Kristina Forsterova

Optimized protocol for gene expression analysis in formalin-fixed, paraffin-embedded tissue using real-time quantitative polymerase chain reaction.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common tissue specimen available after microscopic examination. Molecular methods, such as polymerase chain reaction (PCR) and gene expression examination, serve as a source of diagnostic and prognostic information but require high-quality RNA. However, the increasing application of RNA extracted from FFPE tissue frequently results in very small and degraded quantities of nucleic acid. This study targets gene expression analysis from FFPE specimens using real-time quantitative PCR. The whole protocol consists of several steps, that is, RNA extraction and its quality control, reverse transcription, and fluorescence detection during real-time quantitative PCR. We compared several methods in each step, chose the most effective, and with that combination we successfully examined 95% (62 from 65) FFPE samples for our genes of interest. We reached the best results with RNA isolation by using a commercial kit, carefully interpreted UV spectrophotometric values, and meticulously chose reverse transcriptase and TaqMan fluorescence detection. Our protocol improves the utility of FFPE tissue for molecular profiling studies.

Invasive aspergillosis in patients with hematological malignancies in the Czech and Slovak republics: Fungal InfectioN Database (FIND) analysis, 2005-2009.

OBJECTIVES To evaluate risk factors, diagnostic procedures, and treatment outcomes of invasive aspergillosis (IA) in patients with hematological malignancies. METHODS A retrospective analysis of data from proven/probable IA cases that occurred from 2005 to 2009 at 10 hematology centers was performed. RESULTS We identified 176 IA cases that mainly occurred in patients with acute leukemias (58.5%), mostly those on induction/re-induction treatments (39.8%). Prolonged neutropenia was the most frequent risk factor for IA (61.4%). The lungs were the most frequently affected site (93.8%) and computed tomography detected abnormalities in all episodes; however, only 53.7% of patients had findings suggestive of IA. Galactomannan (GM) detection in serum or bronchoalveolar lavage fluid (positive in 79.1% and 78.8% of episodes, respectively) played a crucial role in IA diagnosis. Neutrophil count and antifungal prophylaxis did not influence the GM positivity rate, but empirical therapy decreased this rate (in serum). Of the IA cases, 53.2% responded to initial antifungal therapy. The combination of voriconazole and echinocandin, even as initial or salvage therapy, did not perform better than voriconazole monotherapy (p=0.924 for initial therapy and p=0.205 for salvage therapy). Neutrophil recovery had a significant role in the response to initial (but not salvage) antifungal therapy. CONCLUSIONS Our retrospective analysis identified key diagnostic and treatment characteristics, and this understanding could improve the management of hematological malignancy patients with IA.

The use of formalin‐fixed, paraffin‐embedded lymph node samples for the detection of minimal residual disease in mantle cell lymphoma

Dejean, L.M., Ryu, S.Y., Martinez-Caballero, S., Teijido, O., Peixoto, P.M. & Kinnally, K.W. (2010) MAC and Bcl-2 family proteins conspire in a deadly plot. Biochimica et Biophysica Acta, 1797, 1231–1238. Gyulkhandanyan, A.V., Mutlu, A., Freedman, J. & Leytin, V. (2013) Selective triggering of platelet apoptosis, platelet activation or both. British Journal of Haematology, 161, 245–254. Gyulkhandanyan, A.V., Mutlu, A., Allen, D.J., Freedman, J. & Leytin, V. (2014) BH3-mimetic ABT-737 induces strong mitochondrial membrane depolarization in platelets but only weakly stimulates apoptotic morphological changes, platelet shrinkage and microparticle formation. Thrombosis Research, 133, 73–79. Kroemer, G., Galluzzi, L. & Brenner, C. (2007) Mitochondrial membrane permeabilization in cell death. Physiological Reviews, 87, 99–163. Leytin, V. (2012) Apoptosis in the anucleate platelet. Blood Reviews, 26, 51–63. Leytin, V., Allen, D.J., Mutlu, A., Gyulkhandanyan, A.V., Mykhaylov, S. & Freedman, J. (2009) Mitochondrial control of platelet apoptosis: effect of cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. Laboratory Investigation, 89, 374–384. Mutlu, A., Gyulkhandanyan, A.V., Freedman, J. & Leytin, V. (2012) Activation of caspases-9, -3 and 8 in human platelets triggered by BH3-only mimetic ABT-737 and calcium ionophore A23187: caspase-8 is activated via bypass of the death receptors. British Journal of Haematology, 159, 565–571. Mutlu, A., Gyulkhandanyan, A.V., Freedman, J. & Leytin, V. (2013) Concurrent and separate inside-out transition of platelet apoptosis and activation markers to the platelet surface. British Journal of Haematology, 163, 377–384. Oltersdorf, T., Elmore, S.W., Shoemaker, A.R., Armstrong, R.C., Augeri, D.J., Belli, B.A., Bruncko, M., Deckwerth, T.L., Dinges, J., Hajduk, P.J., Joseph, M.K., Kitada, S., Korsmeyer, S.J., Kunzer, A.R., Letai, A., Li, C., Mitten, M.J., Nettesheim, D.G., Ng, S., Nimmer, P.M., O’Connor, J.M., Oleksijew, A., Petros, A.M., Reed, J.C., Shen, W., Tahir, S.K., Thompson, C.B., Tomaselli, K.J., Wang, B., Wendt, M.D., Zhang, H., Fesik, S.W. & Rosenberg, S.H. (2005) An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature, 435, 677–681. Peixoto, P.M., Dejean, L.M. & Kinnally, K.W. (2012) The therapeutic potential of mitochondrial channels in cancer, ischemia–reperfusion injury, and neurodegeneration. Mitochondrion, 12, 14–23.

Micafungin as empirical antifungal therapy in hematological patients: a retrospective, multicenter study in the Czech and Slovak Republics.

Abstract The objective of this retrospective, multicenter study was to evaluate the efficacy and safety of micafungin as empirical antifungal therapy during febrile neutropenia (FN) in 73 hematological patients from six centers in two countries. All patients received 100 mg of micafungin/day. The overall favorable response rate (RR) was 64.8% when the resolution of fever during neutropenia was included in the response criteria and 84.5% when excluded. A significantly lower favorable RR in patients with persistent fever and non-specific pulmonary infiltrates compared to patients with persistent fever only (82.8 vs. 52.4%, respectively; p = 0.011) was not found when resolution of fever was not included in the composite endpoint criteria (93.1 vs. 78.6%, respectively; p = 0.180). Breakthrough fungal disease developed in 2.7% of patients. Treatment was discontinued in 16.4% of cases. Only one patient (1.4%) discontinued therapy due to an adverse event. Posaconazole prophylaxis improved favorable RR when defervescence was included as composite endpoint criterion (p = 0.047), but not when it was excluded (p = 0.485). However, neutrophil recovery did not influence favorable RR (p = 0.803 and p = 0.112, respectively). These data suggest that micafungin is safe and effective as an empirical therapy in patients with FN.

Mantle cell lymphoma‐variant Richter syndrome: Detailed molecular‐cytogenetic and backtracking analysis reveals slow evolution of a pre‐MCL clone in parallel with CLL over several years

Richter syndrome represents the transformation of the chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, most frequently the diffuse large B‐cell lymphoma (DLBCL). In this report we describe a patient with CLL, who developed a clonally‐related pleomorphic highly‐aggressive mantle cell lymphoma (MCL) after five cycles of a fludarabine‐based second‐line therapy for the first relapse of CLL. Molecular cytogenetic methods together with whole‐exome sequencing revealed numerous gene alterations restricted to the MCL clone (apart from the canonical t(11;14)(q13;q32) translocation) including gain of one copy of ATM gene or emergence of TP53, CREBBP, NUP214, FUBP1 and SF3B1 gene mutations. Similarly, gene expression analysis revealed vast differences between the MCL and CLL transcriptome, including overexpression of cyclin D1, downregulation of cyclins D2 and D3, or downregulation of IL4R in the MCL clone. Backtracking analysis using quantitative PCR specifically detecting an MCL‐restricted focal deletion of TP53 revealed that the pre‐MCL clone appeared in the bone marrow and peripheral blood of the patient approximately 4 years before the clinical manifestation of MCL. Both molecular cytogenetic and sequencing data support the hypothesis of a slow development of the pre‐MCL clone in parallel to CLL over several years, and thereby exclude the possibility that the transformation event occurred at the stage of the CLL relapse clone by mere t(11;14)(q13;q32) acquisition.

Alternating R‐CHOP and R‐cytarabine is a safe and effective regimen for transplant‐ineligible patients with a newly diagnosed mantle cell lymphoma

Implementation of cytarabine into induction therapy became standard of care for younger patients with mantle cell lymphoma (MCL). On the basis of its beneficial impact, many centers incorporated cytarabine at lower doses also into first‐line treatments of elderly patients. We conducted a multicenter observational study that prospectively analyzed safety and efficacy of alternating 3 + 3 cycles of R‐CHOP and R‐cytarabine for newly diagnosed transplant‐ineligible MCL patients. A total of 73 patients were enrolled with median age 70 years. Most patients had intermediate (39.7%) and high‐risk (50.7%) disease according to MCL international prognostic index. Rituximab maintenance was initiated in 58 patients. Overall response rate reached 89% by positron emission tomography–computed tomography, including 75.3% complete remissions. Two patients (2.7%) did not complete the induction therapy because of toxicity. Three patients (4.1%) were considered nonresponders, which led to therapy change before completion of induction. Estimated progression‐free survival and overall survival were 51.3% and 68.6% at 4 years, respectively. Mantle cell lymphoma international prognostic index, bulky disease (≥ 5 cm), and achievement of positron emission tomography–negativity independently correlated with progression‐free survival. Grade 3 to 4 hematologic and nonhematologic toxicity was documented in 48% and 20.5% patients, respectively. Alternation of R‐CHOP and R‐cytarabine represents feasible and very effective regimen for elderly/comorbid MCL patients. This study was registered at GovTrial (clinicaltrials.gov) NCT03054883.

Cyclin D1 mRNA as a molecular marker for minimal residual disease monitoring in patients with mantle cell lymphoma

Chromosomal translocation t(11;14)(q13;q32) is a characteristic molecular marker of mantle cell lymphoma (MCL) and leads to the fusion of the immunoglobulin heavy chain enhancer-promoter with the cyclin D1 gene. Both aberrant cyclin D1 expression and underlying chromosomal aberration may be used as molecular targets for monitoring minimal residual disease (MRD). The present study aims to assess the usefulness of quantitative cyclin D1 gene expression compared to the standardised but more technologically demanding DNA-based method for immunoglobulin heavy chain (IGH) or t(11;14) clone-specific gene rearrangement quantification in a cohort of bone marrow (BM) and peripheral blood (PB) samples from patients with MCL. We simultaneously evaluated DNA-MRD and cyclin D1 expression levels in 234 samples from 57 patients. We observed that both in DNA-MRD positive and negative BM/PB pairs from the same time points the expression levels of cyclin D1 are lower in PB than in BM (median 19×, BM/PB range 0.41–352). The correlation of cyclin D1 transcript levels with DNA-MRD or with flow cytometry was good only in samples with a very high infiltration. In DNA-MRD-negative BM samples, we observed a significant heterogeneity of cyclin D1 expression (in the range of more than three orders of magnitude). This is in contrast to previous reports demonstrating the usefulness of cyclin D1 for MRD monitoring that did not use DNA-based method as a reference. In PB, the specificity of cyclin D1 expression was better due to a lower physiological background. In conclusion, we show that cyclin D1 is unsuitable for MRD monitoring in BM.

Potential loss of prognostic significance of minimal residual disease assessment after R-CHOP-based induction in elderly patients with mantle cell lymphoma in the era of rituximab maintenance

Rituximab maintenance (RM) prolongs survival of elderly patients with mantle cell lymphoma (MCL). Persistent minimal residual disease (MRD) after induction repeatedly correlated with shorter progression‐free survival (PFS). However, none of the published studies analyzed patients treated with RM. The main purpose was to analyze prognostic significance of MRD in the elderly patients with newly diagnosed MCL treated according to the recently published observational trial protocol (alternation of R‐CHOP and R‐cytarabine, 3 + 3 cycles, GovTrial number NCT03054883) at the centers that implemented RM. Minimal residual disease was evaluated by a EuroMRD standardized real‐time PCR approach after 3 and 6 cycles of the induction therapy. Prognostic significance of MRD was analyzed in a subcohort of patients treated at the centers that implemented RM as a standard approach. Bone marrow proved to be a significantly more sensitive source for MRD detection than peripheral blood. In either compartment MRD (positive versus negative) after 3 or 6 cycles of the induction therapy did not correlate with PFS. The observed loss of prognostic significance of MRD after the R‐CHOP‐based induction appears to be a consequence of RM immune control over the residual lymphoma.