Kátia Cândido Carvalho

Glut1 and Glut3 as potential prognostic markers for oral squamous cell carcinoma

We associated clinical-pathological features of 142 OSCC with the expression pattern of GLUT1 and GLUT3 in order to estimate their prognostic value. Methods: Clinical-pathological features and overall survival data of 142 patients with Oral Squamous Cell Carcinoma (OSCC) were retrospectively reviewed from A.C.Camargo hospital records. A tissue microarray (TMA) was built for the immunohistochemical (IHC) analysis of GLUT 1 and GLUT 3. IHC results were evaluated according to the staining pattern and number of positive cells. Results: GLUT 1 was over expressed in 50.3% of OSSC cases showing membrane staining pattern. However, nuclear expression was observed in 49.7% of the analyzed cases. GLUT 3 over expression was detected in 21.1% of OSCC cases. The pattern of GLUT 1 expression showed significant association with alcohol consumption (p = 0.004). Positive cell membrane GLUT 3 protein expression was associated with advanced clinic-staging of tumours (p = 0.005) as well as with vascular embolization (p = 0.005). Positive expression of GLUT 3 was associated with unfavorable free-disease survival (p = 0.021). Conclusion: GLUT1 and GLUT3 protein expression evaluated by immunohistochemistry are, significantly, indicators of poor prognosis outcome in oral squamous cell carcinoma, probably due to the enhanced glycolytic metabolism of more aggressive neoplastic cells.

GLUT1 expression in malignant tumors and its use as an immunodiagnostic marker

OBJECTIVE: To analyze glucose transporter 1 expression patterns in malignant tumors of various cell types and evaluate their diagnostic value by immunohistochemistry. INTRODUCTION: Glucose is the major source of energy for cells, and glucose transporter 1 is the most common glucose transporter in humans. Glucose transporter 1 is aberrantly expressed in several tumor types. Studies have implicated glucose transporter 1 expression as a prognostic and diagnostic marker in tumors, primarily in conjunction with positron emission tomography scan data. METHODS: Immunohistochemistry for glucose transporter 1 was performed in tissue microarray slides, comprising 1955 samples of malignant neoplasm from different cell types. RESULTS: Sarcomas, lymphomas, melanomas and hepatoblastomas did not express glucose transporter 1. Forty-seven per cent of prostate adenocarcinomas were positive, as were 29% of thyroid, 10% of gastric and 5% of breast adenocarcinomas. Thirty-six per cent of squamous cell carcinomas of the head and neck were positive, as were 42% of uterine cervix squamous cell carcinomas. Glioblastomas and retinoblastomas showed membranous glucose transporter 1 staining in 18.6% and 9.4% of all cases, respectively. Squamous cell carcinomas displayed membranous expression, whereas adenocarcinomas showed cytoplasmic glucose transporter 1 expression. CONCLUSION: Glucose transporter 1 showed variable expression in various tumor types. Its absence in sarcomas, melanomas, hepatoblastomas and lymphomas suggests that other glucose transporters mediate the glycolytic pathway in these tumors. The data suggest that glucose transporter 1 is a valuable immunohistochemical marker that can be used to identify patients for evaluation by positron emission tomography scan. The function of cytoplasmic glucose transporter 1 in adenocarcinomas must be further examined.

Triple-negative and luminal A breast tumors: differential expression of miR-18a-5p, miR-17-5p, and miR-20a-5p

New concepts in epigenetics, microRNAs, and gene expression analysis have significantly enhanced knowledge of cancer pathogenesis over the last decade. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression by base pairing with target messenger RNAs (mRNAs), resulting in the repression of translation or the degradation of mRNA. To compare the carcinogenic process in tumors with different prognoses, we used real-time RT-PCR to evaluate the miRNA expression profiles of 24 triple-negative breast invasive ductal carcinoma, 20 luminal A breast invasive ductal carcinoma, and 13 normal breast parenchyma controls. We extracted total RNA from tissues fixed in formol and embedded in paraffin (FFPE). Results revealed the upregulation of miR-96-5p (9.35-fold; p = 0.000115), miR-182-5p (7.75-fold; p = 0.000033), miR-7-5p (6.71-fold; p = 0.015626), and miR-21-5p (6.10-fold; p = 0.000000) in tumors group. In addition, the expression of miR-125b-5p (4.49-fold; p = 0.000000) and miR-205-5p (4.36-fold; p = 0.006098) was downregulated. When the expression profiles of triple-negative and luminal A tumors were compared, there was enhanced expression of miR-17-5p (4.27-fold; p = 0.000664), miR-18a-5p (9.68-fold; p = 0.000545), and miR-20a-5 (4.07-fold; p = 0.001487) in the triple-negative tumors compared with luminal A. These data suggest that there is a similar regulation of certain miRNAs in triple-negative and luminal A tumors. However, it is possible that differences in the expression of miR-17-92 cluster will explain the phenotypic differences between these molecular tumor subtypes.

MGMT and PTEN as potential prognostic markers in breast cancer

AIM To evaluate the prognostic importance of MGMT and PTEN concerning their correlation with other prognostic factors evaluated by immunohistochemistry (IHC) and the molecular phenotype of breast cancers. METHODS IHC for estrogen and progesterone receptors, HER2, Ki67, p53, p63, e-cadherin, EGFR, CK5, CK14, MGMT and PTEN was performed on 200 breast tumors. Basal-like and luminal breast carcinomas were defined by the IHC evaluation of these markers. Fluorescent in situ hybridization (FISH) was performed for PTEN and HER2 analysis using the Vysis PTEN and HER2 DNA probe kits (Abbott™). RT-PCR was performed to evaluate gene expressions of MGMT and PTEN in frozen tissue of 59/200 cases. RESULTS 147/200 cases were triple-negative (73.5%), 47/147 were basal-like carcinomas (31.9%). 53 cases (26.5%) were luminal-like type A or B. 56 (93.3%) and 46 samples (76.6%) expressed lower levels of MGMT and PTEN mRNA, respectively, compared with normal breast (p<0.001). There was a positive correlation between the IHC results and the RT-PCR values for MGMT and PTEN. Tumors with homozygotic deletion of PTEN expressed little or no mRNA or protein. Positive p53, high Ki67, and basal-like tumors expressed significant lower MGMT and PTEN. CONCLUSIONS We hypothesize that MGMT and PTEN expressions have prognostic significance in breast cancer. Also, based on their predictive value of response to therapy, evaluating MGMT and PTEN and learning to interpret their patterns of immunoexpression will undoubtedly lead to a greater understanding of breast cancer and its treatment.

A clinical, pathologic, and molecular study of p53 and murine double minute 2 in penile carcinogenesis and its relation to prognosis.

Penile carcinoma constitutes up to 16% of male malignancies in developing countries. Changes in the p53 and murine double minute 2 pathway are important events in various cancers. Associate alterations in murine double minute 2 and p53 expression were evaluated by molecular techniques, with the clinical data of 297 cases of penile carcinoma. Automated immunohistochemistry was performed for murine double minute 2 and p53 using the primary antibodies SPM14 and DO7, respectively. Fluorescent in situ hybridization was performed using the probes murine double minute 2 at 12q15 and TP53 at 17p13.1. Slides were digitalized, and bright-field and fluorescent images were analyzed. TP53 was sequenced in 16 cases. The expression of p53 was higher in poorly differentiated, infiltrative border, corpus spongiosum, corpora cavernosa, and invasive urethral carcinomas. Patients who died of disease also expressed higher levels of p53. p53-negative tumors were associated with higher overall survival. Murine double minute 2 showed no difference of expression in any group of tumors, no correlation with p53 expression. No alterations in genes or chromosomes were observed. Mutations in TP53 were observed in 4 of 16 cases: p.T170M, p.L252P, p.C176Y, and the novel c.803_810del8; these changes correlated with p53 expression by immunohistochemistry. Murine double minute 2 is not useful in the prognosis of penile carcinoma by immunohistochemistry. Additional studies on the transcriptional, posttranscriptional, and epigenetic aspects are necessary to understand the interactions between p53 and murine double minute 2 because we did not observe any numeric alterations by fluorescent in situ hybridization. Examining p53 is helpful in identifying patients with more aggressive tumors and may be crucial in selecting the most suitable surgical procedure.

Transcriptional profiles of SHH pathway genes in keratocystic odontogenic tumor and ameloblastoma

BACKGROUND Sonic hedgehog (SHH) pathway activation has been identified as a key factor in the development of many types of tumors, including odontogenic tumors. Our study examined the expression of genes in the SHH pathway to characterize their roles in the pathogenesis of keratocystic odontogenic tumors (KOT) and ameloblastomas (AB). METHODS We quantified the expression of SHH, SMO, PTCH1, SUFU, GLI1, CCND1, and BCL2 genes by qPCR in a total of 23 KOT, 11 AB, and three non-neoplastic oral mucosa (NNM). We also measured the expression of proteins related to this pathway (CCND1 and BCL2) by immunohistochemistry. RESULTS We observed overexpression of SMO, PTCH1, GLI1, and CCND1 genes in both KOT (23/23) and AB (11/11). However, we did not detect expression of the SHH gene in 21/23 KOT and 10/11 AB tumors. Low levels of the SUFU gene were expressed in KOT (P = 0.0199) and AB (P = 0.0127) relative to the NNM. Recurrent KOT exhibited high levels of SMO (P = 0.035), PTCH1 (P = 0.048), CCND1 (P = 0.048), and BCL2 (P = 0.045) transcripts. Using immunolabeling of CCND1, we observed no statistical difference between primary and recurrent KOT (P = 0.8815), sporadic and NBCCS-KOT (P = 0.7688), and unicystic and solid AB (P = 0.7521). CONCLUSIONS Overexpression of upstream (PTCH1 and SMO) and downstream (GLI1, CCND1 and BCL2) genes in the SHH pathway leads to the constitutive activation of this pathway in KOT and AB and may suggest a mechanism for the development of these types of tumors.

Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside.

BackgroundVulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection.Methodsc-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO® A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes–Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients.Resultsc-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009).ConclusionsOur findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative surgery techniques and lymph node dissection in vulvar cancer. So part of the essence of our study is to see the possibility of translating our current results from the bench to the bedside. This will help provide patients a more appropriate, less mutilating treatment, in order to keep the maximum physical and psychic quality as possible to these women.

Differences in neonatal exposure to estradiol or testosterone on ovarian function and hormonal levels

Exposure to an excess of androgen or estrogen can induce changes in reproductive function in adult animals that resemble polycystic ovary syndrome in humans. However, considerable differences exist among several types of animal models. Little is known about the molecular features of steroidogenesis and folliculogenesis in the ovaries of rats exposed to different sex steroids as neonates. Here, we evaluated the impact of androgen and estrogen exposure on the ovaries of adult female rats during their neonatal period in the gene expression of Lhr and Cyp17a1, two key players of steroidogenesis. We also assessed hormone levels, folliculogenesis and the theca-interstitial cell population. The study was performed on the second postnatal day in thirty female Wistar rats that were sorted into the following three intervention groups: testosterone, estradiol and vehicle (control group). The animals were euthanized 90 days after birth. The main outcomes were hormone serum levels, ovary histomorphometry and gene expression of Lhr and Cyp17a1 as analyzed via quantitative real-time PCR. We found that exposure to excess testosterone in early life increased the LH and testosterone serum levels, the LH/FSH ratio, ovarian theca-interstitial area and gene expression of Lhr and Cyp17a1 in adult rats. Estrogen induced an increase in the ovarian theca-interstitial area, the secondary follicle population and gene expression of Lhr and Cyp17a1. All animals exposed to the sex steroids presented with closed vaginas. Our data suggest that testosterone resulted in more pronounced reproductive changes than did estrogen exposure. Our results might provide some insight into the role of different hormones on reproductive development and on the heterogeneity of clinical manifestations of conditions such as polycystic ovary syndrome.

Prognostication of Soft Tissue Sarcomas Based on Chromosome 17q Gene and Protein Status: Evaluation of TOP2A, HER-2/neu, and Survivin

BackgroundTopoisomerase 2 alpha (TOP2A), HER-2/neu, and survivin are genes that lie on chromosome 17 and correlate with the prognosis and prediction of target-driven therapy against tumors. In a previous study, we showed that TOP2A transcripts levels were significantly higher in soft tissue sarcomas (STS) than in benign tumors and desmoid-type fibromatoses (FM). Because these genes have been insufficiently examined in STS, we aimed to identify alterations in TOP2A and HER-2 expression by fluorescent in situ hybridization and immunohistochemistry, as well as that of survivin, and correlate them with clinicopathologic findings to assess their prognostic value.MethodsEighteen FM and 244 STS were included. Fluorescent in situ hybridization and immunohistochemistry were performed on a tissue microarray.ResultsTOP2A and survivin were more highly expressed in sarcomas than in FM. TOP2A was an independent predictor of an unfavorable prognosis; it was combined with formerly established prognostic factors (primarily histologic grade and tumor size at diagnosis) to create a prognostic index that evaluated overall survival. Gene amplification/polysomy (13%) did not correlate with protein overexpression. Survivin and HER-2 expression were not associated with patient outcomes.ConclusionsThese findings might become valuable in the management of patients with STS and possibly in the prospective evaluation of responses to new target-driven therapies.

Best practice for PTEN gene and protein assessment in anatomic pathology.

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<0.001 for all) and with regard to cytoplasmic staining, Y184 RabMAb (Cat#NB110-57441); polyclonal (Cat#22034-1-AP) and 217702 mMAb presented the highest agreement (p<0.001 for all). Our results indicate that several commercially available antibodies do not show reliability of sensitivity and specificity for PTEN evaluation and we propose 6H2.1 mMAb (Cat#ABM-2052) as the antibody of choice for laboratory standardization and best practice in clinical routine, which demonstrated excellent sensitivity for both nuclear and cytoplasmic staining, specificity for PTEN by Western blot and good correlation with PTEN status by FISH with regard to nuclear staining.