Corrigendum: Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

[This corrects the article DOI: 10.3389/fmolb.2023.1082915.].

DNA aneuploidy identifies a subset of Luminal subtype breast carcinoma patients with worse clinical outcome.

AIM: In breast carcinoma (BC), the relationship between the ploidy pattern and molecular subtyping is still unknown. We aim to investigate the prognostic impact of DNA ploidy within molecular subtypes of a large cohort of BC patients. METHODS: The series involved 520 BC patients with no neoadjuvant therapy and a median follow-up of 115.5 months. Immunohistochemical assessment of hormonal receptors, ERBB2 (HER2) status and Ki67 proliferative activity was the basis of the surrogate molecular subtyping. Ploidy was evaluated by DNA flow cytometry in fresh/frozen tumour samples. Kaplan-Meier (K-M) survival estimation was used for prognostic statistical analysis. RESULTS: Luminal A subtype had the lowest prevalence of disease recurrences (23.6 %) and deaths from disease (18.3 %), while Luminal B (42.2 %/37.9 %), Triple-negative (46.4 %/40.6 %), and HER2-positive (55.9 %/50.0 %) subtypes had the highest. The Triple-positive subtype shows an intermediate/low frequency of adverse events (29.4 % of disease recurrences and 17.6 % of deaths from disease). Luminal A tumours were mostly diploid (55.3 %), while Triple-negative and HER2-positive tumours showed a high incidence of aneuploidy (82.6 % and 88.2 %, respectively). Luminal B and Triple-positive tumours had an intermediate percentage of aneuploidy (63.8 % and 70.6 %, respectively). K-M survival curves showed that DNA aneuploidy is significantly associated with shorter disease-free survival and overall survival in Luminal A and Luminal B molecular subtypes. CONCLUSION: DNA aneuploidy identifies a subset of Luminal BC patients with worse clinical outcome, potentially eligible for more aggressive adjuvant therapy.

Adjuvant Chemotherapy De-Escalation with Genomic Assay Protocol in Patients with Early Breast Cancer: A Single-Centre Prospective Cohort Study.

INTRODUCTION: Genomic assays are useful tools for tailoring adjuvant treatment in early breast cancer. We aimed to analyse the role of an institutional protocol of a genomic assay for chemotherapy de-escalation. MATERIAL AND METHODS: Prospective cohort study of all consecutive women diagnosed with hormone receptor-positive and human epidermal growth factor receptor 2-negative early breast cancer, tested with the 21-gene Recurrence Score (RS) assay from August 2015 to July 2018 at a Portuguese cancer centre. For being tested, patients should meet at least one of the pre-defined inclusion criteria: i) luminal A-like, pT2pN0; ii) luminal A-like, 1 - 3 positive nodes and comorbidities with higher risk of chemotherapy-induced toxicity; iii) pT1-2pN0, progesterone receptor ≤ 20% and/or Ki67 14% - 40%. Adjuvant treatment was de-escalated to isolated endocrine therapy if RS was less than 18. We measured the reduction in chemotherapy prescribing and its clinical impact, the RS association with pathologic features, and the protocol feasibility. RESULTS: We tested 154 women with a median age of 61 years old (range: 25 - 79), 69% postmenopausal. Tumours were mainly pT1 (55%), pN0 (82%), invasive ductal (73%), G2 (86%), luminal B-like (69%) and stage IA (85%). We obtained a RS less than 18 in 60% of women, with an overall adjuvant chemotherapy reduction of 65%. Seven (95% confidence interval: 5 - 10) patients needed to be screened with the 21-gene RS assay to prevent one clinically relevant adverse event during the first six months of adjuvant treatment. Considering the currently used RS cut-off, only 9% of node-negative and 11% of node-positive patients had RS over 25. We found no relevant associations between RS and pathologic features. The protocol was feasible and did not compromise the adequate timing for adjuvant treatment. CONCLUSION: These criteria allowed the de-escalation of adjuvant systemic treatment in at least six out of ten women.

Evaluation of PIK3CA mutations in advanced ER+/HER2-breast cancer in Portugal - U-PIK Project.

Background: Around 40% of ER+/HER2-breast carcinomas (BC) present mutations in the PIK3CA gene. Assessment of PIK3CA mutational status is required to identify patients eligible for treatment with PI3Kα inhibitors, with alpelisib currently the only approved tyrosine kinase inhibitor in this setting. U-PIK project aimed to conduct a ring trial to validate and implement the PIK3CA mutation testing in several Portuguese centers, decentralizing it and optimizing its quality at national level. Methods: Eight Tester centers selected two samples of patients with advanced ER+/HER2- BC and generated eight replicates of each (n = 16). PIK3CA mutational status was assessed in two rounds. Six centers used the cobas® PIK3CA mutation test, and two used PCR and Sanger sequencing. In parallel, two reference centers (IPATIMUP and the Portuguese Institute of Oncology [IPO]-Porto) performed PIK3CA mutation testing by NGS in the two rounds. The quality of molecular reports describing the results was also assessed. Testing results and molecular reports were received and analyzed by U-PIK coordinators: IPATIMUP, IPO-Porto, and IPO-Lisboa. Results: Overall, five centers achieved a concordance rate with NGS results (allele frequency [AF] ≥5%) of 100%, one of 94%, one of 93%, and one of 87.5%, considering the overall performance in the two testing rounds. NGS reassessment of discrepancies in the results of the methods used by the Tester centers and the reference centers identified one probable false positive and two mutations with low AF (1-3%, at the analytical sensitivity threshold), interpreted as subclonal variants with heterogeneous representation in the tissue sections processed by the respective centers. The analysis of molecular reports revealed the need to implement the use of appropriate sequence variant nomenclature with the identification of reference sequences (HGVS-nomenclature) and to state the tumor cell content in each sample. Conclusion: The concordance rates between the method used by each tester center and NGS validate the use of the PIK3CA mutational status test performed at these centers in clinical practice in patients with advanced ER+/HER2- BC.

S-phase fraction, lymph node status and disease staging as the main prognostic factors to differentiate between young and older patients with invasive breast carcinoma.

The influence of age on the outcome of patients with invasive breast carcinoma (IBC) has not yet been fully established. The present study investigated two subgroups of patients either side of the age spectrum, and evaluated cytometric, histopathological and molecular characteristics. The series involved 219 patients with IBC that had long-term follow-up, which were divided into two subgroups: Young (≤45 years; n=103) and old patients (≥75 years; n=116). Immunohistochemical evaluation of hormonal receptors, Ki67 index and HER2 status (plus HER2 silver in situ hybridization in equivocal cases) were used as the basis for surrogate molecular subtyping. Ploidy and S-phase fraction (SPF) were analysed by DNA flow cytometry. Differences between the subgroups' characteristics were assessed by the two proportion Z test. Kaplan-Meier estimation and log-rank test were applied for survival analyses. The median age in the subgroups were 40 years (range, 19-45 years) in the young group and 78 years (range, 75-91 years) in the older subgroup. Young patients exhibited higher lymph node involvement, more advanced disease staging, higher SPF tumour proliferative activity, and a trend of lower incidence of Luminal A and higher incidence of Luminal B tumours. The median SPF value was significantly higher in young patients [7.1% (range, 1.5-23.7%) vs. 4.5% (range, 0.7-26.4%)], whereas the ploidy pattern showed no significant difference. In the whole series, as within IBC of no special type, young patients had a higher rate of recurrence (46.6 vs. 22.4%; P<0.001) and deaths from disease (35.9 vs. 20.7%; P=0.030), with a statistically significant difference for disease-free survival. In conclusion, the present study indicated that young patients with IBC exhibited more aggressive disease, with an increased risk of recurrence and shorter disease-free survival. SPF, lymph node status and staging appeared to be the main prognostic factors to differentiate young from older patients with IBC.

Patient-Derived Breast Cancer Tissue Cultures for Anti-Endocrine Drug Assays.

Breast cancer is a complex and heterogeneous pathology, characterized by a variety of histological and molecular phenotypes. The majority of the breast cancers express the estrogen receptor alpha (ER), which plays a pivotal role in the pathobiology of the disease and are therefore classified as ER-positive (ER+). In fact, targeting of the ER signaling pathway is the main therapeutic strategy for ER+ breast cancer. Despite the success of endocrine therapy, intrinsic and acquired resistance are reported in 30-50% of the ER+ breast cancers. However, the mechanisms underlying ER heterogeneity and therapeutic resistance are far from being fully disclosed, and efficacious clinical strategies to overcome resistance are still pending. One of the hurdles in studying ER+ breast cancer resistance is related with the scarcity of experimental models that can recapitulate ER heterogeneity and signaling. This is the case of ER+ breast cancer cell models, typically based on cells derived from metastasis, which also fail to recapitulate tumor complexity. Primary cultures of patient-derived breast cancer cells are difficult to establish, and generally characterized by stromal fibroblasts overgrowth and rapid loss of phenotypic and molecular traits of the tumor cells, including ER expression. Ex vivo cultures of breast cancer tissue have been reported to retain the tissue architecture, with preservation of the tumor microenvironment (TME) and ER expression for short periods of time.Given the cumulating evidence on the role of the TME in sustaining ER+ tumor cells, we hypothesized that TME preservation in culture would favor the long-term retention of ER expression and signaling. We employed alginate encapsulation to provide a supporting scaffold to breast cancer tissue microstructures, coupled to dynamic culture to improve the lifespan of the culture by avoiding diffusional limitations. In this chapter, we provide a detailed description of this culture methodology, which has been previously published by our group (Cartaxo et al., J Exp Clin Cancer Res 39:161, 2020), based on electrostatically driven breast cancer tissue encapsulation in alginate, coupled to culture under agitation in a defined culture medium. We also describe challenge of the ex vivo model with an ER activator and inhibitors (anti-endocrine drugs) and a gene expression endpoint of drug response using reverse transcription PCR-based analysis of three distinct genes downstream of ER.

Male and female breast cancer: the two faces of the same genetic susceptibility coin.

BACKGROUND: Breast cancer (BC) is the most common cancer in women. In contrast, male BC is about 100 times less common than in women, being considered a rare disease. Male BC may be a distinctive subtype of BC and available data seems to indicate that male BC has a higher dependence on genetic variants than female BC. Nevertheless, the same prognostic and predictive markers are used to determine optimal management strategies for both male and female BC. Several studies have assessed the role of genetic polymorphisms (SNPs) in DNA repair genes in female BC susceptibility. However, data on male BC is scarce. Thus, the current study aimed to assess the role of SNPs in XRCC1, MUTYH and TP53 genes in a male cohort of BC, and, in addition, compare the male data with matched results previously genotyped in female BC patients. METHODS: The male BC cohort was genotyped through Real-Time PCR using TaqMan Assays for several SNPs previously analysed in Portuguese female BC patients. RESULTS: The results obtained indicate significant differences in BC susceptibility between males and females for the XRCC1 rs1799782, MUTYH rs3219489 and TP53 rs1042522 and rs8064946 variants. CONCLUSIONS: In males, XRCC1 and TP53 variants, when in heterozygosity, seem to be related with lower susceptibility for BC, contrasting with higher susceptibility for a MUTYH variant in females. These findings may help to explain the difference in incidence of BC between the two sexes.

Male Breast Cancer-Immunohistochemical Patterns and Clinical Relevance of FASN, ATF3, and Collagen IV.

BACKGROUND: Male breast carcinoma (male BC) is an uncommon neoplasia without individualized strategies for diagnosis and therapeutics. Low overall survival (OS) rates have been reported, mostly associated with patients' advanced stage and older age. Intratumoral heterogeneity versus homogeneity of malignant epithelial cells seems to be an important factor to consider for the development of combination therapies with curative intention. OBJECTIVE: In this preliminary study, we aim to provide valuable insight into the distinct clinicopathologic features of male BC. MATERIAL AND METHODS: In a series of 40 male BC patients, we evaluated by immunohistochemistry androgen receptor; activating transcription factor 3 (ATF3); p16; cyclin D1; fatty acid synthase (FASN); fatty acid transport protein 1 (FATP1); ß1, ß3, ß4, and ß6 integrins; collagen I and collagen IV; and their interactions. Kaplan-Meier survival curves and log-rank tests were assessed for statistical analysis. RESULTS: Homogeneous epithelial staining of p16, ATF3, ß6 integrin, FASN, and FATP1 was found to be significantly intercorrelated, and associated with high Ki67. These markers also stained tumor stromal fibroblasts. The prognostic analysis showed statistically significant associations of FASN with disease-free survival (DFS) and OS, as well as of ATF3 with OS and collagen IV with DFS. CONCLUSIONS: This study highlights, as a novel finding, the relevance of FASN, ATF3, and collagen IV immunophenotypes, which may have innovative application in the clinical management of male BC.

A novel culture method that sustains ERα signaling in human breast cancer tissue microstructures.

BACKGROUND: Estrogen receptor α (ERα) signaling is a defining and driving event in most breast cancers; ERα is detected in malignant epithelial cells of 75% of all breast cancers (classified as ER-positive breast cancer) and, in these cases, ERα targeting is the main therapeutic strategy. However, the biological determinants of ERα heterogeneity and the mechanisms underlying therapeutic resistance are still elusive, hampered by the challenges in developing experimental models recapitulative of intra-tumoral heterogeneity and in which ERα signaling is sustained. Ex vivo cultures of human breast cancer tissue have been proposed to retain the original tissue architecture, epithelial and stromal cell components and ERα. However, loss of cellularity, viability and ERα expression are well-known culture-related phenomena. METHODS: BC samples were collected and brought to the laboratory. Then they were minced, enzymatically digested, entrapped in alginate and cultured for 1 month. The histological architecture, cellular composition and cell proliferation of tissue microstructures were assessed by immunohistochemistry. Cell viability was assessed by measurement of cell metabolic activity and histological evaluation. The presence of ERα was accessed by immunohistochemistry and RT-qPCR and its functionality evaluated by challenge with 17-ß-estradiol and fulvestrant. RESULTS: We describe a strategy based on entrapment of breast cancer tissue microstructures in alginate capsules and their long-term culture under agitation, successfully applied to tissue obtained from 63 breast cancer patients. After 1 month in culture, the architectural features of the encapsulated tissue microstructures were similar to the original patient tumors: epithelial, stromal and endothelial compartments were maintained, with an average of 97% of cell viability compared to day 0. In ERα-positive cases, fibers of collagen, the main extracellular matrix component in vivo, were preserved. ERα expression was at least partially retained at gene and protein levels and response to ERα stimulation and inhibition was observed at the level of downstream targets, demonstrating active ER signaling. CONCLUSIONS: The proposed model system is a new methodology to study ex vivo breast cancer biology, in particular ERα signaling. It is suitable for interrogating the long-term effects of anti-endocrine drugs in a set-up that closely resembles the original tumor microenvironment, with potential application in pre- and co-clinical assays of ERα-positive breast cancer.

Analysis of Epigenetic Alterations in Homologous Recombination DNA Repair Genes in Male Breast Cancer.

BACKGROUND: Male breast cancer (BC) is a distinct neoplasm with low but rising incidence, frequently diagnosed as advanced stage disease. Considering the relevance of altered homologous recombination repair (HRR) in male BC, we aimed to explore the biomarker potential of aberrant promoter methylation of ATM, BRCA1, PALB2, RAD51B, and XRCC3. METHODS: Formalin-fixed paraffin-embedded (FFPE) tissue samples from 128 male BC patients, paired adjacent normal tissue and 19 gynecomastia cases were collected and assessed by quantitative methylation-specific PCR (qMSP). Non-parametric tests were used to compare methylation levels between tumor and non-tumor samples and to seek for associations with clinicopathological variables. RESULTS: Only RAD51B and XRCC3 disclosed significant differences between tumor and gynecomastia (p < 0.0001 and p = 0.020, respectively). Assembled in a panel, RAD51B and XRCC3 promoter methylation discriminated male BC from gynecomastia with 91.5% sensitivity, 89.5% specificity, and 91.2% accuracy. Moreover, promoter methylation levels were lower in paired non-tumor tissues, comparing to tumor samples. No associations were found between epigenetic alterations and clinicopathological features, as well as with RAD51 and XRCC3 immunoexpression and methylation levels. CONCLUSION: Quantitative promoter methylation of RAD51B and XRCC3 constitutes a promising and accurate biomarker for male BC. Validation in larger series and in liquid biopsies is warranted to confirm its usefulness in detection and monitoring settings.

Granular cell tumor of the breast: correlations between imaging and pathology findings.

OBJECTIVE: To review the imaging features of granular cell tumors of the breast (on mammography, ultrasound, and magnetic resonance imaging), establishing a pathological correlation, in order to familiarize radiologists with this entity and make them aware of the differential diagnoses, other than malignancy, of lesions with spiculated margins. MATERIALS AND METHODS: We reviewed the medical records (from a clinical-pathology database and picture archiving and communication system) of five patients with a pathologically confirmed diagnosis of granular cell tumor of the breast, treated at the Portuguese Oncology Institute of Lisbon, in the city of Lisbon, Portugal, between January 2012 and December 2018. RESULTS: All five tumors exhibited imaging features highly suggestive of malignancy (BI-RADS 5 lesions), namely spiculated margins, significant depth, and posterior acoustic shadowing (on ultrasound). One tumor showed a kinetic curve indicative of washout on magnetic resonance imaging, two were adherent to the pectoralis muscle, and one was accompanied by skin retraction. Pathology provided the definitive diagnosis in all cases. CONCLUSION: Granular cell tumors of the breast pose a diagnostic challenge because they can present with clinical and imaging features mimicking malignancy, and the diagnosis is therefore provided by pathology. Radiologists should be familiarized with this entity, so they can be aware of the fact that breast lesions with spiculated margins can be indicative of diagnoses other than malignancy.

OBJETIVO: Rever as características de imagem dos tumores de células granulares da mama (mamográficas, ultrassonográficas e de ressonância magnética), estabelecendo uma correlação anatomopatológica, no intuito de proporcionar aos radiologistas uma familiarização com esta entidade e alertar para outros diagnósticos diferenciais de lesões espiculadas além das malignas. MATERIAIS E MÉTODOS: Consulta dos processos clínicos (base de dados clínica, anatomopatológica e sistema de comunicação e arquivamento de imagens) de doentes seguidos no Instituto Português de Oncologia de Lisboa, com diagnóstico anatomopatológico confirmado de tumor de células granulares da mama, de janeiro de 2012 a dezembro de 2018. RESULTADOS: Todos os tumores exibiram características de imagem altamente sugestivas de malignidade (BI-RADS 5), nomeadamente espiculações, crescimento em profundidade e atenuação posterior (ultrassonografia), um mostrou um perfil cinético com washout na ressonância magnética, dois estavam aderentes ao músculo peitoral e um associava retração cutânea. O diagnóstico definitivo foi anatomopatológico. CONCLUSÃO: Os tumores de células granulares da mama constituem um desafio diagnóstico, pois podem apresentar características clínicas e de imagem que mimetizam malignidade, pelo que o diagnóstico é anatomopatológico. Os radiologistas devem estar familiarizados com esta entidade de forma a considerá-la nos diagnósticos diferenciais de lesões espiculadas, além das lesões malignas.

Granular cell tumor of the breast: correlations between imaging and pathology findings / Tumor de células granulares da mama: correlação anatomorradiológica

Abstract Objective: To review the imaging features of granular cell tumors of the breast (on mammography, ultrasound, and magnetic resonance imaging), establishing a pathological correlation, in order to familiarize radiologists with this entity and make them aware of the differential diagnoses, other than malignancy, of lesions with spiculated margins. Materials and Methods: We reviewed the medical records (from a clinical-pathology database and picture archiving and communication system) of five patients with a pathologically confirmed diagnosis of granular cell tumor of the breast, treated at the Portuguese Oncology Institute of Lisbon, in the city of Lisbon, Portugal, between January 2012 and December 2018. Results: All five tumors exhibited imaging features highly suggestive of malignancy (BI-RADS 5 lesions), namely spiculated margins, significant depth, and posterior acoustic shadowing (on ultrasound). One tumor showed a kinetic curve indicative of washout on magnetic resonance imaging, two were adherent to the pectoralis muscle, and one was accompanied by skin retraction. Pathology provided the definitive diagnosis in all cases. Conclusion: Granular cell tumors of the breast pose a diagnostic challenge because they can present with clinical and imaging features mimicking malignancy, and the diagnosis is therefore provided by pathology. Radiologists should be familiarized with this entity, so they can be aware of the fact that breast lesions with spiculated margins can be indicative of diagnoses other than malignancy.

Resumo Objetivo: Rever as características de imagem dos tumores de células granulares da mama (mamográficas, ultrassonográficas e de ressonância magnética), estabelecendo uma correlação anatomopatológica, no intuito de proporcionar aos radiologistas uma familiarização com esta entidade e alertar para outros diagnósticos diferenciais de lesões espiculadas além das malignas. Materiais e Métodos: Consulta dos processos clínicos (base de dados clínica, anatomopatológica e sistema de comunicação e arquivamento de imagens) de doentes seguidos no Instituto Português de Oncologia de Lisboa, com diagnóstico anatomopatológico confirmado de tumor de células granulares da mama, de janeiro de 2012 a dezembro de 2018. Resultados: Todos os tumores exibiram características de imagem altamente sugestivas de malignidade (BI-RADS 5), nomeadamente espiculações, crescimento em profundidade e atenuação posterior (ultrassonografia), um mostrou um perfil cinético com washout na ressonância magnética, dois estavam aderentes ao músculo peitoral e um associava retração cutânea. O diagnóstico definitivo foi anatomopatológico. Conclusão: Os tumores de células granulares da mama constituem um desafio diagnóstico, pois podem apresentar características clínicas e de imagem que mimetizam malignidade, pelo que o diagnóstico é anatomopatológico. Os radiologistas devem estar familiarizados com esta entidade de forma a considerá-la nos diagnósticos diferenciais de lesões espiculadas, além das lesões malignas.

Unraveling FATP1, regulated by ER-ß, as a targeted breast cancer innovative therapy.

The biochemical demands associated with tumor proliferation prompt neoplastic cells to augment the import of nutrients to sustain their survival and fuel cell growth, with a consequent metabolic remodeling. Fatty acids (FA) are crucial in this process, since they have a dual role as energetic coins and building blocks. Recently, our team has shown that FATP1 has a pivotal role in FA transfer between breast cancer cells (BCCs) and non-cancerous cells in the microenvironment. We aimed to investigate the role of FATP1 in BCCs and also to explore if FATP1 inhibition is a promising therapeutic strategy. In patients' data, we showed a higher expression of FATP1/SLC27A1 in TNBC, which correlated with a significant decreased overall survival (OS). In vitro, we verified that FA and estradiol stimulated FATP1/SLC27A1 expression in BCCs. Additionally, experiments with estradiol and PHTPP (ER-ß antagonist) showed that estrogen receptor-ß (ER-ß) regulates FATP1/SLC27A1 expression, the uptake of FA and cell viability, in four BCC lines. Furthermore, the inhibition of FATP1 with arylpiperazine 5k (DS22420314) interfered with the uptake of FA and cell viability. Our study, unraveled FATP1 as a putative therapeutic target in breast cancer (BC).

Loss of HER2 and disease prognosis after neoadjuvant treatment of HER2+ breast cancer.

INTRODUCTION: HER2 overexpression/amplification occurs in 15-20% breast cancers (BC) and is associated with worse prognosis. The addition of anti-HER2 treatment to neoadjuvant chemotherapy significantly improves the pathological complete response (pCR) rate. Changes in HER2 status after neoadjuvant treatment (NAT) have been reported and may affect prognosis. The aim of this study was to assess the efficacy of NAT in patients with HER2+ BC and its influence on HER2 status and associated prognostic impact. METHODS: Retrospective chart review and pathologic evaluation of all consecutive patients with HER2+ BC (defined as IHC 3+ or IHC 2+ confirmed by SISH) submitted to NAT between 2010-2015 in three Portuguese Hospitals. RESULTS: One hundred eight female patients were included; 40 with stage II, 68 with stage III. Hormone receptors were positive in 70. pCR (ypT0/isN0) was achieved in 48 patients (44%). With a median follow-up of 52 months, there were 5 disease free survival (DFS) events among pCR patients and 19 among non-pCR (P = 0.02). Of the 60 patients with residual disease at surgery, 52 remained HER2+ and 8 (13%) lost HER2 overexpression/amplification. 5y-DFS and 5y-OS was 70% and 84%, respectively, for patients whose residual tumors remained HER2+, and 21% and 50% for patients whose residual tumors became HER2 negative (P = 0.02 and < 0.001). DISCUSSION: We confirmed the negative prognostic impact of NAT-induced HER2 loss on residual tumor leading to worse DFS and OS. Despite the retrospective design and small sample size, these results suggest that it is important to retest HER2 after NAT, to better refine patient outcome.

Establishment and characterization of a new patient-derived anaplastic thyroid cancer cell line (C3948), obtained through fine-needle aspiration cytology.

PURPOSE: Anaplastic thyroid cancer (ATC) is among the most aggressive and unresectable tumors, presenting a bad prognosis. A better comprehension of the functional and molecular mechanisms behind the aggressiveness of this cancer, as well as new biomarkers for aggressiveness, prognosis, and response to therapy are required. However, owing to their irresectability, ATC tissue is not always accessible. Here we describe the establishment and characterization of a new patient-derived cell line, obtained from an unresectable ATC through fine-needle aspiration cytology (FNAC). METHODS: The morphology, expression of epithelial and thyroid markers, cytogenetic, mutational and gene expression profiles, doubling time, and drug-resistance profile of the new cell line, designated C3948, were investigated using several methodologies: immunostaining, karyotype analysis, comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), next-generation sequencing (NGS), Sanger sequencing, gene expression microarrays, cell counting, and IC50 determination. RESULTS: Results indicate that C3948 cell line has a histological phenotype representative of original ATC cells and a completely aberrant karyotype with many chromosomal losses and gains; harbors mutated TP53, STK11, and DIS3L2 genes; presents a gene expression profile similar to C643 ATC commercial cell line, but with some unique alterations; has a doubling time similar to C643; and the IC50 profile for paclitaxel, doxorubicin, and cisplatin is similar to C643, although higher for cisplatin. CONCLUSIONS: These observations are consistent with a typical ATC cell profile, supporting C3948 cell line as a novel preclinical model, and FNAC as a useful approach to better study anaplastic thyroid cancer, including testing of new anticancer therapies.

Male breast cancer: Specific biological characteristics and survival in a Portuguese cohort.

Male breast cancer (BC) represents an individual subtype of BC, with therapeutic procedures based on female BC therapy results. The present study evaluated the parameters currently used for the characterization and therapy of male BC, and their association with disease-free (DFS) and overall survival (OS), aiming to obtain a comprehensive basis to improve the personalized care of male BC. A total of 196 patients from March 1970 to March 2018 (mean follow-up, 84.9 months) were profiled, using clinicopathological review, molecular assessment [BRCA1/2, DNA repair associated (BRCA1/2) status, immunohistochemistry, fluorescence in situ hybridization and DNA flow cytometry] and Cox regression statistical analysis. The median age of patients was 66.5 years. At presentation, 39.2% of patients with invasive carcinomas were in anatomic stage (AS) I. Patients exhibited primarily invasive carcinomas of no special type, histological grade 2, estrogen receptor α-(ERα) and progesterone receptor (PR)-positive, receptor tyrosine kinase erbB-2-negative, high Ki-67, Luminal B-like and aneuploid tumors. A total of 13 of the 44 (29.5%) BRCA-evaluated patients exhibited BRCA2 mutations, significantly associated with family history (FH), bilaterality, high Ki-67 expression, absence of PR and Luminal B-like tumors. Bilaterality was associated with the occurrence of non-breast primary neoplasms (NBPN). The 5 and 10-year DFS rates, excluding patients with distant metastasis, NBPN and in situ carcinomas (n=145) were 65.9 and 58.2%, respectively, and the 5 and 10-year OS rates were 77.5 and 59.2%, respectively. In the univariate analysis, Luminal B-like subtype, BRCA2 mutations, high Ki-67 expression, and AS II and III were significantly associated with shorter DFS and OS. In addition, age >70 years was associated with low OS. In the multivariate analysis, FH, AS II and III, and Luminal B-like subtypes were associated with poorer OS. In conclusion, the data from the present study emphasize the high incidence of BRCA2 mutation in male BC, and its association with FH, bilaterality, high Ki-67 expression, negative PR expression and Luminal B-like subtypes, and with shorter DFS and OS in univariate analysis.

Bilateral breast fibromatosis after silicone prosthetics in a patient with classic familial adenomatous polyposis: A case report.

Breast fibromatosis is a benign fibroblastic proliferation accounting for less than 0.2% of breast tumors. It presents sporadically or as a manifestation of familial adenomatous polyposis (FAP). Fibromatosis in FAP may develop in patients with adenomatous polyposis coli (APC) gene mutations at any location through the gene. Notably, there is an increased risk if mutation is downstream codon 1400. The present case report described a 33-year-old woman with recurrent bilateral breast fibromatosis after breast implants in a context of classic FAP. APC mutation (codon-935) was detected at the age of 16. In the same year, a thyroidectomy for a cribriform-morular papillary thyroid carcinoma (pT1) was performed. Seven years later, a prophylactic total colectomy with >100 adenomas without invasive carcinoma was performed and the patient was kept under surveillance. At the age of 30 years old, she underwent breast silicone implantation for cosmetic reasons. One year later, bilateral breast tumors were diagnosed in core biopsy as fibromatosis (nuclear ß-catenin+, estrogen receptors-). After no success with medical treatment with tamoxifen, bilateral mastectomy was performed. The patient relapsed one year later and a fibromatosis lesion in the right thoracic wall was excised again. The patient demonstrated no signs of relapse 24 months after the surgery. This rare case illustrates that the increased risk of developing fibromatosis in patients with FAP, even in the classic form, should be considered before deciding to place breast implants.

The Use of Sentinel Lymph Node Biopsy in BRCA1/2 Mutation Carriers Undergoing Prophylactic Mastectomy: A Retrospective Consecutive Case-Series Study.

INTRODUCTION: Sentinel lymph node biopsy in prophylactic mastectomy is controversial. It avoids lymphadenectomy in occult carcinoma but is associated with increased morbidity. Women with BRCA mutations have a higher incidence of occult carcinoma and our objective was to assess the clinical utility of sentinel lymph node biopsy when these women undergo prophylactic mastectomy. MATERIALS AND METHODS: Seven-year retrospective consecutive case-series study of women, with a BRCA deleterious mutation, admitted to prophylactic mastectomy, at our center. Breast MRI < 6 months before surgery was routine, unless contraindicated. RESULTS: Fifty-seven patients (43% BRCA1; 57% BRCA2) underwent 80 prophylactic mastectomies. 72% of patients had had breast cancer treated before prophylactic mastectomy or synchronously to it. The occult carcinoma incidence was 5%, and half of the cases were invasive. SLNB was performed in 19% of the prophylactic mastectomies; none of these had tumor invasion. Women with invasive carcinoma who had not undergone sentinel lymph node biopsy were followed closely with axillary ultrasound. The median follow-up was 37 months, with no local recurrence; 1 patient died of primary tumor systemic relapse. CONCLUSIONS: Our data do not support this procedure for routine (in agreement with previous literature), in this high risk for occult carcinoma population.

Men seeking counselling in a Breast Cancer Risk Evaluation Clinic.

BACKGROUND: Hereditary breast and ovary cancer syndrome affects both genders but little is known about the uptake of genetic services by men. The objective of this study is to characterise the male population counselled through a multidisciplinary breast/ovarian program. METHODS: Descriptive analysis of male patients counselled from January 2000 to December 2015. Data in this analysis include new cancer diagnoses during prospective follow up. RESULTS: From 4,320 families registered, 362 male patients were identified: 236 (65.2%) from hereditary cancer families (HCF) and 126 (34.8%) from non-HCF. In HCF, 121 patients (51.3%) were mutation carriers (MC): BRCA2 - 102 (84.3%), BRCA1 - 16 (13.2%), CHEK2 - 1 (0.8%) and TP53 - 2 (1.7%). Non-HCF included 126 patients: 85 (67.5%) belonged to families without pathogenic mutations or with variants of unknown clinical significance; 22 (17.5%) refused testing after counselling and 19 (15.0%) did not meet criteria for testing. Both HCF and non-HCF included patients with previous cancer diagnoses: HCF- Breast Cancer (BC) - 18; prostate cancer (PC) - 13; melanoma - 1; others - 7) and non-HCF (BC - 77; PC - 20; gastric cancer (GC) - 1; melanoma - 8; bladder cancer - 1; others - 22). From the 121 MC identified (including the TP53 and CHEK2 carriers), 97 patients (80.2%) adhered to prospective surveillance. With a median follow-up of 36.9 months, 17 cancers were diagnosed in 14 patients, PC being the most frequently diagnosed neoplasia (5 cases). Eleven patients (78.6%) are alive and three patients died of advanced cancer (2 with GC, 1 with disseminated adenocarcinoma). CONCLUSION: We observed a high adherence to counselling, genetic testing and active surveillance by men belonging to hereditary BC families. Male carriers of pathogenic DNA variants are at risk for several cancers and should be included in prospective follow-up studies.