EGFR and KRAS mutational profiling in fresh non-small cell lung cancer (NSCLC) cells

Purpose

Knowledge of tumor mutational status has become a priority for effective NSCLC-tailored treatment. NSCLC diagnosis is more often reached through biopsy; thus, there is a clear need to implement for routine tumor molecular profiling on small cytological samples. This work aims to screen and compare the EGFR and KRAS mutational prevalence in fresh tumor cells and in corresponding routinely processed samples derived from trans-thoracic fine-needle aspiration. The latter currently represents the most appropriate diagnostic procedure in case of peripheral lesions, such as adenocarcinomas, which account for almost 40 % of all NSCLCs and for the highest EGFR mutational rates.

Methods

Two hundred and forty-four patients carrying peripheral lung masses underwent CT-guided aspiration. The obtained material was split, and a part was addressed to conventional histopathological analysis while the remaining one was stored at ?20 °C. In case of confirmation of adenocarcinoma, tumor genomic DNA was extracted from both fresh and fixed material, and EGFR and KRAS sequencing was performed.

Results

We identified 136 adenocarcinomas; from 134, we could recover enough material for the study. A full match was demonstrated between EGFR/KRAS mutational prevalences through the two approaches tested. We found EGFR mutations in 13 patients (9.7 %); 7 were females and 11 never or former smokers. KRAS mutations occurred in 20 (14.9 %) patients. EGFR and KRAS mutations were mutually exclusive.

Conclusions

Mutational screening on fresh cancer cells is an achievable, safe and cost-effective procedure which might allow routinely tumor molecular profiling as powerful integration of conventional histopathological analysis.     

Prognostic relevance of microsatellite instability in pT3N0M0 colon cancer: a population-based study

Although surgery alone represents a curative approach for patients with pT3N0M0 colon cancer, about 15–20 % of these patients develop a relapse of disease. Microsatellite instability (MSI) is one of the most important molecular markers in colorectal cancer. The aim of this study was to investigate the prognostic relevance of MSI in all pT3N0M0 tumors recorded in the Cancer Registry of the Province of Modena—(Northern Italy) within the 2002–2006 period in patients who showed a relapse of disease during the 5-year period of follow-up (59 cases). They were compared to 59 controls similar in clinical and pathological features but with good prognosis. None of the subjects received adjuvant chemotherapy. MSI status was tested using BAT25, BAT26, NR24, and CAT25 fluorescent-labeled mononucleotide markers. The overall prevalence of MSI was 12.7 % (15 of 118 cases). MSI was detected mainly in mucinous adenocarcinoma (p < 0.003), in high-grade tumors (p < 0.008), in right-sided neoplasms (p < 0.005), and in patients with a better prognosis, though the difference was not statistically significant (11/59 patients ?18.6 % vs 4/59 patients ?6.7 %; OR 0.36 CI 95 % 0.11–1.15; p = 0.08). However, in multivariate analysis, MSI status becomes the strongest independent factor associated with relapse (OR 0.21, CI 95 % 0.06–0.81; p = 0.023), together with mucinous histological type (OR 0.40, CI 90 % 0.18–0.92). MSI is a relevant prognostic factor in stage pT3N0M0 colon cancer suitable to discriminate those patients with a high risk of relapse.     

Intracellular detection of interleukin 17 and other cytokines in human bronchoalveolar lavage fluid: a first assessment

Increasing evidence links pulmonary pathology to cytokines determining an inflammatory environment in the lung. Detection of cells secreting specific cytokines in BALF could be helpful as a diagnostic tool but which cytokines to choose among their great variety may be the first question to solve. The aim of this study was to investigate the Th1, Th2 and Th17 cytokine profile in whole cells within the human bronchoalveolar lavage fluid (BALF) by flow cytometry, with a focus on interleukin (IL)-17-producing cells, in order to assess which cytokines might lend themselves as markers of disease in future studies. BALF and paired peripheral blood samples were collected from 52 patients admitted to hospital for pulmonary pathologies. Cells obtained from BALF and peripheral blood were incubated in vitro in the absence or presence of appropriate stimuli and analyzed for intracellular content of IL-4, -10, -12, -17, interferon (IFN)γ and tumor necrosis factor (TNF)α in association to expression of either HLA-DR or CD4. IL-17-secreting cells were further characterized.Production of IL-17 by unstimulated BALF cells could be detected in 2 of the 32 patients that could be examined; upon PMA/IM stimulation in vitro, IL-17 was produced by varying percentages of lymphocytes, mostly memory CD4⁺ cells, in all BALF samples. IL-4 could be detected in a relatively high proportion of unstimulated HLA-DR^+/−, SSC^hi cells, most probably granulocytes; IL-10 could be found mostly in macrophages in a number of the BALF samples analyzed. Finally, IFNγ and TNFα were only produced by lymphocytes after in vitro stimulation.This study shows that T cells producing IL-17 can be found in the lung of respiratory patients in the absence of ex vivo stimulation, making IL-17 a good candidate marker of specific pathologies of the lung. Upon stimulation, IL-17 production was accounted for by CD4⁺ CD45RO⁺ cells. Other cytokines are also discussed. An interesting cytokine secretion profile found in BALF from a patient with rheumatoid lung disease is also reported.     

Serum interleukin-6 (IL-6), IL-10, tumor necrosis factor (TNF) alpha, soluble type II TNF receptor, and transforming growth factor beta levels in human immunodeficiency virus type 1-infected individuals with Mycobacterium avium complex disease

To characterize changes in serum cytokine levels in human immunodeficiency virus type 1 (HIV-1)-infected persons with Mycobacterium avium complex (MAC) bacteremia, the levels of IL-1alpha (interleukin-1alpha), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), soluble type II TNF receptor (sTNF-RII), and transforming growth factor beta (TGF-beta) in serum were measured in two cohorts of HIV-1-infected persons with MAC bacteremia. The first cohort was part of a MAC prophylaxis study. Patients with bacteremia were matched with controls without bacteremia. Elevated IL-6, IL-10, TNF-alpha, sTNF-RII, and TGF-beta levels were noted at baseline for all subjects, a result consistent with advanced HIV-1 disease. IL-1alpha was not detected. No differences in cytokine levels in serum were noted at baseline and at the time of bacteremia between patients with MAC and controls. In the second cohort, subjects had serum samples collected at the time of MAC bacteremia and thereafter while on macrolide therapy. Serum samples at time of bacteremia were collected from HIV-1-infected persons at a time when neither highly active antiretroviral therapy (HAART) nor MAC prophylaxis was used routinely. MAC treatment resulted in decreased levels of IL-6 and TNF-alpha in serum, which were evident for IL-6 by 4 to 6 weeks and for TNF-alpha by 8 to 16 weeks. Thus, antibiotic treatment for MAC results in decreased levels of IL-6 and TNF-alpha in serum in HIV-1-infected persons who are not on HAART.     

Absence of coding mutations in the X-linked genes neuroligin 3 and neuroligin 4 in individuals with autism from the IMGSAC collection.

Neuroligin abnormalities have been recently implicated in the aetiology of autism spectrum disorders (ASD), given the finding of point mutations in the two X-linked genes NLGN3 and NLGN4X and the important role of neuroligins in synaptogenesis. To enquire on the relevance and frequency of neuroligin mutations in ASD, we performed a mutation screening of NLGN3 and NLGN4X in a sample of 124 autism probands from the International Molecular Genetic Study of Autism Consortium (IMGSAC). We identified a new non-synonymous variant in NLGN3 (Thr632Ala), which is likely to be a rare polymorphism. Our data indicate that coding mutations in these genes are very rarely associated to ASD.     

Evaluation of viral load in infants hospitalized with bronchiolitis caused by respiratory syncytial virus

The relationship between viral load, disease severity and antiviral immune activation in infants suffering from respiratory syncytial virus (RSV)-associated bronchiolitis has not been well identified. The main objective of this study was to determine the existence of a correlation between RSV load and disease severity and also between different clinical markers and mRNA levels of the interferon stimulated gene (ISG)56 in infants hospitalized for bronchiolitis. We also evaluated whether viral load tended to be persistent over the course of the RSV infection. The levels of RSV-RNA were quantified in nasopharyngeal washings, collected from 132 infants infected with RSV as a single (90.15%) or as a dual infection with other respiratory viruses (9.85%). Results indicated that viral load was positively related to the clinical severity of bronchiolitis, the length of hospital stay, the levels of glycemia and the relative gene expression of ISG56, whereas an inverse correlation was observed with the levels of hemoglobin. We also found that the RSV load significantly decreased between the first and second nasopharingeal washings sample in most subjects. These results suggest that infants with high RSV load on hospital admission are more likely to have both more severe bronchiolitis and a higher airway activation of antiviral immune response.     

Developmental regulation of type 1 and type 3 interferon production and risk for infant infections and asthma development

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Ala12Ala genotype of the peroxisome proliferator-activated receptor gamma2 protects against atherosclerosis

A mutation in the peroxisome proliferator-activated receptor gamma2 (PPARgamma2) gene with a cytosine to guanine substitution results in an exchange of proline (Pro) with alanine (Ala) in exon B (codon 12) of this gene. This polymorphism has been associated with high insulin sensitivity and low body weight, but no data have been published to date about its effect on early atherosclerosis. We investigated the relationship of the Pro(12)Ala polymorphism to early atherosclerosis, measured by the intima-media thickness (IMT). A total of 622 subjects were included, aged 40-70 yr, who were participants of the RIAD (Risk factors in Impaired glucose tolerance for Atherosclerosis and Diabetes) study and were at risk of developing type 2 diabetes. Altogether, 449 of the subjects had the common genotype (Pro(12)Pro), 162 had the Pro(12)Ala genotype, and 11 the Ala(12)Ala genotype. IMT was significantly decreased in subjects with the Ala(12)Ala genotype compared with subjects with the other two genotypes. Body mass index, free fatty acid levels, and leukocyte count were lower in subjects with the Ala(12)Ala genotype compared with subjects with the Pro(12)Pro or Pro(12)Ala genotypes. In multivariate analysis, the Ala(12)Ala genotype was a significant independent determinant of IMT. Furthermore, we demonstrated specific expression of the PPARgamma2 gene in human atherosclerotic lesions as well as in cultured primary macrophages and foam cells. In conclusion, our data suggest that the Ala(12)Ala genotype of the PPARgamma2 gene may protect from early atherosclerosis in subjects at risk for diabetes.