T cell depletion utilizing CD34 stem cell ... - Wiley Online Library

research paper

T cell depletion utilizing CD34+ stem cell selection and CD3+ addback from unrelated adult donors in paediatric allogeneic stem cell transplantation recipients

Mark B. Geyer,1 Angela M. Ricci,2 Judith S. Jacobson,3 Robbie Majzner,2 Deirdre Duffy,4 Carmella Van de Ven,4 Janet Ayello,4 Monica Bhatia,2 James H. Garvin Jr,2 Diane George,2 Prakash Satwani,2 Lauren Harrison,4 Erin Morris,4 Mildred Semidei-Pomales,4 Joseph Schwartz,5 Bachir Alobeid,5 Lee Ann Baxter-Lowe6 and Mitchell S. Cairo4,7,8,9,10 1

Department of Medicine, Massachusetts General

Hospital, Harvard Medical School, Boston, MA, 2

Department of Pediatrics, Columbia University,

3

Department of Epidemiology, Columbia Univer-

sity, New York, NY, 4Department of Pediatrics, New York Medical College, Valhalla, NY, 5

Department of Pathology and Cell Biology,

Columbia University, New York, NY, 6Department of Surgery, University of California San Francisco, San Francisco, CA, 7Department of Medicine, New York Medical College, 8Department of Pathology, New York Medical College, 9

Department of Microbiology and Immunology, New York Medical College, and 10Cell Biology and Anatomy, New York Medical College,

Summary CD34-selected haploidentical and unrelated donor allogeneic stem cell transplantation (AlloSCT) in paediatric recipients is associated with sustained engraftment and low risk of acute graft-versus-host disease (aGVHD), but limited by delayed immune reconstitution and increased risk of viral and fungal infection. The optimal dose of donor T cells to prevent graft failure and minimize risk of early opportunistic infection and post-transplant lymphoproliferative disorder (PTLD), while avoiding severe aGVHD, remains unknown. We prospectively studied CD34-selected 8–10/ 10 human leucocyte antigen (HLA)-matched unrelated donor (MUD) peripheral blood stem cell transplantation (PBSCT) in a cohort of 19 paediatric AlloSCT recipients with malignant (n = 13) or non-malignant (n = 6) diseases. T cells were added back to achieve total dose 1·0– 2·5 9 105 CD3+/kg. GVHD pharmacoprophylaxis consisted only of tacrolimus. All patients engrafted neutrophils. Probabilities of grade II–IV aGVHD, limited chronic GVHD (cGVHD), and extensive cGVHD were 15·8%, 23·3%, and 0%, respectively. One patient developed PTLD. Oneyear infection-related mortality was 5·6%. T cell immune reconstitution was delayed. One-year overall survival was 82·3%. Five patients with malignant disease ultimately died from progressive disease. CD34-selected MUD PBSCT using a defined dose of T cell add-back resulted in high rates of engraftment and low risk of grade II–IV aGVHD, early transplantationrelated mortality, and extensive cGVHD.

Valhalla, NY, USA

Keywords: paediatrics, T cell depletion, allogeneic stem cell transplantation. Received 14 October 2011; accepted for publication 27 December 2011 Correspondence: Mitchell S. Cairo, Department of Pediatrics, Medicine, Pathology, Microbiology & Immunology and Cell Biology & Anatomy, Maria Fareri Children’s Hospital at Westchester Medical Center, New York Medical College, Munger Pavilion Room 110A, Valhalla, NY 10595, USA. E-mail: [email protected]

Allogeneic haematopoietic stem cell transplantation (AlloSCT) offers the best potential for long-term survival and cure in a subset of patients with advanced haematological malignancies and certain non-malignant diseases. Although recipients of human leucocyte antigen (HLA)-identical ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

sibling donor transplantation have a lower risk of transplantrelated mortality (TRM) than recipients of unrelated donor transplantation, 0·75 9 109/l after AlloSCT, received prophylaxis with foscarnet (90 mg/kg/dose) QOD alternating with ganciclovir (5 mg/kg/dose) QOD until reaching day +100 or 3 months off immunosuppression with CD4 counts >0·2 9 109/l, as have previously described (Shereck et al, 2007). Initial management of CMV reactivation consisted of ganciclovir (5 mg/kg IV q 12 h). Initial management of invasive fungal infections (IFIs) consisted of liposomal amphotericin B (5 mg/kg IV q 24 h) or voriconazole, with or without the addition of an echinocandin.

CD34+ selection and CD3+ add-back Immunomagnetic CD34+ cell selection of the apheresis haematopoietic progenitor cell product (HPC-A) was performed using the Isolex 300i (Baxter, Deerfield, IL, USA) as has been previously described (Firat et al, 1998; Stainer et al, 1998). The technique involved incubation and binding of the CD34+ cells with unconjugated mouse anti-CD34 IgG1 monoclonal antibody followed by a washing step to remove unbound antibody. Magnetic beads coated with polyclonal sheep antimouse IgG1 antibodies were then added, and the mixture incubated to produce rosette formation. The product was then exposed to a magnetic field to positively select the magnetic bead-bound CD34+ cells. After washing steps to remove the unbound non-targeted cells, the CD34+ cells were released from the magnetic beads using an octapeptide (PR34+) releasing agent with a higher affinity for the anti-CD34 monoclonal antibody. After flow cytometric analysis of the positive (CD34+ cell enriched) and negative fractions, a volume of unmanipulated HPC-A product was ‘added back’ to the positive fraction (as applicable) to achieve a target T cell dose of 1·0– 2·5 9 105 CD3+ cells/kg. The resulting CD34+ enriched HPCA product (target 5·0 9 106 CD34+ cell/kg) was then freshly infused. The remaining negative fraction was discarded and any unmanipulated HPC-A product exceeding the total nucleated cell load limit for the Isolex and residual positive fraction above the infused dose (as applicable) was then cryopreserved.

Engraftment and donor chimerism Neutrophil recovery was defined as an ANC of 0·5 9 109/l for three consecutive days. Platelet recovery was defined as a platelet count of 20 9 109/l independent of platelet transfusions for at least 7 d. Primary myeloid graft failure was defined as failure to achieve a donor-derived ANC 0·5 9 109/l by day +42 and/or 50% whole blood donor chimerism by day +60. Donor myeloid and/or lymphoid 207

208

SAA APML b-Thal ALL ALL ALL ALL MAS SAA ALL SAA HTLV-1 adult T cell LL Wolman disease AML AML ALL Plasmacytoid dendritic cell tumor Burkitt lymphoma AML

1 2 3 4 5 6 7 8 9 10 11 12

CR2

SD

SD CR2 CR2 CR1 CR1

SD CR2 SD CR2 CR3 CR1 CR2 SD SD CR3 SD SD

M

M

F M F M M

M F F M M M F M M F F M

Sex

14·8

17·0

16·4 12·0 12·0 10·0 8·7

15·6 20·9 18·4 23·2 10·1 15·3 13·9 10·1 19·1 12·2 15·4 16·2

Age at PBSCT (years)

8/10

8/10

8/10 8/10 8/10 8/10 10/10

9/10 10/10 9/10 9/10 9/10 10/10 10/10 10/10 9/10 9/10 8/10 8/10

HLA match

MAC

MAC

RTC RTC MAC MAC RTC

RTC RTC MAC MAC MAC MAC MAC RTC RTC MAC MAC RTC

Conditioning intensity

Bu/Mel/ATG

TBI/TT/Cy/ATG

Bu/Flu/alemtuz Bu/Flu/alemtuz Bu/Mel/ATG TBI/TT/Cy/ATG Bu/Flu/alemtuz

Bu/Flu/alemtuz Bu/Flu/alemtuz Bu/Mel/ATG TBI/Mel/ATG TBI/Mel/ATG TBI/Mel/ATG Bu/Mel/ATG Bu/Flu Bu/Flu/alemtuz TBI/Mel/ATG Bu/Mel/ATG Bu/Flu/alemtuz

Conditioning regimen

3·0

4·3

7·9 5·9 2·5 11·9 4·8

8·9 5·0 5·9 4·0 5·1 5·8 4·4 13·3 2·0 4·8 5·7 1·3*

CD34+ infused (9106/kg)

1·6

2·0

1·5 1·7 1·6 1·5 4·8

2·2 2·6 2·2 2·0 0·9 1·1 0·1 1·0 1·3 1·3 1·5 2·6

CD3+ infused (9105/kg)

I

0

IV I 0 III 0

0† 0 0 0 I 0 0 0 0 III‡ 0 0

Max aGVHD grade

0

0

Limited 0 0 0 0

0 0 Limited Limited 0 0 0 Limited 0 0 0 0

cGVHD

Alive, 13·4

Alive, 14·8

Deceased, 14·7 Alive, 19·1 Deceased, 7·5 Alive, 15·8 Alive, 15·3

Alive, 69·8 Alive, 54·9 Deceased, 17·6 Deceased, 23·3 Deceased, 2·4 Alive, 45·7 Deceased, 19·2 Alive, 33·5 Alive, 32·7 Deceased, 9·1 Alive, 28·4 Deceased, 18·0

Survival (months)

PBSCT, peripheral blood stem cell transplantation; HLA, human leucocyte antigen; aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; SAA, severe aplastic anaemia; APML, acute promyelocytic leukaemia; b-Thal, beta-thalassaemia; ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; MAS, macrophage activation syndrome; HTLV-1, human Tlymphotrophic virus-1; LL, leukaemia and lymphoma; CR, complete response,; SD, stable disease; MAC, myeloablative conditioning; RTC, reduced toxicity conditioning; TBI, total body-irradiation (1200 cGy); Mel, melphalan (135 mg/m2); ATG, anti-thymocyte globulin (8 mg/kg); Cy, cyclophosphamide (120 mg/kg); Flu, fludarabine (180 mg/m2); Alemtuz, alemtuzumab (54 mg/m2); TT, thiotepa (10 mg/kg); Bu, busulfan (12·8 mg/kg). *Product clotted during the selection process as it had exceeded the product expiration time for processing; excluded from cell selection analyses. †Developed grade II aGVHD on day +385 following donor lymphocyte infusion. ‡Developed grade III aGVHD on day +255 following donor lymphocyte infusion.

19

18

13 14 15 16 17

Diagnosis

Patient no.

Disease status at PBSCT

Table I. Patient demographics, HLA match, cell dose, incidence of GVHD, and survival.

M. B. Geyer et al

ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

T Cell Depletion in Paediatric AlloSCT chimerism was measured on day +30, 60, 100, 180 and 365 after transplant. The percentage of donor chimerism was determined by quantifying fluorescent-labelled polymerase chain reaction (PCR) products from donor and recipient alleles at short tandem repeat loci (Horn et al, 2009). Donor chimerism was determined for whole blood and cell subsets as required by the individual disease protocol. Cell subsets were isolated using Miltenyi magnetic particles (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of each subset was determined by flow cytometry.

Toxicity/definitions Toxicity was graded according to the National Cancer Institute Common Terminology Criteria for ADVERSE EVENTS version 3.0 (http://ctep.cancer.gov/protocolDevelopment/electronic_applica tions/docs/ctcaev3.pdf). Systemic viral infections (SVIs) and IFIs were defined as previously described (Satwani et al, 2009). Patients with refractory malignant disease and/or without a complete response (CR) at the time of MUD PBSCT and/or in CR3 or beyond were classified as poor risk; all others were classified as average risk. Relapse was defined by morphological and/or radiographic evidence of malignant disease in any site and time to relapse was defined as the interval between the day of AlloSCT and relapse, with censoring at death. Day +100 TRM was defined as death due to any cause except relapse in the first 100 d following transplantation. Overall survival (OS) was the time between transplantation and death due to any cause, and was censored if patients were alive at last follow-up.

Immune reconstitution Mononuclear cells (MNC) from patient samples were washed in phosphate-buffered saline supplemented with 1% heat activated fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA) and 1% azide (Sigma-Aldrich). Cells were analysed as previously described (Ayello et al, 2006). Briefly, fluorescent conjugated monoclonal antibodies (CD3, CD16, CD56, CD4, CD8, CD19; BD Biosciences, Franklin Park, NJ, USA) were added to MNCs, incubated in the dark at 4°C for 20– 30 min, washed in azide buffer and fixed with 0·5% paraformaldehyde (Sigma-Aldrich). Samples were analysed on a FACS Calibur cytometer (BD Biosciences) and then analysed using CELLQUEST software (BD Biosciences). The lymphocyte subpopulation was gated and used as a reference for the determination of CD3+, CD3+CD4+ and CD3+CD8+ (T cell) and CD19+ (B cell) and CD3 /16+/56+ [natural killer (NK)cell] subsets at days +100, +180 and +365 (±10 d). Each subset was then analysed for both its light scatter and forward side scatter characteristics and expression of the surface antigens recognized by the monoclonal antibodies mentioned above. Appropriate isotype controls were included and a minimum of 10 000 events was collected for each evaluation. Absolute lymphocyte subset counts for each patient were ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

subsequently assessed as being normal or low according to age-specific reference ranges provided by our institution’s laboratory. Immunoglobulin levels IgG, IgA and IgM, were measured using enzyme-linked immunosorbent assay (ELISA) at days +100, +180 and +365 (±10 d). Plasma or serum from patient samples were used and protein levels measured by quantitative ELISA according to the manufacturer’s kit instructions (R&D systems, Milwaukee, MN, USA; Raybiotec, Norcross, GA, USA). Samples were run in triplicate and summarized as means. Immunoglobulin levels were assessed as being normal or low according to age-specific reference ranges as defined by Lockitch et al (1988).

Statistics All statistical analyses were performed in PRISM 5.03 (GraphPad, La Jolla, CA, USA). The cut-off date for analysis was January 14, 2011. Study objectives were outlined in the protocol and included estimating the incidence of primary and secondary graft failure and PTLD, the incidence and severity of aGVHD and cGVHD, and time to immune reconstitution. Continuous variables were summarized as medians and ranges or as means ± standard error of the mean (SEM) and categorical variables as percentages. Continuous variables were compared between two groups using two-sample t-tests. Probabilities of neutrophil and platelet engraftment, aGVHD, cGVHD, day 100 TRM, SVI, IFI, and OS were estimated using the Kaplan–Meier method; cumulative incidence functions were used to summarize time to engraftment and time to aGVHD and cGVHD (Kaplan & Meier, 1958). Kaplan– Meier curves were compared between groups using log-rank tests for OS. A P-value < 0·05 was considered significant.

Results Demographics Of 19 CD34-selected MUD PBSCT recipients analysed in this report, 13 had a malignant and 6 a non-malignant disease (Table I). Median age at the time of transplantation was 15 years (range 8–23); median follow-up time post-transplant was 547 d (range 72–2124) for all patients and 949 d (408–2124) for survivors.

HLA disparities Five of 19 patients underwent fully (10/10) HLA-matched MUD PBSCT. Six donor–recipient pairs were one HLAmismatched, with disparities at HLA-DRB1 (n = 2), HLA-C (n = 2), HLA-A (n = 1) or HLA-B (n = 1). Eight donor recipient pairs were two antigen-mismatched, with disparities at HLA-B and HLA-C (n = 4), HLA-A and HLA-C (n = 1), HLA-A and HLA-DQB1 (n = 1), HLA-C and HLA-DRB1 (n = 1) or HLA-DRB1 and HLA-DQB1 (n = 1). 209

M. B. Geyer et al

Cell selection Collected HPC-A products contained a median of 9·4 9 106 CD34+/kg (3·3–18·3) prior to CD34+ selection and 5·6 9 106 CD34+/kg (2·0–14·2) following selection; median recovery of CD34+ cells in the selected product was 65·8% (42·9–85·8%) (Fig S1A). Apheresis products contained a median of 4·8 9 108 CD3+/kg (1·8–20·7) prior to CD34+ selection and 2·3 9 104 CD3+/kg (undetectable – 44·7) following selection. CD34+ selection resulted in median CD3+ depletion of 4·2 logs (2·8 logs – undetectable CD3+) (Fig S1B). Median T cell add-back was 1·0 9 105 CD3+/kg (0–2·2). The infused stem cell grafts contained a median of 1·6 9 105 CD3+/kg (0·1 –4·8) and 5·1 9 106 CD34+/kg (2·0–13·3). Seventeen of 19 patients received T cell doses of 0·9–2·6 9 105 CD3+/kg.

(A)

(B)

Haematopoietic reconstitution and donor chimerism All patients engrafted neutrophils at a median of 13 d (9–27) (Fig. 1A). One patient experienced a secondary graft failure but had stable neutrophil engraftment following a stem cell boost. Eighty two percent (82%) of evaluable patients engrafted platelets. Of those who engrafted platelets, the median time to engraftment was 32 d (21–44) (Fig. 1B). Of those who did not engraft platelets, one patient died at day +72 from progressive disease and two received stem cell boosts, including the aforementioned patient with secondary graft failure; neither achieved platelet transfusion independence. Patients achieved mean whole blood chimerism of 98%, 93%, 97%, 96%, and 97% at days 30, 60, 100, 180, and 365 post-transplant respectively (Fig. 1C). Mean CD3+ donor chimerism at these time points was 71%, 70%, 82%, 74%, and 80% respectively (Fig. 1D).

(C)

(D)

Acute and chronic GVHD Three patients developed grade II–IV aGVHD (two grade III, one grade IV), representing a Kaplan–Meier probability of 15·8% [95% confidence interval (CI95): 0·3–55·2] (Fig. 2A). Two patients developed grade II–IV aGVHD (one grade II, one grade III) following donor lymphocyte infusions (DLI) that were not CD34-selected. Four patients developed limited cGVHD, a Kaplan–Meier probability of 23·3% (CI95: 2·0– 58·0) (Fig. 2B). No patients developed extensive cGVHD. In this small study sample, incorporation of alemtuzumab versus ATG in the conditioning regimen was not significantly associated with incidence of grade II–IV aGVHD or cGVHD.

Grade III–IV non-haematological toxicities related to conditioning The patient with Wolman disease developed acute liver failure following conditioning, with transaminitis peaking on day +2 (alanine transaminase 1630 iu/l, aspartate transami210

Fig 1. (A) Probability of myeloid engraftment (first day of three consecutive days with ANC 0·5 9 109/l after the nadir) by Kaplan–Meier method. (B) Probability of platelet engraftment (first day of seven consecutive days with platelet count 20 9 109/l untransfused with platelets) by Kaplan–Meier method. (C) Percent whole blood donor chimerism (mean ± SEM) over time from day +30 to day +365. (D) Percent CD3 donor chimerism (mean ± SEM) over time from day +30 to day +365.

nase 2171 iu/l) and hyperbilirubinaemia peaking on day +18 (total bilirubin 908 lmol/l). She underwent deceased-donor liver transplantation on day +19, with recovery of liver function. Her acid lipase levels normalized following stem cell ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

T Cell Depletion in Paediatric AlloSCT Table II. Viral and fungal infections acquired on-study.

(A)

Systemic viral infections

13

Invasive fungal infections

5

BK virus Adenovirus HHV6 CMV Influenza (H1N1) RSV VZV

4 2 2 2 1 1 1

Candida glabrata Candida krusei Candida parapsilosis Aspergillus spp. Penicillium spp.

1 1 1 1 1

VZV, varicella zoster virus; HHV6, human herpes virus 6; CMV, cytomegalovirus; RSV, respiratory syncytial virus.

(B) hybridization or immunostaining for latent membrane protein 1 (LMP1). His PTLD resolved completely following an unmodified DLI from his original MUD donor (5 9 106 CD3+/kg) on day +372.

Immune reconstitution

Fig 2. (A) Probability of acute graft-versus-host disease (aGVHD) grade II–IV by Kaplan–Meier method. This excludes two patients who received donor lymphocyte infusion and developed aGVHD on days +255 and +385. (B) Probability of limited chronic GVHD (cGVHD) by Kaplan–Meier method.

transplantation, and she experienced no recurrence of her underlying Wolman disease.

Viral and fungal infection SVIs and IFIs are summarized in Table II. Eleven of 19 transplants involved a CMV-positive donor and/or recipient; two patients experienced CMV reactivation. No patients experienced Epstein–Barr virus (EBV) reactivation. The 1-year cumulative incidence of SVI was 42·1%, and that of IFI was 28·0%. The 1-year probability of SVI/IFI-related mortality was 5·6%. Patients undergoing RTC had fewer SVIs per patient (P = 0·03) than MAC recipients but not significantly fewer IFIs.

Post-transplant lymphoproliferative disorder Patient 1 (Table I) developed fevers to 105○F and reduced WBC and platelet counts in the setting of negative microbial blood cultures and viral PCR studies, including EBV, approximately 11·5 months post-transplant. Computerized tomography and positron emission tomography scans were consistent with PTLD and he began therapy with rituximab and methylprednisolone. PTLD was confirmed by lymph node biopsy on day +366; micrographs from the biopsy are displayed in Fig S2. Notably, the specimen showed no evidence of EBV by Epstein–Barr encoded RNA (EBER) in situ ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

Immunophenotypic analysis of T cell (CD3+, CD3+CD4+, CD3+CD8+), B cell (CD19+) and NK-cell (CD3 CD56+) reconstitution at days +100, +180 and +365 is presented as mean lymphocyte subset counts (Figs 3A, C, E and 4A, C). At day +180/365, immune cell subset counts were above the age-specific lower limit of normal in the following percentages of patients: total T cells (11%/25%), helper T cells (0%/ 25%), cytolytic T cells (22%/38%), B cells (56%/75%) and NK-cells (94%/94%) (Figs 3B, D, F and 4B, D). At day +180/365, median immunoglobulin levels were as follows (in g/l): IgG (6·66/7·91), IgM (0·46/0·42), and IgA (0·747/0·593); at day +180/365, immunoglobulin subset counts were above the age-specific lower limits of normal in the following percentages of patients: IgG (47%/53%), IgM (59%/53%) and IgA (59%/47%). Immune cell subset counts and immunoglobulin levels did not differ significantly between recipients of RTC versus MAC.

Malignant relapse and causes of death Of 13 patients with malignant disease, five relapsed posttransplant on days +66, 175, 389, 454, and 623 (Table III). Three of these patients died from complications related to progressive leukaemia at days +72, +585, and +708 posttransplant. One of these patients received re-induction therapy following relapse followed by an infusion of CD34selected PBSCs from her prior donor, due to bone marrow aplasia; although acute myeloid leukaemia (AML) was not evident after this further therapy, she succumbed to fungal pneumonia on day +229. One of these patients received RTC and a second allograft from a new donor (9/10 HLA match) but ultimately died from P. jirovecii and Candida pneumonia. Three patients died despite having no relapse of their underlying disease. One patient suffered grade IV haemor211

M. B. Geyer et al

(A)

(B)

(C)

(D)

(E)

(F)

Fig 3. T cell immune reconstitution by mean absolute cell counts (cells 9 109/l; horizontal lines indicate mean values) (A, C, E) and percentages (B, D, F) of children achieving normal lymphocyte counts at days 100, 180 and 365 post-transplant for CD3+ (A and B), CD3+CD4+ (C and D) and CD3+CD8+ cells (E and F).

rhage related to placement of a tunnelled central venous catheter following accidental pulmonary artery puncture and subsequently died day +534. Another patient received a DLI (1·1 9 107 CD3+/kg) day +225 due to dropping chimerism levels (79% whole blood and 77% CD3 donor chimerism on day +222 after previously achieving 100% donor chimerism in whole blood and CD3+ subsets), developed uncontrolled severe liver GVHD, and died of subsequent multi-organ system failure on day +277. The patient with Wolman disease died suddenly at home on day +448; a post-mortem nasal swab revealed H1N1 influenza and autopsy revealed pneumococcal pneumonia. 212

Day 100 TRM and overall survival Day +100 TRM was 0%. The probability of 1-year OS for all patients enrolled was 82·3% (CI95: 54·0–94·0) and of 2-year OS was 48·2% (CI95: 21·6–70·7). Five patients died more than 1 year following stem cell transplantation as of the time of analysis (Fig. 5).

Discussion T cell depletion prior to AlloSCT effectively reduces risk of aGVHD and cGVHD (Ho & Soiffer, 2001). We observed only ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

T Cell Depletion in Paediatric AlloSCT

(A)

(B)

(C)

(D)

Fig 4. B-lymphocyte and NK-cell reconstitution by mean (±SEM) absolute cell counts (cells 9 109/l) (A, C) and percentages (B, D) of children achieving normal counts at days 100, 180 and 365 post-transplant for CD19+ (A and B) and CD3 CD56+ cells (C and D).

a 15·8% incidence of grade II–IV aGVHD, no extensive cGVHD, and 100% primary engraftment following CD34+ selection and MUD PBSCT with fixed-dose T cell add-back. The incidence of grade II–IV aGVHD following T cell replete fully matched unrelated donor PBSCT was 45% in a cohort of 266 paediatric recipients with malignant diseases (Shaw et al, 2010), and has exceeded 55% in other series of 7–10/10 HLAmatched MUD PBSCT, despite high resolution HLA typing and pharmacoprophylaxis with calcineurin inhibitors and methotrexate (Sedlacek et al, 2006; Kalwak et al, 2010). Significantly decreased incidence of grade II–IV aGVHD (0–24%) and cGVHD (0–19%) has been reported following CD34-selected haploidentical or MUD SCT (Peters et al, 1999; Handgretinger et al, 2001; Ortin et al, 2002; Lang et al, 2003, 2004a,b; Ball et al, 2005, 2007; Klingebiel et al, 2006; Marks et al, 2006; Lang & Handgretinger, 2008). A large randomized study of T cell replete (GVHD prophylaxis using methotrexate and ciclosporin) versus T cell depleted MUD BMT in 405 adult and paediatric patients with haematological malignancies found significantly decreased incidence of grade II–IV aGVHD (39% vs. 63%) in T cell depleted MUD BMT recipients, without significant differences in cGVHD and 3-year disease-free survival (Wagner et al, 2005). Recently, ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

two prospective Phase II trials of MAC and CD34-selected PBSCT demonstrated low rates of aGVHD and extensive cGVHD while maintaining engraftment rates of 100%. Devine et al (2011) reported 22·7% risk of grade II–IV aGVHD and 6·8% risk of extensive cGVHD (19% overall cGVHD) in 44 adults with AML in first (CR1) or second (CR2) complete remission undergoing CD34-selected (median T cell dose 6·6 9 103 CD3+/kg) HLA-identical sibling donor PBSCT. Jakubowski et al (2011) reported 9% risk of grade II –IV aGVHD and 11% risk of extensive cGVHD (29% overall cGVHD) in 35 adults with haematological malignancies undergoing 7–10/10 HLA-matched MUD AlloSCT (most receiving PBSCs) following CD34 selection using the Isolex 300i (median T cell dose 1·5 9 103 CD3+/kg). We observed a 23·3% incidence of cGVHD (all limited). While the proportion of patients with cGVHD in our series is slightly higher than other reports of CD34-selected haploidentical or MUD PBSCT in paediatric recipients, which have generally employed significantly lower T cell doses and had a lower median age, in another series, CD34-selected MUD PBSCT with T cell add-back of 5 9 105 CD3+/kg was associated with a 61·5% probability of cGVHD (all limited) (Bunin et al, 2006). 213

M. B. Geyer et al Table III. Characteristics of patients developing progressive disease on study.

Pt no.

Diagnosis

4

ALL

5

ALL

7

ALL

12

HTLV-1 adult T cell LL

15

AML

Overall risk*

Time to relapse or progression (months)

Early BM relapse (CR1 < 24 months) CR3, 11q23 rearrangement CNS disease at diagnosis, early CNS relapse (CR1 < 20 months) Refractory disease, active disease at time of AlloSCT confirmed on lung biopsy

Average

20·5

M6 subtype

Average

Poor prognostic features

Poor

2·2

Average

14·9

Poor

12·8

5·8

Time to death (months)

Cause of death

Declined further chemotherapy Clofarabine

23·3

Progressive disease

2·4

Progressive disease

Intrathecal topotecan and cytarabine; Cytarabine, etoposide, dexamethasone Pralatrexate, vorinostat, alemtuzumab; RTC (fludarabine, melphalan, alemtuzumab) and 9/10 MUD BMT from new donor Reinduction with fludarabine, cytarabine, idarubicin; Stem cells (CD34+ selected) from same donor infused due to subsequent BM aplasia

19·2

Progressive disease

18·0

P. jirovecii and fungal pneumonia

Therapy following relapse

7·5

Fungal pneumonia

MUD, matched unrelated donor; BMT, bone marrow transplant; ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; HTLV-1, human T-lymphotrophic virus-1; LL, leukaemia and lymphoma, CR1, first complete response; CR3, third complete response; AlloSCT, allogeneic stem cell transplantation; RTC, reduced toxicity conditioning; CNS, central nervous system. *Center for International Blood and Marrow Transplant Research criteria.

Fig 5. Probability of overall survival by Kaplan–Meier method for all patients (solid line) and stratified by myeloablative conditioning (dotted line) versus reduced toxicity conditioning (broken line) (P = 0·14).

Donor T cells may help to prevent graft rejection by elimination of alloreactive host cells. Risk of graft failure is low (30%) have been observed. Median T cell doses infused (0·7–5·6 9 104 CD3+/kg) were lower than those in our series (Peters et al, 1999; Handgretinger et al, 2001; Kremens et al, 2002; Ortin et al, 2002; Lang et al, 2004a; Ball et al, 2005, 2007; Klingebiel et al, 2006; Marks et al, 2006; Lang & Handgretinger, 2008; Kennedy-Nasser et al, 2011). One group observed a 16% incidence of primary graft failure in paediatric patients with malignant and nonmalignant diseases following CD34-selected matched related donor (MRD) or MUD PBSCT, with a median T cell dose of 6 9 103 CD3+/kg (Lang et al, 2003, 2004b). No cases of graft failure were reported among 25 paediatric patients with leukaemia who underwent MAC and CD34-selected MUD PBSCT with a T cell dose of 5 9 105 CD3+/kg (Bunin et al, 2006). T cell add-back, despite the use of ATG and alemtuzumab in conditioning, may have contributed to the prevention of primary graft failure in the series presented by Bunin et al (2006) and in the present study. We observed a moderately high incidence of SVI and IFI and delayed T cell immune reconstitution, consistent with other reports of CD34-selected haploidentical or MUD AlloSCT. In the aforementioned randomized trial of T cell ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

T Cell Depletion in Paediatric AlloSCT depleted versus T cell replete MUD BMT with conventional GVHD prophylaxis, severe CMV infection (28% vs. 17%) and life-threatening or fatal aspergillosis (16% vs. 7%) were observed significantly more frequently among T cell depleted MUD BMT recipients (Wagner et al, 2005). In a prospective study of immune reconstitution in paediatric patients undergoing CD34-selected hapoloidentical SCT, robust recovery of NK cells was observed within 1 month post-transplant, but significant thymus-dependent proliferation of naı¨ve T cells and expansion of the T cell receptor repertoire required 6 months. Normal T and B cell counts and proliferative responses were achieved at a median of 7–8 months (Eyrich et al, 2001). Lang et al (2004a) reported risks of lethal viral and fungal infections of 16% and 6% respectively, all occurring within 6 months following CD34-selected haploidentical SCT, when antigen-specific T cell immunity is most compromised. Similarly, Handgretinger et al (2001) noted 13% and 5% risks of lethal viral and fungal infections in their cohort, respectively. Among the studies reviewed, CMV reactivation was reported in 14–36%, with a 0–7% incidence of lethal infection. Adenovirus infection was reported in 8·5–29%, with lethal infection in 0–18% (Handgretinger et al, 2001; Kremens et al, 2002; Ortin et al, 2002; Lang et al, 2004a; Ball et al, 2005, 2007; Feuchtinger et al, 2005; Marks et al, 2006; Lang & Handgretinger, 2008). Lang et al (2003) noted a 37% risk of fatal SVI or IFI following CD34-selected MUD PBSCT in paediatric recipients with leukaemia, mostly occurring within the first 6 months post-AlloSCT, prior to T cell recovery. In a similar population, Bunin et al (2006) noted TRM of 41·7% by day +85, attributable in part to five deaths due to SVI or IFI. Despite the high incidence of SVI and IFI in our series, the 1-year probability of mortality due to SVI and IFI was only 5·6%. Our institution’s aggressive prophylaxis, monitoring, and treatment for SVIs and IFIs may have helped to limit early morbidity and mortality related to opportunistic infection (Shereck et al, 2007; Roman et al, 2008). Though most RTC regimens in this study incorporated alemtuzumab, which may have contributed to delayed T cell immune reconstitution, only two SVIs arose following alemtuzumab therapy: one case of disseminated adenovirus and one late case of H1N1 influenza, with no alemtuzumab recipients experiencing CMV reactivation as observed at high rates in prior studies (Osterborg et al, 2009; Chakraverty et al, 2010). Peripheral blood (PB) or cord blood (CB)-derived cytotoxic T-lymphocytes directed against viral infection have been successfully used as adoptive cellular immunotherapy in AlloSCT recipients and may further serve to decrease infection-related mortality following T cell depleted AlloSCT. (Feuchtinger et al, 2006; Leen et al, 2006, 2009). PTLD is a recognized complication of AlloSCT, often arising within 1 year of transplantation in the setting of EBVinduced B-lymphocyte proliferation and T cell dysfunction. Selective T cell depletion has been strongly associated with increased incidence of PTLD following AlloSCT; one report ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

noted no cases of PTLD among 65 recipients of T cell replete grafts, but an incidence of PTLD approaching 25% at 180 d post-transplant in those receiving ATG as part of conditioning followed by T cell depleted AlloSCT (van Esser et al, 2001). A recent analysis of 26 901 AlloSCT recipients confirmed that selective T cell depletion [relative risk (RR) 9·4], broad lymphocyte depletion (RR 3·1), ATG use (RR 3·8), and 2 HLA mismatches (RR 3·8, when accompanied by selective T cell depletion or ATG use) were significant risk factors for PTLD in a multivariate model (Landgren et al, 2009). We observed a single case of PTLD in this series, which did not occur until 1-year post-transplant. The use of broad lymphocyte versus T cell-specific depletion, potentially decreasing the number of EBV-infected B cells prior to AlloSCT, and T cell addback may have helped to limit the incidence of early PTLD in our series, though this is difficult to assess in our small sample. Particularly in the combined presence of T cell depletion and ATG use, routine monitoring of EBV titres and early treatment with rituximab remain important tools in effective prevention of life-threatening PTLD. While prior studies of CD34-selected MUD PBSCT in paediatric recipients have employed MAC, eight patients in our series received RTC, which has emerged as an alternative to traditional MAC for selected paediatric patients with malignant and non-malignant diseases (Satwani et al, 2005, 2008; Pulsipher et al, 2009). In 25 paediatric patients with refractory haematological malignancies, RTC consisting of fludarabine, melphalan, thiotepa, and anti-CD3 monoclonal antibody and haploidentical AlloSCT was associated with rapid immune reconstitution and low TRM (Chen et al, 2006). Handgretinger et al (2007) employed a similar conditioning regimen followed by CD3/19-depleted haploidentical PBSCT in 38 children and adults with refractory haematological malignancies or severe aplastic anaemia, with low TRM (3%) but high rates of primary graft failure (17%) observed. Another recent report of alemtuzumab-based T cell depleted MRD or MUD RTC AlloSCT in 168 adults with AML found encouraging 3-year disease-free survival rates of 49% and 42% among patients transplanted in CR1 or CR2/3 respectively, as well as a strong association between post-transplant immune suppression with ciclosporin and risk of relapse, suggesting that a strong graft-versus-leukaemia effect is retained in alemtuzumab-based T cell depleted RTC AlloSCT (Craddock et al, 2010). We observed a moderate incidence of progressive disease among patients with malignant diseases. Two of the five patients experiencing disease relapse had poor risk disease prior to transplantation (Table III). Three patients experienced malignant relapse more than 1 year post-AlloSCT; two of these three had achieved CD8+ levels above the age-specific lower limit of normal prior to relapse. CD34+ selection potentially abrogates the anti-cancer effects of an allograft through T cell depletion. In a randomized trial of T cell replete versus T cell depleted AlloSCT, risk of CML relapse (20% vs. 7%) but not acute leukaemia relapse was observed 215

M. B. Geyer et al among patients undergoing T cell depleted AlloSCT (Wagner et al, 2005). In the aforementioned report (Jakubowski et al, 2011), a low cumulative incidence of relapse (6%) of haematological malignancies (mainly acute leukaemias) was observed despite a median T cell inoculum two logs lower than the T cell dose employed in our study, further arguing against loss of graft-versus-cancer effects following T cell depleted AlloSCT in patients with acute leukaemias. This study has several limitations, including small sample size, an observational design, the lack of a contemporaneously treated control group undergoing T cell replete AlloSCT, the use of a cell selection platform no longer commercially available, a heterogeneous group of primary malignant and non-malignant diseases, and multiple conditioning regimens. While this considerable heterogeneity should inform interpretation of our results, the small number of paediatric patients undergoing AlloSCT for diseases in which CD34-selection is employed suggests that the safety and efficacy of these strategies may sometimes be best assessed by combining together patients with different diseases and receiving different conditioning regimens. Though limitations in technical precision resulted in small, unintentional differences in T cell inocula between patient, graft manipulation remained similar in all patients. In summary, we observed 100% primary engraftment, 0% extensive cGVHD, and low risk of grade II–IV aGVHD and day +100 TRM following CD34-selected MUD PBSCT with T cell add-back in paediatric recipients. These results suggest that T cell depletion of MUD PBSCs followed by fixed dose T cell addback is a feasible strategy in children, adolescents, and young adults lacking a suitable related donor, and that up to two HLA disparities are acceptable. We have obtained an approved Investigational New Drug application (#14359) under which to use the CliniMACS® (Miltenyi Biotech) for CD34+ selection and plan to pursue familial haploidentical and matched unrelated donor PBSCT in children and young adults with malignant and non-malignant diseases, including familial haploidentical CD34-selected PBSCT in poor-risk patients with sickle cell disease lacking an unaffected matched related or fully matched unrelated donor. RTC prior to T cell depleted MUD PBSCT also appears to result in high rates of engraftment and may be a therapeutic option in paediatric recipients unable to tolerate MAC while reducing the risks of GVHD and organ toxicity associated with MAC and mismatched unrelated adult donor transplantation. While umbilical CB transplantation may hasten stem cell procurement for patients unable to wait for the duration of an unrelated adult donor search, CD34-selected MUD AlloSCT offers several potential advantages, including decreased risk of graft failure and increased cell dose, particularly for larger paediatric patients in whom a single CB unit would provide insufficient cell dose (Cairo et al, 2008; Liao et al, 2011). Infusion of donor regulatory T cells prior to CD34selelected familial haploidentical AlloSCT and infusion of 216

conventional T cells may limit GVHD while enhancing immune reconstitution and preserving graft-versus-cancer effects (Di Ianni et al, 2011). Advances in the use of cellular therapies to reduce the risk of GVHD, life-threatening viral and fungal infections, and malignant relapse and to accelerate immune reconstitution warrant further investigation and have the potential to improve outcomes substantially in MUD and familial haploidentical donor PBSCT recipients.

Acknowledgements This work was supported in part by grants from the Pediatric Cancer Research Foundation, National Institute of Arthritis and Musculoskeletal and Skin Diseases (R21 AR49330; MSC), Dreaming for Discovery and Cure Fund, Marisa Fund, Sonia Scaramella Fund, Paul Luisi Foundation, Brittany Barron Fund, and the Doris Duke Charitable Foundation. Presented in part at the BMT Tandem Meetings, February, 2010, Orlando, Florida and the Pediatric Academic Societies Meeting, May, 2010, Vancouver, British Columbia, Canada. The authors would like to thank the patients and their families who participated in these trials and the inpatient and outpatient bedside nurses who provided expert care on a daily basis to these patients at our institution.

Author contributions M.B.G., A.M.R., performed the research, analysed the data and wrote the paper, J.S.J., C.V.V., analysed the data and wrote the paper, R.M., D.D., J.A., M.B., J.H.G., D.G., P.S., L. H., M.S-P., J.S., performed the research, E.M., B.A., L.A.B-L. performed the research and wrote the paper, and M.S.C. designed the research study, performed the research, analysed the data and wrote the paper.

Conflict of interest All authors disclose no conflict of interest.

Supporting Information Additional Supporting Information may be found in the online version of this article: Fig S1. (A) Median CD34+/kg in apheresis products before and after CD34 cell selection (P < 0·0001). (B) Median CD3+/kg in apheresis products before and after CD34+ selection (P < 0·0001). Fig S2. Lymph node biopsy of patient developing PTLD. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 205–219

T Cell Depletion in Paediatric AlloSCT

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