Genetic screening and molecular characterization of <Emphasis Type="Italic">MET</Emphasis> alterations in non-small cell lung cancer

Purpose

Aberrant activation of MET as a result of exon 14-skipping (METex14) mutations or gene amplification is an oncogenic mechanism in non-small cell lung carcinoma (NSCLC) and a potential therapeutic target. The purpose of this study was to characterize MET alterations in a cohort of NSCLC patients treated with surgery.

Methods and patients

157 NSCLCs of various histopathologies, including pulmonary sarcomatoid carcinomas (PSC), were tested for MET alterations. METex14 mutations, MET copy number alterations and the levels of MET protein were determined by Sanger sequencing, fluorescence in situ hybridization and immunohistochemistry, respectively. Concurrent alterations of other important cancer genes and immunostaining of the downstream effector, phopho-S6, were also determined.

Results

METex14 mutations and MET amplification were detected in seven tumors. MET genetic alterations were found predominantly in the lung adenocarcinoma (ADC) and PSC histopathologies. High levels of MET protein were found in most MET-amplified tumors, but not in all METex14-mutated tumors. Strong phopho-S6 staining was observed in about half of the MET-activated tumors. One tumor with METex14 exhibited concurrent ERBB2 amplification.

Conclusions

MET activation, by either METex14 mutations or amplification, is characteristic of a subset of early stage NSCLCs and may coexist with ERBB2 amplification. This may have potential therapeutic implications. The presence of METex14 mutations was associated with low levels of MET protein, which may limit the use of total MET immunostaining as a marker for preselecting patients for MET-targeted therapies.

     

Predictors of favourable outcome in inflammatory Crohn's disease. A retrospective observational study

Background

No studies have specifically searched for predictors of a favourable outcome that would allow a conservative therapeutic approach in adult Crohn's disease (CD).

Aims

To identify predictors of a favourable disease course over time at CD diagnosis.

Methods

We identified and included all patients diagnosed with CD between January 1994 and December 2003, who had CD with an inflammatory pattern and no perianal disease at diagnosis, and who were followed up for at least 5 years. Clinical and therapeutic features until December 2008 and losses to follow-up were identified. We defined a favourable outcome as the absence of stricturing and penetrating complications of the disease (including perianal disease), together with the absence of need for anti-TNF therapy or resectional surgery during follow up.

Results

One hundred and forty-five patients were included and followed up for a median of 96 months (IQR, 79–140). At diagnosis, location was ileal in 39%, colonic in 28%, and ileocolonic in 32%; 50% of the patients were active smokers, and 41% used immunomodulators. Eighty-two patients (57%) met the criteria for a favourable outcome at the end of follow-up. The only factor associated with a favourable outcome was isolated colonic involvement (P = 0.022), with 73% of these patients meeting the criteria for a favourable outcome.

Conclusions

A favourable outcome of initially uncomplicated CD is not easily predicted at disease diagnosis by means of clinical or epidemiologic factors. Nevertheless, patients with isolated colonic disease are less likely to have an aggressive course.     

Branch migration during Rad51-promoted strand exchange proceeds in either direction

The Saccharomyces cerevisiae Rad51 protein is important for genetic recombination and repair of DNA double-strand breaks in vivo and can promote strand exchange between linear double-stranded DNA and circular single-stranded DNA in vitro. However, unlike Escherichia coli RecA, Rad51 requires an overhanging complementary 3′ or 5′ end to initiate strand exchange; given that fact, we previously surmised that the fully exchanged molecules resulted from branch migration in either direction depending on which type of end initiated the joint molecule. Our present experiments confirm that branch migration proceeds in either direction, the polarity depending on whether a 3′ or 5′ end initiates the joint molecules. Furthermore, heteroduplex DNA is formed rapidly, first at the overhanging end of the linear double-stranded DNA’s complementary strand and then more slowly by progressive lengthening of the heteroduplex region until strand exchange is complete. Although joint molecule formation occurs equally efficiently when initiated with a 3′ or 5′ overhanging end, branch migration proceeds more rapidly when it is initiated by an overhanging 3′ end, i.e., in the 5′ to 3′ direction with respect to the single-stranded DNA.     

6-mercaptopurine in patients with inflammatory bowel disease and previous digestive intolerance of azathioprine

OBJECTIVE: Azathioprine and 6-mercaptopurine are useful therapies in inflammatory bowel diseases. Despite their efficacy, their use is limited owing to treatment intolerance or toxicity in 10-15% of patients. It has been suggested that both drugs could be interchangeable. MATERIAL AND METHODS: All patients treated with 6-mercaptopurine because of previous digestive intolerance of azathioprine in four Spanish hospitals were reviewed. Tolerance of 6-mercaptopurine therapy was assessed. RESULTS: Fifteen patients (11 Crohn's disease, 4 ulcerative colitis) were included. Immunosuppressant therapy was prescribed for steroid-dependent disease in 13 cases, and for perianal disease in 2. Main symptoms of digestive intolerance were epigastric pain, nausea and vomiting, which developed within the first weeks of treatment. Acute pancreatitis was ruled out in all the cases. Five patients commenced 6-mercaptopurine immediately after azathioprine discontinuation and 7 patients within the first month. Eleven patients (73.3%) tolerated 6-mercaptopurine and reached the therapeutic goals; only two patients had to discontinue 6-mercaptopurine because of adverse effects. CONCLUSIONS: Treatment with 6-mercaptopurine is a safe alternative in patients with inflammatory bowel diseases and previous digestive intolerance of azathioprine.     

Nosocomial transmission of HCV in the liver unit of a tertiary care center

Despite its medical and legal implications, there are no prospective studies analyzing the incidence and mechanisms involved in the nosocomial transmission of hepatitis C virus (HCV) in liver units. This study prospectively investigates the nosocomial transmission of HCV in the liver unit of a tertiary care center from August 2000 to October 2002. The median prevalence of HCV infection among hospitalized patients was 50%. Anti-HCV-negative patients admitted to the liver unit during the study period were prospectively followed, and serum markers of HCV infection were repeated 6 months after discharge. All known risk factors for HCV transmission (including the physical allocation of HCV-infected and noninfected patients during hospitalization) were recorded. Complete follow-up data were available in 1301 (84.5%) of 1540 patients. Six patients (0.46%) acquired HCV infection (annual incidence: 0.27/100 admissions). Phylogenetic analyses of recovered HCV sequences identified the source of infection as an HCV-infected roommate (3 cases) and a patient receiving care by the same nurse team (1 case). The most relevant risk factors associated with HCV acquisition were duration of hospitalization (>10 days; OR, 35; 95% CI, 1.96-622) and hospitalization with an HCV-infected roommate (>5 days; OR, 12; 95% CI, 1.39-103). In fact, HCV infection occurred in 1.7% of the 357 patients hospitalized longer than 10 days. In conclusion, HCV nosocomial infection appears to occur via patient-to-patient transmission in liver units, particularly in individuals who require long hospitalizations. Continuous reinforcement of universal prevention measures and, when possible, isolation of patients at higher risk might further reduce nosocomial HCV transmission.     

Repositioning chloroquine and metformin to eliminate cancer stem cell traits in pre-malignant lesions

Ideal oncology drugs would be curative after a short treatment course if they could eliminate epithelium-originated carcinomas at their non-invasive, pre-malignant stages. Such ideal molecules, which are expected to molecularly abrogate all the instrumental mechanisms acquired by migrating cancer stem cells (CSCs) to by-pass tumour suppressor barriers, might already exist. We here illustrate how system biology strategies for repositioning existing FDA-approved drugs may accelerate our therapeutic capacity to eliminate CSC traits in pre-invasive intraepithelial neoplasias. First, we describe a signalling network signature that overrides bioenergetics stress- and oncogene-induced senescence (OIS) phenomena in CSCs residing at pre-invasive lesions. Second, we functionally map the anti-malarial chloroquine and the anti-diabetic metformin (“old drugs”) to their recently recognized CSC targets (“new uses”) within the network. By discussing the preclinical efficacy of chloroquine and metformin to inhibiting the genesis and self-renewal of CSCs we finally underscore the expected translational impact of the “old drugs–new uses” repurposing strategy to open a new CSC-targeted chemoprevention era.     

SNP discovery in pooled samples with mismatch repair detection

A targeted discovery effort is required to identify low frequency single nucleotide polymorphisms (SNPs) in human coding and regulatory regions. We here describe combining mismatch repair detection (MRD) with dideoxy terminator sequencing to detect SNPs in pooled DNA samples. MRD enriches for variant alleles in the pooled sample, and sequencing determines the nature of the variants. By using a genomic DNA pool as a template, approximately 100 fragments were amplified and subsequently combined and subjected en masse to the MRD procedure. The variant-enriched pool from this one MRD reaction is enriched for the population variants of all the tested fragments. Each fragment was amplified from the variant-enriched pool and sequenced, allowing the discovery of alleles with frequencies as low as 1% in the initial population. Our results support that MRD-based SNP discovery can be used for large-scale discovery of SNPs at low frequencies in a population.