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Background:Activating epidermal growth factor receptor (EGFR) gene mutations can be successfully treated by EGFR tyrosine kinase inhibitors (EGFR-TKIs), but nearly 50% of all patients' exhibit progression of the disease until treatment because of T790M mutations. It is proposed that this is mostly caused by therapy-resistant tumor clones harboring a T790M mutation. Until now no cost-effective routine-diagnostic method for EGFR-resistance mutation status analysis is available leaving long-time response to TKI treatment to chance. Unambiguous identification of T790M EGFR mutations is mandatory to optimize initial treatment strategies.Materials and Methods:Artificial EGFR T790M mutations and human wild-type gDNA were prepared in several dilution series. Preferential amplification using coamplification at lower denaturation temperature-PCR (COLD-PCR) of the mutant sequence and subsequent HybProbe melting curve detection or pyrosequencing were performed in comparison to normal processing.Results:COLD-PCR-based amplification allowed the detection of 0.125% T790M mutant DNA in a background of wild-type DNA in comparison to 5% while normal processing. These results were reproducible.Conclusions:COLD-PCR is a powerful and cost-effective tool for routine diagnostic to detect underrepresented tumor clones in clinical samples. A diagnostic tool for unambiguous identification of T790M-mutated minor tumor clones is now available enabling optimized therapy.

Background: Lung cancer still remains the leading cause of cancer for men after prostate cancer and breast cancer for women. Angiogenesis is considered a major microenvironment modifier. Material and Methods: Demographic data and study design; The study is based on a collective of twenty representative specimens of each tumour entity (Typical Carcinoid, Atypical Carcinoid, Large-Cell Neuroendocrine Carcinoma, Small Cell Lung Cancer) for mRNA expression analysis. The following methods were performed: RNA Extraction and RNA Integrity Assessment, NanoString CodeSet Design and Expression Quantification, NanoString Data Processing and Statistical Analysis. Results: KDR rendered significant association to aggressiveness of the tumour and decreases with increasing malignancy (p=0.049). A decreased expression of HIF1A and KDR mRNA as associated with a higher risk of tumour invasion in vessels (HIF1A: p=0.034; KDR: p=0.029). FIGF and HIF1A expression levels are significantly associated with progression-free survival (FIGF: p=0.021; HIF1A: p=0.049). CRHR2 and FLT4 are stronger expressed in female than in male patients (CRHR2: p=0.024, FLT4: p=0.004). FIGF expression is still significant between LCNEC and SCLC (p=0.023). FLT4 and KDR show highly significant association to one of the analysed groups (FLT4: p=0.001; KDR: p=0.006). Additionally, HIF1A expression differs significantly between these focus cohorts (p=0.018). Conclusion: We should consider for clinical practice application which factors affect most the tumour growth and distal metastasis, thereafter investigate easy to administer drugs with low side effects. Probably a cluster system of therapy should be established where a drug targets simultaneously different pathways of the same origin.

Aims:TP53 mutations are extremely rare in malignant pleural mesothelioma (MPM). In TP53 wild-type tumors, the functional p53 protein can be inactivated by MDM2. Materials & methods: A total of 61 patient samples were tested for their Mdm2 and p53 protein expression levels via immunohistochemistry. Results: This study demonstrates nuclear Mdm2 expression in three out of four mesothelioma cell lines and 21.3% of the MPM specimens investigated. After silencing of the MDM2 gene by siRNA in MPM cell lines, Mdm2 immunoexpression is lost and cells show changes indicative of severe damage. Mdm2 protein expression in MPM is detected in epithelioid and biphasic subtypes only and is significantly associated with poor survival compared with Mdm2-negative tumors. This may be explained by increased Mdm2 levels possibly leading to an increased ubiquitilation and proteasomal degradation of functional p53 protein. Conclusion: Expression of Mdm2 is a strong prognostic factor associated with shortened overall survival in MPM.

Background and methods: Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. Results: A total of 250 mu g DNA (concentration 5 mu g/mu L) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. Conclusion: We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Fabian D Mairinger,1 Robert FH Walter,2 Claudia Vollbrecht,3 Thomas Hager,1 Karl Worm,1 Saskia Ting,1 Jeremias Wohlschläger,1 Paul Zarogoulidis,4 Konstantinos Zarogoulidis,4 Kurt W Schmid1 1Institute of Pathology, 2Ruhrlandklinik, West German Lung Center, University Hospital Essen, Essen, 3Institute of Pathology, University Hospital Cologne, Cologne, Germany; 4Pulmonary Department, Oncology Unit, G Papanikolaou General Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece Background and methods: Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. Results: A total of 250 µg DNA (concentration 5 µg/µL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. Conclusion: We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA. Keywords: isothermal multiple displacement amplification, isotherma