Vandana G. Abramson

Background: Tumor-infiltrating lymphocytes (TILs), assessed by visual examination, are prognostic and predictive in early-stage triple-negative breast cancer. Computational assessment may provide a complementary approach. We evaluated the prognostic value of TILs by visual examination and computational assessment. Methods: Cisplatin vs Paclitaxel for Triple Negative Breast Cancer (TBCRC030; ClinicalTrials.gov identifier NCT01982448) was a randomized phase 2 trial enrolling patients with BRCA1/2-proficient stage I to III triple-negative breast cancer to receive preoperative cisplatin or paclitaxel. The primary endpoint was pathological response at surgery. The TILs were visually scored on digitized pretreatment biopsies per International TILS Working Group recommendations. Computational assessment used the 4D Path QPOR platform to generate TILs, an immune heterogeneity index, and a combined immune/cell cycle biomarker (CmbI). Predictive performance for residual cancer burden 0/1 was assessed using receiver operating characteristic curves and odds ratios (ORs) with 95% CIs; all statistical tests were 2-sided. Results: Of 139 response-evaluable patients, 121 had matched visual examination and computational assessment data (59 on cisplatin, 62 on paclitaxel). Median visual examination TILs were higher in responders (40.0% vs. 10.0%; P = .002) and predicted response (OR = 1.86, 95% CI = 1.24 to 2.87; area under the curve = 0.69, 95% CI = 0.57 to 0.80). Computational assessment CmbI differed by response group and predicted residual cancer burden 0/1 (OR = 3.20, 95% CI = 1.05 to 11.07; area under the curve = 0.62, 95% CI = 0.51 to 0.73). Computational assessment TILs and immune heterogeneity index were not predictive. Visual examination TILs and computational assessment CmbI predicted response to paclitaxel (OR = 2.91, 95% CI = 1.56 to 6.14; OR = 9.17, 95% CI = 2.01 to 66.39, respectively) but not to cisplatin. Conclusion: Visual examination TILs and computational assessment CmbI were each associated with response to neoadjuvant chemotherapy in triple-negative breast cancer in the overall cohort and the paclitaxel arm. Computational assessment CmbI did not outperform visual assessment. Further validation is needed before clinical implementation of computational approaches.

PURPOSE: Predictive biomarkers to better tailor therapy for patients with early-stage human epidermal growth factor 2 (HER2)-positive breast cancer are a priority. We hypothesized that HER2 and immune-based biomarkers would be predictive of pathologic complete response (pCR) to preoperative trastuzumab/pertuzumab (HP). MATERIALS AND METHODS: Patients with stage II/III, estrogen receptor (ER)-negative, HER2-positive breast cancer received neoadjuvant HP in the TBCRC026 clinical trial. The pCR after receiving HP alone was 22% (18/83). Tumor biopsies were performed at baseline. Secondary correlative objectives were to determine the relationship between HER2-based biomarkers and immune processes with pCR. NanoString code sets BC360 and IO360 were used to compare differential gene expression in baseline tumors that underwent pCR versus no pCR. NanoString GeoMx digital spatial profiling was used to assess immune protein abundance in intraepithelial and stromal segments. Stromal tumor-infiltrating lymphocytes and Ki67 were evaluated by hematoxylin and eosin and immunohistochemistry, respectively. RESULTS: Intraepithelial HER2 protein abundance was significantly associated with pCR ( P = .001). Low HER2 abundance tumors were primarily basal-like, and essentially all (19/20) failed to achieve pCR. High HER2 abundance tumors that achieved pCR (14/51) exhibited a high degree of immune cell activity, whereas high HER2 abundance tumors that failed to achieve pCR tumors (37/51) were enriched for M-phase processes and epidermal growth factor receptor signaling. Baseline Ki67 was significantly higher in nonresponders ( P = .04). CONCLUSION: ER-negative, HER2-positive breast cancer has unique molecular and immunologic features that may predict pCR after neoadjuvant HP. Validation of these potential biomarkers and composite biomarker analyses may guide design of future clinical trials.

PURPOSE: The time required for in-clinic drug administration can substantially affect breast cancer patients' quality of life. Subcutaneous (SC) drug administration, as opposed to intravenous (IV), may reduce this time commitment. This study sought to estimate the difference in time burden between IV and SC administration of trastuzumab and pertuzumab (HP). METHODS: We prospectively enrolled a subcohort of patients participating in the ADEPT trial (ClinicalTrials.gov identifier: NCT04569747 , investigating adjuvant HP plus endocrine therapy for stage I human epidermal growth factor receptor 2–positive breast cancer) to this single-arm crossover time and motion substudy. Patients received two cycles of IV HP followed by two cycles of SC HP. During each cycle, time points in drug preparation and administration were captured. The primary end point was total patient time in the treatment chair. Additional end points included total patient treatment experience time and total pharmacy workflow time. A sample size of 22 patients was estimated to provide 90.7% power with two-sided alpha .05 to detect a difference of 70 minutes in the primary end point by treatment arm (IV v SC). RESULTS: Twenty-two patients were enrolled. The mean total patient time in the treatment chair was 61.8 minutes shorter with SC versus IV HP (22.5 v 84.3 minutes; P < .0001). The mean total patient treatment experience time (incorporating time spent waiting for treatment initiation and time spent in the treatment chair) was 81.8 minutes shorter for SC administration (96 v 177.8 minutes; P < .0001). The pharmacy workflow time was 78.2 minutes shorter for SC versus IV formulation (41 v 119.2 minutes; P < .0001). CONCLUSION: SC administration of HP shortened patient time burden by approximately 1 hour. SC drug administration can facilitate faster workflows for health care professionals and improve patients' breast cancer treatment experience.

PURPOSE: Long-term outcomes of patients with stage I human epidermal growth factor receptor 2 (HER2)–positive breast cancer receiving adjuvant trastuzumab emtansine (T-DM1) remain undefined, and prognostic predictors represent an unmet need. METHODS: In the ATEMPT phase II trial, patients with stage I centrally confirmed HER2-positive breast cancer were randomly assigned 3:1 to adjuvant T-DM1 for 1 year or paclitaxel plus trastuzumab (TH). Coprimary objectives were to compare the incidence of clinically relevant toxicities between arms and to evaluate invasive disease-free survival (iDFS) with T-DM1. Correlative analyses included the HER2DX genomic tool, multiomic evaluations of HER2 heterogeneity, and predictors of thrombocytopenia. RESULTS: After a median follow-up of 5.8 years, 11 iDFS events were observed in the T-DM1 arm, consistent with a 5-year iDFS of 97.0% (95% CI, 95.2 to 98.7). At 5 years, the recurrence-free interval (RFI) was 98.3% (95% CI, 97.0 to 99.7), the overall survival was 97.8% (95% CI, 96.3 to 99.3), and the breast cancer-specific survival was 99.4% (95% CI, 98.6 to 100). Comparable iDFS was observed with T-DM1 irrespective of tumor size, hormone receptor status, centrally determined HER2 immunohistochemical score, and receipt of T-DM1 for more or less than 6 months. Although ATEMPT was not powered for this end point, the 5-year iDFS in the TH arm was 91.1%. Among patients with sufficient tissue for HER2DX testing (n = 187), 5-year outcomes significantly differed according to HER2DX risk score, with better RFI (98.1% v 81.8%, hazard ratio [HR], 0.10, P = .01) and iDFS (96.3% v 81.8%, HR, 0.20, P = .047) among patients with HER2DX low-risk versus high-risk tumors, respectively. CONCLUSION: Adjuvant T-DM1 for 1 year leads to outstanding long-term outcomes for patients with stage I HER2-positive breast cancer. A high HER2DX risk score predicted a higher risk of recurrence in ATEMPT.

Anti-PD-L1 did not induce a proinflammatory immune response in B7-H4+ tumors. CD45+ cells were sorted from EMT6 tumors ± B7-H4 and subjected to NanoString gene expression analysis using the Mouse Pan-Cancer Immune Panel. A, Differentially expressed genes from CD45+ sorted cells from EMT6 control tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. B, Changes in immune gene sets between isotype and anti-PD-L1–treated tumors. C, Differentially expressed genes from CD45+ sorted cells from EMT6-B7-H4+ tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. D, Changes in immune gene sets between treatment groups of EMT6-B7-H4+ tumors. Data were analyzed by Wilcoxon rank-sum test. Genes with log2 fold change >0.5 or <−0.5 and P-value <0.01 were regarded as significant. n = 6 mice per group for all groups.

Supplemental Figure 7. B7-H4 and PD-L1 are not mutually exclusive on tumor cells. Data shown are reverse phase protein array expression data of B7-H4 and PD-L1 (SP142, 22C3, or Atezolizumab) from the I-SPY2 patient cohort. Samples were a mixture of TNBC and ER+ tumors and were treated with chemotherapy ± anti-PD-1. Depending on the antibody clone selected to detect PD-L1 expression, B7-H4 had no correlation, or a positive correlation to PD-L1 expression in these tumors. Data analyzed by spearman correlation and shown with linear regression best-fit line. n = 151 patients.

Anti-PD-L1 did not induce a proinflammatory immune response in B7-H4+ tumors. CD45+ cells were sorted from EMT6 tumors ± B7-H4 and subjected to NanoString gene expression analysis using the Mouse Pan-Cancer Immune Panel. A, Differentially expressed genes from CD45+ sorted cells from EMT6 control tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. B, Changes in immune gene sets between isotype and anti-PD-L1–treated tumors. C, Differentially expressed genes from CD45+ sorted cells from EMT6-B7-H4+ tumors when treated with anti-PD-L1 or isotype control and harvested 7 days after treatment. D, Changes in immune gene sets between treatment groups of EMT6-B7-H4+ tumors. Data were analyzed by Wilcoxon rank-sum test. Genes with log2 fold change >0.5 or <−0.5 and P-value <0.01 were regarded as significant. n = 6 mice per group for all groups.

Supplemental Figure 4. B7-H4 expression did not change quantity of infiltrating tumor immune cells in vivo regardless of anti-PD-L1 treatment. Untreated and anti-PD-L1 treated EMT6 tumors ± B7-H4 were dissociated to single cell suspension and subjected to flow cytometry with a 14 (for myeloid cells) or 17 (for T cells) color panel on a CyTEK Aurora. n = 3/group for control and 8/group for treated samples. Data were analyzed by One-way ANOVA with Sidak’s post-hoc test for multiple comparisons between the EMT6 control anti-PD-L1 and EMT6 B7-H4+ anti-PD-L1 treatment groups. One-way ANOVA was not significant between groups. Data were analyzed in GraphPad Prism v10.

B7-H4 expression is regulated by PI3K signaling. A, Spearman correlations in protein expression from RPPA data collected from patients from the ISPY2 trial. Key positive correlations are called out in blue and key negative correlations are called out in red. B, RPPA data show strong positive correlations between B7-H4 and AKT (pAKT-Ser 473) and strong negative correlations between B7-H4 and PTEN (Ser 380) regardless of tumor hormone receptor status. C, PI3K inhibitor (buparlisib) treatment for 72 hours robustly decreases B7-H4 expression in human MDA-MB-468 breast cancer cells, while MEK inhibitor (trametinib) has no effect. D, Likewise, in MMTV-neu cells, buparlisib reduces B7-H4 expression in dose-dependent manner after 72 hours. Data were analyzed by one-way ANOVA with Dunnet post hoc test for multiple comparisons, P < 0.0001 and P = 0.0005).

B7-H4 (VTCN1) is highly correlated with epithelial gene markers in mouse and human cells unlike other checkpoint ligands. A, In human breast cancer cell lines (CCLE), B7-H4 is the only checkpoint ligand positively correlated with epithelial markers and negatively correlated with mesenchymal markers. Data shown are Spearman correlations between genes. Significant correlations (P < 0.05) are indicated by asterisk. B, Genes downregulated during EMT are positively correlated with VTCN1 in human cell lines (CCLE). C, Murine cancer cell lines express B7-H4. D, B7-H4 is only expressed on epithelial EpCAM+ cells in murine and human cells. E, The MMTV-neu cell line with highest B7-H4 levels is comprised of phenotypically epithelial-like (abbreviated E) and mesenchymal-like (M) cells. F, In MMTV-neu cells, the CD44+ M cells do not express B7-H4 but all the EpCAM+ E cells do. G, MMTV-neu E and M cells express hallmark markers. H, Single-cell RNA sequencing of the heterogenous MMTV-neu cells confirms Vtcn1 is solely expressed in the epithelial cell population. I, A cohort of human tumors including both TNBC and ER+HER2− were stained for B7-H4, EpCAM, and CD44 by mIF. Data shown are log2 of % B7-H4+ tumor cells and include 132 samples with >1% B7-H4 expression (paired t test of transformed data).