Search

Background: The introduction of immune checkpoint inhibitors (ICI) to standard neoadjuvant chemotherapy regimens has been shown to significantly improve outcomes in patients with triple negative breast cancer and is being investigated for high-risk hormone receptor-positive (HR+)/human epidermal growth factor-2 negative (HER2-) breast cancer. Preclinical evidence suggests radiation therapy (RT) can stimulate intra-tumoral T cell infiltration and enhance the expression and immune detection of tumor-specific neoantigens. This phase II pilot randomized study (NCT04443348) aims to evaluate the safety and efficacy of two different doses of preoperative primary tumor RT boost when combined with neoadjuvant pembrolizumab, then followed by standard neoadjuvant chemotherapy. Dual co-primary endpoints include determining the pathologic complete response (pCR) rate in the non-irradiated and pathologically confirmed metastatic axillary lymph node(s) in each treatment arm and quantifying tumor-infiltrating T lymphocytes in on-treatment (C1D14) tumor biopsies. We hypothesize that high-dose RT will increase the proportion of tumors with high T cell infiltration (i.e., top quartile) from 25% to 55%. Secondary endpoints include measuring residual cancer burden, evaluating tolerability of the regimen, and assessing quality of life. Exploratory endpoints include evaluation of treatment-associated changes in the tumor immune microenvironment, circulating immune cell analyses, and circulating tumor DNA kinetics. Methods: The study plans to enroll 128 participants with either triple negative (n=80) or high-risk HR=/HER2- (n=48) breast cancer who will be randomized to receive no, low (9 Gy), or high (24 Gy) dose of preoperative RT boost, after which 24 participants of either breast cancer subtype will be enrolled to an exploratory high dose proton therapy boost cohort. The eligibility criteria include patients who have biopsy-proven, axillary lymph node-positive breast cancer that is either triple negative (defined as ER< 10%, PR< 10%, and HER2-negative) or high-risk HR+/HER2- (grade III or having a high-risk genomic assay score). Study treatment is given in 6-week cycles, with 400 mg Pembrolizumab given on day 1 of each cycle. For those participants randomized to receive a preoperative RT boost, treatment is delivered in 3 fractions (3 × 3 Gy or 3 × 8 Gy) over consecutive business days, where one of the fractions is given on the same day as C1D1 Pembrolizumab. Standard neoadjuvant chemotherapy begins on C1D15 with paclitaxel (plus carboplatin for triple negative) administered weekly for 12 weeks, and then starting on C3D15, dose-dense doxorubicin/cyclophosphamide is administered every 2 weeks for 8 weeks. Following neoadjuvant treatment, participants will receive standard breast surgery (including removal of the pathologically confirmed metastatic lymph node) followed by adjuvant pembrolizumab, radiation therapy, and standard-of-care systemic therapy as clinically indicated. Tissue samples from the primary tumor and biopsy-proven lymph node are taken at baseline, C1D14, and at the time of surgery. There are eleven blood collection timepoints throughout the neoadjuvant and adjuvant settings. Participants will be followed for 2 years after surgery to assess safety and durability of responses. Results: This study has accrued 12 participants to date, including 10 with triple negative breast cancer and 2 with high-risk HR+/HER2- breast cancer. Formal results for this study are forthcoming, as the trial is actively accruing at 6 institutions, with plans to open at 3 more within the year. For persons with a specific interest in this trial, please contact Joseph Connolly, Multi-Center Coordinator, at jconnolly28@mgh.harvard.edu. Citation Format: Joseph J. Connolly, Laura M. Spring, Alphonse G. Taghian, Michele Gadd, Laura Warren, Ana C. Garrido-Castro, Tari King, Elizabeth A. Mittendorf, Jose P. Leone, Dana L. Casey, Lisa Carey, Tiffany A. Traina, Yara Abdou, Atif Khan, George Plitas, Jean Wright, Cesar Augusto Santa-Maria, Lisa Jacobs, Rachel Blitzblau, E Shelley Hwang, Carey Anders, Ian Krop, Antonio C. Wolff, Alastair M. Thompson, Elyssa Denault, Gaorav Gupta, Alice Ho. TBCRC-053: P-RAD: A Randomized Study of Preoperative Chemotherapy, Pembrolizumab and No, Low or High Dose RADiation in Node-Positive, HER2-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 OT3-15-01.

Introduction: Despite the recent overall improvement in survival for patients with breast cancer, racial disparities in outcomes persist. While studies have demonstrated that socioeconomic factors and access to treatment play a role, differences in tumor biology may also contribute. Black women are significantly more likely to develop triple negative breast cancer (TNBC), the deadliest of the breast cancer subtypes, with more TNBC patients progressing to incurable, metastatic disease than patients with any other breast cancer subtype. Studies have demonstrated that TNBC patients who achieve a pathologic complete response (pCR), defined as no residual invasive cancer in the breast or lymph nodes after neoadjuvant chemotherapy (NAC), have improved survival. We hypothesize that rates of pCR and overall survival (OS) in patients with TNBC may differ by race/ethnicity, which may account in part for the disparities in outcomes observed. Methods: Adult female patients with stage I-III TNBC diagnosed in 2010-2019 who received NAC followed by surgery were identified from the National Cancer Database (NCDB). Race/ethnicity was defined as Hispanic (H), Non-Hispanic White (NHW), Non-Hispanic Black (NHB), Non-Hispanic Asian (NHA), and Non-Hispanic Other (NHO). pCR was defined as no invasive cancer in either the breast or axilla (ypT0/is,N0) at surgery. Logistic regression was used to estimate the association of race/ethnicity with achievement of pCR after adjustment for covariates. Unadjusted OS was estimated using the Kaplan-Meier method, and the log-rank test was used to compare groups. Cox Proportional Hazards models were used to estimate the association of race/ethnicity and achievement of pCR with OS after adjustment for covariates. Additional interaction and subgroup analyses were also conducted. Results: Of the 40,890 patients identified, 29.8% (n=12,173) demonstrated pCR after NAC. The unadjusted 5-year OS rates for those who achieved pCR were significantly higher compared to patients with no pCR (0.917, 95% CI 0.911-0.923 vs 0.667, 95% CI 0.661-0.673, log-rank p< 0.001). Hispanic patients were more likely to achieve pCR (OR 1.19, 95% CI 1.08-1.31, p=0.001), and NHB patients were less likely to achieve pCR (OR 0.89, 95% CI 0.83-0.95, p=0.001) compared to NHW, even after adjustment. Unadjusted OS was also notably lower for NHB patients compared to every other race group (5-year OS rate: NHB 0.709 vs NHW 0.746 vs NHO 0.771 vs H 0.772 vs NHA 0.816, log-rank p< 0.001); however, this difference did not persist after adjustment for patient and disease factors, including achievement of pCR. Interval from diagnosis to start of chemotherapy (OR 0.95, 95% CI 0.94-0.96, p< 0.001) and duration after chemotherapy start to surgery (OR 1.02, 95% CI 1.02-1.03, p< 0.001) were associated with the odds of achieving pCR. Overall, the effect of achieving pCR on OS did not differ by race/ethnicity (interaction p=0.10). Discussion: Achieving pCR after NAC in patients with TNBC is associated with a significant improvement in OS. Yet, rates of pCR appear to differ based on race/ethnicity, with NHB patients demonstrating significantly lower rates of pCR than NHW patients, which may contribute to the disparities in survival outcomes observed. In addition to addressing socioeconomic factors and access to treatment, further research examining whether biological differences exist based on race that influence response of TNBC to current standard therapies is essential for improving survival outcomes for this disproportionately affected patient population. Citation Format: Hannah E. Woriax, Samantha M. Thomas, Jennifer K. Plichta, Laura H. Rosenberger, Astrid Botty van de bruele, Akiko Chiba, Gayle DiLalla, Carolyn Menendez, E Shelley Hwang, Maggie L. DiNome. The effect of race on pathologic complete response rates and overall survival in patients with 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 P1-06-02.

Background: Metastasis is the leading cause of cancer related deaths in breast cancer patients. Lymphovascular invasion represents one of the earliest stages of metastasis wherein the cells are introduced to a very different and distinct microenvironment. Methods: We leveraged spatial techniques developed for limited specimens in archival tissue to study patient matched cross-sectional tumor samples from different stages of breast neoplasia including normal breast, ductal carcinoma in situ (DCIS), primary invasive carcinoma (IBC), lymphovascular invasion (LVI) and regional lymph node metastasis. We selected a set of 21 patients with ER+ breast cancer to generate cross-sectional samples of each of these stages, for a total of 331 samples. The areas of LVI were identified by a combination of H&E review and immunohistochemistry for podoplanin. We performed smart-3SEQ for gene expression profiling and light pass whole genome sequencing for DNA copy number alterations. Results: We profiled the spectrum of neoplasia for transcriptome-wide gene expression. Principal component analysis of all 252 DCIS, LVI, IBC, or metastasis samples using the top 500 genes with the highest variance demonstrated that clustering was roughly based on the diagnostic stage (i.e. DCIS, LVI, IBC, or metastasis). Differential gene expression profiling identified thousands of genes increased or decreased in expression across the transitional stages with the largest change in gene expression being the transition from normal breast to DCIS, dominated by gene expression down regulation. We next performed NMF clustering on 62 samples of LVI from 18 cases and identified two patterns of gene expression which define two subgroups. Gene ontology analysis revealed that one cluster was associated with increased proliferation and metabolism, whereas the second cluster was dominated by an immune response. When we analyzed the immune and proliferative LVI subgroups separately, we found that the immune profiles in the patient matched IBC and LVI samples from the LVI Immune cluster were similar, whereas the immune profiles in the patient matched IBC and LVI samples from the Proliferative cluster were significantly different. At the LVI stage, all immune cell populations estimated by CibersortX were decreased in the Proliferative LVI cluster. These changes were validated using immunofluorescence for proliferation (Ki67), T cells (CD3) and macrophages (CD68) on the same samples. Using the LVI centroids, we built a model that could predict the same clusters in the METABRIC IBC. Kaplan-Meier analysis showed a significant difference between groups, with the Proliferative-like IBC group having a worse prognosis than the Immune-like IBC group. Conclusions: We observed a dichotomy at the LVI stage with a more proliferative cluster that may escape the immune response and an immune cluster which has a microenvironment with a similar pattern to its primary IBC. The recognition of two groups of LVI, differing in immune association and proliferation, raises the possibility that the risk of metastasis could be different in these two groups, leading to different biological pathways of progression. Citation Format: Belén Rivero-Gutiérrez, Diego Mallo, Almudena Espín-Pérez, Sujay Vennam, Chunfang Zhu, Sushama Varma, Greg Scott, Joseph Foley, E Shelley Hwang, Carlo Maley, Robert West. Characterization of the lymphovascular invasion microenvironment reveals immune response dichotomy [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 PD2-09.

Ductal carcinoma in situ (DCIS) is characterized by inter-tumor heterogeneity that poses a therapeutic challenge due to its unpredictable recurrence and progression to invasive breast cancer (IBC). Recent publications have implicated the crucial role of the tumor microenvironment particularly stromal differences between DCIS patients who progress to IBC (progressors) and those that do not (non-progressors). However, spatial regulation of the collagen proteome has yet to be investigated in the context of disease progression in DCIS. In this study, we hypothesized that the collagen proteome was significantly altered between DCIS and IBC and that differentiating collagen peptide signatures could be related to clinical outcomes. Our initial studies investigated collagen peptide signatures in lumpectomies (n=13) annotated as DCIS, DCIS and invasive ductal carcinoma (IDC), or IDC only. We leveraged our previously published method for spatial imaging of collagen proteomics on tissue to report collagen types and collagen post-translational modifications including 40 other extracellular matrix (ECM) proteins involved in the regulation of collagen fibers. Over 1000 peaks were found to be linked to annotated pathologies or adjacent regions. Initial comparison of DCIS to IDC lesions demonstrated 63 differentially expressed peaks between these regions by unpaired, two-tailed t-test (p< 0.001). Image segmentation of the 315,541 pixels demonstrated 16 high-level hierarchical groups designating unique spatially localized ECM proteomic groups. Notably, these groups overlaid with histopathological features and pathological annotations. Next, we investigated collagen peptide signatures in a subset of DCIS samples from the Resource of Archival Breast Tissue (RAHBT) (n=37). Samples were histologically diverse within the tissue microarrays, with cribriform, micropapillary, papillary, solid, and comedo necrosis architectural patterns. In our preliminary analysis, we found two peptide peaks that could distinguish the solid subtype (n=22) from comedo necrosis (n=4) and one peak that could discriminate between the cribriform (n=8) and solid subtype (n=22) by area under the receiver operating curve (AUROC)≥0.75 and Wilson/Brown t-test (p< 0.05). Evaluated per clinical outcome, four ECM peptides showed significantly different peak intensities in progressors with IBC recurrence (n=7) compared to non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p< 0.05). One peptide had significantly different peak intensities between progressors with contralateral IBC recurrence and DCIS recurrence (n=5) and non-progressors (n=26) (AUROC≥0.75; Wilson/Brown t-test p< 0.05). Overall, the data suggest that unique collagen signatures in DCIS could be useful for understanding recurrence and progression to IBC. Further investigation of the spatial distribution of the collagen proteome within DCIS pathologies and relative to clinical outcome is warranted. Citation Format: Taylor S. Hulahan, Elizabeth N. Wallace, Siri H. Strand, Graham A. Colditz, E Shelley Hwang, Robert West, Laura Spruill, Jeffrey Marks, Richard R. Drake, Peggi M. Angel. Unique Collagen Peptide Signatures between Ductal Carcinoma in Situ and Invasive Breast Cancer by Mass Spectrometry Tissue Imaging [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 P2-21-03.

Background: Microscopically tumor-free lumpectomy margins are critical for safe breast conserving surgery. With current tools, 15%-25% of lumpectomies have positive margins that require second surgical procedures and increase cost and patient discomfort. Additionally, current lumpectomy margin assessment techniques show poor performance in predicting residual disease at re-excision, with a PPV of 35%. Better detection tools are needed to identify residual cancer during the initial lumpectomy and reduce second operations. LUM015 is a protease-activated fluorescent imaging agent that accumulates in tumor cells and tumor associated macrophages after preoperative intravenous injection. The LUM Imaging System visualizes activated LUM015 in the lumpectomy cavity via a hand-held wide field detector and proprietary tumor detection software. This system has been tested in multiple single-site studies and two prospective multi-site studies enrolling >600 patients, and demonstrated successful detection of residual lumpectomy cavity tumor. Initial studies excluded the approximately 20% of patients receiving neoadjuvant therapy. Patchy tumor cell death with preoperative therapy can leave small, multifocal deposits of tumor invisible on pre-operative imaging and not palpable or visible during surgery. We now evaluate the LUM Imaging System in patients with and without neoadjuvant therapy. Trial Design and Specific Aims: This prospective, multi-center study at 6 US sites tests the LUM Imaging System in lumpectomy surgery after neoadjuvant therapy to evaluate potential impact of treatment-related tissue changes and tumor cell death on tumor detection algorithms. An initial cohort of 10 patients address the objective of algorithm development. A second cohort of 104 patients will further evaluate the feasibility of the LUM Imaging System after neoadjuvant therapy. A third cohort will enroll 208 patients who have not received neoadjuvant therapy. All cohorts are evaluated for safety and for reduction in residual tumor after LUM Imaging System guidance compared to standard of care lumpectomy. After excision of the main lumpectomy specimen, patients are randomized 3:1 to device or control arms. In the device arm, the cavity is imaged and margins with LUM015 signal are excised. Final comprehensive shaved margins are removed in both arms to evaluate extent of residual disease after the use of the LUM Imaging System or after standard lumpectomy. No LUM Imaging is performed in the control arm, however, all patients are injected with LUM015 to evaluate drug safety. Patient reported outcomes assessing re-excision concerns, breast appearance and preferences for treatment type are collected. Eligibility Criteria: This study seeks to enroll women 18 and older with histologically confirmed primary invasive breast cancer (IBC), ductal carcinoma in situ (DCIS) or a combination of IBC/DCIS undergoing a lumpectomy for their breast malignancy who have received any form of neoadjuvant treatment prior to surgery (cohorts 1 and 2) or who have not received any therapy prior to lumpectomy (cohort 3). Patients allergic to polyethylene glycol or intravenous contrast agents are excluded. Use of blue node mapping dyes before imaging with the LUM015 is not allowed per study protocol. Accrual and Study Progress: Cohort 1 has completed enrollment and interim analysis. No new risks specific to the neoadjuvant population were identified. LUM015 fluorescent signals measured in neoadjuvant patients were within the expected range, and no changes to the tumor detection algorithm were required. Cohorts 2 and 3 have enrolled a total of 38 patients. This trial is registered as NCT04440982. The NIH funds this study through a R01 grant issued to Massachusetts General Hospital. Citation Format: Irene L. Wapnir, Kelly K. Hunt, E Shelley Hwang, Kate Smith, Peter Blumencranz, David Carr, Jorge Ferrer, Heidi Santa Cruz, Alexandra Webster, Julia Shanno, Alexander Pogrebinsky, Manna Chang, Barbara L. Smith. Feasibility study to evaluate performance of the LUM Imaging System for intraoperative detection of residual tumor in breast cancer patients receiving and not receiving neoadjuvant therapy [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 OT2-07-01.

PURPOSE To examine incremental values of estrogen receptor (ER) status, body mass index (BMI), menopausal status, and a previously reported multi-gene classifier over commonly used clinical factors (i.e. age, tumor grade, comedonecrosis, surgical margins, and treatment) in predicting risk of any ipsilateral recurrence (IR) event within five years after DCIS diagnosis. METHODS A derivation cohort consisted of participants in the Translational Breast Cancer Research Consortium (TBCRC) 038, a retrospective multicenter cohort study in women undergoing surgical resection for DCIS between 01/01/1998 and 02/29/2016 (n=216). The validation cohort, the Repository of Archival Human Breast Tissue (RAHBT) at Washington University School of Medicine, provided cases meeting the same eligibility criteria as TBCRC038 (n=97). Participants in both cohorts had RNA-seq data and either developed IR 1-5y after initial DCIS diagnosis or were free from subsequent breast events for at least five years. The previously reported 812-gene classifier had been developed from a subset of the TBCRC038 samples using a negative-binomial regression model to identify differentially expressed genes in the primary tumor associated with subsequent recurrence events. This classifier has been shown to be highly correlated with 5-year invasive, DCIS, and all breast cancer events, and validated in the RAHBT cohort. Cox proportional hazards regression was used to estimate hazard ratios (HRs) of IR in the TBCRC038 cohort (76 with IR). The clinical score was developed using clinical predictors (aforementioned clinical factors and ER) and their regression coefficients from the model with the maximum predictive accuracy (e.g. c-index) and the minimum number of predictors; the summary score integrated the clinical score and multi-gene classifier. Predictive performance of both clinical and summary scores was validated in the RAHBT cohort (20 with IR). RESULTS In the TBCRC cohort derivation set, we used a multivariable model based on clinical factors alone (clinical score) and found that ER status, but not BMI or menopausal status, was independently associated with a higher IR risk (HR=2.06, 95% CI 1.18-3.58). Adding the multi-gene classifier to the clinical factors-based model (summary score) in the TBCRC038 test set increased predictive accuracy (c-index 0.68 to 0.70), with the genomic classifier-adjusted HR of 14.96 (95% CI 8.64-25.91). The summary score had higher predictive performance for IR risk than clinical score alone (c-index 0.82 vs. 0.70). In the RAHBT validation samples, model performance was similarly improved using summary scores clinical factors-based model plus multigene classifier as compared to clinical scores along (c-index 0.74 vs. 0.58). CONCLUSION Combining clinical factors and a multigene classifier provided more accurate risk estimates of IR within five years after excision of DCIS than clinical factors alone. Figure 1. Observed and predicted recurrence-free survival in the first five years after initial DCIS diagnosis in the RAHBT validation cohort, by risk groups defined by clinical scores (left) and clinical score plus multigene classifier (right). Citation Format: Ying Liu, Siri H. Strand, Lorraine King, Bryan Harmon, Fergus J. Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F. McAuliffe, Jeffrey Marks, Carlo Maley, Robert West, E Shelley Hwang, Graham A. Colditz. Using clinical characteristics and molecular markers to predict the risk of subsequent ipsilateral breast events after excision of DCIS [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 P1-07-02.

Background: The predictive and prognostic value of the 21-gene recurrence score (RS) has emphasized the importance of tumor biology and minimized the credence of a limited (1-3 positive) nodal burden. The practice changing results of RxPonder demonstrated that post-menopausal women with 1-3 positive lymph nodes (pN1) and a RS of ≤25 did not necessarily benefit from adjuvant chemotherapy. Given that RS influences adjuvant therapy decision-making more significantly than nodal status, it is unclear whether axillary staging with sentinel lymph node biopsy (SLNB) has a continued role in the surgical care of post-menopausal patients otherwise presenting with early stage, clinically node negative (cN0) hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) invasive breast cancer. In this context, the de-escalation of axillary staging, especially in the presence of a low RS, is an area of active investigation. To help elucidate this further, we sought to estimate the association of RS with pathologic nodal stage. Methods: Using the 2004-2019 National Cancer Database (NCDB) Patient User File (2022 release), we evaluated the association of RS with the incidence of pN0, pN1 and pN2-3 disease. Only female patients diagnosed who were age 50 and older with HR+/HER2- invasive breast cancer were eligible, and only those presenting with cT1-T2N0 who underwent upfront surgery with a SLNB comprised our study population. Those with Oncotype DX testing performed with an available RS were included. Given the limitations within the dataset, age 50 and over was selected as a surrogate for post-menopausal status. Categorical variables were compared between RS groups (≤25 vs. >25) using chi-square tests and continuous variables were compared using t-tests. A logistic regression analysis was performed to estimate the association between RS (≤25 vs. >25) and nodal burden (pN2-3 vs pN0-1). Results: There were 151,447 patients with an invasive breast cancer diagnosis between 2015 and 2019 who met inclusion criteria. The average age at diagnosis was 64.1 (IQR 58-69) and almost 75% of tumors displayed ductal histology. There were 130,568 (86.2%) patients with a RS≤25 and 20,879 (13.8%) with a RS >25. On final pathology, 85.2% were pN0 and 14.8% were pN1-3. For those with a RS ≤25, 84.9% were pN0, 14.8% were pN1 and 0.3% were pN2-3. For those with a RS >25, 86.8% were pN0, 12.9% were pN1 and 0.3% were pN2-3. Overall, 14.5% demonstrated pN1 disease, of which 12.3% yielded a RS >25. Of the 461 patients with pN2-3 disease for whom RS was available, 12.4% (57 patients) had RS >25. After adjustment, RS >25 was associated with reduced incidence of pN2-3 compared to pN0-1 (OR=0.64, 95% CI 0.47-0.87, p=0.004). Conclusion: In this population of post-menopausal patients with cT1-T2N0, HR+/HER2- invasive breast cancer and an available RS, almost 86% displayed pN0 or pN1 disease in conjunction with a RS ≤25. Based on the current available literature, less than 5% of cT1-2N0 patients are thought to harbor >pN1 disease. These data add further support, suggesting that this patient population is unlikely to harbor a higher than limited nodal burden given a clinically negative axilla. Though less than 0.5% of the studied patient population demonstrated pN2-3 disease, an important caveat to make is that these patients would not have met criteria for RS, and it’s likely this low number reflects the absence of testing. Given that RS has not been validated for this higher nodal stage, we cannot make recommendations to omit axillary surgery in this cohort of patients. The data presented here provides further rationale for the two large prospective studies addressing whether SLNB could be eliminated in patients with otherwise small HR+/HER2- tumors which are currently ongoing. Table 1: Pathologic Nodal Staging Based on Recurrence Score Citation Format: Astrid Botty van den Bruele, Morgan Paul, Samantha M. Thomas, Sarah L. Sammons, Maggie L. DiNome, Jennifer K. Plichta, Akiko Chiba, Laura H. Rosenberger, E Shelley Hwang. Low 21-Gene Recurrence Score Is Not Associated with a High Axillary Nodal Burden in Post-Menopausal Women Presenting with a Clinically Negative Axilla [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 P3-05-20.

Previous studies have identified racial-ethnic differences in the diagnostic patterns and recurrence outcomes of women with phyllodes tumors (PT). However, these studies are generally limited in size and generalizability. We therefore sought to explore racial-ethnic differences in age, tumor size, subtype, and recurrence in a large US cohort of women with PT.We performed an 11-institution retrospective review of women with PT from 2007 to 2017. Differences in age at diagnosis, tumor size and subtype, and recurrence-free survival according to race-ethnicity.Women of non-White race or Hispanic ethnicity were younger at the time of diagnosis with phyllodes tumor. Non-Hispanic Other women had a larger proportion of malignant PT. There were no differences in recurrence-free survival in our cohort.Differences in age, tumor size, and subtype were small. Therefore, the workup of young women with breast masses and the treatment of women with PT should not differ according to race-ethnicity. These conclusions are supported by our finding that there were no differences in recurrence-free survival.

Background Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome may contribute to matrix metalloprotease (MMP) dysregulation given their key role in invasion, which is the first step of the metastatic process.Methods We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with invasion. We stably disrupted the CCCTC-Binding Factor (CTCF) binding site of a single insulator element in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. Our findings were then also tested in a ductal carcinoma in situ (DCIS) cohort.Results We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was strongly associated with worse relapse-free (HR = 1.57 [1.06 − 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 − 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability. Finally, we observed that the imbalance in the MMP expression predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p Conclusion Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

Supplementary Table 1

Ductal carcinoma in situ (DCIS) is a biologically heterogenous entity with uncertain risk for invasive ductal carcinoma (IDC) development. Standard treatment is surgical resection often followed by radiation. New approaches are needed to reduce overtreatment. This was an observational study that enrolled patients with DCIS who chose not to pursue surgical resection from 2002 to 2019 at a single academic medical center. All patients underwent breast MRI exams at 3- to 6-month intervals. Patients with hormone receptor–positive disease received endocrine therapy. Surgical resection was strongly recommended if clinical or radiographic evidence of disease progression developed. A recursive partitioning (R-PART) algorithm incorporating breast MRI features and endocrine responsiveness was used retrospectively to stratify risk of IDC. A total of 71 patients were enrolled, 2 with bilateral DCIS (73 lesions). A total of 34 (46.6%) were premenopausal, 68 (93.2%) were hormone-receptor positive, and 60 (82.1%) were intermediate- or high-grade lesions. Mean follow-up time was 8.5 years. Over half (52.1%) remained on active surveillance without evidence of IDC with mean duration of 7.4 years. Twenty patients developed IDC, of which 6 were HER2 positive. DCIS and subsequent IDC had highly concordant tumor biology. Risk of IDC was characterized by MRI features after 6 months of endocrine therapy exposure; low-, intermediate-, and high-risk groups were identified with respective IDC rates of 8.7%, 20.0%, and 68.2%. Thus, active surveillance consisting of neoadjuvant endocrine therapy and serial breast MRI may be an effective tool to risk-stratify patients with DCIS and optimally select medical or surgical management.Significance:A retrospective analysis of 71 patients with DCIS who did not undergo upfront surgery demonstrated that breast MRI features after short-term exposure to endocrine therapy identify those at high (68.2%), intermediate (20.0%), and low risk (8.7%) of IDC. With 7.4 years mean follow-up, 52.1% of patients remain on active surveillance. A period of active surveillance offers the opportunity to risk-stratify DCIS lesions and guide decisions for operative management.

Supplementary Methods, Figures 1-10, Tables 1-8 from Leukocyte Complexity Predicts Breast Cancer Survival and Functionally Regulates Response to Chemotherapy

Purpose:We examined the prognostic impact of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) detected at the time of surgery in 742 untreated patients with early breast cancer.Experimental Design:DTCs in bone marrow were enumerated using the EPCAM-based immunomagnetic enrichment and flow cytometry (IE/FC) assay. CTCs in blood were enumerated either by IE/FC or CellSearch. Median follow-up was 7.1 years for distant recurrence-free survival (DRFS) and 9.1 years for breast cancer–specific survival (BCSS) and overall survival (OS). Cox regressions were used to estimate hazard ratios for DRFS, BCSS, and OS in all patients, as well as in hormone receptor–positive (HR-positive, 87%) and HR-negative (13%) subsets.Results:In multivariate models, CTC positivity by IE/FC was significantly associated with reduced BCSS in both all (n = 288; P = 0.0138) and HR-positive patients (n = 249; P = 0.0454). CTC positivity by CellSearch was significantly associated with reduced DRFS in both all (n = 380; P = 0.0067) and HR-positive patients (n = 328; P = 0.0002). DTC status, by itself, was not prognostic; however, when combined with CTC status by IE/FC (n = 273), double positivity (CTC+/DTC+, 8%) was significantly associated with reduced DRFS (P = 0.0270), BCSS (P = 0.0205), and OS (P = 0.0168). In HR-positive patients, double positivity (9% of 235) was significantly associated with reduced DRFS (P = 0.0285), BCSS (P = 0.0357), and OS (P = 0.0092).Conclusions:Detection of CTCs in patients with HR-positive early breast cancer was an independent prognostic factor for DRFS (using CellSearch) and BCSS (using IE/FC). Simultaneous detection of DTCs provided additional prognostic power for outcome, including OS.

Supplementary Figures and Tables except Table S7

Supplementary Data from Protein Acetylation and Histone Deacetylase Expression Associated with Malignant Breast Cancer Progression

Purpose: Excess histone deacetylase (HDAC) activity can induce hypoacetylation of histone and nonhistone protein substrates, altering gene expression patterns and cell behavior potentially associated with malignant transformation. However, HDAC expression and protein acetylation have not been studied in the context of breast cancer progression.Experimental Design: We assessed expression levels of acetylated histone H4 (ac-H4), ac-H4K12, ac-tubulin, HDAC1, HDAC2, and HDAC6 in 22 reduction mammoplasties and in 58 specimens with synchronous normal epithelium, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) components. Differences among groups were tested for significance using nonparametric tests.Results: From normal epithelium to DCIS, there was a marked reduction in histone acetylation (P < 0.0001). Most cases showed similar levels of acetylation in DCIS and IDC, although some showed further reduction of ac-H4 and ac-H4K12 from DCIS to IDC. Expression of HDAC1, HDAC2, and HDAC6 was also significantly reduced but by a smaller magnitude. Greater reductions of H4 acetylation and HDAC1 levels were observed from normal to DCIS in estrogen receptor–negative compared with estrogen receptor–positive, and in high-grade compared with non–high-grade tumors.Conclusion: Overall, there was a global pattern of hypoacetylation associated with progression from normal to DCIS to IDC. These findings suggest that the reversal of this hypoacetylation in DCIS and IDC could be an early measure of HDAC inhibitor activity.

BACKGROUND: Improving diagnosis of ductal carcinoma in situ (DCIS) before surgery is important in choosing optimal patient management strategies. However, patients may harbor occult invasive disease not detected until definitive surgery. PURPOSE: To assess the performance and clinical utility of mammographic radiomic features in the prediction of occult invasive cancer among women diagnosed with DCIS on the basis of core biopsy findings. MATERIALS AND METHODS: In this Health Insurance Portability and Accountability Act–compliant retrospective study, digital magnification mammographic images were collected from women who underwent breast core-needle biopsy for calcifications that was performed at a single institution between September 2008 and April 2017 and yielded a diagnosis of DCIS. The database query was directed at asymptomatic women with calcifications without a mass, architectural distortion, asymmetric density, or palpable disease. Logistic regression with regularization was used. Differences across training and internal test set by upstaging rate, age, lesion size, and estrogen and progesterone receptor status were assessed by using the Kruskal-Wallis or χ(2) test. RESULTS: The study consisted of 700 women with DCIS (age range, 40–89 years; mean age, 59 years ± 10 [standard deviation]), including 114 with lesions (16.3%) upstaged to invasive cancer at subsequent surgery. The sample was split randomly into 400 women for the training set and 300 for the testing set (mean ages: training set, 59 years ± 10; test set, 59 years ± 10; P = .85). A total of 109 radiomic and four clinical features were extracted. The best model on the test set by using all radiomic and clinical features helped predict upstaging with an area under the receiver operating characteristic curve of 0.71 (95% CI: 0.62, 0.79). For a fixed high sensitivity (90%), the model yielded a specificity of 22%, a negative predictive value of 92%, and an odds ratio of 2.4 (95% CI: 1.8, 3.2). High specificity (90%) corresponded to a sensitivity of 37%, positive predictive value of 41%, and odds ratio of 5.0 (95% CI: 2.8, 9.0). CONCLUSION: Machine learning models that use radiomic features applied to mammographic calcifications may help predict upstaging of ductal carcinoma in situ, which can refine clinical decision making and treatment planning. © RSNA, 2022

To design and establish a prospective biospecimen repository that integrates multi-omics assays with clinical data to study mechanisms of controlled injury and healing.Elective surgery is an opportunity to understand both the systemic and focal responses accompanying controlled and well-characterized injury to the human body. The overarching goal of this ongoing project is to define stereotypical responses to surgical injury, with the translational purpose of identifying targetable pathways involved in healing and resilience, and variations indicative of aberrant peri-operative outcomes.Clinical data from the electronic medical record combined with large-scale biological data sets derived from blood, urine, fecal matter, and tissue samples are collected prospectively through the peri-operative period on patients undergoing 14 surgeries chosen to represent a range of injury locations and intensities. Specimens are subjected to genomic, transcriptomic, proteomic, and metabolomic assays to describe their genetic, metabolic, immunologic, and microbiome profiles, providing a multidimensional landscape of the human response to injury.The highly multiplexed data generated includes changes in over 28,000 mRNA transcripts, 100 plasma metabolites, 200 urine metabolites, and 400 proteins over the longitudinal course of surgery and recovery. In our initial pilot dataset, we demonstrate the feasibility of collecting high quality multi-omic data at pre- and postoperative time points and are already seeing evidence of physiologic perturbation between timepoints.This repository allows for longitudinal, state-of-the-art geno-mic, transcriptomic, proteomic, metabolomic, immunologic, and clinical data collection and provides a rich and stable infrastructure on which to fuel further biomedical discovery.

Background. DCIS consists of a molecularly heterogeneous group of premalignant lesions, with variable risk of invasive progression. Understanding biomarkers for invasive progression could help individualize treatment recommendations based upon tumor biology. As part of the NCI Human Tumor Atlas Network (HTAN), we conducted comprehensive genomic analyses on two large DCIS case-control cohorts. Methods. We performed smart3-seq and low-pass whole genome sequencing on two independent, retrospective, longitudinally sampled DCIS case-control cohorts. TBCRC 038 was a multicenter cohort diagnosed with DCIS between 1998 and 2016 at one of the Translational Breast Cancer Research sites; the RAHBT (Resource of Archival Human Breast Tissue) cohort included women identified through the St. Louis Breast Tissue Repository, and the Women’s Health Repository diagnosed between 1997 and 2001. We studied the spectrum of molecular changes present and sought genomic predictors of subsequent ipsilateral breast events (iBEs: DCIS recurrence or invasive progression) in both DCIS epithelium and stroma in formalin fixed paraffin embedded tissue. We generated de novo tumor and stroma-centric subtypes for DCIS that represents fundamental transcriptomic organization. Copy number analysis was performed using low-pass DNA sequencing. Non-negative matrix factorization (NMF) was applied to the RNA expression of all coding genes to identify clusters. A negative-binomial regression model was used to identify differentially expressed genes. Results. We analyzed 677 DCIS samples from 481 patients with 7.1 years median follow-up. In TBCRC samples, we identified three clusters via NMF in TBCRC referred to as ER low, quiescent, and ER high. The ER-low cluster had significantly higher levels of ERBB2 and lower levels of ESR1 compared to quiescent and ER-high clusters. Quiescent cluster lesions were less proliferative and less metabolically active than ER high and ER low subtypes. These findings were replicated in the RAHBT cohort. Focusing on the stromal component of DCIS from laser capture microdissection in RAHBT samples, we identified four distinct DCIS-associated stromal clusters. A “normal-like” stromal cluster with ECM organization and PI3K-AKT signaling; a “collagen-rich” stromal cluster; a “desmoplastic” stromal cluster with high fibroblast and total myeloid abundance, mostly associated with macrophages and myeloid dendritic cells (mDC); and an “immune-dense” stromal cluster. Further, we compared differentially expressed genes in patients with or without subsequent iBEs within 5 years of diagnosis. Hypothesizing that the resulting 812 DE genes (DESeq2) represent multiple routes to subsequent iBEs, we leveraged NMF to identify paths to progression. In both TBCRC and RAHBT cohorts, poor outcome groups exhibited increased ER, MYC signaling, and oxidative phosphorylation, supporting that these pathways are important for DCIS recurrence and progression. Conclusion. Comprehensive genomic profiling in two independent DCIS cohorts with longitudinal outcomes shows distinct DCIS stromal expression patterns and immune cell composition. RNA expression profiles reveal underlying tumor biology that is associated with later iBEs in both cohorts. These studies provide new insight into DCIS biology and will guide the design of diagnostic strategies to prevent invasive progression. Citation Format: Siri H Strand, Belén Rivero-Gutiérrez, Kathleen E Houlahan, Jose A Seoane, Lorraine M King, Tyler Risom, Lunden Simpson, Sujay Vennam, Aziz Khan, Timothy Hardman, Bryan E Harmon, Fergus J Couch, Kristalyn Gallagher, Mark Kilgore, Shi Wei, Angela DeMichele, Tari King, Priscilla F McAuliffe, Julie Nangia, Joanna Lee, Jennifer Tseng, Anna Maria Storniolo, Alastair Thompson, Gaorav Gupta, Robyn Burns, Deborah J Veis, Katherine DeSchryver, Chunfang Zhu, Magdalena Matusiak, Jason Wang, Shirley X Zhu, Jen Tappenden, Daisy Yi Ding, Dadong Zhang, Jingqin Luo, Shu Jiang, Sushama Varma, Cody Straub, Sucheta Srivastava, Christina Curtis, Rob Tibshirani, Robert Michael Angelo, Allison Hall, Kouros Owzar, Kornelia Polyak, Carlo Maley, Jeffrey R Marks, Graham A Colditz, E Shelley Hwang, Robert B West. The Breast PreCancer Atlas DCIS genomic signatures define biology and correlate with clinical outcomes: An analysis of TBCRC 038 and RAHBT cohorts [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS4-07.