Pre-B-cell acute lymphoblastic leukemia with bulk extramedullary disease and chromosome 22 (EWSR1) rearrangement masquerading as Ewing sarcoma

We report a 2-year-old female with a subcutaneous tumor who was initially misdiagnosed as suffering from Ewing sarcoma with a positive EWSR1 rearrangement and EWS/FLI1 transcript. After finding lymphoblasts in peripheral blood, the diagnosis of acute lymphoblastic leukemia was established. This necessitated further analysis of the subcutaneous tumor. The tissue was positive for immature B-cell markers and an immunoglobulin heavy chain gene rearrangement, which confirmed the final diagnosis of common type acute lymphoblastic leukemia with bulk extramedullary disease. The patient was treated with chemotherapy and was in remission 30 months after the diagnosis

Quantity of Salivary Immunoglobulin A, Lysozyme and Magnesium in Patients with Burning Mouth Syndrome and Xerostomia

Nedavne studije izvješćuju o povezanosti između sindroma pečenja usta te količine i sastava sline. Svrha našeg ispitivanja bila je ustanoviti količinu salivarnog imunoglobulina A, lizozima i magnezija u nestimuliranoj i stimuliranoj ukupnoj slini bolesnika sa sindromom pečenja usta (SPU) i kserostomijom. Uzorci sline dobiveni su sijalometrijom. Salivarni IgA je određen radijalnom imunodifuzijom po Manziniju, količina lizozima po metodi Osserman i Lowlor, a magnezij je određen atomskom apsorpcijskom spektrofotometrijom. Količina sIgA i lizozima bila je znatno snižena u stimuliranoj ukupnoj slini bolesnika sa SPU i kserostomijom u usporedbi s nestimuliranom (p<0,001). Količina magnezija nije se mijenjala s obzirom na stimulaciju sline. Rezultati našeg ispitivanja pokazuju da količina sline ima utjecaja na sastav sline te da zajedno mogu biti jedan od mnogobrojnih etioloških čimbenika u nastanku simptoma sindroma pečenja usta.Recent studies suggest a connection between burning mouth syndrome (BMS) and the quantitiy and quality of saliva. The aim of our study was to determine quantities of salivary immunoglobulin A (sIgA), lysozyme and magnesium in unstimulated and stimulated saliva of patients with burning mouth syndrome and xerostomia. Salivary samples were obtained by sialometry. Salivary immunoglobulin A was determined by radial immunodiffusion according to Manzini, lysozyme levels were obtained according to Osserman and Lowlor, and magnesium was determined using atomic absorbance spectrophotometry. Levels of sIgA were decreased in stimulated whole saliva of patients with BMS and xerostomia when compared to those in unstimulated saliva (p<0.001). Lysozyme levels were also lower in stimulated whole saliva in such patients when compared to the levels in unstimulated saliva (p<0.001). Magnesium levels remain unchanged with regard to the salivary stimulation. The results of our study indicate that the quantity and quality of saliva could have an impact on symptoms of burning mouth syndrome

Verification of automated latex-enhanced particle immunoturbidimetric D-Dimer assays on different analytical platforms and comparability of test results

Introduction: The aim of the study was the analytical verification of automated latex-enhanced particle immunoturbidimetric (LPIA) D-Dimer assay INNOVANCE D-dimer on Sysmex CS-5100 and Atellica COAG 360 analysers, and HemosIL D-dimer HS500 on ACL TOP 550, as well as the comparison with the enzyme-linked immunofluorescent assay (ELFA) on the miniVidas analyser. Materials and methods: Verification included assessment of within-run and between-run precision, bias, measurement uncertainty (MU), verification of the cut-off, method comparison between all assessed assays, and the reference commercial ELFA VIDAS D-Dimer Exclusion II. Results: Within-run coefficients of variations (CVs) ranged from 1.6% (Atellica COAG 360) to 7.9% (ACL TOP 550), while between-run CVs ranged from 1.7% (Sysmex CS-5100) to 6.9% (Atellica COAG 360). Spearman’s rank correlation coefficients were > 0.99 between LPIAs and ≥ 0.93 when comparing ELFA with LPIA. Passing-Bablok regression analysis yielded constant and proportional difference for comparison of ACL TOP 550 with both Sysmex CS-5100 and Atellica COAG360, and for miniVidas with Atellica COAG360. Small proportional difference was found between miniVidas and both Sysmex CS-5100 and ACL TOP 550. Calculated MUs using D-dimer HS 500 calibrator were 12.6% (Sysmex CS-5100) and 15.6% (Atellica COAG 360), while with INNOVANCE D-dimer calibrator 12.0% (Sysmex CS-5100), 10.0% (Atellica COAG 360) and 28.1% (ACL TOP 550). Excellent agreement of results was obtained, with occasional discrepancies near the cut-off. The cut-off (0.5 mg/L FEU) was confirmed. Conclusions: The obtained results prove satisfactory analytical performance of LPIAs, their high comparability and almost equal discriminatory characteristics, suggesting them as a valid alternative to ELFA

Monitoring chronic myeloid leukemia patients responding to treatment with tyrosine kinase inhibitors by real-time quantitative polymerase chain reaction

Uvod: Kroz povijest je terapija kronične mijeloične leukemije (engl. chronic myeloidleukemia, CML) imala puno revolucionarnih pomaka, među kojima je najvažniji uvođenje imatinib mesilata (engl. imatinib mesylate, IM). Preciznija procjena terapijskog odgovora na IM i toč no mjerenje stupnja smanjenja prijepisa BCR-ABL, može se postići upotrebom kvantitativne lančane reakcije polimeraze u stvarnom vremenu (engl. real-time quantitative polymerase chain reaction, RQ-PCR). Cilj: Kvantificirati prijepise BCR-ABL kod bolesnika s CML i promatrati odgovor na liječenje inhibitorima tirozin-kinaze. Materijali i metode: U istraživanje je bio uključen 31 bolesnik liječen pomoću IM. Napravljena je RQ-PCR prema protokolu Europe AgainstCancers ABL kao „kućepaziteljskim" genom (engl. housekeepinggene) i izrač unat je omjer BCR-ABL/ABL. Bolesnici su podijeljeni u skupine prema kriterijima Europske mreže za leukemiju (engl. European Leukemia Net) za postizanje značajnog molekularnog odgovora (engl. major molecular response, MMoR). I. skupina sastojala se od 11 bolesnika sa smanjenjem višim od 3 log, II. skupina od 13 bolesnika sa smanjenjem nižim od 3 log, a III. skupina od 7 bolesnika koji su bili manje od 18 mjeseci na kontrolama i praćenju. Rezultati: Bolesnici I. skupine, koji su bili pozitivni ili negativni na prijepis BCR-ABL, odgovarali su na terapiju u vremenu od 2 godine praćenja i kontrola te su ispunili kriterije za povoljnu dugoroč nu prognozu. Bolesnici II. skupine nisu odgovarali na terapiju pomoću IM te su zahtijevali drugačiji terapijski pristup, višu dozu istog inhibitora tirozin-kinaze ili drugu generaciju lijeka. Sedmoro bolesnika III. skupine kojima je nedavno dijagnosticirana bolest, praćeni su manje od 18 mjeseci pa im se MMoR nije mogao procijeniti. Primjenom iste analogije kao kod prve dvije skupine, može se napraviti prognoza tijeka bolesti. Zaključak: Naši rezultati pokazuju da je RQ-PCR obvezna metoda za pažljivo promatranje odgovora na liječenje inhibitorima tirozin-kinaze, radi omogućavanja ispravne terapije te kako bi se odlučilo treba li mijenjati terapiju, a u slučaju da treba, kada to učiniti.Introduction: Historically, many revolutionary advances in therapy for chronic myeloid leukemia (CML) have been achieved over time, among them most important being imatinib mesylate (IM). More precise assessment of response to therapy with IM and an accurate measure of the degree of BCR-ABL transcript reduction can be achieved by using real-time quantitative polymerase chain reaction (RQ-PCR). Aim: To quantitate BCR-ABL transcripts in CML patients and to monitor response to treatment with tyrosine kinase inhibitors. Materials and methods: The study included a 31 patients treated with IM. RQ-PCR was performed according to the Europe Against Cancer protocol, with ABL as a housekeeping gene. BCR-ABL/ABL ratio was calculated. Patients were divided into groups according to the European Leukemia Net criteria for achievement of major molecular response (MMoR). Group I consisted of 11 patients with more than 3 log reduction, group II consisted of 13 patients with less than 3 log reduction, and group III included 7 patients with a follow up of less than 18 months. Results: Group I patients achieved MMoR with detectable or undetectable BCR-ABL transcript in a period of 2 years of follow up and fulfilled the criteria for favorable long term prognosis. Group II patients never achieved MMoR with IM and required different therapy approach, higher dose of the same tyrosine kinase inhibitor or a second generation drug. Seven newly diagnosed CML patients from group III were monitored for less than 18 months and therefore MMoR could not be estimated. Using the same analogy as in the first two groups, prediction of the course of disease could be possible. Conclusion: Study results show that RQ-PCR is mandatory for careful monitoring of therapeutic response to tyrosine kinase inhibitors in order to ensure that an individual patient receives proper treatment and to decide whether and when therapy should be changed

Reevaluation of von Willebrand disease diagnosis in a Croatian paediatric cohort combining bleeding scores, phenotypic laboratory assays and next generation sequencing: a pilot study

This study reevaluated von Willebrand disease (vWD) diagnosis in a Croatian paediatric cohort by combining bleeding scores (BS), phenotypic laboratory testing, and next-generation sequencing (NGS). A total of 25 children (11 males and 14 females, median age 10 years, from 2 to 17) previously diagnosed with vWD were included. BS were calculated using an online bleeding assessment tool. Phenotypic laboratory analyses included platelet count, platelet function analyser closure times, prothrombin time, activated partial thromboplastin time, von Willebrand factor antigen (vWF:Ag), vWF gain-of-function mutant glycoprotein Ib binding activity (vWF:GPIbM), vWF collagen binding activity (vWF:CBA), factor VIII activity (FVIII:C) and multimeric analysis. Next-generation sequencing covered regions of both vWF and FVIII genes and was performed on MiSeq (Illumina, San Diego, USA). Disease-associated variants identified in 15 patients comprised 11 distinct heterozygous vWF gene variants in 13 patients and one novel FVIII gene variant (p.Glu2085Lys) in two male siblings. Four vWF variants were novel (p.Gln499Pro, p.Asp1277Tyr, p.Asp1277His, p.Lys1491Glu). Three patients without distinctive variants had vWF:GPIbM between 30 and 50%. Patients with identified vWF gene variants had statistically significant lower values of vWF:GPIbM (P = 0.002), vWF:Ag (P = 0.007), vWF:CBA (P < 0.001) and FVIII:C (P = 0.002), compared to those without. Correlations between BS and phenotypic laboratory test results were not statistically significant for either of the tests. The applied diagnostic approach confirmed the diagnosis of vWD in 13 patients and mild haemophilia A in two. Limited utility of BS in the paediatric population was evidenced

Clinical significance of T315I ABL kinase domain mutation detection in patients resistant to imatinib mesylate therapy

Uvod: Kronična mijeloična leukemija (engl. chronic myeloid leukemia, CML) je mijeloproliferativna bolest koju karakterizira prisutnost fuzijskog gena bcr-abl i posljedično fuzijskog proteina bcr-abl. Iako je otkriće inhibitora tirozin-kinaze (engl. tyrosine kinase inhibitor, TKI), imatinib mesilata (IM) poboljšalo liječenje bolesnika oboljelih od CML, dio bolesnika razvija rezistenciju na lijek, što dovodi do povišene razine bcr-abl prijepisa. Jedan od mogućih razloga te rezistencije su mutacije u domeni ABL kinaze. Neke se mutacije mogu prevladati povećanjem doze lijeka ili primjenom nove generacije TKI. Jedina mutacija rezistentna na trenutno dostupne TKI jest T315I. Cilj ovog istraživanja bio je otkriti da li je prisutnost T315I kod bolesnika rezistentnih na liječenje imatinib mesilatom povezana s povećanom ili neprekidno visokom razinom bcr-abl prijepisa. Također, cilj je bio procijeniti moguću razliku u razini bcr-abl prijepisa kod bolesnika rezistentnih na liječenje imatinib mesilatom sa i bez T315I mutacije. Ispitanici i metode: U ispitivanje su bila uključena 24 bolesnika oboljela od CML s neodgovarajućim odgovorom na liječenje imatinib mesilatom. Provedena je kvantitativna lančana reakcija polimerazom u stvarnom vremenu (engl. real time quantitative polymesare chain reaction, RQ-PCR) prema protokolu udruženja Europa protiv raka (engl. Europe Against Cancer), a za dokazivanje mutacije T315I rabljena je metoda alel specifične oligonukleotidne PCR (engl. allele specific oligonucleotide PCR, ASO PCR). Rezultati: Kod 4 od 24 bolesnika dokazana je mutacija T315I (17%). Izrač u-nat je i medijan omjera bcr-abl/abl koji je iznosio 19% za bolesnike s T315I mutacijom i 13% za bolesnike bez mutacije, no razlika između tih dviju skupina nije bila statistički značajna (P = 0,394) Zaključak: Dokazivanje prisutnosti mutacije T315I ključno je u terapijskom pristupu bolesnicima s CML, budući daje liječenje izbora za nositelje te mutacije transplantacija matičnih stanica.Background: Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of bcr-abl fusion gene and consequently bcr-abl fusion protein. Although the discovery of tyrosine kinase inhibitor (TKI), imatinib mesylate (IM), improved the treatment of CML patients, a proportion of patients develop resistance to the drug resulting in increased bcr-abl level. One of possible reasons for resistance are the mutations in ABL kinase domain. Some mutations can be overcome by increasing the drug dose or by using the new generation of TKI. The only mutation resistant to currently available TKI is T315I. The aim of this study was to detect if the presence of T315I in patients resistant to imatinib mesylate therapy is associated with the increase or constantly high bcr-abl level. We also aimed to assess the possible difference in bcr-abl level in imatinib-resistant patients with and without T315I mutation. Materials and methods: The study included 24 CML patients with inadequate response to IM therapy. Real time quantitative PCR was performed according to Europe Against Cancer protocol and allele specific oligonucleotide PCR was used for T315I mutation detection. Results:T315I was detected in 4out of 24 patients (17%). Calculated median bcr-abl/abl levels were 19%forT315I positive and 13%forT315I negative patients, but the difference was not statistically significant (P = 0.394). Conclusions: T315I detection is essential in therapy approach for CML patients as the treatment of choice for T315I carriers is stem-cell transplantation

Clinical significance of T315I ABL kinase domain mutation detection in patients resistant to imatinib mesylate therapy

Uvod: Kronična mijeloična leukemija (engl. chronic myeloid leukemia, CML) je mijeloproliferativna bolest koju karakterizira prisutnost fuzijskog gena bcr-abl i posljedično fuzijskog proteina bcr-abl. Iako je otkriće inhibitora tirozin-kinaze (engl. tyrosine kinase inhibitor, TKI), imatinib mesilata (IM) poboljšalo liječenje bolesnika oboljelih od CML, dio bolesnika razvija rezistenciju na lijek, što dovodi do povišene razine bcr-abl prijepisa. Jedan od mogućih razloga te rezistencije su mutacije u domeni ABL kinaze. Neke se mutacije mogu prevladati povećanjem doze lijeka ili primjenom nove generacije TKI. Jedina mutacija rezistentna na trenutno dostupne TKI jest T315I. Cilj ovog istraživanja bio je otkriti da li je prisutnost T315I kod bolesnika rezistentnih na liječenje imatinib mesilatom povezana s povećanom ili neprekidno visokom razinom bcr-abl prijepisa. Također, cilj je bio procijeniti moguću razliku u razini bcr-abl prijepisa kod bolesnika rezistentnih na liječenje imatinib mesilatom sa i bez T315I mutacije. Ispitanici i metode: U ispitivanje su bila uključena 24 bolesnika oboljela od CML s neodgovarajućim odgovorom na liječenje imatinib mesilatom. Provedena je kvantitativna lančana reakcija polimerazom u stvarnom vremenu (engl. real time quantitative polymesare chain reaction, RQ-PCR) prema protokolu udruženja Europa protiv raka (engl. Europe Against Cancer), a za dokazivanje mutacije T315I rabljena je metoda alel specifične oligonukleotidne PCR (engl. allele specific oligonucleotide PCR, ASO PCR). Rezultati: Kod 4 od 24 bolesnika dokazana je mutacija T315I (17%). Izrač u-nat je i medijan omjera bcr-abl/abl koji je iznosio 19% za bolesnike s T315I mutacijom i 13% za bolesnike bez mutacije, no razlika između tih dviju skupina nije bila statistički značajna (P = 0,394) Zaključak: Dokazivanje prisutnosti mutacije T315I ključno je u terapijskom pristupu bolesnicima s CML, budući daje liječenje izbora za nositelje te mutacije transplantacija matičnih stanica.Background: Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of bcr-abl fusion gene and consequently bcr-abl fusion protein. Although the discovery of tyrosine kinase inhibitor (TKI), imatinib mesylate (IM), improved the treatment of CML patients, a proportion of patients develop resistance to the drug resulting in increased bcr-abl level. One of possible reasons for resistance are the mutations in ABL kinase domain. Some mutations can be overcome by increasing the drug dose or by using the new generation of TKI. The only mutation resistant to currently available TKI is T315I. The aim of this study was to detect if the presence of T315I in patients resistant to imatinib mesylate therapy is associated with the increase or constantly high bcr-abl level. We also aimed to assess the possible difference in bcr-abl level in imatinib-resistant patients with and without T315I mutation. Materials and methods: The study included 24 CML patients with inadequate response to IM therapy. Real time quantitative PCR was performed according to Europe Against Cancer protocol and allele specific oligonucleotide PCR was used for T315I mutation detection. Results:T315I was detected in 4out of 24 patients (17%). Calculated median bcr-abl/abl levels were 19%forT315I positive and 13%forT315I negative patients, but the difference was not statistically significant (P = 0.394). Conclusions: T315I detection is essential in therapy approach for CML patients as the treatment of choice for T315I carriers is stem-cell transplantation

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

Spontaneous Perforation of the Small Intestine, a Novel Manifestation of Classical Homocystinuria in an Adult with New Cystathionine b-synthetase Gene Mutations

The clinical picture of classical homocystinuria is diverse. This is the first report of an adult homocystinuric patient with non-traumatic spontaneous small bowel perforation. A 47-year old man presented with abdominal rebound tenderness, hypotension and tachycardia, anemia, and elevated markers of inflammation. Other routine laboratory tests were normal. Abdominal x-ray showed no free air. An emergency laparotomy revealed jejunal perforation in the left upper quadrant. Histologic specimen showed full-thickness nonspecific inflammation of the intestinal wall with granulocytic infiltration, hemorrhage and necrosis. Tuberculosis, actinomycosis and typhus were histologically and clinically excluded. After excluding all known possible causes of perforation, we presumed a causative relationship between homocystinuria and small bowel perforation. It could be hypothesized that connective tissue weakness in homocystinuria is a result of homocysteine interference with recombinant human fibrillin-1 fragments or cross-linking of collagen through permanent degradation of disulfide bridges and lysine amino acid residues in proteins. DNA analysis showed three detectable mutations in the cystathionine beta-synthetase gene, 1278T:c.833T>C, and two new mutations, V372G:c.1133T>G, and D520G:c.1558A>G in the alternatively spliced exon 15