Dvije kvantitativne metode za mjerenje opterećenja mutiranim alelom V617F u genu za JAK2: usporedba rezultata

Mutacija V617F u genu JAK2 glavna je oznaka Philadelphia negativnih mijeloproliferacijskih zloćudnih tumora (MPN). Zbog brojnih različitih kvantitativnih metoda koje nisu davale usporedive rezultate, organizacije European LeukemiaNet i MPN&MPNr-Euronet pokrenule su standardizaciju osiguravši komercijalno dostupne standarde Svjetske zdravstvene organizacije za kvantifikaciju opterećenja mutiranim alelom V617F. Cilj ovog istraživanja bio je usporediti rezultate dobivene s dvjema najčešće korištenim kvantitativnim metodama, QIAGEN Mutaquant kit i interno razvijenom metodom koju su objavili Larsen et al. 2007. Kvantifikacija opterećenja mutiranim alelom provedena je u uzorcima DNK 101 JAK2 V617F pozitivnog bolesnika pomoću JAK2 MutaQuant i interno razvijene RQ‑PCR metode. Usporedba metoda pokazala je da nema statistički značajne razlike između rezultata u svakoj podskupini MPN bolesnika kao ni kad su se rezultati podijelili u četiri različita raspona opterećenja mutiranim alelom V617F. Statistička analiza je pokazala da ne postoji stalna i proporcionalna sustavna pogreška između dviju uspoređenih metoda te da se obje metode mogu koristiti naizmjenično u rutinskoj praksi

Dvije kvantitativne metode za mjerenje opterećenja mutiranim alelom V617F u genu za JAK2: usporedba rezultata

Mutacija V617F u genu JAK2 glavna je oznaka Philadelphia negativnih mijeloproliferacijskih zloćudnih tumora (MPN). Zbog brojnih različitih kvantitativnih metoda koje nisu davale usporedive rezultate, organizacije European LeukemiaNet i MPN&MPNr-Euronet pokrenule su standardizaciju osiguravši komercijalno dostupne standarde Svjetske zdravstvene organizacije za kvantifikaciju opterećenja mutiranim alelom V617F. Cilj ovog istraživanja bio je usporediti rezultate dobivene s dvjema najčešće korištenim kvantitativnim metodama, QIAGEN Mutaquant kit i interno razvijenom metodom koju su objavili Larsen et al. 2007. Kvantifikacija opterećenja mutiranim alelom provedena je u uzorcima DNK 101 JAK2 V617F pozitivnog bolesnika pomoću JAK2 MutaQuant i interno razvijene RQ‑PCR metode. Usporedba metoda pokazala je da nema statistički značajne razlike između rezultata u svakoj podskupini MPN bolesnika kao ni kad su se rezultati podijelili u četiri različita raspona opterećenja mutiranim alelom V617F. Statistička analiza je pokazala da ne postoji stalna i proporcionalna sustavna pogreška između dviju uspoređenih metoda te da se obje metode mogu koristiti naizmjenično u rutinskoj praksi

Oral Mucormycosis and Aspergillosis in the Patient with Acute Leukemia

Bolesnik u dobi od 54 godine s akutnom limfoblastičnom leukemijom upućen je u Zavod za oralnu medicinu. Imao je primarnu refraktornu bolest i liječen je prema protokolima HOVON 71 i HAM. Šesnaest dana nakon početka primjene protokola HAM, bolesniku se pojavila oralna tamnocrvena/smećkasta lezija nepca i maksilarna vestibularna egzofitična lezija. Iz obiju su uzeti uzorci za biopsiju te su mikrobiološkom obradom potvrđeni Aspergillus fumigatus i Rhizopus genus. Liječenje bolesnika sastojalo se od inferiorne maksilektomije te intravenski posakonazola i amfotericina tijekom sljedećih 28 dana. Budući da se koinfekcija tim dvjema gljivicama iznimno rijetko pojavi u usnoj šupljini, česte su dijagnostičke i terapijske dileme.A 54-year-old male patient with acute lymphoblastic leukemia was referred to the Department of Oral Medicine. He had a primary refractory disease and was treated according to HOVON71 and HAM protocol. Sixteen days after the start of the HAM protocol the patient developed palatal dark red/brownish lesion and maxillary vestibular exophytic lesion. Biopsy specimens from oral lesions were taken and microbiologic evaluation confirmed the presence of Aspergillus fumigatus and Rhizopus genus. The treatment of the patient consisted of the inferior maxillectomy and intravenous posaconazole and amphotericine B for the following 28 days. Since the coinfection with Aspergillus and Rhizopus is extremely rarely seen in the oral cavity, a diagnostic and therapeutic dilemma easily presents itself

Prognostic value of aberrant phenotypes in acute myeloid leukemia

Cilj: Dijagnoza akutne mijeloične leukemije (AML) temelji se na nalazu citomorfologije, imunofenotipizacije i citogenetike. Osnovna uloga imunofenotipizacije jest određivanje krvne loze te prepoznavanje aberantnih fenotipova. U ovom članku određena je učestalost i prognostička vrijednost aberantnih fenotipova u bolesnika s AML-om. Metode: U retrospektivnom istraživanju prikupljeni su podaci o dijagnozi i liječenju 123 odrasla bolesnika s novodijagnosticiranom akutnom mijeloičnom leukemijom, koji su liječeni u Zavodu za hematologiju Klinike za unutarnje bolesti KBC-a Zagreb u razdoblju od 2000. do 2007. godine. Rezultati: Učestalost aberantnih imunofenotipova iznosila je 74.8 %. Najčešća je bila asinkrona ekspresija biljega, nađena u 70 % bolesnika, zatim koekspresija limfocitnih biljega u 35 % bolesnika, te neizražaj biljega specifičnih za lozu u 24 % bolesnika. U procjeni odgovora na liječenje nađeno je da bolesnici s aberantnim fenotipom po tipu neizražaja biljega specifičnog za lozu: CD33-CD13+ imaju lošiji odgovor na uvodno liječenje; u analizi preživljenja bolesnici s aberantnim fenotipom po tipu asinkroniciteta: CD117+C34+CD15+/CD14+/CD11b+ imaju kraće ukupno preživljenje i preživljenje bez znakova bolesti. Rasprava i zaključak: Uz dijagnostičku, imunofenotipizacija procjenom prisustva aberantnih imunofenotipova ima i prognostičku vrijednost u bolesnika s AML-om.Aim: Diagnosis of acute myeloid leukemia (AML) is based on cytomorphological, immunophenotypic and cytogenetic findings. The main role of immunophenotyping is lineage assignement and detection of aberrant phenotypes. The objective of this study was to analyze the incidence and the prognostic value of aberrant phenotypes in AML. Methods: Data on diagnosis and treatment of 123 patients with newly diagnosed AML, treated in the University Hospital Center Zagreb in the period from 2000-2007, was retrospectively analyzed. Results: The incidence of aberrant phenotypes was 74.8 %. The most frequent aberrant phenotype was asynchronous expression which was found in 70 % of patients, followed by co-expression of lymphoid markers in 35 % of patients and loss of lineage-specific markers in 24 % of patients. The aberrant phenotype CD33-CD13+ correlates with lower complete remission rate, while the aberrant phenotype CD117+C34+CD15+/CD14+/CD11b+ correlates with shorter overall and disease-free survival. Discussion and conclusion: In addition to its diagnostic role, immunophenotyping of AML offers information about patient prognosis through the detection of aberrant phenotypes

Serum chitotriosidase: a circulating biomarker in polycythemia vera

Objectives: Serum chitotriosidase activity (CHIT1) is a biomarker of macrophage activation with an important role in inflammation-induced tissue remodeling and fibrosis. Macrophages have been described to play a crucial role in regulating pathological erythropoiesis in polycythemia vera (PV). The aim of this study was to evaluate CHIT1 in patients diagnosed with Philadelphia-negative myeloproliferative neoplasms (MPNs). ----- Methods: Using fluorometric assay, we measured CHIT1 in 28 PV, 27 essential thrombocythemia (ET), 17 primary myelofibrosis (PMF), 19 patients with secondary myelofibrosis and in 25 healthy controls. ----- Results: CHIT1 was significantly higher in PV (p < .001) and post-PV myelofibrosis (MF) transformation (post-PV MF) (p = .020), but not in ET (p = .080), post-ET MF transformation (p = .086), and PMF patients (p = .287), when compared to healthy controls. CHIT1 in PV was positively correlated with hemoglobin (p = .026), hematocrit (p = .012), absolute basophil count (p = .030) and the presence of reticulin fibrosis in the bone marrow (p = .023). ----- Discussion: A positive correlation between CHIT1 and these distinct laboratory PV features might imply macrophages closely related to clonal erythropoiesis as cells of CHIT1 origin. In addition, a positive association between CHIT1 and reticulin fibrosis might indicate its potential role in PV progression. ----- Conclusion: CHIT1 might be considered as a circulating biomarker in PV. Additional studies are needed to clarify the role of CHIT1 in promoting disease progression and bone marrow fibrosis in PV

Comparison of branded and generic imatinib plasma concentrations in patients with chronic myelogenous leukemia: unicentric study

INTRODUCTION: For over a decade, imatinib has been the first-line treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML). Doubts on the bioequivalence and bioavailability of emerging generic compounds have been expressed. Adequate imatinib plasma concentration ([IPC] ≥1000 μmol/L) is associated with a better chance of optimal treatment response in patients with CML. In this study, we compared the achieved IPCs between the branded compound and its 2 generic forms. ----- PATIENTS AND METHODS: IPCs were compared in 24 consecutive patients with CML in the first chronic phase who changed from branded to generic imatinib. The median age was 49 years (range, 22-76 years). Fifteen of them were male. Six patients were switched to Neopax, 13 to Imakrebin, and 5 patients received both generics consecutively. All compounds were used in an equivalent dose of 400 mg orally once daily for at least 1 month before plasma concentrations were measured. High-performance liquid chromatography was used to determine imatinib plasma concentration from a specimen collected 21 to 24 hours after the last dose. ----- RESULTS: The median IPC achieved with branded imatinib was 1454 μmol/L (range, 485-2707 μmol/L) with 18 patients (75%) having IPC ≥ 1000 μmol/L. For Neopax and Imakrebin, median IPCs were 1717 μmol/L (range, 1249-3630 μmol/L) and 1458 μmol/L (range, 707-880 μmol/L), respectively, with 11 of 11 (100%) and 16 of 18 (89%) patients having IPC ≥ 1000 μmol/L. No significant difference in measured IPCs between all 3 compounds was found (P > .257). ----- CONCLUSION: When taken at equivalent doses, imatinib generics are bioequivalent and comparable in clinical efficacy and have the potential for substantial savings in the treatment cost for CML

Influence of blood count, cardiovascular risks, inherited thrombophilia, and JAK2 V617F burden allele on type of thrombosis in patients with Philadelphia chromosome negative myeloproliferative neoplasms

Introduction: Thrombosis is the most common complication in Philadelphia chromosome negative (Ph-) myeloproliferative neoplasms patients. ----- Patients and methods: In a cohort of 258 Ph- myeloproliferative neoplasm patients, the difference between patients with and without thrombosis was analyzed according to genetic thrombophilia factors, JAK2 V617F status and burden allele, blood count, cardiovascular risk factors and age. Patients were also divided in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) subgroups as well as by the type of thrombosis. ----- Results: Analysis of cardiovascular risk factors regarding arterial thrombosis showed that PV patients with thrombosis had higher incidence of diabetes (P = .030), ET patients more often had hypertension (P = .003) and hyperlipidemia (P = .005), while PMF patients had hyperlipidemia (P = .046) and at least one cardiovascular risk factor (P = .044). Moreover, leukocytes > 18 × 109/L and V617F burden allele > 25.7% were statistically significantly different in PV patients (P = .019 and borderline significant at P = .055, respectively), while in ET patients leukocytes > 9.2 × 109/L (P 55 years were statistically significantly different (P = .002). PMF patients with V617F burden allele ≤ 34.8% were more prone to thrombosis (P = .032). When comparing patients with and without venous thrombosis, cutoff value of V617F burden allele > 90.4% was significant for PV patients with thrombosis (P = .036), as was > 56.7% for PMF patients with thrombosis (P = .046). Platelets ≤ 536 × 109/L and age at diagnosis > 54 years showed statistically significant difference for ET patients with thrombosis (P = .015 and P = .041, respectively). ----- Conclusion: On the basis of our results, a new scoring system for thrombosis risk in PV could be made, while PMF prognostic model may be expanded for better recognition of potential thrombotic risk factors

Use of bendamustin instead of carmustin in autologous stem cell transplantation conditioning – toxicity and infectious complications comparison

Unatrag nekoliko godina u hematologiji i onkologiji globalno sve češći problem postaje prikladna opskrba „starijim i manje zanimljivim“ kemoterapeuticima. Zbog povremene nestašice karmustina, jednog od osnovnih kemoterapeutika pri kondicioniranju prije autologne transplantacije krvotvornih matičnih stanica (ATK S) u oboljelih od limfoma, u našem se centru od 2016. godine on zamjenjuje bendamustinom. U ovom radu retrospektivno analiziramo tijek ATK S-a u 41 bolesnika koji su primili bendamustin u sklopu protokola BeEA M te ga uspoređujemo s tijekom ATK S-a u 40 bolesnika koji su primili karmustin u sklopu protokola BEA M. Medijan oporavka vrijednosti neutrofila (> 0,5 × 109/l) u skupini koja je primila bendamustin iznosio je 11 dana, dok je u skupini kondicioniranoj karmustinom iznosio 10 dana. Medijan oporavka vrijednosti trombocita (> 20 × 109/l) bio je duži kod skupine koja je primala bendamustin (16 prema 13 dana) te su ti bolesnici bili duže ovisni o transfuzijama eritrocita (7 prema 5 dana). Infektivne komplikacije nisu bile češće nakon primjene bendamustina, ali smo nakon primjene karmustina imali veću pojavu mukozitisa II. – III. stupnja (35% prema 12%). Nakon primjene bendamustina zabilježen je jedan slučaj nefrotoksičnosti i kardiotoksičnosti terapije, dok kod primjene karmustina te komplikacije nisu zabilježene. Pri upotrebi bendamustina kod kondicioniranja u naših bolesnika u ovom trenutku nije utvrđena znatnija hematološka toksičnost u odnosu prema karmustinu, ali su prisutni dulji period oporavka vrijednosti trombocita te niža incidencija mukozitisa.Inadequate supply of „old and less interesting“ chemotherapeutic agents is becoming a global issue in hemato-oncology today. In 2016 we were faced with occasional carmustin shortage, one of the most commonly used in autologous transplant conditioning regimens for lymphoma in our centre, so we decided to use bendamustin instead. We performed a retrospective analysis of 41 patients treated at our centre who had received bendamustin within BeEA M protocol and compared them with 40 patients who had received carmustin within BEA M protocol. Both protocols were used as conditioning protocols before autologous stem cell transplantation. Neutrophil recovery median following transplantation (AN C>0,5x109/l) was 11 days in the bendamustin group in comparison to 10 days in the carmustin group.Platelets recovery median following transplantation (PLT>20x109/l) was longer in the bendamustin group (16 vs.13 days) as was blood transfusion dependency (7 vs. 5 days). Infectious complications were not more frequent after bendamustin, but grade II–III mucositis was more frequent in patients who received carmustin (35% vs.12%). Following bendamustin we had one reported case of nephrotoxicity and cardiac toxicity, not reported with carmustin. Bendamustin has shown similar hematologic toxicity compared to carmustin but a longer platelet recovery period and a lower mucositis incidence

Serum procalcitonin in Philadelphia-negative myeloproliferative neoplasms

Philadelphia-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF), are rare clonal hematopoietic stem cell disorders accompanied by a strong inflammatory milieu, which is directly responsible for constitutional symptoms associated with the disease, such as fever, weight loss or night sweats. Non-hematologists sometimes (and often wrongly) consider the fever in MPN patients to be a symptom of an underlying disease, which may have devastating consequences. Serum procalcitonin (PCT) is a circulating biomarker commonly used to improve the diagnostic accuracy of bacterial infections and to guide antibiotic therapy. The aim of this study was to test whether PCT could aid the clinician in the early diagnosis of bacterial infections in MPNs. This study investigated PCT in 41 ambulatory MPN patients (13 ET, 13 PV and 15 MF) who had no signs of infection and compared it to 10 MPN patients with microbiologically and/or serologically documented bacterial infections. Median PCT in MPN patients was 0.02 ng/mL (range 0.01-0.09 ng/mL). No difference in PCT was found between ET, PV and MF patients (p = 0.993), whereas MPN patients with documented bacterial infections had significantly higher PCT (median PCT 2.45, range 0.90-5.40 ng/mL) when compared to MPN patients with (median PCT 0.03 ng/mL) or without constitutional symptoms (median PCT 0.02 ng/mL; p < 0.001 for both analyses). These results clearly show that PCT should not be considered as a disease biomarker in MPNs and careful clinical assessment for the signs of infection is needed when MPN patients present with fever and high PCT

Circulating YKL-40 in Philadelphia-negative myeloproliferative neoplasms

Objectives: Philadelphia-negative chronic myeloproliferative neoplasms (MPNs), essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF), are characterized by clonal myeloproliferation and a strong inflammatory atmosphere. YKL-40, expressed in granulocytes, macrophages, megakaryocytes and malignant cells, is an acute phase reactant with an important role in tissue remodeling and atherosclerotic inflammation. The aim of this study was to investigate serum YKL-40 levels in MPNs and to assess its clinical correlations. ----- Methods: ELISA test was used to measure serum YKL-40 levels in 111 MPN patients and in 32 healthy controls. ----- Results: Serum YKL-40 levels were higher in ET, post-ET MF, PV, post-PV MF and primary MF patients, when compared to healthy controls (p < 0.001). Higher serum YKL-40 levels were associated with parameters indicative of the increased inflammatory state (higher C-reactive protein, poor performance status, presence of constitutional symptoms and cardiovascular risk factors). Additionally, higher serum YKL-40 levels in MF patients were associated with blast phase disease, lower hemoglobin and higher Dynamic International Prognostic Scoring System score. In the multivariate Cox regression models, higher serum YKL-40 levels in ET and PV patients were independently associated with an increased risk of thrombosis (HR 4.64, p = 0.031) and impaired survival in MF patients (HR 4.31, p = 0.038). ----- Conclusion: These results indicate that higher circulating YKL-40 levels in MPNs might have a pathophysiological role in disease progression and thrombosis development. Assessing circulating YKL-40 could help in identification of ET and PV patients at a high risk of future cardiovascular events and has a good potential for improving prognostication of MF patients