Marcilei Eliza Cavicchioli Buim

Primary oral mucosal melanoma: a series of 35 new cases from South America.

Oral mucosal melanoma is rare and reported to be more aggressive than its cutaneous counterpart. Due to the rarity of this entity, data on epidemiology, tumor behavior, treatment, follow-up, and survival of patients are mainly based on single case reports. The few existing series of patients show that oral mucosa melanoma has its peak between 41 and 60 years of age, and male to female ratio is 2:1. Preferred oral sites include hard palate and maxillary alveolar crests. Risk factors have not been clearly identified, and surgical treatment is still the treatment of choice for oral mucosal melanomas. The authors retrospectively studied 35 patients with primary melanoma of the oral cavity to report their clinical and pathological features, such as age, sex, site of the tumor, metastasis, treatment, response to therapy, and outcome. We found no significant sex predominance, and the mean age of the patients was 60.6 years, with a range from 9 to 91 years. The majority of the patients (71.42%) had palate commitment, and invasive histopathological aspect was observed in 80% of the specimens (grade 3). Long-distance metastasis was found in 60% of the cases. Fourteen patients were submitted to wide surgical resections, with local relapse being observed in 11 of them (78.5%). The authors suggest that improved outcome in oral malignant melanoma requires the development of new therapies and the prevention of distant metastasis.

Human salivary gland branching morphogenesis: morphological localization of claudins and its parallel relation with developmental stages revealed by expression of cytoskeleton and secretion markers

Development of salivary glands is a highly complex and dynamic process termed branching morphogenesis, where branched structures differentiate into mature glands. Tight junctions (TJ) are thought to play critical roles in physiological functions of tubular organs, contributing to cell polarity and preventing lateral movement of membrane proteins. Evidence demonstrated that claudins are directly involved in TJ formation and function. Using immunohistochemistry and immunofluorescence we have mapped the distribution of claudins-1, 2, 3, 4, 5, 7 and 11 and compared it with the expression of differentiation markers in human salivary glands obtained from foetuses ranging from weeks 4 to 24 of gestation. Expression of all claudins, except claudin-2 was detected in the various phases of human salivary gland development, up to fully mature salivary gland. The expression of all claudins increased according to the progression of salivary gland maturation evidenced by the classical markers—cytokeratin 14, cytokeratin low molecular weight, smooth muscle actin and human secretory component. Tight junction proteins—claudins appear to be important in the final shape and physiological functions of human salivary glands and are parallel related with markers of salivary gland differentiation.

Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers

Ianez R F, Buim M E, Coutinho‐Camillo C M, Schultz R, Soares F A & Lourenço S V
(2010) Histopathology 57, 410–417
Human salivary gland morphogenesis: myoepithelial cell maturation assessed by immunohistochemical markers

Isolation, detection, and immunomorphological characterization of circulating tumor cells (CTCs) from patients with different types of sarcoma using isolation by size of tumor cells: a window on sarcoma-cell invasion

Background Sarcomas are rare and heterogeneous neoplasms with poor prognosis that are thought to spread to distant organs mainly by hematogenous dissemination. However, circulating tumor cells (CTCs) have never been visualized in sarcomas. Objectives To investigate the feasibility of using isolation by size of tumor cells (ISET) for isolation, identification, and characterization of CTCs derived from patients with high-grade and metastatic sarcomas. Patients and methods We studied eleven patients with metastatic/recurrent or locally advanced soft-tissue sarcomas (STSs), six of whom had synovial sarcomas. Blood samples (8 mL) were collected from patients with advanced STS and treated by ISET, a marker- independent approach that isolates intact CTCs from blood, based on their larger size compared with leukocytes. CTCs were identified by cytomorphology and characterized by dual-color immunocytochemistry using antivimentin or anti-Pan CK, and anti-CD45. Results All patients with STS included in this study showed CTCs, with numbers ranging from two to 48 per 8 mL of blood. Conclusion This study shows the feasibility of isolating, identifying, and characterizing CTCs from patients with different types of sarcomas and the presence of circulating sarcoma cells in all the tested patients. Our results set the basis for further studies aimed at exploring the presence, number, and immunomolecular characteristics of CTCs in different types of sarcoma, and bring more light to the mechanisms of tumor invasion for these tumors.

Thymidylate synthase expression in circulating tumor cells: A new tool to predict 5‐fluorouracil resistance in metastatic colorectal cancer patients

Thymidylate synthase (TYMS) is an important enzyme for 5‐fluorouracil (5‐FU) metabolism in metastatic colorectal cancer (mCRC) patients. The search for this enzyme in circulating tumor cells (CTCs) can be a powerful tool to follow‐up cancer patients. mCRC patients were enrolled before the beginning of 5‐FU‐based chemotherapy. The blood was filtered on Isolation by Size of Epithelial Tumor Cells (ISET), and the analysis of TYMS expression in CTCs was made by immunocytochemistry. Additionally, we verified TYMS staining in primary tumors and metastases from the same patients. There were included 54 mCRC patients and 47 of them received 5‐FU‐based chemotherapy. The median CTCs number was 2 per mL. We were not able to analyze immunocytochemistry in 13 samples (9 patients with absence of CTCs and 4 samples due to technical reasons). Therefore, TYMS expression on CTCs was analyzed in 34 samples and was found positive in 9 (26.5%). Six of these patients had tumor progression after treatment with 5‐FU. We found an association between CTC TYMS staining and disease progression (DP), although without statistical significance (P = 0.07). TYMS staining in primary tumors and metastases tissues did not have any correlation with disease progression (P = 0.67 and P = 0.42 respectively). Patients who had CTC count above the median (2 CTCs/mL) showed more TYMS expression (P = 0.02) correlating with worse prognosis. Our results searching for TYMS staining in CTCs, primary tumors and metastases suggest that the analysis of TYMS can be useful tool as a 5‐FU resistance predictor biomarker if analyzed in CTCs from mCRC patients.

Activation of sonic hedgehog signaling in oral squamous cell carcinomas: A preliminary study

Sonic hedgehog signaling is important for human development, and aberrant regulation of this pathway can result in the development of tumors. The aim of this study was to examine the expression of sonic hedgehog signaling molecules in oral squamous cell carcinoma. By quantitative real-time polymerase chain reaction, the expression of SHH, SMO, PTCH-1, and GLI-1 was analyzed in 30 oral squamous cell carcinoma cases and 8 samples of nonneoplastic oral mucosa and associated to clinical pathologic features. The expression of β-catenin, cyclin D1, Wnt-1, and Egfr was evaluated by immunohistochemistry in 26 available cases of oral squamous cell carcinoma. Normal oral mucosa from healthy individuals was negative for all genes that were evaluated. SHH, PTCH-1, SMO, and GLI-1 were not expressed in nonneoplastic oral mucosa, and low levels of GLI-1 were observed in nonneoplastic oral mucosa that was adjacent to the tumor. All oral squamous cell carcinoma cases expressed high levels of PTCH-1, SMO, and GLI-1 and were devoid of SHH. The expression of SMO was associated with clinical stage (P = .022) and a borderline association in cervical lymph node metastasis (P = .053). PTCH-1 expression showed a strong correlation with SMO (rs = 0.64; P < .001) and GL-1 (rs = 0.70; P < .001); SMO and GLI-1 also correlated with each other (rs, 0.55; P < .001). All proteins evaluated were expressed as cyclin D1 (92% of samples), β-catenin (73%), Egfr (46%), or Wnt-1 (32%). Our data demonstrate that sonic hedgehog signaling is activated in oral squamous cell carcinoma and suggest that this pathway mediates its tumorigenesis.

Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma

Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p=0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p=0.010 and p=0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC.

Oral squamous cell carcinoma: status of tight junction claudins in the different histopathological patterns and relationship with clinical parameters. A tissue-microarray-based study of 136 cases

Aims Claudins are integral transmembrane proteins of the tight junctions, critical for maintaining cell adhesion and polarity. Alterations in the expression of individual claudins have been detected in carcinomas and appear to correlate with tumour progression. Methods In this study, a panel of anti-claudin antibodies (anti-claudins 1, 2, 3, 4, 5 and 7) was employed to map claudin expression in 136 cases of oral squamous cell carcinoma (OSCC) organised in a tissue microarray. Results Claudins were expressed in a reticular pattern up to the prickle layer in normal mucosal epithelium. In OSCC, claudins were strongly present in well-differentiated tumours, they presented mild and low expression in moderately differentiated OSCC, and were negative in poorly differentiated OSCC; the absences of claudin 1 (p=0.002) and claudin 4 (p<0.001) were associated with moderately/poorly differentiated tumours. Strong expression of claudin 4 was associated with decreased perineural infiltration (p=0.024). Claudins 5 and 7 were mostly negative or weakly expressed in all cases studied. Expression of claudin 7 was associated with the early clinical stages of the disease, whereas loss of claudin 7 tended to be more frequent in advanced stages of OSCC (p=0.054). Absence of claudin 7 was also associated with absent vascular infiltration (p=0.045) and with presence of recurrence (p=0.052). Conclusions Claudin expression patterns showed a strong correlation with histological type of OSCC; claudin expression was decreased in areas of invasion, and negative in poorly differentiated tumours. This pattern may be related to evolution and prognosis of these tumours, especially in the case of claudin 7, which seems to be associated with a poor prognosis in OSCC.

The Transcripts of SFRP1‚ CEP63 and EIF4G2 Genes Are Frequently Downregulated in Transitional Cell Carcinomas of the Bladder

Objective: The aim of the present study was to identify differentially expressed genes that might be associated with the phenotype of superficial and invasive bladder cancer. Methods: Differential display reverse transcriptase PCR (DDRT-PCR) was used to compare the expression pattern between normal bladder tissue and 4 groups of transitional cell carcinomas of the bladder regarding clinical stage and grade. Results: We were able to identify 72 different transcripts, of which 57 (79%) showed homology to known genes, 12 (17%) to hypothetical proteins and 3 (4%) to human expressed sequence tags. Among the differentially expressed genes, SFRP1,CEP63 and EIF4G2 were further validated by quantitative RT-PCR in a series of 50 transitional cell carcinomas. Overall, the transcripts of these three genes were shown to be downregulated in the bladder tumors analyzed. In accordance with the DDRT-PCR results, the SFRP1 transcripts were shown to be downregulated in 90% (45/50) of the bladder tumors as compared with the normal bladder tissue. Although EIF4G2 and CEP63 transcripts exhibited three different expression patterns, downregulation was found in about 50% of the cases analyzed. In addition, downregulation of both CEP63 and EIF4G2 gene transcription was associated with invasive tumors. Conclusion: The use of DDRT-PCR analysis to compare expression patterns among different subgroups of bladder tumors allowed us to identify a significant number of genes implicated in different cellular pathways that, when up- or downregulated, might play a role in the tumorigenic process of the bladder.

Detection of KRAS mutations in circulating tumor cells from patients with metastatic colorectal cancer.

Background: Quantification of Circulating Tumor Cells (CTCs) as a prognostic marker in metastatic colorectal cancer (mCRC) has already been validated and approved for routine use. However, more than quantification, qualification or characterization of CTCs is gaining importance, since the genetic characterization of CTCs may reflect, in a real time fashion, genetic profile of the disease. Objective: To characterize KRAS mutations (codon 12 and 13) in CTCs from patients with mCRC and to compare with matched primary tumor. Additionally, correlate these mutations with clinical and pathological features of patients. Methods: Blood samples were collected from 26 patients with mCRC from the AC Camargo Cancer Center (São Paulo-Brazil). CTCs were isolated by ISET technology (Isolation by Size of Epithelial Tumors; Rarecells Diagnostics, France) and mutations analyzes were performed by pyrosequencing (QIAGEN). Results: KRAS mutation was detected in 7 of the 21 cases (33%) of samples from CTCs. In matched primary tumors, 9 of the 24 cases (37.5%) were found KRAS mutated. We observed that 5 of the 9 samples with KRAS mutation in their primary tumor had also KRAS mutation in CTCs, meaning a concordance of 71% of matched cases (P = 0.017). KRAS mutation neither on primary tumor nor in CTCs was associated with clinical-pathological parameters analyzed. Conclusion: Faced with a polyclonal disease like colorectal cancer, which is often treated with alternating and successive lines of chemotherapy, real time genetic characterization of CTCs, in a fast and feasible fashion, can provide important information to clinical management of metastatic patients. Although our cohort was limited, it was possible to show a high grade of concordance between primary tumor and CTCs, which suggests that CTCs can be used as surrogate of primary tumors in clinical practice, when the knowledge of mutation profile is necessary and the primary tumor is not available.