Gillian L. Hirst

Dynamic biomarkers of therapy response are critical for precision oncology but often rely on serial tissue biopsies, which are invasive and not always feasible. In contrast, peripheral blood offers a minimally invasive, dynamic window into the evolving systemic immune landscape. Leveraging this, we performed RNA sequencing on 546 peripheral blood samples from 160 patients with high-risk stage II/III human epidermal growth factor receptor 2 (HER2)–negative breast cancer treated with either chemotherapy alone or in combination with immunotherapy (chemoimmunotherapy). Our analysis uncovered immune correlates of tumor subtype and treatment response. For example, samples from patients with triple-negative breast cancer exhibited elevated T cell receptor clonality and robust immune activation profiles. Among patients receiving chemoimmunotherapy, early responders demonstrated high baseline T cell receptor diversity, followed by rapid clonal expansion and activation of T cells after just one treatment cycle. We developed a multiparametric peripheral immune biomarker that integrated baseline and early on-treatment features to predict response to pembrolizumab, which was successfully validated in an independent cohort of 59 patients with breast cancer treated with neoadjuvant dostarlimab. These findings reveal the potential of blood-based immune monitoring to predict immunotherapy benefit, offering an accessible tool for tailoring treatment strategies in breast cancer.

Early-stage breast cancers resistant to neoadjuvant therapy (NAT), characterized by high residual cancer burden (RCB) after treatment, have an increased risk of metastatic recurrence. Here, we show that circulating tumor DNA (ctDNA) detected using a tumor-informed test (1) can improve risk stratification of patients with NAT-resistant tumors (RCB-II/RCB-III) and (2) predict response to NAT. Stratification using ctDNA status at pretreatment or post-NAT and ctDNA dynamics identified NAT-resistant tumors with a significantly decreased risk of metastatic recurrence. ctDNA clearance as early as week 3 across receptor subtypes predicted favorable responses to NAT, including immunotherapies. Interestingly, less than a fifth of patients with NAT-resistant tumors were ctDNA-positive post-NAT. Serial mutation profiling of NAT-resistant tumors revealed that patient-specific ctDNA assay variants remained detectable over time, including in tumors of patients ctDNA-negative post-NAT. Refining risk stratification for NAT-resistant tumors using ctDNA and understanding ctDNA shedding in these tumors could guide treatment decisions to prevent or delay metastatic recurrence.

Purpose:Pretreatment specimens from patients treated on the I-SPY2 neoadjuvant breast cancer trial were studied to identify prespecified biomarkers associated with response to the regimen of paclitaxel, the anti–type I insulin-like growth factor receptor (IGF-1R) antibody ganitumab, and metformin (PGM) followed by doxorubicin and cyclophosphamide (AC) compared with control therapy (paclitaxel followed by AC). The primary endpoint of this trial is pathologic complete response (pCR).Experimental Design:One hundred six patients treated with PGM and 119 contemporary controls were evaluated using laser capture microdissection and reverse-phase protein array to evaluate 32 prespecified potential predictive biomarkers in the IGF-1R pathway and 109 additional exploratory endpoints.Results:Total levels of IGF-1R were poorly correlated with phosphorylated IGF-1R/insulin receptor (IR). Higher levels of phosphorylated IGF-1R/IR were associated with an increased likelihood of obtaining pCR, especially in the hormone receptor (HR)–positive subgroup. Markers of immune response also showed an association with pCR but differed between HR+ and HR− subgroups. In HR− tumors, phospho-STAT1 Y701 and low levels of phospho-p27 associated with pCR. These relationships were not observed in patients treated with control chemotherapy.Conclusions:Activation status of IGF-1R/IR associated with increased pCR to PGM in HR+ breast cancers. Immune activation markers were also associated with response in HR+ and HR− subgroups. Thus, IGF-1R may directly regulate tumor biology and associate with immune response to therapy.

Supplementary Tables 1 from Immune and Growth Factor Signaling Pathways Are Associated with Pathologic Complete Response to an Anti–Type I Insulin-like Growth Factor Receptor Regimen in Patients with Breast Cancer

Supplementary Figure 1 from Immune and Growth Factor Signaling Pathways Are Associated with Pathologic Complete Response to an Anti–Type I Insulin-like Growth Factor Receptor Regimen in Patients with Breast Cancer

Purpose: Pretreatment specimens from patients treated on the I-SPY2 neoadjuvant breast cancer trial were studied to identify prespecified biomarkers associated with response to the regimen of paclitaxel, the anti–type I insulin-like growth factor receptor (IGF-1R) antibody ganitumab, and metformin (PGM) followed by doxorubicin and cyclophosphamide (AC) compared with control therapy (paclitaxel followed by AC). The primary endpoint of this trial is pathologic complete response (pCR). Experimental Design: One hundred six patients treated with PGM and 119 contemporary controls were evaluated using laser capture microdissection and reverse-phase protein array to evaluate 32 prespecified potential predictive biomarkers in the IGF-1R pathway and 109 additional exploratory endpoints. Results: Total levels of IGF-1R were poorly correlated with phosphorylated IGF-1R/insulin receptor (IR). Higher levels of phosphorylated IGF-1R/IR were associated with an increased likelihood of obtaining pCR, especially in the hormone receptor (HR)–positive subgroup. Markers of immune response also showed an association with pCR but differed between HR+ and HR− subgroups. In HR− tumors, phospho-STAT1 Y701 and low levels of phospho-p27 associated with pCR. These relationships were not observed in patients treated with control chemotherapy. Conclusions: Activation status of IGF-1R/IR associated with increased pCR to PGM in HR+ breast cancers. Immune activation markers were also associated with response in HR+ and HR− subgroups. Thus, IGF-1R may directly regulate tumor biology and associate with immune response to therapy.

This study investigates the association between peripheral blood immunological features and immunotherapy response in breast cancer. We generated and analyzed RNAseq data from 546 blood samples of patients with high-risk stage II/III HER2-negative breast cancer enrolled in the I-SPY2 trial and identified peripheral immune signatures associated with tumor characteristics and immunotherapy response. Triple negative breast cancer (TNBC) patients showed higher T cell receptor (TCR) clonality and immune activation signatures. Responders to the chemotherapy + pembrolizumab regimen had high baseline TCR diversity, with TNBC responders experiencing T cell clonal expansion and activation after treatment. A logistic regression model based on immunological features before and early-on-treatment predicted response to pembrolizumab. The model was validated in an independent cohort of patients treated with dostarlimab in the neoadjuvant setting. We report the potential of peripheral blood-derived gene expression tests to predict immunotherapy benefit, guiding personalized treatment in breast cancer with a minimally invasive approach.

Ductal carcinoma in situ (DCIS) incidence has risen rapidly with the introduction of screening mammography, yet it is unclear who benefits from both the amount and type of adjuvant treatment (radiation therapy, endocrine therapy) versus what constitutes over-treatment. Our goal was to identify the effects of adjuvant radiation therapy, or endocrine ± radiation therapy versus breast conservation surgery alone in a large multi-center registry of retrospective DCIS cases (N = 1916) with median follow up of 7.0 (IQR: 8.43) years. We show that patients with DCIS who took less than 2 years of adjuvant endocrine therapy alone have a similar second event rate as breast conservation surgery. However, patients who took more than 2 years of endocrine therapy show a significantly reduced second event rate, similar to those who received either radiation or combined endocrine + radiation therapy, which was independent of age, tumor size, grade, or period of diagnosis. This highlights the importance of endocrine therapy duration for risk reduction.

PURPOSE: Neoadjuvant immunotherapy (IO) has become the standard of care for early-stage triple-negative breast cancer (TNBC), but not yet for other subtypes. We previously developed a clinical-grade mRNA-based immune classifier (ImPrint) predicting response to IO that is now being used in I-SPY2.2 as part of the response predictive subtypes. We report the performance of ImPrint in hormone receptor–positive and human epidermal growth factor receptor 2–negative (HR+HER2–) patients from five IO arms. METHODS: A total of 204 HR+HER2– (MammaPrint high-risk) patients from five IO arms (anti–PD-1, anti–PD-L1/poly [ADP-ribose] polymerase inhibitor combination, anti–PD-1/toll-like receptor 9 dual-IO combination, and anti–PD-1 ± lymphocyte activation gene 3 dual-IO combination) and 191 patients from the chemotherapy-only control arm were included in this analysis. Patients were classified as ImPrint+ (likely sensitive) versus ImPrint– (likely resistant), using pretreatment mRNA. Performance of ImPrint for predicting pathologic complete response (pCR) to IO-containing arms was characterized and compared with tumor grade (III), MammaPrint (ultra) High2 risk (MP2), and estrogen receptor (ER)-low (ER ≤ 10%). RESULTS: Overall, the pCR rate across the five IO arms was 33%. 26% of HR+HER2– patients were ImPrint+, and pCR rates with IO were 75% in ImPrint+ versus 17% in ImPrint–, with the highest pCR rate >90% in a dual-IO arm. In the control arm, pCR rates were 33% in ImPrint+ and 8% in ImPrint–. Tumor grade (III), MP2, and ER-low showed pCR rates in IO of 45%, 56%, and 63%, respectively, with lower pCR odds ratios (OR < 7.5) compared with ImPrint (OR = 14.5). CONCLUSION: Using an accurate selection strategy, HR+HER2– patients could achieve pCR rates similar to what is seen with best neoadjuvant therapies in TNBC and HER2+ (ie, pCR rate >65%-70%). ImPrint, an Food and Drug Administration IDE-enabled assay, may represent a way to identify HR+HER2– patients for IO that best balances likely benefit versus risk of serious immune-related adverse events.

Purpose:The MammaPrint (MP) prognostic assay categorizes breast cancers into high- and low-risk subgroups, and the high-risk group can be further subdivided into high-1 (MP-H1), and very high-risk high-2 (MP-H2). The aim of this analysis was to assess clinical and molecular differences between the hormone receptor–positive (HR+)/HER2-negative MP-H1, -H2, and triple-negative (TN) MP-H1 and -H2 cancers.Experimental Design:Pretreatment gene expression data from 742 HER2-negative breast cancers enrolled in the I-SPY2 neoadjuvant trial were used. Prognostic risk categories were assigned using the MP assay. Transcriptional similarities across the four receptor and prognostic groups were assessed using principal component analyses and by identifying differentially expressed genes. We also examined pathologic complete response rates and event-free survivals by risk group.Results:Principal component analysis showed that HR+/MP-H2 tumors clustered with TN/MP-H2 cancers. Only 125 genes showed differential expression between the HR+/MP-H2 and TN/MP-H2 cancers, whereas 1,465 genes were differentially expressed between HR+/MP-H2 and -H1. Gene set analysis revealed similarly high expression of cell cycle, DNA repair, and immune infiltration–related pathways in HR+/MP-H2 and TN/MP-H2 cancers. HR+/MP-H2 cancers also showed low estrogen receptor–related gene expression. Pathologic complete response rates were similarly high in TN/MP-H2 and HR+/MP-H2 cancers (42% vs. 30.5%; P = 0.11), and MP-H2 cancers with residual cancer had similarly poor event-free survival regardless of estrogen receptor status.Conclusions:In conclusion, HR+/MP-H2 cancers closely resemble TN breast cancers in transcriptional and clinical features and benefit from similar treatment strategies.