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ObjectiveTo better understand the immune microenvironment of pancreatic ductal adenocarcinomas (PDACs), here we explored the relevance of T and B cell compartmentalisation into tertiary lymphoid structures (TLSs) for the generation of local antitumour immunity.DesignWe characterised the functional states and spatial organisation of PDAC-infiltrating T and B cells using single-cell RNA sequencing (scRNA-seq), flow cytometry, multicolour immunofluorescence, gene expression profiling of microdissected TLSs, as well as in vitro assays. In addition, we performed a pan-cancer analysis of tumour-infiltrating T cells using scRNA-seq and sc T cell receptor sequencing datasets from eight cancer types. To evaluate the clinical relevance of our findings, we used PDAC bulk RNA-seq data from The Cancer Genome Atlas and the PRINCE chemoimmunotherapy trial.ResultsWe found that a subset of PDACs harbours fully developed TLSs where B cells proliferate and differentiate into plasma cells. These mature TLSs also support T cell activity and are enriched with tumour-reactive T cells. Importantly, we showed that chronically activated, tumour-reactive T cells exposed to fibroblast-derived TGF-β may act as TLS organisers by producing the B cell chemoattractant CXCL13. Identification of highly similar subsets of clonally expandedCXCL13+tumour-infiltrating T cells across multiple cancer types further indicated a conserved link between tumour-antigen recognition and the allocation of B cells within sheltered hubs in the tumour microenvironment. Finally, we showed that the expression of a gene signature reflecting mature TLSs was enriched in pretreatment biopsies from PDAC patients with longer survival after receiving different chemoimmunotherapy regimens.ConclusionWe provided a framework for understanding the biological role of PDAC-associated TLSs and revealed their potential to guide the selection of patients for future immunotherapy trials.

APOBEC3A and APOBEC3B cytidine deaminases have been implicated in the pathogenesis of multiple cancers, including breast cancer (BC). A germline deletion linking APOBEC3A and APOBEC3B loci (A3A/B) has been associated with higher APOBEC-mediated mutational burden, but its association with BC risk have been controversial. Therefore, this study investigated the association between A3A/B and BC susceptibility and clinical presentation in a Brazilian cohort. A3A/B deletion was evaluated through allele-specific PCR in 341 BC patients and 397 women without familial or personal history of neoplasia from Brazil and associations with susceptibility to BC subtypes were tested through age-adjusted logistic models while correlations with clinicopathological parameters were tested using Kendall’s tests. No association was found between A3A/B and BC susceptibility; however, in Luminal-A BCs, it was positively correlated with tumor size (Tau-c = 0.125) and Ki67 (Tau-c = 0.116) and negatively correlated with lymph node metastasis (LNM) (Tau-c = − 0.162). The negative association between A3A/B with LNM in Luminal-A BCs remained significant even after adjusting for tumor size and Ki67 in logistic models (OR = 0.22; p = 0.008). These results show that although A3A/B may not modify BC susceptibility in Brazilian population, it may affect clinicopathological features in BC subtypes, promoting tumor cell proliferation while being negatively associated with LNM in Luminal-A BCs.

Background: The high infectivity rates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the prolonged duration of coronavirus disease 2019 (COVID-19) pandemics have contributed to the emergence of viral variants endowed with evolutionary advantages, leading to enhanced infectivity. The tracking of these lineages is urgent. However, the need to sequence whole-viral genomes through next-generation sequencing (NGS) represents a barrier hampering the massive identification of these variants. Therefore, in the current study, we developed Sanger-sequencing approaches targeting regions of interest containing vast lineage-defining mutations in the SARS-CoV-2 S gene and ORF8 region, allowing for unambiguous identification of all SARS-CoV-2 variants of concern (VOCs) and of interest (VOIs).Methods and results: Primers were designed for polymerase chain reaction (PCR) and nested-PCR to amplify and sequence samples with a low-viral burden. The primers’ annealing sites conservancy were checked in a large group of sequences. Amplification protocols were standardized, and sequencing reactions were performed in a cohort of samples for validation. The primers were highly efficient and sequencing of the targeted regions matched those generated by NGS in the same samples. The sequencing results allowed for the unambiguous identification of B.1.1.7, P.1 and P.2 samples, and would also allow the identification of B.1.617.2, B.1.351 and B.1.427/B.1.429 lineages, which were absent in our cohort.Conclusion: Implementing Sanger-sequencing-based approaches to identify SARS-CoV-2 lineages may represent an alternative to tracking these variants by more laboratories around the world and providing valuable molecular and epidemiologic information to inform health systems.

Background TGFβ signaling exerts context-specific effects in breast cancer (BC) pathogenesis and single nucleotide polymorphisms (SNPs) in TGFβ-signaling components play a role in the genetic control of their expression and in BC susceptibility and clinical presentation. However, studies investigating the association between the TGFβ-signaling molecules and BC prognosis rarely considered disease subtypes and SNPs. Therefore, the present study aimed to evaluate the expression of TGFβ-signaling components in BC tissue from patients with available data regarding TGFB1 and TGFBR2 SNPs and plasmatic TGFβ1 levels. Methods Immunostaining for TGFβ1, TGFβRII and phosphorylated (p)-SMAD2/3 was investigated in primary tumor tissue from 34 patients with luminal-B-HER2+ (LB-HER2), HER2-enriched (HER2) and triple negative (TN) BC subtypes genotyped for TGFB1 (rs1800468, rs1800469, rs1800470 and rs1800471) and TGFBR2 (rs3087465) SNPs. Results Strong positive correlations were observed between TGFβ1, TGFβRII and p-SMAD2/3 in tumor tissue, and an inverse correlation was observed between intratumor and plasmatic TGFβ1 levels in TN BCs. In LB-HER2+ tumors, p-SMAD2/3 was associated with older age at diagnosis and inversely correlated with p53 staining and lymph-node metastasis, while tumor-size negatively correlated with TGFβ1 and TGFβRII in this BC subgroup. Also, in p53-negative BCs, tumor size and Ki67 negatively correlated with both TGFβ1, TGFβRII and p-SMAD2/3. No correlation was found between SNPs and TGFβ1-signaling components expression. Conclusion TGFβ1 canonical signaling is activated in approximately half of BCs, and correlation between TGFβ components indicate a paracrine activation, which may exert tumor suppressor effects in p53-negative or Luminal-B-HER2+ subgroups.

Made available in DSpace on 2022-04-29T08:32:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-09-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Background: Obesity is considered a chronic inflammatory disease and transforming growth factor beta 1 (TGFβ1) might exert important roles in disease pathogenesis regulating adipocyte differentiation and immune-inflammatory environment. However, the role of this cytokine as a biomarker in obesity is poorly addressed. Therefore, the present study aimed to evaluate the impact of TGFB1 polymorphisms and TGFβ1 plasmatic levels in obesity Methods and results: TGFB1 promoter region polymorphisms (rs1800468, G-800A and rs1800469, C-509 T) were evaluated in 75 obese patients and 45 eutrophic patients through PCR–RFLP and plasmatic TGFβ1 was quantified through ELISA from 37 of the obese patients, and correlations with clinical and biochemical parameters were tested. Despite no association was found between TGFB1 polymorphisms and obesity susceptibility, several correlations with clinical data were noted. Among others, AC haplotype negatively correlated with plasmatic TGFβ1, while plasmatic TGFβ1 negatively correlated with C-reactive protein and positively correlated with liver abnormalities on ultrasound and, specifically, with steatosis presence and degree. Conversely, GT haplotype, which associates with higher TGFβ1 production, was also positively correlated with the same parameters of liver abnormalities. Further, plasmatic vitamin D negatively correlated with TGFβ1, while positively correlated with AC haplotype. Conclusion: Overall, the results indicate that TGFβ1 might exert important roles in obesity pathophysiology and correlate with biochemical and clinical parameters both at systemic protein as well as at genetic level. Importantly, the consistent positive correlation at both levels with steatosis might suggest this cytokine as a biomarker for this hepatic abnormality in obese patients. Department of General Biology Biological Sciences Center Londrina State University (UEL) Department of Pathological Sciences Biological Sciences Center Londrina State University (UEL) Clinical Surgery Department Federal University of Parana (UFPR) School of Medicine Department of Pathology São Paulo State University (UNESP) Laboratory of DNA Polymorphisms and Immunology Department of Pathological Sciences Biological Sciences Center State University of Londrina, PR445, Km 380 Celso Garcia Cid highway School of Medicine Department of Pathology São Paulo State University (UNESP) CNPq: 152170/2019-7 CNPq: 306386/2017-8

Transforming growth factor beta (TGFβ) has paradoxical effects in breast cancer (BC), inhibiting initial tumors while promoting aggressive ones. A polymorphism on TGFBR2 promoter region (G-875A, rs3087465) increases TGFβ type II receptor expression and is protective against cancer. Previously, we have shown that TGFB1 variants have subtype-specific roles in BC. This work sought to investigate the association between TGFBR2 and susceptibility and clinicopathological features in BC subgroups. TGFBR2 G-875A was analyzed through PCR-RFLP in 388 BC patients and 405 neoplasia-free women. Case–control analyses as well as interaction with TGFB1 haplotypes previously associated with BC were tested through age-adjusted logistic regression. Correlations between G-875A and clinicopathological parameters were assessed through Kendall’s Tau-b test. All statistical tests were two-tailed (α = 0.05). TGFBR2 G-875A was protective against BC in additive, genotypic, and dominant models. In subgroup-stratified analyses, these effects were greater in hormonal receptor-positive and luminal-A tumors, but were not significant in other subgroups. Logistic models including TGFB1 variants showed that in luminal-A tumors, G-875A retained its significance while TGFB1 haplotype showed a trend towards significance; otherwise, in HER2+ tumors TGFB1 variants remained significant while TGFBR2 showed a trend for association. There was no interaction between these genes. In correlation analyses, G-875A positively correlated with histopathological grade in total sample, and a trend towards significance was observed in triple-negative BCs. These results indicate that G-875A is a protective factor against BC, especially from luminal-A subtype, but may promote anaplasia in established tumors, consistent with TGFβ signaling roles in BC.

Obesity is considered a chronic inflammatory disease and transforming growth factor beta 1 (TGFβ1) might exert important roles in disease pathogenesis regulating adipocyte differentiation and immune-inflammatory environment. However, the role of this cytokine as a biomarker in obesity is poorly addressed. Therefore, the present study aimed to evaluate the impact of TGFB1 polymorphisms and TGFβ1 plasmatic levels in obesity METHODS AND RESULTS: TGFB1 promoter region polymorphisms (rs1800468, G-800A and rs1800469, C-509 T) were evaluated in 75 obese patients and 45 eutrophic patients through PCR-RFLP and plasmatic TGFβ1 was quantified through ELISA from 37 of the obese patients, and correlations with clinical and biochemical parameters were tested. Despite no association was found between TGFB1 polymorphisms and obesity susceptibility, several correlations with clinical data were noted. Among others, AC haplotype negatively correlated with plasmatic TGFβ1, while plasmatic TGFβ1 negatively correlated with C-reactive protein and positively correlated with liver abnormalities on ultrasound and, specifically, with steatosis presence and degree. Conversely, GT haplotype, which associates with higher TGFβ1 production, was also positively correlated with the same parameters of liver abnormalities. Further, plasmatic vitamin D negatively correlated with TGFβ1, while positively correlated with AC haplotype.Overall, the results indicate that TGFβ1 might exert important roles in obesity pathophysiology and correlate with biochemical and clinical parameters both at systemic protein as well as at genetic level. Importantly, the consistent positive correlation at both levels with steatosis might suggest this cytokine as a biomarker for this hepatic abnormality in obese patients.

Background: Breast cancer (BC) is a complex disease in which susceptibility and clinical course depend on multiple factors. Evidence suggests that a mouse mammary tumor virus (MMTV)-homolog may be present in human BCs; however, little is known about its clinical implications. Methods: MMTV-like env nucleotide-sequence was searched in tumor and tumor-adjacent tissues from 217 Brazilian BC patients through nested-PCR and confirmed through PCR-sequencing. Blood samples were also tested for patients with MMTV-like env gene-positive tumors. Correlations with clinicopathological parameters were evaluated. Results: MMTV-like env sequence was detected in tumor and tumor-adjacent tissue samples from 41/217 and 30/196 patients, respectively. In blood, MMTV-like was detected in 17/32 patients. In Luminal-B tumors, MMTV-like in tumor tissue was negatively correlated with tumor size and disease stage, whereas in HER2 tumors it anti-correlated with lymph node metastasis (LNM) and disease stage. Considering blood, MMTV-like env gene positivity negatively correlated with age in general BC, while in Luminal-A tumors it positively correlated with Ki67 but negatively correlated with age and LNM. The associations with decreased LNM frequency were independent of other prognostic factors. Conclusion: MMTV-like env positivity is associated with better prognostic parameters in BC subtypes, which might be explainable by its anti-metastatic potential and by putative activation of immune milieu.

O câncer de mama (CM) é a neoplasia mais frequente e maior causa de mortalidade por câncer em mulheres no mundo, e apresenta-se como uma doença complexa e heterogênea, composta por diversos subtipos moleculares de relevância clínica. Sua evolução depende de interações com o estroma, incluindo células imunológicas, e polimorfismos genéticos modulando o estroma do câncer de mama já foram associados à doença. O presente trabalho contou com um total de 388 pacientes com CM e 405 mulheres livres de neoplasia que foram avaliadas quanto a polimorfismos funcionais nos genes IL7RA, TGFB1, TGFBR2 e APOBEC3B. Além disso, os níveis plasmáticos da citocina TGFβ1 foram quantificados em 113 pacientes com CM livres de tratamento, 44 pacientes com CM tratadas e em 184 pacientes livres de neoplasia e a expressão de TGFβ1, TGFβRII e pSMAD2/3 no tecido tumoral foi avaliada por imunohistoquímica em 34 pacientes, todas genotipadas para os polimorfismos nos genes TGFB1 e TGFBR2. Os resultados demonstraram que o polimorfismo rs6897932 (Thr244Ile) de IL7RA foi associado à susceptibilidade a cânceres do subtipo triplo negativos, enquanto indicou menor estadiamento da doença nesse mesmo subgrupo; esse polimorfismo também foi associado a parâmetros de pior prognóstico em tumores HER2+ (maior proliferação celular, grau histopatológico e metástase em linfonodos). Com relação ao TGFB1, quatro variantes funcionais afetando a produção da citocina TGFβ1 foram avaliadas (rs1800468, rs1800469, rs1800470 e rs1800471) e suas estruturas haplotípicas inferidas; haplótipos associados a maior produção de TGFβ1 foram associadas a maior susceptibilidade a tumores do subtipo HER2+ e correlacionaram-se com parâmetros de pior prognóstico nesse mesmo subgrupo e em tumores triplo-negativos, mas indicaram menor proliferação celular em tumores luminal-A, enquanto variantes de baixa produção demonstraram o padrão oposto. Com relação ao polimorfismo rs3087465 (G-875A) do TGFBR2 os resultados demonstraram uma forte associação protetora contra o CM, especialmente do subtipo luminal-A, e uma correlação positiva com o grau histopatológico. O haplótipo ACTG de TGFB1 indicou menores níveis de TGFB1 quando comparado ao haplótipo de referência (GCTG) em controles saudáveis, mas não em pacientes com CM. Já a expressão de componentes da via de sinalização de TGFβ no tecido tumoral não foi associada aos polimorfismos analisados, mas se associou a parâmetros de melhor prognóstico em tumores Luminal-B e sem mutação em p53. Tais resultados são consistentes com os efeitos paradoxais descritos para a sinalização pelo TGFβ em CM atuando como supressor tumoral em tumores iniciais e pouco agressivos (como os luminais), mas induzindo a progressão de tumores mais agressivos (como os HER2+ e os triplo-negativos). Com relação à deleção de APOBEC3B não foi observada associação com a susceptibilidade a nenhum subtipo da doença, mas a deleção desse gene foi um fator protetor independente contra metástases em linfonodo no subtipo luminal-A. Assim, os resultados descritos indicam associações subtipo-específicas para diversos genes relacionados ao sistema imune com a susceptibilidade e apresentação clínica do CM, e revelam marcadores promissores nessa doença. Breast cancer (BC) is the most common cancer and the leading cause of cancer mortality in women worldwide, and shows up as a complex and heterogeneous disease, composed of several molecular subtypes of clinical relevance. Its evolution depends on interactions with tumor stroma, which includes immune cells, and genetic polymorphisms modulating breast cancer microenvironment were associated with this disease. The present thesis evaluated a total of 388 breast cancer patients and 405 cancer-free women for functional polymorphisms in the IL7RA, TGFB1, TGFBR2 and APOBEC3B genes, selected based on their relevance to breast cancer pathogenesis and on their potential to act both on neoplastic cells and modulate local immune microenvironment. In addition, TGFβ1 plasma levels were quantified in 113 treatment-free breast cancer patients, 44 patients with treated breast cancer, and 184 cancer-free patients, and the protein expression of TGFβ1, TGFβRII and pSMAD2/3 in tumor tissue was evaluated by immunohistochemistry in 34 patients, all genotyped for TGFB1 and TGFBR2 polymorphisms. Results have shown that IL7RA rs6897932 (Thr244Ile) polymorphism was associated with susceptibility to triple-negative BCs, while indicating lower disease staging in this same subgroup; this polymorphism was also associated with worse prognosis parameters in HER2+ tumors (higher cell proliferation, histopathological grade and presence of lymph node metastasis). Regarding TGFB1, four functional variants affecting TGFβ1 production were evaluated (rs1800468, rs1800469, rs1800470 and rs1800471) and their haplotypic structures were inferred; haplotypes associated with higher TGFβ1 production were associated with increased susceptibility to HER2+ cancers and correlated with worse prognosis parameters in this same subgroup and in triple-negative tumors, but indicated lower cell proliferation in luminal-A tumors; otherwise, low-production variants showed the opposite pattern. Regarding the TGFBR2 rs3087465 (G-875A) polymorphism, a strong protective association against breast cancer was found, especially for luminal-A subtype, and a positive correlation with histopathological grade was noted. The TGFB1 ACTG haplotype was associated with lower TGFβ1 levels when compared to the reference haplotype (GCTG) in healthy individuals, but not in BC patients. The expression of TGFβ1-signaling components in BC tissue otherwise was not associated with any polymorphism analyzed, but was associated with better prognostic parameters in Luminal-B and tumors without p53 mutations. These results are largely consistent with the paradoxical effects of TGFβ signaling in breast cancer, acting as a tumor suppressor in initial and mildly aggressive tumors (such as luminal BCs), but inducing progression of more aggressive cancers (such as HER2 + and triple-negative). Regarding APOBEC3B deletion, it was not associated with susceptibility to any breast cancer subtype. However, it was an independent protective factor against lymph node metastases in luminal-A subtype. Therefore, the results shown here point to subtype-specific associations between polymorphisms in immune-related genes and BC susceptibility and clinical presentation, and reveal promising markers for this disease.

Transforming growth factor beta 1 (TGFβ1) is a pleiotropic cytokine that acts in a context-dependent manner. In breast cancer (BC) this cytokine exerts subtype- and stage-specific roles, inhibiting poorly aggressive tumors while enhances the invasive potential of highly aggressive cancers. Single-nucleotide polymorphisms (SNPs) affecting TGFβ1 production largely reflect this pattern of association, but studies investigating systemic TGFβ1 levels in BC patients and their association with clinical features or SNPs produced conflicting conclusions. Therefore, the present work investigated plasmatic TGFβ1 levels through enzyme linked immunosorbent assay (ELISA) in 341 individuals previously genotyped for four TGFB1 SNPs [G-800A (rs1800468), C-509T (rs1800469), T29C (rs1800470) and G74C (rs1800471)], encompassing 184 neoplasia-free women with clinical information regarding health status, 113 treatment-free pre-surgery BC patients and 44 treated BC patients. Results have shown that TGFβ1 levels varied greatly in function of health status in neoplasia-free women, and disease-free individuals had higher TGFβ1 levels than both treatment-free or treated BC patients. There was no correlation between TGFβ1 with clinicopathological features in treatment-free BC general group, but it was negatively correlated with tumor size in luminal-B-HER2+ patients and with histopathological grade in triple-negative group. Also, TGFB1 ACTG haplotype (from G-800A to G74C) was associated with decreased TGFβ1 levels compared to the reference GCTG haplotype, and regression analyses showed that this association was independent of age, health status or BC diagnosis. In conclusion, several factors may influence TGFβ1 levels, and ACTG haplotype seems to be an important factor regulating TGFβ1 production.

Interleukin-7 (IL-7) exerts crucial functions on lymphoid cells’ development and maintenance. In breast cancer (BC), IL-7 promotes growth of tumor cells in culture through the activation of JAK1/3-STAT5 and PI3K/AKT pathways, and expression of IL-7 signaling components was associated with worst prognosis. A C>T polymorphism (rs6897932; Thr244Ile) at exon 6 of IL-7 receptor alpha (IL-7Rα) gene (IL7RA) shifts the balance between the membrane-bound and soluble IL-7Rα splicing variants and was previously associated with autoimmune diseases, but has not been studied in cancer, including BC, so far. Therefore, the present study aimed to investigate the possible association of this polymorphism with the susceptibility and clinicopathological parameters of BC subgroups. IL7RA Thr244Ile was genotyped through PCR-RFLP in 403 women without neoplasia, no personal history of malignancy or family history of BC and in 338 BC patients with clinicopathological data available. BC patients were stratified according to their positivity for estrogen (ER) and/or progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Age-adjusted logistic regression was performed for case-control analyses, and correlations with clinicopathological parameters were assessed through Kendall’s Tau-b coefficient. All analyses were two-tailed and had 95% confidence interval. In ER−PR−HER2− BCs, TT genotype was associated with increased susceptibility both in genotypic (TT vs. CC: OR = 3.07; CI = 1.01–9.38; p = 0.05) and recessive (TT vs. CC + CT: OR = 3.59; CI = 1.19–10.85; p = 0.02) models and negatively correlated with disease stage (Tau-b = −0.27; p = 0.05). Whereas T allele was positively correlated with histopathological grade (Tau-b = 0.29; p = 0.03) and lymph node metastasis (Tau-b = 0.35; p = 0.02) in ER/PR+HER2+BCs and with Ki67 (Tau-b = 0.51; p = 0.008) in ER−PR−HER2+ subgroup. These data indicate that IL-7Rα is involved in BC, and that IL7RA polymorphism may play distinct roles in breast carcinogenesis according to BC subtype, pointing this genetic variant as an interesting marker for breast carcinogenesis to be validated by further mechanistic and prospective studies with larger samples.

Despite the documented dual role of TGFβ1 in breast cancer (BC) pathogenesis, the subtype-specific influences of its polymorphisms remain undocumented. The present study investigated the effects of the TGFB1 promoter region (rs1800468 or G-800A and rs1800469 or C-509T) and signal peptide (rs1800470 or C29T and rs1800471 or G74C) single nucleotide polymorphisms (SNPs) and their haplotype structures on the susceptibility and clinicopathological presentation of BC subtypes. TGFB1 genotypes were assessed by PCR-RFLP and haplotype structures were inferred for 323 BC patients and 405 neoplasia-free women, and case–control analyses were performed by logistic regression adjusted by age. Clinicopathological parameters (age at diagnosis, tumor size, histopathological grade, lymph node metastasis, proliferation index and disease stage) were tested for correlation with TGFB1 variants. All statistical analyses were two-tailed with an alpha level of 0.05. Variants related to increased TGFβ1 production (C-509T SNP and GTCG haplotype) were associated with increased susceptibility to HER2+ tumors and correlated with worse prognostic parameters in HER2+ and triple-negative (TN) BCs, but correlated negatively to Ki67 in ER/PR+HER2− tumors. Conversely, low TGFβ1 production variants (C29T SNP and GCTG haplotype) were protective against HER2+ tumors and correlated negatively with prognostic parameters in HER2+ and TN BCs, while indicating higher proliferation rates in ER/PR+HER2− tumors. Furthermore, the GCCG haplotype was associated with decreased susceptibility to ER/PR+HER2− tumors, but correlated positively with Ki67 in this subgroup. The present study indicates that TGFB1 variants have subtype-specific roles in BC and may switch from tumor suppressor to promoter during tumor development, consistent with TGFβ1 dual role in BC pathogenesis.

Breast cancer represents a complex and heterogeneous disease that comprises distinct disease conditions, histological features, and clinical outcome. Since many years, it has been demonstrated as an association between HER2 amplification and poor prognosis, because its overexpression is associated with an aggressive phenotype of breast tumor cells. A significant proportion of cases have developed resistance to the current therapies available. Consequently, new prognostic markers are urgently needed to identify patients who are at the highest risk for developing metastases. During the past decade, new insights provided valuable knowledge regarding mechanisms underlying the dynamic interplayed between immune cells and tumor progression. It has been shown that the presence of a lymphocytic infiltrate, particularly of regulatory T cells, in cancer tissue, is associated with clinical outcome promoting rather than inhibiting cancer development and progression. It has been also verified that the clinical value of lymphocytic infiltration in breast cancers could be subtype-dependent, including the HER2-enriched subtype. In this context, this work summarizes proposed to discuss the prognostic value of regulatory T cell infiltration in microenvironment of HER2-enriched breast cancer.

Breast cancer (BC) is the main worldwide neoplasia in women. The metabolic balance between xenobiotic absorption and elimination rates plays an important role in preventing DNA damage and, consequently, tumor development. The glutathione S-transferases (GSTs), such as GSTM1 and GSTT1, and the NAD(P)H quinone oxidoreductase are important enzymes involved in phase II detoxification reactions. Deletions in GSTM1 and GSTT1, and single-nucleotide polymorphism (SNP) in NQO1 (rs1800655) have been investigated in cancer context, revealing conflicting results. The present study analyzed these genetic polymorphisms in 121 BC patients and 151 BC-free controls in order to verify if they could act as susceptibility modifiers and/or prognostic factors. Binary logistic regressions adjusted by age were performed to assess associations between allelic variants and interactions in polymorphisms combination with BC susceptibility, but no significant association was found. Genotypes distribution was also compared between BC subtypes, but no significant difference was observed (p > 0.05). GSTM1 deletion was significantly associated with histopathological grade, with a greater proportion of patients presenting grade III tumors (p = 0.007). Univariate analysis identified tumor size as the only clinicopathological parameter potentially associated with recurrence risk in patients that received adjuvant chemotherapy (p

Neuroblastoma (NB) is a heterogeneous and particularly malignant childhood neoplasm in its higher stages, prone to form metastasis in selected organs and for which there is still no efficient treatment available beyond surgery. Evidence indicates that chemokines and their receptors present involvement as mediators of neuroinflammation and have a neurophysiological role. In the present study, we aimed to verify if CCR5 (rs333) and CXCL12 (rs1801157) allelic variants were associated with NB. For CCR5 (rs333) D32 carriers (OR: 5.96, IC: 2.21-16.06) and for CXCL12 genotype 3'A/3'A (OR:26.18, IC:6.15-111.4) there were statistically significant differences as well to allelic frequency (OR:4.20, IC: 2.19-8.03). Although no correlation was verified regarding prognostic parameters for both CCR5 and CXCL12 polymorphic variants, these polymorphisms may be associated with NB susceptibility which deserve attention for future investigations.

Medulloblastoma (MB) is the most common malignant brain tumor occurring in children, and although high long-term survival rates have been reached with current therapeutic protocols, several neurological injuries are still observed among survivors. It has been shown that the development of MB is highly dependent on the microenvironment surrounding it and that the CXCL12 chemokine and its receptor, CXCR4 and the Sonic Hedgehog (SHH) pathway are crucial for cerebellar development, coordinating proliferation and migration of embryonic cells and malfunctions in these axes can lead to MB development. Indeed, the concomitant overactivation of these axes was suggested to define a new MB molecular subgroup. New molecules are being studied, aiming to inhibit either CXCR4 or the SHH pathways and have been tested in preclinical settings for the treatment of cancers. The use of these molecules could improve MB treatment and save patients from aggressive surgery, chemotherapy and radiotherapy regimens, which are responsible for severe neurological consequences. This review aims to summarize current data about the experimental inhibition of CXCR4 and SHH pathways in MB and its potential implications in treatment of this cancer.

Background The HER2 (human epidermal growth factor receptor-2) Ile655Val (rs1136201) genetic polymorphism can alter the receptor structure and its auto-activation, which can modify the signal transduction and, consequently, the cell cycle regulation. For this reason, this polymorphism has been extensively investigated as a candidate marker for breast cancer (BC). In this context, the aim of this study was to evaluate the possible influence of HER2 Ile655Val in BC susceptibility and prognostic factors in a Brazilian population. Methods Polymorphism genotype was assessed through RFLP-PCR in 107 BC patients with clinicopathological data available and in 150 women with no evidence of neoplasia and with no familial history of BC as control group. Association between this polymorphism and BC susceptibility and clinical parameters was evaluated through odds ratio (OR) and chi-squared or Fisher's exact test, respectively. Results A significant negative association between valine allele and BC susceptibility in dominant model was found (OR 0.5; 95% CI 0.27-0.93, P = .036). No significant association was found in relation to BC clinicopathological features (tumor size, lymph nodes commitment, histological grade, HER2 overexpression, hormonal receptors, p53, and Ki-67). Conclusion Although this polymorphism did not demonstrate potential as a prognostic marker, it may be a suitable susceptibility marker for BC.

Hematological malignancies (HM) are a group of neoplastic diseases that arise from hematologic cell lineages. Transforming growth factor beta 1 (TGFβ1) is shown to negatively regulate normal and malignant hematopoiesis and, in immunological context, to promote T cell exhaustion and generation of regulatory T cells, which are shown to be deleterious in cancer, by the induction of transcription factor FOXP3 expression. The present study aimed to evaluate TGFB1 exon-1 rs1800470 and FOXP3 intron-1 rs2232365 polymorphisms in relation to HM susceptibility. DNA was extracted from blood samples of 43 HM patients and 142 neoplasia-free individuals and polymorphisms were analyzed by allelic-specific PCR. Association analysis was assessed by the Odds Ratio (OR) with significance level of 5%. Regarding FOXP3 polymorphism, no significant differences were observed in genotype or allele distribution among the patients and controls. However, there was a positive association between TGFB1 TT genotype and HM susceptibility (OR = 4.07; CI95% = 1.94 - 8.52). In the combined analysis, a positive association was also observed for TGFB1 TT and FOXP3 GG genotypes (OR = 4.00; CI95% = 1.54 - 10.41) in relation to HM susceptibility. Our results indicated promising new markers to be further investigated in hematological malignancies.

CXCR4genetic polymorphisms, as well as their expression level, have been associated with cancer development and prognosis. The present study aimed to investigate the influence ofCXCR4rs2228014 polymorphism on its mRNA and protein expression in breast cancer samples. It was observed that patients presented higherCXCR4mRNA relative expression (5.7-fold) than normal mammary gland, but this expression was not correlated with patients clinicopathological features (nuclear grade, nodal status, ER status, PR status, p53 staining, Ki67 index, and HER-2 status). Moreover,CXCR4mRNA relative expression also did not differ regarding the presence or absence of T allele (p=0.301). In the immunohistochemical assay, no difference was observed for CXCR4 cytoplasmic protein staining in relation to different genotypes (p=0.757); however, high cytoplasmic CXCR4 staining was verified in invasive breast carcinoma (p<0.01). All in all, the results from present study indicated that rs2228014 genetic variant does not alterCXCR4mRNA or protein expression. However, this receptor was more expressed in tumor compared to normal tissue, in both RNA and protein levels, suggesting its promising applicability in the general context of mammary carcinogenesis.

A subgroup of tumor that has received attention is triple-negative breast cancer (TNBC), which presents phenotype of negative estrogen receptor, negative progesterone receptor and has no overexpression of HER2. TP53 acts as a tumor suppressor limiting the proliferation of damaged cells. A polymorphic site (rs1042522) of TP53 encodes either an arginine or a proline amino acid, but its biological significance remains unclear. This study aimed to investigate this variant and its expression in search for a possible involvement in TNBC susceptibility and clinical outcome. Genetic polymorphism was evaluated in 50 patients and 115 controls by PCR based methodology and immunohistochemistry was done with monoclonal antibody. Case-control study showed no positive or negative association (OR= 0.95; CI95%= 0.48-1.89). Comparison of genotypes and clinical outcome showed no significant results. Despite most of patients presented p53 positive staining by immunohistochemistry, there was no significant association in relation to prognostic parameters. Results demonstrated a lack of association between codon 72 polymorphism, susceptibility and prognosis of TNBC. Immunohistochemistry analysis should be done more carefully, since most of the patients had the somatic mutation of p53, which could be an indicator of prognostic value in TNBC.

Breast cancer (BC) is a complex and heterogeneous disease whose evolution depends on the tumor-host interaction. This type of cancer occurs when the mammary cells begin to grow wildly and become able to invade nearby tissues and/or promote metastases. Mouse mammary tumor virus (MMTV) is the accepted etiological agent of mammary tumors in mice. The identification of MMTV-like sequences and antigens in human mammary carcinoma has supported the theory that a virus homologous to MMTV (namely, HMTV) may be involved in human BC, but the role of retroviral elements in this disease remains elusive, as results from different research groups were contradictory. In the present review we present works for and against the involvement of HMTV in BC and discuss possible causes of divergences among studies. In the final section we fit current data regarding this issue to stablished causality criteria. We conclude that there is convincing data supporting the association of HMTV with BC, however there is still a need for epidemiological and basic research studies focusing on carcinogenic mechanisms for this virus in humans to fully understand its role in BC. This knowledge may open the way for the development of new preventive and therapeutic approaches in human BC.

FOXP3genetic polymorphisms have been associated with cancer development and prognosis. In this context, the present study aimed to evaluate the g.10403A>G (rs2232365) polymorphisms and g.8048A>C (rs3761548), in aggressive breast cancer (BC) subtypes, including, Luminal B HER2+ (LB), HER2-enriched (HER2+), and triple-negative (TN). Polymerase chain reaction followed by enzymatic restriction was performed to genotyping 117 BC samples and 300 controls. A significant association of AA genotype (g.10403A>G) in relation to BC susceptibility (OR = 1.93; 95% CI = 1.01–3.66;p=0.046) was observed. The GG (g.10403A>G) genotype was correlated with higher proliferation index (Ki-67) in HER2+ subtype (τ = 0.47;p=0.019) and advanced TNM staging in TN (τ = 0.23;p=0.032). A correlation of AA genotype (g.8048A>C) with higher Ki-67 (τ = −0.47;p=0.018) and lower histological grade (τ = 0.39;p=0.026) in HER2+ was also found. GA haplotype was correlated with lower histological grade (τ = −0.15;p=0.009) and higher Ki-67 (τ = 0.43;p=0.036) in HER2+ and advanced staging in TN (τ = 0.29;p=0.044). On the other hand, AC haplotype was correlated with lower Ki-67 (τ = −0.54;p=0.005) and staging (τ = −0.29;p=0.027) in HER2+ and TN respectively. Results showed thatFOXP3influence regarding clinical outcome depends greatly on the BC subtype and indicated this transcription factor as a promising marker in aggressive BC subtypes.

Acute lymphoblastic leukemia (ALL) is a malignant disorder that originates from one single hematopoietic precursor committed to B- or T-cell lineage. Ordinarily, these cells express CCR5 chemokine receptor, which directs the immune response to a cellular pattern and is involved in cancer pathobiology. The genetic rs333 polymorphism of CCR5 (Δ32), results in a diminished receptor expression, thus leading to impaired cell trafficking. The objective of the present study was to investigate the effect ofCCR5chemokine receptor rs333 polymorphism in the pathogenesis of ALL. The genotype distribution was studied in 79 patients and compared with 80 control subjects, in a childhood population of Southern Brazil. Genotyping was performed using DNA samples amplified by polymerase chain reaction with sequence-specific primers (PCR-SSP). The homozygous (Δ32/Δ32) deletion was not observed in any subject involved in the study. Heterozygous genotype was not associated with ALL risk (OR 0.7%; 95% CI 0.21–2.32;P>0.05), nor recurrence status of ALL (OR 0.86; 95% CI 0.13–5.48;P>0.05). This work demonstrated, for the first time, no significant differences in the frequency of the CCR5/Δ32 genotype between ALL and control groups, indicating no effect of this genetic variant on the ALL susceptibility and recurrence risk.

CXCL12/CXCR4 signaling has been implicated in breast carcinogenesis, and genetic polymorphisms in these molecules have been associated with different types of cancer. The present study analyzed genetic polymorphisms in CXCL12 (rs1801157, G > A) and CXCR4 (rs2228014, C > T) and CXCR4 immunostaining in tumor tissues from patients with triple negative breast cancer (TNBC) aiming to evaluate their possible role in its’ susceptibility and prognosis. Genetic polymorphisms were analyzed in 59 TNBC patients and 150 control women; age-adjusted logistic regression showed no association when variants were considered in isolation; however, a statistically significant interaction was noted for heterozygosis for both allelic variants increasing the odds for TNBC (CXCL12-GA by CXCR4-CT: OR 7.23; 95% CI 1.15–45.41; p = 0.035). CXCL12 polymorphism was correlated negatively with proliferation index (Ki67) (Tau-b = − 0.406; p = 0.006). CXCR4 immunostaining was evaluated in 37 TNBC patients (22 with paired tumor-normal adjacent tissue). CXCR4 was detected more intensely in cell cytoplasm than in membrane, and was more expressed in tumor than in normal adjacent tissues, although not statistically significant. CXCR4 expression on the membrane of tumor cells was correlated positively with histopathological grade (Tau-b = 0.271; p = 0.036) and negatively with lymph node metastasis (Tau-b = − 0.478; p = 0.036). The present study indicates that CXCL12 and CXCR4 polymorphisms and CXCR4 immunostaining might have susceptibility and prognostic roles in TNBC pathogenesis.

O câncer de mama, neoplasia mais frequente e com a maior taxa de mortalidade entre as mulheres no mundo todo, é uma doença heterogênea, composta por ao menos seis subtipos moleculares que diferem na apresentação clínica e no valor prognóstico e resposta terapêutica. O Fator de Crescimento Transformador β1 (TGFβ1) é uma citocina pleiotrópica, que desempenha funções célula- e contexto-específicas. No câncer de mama essa citocina inibe o crescimento de células pouco invasivas, porém estimula a proliferação e invasão de carcinomas mais agressivos. Apesar desse efeito paradoxal, pouco se conhece sobre os possíveis efeitos subtipo-específicos de polimorfismos do gene TGFB1 em relação à susceptibilidade e apresentação clínica de carcinomas mamários. Dessa forma, o presente estudo objetivou avaliar a influência dos polimorfismos rs1800468 (G-800A), rs1800469 (C-509T), rs1800470 (C29T) e rs1800471 (G74C) do TGFB1 e de suas estruturas haplotípicas na susceptibilidade e apresentação clínica de carcinomas mamários em análises estratificadas por subtipos tumorais. Os genótipos foram analisados por PCR-RFLP em 320 pacientes com câncer de mama e 405 mulheres livres de neoplasia. As análises de associação foram feitas por regressão logística binária controlada pela idade. O polimorfismo C-509T aumentou o risco para câncer de mama de forma dominante (OR=1,5; IC95%=1,09-2,04), especialmente para o subgrupo HER2+ (OR=2,16; IC95%=1,16-4,01); o polimorfismo G74C também foi um fator de risco para cânceres HER2+ recessivamente (OR=11,37; IC95%=1,65-78,2), enquanto o polimorfismo C29T foi protetor contra esses tumores no modelo recessivo (OR=0,39; IC95%=0,18-0,84). Reciprocamente, o haplótipo GTCG foi um fator de risco no modelo dominante para o câncer de mama geral (OR=1,41; IC95%=1,03-1,92), particularmente para cânceres HER2+ (OR=1,89; IC95%=1,07-3,35); o haplótipo GCTC também foi um fator de risco para tumores HER2+ (OR=17,9; IC95%=1,86-171,3), enquanto o haplótipo GCTG mostrou-se um fator protetor para esses tumores tanto no modelo recessivo (OR=0,35; IC95%=0,13-0,9) quanto no modelo dominante (OR=0,51; IC95%=0,29-0,9); o haplótipo GCCG foi um fator protetor de forma geral (OR=0,52; IC95%=0,29-0,94), mas especialmente os do subtipo luminal A (LA) (OR=0,49; IC95%=0,24-0,97). O polimorfismo C29T e o haplótipo GCTG correlacionaram-se negativamente com parâmetros clínicos de agressividade (tamanho tumoral, metástase em linfonodo e grau histopatológico) em cânceres HER2+ e triplo negativos (TN); no entanto, o haplótipo GCTG correlacionou-se positivamente com o índice de proliferação celular (Ki67) em tumores LA; por outro lado, o haplótipo GTCG indicou pior prognóstico em cânceres HER2+ e TN, mas correlacionou-se negativamente com o a marcação de Ki67 em tumores LA. Assim, o presente trabalho demonstra, pela primeira vez, que variantes alélicas no gene TGFB1 envolvidas na produção da respectiva citocina apresentam papéis subtipo-específicos na patogênese tumoral mamária, o que é consistente com o papel paradoxal desse marcador, inibindo tumores iniciais e pouco agressivos e promovendo aqueles de fenótipo mais agressivos. Breast cancer (BC), the most common cancer and the leading cause of cancer death in women worldwide, is a heterogeneous disease, composed of at least six molecular subtypes differing in clinical presentation and prognostication. Transforming Growth Factor β1 (TGFβ1) is a pleiotropic cytokine, exerting different functions in a cell- and context-specific manner. In BC this cytokine suppresses the growth of normal or poorly invasive cells, but stimulates proliferation and invasion in aggressive carcinomas. Despite this dual role, little is known about possible subtype-specific influences of TGFB1 polymorphisms on BC susceptibility and clinical outcome. Thus, the present study aimed to investigate the effect of the TGFB1 polymorphisms rs1800468 (G-800A); rs1800469 (C-509T); rs1800470 (C29T); rs1800471 (G74C) and their haplotype structures on the susceptibility and clinical presentation of BC in subtype-stratified analyses. TGFB1 genotypes were assessed by RFLP-PCR in 320 BC patients and 405 control women. Association analyses were performed by binary logistic regression controlled by age. C-509T dominantly increased the risk for BC (OR=1.5; CI95%=1.09-2.04), especially for HER2+ BCs (OR=2.16; CI95%=1.16-4.01); G74C also increased the risk for HER2+ BC recessively (OR=11.37; CI95%=1.65-78.2), while C29T recessively protected against these tumors (OR=0.39; CI95%=0.18-0.84). Conversely, GTCG haplotype dominantly increased the risk for BC (OR=1.41; CI95%=1.03-1.92), especially HER2+ BCs (OR=1.89; CI95%=1.07-3.35); GCTC haplotype also increased the risk for this subtype (OR=17.9; CI95%=1.86-171.3) while GCTG was protective for these tumors both recessively (OR=0.35; CI95%=0.13-0.9) and dominantly (OR=0.51; CI95%=0.29-0.9); GCCG haplotype was protective against BC (OR=0.52; CI95%=0.29-0.94), especially for luminal A (LA) subtype (OR=0.49; CI95%=0.24-0.97). C29T polymorphism and GCTG haplotype also correlated negatively with clinical parameters (tumor size, lymph node metastasis and histopathological grade) on HER2+ and triple negative breast cancers (TNBCs); however, GCTG haplotype positively correlated with proliferation index (Ki67) in LA tumors, while GTCG indicated worst prognosis in HER2+ and TNBC, but were negatively correlated with Ki67 in LA tumors. Thus, the present work indicate for the first time that TGFB1 variants involved in TGFβ1 production have subtype-specific roles in BC, consistent with TGFβ1 dual role in inhibiting initial and poorly aggressive tumors while promoting highly aggressive cancers.

Purpose: Chronic myeloid leukemia (CML) treatment has improved significantly in the last decade with the introduction of tyrosine kinase inhibitors (TKIs), which target BCR/ABL oncoprotein. However, a large proportion of patients develop resistance to this treatment protocol, with frequent relapse cases. CXCR4 up-regulation in leukemic cells induced by TKIs has been reported as a mechanism of chemoresistance by promoting migration of these cells to bone marrow, where they receive pro-survival signals and persist as quiescent cells. In the present study, we investigated the possible influence of treatment on the expression of CXCR4 and CXCL12 in peripheral blood cells of CML patients. Methods: Relative expressions (RE) were calculated from mRNA obtained from leukocytes of 21 patients in chronic phase, under treatment, and 54 healthy individuals, used as controls.Results: CXCR4 expression was increased in CML patients compared to controls (RE: 1.931; p = 0.006). In CML patients, CXCR4 and CXCL12 expressions were correlated (r = 0.631; p = 0.002), and no differences in the expression of these genes were observed among different treatment protocols. However, CXCR4 expression was positively correlated with imatinib treatment period duration of treatment (r = 0.56; p = 0.02). Conclusion: This data has pointed peripheral blood CXCR4 expression as a possible marker for treatment monitoring, which may be useful to predict which patients would be benefit from newly developed treatments targeting CXCR4 and BCR/ABL concomitantly.