The Potential Clinical Implications of Circulating Tumor Cells and Circulating Tumor Microemboli in Gastric Cancer.

BACKGROUND: Gastric adenocarcinoma (GAC) is the third deadliest malignant neoplasm worldwide, mostly because of late disease diagnosis, low chemotherapy response rates, and an overall lack of tumor biology understanding. Therefore, tools for prognosis and prediction of treatment response are needed. Quantification of circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) and their expression of biomarkers has potential clinical relevance. Our aim was to evaluate CTCs and CTM and their expression of HER2 and plakoglobin in patients with nonmetastatic GAC, correlating the findings to clinicopathological data. MATERIALS AND METHODS: CTC enrichment was performed with isolation by size of epithelial tumor cells, and the analysis was performed with immunocytochemistry and microscopy. Two collections were made: one at diagnosis (55 samples before neoadjuvant treatment) and one after surgery and before adjuvant therapy (33 samples). RESULTS: A high detection rate of CTCs (90%) was observed at baseline. We evaluated HER2 expression in 45/55 biopsy samples and in 42/55 CTC samples, with an overlap of 36 subjects. Besides the good agreement observed for HER2 expression in primary tumors and paired CTCs for 36 cases (69.4%; κ = 0.272), the analysis of HER2 in CTCs showed higher positivity (43%) compared with primary tumors (11%); 3/5 patients with disease progression had HER2-negative primary tumors but HER2-positive CTCs. A significant CTC count drop in follow-up was seen for CTC-HER2-positive cases (4.45 to 1.0 CTCs per mL) compared with CTC-HER2-negative cases (2.6 to 1.0 CTCs per mL). The same was observed for CTC-plakoglobin-positive cases (2.9 to 1.25 CTCs per mL). CONCLUSION: CTC analysis, including their levels, plakoglobin, and HER2 expression, appears to be a promising tool in the understanding the biology and prognosis of GAC. IMPLICATIONS FOR PRACTICE: The analysis of circulating tumor cell levels from the blood of patients with gastric adenocarcinoma, before and after neoadjuvant treatment, is useful to better understand the behavior of the disease as well as the patients more likely to respond to treatment.

EGFR expression in circulating tumor cells from high-grade metastatic soft tissue sarcomas.

INTRODUCTION: Soft tissue Sarcomas (STS) are rare malignances, with high mortality rates. Half of patients develop metastasis. The presence of isolated Circulating Tumor Cells (CTCs) and Circulating Tumor Microemboli (CTM) in the blood may be early markers of tumor invasion. Epidermal Growth Factor (EGF) family receptors can also influence this process. OBJECTIVES: to quantify CTCs and identify CTM as well as the EGF Receptor (EGFR) protein expression in these cells and correlate with clinical outcome in metastatic STS. MATERIALS AND METHODS: Approximately 8mL of blood was prospectively collected from patients with different types of high-grade STS, before the beginning of chemotherapy. The samples were processed and filtered by ISET (Rarecells, France) for the isolation and quantification of CTCs and CTMs. EGFR expression was analyzed by immunocytochemistry (ICC) on CTCs/ CTMs. RESULTS: We analyzed 18 patients with median age of 49 years (18-77 y). The positivity for EGFR protein expression in CTCs was observed in 93.75% of the patients. This result shows that targeting EGFR positive CTCs from STS origen can be translated in clinical benefit for some patients. In addition, if target therapy is chosen, the EGFR expression in CTCs can be used in follow-up to measure treatment effectiveness. CONCLUSIONS: This is the first study to demonstrate the expression of EGFR protein in CTCs from sarcoma patients. It may open an area for future investigations. The next step is to characterize CTCs in a larger cohort of patients to better understand the role of EGFR in sustaining tumor metastasis in sarcomas.

Reflectance confocal microscopy features of BRAF V600E mutated thin melanomas detected by immunohistochemistry.

The classification of melanoma into four histological subtypes has been questioned regarding its clinical validity in providing relevant information for treatment for metastatic tumors. Specific genetic alterations are associated with particular clinical and histopathological features, suggesting that these could be helpful in refining existing melanoma classification schemes. We analyzed BRAF V600E mutated melanomas to explore the Reflectance confocal microscopy (RCM) utility as a screening aid in the evaluation of the most appropriate patients for genetic testing. Thus, 32 melanomas were assessed regarding their BRAF V600E mutational status. Experts blinded to dermoscopic images and V600E immunohistochemistry results evaluated RCM images regarding previously described melanoma features. BRAF positive melanomas were related to younger age (p = 0.035), invasive melanomas (p = 0.03) and to the presence of hiporreflective cells (p = 0.02), epidermal nests (p = 0.02), dermal-epidermal junction nests (p = 0.05), edged papillae (p = 0.05), and bright dots (p = 0.05), and to absence of junctional thickening due to isolated cells (p = 0.01) and meshwork (p = 0.02). This study can not characterize other mutations in the BRAF, because the immunohistochemistry is specific to the type V600E. The findings should encourage the genetic evaluation of BRAF mutation. This study highlights the potential of RCM as a supplementary tool in the screening of BRAF-mutated melanomas.

Análise comparativa do perfil molecular do status mutacional dos genes BRAF, NRAS, e C-Kit em melanomas esporádicos primários e metastáticos / Comparative analysis of molecular profiling of themutational status of BRAF, NRAS and C-Kit gene in primary and metastatic sporadic melanomas

INTRODUÇÃO: A ativação anormal da via MAP Quinase está presente em mais de 80% dos melanomas primários. As mutações têm sido documentadas em todos os subtipos de melanoma, incluindo os cutâneos, nos quais as mutações em BRAF e NRAS e KIT são mais comuns. OBJETIVOS: Identificar a frequência de mutações do gene BRAF (V600E, V600K), NRAS (códons 12, 13 e 61) e KIT (éxons 11, 13 e 17) em melanomas cutâneos primários e em suas respectivas metástases e avaliar sua relação com fatores prognósticos clínicos e anatomopatológicos para caracterizar sua relação com a progressão tumoral e seus efeitos na evolução da doença. MÉTODOS: Foi realizado um estudo de coorte. retrospectivo com 98 casos pareados de melanoma esporádico primário e metastático matriculados no Departamento de Câncer de Pele e Dermatologia do A.C.Camargo Cancer Center e atendidos de 2000 a 2013.. Para análise de BRAF e NRAS foi utilizado o método de pirosequenciamento e para KIT foi feito sequenciamento de Sanger, foram realizados também. imunoistoquímica de C-Kit e imunoistoquímica de anti-VE1. RESULTADOS: A maioria dos pacientes era caucasiana (93.9%) do sexo masculino (66.3%).. O maior grupo etário teve 60 anos ou mais (45.9%), com média de 57.36 anos (SD = 16.87). O local de maior incidência do primário foi tronco (29.6%). A maioria dos casos foi Extensivo superficial (45.9%), com ulceração (62.2%). A média de Breslow foi de 4.63 mm (SD = 3.7). A média do índice mitótico foi de 5.37 mitoses/mm² (SD = 6.08). Todos os pacientes tiveram ao menos uma metástase, sendo o linfonodo regional o local mais acometido. No fim do estudo a maioria foi estádio IV (68.4%). A média de seguimento foi de 37,29 meses (SD = 32,68) e 67.3% teve óbito por. melanoma. Para os tumores primários houve 53.1% de mutações em BRAF,10.2% em NRAS e 32.7% de mutações em KIT. Para as metástases encontrou-se 46.9% de mutações em BRAF, 9.2% de mutações em NRAS e 29.6% de mutações em KIT. CONCLUSÕES: As análises de concordância para BRAF ou NRAS mostraram-se substanciais e reprodutíveis Para KIT foi encontrada apenas ligeira concordância para o éxon 17. Análise de BRAF do tumor primário apresentou relação com localização do tumor (p = 0.03) e tipo histológico (p = 0,016). Análise de BRAF na metástase foi relacionada com idade (p = 0.033); local (p = 0.003) e tipo histológico (p = 0.011). NRAS no tumor primário apresentou relação com idade (p = 0.01) e infiltração peritumoral (p = 0,030). NRAS na metástase foi correlacionada com idade (p = 0,017) e gênero (p = 0.037). A análise de KIT nas metástases apresentou relação com ulceração (p = 0.020). Apenas um caso apresentou heterogeneidade tumoral em baixa frequência para NRAS. A imunoistoquímica de C-Kit não apresentou concordância com os achado de KIT em nenhum dos três éxons. O anticorpo anti-BRAF V600E (VE1)correlacionou-se perfeitamente aos achados encontrados nas análises por pirosequenciamento.

INTRODUCTION: The abnormal activation of MAP Kinase pathway is present in over 80% of primary melanomas. These mutations have been documented at all melanoma subtypes, especially cutaneous melanomas, in which mutations in BRAF and NRAS and KIT are more common. OBJECTIVES: To analyze the incidence of mutations in BRAF (V600E, V600K), NRAS (codons 12, 13 and 61) and KIT gene (exons 11, 13 and 17) in primary cutaneous melanomas and in their metastases and evaluate its relationship with clinical and pathological prognostic factors to characterize their relationship with tumor progression and its effects on disease progression. METHODS: We conducted a retrospective cohort study of 98 Lmatched cases of primary and metastatic melanoma registered at Department of Dermatology and Skin Cancer A.C.Camargo Cancer Center and catered from 2000 to 2013. BRAF and NRAS analysis was performed using the method of pyrosequencing and KIT was done by Sanger sequencing were also performed immunohistochemical C-Kit and anti-VE1 immunohistochemistry. RESULTS: The majority of patients were Caucasian (93.9%) and male (66.3%). The largest age group was 60 or older (45.9%) with a mean of 57.36 years (SD = 16.87). The site with the highest incidence of the primary tumor was trunk (29.6%). Most cases was superficial spreading (45.9%), with ulceration (62.2%). The mean Breslow thickness was 4.63 mm (SD = 3.7). The average mitotic index was 5.37 mitoses / mm² (SD = 6.08). All patients had at least one metastasis, and the regional lymph nodes most commonly affected. At the end of the study most of the patients were stage IV (68.4%). The average follow-up was 37.29 months (SD = 32.68) and 67.3% had died of melanoma. Primary tumors had 53.1% of mutations in BRAF, 10.2% of mutations in NRAS and 32.7% of mutations in KIT. For metastasis 46.9% had mutations in BRAF, 9.2% had mutations in NRAS and 29.6% had mutations in KIT. CONCLUSIONS: The concordance analysis for BRAF and NRAS proved to be substantial and reproducible. Regarding KIT was found only slight agreement for exon 17. BRAF primary tumor was correlated with site (p = 0.03) and histological type (p = 0.016). BRAF metastasis was correlated with age (p = 0.033); site (p = 0.003); histological type (p = 0.011). NRAS primary tumor was correlated with age (p = 0.01) and lymphocite infiltration (p = 0.030). NRAS metastasis was correlated with age (p = 0.017) and gender (p = 0.037). The KIT analysis in metastases were related to ulceration (p = 0.020). Only one case had tumor heterogeneity at low frequency for NRAS. C-Kit immunohistochemistry has not provided correlation with the KIT found in any of the three exons. The znti-BRAF V600E antibody (VE1) correlated perfectly to the findings in the analysis by pyrosequencing.