Frequency of CDH1 germline variants and contribution of dietary habits in early age onset gastric cancer patients in Brazil.

INTRODUCTION: The contribution of CDH1 germline variants to gastric cancer burden among young adults is unknown in Brazil. We aimed to evaluate the frequency of CDH1 germline variants and the diet/lifestyle habits in early age onset gastric cancer (EOGC, ≤ 55 years old) patients. METHODOLOGY: From 2013 to 2015, a total of 88 unrelated and consecutive patients diagnosed with EOGC were enrolled. All CDH1 exons and intronic boundaries were sequenced, and large genomic rearrangements were screened by MLPA. CDH1 transcription analysis was performed for variants that could potentially induce an effect on splicing. The diet and lifestyle habits of EOGC patients were compared to Brazilian population diet and lifestyle, obtained from governmental databases. RESULTS: Of 88 patients, the mean age at EOGC diagnosis was 39 years and 55% fulfilled the criteria for hereditary diffuse gastric cancer. The majority of the tumors were diffuse (74%) and poorly differentiated (80%). In total, 4 novel missense variants of uncertain significance (VUS) were identified: c.313T>A, c.387G>T, c.1676G>A, and c.1806C>A. The MLPA results revealed no rearrangements and CDH1 transcription analysis for variants of interest were inconclusive. EOGC patients had a higher red (OR:2.6, 95%CI:1.4-4.9) and processed (OR:3.1, 95%CI:1.6-6.0) meat intake and higher fruit consumption (OR:0.4, 95%IC:0.3-0.7) compared to eating habits of the Brazilian population. CONCLUSIONS: No unequivocal pathogenic germline CDH1 variants were identified in Brazilian EOGC patients. Dietary habits may be associated with the EOGC development.

Germline mutations in BRCA1 and BRCA2 in epithelial ovarian cancer patients in Brazil.

BACKGROUND: Approximately 8-15% epithelial ovarian cancer patients are BRCA1 or BRCA2 germline mutation carriers. Brazilian inhabitants may have peculiar genetic characteristics associated with ethnic diversity, and studies focusing on the entire BRCA1/BRCA2 gene sequencing in Brazilian ovarian cancer patients are still lacking. The aim of this study was to evaluate BRCA1/2 mutations, through entire gene sequencing, in a Brazilian population of women with epithelial ovarian cancer. METHODS: In a cross sectional study performed in one reference centre for cancer treatment in São Paulo, Brazil, 100 patients diagnosed with epithelial ovarian cancer unselected for family history of breast and/or ovarian cancer were included. The complete coding sequence of BRCA1/2 genes was evaluated through Next-Generation or capillary sequencing. Large deletions were investigated through Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: Nineteen pathogenic mutations (BRCA1: n = 17 and BRCA2: n = 2) featuring 14 different mutations, including two large deletions in BRCA1 (exon 1-2 deleted and exon 5-7 deleted) were identified. Three mutations were detected more than once (c.3331_3334delCAAG, c.5266dupC and c.4484G > T). Two novel frameshift mutations were identified, one in BRCA1 (c.961_962delTG) and one in BRCA2 (c.1963_1963delC). BRCA1/2 mutations were seen in 35.5% of the patients with first and/or second-degree relatives with breast and/or ovarian cancer. Nineteen variants of uncertain significance (VUS) were detected (BRCA1: n = 2 and BRCA2: n = 17), including five distinct missense variants (BRCA1: c.5348 T > C; BRCA2: c.2350A > G, c.3515C > T, c.7534C > T, and c.8351G > A). CONCLUSIONS: Among epithelial ovarian cancer patients unselected for family history of cancer, 19% were BRCA1/2 germline mutation carriers. Almost ¾ of the BRCA mutations, including two large deletions, were detected only once. Our work emphasizes the need of entire gene sequencing and MLPA screening in Brazil.

Custom target-sequencing in triple-negative and luminal breast cancer from young Brazilian patients.

OBJECTIVES: To identify somatic mutations in tumors from young women with triple-negative or luminal breast cancer, through targeted sequencing and to explore the cancer driver potential of these gene variants. METHODS: A customized gene panel was assembled based on data from previous sequencing studies of breast cancer from young women. Triple-negative and luminal tumors and paired blood samples from young breast cancer patients were sequenced, and identified gene variants were searched for their driver potential, in databases and literature. Additionally, the authors performed an exploratory analysis using large, curated databases to evaluate the frequency of somatic mutations in this gene panel in tumors stratified by age groups (every 10 years). RESULTS: A total of 28 young women had their tumoral tissue and blood samples sequenced. Using a customized panel of 64 genes, the authors could detect cancer drivers in 11/12 (91.7 %) TNBC samples and 11/16 (68.7 %) luminal samples. Among TNBC patients, the most frequent cancer driver was TP53, followed by NF1, NOTCH1 and PTPN13. In luminal samples, PIK3CA and GATA3 were the main cancer drivers, and other drivers were GRHL2 and SMURF2. CACNA1E was involved in both TN and luminal BC. The exploratory analysis also indicated a role for SMURF2 in luminal BC development in young patients. CONCLUSIONS: The data further indicates that some cancer drivers are more common in a specific breast cancer subtype from young patients, such as TP53 in TNBC and PIK3CA and GATA3 in luminal samples. These results also provide additional evidence that some genes not considered classical cancer-causing genes, such as CACNA1E, GRHL2 and SMURF2 might be cancer drivers in this age group.

Prevalence of germline variants in Brazilian pancreatic carcinoma patients.

We evaluated the prevalence of pathogenic/likely pathogenic germline variants (PGV) in Brazilian pancreatic adenocarcinoma (PC) patients, that represent a multiethnic population, in a cross-sectional study. We included 192 PC patients unselected for family history of cancer. We evaluated a panel of 113 cancer genes, through genomic DNA sequencing and 46 ancestry-informative markers, through multiplex PCR. The median age was 61 years; 63.5% of the patients presented disease clinical stages III or IV; 8.3% reported personal history of cancer; 4.7% and 16.1% reported first-degree relatives with PC or breast and/or prostate cancer, respectively. Although the main ancestry was European, there was considerable genetic composition admixture. Twelve patients (6.25%) were PGV carriers in PC predisposition genes (ATM, BRCA1, BRCA2, CDKN2A, MSH2, PALB2) and another 25 (13.0%) were PGV carriers in genes with a limited association or not previously associated with PC (ACD, BLM, BRIP1, CHEK2, ERCC4, FANCA, FANCE, FANCM, GALNT12, MITF, MRE11, MUTYH, POLE, RAD51B, RAD51C, RECQL4, SDHA, TERF2IP). The most frequently affected genes were CHEK2, ATM and FANC. In tumor samples from PGV carriers in ACD, BRIP1, MRE11, POLE, SDHA, TERF2IP, which were examined through exome sequencing, the main single base substitutions (SBS) mutational signature was SBS1+5+18, probably associated with age, tobacco smoking and reactive oxygen species. SBS3 associated with homologous repair deficiency was also represented, but on a lower scale. There was no difference in the frequency of PGV carriers between: (a) patients with or without first-degree relatives with cancer; and (b) patients with admixed ancestry versus those with predominantly European ancestry. Furthermore, there was no difference in overall survival between PGV carriers and non-carriers. Therefore, genetic testing should be offered to all Brazilian pancreatic cancer patients, regardless of their ancestry. Genes with limited or previously unrecognized associations with pancreatic cancer should be further investigated to clarify their role in cancer risk.

Breast cancer tissue slices as a model for evaluation of response to rapamycin.

Rapamycin is a selective inhibitor of the mammalian target of rapamycin (mTOR), a regulator kinase that integrates growth factors signaling via the phosphoinositide-3-kinase pathway and that has emerged as a novel therapeutic modality in breast cancer (BC). We propose a pre-clinical "ex-vivo" personalized organotypic culture of BC that preserves the microenvironment to evaluate rapamycin-mediated gene expression changes. Freshly excised ductal invasive BC slices, 400 µm thick (n=30), were cultured in the presence or absence (control) of rapamycin (20 nM) for 24 h. Some slices were formalin-fixed for immunohistochemical determinations and some were processed for microarray analysis. Control slices in culture retained their tissue morphology and tissue viability (detected by BrdU uptake). The percentage of proliferating cells (assessed by Ki67) did not change up to 24 h of treatment. Immunohistochemical evaluation of p-AKT, p-mTOR, p-4EBP1 and p-S6K1 indicated that AKT/mTOR pathway activation was maintained during cultivation. For microarray analysis, slices were divided into two groups, according to the presence/absence of epidermal growth factor receptor-type 2 and analyzed separately. Limited overlap was seen among differentially expressed genes after treatment (P<0.01) in both groups suggesting different responses to rapamycin between these BC subtypes. Ontology analysis indicated that genes involved in biosynthetic processes were commonly reduced by rapamycin. Our network analysis suggested that concerted expression of these genes might distinguish controls from treated slices. Thus, breast carcinoma slices constitute a suitable physiological tool to evaluate the short-term effects of rapamycin on the gene profile of individual BC samples.

Transcriptional effects of 1,25 dihydroxyvitamin D(3) physiological and supra-physiological concentrations in breast cancer organotypic culture.

BACKGROUND: Vitamin D transcriptional effects were linked to tumor growth control, however, the hormone targets were determined in cell cultures exposed to supra physiological concentrations of 1,25(OH)(2)D(3) (50-100nM). Our aim was to evaluate the transcriptional effects of 1,25(OH)(2)D(3) in a more physiological model of breast cancer, consisting of fresh tumor slices exposed to 1,25(OH)(2)D(3) at concentrations that can be attained in vivo. METHODS: Tumor samples from post-menopausal breast cancer patients were sliced and cultured for 24 hours with or without 1,25(OH)(2)D(3) 0.5nM or 100nM. Gene expression was analyzed by microarray (SAM paired analysis, FDR≤0.1) or RT-qPCR (p≤0.05, Friedman/Wilcoxon test). Expression of candidate genes was then evaluated in mammary epithelial/breast cancer lineages and cancer associated fibroblasts (CAFs), exposed or not to 1,25(OH)(2)D(3) 0.5nM, using RT-qPCR, western blot or immunocytochemistry. RESULTS: 1,25(OH)(2)D(3) 0.5nM or 100nM effects were evaluated in five tumor samples by microarray and seven and 136 genes, respectively, were up-regulated. There was an enrichment of genes containing transcription factor binding sites for the vitamin D receptor (VDR) in samples exposed to 1,25(OH)(2)D(3) near physiological concentration. Genes up-modulated by both 1,25(OH)(2)D(3) concentrations were CYP24A1, DPP4, CA2, EFTUD1, TKTL1, KCNK3. Expression of candidate genes was subsequently evaluated in another 16 samples by RT-qPCR and up-regulation of CYP24A1, DPP4 and CA2 by 1,25(OH)(2)D(3) was confirmed. To evaluate whether the transcripitonal targets of 1,25(OH)(2)D(3) 0.5nM were restricted to the epithelial or stromal compartments, gene expression was examined in HB4A, C5.4, SKBR3, MDA-MB231, MCF-7 lineages and CAFs, using RT-qPCR. In epithelial cells, there was a clear induction of CYP24A1, CA2, CD14 and IL1RL1. In fibroblasts, in addition to CYP24A1 induction, there was a trend towards up-regulation of CA2, IL1RL1, and DPP4. A higher protein expression of CD14 in epithelial cells and CA2 and DPP4 in CAFs exposed to 1,25(OH)(2)D(3) 0.5nM was detected. CONCLUSIONS: In breast cancer specimens a short period of 1,25(OH)(2)D(3) exposure at near physiological concentration modestly activates the hormone transcriptional pathway. Induction of CYP24A1, CA2, DPP4, IL1RL1 expression appears to reflect 1,25(OH)(2)D(3) effects in epithelial as well as stromal cells, however, induction of CD14 expression is likely restricted to the epithelial compartment.

Detection of germline variants in Brazilian breast cancer patients using multigene panel testing.

Genetic diversity of germline variants in breast cancer (BC) predisposition genes is unexplored in miscegenated populations, such those living in Latin America. We evaluated 1663 Brazilian BC patients, who underwent hereditary multigene panel testing (20-38 cancer susceptibility genes), to determine the spectrum and prevalence of pathogenic/likely pathogenic (P/LP) variants and variants of uncertain significance (VUS). Associations between P/LP variants and BC risk were estimated in a case-control analysis of BC patients and 18,919 Brazilian reference controls (RC). In total, 335 (20.1%) participants carried germline P/LP variants: 167 (10.0%) in BRCA1/2, 122 (7.3%) in BC actionable non-BRCA genes and 47 (2.8%) in candidate genes or other cancer predisposition genes. Overall, 354 distinctive P/LP variants were identified in 23 genes. The most commonly mutated genes were: BRCA1 (27.4%), BRCA2 (20.3%), TP53 (10.5%), monoallelic MUTYH (9.9%), ATM (8.8%), CHEK2 (6.2%) and PALB2 (5.1%). The Brazilian variant TP53 R337H (c.1010G>A, p.Arg337His), detected in 1.6% of BC patients and 0.1% of RC, was strongly associated with risk of BC, OR = 17.4 (95% CI: 9.4-32.1; p < 0.0001); monoallelic MUTYH variants c.1187G>A and c.536A>G, detected in 1.2% (0.9% RC) and 0.8% (0.4% RC) of the patients, respectively, were not associated with the odds of BC, the former with OR = 1.4 (95% CI: 0.8-2.4; p = 0.29) and the latter with OR = 1.9 (95% CI: 0.9-3.9; p = 0.09). The overall VUS rate was 46.1% for the entire patient population. Concluding, the use of multigene panel testing almost doubled the identification of germline P/LP variants in clinically actionable predisposition genes in BC patients. In Brazil, special attention should be given to TP53 P/LP variants.

Influence of the interaction between nodal fibroblast and breast cancer cells on gene expression.

Our aim was to evaluate the interaction between breast cancer cells and nodal fibroblasts, by means of their gene expression profile. Fibroblast primary cultures were established from negative and positive lymph nodes from breast cancer patients and a similar gene expression pattern was identified, following cell culture. Fibroblasts and breast cancer cells (MDA-MB231, MDA-MB435, and MCF7) were cultured alone or co-cultured separated by a porous membrane (which allows passage of soluble factors) for comparison. Each breast cancer lineage exerted a particular effect on fibroblasts viability and transcriptional profile. However, fibroblasts from positive and negative nodes had a parallel transcriptional behavior when co-cultured with a specific breast cancer cell line. The effects of nodal fibroblasts on breast cancer cells were also investigated. MDA MB-231 cells viability and migration were enhanced by the presence of fibroblasts and accordingly, MDA-MB435 and MCF7 cells viability followed a similar pattern. MDA-MB231 gene expression profile, as evaluated by cDNA microarray, was influenced by the fibroblasts presence, and HNMT, COMT, FN3K, and SOD2 were confirmed downregulated in MDA-MB231 co-cultured cells with fibroblasts from both negative and positive nodes, in a new series of RT-PCR assays. In summary, transcriptional changes induced in breast cancer cells by fibroblasts from positive as well as negative nodes are very much alike in a specific lineage. However, fibroblasts effects are distinct in each one of the breast cancer lineages, suggesting that the inter-relationships between stromal and malignant cells are dependent on the intrinsic subtype of the tumor.

Somatic Mutational Profile of High-Grade Serous Ovarian Carcinoma and Triple-Negative Breast Carcinoma in Young and Elderly Patients: Similarities and Divergences.

BACKGROUND: Triple-negative breast cancer (TNBC) and High-Grade Serous Ovarian Cancer (HGSOC) are aggressive malignancies that share similarities; however, different ages of onset may reflect distinct tumor behaviors. Thus, our aim was to compare somatic mutations in potential driver genes in 109 TNBC and 81 HGSOC from young (Y ≤ 40 years) and elderly (E ≥ 75 years) patients. METHODS: Open access mutational data (WGS or WES) were collected for TNBC and HGSOC patients. Potential driver genes were those that were present in the Cancer Gene Census-CGC, the Candidate Cancer Gene Database-CCGD, or OncoKB and those that were considered pathogenic in variant effect prediction tools. RESULTS: Mutational signature 3 (homologous repair defects) was the only gene that was represented in all four subgroups. The median number of mutated CGCs per sample was similar in HGSOC (Y:3 vs. E:4), but it was higher in elderly TNBC than it was in young TNBC (Y:3 vs. E:6). At least 90% of the samples from TNBC and HGSOC from Y and E patients presented at least one known affected TSG. Besides TP53, which was mutated in 67-83% of the samples, the affected TSG in TP53 wild-type samples were NF1 (yHGSOC and yTNBC), PHF6 (eHGSOC and yTNBC), PTEN, PIK3R1 and ZHFX3 (yTNBC), KMT2C, ARID1B, TBX3, and ATM (eTNBC). A few samples only presented one affected oncogene (but no TSG): KRAS and TSHR in eHGSOC and RAC1 and PREX2 (a regulator of RAC1) in yTNBC. At least ⅔ of the tumors presented mutated oncogenes associated with tumor suppressor genes; the Ras and/or PIK3CA signaling pathways were altered in 15% HGSOC and 20-35% TNBC (Y vs. E); DNA repair genes were mutated in 19-33% of the HGSOC tumors but were more frequently mutated in E-TNBC (56%). However, in HGSOC, 9.5% and 3.3% of the young and elderly patients, respectively, did not present any tumors with an affected CGC nor did 4.65% and none of the young and elderly TNBC patients. CONCLUSION: Most HGSOC and TNBC from young and elderly patients present an affected TSG, mainly TP53, as well as mutational signature 3; however, a few tumors only present an affected oncogene or no affected cancer-causing genes.

Analysis of RPL37A, MTSS1, and HTRA1 expression as potential markers for pathologic complete response and survival.

BACKGROUND: Non-metastatic locally advanced breast carcinoma (LABC) treatment involves neoadjuvant chemotherapy (NCT). We evaluated the association of clinical-pathological data and immunoexpression of hormone receptors, HER2 and Ki67, and new biomarkers, RPL37A, MTSS1 and HTRA1, with pathological complete response (PCR) or tumour resistance (stable disease or disease progression), disease-free survival (DFS) and cancer-specific survival (CSS). METHODS: This is a retrospective study of 333 patients with LABC who underwent NCT. Expression of MTSS1, RPL37A and HTRA1/PRSS11 was evaluated by immunohistochemistry in TMA slides. Cutoff values were established for low and high tumour expression. ROC plotter evaluated response to NCT. Chi-square test for factors related to PCR, and Kaplan-Meier test and Cox model for factors related to DFS and CSS were prformed. RESULTS: The mean follow-up was 70.0 months and PCR rate was 15.6%. At 120 months, DFS rate was 32.5% and CSS rate was 67.1%. In multivariate analysis, there was an association between: (1) necrosis presence, intense inflammatory infiltrate, ER absence, HER2 molecular subtype and high RPL3A expression with increased odds of PCR; (2) lymph node involvement (LNI), high Ki67, low RPL37A and high HTRA1 expression with increased risk for NCT non-response; (3) LNI, high proliferation, necrosis absence, low RPL37A and high HTRA1 expression with increased recurrence risk; (4) advanced LNI, ER negative tumours, high HTRA1, low RPL37A expression and desmoplasia presence with higher risk of cancer death. CONCLUSION: RPL37A is a potential biomarker for response to NCT and for prognosis. Additional studies evaluating HTRA1 and MTSS1 prognostic value are needed.

Survival analysis of young adults from a Brazilian cohort of non-small cell lung cancer patients.

BACKGROUND: The influence of age at diagnosis in non-small cell lung cancer (NSCLC) prognosis is unclear. OBJECTIVES: To compare in a Brazilian cohort of NSCLC patients of different age groups: 1) The overall survival; 2) Clinical features and treatment options. METHODS: This is a retrospective cohort study using a hospital-based registry, for NSCLC patients registered in years 2000-2009. Patients were grouped into three age groups: Young adults (YA: < 40 years), middle-aged (MA: 40-64 years) and elderly (E: ≥ 65 years). Kaplan-Meier was used to estimate overall survival and Cox regression for hazard ratios (HRs) and 95% confidence intervals. RESULTS: 17,422 NSCLC patients were included: 370 YA (2.1%), 8,697 MA (49.9%) and 8,355 E (48.0%). Compared with older age groups, the YA group had a higher proportion of females, patients diagnosed with adenocarcinoma and metastatic disease (63.2%). Overall survival was longer in YA in the entire cohort and in all clinical stages (CSs) (p < 0.001). For YA, higher education level was a good prognosis factor (compared with illiterate and incomplete elementary); advanced or metastatic disease (compared with early-stage disease) and treatment based in radiotherapy or chemotherapy (CT) (without surgery), compared with treatment combinations with surgery, were poor prognostic factors. Young men (but not women) had lower HR of death compared with older groups; YA had lower HR of death in all CSs compared with patients from older groups. A higher percentage of YA were treated with surgery or CT in early-stage disease compared with older groups. Besides that, YA and MA patients treated with surgery or CT had a better prognosis than elderlies. Conclusions: In this Brazilian cohort of NSCLC patients, most young individuals were diagnosed with metastatic disease. YA presented longer survival than older age groups in all CSs, but mainly in CS I/II and III, where some patients may achieve long remissions or cure.

Germline and Somatic mutations in postmenopausal breast cancer patients.

OBJECTIVES: In breast cancer (BC) patients, the frequency of germline BRCA mutations (gBRCA) may vary according to the ethnic background, age, and family history of cancer. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) is the second most common somatic mutated gene in BC; however, the association of mutations in both genes with cancer has not been thoroughly investigated. Thus, our aims were to investigate gBRCA mutation frequency in a cohort of postmenopausal Brazilian BC patients and the association of gBRCA1/BRCA2 and PIK3CA somatic mutations. METHODS: Forty-nine postmenopausal (>55 years) and forty-one young (≤35 years) BC patients were included in this study. The postmenopausal group included patients who reported a positive family history of cancer. For these patients, gBRCA1/BRCA2 were sequenced using next-generation sequencing (NGS) or Sanger sequencing. Data for gBRCA in young patients were already available from a previous study. DNA from formalin-fixed, paraffin-embedded (FFPE) tumors was obtained from 27 postmenopausal and 41 young patients for analyzing exons 9 and 20 of PIK3CA. The association between gBRCA1/BRCA2 and somatic mutations in PIK3CA was investigated. RESULTS: The overall frequency of gBRCA1/BRCA2 among the 49 postmenopausal patients was 10.2%. The frequencies of somatic mutations in PIK3CA in the postmenopausal and young patients were 37% and 17%, respectively (ns). The most common PIK3CA mutation was found to be E454A. Nonsense and frameshift mutations, which may counteract the oncogenic potential of PIK3CA were also detected. Regardless of age, 25% of BRCA1/BRCA2 mutation carriers and non-carriers , each, had PIK3CA somatic mutations. CONCLUSIONS: Data obtained indicate that BRCA1/BRCA2 gene testing may be considered for postmenopausal patients with BC who have a family history of cancer. Although some of them are not considered pathogenic, somatic variants of PIK3CA are frequently observed in BC patients, especially in postmenopausal patients.

Reciprocal changes in gene expression profiles of cocultured breast epithelial cells and primary fibroblasts.

The importance of epithelial-stroma interaction in normal breast development and tumor progression has been recognized. To identify genes that were regulated by these reciprocal interactions, we cocultured a nonmalignant (MCF10A) and a breast cancer derived (MDA-MB231) basal cell lines, with fibroblasts isolated from breast benign-disease adjacent tissues (NAF) or with carcinoma-associated fibroblasts (CAF), in a transwell system. Gene expression profiles of each coculture pair were compared with the correspondent monocultures, using a customized microarray. Contrariwise to large alterations in epithelial cells genomic profiles, fibroblasts were less affected. In MDA-MB231 highly represented genes downregulated by CAF derived factors coded for proteins important for the specificity of vectorial transport between ER and golgi, possibly affecting cell polarity whereas the response of MCF10A comprised an induction of genes coding for stress responsive proteins, representing a prosurvival effect. While NAF downregulated genes encoding proteins associated to glycolipid and fatty acid biosynthesis in MDA-MB231, potentially affecting membrane biogenesis, in MCF10A, genes critical for growth control and adhesion were altered. NAFs responded to coculture with MDA-MB231 by a decrease in the expression of genes induced by TGFbeta1 and associated to motility. However, there was little change in NAFs gene expression profile influenced by MCF10A. CAFs responded to the presence of both epithelial cells inducing genes implicated in cell proliferation. Our data indicate that interactions between breast fibroblasts and basal epithelial cells resulted in alterations in the genomic profiles of both cell types which may help to clarify some aspects of this heterotypic signaling.

Stromal Cell Signature Associated with Response to Neoadjuvant Chemotherapy in Locally Advanced Breast Cancer.

Breast cancer stromal compartment, may influence responsiveness to chemotherapy. Our aim was to detect a stromal cell signature (using a direct approach of microdissected stromal cells) associated with response to neoadjuvant chemotherapy (neoCT) in locally advanced breast cancer (LABC). The tumor samples were collected from 44 patients with LABC (29 estrogen receptor (ER) positive and 15 ER negative) before the start of any treatment. Neoadjuvant chemotherapy consisted of doxorubicin and cyclophosphamide, followed by paclitaxel. Response was defined as downstaging to maximum ypT1a-b/ypN0. The stromal cells, mainly composed of fibroblast and immune cells, were microdissected from fresh frozen tumor samples and gene expression profile was determined using Agilent SurePrint G3 Human Gene Expression microarrays. Expression levels were compared using MeV (MultiExperiment Viewer) software, applying SAM (significance analysis of microarrays). To classify samples according to tumor response, the order of median based on confidence statements (MedOr) was used, and to identify gene sets correlated with the phenotype downstaging, gene set enrichment analysis (GSEA). Nine patients presented disease downstaging. Eleven sequences (FDR 17) were differentially expressed, all of which (except H2AFJ) more expressed in responsive tumors, including PTCHD1 and genes involved in abnormal cytotoxic T cell physiology, TOX, LY75, and SH2D1A. The following four pairs of markers could correctly classify all tumor samples according to response: PTCHD1/PDXDC2P, LOC100506731/NEURL4, SH2D1A/ENST00000478672, and TOX/H2AFJ. Gene sets correlated with tumor downstaging (FDR < 0.01) were mainly involved in immune response or lymphocyte activation, including CD47, LCK, NCK1, CD24, CD3E, ZAP70, FOXP3, and CD74, among others. In locally advanced breast cancer, stromal cells may present specific features of immune response that may be associated with chemotherapy response.

Clinical stage and histological type of the most common carcinomas diagnosed in young adults in a reference cancer hospital.

OBJECTIVES: Cancer in young adults represents a great challenge, both biologically and socially, and understanding the unique characteristics of neoplasms in this age group is important to improving care. We aimed to evaluate the most common carcinomas and their characteristics, such as histological type and clinical stage, in young adults in the largest cancer hospital in Latin America. METHODS: The hospital registry was consulted for the period between 2008 and 2014. Young adults were defined as individuals aged 18 to 39 years, and older adults were defined as individuals aged 40 years and older. Differences between age groups were assessed through chi-square tests. RESULTS: Of the 39,389 patients included, 3,821 (9.7%) were young adults. Among the young adults, the most frequent cancer types were the following: breast, lymph node, colorectal, thyroid, testicle, hematopoietic and reticuloendothelial, uterine cervix, brain, soft tissue and stomach; these sites accounted for 74.5% of the observed tumors. Breast, colorectal and stomach cancers were more frequently diagnosed at advanced stages in young adults than in older adults (p<0.001). The most common histological types were infiltrating ductal carcinoma (86.12%) for breast cancer, adenocarcinomas not otherwise specified (45.35%) for colorectal cancer, squamous cell carcinoma not otherwise specified (65.26%) for uterine cervix cancer, signet ring cell adenocarcinomas (49.32%) for stomach cancer and adenocarcinomas not otherwise specified (50.79%) for lung cancer. CONCLUSION: Young adults are diagnosed with cancer at more advanced stages, indicating that health professionals should be aware of cancer incidence in this age group. It is necessary to develop a better understanding of cancer in young adults and to implement dedicated health care strategies for these patients.

Survival and prognosis of young adults with gastric cancer.

OBJECTIVES: Survival data for young adults (YA) with gastric cancer is conflicting and scarce in Brazil. The aim of this study was to compare the clinicopathological factors and survival rates of younger and older patients with gastric cancer. METHODS: Hospital registries for 294 gastric cancer patients from a reference cancer hospital in São Paulo, Brazil, were consulted for the retrieval of clinicopathological information and follow-up time. Patients were placed into the following groups: YA (≤40 years; N=71), older adult (OA: 41 to 65 years; N=129) and elderly (E: ≥66 years; N=94). Differences were assessed through Pearson's χ2 test, Kaplan-Meier analysis, Log rank test and Cox regression. RESULTS: More YA were diagnosed with advanced disease (clinical stage III/IV: 86.7% YA, 69.9% OA, and 67% E); however, fewer E patients underwent surgery (64.3% YA, 72.7% OA, and 52.4% E). The median overall survival among all patients was 16 months, and the overall survival rate was not significantly different among the age groups (p=0.129). There were no significant differences in the disease-free survival rate. Metastatic disease at diagnosis (HR=4.84; p<0.01) was associated with an increased hazard of death for YA. CONCLUSION: Overall survival was similar among age groups. Metastatic disease at diagnosis was the only factor associated with a poorer prognosis in YA. These results suggest that younger patients deserve special attention regarding the detection of early stage disease.

Cost effectiveness of the cancer prevention program for carriers of the BRCA1/2 mutation.

OBJECTIVE: To analyze the cost effectiveness of the diagnostic program for the germline mutation in BRCA1/2 genes and of preventative strategies for the relatives of patients diagnosed with ovarian cancer associated with this mutation. METHODS: The study analyzed the cost effectiveness by developing an analysis of the Markov decision process from the perspective of the National Health System. The strategies compared reflect upon the adoption of genetic testing and preventative strategies for relatives or the usual care currently proposed. The incremental cost-effectiveness ratio was expressed in terms of cost per case avoided. The sensitivity analysis was performed in a univariate and deterministic manner. RESULTS: The study showed increments for effectiveness and for costs when performing genetic testing and adopting prophylactic measures for family members. The incremental cost-effectiveness ratio was estimated at R$908.58 per case of cancer avoided, a figure considered lower than the study's cost-effectiveness threshold (R$7,543.50). CONCLUSIONS: The program analyzed should be considered a cost-effective strategy for the national situation. Studies in various other countries have reached similar conclusions. One possible ramification of this research might the need to perform a budgetary-impact analysis of making the program one of the country's health policies.

Breast-conserving surgery in locally advanced breast cancer submitted to neoadjuvant chemotherapy. Safety and effectiveness based on ipsilateral breast tumor recurrence and long-term follow-up.

OBJECTIVE:: To evaluate ipsilateral breast tumor recurrence after breast-conserving surgery for locally advanced breast cancer. METHODS:: A retrospective observational cohort study was performed in patients with locally advanced breast cancer submitted to breast-conserving surgery after neoadjuvant chemotherapy based on an adriamycin-cyclophosphamide-paclitaxel regimen. We evaluated the clinical, pathologic, immunohistochemistry, and surgical factors that contribute to ipsilateral breast tumor recurrence and locoregional recurrence. A Kaplan-Meier analysis and Cox model were used to evaluate the main factors related to disease-free survival. RESULTS:: Of the 449 patients who received neoadjuvant chemotherapy, 98 underwent breast-conserving surgery. The average diameter of the tumors was 5.3 cm, and 87.2% reached a size of up to 3 cm. Moreover, 86.7% were classified as clinical stage III, 74.5% had T3-T4 tumors, 80.5% had N1-N2 axilla, and 89.8% had invasive ductal carcinoma. A pathologic complete response was observed in 27.6% of the tumors, and 100.0% of samples had free margins. The 5-year actuarial overall survival rate was 81.2%, and the mean follow-up was 72.8 months. The rates of ipsilateral breast tumor recurrence and locoregional recurrence were 11.2% and 15.3%, respectively. Multifocal morphology response was the only factor related to ipsilateral breast tumor recurrence disease-free survival (p=0.04). A multivariate analysis showed that the pathologic response evaluation criteria in solid tumors (RECIST)-breast cutoff was the only factor related to locoregional recurrence disease-free survival (p=0.01). CONCLUSIONS:: Breast-conserving surgery is a safe and effective therapy for selected locally advanced breast tumors.

25(OH)D3 and 1,25(OH)2D3 serum concentration and breast tissue expression of 1alpha-hydroxylase, 24-hydroxylase and Vitamin D receptor in women with and without breast cancer.

1,25(OH)2D3 is an antiproliferative agent that may inhibit proliferation of breast cancer (BC) cells in vitro and BC development in animals. Epidemiological studies have shown a high incidence of BC in people less exposed to solar rays. To unravel the role of Vitamin D3 in BC patients, we have investigated serum levels of 25(OH)D3 and its active form 1,25(OH)2D3 as well as tissue expression of 1alpha-hydroxylase, 24-hydroxylase, and Vitamin D-receptor (VDR), determined by semiquantitative RT-PCR, in 88 Brazilian BC patients and 35 women without cancer (submitted to mammoplasties or resection of benign lesions). Median age of women with and without cancer was 51 and 46 years, respectively, and the majority of BC patients were classified as clinical stage II (67%). Although no differences in 25(OH)D3 serum concentration were found, 1,25(OH)2D3 (40+/-21 pg/ml) levels in BC patients were lower than in women without cancer (53+/-23). Our results indicate that 24-hydroxylase, VDR and 1alpha-hydroxylase mRNA tissue expression is similar in both groups and no correlation between 24-hydroxylase, 1alpha-hydroxylase, and VDR expression in breast tumors was found. A low 1,25(OH)2D3 serum concentration seems to be associated to breast cancer, however, the mechanism involved in this regulation is still unclear.

Gene expression profile associated with response to doxorubicin-based therapy in breast cancer.

PURPOSE: This study was designed to identify genes that could predict response to doxorubicin-based primary chemotherapy in breast cancer patients. EXPERIMENTAL DESIGN: Biopsy samples were obtained before primary treatment with doxorubicin and cyclophosphamide. RNA was extracted and amplified and gene expression was analyzed using cDNA microarrays. RESULTS: Response to chemotherapy was evaluated in 51 patients, and based on Response Evaluation Criteria in Solid Tumors guidelines, 42 patients, who presented at least a partial response (> or =30% reduction in tumor dimension), were classified as responsive. Gene profile of samples, divided into training set (n = 38) and independent validation set (n = 13), were at first analyzed against a cDNA microarray platform containing 692 genes. Unsupervised clustering could not separate responders from nonresponders. A classifier was identified comprising EMILIN1, FAM14B, and PBEF, which however could not correctly classify samples included in the validation set. Our next step was to analyze gene profile in a more comprehensive cDNA microarray platform, containing 4,608 open reading frame expressed sequence tags. Seven samples of the initial training set (all responder patients) could not be analyzed. Unsupervised clustering could correctly group all the resistant samples as well as at least 85% of the sensitive samples. Additionally, a classifier, including PRSS11, MTSS1, and CLPTM1, could correctly distinguish 95.4% of the 44 samples analyzed, with only two misclassifications, one sensitive sample and one resistant tumor. The robustness of this classifier is 2.5 greater than the first one. CONCLUSION: A trio of genes might potentially distinguish doxorubicin-responsive from nonresponsive tumors, but further validation by a larger number of samples is still needed.