Preamplification techniques for real-time RT-PCR analyses of endomyocardial biopsies

<p>Abstract</p> <p>Background</p> <p>Due to the limited RNA amounts from endomyocardial biopsies (EMBs) and low expression levels of certain genes, gene expression analyses by conventional real-time RT-PCR are restrained in EMBs. We applied two preamplification techniques, the TaqMan^®PreAmp Master Mix (T-PreAmp) and a multiplex preamplification following a sequence specific reverse transcription (SSRT-PreAmp).</p> <p>Results</p> <p>T-PreAmp encompassing 92 gene assays with 14 cycles resulted in a mean improvement of 7.24 ± 0.33 Ct values. The coefficients for inter- (1.89 ± 0.48%) and intra-assay variation (0.85 ± 0.45%) were low for all gene assays tested (<4%). The PreAmp uniformity values related to the reference gene CDKN1B for 91 of the investigated gene assays (except for CD56) were -0.38 ± 0.33, without significant differences between self-designed and ABI inventoried Taqman^®gene assays. Only two of the tested Taqman^®ABI inventoried gene assays (HPRT-ABI and CD56) did not maintain PreAmp uniformity levels between -1.5 and +1.5. In comparison, the SSRT-PreAmp tested on 8 self-designed gene assays yielded higher Ct improvement (9.76 ± 2.45), however was not as robust regarding the maintenance of PreAmp uniformity related to HPRT-CCM (-3.29 ± 2.40; p < 0.0001), and demonstrated comparable intra-assay CVs (1.47 ± 0.74), albeit higher inter-assay CVs (5.38 ± 2.06; p = 0.01). Comparing EMBs from each 10 patients with dilated cardiomyopathy (DCM) and inflammatory cardiomyopathy (DCMi), T-PreAmp real-time RT-PCR analyses revealed differential regulation regarding 27 (30%) of the investigated 90 genes related to both HPRT-CCM and CDKN1B. Ct values of HPRT and CDKN1B did not differ in equal RNA amounts from explanted DCM and donor hearts.</p> <p>Conclusion</p> <p>In comparison to the SSRT-PreAmp, T-PreAmp enables a relatively simple workflow, and results in a robust PreAmp of multiple target genes (at least 92 gene assays as tested here) by a mean Ct improvement around 7 cycles, and in a lower inter-assay variance in RNA derived from EMBs. Preliminary analyses comparing EMBs from DCM and DCMi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that T-PreAmp is a suitable tool to perform gene expression analyses in EMBs, expanding gene expression investigations with the limited RNA/cDNA amounts derived from EMBs. CDKN1B, in addition to its function as a reference gene for the calculation of PreAmp uniformity, might serve as a suitable housekeeping gene for real-time RT-PCR analyses of myocardial tissues.</p

Immunochemotherapy for Richter syndrome: current insights

Bartosz Puła,1 Aleksander Salomon-Perzyński,1 Monika Prochorec-Sobieszek,2,3 Krzysztof Jamroziak1 1Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 2Department of Diagnostic Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland; 3Department of Pathology and Laboratory Medicine, Maria Sklodowska-Curie Institute &ndash; Oncology Center, Warsaw, Poland Abstract: Richter syndrome (RS) is recognized as the development of a secondary and aggressive lymphoma during the clinical course of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Most of such histological transformations are from RS to diffuse large B-cell lymphoma (DLBCL-RS, 90%) and Hodgkin&rsquo;s lymphoma (HL-RS, 10%). &shy;Histopathological examination is a prerequisite for diagnosis. It is crucial to assess the relationship between the RS clone and the underlying CLL/SLL because clonally related DLBCL-RS has a poor outcome, while clonally unrelated cases have a prognosis similar to de novo DLBCL. An anti-CD20 antibody-based immunochemotherapy is hitherto the frontline treatment of choice for DLBCL-RS; nonetheless, the results are unsatisfactory. Allogeneic stem cell transplantation should be offered to younger and fit patients as a consolidative treatment; however, the majority of the patients may not be qualified for this procedure. The HL-RS transformation has better outcomes than those of DLBCL-RS and can effectively be treated by the adriamycin, bleomycin, vinblastine, and dacarbazine regimen. Although novel agents are currently being investigated for RS, immunochemotherapy nevertheless remains a standard treatment for DLBCL-RS. Keywords: Richter syndrome, Richter transformation, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, Hodgkin&rsquo;s lymphom

Diagnostyka z wykorzystaniem sekwencjonowania następnej generacji (NGS) w mięsakach — rekomendacje

Soft tissue and bone sarcomas are a group of rare, heterogeneous, high-mortality cancers, each with a different biology and clinical course. They constitute 20% of all childhood cancers and about 1% of all adult malignancies. Advanced pathomorphological-molecular diagnostics using classic methods is often insufficient to make the final diagnosis, and thus implement the right therapy. In these cases, next generation sequencing technology (NGS) is helpful, which allows for analysis of many genes and classes of mutations in one assay with minimal consumption of tissue material. Wide genetic profiling enables, in many cases, not only to make the correct diagnosis, but also to identify potential therapeutic goals for modern targeted therapies in oncology. In this paper, we present recommendations on the use of the NGS method in advanced genetic diagnosis of sarcomas in adults, children and adolescents.Mięsaki tkanek miękkich i kości to grupa rzadkich, heterogennych nowotworów o wysokim stopniu śmiertelności, z których każdy ma inną biologię i przebieg kliniczny. Stanowią 20% wszystkich nowotworów wieku dziecięcego i około 1% wszystkich nowotworów złośliwych występujących u dorosłych. Zaawansowana diagnostyka patologiczno-molekularna z zastosowaniem klasycznych metod często jest niewystarczająca w ustaleniu ostatecznego rozpoznania, co związane jest z wdrożeniem właściwej terapii. W takich przypadkach pomocna staje się technologia sekwencjonowania następnej generacji (NGS), która umożliwia przeanalizowanie wielu genów oraz klas mutacji w pojedynczym oznaczeniu, przy minimalnym zużyciu materiału tkankowego. Szerokie profilowanie genetyczne umożliwia, w wielu przypadkach, nie tylko postawienie prawidłowej diagnozy, ale również identyfikację potencjalnych celów terapeutycznych dla nowoczesnych terapii celowanych w onkologii. W niniejszej pracy przedstawiono rekomendacje dotyczące zastosowania metody NGS w zaawansowanej diagnostyce genetycznej mięsaków występujących u dorosłych, dzieci i młodzieży

Distribution of lymphomas in Poland according to World Health Organization classification : analysis of 11718 cases from National Histopathological Lymphoma Register project - the Polish Lymphoma Research Group study

Most national lymphoma registers rely on broad classifications which include Hodgkin and non-Hodgkin lymphomas (NHL), multiple myeloma and leukaemia. In Poland the National Histopathological Lymphoma Register project (NHLR) was implemented by hematopathologists in accordance with the 2008 WHO classification into haematopoietic and lymphoid tissues. We present the NHLR data and compare lymphoma distribution in Poland, Europe, as well as in North Central and South America. Records of 11718 patients diagnosed in 24 pathology departments from all over the country were retrieved and reclassified into indolent and aggressive lymphomas according to the 2008 revised WHO classification system. DLBCL (32.9%; 2587), CLL/SLL (31.84%; 2504) and MCL (9.04%; 711) were the three most frequent NHL. The ratio of indolent to aggressive NHL was 1.72; 63.25% (4809) to 36.25% (2794) of cases respectively. Multiple myeloma was less frequent as compared to the data from population-based national cancer register (13.32% vs. 28.94%). Major differences between NHLR and European and American data on NHL subtypes concered: higher incidence of aggressive B-cell lymphomas including DLBCL, lower FL and MALT incidence rate. The percentage of unclassified lymphomas in the study was minimal due to participation of hematopathologists