Objective: This study was done to determine the relationship between donor chimerism and the presence of bcr-abl gene in chronic myeloid leukaemia (CML) patients post-transplantation. Methods: The study population consisted of all CML patients who had undergone non-myeloablative peripheral blood stem cell transplant in Hospital Universiti Kebangsaan Malaysia (HUKM) during the study period. All patients had their bone marrow aspiration done at diagnosis and day 30, 60, 100, 130, 160 and 190 post-transplantation. The samples were analysed for bcr-abl transcript as well as chimerism status. Results: A total of nine cases underwent non-myeloablative peripheral blood stem cell transplant. All patients were transplanted during the chronic phase. One patient was found to show mixed chimerism at day 30 post-transplant coinciding with bcr-abl transcript disappearance. Six patients showed that full donor chimerism correlated with bcr-abl transcript disappearance. In one patient, chronic myeloid leukaemia transformed into acute myeloid leukaemia. Another patient had a graft failure. Conclusion: This observational cohort study showed that full chimerism is required for disappearance of bcr-abl transcript but one case showed disappearance of bcr-abl transcript at day 30 while full chimerism was not achieved

Cheong, Soon Keng

Hussin, Noor Hamidah

Leong, Chooi Fun

Othman, Ainoon

Seman, Zainina

Universiti Putra Malaysia Institutional Repository

D on o r C h im er ism an d Bcr-Abl G en e S ta tu s F o llow in gN on -M ye lo a b la t iv e P er ip h era l B lo o d S tem C e ll T ran sp la n ta t io nin C h ron ic M ye lo id L eu k a em ia P a tien ts in H U K MIS Zainina, 2CF Leong, 2NH Hamidah, 20 Ainoon & 3SK CheongJDepa r tment of P a thology, F a culty of Medicine and Hea lth SciencesUniver siti P utra Ma laysia , 4 3 4 0 0 UPM Serdang, Selangor , Ma laysia2Depa r tment of P a thology, Hospita l Univer siti Kebangsa an Ma laysiaJa la n Yakob La tif, Chera s, Kua la Lumpur , Ma laysiaJDepa r tment of Medicine, Clinica l School, Interna tiona l Medica l Univer sitySeremban, Neger i Sembila n, Ma laysiaABSTRACTObjective: This study was done to determine the relationship between donor chimerismand the presence of her -abl gene in chronic myeloid leukaemia (CML) patients post-transplantation. Methods: The study population consisted of all CML patients who hadundergone non-myeloablative peripheral blood stem cell transplant in Hospital UniversitiKebangsaan Malaysia (HUKM) during the study period. All patients had their bonemarrow aspiration done at diagnosis and day 30, 60, 100, 130, 160 and 190 post-transplantation. The samples were analysed for her -abl transcript as well as chimerismstatus. Results: A total of nine cases underwent non-myeloablative peripheral blood stemcell transplant. All patients were transplanted during the chronic phase. One patient wasfound to show mixed chimerism at day 30 post-transplant coinciding with her -abl transcriptdisappearance. Six patients showed that full donor chimerism con'elated with her -abltranscript disappearance. In one patient, chronic myeloid leukaemia transformed intoacute myeloid leukaemia. Another patient had a graft failure. Conclusion: Thisobservational cohort study showed that full chimerism is required for disappearance ofher -abl transcript but one case showed disappearance of her -abl transcript at day 30 whilefull chimerism was not achieved.IN T R O D U C T IO NChronic myeloid leukaemia (CML) was first recognised as early as in 1845 when severalcases presented with splenomegaly, anaemia and massive granulocytosisJ1.2] It was thediscovery of Philadelphia (Ph) chromosome in 1960 that led to a better understanding of thepathogenesis of the disease. It took another thirteen years before the Ph chromosome wasshown to be generated by a specific translocation involving chromosomes 9 and 2 2 . In the1980s, the bcr -abl fusion oncogene was described, and found to be transcribed and translatedinto a functional protein. Finally, in 1990, the first definitive evidence of the ability of bcr -abl protein to transform primary myeloid cells and induce a CML-like disease in mice waspublished. [ I)Conclusi ve evidence of CML relies on cytogenetic and molecular studies to detect thet(9; 22) (q34; q II) translocation. The Ph chromosome is an acquired cytogenetic abnom1alitythat characterises all leukaemic cells in CML. However, it is not pathognomonic for CML asit is present in 25-50% of adult patients with acute lymphoblastic leukaemia and in 2% withacute myelogenous leukaemiaYl It was the first chromosomal abnormality associated witha specific malignancy. [3 )In CML transplantation, three strategies have been developed that include the use ofreduced intensity preparative regimens, the induction of tolerance between host and donorimmune systems by creating a mixed chimaeric state after transplant and optimising graftversus leukaemic effect by increasing the speed and completeness of donor immune recovery.These approaches are collectively grouped under the term 'non-myeloablative stem celltransplant' or called as 'minitransplant'.P a tient SampleBone marrow samples was collected with patients' consent from the haematology wards ofHUKM. Ethical committee approval was obtained from HUKM. Selection criterion wasCML patients who had undergone minitransplant. An exclusion criterion was CML patientswho only received medications. Nine patients had their bone marrow aspiration done atdiagnosis, day 30 post-transplant, day 60 post-transplant, day 100 post-transplant, day 130post-transplant and day 160 post-transplant. The samples were analysed for bcr -abltranscripts as well as chimerism status.Mononuclea r Cell Extra ction from Bone Ma r row Aspira tionMononuclear cell isolation was performed by gradient density centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech Ab, Uppsala, Sweden). Cells were washed toremove excess Ficoll-Hypaque. The cells were spun at 1000 rpm for 10 minutes to obtain cellpellet. The supematant was decanted.Chimer ism Assessment by Shor t Tandem Repea tsDNA extraction200 .ul binding buffer and 40 .ul proteinase K were added to 200 .ul of the bone marrowspecimen. The mixture was mixed immediately and incubated for 10 minutes at n ° e . 100.u1of isopropanol was added and the mixture was applied to a h i g h pure filter tube and centrifugedfor I minute at 8000 rpm. Then 500 .ul of wash buffer was added and centrifuged for I minuteat 8000 rpm. A new tube and 200 .ul of elution buffer were added before centrifuging themixture for I minute at 8000 rpm. The micro centrifuge tube then contained eluted nucleicacid.DNA quantificationThe purity and quantity of DNA were determined by spectrophotometer. The ratio ofwavelength reading at 260:280 nm was between 1.8-2.0. The DNA concentration wasestimated by measuring its absorbance at 260 nm.DNA amplificationThe master mix was prepared by using 21 p i ofthe reaction mix, 11 p i of the primer set and 1.0p i of the Ampli Taq Gold (Applied Biosystems, Foster City,Canada). The mixture wasvortexed for 5 seconds and 30 p I was pipetted into each PCR tube. 20 p i of DNA andpositive control were added into the PCR tubes respectively. In the negative control tube,20 p i of the TE buffer was added. All tubes were placed into the GeneAmp PCR system 9700(Applied Biosystems, Foster City, Canada) instrument.Acrylamide gel electrophoresisAcrylamide gel electrophoresis was done by using the ABI Prism 377 instrument. Once thegel temperature reached 51°C, 1.5 pi of DNA sample was injected using the flat tip into eachof the sample loading areas.Bcr-abl Sta tus Assessment by RT-PCRRNA was extracted according to the manufacturer's instructions as described before.Following that, the RNA was converted to cDNA. 8 p i of cDNA sample and I p i of TaqPolymerase were mixed with the PCR mix which contained 2.4 p i of PCR buffer, 0.9 p i ofMgCl2, 0.48 pi of dTPs, 0.112,ul ofCA3, 0.106,ul ofC5e, 0.100 ,ulofB2B, 0.088,ul ofBCR-C and11.812 pi of distilled water. The mixture was amplified using thermal cycle 480 and theproduct was run onto agarose gel for electrophoresis. The band was visualised usingultraviolet light.Clinica l F ea turesA total of nine cases of CML underwent non-myeloablative peripheral blood stem celltransplant in Hospital Universiti Kebangsaan Malaysia during the study period (Table 1).All patients presented and were transplanted during the chronic phase. Eight patients weretransplanted within one year of diagnosis of CML. However, one patient (CML 9) wastransplanted 4 years after he had been diagnosed to have CML. All except one were males.The median age was 32 years (range 15-49 years). Four were Malays while five wereChinese.All patients had high total white cell count at presentation, with the lowest being16.0xl09/1 and the highest 231.0x109/1. CML Patient 6 had the biggest spleen size at28 S Za inina , CF Leong, NH Hamidah, 0 Ainoon & SK CheongT ab le 1 . Patients characteristicsPatient Age Gender Ethnic Total white Spleen Liver Ber -abi atgroup blood cell diagnosisCMLI 23 Male Malay 60.0x 109/L 3cm 4cm PositiveCML2 28 Male Chinese 231.0 x 109Jl 15cm 6cm PositiveCML3 37 Male Chinese 36.6 x 109Jl 2cm nil PositiveCML4 49 Male Malay 45.6 x 109Jl 7cm lcm PositiveCML5 17 Male Malay 63.2 x 109/1 12cm 3cm PositiveCML6 35 Female Malay 150.0x 109Jl 16cm 6cm PositiveCML7 40 Male Chinese 23.4 x 109/1 5cm 2cm PositiveCML8 15 Male Chinese 16.0 x 109/1 3cm lcm PositiveCML9 49 Male Chinese 47,5 x 109/1 4cm nil Positivepresentation and she also had a hepatomegaly of 6 cm. All patients exhibited ber -abi genetranscripts positivity pre-transplant.Chimer ism Sta tus and bcr-abl Ana lysisAll patients had ber -abl analysis done pre-transplant and all were found to be positive.Both ber -abi and chimerism study were performed on day 30, 60, 100, 130, 160 and day 190post -transplantation.CML Patient I attained mixed chimerism at day 60 post-transplant, however ber -abistill showed positivity. He only attained complete chimerism at day-130 post-transplantand simultaneous disappearance of the ber -obl transcripts. The same findings were observedfor CML Patient 2. In the case of CML Patient 3, he attained complete chimerism anddisappearance of the ber -obl transcripts at day 100 post transplant. In CML Patient 4, afterday-IOO post transplant, analysis still showed no chimerism; he received donor lymphocyteinfusion. Subsequently, only at day 160 post transplant did he achieve complete chimerismtogether with the disappearance of the ber -abl transcripts. He also did not experience anygraft versus host disease (GVHD) which may delay ber -abi transcripts clearance. WhereasCML Patient 5 showed graft failure signs at day 60 post transplant and subsequentlyreceived donor lymphocyte infusion. Further, at day 100 post transplant, he did not achieveany chimerism status. Only at day 130 post transplant, did he show complete chimerism aswell as disappearance of the ber -abl transcripts. CML Patient 6 achieved transient mixedchimerism at day 30 post transplant, but reverted to no chimerism status for the subsequentanalysis. She received donor lymphocyte infusion two times, at day 120 and day 180 post-transplant; however, her leukaemic cells failed to clear. Patient CML 7 achieved completechimerism status and disappearance of the ber -abl transcripts faster than the other patientsdescribed before and this status persisted throughout the study period. Patient CML 8 wasthe unique patient; at day 30 post-transplant, he showed mixed chimerism as well asdisappearance of the ber -abl transcripts. Subsequently, he achieved complete chimerismwhich persisted. Patient CML 9 showed mixed chimerism and persistently positive ber -ablCC - Complete chimerismNC - No chimerismMC - Mixed chimerismtranscripts. At day 100 post-transplant, he showed signs oftransforrnation to acute leukaemiawith his peripheral blood showing presence of blast cells. He immediately received donorlymphocyte infusion but it failed to kill the leukaemic cells. Subsequent analysis showedno chimerism and presence of the bcr - abl transcripts.D IS C U S S IO NIn the CML patients studied, 55.5% were Chinese whereas 44.4% were Malays. There wasa marked gender difference in this group of patients with 88.8% being male and 11.1% beingfemale. The median age at presentation was 32 (range 15-49 year old) compared to SC Nget a l. 141 who found the age at presentation to be 34.9 years while Hamidah et a l. [51 reportedthe mean age at presentation to be 34.8 years. It appears that Malaysian CML population isyounger compared to the West. The reason is not clear but could be due to the loweraverage age of the Malaysian population compared to the population in the West.[4]In this study, the chronic leukaemia of CML Patient 9 transformed into acute leukaemiaafter day 90 post-transplantation. Though initially, he attained mixed chimerism, promisingfor engraftment, the mixed chimerism failed to sustain. At the time he showed haematologicaltransformation into acute leukaemia, he had received donor lymphocyte infusion (DLI).Donor lymphocyte can restore complete remission but is only effective in about 70% ofcases with a failure rate of 30%.16]Some studies indicate that mixed chimeras may be at higher risk of relapse [7.8,9Jwhileother studies do not corroborate this finding.[9,IO,'I]No conclusion can be drawn from oursmall series. To date, many investigators believed that CML patients need complete donorchimerism to eliminate the ber -abl transcripts. Our study appears to support this belief ascomplete chimerism was observed to correlate with clearance of ber -abl transcripts. Sevenout of nine patients attained complete chimerism simultaneously with the disappearance ofthe ber -abl transcripts. In one of the studies carried out[121, the authors found immunereconstitution after transplantation showing a complete chimerism state had significantlyhigher subsets ofCD2, CD3, CD4, CDS and CD6 cells than that of the mixed chimeric state.In our series, one patient (CML 8) managed to eliminate his ber -abl transcripts while hewas only at mixed chimerism state. The reasons for such a finding could be as follows:1 . NK cells after transplant are higher in a mixed chimeric state [121.NK cells are capable ofcytolytic activity against residual tumour cells. So this could be one of the possibilitieswhy his mixed chimerism state appeared to eliminate his ber -abl transcripts.2. The persistence of recipient cells in a mixed chimeric state results in the continuedpresence of allo-antigens (e.g. minor histo-compatibility antigens) unfamiliar to the donororigin of T cell precursors [131. Evidence suggests that such minor histo-compatibilityplays an important role in the OVHD and graft versus leukaemia (OVL) effect.[13] Anincreasing number of minor histo-compatibility antigens have been identified and it hasbeen shown that the expression of some minor his to-compatibility antigens is restrictedto haematopoietic cells and leukaemia cells.[14]3. It could be that the residual disease in this patient is more sensitive and fragile toimmune control by the graft compared to the other patients.4. The last reason could be that he underwent the transplantation at a stage where hisresidual disease is very low and this facilitated clearance of ber -abl transcripts by thegraft.C O N C L U S IO NThis observational cohort study showed that full chimerism is essential for disappearanceof ber -abl transcripts. However, one patient did not require full chimerism state to clear thedisease. Further studies need to be carried out to elucidate the mechanisms of tumourclearance without full chimerism.R E F E R E N C E S[I] Holyoake, Dr Tessa L. Recent advances in the molecular and cellular biology of chronic myeloidleukaemia: lessons to be learnt from the laboratory. Br J Haematol 200 I; 113: 11-23.[4] SC Ng, P Kuperan. Correspondence: age at diagnosis of chronic myeloid leukaemia. MalaysianJ Pathol 1990; 12(2): 111[5] NH Hamidah, SK Cheong, ° Ainoon. Correspondence: age at diagnosis of chronic myeloidleukaemia. Malaysian J Pathol. 1999; 13(2): 123.[6] Kolb, Schattenberg, Goldman JM, Hertenstein B, Jacobsen N, Arcese W, Ljungman P, FerrantA, Verdock L, Niederwieser D, Van Rhee F, Mittermuller J, De Witte T, Holl E, Anari H. Graftversus leukaemia effect of donor lymphocyte infusions in marrow grafted patients. Blood1995; 86: 2041-7.[7] Lawler SD, Baker MC, Harris B, Morgenstern GR. Cytogenetics study on recipients ofallogeneic bone marrow using the sex chromosome as a markers of cellular origin. Br J Haematol1984:56:431-42.[8] Walker H , Singer CRJ, Pattereson J, Goldstone AH. The significance of the host haemopoieticcells detected by cytogenetic analysis of bone marrow from recipients of bone marrow transplant.Br J Haematol 1986;62: 385-95.[9] Bretagne S, Vidaud M, Kuentz M, Cordonnier C. Mixed blood chimerism in T cell depletedmarrow transplant recipients: evaluation using DNA polymorphism. Blood 1987;70: 1692-709.[10] Schimitz M, Godde Salz E, Lofhler H. Cytogenetics study on recipients of allogeniec bonemarrow transplant after fractionated total body irradiation. Br J Haematol 1985;60: 239-52.[II] Schattenberg A. DeWitte T, Salden M, Vet J. Mixed haemopoietic cimerism after allogeneictransplant with lymphocyte depleted bone marrow not associated with a higher incidence ofrelapse. Blood 1989;73:1367-78.[12] Denis C, Ramana T, Christine M, Keith D, Barbara G, Kenneth C, Anderson A, Lee M, JeromeR. Natural history of mixed chimerism after bone marrow transplantation with CD6 depletedmarrow: a stable equilibrium. Blood 1990;75(1): 296-304.[13] Chaterine J, Antoinette C, Edwin P,Enrica 0 , Donna N, Robert J, Jerome R. Reconstitution ofT cell receptor repertoire diversity following T cell depleted allogeneic bone marrowtransplantation related to haematopoietic chimerism. Blood 2000;95(1):352-9.[14] Warren EH, Greenberg PD, Riddel SR. Cytotoxic T lymphocyte define human histocompatibilityantigens with a restricted tissue distribution. Blood 1998; 91:2197-207.

Donor chimerism and bcr-abl gene status following non-myeloablative peripheral blood stem cell transplantation in chronic myeloid leukaemia patients in HUKM

http://psasir.upm.edu.my/14132/1/Donor%20chimerism.pdf

Donor chimerism and bcr-abl gene status following non-myeloablative peripheral blood stem cell transplantation in chronic myeloid leukaemia patients in HUKM

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