Dorota Wojcik

Aberrant DNA methylation characterizes juvenile myelomonocytic leukemia with poor outcome

Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of cancer, including myeloid leukemia. We hypothesized that CpG island hypermethylation also occurs in juvenile myelomonocytic leukemia (JMML) and investigated whether it is associated with clinical, hematologic, or prognostic features. Based on quantitative measurements of DNA methylation in 127 JMML cases using mass spectrometry (MassARRAY), we identified 4 gene CpG islands with frequent hypermethylation: BMP4 (36% of patients), CALCA (54%), CDKN2B (22%), and RARB (13%). Hypermethylation was significantly associated with poor prognosis: when the methylation data were transformed into prognostic scores using a LASSO Cox regression model, the 5-year overall survival was 0.41 for patients in the top tertile of scores versus 0.72 in the lowest score tertile (P = .002). Among patients given allogeneic hematopoietic stem cell transplantation, the 5-year cumulative incidence of relapse was 0.52 in the highest versus 0.10 in the lowest score tertile (P = .007). In multivariate models, DNA methylation retained prognostic value independently of other clinical risk factors. Longitudinal analyses indicated that some cases acquired a more extensively methylated phenotype at relapse. In conclusion, our data suggest that a high-methylation phenotype characterizes an aggressive biologic variant of JMML and is an important molecular predictor of outcome.

Hematopoietic stem cell transplantation for advanced myelodysplastic syndrome in children: results of the EWOG-MDS 98 study

We report on the outcome of children with advanced primary myelodysplastic syndrome (MDS) transplanted from an HLA-matched sibling (MSD) or an unrelated donor (UD) following a preparative regimen with busulfan, cyclophosphamide and melphalan. Ninety-seven patients with refractory anemia with excess blasts (RAEB, n=53), RAEB in transformation (RAEB-T, n=29) and myelodysplasia-related acute myeloid leukemia (MDR-AML, n=15) enrolled in the European Working Group of MDS in Childhood (EWOG-MDS) 98 study and given hematopoietic stem cell transplantation (HSCT) were analyzed. Median age at HSCT was 11.1 years (range 1.4–19.0). Thirty-nine children were transplanted from an MSD, whereas 58 were given the allograft from a UD (n=57) or alternative family donor (n=1). Stem cell source was bone marrow (n=69) or peripheral blood (n=28). With a median follow-up of 3.9 years (range 0.1–10.9), the 5-year probability of overall survival is 63%, while the 5-year cumulative incidence of transplantation-related mortality (TRM) and relapse is 21% each. Age at HSCT greater than 12 years, interval between diagnosis and HSCT longer than 4 months, and occurrence of acute or extensive chronic graft-versus-host disease were associated with increased TRM. The risk of relapse increased with more advanced disease. This study indicates that HSCT following a myeloablative preparative regimen offers a high probability of survival for children with advanced MDS.

Genotype-phenotype correlation in cases of juvenile myelomonocytic leukemia with clonal RAS mutations

To the editor: In a recent issue of Blood , Matsuda et al reported 11 children with juvenile myelomonocytic leukemia (JMML) and clonal NRAS or KRAS mutations.[1][1] Three patients showed improvement of various clinical and laboratory features over a 2- to 4-year period without chemotherapy or

Additional genetic risk factor for death in children with acute lymphoblastic leukemia: A common polymorphism of the MTHFR gene

The presence of metabolically important genetic polymorphisms may affect treatment efficacy in patients with malignancies. The objective of this prospective multicenter study was to evaluate the role of selected polymorphisms of genes associated with metabolism of chemotherapeutic drugs as prognostic markers in children with acute lymphoblastic leukemia.

JAK2 V617F mutation is a rare event in juvenile myelomonocytic leukemia

our results consistently show that in pediatric patients, NPM1 mutations are less common than in adults and tend to affect older patients. Preliminary data also indicate that NPM1 mutations might be associated with favorable outcome, which warrants further studies to address this question. In addition, our results point to an effect of age on the prevalence of different NPM1-mutations, with non-typical (i.e. non-type A) mutations being most prevalent in children and younger adults. The reason for this association is unknown, but might refer to different molecular mechanisms involved in the development of this abnormality, a process which is currently largely unclear. This finding has also important implications for the MRD analysis and molecular follow-up of pediatric cases with NPM1þ AML. Whereas in adults, type A mutations predominate and assays focusing on this type will be suitable in most cases, in pediatric patients mutations should always be analyzed by sequencing. In these cases, a recently reported LNA-based procedure might be advantageous for follow-up of residual disease after treatment. C Thiede, E Creutzig, D Reinhardt, G Ehninger and U Creutzig Medizinische Klinik und Poliklinik 1, Universitätsklinikum Carl Gustav Carus, Dresden, Germany; Department of Pediatric Hematology and Oncology, Medical High School Hannover, Hannover, Germany and Department of Pediatric Hematology and Oncology, University Children’s Hospital, Muenster, Germany E-mail: christian.thiede@uniklinikum-dresden.de References

Efficacy and safety of Voriconazole in immunocompromised patients – single centre experience

Summary Background Data on epidemiology and survival after fungal infections in patients with cancer are primarily based on studies in adults, whereas few data are available on children. Aim The objective of this study was to determine the safety and tolerability of the oral and i.v. formulations of voriconazole Vfend®, previously not reported in Poland. Materials/Methods Twenty consecutive children and young adults aged 1 to 25 years, submitted for blood and marrow transplantation (BMT), were recruited during 10 months. When suspected or verified breakthrough systemic fungal infection occurred while kept on primary prophylactic fluconazole regimen, all patients received two loading doses of i.v. or oral voriconazole 6–9 mg/kg every 12 hrs, followed by a maintenance dose of 3 mg/kg every 12 hrs for five doses. If well tolerated, the voriconazole dosage was increased to 4 mg/kg every 12 hrs thereafter. Results Fungal infection was possible in 15/20, organ involvement with pulmonary process in 13 pts; CNS changes in 2 pts. Fungal aetiology was proven in 3 children (pulmonary Aspergillosis in 2 and Candida krusei in 1 patient), and considered as probable in 2 patients, having lung (2) and CNS (1) involvement. Four patients died. Clinical and radiological improvement was obtained in 14 patients, while uncontrolled, progressive disease occurred in 1 patient. Transient visual disturbances occurred in one patient. Major caution is warranted due to drug interaction via the cytochrome P-450 system which may lead to unexpected toxicity of the coadministered drugs (e.g. Vinca alkaloids, cyclosporine, rifampicin, erythromycin, etc). Conclusions Voriconazole is an advantageous new azole well tolerated in 20 patients. Outcome in BMT breakthrough infections was good.

Analiza wybranych czynników ryzyka zaburzeń dojrzewania płciowego u dzieci po leczeniu hematoonkologicznym z procedurą allogenicznego przeszczepu krwiotwórczych komórek hematopoetycznych (HSCT). Badanie pilotażowe

Cel Celem pracy byla ocena wybranych czynnikow ryzyka mających wplyw na przebieg procesu dojrzewania u dzieci po alogenicznym przeszczepie krwiotworczych komorek macierzystych (allo-HSCT). Material i metody Prospektywnym badaniom poddano grupe 21 pacjentow (13 dziewczynek, 8 chlopcow) w wieku 10–20 lat (mediana 14). Dzieci leczone byly z powodu: ostrej bialaczki limfoblastycznej (ALL) – 5 pacjentow, ostrej bialaczki nielimfoblastycznej (ANNL) – 4 pacjentow, przewleklej bialaczki szpikowej (CML) – 5 pacjentow, zespolu mielodysplastycznego (MDS) – 2 pacjentow, chloniaka nieziarniczego (NHL) – 1 pacjent, mlodzienczej bialaczki mielomonocytowej (JMML) – 1 pacjent, ciezkiej postaci anemii plastycznej (SAA) – 1 pacjent, niedokrwistości Blackfana-Diamonda – 1 pacjent oraz miesaka prązkowanokomorkowego (RMS) – 1 pacjent. Typ zastosowanego przeszczepu: HLA ID SIB (11), HLA MM REL (3), MUD-HSCT (7). Najcześciej stosowano wysokodawkowaną chemioterapie w postaci: busulfan/melfalan (BU/MEL-2); busulfan/cyklofosfamid/etopozyd(BU/CY/VP-5); busulfan/cyklofosfamid/ATG (BU/CY/ATG 4); 3 dzieci poddano napromienianiu calego ciala (total body irradiation–TBI). Napromienianie OUN przeprowadzono u 5 pacjentow: 18 Gy (4), 24 Gy (1). U 7 dzieci zastosowano kortykoterapie >28 dni przed przeszczepem i u 10 po przeszczepie. Analizie poddano wyniki nastepujących badan: stezenia gonadotropin – hormon folikulotropowy (FSH), hormon luteinotropowy (LH); prolaktyny (PRL); estradiolu, testosteronu, wolnej 3,5,3’-trijodotyronine (fT3), wolną tyroksyne (fT4), przeciwciala przeciw peroksydazie tarczycowej (ATPO), testy: z tyreoliberyną (TRH) i z gonadoliberyną (GnRH). Wyniki U 12 dzieci (9 dziewczynek, 3 chlopcow) stwierdzono nieprawidlową funkcje gonad od 10 do 72 miesiecy (średnio 28 miesiecy po przeszczepie): FSH 17,9–170 mIU/ml (średnio 39,89 mIU/ml), LH 6–40,1 mIU/ml (średnio 12,78 mIU/ml), estradiol Wnioski Nieprawidlowa funkcja gonad jest czestym nastepstwem stosowania chemioterapii wysokodawkowanej: cyklofosfamidu i busulfanu. Nasze badania nie wykazaly wplywu melfalanu i TBI na wystąpienie zaburzen dojrzewania plciowego, w odroznieniu od doniesien innych autorow.

2. Late onset idiopathic thrombocytopenic purpura carrelates with rapid B cell recovery in children after allogeneic transplants from alternative donors. Single centre experience

Late onset steroid-resistant idiopathic thrambocytopenic purpura (ITP) after allogeneic transplant remains rare yet important clinical problem. Two cases of ITP late post transplant are reported and discussed. Patient UPN 176. 16-year old girl with MDS-RA underwent allogeneic PBSCT fram male matched unrelated donar in August 2000. She was conditioned with Bu 16 mg/kg, Cy 200 mg/kg and ATG 20 mg/kg. GvHD prophylaxis consisted of CsA, MTX and methylprednisolone (MP). Hematological recovery was adequate: ANC > 0.5 G/l on day +16, platelets > 50 G/l on day +33. Mild aGvHD l° resolved quickly. No complications were observed until January 2001 (147 days after transplantation), when she developed ITP with a platelet count of 7 G/I and numerous petechiae. BM biopsy confirmed trilineage engratfment with almost 100% donor chimaerism. Megakaryocytes were abundant and appeared normal. Analysis of lymphocyte subsets revealed rapid recovery of CD19+ B cells (131/μl, incl. CD5+CD19+ cells) in comparison with the previous examination a month ago (9/μl) and substantial number of CD3+CD4+ Th cells (> 100/μl). Steroid-treatment was ineffective and she received high-dose (HD) IVIG (2 g/kg over 4 days) with sustained curative effect. She remains now in CR of both MDS and ITP with 100% donor chimaerism (XY-FISH). Patient UPN 142. 10-month-old boy with Omenn Syndrome underwent allogeneic haploidentical T-cell depleted (TCD) PBSCT from HLA-mismatched mother in October 1999. He was conditioned with Bu 20 mg/kg, TT 10 mg/kg, Flu 200 mg/m 2 and OKT-3. GvHD prophylaxis consisted of TCD (CliniMACS ® ), CsA, MP and MMF. Hematological recovery was prompt: ANC > 0.5 G/l on day +9, platelets > 50 G/l on day +30. Neither GvHD nor other serious complications were observed until May 2000 (217 days after transplantation), when he developed ITP. with a platelet count of 31 G/l. Chimaerism analysis confirmed 100% donor engraftment. Immune studies revealed rapid recovery of CD19+ B cells (324/ μl, incl. CD5+CD19+ cells) in comparison with the previous examination two months ago (57/μl) and peripheral expansion of CD3+CD4+ cells (> 400/μl) incl. CD4+CD69+ cells. Different modalities for treatment of ITP were tried with transient success. Prednisone (4 mg/kg) together with IVIG (O 4 g/kg) resulted in ITP resolution lasting 2 months. Platelet count dropped again to 51 G/l in August 2000 and HD IVIG (2g/kg) was administered with good yet transient effect. Four more HD IVIG courses every month were required to attain final ITP resolution in January 2001 – 14 months post transplant. The child remains now alive and well with platelet count above 200 G/l. Conclusions Rapid B cell recovery correlates with the occurrence of late onset steroid-resistant ITP in recipients of alternative transplants. Th (CD3+CD4+) cell recovery might also trigger autoimmunity by IL-2 mediated non-specific immune activation. The treatment of choice remains HD IVIG. Anti-D immunoglobulin might be used in Rh-positive patients. A novel highly effective option might be therapy with chimeric monoclonal anti-CD 20 antibody (rituximab – Mabthera ® ). supported by grant 687 from University of Medicine Wroclaw, Poland