Sequential molecular monitoring of chimerism in chronic myeloid ...

Bone Marrow Transplantation, (1997) 19, 703–707  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

Sequential molecular monitoring of chimerism in chronic myeloid leukemia patients receiving donor lymphocyte transfusion for relapse after bone marrow transplantation MC Rapanotti, W Arcese, S Buffolino, AP Iori, A Mengarelli, MR De Cuia, A Cardillo and G Cimino Hematology, Department of Human Biopathology, University ‘La Sapienza’ of Rome, Italy

Summary: Recent observations of chimerism in patients relapsed following an allotransplant suggest the persistence of immunotolerance, thus offering a biologic rationale for the use of donor lymphocyte transfusion (DLT). In this study, we have analyzed by PCR amplification of several VNTR regions, sequential bone marrow and peripheral blood DNA samples in four patients who received DLT for CML relapse after bone marrow transplantation. Prior to DLT, all patients showed mixed chimerism in peripheral blood cells while two had mixed chimerism and two no chimerism in the BM. None of these four patients showed evidence of chimerism at the cytogenetic level (all had 100% +ve metaphases). After DLT, a complete hematologic and molecular remission (ie disappearance of the BCR/ABL fusion transcript) was obtained in the two patients who had bone marrow mixed chimerism prior to DLT. The two patients without evidence of marrow chimerism prior to DLT converted to a pattern of mixed chimerism after DLT, but both developed a severe bone marrow aplasia occurring at day 56 and 36, respectively. With regard to the sequential analysis of bone marrow chimerism after DLT we observed that: (1) the disappearance of BCR/ABL +ve cells paralleled the conversion to a pattern of full donor chimerism; and (2) the time interval to achieve CR was inversely correlated with the percentage of donor DNA in bone marrow. In conclusion, we have shown here that the assessment of bone marrow pre-DLT chimerism by PCR analysis might predict the response in patients with favorable characteristics, and also might identify patients at high risk of developing severe myelosuppression. Keywords: chimerism; donor lymphocytes transfusion; CML relapse

chronic myeloid leukemia (CML) relapsed after allogeneic bone marrow transplantation (BMT). 1–5 Chimerism studies in patients who relapsed following an allotransplant suggest the persistence of immunotolerance thus offering a biologic rationale for the use of DLT.6 Several methods with different sensitivities are useful to evaluate the chimerism status in transplanted patients. These include cytogenetics, Y body detection, protein polymorphism, and red cell phenotyping.7–9 More recently, DNAbased methodology has provided more sensitive techniques. In particular, polymerase chain reaction (PCR) amplification of individual specific genetic loci, such as the variable tandem repeats (VNTR) and the short tandem repeats (STR), have increased the sensitivity of the analysis up to 1–0.1% and 0.1–0.01%, respectively.10,11 Using these assays, a condition of mixed chimerism (MC) has been shown in 80% of CML patients receiving T cell-depleted allografts.11 With regard to CML patients treated with DLT for relapse after BMT, Mackinnon et al12 have analyzed the chimerism status of T lymphocytes by PCR and shown that a MC was present in almost every patient prior to DLT. While this observation encourages the use of adoptive immunotherapy in these patients, it provides no prognostic information on the post-DLT clinical outcome. In this study, we have analyzed by PCR amplification of several VNTR regions sequential bone marrow and peripheral blood DNA samples in four patients who received DLT for CML relapse after BMT. We show that bone marrow chimerism pre-DLT is prognostically important, since it may predict either response to treatment or the occurrence of severe aplasia.

Materials and methods Patients

Donor lymphocyte transfusion (DLT) has been successfully employed as adoptive immunotherapy in patients with Correspondence: Dr G Cimino, Hematology, Department of Human Biopathology, University ‘La Sapienza’, Via Benevento 6, 00161, Rome, Italy Received 1 August 1996; accepted 30 November 1996

Chimerism was evaluated in four patients (three males, one female) with Ph +ve CML undergoing DLT for relapse after an allogeneic HLA-identical BMT. The median age of the patients was 35 years (range 20–47). Marrow donors were sex mismatched in one case and ABO incompatible in two. At the time of transplant, three patients (UPN 034, 036 and 044) were grafted in first and one patient (UPN 007) in second chronic phase (CP).

Molecular monitoring of chimerism after DLT MC Rapanotti et al

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Prior to BMT all patients were conditioned by the association of 12 Gy fractionated total body irradiation (TBI) and cyclophosphamide combined with high-dose Ara-C in three cases. For GVHD prophylaxis, all patients received a T celldepleted graft and cyclosporine was added in three of them. No patient developed acute or chronic GVHD. The first evidence of relapse was cytogenetic in two patients (UPN 034, 036), with 7% and 50% of Ph +ve cells in the marrow, and hematologic (one CP, and one accelerated phase (AP)) in the other two (UPN 007, 044). Interferon, alone or combined with hydroxyurea was used to treat patients with cytogenetic or hematologic relapse, respectively. The interval between the first evidence of relapse and DLT was 92 (UPN 034), 93 (UPN 036), 14 (UPN 044) and 108 (UPN 007) months. At the time of DLT, all patients showed 100% of Ph +ve cells in the marrow. Two cases received DLT for cytogenetic relapse and two for hematological relapse (one in CP and one in AP). Donor lymphocyte transfusion Peripheral blood mononuclear cells were collected from the donor by two or three leukaphereses using the Cobe Spectra Apheresis System (Lakewood, CO, USA). Collections were performed over 2 or 3 consecutive days. The number of transfused mononuclear cells ranged from 2.0 to 3.01 × 108 per kg of recipient body weight. Molecular analysis of chimerism status Cell nuclei were prepared from whole BM and PB by a Triton-X-100 based method.13 High molecular weight DNA was extracted according to the salting-out method described by Miller et al.14 To assess the chimerism status we amplified by PCR the following VNTR regions: (1) D1S80 (defined by the probe pMCT118);15 (2) D4S95 (defined by the probe BS674E-D);16 (3) apolipoprotein B locus (located on chromosome 2 in the 3′ hypervariable region);17 and (4) the D17S30 (defined by the probe pYNZ22).18 The nucleotide sequences of the primers used are the following: D1S80: 5′-GAAACTGGCCTCCAAACACTGCCCGCCG3′ and 5′-GTCTTGTTGGAGATGCACGTGCCCCTTGC3′; D4S95: 5′-GCATAAAATGGGGATAACAGTAC-3′ and 5′-GACATTGCTTTATAGCTGTGCCTCAGTTT-3′; ApoB 5′-AAACGGAGAAATTATGGAGGGA-3′ and 5′CCTGAGATCAATAACCTCGT-3′; YNZ22 5′-CGAAGAGTGAAGTGCACAGG-3′ and 5′-CACAGTCTTTATTCTTCAGCG-3′. The minimum amount of genomic DNA amplified was 200 ng for Apo B and D17S30 VNTR loci, 350 ng for D4S95 and 700 ng for D1S80. PCR tests were performed in a final volume of 100 ml containing 1 × PCR-gelatin buffer (50 mm KCl, 10 mm Tris-HCl pH 8.4, 1.5 mm MgCl2, and 200 mg/ml gelatin). For each reaction we used 30 pmols of each primer, 200 mm dNTPs, 0.2 U AmpliTaq (Thermus aquaticus DNA polymerase; Perkin-Elmer Cetus, Norwalk, CT, USA). For each VNTR region 30 cycles of DNA amplification were performed using a thermal cycler Perkin-Elmer 9600 (PerkinElmer Cetus), according to the following cycling parameters: 1 min at 94°C, 2 min at 60°C for the VNTR Apo

B and D4S95; 1 min at 94°C, 5 min at 65°C for the D1S80; 1 min at 94°C, 1 min at 55°C and 1 min at 72°C for the D17S30. An aliquot of each amplified reaction mixture was electrophoresed on a 8% polyacrylamide gel (acrylamide/ bis-acrylamide ratio (37.5:1), and then stained with ethidium bromide and visualized under a UV lamp. In all cases, the size of the PCR products was in the range expected for each VNTR locus.15–18 Negative controls consisting of all reagents plus water and no DNA were included in all experiments to detect DNA contaminations. PCR detection of BCR/ABL fusion transcript RNA prepared from BM cells after stratification on a Ficoll–Hypaque density gradient, was extracted by the acid guanidium/phenol-chloroform method.19 To prepare cDNA, between 2 and 5 mg of total RNA was added to a total volume of 20 ml reverse transcriptase reaction containing 1 × reverse transcriptase buffer (50 mm Tris-HCl pH 8.3, 75 mm KCl, 3 mm MgCl2), 10 mm dithiotreitol (DTT), 1 mm each dNTPs, RNase inhibitor 1 U/ml, murine leukemia virus reverse transcriptase 2.5 U/ml (Perkin-Elmer Cetus) and 100 pmols of the following primer: 5′TGTGATTATAGCCTAAGACCCGGAG-3′. Reactions were incubated at 42°C for 20 min, followed by 5 min at 99°C and 5 min at 4°C to inactivate the reverse transcriptase. To amplify the specific p210 BCR/ABL chimeric transcript, a nested PCR technique was used as previously described.20 The amplified product was analyzed on ethidium-bromide 2% agarose gel and visualized under UV light. Negative and positive controls were performed to check each step of the RNA extraction and cDNA amplification. Results To test polymorphic variations in BM and PB DNA samples of the four donor–recipient pairs we used four minisatellite markers. Of these, the VNTR D1S80 was informative in patients UPN 007, 034 and 036, while the Apo B was informative in patients UPN 044 and UPN 036. Serial dilution experiments performed by mixing known amounts of host and donor DNA demonstrated that PCR amplification of D1S80 and Apo B VNTR regions allows the detection of 0.5% individual specific DNA (Figure 1). Table 1 shows the chimeric status prior to DLT according to BM karyotype, and DNA analysis of either BM or PB. None showed evidence of chimerism at cytogenetic analysis (all had 100% Ph +ve metaphases). With regard to DNA studies, all patients showed a MC in PB, while two (UPN 036 and 044) had MC and two (UPN 007 and 034) had no chimerism in the BM. Table 2 shows the response to treatment, therapy-related complications and clinical and biological follow-up according to chimerism status. All patients developed acute grade I–II GVHD while three of them had chronic GVHD. A complete hematologic and molecular remission (ie disappearance of the BCR/ABL fusion transcript) was obtained in UPN 036 and


Table 1

Patient pre-DLT chimerism status according to type of relapse and time intervals from BMT to relapse and from relapse to DLT

UPN

Chimerism status prior to DLT Karyotype (% Ph)

007 034 036 044

BM-DNA

100 100 100 100

Type of relapse

Time from BMT to relapse (months)

Time from relapse to DLT (months)

AP Cytogenetic Cytogenetic CP

24 6 6 62

108 92 93 14

PB-DNA

No chimerism No chimerism MC MC

MC MC MC MC

MC = mixed chimerism; AP = accelerated phase; CP = chronic phase.

Table 2 Patient pre-DLT BM DNA chimerism according to therapeutic response, occurrence of severe myelosuppression and GVHD, and patient clinical outcome after DLT UPN

Pre-DLT BM DNA chimerism (% donor DNA)

007 034 036 044

GVHD

No chimerism No chimerism Mixed chimerism (5%) Mixed chimerism (70%)

Acute

Chronic

I II II II

No Yes Yes Yes

Severe myelosuppression

Therapeutic response to DLT

Clinical outcome (months)

Severe Severe Transient No

NE CR a CR CR

Died (3.5) CCR (23) CCR (22) CCR (38)

a

CR after PBSC infusion (see text). NE = not evaluable; CR = complete response; CCR = complete continuous remission.

M

H

D

50

60

70

80

90

95 97.5 99

99.5

50

40

30

20

10

5

0.5

2.5

1

P

M

Figure 1 A dilution experiment demonstrating the sensitivity of minisatellite PCR amplification. H, host; D, donor; lane 3, 50% H: 50% D; lane 4, 60% H: 40% D; lane 5, 70% H: 30% D; lane 6, 80% H: 20% D; lane 7, 90% H: 10% D; lane 8, 95% H: 5% D; lane 9, 97.5% H: 2.5% D; lane 10, 99% H: 1% D; lane 11, 99.5% H: 0.5% D; lane 12, BM pre-DLT chimerism assessed in patient UPN 036. The amount of donor DNA was estimated comparing the intensity of PCR products obtained in pre-DLT BM samples with those achieved diluting in vitro pre-fixed amounts of donor/host DNA.

044 who had shown a marrow MC prior to DLT. As illustrated in Figure 2, the disappearance of BCR/ABL fusion transcript was more rapid (30 days post-DLT) in case UPN 044 than in case UPN 036 (425 days post-DLT). In these two patients, the amount of BM pre-DLT donor DNA, estimated comparing the intensity of patient BM pre-DLT PCR products with those achieved amplifying pre-fixed known amounts of ‘in vitro’ mixed donor/host DNA (example in Figure 1), was 70% and 5%, respectively. Patients UPN 007 and 034 who had no evidence of MC prior to DLT converted to a pattern of MC after DLT, but both developed a severe BM aplasia at day 56 and 36, respectively (Figure 3). Case UPN 034 was reinfused at day 60 after DLT with donor peripheral blood stem cells (PBSC) mobilized by GCSF, achieving a prompt resolution of the myelosuppres-

sion together with the disappearance of BCR/ABL fusion transcript and the conversion to a complete full donor chimerism (Figure 3a), which persists at the last follow-up control (day 365 post-DLT). Moreover, in the responders the achievement of a molecular remission corresponded to the conversion to a pattern of full donor chimerism. Owing to donor refusal, PBSC reinfusion was not possible in case UPN 007 who died in aplasia at day 90 after DLT. Discussion In this study, we show that the assessment of BM chimerism in CML patients receiving DLT for relapse after BMT may be relevant as a predictor of either the response to treatment or the occurrence of severe myelosuppression. Using a non-radioactive PCR-based approach to amplify patient and donor VNTR, we identified informative DNA polymorphisms which were also evaluated during the follow-up, in conjunction with the specific leukemia marker (ie BCR/ABL fusion transcript). As demonstrated in serial dilution experiments, the amplification of these VNTR sequences identified up to 0.5–1% of individual specific DNA, thereby showing a sensitivity level comparable to that reported by using other methods including radioactive and silver-staining detection systems.10,11 Furthermore, our analysis allows us to detect differences between PB and BM samples. In fact, mixed chimerism was detected in PB of all four patients and in BM of two of the four patients. This implies that in the two patients in whom no BM chimerism was detected, the small amount of donor PB cells contaminating the marrow is diluted below the level of detectability according to the sensitivity of our test.

705


36

Do no r

5d .

d.

PB +

+3

65

0d .

BM

+6

0d .

pre -D LT

+3

BM

M

BM

a

BM

M

Ho st

pre -D LT +3 0d PB +3 0d BM +3 65 d. PB +3 65 d. BM +4 25 d. PB +4 25 d. Do no r BM

M

BM

a

Ho st

706

M

VNTR D1S80 VNTR D1S80

N.D.

N.D.

N.D.

N.D.

el.

BCR/ABL

d.

. r no Do

BM

+6

0d

0d +3 BM

BM

M

b

Ho

st

M

pre

-D

.

LT

r no Do

+3 BM

0d +3

65

.

LT pre

-D BM

M

BM

b

BM

po

st-

BM

Tr

BCR/ABL

M

VNTR ApoB VNTR D1S80 N.E.

BCR/ABL

Figure 2 Sequential analysis of chimerism and BCR/ABL fusion transcript in patients UPN 036 (a) and UPN 044 (b). Chimerism status was evaluated by PCR using primers for the D1S80 VNTR in case UPN 036 and primers for the Apo B VNTR in case UPN 044. In this latter patient pre-transplant host DNA was not available. However, mixed chimerism (ie three allelic pattern) was demonstrated both at relapse and prior to DLT (lanes 2 and 3 b). The analysis of BCR/ABL fusion transcript performed at the same time intervals from DLT is shown below.

Recent clinical trials have reported that CML patients relapsed after BMT can be reinduced into hematologic and molecular CR by DLT, with relatively low toxicity.1–5 Chimerism status has been recently investigated in CML patients receiving allogeneic BMT.6,10,11,21–23 Using PCRbased methods, Mackinnon et al12 have reported the longterm persistence of donor memory T cells in all relapsed patients. While offering a biologic rationale for the use of DLT, this observation provides no prognostic information with regard to the clinical outcome. The result of our study suggests that assessment of chimerism in the BM appears to provide some clinically relevant information. In fact, persistence of PCR detectable donor cells in BM prior to DLT was associated with hematologic and molecular remission without risk of severe aplasia, whereas the absence of BM chimerism correlated with the occurrence of severe myelosuppression.

BCR/ABL

Figure 3 Sequential analysis of chimerism and BCR/ABL fusion transcript in patients UPN 034 (a) and UPN 007 (b). Chimerism analysis was performed on donor, host, and post-DLT sequential DNA samples using for PCR amplification the primers for the D1S80 VNTR. In (a) the arrow indicates the time in which donor PBSCs were transfused to patients UPN 034 (see text). The parallel study of BCR/ABL fusion transcript done in patients’ BM cells at the same time intervals from DLT is shown below the chimerism analysis.

Two studies have reported the assessment of pre-DLT BM chimerism in CML patients relapsed after BMT. Using Southern blot RFLP analysis, erythrocyte phenotype and cytogenetics, these studies did not report correlations between chimerism, response to DLT therapy and severe myelosuppression.2,4 We believe that such discordant results could be related to the inferior sensitivity of these techniques compared to PCR DNA analysis.10,22 In our experience, a condition of MC could be detected by PCR analysis in two patients who had shown absence of chimerism by cytogenetics. BM aplasia is a major DLT-related complication which may occur in up to 50% of patients. These patients can be successfully rescued with donor BM or PBSC reinfu-


sion.1–5,12 In our series, patient UPN 034 developed myelosuppression despite receiving DLT for a clinical favorable situation as a cytogenetic relapse; it is noteworthy that DNA analysis of chimerism did not detect in the BM of this patient pre-DLT donor DNA. Also, in the other patient who developed a BM aplasia, we could document the pre-therapy absence of BM chimerism. With regard to the sequential analysis of BM chimerism after DLT we observed that: (1) the disappearance of BCR/ABL +ve cells paralleled the conversion to a pattern of full donor chimerism (Figure 2a and b); and (2) the time interval to achieve CR was shorter in UPN 044 with .70% donor DNA compared with patient UPN 036 who showed only ,5% donor DNA, suggesting that the amount of donor hemopoiesis is inversely correlated with time to CR. In conclusion, we have shown here that PCR analysis of VNTR regions can provide relevant clinical information in the setting of DLT therapy. The assessment of BM preDLT chimerism might not only predict the response in patients with favorable characteristics, but also identify patients with no evidence of BM chimerism who are at high risk of developing severe myelosuppression. To avoid such complications a combination of DLT and PBSC should be considered in treating these patients. Acknowledgements We thank Dr Robin Foa for critical reading of the manuscript. This work was partially supported by ROMAIL.

References 1 Kolb HJ, Mittermuller J, Clemm C et al. Donor leukocyte transfusion for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: 2462–2466. 2 Ba¨r BMAM, Schattenberg A, Mensink EJBM et al. Donor leukocyte infusion for chronic myeloid leukemia relapsed after allogeneic bone marrow transplantation. J Clin Oncol 1993; 11: 513–519. 3 Helg C, Roux E, Beris P et al. Adoptive immunotherapy for recurrent CML after BMT. Bone Marrow Transplant 1993; 12: 125–129. 4 Drobysky WR, Keever CA, Roths MS et al. Salvage immunotherapy using donor leukocyte infusion as treatment for relapsed chronic myelogenous leukemia after bone marrow transplantation: efficacy and toxicity of a defined T-cell dose. Blood 1993; 82: 2310–2318. 5 Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versusleukemia effect of donor lymphocyte transfusion in marrow grafted patients. Blood 1995; 86: 2041–2050. 6 Mackinnon S, Barnett S, Heller G, O’Reilly RJ. Minimal residual disease is more common in patients who have mixed T-cell chimerism after bone marrow transplantation for chronic myelogenous leukemia. Blood 1994; 83: 3409–3416. 7 Sparkes MC, Crist ML, Sparkes RS et al. UCLA Transplantation Group. Gene markers in human marrow transplantation. Vox Sang 1977; 33: 202–205.

8 Ba¨r BMAM, Schattenberg A, Van Dijk BA et al. Host and donor erythrocytes repopulation patterns after allogenic bone marrow transplantation analysed with antibody-coated fluorescent microspheres. Br J Haematol 1989; 72: 239–245. 9 De Man AJM, Foolen WJG, Van Dijk BA et al. A fluorescent microsphere method for the investigation of erythrocyte chimerism after allogenic bone marrow transplantation using antigenic differences. Vox Sang 1988; 55: 37–41. 10 Uguzzoli L, Yam P, Petz LD et al. Amplification by the polymerase chain reaction of hypervariable regions of human genome for the evaluation of chimerism after bone marrow transplantation. Blood 1991; 77: 1607–1615. 11 Lawler M, Humphries P, McCann SR. Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction. Blood 1991; 77: 2504–2514. 12 Mackinnon S, Papadopoulos EB, Carabasi MH et al. Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus host-disease. Blood 1995; 86: 1261–1268. 13 Higuchi R. Simple and rapid preparation of sample for PCR. In: Erlich HA (ed). PCR Technology. Principles and Applications for DNA Amplification. Stockton: New York, 1989, p 31. 14 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215. 15 Sajantila A, Budowle B, Stro¨m M et al. PCR amplification of alleles at D1S80 locus: comparison of a Finnish and North American, Caucasian population sample, and forensic casework evaluation. Am J Hum Genet 1992; 50: 816–825. 16 Allitto BA, Horn GT, Altherr MR et al. Detection by PCR of the VNTR polymorphism at D4S95. Nucleic Acids Res 1991; 9: 4015. 17 De Corte R, Cuppens H, Marynen P, Cassiman JJ. Rapid detection of hypervariable regions by the polymerase chain reaction technique. DNA Cell Biol 1990; 9: 461–469. 18 Horn GT, Richards B, Klinger KW. Amplification of a highly polymorphic VNTR segment by the polymerase chain reaction. Nucleic Acids Res 1989; 17: 2140. 19 Chomczynski P, Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate-chloroform extraction. Anal Biochem 1987; 162: 156–159. 20 Lo Coco F, Mandelli F, Diverio D et al. Therapy-induced Ph′ suppression in chronic myeloid leukemia. Molecular and cytogenetic studies in patients treated with alpha-2b interferon, high dose chemotherapy and autologous stem cell infusion. Bone Marrow Transplant 1990; 6: 253–258. 21 Minn GL, Hibbin J, Arthur C et al. Use of minisatellite DNA probes for recognition and characterization of relapse after allogeneic bone marrow transplantation. Br J Haematol 1988; 68: 195–201. 22 Lawer M, McCann SR, Conneally E, Humphries P. Chimerism following allogeneic bone marrow transplantation: detection of residual host cells using the polymerase chain reaction. Br J Haematol 1989; 73: 205–210. 23 Chalmers EA, Sprul AM, Mills KI et al. Use of the polymerase chain reaction to monitor engraftment following allogeneic bone marrow transplantation. Bone Marrow Transplant 1990; 6: 399–403.

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