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PDF file - 58K, Describes the types of infecting HPV among the 20 positive genotyped selected samples

The study of the tumor microenvironment (TME) in follicular lymphoma (FL) has produced conflicting results due to assessment of limited TME subpopulations, and because of heterogeneous treatments among different cohorts. Also, important genetic determinants of immune response, such as single-nucleotide polymorphisms (SNPs), remain underexplored in this disease. We performed a detailed study of the TME in 169 FL biopsies using immunohistochemistry, encompassing lymphocytes, macrophages, and cytokines. We also genotyped 16 SNPs within key immune-response genes (IL12A, IL2, IL10, TGFB1, TGFBR1, TGFBR2, IL17A, and IL17F) in 159 patients. We tested associations between SNPs, clinicopathological features and TME composition, and proposed survival models in R-CHOP/R-CVP-treated patients. Presence of the IL12A rs568408 “A” allele associated with the follicular pattern of FOXP3+ cells. The IL12A AA haplotype included rs583911 and rs568408 and was an independent predictor of worse survival, together with the follicular patterns of T-cells (FOXP3+ and CD8+) and high IL-17F tumor levels. The patterns of CD3+, CD4+ and CD8+ cells, displayed as a principal component, also associated with survival. Hierarchical clustering of the TME proteins demonstrated a cluster that was associated with worse prognosis (tumors enriched in IL-17A, IL-17F, CD8, PD1, and Ki-67). The survival of FL patients who were treated in the rituximab era shows a strong dependence on TME signals, especially the T-cell infiltration patterns and IL-17F tumor levels. The presence of the AA haplotype of IL12A in the genome of FL patients is an additional prognostic factor that may modulate the composition of T-reg cells in this disease.

Made available in DSpace on 2020-12-10T20:13:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Conselho Nacional Cientifico e Tecnologico Fundacao de Apoio a Pesquisa e Extensao de Sao Jose do Rio Preto Mammary neoplasms are a heterogeneous form of disease, and in order to determine its course and biological features with more accuracy, investigations based on tumor phenotypes are required. The aim of the present study was to propose and validate a phenotypic classification for canine mammary tumors and to assess any association between clinicopathological characteristics, survival and prognosis. For the immunohistochemistry analysis, the primary antibodies against estrogen receptor-alpha, progesterone receptor, human epidermal growth factor receptor 2 (HER-2)/neu and E-cadherin were used. A total of 110 canine mammary tumors were investigated; 42 tumors were classified as luminal A, 41 as luminal B, 17 as triple-negative and 10 as HER-2-positive. The luminal A and B phenotypes were associated with improved prognosis, whereas HER-2positive and triple-negative tumors were more aggressive, and exhibited a significant association with the occurrence of metastasis, a worse Tumor-Node-Metastasis classification and shorter survival time (P

The role and prognostic value of tetraspanins (TSPANs) in vulvar squamous cell carcinoma (VSCC) remain poorly understood. We sought to primarily determine, at both the molecular and tissue level, the expression profile of the TSPANs CD9, CD63, CD81, and CD82 in archived VSCC samples (n = 117) and further investigate their clinical relevance as prognostic markers. Our studies led us to identify CD63 as the most highly expressed TSPAN, at the gene and protein levels. Multicomparison studies also revealed that the expression of CD9 was associated with tumor size, whereas CD63 upregulation was associated with histological diagnosis and vascular invasion. Moreover, low expression of CD81 and CD82 was associated with worse prognosis. To determine the role of TSPANs in VSCC at the cellular level, we assessed the mRNA levels of CD63 and CD82 in established metastatic (SW962) and non-metastatic (SW954) VSCC human cell lines. CD82 was found to be downregulated in SW962 cells, thus supporting its metastasis suppressor role. However, CD63 was significantly upregulated in both cell lines. Silencing of CD63 by siRNA led to a significant decrease in proliferation of both SW954 and SW962. Furthermore, in SW962 particularly, CD63-siRNA also remarkably inhibited cell migration. Altogether, our data suggest that the differential expression of TSPANs represents an important feature for prognosis of VSCC patients and indicates that CD63 and CD82 are likely potential therapeutic targets in VSCC.

Background There is evidence to consider that the tumor microenvironment (TME) composition associates with antitumor immune response, and may predict the outcome of various non-Hodgkin lymphoma subtypes. However, in the case of mantle cell lymphoma (MCL), a rare and aggressive disease, there is lacking a detailed study of the TME components, as well as an integrative approach among them in patients’ samples. Also, from the genetic point of view, it is known that single nucleotide variants (SNVs) in immune-response genes are among important regulators of immunity. At present, it is uncertain whether SNVs in candidate immune-response genes and the TME composition are able to alter the prognosis in MCL. Methods We assessed a detailed TME composition in 88 MCL biopsies using immunohistochemistry, which was automatically analyzed by pixel counting (Aperio system). We also genotyped SNVs located in candidate immune-response genes (IL12A, IL2, IL10, TGFB1, TGFBR1, TGFBR2, IL17A, IL17F) in 95 MCL patients. We tested whether the SNVs could modulate the respective protein expression and TME composition in the tumor compartment. Finally, we proposed survival models in rituximab-treated patients, considering immunohistochemical and SNV models. Results High FOXP3/CD3 ratios (p = 0.001), high IL17A levels (p = 0.003) and low IL2 levels (p = 0.03) were individual immunohistochemical predictors of poorer survival. A principal component, comprising high quantities of macrophages and high Ki-67 index, also worsened outcome (p = 0.02). In the SNV model, the CC haplotype of IL10 (p IL2 rs2069762 (p = 0.02) and the AA+AG genotypes of TGFBR2 rs3087465 (p TGFB1 rs6957 associated with lower tumor TGFβ levels (p = 0.03) and less CD163+ macrophages (p = 0.01), but did not modulate patients’ survival. Conclusions Our results indicate that the TME composition has relevant biological roles in MCL. In this setting, immunohistochemical detection of T-reg cells, IL17A and IL2, coupled with SNV genotyping in IL10, TGFBR2 and IL2, may represent novel prognostic factors in this disease, following future validations.

Molecular phenotyping and tissue microarray (TMA) studies have identified distinct invasive breast carcinoma subtypes: Luminal A, luminal B, enriched with overexpressed human epidermal growth factor receptor 2 (HER-2) and triple-negative, i.e., negative for HER-2, as well as for estrogen and progesterone receptor (ER and PR, respectively) expression. These subtypes are useful in clinical management, since they bear distinct prognoses and predictive responses to targeted therapy. However, although molecular profiling provides important prognostic indicators, breast cancer risk stratification remains a challenge in triple-negative cases. What is referred to as claudin-low subtype was identified as a triple-negative subset that is associated with more aggressive tumor behavior and worse prognosis. However, the immunohistochemical expression of claudins has not yet been standardized. Our objective was to verify whether the immunoexpression of claudins 4 and 7 (the main claudins specifically expressed in human breast tissue) in TMA is associated with survival and prognosis in luminal A, HER-2 and triple-negative molecular subtypes. In this diagnostic study, we investigated ER/PR receptor status, HER-2, claudin 4 and 7 expression and stem cell CD44/24 profiles, and verified the association with prognosis and survival outcomes in 803 invasive breast carcinoma cases arranged in four TMAs. Among these, 503 (62.6%) were positive for claudin 4 and 369 (46.0%) for claudin 7. Claudin 4 exhibited the lowest expression in luminal A and triple-negative subtypes, and the highest frequency of expression in HER-2-enriched subtypes, whereas claudin 7 staining was not associated with any subtype. The stem cell phenotype was not associated with subgroups or claudins 4 and 7. Claudin immunoexpression profile was not able to distinguish between patients with better or worse prognosis, and it was not correlated to triple-negative cases. Therefore, it may be concluded that the immunoexpression of claudins 4 and 7, individually or within the usual immunohistochemical context (ER, PR and HER-2), does not provide additional prognostic information on breast cancer subtypes.

Anterior gradient 2 protein belongs to a family of chaperone‐like proteins, namely protein disulfide isomerase. Generally, AGR2 is highly expressed in mucus‐secreting cells and endocrine organs, and in this study, we aimed to evaluate AGR2 and cell cycle molecules in epithelial ovarian cancer and its implications on prognosis. One hundred seventy‐five patient's samples that were diagnosed with primary epithelial ovarian carcinoma were selected. All the patients were treated with platinum‐taxane standard chemotherapy after surgery and CA125 serum levels were routinely determined. Four‐micrometer‐thick sections were processed by immunohistochemistry using an automated immunostainer, Ventana BenchMark AutoStainer with AGR2, cyclin D1, p21WAF1, and p53. Forty‐nine of 167 cases (29.3%) showed strong to moderate cytoplasmic marking of AGR2, and 118 (70.7%) had weak to negative expression. The absence of the AGR2 protein was observed in high‐grade serous carcinoma (P

Molecular phenotyping has improved the understanding of a wide range of breast cancer disease. Recently, a new molecular subtype denominated “claudin-low” (CL) was described in breast cancer and correlated to worse prognosis and to CD44+/24- stem cell profile. Among 19 known claudin proteins, isotypes 4 (CL4) and 7 (CL7) are the most common in the breast biology. Objective: To verify differences in CL4 and CL7 immunoexpression between Luminal A, HER-2, and triple negative breast cancer phenotypes; and their association to CD44/24 status and tumor prognosis. Design: Estrogen and progesterone receptor status (ER/PR), HER-2, CL4 and CL7 expression and CD44/24 profiles were evaluated in 803 invasive ductal breast carcinomas arranged into four tissue microarrays (TMA) and results were correlated with prognosis and important clinical data. Results: 503 (62.6%) cases were positive for CL4 and 369 (46.0%) cases for CL7. The majority (199/283, 70.3%) of CL4 negative cases were clustered in the luminal A subtype whereas 63 (22.3%) showed triple negative profile and the remaining 21 cases (7.4%) exhibited positive HER-2 expression (p Citation Format: Logullo AF, Rocha RM, Stiepcich M, Soares FA, Pasini FS, Nonogaki S, Brentani MM. Prognostic relevance of claudins 4 and 7 in invasive breast carcinoma (NOS) subtypes: A large tissue microarray study [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-08.

Objective: To evaluate immunohistochemical erythropoietin (EPO) expression in clear cell renal cell carcinoma (ccRCC), its association with major clinicopathological variables and its prognostic impact. Methods: A total of 220 patients with renal cell carcinoma (RCC) surgically treated between 1989 and 2009 were evaluated in this multi-institutional study. All the cases were reviewed by a single pathologist and the immunohistochemical reactivity to EPO was analysed using tissue microarray. Results: A total of 176 patients with ccRCC were considered, with an average of 48 months of follow-up. Of the tumours evaluated, 47 (26.7%) were negative for EPO expression, and 129 (73.3%) were positive. EPO expression was associated with incidental tumour (p = 0.016), tumour size (p = 0.015), Karnofsky Performance Score (KPS) (p = 0.016), blood transfusion (p = 0.009) and adrenal involvement (p = 0.038). The median ages of the patients with positive and negative EPO expression were 56.2 years and 66.6 years. Immunohistochemical EPO expression affected overall survival (OS) and disease-specific survival (DSS) rates. The DSS rates of the patients whose tissue was positive and negative for EPO expression were 85.3% and 76.1%, respectively (p = 0.044). In a multivariate analysis, the absence of EPO expression proved to be a bad prognostic factor and negatively affected the OS (p

This study sought to understand the role of breast carcinoma-associated fibroblasts in the progression of cancer cells into lymph nodes. We compared fibroblasts of primary tumors and matched the involved lymph nodes to select fibroblast activation markers, namely α-smooth muscle actin (α-SMA), S100A4, and vimentin, as well as to determine the frequency of transforming growth factor β1, a pleiotropic cytokine that induces the differentiation of fibroblasts to myofibroblasts, and its downstream effectors: CXCR4 and p-AKT. We disposed samples of 80 primary invasive ductal carcinomas and matched the involved lymph nodes from 43 cases into 3 tissue microarrays, and analyzed stromal and tumor epithelial cells separately by immunohistochemistry. Control uninvolved lymph nodes were analyzed by whole-tissue sections. Cancer-associated fibroblast in lymph nodes with macrometastasis expressed similar profiles of vimentin, α-SMA, and S100A4 as those found in primary tumors. Cancer-associated fibroblast were uniformly estrogen receptor, progesterone receptor, HER-2, Ki-67, and p53 negative, but expressions of transforming growth factor β1 (TGFβ1), CXCR4, and p-AKT staining (62.3%, 52.4%, 65%, respectively) were equivalent between primary and lymph node metastasis (LNM) fibroblasts. A significant coexpression of TGFβ1 with p-AKT and CXCR4 in LNMs suggested the involvement of these proteins with TGFβ1 signaling. These biomarkers, including α-SMA and S100A4, were negative in fibroblasts of cancer-free lymph nodes, with the exception of vimentin. Our finding that expressions of biological markers were similar in fibroblasts of the primary tumors and in matched LNMs, but were absent in cancer-free lymph nodes, supports the assumption that the lymph node stroma mimics the microenvironment observed in primary tumors.

Background: Xeroderma pigmentosum (XP) patients present a high risk of developing skin cancer and other complications at an early age. This disease is characterized by mutations in the genes related to the DNA repair system. Objectives: To describe the clinical and molecular findings in a cohort of 32 Brazilian individuals who received a clinical diagnosis of XP. Methods: Twenty-seven families were screened for germline variants in eight XP-related genes. Results: All patients (N = 32) were diagnosed with bi-allelic germline pathogenic or potentially pathogenic variants, including nine variants previously undescribed. The c.2251-1G>C XPC pathogenic variant, reported as the founder mutation in Comorian and Pakistani patients, was observed in 15 cases in homozygous or compound heterozygous. Seven homozygous patients for POLH/XPV variants developed their symptoms by an average age of 7.7 years. ERCC2/XPD, DDB2/XPE and ERCC5/XPG variants were found in a few patients. Aside from melanoma and non-melanoma skin tumours, a set of patients developed skin sebaceous carcinoma, leiomyosarcoma, angiosarcoma, mucoepidermoid carcinoma, gastric adenocarcinoma and serous ovarian carcinoma. Conclusions: We reported a high frequency of XPC variants in 32 XP Brazilian patients. Nine new variants in XP-related genes, unexpected non-skin cancer lesions and an anticipation of the clinical manifestation in POLH/XPV cases were also described.

The objective of this case report was to describe histopathological and immunohistochemical characteristics of the first reported basaloid carcinomas in the canine mammary gland. Two bitches were treated for tumors in the mammary gland and underwent mastectomy. Microscopic evaluation of these tumors revealed epithelial cells arranged in a predominantly solid pattern with hyperchromatic peripheral cells arranged in a palisade pattern. Metastases in regional lymph nodes were found in both animals, and one bitch exhibited pulmonary metastasis. Immunohistochemistry revealed positive labeling for the basal cell markers cytokeratin 14 and p63. Histopathological and immunohistochemical findings led to diagnoses of basaloid carcinoma of the canine mammary gland with regional and distant metastasis. RESUMO O objetivo deste relato de caso é descrever as características histopatológicas e imuno-histoquímicas do primeiro relato de carcinoma basaloide na glândula mamária canina. Duas cadelas foram atendidas com tumores na glândula mamária e foram submetidas à mastectomia. A avaliação microscópica demonstrou células epiteliais arranjadas em um padrão predominantemente sólido, com células periféricas hipercromáticas, dispostas em paliçada. As duas apresentaram metástase em linfonodos regionais e uma delas metástase pulmonar. A imuno-histoquímica revelou marcação positiva para citoqueratina 14 e p63, marcadores de células basais. Achados histopatológicos e imuno-histoquímicos levaram ao diagnóstico de carcinoma basaloide da glândula mamária canina com metástase regional e a distância.

The E-cadherin loss has frequently been associated with transcriptional repression mediated by transcription factors, such as the Zinc Finger E-Box Binding Homeobox-2 (ZEB2). Invasive micropapillary carcinomas (IMPCs) of the breast are aggressive neoplasms frequently related to lymph node metastasis and poor overall survival. In the canine mammary gland, IMPCs has just been reported and, based on its behavioral similarity with the human IMPCs, appears to be a good spontaneous model to this human entity. This study aimed to evaluate the relationship between E-cadherin and ZEB2 in a spontaneous canine model of invasive micropapillary carcinoma of the mammary gland. The correlation among gene expression (ZEB2 and CDH1) and clinicopathological findings was also explored. Nineteen cases of IMPC of the canine mammary gland were obtained, protein and mRNA expression were investigated through immunohistochemistry and RNA In Situ Hybridization, respectively. To better understand the relationship between E-cadherin and ZEB2, immunofluorescence was performed in canine IMPCs. Immunohistochemically, most of IMPCs showed 1+ (14/19, 73.7%) for E-cadherin; and positivity for ZEB2 was diagnosed in 47.4% of the IMPCs. Regarding the RNA In Situ Hybridization (ISH), most of IMPCs showed 4+ and 0+ for E-cadherin (CDH1) and ZEB2 respectively. Through immunofluorescence, the first and second more frequent combinatorial group were E-cadherin+ZEB2- and E-cadherin+ZEB2+; neoplastic cells showing concomitantly weak expression for E-cadherin and positivity for ZEB2 were frequently observed. A negative correlation was observed between E-cadherin and progesterone receptor expression in IMPCs. Based on these results, canine mammary IMPCs show E-cadherin lost and, at times reveals nuclear positivity for the transcription factor ZEB2 that seems to exert transcriptional repression of the CDH1.

Vulvar squamous cell carcinoma (VSCC) is a rare disease that has a high mortality rate (∼40%). However, little is known about its molecular signature. Therefore, an integrated genomics approach, based on comparative genome hybridization (aCGH) and genome-wide expression (GWE) array, was performed to identify driver genes in VSCC. To achieve that, DNA and RNA were extracted from frozen VSCC clinical specimens and examined by aCGH and GWE array, respectively. On the basis of the integration of data using the CONEXIC algorithm, PLXDC2 and GNB3 were validated by RT-qPCR. The expression of these genes was then analyzed by IHC in a large set of formalin-fixed paraffin-embedded specimens. These analyses identified 47 putative drivers, 46 of which were characterized by copy number gains that were concomitant with overexpression and one with a copy number loss and downregulation. Two of these genes, PLXDC2 and GNB3, were selected for further validation: PLXDC2 was downregulated and GNB3 was overexpressed compared with non-neoplastic tissue. By IHC, both proteins were ubiquitously expressed throughout vulvar tissue. High expression of GNB3 and low PLXDC2 immunostaining in the same sample was significantly associated with less lymph node metastasis and greater disease-free survival. On the basis of a robust methodology never used before for VSCC evaluation, two novel prognostic markers in vulvar cancer are identified: one with favorable prognosis (GNB3) and the other with unfavorable prognosis (PLXDC2). Implications: This genomics study reveals markers that associate with prognosis and may provide guidance for better treatment in vulvar cancer. Mol Cancer Res; 14(8); 720–9. ©2016 AACR.

// Beatriz de Melo Maia 1, 2 , Iara Santana Rodrigues 1 , Erica Mie Akagi 1 , Nayra Soares do Amaral 1 , Hui Ling 2 , Paloma Monroig 2 , Fernando Augusto Soares 1 , George Adrian Calin 2, 3 , Rafael Malagoli Rocha 4 1 Molecular Morphology Laboratory, Anatomic Pathology Department, AC Camargo Cancer Center, Sao Paulo, Brazil 2 Department of Experimental Therapeutics, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA 3 The Center for RNA Interference and Non-Coding RNAs, The University of Texas, MD Anderson Cancer Center, Houston, TX, USA 4 Gynecology Laboratory, Gynecologic Department Federal University of Sao Paulo, Sao Paulo, Brazil Correspondence to: Rafael Malagoli Rocha, email: rafael.malagoli@gmail.com Keywords: vulvar cancer, microRNAs, cellular assays, hsa-miR-223-5p, TP63 Received: January 11, 2016 Accepted: May 08, 2016 Published: June 23, 2016 ABSTRACT MiR-223-5p has been previously mentioned to be associated with tumor metastasis in HPV negative vulvar carcinomas, such as in several other tumor types. In the present study, we hypothesized that this microRNA would be important in vulvar cancer carcinogenesis and progression. To investigate this, we artificially mimicked miR-223-5p expression in a cell line derived from lymph node metastasis of vulvar carcinoma (SW962) and performed in vitro assays. As results, lower cell proliferation ( p < 0.01) and migration ( p < 0.001) were observed when miR-223-5p was overexpressed. In contrast, increased invasive potential of these cells was verified ( p < 0.004). In silico search indicated that miR-223-5p targets TP63 , member of the TP53 family of proteins, largely described with importance in vulvar cancer. We experimentally demonstrated that this microRNA is capable to decrease levels of p63 at both mRNA and protein levels ( p < 0.001, and p < 0.0001; respectively). Also, a significant inverse correlation was observed between miR-223-5p and p63 expressions in tumors from patients ( p = 0.0365). Furthermore, low p63 protein expression was correlated with deeper tumor invasion ( p = 0.0491) and lower patient overall survival ( p = 0.0494). Our study points out miR-223-5p overexpression as a putative pathological mechanism of tumor invasion and a promising therapeutic target and highlights the importance of both miR-223-5p and p63 as prognostic factors in vulvar cancer. Also, it is plausible that the evaluation of p63 expression in vulvar cancer at the biopsy level may bring important contribution on prognostic establishment and in elaborating better surgical approaches for vulvar cancer patients.

Dysregulation of microRNAs has been studied thoroughly, and has been observed in a variety of tumors including vulvar carcinomas, a rare type of gynecological tumor with increasing incidence. However, very few therapeutic alternatives have reached the clinical setting, and there is an urgent unmet need to develop novel strategies for patients with this tumor type. Thus, a microRNA (miRNA) sponge for the miR-17 miRNA family was designed, synthesized and validated in vitro in order to explore a new therapeutic strategy based on inhibiting this oncogenic miRNA family in vulvar cancer. Members of the miR-17 family were evaluated for expression in a vulvar tumor cell line (SW954) and 20 HPV negative formalin-fixed paraffin-embedded (FFPE) samples by quantitative real-time PCR (qRT-PCR). Six in tandem, bulged sequences that were complementary to these miRNAs were designed, synthesized, cloned, and transfected into SW954 cells. A luciferase reporter assay with a psiCheck2 vector was used to test the specificity of the sponge sequences for miR-17 family miRNA binding. Taqman qRT-PCR was used to test how the sponges affected miRNA expression. In FFPE samples, higher expression of miR-20a and miR-106a correlated with deeper tumor invasion (P = 0.0187 and P = 0.0404, respectively). The luciferase reporter assay validated the specificity of the sponge for miR-17 family members. Using qRT-PCR, we confirmed this specificity with decreased expression in 5 (out of six) miRNAs of the miR-17 family in SW954 cells. Although our results are preliminary, these results demonstrate that these miRNA sponges are potent inhibitors of the miR-17 family of miRNAs in SW954. Therefore, this miRNA-specific sponge may be developed into a novel therapeutic treatment for patients with vulvar cancer.

To identify biomarkers that could predict response or lack of response to conventional chemotherapy at the time of diagnosis of high‑grade serous ovarian carcinoma (HGSOC), the present study compared large‑scale gene expression from patients with short or long disease‑free survival times, according to the last cycle of chemotherapy, and validated these findings using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and conventional immunohistochemical (IHC) analysis. Samples were selected for microarray evaluation, at the time of diagnosis, using the following criteria: Identical debulking primary surgery, International Federation of Gynaecology and Obstetrics staging, histological subtype and grade. These were divided into 2 groups, regarding the outcome after 2 years of follow-up. Prostaglandin D2 synthase 21 kDa (brain) (PTGDS) was found to be expressed at a significantly higher level in the tumours of patients with a short disease‑free survival time, and this was validated by RT‑qPCR in all samples. Furthermore, the study evaluated PGD2, the protein product of the PTGDS gene, in a large cohort of 114 HGSOC patients using the Ventana Benchmark automated platform, and IHC positivity was correlated with clinicopathological data and outcome. The global gene expression analysis identified 1,149 genes that were differentially expressed in microarray data, according to the patient outcome. Further analysis RT‑qPCR validated PTGDS gene expression in the same samples (r=0.945; P

We assessed the co-expression of cell cycle-related biomarkers in a series of 121 consecutive cases of high-grade ductal carcinoma in situ (DCIS), pure or associated with invasive carcinoma, and their associations with the different immunoprofiles of DCIS. Cases were identified from the histopathology files of the Breast Pathology Laboratory, Federal University of Minas Gerais, Brazil, from 2003 to 2008. The expression of estrogen receptor, progesterone receptor, HER2 overexpression, cytokeratin 5, epidermal growth factor receptor 1, cyclooxygenase-2, p16 and Ki67 were assessed. Tumors were placed into five subgroups according to their immunohistochemical profile: luminal A, luminal B, HER2, basal-like and “not classified”. We found that the basal phenotype was associated with a higher frequency of p16-positive cases (83%) and the luminal A phenotype showed a higher frequency of p16-negative cases (93%; p=0.000). The association of biomarkers p16+/Ki67+/COX2+ was expressed in 02/06 cases (33.3%) of the basal phenotype but in only 01/70 cases (1.4%) of the luminal A phenotype ( p=0.01). The co-expression of p16+/Ki67+/COX2- was associated with a basal phenotype ( p=0.004). P16 expression, p16+/Ki67+/COX2+ and p16+/Ki67+/COX2- co-expression showed significant associations with the basal phenotype and these profiles could be used to guide more aggressive treatment strategies in patients with high-grade DCIS.

Whole slide imaging (WSI) technology has been used for training, teaching, researching, and remote consultation. Few studies compared HER2 expression using optical microscopy (OM) and WSI evaluations in breast carcinomas. However, no consensus has been achieved comparing both assessments. Material and methods Sections from tissue microarray containing 200 preselected invasive breast carcinomas were submitted to immunohistochemistry applying three anti-HER2 antibodies (HercepTest™, CB11, SP3) and in situ hybridization (DDISH). Slides were evaluated using OM and WSI (Pannoramic MIDI and Viewer, 3DHISTECH). Sensitivity and specificity were calculated comparing the anti-HER2 antibodies and DDISH. Results WSI and OM HER2 evaluations agreement was considered good (SP3, k = 0.80) to very good (CB11 and HercepTest™, k = 0.81). WSI evaluation led to higher sensitivity (ranging from 100 of SP3 and HercepTest™ to 97 of CB11) and lower specificity (ranging from 86.4 of SP3 to 89.4 of HercepTest™) compared to OM evaluation (sensitivity ranged from 92.1 of CB11 to 98 of SP3 and specificity ranged from 95.2 of SP3 and HercepTest™ to 97.1 of CB11 and SP3). Conclusion High agreement was achieved between WSI and OM evaluations. All three antibodies were highly sensitive and specific using both evaluations. WSI can be considered a useful tool for HER2 immunohistochemical assessment.

Cancer cells can divert metabolites into anabolic pathways to support their rapid proliferation and to accumulate the cellular building blocks required for tumour growth. However, the specific bioenergetic profile of invasive and metastatic cancer cells is unknown. Here we report that migratory/invasive cancer cells specifically favour mitochondrial respiration and increased ATP production. Invasive cancer cells use the transcription coactivator peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1A, also known as PGC-1α) to enhance oxidative phosphorylation, mitochondrial biogenesis and the oxygen consumption rate. Clinical analysis of human invasive breast cancers revealed a strong correlation between PGC-1α expression in invasive cancer cells and the formation of distant metastases. Silencing of PGC-1α in cancer cells suspended their invasive potential and attenuated metastasis without affecting proliferation, primary tumour growth or the epithelial-to-mesenchymal program. Inherent genetics of cancer cells can determine the transcriptome framework associated with invasion and metastasis, and mitochondrial biogenesis and respiration induced by PGC-1α are also essential for functional motility of cancer cells and metastasis.

Cytokeratins (CKs) constitute the cytoskeletal network and are regulated by post-translational modifications, acting not only as a mechanical support, but also in cell signaling and regulatory processes. Signaling is mediated by CK-associated proteins, such as Annexin A1 (ANXA1), a ligand of the CK18/CK8 complex. ANXA1 has a pivotal role in cellular and immunological responses, and together with CK18 have been implicated in several processes related to malignant transformation in breast cancer (BC). Our aim was to demonstrate how their interaction might be linked to BC development. We investigated transcript levels, protein expression and distribution for both targets in breast tissues of 92 patients (42 BCs and 50 benign diseases) using qPCR and immunohistochemistry, respectively. ANXA1 and CK18 mRNAs were inversely correlated, and their ratio in each TNM stage significantly differentiated BC from benign diseases (OR = 5.62). These differences did not mirror tissue protein levels, but a significant dichotomous protein distribution in tumor tissues was observed, differing from the expected co-localization observed during cell homeostasis. The disequilibrium of transcriptional levels between ANXA1/CK18 and alterations in their tissue distribution are present either in initial events or tumor progression, which suggest a critical event in BC. The broken dialog between ANXA1 and CK18 in normal breast tissues may play a critical role in BC development, and together may be used as combined targets for BC diagnostics.

Atypical vascular lesions (AVL) that occur in the field of prior radiation therapy for breast carcinoma are placed within the differential diagnosis with low grade angiosarcoma and other benign vascular lesions. Although considered a benign entity, the exact biological behavior of AVLs is not fully established because of the small number of cases reported in the literature. We aim to further characterize these lesions clinically and histopathologically, and to study their behavior. We report a series of 30 patients with AVL of the breast occurring after radiation exposure, diagnosed and treated at the European Institute of Oncology, Italy. Immunohistochemical study was performed in all cases, using CD31, D2-40, CD105, and Ki-67 antibodies. Twenty-seven patients were treated with standard doses of conventional adjuvant radiation therapy for the prior breast carcinoma. Three patients were treated with intraoperative radiotherapy with electrons. The post-radiation latency interval from breast carcinoma to AVL was 48.5 months (ranged from 1 to 146 months). Most of the lesions were classified as lymphatic type (78.6 %) based on D2-40 positivity. No extension into subcutaneous tissue or significant atypia was noted in all cases. Despite the fact that the AVL of our series have shown benign behavior in 93.3 %, one patient developed local recurrence of AVL, and two cases progressed to angiosarcoma at the previous AVL site. Further studies should be conducted to better understand the clinical behavior and to propose additional histopathologic diagnostic criteria to distinguish AVL from low grade angiosarcoma and those AVL with increased risk for malignant progression. Concerning current treatments of AVL, we recommend complete excision with free surgical margins and close follow up.

Epidermal growth factor receptor (EGFR) protein expression was assessed by immunohistochemistry (IHC) in 150 cases of invasive vulvar squamous cell carcinoma. In addition, gene copy number status by fluorescence in situ hybridization was performed in a smaller set of samples. Results were correlated with patient's clinical data and prognostic factors. EGFR overexpression (2+ and 3+) was observed on the membrane in 24.66% and 21.33% of all cases, respectively. Higher EGFR expression was associated with depth of invasion (P = .0409) and disease recurrence (P = .0401). Cytoplasm staining was found in 21.33% of the cases and was associated with absence of nodal metastasis (P = .0061) and better survival (P = .0199). Intratumor heterogeneity of EGFR IHC staining was frequently observed (55.33%) and was associated with the presence of nodal metastasis (P = .0207) and tumor invasion (P = .0161). Worse survival outcomes have been demonstrated in tumors with EGFR heterogeneity (P = .0434). EGFR gene status evaluated by fluorescence in situ hybridization did not correlate with protein expression evaluated by IHC. In conclusion, EGFR cytoplasm staining has no link with poorer outcome; still, this pattern of staining is even more related to better prognosis. EGFR heterogeneity of staining correlated with more aggressive tumors, and presented to be an important marker of poor prognosis in vulvar squamous cell carcinoma. The usage of small biopsies or even tissue microarrays for vulvar cancer evaluation should be carefully reconsidered for the assessment of EGFR as the results may be misleading. Protein overexpression may be independent on gene amplification, showing that other molecular mechanisms than copy number variation may regulate protein expression of EGFR in vulvar cancer.

Surgical treatment of vulvar cancer has been shifted from ultraradical procedures associated with huge morbidity to less extensive surgery with better psychosexual result and less morbidity, without compromising survival. The authors review and discuss the recent literature regarding the surgical management of vulvar squamous cell carcinoma.Surgery remains the cornerstone in the treatment of vulvar cancer. Radical vulvectomy with inguinofemoral lymphadenectomy has been replaced by radical local excision with sentinel node procedure for early disease. However, the role and distance of pathological margins are still on debate. Recent results from a large prospective trial corroborate the safety of sentinel node biopsy for early disease, even after primary tumor resection. An experienced team should perform sentinel node procedure using combined technique (blue dye and lymphoscintigraphy) and ultrastaging pathology. Moreover, midline tumors still need lymph node biopsy from both groins.Primary vulvar cancer may be safely treated with radical/wide local resection. In case of other suspicious lesion or multifocal disease, radical vulvectomy is performed. Patients with unifocal disease, tumor size less than 4 cm, and clinically negative groins are candidates to sentinel node procedure. In the case of clinically positive node or sentinel node metastasis, a systematic inguinofemoral lymphadenectomy should be performed.

The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies.

BACKGROUND: Ataxia telangiectasia-mutated (ATM) gene downexpression has been reported in sporadic breast carcinomas (BC); however, the prognostic value and mechanisms of ATM deregulation remain unclear.PATIENTS AND METHODS: ATM and miRNAs (miR-26a, miR-26b, miR-203, miR-421, miR-664, miR-576-5p and miR-18a) expression levels were evaluated by quantitative real-time PCR (RT-qPCR) in 52 BC and 3 normal breast samples. ATM protein expression was assessed by immunohistochemistry in 968 BC and 35 adjacent normal breast tissues. ATM copy number alteration was detected by array comparative genomic hybridization (aCGH) in 42 tumours.RESULTS: Low ATM levels were associated with tumour grade. Absence of ATM protein expression was associated with distant metastasis (P < 0.001), reduced disease-free survival (DFS, P < 0.001) and cancer-specific survival (CSS, P < 0.001). Multivariate analysis indicated ATM protein expression as an independent prognostic marker for DFS (P = 0.001, HR = 0.579) and CSS (P = 0.001, HR = 0.554). ATM copy number loss was detected in 12% of tumours and associated with lower mRNA levels. miR-421 over-expression was detected in 36.5% of cases which exhibit lower ATM transcript levels (P = 0.075, r = -0.249).CONCLUSIONS: The data suggest that ATM protein expression is an independent prognostic marker in sporadic BC. Gene copy number loss and miR-421 over-expression may be involved in ATM deregulation in BC.

There is a lack of standardization of a best practice protocol for Phosphatase and Tensin Homolog (PTEN) assessment by immunohistochemistry in anatomic pathology routine practice. We performed immunohistochemistry for 19 antibodies against PTEN, eleven of which were excluded during the standardization step. Immunohistochemistry of the remaining eight antibodies was performed on a Tissue Microarray containing 55 prostate and 40 renal carcinoma samples. Fluorescent in situ hybridization (FISH) was used as reference standard for immunohistochemistry specificity evaluation. Concerning nuclear staining, polyclonal (Cat#22034-1-AP); 6H2.1 mMAb (Cat#ABM-2052), Y184 RabMAb (Cat#NB110-57441) and 217702 mMAb antibodies presented the highest agreement with fluorescent in situ hybridization (p