*Patrycja Zielińska, Agata Wieczorkiewicz-Kabut, Monika Dzierżak-Mietła, Anna Koclęga, Krzysztof Białas, Iwona Grygoruk-Wiśniowska, Krystyna Jagoda, Agnieszka Karolczyk, Małgorzata Krawczyk-Kuliś, Sławomira Kyrcz-Krzemień
Wczesna ocena chimeryzmu w komórkach CD34+ dodatnich u chorych z ostrą białaczką szpikową/zespołem mielodysplastycznym poddanych allogenicznej transplantacji krwiotwórczych komórek macierzystych – doniesienie wstępne
Early assessment of donor CD34+ positive cells chimerism after allogeneic stem cell transplantation in acute myeloid leukemia/myelodysplastic syndrome patients – pilot study
Department of Hematology and Bone Marrow Transplantation, Medical University of Silesia in Katowice
Acting Head of the Department: Małgorzata Krawczyk-Kuliś, MD, PhD
Streszczenie
Wstęp. W przypadku pacjentów z wysokiego ryzyka ostrą białaczką szpikową/zespołem mielodysplastycznym, poddawanych allogenicznej transplantacji krwiotwórczych komórek macierzystych, wznowa choroby zasadniczej pozostaje najczęstszą przyczyną niepowodzenia. Ocena chimeryzmu specyficznego liniowo jest ważnym narzędziem w monitorowaniu chorych w okresie poprzeszczepowym.
Cel pracy. Celem pracy była wczesna ocena chimeryzmu specyficznego liniowo w grupie chorych poddanych allogenicznej transplantacji krwiotwórczych komórek macierzystych.
Materiał i metody. Do badania włączono 55 chorych ze zdiagnozowaną ostrą białaczką szpikową lub zespołem mielodysplastycznym, którzy poddani zostali allogenicznej transplantacji krwiotwórczych komórek macierzystych. Analizie cytometrycznej oraz sortowaniu poddano próbki szpiku pobrane w dobie +30 po alloSCT. Szczegółowej analizie poddano komórki o fenotypie CD34+CD19-, w których oznaczono chimeryzm metodą STR.
Wyniki. W analizowanej grupie chorych, niższy chimeryzm w komórkach CD34+ stwierdzono w grupie chorych, którzy doświadczyli wznowy choroby zasadniczej. Chimeryzm w komórkach CD34+ w grupie chorych, którzy wznowili, wynosił 14,5% (mediana; zakres 0-51%), podczas gdy w grupie chorych, którzy pozostali w remisji przez okres obserwacji, chimeryzm w komórkach CD34+ nie spadł poniżej 97% (mediana 100%, zakres 97-100%). Wszystkie wznowy były obserwowane w ciągu pierwszego roku po alloSCT. Mediana czasu do wznowy wynosiła 107 dni (zakres 28-323).
Wnioski. Uzyskane wyniki wskazują, że wczesna ocean chimeryzmu w komórkach CD34+ sortowanych ze szpiku kostnego stanowi czułą metodę monitorowania statusu choroby zasadniczej w okresie poprzeszczepowym. Wydaje się, że oznaczanie chimeryzmu dawcy w komórkach CD34+ w dobie +30 po alloSCT może stanowić ważny element w opiece potransplantacyjnej chorych, zwłaszcza chorych wysokiego ryzyka.
Summary
Introduction. In high-risk acute myeloid leukaemia/myelodysplastic syndrome patients, relapse remains the major cause of treatment failure after allogeneic stem cell transplantation (alloSCT). The investigation of lineage-specific chimerism has become an important tool in the management of patients during the post-transplant period.
Aim. Early assessment of lineage specific chimerism in patients who underwent allogeneic hematopoietic stem cell transplantation.
Material and methods. 55 patients with acute myeloid leukemia and myelodysplastic syndrome who underwent alloSCT were included in the study. Flow cytometric analysis and cell sorting was performed in bone marrow collected at day 30 after alloSCT. For the purpose of this study we analyzed sorted immature progenitor cells (CD34+CD19-) using STR method.
Results. All patients who relapsed presented with lower donor chimerism in CD34+ positive cells in comparison to the group of patients in remission of the underlying disease. Median value of chimerism in CD34+ positive cells in the group of patients who relapsed was 14.5% (range 0-51%), whereas in patients who remained in remission of the underlying disease, chimerism in CD34+ never fell below 97% (median 100%, range 97-100%). All relapses occurred during the first year after alloSCT. Median time to relapse was 107 days (range 28-323).
Conclusions. Early assessment of chimerism in CD34+ cells sorted out of bone marrow is a sensitive technique to detect residual or reoccurring disease after allogeneic SCT. The assessment of donor chimerism in CD34+ cells in day +30 after alloSCT seems to be relevant in post-transplant care of high risk patients.
Introduction
In high-risk acute myeloid leukemia and myelodysplastic syndrome, relapse remains the major cause of treatment failure after allogeneic stem cell transplantation (alloSCT). Treatment of post-transplant relapse is difficult and most therapeutic approaches including donor lymphocyte infusions and second allogeneic transplantation usually present limited efficacy in the majority of cases. Following hematopoietic stem cell transplantation, monitoring the proportion of donor and recipient hematopoiesis in the patient is an influential tool in directing further treatment decisions (1, 2). The investigation of lineage-specific chimerism has therefore become an important tool for the management of patients during the post-transplant period.
Aim
The aim of the study was early evaluation of donor CD34+ positive cells chimerism after allogeneic hematopoietic stem cell transplantation. The study design was approved by the local Bioethical Committee, Medical University of Silesia on June 21, 2011 and on June 30, 2014 (continued). Written informed consent was collected from each patient included into the study. This research was supported by Medical University of Silesia.
Material and methods
A total of 55 patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) who underwent alloSCT were included in the study (median age 36 years, range: 19-67 years). Only patients with high risk AML and MDS whose disease had been shown to express CD34+ by the time of initial diagnosis or at relapse were eligible for the study. Disease categories were as follows: AML (n = 51), MDS and therapy related leukemia (n = 4). All patients had received peripheral blood grafts from sibling (n = 12) or unrelated donors (n = 43) after intensive (n = 23) or reduced-intensity (n = 32) conditioning. The study group consisted of 30 males and 25 females. The data of the study group are summarized in table 1.
Tab. 1. Patients characteristics
| Patient and donor characteristics | Number (%) |
Acute myeloid leukemia (risk classification according to ELN)
Favorable
Intermediate I
Intermediate II
Unfavorable | N = 50
3
31
3
13 |
Myelodysplastic syndrome – MDS RAEB2
IPSS low
IPSS intermediate I
IPSS intermediate II
IPSS high | N = 5
0
2
1
2 |
Female
Male | 25
30 |
| Patient age | 36 (19-67) |
Disease status prior to SCT
CR1
CR2
NR | N = 50
33
7
10 |
Donor
HLA identical family donor
HLA identical unrelated donor
HLA mismatched unrelated donor | N = 55
12
29
14 |
Blood group incompatibility
Major
Minor
Bi-directional | N = 43
24
10
9 |
| Donor age | 22 (19-49) |
Donor-recipient sex
Female donor to male recipient
Other |
19
36 |
Donor/recipient CMV status
Positive-negative
Positive-positive
Negative-negative |
12
35
8 |
The major AB0 group mismatch was detected in 24 cases, in 10 – minor, and in 9 donor-recipient pairs major and minor AB0 group incompatibility (bi-directional mismatch) was present. In 12 cases the blood groups between the donor and the recipient showed no difference. Cyclosporine at adjusted doses to concentration in serum and short course of methotrexate were used routinely as an immunosuppressive treatment in all patients. All patients transplanted from unrelated donor received either anti-lymphocyte globulin (ATG) at dose 15 mg/kg on three consecutive days preceding the transplantation or Thymoglobuline at dose 7.5 mg/kg (in 3 cases only).
Flow cytometric analysis and cell sorting was performed in bone marrow collected at day 30 after alloSCT. For the purpose of this study we analyzed immature progenitor cells (CD34+CD19-). Lineage specific donor chimerism was assessed using STR method and was calculated following the defined genetic profiles of the donor and the recipient. The methods for DNA isolation, cell sorting and chimerism analysis have been described in detail previously (3).
Results
Clinical data: All but one patient engrafted. The infused allograft contained a median of 4.56 × 106CD34+ cells/kg (range 1.7-11.91) and 22.31 × 108CD3+ cells/kg (range 7.8-33.87). Hematological recovery was as follows: neutrophils recovered to > 500/μL at a median of 13 (range 12-28) days posttransplant. All patients received IV antibiotics to treat febrile neutropenia. The median time to a platelet count > 50,000/μL was 15 days (range 11-21) posttransplant with 7 patients never dropping their platelet count below 20,000/μL. Patients were not given prophylactically hematopoietic growth factors to enhance engraftment.
The diagnosis of GVHD was based on physical examination and laboratory tests. Viral, allergic, drug-related causes of symptoms were ruled out. Acute GVHD was graded according to the modified Seattle Glucksberg criteria. In 39 patients acute GVHD symptoms developed till day +30, most of the patients presented grade I. Skin involvement was noted most often. In 4 patients severe GVHD symptoms (grade III) were seen, with gastrointestinal tract and liver involvement. Topical steroids were applied in 36 patients and systemic steroid therapy was needed in 24 cases (the maximum dose of methylprednisolone was 2 mg/kg of body weight/day).
Donor lineage-specific chimerism assessment at day +30 after alloSCT: All patients underwent assessment at day +30 after SCT. The median follow-up for all patients was 23 months (range 2-47 months). The analysis of chimerism in the cell subsets of interest was done in most patients. Some of the measurements were not possible, owing to technical reasons, including poor quality of clinical samples and low cell count.
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Piśmiennictwo
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Zielińska P, Markiewicz M, Dzierżak-Mietła M et al.: Assessment of lineage-specific chimerism after allogeneic stem cell transplantation. Acta Haematol Pol 2014; 45(4): 360-369.
Mattsson J, Uzunel M, Tammik L et al.: Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation. Leukemia 2001; 15: 1976-1985.
Rosenow F, Berkemeier A, Krug U et al.: CD34+ lineage specific donor cell chimerism for the diagnosis and treatment of impending relapse of AML or myelodysplastic syndrome after alloSCT. Bone Marrow Transplant 2013 Aug; 48(8): 1070-1076.
Lange T, Hubmann M, Burkhardt R et al.: Monitoring of WT1 expression in PB and CD34+ donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning. Leukemia 2011; 25(3): 498-505.
Bornhäuser M, Oelschlaegel U, Platzbecker U et al.: Monitoring of donor chimerism in sorted CD34+ peripheral blood cells allows the sensitive detection of imminent relapse after allogeneic stem cell transplantation. Haematologica 2009; 94(11): 1613-1617. doi: 10.3324/haematol. 009.007765.
Thiede C, Lutterbeck K, Oelschlagel U et al.: Detection of relapse by sequential monitoring of circulating CD34+ cells. Ann Hematol 2002; 81: S27-S28.
