Justyna Jółkowska

The EVI-1 gene — its role in pathogenesis of human leukemias

EVI-1 (ecotropic virus integration site-1) was at first identified as an integration site of the murine leukemia retrovirus in murine myeloid leukemias. It is involved in pathogenesis of mouse and human leukemias. EVI-1 expression may be activated by retroviral insertion or is caused by chromosomal translocations. EVI-1 gene is located on human chromosome 3, spans over 100 kb and contains 12 exons with ten coding exons. EVI-1 gene encodes 1051 amino acids DNA binding protein containing ten zinc finger repeats organized in two domains. The 145 kDa EVI-1 protein is localized in the nucleus. The structure of the EVI-1 protein indicates that it functions as a transcriptional factor of the zinc finger family. The role of this transcription factor in myeloid cell transformation and the target genes of EVI-1 is still unknown. Occurrence of a few EVI-1 fusion transcripts was shown. The role of this fusion proteins is still unclear. Mouse and human sequences of the gene show a high degree of homology; 91% in nucleotide sequence and 94% in amino acid sequence.

Methods of minimal residual disease (MRD) detection in childhood haematological malignancies

The appropriate management of haematological disorders must rely on a precise and long-term monitoring of the patient’s response to chemotherapy and radiotherapy. Clinical data are not sufficient and that is why in the last decade it became the most important to improve the knowledge of haematological diseases on the basis of molecular techniques and molecular markers. The presence of residual malignant cells among normal cells is termed minimal residual disease (MRD). Nowadays a great progress has been made in the treatment of malignant diseases and in the development of reliable molecular techniques, which are characterised by high sensitivity (10−3–10−6) and ability to distinguish between normal and malignant cells at diagnosis and during follow-up. Especially, MRD data based on quantitative analysis (RQ-PCR, RT-RQ-PCR) appear to be crucial for appropriate evaluation of treatment response in many haematological malignancies. Implementation of standardized approaches for MRD assessment into routine molecular diagnostics available in all oncohaematological centres should be regarded nowadays a crucial point in further MRD study development.

Implementation of the standard strategy for identification of Ig/TCR targets for minimal residual disease diagnostics in B-cell precursor ALL pediatric patients: Polish experience

Introduction:Minimal residual disease (MRD), detected based on immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements as markers of residual leukemic cells, is currently the most reliable prognostic factor in acute lymphoblastic leukemia (ALL). A feasibility study is presented of the standard strategy for the identification of Ig/TCR targets for MRD diagnostics in Polish ALL patients by identifying Ig/TCR gene rearrangement pattern using standard primer sets and protocols.Materials and Methods:The PCR-heteroduplex approach based on BIOMED-1 and BIOMED-2 protocols (recommended as the European standard) was used to detect IGH, IGK-Kde, TCRD, TCRG, and TCRB rearrangements in 58 Polish B-cell precursor ALL patients. Sequencing and homology analysis between the obtained and germline Ig/TCR sequences enabled identification of the rearrangements. The U-Gauss test was used for statistical analysis of the Ig/TCR rearrangement pattern in Polish patients compared with relevant data on other nationalities.Results:The following pattern was identified: IGH: 83% (VH-JH: 74%, DH-JH: 9%), IGK-Kde: 41%, TCRD: 78% (incomplete TCRD: 55%, Vδ2-Dδ3: 45%, Dδ2-Dδ3: 21%, Vδ2-Jα: 35%), TCRG: 50%, and TCRB: 13%. Considerable convergence of the Ig/TCR pattern in Polish patients and those of other nationalities (mainly West Europeans) was demonstrated. Statistically relevant differences were only found between the incidence of DH-JH in Polish (9%) and Dutch patients (24%; p<0.05) and Polish and Italian patients (19%; p<0.05), VH-JH in Polish (74%) and Chilean patients (100%; p<0.05), and TCRG in Polish (50%) and Brazilian patients (69%; p<0.05).Conclusions:The convergence of Ig/TCR patterns in Polish and European patients indicates that the strategy for Ig/TCR target identification based on standard primers and protocols might be directly used for the construction of Polish standards and recommendations for MRD diagnostics.

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.

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