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Introduction: Ductal carcinoma in situ (DCIS) of the breast is a non-obligate precursor of invasive breast cancer (IBC). Many DCIS patients are either undertreated or overtreated. The overarching goal of the study described here is to facilitate detection of patients with DCIS at risk of IBC development. Here, we propose to use risk factor data and formalin-fixed paraffin-embedded (FFPE) DCIS tissue from a large, ethnically diverse, population-based cohort of 8175 women with a first diagnosis of DCIS and followed for subsequent IBC to: identify/validate miRNA expression changes in DCIS tissue associated with risk of subsequent IBC; evaluate ipsilateral IBC risk in association with two previously identified marker sets (triple immunopositivity for p16, COX-2, Ki67; Oncotype DX Breast DCIS score); examine the association of risk factor data with IBC risk., Methods and Analysis: We are conducting a series of case-control studies nested within the cohort. Cases are women with DCIS who developed subsequent IBC; controls (2/case) are matched to cases on calendar year of and age at DCIS diagnosis. We project 485 cases/970 controls in the aim focused on risk factors. We estimate obtaining FFPE tissue for 320 cases/640 controls for the aim focused on miRNAs; of these, 173 cases/346 controls will be included in the aim focused on p16, COX-2 and Ki67 immunopositivity, and of the latter, 156 case-control pairs will be included in the aim focused on the Oncotype DX Breast DCIS score®. Multivariate conditional logistic regression will be used for statistical analyses., Ethics and Dissemination: Ethics approval was obtained from the Institutional Review Boards of Albert Einstein College of Medicine (IRB 2014-3611), Kaiser Permanente Colorado, Kaiser Permanente Hawaii, Henry Ford Health System, Mayo Clinic, Marshfield Clinic Research Institute and Hackensack Meridian Health, and from Lifespan Research Protection Office. The study results will be presented at meetings and published in peer-reviewed journals., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: The 21-gene expression profile [Oncotype DX Recurrence Score (RS)] stratifies benefit from adjuvant chemotherapy in hormone receptor (HR)-positive, HER2/neu-negative, node-negative breast cancer. It is not routinely applied to predict neoadjuvant chemotherapy (NACT) response; data in diverse patient populations also are limited. We developed a statistical model based on standard clinicopathologic features to identify high-risk cases (RS > 30) and then evaluated ability of predicted high RS to predict for NACT downstaging., Methods: Primary surgery patients with Oncotype DX RS testing 2012-2016 were identified from a prospectively-maintained database. A RS predictive model was created and applied to a dataset of comparable NACT patients. Response was defined as tumor size decrease ≥ 1 cm., Results: Of 394 primary surgery patients-60.4% white American; 31.0% African American-RS distribution was similar for both groups. No single feature reliably identified high RS patients; however, a model accounting for age, HR expression, proliferative index (MIB1/Ki67), histology, and tumor size was generated, with receiver operator area under the curve 0.909. Fifty-six NACT patients were identified (25 African American). Of 21 cases with all relevant clinicopathology, 14 responded to NACT and the model generated high-risk RS in 14 (100%); conversely, of 16 cases generating high-risk RS, only 2 did not respond., Conclusions: Predictive modelling can identify high RS patients; this model also can identify patients likely to experience primary tumor downstaging with NACT. Until this model is validated in other datasets, we recommend that Oncotype-eligible patients undergo primary surgery with decisions regarding chemotherapy made in the adjuvant setting.

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

Background: Breast cancer sentinel lymph nodes (SLNs) with metastases (mets) are often palpably enlarged. We hypothesized that the volume of the SLN and the size of mets are directly related. SLNs harboring mets are often firm, with increased intra-nodal pressure (INP), and we hypothesized that SLN volume, as well as INP, would correlate directly with SLN metastasis size., Methods: The SLN volume, INP and met size were measured in 296 SLNs and compared using linear regression analysis. The SLNs were subsequently grouped based upon pN stage. SLN INP and volume were compared between these resultant groups., Results: Increased SLN volume significantly predicted increased SLN met size on univariate and multivariate analysis (p = 0.001 and p = 0.011, respectively). SLN met size predicted increased SLN INP on both univariate and multivariate analysis (both p = 0.001). SLN volume only significantly correlated with increased SLN INP on univariate analysis (p = 0.001). On subgroup analysis of nodal disease, pN1/2/3 nodes (SLN met sizes >2 mm) were significantly larger (p = 0.039 and p = 0.003, respectively) than pN0 and pN1(mi) nodes, and had significantly increased INP (all p = 0.001) as compared to pN0, pN0(i+), and pN1(mi) nodes., Conclusions: SLN volume and INP increased with increasing SLN met size. The threshold met size for this increase was >2 mm (pN1 disease)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The objective of this work was to demonstrate that autoantibodies in breast cancer sera are not epiphenomena, and exhibit unique immunologic features resembling the rheumatic autoimmune diseases., Methods: We performed a comprehensive study of autoantibodies on a collection of sera from women with breast cancer or benign breast disease, undergoing annual screening mammography. All women in this study had suspicious mammography assessment and underwent a breast biopsy. We used indirect immunofluorescence, the crithidia assay for anti-dsDNA antibodies, and multiple ELISAs for extractable nuclear antigens., Results: Autoantibodies were detected in virtually all patients with breast cancer, predominantly of the IgG1 and IgG3 isotypes. The profile detected in breast cancer sera showed distinctive features, such as antibodies targeting mitochondria, centrosomes, centromeres, nucleoli, cytoskeleton, and multiple nuclear dots. The majority of sera showing anti-mitochondrial antibodies did not react with the M2 component of pyruvate dehydrogenase, characteristic of primary biliary cirrhosis. Anti-centromere antibodies were mainly anti-CENP-B. ELISAs for extractable nuclear antigens and the assays for dsDNA were negative., Conclusions: The distinctive autoantibody profile detected in BC sera is the expression of tumor immunogenicity. Although some of these features resemble those in the rheumatic autoimmune diseases and primary biliary cirrhosis, the data suggest the involvement of an entirely different set of epithelial antigens in breast cancer. High titer autoantibodies targeting centrosomes, centromeres, and mitochondria were detected in a small group of healthy women with suspicious mammography assessment and no cancer by biopsy; this suggests that the process triggering autoantibody formation starts in the pre-malignant phase and that future studies using validated autoantibody panels may allow detection of breast cancer risk in asymptomatic women. Autoantibodies developing in breast cancer are not epiphenomena, but likely reflect an antigen-driven autoimmune response triggered by epitopes developing in the mammary gland during breast carcinogenesis. Our results support the validity of the multiple studies reporting association of autoantibodies with breast cancer. Results further suggest significant promise for the development of panels of breast cancer-specific, premalignant-phase autoantibodies, as well as studies on the autoantibody response to tumor associated antigens in the pathogenesis of cancer.

Background: Lymphatic invasion is necessary for regional lymph node (RLN) metastasis in breast cancer (BC), while systemic metastasis requires blood vessel (BV) invasion. The site of BV invasion could be at the primary BC site or through lymphovascular anastomoses. The vague pathologic term "lymphovascular invasion" (LVI) encourages the belief that peri/intratumoral BV invasion may be common. We investigated the relative contribution of RLN metastasis to systemic metastasis by studying the relationship among LVI and RLN and/or systemic metastasis in a population-based cohort of breast cancer patients., Methods: Fisher's exact test was done to assess global associations among LVI and RLN and/or systemic metastasis in a prospective database of breast cancer patients undergoing RLN biopsy. Logistic regression was used to determine multivariable contributions of LVI to metastasis when controlling for available demographic, radiologic, and pathologic variables., Results: Of 1668 patients evaluated 25.4% were RLN positive and 10.4% had LVI. RLN metastasis was predicted by tumor size (P < .0001), HER-2/neu overexpression (P = .0022) and the interaction between LVI positive and HER-2/neu positive tumors (< .0001). Patients with LVI/HER-2-neu positive were 3 times as likely to have positive RLNs compared with patients LVI/HER-2-neu negative. Systemic metastasis was significantly (P = .0013) associated with LVI/RLN positive, but not with LVI positive/RLN negative patients (P = .137)., Conclusions: LVI predicted RLN metastasis. LVI also significantly predicted systemic metastasis, but only when the RLN was also positive. Since RLN requires lymphatic invasion, these data support the hypothesis that primary breast cancers often invade lymphatics to gain access to the systemic circulation.

Background: A benign diagnosis in a core needle biopsy (CNBx) of the breast performed for a clinically and/or radiologically suspicious abnormality is often due to a nonrepresentative sample. However, the discordance may not be recognized, resulting in a logistic delay in the diagnosis., Methods: Twenty-seven false-negative CNBxs were identified in 952 consecutive CNBxs of the breast (653 benign, 266 malignant, and 33 atypical) performed during a 1-year period. Biopsies were analyzed with respect to clinical and radiologic findings, biopsy type, type of malignancy, and interval between the original CNBx and final diagnosis. Four hundred thirty-eight (67%) of the patients with a benign CNBx diagnosis either underwent excision or had a minimum of 1-year follow-up (mean, 35.6 months; median, 36 months)., Results: The cancers missed on CNBx included 6 ductal carcinomas in situ, 17 invasive ductal carcinomas, 3 invasive lobular carcinomas, and 1 non-Hodgkin lymphoma. The overall false-negative rate was 9.1%. For palpable lesions, ultrasound-guided CNBx had a lower rate of missed cancer (3.6%) compared with CNBx without image guidance (13.3%). The false-negative rate for vacuum assisted CNBx biopsy was 7.6% (3.3% for the 11-gauge needle, 22.2% for the 14-gauge needle; 5.6% for nonpalpable mass lesions, 8.2% for microcalcifications). In all seven false-negative CNBxs performed by radiologists, the discordance between the radiologic and pathologic findings was promptly recognized due to their standard follow-up protocol. The discordance between the degree of clinical suspicion, radiologic impression, and the pathologic findings was not immediately recognized in 5 of 20 false-negative CNBxs performed by surgeons (4 without radiologic guidance and 1 with ultrasound guidance), resulting in a delay in the diagnosis ranging from 112-336 days., Conclusions: A false-negative diagnosis of breast carcinoma was found to be more common in CNBx performed without image guidance but occurred to a lesser degree in image-guided biopsies. A delay in diagnosis can be avoided by establishing a standard post-CNBx follow-up protocol., (Copyright 2003 American Cancer Society.)