Agata Gizycka

[Programmed necrosis and necroptosis - molecular mechanisms].

Programmed necrosis has been proven vital for organism development and homeostasis maintenance. Its regulatory effects on functional activity of the immune system, as well as on pathways regulating the death mechanisms in cells with diminished apoptotic activity, including malignant cells, have been confirmed. There is also increasing evidence indicating necrosis involvement in many human pathologies. Contrary to previous beliefs, necrosis is not only a passive, pathological, gene-independent process. However, the current knowledge regarding molecular regulation of programmed necrosis is scarce. In part this is due to the multiplicity and complexity of signaling pathways involved in programmed necrosis, as well as the absence of specific cellular markers identifying this process, but also the ambiguous and imprecise international terminology. This review presents the current state of the art on molecular mechanisms of programmed necrosis. In particular, its specific and frequent form, necroptosis, is discussed. The role of RIP1 and RIP3 kinases in this process is presented, as well as the diverse pathways induced by ligation of tumor necrosis factor α, to its receptor, TNFR1, i.e. cell survival, apoptosis or necroptosis.

RT-PCR and IHC as the alternative methods for detection of ALK aberrations in NSCLC samples - The feasibility study

IHC assay based on anti-ALK D5F3 antibodies clone was reported as reliable, time- and cost-effective diagnostic alternative to FISH 9gold standard9 method for detection of ALK aberrations in NSCLC. We aimed at evaluating the potential applicability of RT-PCR assay for ALK gene rearrangement identification. Reliability of two RT-PCR methods detecting relative expression of 39 ALK mRNA transcript was estimated (method by Wang (I) and/or by Gruber (II) [1,2] and compared to D5F3 antibody-based ALK IHC assay. ALK IHC-positive materials were reassessed with FISH. Total of 89 fresh-frozen and/or FFPE samples from 65 NSCLC patients were analysed. In FFPE samples, viable results were produced only by RT-PCR (II) assay based on shorter amplicons [2]. Still, 11/24 of FFPE samples (46%) were not eligible for diagnostics due to technical reasons. RT-PCR (II) and IHC presented low conformity of results (Cohen9s Kappa=0.262, 95%CI=0.23-0.294; Overall Percent Agreement (OPA) = 81.1% (95%CI=68.5%-93.7%). 37 fresh-frozen and 4 FFPE materials analyzed by both RT-PCR assays presented low Positive Percent Agreement (PPA) =57% (95%CI=20.5%-93.8%) and Cohen9s Kappa=0.429 (95%CI=0,395-0.464). There were 7/63 ALK IHC positive results (11%), 4 of which (57%) were confirmed and other 3 technically unevaluable by FISH. RT-PCR assay presented low reliability and reproducibility, particularly in FFPE materials. Meanwhile, IHC assay with ALK D5F3 antibodies proved robust, provided high conformity with reference method (FISH) and lower rate of unevaluable results in FFPE NSCLC samples. [1] Wang et al., Clin Cancer Res, 2012 [2] Gruber et al., JTO, 2014.

P21. Immunohistochemistry versus DNA testing for detection of EGFR mutations and ALK rearrangements in lung adenocarcinoma patients

Background The molecular diagnostics of epidermal growth factor receptor (EGFR) mutations and ALK rearrangements in non-small cell lung cancer (NSCLC) is currently based on DNA assays. We evaluated the diagnostic effectiveness of immunohistochemistry (IHC) with monoclonal antibodies specific for mutant EGFR and ALK proteins in lung adenocarcinoma samples.

O10. Allele-specific real-time PCR detection of EGFR exon 19 and 21 mutations in various clinical non-small cell lung cancer specimens.

Background The aim of our study was to evaluate the effectiveness of epidermal growth factor receptor (EGFR) gene mutation detection in diverse materials from 707 non-small cell lung cancer (NSCLC) patients using the two ultra-sensitive allele-specific real-time polymerase chain reaction (PCR) assays in the routine diagnostic procedure.

Apoptosis of alveolar lymphocytes. Part 1: pathways of lymphocyte apoptosis

Apoptosis is a form of programmed cell death essential for maintaining homeostasis, including onset, progress and resolution of immune reactions. Two major apoptosis pathways: extrinsic (mediated by death receptors) and intrinsic (mitochondrial), were distinguished. Lymphocytes with cytotoxic activity may also initiate apoptosis of target cells by granzyme/perforin (pseudoreceptor) pathway. The specific apoptotic processes, i.e. activation induced cell death (AICD) and neglect induced death (NID), are types of extrinsic and intrinsic pathways, respectively. They both seem to be crucial in apoptosis of antigen-primed T cells during the contraction phase of inflammation. Alveolar lymphocytes (AL) are almost exclusively T effector cells, engaged in interstitial lung disease (ILD) pathophysiologies. The AL numbers in lower airways depends on recruitment to the lung, proliferation and local apoptosis. According to the references, it should be noted that AL usually do not proliferate in alveoli; their apoptosis rate accounts, on average, for 1% of cells in healthy subjects, and this is significantly decreased in disorders with lymphocytic alveolitis such as sarcoidosis and extrinsic allergic alveolitis (EAA). The mechanisms of AL apoptosis have not been completely explained. However, it is the NID process that is probably critical for the culling of T-cell response, as in EAA or sarcoidosis remission, with AICD as an auxiliary and/or modulating mechanism only. It should be emphasised that many ILDs are chronic disorders with no remission or improvement, and it is difficult to describe the AL response in terms of immune expansion/contraction.