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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.

Despite a wide spectrum of clinical presentations, including primary or secondary, most angiosarcomas are considered high grade. One exception is primary breast angiosarcoma, where historically, histologic grading has shown to predict outcome using the Rosen 3-tier system. However, more recent studies have challenged this concept suggesting that even in this specific clinical context angiosarcomas should be considered high grade. This study aimed to critically reevaluate the impact of histologic grade in a clinically uniform cohort managed at a single institution using a newly proposed grading system. Our study included 49 primary breast angiosarcomas diagnosed during 1994 to 2022 (median follow-up: 33 mo), classified as low grade (29%), intermediate grade (20%), and high grade (51%), based on mitotic count, extent of solid components, and necrosis. At last follow-up, 22% patients developed locoregional recurrences, 63% distant metastases, and 47% patients died of disease. As patients with low and intermediate-grade angiosarcomas had relatively similar outcomes, our cohort was further analyzed using a 2-tier system (low grade and high grade). Targeted-DNA next-generation sequencing (505 cancer gene panel) performed in 11 cases found KDR mutations in 78% and PIK3CA mutations in 44% of high-grade lesions. Histologic grade, by either 3-tier or 2-tier grading systems, had a strong impact on survival, with the 2-tier system being an independent predictor of disease-specific survival and overall survival. Based on 2-tier system, the 5-year overall survival was 38% for high-grade angiosarcoma and 74% for low-grade angiosarcoma. PIK3CA mutations alone or concurrent with KDR alterations were identified in angiosarcomas with worse prognosis.

Spatially-resolved gene expression profiling provides valuable insight into tissue organization and cell-cell crosstalk; however, spatial transcriptomics (ST) lacks single-cell resolution. Current ST analysis methods require single-cell RNA sequencing data as a reference for a rigorous interpretation of cell states and do not utilize associated histology images. Significant sample variation further complicates the integration of ST datasets, which is essential for identifying commonalities across tissues or altered cellular wiring in disease. Here, we present Starfysh, the first comprehensive computational toolbox for joint modeling of ST and histology data, dissection of refined cell states, and systematic integration of multiple ST datasets from complex tissues. Starfysh uses an auxiliary deep generative model that incorporates archetypal analysis and any known cell state markers to avoid the need for a single-cell-resolution reference in characterizing known or novel tissue-specific cell states. Additionally, Starfysh improves the characterization of spatial dynamics in complex tissues by leveraging histology images and enables the comparison of niches as spatial “hubs” across tissues. Integrative analysis of primary estrogen receptor-positive (ER+) breast cancer, triple-negative breast cancer (TNBC), and metaplastic breast cancer (MBC) tumors using Starfysh led to the identification of heterogeneous patient- and disease-specific hubs as well as a shared stromal hub with varying spatial orientation. Our results show the ability to delineate the spatial co-evolution of tumor and immune cell states and their crosstalk underlying intratumoral heterogeneity in TNBC and revealed metabolic reprogramming shaping immunosuppressive hubs in aggressive MBC. Starfysh is publicly available (https://github.com/azizilab/starfysh).

STARFYSH model v 1.0.0 : Spatial Transcriptomic Analysis using Reference-Free auxiliarY deep generative modeling and Shared Histology

Metaplastic breast carcinoma (MBC) is a rare, aggressive subtype of breast cancer associated with poorer overall survival than other triple-negative breast cancers. This study sought to compare survival outcomes among histologic subtypes of MBC with those of non-metaplastic triple-negative breast cancer. Clinicopathologic and treatment data for all patients with non-metastatic, pure MBC undergoing surgery from 1995 to 2017 and for a large cohort of patients with other types of triple-negative breast cancer during that period were collected from an institutional database. The MBC tumors were classified as having squamous, spindle, heterologous mesenchymal, or mixed histology. Survival outcomes were compared using the Kaplan-Meier method. Of 132 MBC patients, those with heterologous mesenchymal MBC (n = 45) had the best 5-year overall and breast cancer-specific survival (BCSS, 88%; 95% confidence interval [CI], 0.78–0.99), whereas those with squamous MBC had the worst survival (BCSS, 56%; 95% CI, 0.32–0.79). Overall survival, BCSS, and recurrence-free survival were worse for the patients with MBC than for the patients who had non-MBC triple-negative breast cancer, with a clinicopathologically adjusted recurrence hazard ratio of 2.4 (95% CI, 1.6–3.3; p

Neoadjuvant chemotherapy (NAC) is increasingly used for clinically node-positive (cN+) tumors with intact primary breast cancer (IPBC) to downstage the axilla, and those who convert to cN0 may be eligible for sentinel lymph node biopsy (SLNB). Rates of axillary downstaging in occult primary breast cancer (OPBC) are unknown. The aim of this study was to determine the frequency of nodal pathologic complete response (pCR) following NAC in a cohort of patients with OPBC. Twenty-eight patients with stage II/III OPBC treated between January 2008 and December 2019 were identified. Twenty patients had cN1-3 OPBC, pretreatment lymph node needle biopsy, and received NAC; these constituted the study population. Treatment factors and nodal pCR rates were summarized by tumor subtype. Median age at diagnosis was 54 years. Most patients presented with cN1 disease (75%) and ductal histology (80%). Nodal pCR was seen in 16/20 (80%) patients. Eight (40%) patients were triple negative, 6 (30%) were estrogen receptor-positive/human epidermal growth factor receptor 2-negative (ER +/HER2 −), and 6 (30%) were HER2 positive, with pCR rates of 88%, 50%, and 100%, respectively. Among the 15 patients who presented as cN1, 14 (93%) converted to cN0 following NAC. Of these, nine underwent SLNB and all achieved nodal pCR (100%). In this small series, 80% of OPBC patients achieved nodal pCR following NAC. pCR rates varied by receptor profile, being lowest in the ER positive/HER2 negative group and highest in the HER2 positive group (50–100%); however, these rates are excellent and numerically exceed those in the literature for IPBC. Given the pCR rate, SLNB may be an option in select OPBC patients who downstage following NAC.

BACKGROUND: Whether routinely prescribed opioids are necessary for pain control after discharge among lumpectomy/sentinel node biopsy (Lump/SLNB) patients is unclear. We hypothesized that Lump/SLNB patients could be discharged without opioids with a failure rate of

The immune system has evolved complex effector mechanisms to protect the host against a diversity of pathogenic organisms and regulatory adaptations that can curtail pathological sequelae of inflammatory events, prevent autoimmunity, and assist in tissue repair. Cancers, by virtue of their local manifestations of tissue dysfunction and destruction, inflammation, and genomic instability, can evoke these protective mechanisms, which support the progression of tumors and prevent their immune eradication. Central to these processes is a subset of CD4+ T cells, known as regulatory T (Treg) cells, that express the X chromosome–linked transcription factor FOXP3. In addition to their critical role in controlling autoimmunity and suppressing inflammatory responses in diverse biological settings, Treg cells are ubiquitously present in the tumor microenvironment where they promote tumor development and progression by dampening antitumor immune responses. Furthermore, Treg cells can directly support the survival of transformed cells through the elaboration of growth factors and interacting with accessory cells in tumors such as fibroblasts and endothelial cells. Current insights into the biology of tumor-associated Treg cells have opened up opportunities for their selective targeting in cancer, with the goal of alleviating their suppression of antitumor immune responses while maintaining overall immune homeostasis.

In the ACOSOG (American College of Surgeons Oncology Group) Z0011 trial and the AMAROS (After Mapping of the Axilla: Radiotherapy or Surgery?) trial, matted nodes with gross extracapsular extension (ECE), a risk factor for locoregional recurrence, were an indication for axillary lymph node dissection (ALND), but the effect of microscopic ECE (mECE) in the sentinel lymph nodes (SLNs) on recurrence was not examined. Between 2010 and 2017, 811 patients with cT1-2N0 breast cancer and SLN metastasis were prospectively managed according to Z0011 criteria, with ALND for those with more than two positive SLNs or gross ECE. Management of mECE was not specified. In this study, we compare outcomes of patients with one to two positive SLNs with and without mECE, treated with SLN biopsy alone (n = 685). Median patient age was 58 years, and median tumor size was 1.7 cm. mECE was identified in 210 (31%) patients. Patients with mECE were older, had larger tumors, and were more likely to be hormone receptor positive and HER2 negative, have two positive SLNs, and receive nodal radiation. At a median follow-up of 41 months, no isolated axillary failures were observed. There were 11 nodal recurrences; two supraclavicular ± axillary, four synchronous with breast, and five with distant failure. The five-year rate of any nodal recurrence was 1.6% and did not differ by mECE (2.3% vs. 1.3%; p = 0.84). No differences were observed in local (p = 0.08) or distant (p = 0.31) recurrence rates by mECE status. In Z0011-eligible patients, nodal recurrence rates in patients with mECE are low after treatment with SLN biopsy alone, even in the absence of routine nodal radiation. The presence of mECE should not be considered a routine indication for ALND.

Importance Prospective trials have demonstrated sentinel lymph node (SLN) false-negative rates of less than 10% when 3 or more SLNs are retrieved in patients with clinically node-positive breast cancer rendered clinically node-negative with neoadjuvant chemotherapy (NAC). However, rates of nodal recurrence in such patients treated with SLN biopsy (SLNB) alone are unknown because axillary lymph node dissection (ALND) was performed in all patients, limiting adoption of this approach. Objective To evaluate nodal recurrence rates in a consecutive cohort of patients with clinically node-positive (cN1) breast cancer receiving NAC, followed by a negative SLNB using a standardized technique, and no further axillary surgery. Design, Setting, and Participants From November 2013 to February 2019, a cohort of consecutively identified patients with cT1 to cT3 biopsy-proven N1 breast cancer rendered cN0 by NAC underwent SLNB with dual tracer mapping and omission of ALND if 3 or more SLNs were identified and all were pathologically negative. Metastatic nodes were not routinely clipped, and localization of clipped nodes was not performed. The study was performed in a single tertiary cancer center. Intervention Omission of ALND in patients with cN1 breast cancer after NAC if 3 or more SLNs were pathologically negative. Main Outcome and Measures The primary outcome was the rate of nodal recurrence among patients with cN1 breast cancer treated with SLNB alone after NAC. Results Of 610 patients with cN1 breast cancer treated with NAC (median [IQR] age, 49 [40-58] years), 555 (91%) converted to cN0 and underwent SLNB; 234 (42%) had 3 or more negative SLNs and had SLNB alone. Median age was 49 years. Median tumor size was 3 cm; 144 (62%) wereERBB2(formerlyHER2)-positive, and 43 (18%) were triple negative. Most (212 [91%]) received doxorubicin-based NAC, 205 (88%) received adjuvant radiotherapy (RT), and 164 (70%) also received nodal RT. At a median follow-up of 40 months, there was 1 axillary nodal recurrence synchronous with local recurrence in a patient who refused RT. Among patients who received RT (n = 205), there were no nodal recurrences. Conclusions and Relevance This cohort study found that in patients with cN1 disease rendered cN0 with NAC, with 3 or more negative SLNs with SLNB alone, nodal recurrence rates were low, without routine nodal clipping. These findings potentially support omitting ALND in such patients.

Metaplastic breast carcinoma (MpBC) is a rare special histologic subtype of breast carcinoma characterized by the presence of squamous and/or mesenchymal differentiation. Most MpBCs are of triple-negative phenotype and neoadjuvant chemotherapy (NAC) is frequently utilized in patients with MpBC. The aim of this study was to evaluate response to NAC in a retrospective cohort of MpBCs. We identified 44 patients with MpBC treated with NAC at our center between 2002 and 2018. Median age was 48 years, 86% were clinical stage II–III, and 36% were clinically node-positive. Most (80%) MpBCs were triple-negative or low (1–10%) hormonal receptor positive and HER2 negative on pre-NAC biopsy. While on NAC, 49% showed no clinical response or clinico-radiological progression. Matrix-producing subtype was associated with clinico-radiological response (p = 0.0036). Post NAC, two patients initially ineligible for breast-conserving surgery (BCS) were downstaged to be eligible for BCS, whereas three patients potentially eligible for BCS before treatment became ineligible due to disease progression. Only one (2%) patient had a pathologic complete response (pCR). Among the 16 patients presenting with biopsy-proven clinical node-positive disease, 3 (19%) had nodal pCR. Axillary lymph node dissection was avoided in 3 (19%) patients who had successful axillary downstaging. Residual cancer burden (RCB) was assessed in 22 patients and was significantly associated with disease-free survival and overall survival. We observed a poor response or even disease progression on NAC among patients with MpBC, suggesting that NAC should be reserved for patients with inoperable MpBC.

Aims Radiation-associated angiosarcomas (RT-ASs) of the breast are rare tumours with a poor prognosis. MYC gene amplification is considered to be the hallmark of RT-AS, and is sometimes used as a diagnostic tool to distinguish it from other radiation-associated vascular lesions. However, a small subset of RT-ASs lacks MYC amplification, and this may be associated with better outcome. Loss of trimethylation at lysine 27 of histone 3 (H3K27me3) expression by immunohistochemistry (IHC) has been recently postulated as an additional diagnostic marker for RT-AS. The aims of this study were to evaluate the impact of MYC amplification as detected by fluorescence in-situ hybridisation and/or next-generation sequencing on clinicopathological features and outcome in a large cohort of RT-ASs, compare outcome with those of radiation-associated sarcomas (RT-Ss) of the breast other than angiosarcoma, and evaluate expression of H3K27me3 IHC in these groups. Methods and results Eighty-one RT-ASs were identified, including 73 that were MYC-amplified and 8 (10%) that were MYC-non-amplified. MYC-amplified RT-ASs were diagnosed in older patients (median age, 69 years versus 61 years). The 5-year disease-specific survival and 5-year overall survival rates were 56% and 47%, respectively. Older age, larger tumour size, positive margin and MYC amplification were associated with worse prognosis. None of the RT-ASs showed complete loss of H3K27me3 IHC expression. All 18 RT-Ss were MYC-non-amplified, and complete loss of H3K27me3 expression was seen in 2 cases. We found no difference in prognosis between RT-AS and RT-S. Conclusions RT-AS of the breast is associated with a poor prognosis. Older age at diagnosis, larger tumour size, positive margin at excision and MYC amplification are associated with worse prognosis.

Treatment with immune checkpoint inhibitors (ICI) has demonstrated clinical benefit for a wide range of cancer types. Because only a subset of patients experience clinical benefit, there is a strong need for biomarkers that are easily accessible across diverse practice settings. Here, in a retrospective cohort study of 1714 patients with 16 different cancer types treated with ICI, we show that higher neutrophil-to-lymphocyte ratio (NLR) is significantly associated with poorer overall and progression-free survival, and lower rates of response and clinical benefit, after ICI therapy across multiple cancer types. Combining NLR with tumor mutational burden (TMB), the probability of benefit from ICI is significantly higher (OR = 3.22; 95% CI, 2.26-4.58; P, There is an unmet clinical need for simple, accessible biomarkers to select patients who are more likely to respond to immune checkpoint therapy. Here the authors show that a lower neutrophil-to-lymphocyte ratio is associated with better overall and progressive-free survival, as well as higher rate of response, in a multi-cancer cohort of patients treated with immune checkpoint inhibitors.

The immune system mediates powerful effector mechanisms to protect against a diversity of pathogens and equally as important regulatory functions, to limit collateral damage of inflammation, prevent misguided immune responses to "self", and promote tissue repair. Inadequate regulatory control can lead to a variety of inflammatory disorders including autoimmunity, metabolic syndrome, allergies, and progression of malignancies. Cancers evolve complex mechanisms to thwart immune eradication including coopting normal host regulatory processes. This is most evident in the analysis of tumor infiltrating lymphocytes (TILs), where a preponderance of immunosuppressive immune cells, such as regulatory T (Treg) cells are found. Treg cells express the X-chromosome linked transcription factor Foxp3 and play a crucial role in maintaining immune homeostasis by suppressing inflammatory responses in diverse biological settings. Treg cells in the tumor microenvironment promote tumor development and progression by dampening anti-tumor immune responses, directly supporting the survival of transformed cells through elaboration of growth factors, and interacting with accessory cells in tumors such as fibroblasts and endothelial cells. Current insights into the phenotype and function of tumor associated Treg cells have opened up opportunities for their selective targeting in cancer with the goal of alleviating their suppression of anti-tumor immune responses while maintaining overall immune homeostasis. Here, we review Treg cell biology in the context of the tumor microenvironment (TME), and the important role they play in cancer immunotherapy.

Key Points Question Following resection of a localized breast cancer, why does adjuvant regional nodal irradiation (RNI) reduce distant recurrences to a greater extent than regional recurrences, despite only targeting the regional lymph nodes? Findings In this cohort study of 597 patients with de novo metastatic breast cancer, 85.8% harbored regional disease at the time of metastatic presentation. The consistent detection of concurrent regional and metastatic disease, along with the reduction in distant recurrences observed following RNI, is consistent with the hypothesis that regional nodal disease may precede metastatic dissemination. Meaning These findings suggest that RNI may reduce distant recurrences by sterilizing occult regional nodal disease prior to systemic seeding., This cohort study examines whether and why regional node irradiation in patients with de novo metastatic breast cancer has different levels of effectiveness for preventing recurrences of cancer in regional nodes and distant mestastases., Importance Regional nodal irradiation (RNI) for node-positive breast cancer reduces distant metastases and improves survival, albeit with limited reduction in regional nodal recurrences. The mechanism by which RNI robustly reduces distant metastases while modestly influencing nodal recurrences (ie, the presumed target of RNI) remains unclear. Objective To determine whether some distant metastases putatively arise from occult regional nodal disease and whether regional recurrences otherwise remain largely undetected until an advanced cancer presentation. Design, Setting, and Participants This cohort study examined patients presenting with de novo stage IV breast cancer to the Memorial Sloan Kettering Cancer Center in New York, New York, from 2006 to 2018. Medical records were reviewed to ascertain clinicopathological parameters, including estrogen receptor status and survival. Pretreatment positron emission tomography–computed tomography (PET-CT) imaging was reviewed to ascertain the extent of regional nodal involvement at metastatic diagnosis using standard nodal assessment criteria. A subset underwent regional lymph node biopsy for diagnostic confirmation and served to validate the radiographic nodal assessment. Data analysis was performed from October 2019 to February 2020. Exposures Untreated metastatic breast cancer. Main Outcome and Measures The primary outcome was the likelihood of regional nodal involvement at the time of metastatic breast cancer presentation and was determined by reviewing pretreatment PET-CT imaging and lymph node biopsy findings. Results Among 597 women (median [interquartile range] age, 53 [44–65] years) with untreated metastatic breast cancer, 512 (85.8%) exhibited regional lymph node involvement by PET-CT or nodal biopsy, 509 (85%) had involvement of axillary level I, 328 (55%) had involvement in axillary level II, 136 (23%) had involvement in axillary level III, 101 (17%) had involvement in the supraclavicular fossa, and 96 (16%) had involvement in the internal mammary chain. Lymph node involvement was more prevalent among estrogen receptor–negative tumors (92.4%) than estrogen receptor–positive tumors (83.6%). Nodal involvement at the time of metastatic diagnosis was not associated with overall survival. Conclusions and Relevance These findings suggest that a majority of patients with de novo metastatic breast cancer harbor regional lymph node disease at presentation, consistent with the hypothesis that regional involvement may precede metastatic dissemination. This is in alignment with the findings of landmark trials suggesting that RNI reduces distant recurrences. It is possible that this distant effect of RNI may act via eradication of occult regional disease prior to systemic seeding. The challenges inherent in detecting isolated nodal disease (which is typically asymptomatic) may account for the more modest observed benefit of RNI on regional recurrences. Alternative explanations of nodal involvement that arises concurrently or after metastatic dissemination remain possible, but do not otherwise explain the association of RNI with distant recurrence.

Metaplastic breast carcinoma (MBC) is a rare, aggressive subtype of breast cancer associated with poorer overall survival than other triple-negative breast cancers. This study sought to compare survival outcomes among histologic subtypes of MBC with those of non-metaplastic triple-negative breast cancer.Clinicopathologic and treatment data for all patients with non-metastatic, pure MBC undergoing surgery from 1995 to 2017 and for a large cohort of patients with other types of triple-negative breast cancer during that period were collected from an institutional database. The MBC tumors were classified as having squamous, spindle, heterologous mesenchymal, or mixed histology. Survival outcomes were compared using the Kaplan-Meier method.Of 132 MBC patients, those with heterologous mesenchymal MBC (n = 45) had the best 5-year overall and breast cancer-specific survival (BCSS, 88%; 95% confidence interval [CI], 0.78-0.99), whereas those with squamous MBC had the worst survival (BCSS, 56%; 95% CI, 0.32-0.79). Overall survival, BCSS, and recurrence-free survival were worse for the patients with MBC than for the patients who had non-MBC triple-negative breast cancer, with a clinicopathologically adjusted recurrence hazard ratio of 2.4 (95% CI, 1.6-3.3; p 0.001). Of the 10 MBC patients who received neoadjuvant chemotherapy, 4 progressed while receiving treatment, and 3 had no response.Metaplastic breast carcinoma is associated with worse survival than other triple-negative breast cancers. The heterologous mesenchymal subtype is associated with the best survival, whereas the squamous subtype is associated with the worst survival. These data call for research to identify therapies tailored to MBC's unique biology.

Immunotherapy has been incorporated into the standard of care for a wide range of malignancies. The study of tumor-infiltrating lymphocytes has emphasized the importance of the host anti-tumor immune response in the natural history of breast cancer. Recent clinical trials have used immunotherapeutic approaches to augment this response and improve outcomes for patients with breast cancer. Here, we review several current clinical trial data that indicate checkpoint blockade may mediate clinically significant responses.

Background Opioid-induced immunomodulation may be of particular importance in triple-negative breast cancer (TNBC) where an immune response is associated with improved outcome and response to immunotherapy. We evaluated the association between intraoperative opioids and oncological outcomes and explored patterns of opioid receptor expression in TNBC. Methods Consecutive patients with stage I–III primary TNBC were identified from a prospectively maintained database. Opioid receptor expression patterns in the tumour microenvironment were analysed using publicly available bulk and single-cell RNA-seq data. Results A total of 1143 TNBC cases were retrospectively analysed. In multivariable analysis, higher intraoperative opioid dose was associated with favourable recurrence-free survival, hazard ratio 0.93 (95% confidence interval 0.88–0.99) per 10 oral morphine milligram equivalents increase (P=0.028), but was not significantly associated with overall survival, hazard ratio 0.96 (95% confidence interval 0.89–1.02) per 10 morphine milligram equivalents increase (P=0.2). Bulk RNA-seq analysis of opioid receptors showed that OPRM1 was nearly non-expressed. Compared with normal breast tissue OGFR, OPRK1, and OPRD1 were upregulated, while TLR4 was downregulated. At a single-cell level, OPRM1 and OPRD1 were not detectable; OPRK1 was expressed mainly on tumour cells, whereas OGFR and TLR4 were more highly expressed on immune cells. Conclusions We found a protective effect of intraoperative opioids on recurrence-free survival in TNBC. Opioid receptor expression was consistent with a net protective effect of opioid agonism, with protumour receptors either not expressed or downregulated, and antitumour receptors upregulated. In this era of personalised medicine, efforts to differentiate the effects of opioids across breast cancer subtypes (and ultimately individual patients) should continue.

Background: Treatment with immune checkpoint inhibitors (ICI) has demonstrated clinical benefit for a wide range of cancer types. Because only a subset of patients benefit, there is a strong need for predictive biomarkers. The aim of this study was to analyze the predictive capacity of neutrophil-to-lymphocyte ratio (NLR) in cancer patients treated with ICI, and to analyze the predictive value of combining NLR with tumor mutational burden (TMB). Methods: Retrospective cohort study of patients treated during 2015-2018. A total of 1714 patients with 16 different cancer types, who had complete blood count within 30 days prior to first ICI infusion, and whose tumors underwent next-generation sequencing were included. Overall survival, progression-free survival, response to ICI, and clinical benefit (response or stable disease ≥6 months) were the primary endpoints. Findings: Patients with NLR in the top 20th percentile within cancer type had significantly poorer overall survival (HR = 2·17; 95% CI, 1·89-2·50; P

Introduction Chemotherapy (CT) is the standard of care for most triple negative breast cancer (TNBC). Chemotherapy is less commonly recommended in older than younger patients. We aim to explore the frequency of CT delivered in elderly patients when compared to young patients. Methods Patients ≤50yrs and ≥70 yrs with stage I-III TNBC defined as ER Results We identified 901 pts with TNBC; 664 (73.7%) were ≤50yrs and 237 (26.3%) were ≥70 yrs. Median followup is 7 yrs (range, 0-16.8yrs). Younger women diagnosed with TNBC were more likely to have stronger family history of breast cancer (p The 5 year rates of DOD were similar between both groups at 10.6% (range, 8.3-13.2) for pts ≤50yrs and 10.8% (range, 7.0-15.4) (p=0.52) for the older group; meanwhile, the 5 year OS rates were significantly different between both groups at 87.5% (range, 84.7-90.0) for pts ≤50yrs and 74.3% (range, 68.2-80.0) (p CT was given to 115 (49%) patients of the ≥70 yrs cohort with a selection biased by larger tumors (p When categorized based on age and receipt of CT, in the 662 pts ≤ 50 yrs, 609 (92%) and 53 (8%) received CT vs no CT respectively; in the 233 pts ≥70 yrs, 115 (49%) and 118 (50%) received CT vs no CT; the cumulative incidence curves for DOD were not statistically different for the four groups (p=0.85) at 5 years. Conclusions In our series, CT was given to 92% of patients ≤ 50 yrs of age. In the elderly pts ≥ 70 yrs of age, CT was limited to 50% of patients, namely those with worse clinicopathologic features. Citation Format: Muhsen S, Dang C, Plitas G, Knezevic A, Stempel M, Patil S, Morrow M, El-Tamer M. Frequency of delivery of systemic chemotherapy in elderly versus younger patients with triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-13-05.

Introduction Trastuzumab with systemic chemotherapy has shown an improvement in outcomes for patients (pts) with HER2 amplified/overexpressed (HER2+) breast cancer. Pts enrolled onto trials were young with a minority of pts at ≥65 years (yrs) of age. Herein, we report the administration of systemic treatment (ST) (chemotherapy and/or trastuzumab) verus no treatment in elderly pts at a single center. Methods Patients ≥65 yrs with stage I-III HER2+ (defined as IHC 3+ or FISH >2.0) breast cancer, treated at Memorial Sloan Kettering Cancer Center between 2000-2012, were retrospectively identified from our database. Clinicopathologic features were retrieved and co-morbidity indexes (CI) were calculated. Pts were divided by hormone receptor (HR) (defined as ER >10% and/or PR >10%) status into HER2+HR- and HER2+HR+. Each group was further divided by use of ST into: chemotherapy and trastuzumab (CT+T), chemotherapy alone (CT) or no systemic treatment (No Rx). Patients receiving neoadjuvant ST or trastuzumab only as ST were excluded from the KM analysis. Primary objective was to identify patterns of treatment recommendation in the elderly population. We explored disease-free survival (DFS) as estimated using the Kaplan-Meier (KM) method. Results We identified 300 pts ≥65 yrs with HER2+ tumors. 128 (42.7%) were HER2+HR- and 172 (57.3%) were HER2+HR+. The median follow-up for all patients was 6.1 years (range, 0.07-16.7). In the HER2+HR- group, 63 (49.2%) patients received CT+T, 25 (19.5%) CT alone, and 40 (31.3%) had no Rx. Anthracycline based chemotherapy was administered to 57/88 (65%) of patients on CT. Women receiving chemotherapy with or without trastuzumab were younger (65-70 vs >70 years of age) (p=.002) and had more advanced tumor stages (p=.003). Their respective 5-yr DFS KM estimates were 0.84, 0.80, and 0.61 (logrank p=0.06). In the HER2+HR+ group, 77 (44.8%) patients received CT+T, 22 (12.8%) CT alone, and 73 (42.2%) had no Rx. Anthracycline based chemotherapy was administered to 51/99 (51%) of patients on CT. Endocrine therapy was given to 153/172 (89%) of the total cohort. Women receiving chemotherapy with or without trastuzumab were younger (p Conclusions At a single center, in the elderly populations at ≥65 years of age with HER2+ HR- and HER2+HR+ breast cancer, pts who received systemic treatment were younger and had higher stage of disease than those who received no treatment. In an exploratory analysis, there appeared to be a benefit of systemic treatment in pts in the HER+HR- group. Citation Format: Muhsen S, Dang C, Plitas G, Seier K, Stempel M, Patil S, Morrow M, El-Tamer M. Chemotherapy with and without trastuzumab or no treatment in elderly patients with HER2 amplified breast cancer at a single center [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-13-07.

Background: Four prospective multi-institutional trials have demonstrated that clinically node-positive patients (cN1) who receive neoadjuvant therapy (NAC) and convert to cN0 can be reliably staged with sentinel lymph node biopsy (SLNB) with false-negative rates (FNRs) of < 10%, when ≥ 3 SLNs are retrieved. Since study patients all had axillary lymph node dissection (ALND), the rate of axillary recurrence after SLNB alone is unknown. Of concern is the possibility that residual chemotherapy-resistant axillary disease could lead to higher recurrence rates than seen in the primary surgery setting for cN0 patients where SLN FNRs of 5-10% result in axillary recurrence in < 1% of cases. Here we report regional recurrence rates in a prospectively defined cohort of cN1 patients receiving NAC, followed by a negative SLNB using a standardized technique, and no further axillary surgery. Methods: From 06/2014 to 02/2019, patients with cT1-3 biopsy-proven cN1 breast cancer who received NAC and converted to cN0 by physical exam were prospectively managed with SLNB with dual tracer mapping and omission of ALND if ≥ 3 SLNs were pathologically negative. Nodes were not routinely clipped, and retrieval of clipped metastatic nodes was not required. Pathologically negative SLNs were defined as the absence of any metastases including isolated tumor cells. Results: Of 610 cN1 patients treated with NAC, 555 (91%) converted to cN0 and had SLNB; 234 (42%) had ≥ 3 negative SLNs and were treated with SLNB alone. Median patient age was 49 years and median tumor size at presentation was 3 cm; 61% were HER2+ and 18% triple negative. Most (91%) received doxorubicin-based NAC and 88% received adjuvant radiotherapy (RT), with 80% (n = 164) of RT patients receiving nodal RT (Table). At a median follow-up of 35 months, there was only 1 (0.4%) axillary recurrence for the entire cohort, synchronous with a breast recurrence, in a patient who refused RT. Among patients who received RT (n = 205), there were no axillary recurrences. The 4-year rate of distant recurrence for all patients was 6.1% (95% CI, 3.4-10.7%) and 4-year overall survival was 93.9% (95% CI, 87.6-97.1%). Conclusion: In cN1 patients treated with NAC, rates of axillary recurrence in patients with ≥ 3 pathologically negative SLNs treated with SLNB alone were low, without routine nodal clipping. Although further follow-up is needed, multiple studies have shown that nodal recurrence is an early event, particularly in HER2+ and triple negative patients, who comprised the majority of the population. Our findings support omitting ALND in cN1 patients after NAC when the SLNs are negative using an optimal SLNB technique. Table. Patient PopulationOverall cohort (n = 234)Age, years (median, IQR)49 (40, 58)Tumor size at presentation, cm (median, IQR)3.0 (2.2, 5.0)Number SLNs retrieved (median, IQR)4 (3, 5)Palpable nodes at presentation (n, %)179 (76%)HistologyDuctal211 (90%)Lobular and mixed7 (3%)Micropapillary and mixed10 (4%)Other3 (1%)Occult3 (1%)DifferentiationWell1 (0.5%)Moderate36 (15%)Poor196 (84%)Unknown1 (0.5%)Receptor StatusHR+/HER2-47 (20%)HR+/HER2+80 (34%)HR-/HER2+64 (27%)HR-/HER2-43 (18%)Breast SurgeryBCS118 (50%)Mastectomy116 (50%)Breast pCR¥Yes161 (70%)No70 (30%)NAC regimenAC-T197 (84%)AC-T + carbo15 (6.4%)TC8 (3.4%)Other14 (6%)Neoadjuvant anti-HER2 treatmentHP (dual-therapy)144 (100%)Adjuvant RTYes205 (88%)No*29 (12%)¥3 patients had occult primary breast cancer and were not included in breast pCR calculation; *6/29 patients who did not receive RT enrolled in NSABP B-51 Citation Format: Andrea V Barrio, Giacomo Montagna, Anita Mamtani, Varadan Sevilimedu, Hiram S Cody, III, Mahmoud El-Tamer, Mary L Gemignani, Alexandra S Heerdt, Tracy-Ann Moo, Melissa Pilewskie, George Plitas, Lisa Sclafani, Kimberly J Van Zee, Monica Morrow. Axillary recurrence is a rare event in node-positive patients. treated with sentinel node biopsy alone after neoadjuvant chemotherapy: Results of a prospective study [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD4-05.

Neoadjuvant chemotherapy (NAC) is used to convert patients with inoperable locally advanced breast cancer (LABC) to operability, but has not traditionally been used to avoid mastectomy or axillary dissection in this subset. The purpose of this study was to determine the rates of pathologic complete response (pCR) in LABC patients, and identify factors predictive of pCR to determine if responding patients might be suitable for limited surgery. From 2006 to 2016, 1522 patients received NAC followed by surgery; 321 had advanced disease in the breast (cT4) and/or in the nodes (cN2/N3). pCR rates were assessed by T and N stage, and receptor subtype. Of 321 LABC patients, 223 were cT4, 77 were cN2, and 82 were cN3. Forty-three percent were hormone receptor (HR) positive/human epidermal growth factor receptor 2 (HER2) negative (HR+/HER2−), 23% were triple negative, and 34% were HER2+. The overall pCR rate was 25% and differed by receptor subtype (HR+/HER2− 7%, triple negative 23%, HER2+ 48%; p

Tissue resident memory CD8+ T cells (Trm) are poised for immediate reactivation at sites of pathogen entry and provide optimal protection of mucosal surfaces. The intestinal tract represents a portal of entry for many infectious agents; however, to date specific strategies to enhance Trm responses at this site are lacking. Here, we present TMDI (Transient Microbiota Depletion-boosted Immunization), an approach that leverages antibiotic treatment to temporarily restrain microbiota-mediated colonization resistance, and favor intestinal expansion to high densities of an orally-delivered Listeria monocytogenes strain carrying an antigen of choice. By augmenting the local chemotactic gradient as well as the antigenic load, this procedure generates a highly expanded pool of functional, antigen-specific intestinal Trm, ultimately enhancing protection against infectious re-challenge in mice. We propose that TMDI is a useful model to dissect the requirements for optimal Trm responses in the intestine, and also a potential platform to devise novel mucosal vaccination approaches., Tissue resident CD8 + T cells present at mucosal surfaces are poised to elicit function in situ, however approaches to boost their number in the gastrointestinal mucosa has been limited. Here the authors combine the use of Listeria monocytogenese and transient depletion of the intestinal microbiome to boost the resident CD8 + T cell response.

A breast mass is the most common presenting symptom among patients in a breast clinic. The presence of a breast mass can cause a great deal of anxiety in women, as well as their physicians. The differential diagnosis of a palpable breast abnormality is broad, although the majority of breast masses are benign. The responsibility of the physician who is evaluating a breast mass is to exclude the presence of malignancy. Once cancer is ruled out, the physician should then attempt to provide an accurate diagnosis, appropriate treatment, and reassurance to the patient. This chapter discusses the assessment of normal breast physiology, identification of a breast mass, evaluation of the various classifications of breast mass (e.g., dominant mass with clinically benign features and dominant mass with suspicious features), differential diagnosis and management of common benign breast masses (e.g., cysts, fibroadenomas, phyllodes tumors, hamartomas, fat necrosis), and the risk of breast cancer associated with benign breast lesions. The chapter also discusses the diagnosis and management of a breast mass in male patients. Tables outline breast lesions that may present as a palpable abnormality, factors used for the assessment of breast cancer risk, physical characteristics of benign and malignant breast masses, the accuracy of fine-needle aspiration, and benign breast lesions by category. Figures illustrate diagnostic procedures, the anatomy of the human breast, visual inspection of the breasts, physical examination of the breasts, breast palpation technique, the evaluation and management of a new breast mass, and the identification of cysts. This review contains 10 figures, 14 tables, and 64 references. Keywords: breast mass, lobuloalveolar development, subareolar nodularity, parenchyma (glandular elements), stromal tissue, ovarian graafian follicles

The immune system of vertebrate animals has evolved to mount an effective defense against a diverse set of pathogens while minimizing transient or lasting impairment in tissue function that could result from the inflammation caused by immune responses to infectious agents. In addition, misguided immune responses to “self” and dietary antigens, as well as to commensal microorganisms, can lead to a variety of inflammatory disorders, including autoimmunity, metabolic syndrome, allergies, and cancer. Regulatory T cells expressing the X chromosome–linked transcription factor Foxp3 suppress inflammatory responses in diverse biological settings and serve as a vital mechanism of negative regulation of immune-mediated inflammation. Cancer Immunol Res; 4(9); 721–5. ©2016 AACR.

542 Background: Opioid-induced immunomodulation may be of particular importance in triple negative breast cancer (TNBC) where an immune response is associated with improved outcome and response to immunotherapy. We evaluated the association between intraoperative opioids and outcomes in a large TNBC cohort. Methods: Consecutive patients with stage I-III primary TNBC treated between 03/2010 and 12/2016 were identified from our prospectively maintained database. Total intraoperative opioid dose was extracted from anesthetic records and converted to oral morphine milligram equivalents (MME) (10 MME = 50 mcg fentanyl IV). Univariable and multivariable (MVA) Cox proportional hazards analysis (adjusting for relevant clinicopathological features, (neo)adjuvant therapy, anesthesia technique and morbidity score), were performed to quantify the association between opioid exposure and recurrence-free survival (RFS) and overall survival (OS). Results: 1143 patients were included. Median age was 54 years (IQR 45, 64). 911 (80%) had ductal histology, 359 (31%) had nodal metastases, and 1070 (94%) were poorly differentiated. 781 (68%) received adjuvant chemotherapy and 313 (27%) received neoadjuvant chemotherapy. 525 (46%) received total intravenous anesthesia and 618 (54%) had general anesthesia. Median intraoperative opioid dose was 30 MME (IQR 20, 60). 5-year RFS was 81% (95% CI 79%-84%), 5-year OS was 86% (95% CI 84%-88%). In MVA, higher opioid administration was associated with favorable RFS but did not significantly affect OS (Table). Conclusions: Our study is the first to directly evaluate intraoperative opioid administration in TNBC and suggests a protective effect on RFS. Future work will focus on elucidating the underlying mechanism for this effect, including possible modulation of endogenous opioid signaling pathways and immunologic correlates. [Table: see text]

Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

SUMMARYKnowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We created an immune map of breast cancer using single-cell RNA-seq data from 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph node. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous tumor-specific phenotypic expansions driven by environmental cues. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer, with important implications for characterizing tumor-infiltrating immune cells.

To determine rates of axillary dissection (ALND) and nodal recurrence in patients eligible for ACOSOG Z0011.Z0011 demonstrated that patients with cT1-2N0 breast cancers and 1 to 2 involved sentinel lymph nodes (SLNs) having breast-conserving therapy had no difference in locoregional recurrence or survival after SLN biopsy alone or ALND. The generalizability of the results and importance of nodal radiotherapy (RT) is unclear.Patients eligible for Z0011 had SLN biopsy alone. Prospectively defined indications for ALND were metastases in ≥3 SLNs or gross extracapsular extension. Axillary imaging was not routine. SLN and ALND groups and radiation fields were compared with chi-square and t tests. Cumulative incidence of recurrences was estimated with competing risk analysis.From August 2010 to December 2016, 793 patients met Z0011 eligibility criteria and had SLN metastases. Among them, 130 (16%) had ALND; ALND did not vary based on age, estrogen receptor, progesterone receptor, or HER2 status. Five-year event-free survival after SLN alone was 93% with no isolated axillary recurrences. Cumulative 5-year rates of breast + nodal and nodal + distant recurrence were each 0.7%. In 484 SLN-only patients with known RT fields (103 prone, 280 supine tangent, 101 breast + nodes) and follow-up ≥12 months, the 5-year cumulative nodal recurrence rate was 1% and did not differ significantly by RT fields.We confirm that even without preoperative axillary imaging or routine use of nodal RT, ALND can be avoided in a large majority of Z0011-eligible patients with excellent regional control. This approach has the potential to spare substantial numbers of women the morbidity of ALND.

Regulatory T (Treg) cells reside in lymphoid organs and barrier tissues where they control different types of inflammatory responses. Treg cells are also found in human cancers, and studies in animal models suggest that they contribute to cancer progression. However, properties of human intratumoral Treg cells and those present in corresponding normal tissue remain largely unknown. Here, we analyzed features of Treg cells in untreated human breast carcinomas, normal mammary gland, and peripheral blood. Tumor-resident Treg cells were potently suppressive and their gene-expression pattern resembled that of normal breast tissue, but not of activated peripheral blood Treg cells. Nevertheless, a number of cytokine and chemokine receptor genes, most notably CCR8, were upregulated in tumor-resident Treg cells in comparison to normal tissue-resident ones. Our studies suggest that targeting CCR8 for the depletion of tumor-resident Treg cells might represent a promising immunotherapeutic approach for the treatment of breast cancer.

592 Background: Checkpoint inhibition (CPI) combined with local strategies that cause local tumor destruction, such as cryo may augment tumor specific immunity and improve survival. We previously demonstrated in 18 ESBC patients (pts) that pre-operative (pre-op) cryo with ipilimumab (ipi) is not only safe but also generates robust local and systemic immune responses (NCT01502592). Given the added activity of dual CPI in other tumors, we undertook a second pilot study of pre-op ipi/nivolumab (nivo)/cryo to confirm the safety of this combination and the impact on immune biomarkers. Methods: In both pilot studies, eligible pts had operable ≥1.5cm invasive HER2 negative ESBC. CPI was administered 8-15d prior to, and cryo was performed 7-10d prior to, standard-of-care (SOC) surgery. Toxicity evaluation continued for 12wks after drug administration. Blood for immune correlates was obtained at baseline, cryo, surgery and 2-4 weeks thereafter. Tumor samples were obtained at cryo and surgery. Flow-cytometry of peripheral lymphocytes was compared to previously reported ipi/cryo responses. Results: After a median follow-up of 66 months all 18 ESBC ipi/cryo pts, including 3 TNBC pts, are recurrence free. In the ipi/nivo/cryo study, the safety primary endpoint was met when 5 pts underwent SOC surgery without delay. Ipi/nivo/cryo was well tolerated overall. One pt on an aromatase inhibitor had grade 4 liver toxicity 8 weeks after surgery. One pt, 3 weeks after her SOC surgery, developed grade 1 hyperthyroidism, preventing a secondary axillary dissection from proceeding as scheduled. Robust activation of peripheral CD4+ and CD8+ T cells peaked at week 2 post ipi/nivo with the majority of activated CD8+ T cells expressing PD1. Comparing the correlatives of the ipi/nivo/cryo study with the prior ipi/cryo study, we observed higher expression of activation markers (Ki-67, ICOS, CTLA-4, LAG-3) on peripheral T cells and downregulation of suppressor cells. Conclusions: Ipi/cryo-treated pts, including 3 TNBC pts, remain recurrence free after > 5y. Combining cryo with ipi/nivo preop is feasible, safe, and associated with greater T cell activation than ipi/cryo alone. These results informed an ongoing randomized phase 2 study of pre-op ipi/nivo/cryo versus SOC in women with residual TNBC after neoadjuvant chemotherapy (NCT03546686). Clinical trial information: NCT02833233.

Background In ACOSOG Z0011 and AMAROS, matted nodes with gross extracapsular extension (ECE)—a risk factor for locoregional recurrence—were an indication for axillary dissection (ALND), but the effect of microscopic ECE (mECE) in the sentinel nodes (SLNs) on recurrence was not examined. Methods Between 2010-2017, 815 patients with cT1-2N0 breast cancer and SLN metastasis were prospectively managed according to Z0011 criteria, with ALND for those with >2 positive SLNs. Management of mECE was not specified. Here we report outcomes of patients with 1-2 positive SLNs treated with SLN biopsy alone (n=685) and evaluate the impact of mECE on nodal recurrence. Outcomes of the 118 patients treated with ALND, of which 70% had >2 positive SLNs, are provided for comparison. Results Median patient age was 58 years and median tumor size was 1.7 cm. In the SLN group, 210 (31%) had mECE. Patients with mECE were older, had larger tumors, were more likely to be hormone receptor positive (HR+) and HER2-, have 2 positive SLNs, and to receive nodal radiation (Table). At a median follow-up of 41 months, no isolated axillary failures were observed. There were 11 nodal recurrences; 2 isolated, 4 synchronous with breast, and 5 with distant failure. The 5-year rate of any nodal recurrence was 1.6% and did not differ by mECE (2.3% vs 1.3%, p=0.84). No differences were observed in local (0% mECE vs. 1.9% no mECE, p=0.08) or distant (1.2% mECE vs. 4.6% no mECE, p=0.31) recurrence rates by mECE status. In comparison, in the 118 patients having ALND, 101 (86%) had mECE, and 1 combined nodal and distant recurrence was seen. Patient Characteristics Overall n=685No mECE n=475mECE n=210P valueAge, years (median, range)58 (30, 92)57 (30, 85)61 (34, 92)0.0002Pathologic tumor size, cm (median, range)1.7 (0.1, 5.2)1.6 (0.1, 5.2)1.8 (0.4, 5.2)0.008Histology 0.24Ductal598 (87%)421 (89%)177 (84%) Lobular59 (9%)34 (7%)25 (12%) Mixed24 (4%)17 (4%)7 (3%) Other4 (2mmNANA93 (44%) Systemic therapy* 0.03Chemotherapy + endocrine424 (62%)284 (60%)140 (67%) Chemotherapy only67 (10%)55 (12%)12 (5%) Endocrine only172 (25%)118 (25%)54 (26%) Unknown§22 (3%)18 (4%)4 (2%) Radiation field* Conclusions In Z0011-eligible patients, rates of nodal recurrence in patients with mECE are low after treatment with SLN biopsy alone, even in the absence of routine nodal radiation. The presence of mECE should not be considered a routine indication for ALND. Citation Format: Barrio AV, Downs-Canner S, Cody HS, Van Zee KJ, Gemignani ML, Pilewskie M, Plitas G, El-Tamer M, Kirstein L, Capko D, Patil S, Morrow M. Microscopic extracapsular extension in sentinel lymph nodes does not mandate axillary dissection in Z0011-eligible patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD8-01.

Transient ablation of regulatory T cells in a murine model of breast carcinogenesis inhibits primary tumor and lung metastatic growth and enhances the therapeutic effect of radiotherapy, but not immune checkpoint blockade., Rational combinatorial therapeutic strategies have proven beneficial for the management of cancer. Recent success of checkpoint blockade in highly immunogenic tumors has renewed interest in immunotherapy. Regulatory T (T reg) cells densely populate solid tumors, which may promote progression through suppressing anti-tumor immune responses. We investigated the role of T reg cells in murine mammary carcinogenesis using an orthotopic, polyoma middle-T antigen-driven model in Foxp3DTR knockin mice. T reg cell ablation resulted in significant determent of primary and metastatic tumor progression. Importantly, short-term ablation of T reg cells in advanced spontaneous tumors led to extensive apoptotic tumor cell death. This anti-tumor activity was dependent on IFN-γ and CD4+ T cells but not on NK or CD8+ T cells. Combination of T reg cell ablation with CTLA-4 or PD-1/PD-L1 blockade did not affect tumor growth or improve the therapeutic effect attained by T reg cell ablation alone. However, T reg cell targeting jointly with tumor irradiation significantly reduced tumor burden and improved overall survival. Together, our results demonstrate a major tumor-promoting role of T reg cells in an autochthonous model of tumorigenesis, and they reveal the potential therapeutic value of combining transient T reg cell ablation with radiotherapy for the management of poorly immunogenic, aggressive malignancies.

The surgical management of the regional lymph node basin of melanoma has undergone significant changes in the past 2 decades, most of which have been guided by prospective randomized trials. Historically, routine elective lymph node dissection was recommended for the management of melanoma regardless of clinical nodal involvement. Subsequent randomized trials failed to show a clear benefit for all patients, and sentinel lymph node (SLN) biopsy emerged as an alternative. Although the prognostic value of SLN biopsy in intermediate-thickness melanoma is well accepted, its value for patients with thin and thick lesions is debated. The therapeutic advantage of removing an involved SLN, and the need for a completion lymph node dissection after the identification of a positive SLN, are areas of continued controversy. This article discusses these issues in the management of the regional lymph node basin in patients with melanoma.

Regulatory T (Treg) cells, which suppress autoimmunity and other inflammatory states, are characterized by a distinct set of genetic elements controlling their gene expression. However, the extent of genetic and associated epigenetic variation in the Treg cell lineage and its possible relation to disease states in humans remain unknown. We explored evolutionary conservation of regulatory elements and natural human inter-individual epigenetic variation in Treg cells to identify the core transcriptional control program of lineage specification. Analysis of single nucleotide polymorphisms in core lineage-specific enhancers revealed disease associations, which were further corroborated by high-resolution genotyping to fine map causal polymorphisms in lineage-specific enhancers. Our findings suggest that a small set of regulatory elements specify the Treg lineage and that genetic variation in Treg cell-specific enhancers may alter Treg cell function contributing to polygenic disease. DOI: http://dx.doi.org/10.7554/eLife.07571.001, eLife digest The immune system protects the body from infection. Key to this protection is the ability to mount an immune response that is sufficient to deal with the threat, but is not so large that the damage it causes to the body exceeds its immediate benefit. Immune cells called regulatory T cells (or Treg cells for short) help to shut down the immune response after a threat has been successfully destroyed. They also prevent the immune system from attacking the body's own cells, a phenomenon known as autoimmunity. All cells in the body carry the same set of genes, but the activity of these genes varies between cell types to enable the cells to perform their different jobs. This is possible because our DNA contains regions called regulatory elements that can control the expression of particular genes. These regions can be activated in specific types of cells, which results in specific chemical modifications to DNA that only affect gene activity in those cells. The DNA sequences of these regulatory elements vary between individuals. This ‘genetic variation’ can lead to differences in the chemical modifications that occur to DNA, which is known as epigenetic variation. This means that Treg cells in one person may work in a different way to those in another individual, which could make some individuals more susceptible to autoimmune diseases than others. However, it was not clear how much genetic and epigenetic variation exists in Treg cells. Here, Arvey et al. examined Treg and other immune cells from human and mouse donors. The experiments show that some of the epigenetic modifications present in many individuals only in Treg cells, which indicates that they may be important for the activity of the Treg cells. Unexpectedly, most of the epigenetic modifications were specific to either mice or humans, but Arvey et al. identified a core set of genes that had been modified only in Treg cells in both species. In the human cells, Arvey et al. also identified genetic differences in regulatory elements that are associated with autoimmune diseases. Arvey et al.'s findings suggest that a small set of regulatory elements are crucial to the role of Treg cells in the immune system. Furthermore, genetic variation in these elements can lead to epigenetic changes in Treg cells that contribute to autoimmune diseases in humans. Further study may lead to the development of new treatments for these diseases. DOI: http://dx.doi.org/10.7554/eLife.07571.002

TLRs are recognized as promoters of tissue damage, even in the absence of pathogens. TLR binding to damage-associated molecular patterns (DAMPs) released by injured host cells unleashes an inflammatory cascade that amplifies tissue destruction. However, whether TLRs possess the reciprocal ability to curtail the extent of sterile inflammation is uncertain. Here, we investigated this possibility in mice by studying the role of conventional DCs (cDCs) in liver ischemia/reperfusion (I/R) injury, a model of sterile inflammation. Targeted depletion of mouse cDCs increased liver injury after I/R, as assessed by serum alanine aminotransferase and histologic analysis. In vitro, we identified hepatocyte DNA as an endogenous ligand to TLR9 that promoted cDCs to secrete IL-10. In vivo, cDC production of IL-10 required TLR9 and reduced liver injury. In addition, we found that inflammatory monocytes recruited to the liver via chemokine receptor 2 were downstream targets of cDC IL-10. IL-10 from cDCs reduced production of TNF, IL-6, and ROS by inflammatory monocytes. Our results implicate inflammatory monocytes as mediators of liver I/R injury and reveal that cDCs respond to DAMPS during sterile inflammation, providing the host with protection from progressive tissue damage.