KRAS insertions in colorectal cancer: what do we know about unusual KRAS mutations?

INTRODUCTION: KRAS mutations are negative predictors of the response to anti-EGFR therapy in colorectal carcinomas (CRCs). Point mutations in codons 12, 13, and 61 are the most common KRAS mutations in CRC. There are few reports on insertions in KRAS, and little is known about its ability to activate the RAS pathway. The scarcity of data regarding insertion frequencies and nucleotide additions in KRAS impedes the management of patients with such mutations. We present data on KRAS insertions in CRC and discuss a case. MATERIALS AND METHODS: Pyrosequencing and Sanger sequencing were performed to identify KRAS and BRAF mutations in paraffin-embedded samples of CRC. Expression of mismatch repair proteins was examined by immunohistochemistry. RESULTS: We detected a GGT insertion between codons 12 and 13 (c.36_37insGGT;p.G12_G13insG) in a CRC patient. We found that insertions in KRAS is very rare in CRC and that the most frequent type of insertion is c.36_37insGGT. CONCLUSIONS: KRAS gene insertions represent a diagnostic and clinical challenge due to the difficult and unusual pyrosequencing findings and the lack of information regarding its clinical impact.

Comprehensive Analysis of DNA Methylation and Prediction of Response to NeoadjuvantTherapy in Locally Advanced Rectal Cancer.

The treatment for locally advanced rectal carcinomas (LARC) is based on neoadjuvant chemoradiotherapy (nCRT) and surgery, which results in pathological complete response (pCR) in up to 30% of patients. Since epigenetic changes may influence response to therapy, we aimed to identify DNA methylation markers predictive of pCR in LARC patients treated with nCRT. We used high-throughput DNA methylation analysis of 32 treatment-naïve LARC biopsies and five normal rectal tissues to explore the predictive value of differentially methylated (DM) CpGs. External validation was carried out with The Cancer Genome Atlas-Rectal Adenocarcinoma (TCGA-READ 99 cases). A classifier based on three-CpGs DM (linked to OBSL1, GPR1, and INSIG1 genes) was able to discriminate pCR from incomplete responders with high sensitivity and specificity. The methylation levels of the selected CpGs confirmed the predictive value of our classifier in 77 LARCs evaluated by bisulfite pyrosequencing. Evaluation of external datasets (TCGA-READ, GSE81006, GSE75546, and GSE39958) reproduced our results. As the three CpGs were mapped near to regulatory elements, we performed an integrative analysis in regions associated with predicted cis-regulatory elements. A positive and inverse correlation between DNA methylation and gene expression was found in two CpGs. We propose a novel predictive tool based on three CpGs potentially useful for pretreatment screening of LARC patients and guide the selection of treatment modality.

Oral ulcerative lesions in a post-liver-transplantation patient.

Oral involvement is rarely found in histoplasmosis, except in its disseminated form, which is mostly observed in the severely immunocompromised host. Herein, we presented the case of a 36-year-old female with a previous history of liver transplant, who was hospitalized due to fever, chills, night sweats, diarrhea, and painful oral lesions over the last 3 days. The oral examination revealed the presence of painful shallow ulcers lined by a pseudomembrane in the gingiva and the soft and hard palate. The initial working diagnosis comprised cytomegalovirus reactivation or herpes simplex virus infection. The diagnostic work-up included incisional biopsies of the gingiva and the sigmoid colon. Both biopsies confirmed the diagnosis of histoplasmosis. Intravenous itraconazole was administered with significant improvement after 7 days. Although oral involvement is rare, histoplasmosis should be included in the differential diagnosis of oral lesions, particularly when the patient is immunosuppressed. This study reports a rare presentation of histoplasmosis involving the mucosa of the oral cavity and the colon.

Increased Levels of Genomic Instability and Mutations in Homologous Recombination Genes in Locally Advanced Rectal Carcinomas.

Pre-operative 5-fluoracil-based chemoradiotherapy (nCRT) is the standard treatment for patients with locally advanced rectal cancer (LARC). Patients with pathological complete response (pCR-0% of tumor cells in the surgical specimen after nCRT) have better overall survival and lower risk of recurrence in comparison with incomplete responders (pIR). Predictive biomarkers to be used for new therapeutic strategies and capable of stratifying patients to avoid overtreatment are needed. We evaluated the genomic profiles of 33 pre-treatment LARC biopsies using SNP array and targeted-next generation sequencing (tNGS). Based on the large number of identified genomic alterations, we calculated the genomic instability index (GII) and three homologous recombination deficiency (HRD) scores, which have been reported as impaired DNA repair markers. We observed high GII in our LARC cases, which was confirmed in 165 rectal cancer cases from TCGA. Patients with pCR presented higher GII compared with pIR. Moreover, a negative correlation between GII and the fraction of tumor cells remaining after surgery was observed (ρ = -0.382, P = 0.02). High HRD scores were detected in 61% of LARC, of which 70% were incomplete responders. Using tNGS (105 cancer-related genes, 13 involved in HR and 5 in mismatch repair pathways), we identified 23% of cases with mutations in HR genes, mostly in pIR cases (86% of mutated cases). In agreement, the analysis of the TCGA dataset (N = 145) revealed 21% of tumors with mutations in HR genes. The HRD scores were shown to be predictive of better response to PARP-inhibitors and platinum-based chemotherapy in breast and ovarian cancer. Our results suggest that the same strategy could be applied in a set of LARC patients with HRD. In conclusion, we identified high genomic instability in LARC, which was related to alterations in the HR pathway, especially in pIR. These findings suggest that patients with impaired HRD would clinically benefit from PARP-inhibitors and platinum-based therapy.

Locally advanced rectal cancer transcriptomic-based secretome analysis reveals novel biomarkers useful to identify patients according to neoadjuvant chemoradiotherapy response.

Most patients with locally advanced rectal cancer (LARC) present incomplete pathological response (pIR) to neoadjuvant chemoradiotherapy (nCRT). Despite the efforts to predict treatment response using tumor-molecular features, as differentially expressed genes, no molecule has proved to be a strong biomarker. The tumor secretome analysis is a promising strategy for biomarkers identification, which can be assessed using transcriptomic data. We performed transcriptomic-based secretome analysis to select potentially secreted proteins using an in silico approach. The tumor expression profile of 28 LARC biopsies collected before nCRT was compared with normal rectal tissues (NT). The expression profile showed no significant differences between complete (pCR) and incomplete responders to nCRT. Genes with increased expression (pCR = 106 and pIR = 357) were used for secretome analysis based on public databases (Vesiclepedia, Human Cancer Secretome, and Plasma Proteome). Seventeen potentially secreted candidates (pCR = 1, pIR = 13 and 3 in both groups) were further investigated in two independent datasets (TCGA and GSE68204) confirming their over-expression in LARC and association with nCRT response (GSE68204). The expression of circulating amphiregulin and cMET proteins was confirmed in serum from 14 LARC patients. Future studies in liquid biopsies could confirm the utility of these proteins for personalized treatment in LARC patients.

Oral ulcerative lesions in a post-liver-transplantation patient

Oral involvement is rarely found in histoplasmosis, except in its disseminated form, which is mostly observed in the severely immunocompromised host. Herein, we presented the case of a 36-year-old female with a previous history of liver transplant, who was hospitalized due to fever, chills, night sweats, diarrhea, and painful oral lesions over the last 3 days. The oral examination revealed the presence of painful shallow ulcers lined by a pseudomembrane in the gingiva and the soft and hard palate. The initial working diagnosis comprised cytomegalovirus reactivation or herpes simplex virus infection. The diagnostic work-up included incisional biopsies of the gingiva and the sigmoid colon. Both biopsies confirmed the diagnosis of histoplasmosis. Intravenous itraconazole was administered with significant improvement after 7 days. Although oral involvement is rare, histoplasmosis should be included in the differential diagnosis of oral lesions, particularly when the patient is immunosuppressed. This study reports a rare presentation of histoplasmosis involving the mucosa of the oral cavity and the colon.

Estudo da frequência da infecção pelo vírus de Epstein-Barr em adenocarcinomas gástricos, sua relação com o ciclo celular e sintases do óxido nítrico / Study of the frequency of infection by the Epstein-Barr virus in gastric adenocarcinomas, their relationship with the cell cycle and synthases of nitric oxide

O vírus de Epstein-Barr (EBV) está diretamente implicado na patogênese de uma variedade de neoplasias principalmente em alguns tipos de linfomas. Recentemente a associação de infecção pelo EBV e carcinomas tem sido demonstrada na literatura e a associação de EBV e carcinomas gástricos tem sido relatada. O EBV é um vírus da família dos herpesvirus que infecta linfócitos B e células epiteliais, conferindo às células um estado de latência, estabelecendo uma infecção persistente no hospedeiro. A associação entre a infecção pelo EBV e expressão de proteínas associadas à apoptose nos carcinomas gástricos já foi relatada na literatura, porém os resultados entre os diversos autores ainda são controversos. O óxido nítrico (NO, do inglês nitric oxide) é um importante agente bioativo e uma molécula sinalizante mediadora de diversas funções como vasodilatação, neurotransmissão e metabolismo do ferro. Na biologia tumoral, os seus efeitos são complexos e ambíguos. A atuação do NO e de suas sintases (NOS, do inglês nitric oxide synthases) tem sido demonstrada em vários tumores com implicação na carcinogênese, resposta imune, citotoxidade e angiogênese. Em nosso estudo, a presença de EBV, através de hibridação in situ foi pesquisada em 118 casos de carcinomas gástricos, utilizando-se transcritos de RNA (EBER-1). A comparação entre carcinomas gástricos EBV positivos e EBV negativos em relação à pesquisa de proteína de membrana latente (LMP-1), proteínas da família do Bcl-2 (Bcl-2, Bcl-x, Bak e Bax) e sintases do óxido nítrico (NOS1, NOS2 e NOS3) foi realizada. A presença de EBER-1 foi detectada em 9 dos 118 (7%) casos. A positividade para a LMP1 foi detectada em 5 dos 9 casos EBER-1 positivos. A expressão das proteínas associadas à apoptose, p53 e sintases do óxido nítrico (NOS 1, NOS2 e NOS3) estavam aumentadas de maneira geral nos carcinomas gástricos, mas não mostraram diferenças de expressão nos grupos de carcinomas gástricos EBV+ e EBV-. Em relação às características demográficas e histopatológicas, os carcinomas gástricos EBV+ apresentaram maior freqüência no sexo masculino, com localização na região da cárdia, sendo comuns no tipo difuso, acompanhados por infiltrado inflamatório linfocitário de moderada a intensa quantidade. Este estudo, portanto demonstrou a freqüência de infecção por EBV nos carcinomas gástricos, correlacionando-se com algumas características demográficas e histopatológicas que permitissem uma melhor distinção entre os grupos de carcinomas EBV+ e EBV-. A pesquisa de marcadores imuno-histoquímicos de proteínas associadas à apoptose, ciclo celular e sintases do óxido nítrico, mostrou que embora os grupos exibissem expressões semelhantes destas proteínas, pequenas diferenças de expressão puderam ser observadas. Estas diferenças poderiam representar um mecanismo de escape do controle da divisão celular e apoptose que permitiriam uma disseminação tumoral e resistência ao tratamento.

Epstein- Barr virus (EBV) is directly implicated in the pathogenesis of a variety of undifferentiated carcinomas and has also been identified in convencional adenocarcinomas of the stomach. EBV is an umbiquitous herpesvirus that infects and stablishes a persistent infection in the host. There have been several studies that compare the ocorrence of EBV in gastric carcinomas with apoptotic molecules, with disparated results. However, the roles of these apoptotic molecules in EBV+ gastric tumors need to been elucited. Nitric oxide (NO) is an important bioactive agent and signaling molecule that mediates a diverse array of actions such as vasodilatation, neurotransmission, and iron metabolism. There has been mounting evidence that NO acts as a carcinogen. Recent studies have examined the expression and activity of the NOS isoforms in human cancer and its relationship with angiogenesis, apoptosis and cell proliferation. In this study, 118 cases of gastric carcinomas were evaluated for the presence of EBV using a sensitive in situ hybridization assay targeting Epstein-Barr virus- encoded RNA 1 (EBER 1) transcripts. lmmunohistochemistry was performed to evaluate the expression of latent membrane protein-1 (LMP-1 ), proteins of the Bcl-2 family (Bcl-2, Bcl-x, Bax and Bak), p53 and NOS. Five cases of 9 EBER-1+ were positive for LMP1. EBER1 was detected in 9 of 118 (7o/o} of cases. The expression of apoptosis- associated proteins, p53 and NOS showed no correlation with EBV infection, except the expression of Bak, that was lower in cases EBER 1- positive than EBER 1- negative. Clinicopathological data of EBV+ showed male predominance, preferentially localization in the cardiac region, diffuse patterns accompany relatively strong lymphocyte infiltration. In conclusion, it is clear that EBV is associated with some cases of gastric carcinomas. EBER 1-positive carcinomas were characterized by male e dominance, preferential location in cardia region, prevalence in the diffuse type with intense lymphoid infiltrate. No association was showed with proteins of apoptosis (Bcl-2, Bcl-x , Bak and Bax). Expression of NOS and p53 also showed no correlation with EBV positivity. Some cases reveled immunoposivity cells for LMP1 latent membrane antigen. This suggests that the EBV-positive gastric carcinomas may have on oncogenic pathway similar to that of other EBV-associated carcinomas and is distinct from the EBVnegative gastric carcinomas.

Estudo da frequ�ncia da infec��o pelo v�rus de Epstein-Barr em adenocarcinomas g�stricos, sua rela��o com o ciclo celular e s�ntases do �xido n�trico / Study of frequency of infection with Epstein-Barr virus in gastric adenocarcinomas, its relationship with the cell cycle and nitric oxide synthase

O v�rus de Epstein-Barr (EBV) est� diretamente implicado na patog�nese de uma variedade de neoplasias como alguns tipos de carcinomas, principalmente os tipos indiferenciados, incluindo os carcinomas g�stricos. O EBV � um v�rus da fam�lia dos herpesvirus que infecta e estabelece uma infec��o persistente no hospedeiro. Recentemente, alguns estudos tem demonstrado a correla��o da presen�a do EBV em carcinomas g�stricos com mol�culas associadas � apoptose, por�m com resultados discordantes. O �xido n�trico (NO, do ingl�s nitric oxide) � um importante agente bioativo e uma mol�cula sinalizante mediadora de diversas fun��es como vasodilata��o, neurotransmiss�o e metabolismo do ferro. Na biologia tumoral, os seus efeitos s�o complexos e amb�guos. A atua��o do NO e de suas sintases (NOS, do ingl�s nitric oxide synthases) tem sido demonstrada em v�rios tumores com implica��o na carcinog�nese, resposta imune, citotoxidade e angiog�nese. Em nosso estudo, a presen�a de EBV, atrav�s de hibridiza��o in situ foi pesquisada em 118 casos de carcinomas g�stricos, utilizando-se transcritos de RNA (EBER-1). A compara��o entre carcinomas g�stricos EBV positivos e EBV negativos em rela��o � pesquisa de prote�na de membrana latente (LMP-1), prote�nas da fam�lia do Bcl-2 (Bcl-2, Bcl-x, Bak e Bax) e sintases do �xido n�trico (NOS1, NOS2 e NOS3) foi realizada. A presen�a de EBER-1 foi detectada em 9 dos 118 (7%) casos. A positividade para a LMP1 foi detectada em 6 dos 9 casos EBER-1 positivos. A express�o das prote�nas associadas � apoptose, p53 e sintases do �xido n�trico (NOS 1, NOS2 e NOS3) n�o mostraram associa��o com a infeC��O? pelo EBV, com exce��o da express�o de Bak, que mostrou menor express�o nos casos EBV positivos em rela��o aos casos EBV negativos. Em rela��o aos achados cl�nicos e histol�gicos, os casos EBV+ apresentaram maior freq��ncia no sexo masculino, localiza��o na regi�o da c�rdia, sendo do tipo difuso e acompanhados por infiltrado inflamat�rio linfocit�rio de moderada a intensa quantidade...(AU)