Yolanda Madarnas

Breast cancer in pregnancy: a literature review

PurposeBreast cancer in pregnancy is a clinically challenging situation for patients and their physicians. A review of the literature was performed to help identify optimal treatment strategies.MethodsA Medline search between 1966 to the present using the keywords “breast”, “carcinoma”, and “pregnancy” revealed numerous hits, from which English-language articles including epidemiologic studies, case series, and general summaries were reviewed.ResultsThere is a paucity of prospective studies regarding diagnosis and treatment of breast cancer in pregnancy due to its rarity. However a general review of the literature database reveals that women diagnosed with breast cancer during pregnancy have similar disease characteristics to age-matched controls. Surgery remains the mainstay of treatment of breast cancer during pregnancy, and in some circumstances breast-conserving surgery is an acceptable option. Adjuvant treatment can proceed with some modifications that minimize harm to the fetus, namely limiting radiation exposure and timing chemotherapy properly. Post-partum decisions regarding lactation and future fertility should be addressed on a per-patient basis.ConclusionBreast cancer in pregnancy is an uncommon phenomenon but one which poses dilemmas for patients and their physicians. A multi-disciplinary approach is recommended for optimal clinical-decision making.

A novel role for ezrin in breast cancer angio/lymphangiogenesis

Recent evidence suggests that tumour lymphangiogenesis promotes lymph node metastasis, a major prognostic factor for survival of breast cancer patients. However, signaling mechanisms involved in tumour-induced lymphangiogenesis remain poorly understood. The expression of ezrin, a membrane cytoskeletal crosslinker and Src substrate, correlates with poor outcome in a diversity of cancers including breast. Furthermore, ezrin is essential in experimental invasion and metastasis models of breast cancer. Ezrin acts cooperatively with Src in the regulation of the Src-induced malignant phenotype and metastasis. However, it remains unclear if ezrin plays a role in Src-induced tumour angio/lymphangiogenesis. The effects of ezrin knockdown and mutation on angio/lymphangiogenic potential of human MDA-MB-231 and mouse AC2M2 mammary carcinoma cell lines were examined in the presence of constitutively active or wild-type (WT) Src. In vitro assays using primary human lymphatic endothelial cells (hLEC), an ex vivo aortic ring assay, and in vivo tumour engraftment were utilized to assess angio/lymphangiogenic activity of cancer cells. Ezrin-deficient cells expressing activated Src displayed significant reduction in endothelial cell branching in the aortic ring assay in addition to reduced hLEC migration, tube formation, and permeability compared to the controls. Intravital imaging and microvessel density (MVD) analysis of tumour xenografts revealed significant reductions in tumour-induced angio/lymphangiogenesis in ezrin-deficient cells when compared to the WT or activated Src-expressing cells. Moreover, syngeneic tumours derived from ezrin-deficient or Y477F ezrin-expressing (non-phosphorylatable by Src) AC2M2 cells further confirmed the xenograft results. Immunoblotting analysis provided a link between ezrin expression and a key angio/lymphangiogenesis signaling pathway by revealing that ezrin regulates Stat3 activation, VEGF-A/-C and IL-6 expression in breast cancer cell lines. Furthermore, high expression of ezrin in human breast tumours significantly correlated with elevated Src expression and the presence of lymphovascular invasion. The results describe a novel function for ezrin in the regulation of tumour-induced angio/lymphangiogenesis promoted by Src in breast cancer. The combination of Src/ezrin might prove to be a beneficial prognostic/predictive biomarker for early-stage metastatic breast cancer.

Automated Quantitative Analysis of p53, Cyclin D1, Ki67 and pERK Expression in Breast Carcinoma Does Not Differ from Expert Pathologist Scoring and Correlates with Clinico-Pathological Characteristics

There is critical need for improved biomarker assessment platforms which integrate traditional pathological parameters (TNM stage, grade and ER/PR/HER2 status) with molecular profiling, to better define prognostic subgroups or systemic treatment response. One roadblock is the lack of semi-quantitative methods which reliably measure biomarker expression. Our study assesses reliability of automated immunohistochemistry (IHC) scoring compared to manual scoring of five selected biomarkers in a tissue microarray (TMA) of 63 human breast cancer cases, and correlates these markers with clinico-pathological data. TMA slides were scanned into an Ariol Imaging System, and histologic (H) scores (% positive tumor area x staining intensity 0–3) were calculated using trained algorithms. H scores for all five biomarkers concurred with pathologists’ scores, based on Pearson correlation coefficients (0.80–0.90) for continuous data and Kappa statistics (0.55–0.92) for positive vs. negative stain. Using continuous data, significant association of pERK expression with absence of LVI (p = 0.005) and lymph node negativity (p = 0.002) was observed. p53 over-expression, characteristic of dysfunctional p53 in cancer, and Ki67 were associated with high grade (p = 0.032 and 0.0007, respectively). Cyclin D1 correlated inversely with ER/PR/HER2-ve (triple negative) tumors (p = 0.0002). Thus automated quantitation of immunostaining concurs with pathologists’ scoring, and provides meaningful associations with clinico-pathological data.

Information for decision making by post-menopausal women with hormone receptor positive early-stage breast cancer considering adjuvant endocrine therapy

PURPOSE To identify the information that post-menopausal women with hormone-receptor positive, early-stage breast cancer want, to help them decide among six treatment options for adjuvant-endocrine therapy. METHODS We surveyed women with early-stage breast cancer who were eligible for adjuvant endocrine-therapy 3-18 months earlier. Participants rated the importance of getting each of 95 questions answered before this decision is made (options: essential/desired/not important/avoid). For questions rated essential or desired, participants identified the purpose(s) for having the question answered: to help them understand, make the decision, plan, or other. Participants indicated the role they played in their actual decision and the role they would prefer if the decision was made today. They also indicated whether they felt they had had a choice of endocrine therapy treatments. RESULTS 188 of 343 questionnaires were returned (response rate 55%). Mean age was 67 yr (range 38-88 yr); 76% were married, and 39% had secondary school education or less. On average, respondents rated 18 questions (range 0-94) essential for decision making. Each question was rated essential for decision making by ≥ 7% of participants but only 1 question by >50%. Regarding roles, 89% of respondents had participated in their actual decision and would want to again; an additional 9% had not participated in their actual decision but would want to at the time of the survey. The percentage of respondents who felt they had no choice of endocrine therapy treatments varied between centres, 25% vs 41% and 49%. CONCLUSIONS Most patients want to participate in the decision but they vary widely in the amount and which specific details they want to help them make the decision. IMPLICATION The wide variation in questions considered important means the support should be tailored to the needs of the individual patient.

Immunohistochemical Assessment of Expression of Centromere Protein—A (CENPA) in Human Invasive Breast Cancer

Abnormal cell division leading to the gain or loss of entire chromosomes and consequent genetic instability is a hallmark of cancer. Centromere protein –A (CENPA) is a centromere-specific histone-H3-like variant gene involved in regulating chromosome segregation during cell division. CENPA is one of the genes included in some of the commercially available RNA based prognostic assays for breast cancer (BCa)—the 70 gene signature MammaPrint® and the five gene Molecular Grade Index (MGISM). Our aim was to assess the immunohistochemical (IHC) expression of CENPA in normal and malignant breast tissue. Clinically annotated triplicate core tissue microarrays of 63 invasive BCa and 20 normal breast samples were stained with a monoclonal antibody against CENPA and scored for percentage of visibly stained nuclei. Survival analyses with Kaplan–Meier (KM) estimate and Cox proportional hazards regression models were applied to assess the associations between CENPA expression and disease free survival (DFS). Average percentage of nuclei visibly stained with CENPA antibody was significantly higher (p = 0.02) in BCa than normal tissue. The 3-year DFS in tumors over-expressing CENPA (>50% stained nuclei) was 79% compared to 85% in low expression tumors (<50% stained nuclei). On multivariate analysis, IHC expression of CENPA showed weak association with DFS (HR > 60.07; p = 0.06) within our small cohort. To the best of our knowledge, this is the first published report evaluating the implications of increased IHC expression of CENPA in paraffin embedded breast tissue samples. Our finding that increased CENPA expression may be associated with shorter DFS in BCa supports its exploration as a potential prognostic biomarker.

Novel prognostic and predictive microRNA targets for triple-negative breast cancer

Triple‐negative breast cancers (TNBCs) account for ~25% of all invasive carcinomas and represent a large subset of aggressive, high‐grade tumors. Despite current research focused on understanding the genetic landscape of TNBCs, reliable prognostic and predictive biomarkers remain limited. Although dysregulated microRNAs (miRNAs) have emerged as key players in many cancer types, the role of miRNAs in TNBC disease progression is unclear. We performed miRNA profiling of 51 TNBCs by next‐generation sequencing to reveal differentially expressed miRNAs. A total of 228 miRNAs were identified. Three miRNAs (miR‐224–5p, miR‐375, and miR‐205–5p) separated the tumors based on basal status. Six miRNAs (high let‐7d‐3p, miR‐203b‐5p, and miR‐324–5p; low miR‐30a‐3p, miR‐30a‐5p, and miR‐199a‐5p) were significantly associated with decreased overall survival (OS) and 5 miRNAs (high let‐7d‐3p; low miR‐30a‐3p, miR‐30a‐5p, miR‐30c‐5p, and miR‐128–3p) with decreased relapse‐free survival (RFS). On multivariate analysis, high expression of let‐7d‐3p and low expression of miR‐30a were independent predictors of decreased OS and RFS. High expression of miR‐95–3p was significantly associated with decreased OS and RFS in patients treated with anthracycline‐based chemotherapy. Five miRNAs (let‐7d‐3p, miR‐30a‐3p, miR‐30c‐5p, miR‐128–3p, and miR‐95–3p) were validated by quantitative RT‐PCR. Our findings unveil novel prognostic and predictive miRNA targets for TNBC, including a miRNA signature that predicts patient response to anthracycline‐based chemotherapy. This may improve clinical management and/or lead to the development of novel therapies.—Turashvili, G., Lightbody, E. D., Tyryshkin, K., SenGupta, S. K., Elliott, B. E., Madarnas, Y., Ghaffari, A., Day, A., Nicol, C. J. B. Novel prognostic and predictive microRNA targets for triple‐negative breast cancer. FASEB J. 32, 5937–5954 (2018). www.fasebj.org

Abstract P5-01-01: Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer

Lymph node (LN) metastasis is a key driver of recurrence and survival in breast cancer (BC) patients. However, the mechanisms of metastatic dissemination of tumour cells from LNs to distant sites and their predictors of response to systemic therapy remain poorly understood, mainly due to a lack of non-invasive in vivo imaging models. We have recently described ezrin, a pro-metastatic crosslinker protein, as a regulator of tumour lymphangiogenesis and metastasis in BC (Breast Cancer Res. 2014; 16(5): 438). Furthermore, we demonstrated significant association of high ezrin expression with lymphovascular invasion in a cohort (n=63) of premenopausal patients with invasive BC (p =0.024). These findings prompted us to examine the role of ezrin in migration and invasion of metastatic tumour cells in LNs and their response to ezrin-targeted therapy. Using a locally accrued LN positive patient cohort (n=94), we demonstrated a significant association between high ezrin levels and reduced recurrence-free survival (univariate Log-rank test, p=0.033), suggesting that ezrin is a potential predictor of relapse in LN positive BC. To address the mechanistic role of ezrin in LN metastasis, we developed a novel intravital imaging model using a lymphatic reporter transgenic mouse (B6-prox1-mOrange2-pA-BAC) to examine the response of tumour-draining LN to anti-ezrin systemic therapy in real time. Next, we tested the effects of a small molecule ezrin inhibitor (NSC668394) in vitro and observed significant suppression of ezrin activation (p-T567) and cancer cell invasive phenotype. Intravital imaging of inguinal LN metastases, derived from subcutaneously implanted breast adenocarcinoma E0771-LMV (lung metastatic variant) cells, demonstrated significant reduction in mobility and invasiveness (Mann Whitney, p (Supported by CRS, CIHR, CBCF, BCAK, Queen9s SRC). Citation Format: Ghaffari A, Hoskin V, Mullins G, Greer P, Kiefer F, Madarnas Y, SenGupta S, Elliott B. Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-01-01.

Abstract 3207: Automated quantitative analysis of p53, cyclin D1 and pErk expression in breast carcinoma does not differ from expert pathologist scoring and correlates well with clinico-pathological characteristics

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Prognosis and risk assessment of breast cancer patients are currently driven by TNM stage, ER/PR/HER2 expression, tumor grade and lymphovascular invasion (LVI). However there is critical need for improved biomarker assessment platforms to better predict systemic treatment response. One roadblock is the lack of semi-quantitative methods to reliably measure expression, activity and localization of biomarkers in formalin-fixed tumor specimens. The present study assesses reliability of automated IHC scoring compared to manual scoring of routine and non-routine biomarkers (HER2, cyclin D1, p53 and phospho(p)-ERK) on a human breast cancer tissue microarray according to REMARK guidelines, and correlates these markers with clinical-pathological data. Using a triplicate core TMA of formalin-fixed paraffin embedded tissues, we investigated 63 primary invasive breast cancers, for which ER/PR/HER2 status, LVI, grade and recurrence status were recorded. IHC was performed on the TMA for the above biomarkers (pH 6 citrate buffer conditions). Histologic (H) scores (% positive tumor area × staining intensity 0-3) were determined manually by two independent evaluators with resolution of discordant cases by a senior pathologist. Excellent replicability was observed between H scores for each marker compared on replicate slides, as determined by Spearman correlations (0.79-0.82). Each TMA slide was then scanned into the Ariol Imaging System, algorithms were trained for each marker, and H scores were calculated. Pearson correlation coefficients (with data left as continuous) and Kappa statistics (with dichotomized data) were used for inter-method comparisons. Associations between biomarker positivity and clinical data were assessed by Fishers exact test. Excellent concordance between manual and automated Ariol scores was observed for all four markers based on Kappa statistics (0.667-0.813) and Pearson correlation coefficients (0.790-0.885). Distinct proportions of tumor cases showed any positive staining for membranous HER2 (19/63), nuclear p53 (16/56), cyclin D1 (26/57) and pERK (32/59). A statistically significant association of pERK positivity with absence of LVI (p=0.0025) and lymph node negativity (p=0.0006) was observed. In contrast, pERK positivity was associated with high-grade tumors (p=0.0040), consistent with a role of pERK in poorly differentiated high-grade primary tumors. p53 over-expression, characteristic of dysfunctional p53 in breast cancer, was also associated with high tumor grade (p=0.0074). Thus automated quantitation of immunostaining yields objective results that do not differ from pathologists’ scoring, and provide meaningful associations with clinico-pathological data. (Supported by CIHR, PSI, and Queens Dept. Pathol. & Mol. Med.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3207. doi:10.1158/1538-7445.AM2011-3207

Abstract P2-06-11: Ramifications of HER2/ER/PR Guidelines from ASCO/CAP for Translational Cancer Research Using a Cohort from a Tertiary Care Centre in Ontario

Background: A transdisciplinary team from basic science, pathology, clinical and biostatistics was assembled to establish a framework with which to take novel laboratory biomarkers and targets to clinical validation. Human epidermal growth factor receptor (HER2), estrogen (ER) and progesterone (PR) receptor are of important prognostic and predictive value and drivers of systemic therapy for breast cancer (BC). As a first step, the current ASCO/CAP guidelines were used to re-assign centrally reviewed tumour specimens and compare to the clinically assigned scores for ER/PR and HER2. Methods: With REB approval, a cohort of 62 cases of non-metastatic invasive BC with banked tumour specimens was assembled between 2005 and 2007. Clinico-pathological information for each case was retrospectively obtained from the medical file and entered into an anonymized database. Full section slides were originally stained by routine immunohistochemistry (IHC). Categorical clinical scores for ER/PR (negative-neg/weak/positive-pos) were compared to the continuous scores assigned in a blinded fashion using ASCO/CAP criteria (% pos/H-score). Categorical clinical scores obtained with duplicate IHC antibody staining of full sections for HER2 (neg/equivocal-eq/pos) were compared to those obtained from IHC assessments of triplicate 6mm cores in a tissue microarray (TMA) that were assigned to be neg/eq/pos using ASCO/CAP criteria. A senior breast pathologist adjudicated discordant specimens. Exact Fisher tests were used to compare the two sets of categorical assessments. Results: Mean age was 43.5 years, (range 29-49). The majority of the cohort (59.7%) had N0 disease and received adjuvant chemotherapy (74.2%); 72.6% of the cohort was alive at the time of this analysis. Score means and ranges of ER/PR are displayed below. Two of 16 clinically ER neg cases (12.5%) were rescored as pos and 0/43 clinically ER pos cases were rescored as neg, P ER/PR scores Conclusions: Systemic therapy recommendations could be impacted in a small but substantive number of cases by the methodology used for biomarker assessment and scoring, particularly near threshold values. This study illustrates that the scoring criteria used may be an important contributor to variability in correlative biomarker studies. Consideration should be given to routine systematic reassessment with continuous scoring for biomarker data proposed for use in correlative science studies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-11.