Use of unrelated marrow grafts compensates for reduced graft-versus-leukemia reactivity after T-cell-depleted allogeneic marrow transplantation for chronic myelogenous leukemia

1995 86: 3987-3996

and WR DrobyskiMJ Hessner, DJ Endean, JT Casper, MM Horowitz, CA Keever-Taylor, M Roth, N Flomenberg marrow transplantation for chronic myelogenous leukemiagraft-versus- leukemia reactivity after T-cell-depleted allogeneic Use of unrelated marrow grafts compensates for reduced

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Use of Unrelated Marrow Grafts Compensates for Reduced Graft-Versus- Leukemia Reactivity After T-Cell-Depleted Allogeneic Marrow

Transplantation for Chronic Myelogenous Leukemia

By Martin J. Hessner, Debra J. Endean, James T. Casper, Mary M. Horowitz, Carolyn A. Keever-Taylor, Mark Roth, Neal Flomenberg, and William R. Drobyski

The effect of donorlrecipient histocompatibility on relapse in patients receiving T-cell-depleted (TCD) grafts for chronic myelogenous leukemia (CML) was evaluated. Specifically. we sought to determine whether TCD results in an attenuation of the graft-versus-leukemia (GVL) effect in recipients of unrelated marrow grafts similar to that observed in HLA- identical sibling marrow transplantations. This question was addressed by comparative analysis of the relapse rates in recipients of HLA-identical sibling versus unrelated donor marrow grafts who otherwise received identical preparative regimens and graft-versus-host disease (GVHD) prophylaxis schedules (T-cell depletion with T& monoclonal antibody and complement plus posttransplant cyclosporine) and by serial molecular analyses using the polymerase chain reaction (PCR) to detect the bcrlabl RNA transcript in patients transplanted with unrelated donor grafts. Patients transplanted with advanced disease (accelerated phase or blast crisis) had equally high relapse rates, regardless of whether they received HLA-identical sibling (56%; 95% confidence in- terval [Cl], 29% to 82%) or unrelated marrow grafts (39%; 95% Cl, 16% t o 66%; P = .37). Conversely, patients transplanted in chronic phase with unrelated marrow grafts (8%; 95% Cl, 0% t o 28%) had a significantly lower incidence of relapse than did patients transplanted with HLA-identical marrow grafts (47%; 95% Cl, 23% t o 71%; P = .002). Because all patients were similarly treated, these data indicate that

LLOGENEIC BONE marrow transplantation (BMT) is thought to be curative in part because of a graft-versus-

leukemia (GVL) effect derived from the adoptive transfer of immunocompetent cells in the donor graft. This GVL effect appears to be mediated by multiple effector populations, including T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells, which exert an antileukemia effect either directly through lytic mechanisms or indirectly through cytokine production or activation of secondary effector populations.‘” Whereas GVL reactivity is typically coexpressed with graft-versus-host disease (GVHD),3 epidemiologic analyses in humans4 and experi- mental studies in a n i r n a l ~ ~ . ~ provide support for an antileukemia effect that is independent of clinically evident GVHD. Other factors such as the immunogenetic relationship between donor and host?’ the relative expression of target antigens on host and leukemia cells>-” and the pretransplant manipulation of the donor r n a r r ~ w ’ ~ ~ ’ ~ also appear to modulate the intensity and specificity of the GVL effect. The relative importance of each of these variables is dependent on the specific disease entity for which allogeneic marrow transplantation is performed.

In chronic myelogenous leukemia (CML), retention of a GVL effect has been shown to be largely dependent on the development of GVHD and the presence of mature T cells in the donor graft! Thus, patients transplanted with unmodified grafts from HLA-identical sibling donors have a low rate of disease re1ap~e.I~ Conversely, use of T-cell depletion (TCD) as a GVHD prophylaxis strategy has been almost uniformly

A

Blood, Vol86, No 10 (November 15). 1995: pp 3987-3996

the lower relapse rate in these unrelated patients was caused by an augmented GVL effect that was most likely attributable to increased HLA disparity between donor and recipient. The probability of developing both acute and chronic GVHD was significantly increased in chronic-phase recipients of unrelated marrow grafts, suggesting that the enhanced GVL effect was a t least partly GVHD-associated. The lack of such a finding in advanced disease patients receiving unrelated marrow grafts raises the possibility that a clinically significant GVL effect after TCD marrow transplantation was limited and confined to patients with more indolent disease. Serial PCR analyses for the presence of the bcrlabl RNA transcript showed that the vast majority of patients transplanted in chronic phase with unrelated marrow grafts were persistently PCR-negative, indicating that the GVL effect was durable in these patients. Most of these patients were observed to become PCR negative within 1 to 2 months after transplantation, showing that early eradication of leukemia was possible with TCD marrow grafts. This study shows that the use of unrelated marrow grafts compensates for reduced GVL reactivity associated with TCD in patients transplanted for CML. Furthermore, these data indicate that, in selected patient populations with CML, TCD can be used to reduce GVHD without a commensurate com- promise in the GVL effect. 0 1995 by The American Society of Hematology.

associated with higher relapse rates and poorer disease-free survival in patients receiving HLA-identical sibling marrow transplant^.'^ In unrelated marrow transplantation for CML, GVHD is a more significant problem and a major cause of transplant-related mortality.I6 The salutory antileukemia effect conferred by mature T cells in the donor graft is therefore partially offset by enhanced GVHD-related toxicity. Moreover, concerns about GVHD have limited the degree of HLA disparity between donor and host that can be toler- ated in recipients of unmodified transplants. A requirement for effective GVHD prophylaxis strategies is more compel- ling in this setting, but the effect of approaches such as TCD on GVL reactivity is unknown.

From The Blood Center of Southeastern Wisconsin, Milwaukee, WI; The Bone Marrow Transplant Program, Departments of Medi- cine and Pediatrics, Medical College of Wisconsin, Milwaukee, WI: and The Department of Medicine, University of Michigan, Ann Arbor, MI.

Submitted May 8, 1995; accepted July 12, 1995. This study is dedicated to the memory of Mark Roth, MD. Address reprint requests to William R. Drobyski, MD, Bone Mar-

row Transplant Program, Box 176, 8700 W Wisconsin Ave, Milwau- kee, WI 53226.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

0 1995 by The American Society of Hematology. 0006-4971/95/8610-0010$3.00/0

3987




3988 HESSNER ET AL

Table 1. Patient Characteristics

HLA-Identical Sibling Unrelated PValue

Sex (M/F) 16/14 31/17 .32 Age (yr)

Median 40.1 31.7 ,0019 Range 21.6-57.7 5.4-53.0

First chronic phase 15 (50%) 27 (56%) Accelerated phase 14(47%) 17 (36%) .49 Blast crisis 1 (3%) 4 (8%)

Disease status, n (%)

Donor/recipient histocompatibility, n (%)

Matched 20 (42%) Mismatched 28 (58%)

GM-CSF therapy posttransplantation 7 (23%) 18 (38%) .l9

In this study, we sought to examine the impact of donor1 recipient histocompatibility on relapse in patients receiving TCD marrow grafts for CML. We specifically sought to define whether TCD results in an attenuation of the GVL effect in recipients of unrelated marrow grafts similar to that observed in HLA-identical sibling marrow transplants. This question was addressed first by examining the relapse rates in recipients of HLA-identical sibling and unrelated marrow grafts who otherwise received identical preparative regimens and GVHD prophylaxis schedules and secondly by per- forming longitudinal molecular analyses for the presence of the bcr/abl RNA transcript in patients receiving unrelated marrow grafts.

MATERIALS AND METHODS

Patient population. The population was composed of consecu- tive patients who received TCD allogeneic BMTs from either HLA- identical sibling or unrelated marrow donors for the treatment of CML between January 1988 and June 1993 at the Milwaukee County Medical Complex and Children’s Hospital of Wisconsin. The patients receiving transplants from unrelated donors have been described in detail.” The disease status and other demographic data from each of the patient cohorts are shown in Table 1. CML was classified as accelerated if one or more of the following criteria were present: (1) progressive leukocytosis, thrombocytosis, or splenomeg- aly unresponsive to therapy with hydroxyurea, busulfan, or inter- feron; (2) 220% basophils plus eosinophils in the peripheral blood (PB); (3) 2 10% blasts in the PB or BM; (4) chromosomal abnormalities in addition to the Philadelphia chromosome; (5) persistent ane- mia or thrombocytopenia unrelated to cytotoxic therapy; and/or (6) 220% blasts plus promyelocytes in the PB or BM. Blast crisis was defined as 230% blasts plus promyelocytes in the PB or BM. In- formed consent was obtained from each patient (or their guardians), and all treatment was administered under protocols approved by the institutional review committees of the Medical College of Wiscon- sin.

Donor selection and histocompatibiliv testing. Each recipient and donor candidate for either HLA-identical sibling or unrelated marrow transplantation underwent extended serotyping for HLA-A, B, DR, and DQ by standard microcytotoxicity assays to detect all specificities currently recognized by the World Health Organization (WHO).” In unrelated marrow transplants, all recipients and donors deemed to be suitably matched based on screening serologic and

mixed lymphocyte culture assays underwent further prospective oligotyping of HLA-DRB1.I9 Prospective oligotyping for HLA-DQB 1 was begun in January 1990, as previously described.” Donodrecipi- ent pairs from transplants performed before these dates were retro- spectively analyzed so that the degree of HLA-DR and DQ compatibility of the entire unrelated patient population was determined. Oligotyping of patient and donor samples was performed using the polymerase chain reaction (PCR) in conjunction with sequence-specific oligonucleotide probe hybridization. The assignment of DR and DQ specificities was made according to the nomenclature presently recognized by the WHO.“ Any donodrecipient disparity that was detectable by serology or molecular genetic analyses was considered a mismatch. Recipients of matched and mismatched unrelated marrow grafts were similar with respect to age, disease status, and recipient cytomegalovirus (CMV) status.I7

Preparative regimen, GVHD prophylaxis, and supportive care. Patients were treated in laminar air flow or high-efficiency particulate air-filtered rooms. All patients in each cohort received a standardized pretransplant conditioning regimen of high-dose cytosine arabino- side, cyclophosphamide, and methylprednisolone, followed by frac- tionated total body irradiation (TBI) to a total dose of 14 Gy, as previously described.** Seven children (< l6 years of age) who received unrelated marrow grafts also received oral busulfan (4 mg/ kg/d). GVHD prophylaxis consisted of ex vivo TCD with the ap T-cell receptor antibody T,& and complement plus posttransplantation cyclosporine.*3 All patients received prophylactic acyclovir and trimethoprim-sulfamethoxazole both before and after transplantation. CMV-seronegative patients received blood components from CMV-seronegative donors. Prophylactic intravenous Ig was administered to all patients as previously described.” Granulocyte-macro- phage colony-stimulating factor (GM-CSF; 250 pglm2) was administered to 18 patients who received unrelated grafts and 7 patients receiving HLA-identical sibling marrow grafts, beginning on the day of transplantation and continuing for 14 to 28 days to accelerate myeloid engraftment. Patients transplanted with HLA-identical sibling marrow grafts who were treated with GM-CSF were part of a randomized study designed to assess the effect of GM-CSF on engraftment.24

Assessment of GVHD and relapse. Acute GVHD was graded as 0 to IV according to criteria of Glucksberg et al.’’ Chronic GVHD was defined as none, limited, or extensive.*‘ Patients who had evidence of engraftment were evaluable for acute GVHD, whereas patients who engrafted and also survived more than 100 days were evaluable for chronic GVHD. Relapse was defined as morphologic evidence of leukemia in the PB, BM, or extramedullary sites or by the recurrence and sustained presence of the Philadelphia chromosome or other pretransplant karyotypic abnormalities.

Preparation of RNA. Total cellular RNA was prepared from PB, PB buffy coats, or PB mononuclear cells isolated by ficoll-hypaque, BM, or cultured B cells and K562 (Phl-positive) cells. Cells were either viably frozen or directly added to 4 mom guanidinium iso- thiocyanate. RNA was prepared from 3 to 5 million cells as previously reported with minor modification^.^^ Electrophoretic separation of sample RNA preparations through 2% agarose ethidium bromide stained gels showed that isolations were not free of DNA; however, the relative amount of contaminating DNA was consistent from sample to sample.

Reverse transcription and PCR amplification. One microgram of total cellular RNA was reverse transcribed for 1 hour at 42°C in 50 mmoVL Tris-HC1, pH 8.3, 50 mmoUL KCI, 8 mmoVL MgC12, 10 mmoVL dithiothreitol (Dm) , 0.5 mmoVL dNTPs, and 2 pg A N - RT primers using 30 U AMV-RT (Seikagaku America, Rockville, MD) in a total volume of 50 pL. PCR was performed directly on 10 pL of this material in 10 mmol/L Tris-HC1, pH 8.3, 50 mm0U

L KC1, 1.6 m m o n MgC 1 2r 2 mmom D m , 0.2 mmom dNTPs,




GVL REACTIVITY AFTER UNRELATED BMT FOR CML 3989

U

Fig 1. Diagram illustrating the processing of individual PB or BM samples for PCR analysis of the bcrl ab1 RNA transcript. A and B denote duplicate RNA samples.

PCR b1.M

and 0.2 pg each of the primers ABL-1 and BCR-I using 1 U of Taq polymerase in a total reaction volume of 50 pL. Thirty-five cycles were performed consisting of 1.25 minutes at 94°C and 1 .S minutes at 72°C in a Perkin-Elmer 480 thermalcycler. All PCRs were termi- nated after 10 minutes of extension at 72°C. Second-round PCR was performed using the internally nested primers (BCR-I and ABL-2) with 1 pL of the first-round PCR product as template for an additional 35 cycles. The sequences of the oligonucleotide primers BCR- 1 , BCR-2, ABL-I, ABL-2, and ABL-3 have been previously described.** Fifteen microliters of each amplified PCR product was analyzed by electrophoresis through 2% agarose gels and stained with ethidium bromide.

Assay control. Figure 1 depicts the methodologic approach used to insure accuracy, establish reproducibility, and limit the possibility of false-positivity for testing of individual samples. All RNA isolations, cDNA synthesis, and set-ups (except for introduction of second round PCR template) were performed in a positive pressure high- efficiency particulate air-filtered clean room to prevent the introduction of previously amplified material. The evaluation of RNA integ- rity and adequate cDNA synthesis from samples was accomplished through the detection of the constitutively expressed ABL mRNA from the same reverse transcription reaction. The ABL cDNA was amplified using the primers ABL-1 and ABL-3 (which targets ABL exon la). Assay sensitivity was monitored through the addition and detection of 0.05 ng K562 RNA mixed within a replicate assay for each unknown sample.29 This quantity of RNA represents nucleic acid isolated from approximately 5 K562 cells, which requires two rounds of nested primer PCR for detection. Failure to detect the spiked K562 RNA could indicate the presence of inhibitors or ribo- nucleases within the unknown RNA sample. Mock RNA preparations were included in every assay as negative controls to survey for the possibility of false-positives due to the introduction of previously amplified DNA. All samples and controls were analyzed in duplicate, beginning with independent RNA isolations. To be deemed acceptable, samples had to meet the following criteria: (1) the abUabl message was detectable after a single round of reverse transcription PCR, (2) the spiked K562 RNA sample was detectable only after nested primer RT PCR and not after single round amplification, and ( 3 ) duplicate samples yielded concordant results. When discordance was observed between duplicate samples, a new RNA isolation was performed from another sample and three of four samples had to be concordant to be deemed a valid result.

Statistical analysis. Endpoints were calculated at the date of last contact, with the date of latest follow-up being December 31, 1994. The median duration of follow-up was 36 months (range, 12 to 73

months) for patients receiving HLA-identical sibling marrow grafts and 38 months (range, 10 to 79 months) for patients receiving unrelated marrow grafts. Patients in either accelerated phase or blast crisis at the time of transplant were analyzed together as advanced disease. Cumulative probabilities of relapse and acute and chronic GVHD were calculated using the Kaplan-Meier method.30 For calculating the probabilities of acute and chronic GVHD, patients were censored at last follow-up or at death without development of these endpoints. For calculating the probability of relapse, patients in con- tinuous complete remission were censored at death or last follow- up. Confidence limits for the Kaplan-Meier estimate were based on arcsine transformation using Greenwood's formula for the standard error of their survival e~timator.~' Comparisons of time to event distribution were made using the log rank test.

RESULTS

Comparative risk of relapse afer unrelated and HLA- identical sibling marrow transplantation. The effect of donorhecipient histocompatability on relapse after TCD allogeneic marrow transplantation for CML was assessed by comparing relapse rates in patients receiving either HLA- identical sibling or unrelated marrow grafts. The 3-year probability of relapse after transplantation for chronic-phase CML was 8% (95% CI, 0% to 28%) for recipients of unrelated marrow grafts versus 47% (95% CI, 23% to 71%) for patients transplanted with HLA-identical marrow grafts ( P = .002; Fig 2). When patients were transplanted for advanced phase of CML, there was no statistically significant difference in relapse between patients receiving unrelated (39%; 95% CI, 16% to 66%) versus HLA-identical sibling (56%; 95% CI, 29% to 82%) marrow grafts (P = .37; Fig 3) . Of note, the relapse rates for unrelated marrow transplant recipients in either chronic phase or with advanced disease were not altered when the seven pediatric patients who received additional busulfan (2 in chronic phase and 5 in advanced phase) were excluded from the analysis. The relapse rate in patients receiving matched or mismatched unrelated grafts was examined to assess whether increased HLA disparity was associated with a lower disease relapse. The 3- year probability of relapse was not significantly different for patients receiving mismatched grafts (20%; 95% CI, 4% to 44%; N = 28) when compared with that of patients receiving




3990 HESSNER ET AL

P-OaQU

i : 6

EFLA'IED m-15)

$ M _r

8 E 0.2.

umm..mm m-zn c Fig 2. Actuarial probability of leukemia relapse

for patients who received either HLA-id.nticd sibling or unrelated marrow grafts for treatment of chronic-

60 phase CML. Tick marks represent censored observations.

ao\::: ! : : : , : 0 12 U 86 18

MONT88 " E R TBAN(IpLANT

phenotypically matched grafts (38%; 95% CI, 13% to 67%; N = 20; P = .20).

Patients transplanted in chronic phase with unrelated and HLA-identical sibling marrow grafts had an overall 3-year probability of survival of 55% (95% CI, 37% to 73%) and 100% and a disease-free survival of 51% (95% CI, 32% to 70%) and 53% (29% to 77%), respectively. Seven of eight patients in chronic phase who relapsed after receiving an HLA-identical sibling graft were salvaged with donor leuko- cyte infusions and are in remission. Overall survival for advanced-disease patients who received unrelated grafts was 51% (95% CI, 29% to 73%) and for patients who received HLA-identical sibling grafts was 53% (95% CI, 27% to 79%). Disease-free survival was 27% (95% CI, 10% to 48%) and 27% (95% CI, 8% to 51%), respectively.

Ten recipients of HLA-identical sibling transplants and 19 recipients of unrelated donor transplants were in clinical remission at last contact. Cytogenetic data were available at the time of last contact for 14 of these patients (7 recipients

8

Fig 3. Actuarial probability of leukemia relapse for patients who received either HlA-identical sibling or unrelated marrow grafts for treatment of advanced-phase CML. Tick marks represent censored observations.

"1

of HLA-identical sibling marrow grafts and 7 recipients of unrelated marrow grafts). Additionally, cytogenetic data from a 3-year evaluation were available in 2 recipients who were not evaluable cytogenetically at last contact. All of these patients are currently in cytogenetic remission.

Incidence and severity of GVHD. Because GVHD is known to affect the probability of relapse after allogeneic BMT for CML?'' the incidence of acute and chronic GVHD in both patient populations was assessed. Forty-seven of 48 recipients of unrelated transplants and 29 of 30 recipients of HLA-identical sibling transplants were evaluable for acute GVHD. There was a higher incidence of grades I1 to IV acute GVHD in unrelated (43%; 95% CI, 29% to 57%) as opposed to HLA-identical sibling recipients (14%; 95% CI, 4% to 28%; P = .009; Fig 4). The incidence of grade I11 or IV acute GVHD, however, was similar in recipients of unrelated marrow grafts (8.5%; 95% CI, 2% to 18%) when compared with that of patients receiving HLA-identical grafts (3.4%; 95% CI, 0% to 13%; P = .40). Thirty-nine of




GVL REACTIVITY AFTER UNRELATED BMT FOR CML

Lo'

as-

E l

3991

a $ OA-

E 02. RELATF.D W-29)

Fig 4. Actuarial probability of developing grades Il-IV acute GVHD for patients who received either ao\ HLA-identical sibling or unrelated marrow grafts. 0 Tick marks represent censored observations.

48 receiving unrelated donor transplants and 28 of 30 patients receiving HLA-identical sibling marrow grafts survived at least 100 days and were evaluable for chronic GVHD. Patients receiving unrelated marrow grafts had a higher probability of developing chronic GVHD (74%; 95% CI, 60% to 87%) than those receiving HLA-identical sibling transplants (37%; 95% CI, 20% to 56%; P = .0005; Fig 5). In the 29 patients transplanted with unrelated donor grafts who developed chronic GVHD, limited disease occurred in 25 and extensive chronic GVHD in 4 recipients. Of the 10 patients transplanted with HLA-identical sibling grafts who developed chronic GVHD, 9 had limited and 1 had extensive disease. The 3-year probability of developing extensive chronic GVHD was 4% (95% CI, 0% to 14%) in HLA- identical sibling and 13% (95% CI, 4% to 28%) in unrelated marrow transplant recipients (P = .31).

The incidence of grade 11-IV acute GVHD was also assessed by disease status. Patients transplanted in the chronic phase with unrelated marrow grafts had a significantly higher

20 U) 60 80 Km DAY8 TBANLlpLANT

incidence of GVHD (45%; 95% CI, 26% to 64%) than similarly staged patients transplanted with HLA-identical sibling grafts (0%; P = .003). Conversely, there was no difference in grade 11-IV acute GVHD for patients with advanced disease who received unrelated (40%; 95% CI, 20% to 62%) versus related marrow grafts (29%; 95% CI, 9% to 54%; P = 4). Patients transplanted in chronic phase with unrelated marrow grafts were also observed to have a higher incidence of chronic GVHD (67%; 95% CI, 45% to 85%) than comparable patients transplanted with HLA-identical sibling marrow grafts (20%; 95% CI, 4% to 43%; P = .003). A similar finding was noted in patients transplanted with advanced disease from unrelated donors (83%; 95% CI, 63% to 96%) versus HLA-identical sibling donors (54%; 95% CI, 30% to 85%; P = .04).

PCR results in unrelated marrow transplant recipients. Thirty-seven of 48 patients who received unrelated marrow grafts for CML were serially analyzed for detection of the bcr/abl RNA transcript. Eleven patients who failed to survive

P-O.OOM Fig 5. Actuarial probability of developing chronic - GVHD for patients who received either HLA-identical

l2 U I 48 sibling or unrelated marrow grafts. Tick marks repre- MYSAFllEBllUh%PLMT sent censored observations.




209 217 222 243 283 284 29 1 315 323 3.25 321 328 363 369 377 385 492 509 534 551

HESSNER ET AL

Pntient Statla aGVHD

deceased d548 I1 remission I remission I1 remission I remission I decacedd309 I decaced d284 I1 rel.psed d795 I remission I1 remission I1 remission I remission I dsersedd247 I1 remission I remission U dsocrsed dl68 I remission I remission I r e m i s s i o n I1 remission none

cGVHD

E none E L L L E L L L

m e none L L

none L

none L

none none

Fig 6. Serial PCR analysis for patients transplanted with unrelated marrow grafts for the treatment of CML in the chronic phase. (0) Patients testing PCR-positive for presence of the bcrlabl RNA tmnacript. (01 Patients who were PCR-negative. Clinical status at date of last contact is shown under patient status. Arrows indicate time of relapse posttransplant. Patients reported as deceased died without evidence of leukemia.

at least 100 days posttransplantation were excluded from this analysis. A total of 189 RNA samples (36 pretransplantation and 153 posttransplantation) were deemed to be acceptable using criteria detailed in the Materials and Methods and were analyzed for the presence of the bcr/abl transcript. Pretrans- plantation samples were obtained from the marrow in 17 patients and the PB in 19 patients. Sixty-two of 153 posttransplant samples were collected from the BM and 91 from the PB. The average number of posttransplant samples assayed per patient was 4.2 (range, 2 to 10). Thirty-six of 37 patients had detectable bcr/abl RNA transcripts after one round of amplification before transplantation. The bcr exon IIVabl exon I1 fusion product was documented in 32 patients, whereas the bcr exon IVabl exon I1 product was detected in 4 patients. In 1 patient, no pretransplant sample was available for testing. No patient had both transcripts detected.

Of the 37 patients who were longitudinally followed-up, 11 were PCR-positive on at least one occasion after marrow transplantation (1 1 of 37 [30%]). Overall, only 19 of 153 (12%) posttransplant samples were positive by PCR. Post- transplant bcr/abl transcript(s) sizes were concordant with pretransplant results in all patients (ie, no patient relapsed with a different bcr/abl transcript). Twenty of 27 patients transplanted in chronic phase survived more than 100 days posttransplantation. Serial PCR results and the clinical status of these patients are shown in Fig 6. Fifteen of these 20 patients were PCR-negative at all time points. All but 1 patient were in clinical remission at the time of last contact. The remaining 5 patients were noted to be PCR positive on at least one occasion. In 4 of these 5 patients, a positive PCR result was observed on only one occasion; a subsequent assay was negative in 3 of the 4 recipients. The fifth patient from whom earlier samples were unavailable was noted to be PCR-positive at the time of cytogenetic relapse. Seventeen of

21 patients transplanted with advanced disease survived for more than 100 days. The PCR results and clinical outcome are shown in Fig 7. Six of 17 patients were PCR-positive on at least one occasion. One of these patients was positive at the time of clinical relapse. Four of the remaining 5 patients relapsed subsequently after a positive PCR result. Eleven patients were PCR negative at all time points. One of these patients, whose last PCR assay at 475 days was negative, relapsed at day 1,066.

Prior studies have shown that patients transplanted with TCD HLA-identical sibling marrow grafts are often PCR positive early after BMT,32-3s suggesting incomplete eradication of the leukemic clone. We therefore examined the incidence and prognostic significance of early PCR positivity in patients receiving unrelated marrow grafts to determine if the GVL effect was operative in the immediate posttransplantation period. Twenty-three patients who had at least one assay performed within the first 100 days posttransplantation were assessed. Four of 23 patients were observed to be PCR- positive before day 100; 3 of these 4 patients eventually relapsed. Of the remaining 19 patients, 17 remain in clinical remission or were in clinical remission at the time of death. Eight of these 17 patients were serially followed-up for more than 1 year. Thus, in the majority of tested patients, eradication of leukemia as determined by PCR occurred early after marrow transplantation and was predictive of a durable antileukemia response.

DISCUSSION

The efficacy of allogeneic marrow transplantation in the treatment of CML has been shown to be highly dependent on the disease status at the time of tran~p1antation.l~ This is due to the fact that the probability of leukemia relapse in- creases as disease status becomes more advanced. When




GVL REACTIVITY AFTER UNRELATED BMT FOR CML

213 218 258 265 268 211 288 367 380 382 434 443 4 6 1 470 515 528 559

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Patient Status aCVHD

ramission I dscusd dl478 I rrLpsd d752 I1 dscuedd175 I1

I leulimion I mmiuion I d s a r d d 1 9 1 I duxascdd218 I1 nmiuion I dscusd dl55 I1 r e l a p s e d dl066 I1 remission 111 rSLpsedd306 I1 nllposdd559 I deesusdd273 I relapsed dl78 none

3993

CGVHD

L L L L L

non8 L L L L L L E L

nono L

none

Fig 7. Serial PCR analysis for patients who received unrelated marrow grafts for treatment of advanced-phase CML. (0) Patients testing PCR-positive for presence of the bcrlabl RNA transcript. (01 Patients who were PCR-negative. Clinical status at date of last contact is shown under patient status. Arrows indicate time of relapse posttransplant. Current clinical status is shown in the last column. Patients reported as deceased died without evidence of leukemia.

TCD of donor marrow is used as GVHD prophylaxis in recipients of HLA-identical sibling marrow grafts, the risk of relapse is further enhanced in patients transplanted in chronic or accelerated phase," indicating that T cells play a major role in mediating a GVL effect. The observation that patients in blast crisis transplanted either with TCD or unmodified marrow grafts have similar relapse rates, however, is evidence that the antileukemia effect of T cells is limited in patients with biologically aggressive disease." The impli- cation of these data is that the clinical expression of the GVL effect in CML is dependent on the number of alloreactive donor T cells and the biologic characteristics of the leukemia cell population.

we observed high relapse rates in patients receiving TCD HLA-identical sibling marrow grafts. The disease status of patients at the time of transplantation did not influence the probability of relapse because equally high rates were seen in patients with chronic or advanced-phase CML. However, in patients transplanted with unrelated marrow grafts, discor- dant results were seen. Patients transplanted in accelerated phase or blast crisis had equally high relapse rates when compared with similar patients receiving HLA-identical sibling marrow grafts. Patients transplanted in chronic phase, on the other hand, had a significantly lower probability of relapse, which was similar to that reported for patients receiving unmodified grafts from HLA-identical ~ib1ings.I~ Be- cause all patients were similarly conditioned and received identical GVHD prophylaxis schedules, the lower relapse rate in this group of patients compared with HLA-identical sibling transplants was most likely attributable to transplantation with an unrelated marrow graft. Thus, these data are consistent with our interpretation that use of an unrelated marrow graft was able to compensate for reduced GVL reactivity associated with TCD in patients transplanted in chronic phase. The fact that patients transplanted with unrelated and

In the present study, consistent with previous

HLA-identical sibling marrow grafts for advanced disease had a similar probability of relapse raises the possibility that use of TCD unrelated marrow grafts may have limited GVL reactivity in patients with the degree of donorhecipient histocompatibility noted in this study. However, more patients will be necessary to definitively address this question. Whether more significant HLA disparities between donor and recipient would confer an enhanced GVL effect in advanced-phase patients is not resolved by this study.

Prior studies have shown that recipients of TCD HLA- identical marrow grafts have a reduced incidence of relapse if they develop acute andor chronic GVHD in the posttransplantation period." However, these patients still have a higher risk of relapse when compared with recipients of unmodified grafts who do not develop GVHD, indicating that GVHD cannot completely compensate for the adverse impact of TCD on relapse. The assumption from these data is that a component of the GVL effect in CML is independent of GVHD and is altered by TCD. In this study, GVHD may have been partially responsible for the lower relapse rate observed in chronic-phase patients receiving unrelated marrow grafts. The incidence and severity of acute GVHD was noted to be significantly higher in recipients of unrelated marrow grafts when compared with patients receiving HLA- identical sibling grafts. Similarly, chronic GVHD was also more prevalent in this patient population. Whereas the incidence of relapse in chronic-phase patients transplanted with unrelated donor grafts was reduced, it remained high in advanced-stage patients, despite the fact that the incidence of grade 11-IV acute GVHD (45% v 40%, P = .98) and chronic GVHD (67% v 83%, P = .35) between the two groups did not significantly differ. This indicates that, if there was a GVHD-associated antileukemia effect, it was clinically significant only in patients with more indolent disease. The fact that the relapse rate in chronic-phase patients receiving TCD unrelated grafts was comparable to that reported in similar




3994 HESSNER ET AL

patients receiving unmodified grafts from HLA-identical sib- l i n g ~ , ~ , ' ~ despite a much reduced incidence of GVHD, sug- gests that a component of the observed GVL effect was GVHD-independent. This premise is further supported by the observation that some patients with durable remissions had minimal (grade I only) or no evidence of clinical GVHD, indicating that the GVL effect in these patients may have been due to an allogeneic effect independent of GVHD (Figs 6 and 7).

Studies in animal models and in humans have provided evidence that T cells are a major effector of GVL reactivity after allogeneic BMT. Direct evidence for a T-cell role has come from murine studies that have shown that cytotoxic T-cell lines with specificity for either leukemia antigens alone or for shared host and leukemia antigens can eradicate disease when administered to leukemia-bearing host^.^^.'^ In humans, most of the evidence supporting a role for T cells in GVL reactivity has been indirect and derived from the identification of T-cell clones with leukemia specificity in the PB of BMT re~ipients.~~.~' These clones, for the most part, have been isolated from recipients of HLA-identical sibling grafts and have been shown to be HLA-restricted minor histocompatibility antigen specific, recognizing either non-sex-linked minor histocompatibility antigens (eg, HA- 1) or the male minor antigen, H-Y."' In certain instances, similar to what has been reported in animal studies, clones with specificity for both leukemia and host antigens have been identified as have clones with relative specificity for leukemia cells only. In the present analysis, one explanation for the lower relapse rate observed in chronic-phase patients could be the higher frequency of alloreactive donor T-cell clones recognizing shared host and leukemia allodetermi- nants. Several studies have shown that the cytotoxic T-lymphocyte precursor and helper T-cell precursor frequencies directed against host cells are increased in unrelated as opposed to HLA-identical sibling donor/recipient pairs.42 In patients receiving mismatched marrow grafts, these allode- terminants could be either major or minor histocompatibility antigens, whereas in phenotypically matched unrelated BMT recipients, minor histocompatibility antigens are the most likely target antigen. This would seem to be a plausible explanation for the enhanced GVL reactivity given the fact that all patient grafts were TCD in the same fashion and, therefore, the frequency of potential leukemia-specific T cells in the marrow grafts should have been comparable between populations. Moreover, we would not have expected the antigenic characteristics of the leukemia cell populations, which could serve as targets for T cells, to have varied between the respective patient cohorts, because there was no difference in disease status at the time of transplant between these two groups.

To assess the remission status in patients transplanted with unrelated marrow grafts, we performed PCR analyses of PB and BM samples for the presence of the bcr/abl RNA transcript. All samples were assayed in duplicate and rigor- ous quality controls were incorporated into the assay to insure accuracy and reproducibility. Prior studies that have serially followed-up recipients of HLA-identical sibling marrow grafts for evidence of molecular relapse have shown

that most patients receiving TCD marrow grafts remain inter- mittently or persistently PCR-positive after transplant.'*.'' The inference from these data is that these patients have a persistent low-level disease burden and are at higher risk for subsequent disease relapse, reflecting a suboptimal GVL response. Although we observed a low incidence of relapse in chronic-phase patients transplanted with unrelated marrow grafts, the presence of detectable bcr/abl transcripts by PCR in patients presently in clinical remission would identify a group of patients at potential risk for subsequent relapse. However, analysis of these data showed that nearly all chronic-phase patients in hematologic andor cytogenetic remission were persistently PCR-negative. The majority of these patients became PCR-negative within 1 to 2 months after transplantation, indicating that early eradication of leukemia was associated with a durable antileukemia response. Of note, most long-term survivors transplanted with advanced disease also remained PCR-negative, although the overall percentage of patients remaining in remission in this group was substantially lower. The incidence of PCR-positivity in recipients of HLA-identical sibling marrow grafts was not evaluated, because the probability of clinical relapse in this group was substantial. Thus, there were already con- vincing data indicating that the GVL effect in this patient population was suboptimal and that no need for confirmation of this fact by further PCR studies was necessary. The observation that patients receiving unrelated marrow transplants in chronic phase remained PCR-negative supports the premise that retention of the GVL effect after TCD-unrelated marrow transplantation can be achieved and provides evidence that the GVL effect in this patient population is quanti- tatively different from that observed after HLA-identical sibling transplants.

It should be emphasized that the data and conclusions in this study were derived from a patient population transplanted using a specific TCD technique. TCD of donor marrow with the monoclonal antibody T10B9 plus complement results in an approximate 1.7 2 0.35 log depletion of mature T cells (C. Keever-Taylor, unpublished observations). Fur- thermore, complement-mediated lysis using this monoclonal antibody has preferential specificity for the ap T-cell recep- to?3 and results in relative preservation of yS T cell, NK cell, and LAK cell population^.^^ This TCD approach is, therefore, distinctly different both qualitatively and quantita- tively from other clinically used TCD strategies. It is, therefore, conceivable that other TCD techniques may modulate GVL reactivity differently and that the conclusions of this study may be unique to this particular approach. The observation that a clinically significant GVL effect persists after unrelated marrow transplantation will, therefore, require confirmation. Large-scale registry analyses may be the opti- mal methodologic approach to address this question.

In summary, this study shows that transplantation of unrelated marrow grafts can compensate for reduced GVL reactivity associated with T-cell depletion in patients with CML. Retention of the GVL effect was most likely attributable to increased HLA disparity between donor and recipient, suggesting that effector populations recognizing shared antigens on host and leukemia cells mediated this effect. While




GVL REACTIVITY AFTER UNRELATED BMT FOR CML 3995

preservation of GVL reactivity was restricted to patients transplanted in chronic phase, the GVL effect in these patients was found to be quite potent, as evidenced by absence of the bcr/abl RNA transcript in longitudinal studies. Finally, these data indicate that strategies designed to reduce GVHD in unrelated marrow transplantation can be used without a commensurate increase in disease relapse in selected patient populations with CML.

ACKNOWLEDGMENT

We thank Susan Cadwallader, Kathy Sobocinski, and Melodee Nugent for assistance in data analysis and retrieval. We also thank Thomas Wank for assistance in procuring stored patient samples.

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Use of unrelated marrow grafts compensates for reduced graft-versus-leukemia reactivity after T-cell-depleted allogeneic marrow transplantation for chronic myelogenous leukemia