Comparison of next generation sequencing, SNaPshot assay and real-time polymerase chain reaction for lung adenocarcinoma EGFR mutation assessment.

BACKGROUND: The epidermal growth factor receptor (EGFR) mutation status assessment has become increasingly important given the significant impact of tyrosine kinase inhibitors in lung cancer management. Our aim was to compare real life operational characteristics for three EGFR mutation assays - two targeted approaches and a next generation sequencing (NGS) technique. METHODS: EGFR mutation status was assessed for lung adenocarcinoma samples (formalin fixed- paraffin embedded samples) using qPCR, SNaPshot and NGS (Ion Torrent™) techniques. RESULTS: A total of 15 high clinical significance mutations were identified by at least one technique from the total of 64 samples. All mutations were identified by the TaqMan qPCR technique while SNaPshot in conjunction with fragment analysis identified 11 EGFR mutations. The NGS approach followed by an automatic analysis using the default calling parameters identified 10 mutations from the SNaPshot/qPCR panel and other three insertions, five point mutations and 58 silent variants; manual data review identified all 15 high significance mutations. CONCLUSIONS: Performance was similar for high tumor content samples but careful data analysis and post hoc variant calling filter alterations were necessary in order to obtain robust results from low tumor content samples by NGS. NGS is able to generate a comprehensive mutational profile albeit at a higher cost and workload. Result interpretation should take into account not only general run parameters such as mean read depth but also relative coverage and read distribution; currently there is an acute need to define firm recommendations/standards concerning NGS data interpretation and quality control.

p53 dependent cell-cycle arrest triggered by chemotherapy in xenografted breast tumors.

The major long-term prognostic factor for breast cancer patients treated by first-line chemotherapy is response to treatment. We have previously shown that complete responses to high doses epirubicin-cyclophosphamide were observed only in human tumors bearing a TP53 mutation. Three xenografted human breast tumors, 2 of them with a TP53 mutation and one of them without, were studied for their immediate response to this drug association. Cell cycle, cellular senescence and cell death were characterized and quantified on tissue section before and after treatment. The TP53 wild-type tumor showed a strong early induction of senescence-like phenotype with overexpression of SA-beta-gal and p21(CIP1). In contrast both TP53 mutated tumors showed no sign of cell cycle arrest or senescence. Conversely, abnormal mitoses strongly increased, only in TP53 mutated tumors. Thus, in these in vivo models, epirubicin-cyclophosphamide treatment induces senescence-like features in TP53 wild-type tumor, likely accounting for cell cycle arrest and subsequent resistance to treatment. Conversely in TP53 mutated tumors, chemotherapy induces mitotic catastrophe and tumor death, accounting for complete response to this association exclusively in patients with TP53 mutated tumors.

[Matrix metalloproteinase 9 presence in adhesive intraperitoneal processes]. / Studiul prezentei metaloproteinazei 9 la nivelul proceselor aderentiale cronice intraperitoneale.

UNLABELLED: Matrix metalloproteinases (MMP) represents a zinc-dependent proteolytic enzyme multigenic family, directly involved in embryogenesis, some physiological and pathological processes, occurring in the adult organism. For physiological processes, MMP play roles in proliferation, tissue remodeling, wound healing, angiogenesis. AIM: The aim of the study was to analyze the MMP9 presence in chronic peritoneal adherences, following the correlation between the presence of this metalloproteinase and the evolution of the connective tissue remodeling process from intraperitoneal adherences. MATERIAL AND METHODS: 6 women formed the study group: 5 operated for gynecological pathologies (with harvested tissue fragments from the adherences) and one patient with harvested tissue from normal peritoneum. Fragments were fixed in 10% formalin and processed for the microscopic exam in routine staining and immunohistochemistry, using anti-MMP9 antibodies and a technique based on peroxidaze-antiperoxidase system. The immunohistochemical reaction was quantified by using image analysis software. RESULTS: The morphological structure of the adherences was mainly represented by fibrous tissue, sometimes associated with elements of muscle and adipose tissues. For the fibrous connective tissue, the collagen component was characterized by a fascicular organization with different orientations and with fibrillar aspect. Semiquantitative analysis showed increased levels for MMP9 in adherences by comparison with normal peritoneum (mean ration for the MMP9-positive area: 26.5% for the adherences versus 15.93% normal peritoneum). CONCLUSION: The intraperitoneal adherences with more than 1 year of evolution show an extension of the remodeling processes that can lead to the possible resolution.

[Comparative evaluation between MB/BacT system and Löwenstein-Jensen solid culture media for mycobacterial isolation]. / Evaluare comparativa a sistemului MB/BacT cu mediul Löwenstein-Jensen pentru izolarea micobacteriilor.

Tuberculosis is a public health issue in both developed and developing countries. Success of the treatment depend on the identification of patients with positive sputum smears, rapid confirmation of diagnosis in patients with negative microscopy and identification of mycobacterial strains with altered drug susceptibility. Data from the literature show that liquid culture media have a higher sensitivity for isolation mycobacteria than solid culture media as Löwenstein-Jensen. In our series inoculation on liquid media resulted in retrieval of a significant higher number of mycobacterial strains than on solid media (435 vs 250). The time needed to obtain a positive culture was also lower for liquid media (15.89 +/- 9 days, mean +/- standard deviation) than for solid media (27.77 +/- 10.13 days), p < 0.001. These differences were seen in both smear negative and smear positive cases. Culture in liquid media isolated more strains with altered drug susceptibility but this difference was not statistically significant.

[Arrhythmogenic right ventricular dysplasia]. / Displazia aritmogena a ventriculului drept.

Arrhythmogenic right ventricular dysplasia (ARVD) is a new form of cardiomyopathy probably more frequent than commonly reported. The incidence is unknown. ARVD is a heart muscle disorder of unknown cause that is characterised pathologically by fibro-fatty replacement of the right ventricular myocardium. It is a rare but important cause of sudden cardiac death in young, otherwise healthy persons. The right ventricle should be extensively sampled histologically in all cases of sudden unexpected death, especially those that are exercise related. The disease is often familial (about 30%) with an autosomal dominant inheritance.

[Hypertrophic cardiomyopathy--cause of sudden death]. / Cardiomiopatia hipertrofica--cauza de moarte subita.

Hypertrophic cardiomyopathy (CMH) is defined by the absence of left ventricular dilatation and the presence of myocardial hypertrophy that is not due to another recognised cause of hypertrophy such as systemic hypertension or aortic stenosis. The bizarre histological appearance of the myocardium is the hallmark of CMH. Myocytes and bundles of myocytes are malaligned and fibrosis may be extensive. Disarray is maximal in areas of macroscopic wall thickening. The condition is most easily recognized in a series of transverse, short axis slices across both ventricles. Histological examination of sections taken in this transverse plane at all three levels (high, mid, apical) including septum, anterior, posterior and lateral walls are needed. The authors report a case of cardiac sudden death to a 45 years old white man. The diagnosis was revealed intra vitam and it was confirmed post mortem.

[Primary pulmonary hypertension--morphologic study]. / Hipertensiunea pulmonara primara--studiu morfologic.

Primary pulmonary hypertension is characterized by elevation of pulmonary arterial pressure over 25 mm Hg with increasing of pulmonary vascular resistance. Primary pulmonary hypertension is the result of idiopathic narrowing of pulmonary arteries and extensive remodeling of the pulmonary vasculature in case of lack of the pulmonary interstitial disease, cardiac diseases, and pulmonary thromboembolism. The morphological study revealed the histological vascular elements characteristic in arterial hypertension (intimal proliferation, medial hypertrophy, plexiform and dilatative lesions) associated with recent and organized arterial thrombosis secondary to intimal lesions through a recent infectious process. Our study revealed the necessity of morphological diagnosis in the view of accurate diagnosis and adequate cure. After all, the prognosis of primary pulmonary hypertension remain poor.