Adrada, Miral Patel, Frances Perez, Rosalind Candelaria, Wei Yang, Jia Sun, Rania M. Mohamed, Medine Boge, H. T. Carisa Le-Petross, Jessica Leung, Gary J. Whitman, Deanna L. Lane, Marion E. Scoggins, Tanya Moseley, Benjamin Musall, Jason White, Sanaz Pashapoor, Peng Wei, Jong Bum Son, Ken-Pin Hwang, Bikash Panthi, Mark Pagel, Lei Huo, Kelly K. Hunt, Elizabeth Ravenberg, Alastair M. Thompson, Jennifer K. Litton, Vicente Valero, Debu Tripathy, Stacy Moulder, Clinton Yam, Jingfei Ma, Gaiane Rauch. DCE-MRI for early prediction of excellent response versus chemoresistance in triple negative breast cancer [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 P1-05-15.

High stromal tumor-infiltrating lymphocytes (sTILs) are associated with improved pathologic complete response (pCR) in triple-negative breast cancer (TNBC). We hypothesize that integrating high sTILs and additional clinicopathologic features associated with pCR could enhance our ability to predict the group of patients on whom treatment de-escalation strategies could be tested. In this prospective early-stage TNBC neoadjuvant chemotherapy study, pretreatment biopsies from 408 patients were evaluated for their clinical and demographic features, as well as biomarkers including sTILs, Ki-67, PD-L1 and androgen receptor. Multivariate logistic regression models were developed to generate a computed response score to predict pCR. The pCR rate for the entire cohort was 41%. Recursive partitioning analysis identified ≥20% as the optimal cutoff for sTILs to denote 35% (143/408) of patients as having high sTILs, with a pCR rate of 59%, and 65% (265/408) of patients as having low sTILs, with a pCR rate of 31%. High Ki-67 (cutoff > 35%) was identified as the only predictor of pCR in addition to sTILs in the training set. This finding was verified in the testing set, where the highest computed response score encompassing both high sTILa and high Ki-67 predicted a pCR rate of 65%. Integrating Ki67 and sTIL may refine the selection of early stage TNBC patients for neoadjuvant clinical trials evaluating de-escalation strategies.

Triple-negative breast cancer (TNBC) is persistently refractory to therapy, and methods to improve targeting and evaluation of responses to therapy in this disease are needed. Here, we integrate quantitative MRI data with biologically based mathematical modeling to accurately predict the response of TNBC to neoadjuvant systemic therapy (NAST) on an individual basis. Specifically, 56 patients with TNBC enrolled in the ARTEMIS trial (NCT02276443) underwent standard-of-care doxorubicin/cyclophosphamide (A/C) and then paclitaxel for NAST, where dynamic contrast-enhanced MRI and diffusion-weighted MRI were acquired before treatment and after two and four cycles of A/C. A biologically based model was established to characterize tumor cell movement, proliferation, and treatment-induced cell death. Two evaluation frameworks were investigated using: (i) images acquired before and after two cycles of A/C for calibration and predicting tumor status after A/C, and (ii) images acquired before, after two cycles, and after four cycles of A/C for calibration and predicting response following NAST. For Framework 1, the concordance correlation coefficients between the predicted and measured patient-specific, post-A/C changes in tumor cellularity and volume were 0.95 and 0.94, respectively. For Framework 2, the biologically based model achieved an area under the receiver operator characteristic curve of 0.89 (sensitivity/specificity = 0.72/0.95) for differentiating pathological complete response (pCR) from non-pCR, which is statistically superior (P < 0.05) to the value of 0.78 (sensitivity/specificity = 0.72/0.79) achieved by tumor volume measured after four cycles of A/C. Overall, this model successfully captured patient-specific, spatiotemporal dynamics of TNBC response to NAST, providing highly accurate predictions of NAST response. Significance: Integrating MRI data with biologically based mathematical modeling successfully predicts breast cancer response to chemotherapy, suggesting digital twins could facilitate a paradigm shift from simply assessing response to predicting and optimizing therapeutic efficacy.

Purpose: The immunological profile of early-stage breast cancer treated with neoadjuvant PARP inhibitors has not been described. The aim of this study was to delineate the changes in the tumor immune microenvironment (TiME) induced by talazoparib. Patients and Methods: Patients with operable germline BRCA1/2 pathogenic variant (gBRCA1/2+) breast cancer were enrolled in a feasibility study of neoadjuvant talazoparib. Thirteen patients who received 8 weeks of neoadjuvant talazoparib were available for analysis, including 11 paired pre- and post-talazoparib core biopsies. Treatment-related changes in tumor-infiltrating lymphocytes were examined and immune cell phenotypes and their spatial distribution in the TiME were identified and quantified by multiplex immunofluorescence using a panel of 6 biomarkers (CD3, CD8, CD68, PD-1, PD-L1, and CK). Results: Neoadjuvant talazoparib significantly increased infiltrating intratumoral and stromal T-cell and cytotoxic T-cell density. There was no difference in PD-1 or PD-L1 immune cell phenotypes in the pre- and post-talazoparib specimens and PD-L1 expression in tumor cells was rare in this cohort. Spatial analysis demonstrated that pre-talazoparib interactions between macrophages and T cells may correlate with pathologic complete response. Conclusions: This is the first study with phenotyping to characterize the immune response to neoadjuvant talazoparib in patients with gBRCA1/2+ breast cancer. These findings support an emerging role for PARP inhibitors in enhancing tumor immunogenicity. Further investigation of combinatorial strategies is warranted with agents that exploit the immunomodulatory effects of PARP inhibitors on the TiME.

Early-phase trials targeting the T-cell inhibitory molecule programmed cell death ligand 1 (PD-L1) have shown clinical efficacy in cancer. This study was undertaken to determine whether PD-L1 is overexpressed in triple-negative breast cancer (TNBC) and to investigate the loss of PTEN as a mechanism of PD-L1 regulation. The Cancer Genome Atlas (TCGA) RNA sequencing data showed significantly greater expression of the PD-L1 gene in TNBC (n = 120) compared with non-TNBC (n = 716; P < 0.001). Breast tumor tissue microarrays were evaluated for PD-L1 expression, which was present in 19% (20 of 105) of TNBC specimens. PD-L1+ tumors had greater CD8+ T-cell infiltrate than PD-L1− tumors (688 cells/mm vs. 263 cells/mm; P < 0.0001). To determine the effect of PTEN loss on PD-L1 expression, stable cell lines were generated using PTEN short hairpin RNA (shRNA). PTEN knockdown led to significantly higher cell-surface PD-L1 expression and PD-L1 transcripts, suggesting transcriptional regulation. Moreover, phosphoinositide 3-kinase (PI3K) pathway inhibition using the AKT inhibitor MK-2206 or rapamycin resulted in decreased PD-L1 expression, further linking PTEN and PI3K signaling to PD-L1 regulation. Coculture experiments were performed to determine the functional effect of altered PD-L1 expression. Increased PD-L1 cell surface expression by tumor cells induced by PTEN loss led to decreased T-cell proliferation and increased apoptosis. PD-L1 is expressed in 20% of TNBCs, suggesting PD-L1 as a therapeutic target in TNBCs. Because PTEN loss is one mechanism regulating PD-L1 expression, agents targeting the PI3K pathway may increase the antitumor adaptive immune responses. Cancer Immunol Res; 2(4); 361–70. ©2014 AACR.

PDF file - 78K, Supplementary figure 2. T cell Proliferation is decreased after co-culture with MDA-MB-157 breast cancer cells following PTEN knockdown. (A) To determine the effect of the increased PD-L1 cell surface expression following PTEN knockdown on T cell proliferation, peripheral blood mononuclear cells (PBMC) from healthy donors were labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with MDA-MB-231 breast cancer cells that were transduced with PTEN shRNA (i.e. increase surface PD-L1) or negative control groups including parental MDA-MB-231 cells or MDA-MB-231 cells transduced with non-targeting shRNA.. After stimulation with anti-CD3/CD28, proliferation was measured using flow cytometry. CFSE staining of CD4+ or CD8+T cells is shown in representative histograms from one replicate from one experiment. The experiment was performed three times in triplicate and the average percent proliferation ? SD for one experiment is shown in panel B. Unstimulated and anti-CD3/CD28 stimulated PBMC represent PBMC groups lacking MDA-MB-231 cells and were used as negative and positive controls, respectively. Significance was determined using an ANOVA test (*P

PDF file - 76K, Supplementary figure 3. T cell Proliferation is decreased after co-culture with MDA-MB-157 breast cancer cells following PTEN knockdown. In addition to MDA-MB-231 cells, PTEN knockdown induced PD-L1 expression in MDA-MB-157 breast cancer cell line also decreases T cell proliferation. (A) Representative histogram of carboxyfluorescein succinimidyl ester (CFSE) assays shows decreased T cell proliferation when PBMC were co-cultured with MDA-MB-157 breast cancer cells transduced with PTEN shRNA in comparison with co-culture with parental MDA-MB-157 cells or MDA-MB-157 cells transduced with non-targeting shRNA. Unstimulated PBMC and stimulated PBMC alone were used as negative controls. CFSE analysis was performed on CD4+ or CD8+ cells. (B) Graphical analysis of CFSE data from a representative experiment performed in triplicate. Significance was determined using an ANOVA test (*P

PDF file - 110K, Supplementary figure 4. T cell's undergo apoptosis after co-culture with MDA-MB-231 breast cancer cells following PTEN knockdown To determine the effect of increased PD-L1 cell surface expression on apoptosis, standard annexin V assays were performed. Anti-CD3/CD28-stimulated PBMC were co-cultured with breast cancer cells for 20 hours and then stained with anti-CD4 and anti-CD8 antibodies and resuspended in Annexin binding buffer. Analysis was performed using flow cytometry gating on CD4+ or CD8+ T cells. A representative histogram from one experiment is shown in panel A. The experiment was repeated three times in triplicate and the percent Annexin V positive CD4+ and CD8+ T cells from one experiment is shown in panel B.

Supplementary Tables

Adverse Events

Supplementary Figure S2

Clinicopathologic characteristics of randomized triple-negative breast cancer patients

HER2 FISH BCX.017

Purpose:Preclinical data provide evidence for synergism between HER2-targeted peptide vaccines and trastuzumab. The efficacy of this combination was evaluated in patients with HER2 low-expressing breast cancer in the adjuvant setting.Patients and Methods:A phase IIb, multicenter, randomized, single-blinded, controlled trial enrolled disease-free patients after standard therapy completion (NCT01570036). Eligible patients were HLA-A2, A3, A24, and/or A26+, and had HER2 IHC 1+/2+, FISH nonamplified breast cancer, that was node positive and/or hormone receptor–negative [triple-negative breast cancer (TNBC)]. Patients received trastuzumab for 1 year and were randomized to placebo (GM-CSF, control) or nelipepimut-S (NPS) with GM-CSF. Primary outcome was 24-month disease-free survival (DFS). Secondary outcomes were 36-month DFS, safety, and immunologic response.Results:Overall, 275 patients were randomized; 136 received NPS with GM-CSF, and 139 received placebo with GM-CSF. There were no clinicopathologic differences between groups. Concurrent trastuzumab and NPS with GM-CSF was safe with no additional overall or cardiac toxicity compared with control. At median follow-up of 25.7 (interquartile range, 18.4–32.7) months, estimated DFS did not significantly differ between NPS and control [HR, 0.62; 95% confidence interval (CI), 0.31–1.25; P = 0.18]. In a planned exploratory analysis of patients with TNBC, DFS was improved for NPS versus control (HR, 0.26; 95% CI, 0.08–0.81, P = 0.01).Conclusions:The combination of NPS with trastuzumab is safe. In HER2 low-expressing breast cancer, no significant difference in DFS was seen in the intention-to-treat analysis; however, significant clinical benefit was seen in patients with TNBC. These findings warrant further investigation in a phase III randomized trial.

Correlations to TNBCtypes

Supplementary Data from Determinants of Response to Talazoparib in Patients with HER2-Negative, Germline BRCA1/2-Mutated Breast Cancer

Clinical and pathological characteristics of 53 triple negative breast carcinoma patients

PARP inhibitors (PARPi) benefit only a fraction of breast cancer patients. Several of those patients exhibit intrinsic/acquired resistance mechanisms that limit efficacy of PARPi monotherapy. Here we show how the efficacy of PARPi in triple-negative breast cancers (TNBC) can be expanded by targeting MYC-induced oncogenic addiction. In BRCA-mutant/sporadic TNBC patients, amplification of the MYC gene is correlated with increased expression of the homologous DNA recombination enzyme RAD51 and tumors overexpressing both genes are associated with worse overall survival. Combining MYC blockade with PARPi yielded synthetic lethality in MYC-driven TNBC cells. Using the cyclin-dependent kinase inhibitor dinaciclib, which downregulates MYC expression, we found that combination with the PARPi niraparib increased DNA damage and downregulated homologous recombination, leading to subsequent downregulation of the epithelial–mesenchymal transition and cancer stem-like cell phenotypes. Notably, dinaciclib resensitized TBNC cells, which had acquired resistance to niraparib. We found that the synthetic lethal strategy employing dinaciclib and niraparib was also highly efficacious in ovarian, prostate, pancreatic, colon, and lung cancer cells. Taken together, our results show how blunting MYC oncogene addiction can leverage cancer cell sensitivity to PARPi, facilitating the clinical use of c-myc as a predictive biomarker for this treatment.Significance: Dual targeting of MYC-regulated homologous recombination and PARP-mediated DNA repair yields potent synthetic lethality in triple-negative breast tumors and other aggressive tumors characterized by MYC overexpression. Cancer Res; 78(3); 742–57. ©2017 AACR.

Supplementary Figures Legends

This file contains figure legends pertaining to the supplementary figures.

The toxicity profiles were similar between the treatment arms. (A) There was no difference in the total number of graded adverse events (AE) between arms of the trial. (B) There was no significant difference in the maximum local and systemic toxicity experienced by each patient between arms of the trial.

Purpose:On the basis of strong preclinical rationale, we sought to confirm recommended phase II dose (RP2D) for olaparib, a PARP inhibitor, combined with the AKT inhibitor capivasertib and assess molecular markers of response and resistance.Patients and Methods:We performed a safety lead-in followed by expansion in endometrial, triple-negative breast, ovarian, fallopian tube, or peritoneal cancer. Olaparib 300 mg orally twice daily and capivasertib orally twice daily on a 4-day on 3-day off schedule was evaluated. Two dose levels (DL) of capivasertib were planned: 400 mg (DL1) and 320 mg (DL-1). Patients underwent biopsies at baseline and 28 days.Results:A total of 38 patients were enrolled. Seven (18%) had germline BRCA1/2 mutations. The first 2 patients on DL1 experienced dose-limiting toxicities (DLT) of diarrhea and vomiting. No DLTs were observed on DL-1 (n = 6); therefore, DL1 was reexplored (n = 6) with no DLTs, confirming DL1 as RP2D. Most common treatment-related grade 3/4 adverse events were anemia (23.7%) and leukopenia (10.5%). Of 32 evaluable subjects, 6 (19%) had partial response (PR); PR rate was 44.4% in endometrial cancer. Seven (22%) additional patients had stable disease greater than 4 months. Tumor analysis demonstrated strong correlations between response and immune activity, cell-cycle alterations, and DNA damage response. Therapy resistance was associated with receptor tyrosine kinase and RAS-MAPK pathway activity, metabolism, and epigenetics.Conclusions:The combination of olaparib and capivasertib is associated to no serious adverse events and demonstrates durable activity in ovarian, endometrial, and breast cancers, with promising responses in endometrial cancer. Importantly, tumor samples acquired pre- and on-therapy can help predict patient benefit.

Supplementary Tables S1-5 and Supplementary Fig. S1

Triple-negative breast cancer (TNBC) is persistently refractory to therapy, and methods to improve targeting and evaluation of responses to therapy in this disease are needed. Here, we integrate quantitative MRI data with biologically based mathematical modeling to accurately predict the response of TNBC to neoadjuvant systemic therapy (NAST) on an individual basis. Specifically, 56 patients with TNBC enrolled in the ARTEMIS trial (NCT02276443) underwent standard-of-care doxorubicin/cyclophosphamide (A/C) and then paclitaxel for NAST, where dynamic contrast-enhanced MRI and diffusion-weighted MRI were acquired before treatment and after two and four cycles of A/C. A biologically based model was established to characterize tumor cell movement, proliferation, and treatment-induced cell death. Two evaluation frameworks were investigated using: (i) images acquired before and after two cycles of A/C for calibration and predicting tumor status after A/C, and (ii) images acquired before, after two cycles, and after four cycles of A/C for calibration and predicting response following NAST. For Framework 1, the concordance correlation coefficients between the predicted and measured patient-specific, post-A/C changes in tumor cellularity and volume were 0.95 and 0.94, respectively. For Framework 2, the biologically based model achieved an area under the receiver operator characteristic curve of 0.89 (sensitivity/specificity = 0.72/0.95) for differentiating pathological complete response (pCR) from non-pCR, which is statistically superior (P < 0.05) to the value of 0.78 (sensitivity/specificity = 0.72/0.79) achieved by tumor volume measured after four cycles of A/C. Overall, this model successfully captured patient-specific, spatiotemporal dynamics of TNBC response to NAST, providing highly accurate predictions of NAST response.Significance:Integrating MRI data with biologically based mathematical modeling successfully predicts breast cancer response to chemotherapy, suggesting digital twins could facilitate a paradigm shift from simply assessing response to predicting and optimizing therapeutic efficacy.

Supplementary materials

Supplementary Figure from Molecular Characterization and Prospective Evaluation of Pathologic Response and Outcomes with Neoadjuvant Therapy in Metaplastic Triple-Negative Breast Cancer

Purpose:PARP inhibitors (PARPi) have demonstrated efficacy in tumors with germline breast cancer susceptibility genes (gBRCA) 1 and 2 mutations, but further factors influencing response to PARPi are poorly understood.Experimental Design:Breast cancer tumor tissue from patients with gBRCA1/2 mutations from the phase III EMBRACA trial of the PARPi talazoparib versus chemotherapy was sequenced using FoundationOne CDx.Results:In the evaluable intent-to-treat population, 96.1% (296/308) had ≥1 tumor BRCA (tBRCA) mutation and there was strong concordance (95.3%) between tBRCA and gBRCA mutational status. Genetic/genomic characteristics including BRCA loss of heterozygosity (LOH; identified in 82.6% of evaluable patients), DNA damage response (DDR) gene mutational burden, and tumor homologous recombination deficiency [assessed by genomic LOH (gLOH)] demonstrated no association with talazoparib efficacy.Conclusions:Overall, BRCA LOH status, DDR gene mutational burden, and gLOH were not associated with talazoparib efficacy; however, these conclusions are qualified by population heterogeneity and low patient numbers in some subgroups. Further investigation in larger patient populations is warranted.

Supplementary Data from MRI-Based Digital Models Forecast Patient-Specific Treatment Responses to Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer

Supplementary Figure 9 The effect of treatment with c-myc inhibitor 10058-F4 in combination with niraparib Supplementary Figure 10 Screening of known MYC-inhibiting drugs. Supplementary Figure 11 Synergistic assessment of combination treatment with dinaciclib and PARPi on TNBC in vitro Supplementary Figure 12 Immunofluorescent analysis of breast cancer cells treated for 72-h with DMSO (control), niraparib (5 μM), dinaciclib (10 nM), or niraparib and dinaciclib (Combo).

Supplementary Figure 13 Effects of treatment with Dinaciclib and niraparib on TNBC in vivo. Supplementary Figure 14 Niraparib resistance of breast cancer cells. (a-d) Knockdown of 53BP1 expression in HCC1806 cells. Supplementary Figure 15 Analysis of MYC mRNA expression in different TCGA cancer patient cohorts. Supplementary Figure 16 Evaluation of TCGA cohort of 82 patients with TNBC, separated according to MYC expression and showing correlation with HR mutation status. Molecular analysis of the patients' tumors predicted their response to PARPi-targeted therapy.

Supplementary Table from Tumor Immune Microenvironment Changes by Multiplex Immunofluorescence Staining in a Pilot Study of Neoadjuvant Talazoparib for Early-Stage Breast Cancer Patients with a Hereditary BRCA Mutation

Supplementary Figure from Tumor Immune Microenvironment Changes by Multiplex Immunofluorescence Staining in a Pilot Study of Neoadjuvant Talazoparib for Early-Stage Breast Cancer Patients with a Hereditary BRCA Mutation

Purpose:Metaplastic breast cancer (MpBC) is a rare subtype of breast cancer that is commonly triple-negative and poorly responsive to neoadjuvant therapy in retrospective studies.Experimental Design:To better define clinical outcomes and correlates of response, we analyzed the rate of pathologic complete response (pCR) to neoadjuvant therapy, survival outcomes, and genomic and transcriptomic profiles of the pretreatment tumors in a prospective clinical trial (NCT02276443). A total of 211 patients with triple-negative breast cancer (TNBC), including 39 with MpBC, received doxorubicin-cyclophosphamide–based neoadjuvant therapy.Results:Although not meeting the threshold for statistical significance, patients with MpBCs were less likely to experience a pCR (23% vs. 40%; P = 0.07), had shorter event-free survival (29.4 vs. 32.2 months, P = 0.15), metastasis-free survival (30.3 vs. 32.4 months, P = 0.22); and overall survival (32.6 vs. 34.3 months, P = 0.21). This heterogeneity is mirrored in the molecular profiling. Mutations in PI3KCA (23% vs. 9%, P = 0.07) and its pathway (41% vs. 18%, P = 0.02) were frequently observed and enriched in MpBCs. The gene expression profiles of each histologically defined subtype were distinguishable and characterized by distinctive gene signatures. Among nonmetaplastic (non-Mp) TNBCs, 10% possessed a metaplastic-like gene expression signature and had pCR rates and survival outcomes similar to MpBC.Conclusions:Further investigations will determine if metaplastic-like tumors should be treated more similarly to MpBC in the clinic. The 23% pCR rate in this study suggests that patients with MpBC should be considered for NAT. To improve this rate, a pathway analysis predicted enrichment of histone deacetylase (HDAC) and RTK/MAPK pathways in MpBC, which may serve as new targetable vulnerabilities.

Purpose:Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR.Experimental Design:We obtained pretreatment core-needle biopsies from 105 patients with stage I–III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT.Results:The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 μm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage.Conclusions:In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

Supplementary Figure 1 Comparison of 24h, 72-h and continuous HTSA in PARPi treated cells Supplementary Figure 2 HTSA versus MTT proliferation assays Supplementary Figure 3 PARPi sensitivity and resistance in breast cancer cells Supplementary Figure 4 Cell-cycle changes following PARP inhibitor treated sensitive (MB436, HCC1806) versus resistant (MB231, SUM149) breast cancer cells.

Purpose:The immunological profile of early-stage breast cancer treated with neoadjuvant PARP inhibitors has not been described. The aim of this study was to delineate the changes in the tumor immune microenvironment (TiME) induced by talazoparib.Patients and Methods:Patients with operable germline BRCA1/2 pathogenic variant (gBRCA1/2+) breast cancer were enrolled in a feasibility study of neoadjuvant talazoparib. Thirteen patients who received 8 weeks of neoadjuvant talazoparib were available for analysis, including 11 paired pre- and post-talazoparib core biopsies. Treatment-related changes in tumor-infiltrating lymphocytes were examined and immune cell phenotypes and their spatial distribution in the TiME were identified and quantified by multiplex immunofluorescence using a panel of 6 biomarkers (CD3, CD8, CD68, PD-1, PD-L1, and CK).Results:Neoadjuvant talazoparib significantly increased infiltrating intratumoral and stromal T-cell and cytotoxic T-cell density. There was no difference in PD-1 or PD-L1 immune cell phenotypes in the pre- and post-talazoparib specimens and PD-L1 expression in tumor cells was rare in this cohort. Spatial analysis demonstrated that pre-talazoparib interactions between macrophages and T cells may correlate with pathologic complete response.Conclusions:This is the first study with phenotyping to characterize the immune response to neoadjuvant talazoparib in patients with gBRCA1/2+ breast cancer. These findings support an emerging role for PARP inhibitors in enhancing tumor immunogenicity. Further investigation of combinatorial strategies is warranted with agents that exploit the immunomodulatory effects of PARP inhibitors on the TiME.

Supplementary Table from Molecular Characterization and Prospective Evaluation of Pathologic Response and Outcomes with Neoadjuvant Therapy in Metaplastic Triple-Negative Breast Cancer

This file contains all the methods pertaining to the supplemental figures