Anna Pieczonka

Infant acute bilineal leukemia

Most cases of acute leukemia can be assigned to the myeloid, B or T lineage. There are rare cases of acute leukemia, which cannot be clearly classified, because either blasts express antigens of more than one lineage (acute biphenotypic leukemias) or distinct blast populations of two lineages co-exist (acute bilineal leukemias, aBLL). We present a 10-month-old infant with de novo aBLL, characterized by blasts of monocytic and B-cell precursor lineages. All leukemic cells harbored identical complex MLL gene rearrangement. Despite poor initial response, both to acute lymphoblastic leukemia (ALL) induction treatment and acute myeloid leukemia induction blocks, the child reached complete clinical remission with minimal residual disease negative status and was transplanted. Unfortunately, 16 months from HSCT the patient experienced BM relapse with all blasts characterized by pro-B-ALL immunophenotype. This case report illustrates that aBLL is a very aggressive type of acute leukemia that should be individually treated and monitored, particularly in children less than 1 year of age.

Hematopoietic chimerism after allogeneic stem cell transplantation: a comparison of quantitative analysis by automated DNA sizing and fluorescent in situ hybridization

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed mainly in patients with high-risk or advanced hematologic malignancies and congenital or acquired aplastic anemias. In the context of the significant risk of graft failure after allo-HSCT from alternative donors and the risk of relapse in recipients transplanted for malignancy, the precise monitoring of posttransplant hematopoietic chimerism is of utmost interest. Useful molecular methods for chimerism quantification after allogeneic transplantation, aimed at distinguishing precisely between donors and recipients cells, are PCR-based analyses of polymorphic DNA markers. Such analyses can be performed regardless of donors and recipients sex. Additionally, in patients after sex-mismatched allo-HSCT, fluorescent in situ hybridization (FISH) can be applied.MethodsWe compared different techniques for analysis of posttransplant chimerism, namely FISH and PCR-based molecular methods with automated detection of fluorescent products in an ALFExpress DNA Sequencer (Pharmacia) or ABI 310 Genetic Analyzer (PE). We used Spearman correlation test.ResultsWe have found high correlation between results obtained from the PCR/ALF Express and PCR/ABI 310 Genetic Analyzer. Lower, but still positive correlations were found between results of FISH technique and results obtained using automated DNA sizing technology.ConclusionsAll the methods applied enable a rapid and accurate detection of post-HSCT chimerism.

Ph‐negative isolated myeloid sarcoma with NPM1 gene mutation in adolescent with Ph‐positive chronic myeloid leukemia in remission after treatment with allogeneic bone marrow transplantation and imatinib mesylate

Few patients in remission of Ph‐positive chronic myelogenous leukemia (CML) develop Ph‐negative MDS/AML, usually with clonal cytogenetic abnormalities. Isolated Ph‐negative myeloid sarcoma (MS) is presented here as a form of such disorder, different from Ph‐positive MS establishing CML relapse in blastic phase. We describe 11‐year‐old male who developed Ph‐negative isolated MS with NPM1 mutation, remaining in complete molecular remission of Ph‐positive chronic myeloid leukemia treated with allo‐HSCT in first chronic phase and with imatinib and donor lymphocyte infusion in molecular relapse. The possible mechanisms of the tumor formation are reviewed with stress on importance of comprehensive molecular/cytogenetic evaluations. Pediatr Blood Cancer 2015;62:1070–1071.

Allogeneic bone marrow transplantation in children with acute lymphoblastic leukaemia in the first and second complete remission conditioned with fractionated total body irradiation and cyclophosphamide or etoposide

Patients and methods: From 1993 to 2001 thirty-two children underwent bone marrow transplantation (BMT) for acute lymphoblastic leukaemia (ALL) (12 in I complete remission /I CR/ of high-risk /HR/ ALL, and 20 in II CR after early bone marrow or combined bone marrow/organ relapse). Except for two syngeneic all others were matched sibling donor transplants. All patients (pts) were conditioned with fractionated total body irradiation (FTBI) at a total dose of 12,6 Gy, given in 8 fractions during 4 days with lung shielding (9,4 Gy) and cyclophosphamide (CY) 60 mg/kg i.v for 2 days (total dose 120 mg/kg) (n = 1 in I CR and n = 11 in II CR) or etoposide (VP) 60 mg/kg i.v (n = 11 in I CR and n = 9 in II CR). Patients in I CR were given 1,1–4,9x10 8 nucleated cells /kg (med. 2,7x10 8 /kg), while pts

Development and current use of in hematopoietic stem cell transplantation in children and adolescents in Poland: Report of the Polish pediatric study group for hematopoietic stem cell transplantation of the Polish society for pediatric oncology and hematology

The purpose of the survey was to evaluate the development and current use of hematopoietic stem cell transplantation (HSCT) in Poland between 1989-2016. The data for analysis (indication, number of performed HSCT, HSCT type, donor type, and stem cell source, year) have been collected annually using a standardized form. In Poland, between 1989-2016, the number of pediatric transplant beds grew from one to 40 and number and rate of transplants increased annually from 1/year (0.8/10 million) to 186/year (248/10 million). During the analyzed time period 2506 HSCTs were performed, including 1718 (68.6%) allogeneic transplants (allo-HSCT) with142 in 2016 and 788 (31.4%) autologous transplants (auto-HSCT) with 44 in 2016. Among 1718 allo-HSCT, 74% were performed for malignancy (ALL 47.2%, AML 26.2%, MDS 10.8%, CML 8.1%, NHL/HD 6.1%, others 2.5%), and 26% for non-malignant disorders (SAA 41%, congenital immunodeficiencies 35.4%, hereditary bone marrow failure 16%, metabolic disorders 7%). Among 788 auto-HSCTs, 30.8% were done for hematological malignancy (NHL 41.2%, AML 23.9%, HD 17.7%, ALL 15.6%, other 1.5%), while the remaining 69.2% for solid tumors (neuroblastoma 59.8%, Ewings sarcoma 20.4%, other 19.8%). In Poland, between 1989-2016, the infrastructure indispensable to perform HSCT in every child with indication for this therapeutic procedure was created, and HSCT became an important part of pediatric treatment, especially in pediatric oncology, hematology, and in primary immunodeficiencies.

HLA-inferred extended haplotype disparity level is more relevant than the level of HLA mismatch alone for the patients survival and GvHD in T cell-replate hematopoietic stem cell transplantation from unrelated donor

Serious risks in unrelated hematopoietic stem cell transplantation (HSCT) including graft versus host disease (GvHD) and mortality are associated with HLA disparity between donor and recipient. The increased risks might be dependent on disparity in not-routinely-tested multiple polymorphisms in genetically dense MHC region, being organized in combinations of two extended MHC haplotypes (Ehp). We assessed the clinical role of donor-recipient Ehp disparity levels in N = 889 patients by the population-based detection of HLA allele phase mismatch. We found increased GvHD incidences and mortality rates with increasing Ehp mismatch level even with the same HLA mismatch level. In multivariate analysis HLA mismatch levels were excluded from models and Ehp disparity level remained independent prognostic factor for high grade acute GvHD (p = 0.000037, HR = 10.68, 95%CI 5.50-32.5) and extended chronic GvHD (p < 0.000001, HR = 15.51, CI95% 5.36-44.8). In group with single HLA mismatch, patients with double Ehp disparity had worse 5-year overall survival (45% vs. 56%, p = 0.00065, HR = 4.05, CI95% 1.69-9.71) and non-relapse mortality (40% vs. 31%, p = 0.00037, HR = 5.63, CI95% 2.04-15.5) than patients with single Ehp disparity. We conclude that Ehp-linked factors contribute to the high morbidity and mortality in recipients given HLA-mismatched unrelated transplant and Ehp matching should be considered in clinical HSCT.

Allogeneic Stem Cell Transplantation From HLA-Mismatched Donors for Pediatric Patients with Acute Lymphoblastic Leukemia Treated According to the 2003 BFM and 2007 International-BFM Studies: Impact of Disease Risk on Outcomes.

Allogeneic hematopoietic stem cell transplantation (HSCT) is beneficial for pediatric patients with relapsed or (very) high-risk acute lymphoblastic leukemia (ALL) in remission. A total of 1115 consecutive patients were included in the ALL SCT 2003 BFM study and the ALL SCT 2007 I-BFM study and were stratified according to relapse risk (standard versus high versus very high risk of relapse) and donor type (matched sibling versus matched donor versus mismatched donor). A total of 148 patients (60% boys; median age, 8.7 years; B cell precursor ALL, 75%) were transplanted from mismatched donors, which was defined as either less than 9/10 HLA-compatible donors or less than 5/6 unrelated cord blood after myeloablative conditioning regimen (total body irradiation based, 67%) for high relapse risk (HRR; n = 42) or very HRR (VHRR) disease (n = 106). The stem cell source was either bone marrow (n = 31), unmanipulated peripheral stem cells (n = 28), T cell ex vivo depleted peripheral stem cells (n = 59), or cord blood (n = 25). The median follow-up was 5.1 years. The 4-year rates of overall survival (OS) and event-free survival were 56% ± 4% and 52% ± 4%, respectively, for the entire cohort. Patients transplanted from mismatched donors for HRR disease obtained remarkable 4-year OS and event-free survival values of 82% ± 6% and 80% ± 6%, respectively, whereas VHRR patients obtained values of 45% ± 5% and 42% ± 5% (P < .001), respectively. The cumulative incidence of relapse was 29% ± 4% and that of nonrelapse mortality 19% ± 3%. The cumulative incidence of limited and extensive chronic graft-versus-host disease was 13% ± 3% and 15% ± 4%, respectively, among the 120 patients living beyond day 100. Multivariate analysis showed that OS was lower for transplanted VHRR patients (P = .002; hazard ratio [HR], 3.62; 95% confidence interval [CI], 1.60 to 8.20) and for patients beyond second complete remission (CR2) versus first complete remission (P < .001; HR, 3.68; 95% CI, 1.79 to 7.56); relapse occurred more frequently in patients with VHRR disease (P = .026; HR, 3.30; 95% CI, 1.16 to 9.60) and for those beyond CR2 (P = .005; HR, 4.16; 95% CI, 1.52 to 10.59). Nonrelapse mortality was not significantly higher for cytomegalovirus-positive recipients receiving cytomegalovirus-negative grafts (P = .12; HR, 1.96; 95% CI, .84 to 4.58). HSCT with a mismatched donor is feasible in pediatric ALL patients but leads to inferior results compared with HSCT with better matched donors, at least for patients transplanted for VHRR disease. The results are strongly affected by disease status. The main cause of treatment failure is still relapse, highlighting the urgent need for interventional strategies after HSCT for patients with residual leukemia before and/or after transplantation.

10. Treosulfan with fludarabine and melphalan as conditioning regimen for second allogeneic BMT in a child with post-transplant MDS relapse resistent to adoptive immunotherapy – a case report

We report a 10-year old girl with a diagnosis of myelodysplastic syndrome, who after first bone marrow transplantation (HLA – matched sibling donor – May 1999) suffered from relapse (Dec 2001) and subsequently was treated by donor lymphocyte infusions (twice) with no response. We decided to perform a second transplantation from the same donor but with the use of different conditioning regimen. It consisted of: treosulfan (10 g/m2/day for 3 days, fludarabine (30 mg/kg/day for 5 days) and melphalan (140 mg/kg/day single dose). On day „0,, (30 May 2001) she was infused with bone marrow from her 8-year old, HLA-identical sister (20 × 106 CD34 cells/kg). As acute graft versus host disease prophylaxis she received cyclosporin A (1,5 mg/kg from day – 1). From day+16 we observed aGVHD II0 in the skin and gastrointestinal tract. Administration of metylprednisolon in max. dose of 1,5 mg/kg proved effecttive. No toxicity attributable to the conditioning regimen was noted. She was discharged on day+36. Complete donor chimerism was reached by day+14 and has not changed since then. On day+183 the girl is doing well with stable haematopoiesis of donor origin. We conclude that preparative regimen containing treosulfan is a reasonable option in heavily treated children undergoing bone marrow transplantation.

8. Monitoring of minimal residual disease (MRD) and hematopoietic chimerism with several complementary genetic methods in children treated with adoptive immunotherapy for post-transplant hematological CML relapse

The aim of this study was to compare results of MRD and hematopoietic chimerism obtained by several molecular methods (FISH, RT-PCR, STR-PCR) in children with post-transplant CML relapse treated with adoptive immunotherapy. Genomic DNA was isolated from frozen whole peripheral blood by saltingout method, amplified with fluorescent polymerase chain reaction primers (specific for STR marker loci: FGA, VWA, TH01, F13A1, D21S11) and analysed using quantitative automated DNA sizing technology. FISH was carried out on interphase nuclei with the use of probes specific for X and Y-chromosomes (DXZ 1 and DYZ 1 loci). Results obtained with the use of FISH, RT-PCR and STR-PCR were shown in table. CML Patient A.S. (UPN 26) Patient M.L. (UPN 45) Patient E. M. (UPN 18) Alla-BMT - 8.11.1994 Alla-BMT -15.11.1996 Allo-BMT - 24.08.1993 FISH Bcr-abl (+) RT-PCR Chimerism* CML FISH Bcr-abl (+) RT-PCR Chimerism* FISH X,Y CML FISH Bcr-abl (+) RT-PCR Chimerism* Relapse 11.03.99 Relapse 07.10.98 Relapse 01.02.00 16.03.99 50% Recipients genotype 20.10.98 60-80 % R 08.02.00 9,9 % Mixed 12-24.03.99 HU 21.10.98 DLI I 29.02.00 23,1% Recipients genotype 24.03.99 -11.01.00 INF-alfa HU 7.1<H;.11.98 I DLI 14.03.00 Mixed 28.10.99 2,92% b3a2 100 % D INF-alfa 02.11.- 02.12.98 12.04.00 37,3 % b3a2 Mixed 04.11.99 b3a2 18.01.99 b2a2 60–80 % R 26.04.00 51,3% 16.02.00 2% 01.03.99 b2a2 20–40 % R II DLI 05.05.00 14.06.00 100 % D 23.03.99 b2a2 10–20 % R 06.06.00 2,2% b3a2 Mixed 27.06.00 100 % D 29.03.99 2% b2a2 100 % D 04.07.00 b3a2b2a2 100 % D 26.07.00 100 % D 19.04.99 21.04.99 20% 23% b2a2 100 % D 23% XX 77% XY 10.08.00 100 % D I DLI 28.06.00 100 % D 05.07.99 17% b2a2 100 % D 14.11.00 b2a2 100 % D 30.08.00 3% 100 % D 22.09.99 6% b2a2 100 % D 8% XX 10.01.01 b3a2b2a2 07.12.00 0% b2a2 100 % D 28.09.99 b2a2 07.02.01 b3a2 25.04.01 22% b2a2 22.12.99 5% b2a2 09.04.01 - 26.06.01 10% b2a2 29.03.00 1,97% b2a2 100 % D 27.06.01 - 100 % D 28.08.01 21% b2a2 100 % D 06.06.00 b2a2 100 % D 29.09.01 10% b2a2 06.09.00 0% - 100 % D 100% XY 30.11.00 b2a2 100 % D 03.01.00 - 100 % D 22.03.01 0% b2a2 100 % D 100% XY 12.09.01 7% b2a2 100 % D 09.11.01 23% b2a2 100 % D 77% XY * examinations of chimerism status with the use of STR-PCR were performed without recipients control sample before BMT. Full-size table Table options View in workspace Download as CSV Conclusion : Combined evaluation of the minimal residual disease by FISH, RT-PCR and hematopoietic chimerism by STR-PCR method gives more informative details concerning response to adoptive immunotherapy in post-transplant relapsed CML patients with BCR/ABL fusion gene. This work was supported by the State Committee for Scientific Research – Projects NO. 4 POSE 07419, NO. 6 P05E 035 20 and No 4 P05E 108 18.

7. Adoptive immunotherapy of post–transplant hematological CML relapse with donor lymphocyte infusion and interferon-α

Purpose To evaluate the preliminary results obtained with donor CD3 lymphocyte infusion (DLI) and interferon-α (INF-α) in children with hematological CML relapse occurring after allogeneic bone marrow transplantation (allo-BMT). Patients and methods Between 1998–2000 three children with CML underwent allo-BMT from HLA-identical sibling and developed hematological leukemia relapse, which has been treated with DLI and INF-α. Conclusion DLI + INF-α seems to offer relatively effective and safe therapeutic option for advanced types of CML relapse occurring after allo-BMT in children, but further long lasting follow-up is necessary to define its place in management of post-transplant leukemia relapse. This work was supported by the State Committee for Scientific Research – Project No 6 POSE 050 21 . UPN 18 UPN 26 UPN 45 CML phase pre-BMT II CP I CP I CP Donor/recipient sex F/F M/M M/F Conditioning regiment for BMT Bu16+Vp30+Cy120 BU16+Vp30+Cy120 Bu16+Cy120 GvHD prophylaxis Csa + MTX Csa + MTX Csa + MTX GvHD occurrences after BMT Not observed Not observed II° (skin) Relapse after BMT +76 m-c Hematological - AP +52 m-c Hematological - AP +23 m-c Hematological - CP Relapse therapy prior DLI HU HU + INF-α HU DLI (donor CD3 dose/kg I 1,0 × 10 8 II 1,0 ×10 8 I 3,0 × 10 7 I 0,5 × 10 8 II 0,5 × 10 8 III 1,0 × 10 8 INF-α after DLI (+) (+) (+) Evaluation of efficacy and modification of the treatment have been based on results of FISH for Philadelphia chromosome detection and cellular chimerism in case of sex-mismatch. Results: UPN 18 UPN 26 UPN 45 GvHD not observed not observed not observed Pancytopenia observed not observed observed Time from 1st DLI to cytogenetic remision - + 162 +618 Disease status at last follow up Hematol. remission (10%)Ph+(FISH) from day +665 22 months Hematol. remission (10%) Ph+(FISH) from day +532 17,7 months Hematol. remission (7%) Ph+ (FISH) from day +1108 37 months