Comprehensive immunohistochemical analysis of RET, BCAR1, and BCAR3 expression in patients with Luminal A and B breast cancer subtypes.

PURPOSE: Breast cancer (BC) is considered a heterogeneous disease composed of distinct subtypes with diverse clinical outcomes. Luminal subtype tumors have the best prognosis, and patients benefit from endocrine therapy. However, resistance to endocrine therapies in BC is an obstacle to successful treatment, and novel biomarkers are needed to understand and overcome this mechanism. The RET, BCAR1, and BCAR3 genes may be associated with BC progression and endocrine resistance. METHODS: Aiming to evaluate the expression profile and prognostic value of RET, BCAR1, and BCAR3, we performed immunohistochemistry on tissue microarrays (TMAs) containing a cohort of 361 Luminal subtype BC. RESULTS: Low expression levels of these three proteins were predominantly observed. BCAR1 expression was correlated with nuclear grade (p = 0.057), and BCAR3 expression was correlated with lymph node status (p = 0.011) and response to hormonal therapy (p = 0.021). Further, low expression of either BCAR1 or BCAR3 was significantly associated with poor prognosis (p = 0.005; p = 0.042). Pairwise analysis showed that patients with tumors with low BCAR1/low BCAR3 expression had a poorer overall survival (p = 0.013), and the low BCAR3 expression had the worst prognosis with RET high expression stratifying these patients into two different groups. Regarding the response to hormonal therapy, non-responder patients presented lower expression of RET in comparison to the responder group (p = 0.035). Additionally, the low BCAR1 expression patients had poorer outcomes than BCAR1 high (p = 0.015). CONCLUSION: Our findings suggest RET, BCAR1, and BCAR3 as potential candidate markers for endocrine therapy resistance in Luminal BC.

Molecular mechanisms linking high body mass index to breast cancer etiology in post-menopausal breast tumor and tumor-adjacent tissues.

PURPOSE: In post-menopausal women, high body mass index (BMI) is an established breast cancer risk factor and is associated with worse breast cancer prognosis. We assessed the associations between BMI and gene expression of both breast tumor and adjacent tissue in estrogen receptor-positive (ER+) and estrogen receptor-negative (ER-) diseases to help elucidate the mechanisms linking obesity with breast cancer biology in 519 post-menopausal women from the Nurses' Health Study (NHS) and NHSII. METHODS: Differential gene expression was analyzed separately in ER+ and ER- disease both comparing overweight (BMI ≥ 25 to < 30) or obese (BMI ≥ 30) women to women with normal BMI (BMI < 25), and per 5 kg/m2 increase in BMI. Analyses controlled for age and year of diagnosis, physical activity, alcohol consumption, and hormone therapy use. Gene set enrichment analyses were performed and validated among a subset of post-menopausal cases in The Cancer Genome Atlas (for tumor) and Polish Breast Cancer Study (for tumor-adjacent). RESULTS: No gene was differentially expressed by BMI (FDR < 0.05). BMI was significantly associated with increased cellular proliferation pathways, particularly in ER+ tumors, and increased inflammation pathways in ER- tumor and ER- tumor-adjacent tissues (FDR < 0.05). High BMI was associated with upregulation of genes involved in epithelial-mesenchymal transition in ER+ tumor-adjacent tissues. CONCLUSIONS: This study provides insights into molecular mechanisms of BMI influencing post-menopausal breast cancer biology. Tumor and tumor-adjacent tissues provide independent information about potential mechanisms.

BRCA1 deficiency is a recurrent event in early-onset triple-negative breast cancer: a comprehensive analysis of germline mutations and somatic promoter methylation.

PURPOSE: BRCA1 germline mutation is closely associated with triple-negative breast cancer. BRCA deficiency leads to impaired DNA repair and tumor development, and understanding this deficiency, in both hereditary and sporadic scenarios, is of great clinical and biological interest. Here, we investigated germline or somatic events that might lead to BRCA1 impairment in triple-negative breast cancer. We also analyzed the clinical implications associated with BRCA deficiency. METHODS: Next-generation sequencing for the BRCA1/2 genes and multiplex ligation-dependent probe amplification (MLPA) for the BRCA1 gene were performed for mutation screening. A customized bisulfite next-generation sequencing approach was used for assessing BRCA1 promoter methylation status in tumor tissue. RESULTS: A total of 131 triple-negative cases were assessed, and germline pathogenic variants were detected in 13.0% of all cases and in 26% of cases diagnosed in young women. Most germline pathogenic variants (88.2%) occurred in the BRCA1 gene. BRCA1 promoter hypermethylation was detected in 20.6% of tumors; none of these tumors were in BRCA1/2 pathogenic variant carriers. BRCA1 impairment by either germline or somatic events was significantly more frequent in young women (55% in those ≤ 40 years; 33% in those 41-50 years; 22% in those > 50 years of age) and associated with better overall and disease-free survival rates in this group of patients. CONCLUSIONS: BRCA1 deficiency was recurrent in early-onset triple-negative breast cancer in Brazilian patients and associated with improved survival. With the new treatment modalities being investigated, including poly (ADP-ribose)-polymerase (PARP) inhibitor therapy, our results suggest that a significant proportion of young women with this subtype of tumor might benefit from PARP inhibitor treatment, which warrants further investigation.

Identification of novel biomarkers associated with poor patient outcomes in invasive breast carcinoma.

Breast carcinoma (BC) corresponds to 23 % of all cancers in women, with 1.38 million new cases and 460,000 deaths worldwide annually. Despite the significant advances in the identification of molecular markers and different modalities of treatment for primary BC, the ability to predict its metastatic behavior is still limited. The purpose of this study was to identify novel molecular markers associated with distinct clinical outcomes in a Brazilian cohort of BC patients. We generated global gene expression profiles using tumor samples from 24 patients with invasive ductal BC who were followed for at least 5 years, including a group of 15 patients with favorable outcomes and another with nine patients who developed metastasis. We identified a set of 58 differentially expressed genes (p ≤ 0.01) between the two groups. The prognostic value of this metastasis signature was corroborated by its ability to stratify independent BC patient datasets according to disease-free survival and overall survival. The upregulation of B3GNT7, PPM1D, TNKS2, PHB, and GTSE1 in patients with poor outcomes was confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in an independent sample of patients with BC (47 with good outcomes and eight that presented metastasis). The expression of BCL2-associated agonist of cell death (BAD) protein was determined in 1276 BC tissue samples by immunohistochemistry and was consistent with the reduced BAD mRNA expression levels in metastatic cases, as observed in the oligoarray data. These findings point to novel prognostic markers that can distinguish breast carcinomas with metastatic potential from those with favorable outcomes.

Lymphovascular invasion and histologic grade are associated with specific genomic profiles in invasive carcinomas of the breast.

Lymphovascular invasion (LVI) and histologic grade are clinical parameters of high prognostic value in breast cancer and indicate the level of tumor aggressiveness. Many studies have focused on the association of breast cancer subtypes with gene expression and chromosomal profiles, but considerably less genomic information is available regarding traditional prognostic factors such as histologic grade and LVI. We studied by array-CGH a group of 57 invasive ductal carcinomas of the breast to outline the DNA copy number aberration (CNA) profile linked to high histologic grades and LVI. Selected CNAs were validated using real-time quantitative PCR (qPCR). Furthermore, gene expression analysis was performed in a subset of 32 of these tumors, and findings were integrated with array-CGH data. Our findings indicated an accumulation of genomic alterations in high-grade breast tumors compared to low-grade samples. Grade III tumors showed higher number of CNAs and larger aberrations than low-grade tumors and displayed a wide range of chromosomal aberrations, which were mainly 5p, 8q, 10p, 17q12, and 19 gains, and 3p, 4, 5q proximal, 9p, 11p, 18q, and 21 losses. The presence of LVI, a well-established prognostic marker, was not significantly associated with increased genomic instability in comparison to breast tumors negative for LVI, considering the total number of chromosomal alterations. However, a slightly increase in the frequency of specific alterations could be detected in LVI-positive group, such as gains at 5p, 16p, 17q12, and 19, and losses at 8p, 11q, 18q, and 21. Three newly reported small-scale rearrangements were detected in high-risk tumors (LVI-positive grade III) harboring putative breast cancer genes (amplicons at 4q13.3 and 11p11.2, and a deletion at 12p12.3). Furthermore, gene expression analysis uncovered networks highlighting S100A8, MMP1, and MED1 as promising candidate genes involved in high-grade and LVI-positive tumors. In summary, a group of genomic regions could be associated with high-risk tumors, and expression analysis pinpointed candidate genes deserving further investigation. The data has shed some light on the molecular players involved in two highly relevant prognostic factors and may further add to the understanding of the mechanisms of breast cancer aggressiveness.

Interleukin-6 Signaling in Triple Negative Breast Cancer Cells Elicits the Annexin A1/Formyl Peptide Receptor 1 Axis and Affects the Tumor Microenvironment.

Annexin A1 (AnxA1) is a pleiotropic protein that exerts essential roles in breast cancer (BC) growth and aggressiveness. In our previous work, we described the autocrine signaling of AnxA1 through formyl peptide receptor 1 (FPR1) in the triple-negative (TN) BC cell line, MDA-MB-231. Here, we aimed to describe the interaction between the AnxA1/FPR1 and the Interleukin-6 (IL-6) signaling pathways and their role in the tumor microenvironment (TME). First, we demonstrated that AnxA1 and IL-6 expression levels are correlated in BC tissue samples. In three TNBC cell lines, overexpression of both AnxA1 and IL-6 was also identified. Next, we inhibited FPR1, the IL-6 receptor and STAT3 in both MDA-MB-231 and MDA-MB-157 cells. The FPR1 inhibition led to increased levels of IL-6 and secreted AnxA1 in both cell lines. On the other side, inhibition of the IL-6 receptor or STAT3 led to the impairment of AnxA1 secretion, suggesting the essential role of the IL-6 signaling cascade in the activation of the AnxA1/FPR1 autocrine axis. Finally, we described the interaction between IL-6 and the AnxA1/FPR1 pathways and their role on the TME by analyzing the effect of supernatants derived from MDA-MB-231 and MDA-MB-157 cells under the inhibition of FPR1 or IL-6 signaling on fibroblast cell motility.

Priorities for cancer research in low- and middle-income countries: a global perspective.

Cancer research currently is heavily skewed toward high-income countries (HICs), with little research conducted in, and relevant to, the problems of low- and middle-income countries (LMICs). This regional discordance in cancer knowledge generation and application needs to be rebalanced. Several gaps in the research enterprise of LMICs need to be addressed to promote regionally relevant research, and radical rethinking is needed to address the burning issues in cancer care in these regions. We identified five top priorities in cancer research in LMICs based on current and projected needs: reducing the burden of patients with advanced disease; improving access and affordability, and outcomes of cancer treatment; value-based care and health economics; quality improvement and implementation research; and leveraging technology to improve cancer control. LMICs have an excellent opportunity to address important questions in cancer research that could impact cancer control globally. Success will require collaboration and commitment from governments, policy makers, funding agencies, health care organizations and leaders, researchers and the public.

Annexin A1 promotes the nuclear localization of the epidermal growth factor receptor in castration-resistant prostate cancer.

Epidermal growth factor receptor is a cancer driver whose nuclear localization has been associated with the progression of prostate cancer to the castration-resistant phenotype. Previous reports indicated a functional interaction between this receptor and the protein Annexin A1, which has also been associated with aggressive tumors. The molecular pathogenesis of castration-resistant prostate cancer remains largely unresolved, and herein we have demonstrated the correlation between the expression levels and localization of the epidermal growth factor receptor and Annexin A1 in prostate cancer samples and cell lines. Interestingly, a higher expression of both proteins was detected in castration-resistant prostate cancer cell lines and the strongest correlation was seen at the nuclear level. We verified that Annexin A1 interacts with the epidermal growth factor receptor, and by using prostate cancer cell lines knocked down for Annexin A1, we succeeded in demonstrating that Annexin A1 promotes the nuclear localization of epidermal growth factor receptor. Finally, we showed that Annexin A1 activates an autocrine signaling in castration-resistant prostate cells through the formyl peptide receptor 1. The inhibition of such signaling by Cyclosporin H inhibits the nuclear localization of epidermal growth factor receptor and its downstream signaling. The present work sheds light on the functional interaction between nuclear epidermal growth factor receptor and nuclear Annexin A1 in castration-resistant prostate cancer. Therefore, strategies to inhibit the nuclear localization of epidermal growth factor receptor through the suppression of the Annexin A1 autocrine loop could represent an important intervention strategy for castration-resistant prostate cancer.

Breast cancer in the elderly: a histological assessment.

AIMS: To understand the correlation between the expression status of different biological markers in breast cancers in the elderly. METHODS AND RESULTS: Three hundred and ninety-seven cases were evaluated for expression of hormone receptors [oestrogen receptors (ER) alpha and beta, progesterone receptor (PR)], basal markers [p63, cytokeratin (CK) 5/6 and CK14] and others (HER2/neu, synaptophysin and chromogranin). The expression rates were 60, 29, 25, 6, 14, 8, 28, 17 and 5%, respectively, for these markers. The expression of ER alpha and beta, PR, synaptophysin and chromogranin at any level correlated with low nuclear or tumour grades, whereas the expression of HER2/neu, CK5/6 and CK14 at any level correlated with high nuclear grade. By using hierarchical clustering, groups of HER2, luminal and basal types were identified. In addition, a neuroendocrine group was also identified, being characterized by expression of synaptophysin, chromogranin, ER and PR, but not HER2/neu, and other basal cytokeratins. This group was associated with lower nuclear grade, and hence better prognosis. CONCLUSIONS: Breast cancer in the elderly shows similar molecular groupings as other breast cancers, with an additional neuroendocrine group that is associated with a favourable biological marker profile.

DNA Methylation-Based Method to Differentiate Malignant from Benign Thyroid Lesions.

Background: The differential diagnosis of thyroid nodules using fine-needle aspiration biopsy (FNAB) is challenging due to the inherent limitation of the cytology tests. The use of molecular markers has potential to complement the FNAB-based diagnosis and avoid unnecessary surgeries. In this study, we aimed to identify DNA methylation biomarkers and to develop a diagnostic tool useful for thyroid lesions. Methods: Genome-wide DNA methylation profiles (Illumina 450K) of papillary thyroid carcinoma (PTC = 60) and follicular thyroid carcinoma (FTC = 10) were compared with non-neoplastic thyroid tissue samples (NT = 50) and benign thyroid lesions (BTL = 17). The results were confirmed in publicly available databases from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) using the same DNA methylation platform. Two classifiers were trained to discriminate FTC and PTC from BTL. To increase the applicability of the method, six differentially methylated CpGs were selected and evaluated in 161 thyroid tumors and 69 BTL postsurgical specimens and 55 prospectively collected FNAB using bisulfite-pyrosequencing. Results: DNA methylation analysis revealed 2130 and 19 differentially methylated CpGs in PTC and FTC, respectively. The CpGs confirmed by GEO and TCGA databases showing high areas under the receiver operating characteristic curve in all sample sets were used to train our diagnostic classifier. The model based on six CpGs was able to differentiate benign from malignant thyroid lesions with 94.3% sensitivity and 82.4% specificity. A similar performance was found applying the algorithm to TCGA and GEO external data sets (91.3-97.4% sensitivity and 87.5% specificity). We successfully evaluated the classifiers using a bisulfite-pyrosequencing technique, achieving 90.7% sensitivity and 75.4% specificity in surgical specimens (five of six CpGs). The study comprising FNAB cytology materials corroborated the applicability and performance of the methodology, demonstrating 86.7% sensitivity and 89.5% specificity in confirmed malignant tumors, and 100% sensitivity and 89% specificity in cases with indeterminate cytology. Conclusions: A novel diagnostic tool with potential application in preoperative screening of thyroid nodules is reported here. The proposed protocol has the potential to avoid unnecessary thyroidectomies.

Invasive micropapillary carcinoma of the mammary gland in humans and canines: Clinicopathological, immunophenotypical and survival approaches.

Invasive micropapillary carcinoma (IMPC) is a breast cancer with a proclivity for lymph node metastasis that affects women. In canines, this carcinoma has only recently been reported and appears to have similar histological aspects as its human counterpart. Thus, the aim of the present study was to compare clinicopathological, immunohistochemical and prognostic characteristics of mammary IMPC between humans and canines. In canines, regional metastasis was more frequently observed. Histopathologically, humans and canines predominantly showed a moderate histological grade. The pure subtype and neoplastic emboli were more frequently observed in canines. Regarding immunohistochemical evaluation, most canine and human IMPCs were positive for the estrogen and progesterone receptors. A reversed pattern of epithelial membrane antigen expression and a high proliferation index predominated in both species. The mortality due to the neoplastic disease was more frequently observed in canines (94%) than in humans (4%). Thus, canine IMPCs show a larger tumor size and higher rates of the pure subtype, regional metastasis and mortality than their human counterparts and appear to provide a good spontaneous model for achieving a better understanding of the biological behavior of human IMPCs.

Infections with multiple high-risk HPV types are associated with high-grade and persistent low-grade intraepithelial lesions of the cervix.

BACKGROUND: Infections with multiple human papillomavirus (HPV) types (mHPV) in Papanicolaou tests have been reported but the histologic correlation and clinical meaning remains debatable. METHODS: The authors prospectively tested 37 HPV types using the Linear Array HPV Genotyping Test and correlated the results to cytology and histology findings in 260 women evaluated from June 2009 to October 2011 and followed for up to 60 months. RESULTS: HPV was detected in 148 of 235 samples (63%) and high-risk HPV was detected in 132 samples (56%). mHPV infection was found to be twice as common as single HPV (sHPV) infection and was detected more frequently in low-grade squamous intraepithelial lesion (LSIL) (48 of 83 samples [58%]) and high-grade squamous intraepithelial lesion or invasive carcinoma (HSIL + (26 of 47 samples [55%]) compared with other categories (P<.001). Of 34 LSIL/cervical intraepithelial neoplasia 1 (CIN1) index cases, 13 of 21 patients with mHPV (61.9%) persisted on CIN1, whereas no histologic abnormality was detected during follow-up in all 12 patients with sHPV infection (high risk or low risk) (P<.001). Eighteen of 20 patients with HSIL/cervical intraepithelial neoplasia 2 (CIN2) (90%) and high-risk mHPV persisted on HSIL+/CIN2 + whereas 6 of 11 patients with sHPV infection did not demonstrate HSIL+/CIN2 + on follow-up (54.5%) (P = .066). Approximately 40% of women with HSIL were infected by high-risk HPV types other than types 16 or 18. CONCLUSIONS: High-risk mHPV infection identified patients with persistent LSIL/CIN1 and may to help identify patients at higher risk of disease progression to HSIL+/CIN2+. Longer follow-up will clarify the role of mHPV testing in patient care. Cancer Cytopathol 2017;125:138-143. © 2016 American Cancer Society.

[Immunophenotype and evolution of breast carcinomas: a comparison between very young and postmenopausal women]. / Imunofenótipo e evolução de câncer de mama: comparação entre mulheres muito jovens e mulheres na pós-menopausa.

PURPOSE: The objective of this study was to evaluate the clinical, pathological and molecular characteristics in very young women and postmenopausal women with breast cancer. METHODS: We selected 106 cases of breast cancer of very young women (<35 years) and 130 cases of postmenopausal women. We evaluated clinical characteristics of patients (age at diagnosis, ethnic group, family history of breast cancer, staging, presence of distant metastases, overall and disease-free survival), pathological characteristics of tumors (tumor size, histological type and grade, axillary lymph nodes status) and expression of molecular markers (hormone receptors, HER2, p53, p63, cytokeratins 5 and 14, and EGFR), using immunohistochemistry and tissue microarray. RESULTS: When comparing clinicopathologic variables between the age groups, younger women demonstrated greater frequency of nulliparity (p=0.03), larger tumors (p<0.000), higher stage disease (p=0.01), lymph node positivity (p=0.001), and higher grade tumors (p=0.004). Most of the young patients received chemotherapy (90.8%) and radiotherapy (85.2%) and less tamoxifen therapy (31.5%) comparing with postmenopausal women. Lower estrogen receptor positivity 49.1% (p=0.01) and higher HER2 overexpression 28.7% (p=0.03) were observed in young women. In 32 young patients (29.6%) and in 20% of the posmenopausal women, the breast carcinomas were of the triple-negative phenotype (p=0.034). In 16 young women (50%) and in 10 postmenopausal women (7.7%), the tumors expressed positivity for cytokeratin 5 and/or 14, basal phenotype (p=0.064). Systemic metastases were detected in 55.3% of the young women and in 39.2% of the postmenopausal women. Breast cancer overall survival and disease-free survival in five years were, respectively, 63 and 39% for young women and 75 and 67% for postmenopausal women. CONCLUSIONS: Breast cancer arising in very young women showed negative clinicobiological characteristics and more aggressive tumors.

Diphyllobothriasis, Brazil.

Cases of human diphyllobothriasis have been reported worldwide. Only 1 case in Brazil was diagnosed by our institution from January 1998 to December 2003. By comparison, 18 cases were diagnosed from March 2004 to January 2005. All patients who became infected ate raw fish in sushi or sashimi.