Invasive Breast Tumors Research Articles

EIF3H Orchestrates Hippo Pathway-Mediated Oncogenesis via Catalytic Control of YAP Stability.

EIF3H is presumed to be a critical translational initiation factor. Here, our unbiased screening for tumor invasion factors has identified an unexpected role for EIF3H as a deubiquitylating enzyme that dictates breast tumor invasion and metastasis by modulating the Hippo-YAP pathway. EIF3H catalyzed YAP for deubiquitylation, resulting in its stabilization. Structure-based molecular modeling and simulations coupled with biochemical characterization unveiled a unique catalytic mechanism for EIF3H in dissociating polyubiquitin chains from YAP through a catalytic triad consisting of Asp90, Asp91, and Gln121. Trp119 and Tyr 140 on EIF3H directly interacted with the N-terminal region of YAP1, facilitating complex formation of EIF3H and YAP1 for YAP1 deubiquitylation. Stabilization of YAP via elevated EIF3H promoted tumor invasion and metastasis. Interference of EIF3H-mediated YAP deubiquitylation blocked YAP-induced tumor progression and metastasis in breast cancer models. These findings point to a critical role for YAP regulation by EIF3H in tumor invasion and metastasis. SIGNIFICANCE: This work demonstrates that EIF3H is a novel bona fide deubiquitinase that counteracts YAP ubiquitylation and proteolysis, and stabilization of YAP by EIF3H promotes tumor invasion and metastasis.

Racial differences in CD8+ T cell infiltration in breast tumors from Black and White women

BackgroundAfrican American/Black women with breast cancer have poorer survival than White women, and this disparity persists even after adjusting for non-biological factors. Differences in tumor immune biology have been reported between Black and White women, and the tumor immune milieu could potentially drive racial differences in breast cancer etiology and outcome.MethodsWe examined the association of CD8+ cytotoxic T cells with clinical-pathological variables in the Women’s Circle of Health Study (WCHS) population of predominantly Black breast cancer patients. We evaluated 688 invasive breast tumor samples (550 Black, 138 White) using immunohistochemical staining of tissue microarray slides. CD8+ T cells were scored for each patient tumor sample with digital image analysis.ResultsBlack women had a significantly higher percentage of high-grade, estrogen receptor (ER)-negative, and triple-negative tumors than White women and significantly higher CD8+ T cell density (median 87.6/mm2 vs. 53.1/mm2; p < 0.001). Within the overall population and in the population of Black women only, CD8+ T cell density was significantly higher in younger patients and patients with high-grade and ER/PR-negative tumors. No significant associations were observed between CD8+ T cell density and overall survival or breast cancer-specific survival in the overall population, or when Black patients were analyzed as a separate group. However, when stratified by subtype, Black women with triple-negative breast cancer and high CD8+ T cell density showed a trend towards better overall survival in comparison with patients with low CD8+ T cell density (HR = 0.51; 95% CI 0.25–1.04).ConclusionsOur data raise the possibility that distinct mechanisms of immune cell action may occur in different racial groups.

Abstract C073: The association between 27-hydroxycholesterol metabolizing enzymes, CYP27A1 and CYP7B1, expression and mortality in a multiethnic U.S. population of breast cancer patients

Abstract Breast cancer is the most common cancer and the second leading cause of cancer death among women in the U.S. Obesity is one of the most significant risk factors for breast cancer in postmenopausal women and is a key predictor for poor prognosis. Postulated mechanisms underlying the association between obesity and post-menopausal breast cancer include higher levels of estradiol, hypercholesterolemia, and inflammation. 27-hydroxychlolesterol (27HC) is produced when cholesterol is metabolized by the enzyme, sterol 27-hydroxylase (CYP27A1), a conversion that is reversed by the catabolizing enzyme, oxysterol 7α-hydroxylase (CYP7B1). Laboratory studies established that 27HC is an endogenous selective estrogen receptor modulator (SERM) capable of promoting breast cancer growth by binding to the estrogen receptor (ER) and by increasing the metastatic potential of breast tumors through the activation of the liver X receptor (LXR). However, there are significant gaps in the understanding of 27HC’s complex role in breast cancer pathobiology and breast cancer survival, particularly in multiethnic populations. We examined the association between protein expression profiles for CYP27A1 and CYP7B1 with breast cancer-specific and overall mortality in a multiethnic U.S. population by conducting immunohistochemical analyses, utilizing commercially available antibodies to CYP27A1 and CYP7B1, on a total of 787 invasive breast tumors included in a population-based tissue microarray. Based on Cox proportional hazards regression analysis with adjustment for age, stage, ER and progesterone receptor (PR) status, and first course treatment, we did not observe significant associations between expression of CYP27A1 or CYP7B1 and mortality across all cases. However, in subgroup analyses within major racial/ethnic groups (Caucasian, Japanese, and Native Hawaiian) we observed a significant negative association for CYP7B1 expression for both breast cancer-specific (HR=0.057, 95% CI: 0.012-0.264) and overall mortality (HR=0.119, 95% CI: 0.028-0.509) among Native Hawaiian women, but not among Caucasian or Japanese women. This is the first report to demonstrate racial/ethnic differences in the association between the levels of these cholesterol regulating enzymes in breast tumors and survival in a multiethnic population of breast cancer patients. Citation Format: Lenora WM Loo, Yurii B Shvetsov, Iona Cheng, Brenda Y Hernandez. The association between 27-hydroxycholesterol metabolizing enzymes, CYP27A1 and CYP7B1, expression and mortality in a multiethnic U.S. population of breast cancer patients [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C073.

Lipid-weighted intraoperative photoacoustic tomography of breast tumors: Volumetric comparison to preoperative MRI

With a lifetime risk of 1 in 8, breast cancer continues to be a major concern for women and their physicians. The optimal treatment of the disease depends on the stage of the cancer at diagnosis, which is typically assessed using medical imaging. However, currently employed imaging systems for breast tumor measurement rarely agree perfectly.Our group developed an Intraoperative Photoacoustic Screening (iPAS) soft tissue scanner featuring high bulk tissue sensitivity, a clinically compatible scan-time of 6 min, imaging depths greater than 2 cm and the capability to visualize whole breast tumors based on their lipid, rather than hemoglobin, profile. Here, we report on the first clinical experience with breast cancer patients by comparing tumor-measurement using iPAS, preoperative dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) and gold-standard pathology. Tumor size was measured volumetrically for iPAS and DCE-MRI, and separately using maximum diameters for pathology, DCE-MRI and iPAS. Comparisons were performed using Pearson’s correlation coefficients, and the non-parametric Wilcoxon signed-rank test.Twelve consecutive patients were included in the study, contingent on pathologically documented invasive carcinoma. iPAS volumetric tumor size was positively correlated to DCE-MRI (Pearson’s r = 0.78, p = 0.003) and not significantly different (Wilcoxon, p = 0.97). In comparison to pathology, tumor diameters given by iPAS were positively correlated (Pearson’s r = 0.87, p = 0.0002) and significantly different (Wilcoxon, p = 0.0015).The results indicated that volumetric-measurement of invasive breast tumors with iPAS is similar to that of DCE-MRI. On the other hand, tumor diameter measurements were less reliable. Beyond enhancing surgical specimen examination, an extension of this technology to diagnostic imaging promises a new perspective on tumor assessment, potentially improving our current understanding and treatment of breast cancer.

Papillary carcinoma breast: A tertiary care institute experience

Introduction: Breast cancer is the most common cancer in women globally and in India. Papillary lesions of the breast constitute around 5% of all the breast lesions. They encompasses a morphologically heterogeneous group of lesions, all of which share a growth pattern characterised by the presence of arborescent fibrovascular stalks lined by epithelial cells with or without a layer of myoepithelial cells. Invasive papillary carcinoma is an invasive adenocarcinoma which has predominant papillary morphology >90% in the invasive component. Invasive micropapillary carcinoma is a rare type accounting for only 0.9 to 2.0% of all invasive breast tumours. Encapsulated papillary carcinoma consists of fibrous capsule surrounding a nodule composed of delicate fibrovascular stalks, covered by monomorphic population of neoplastic epithelial cells. Micropapillary carcinoma is characterized by higher incidence of lymph node metastasis and lymphovascular invasion resulting in poor prognosis. The purpose of this study is to share the last 13 years experience regarding papillary lesions of breast in the department of pathology in our institute. Material and Methods: A 13-year (2006-2019) retrospective review of database of patients diagnosed with breast cancer was performed. The medical records of 8 patients with papillary breast cancer who underwent surgery were retrieved and their clinical data, surgical treatment and pathological findings were reviewed. Results: A total of 884 patients underwent mastectomy for invasive breast cancer during the 13 year period. Out of 884 patients, eight were diagnosed with papillary carcinoma breast (0.90%). Amongst the eight, three were diagnosed as invasive papillary carcinoma, two as encapsulated papillary carcinoma and rest three as invasive micropapillary carcinoma. The mean age of presentation was 63.3 (range 45-80) years. The tumour size ranged between 3.5 to 6cm in its greatest dimension. Two out of eight patients had lymph node metastasis with no distant metastasis. Conclusion: Papillary carcinoma of the breast is a rare entity, the reported incidence of pure micropapillary carcinomas is 0.9 to 2% of all breast cancers. It is associated commonly with lymph node metastasis at the time of presentation and have relatively poor prognosis. Much lower prevalence of papillary carcinoma breast was seen in our institutional study.

Time-to-enhancement at ultrafast breast DCE-MRI: potential imaging biomarker of tumour aggressiveness.

This study was conducted in order to investigate whether there is a correlation between the time-to-enhancement (TTE) in ultrafast MRI and histopathological characteristics of breast cancers. Between January and August 2017, 274 consecutive breast cancer patients (mean age, 53.5years; range, 25-80years) who underwent ultrafast MRI and subsequent surgery were included for analysis. Ultrafast MRI scans were acquired using TWIST-VIBE or 4D TRAK-3D TFE sequences. TTE and maximum slope (MS) were derived from the ultrafast MRI. The repeated measures ANOVA, Mann-Whitney U test and Kruskal-Wallis H test were performed to compare the median TTE, MS and SER according to histologic type, histologic grade, ER/PR/HER2 positivity, level of Ki-67 and tumour subtype. For TTE calculation, intraclass correlation coefficient (ICC) was used to evaluate interobserver variability. The median TTE of invasive cancers was shorter than that of in situ cancers (p < 0.001). In invasive cancers, large tumours showed shorter TTE than small tumours (p = 0.001). High histologic/nuclear grade cancers had shorter TTE than low to intermediate grade cancers (p < 0.001 and p < 0.001). HER2-positive cancers showed shorter TTE than HER2-negative cancers (p = 0.001). The median TTE of cancers with high Ki-67 was shorter than that of cancers with low Ki-67 (p < 0.001). ICC between two readers showed moderate agreement (0.516). No difference was found in the median MS or SER values according to the clinicopathologic features. The median TTE of breast cancer in ultrafast MRI was shorter in invasive or aggressive tumours than in in situ cancer or less aggressive tumours, respectively. • Invasive breast tumours show a shorter TTE in ultrafast DCE-MRI than in situ cancers. • A shorter TTE in ultrafast DCE-MRI is associated with breast tumours of a large size, high histologic or nuclear grade, PR negativity, HER2 positivity and high Ki-67 level.

Tumor Proliferation and Invasiveness Derived From Ultrasound Appearances of Invasive Breast Cancers: Moving Beyond the Routine Differential Diagnosis.

To investigate the correlation between ultrasound (US) appearances of invasive breast cancers and tumor proliferation and invasiveness measured according to the histologic grade, Ki-67 expression, axillary lymph node metastasis (ALNM), and lymphovascular invasion (LVI). This study evaluated 676 patients who underwent primary surgical treatment of invasive breast cancers. The preoperative US reports and postoperative pathologic and immunohistochemical results of the patients were retrospectively reviewed. Ultrasound characteristics were evaluated according to the American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) lexicon. Logistic regression analyses were used to identify independent predictive US features that were correlated with tumor proliferation and invasiveness of breast cancers. Odds ratios (ORs) were calculated. Posterior acoustic enhancement and calcifications on US images were independent predictive factors of a higher histologic grade and a higher Ki-67 level (OR, 1.69-6.54; P < .05). Meanwhile, a noncircumscribed margin (OR, 2.61; P < .05) and posterior acoustic shadow (OR, 1.62; P < .05) were independent predictors of ALNM. An irregular shape (OR, 2.13; P < .05) and calcifications (OR, 1.69; P < .05) were independent risk factors for LVI. Infiltrative breast cancers scored as BI-RADS category 5 had higher probability to be associated with ALNM (OR, 3.33; P < .0005) and LVI (OR, 2.87; P < .0005). Ultrasound features of invasieve breast cancers might have a predictive value for tumor proliferation and invasiveness. The US features correlated with a high cellular proliferation rate were different from those associated with ALNM. The tumor shape, margin, posterior acoustic pattern, and calcifications at US are suggested to be considered by clinicians when making clinical decisions.

Roles and correlation of FOXA1 and ZIC1 in breast cancer

The aim of this study was to evaluate the prognostic role of Forkhead box A1 (FOXA1) in breast cancer and determine the relationship between FOXA1 and zinc finger of the cerebellum 1 (ZIC1). BCIP, GEPIA, and Oncomine databases were used to detect expression of FOXA1 and assess prognostic roles of FOXA1 and ZIC1 in invasive breast tumors. A total of 113 female invasive breast cancer cases were collected to investigate FOXA1 and ZIC1 expression via immunohistochemistry. Twenty pairs of frozen-thawed tumors were used to select reliable indicators via western blotting and real-time quantitative polymerase chain reaction. In addition, Kaplan-Meier curves and Cox regression analysis were performed to analyze the overall survival (OS) and relapse-free survival. Multiple databases showed that FOXA1 expression was elevated in invasive breast cancer and negatively related to ZIC1. BCIP database also displayed a poor prognosis of high FOXA1 and low ZIC1. FOXA1 was positively associated with tumor size, grading, lymph node metastasis, and Tumor Node Metastasis (TNM) staging, while ZIC1 expression was negatively related to grading, lymph node metastasis, and TNM staging. In Kaplan-Meier and Cox regression analysis, FOXA1 negative group and ZIC1 positive group had better OS rate and recurrence-free survival rate. In addition, a joint evaluation showed that “FOXA1- ZIC1+” had the highest OS and relapse-free survival, but “FOXA1+ ZIC1-” had the lowest ones. FOXA1 was negatively related to ZIC1 in breast cancer and they had different roles in clinicopathology and prognosis. Combined examination of FOXA1 and ZIC1 could bring more benefit to breast cancer patients.

Abstract P1-18-19: Phase II, open label, randomized, biomarker study of immune-mediated mechanism of action of neoadjuvant subcutaneous trastuzumab in patients with operable, locally advanced, or inflammatory HER2-positive breast cancer. ImmunHER trial on behalf of the Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC)

Abstract Background: Tumor-infiltrating lymphocytes (TILs) have been reported to be associated with increased therapeutic efficacy of trastuzumab/pertuzumab-based neoadjuvant therapy (NT) in patients (pts) with HER2-positive breast cancer (BC). Subcutaneous (SC) trastuzumab has non-inferior efficacy to intravenous (IV) administration, with a similar safety profile. Interestingly, SC trastuzumab has been observed to be more immunogenic than IV trastuzumab and act at different immunologic levels. Therefore, by modifying the modality of administration of trastuzumab, it could be possible to interfere with different immune pathways and exert a favorable immunomodulation in HER2-positive BC. Methods: In this non-comparative, phase II, neoadjuvant, randomized study, pts were eligible if they had previously untreated, histologically confirmed, locally advanced, inflammatory, or early-stage HER2-positive BC. Pts were treated with FEC (fluorouracil 500 mg/m2; epirubicin 75 mg/m2; cyclophosphamide 500 mg/m2) q21 × 3 cycles. Then, they were randomly assigned (1:1) to receive: docetaxel (75 mg/m2) plus pertuzumab (840 mg loading dose (LD), then 420 mg) plus IV trastuzumab (8 mg/kg LD, then 6 mg/kg) q21 × 4 cycles (arm A) or, docetaxel plus pertuzumab plus SC trastuzumab (fixed dose of 600 mg) q21 × 4 cycles (arm B). After surgery, pts received trastuzumab q21 × 14 cycles using the same formulation (SC or IV) of the preoperative phase. The primary endpoint was the rate of stromal TILs (sTILs) on residual disease after surgery. Tumor biopsy and posttreatment surgical samples were centrally analyzed for TILs. Blood samples were also collected during NT for tumor-specific lymphocyte cell activity analysis. Feasibility, efficacy and safety were also evaluated. ClinicalTrials.gov: NCT03144947. Results: Between November 2016 and September 2017, according to an adaptive Simon's two-stage optimal design, we enrolled 65 pts, of whom two were deemed ineligible for the study. Thus, 63 pts (31 in arm A and 32 in arm B) were assessed for the primary and secondary endpoints. The pathologic complete response (pCR; no invasive tumor in breast and axilla) rates were 64.5% (95% CI, 47-81) in arm A, and 59.4% (95% CI, 42-76) in arm B. The most common adverse events of grade 3 or higher were neutropenia (15 [48.4%] pts in arm A, and 11 [34.4%] in arm B), neurotoxicity (1 [3.2%], and 2 [6.2%], respectively), and diarrhea (1 [3.2%], and 1 [3.1%], respectively). There were no events of congestive heart failure. At surgery, 11 pts in arm A and 13 pts in arm B were evaluable for TIL analysis. The median value of sTILs (7.5%) on pre-treatment tumor biopsies was used as the cut-off value, and high sTIL levels were observed in 27.3% and in 46.1% of residual tumors after treatment arm A and B, respectively. There was a positive correlation between pretreatment sTILs and PD-L1 expression on stromal immune cells (Kendall’s τ =0.80). Interestingly, a significant inverse correlation was observed between PD-L1 expression on pretreatment sTILs and the T cell co-receptor CD3 expressed on posttreatment sTILs (Pearson’s ρ = -0.70). This finding was particularly evident in the arm B group (ρ = -0.85). Conclusions: NT with either SC or IV trastuzumab in combination with pertuzumab and chemotherapy had a significant effect on sTIL expression at surgery. In particular, the SC trastuzumab-based arm exerted the most relevant enrichment of sTILS in posttreatment residual tumors. These findings suggest a role for the SC administration of trastuzumab in determining favorable variations of host immune response parameters among pts with HER2-positive early BC who had residual disease after NT. Citation Format: Antonino Musolino, Stefania Gori, Elisabetta Cretella, Alessandra Marabese, Luigi Cavanna, Antonio Frassoldati, Giancarlo Bisagni, Chiara Casarini, Emilio Bria, Luisa Carbognin, Elena Fiorio, Alba A Brandes, Claudio Zamagni, Lorenzo Gianni, Alberto Zambelli, Filippo Montemurro, Michele Tognetto, Renata Todeschini, Giuseppe Maglietta, Gabriele Missale, Enrico M Silini. Phase II, open label, randomized, biomarker study of immune-mediated mechanism of action of neoadjuvant subcutaneous trastuzumab in patients with operable, locally advanced, or inflammatory HER2-positive breast cancer. ImmunHER trial on behalf of the Gruppo Oncologico Italiano di Ricerca Clinica (GOIRC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-19.

Abstract P1-18-28: Myocet or carboplatin as part of chemotherapy in HER2-positive operable breast cancer treated with trastuzumab and pertuzumab in the neoadjuvant setting: Efficacy data and cardiotoxicity in 60 patients diagnosed from 2016-2019 at a single institution

Abstract Objectives: To evaluate the efficacy and cardiotoxicity profile of liposomal anthracycline or Carboplatin as part of neoadjuvant therapy added to taxanes and dual blockade in HER2-positive operable breast cancer patients. Methods: A total of 60 patients diagnosed from August 2016 to May 2019 were included in the study. The treatment consisted of a sequential regimen of taxanes and non- pegilated liposomal anthracycline (Myocet) plus trastuzumab and pertuzumab, or taxanes in combination with carboplatin plus trastuzumab and pertuzumab. The clinical and pathologic responses were evaluated and correlated with clinical and biological factors. Pathologic complete response was defined as the total absence of invasive tumor in both breast and axillary nodes (ypT0/is ypN0).The cardiotoxicity profile was also analyzed. Results: The median age was 52(26-77) years and 5%, 62% and 33% of patients had stage I, II and III breast cancer, respectively, while 7% had inflammatory breast cancer at diagnosis. Hormone receptor (HR) status was negative in 48%, and 65% and 50% had grade III and ki-67 value up to 35%, respectively breast cancer. 65% of patients received weekly paclitaxelx12-trastuzumab-pertuzumab followed by Myocet-cyclophosphamide with -trastuzumab x 4 cycles or this sequence but beginning with the anthracycline, 23% of patients the same regimen but with pertuzumab concurrently with all chemotherapy treatment. The remaining 12% received docetaxel-carboplatin x6 cycles plus trastuzumab and pertuzumab. All treatment was administered previous surgery. The clinical complete response rate, was 57% and 63%, as assessed using ultrasound and MRI, respectively, and this allowed a high rate of conservative surgery (60%). The pathologic complete response (pCR) rate was 52%, and it was higher in HR-negative (61%) than in HR-positive (39%), in grade 3 (71%) than in grade 2(29%) tumors. There were not statistical significant differences in pCR between different chemotherapy treatments. Five patients showed a 10% decrease in left ventricular ejection fraction (LVEF) to below 50% at the end of neoadjuvant treatment. Asymptomatic drops in 2 patients and with heart failure symptoms in 3 of them. Myocet was part of chemotherapy in all of them. All patients recovered after discontinuation of adjuvant trastuzumab and heart medication. Two of these patients did not complete all preplanned adjuvant trastuzumab doses. Conclusion: A sequential regimen of taxanes and non-pegilated liposomal anthracycline or carboplatin plus trastuzumab and pertuzumab was effective, with high pCR rates and a good cardiotoxicity profile. Citation Format: Silvia Antolin, Cristina Reboredo, Rocío Lesta, Aurea Molina, Joaquin Mosquera, Patricia Cordeiro, Eva Pérez, Lourdes Calvo. Myocet or carboplatin as part of chemotherapy in HER2-positive operable breast cancer treated with trastuzumab and pertuzumab in the neoadjuvant setting: Efficacy data and cardiotoxicity in 60 patients diagnosed from 2016-2019 at a single institution [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-28.

Abstract OT3-09-04: A randomized phase II study comparing surgical excision versus NeOadjuvant Radiotherapy followed by delayed surgical excision of Ductal carcinoma In Situ (NORDIS)

Abstract A randomized phase II study comparing surgical excision versus NeOadjuvant Radiotherapy followed by delayed surgical excision of Ductal carcinoma In Situ (NORDIS) Breast radiotherapy (RT) for DCIS has been studied only in the adjuvant setting, following lumpectomy surgery. Adjuvant RT reduced invasive recurrences by 52% in the combined analysis of the two largest DCIS trials NSABP B-17/B-24, and reduced recurrences even for low or intermediate grade DCIS in the RTOG 9804 trial (recurrence 0.9% with RT versus 6.7% without RT). The mechanism of action is hypothesized to be elimination of occult disease and/or inhibition of neoplastic transformation in normal breast tissue. Trial Design To understand the ablative effects of RT on pure DCIS, we are conducting a prospective randomized trial comparing histopathological findings of surgical excision (Arm 1) to neoadjuvant partial breast irradiation (neoRT) followed by delayed surgical excision (Arm 2) for patients with core needle biopsy proven DCIS. Arm 1 will provide a reference group for upstaging to invasive cancer, molecular markers and pathological-radiological correlations. Arm 2 participants will receive 6 Gy daily x 5 to the intact tumor with a 0.5 cm margin of normal tissue. Surgical excision will be delayed 12-16 weeks to allow for the radioablative effects to occur and radiation-induced inflammation to subside. Specific Aims To determine if neoRT can completely ablate at least 30% of pure DCISTo determine if DCIS subtypes exhibit differential sensitivity to neoRTTo determine the radiation-induced treatment effects; wound complication rates; radiological Eligibility Women 18 years of age or older with DCIS diagnosed on vacuum assisted core needle biopsy who intend to receive breast conserving surgery are eligible, excluding those with a prior history of ipsilateral breast cancer. The imaging abnormality must be mammographic microcalcifications and/or MRI non-mass enhancement measuring &amp;lt;3 cm and not completely removed by core needle biopsy. Statistics A total of 50 patients will be recruited. Upstaging to invasive cancer is anticipated to be approximately 10% in the reference group. With 25 subjects in each arm, we will have 80% power to detect a 30% or higher improvement in the DCIS complete response rate following neoRT. Accrual Opened to accrual June 1, 2019 Contact wapnir@stanford.edu Wapnir IL, Dignam JJ, Fisher B, et al. Long-term outcomes of invasive ipsilateral breast tumor recurrences after lumpectomy in NSABP B-17 and B-24 randomized clinical trials for DCIS. J Natl Cancer Inst. 2011;103(6):478-488. doi:10.1093/jnci/djr027. McCormick B, Winter K, Hudis C, et al. RTOG 9804: a prospective randomized trial for good-risk ductal carcinoma in situ comparing radiotherapy with observation. J Clin Oncol. 2015;33(7):709-715. doi:10.1200/JCO.2014.57.9029. Citation Format: Irene Wapnir, Wendy DeMartini, Kimberly Allison, Kimberly Stone, Frederick Dirbas, Carol Marquez, Debra Ikeda, Sunita Pal, Jacqueline Tsai, Rachel Yang, Robert West, Alex McMillan, Melinda Telli, Kathleen Horst. A randomized phase II study comparing surgical excision versus NeOadjuvant Radiotherapy followed by delayed surgical excision of Ductal carcinoma In Situ (NORDIS) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr OT3-09-04.

Abstract P5-01-25: Potential prognostic value of exosomal Beta Protein 1 in breast cancer

Abstract Background: Beta protein 1 (BP1), discovered in our lab, is an isoform protein of the DLX4 gene that is a member of the homeobox gene family. Previous work showed BP1 mRNA was activated in 80% of invasive ductal breast (IDC) tumors, where 100% of estrogen receptor (ER) negative tumor tissues and 73% of ER positive tumor tissues were BP1 positive. Moreover, high BP1 levels were associated with tumor cell survival, breast cancer aggressiveness and metastasis. BP1 overexpression was found to stimulate known oncogenes including BCL2 and c-Myc. Exosomes are nano-sized membrane-bound vesicles released by various cells and play important roles in intercellular communication. In addition, researchers found that exosomal proteins have unique characteristics compared to traditional biomarkers for carcinoma diagnosis and prognosis. Therefore, we hypothesized that BP1 protein may be packed in exosomes, and analysis of exosomal BP1 protein could provide a novel biomarker for diagnosis or prognosis of breast cancer. Materials and Methods: Exosomes were isolated using the commercially available Exosome Precipitation Solution. In cell line experiments, fluorescent immunohistochemistry was used to detect the location of BP1 protein. Levels of BP1 protein were determined in cell extracts (CE) and conditioned media (CM) using Western Blot analysis. The hypothesis that exosomal BP1 protein may be related to the development of breast cancer was investigated with clinical serum samples purchased from Capital Bioscience. Total exosomal protein levels in the serum were analyzed using BCA assay, and exosomal BP1 protein levels were determined using Western blot. In order to develop a method that could be easily used in the clinic, an ELISA assay was also designed to assess exosomal BP1 protein concentrations in the serum from metastatic breast cancer patients and normal controls. Results: It was observed that BP1 protein is localized to the nucleus, the cytoplasm and the conditioned media of MCF-7 cells. In addition, experiments with cell lines showed that the secreted BP1 protein could be internalized by cells and exhibited mitogenic activity, which is related to cancer metastasis. Moreover, it was observed that exosomal BP1 is included in secreted BP1 protein in the conditioned media. Experiments with serum samples demonstrated that the total exosomal protein levels have no significant differences between that in breast cancer patients and in normal controls. However, the results of Western blot and ELISA both showed exosomal BP1 protein levels were significantly higher in breast cancer samples compared to normal controls (P &amp;lt; 0.05). Moreover, Western blot results suggested that ER positive patients have much more (P &amp;lt; 0.05) exosomal BP1 protein in the serum compared to ER negative patients. Conclusion: It was shown for the first time that BP1 protein is exosomally packaged in breast cancer patients’ serum. The significant difference in the exosomal BP1 protein levels between the serum from women with metastatic breast cancer and that from normal controls indicated that exosomal BP1 protein in the serum could be a potential prognostic biomarker for breast cancer. Furthermore, the ELISA assay, designed to be easily used for exosomal BP1 protein quantification, may have many uses in the clinic through aiding in prognosis, monitoring therapy and early detection. Citation Format: Yaoxian Lou, Paul Goldsmith, Jinguen Rheey, Patricia E. Berg. Potential prognostic value of exosomal Beta Protein 1 in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-25.

Computational Detection of Breast Cancer Invasiveness with DNA Methylation Biomarkers.

Breast cancer is the most common female malignancy. It has high mortality, primarily due to metastasis and recurrence. Patients with invasive and noninvasive breast cancer require different treatments, so there is an urgent need for predictive tools to guide clinical decision making and avoid overtreatment of noninvasive breast cancer and undertreatment of invasive cases. Here, we divided the sample set based on the genome-wide methylation distance to make full use of metastatic cancer data. Specifically, we implemented two differential methylation analysis methods to identify specific CpG sites. After effective dimensionality reduction, we constructed a methylation-based classifier using the Random Forest algorithm to categorize the primary breast cancer. We took advantage of breast cancer (BRCA) HM450 DNA methylation data and accompanying clinical data from The Cancer Genome Atlas (TCGA) database to validate the performance of the classifier. Overall, this study demonstrates DNA methylation as a potential biomarker to predict breast tumor invasiveness and as a possible parameter that could be included in the studies aiming to predict breast cancer aggressiveness. However, more comparative studies are needed to assess its usability in the clinic. Towards this, we developed a website based on these algorithms to facilitate its use in studies and predictions of breast cancer invasiveness.

Immediate breast reconstruction with latissimus dorsi flap for patients with local recurrence of breast cancer

BackgroundIpsilateral breast cancer recurrence (IBTR) occurs in about 7% of patients with primary invasive breast tumor. Salvage mastectomy and breast reconstruction are often discussed and latissimus dorsi (LD) flap is frequently proposed. MethodsWe retrospectively investigated 111 consecutive locally relapsing patients who underwent salvage mastectomy and immediate LD reconstruction. All included patients with IBTR previously underwent conserving surgery for BC, and received a postoperative irradiation. Primary endpoints were disease free survival and overall survival. Secondary endpoints were surgical complications and re-interventions. ResultsInvasive ductal cancer was the most frequent histotype (60.4%) of breast cancer reappearance. rpT1, rpT2 and rpT3 were observed respectively in 50.5%, 20,7% and 3,6% of the patients. rpTis occurred in 11,7% of cases. Positive axillary nodes were observed in 9,9% of patients at reappearance. Post-operative complication other than seroma occurred in 17,1% of patients, while seroma at the donor site was observed in 61.3% of cases. At 5-year after surgery overall survival was 92% (95% CI: 85%–96%) and disease free survival was 78% (95% CI: 69%–85%). ConclusionsImmediate latissimus dorsi flap reconstruction in selected patients with isolated breast tumor recurrence, which occurred after breast irradiation, provides an effective treatment with a satisfactory outcome.

Interplay of ERα binding and DNA methylation in the intron-2 determines the expression and estrogen regulation of cystatin A in breast cancer cells

Despite advances in early detection and treatment, invasion and metastasis of breast tumors remains a major hurdle. Cystatin A (CSTA, also called stefin A), an estrogen-regulated gene in breast cancer cells, is an inhibitor of cysteine cathepsins, and a purported tumor suppressor. Loss of CSTA expression in breast tumors evidently shifts the balance in favor of cysteine cathepsins, thereby promoting extracellular matrix remodeling, tumor invasion and metastasis. However, the underlying mechanism behind the loss of CSTA expression in breast tumors is not known. Here, we have analyzed CSTA expression, and methylation of upstream and intron-2 CpG sites within the CSTA locus in human breast cancer cell lines and breast tumors of the TCGA cohort. Results showed an inverse relationship between expression and methylation. Sequence analysis revealed a potential estrogen response element (ERE) in the intron-2. Analysis of ChIP-seq data (ERP000380) and our own ChIP experiments showed that 17β-estradiol (E2) enhanced ERα binding to this ERE in MCF-7 cells. This ERE was located amidst the differentially methylated intron-2 CpG sites, which provoked us to examine the possible conflict between estrogen-regulation of CSTA and DNA methylation in the intron-2. We analyzed the expression of CSTA and its regulation by E2 in MDA-MB-231 and T47D cells subjected to global demethylation by 5-azacytidine (5-aza). 5-aza significantly demethylated intron-2 CpGs, and enhanced estrogen-induced ERα occupancy at the intron-2 ERE, leading to restoration of estrogen-regulation. Taken together, our results indicate that DNA methylation-dependent silencing could play a significant role in the loss of CSTA expression in breast tumors. The potential of DNA methylation as an indicator of CSTA expression or as a marker of tumor progression can be explored in future investigations. Furthermore, our results indicate the convergence of ERα-mediated estrogen regulation and DNA methylation in the intron-2, thereby offering a novel context to understand the role of estrogen-ERα signaling axis in breast tumor invasion and metastasis.

PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness.

Clinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis.Methods: We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses.We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence.Results: The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. In vivo, the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation.Conclusions: These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization.

Adenomastectomy with immediate reconstruction for the treatment of invasive tumors: complications and oncologic safety

Introduction: Adenomastectomy, also called nipple-sparing mastectomy (NSM), consists of the surgical excision of all mammary gland tissue, with preservation of the skin and the nipple-areolar complex (NAC). In the past few years, different authors have been showing oncologic safety with the use of NSM, with similar recurrence and survival rates when compared to the total mastectomy technique. Besides, patients submitted to NSM present better aesthetic results and quality of life. This study aims at assessing the postoperative complications and local recurrence rates of patients with invasive breast cancer submitted to NSM with immediate reconstruction. Methods: This retrospective study collected data from patients with invasive tumors, submitted to NSM with immediate reconstruction, performed by the authors between 2001 and 2017 in Hospital São Lucas, at PUCRS (HSL), Hospital Moinhos de Vento (HMV), and Hospital Israelita Albert Einstein (HIAE). Results: a total of 198 patients diagnosed with invasive breast tumors were submitted to 374 NSM. Bilateral surgeries were performed in 176 patients, being 7 (4%) patients diagnosed with bilateral invasive tumors; 8 (4.5%), with ductal carcinoma in situ (DCIS) in the contralateral breast; 15 8.5%) with atypia and/or lobular carcinoma in situ (LCIS); and 146 (83%) patients who underwent risk-reducing surgery in the contralateral breast. Mean age of the patients was 45.3 years. Sentinel lymph node biopsy (SLNB) was performed in 143 (72.2%) patients, and 25 (17.5%) presented with positive lymph nodes. Twenty (10.1%) patients were submitted to SLNB, followed by axillary lymph node dissection, and 35 (17.7%) underwent axillary lymph node dissection. Assessing the molecular subtypes, 65 (32.9%) tumors were luminal A; 71 (35.8%), luminal B; 20 (10.1%), luminal B/positive Her2; 15 (7.6%), positive Her2; and 27 (13.6%), triple negative. In the 374 performed NS, it was possible to observe 25 (6.6%) postoperative complications, 10 (2.6%) infections, 2 (0.5%) hematomas, 5 (1.3%) partial necrosis of the NAC, 8 (2.2%) cases of dehiscence. After a mean follow-up time of 50 months, of the 198 patients who underwent NSM to treat cancer, 11 (5.5%) presented local recurrence, being only 3 (1.5%) recurrences in the NAC. Conclusions: After assessing all NSMs with immediate reconstruction carried out for the treatment of invasive cancer, only 6.6% presented postoperative complications. The rates of partial necrosis of the NAC (1.3%) and infection (2.6%) were low in comparison to findings from other authors. Besides, the local recurrence rates of 5.5% and 1.5% in NAC in patients submitted to NSM with immediate reconstruction for the treatment of invasive breast cancer are in accordance with the previous literature. These results show the oncologic safety of this procedure as a surgical therapeutic choice for the selected patients, demonstrating even lower rates of partial NAC necrosis.

Associations between the expression of mucins (MUC1, MUC2, MUC5AC and MUC6) and clinicopathologic parameters of human breast carcinomas.

The aim of this study is to evaluate the relationships between the expression of mucins in invasive breast carcinomas and clinicopathologic parameters. We examined 150 cases of invasive breast carcinoma, using the 2012 World Health Organization (WHO) classification of the tumors of the breast. We studied the expression of MUC1, MUC2, MUC5AC, and MUC6 by immunohistochemistry. We also evaluated normal breast tissue and ductal carcinoma in situ (DCIS) lesions in nearby invasive tumor areas. In invasive breast carcinomas, MUC1, MUC2, MUC5AC, and MUC6 were expressed in 98.6%, 11.3%, 9.9, and 8.5% of cases, respectively. MUC2, MUC5AC, and MUC6 were overexpressed in invasive tumors and DCIS lesions were compared with normal breast tissue. The apical pattern of MUC1 was correlated with low grade and ER expression. MUC2 was correlated with mucinous carcinoma and an inverse association with invasive ductal carcinoma, not otherwise specified (NOS). MUC6 expression was associated with lymphovascular invasion. Most invasive breast tumors express MUC1 and the apical pattern of MUC1 is correlated with low grade and ER expression. MUC6 expression is associated with indicators of poor prognosis. Further comprehensive studies need to evaluate the role of mucins as a potential biomarker and to be used as a specific therapeutic target against breast cancer.

Nghiên cứu tương tác của vorinostat với enzyme HDAC8 (1T67) bằng Autodock

Vorinostat is a histone deacetylase inhibitor which was approved by the US FDA in 2006 for the treatment of cutaneous manifestations in patients with cutaneous T-cell lymphoma. Among 18 HDAC enzymes, vorinostat is a potent inhibitor of the activity of HDAC1, HDAC2, HDAC3 and HDAC6. However, there have not been many published papers on the inhibitory capacity against HDAC8 (1T67) of vorinostat. In this study, the interactions of vorinostat with the enzyme HDAC8 (1T67) were performed and described by docking vorinostat into the active zone of the HDAC8 enzyme using Autodock. HDAC8 is a class I histone deacetylase implicated as a therapeutic target in various diseases, including cancer, parasitic infections and Cornelia de Lange syndrome. In invasive breast tumor cells, HDAC8 is among the three HDAC family members that are upregulated and driving invasiveness. The docking analysis shows vorinostat’s interactions with Zn+2 ion, Gly151, Gly304, Asp178, Tyr306, Phe207, Met274 and other less interacting residues. Therefore, the results could act as a momentum for further studies on the design of new isozyme-selective HDAC8 inhibitors.

Genome-wide characterization of cytosine-specific 5-hydroxymethylation in normal breast tissue

ABSTRACTDespite recent evidence that 5-hydroxymethylcytosine (5hmC) possesses roles in gene regulation distinct from 5-methylcytosine (5mC), relatively little is known regarding the functions of 5hmC in mammalian tissues. To address this issue, we utilized an approach combining both paired bisulfite (BS) and oxidative bisulfite (oxBS) DNA treatment, to resolve genome-wide patterns of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC was differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity, such as heterochromatin and repressed Polycomb regions, were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. Furthermore, genes containing abundant 5hmC were enriched among those actively transcribed in normal breast tissue. Finally, in independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Primarily, our findings identify genomic loci containing abundant 5hmC in breast tissues and provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue. Additionally, these data suggest 5hmC may participate in gene regulatory programs that are dysregulated during breast-related carcinogenesis.