KT Godder, J Metha, KY Chiang, S Adams, F van Rhee, S Singhal, K Higgins-Smith, W O Neal, S DeRienzo and JP Henslee-Downey.

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1 (2001) 28, Nature Publishing Group All rights reserved /01 $ Acute myeloid leukaemia Partially mismatched related donor bone marrow transplantation as salvage for patients with AML who failed autologous stem cell transplant KT Godder, J Metha, KY Chiang, S Adams, F van Rhee, S Singhal, K Higgins-Smith, W O Neal, S DeRienzo and JP Henslee-Downey Division of Transplantation Medicine, South Carolina Cancer Center Columbia, SC, USA Summary: Treatment options for patients who relapse are limited and the outcome is dismal. Between August 1993 and January 1999, 17 patients, median age 26 (4 44) years, underwent T cell depleted bone marrow transplant from partially mismatched related donors (PMRD), as a salvage for AML relapsing after an autograft. The median time from auto-transplant to relapse was 7 months (1.5 24) and the interval between transplants was 10 months (3 30). All patients had active leukemia at time of transplant. Donors were siblings (n 8), parents (n 2), daughters (n 4) and others (n 3), and 82% were 2 major HLA antigen mismatched with the recipient. The conditioning therapy included total body irradiation in 14 patients and was busulfan-based in three. Graft-versus-host disease (GVHD) prophylaxis consisted of partial T cell depletion along with posttransplant immunosuppression. Median day to engraftment was 16 days (12 20). Acute GVHD was seen in six patients, and chronic GVHD in four of 13 surviving beyond 100 days. Ten patients died of nonrelapse causes, at (median 77) days. Two patients relapsed at 3 and 4 months. Five patients (29%) are surviving leukemia-free months post transplant (median 68 months). A short interval between transplants was predictive of early relapse but not mortality. Age 18 and 2 organ toxicities were marginally predictive of better survival. We conclude that BMT from PMRD is a reasonable option for patients with refractory AML post autograft. (2001) 28, Keywords: BMT; mismatched related donor; second transplant; relapsed AML; autologous Autologous stem cell transplant (auto) has been shown to be effective and perhaps superior to chemotherapy in Correspondence: Dr K Godder, Pediatric Stem Cell Program, Department of Pediatrics, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Mail Code CDRCP, Portland, OR 97219, USA Received 4 October 2000; accepted 13 September 2001 patients with acute myelogenous leukemia (AML) in first, or subsequent remissions. 1 5 Patients who relapse after autograft, even if they achieve remission, which is usually of short duration, eventually succumb to their disease within a few months. 6 Those with good performance status have the option to receive either intensive therapy followed by a second auto, or an allogeneic (allo)-bone marrow transplant. Second auto-transplant is relatively well tolerated. However, the main problem remains the high relapse rate; 58% relapse at 3 years was reported in 18 patients from Spain. 7 Of the patients undergoing second transplant at The Royal Marsden Hospital, four of seven who received a second auto, and all three receiving syngeneic graft relapsed. 8 Allogeneic-transplant from a matched sibling donor (MSD) is an option available to some patients, but most do not have a donor and are candidates for alternative donor transplant. No prospective study today compares first or second auto to alternative donor transplant. In a large matched pair analysis of patients undergoing first transplant, Ringden found no survival benefit for those who received alternative donor compared to auto. 9 In the current study, we report on the use of partially mismatched related donor (PMRD) bone marrow transplant (BMT) to salvage patients with AML who relapsed following autologous stem cell transplant and did not have a MSD. We show no engraftment failure and an acceptable incidence of graftversus-host disease (GVHD). We further discuss factors that may affect survival and relapse. This report is an update on an earlier publication. 10 Patients, donors and methods Patients Between August 1993 and January 1999, 17 consecutive patients who were referred to our center with AML, who relapsed following auto, underwent a T cell-depleted BMT from PMRD. Patients ages were 7 44 years (median 26) and their other characteristics are listed in Table 1. FAB morphology was M0 in three patients, M in four, M2 in one, M4 in eight and M5 in one. Median time to relapse post auto-transplant was 7 months (2 24), and between auto and allo was 10 months (3 31). All patients had active dis-

2 1032 Table 1 Patients, donors and transplant information UPN Age Disease Auto to Auto Donor Donor s Sex Conditioning Outcome COD /Gender state relapse allo/bmt relation age MM regimen a (years) (months) (months) (years) 27 36/F 2nd rel res 7 10 half-sister 28 no A alive 28 6/M 2nd rel res 9 19 sister 8 yes A alive 40 13/M 1st rel 7 10 father 44 no A dead pul hem 97 21/M 1st rel untr 4 5 brother 26 no B alive /M 1st rel res 2 6 brother 28 no A dead relapse /M 3rd rel res 9 20 daughter 18 yes A dead acute GVHD /F 1st rel sen 3 9 mother 50 no B dead sec malig /F 1st rel res 6 9 daughter 9 no C dead viral inf /F 2nd rel res 9 16 uncle 45 yes B dead sepsis 254 9/F 2nd rel half-sister 17 no D alive /M 1st rel res daughter 10 yes D dead sepsis /F 4th rel unk daughter 21 no B dead fungal inf /F 2nd rel unk 2 3 sister 36 no B dead relapse /M 2nd rel res 8 10 brother 11 no B alive /M 2nd rel res 4 7 brother 24 no B dead fungal inf /M 1st rel res 4 6 brother 22 no B dead pul toxicity /M 1st rel sen brother 33 no B dead acute GVHD rel relapse; res resistant; sen sensitive; unk unknown; untr untreated; TBI total body irradiation; VP etoposide; ARAC cytosine arabinoside; Cytoxan cyclophosphamide; pul hem pulmonary hemorrhage; sec malig secondary malignancy; inf infection; A TBI, VP, ARA- C, cytoxan, steroids; B TBI, VP, ARAC, cytoxan, steroids, ATG; C busulfan, thiotepa, melphalan, steroids; D busulfan, thiotepa, cytoxan, steroids. ease at time of allo-transplant: eight in first relapse, and seven in second relapse, one in third and forth relapse each. Attempted induction in 16 patients did not result in remission. Donors Donors were readily available from within the family and included eight siblings, two half-siblings, two parents, four offspring (daughters) and one uncle (Table 1), their median age was 24 (8 50), and only four were sex mismatched with the recipient. Patient-donor pairs HLA antigens were mismatched in the donor (GVH) direction in one antigen (n 3), two antigen (n 9) or three antigens (n 5) and in the recipient (HVG) direction in three, 10 and four antigens, respectively. Treatment protocol After signing an informed consent form, five patients were treated on PRMH-protocol No which included TBI, etoposide, cytosine-arabinoside, cytoxan and steroids, as previously described. 11 Another nine patients were treated on a similar protocol (No ) that includes the addition of pre-transplant anti-thymocyte globulin (ATG). 12 The remaining three patients were treated with a busulfan-based protocol. 9 GVHD prophylaxis was similar for patients on all protocols, and consisted of partial T cell depletion of the graft combined with post-transplant immunosuppression using cyclosporine, methylprednisolone and a course of ATG T cell depletion employed the use of monoclonal antibody followed by rabbit complement, with either T10B9 (n 5) or OKT3 (Ortho Biotech, Raritan, NJ, USA) (n 12) as previously described. 14,15 Pre-emptive donor leukocyte infusion (DLI) was given to six patients. Statistical considerations Engraftment day was defined as the first of 3 consecutive days for which the WBC count exceeded 1000/mm 3. Engraftment and remission status were confirmed by histology, cytogenetic analysis and RFLP (or VNTR) evaluation. GVHD was graded using Glucksberg s criteria and toxicity followed Bearman criteria. Patients were evaluated for engraftment and GVHD analysis if they survived beyond day 17 post transplant, the median day for engraftment on those protocols. Only patients who survived beyond day 28 were evaluated for relapse. All patients were eligible for survival analysis. Kaplan Meier estimates were used for survival and leukemia-free survival (LFS) analysis, and to assess the impact of various prognostic factors on survival and relapse. The Cox proportional hazard model was used for multivariate analysis. Cumulative incidence was used for assessment of GVHD, relapse and nonrelapse mortality. Results Bone marrow grafts Patients were infused with a median of ( ) nucleated cells/kg, containing ( ) T cells/kg recipient. The median CD34 dose (available in seven patients) was ( ) cells/kg. Four patients received pre-emptive donor leukocyte infusion at a median of 54 days post transplant ( 28 to 95). Cells were given in one to three infusions, and median total CD3 cell/kg for patient was ( ).

3 Engraftment All 14 patients who survived beyond the second week of transplant, achieved engraftment. The median time to WBC of 500/mm 3 and 1000/mm 3 was 16 (12 20) days. None of the patients experienced late graft failure. Regimen-related toxicity (RRT) Severe RRT (grade 3 4) of the pulmonary system occurred in three patients, kidney in nine patients (none required hemodialysis), liver in nine patients and cardiovascular system in 11 patients. The cardiovascular toxicity manifested as hypertension, secondary to cyclosporine and methylprednisolone used for GVHD prophylaxis. Veno-occlusive disease (VOD) developed in six patients, but was severe in only one patient. Despite the high toxicity, most patients recovered without long-term sequelae. Graft-versus-host disease Acute GVHD (agvhd) grade 1 developed in two patients, grade 2 in two patients, and grade 3 in one patient (Figure 1). Chronic GVHD (cgvhd) was seen in four of 13 patients surviving beyond day 100, and was graded as limited cgvhd in three. Severe and lethal GVHD developed only in patients who received DLI: two of six patients who received pre-emptive DLI succumbed to GVHD (on days 49 and 95, respectively). Relapse Only two patients relapsed, at 3 and 4 months following allo-transplant. Time to relapse post auto-transplant of 3 months was predictive of post-allo relapse; half of the patients who relapsed within 3 months post auto, but none of those who relapsed later, relapsed post allo (P 0.056). Relapse beyond 6 months, disease sensitivity, and patient s stage of disease (1st vs 1st relapse) were not predictive of relapse post allo-transplant (P 0.669, 0.123, 0.515, respectively). Leukemia-free survival (LFS) and survival Five patients are alive and disease free at a median of 57 months (30 73) post BMT, with good quality of life: Karnofsky/Lansky scores of 100 in four patients and 90 in one patient. They maintain longer remissions than following their auto-transplant, with a 29% Kaplan Meier estimate of survival (Figure 2). One patient who developed Hodgkin s disease 5 years after transplant, was treated with chemotherapy and bone marrow boost, and is now diseasefree 16 months after completion of therapy. Ten patients died of non-relapse causes (Table 1). The most common primary cause of death was infection: two patients died of early sepsis, two from fungal and one from viral infection. Factors affecting outcome Post-transplant toxicity ( 2 organs) and patient s age ( 18 years old) were the most significant (although marginal) factors in favor of long-term survival (P 0.09 and P 0.08, respectively in a univariate analysis). Time from autotransplant to relapse, auto to allo-transplant, antigen mismatch with the donor, donor s age, sex mismatch, disease sensitivity, conditioning protocol, T cell depletion agent, liver toxicity, VOD and GVHD were not shown to affect survival (Table 2). Multivariate analysis did not discriminate any factor to be significant in outcome. Discussion In this report we describe our experience with the use of PMRDs for BMT in AML patients who relapsed after autotransplant and did not have a MSD. To our knowledge, this is the largest study using PMRDs for those patients, employing a relatively uniform transplant regimen. Our study demonstrated a high rate of sustained engraftment, and acceptable incidence of GVHD; the cumulative incidence of grade 2 3 agvhd was only 18%, similar to patients who received PMRD as their first transplant, in our institution. 11,12 The Kaplan Meier estimate of LFS and overall survival at 3 years was 29% and with a good quality of life, despite all patients having active disease at time of transplant. Our low incidence of GVHD is especially significant in view of a recent report on the use of allo following autotransplant in lymphoma and AML. 16 This study reports a very high rate of GVHD and discourages the use of allo 1033 Cumulative incidence % Days Figure 1 Cumulative incidence of agvhd in AML patients who relapsed after autograft and were salvaged with PMRD-BMT. Probability Time (days) Figure 2 Probability of leukemia-free survival of AML patients who relapsed after autograft and were salvaged with PMRD-BMT.

4 1034 Table 2 Effect of risk factors on survival (using Kaplan Meier estimates of survival) Risk factor Surviving P value patients a Age at transplant (years) 18 3/ /13 Time to relapse (post auto) (months) 6 1/6 NS 6 4/11 Time between transplants (months) 6 1/4 NS 6 4/13 Disease sensitivity Sen 0/3 NS Res/unk 5/14 Antigen MM 3 3/11 NS 3 2/6 Donor s age (years) 20 3/6 NS 20 2/11 Donor patient sex-mismatch Yes 1/4 NS No 4/13 Conditioning therapy TBI 4/14 NS Non-TBI 1/3 Grade 3 4 toxicity b No. (organs) 2 3/ /13 Liver toxicity Yes 2/9 NS No 3/8 VOD Yes 0/ No 5/12 GvHD (grade 2) Yes 1/3 NS No 4/14 a Entries are number of patients surviving/number of patients. b Systems involved: pulmonary, gastrointestinal, central nervous system, cardiovascular, renal, hepatic. post auto altogether. In this series, 15 patients out of 20 who received allo underwent MSD-BMT. Eighty percent of those developed grade 2 4 GVHD. Five of the six patients with AML (four underwent MSD, one MUD and one PMRD) developed grade 3 4 GVHD. Our relatively low incidence of agvhd can be explained by the aggressive prevention methods including partial T cell depletion of the graft along with post-transplant immunosuppression. Mortality from GVHD in our cohort did not occur, except in patients who received pre-emptive DLI. One of them received DLI prior to day 100, during the time considered to be high risk for development of GVHD. Also, the cell dose was higher than 10 7 /kg, the level shown to be associated with increased risk of GVHD in PMRD-BMT patients. 17 The other patient developed GVHD and died of secondary CMV infection. In our study of allo following auto, we have not seen decreased relapse rates in patients who developed GVHD, 18,19 perhaps because of the small numbers of patients who developed GVHD or experienced relapse. The main cause of death in our study was non-relapse related. Data on patients undergoing a second allotransplant in general have shown 40 52% non-relapse mortality specifically due to VOD. We observed a relatively low incidence of VOD, perhaps as a result of using low molecular weight heparin for prophylaxis. On the other hand, the main cause of death in our series was infection, reflecting the prolonged time to immune reconstitution in patients who receive T cell-depleted alternative donor transplant. 22,23 Previously, we and others have also demonstrated an increased risk of transplant-related mortality if more than one organ with grade 3 4 toxicity was involved. 10,18,24 Unlike in second auto, and comparable to other second allo reports, 16,24 only two patients relapsed, with a cumulative incidence of relapse of 14%. Considering that this is a small series from a single institution, we refrain from inferring that the low relapse rate is significant. Nevertheless, these results suggest that the allogeneic effect of the grafted cells can be important in disease control, and justify its use in patients whose disease is refractory to chemotherapy. As expected, the interval between transplants did affect relapse but not survival. Patients whose allo was within 3 months of auto were more likely to relapse post allo. The Seattle and the Italian groups reporting on allo post auto did not show an impact of time between transplants using 1 year vs 1 year. 24,25 Others, (using the median 566 days vs 566, 18 or 6 vs 6 12 vs 12 months, 20 respectively) did demonstrate an effect on survival and relapse. The differences may be related to the diversity of diseases, disease states and time intervals. Considering the length of remission following auto-transplant, it was not significant in predicting outcome (relapse or survival) in our cohort. This is in contrast to patients who received a second auto (52% vs 20% P 0.02). 8 As to LFS and overall survival, although there are numerous reports on second transplant, only two series describe patients with AML who received allo post auto. The first is a multi-institutional trial reported by Tsai et al, 16 where only one of six AML patients survived, all dying from non-relapse causes. The second is a study from Seattle, reporting on 24 patients with AML, of whom 30% died of non-relapse mortality and 25% of relapsed disease, and 10 patients (46%) survived. 24 The difference between our and the Seattle group results, may be related to patient selection, as our patients were older and all of them were in relapse. Moreover, having an easily available donor may have biased our patients towards the sicker population, since those who were able to survive the waiting period for an unrelated donor search, as in the Seattle group, could have been the better risk patients. Patient age is the primary factor associated with survival in our and other studies. We showed that patients younger than 18 did marginally better than older patients. Those results were not significant in the multivariate analysis, perhaps because of sample size and its low power. Age has also been observed as an important factor in many other studies of second transplant, affecting mostly non-

5 transplant mortality. Mrsic et al, 20 studying second allotransplant, compared patients above and below the median age (26 years), and found similar results. Significantly improved outcome in young patients is related in part to their being devoid of other co-morbid conditions, but also, may be related to their faster immune reconstitution, and subsequent ability to fight infection. 23 As most patients do not have a MSD, using PMRDs is attractive, especially in refractory patients as these donors are easily accessible, and treatment can be initiated immediately. Most reports on second allo transplant show no survival benefit when using alternative donor 16,26 due to high regimen-related toxicity. The recent Seattle report, using alternative donors in 45 transplants and ours with 17 PMRDs suggest that with the advancement in support care, those transplants are more feasible. Another attractive approach to reducing toxicity is using non-myeloablative regimens. It is still to be determined whether patients with aggressive disease will benefit from this type of transplant. Moreover, difficulties in crossing HLA barriers may hamper those attempts in PMRD compared to those with MSD or MUD transplants. In summary, our study shows that PMRD-BMT is a reasonable option for patients with active AML who relapse post auto and do not have a MSD. The study also suggests that patients should not undergo allo within 3 months of their auto-transplant. As the relapse rate was low, our study proposes that improvement in support care, along with attempts to enhance immune reconstitution should improve overall survival in AML patients who relapsed post autotransplant and receive PMRD-BMT. References 1 Zittoun RA, Mandelli F, Willemze R et al. Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia. New Engl J Med 1995; 332: Miggiano MC, Gherlinzoni F, Rosti G et al. 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