Validation of Pyrosequencing for the Analysis of KRAS Mutations in Colorectal Cancer

The use of antibodies against epidermal growth factor receptor( EGFR) in conjunction with conventionalchemotherapy for metastatic colorectal cancer (CRC) in patients with KRAS wild-type tumors has beenproven to be efficacious. Recently, KRAS testing prior to anti-EGFR therapy has become mandatory formetastatic CRC patients. Although newly developed pyrosequencing is expected to be one of the highthroughput procedures detecting such mutations, the accuracy of the procedure has not been well evaluated.In the present study, we aimed to validate the accuracy, especially the potential for a false-negative result,in detecting KRAS mutations by pyrosequencing using cultured tumor cells. DNA extracted from culturedìNOZî gallbladder cancer cells( known to contain KRAS mutation G12V) at concentrations of 1%, 5%, 10%, and 25%, as well as 2 DNA samples extracted from a resected CRC specimen( known to contain anotherKRAS mutation, G12C) at concentrations of 5% and 25%, were prepared. We analyzed KRAS mutationalstatus and nonexistent and/or nonfunctional mutations of these 6 samples using pyrosequencing. TheKRAS mutation detection rates in the 4 NOZ samples( 1%, 5%, 10%, and 25%) were 0.37%, 2.79%, 5.28%,and 13.85%, respectively. Some artifacts of KRAS mutations unlikely to be present were detected in 1%samples of NOZ at a rate similar to that of the G12V mutation( G12C, 0.29%;G13C, 0.42%). Although theKRAS mutation G12C was detected at rates of 1.26% and 6.49% in samples with 5% and 25% DNA extractedfrom resected CRC specimen, respectively, no other type of KRAS mutation was detected in suchsamples. Pyrosequencing could not detect KRAS mutations correctly in the sample containing 1% DNA.This might cause false negatives. A sample mutated DNA concentration of at least 5% was necessary forprecise analyses by this procedure

Cavin‐2 promotes fibroblast‐to‐myofibroblast trans‐differentiation and aggravates cardiac fibrosis

Abstract Aims Transforming growth factor β (TGF‐β) signalling is one of the critical pathways in fibroblast activation, and several drugs targeting the TGF‐β/Smad signalling pathway in heart failure with cardiac fibrosis are being tested in clinical trials. Some caveolins and cavins, which are components of caveolae on the plasma membrane, are known for their association with the regulation of TGF‐β signalling. Cavin‐2 is particularly abundant in fibroblasts; however, the detailed association between Cavin‐2 and cardiac fibrosis is still unclear. We tried to clarify the involvement and role of Cavin‐2 in fibroblasts and cardiac fibrosis. Methods and results To clarify the role of Cavin‐2 in cardiac fibrosis, we performed transverse aortic constriction (TAC) operations on four types of mice: wild‐type (WT), Cavin‐2 null (Cavin‐2 KO), Cavin‐2flox/flox, and activated fibroblast‐specific Cavin‐2 conditional knockout (Postn‐Cre/Cavin‐2flox/flox, Cavin‐2 cKO) mice. We collected mouse embryonic fibroblasts (MEFs) from WT and Cavin‐2 KO mice and investigated the effect of Cavin‐2 in fibroblast trans‐differentiation into myofibroblasts and associated TGF‐β signalling. Four weeks after TAC, cardiac fibrotic areas in both the Cavin‐2 KO and the Cavin‐2 cKO mice were significantly decreased compared with each control group (WT 8.04 ± 1.58% vs. Cavin‐2 KO 0.40 ± 0.03%, P < 0.01; Cavin‐2flox/flox, 7.19 ± 0.50% vs. Cavin‐2 cKO 0.88 ± 0.44%, P < 0.01). Fibrosis‐associated mRNA expression (Col1a1, Ctgf, and Col3) was significantly attenuated in the Cavin‐2 KO mice after TAC. α1 type I collagen deposition and non‐vascular αSMA‐positive cells (WT 43.5 ± 2.4% vs. Cavin‐2 KO 25.4 ± 3.2%, P < 0.01) were reduced in the heart of the Cavin‐2 cKO mice after TAC operation. The levels of αSMA protein (0.36‐fold, P < 0.05) and fibrosis‐associated mRNA expression (Col1a1, 0.69‐fold, P < 0.01; Ctgf, 0.27‐fold, P < 0.01; Col3, 0.60‐fold, P < 0.01) were decreased in the Cavin‐2 KO MEFs compared with the WT MEFs. On the other hand, αSMA protein levels were higher in the Cavin‐2 overexpressed MEFs compared with the control MEFs (2.40‐fold, P < 0.01). TGF‐β1‐induced Smad2 phosphorylation was attenuated in the Cavin‐2 KO MEFs compared with WT MEFs (0.60‐fold, P < 0.01). Heat shock protein 90 protein levels were significantly reduced in the Cavin‐2 KO MEFs compared with the WT MEFs (0.69‐fold, P < 0.01). Conclusions Cavin‐2 loss suppressed fibroblast trans‐differentiation into myofibroblasts through the TGF‐β/Smad signalling. The loss of Cavin‐2 in cardiac fibroblasts suppresses cardiac fibrosis and may maintain cardiac function

Recurrent malignant peritoneal mesothelioma treated by a second resection: A case report

Key Clinical Message Malignant peritoneal mesothelioma, a rare and poor prognosis disease, is seldom treated surgically, especially for recurrence. However, early diagnosis and aggressive treatment of primary and recurrent tumors can achieve long‐term patient survival. Abstract Malignant peritoneal mesothelioma (MPM) is a rare and aggressive tumor, and rarely indicated for surgery, especially for recurrence. In the present case, we report a rare case who could survive long‐term after two surgeries in 4 years for MPM