Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.

Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior.

Current and new frontiers in hereditary cancer surveillance: Opportunities for liquid biopsy.

At least 5% of cancer diagnoses are attributed to a causal pathogenic or likely pathogenic germline genetic variant (hereditary cancer syndrome-HCS). These individuals are burdened with lifelong surveillance monitoring organs for a wide spectrum of cancers. This is associated with substantial uncertainty and anxiety in the time between screening tests and while the individuals are awaiting results. Cell-free DNA (cfDNA) sequencing has recently shown potential as a non-invasive strategy for monitoring cancer. There is an opportunity for high-yield cancer early detection in HCS. To assess clinical validity of cfDNA in individuals with HCS, representatives from eight genetics centers from across Canada founded the CHARM (cfDNA in Hereditary and High-Risk Malignancies) Consortium in 2017. In this perspective, we discuss operationalization of this consortium and early data emerging from the most common and well-characterized HCSs: hereditary breast and ovarian cancer, Lynch syndrome, Li-Fraumeni syndrome, and Neurofibromatosis type 1. We identify opportunities for the incorporation of cfDNA sequencing into surveillance protocols; these opportunities are backed by examples of earlier cancer detection efficacy in HCSs from the CHARM Consortium. We seek to establish a paradigm shift in early cancer surveillance in individuals with HCSs, away from highly centralized, regimented medical screening visits and toward more accessible, frequent, and proactive care for these high-risk individuals.

3D chromatin remodeling potentiates transcriptional programs driving cell invasion.

The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.

Preoperative factors that predict pathologic nodal involvement in early-stage HER2+ breast cancer: selecting cT1cN0 patients for treatment with neoadjuvant chemotherapy versus upfront surgery.

PURPOSE: The goal of this study was to identify the preoperative predictors of pathologic nodal metastases (pN+) in cT1cN0 HER2+ breast cancer undergoing upfront surgery. METHODS: We retrospectively reviewed data from women with cT1-T2N0 HER2+ breast cancer treated with neoadjuvant therapy (NAC) or upfront surgery at our institution between 2012 and 2023. Factors associated with management strategy were evaluated, and in those undergoing upfront surgery, univariate analyses were performed to identify the clinicopathologic factors associated with nodal metastases. RESULTS: Overall, 255 women with cT1-T2N0 HER2+ breast cancer met inclusion criteria, including 170 (68.6%) upfront surgery patients and 85 (31.4%) who underwent NAC. The median age at diagnosis was 59 years (range, 27-90 years). Younger age, larger clinical tumor size, high-grade disease, ER-PR-HER2+ subtype, and year of diagnosis after 2019 were significantly associated with receipt of NAC (p < 0.05). In those undergoing upfront surgery, 25.3% were pN+ , including 32.5% of cT1cN0 tumors. Factors associated with nodal involvement included age under 50, larger clinical tumor size, lymphovascular invasion (LVI), multifocality/multicentricity, and abnormal lymph nodes on axillary ultrasound (p < 0.05). In subset analysis of cT1cN0 HER2+ cases, LVI remained the strongest predictor of pN + disease (73.3% vs. 22.6%, p < 0.001). Patients with cT1cN0 HER2+ breast cancer under 50 years had a 47.1% likelihood of pN+ disease. CONCLUSION: Patients with cT1cN0 breast cancer have a 32.5% likelihood of nodal metastases, with higher incidence with younger age, LVI, multifocality/multicentricity, and abnormal axillary ultrasound. The presence of these factors may identify the patients who would benefit from treatment with neoadjuvant chemotherapy.

Awareness and Candidacy for Endocrine Prevention and Risk Reducing Mastectomy in Unaffected High-Risk Women Referred for Breast Cancer Risk Assessment.

INTRODUCTION: Primary prevention of breast cancer in women at elevated risk includes several strategies such as endocrine prevention and risk-reducing mastectomy (RRM). The objective of this study was to evaluate awareness of different preventive strategies across high-risk subgroups. PATIENTS AND METHODS: Women referred for high risk evaluation between 2020 and 2023 completed an initial risk-assessment questionnaire that included questions around perceived lifetime risk and consideration of preventive strategies. One-way analysis of variance (ANOVA) and chi-squared tests were used to compare differences across different high-risk subgroups. RESULTS: 482 women with a median age of 43 years (20-79 years) met inclusion criteria; 183 (38.0%) germline pathogenic variant carriers (GPV), 90 (18.7%) with high-risk lesions (HRL) on breast biopsy, and 209 (43.4%) with strong family history (FH) without a known genetic predisposition. Most high-risk women reported that they had considered increased screening and surveillance (83.7%) and lifestyle strategies (80.6%), while fewer patients had considered RRM (39.8%) and endocrine prevention (27.0%). Prior to initial consultation, RRM was more commonly considered in GPV carriers (59.4%) relative to those with HRL (33.3%) or strong FH (26.3%, p < 0.001). Based on current guidelines, 206 (43%) patients were deemed eligible for endocrine prevention, including 80.5% with HRL and 39.0% with strong FH. Prior consideration of endocrine prevention was highest in patients with HRL and significantly lower in those with strong FH (47.2% HRL versus 31.1% GPV versus 18.7% FH, p = 0.001). CONCLUSIONS: Endocrine prevention is the least considered preventive option for high-risk women, despite eligibility in a significant proportion of those presenting with HRL or strong FH.

Surgical Decision Making in Genetically High-Risk Women: Quantifying Postoperative Complications and Long-Term Risks of Supplemental Surgery After Risk-Reducing Mastectomy.

BACKGROUND: Risk-reducing mastectomy (RRM) helps prevent breast cancer in high-risk women but also carries a risk of unanticipated supplemental surgeries. We sought to determine the likelihood of supplemental surgeries following RRM. METHODS: We performed a retrospective cohort study of female patients with a confirmed germline pathogenic variant (GPV) in a breast cancer susceptibility gene (BRCA1/2, PALB2 and others) who underwent bilateral or contralateral RRM at our institution between 2006 and 2022. Supplemental surgeries were defined as any operation requiring general or local anesthesia performed outside of the initially planned procedure(s). The Kaplan-Meier method was used to estimate the 5-years cumulative incidence of supplemental surgery. RESULTS: Of 560 GPV carriers, RRMs were performed in 258 (46.1%) women. The median age of the cohort was 44 years (interquartile range 37-52 years), with 33 (12.8%) patients undergoing RRM without reconstruction and 225 (87.2%) undergoing RRM with reconstruction. Following surgery, 34 patients (13.2%) developed early (< 30 days) postoperative complications, including infection, hematoma, seroma, loss of the nipple areola complex, flap necrosis, implant exposure and/or prosthesis removal. At a median follow-up of 3.8 years, 94 (36.4%) GPV carriers underwent at least one reoperation. Participants who experienced an early postoperative complication had the highest rate of reoperation (85.3% vs. 29.0%; p < 0.001) and a significantly higher likelihood of multiple additional surgical interventions (41.2% vs. 10.7%; p < 0.001). The 5-years rate of supplemental surgery was 39.2% [95% confidence interval (CI) 32.7-46.5] in the overall cohort and 31.5% (95% CI 24.9-39.3) in patients without an early postoperative complication. CONCLUSIONS: Unanticipated supplemental surgeries occur in 40% of GPV carriers following RRM and in nearly one-third of patients without early postoperative complications.

Histology agnosticism: Infra-molecularizing disease?

The term "molecularization" has been used by historians and sociologists of science to describe the transition from an anatomic view of the body to a submicroscopic one, where health and illness, indeed life itself, are increasingly defined in terms of an individual's "genetic landscape." Here we introduce the notion of the infra-molecular as a way of extending and nuancing the molecularization trope as it applies to the domain of (post)genomic oncology. In particular we look at how infra-molecularity is enacted in practice as part of the so-called "histology-agnostic" turn in clinical cancer research and care. Drawing on fieldwork in North American oncology settings, we analyze how histology agnosticism partially reconfigures knowledge and practice across the linked domains of drug development and clinical trials, therapeutic decision making, and regulation, and the implications of this for an ongoing revision of how we understand the biopathology and temporality of cancer. We show how, in practice, the inframolecular gaze entails a "return" of histology as a modulator of histology-agnostic drugs and background for interpretation of mutational complexity.

Evaluation of a 'plug and play' nanoflow liquid chromatography system for MS-based proteomic characterization of clinical FFPE specimens.

INTRODUCTION: Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tumor tissue specimens has gained interest in the last 5 years due to technological advances and improved sample collection, as well as biobanking for clinical trials. The real-world implementation of clinical proteomics to these specimens, however, is hampered by tedious sample preparation steps and long instrument acquisition times. AREAS COVERED: To advance the translation of quantitative proteomics into the clinic, we are comparing the performance of the leading commercial nanoflow liquid chromatography (nLC) system (based on literature reviews), the Easy-nLC 1200 (Thermo Fisher Scientific, Waltham, MA, U.S.A.), to the Evosep One HPLC (Evosep Biosystems, Odense, Denmark). We measured FFPE-tissue digests from 21 biological replicates with a similar gradient on both of the LC systems while keeping the on-column amount (1 µg total protein) and the single-shot data-dependent acquisition-based MS/MS method constant. EXPERT OPINION: Overall, the Evosep One facilitates robust and sensitive high-throughput sample acquisition, making it suitable for clinical MS. We found the Evosep One to be a useful platform for positioning mass spectrometry-based proteomics in the clinical setting. The clinical application of nLC/MS will inform clinical decision-making in oncology and other diseases.

Margin Status and Local Recurrence in Phyllodes Tumours of the Breast: A Canadian Series.

BACKGROUND: Phyllodes tumours of the breast are rare fibroepithelial neoplasms with a propensity for recurrence. While surgical excision remains the standard of care, the optimal margin width is an area of active investigation. Recent studies have questioned the necessity for wide, local excision. METHODS: We conducted a retrospective, cohort study of patients with phyllodes tumours treated at our institution between 2003 and 2021. Demographic, histopathological, and recurrence data were captured; malignant phyllodes were excluded. Cox proportional hazard models were used to identify covariates associated with local recurrence. RESULTS: Of 187 patients with phyllodes tumours, 82.9% (n = 155) were classified as benign while 17.1% (n = 32) were borderline. Initial surgical margins were positive in 26.2% (n = 49), < 2 mm in 50.8% (n = 95), and ≥ 2 mm in 23% (n = 43) patients. Among patients with positive margins, 61.2% (n = 30) underwent margin revision. At a median follow-up of 2.9 years, the recurrence rate was 3.7%. On univariate analysis, only a positive margin at the time of initial surgery and not margin width was significantly associated with a higher rate of disease recurrence (hazard ratio [HR] 9.52, 95% confidence interval [CI] 1.85-49.2), as was a size greater than 4 cm on preoperative imaging (HR 10.78, 95% CI 0.97-120.1). Revision of an initially positive margin was not significantly associated with decreased local recurrence (p = 1). CONCLUSIONS: In this large cohort of benign and borderline phyllodes tumours, positive resection margins and not margin width at the initial surgery were associated with a increased recurrence. Individualization of decisions regarding margin reexcision is important.

Clinicopathologic features of breast cancers diagnosed in women treated with prior radiation therapy for Hodgkin lymphoma: Results from a population-based cohort.

BACKGROUND: Childhood and young adult survivors of Hodgkin lymphoma (HL) are at elevated risk of developing breast cancer, yet little data exist on the tumor characteristics that develop in this high-risk patient population. METHODS: The National Cancer Institute's Surveillance, Epidemiology, and End Results database was used to identify breast cancers diagnosed between 1990 and 2016 in women who had received prior radiation therapy for HL at age 30 years or younger. Clinicopathologic features of subsequent breast cancers (breast cancer after radiation therapy for HL [BC-HL]) were examined and compared with breast cancers diagnosed in women who had no prior malignancy (breast cancer with no prior malignancy [BC-NPM]). RESULTS: In total, 321 breast cancers were identified in 257 women who had a history of radiation therapy for HL. The median age at HL diagnosis was 22 years (interquartile range, 18-26 years), and nearly all patients in the BC-HL group (97.9%) were diagnosed ≥8 years after radiation therapy. Overall, 56 patients in the BC-HL group (21.8%) developed bilateral breast cancer. Compared with women who had BC-NPM, those who had BC-HL were younger (43 vs 60 years; P < .001) and were less likely to present with ductal carcinoma in situ (8.4% vs 14.9%; P = .001). On multivariable analysis that included adjustment for age, invasive BC-HL was associated with smaller (≤2 cm) tumor size (odds ratio, 1.64; 95% CI, 1.25-2.15) and upper outer quadrant tumors (odds ratio, 1.37; 95% CI, 1.04-1.81) compared with BC-NPM. In a subset analysis of 102 women who had HER2/neu status available, the distribution of biologic subtype was not significantly different between BC-HL and BC-NPM (P = .16). CONCLUSIONS: Breast cancers in women who previously received radiation therapy for HL are characterized by earlier onset disease, although most remain estrogen receptor-positive and have early stage disease at presentation. LAY SUMMARY: Women who have had radiation therapy for Hodgkin lymphoma at a young age are at increased risk of developing early onset breast cancer; however, most of these breast cancers are sensitive to hormones (estrogen receptor-positive) and are diagnosed at early stages. Because these breast tumors are estrogen receptor-positive, medications that prevent breast cancer by blocking the effect of or lowering hormone levels (also termed endocrine prevention) may be useful in this group of high-risk women.

"Game Changer": Health Professionals' Views on the Clinical Utility of Circulating Tumor DNA Testing in Hereditary Cancer Syndrome Management.

BACKGROUND: We explored health professionals' views on the utility of circulating tumor DNA (ctDNA) testing in hereditary cancer syndrome (HCS) management. MATERIALS AND METHODS: A qualitative interpretive description study was conducted, using semi-structured interviews with professionals across Canada. Thematic analysis employing constant comparison was used for analysis. 2 investigators coded each transcript. Differences were reconciled through discussion and the codebook was modified as new codes and themes emerged from the data. RESULTS: Thirty-five professionals participated and included genetic counselors (n = 12), geneticists (n = 9), oncologists (n = 4), family doctors (n = 3), lab directors and scientists (n = 3), a health-system decision maker, a surgeon, a pathologist, and a nurse. Professionals described ctDNA as "transformative" and a "game-changer". However, they were divided on its use in HCS management, with some being optimistic (optimists) while others were hesitant (pessimists). Differences were driven by views on 3 factors: (1) clinical utility, (2) ctDNA's role in cancer screening, and (3) ctDNA's invasiveness. Optimists anticipated ctDNA testing would have clinical utility for HCS patients, its role would be akin to a diagnostic test and would be less invasive than standard screening (eg imaging). Pessimistic participants felt ctDNA testing would add limited utility; it would effectively be another screening test in the pathway, likely triggering additional investigations downstream, thereby increasing invasiveness. CONCLUSIONS: Providers anticipated ctDNA testing will transform early cancer detection for HCS families. However, the contrasting positions on ctDNA's role in the care pathway raise potential practice variations, highlighting a need to develop evidence to support clinical implementation and guidelines to standardize adoption.

Surgical Management and Contralateral Breast Cancer Risk in Women with History of Radiation Therapy for Hodgkin Lymphoma: Results from a Population-Based Cohort.

BACKGROUND: Women with history of chest irradiation for Hodgkin lymphoma are at increased risk of developing bilateral breast cancer, although contralateral breast cancer risk estimates in this population remain undefined. METHODS: We queried the SEER database for women treated with radiation therapy for Hodgkin lymphoma prior to age 30 years and were diagnosed with a subsequent breast cancer between 1990-2016. Trends in surgical management and the 5- and 10-year cumulative incidence of contralateral breast cancer were evaluated. RESULTS: The cohort included 295 women with a median age of 22 years (range 8-30 years) at Hodgkin lymphoma diagnosis, and 42 years (range 22-65 years) at breast cancer diagnosis. Overall, 263 (89.2%) presented with unilateral breast cancer, while 32 (10.8%) presented with synchronous bilateral breast cancer. Breast-conserving surgery was performed in 17.3% of patients, while mastectomy was performed in 82.7%. In 263 patients presenting with unilateral breast cancer, 50 (19.0%) underwent breast-conserving surgery and 213 (81.0%) underwent mastectomy. Subgroup analysis of mastectomy patients demonstrated a 40.5% bilateral mastectomy rate. The 5-year incidence of contralateral breast cancer in women who underwent unilateral surgery was 9.4% [95% confidence interval (CI), 5.6-15.4%], increasing to 20.2% (95% CI, 13.7-29.2%) at 10-year and 29.9% (95% CI, 20.8-41.9%) at 15-year follow-up. CONCLUSIONS: Women with a history of prior chest radiation for Hodgkin lymphoma with a diagnosis of breast cancer have a 10-year contralateral breast cancer risk of 20%. These findings support consideration of contralateral prophylactic mastectomy during surgical decision-making for management of this high-risk patient population.

Incidence of Occult Breast Cancer in Carriers of BRCA1/2 or Other High-Penetrance Pathogenic Variants Undergoing Prophylactic Mastectomy: When is Sentinel Lymph Node Biopsy Indicated?

BACKGROUND: This study sought to determine the likelihood of occult malignancy during risk-reducing mastectomy in high-penetrance pathogenic variant carriers to help refine axillary staging recommendations. METHODS: The authors performed a retrospective cohort study analyzing all female carriers of pathogenic variants in BRCA1/2, PALB2 or other genes who underwent prophylactic surgery at their institution between 2006 and 2021. Occult breast cancer was defined as the unanticipated presence of in situ or invasive malignancy on pathologic evaluation of prophylactic mastectomy specimens. RESULTS: Of 523 women, 243 carriers met the inclusion criteria for the study including 124 BRCA1 (51.0%), 108 BRCA2 (44.4%), and 11 PALB2, TP53, CDH1, or PTEN (4.6%) carriers. The median age was 44 years (interquartile range, 37-52 years). Overall, 128 women (52.7%) underwent bilateral prophylactic mastectomies, and 115 (47.3%) underwent contralateral prophylactic mastectomy. In the 371 mastectomies performed, 16 (4.3%) occult malignancies were diagnosed. Most of the occult malignancies were ductal carcinoma in situ (13 mastectomies, 3.5%), whereas 3 mastectomies (0.8%) contained invasive breast cancer. If Breast Imaging Reporting and Data System (BIRADS) 1-2 or BIRADS 3 findings were reported on preoperative magnetic resonance imaging (MRI), the rate of occult malignancy decreased to 3.0 and 2.8%, respectively, per mastectomy. The patient-level factors associated with a likelihood of occult breast cancer greater than 10% included a history of prior breast cancer, age exceeding 60 years, and BIRADS 4 findings on preoperative imaging. CONCLUSIONS: Occult invasive malignancy was detected in less than 1% of the risk-reducing mastectomies performed for women with BRCA1/2 or PALB2 pathogenic variants. Sentinel lymph node biopsy can be safely avoided when BIRADS 1-3 findings are reported on preoperative MRI.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Precise Quantitation of PTEN by Immuno-MRM: A Tool To Resolve the Breast Cancer Biomarker Controversy.

The tumor suppressor PTEN is the main negative regulator of PI3K/AKT/mTOR signaling and is commonly found downregulated in breast cancer (BC). Conflicting data from conventional immunoassays such as immunohistochemistry (IHC) has sparked controversy about PTEN's role as a prognostic and predictive biomarker in BC, which can be largely attributed to the lack of specificity, sensitivity, and interlaboratory standardization. Here, we present a fully standardized, highly sensitive, robust microflow immuno-MRM (iMRM) assay that enables precise quantitation of PTEN concentrations in cells and fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) tissues, down to 0.1 fmol/10 µg of extracted protein, with high interday and intraday precision (CV 6.3%). PTEN protein levels in BC PDX samples that were determined by iMRM correlate well with semiquantitative IHC and WB data. iMRM, however, allowed the precise quantitation of PTEN-even in samples that were deemed to be PTEN negative by IHC or western blot (WB)-while requiring substantially less tumor tissue than WB. This is particularly relevant because the extent of PTEN downregulation in tumors has been shown to correlate with severity. Our standardized and robust workflow includes an 11 min microflow LC-MRM analysis on a triple-quadrupole MS and thus provides a much needed tool for the study of PTEN as a potential biomarker for BC.

Population-based analysis of non-operative management and treatment patterns in older women with estrogen receptor-positive breast cancer.

PURPOSE: To examine the proportion of older women with ER + HER2- breast cancer receiving non-operative management versus surgery, and to evaluate the use of axillary staging and adjuvant radiation in this population. METHODS: We queried the SEER database to identify all women aged 70 years or older with stage I-III ER + HER2- invasive breast cancer diagnosed between 2010 and 2016. We evaluated trends in non-operative management, breast surgery, axillary staging, and adjuvant radiation according to age at diagnosis. RESULTS: We identified 57,351 older women with ER + HER2- disease. Overall, 3538 (6.2%) of the cohort underwent non-operative management, 38,452 (67.0%) underwent breast-conserving surgery (BCS), and 15,361 (26.8%) underwent mastectomy. The proportion of patients undergoing non-operative management increased from 2.8% among 70-74-year-old women to 30.1% in those ≥ 90 years old (p < 0.001). In 53,813 women who underwent surgery, 36,850 (68.5%) underwent sentinel lymph node biopsy, while 10,861 (20.2%) underwent axillary lymph node dissection. Subgroup analysis of 29,032 older women undergoing BCS for stage I ER + HER2- breast cancer revealed a 14.2% rate of omission of axillary staging, increasing from 5.3% in those 70-74 years to 67.6% in those ≥ 90 years old (p < 0.001). Receipt of adjuvant radiation occurred in 63.3% of older women following BCS and 18% post-mastectomy, with similar trends towards omission in older age groups. CONCLUSION: Primary breast surgery remains the dominant management strategy for the majority of older women with ER + HER2- breast cancer. Omission of axillary staging and adjuvant radiation are used in a minority of eligible women undergoing breast conservation for early-stage disease.

A qualitative study to evaluate physician attitudes regarding omission of surgery among exceptional responders to neoadjuvant systemic therapy for breast cancer (NRG-CC006).

PURPOSE: Accrual to clinical trials that challenge well-established treatment paradigms represents a unique challenge. Physician opinions on investigation of a novel approach to breast cancer treatment, in which patients with complete response to neoadjuvant chemotherapy are offered omission of lumpectomy, are unknown. NRG-CC006 sought to describe physician attitudes toward a novel approach to breast cancer treatment. METHODS: We recruited 18 participants in the fields of surgery, medical oncology, and radiation oncology to participate in the semi-structured telephone interviews. Main outcomes are qualitative themes associated with omission of surgery. RESULTS: Of 18 interview participants, specialty and gender were evenly represented across surgery, medical oncology, and radiation oncology. Qualitative themes included general attitudes toward treatment de-escalation, stakeholder considerations, and trial/protocol considerations. The vast majority of participants expressed interest in investigation of omission of surgery, with all participants endorsing need for further investigation into treatment de-escalation. Stakeholder considerations in opening such a trial emphasized need for multidisciplinary involvement and, particularly, the unique role of surgeons as gatekeepers in breast cancer treatment. Finally, participants endorsed a need for further foundational studies to develop ways to predict complete pathologic response to chemotherapy without surgical intervention. CONCLUSIONS: Physicians expressed interest in investigating a novel approach to breast cancer treatment that would omit surgery in complete responders to neoadjuvant chemotherapy. Multidisciplinary input, and specifically surgeon engagement, will be key to the success of future investigations. Ongoing work to develop approaches to predict pathologic complete response accurately is needed to achieve the promise of this idea. ClinTrials #: BR005: NCT03188393 June 13, 2017.

Oncologic Safety of Sentinel Lymph Node Biopsy Alone After Neoadjuvant Chemotherapy for Breast Cancer.

BACKGROUND: The oncologic safety of sentinel lymph node biopsy (SLNB) alone for clinically node-positive (cN1-2) patients who convert to pathologic node-negativity (ypN0) after neoadjuvant chemotherapy (NAC) is not well established. METHODS: This study retrospectively identified 244 consecutive patients with a diagnosis of cT1-3cN0-2 breast cancer who underwent NAC followed by SLNB at the authors' institution between 2013 and 2018. The patients were categorized as clinically node-negative (cN0) or cN1-2 before the onset of NAC, and the Kaplan-Meier method was used to compare locoregional and distant recurrence rates after SLNB alone for ypN0 patients. RESULTS: Among 244 patients who underwent NAC followed by surgery with SLNB for axillary staging, 112 (45.9%) were cN0 at presentation, whereas 132 (54.5%) had biopsy-proven cN1-2 disease and converted to cN0 after treatment. Of the patients presenting with cN0 disease, 102 (91.1%) were ypN0 on SLNB pathology compared with 60 cN1/2 patients (45.5%; p < 0.001). Regional nodal irradiation was administered to 5% of the cN0/ypN0 patients compared with 70.7% of the cN1-2/ypN0 patients (p < 0.001). Overall, 211 patients were treated with SLNB alone and had a median follow-up period of 36 months (interquartile range [IQR], 24-53 months). For 101 cN0/ypN0 patients who underwent SLNB alone, the 5-year local and regional recurrence rates were respectively 5.7% (95% confidence interval [CI], 2.4-13.8) and 1% (95% CI 0.1-7.0). For 58 cN1-2/ypN0 patients who underwent SLNB alone, the 5-year local and regional recurrence rates were respectively 4.1% (95% CI 1.0-15.5) and 0%, with no axillary recurrences noted. CONCLUSION: For ypN0 patients, SLNB alone after NAC is associated with low and acceptable short-term axillary recurrence rates. Additional follow-up data from prospective clinical trials are needed to confirm long-term oncologic safety and define optimal local therapy recommendations.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

Using Two Peptide Isotopologues as Internal Standards for the Streamlined Quantification of Low-Abundance Proteins by Immuno-MRM and Immuno-MALDI.

Mass spectrometry (MS), particularly targeted proteomics, is increasingly being used for quantifying specific proteins and peptides in clinical specimens. The coupling of immuno-enrichment of proteotypic peptides with MS [e.g., immuno-multiple reaction monitoring (MRM) and immuno-matrix-assisted laser desorption ionization (MALDI)] enables the development of highly sensitive and specific assays for low-abundance signaling proteins. By incorporating stable isotope-labeled standards, these workflows allow the determination of endogenous protein concentrations. This is typically achieved through external calibration, often using surrogate matrices, which has inherent limitations for the analysis of clinical specimens as there are often substantial variations in the sample matrix, and sample amounts are typically limited. We have previously introduced the use of two peptide isotopologues for generating external calibration curves in plasma. Here, we present a two-point internal calibration (2-PIC) strategy using two isotopologues for immuno-MS assays and demonstrate its flexibility and robustness. Quantification of the tumor suppressor PTEN in Colo-205 cells by immuno-MRM and immuno-MALDI using 2-PIC and external calibration yielded very similar results (relative standard deviation between 2-PIC and external calibration: 4.9% for immuno-MRM; 1.1% for immuno-MALDI), without the need for a surrogate matrix or additional patient material for calibration, while concurrently reducing the instrument time and cost. Although our PTEN immuno-MRM and immuno-MALDI assays can be considered to be orthogonal as they utilized entirely different sample preparation and MS analysis workflows, targeted different PTEN peptides, and were performed in different laboratories, the endogenous Colo-205 PTEN levels determined with 2-PIC showed a good correlation (r2 = 0.9966) and good agreement (0.48 ± 0.01 and 0.29 ± 0.02 fmol/µg of total protein) between immuno-MRM and immuno-MALDI.