Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases

ARTICLE IN PRESS Biol Blood Marrow Transplant 000 (2019) 1
13

Biology of Blood and Marrow Transplantation journal homepage: www.bbmt.org

Prospective Study of a Novel, Radiation-Free, Reduced-Intensity Bone Marrow Transplantation Platform for Primary Immunodeficiency Diseases Dimana Dimitrova1, Juan Gea-Banacloche2, Seth M. Steinberg3, Jennifer L. Sadler1, Stephanie N. Hicks1, Ellen Carroll1, Jennifer S. Wilder4, Mark Parta4, Lauren Skeffington1, Thomas E. Hughes5, Jenny E. Blau6, Miranda M. Broadney7, Jeremy J. Rose1, Amy P. Hsu8, Rochelle Fletcher1, Natalia S. Nunes1, Xiao-Yi Yan1, William G. Telford1, Veena Kapoor1, Jeffrey I. Cohen9, Alexandra F. Freeman8, Elizabeth Garabedian10, Steven M. Holland8, Andrea Lisco11, Harry L. Malech8, Luigi D. Notarangelo8, Irini Sereti11, Nirali N. Shah12, Gulbu Uzel8, Christa S. Zerbe8, Daniel H. Fowler1, Ronald E. Gress1, Christopher G. Kanakry1, Jennifer A. Kanakry1,* 1

Experimental Transplantation and Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland Mayo Clinic, Phoenix, Arizona 3 Biostatistics and Data Management Section, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland 4 Clinical Research Directorate/Clinical Monitoring Research Program, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Bethesda, Maryland 5 National Institutes of Health Clinical Center, Bethesda, Maryland 6 Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 7 Section on Growth and Obesity, Program in Endocrinology, Metabolism and Genetics, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 8 Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 9 Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 10 Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 11 Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 12 Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 2

Article history: Received 11 June 2019 Accepted 28 August 2019 Key Words: Primary immunodeficiency Bone marrow transplantation Reduced-intensity conditioning Post-transplantation cyclophosphamide

A B S T R A C T Allogeneic blood or marrow transplantation (BMT) is a potentially curative therapy for patients with primary immunodeficiency (PID). Safe and effective reduced-intensity conditioning (RIC) approaches that are associated with low toxicity, use alternative donors, and afford good immune reconstitution are needed to advance the field. Twenty PID patients, ranging in age from 4 to 58 years, were treated on a prospective clinical trial of a novel, radiation-free and serotherapy-free RIC, T-cell-replete BMT approach using pentostatin, low-dose cyclophosphamide, and busulfan for conditioning with post-transplantation cyclophosphamide-based graft-versus-host-disease (GVHD) prophylaxis. This was a high-risk cohort with a median hematopoietic cell transplantation comorbidity index of 3. With median follow-up of survivors of 1.9 years, 1-year overall survival was 90% and grade III to IV acute GVHD-free, graft-failure-free survival was 80% at day +180. Graft failure incidence was 10%. Split chimerism was frequently observed at early post-BMT timepoints, with a lower percentage of donor T cells, which gradually increased by day +60. The cumulative incidences of grade II to IV and grade III to IV acute GVHD (aGVHD) were 15% and 5%, respectively. All aGVHD was steroid responsive. No patients developed chronic GVHD. Few significant organ toxicities were observed. Evidence of phenotype reversal was observed for all engrafted patients, even those with significantly mixed chimerism (n = 2) or with unknown underlying genetic defect (n = 3). All 6 patients with pre-BMT malignancies or lymphoproliferative disorders remain in remission. Most patients have discontinued immunoglobulin replacement. All survivors are off immunosuppression for GVHD prophylaxis or treatment. This novel RIC BMT approach for patients with PID has yielded promising results, even for high-risk patients. Published by Elsevier Inc. on behalf of the American Society for Transplantation and Cellular Therapy.

Financial disclosure: See Acknowledgments on page 11. * Correspondence and reprint requests: Jennifer A. Kanakry, MD, Center for Cancer Research, NCI, 10 Center Drive, Building 10, Room 4-3132, Bethesda, MD 20892. E-mail address: [email protected] (J.A. Kanakry).

INTRODUCTION Allogeneic blood or marrow transplantation (BMT) has long served as a potentially curative therapy for patients with primary immune deficiencies (PIDs). However, the landscape of

https://doi.org/10.1016/j.bbmt.2019.08.018 1083-8791/Published by Elsevier Inc. on behalf of the American Society for Transplantation and Cellular Therapy.

ARTICLE IN PRESS 2

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

PIDs has expanded markedly since the first PID transplants were performed, with >350 PIDs genetically and phenotypically now recognized and many characterized by immune dysregulation rather than solely immune deficiency [1]. Increasingly, patients with PIDs of variable natural history, hypomorphic variants, or somatic reversion are diagnosed later in life and only considered for BMT after incurring significant comorbidities or refractory disease sequelae [2-4]. Family members may also be affected, limiting donor options [5]. Furthermore, in patients without PID-associated malignancy, the benefits of conditioning extend only so far as ensuring engraftment, and graft-versus-host disease (GVHD) is not beneficial. Thus, BMTs for PIDs entail unique challenges regarding the best approaches to achieve engraftment and phenotype reversal with as little short- and long-term toxicity as possible. We sought to evaluate a novel, reduced-intensity conditioning (RIC) approach to BMT for PIDs, a platform designed with the goals of reducing regimen-related toxicities and complications such as GVHD, using alternative donors, and achieving successful engraftment and robust immune reconstitution. An approach to lymphodepletion-centered RIC developed at the National Institutes of Health (NIH) [6,7] was integrated with post-transplantation cyclophosphamide (PTCy) for GVHD prophylaxis, given the low rates of severe acute and chronic GVHD and the comparable outcomes between alternative donors and HLA-matched sibling donors (MSDs) observed with PTCy-based platforms [8-13]. Pentostatin with low-dose cyclophosphamide was used as the T-cell-suppressing backbone of conditioning, based on data demonstrating pentostatin plus cyclophosphamide to result in greater T cell functional impairment compared wth fludarabine plus cyclophosphamide, with similar levels of host T cell lymphodepletion [7]. Herein, we present the results of a prospective trial of a novel, radiation- and serotherapy-free RIC, PTCy-based, T-cell-replete BMT platform for children and adults with PIDs. METHODS After institutional review board approval, this single-center prospective clinical trial (NCT02579967, clinicaltrials.gov) was conducted at the NIH Clinical Center. The results of 20 consecutive BMTs performed on the fully accrued RIC arm, which enrolled patients from November 19, 2015, to October 18, 2017, are presented. Eligible patients were aged 4 to 75 years with PID of sufficient severity to warrant BMT. The conditioning regimen was an 11-day, reduced-intensity approach consisting of pentostatin, low-dose cyclophosphamide, and 2 days of pharmacokinetically dosed busulfan with an area under the curve target of 4600 mmol*min/d. GVHD prophylaxis consisted of high-dose PTCy on days +3 and +4, followed by sirolimus and mycophenolate mofetil. All grafts were T-cell-replete bone marrow. The BMT platform, including specific drug doses and time, as well as supportive care measures, are outlined in Table 1. The primary endpoint was to estimate the grade III to IV acute GVHDfree, graft-failure-free survival (GGFS) at day +180 post-BMT; events were grade III- to IV acute GVHD (aGVHD) not responsive to 7 days of high-dose steroids, primary or secondary graft failure, or death from any cause. Acute and chronic GVHD were diagnosed and graded using standard criteria [14,15]. T, B, and natural killer (NK) cell subsets were assessed by peripheral blood flow cytometry weekly through day +100 and at study timepoints. Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6, adenovirus, and BK virus were evaluated weekly in blood and, for BK virus, also urine by quantitative PCR. CMV monitoring and preemptive treatment were performed as previously published [16]. Details regarding study design and definitions, recipient eligibility, donor selection, and monitoring evaluations are listed in the online supplemental methods. Statistical Analysis Data were locked for analysis on January 23, 2019. Survival endpoint probabilities were estimated using the Kaplan-Meier method with 95% confidence intervals (CIs). Cumulative incidences (CuIs) of transplant-related mortality (TRM), GVHD, count recovery, and graft failure were estimated by competing-risk analysis using Gray’s method. Death was a competing risk for

all analyses except TRM; relapse of pre-BMT malignancy was a competing risk for TRM. Graft failure was a competing risk for GVHD. Data were analyzed with R, version 3.3.3 (R Foundation for Statistical Computing, Vienna, Austria) and Prism version 8.0 (GraphPad Software, La Jolla, CA).

RESULTS Patient and Graft Characteristics Table 2 shows recipient and graft characteristics. This was a high-risk cohort of children and adults, with a median hematopoietic cell transplantation-comorbidity index (HCT-CI) [17] score of 3 (range, 0 to 11) and a median follow-up of survivors of 1.9 years (range, 1.3 to 3.1 years). Thirty-five percent of patients received haploidentical donor grafts, 40% matched unrelated, and the rest MSD. Survival Endpoints The primary endpoint of GGFS was 80% at day +180 (95% CI, 55% to 92%), with overall survival at 1 year of 90% (95% CI, 66% to 97%) (Figure 1). There were 2 early deaths, both infectious, at day +44 (P8) and day +110 (P10). Contributing to P8’s death was severe epistaxis leading to hemorrhagic shock, intubation for airway protection, and emergent maxillary artery embolization, now believed to represent a bleeding diathesis previously unappreciated in Magnesium transporter protein 1 (MAGT1) deficiency [18]. An additional patient (P7) had an accidental death on day +613 in the setting of good graft function, complete phenotype reversal including ongoing remission of his EBV+ lymphoma, and no GVHD, organ dysfunction, or infectious issues. Tables 3 and 4 summarize outcomes for the cohort and for individual patients, respectively. Engraftment and Chimerism Neutrophil recovery occurred at median day +17 post-BMT (range, +14 to 42) for all patients. The median duration of neutropenia was 9 days (range, 6 to 36). Platelet recovery occurred at median day +30 post-BMT (range, +16 to 45). The CuI of graft failure was 10%. P14 (primary graft failure at day +26) had brisk recovery of autologous, trilineage hematopoiesis (neutrophils on day +14, platelets on day +20), and P12 with secondary graft failure at day +159 had concomitant autologous recovery of trilineage hematopoiesis. Only P12 developed engraftment syndrome, not requiring systemic steroids. Trends in donor cell engraftment kinetics were observed across all engrafted patients and did not vary by donor type (Figure 2A). Split chimerism, with donor T cell chimerism markedly lower than myeloid, was observed early post-BMT, with only 5 patients (2 HLA-haploidentical donors, 2 matched unrelated donors, 1 MSD) having predominantly/completely donor cells in both compartments at day +28. At day +28, median donor myeloid chimerism was 96% (range, 78% to 100%, n = 19), remaining high on follow-up, whereas at day +28, median donor T cell chimerism was 41% (range, 9% to 100%, n = 18; P12, 5%), rising to a median of 86% (range, 38% to 100%, n = 18) at day +42. Similar kinetics were observed for CD4+ and CD8+ T cell chimerism (Figure 2B). Stopping immunosuppression was associated with subsequent conversion to
95% donor T cell chimerism in 7 of 9 cases where T cell chimerism was <95% donor while on immunosuppression; for 7 patients, T cell chimerism was
95% donor while on immunosuppression, remaining so off immunosuppression. Two patients remain with stable mixed chimerism in both T cell (85% and 21% donor cells, respectively) and myeloid (53% and 65% donor cells, respectively) subsets, both off immunosuppression for 2.5 years

ARTICLE IN PRESS D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

3

Table 1 BMT Platform and Supportive Care Measures Day

Drug/Intervention

Adult Dosing

Pediatric Dosing

Prior to day
11

Busulfan test dose

0.8 mg/kg i.v. to calculate AUC*

0.8 mg/kg i.v. to calculate AUCy

Day
11 and day
7

Pentostatin

4 mg/m2/d i.v.

Days
11 through
8

Cyclophosphamide

3 mg/kg/d orally or i.v., dosage cap of 200 mg/d

Days
7 through
4

Cyclophosphamide

If ALC on day
7 >100 cells/mL, escalation to 5 mg/kg/d orallyz; dosage cap of 400 mg/d

Days
3 and
2

Busulfan

Pharmacokinetically dosed, with targeted daily systemic exposure of 4600 mmolmin based on test dose AUC; default dose of 3.2 mg/kg/d if test dose cannot be performed

Day
1

Day of rest

Day 0

Fresh infusion of T-cell-replete marrow Target dose 4.5 £ 108 TNC/kg recipient IBW

Days +3 and +4

Cyclophosphamidex

50 mg/kg/d i.v.║

Mesna

50 mg/kg/d i.v.║

Sirolimus loading dose**

6 mg orally

Day +5

3 mg/m2 orally{; maximum initial dose 6 mg

Days +5 through +35

MMF

15 mg/kg orally or i.v. 3 times daily; maximum 1000 mg/dose

Starting day +6, continued through day +180, then stopped without taper#

Sirolimus maintenance dose**

2 mg orally; goal trough of 5-12 ng/mL

1 mg/m2 orally{; maximum initial dose 2 mg; goal trough of 5-12 ng/mL

Day

Supportive Care Measure

Adult Dosing

Pediatric Dosing

Prior to day
11

Leuprolide for ovarian suppression

Menstruating females

Prior to day
11

Ivermectin, if from area with endemic Strongyloides

200 mcg/kg orally daily for 2 consecutive days


11 through
1

PJP prophylaxis: trimethoprim-sulfamethoxazole or alternative


4 through
1

Clonazepam for seizure prophylaxis

0.5 mg orally twice daily

<10 years old or <30 kg: 0.005-0.015 mg/kg/dose twice daily, rounded to tablet sizes


4 through
1

Levetiracetam for seizure prophylaxis

500 mg orally twice daily

10 mg/kg orally or i.v. twice daily, maximum dose 500 mg


11 through +100

Ursodiol for hepatic protection

<90 kg: 300 mg orally twice daily
90 kg: 300 mg each morning and 600 mg each evening, orally

<40 kg: 300 mg orally twice daily


11 through +100 or longer

Antifungal prophylaxis

Agent chosen as indicated and tolerated by organ function and drug interactions; end of therapy often chosen to coincide with cessation of sirolimus given drug interactions


11 through +100

Weekly whole-blood monitoring by quantitative PCR for CMV, EBV, adenovirus, and HHV6; pre-emptive treatment strategy used for CMV (letermovir not approved yet during study period for prophylaxis)


11 through +2 years

HSV/VZV prophylaxis: acyclovir

800 mg orally twice daily

20 mg/kg orally twice daily, maximum dose 800 mg

Upon count recovery through +1 year

PJP prophylaxis: trimethoprim-sulfamethoxazole or alternative

0 until PJP prophylaxis resumed

Twice-weekly whole-blood qualitative PCR for toxoplasmosis in seropositive individuals

0 through ANC recovery

Antibacterial prophylaxis: levofloxacin or alternative as appropriate

0 through +5

Avoidance of corticosteroids; hydrocortisone permitted for adrenal insufficiency

+2 through +5

Normal saline for uroprotection

90 mL/m2/hr i.v.

+5 until ANC
1000/mL for 3 days

Filgrastim

5 mcg/kg/d s.c. or i.v.

Postengraftment, as indicated

Immunoglobulin replacement

+180 onward

Post-BMT immunizations

AUC indicates area under the curve; ALC, absolute lymphocyte count; TNC, total nucleated cells; IBW, ideal body weight; MMF, mycophenolate mofetil; PJP, Pneumocystis jiroveci pneumonia; HHV6, human herpesvirus 6; HSV, herpes simplex virus; VZV, varicella zoster virus; ANC, absolute neutrophil count. * For recipients older than age 16 years, busulfan test dose was based on IBW or actual body weight (ABW), whichever is lower, unless recipient was > 120% of IBW, in which case adjusted ideal body weight (IBW+ 25% of difference between IBW and ABW) was used. y For children age 4 to 16 years, busulfan test dose was based on ABW. Only 1 patient, P14, could not undergo busulfan pharmacokinetics. z All patients received this dose increase. x Post-transplantation cyclophosphamide infusion on day +3 began 60 to 72 hours after graft infused (72 hours preferred). ║ Dosed according to IBW, unless recipient weighed less than IBW, in which case ABW was used. { Based on ABW. # Sirolimus could be stopped sooner for toxicities or mixed chimerism, either with or without an alternative agent depending on the clinical situation. ** Sirolimus loading and maintenance doses individualized as necessary based on each patient’s concomitant medications and potential drug interactions, particularly the use of azoles, where coadministration was not considered contraindicated but required significant sirolimus dose reductions.

and 1 year, respectively, with phenotype reversal (Figure 2C). Lineage-specific chimerism trends are shown in Supplemental Figure S1. Post-BMT donor cell infusions were given to 7 (35%) patients for varied indications and with varied efficacy, detailed in Table 5.

GVHD The CuI of aGVHD was 20% (95% CI, 6% to 40%), with CuI of grade II to IV aGVHD of 15% (95% CI, 4% to 34%) and grade III to IV aGVHD of 5% (95% CI, 0% to 21%). No patients had steroiddependent or steroid-refractory aGVHD associated with this BMT platform. P14 underwent second BMT using a different

ARTICLE IN PRESS 4

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

Table 2 Recipient, Donor, and Graft Characteristics (n = 20) Characteristic

Value

Male, n (%)

13 (65)

Age at BMT, median (range), yr Aged >18 years, n (%)

18 (4-58) 10 (50)

Diagnosis, n MAGT1 deficiency (XMEN)

3

PIK3CD gain of function (PASLI)

3

STAT3 deficiency (hyper-IgE syndrome)

3

Unknown PID

3

IFNGR1 deficiency, autosomal dominant

1

IFNGR1 deficiency, autosomal recessive

1

RAG1 deficiency, hypomorphic

1

RAG2 deficiency, hypomorphic

1

Wiskott-Aldrich syndrome, somatic revertant

1

IL2RG deficiency (X-SCID), somatic revertant

1

IL10R1 deficiency

1

NFKB1 haploinsufficiency

1

Active disease manifestations leading into BMT, n (%) Active infection at BMT

8 (40)

Lymphoma/lymphoproliferative disorder

6 (30)

Autoimmunity

8 (40)

Chronic immunosuppression requirement

7 (35)

HCT-CI, median (range)

3 (0-11)

Donor age, median (range), yr

27 (15-56)

Female donor for male recipient, n (%)

2 (10)

Donor source, n (%) HLA-matched-unrelated

8 (40)

HLA-haploidentical

7 (35)

HLA-matched-sibling

5 (25)

ABO, n (%) Matched

12 (60)

Minor mismatch

4 (20)

Major mismatch

2 (10)

Major and minor mismatch

2 (10)

Graft dose, median (range), cells/kg TNC, £ 108

4.54 (2.42-9.30)

CD3+, £ 107

5.06 (1.84-10.00)

CD34+, £ 106

5.31 (2.52-14.20)

CMV serostatus D/R, n (%)
/Unknown*

7 (35)

+/Unknown*

5 (25)


/


4 (20)

+/+

3 (15)

+/


1 (5)

EBV serostatus D/R, n (%) +/+

13 (65)

+/Unknown*

5 (25)

+/


2 (10)

XMEN indicates X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia; PASLI, p110delta-activating mutation causing senescent T cells, lymphadenopathy, and immunodeficiency; D/R, donor/recipient. * Serostatus for CMV and EBV was unknown for recipients on chronic immunoglobulin replacement therapy who did not have a documented history of the virus detected in blood or tissue to indicate a positive serostatus.

BMT platform and did experience grade III, steroid-refractory aGVHD of the skin, liver, and gut after following that second BMT and donor lymphocyte infusion (DLI). No patients developed chronic GVHD.

Infectious Complications Of 9 at-risk patients, 4 (44%) developed CMV reactivation; no patients developed CMV disease following reactivation. Donor-derived CMV-specific cytotoxic T lymphocytes were administered to P3 on day +69 given intolerance of antiviral agents, with resultant prompt CMV control and no associated toxicity. Primary CMV infections were identified in 4 patients, all children themselves and/or having regular direct contact with young children. Three tolerated the infection with minor or no symptoms at 7 months, 1 year, and 3 years post-BMT while the fourth, P13, developed CMV pneumonitis 7 months post-BMT. Given significant lymphopenia in the setting of CMV pneumonitis, P13 received DLI in hopes of numerically augmenting CMV-specific response, although donor remained CMV seronegative at the time of DLI; subsequent studies demonstrated abundant CMV-specific T cells. Severe viral infection occurred in only 1 other patient (P10), who died of rhinovirus/ enterovirus lower respiratory tract infection and suspected herpes simplex virus encephalitis on day +110. Clinically significant BK virus-associated hemorrhagic cystitis occurred in 14 (70% of all patients and 82% known at-risk patients) at a median 23 days post-BMT (range, 15 to 74) with median duration of symptoms of 17 days (range, 6 to 124). Therapy with fluoroquinolones or cidofovir was not used. No BK nephropathy occurred. EBV was frequently detected in whole blood post-BMT, but of 20 at-risk patients, none required preemptive therapy or developed post-transplantation lymphoproliferative disorder. Severe bacterial complications included sepsis and death on day +44 due to health care-associated infection with multidrug-resistant Sphingomonas koreensis (P8), published in detail elsewhere [19], and pneumococcal sepsis at 11 months postBMT leading to postinfectious cryptogenic organizing pneumonia (P1). Three patients had uncomplicated secondary bacterial pneumonia following viral upper respiratory infection 9 to 12 months post-BMT, treated as outpatients. Bronchiectasis exacerbations occurred in P7 and P13, both with severe structural abnormalities, in the setting of suboptimal/absent pulmonary hygiene. Post-BMT fungal infections were infrequent. P1, who had an Aspergillus-colonized pneumatocele pre-BMT, developed an invasive pulmonary aspergillosis at day +28 with associated eosinophilic pneumonia, requiring systemic steroids and tocilizumab along with ongoing posaconazole to prevent Aspergillus resurgence. She had pneumatocele rupture with persistent pneumothorax requiring lobectomy (day +96) and pleurodesis (day +242). P8 was presumed to have disseminated aspergillosis shortly before his death. Fungal infections requiring oral therapy (n = 2) included superficial Trichophyton rubrum and mucocutaneous candidiasis. Organ Toxicities This BMT platform has generally not seemed to have an adverse impact on cardiac or pulmonary function (Supplemental Figure S2). P6 developed idiopathic pneumonia syndrome, which responded to steroids and etanercept, with no clinically significant lung function impairment long term. Renal injury was infrequent and transient in most cases. Two patients developed ongoing, clinically significant chronic kidney disease. P1 has renal parenchymal echogenicity and requires dual-agent antihypertensive therapy. P13 developed focal segmental glomerulosclerosis (FSGS), collapsing variant, of unclear etiology and is currently maintained on immunosuppression. Interestingly, her otherwise healthy donor has ongoing proteinuria and is of a high-risk race and sex for FSGS,

ARTICLE IN PRESS D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

5

Figure 1. Kaplan-Meier curves of overall survival and acute grade III to IV GVHD-free, graft-failure-free survival for the cohort, n = 20.

Table 3 Overview of Outcomes Related to BMT Platform (n = 20) Characteristic

n (%)

Graft failure

2 (10)

Primary graft failure

1 (5)

Secondary graft failure

1 (5)

1-year transplant-related mortality

2 (10)

Acute GVHD Any grade

4 (20)

Grades II-IV

3 (15)

Grades III-IV

1 (5)

Chronic GVHD

0

Relapse of pre-BMT lymphoproliferative disorder/lymphoma, n = 6

0

Sinusoidal obstructive syndrome*

1 (5)

Idiopathic pneumonia syndrome

1 (5)

Post-BMT donor CD3+ T-lymphocyte infusion

4 (20)

For mixed chimerism or slow engraftment

3 (15)

For CMV disease

1 (5)

Second transplant

1 (5)

Freedom from transfusion, n = 18

18 (100)

Freedom from post-BMT immunosuppression, n = 18

17 (94)

Freedom from Ig replacement (n = 13 survivors on pre-BMT Ig replacement)

10 (77)

Evidence of phenotype reversal among those with unknown genetic defect, n = 3

3 (100)

Evidence of phenotype reversal among patients with significantly mixed chimerism, n = 2

2 (100)

Ig indicates immunoglobulin. * P10 developed sinusoidal obstructive syndrome without reversal of flow on ultrasound or fulfillment of Baltimore criteria but histologically proven on day +22 liver biopsy performed for hyperbilirubinemia that additionally showed nodular regenerative hyperplasia (presumed to have been present pre-BMT due to prior 6-mercaptopurine treatment), cholestasis of sepsis, and mild hemosiderosis.

suggesting a previously unappreciated alloimmune/donorrelated factor may exist. One patient (P10) developed histologic evidence of sinusoidal obstructive syndrome (SOS) in the setting of other significant liver pathology. Endocrine function was evaluated pre- and post-BMT. Three patients developed adrenal insufficiency in the setting of corticosteroid use and posaconazole prophylaxis, now resolved in all but P1. Only 1 child exhibited suboptimal linear growth post-BMT, also associated with systemic corticosteroids. Post-BMT bone mineral density loss was noted in 6 patients, and 2 received bisphosphonate therapy. P18 had a previous diagnosis of hypergonadotropic hypogonadism for which he received testosterone replacement pre-BMT. PostBMT, testosterone remained in the low-normal range, without evidence of bone loss or need for therapy. At 3 years postBMT, P2 now has regained ovarian function and has return of menstruation. Transient ovarian insufficiency recovered by +1.5 years in P15, aged 11.7 years at time of BMT. Two other adolescent females (P5, P13) have not yet regained ovarian function as of last follow-up. Immune Reconstitution and Phenotype Reversal All survivors are off immunosuppression for GVHD prophylaxis or treatment by 6 months post-BMT, although P13 receives immunosuppression for FSGS. Lymphocyte subset reconstitution is shown in Figure 3, with additional details in Supplemental Figures S3 and S4. NK cells recovered first, with median NK cell numbers within normal range 6 to 7 weeks post-BMT across age groups. Median total T cell numbers reached normal age-specific range by day +180 in adults, 1 year in adolescents, and 2 years in younger children, the latter perhaps mediated by CMV-seronegative status of all recipients <12 years old and thus lack of CMV-driven CD8+ T cell expansion, as has been shown to be a driving factor in CD8+ T cell recovery previously [20,21]. The potential to recover recent thymic emigrants decreased with recipient age, as expected. Total B cell numbers normalized by day +180 in adolescents

6

Table 4 Details of Individual Patient and Donor Demographics, Graft Doses, and Outcomes Patient

PID

Recipient Age (yr) at BMT, Sex

Donor Source, Age (yr), Sex

HCT-CI

Bone Marrow Graft Dose TNC(£ 108 Cells/kg IBW)

CD3+(£ 107 Cells/kg IBW)

CD34+ (£ 106 Cells/kg IBW)

Graft Failure

Subsequent Cell Infusion

Acute GVHD

Chronic GVHD

Most Recent CD3+ Chimerism (% Donor)

Most Recent Myeloid Chimerism (% Donor)

IgG Replacement Pre-BMT/ PostBMT

Follow-up

7, F

Haplo, 33, F

1

5.2

5.1

8.7

No

No

No

No

98

100

+/+

A&W, off IS, +3.1 yr

P2

PIK3CD GOF

19, F

MUD, 20, F

4

3.5

4.5

3.3

No

No

No

No

85

53

+/


A&W, off IS, +3.0 yr

P3

Unknown

52, M

MSD, 56, M

4

4.1

1.8

3.1

No

CMV-specific CTLs

No

No

100

100

+/


A&W, off IS, +3.0 yr

P4

Complete IFNGR1 deficiency

6, M

MUD, 28, M

5

9.3

10.0

12.9

No

No

No

No

100

69

+/


A&W, off IS, +2.8 yr

P5

STAT3 deficiency

15, F

MSD, 20, F

1

5.5

6.2

13.3

No

No

No

No

100

100

+/


A&W, off IS, +2.7 yr

P6

Partial IFNGR1 deficiency

58, F

MSD, 54, F

3

4.5

4.0

7.7

No

No

Grade III, gut, ste- No roid responsive

100

100


/


A&W, off IS, +2.5 yr

P7

Unknown

29, M

MUD, 26, M

3

3.2

2.5

2.9

No

No

Grade I, skin, resolved without treatment

No

100

100


/


Accidental death, no EOD and off IS, day +613

P8

MAGT1 deficiency

19, M

Haplo, 17, M

6

4.3

5.4

4.2

No

DLI for slow engraftment

No

NE

NE

97

+/


Infectious TRM, day +44

P9

MAGT1 deficiency

29, M

MUD, 29, M

11

4.8

6.3

10.1

No

No

No

No

100

100

+/


A&W, off IS, +2.3 yr

P10

IL10R1 deficiency

28, M

Haplo, 52, F*

8

3.3

5.3

4.7

No

Granulocytes during aplasia

Grade II, gut, steroid-responsive

NE

100

100

+/


Infectious TRM, day +110

P11

MAGT1 deficiency

17, M

MUD, 22, M

1

3.4

3.6

4.1

No

No

No

No

100

100

+/


A&W, off IS, +2.0 yr

P12

NFKB1 haploinsufficiency

8, F

Haplo, 36, M

1

6.1

5.1

7.2

Secondary

DLI £ 2 for fall- No ing chimerism

No

0

3

+/+

Alive with improved disease control, off IS, +1.8 yr

P13

PIK3CD GOF

16, F

Haplo, 17, M

4

2.4

1.9

5.5

No

DLI for CMV disease

No

No

100

100

+/sporadic

Alive with complications, on IS for FSGS, +1.7 yr

P14

PIK3CD GOF

4, M

Haplo, 27, M

0

7.2

7.3

9.2

Primary

Second BMT, followed by DLI for falling chimerism

After second BMT No & DLI: grade III, skin/gut/liver, steroid refractory

100

100

+/+

A&W, off IS, +1.5 yr

P15

Unknown

11, F

MUD, 31, F

0

9.3

9.5

10.7

No

DLI £ 2 for sta- No ble, mixed chimerism

No

21

65

+/


A&W, off IS, +1.5 yr

P16

STAT3 deficiency

12, M

MSD, 15, M

2

5.0

5.0

8.5

No

No

No

No

87

100


/


A&W, off IS, +1.5 yr

P17

RAG2 deficiency, hypomorphic

37, M

MUD, 22, M

1

2.8

1.9

2.5

No

No

Grade II, gut, steroid responsive

No

96

91

+/


A&W, off IS, +1.5 yr

P18

Wiskott-Aldrich syndrome

54, M

MUD, 18, M

0

3.1

3.0

3.9

No

No

No

No

100

100

+/


A&W, off IS, +1.3 yr

(continued)

ARTICLE IN PRESS

STAT3 deficiency

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

P1

ARTICLE IN PRESS

IL2RG, revertant P20

Haplo indicates HLA-haploidentical; F, female; A&W, alive and well; IS, immunosuppression; GOF, gain of function; MUD, 10/10 HLA-matched-unrelated donor; M, male; MSD, HLA-matched-sibling donor; CTL, cytotoxic T lymphocyte; EOD, evidence of disease; DLI, donor lymphocyte infusion; NE, not evaluable; FSGS, focal segmental glomerulosclerosis. * Donor was a heterozygous carrier. For P20, who had X-linked underlying defect, skewed X-inactivation was confirmed in the donor.

A&W, off IS, +1.2 yr
/
6

RAG1 deficiency, hypomorphic P19

26, M

MSD, 24, F*

4.6

2.6

4.2

No

No

No

No

100

100

A&W, off IS, +1.3 yr +/
100 100 No No No 14.2 6.5 2 Haplo, 33, M* 4, M

7.2

CD34+ (£ 106 Cells/kg IBW) CD3+(£ 107 Cells/kg IBW) TNC(£ 108 Cells/kg IBW)

HCT-CI Donor Source, Age (yr), Sex Recipient Age (yr) at BMT, Sex PID Patient

Table 4 (Continued)

Bone Marrow Graft Dose

Graft Failure

Subsequent Cell Infusion

No

Acute GVHD

Chronic GVHD

Most Recent CD3+ Chimerism (% Donor)

Most Recent Myeloid Chimerism (% Donor)

IgG Replacement Pre-BMT/ PostBMT

Follow-up

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

7

and adults and by 1 year in younger children, with memory B cells detectable in varying numbers in all evaluable patients by 1 year post-BMT, comparable to the timing of recovery demonstrated in prior reports of PTCy-based approaches [20]. All engrafted survivors, even those with mixed chimerism or unknown genetic defect, showed evidence of phenotype reversal (Figure 2C). With median follow-up of 1.9 years, humoral recovery in some patients is still in progress, but in all patients, the PID manifestations that led to BMT referral have either resolved or improved remarkably, even if full phenotype reversal is still evolving. Postengraftment inflammatory phenomena were observed in several patients, in response to disseminated mycobacteria (n = 3) [22], Demodex (n = 2), and Aspergillus (n = 1), one of whom required systemic steroids to dampen the inflammatory immune response and one of whom had dramatic improvement upon receiving steroids for aGVHD. Except asplenia prophylaxis, all patients with chronic or recurrent bacterial infections pre-BMT requiring continuous antibiotic use (n = 7) have been able to discontinue or, in the case of patients with a high burden of disseminated mycobacterial disease, gradually reduce antimicrobials, without resurgence of infection. All patients with poor EBV control pre-BMT demonstrated normalization of EBV in the blood (n = 12 of 12) and remission from EBV-lymphoproliferative disorder (LPD) or EBV-associated lymphoma (n =6 of 6), with over 1 year of follow-up. No patients experienced relapse after entering BMT with active malignancy (n = 2) or in first remission (n = 3). All patients with lymphoma who entered BMT in first complete remission received only 2 to 3 cycles of chemotherapy as a bridge to BMT. P2, who had a long history of human papillomavirus (HPV)associated cervical dysplasia, has a normal Pap smear with no detectable HPV 3 years post-BMT, while P20, who had extensive full-body condylomatous disease and recurrent HPV-associated nasal squamous cell carcinoma, has only a few remaining condylomata with no malignancy recurrence. Of 13 engrafted survivors who required immunoglobulin replacement pre-BMT, 2 (P1, P14) continue it at 3 years and 15 months post-BMT, respectively, with plans to stop after completion of vaccine series. Another (P13) has maintained normal IgG, IgA, and IgM in the setting of sporadic noncompliance, in contrast to a pattern of low IgG and IgA with high IgM pre-BMT. Hypogammaglobulinemia persists in P4 (suspected to be due to protein-losing enteropathy), P9, and P18, but all demonstrate adequate vaccine responses. All patients with known poor specific antibody titers before BMT have demonstrated adequate response to vaccines received to date or, if too early in vaccine series to evaluate, have normal immunoglobulins, along with class-switched memory B cells. DISCUSSION This prospective study demonstrates promising outcomes with a novel RIC-BMT platform, enabling the use of alternative donors and unmanipulated, T-cell-replete grafts across a range of diseases in high-risk PID patients, with partial to complete phenotype reversal in all surviving patients, overall low toxicity, indication that thymic function and gonadal function may be preserved, and markedly low rates of GVHD. The platform and patient population differ from previously published, largely retrospective reports of other promising RIC-BMT approaches for PID [23-28] in that our conditioning was radiation free, there was no in vivo T cell depletion with serotherapy or ex vivo graft manipulation, and both pediatric and adult

ARTICLE IN PRESS 8

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

Figure 2. Chimerism and phenotype reversal. (A) Chimerism in myeloid and CD3+ T cell subsets, expressed as the percentage of donor cells. Black lines show the median and interquartile range of myeloid (solid line) and CD3+ (dashed line) chimerism for the RIC arm cohort (n = 18), excluding the patients with graft failure. Blue lines indicate the percentage of myeloid and CD3+ donor chimerism for P12 with secondary graft failure. P14 with primary graft failure is represented as a single, separate data plot (black star) for myeloid and CD3+ chimerism of 0% on day +28. Most recent chimerism data from patients with follow-up beyond 1 year are listed in Table 4. (B) Chimerism in CD4+ (solid line) and CD8+ (dashed line) T cell subsets, shown as subsets shown in panel A for the cohort (n = 18, in black), P12 (in blue), and P14 (black star). (C) Phenotype reversal in engrafted survivors. For each patient, the left column shows pre-BMT phenotype and the right column post-BMT clinical status. Shading indicates the severity of disease manifestations: black, severe; dark gray, moderate; light gray, mild; white, absent. An X indicates that this disease manifestation could not be assessed, such as due to concomitant immunomodulatory immunoglobulin therapy. The dark gray/“moderate” category also includes historical disease manifestations that were inactive at the time of BMT but represent a significant part of the disease phenotype and indication for transplant, for example, splenectomy for immune cytopenias (P2, P13), chemotherapy/radiation for lymphoma/lymphoproliferative disorder (P9), and Guillain-Barre syndrome (P11). Viral mucocutaneous infections included HPV, herpes simplex virus, varicella zoster virus, and molluscum. P13 had significant post-BMT complications, including primary CMV infection resulting in pneumonitis. She also developed focal segmental glomerulosclerosis requiring immunosuppressive therapy. She has 100% donor chimerism across lineages and an underlying PID where full-donor chimerism should result in full phenotype reversal. Therefore, the post-BMT grading for phenotype reversal in P13 reflects events likely not related to failure to reverse her disease phenotype but rather complications in the setting of poor/slow immune reconstitution post-BMT.

patients were included. The rates of graft failure, GVHD, and TRM seen here are lower than other reports of RIC-BMT outcomes for PID where 1 or more of these complications were more frequent [29-31]. The relatively uniform achievement of high myeloid chimerism early post-BMT is notable, and we postulate this to be related to a potent and early graft-versusmarrow effect, as the preparative regimen was not myeloablative. The use of DLI was not infrequent in this study, as similarly reported with other RIC-BMT approaches and best captured in a multicenter, phase II trial of RIC-BMT for PID by Allen and colleagues [27] in which 50% of patients had graft failure or required DLI for falling/low donor chimerism and the 1-year probability of being alive with sustained engraftment (>5% donor cells in whole blood) without intervention was 39%, with 61% alive with sustained engraftment with or without intervention. Using the same engraftment definitions, our platform compares favorably, with 1-year probability of survival with sustained engraftment without intervention of 75% and with or without intervention of 85%; most patients became full-donor chimeras without intervention, often following the cessation of GVHD prophylaxis with sirolimus. The ability of unconditioned DLI to improve mixed chimerism after

BMT may ultimately be low, but data presented here and by others suggest that mixed chimerism may not require intervention with DLI, given that it can be stable over time and sufficient for phenotype reversal for some PIDs [23,24,27,32]. Although PTCy is known to decrease chronic GVHD incidence effectively, including in PID patients [23,25,33-35], the complete absence of chronic GVHD in this study, confirmed on serial, comprehensive evaluations, is one of the most clinically important and promising findings. The low incidence of severe acute and chronic GVHD may be partly related to the gradual increase in donor T cells noted in most patients, allowing for early mixed chimerism, which may be tolerogenic. Even so, our GVHD outcomes stand in contrast to non-PTCy-based approaches to RIC-BMT for PID, even ones that result in potentially protective mixed chimerism [27,28,36]. Notably, the low incidence of GVHD did not compromise control of malignancy and LPD, with all patients with pre-BMT malignancy/LPD remaining in remission, or of pre-BMT immune cytopenias, which also resolved. The immune reconstitution demonstrated herein is in line with previously published immune reconstitution data in the PTCy setting [20]. Furthermore, this report provides the first in-depth evaluation of immune reconstitution in

Table 5 Post-BMT Donor Cell Infusions Patient

DCI Dose

Reason for DCI

P3

Donor-derived trivirus-specific CTLs

Unknown

CMV infection

P8

Unconditioned DLI

5 £ 105 CD3+/kg

P10

Third-party granulocytes

P12

Timing of DCI

Chimerism Pre-DCI (% Donor)

Myeloid

Chimerism Post-DCI (% Donor) T Cell

+69

100%

100%

100%

100%

No

Control of CMV infection

Slow engraftment

+35

Whole blood: 50%

97%

NE

No

Engraftment of neutrophils on day +40, but TRM day +44

N/A

Planned, during aplasia given active bacterial & fungal infections

+6, +8, +11, +14, +17, +21

NE

NE

91%

98%

Yes, but not related

Unclear benefit; developed cholestasis of sepsis, required abscess drainage, respiratory compromise

Unconditioned DLI Unconditioned DLI

1 £ 106 CD3+/kg 5 £ 106 CD3+/kg

Falling T-cell and myeloid chimerism

+68 +159

41% 5%

0% 0%

28% 3%

0% 0%

No No

Brief fevers, abdominal pain—similar but milder to symptoms during initial engraftment Very brief fevers, graft loss

P13

Unconditioned DLI

1 £ 105 CD3+/kg

CMV disease— attempt to augment CMV-specific response

+235

99%

98%

100%

100%

No

CD8+ T-lymphocytosis with elevated levels of CMV-specific T cells in the blood. Recovery from CMV pneumonitis.

P14

Second BMT* Unconditioned DLI*

Haplo PBSC graft (same donor) TNC 7.2 £ 108/kg CD3+ 7.3 £ 107/ kg CD34+ 9.2 £ 106/ kg 5 £ 106 CD3+/kg

Primary graft failure Falling T cell and myeloid chimerism

+95 +79 after second BMT

0% 100%

0% 40%

100% 100%

93% 100%

No Yes

Engrafted, but with mixed T cell chimerism and decreasing blood counts over time Full-donor chimerism, steroid-refractory acute GVHD, grade III, now resolved and off all immunosuppression

P15

Unconditioned DLI Unconditioned DLI

2 £ 106 CD3+/kg 1 £ 107 CD3+/kg

Mixed myeloid and lymphoid chimerism

+227 +304

68% 66%

28% 23%

69% 63%

25% 22%

No No

Stable chimerism, remained clinically well Stable chimerism, remained clinically well

DCI indicates donor cell infusion; DLI, donor lymphocyte infusion; PBSC, peripheral blood stem cell. * P14 underwent nonemergent second BMT per a published regimen [59] after primary graft failure with autologous recovery. He then had threatened secondary graft failure with declining chimerism and blood counts after the second BMT and received an unconditioned DLI post-second BMT with subsequent aGVHD but also achievement of 100% donor chimerism.

ARTICLE IN PRESS

T Cell

Acute GVHD after DCI

Outcome

Myeloid

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

Donor Cell Infusion Type

9

ARTICLE IN PRESS 10

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

Figure 3. Post-BMT lymphocyte subset reconstitution, with box-and-whisker plot denoting median and range. Upper and lower limits of institution-specific adult normal range are demarcated for each subset. Patients who experienced graft failure are excluded from this analysis. Of evaluable patients, 47% attained >300 total T cells/mL and 65% attained >50 CD8+ T cells/mL by day +100, whereas 6% and 44%, respectively, attained >500 CD4+ T cells/mL by 6 months and 1 year post-BMT; these markers shown to predict survival and graft failure outcomes in young infants and children with severe combined immunodeficiency may not be applicable in an older more heterogeneous population.

patients with PID who underwent transplantation using serotherapy-free, PTCy-based approaches and is notably unhindered in this assessment given the low rates of GVHD and freedom from immunosuppression by day +180 after BMT. However, the mixed T cell chimerism seen here early after BMT may not be tolerated in all patients, depending on the underlying disease-causing mutation and if host immune cells have a selective advantage over donor cells. As published by others and demonstrated here, the engraftment of patients with gain of function (GOF) mutations, such as in Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD), is variable, and we have deemed the BMT approach herein to require additional modifications to decrease graft failure rates for these patients [37,38]. Sirolimus, a mammalian target of rapamycin (mTOR) inhibitor that can rescue the host T cell defects in patients with PIK3CD GOF mutations [39], may hinder engraftment of donor T cells when used as GVHD prophylaxis in patients with PIDs, in whom the causative mutation drives hyperactivation of the AKT-mTOR pathway. As a result, we have stopped using mTOR inhibitors as part of GVHD prophylaxis in patients with PIK3CD GOF mutations. As we continue to focus on RIC approaches to transplant but also recognize that this RIC approach may not be optimal for all patients with PID, we have subsequently tailored this approach for patients with PIK3CD GOF mutations or other diseases of T cell dysregulation and/or proliferation by using peripheral blood stem cell grafts and adding distal serotherapy for augmented host lymphodepletion. The incidence of graft failure was respectably low for a RICBMT approach in nonmalignant diseases, and patients with graft failure had immediate autologous reconstitution. Both

patients with graft failure did receive HLA-haploidentical grafts, which may represent part of the barrier to engraftment. However, the engraftment kinetics and chimerism for the 5 other recipients of HLA-haploidentical grafts in this study were on par with that of the recipients of HLA-matched grafts. Thus, our assessment is that the graft failure events relate much more to underlying disease features, disease activity in the periengraftment period, and, in P14, underdosing of his busulfan due to the inability to perform pharmacokinetics and use sirolimus in a PIK3CD GOF patient, although more transplants will need to be performed across donor types using this platform to gain more clarity on the factors contributing to graft failure. At present, there are no published PTCy-based haplo transplant approaches for patients with PID that similarly avoid serotherapy, radiation, and/or myeloablation for direct comparison to this platform. Approaches that do include 1 or more of these factors have resulted in high engraftment rates with haplo donors in patients with PID [23,35,40,41]. Regardless, further improving engraftment without sacrificing the favorable aspects of this platform is desirable. Across all hard-to-engraft diseases, augmented host lymphodepletion, such as with intermediate-timed serotherapy [42], may improve engraftment without escalating conditioning to myeloablative levels. In this study, marrow grafts were used, although peripheral blood stem cell grafts could be considered another measure to decrease the risk of graft failure, albeit potentially with a higher risk of GVHD. Myeloablative conditioning (MAC) is another approach to decrease graft failure rates in PIDs but needs to be considered in view of the guaranteed and potential short- and long-term toxicities. Most patients in this study entered BMT with significant

ARTICLE IN PRESS D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

comorbidities, particularly lung and/or liver dysfunction. Half had HCT-CI scores of
3; similar rates of high-risk patients requiring BMT for PID have been published by others [36]. In a registry analysis of HCT-CI in nonmalignant diseases, HCT-CI scores
3 were associated with a significant decrease in survival [43] and, because severe combined immunodeficiency represented nearly half of PID patients in that study, the extent of comorbidities seen in older patients with PIDs requiring BMT was almost certainly underrepresented. Although reduced-toxicity, treosulfan-containing MAC regimens for PID have yielded very good outcomes, treosulfan is not available worldwide, rates of long-term toxicities such as infertility are not well defined, and SOS risk is not eliminated [44-50]. Additionally, underlying liver pathology may not always be identified pre-BMT, potentially making this patient population more vulnerable to SOS. Thus, MAC-based approaches may not be clinically feasible or desired for many patients with PID referred for BMT. Serious infectious complications were overall modest for this high-risk cohort, suggesting favorable immune reconstitution and likely due in large part to the freedom from immunosuppression afforded by low GVHD rates. Significant bacterial and fungal complications were largely prevented with standard approaches to prophylaxis and empiric therapy. Although detection of respiratory viruses was common, lower respiratory tract infections were rare. No patients had clinically significant EBV-associated complications, consistent with outcomes previously published with PTCy-based approaches [51]. CMV reactivation was controlled with pre-emptive therapy alone in all but 1 patient, which could in part be associated with the use of sirolimus [16,52]. Notably, post-BMT primary CMV infection was not uncommon; all cases occurred after weekly CMV monitoring had stopped, and 1 case progressed to CMV disease. BK-associated hemorrhagic cystitis did not lead to any long-term sequelae, but it posed significant transient morbidity for patients. The high rates of BK-associated hemorrhagic cystitis are thought to be related to the combination of (1) a high-risk patient population with frequent BK virus detection pre-BMT conferring higher risk of symptomatic cystitis postBMT, (2) the use of PTCy, and (3) the use of busulfan [53], with busulfan believed to be a significant cofactor given that we have not observed BK-associated hemorrhagic cystitis in our patients with PID who receive a lower-intensity transplant approach that is identical except that it does not contain busulfan. Of note, BK-associated hemorrhagic cystitis incidence did not vary with donor type, as has been shown elsewhere [54]. The outcomes presented here, coupled with recently reported impressive BMT outcomes by others [23,36,55], support the safety and efficacy of RIC-BMT for patients across a broad range of PIDs, even those with significant comorbidities, older age, active infection/malignancy, only alternative donor options, and immune-dysregulation phenotypes. The spectrum of PIDs continues to expand, and the present era is one far different from when BMT for PID was used pre-emptively in very young children with diseases uniformly associated with early mortality. The cohort of patients represented here, with no patient younger than 4 years, is distinctly different from patients with severe combined immunodeficiency who require immediate transplant in infancy, and thus the approach, outcomes, and discussion should not be generalized to that patient population. RIC-BMT approaches for PID will likely remain at the forefront of the field moving forward, as patients will be referred for BMT when their disease-related comorbidities prohibit MAC, or earlier in their disease course when the risk of MAC-

11

associated morbidity and mortality is difficult to justify. Thus, continued investigation into how to modify promising RIC-BMT approaches further to optimize outcomes, tailoring them as we gain greater understanding of the challenges related to graft failure, organ toxicity, and immune reconstitution that may lie within specific PIDs or disease phenotypes, is essential to further the application of this potentially curative therapy to all who require it [56,57]. Investigation into the performance of this novel platform in other nonmalignant diseases is also of interest. The worldwide availability of the platform presented here offers an alternative to approaches that use treosulfan, alemtuzumab, or graft manipulation techniques such as a/b T cell depletion, which are not globally available, are costlier, and/or require specialized technology and expertise [58]. Longterm follow-up on the relationship between mixed chimerism and both phenotype reversal and late graft failure, as well as the potential for RIC to afford patients preserved fertility and fewer late toxicities, will be critical to gauge the true, durable success of this approach. Ongoing efforts at NIH include modifications to this platform to help engraftment for PID patients with more difficult-to-engraft diseases and shortening of GVHD prophylaxis to improve immune reconstitution and viral control.

ACKNOWLEDGMENTS The authors thank the patients, their families, and the donors who participated in this trial. They thank the providers of the Experimental Transplantation and Immunology Branch and consultants throughout the NIH Clinical Center who cared for the patients, Sharon Adams and staff of the HLA lab, the Department of Transfusion Medicine and Cell Processing Section, Dr. Alina Dulau-Florea and the staff of the Chimerism Lab, Frances T. Hakim and the staff of the Preclinical Development and Clinical Monitoring Facility and the Experimental Transplantation and Immunology Branch Flow Cytometry Core, and the referring doctors who assisted in ongoing post-transplant care of patients upon return home. The authors thankfully acknowledge the generosity of the Mackenzie family in their donation to support the research of transplant for children with primary immunodeficiency diseases. Financial disclosure: This project has been funded in whole or in part with federal funds from the National Cancer Institute, NIH, under contract number HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US government. Conflict of interest statement: There are no conflicts of interest to report. Authorship statement: J.A.K. designed the study with contributions from S.M.S., D.H.F., R.E.G., and C.G.K. S.M.S. guided the statistical design of the study. All authors participated to varying degrees in the multifaceted aspects of performing the study. Data analysis was primarily performed by D.D., S.M.S., and J.A.K. (clinical outcomes); C.G.K, J.J.R., X.Y., R.F., and N.S.N. (flow cytometric immune reconstitution); J.E.B. and M.M.B. (endocrine data); and A.P.H. (mutation sequencing, X-inactivation studies of carriers), with raw data available to all authors. D.D. and J.A.K. wrote the manuscript with review, edits, and final manuscript approval from all authors.

SUPPLEMENTARY MATERIALS Supplementary material associated with this article can be found in the online version at doi:10.1016/j.bbmt.2019.08.018.

ARTICLE IN PRESS 12

D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

REFERENCES 1. Picard C, Bobby Gaspar H, Al-Herz W, et al. International Union of Immunological Societies: 2017 Primary Immunodeficiency Diseases Committee Report on Inborn Errors of Immunity. J Clin Immunol. 2018;38:96–128. 2. Rosenberg E, Dent PB, Denburg JA. Primary immune deficiencies in the adult: a previously underrecognized common condition. J Allergy Clin Immunol Pract. 2016;4:1101–1107. 3. Kobrynski L, Powell RW, Bowen S. Prevalence and morbidity of primary immunodeficiency diseases, United States 2001-2007. J Clin Immunol. 2014;34:954–961. 4. Connelly JA. Hematopoietic stem cell transplant for a new primary immunodeficiency disorder: a voyage where no transplant physician has gone before. Biol Blood Marrow Transplant. 2017;23:863–864. 5. Acevedo MJ, Wilder JS, Adams S, et al. Outcomes of related and unrelated donor searches among patients with primary immunodeficiency diseases referred for allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant. 2019;25:1666–1673. 6. Mossoba ME, Halverson DC, Kurlander R, et al. High-dose sirolimus and immune-selective pentostatin plus cyclophosphamide conditioning yields stable mixed chimerism and insufficient graft-versus-tumor responses. Clin Cancer Res. 2015;21:4312–4320. 7. Mariotti J, Taylor J, Massey PR, et al. The pentostatin plus cyclophosphamide nonmyeloablative regimen induces durable host T cell functional deficits and prevents murine marrow allograft rejection. Biol Blood Marrow Transplant. 2011;17:620–631. 8. McCurdy SR, Kasamon YL, Kanakry CG, et al. Comparable composite endpoints after HLA-matched and HLA-haploidentical transplantation with post-transplantation cyclophosphamide. Haematologica. 2017;102:391–400. 9. How J, Slade M, Vu K, et al. T cell-replete peripheral blood haploidentical hematopoietic cell transplantation with post-transplantation cyclophosphamide results in outcomes similar to transplantation from traditionally matched donors in active disease acute myeloid leukemia. Biol Blood Marrow Transplant. 2017;23:648–653. 10. Ghosh N, Karmali R, Rocha V, et al. Reduced-intensity transplantation for lymphomas using haploidentical related donors versus HLA-matched sibling donors: a center for international blood and marrow transplant research analysis. J Clin Oncol. 2016;34:3141–3149. 11. Moiseev IS, Pirogova OV, Alyanski AL, et al. Risk-adapted GVHD prophylaxis with post-transplantation cyclophosphamide in adults after related, unrelated, and haploidentical transplantations. Eur J Haematol. 2018;100:395–402. 12. Raiola AM, Dominietto A, di Grazia C, et al. Unmanipulated haploidentical transplants compared with other alternative donors and matched sibling grafts. Biol Blood Marrow Transplant. 2014;20:1573–1579. 13. DeZern AE, Zahurak M, Symons H, Cooke K, Jones RJ, Brodsky RA. Alternative donor transplantation with high-dose post-transplantation cyclophosphamide for refractory severe aplastic anemia. Biol Blood Marrow Transplant. 2017;23:498–504. 14. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus conference on acute GVHD grading. Bone Marrow Transplant. 1995;15:825–828. 15. Jagasia MH, Greinix HT, Arora M, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graftversus-host disease: I. The 2014 Diagnosis and Staging Working Group report. Biol Blood Marrow Transplant. 2015;21:389–401. e1. 16. Melendez-Munoz R, Marchalik R, Jerussi T, et al. Cytomegalovirus infection incidence and risk factors across diverse hematopoietic cell transplantation platforms using a standardized monitoring and treatment approach: a comprehensive evaluation from a single institution. Biol Blood Marrow Transplant. 2019;25:577–586. 17. Sorror M, Storer B, Sandmaier BM, et al. Hematopoietic cell transplantationcomorbidity index and Karnofsky performance status are independent predictors of morbidity and mortality after allogeneic nonmyeloablative hematopoietic cell transplantation. Cancer. 2008;112:1992–2001. 18. Dimitrova D, Rose JJ, Uzel G, et al. Successful bone marrow transplantation for XMEN: hemorrhagic risk uncovered. J Clin Immunol. 2019;39:1–3. 19. Johnson RC, Deming C, Conlan S, et al. Investigation of a cluster of Sphingomonas koreensis infections. N Engl J Med. 2018;379:2529–2539. 20. Kanakry CG, Coffey DG, Towlerton AM, et al. Origin and evolution of the T cell repertoire after posttransplantation cyclophosphamide. JCI Insight. 2016;1:e86252. 21. Ogonek J, Varanasi P, Luther S, et al. Possible impact of cytomegalovirusspecific CD8(+) T cells on immune reconstitution and conversion to complete donor chimerism after allogeneic stem cell transplantation. Biol Blood Marrow Transplant. 2017;23:1046–1053. 22. Manion M, Dimitrova D, Pei L, et al. IRIS as a post-transplantation complication in primary immunodeficiency with disseminated M. avium. Clin Infect Dis. 2019. https://doi.org/10.1093/cid/ciz507. [e-pub ahead of print]. 23. Klein OR, Chen AR, Gamper C, et al. Alternative-donor hematopoietic stem cell transplantation with post-transplantation cyclophosphamide for nonmalignant disorders. Biol Blood Marrow Transplant. 2016;22:895–901. 24. Marsh RA, Rao MB, Gefen A, et al. Experience with alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning hematopoietic cell transplantation in patients with nonmalignant diseases reveals good outcomes and that the risk of mixed chimerism depends on underlying

25.

26. 27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

disease, stem cell source, and alemtuzumab regimen. Biol Blood Marrow Transplant. 2015;21:1460–1470. Thakkar D, Katewa S, Rastogi N, Kohli S, Nivargi S, Yadav SP. Successful reduced intensity conditioning alternate donor stem cell transplant for Wiskott-Aldrich syndrome. J Pediatr Hematol Oncol. 2017;39:e493–e496. Ono S, Okano T, Hoshino A, et al. Hematopoietic stem cell transplantation for XIAP deficiency in Japan. J Clin Immunol. 2017;37:85–91. Allen CE, Marsh R, Dawson P, et al. Reduced-intensity conditioning for hematopoietic cell transplant for HLH and primary immune deficiencies. Blood. 2018;132:1438–1451. Marsh RA, Vaughn G, Kim MO, et al. Reduced-intensity conditioning significantly improves survival of patients with hemophagocytic lymphohistiocytosis undergoing allogeneic hematopoietic cell transplantation. Blood. 2010;116:5824–5831. Wehr C, Gennery AR, Lindemans C, et al. Multicenter experience in hematopoietic stem cell transplantation for serious complications of common variable immunodeficiency. J Allergy Clin Immunol. 2015;135:988–997. e6. Grossman J, Cuellar-Rodriguez J, Gea-Banacloche J, et al. Nonmyeloablative allogeneic hematopoietic stem cell transplantation for GATA2 deficiency. Biol Blood Marrow Transplant. 2014;20:1940–1948. Horwitz ME, Barrett AJ, Brown MR, et al. Treatment of chronic granulomatous disease with nonmyeloablative conditioning and a T-cell-depleted hematopoietic allograft. N Engl J Med. 2001;344:881–888. Cooper N, Rao K, Goulden N, Webb D, Amrolia P, Veys P. The use of reduced-intensity stem cell transplantation in haemophagocytic lymphohistiocytosis and Langerhans cell histiocytosis. Bone Marrow Transpl. 2008;42:S47–S50. Shah NN, Freeman AF, Su H, et al. Haploidentical related donor hematopoietic stem cell transplantation for dedicator-of-cytokinesis 8 deficiency using post-transplantation cyclophosphamide. Biol Blood Marrow Transplant. 2017;23:980–990. Rastogi N, Katewa S, Thakkar D, Kohli D, Nivargi S, Yadav SP. Reduced-toxicity alternate-donor stem cell transplantation with posttransplant cyclophosphamide for primary immunodeficiency disorders. Pediatr Blood Cancer. 2018;65. https://doi.org/10.1002/pbc.26783. [e-pub ahead of print]. Parta M, Shah NN, Baird K, et al. Allogeneic hematopoietic stem cell transplantation for GATA2 deficiency using a busulfan-based regimen. Biol Blood Marrow Transplant. 2018;24:1250–1259. Fox TA, Chakraverty R, Burns S, et al. Successful outcome following allogeneic hematopoietic stem cell transplantation in adults with primary immunodeficiency. Blood. 2018;131:917–931. Nademi Z, Slatter MA, Dvorak CC, et al. Hematopoietic stem cell transplant in patients with activated PI3K delta syndrome. J Allergy Clin Immunol. 2017;139:1046–1049. Okano T, Imai K, Tsujita Y, et al. Hematopoietic stem cell transplantation for progressive combined immunodeficiency and lymphoproliferation in patients with activated phosphatidylinositol-3-OH kinase delta syndrome type 1. J Allergy Clin Immunol. 2019;143:266–275. Lucas CL, Kuehn HS, Zhao F, et al. Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110delta result in T cell senescence and human immunodeficiency. Nat Immunol. 2014;15:88–97. Neven B, Diana JS, Castelle M, et al. Haploidentical hematopoietic stem cell transplantation with post-transplant cyclophosphamide for primary immunodeficiencies and inherited disorders in children. Biol Blood Marrow Transplant. 2019;25:1363–1373. Kurzay M, Hauck F, Schmid I, et al. T-cell replete haploidentical bone marrow transplantation and post-transplant cyclophosphamide for patients with inborn errors. Biol Blood Marrow Transplant. 2019. https://doi.org/ 10.3324/haematol.2018.215285. [e-pub ahead of print]. Marsh RA, Kim MO, Liu CY, et al. An intermediate alemtuzumab schedule reduces the incidence of mixed chimerism following reduced-intensity conditioning hematopoietic cell transplantation for hemophagocytic lymphohistiocytosis. Biol Blood Marrow Transplant. 2013;19:1625–1631. Thakar M, Broglie L, Logan B, et al. The Hematopoietic Cell Transplant Comorbidity Index predicts survival after allogeneic transplant for nonmalignant diseases. Blood. 2019;133:754–762. Morillo-Gutierrez B, Beier R, Rao K, et al. Treosulfan-based conditioning for allogeneic HSCT in children with chronic granulomatous disease: a multicenter experience. Blood. 2016;128:440–448. Slatter MA, Boztug H, Potschger U, et al. Treosulfan-based conditioning regimens for allogeneic haematopoietic stem cell transplantation in children with non-malignant diseases. Bone Marrow Transplant. 2015;50:1536–1541. Slatter MA, Rao K, Amrolia P, et al. Treosulfan-based conditioning regimens for hematopoietic stem cell transplantation in children with primary immunodeficiency: United Kingdom experience. Blood. 2011;117:4367–4375. Aydin SE, Freeman AF, Al-Herz W, et al. Hematopoietic stem cell transplantation as treatment for patients with DOCK8 deficiency. J Allergy Clin Immunol Pract. 2019;7:848–855. Haskologlu S, Kostel Bal S, Islamoglu C, et al. Outcome of treosulfan-based reduced-toxicity conditioning regimens for HSCT in high-risk patients with primary immune deficiencies. Pediatr Transplant. 2018;22:e13266. Faraci M, Bertaina A, Luksch R, et al. Gonadal function after busulfan compared with treosulfan in children and adolescents undergoing allogeneic

ARTICLE IN PRESS D. Dimitrova et al. / Biol Blood Marrow Transplant 00 (2019) 1
13

50.

51.

52.

53.

54.

hematopoietic stem cell transplant. Biol Blood Marrow Transplant. 2019;25:1786–1791. https://doi.org/10.1016/j.bbmt.2019.05.005. Faraci M, Bertaina A, Luksch R, et al. Sinusoidal obstruction syndrome/ veno-occlusive disease after autologous or allogeneic hematopoietic stem cell transplantation in children: a retrospective study of the Italian Hematology-Oncology Association-Hematopoietic Stem Cell Transplantation Group. Biol Blood Marrow Transplant. 2019;25:313–320. Kanakry JA, Kasamon YL, Bolanos-Meade J, et al. Absence of post-transplantation lymphoproliferative disorder after allogeneic blood or marrow transplantation using post-transplantation cyclophosphamide as graftversus-host disease prophylaxis. Biol Blood Marrow Transplant. 2013;19: 1514–1517. Guglieri-Lopez B, Perez-Pitarch A, Garcia-Cadenas I, et al. Effect of sirolimus exposure on the need for preemptive antiviral therapy for cytomeglovirus infection after allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2019;25:1022–1030. Gilis L, Morisset S, Billaud G, et al. High burden of BK virus-associated hemorrhagic cystitis in patients undergoing allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant. 2014;49:664–670. Copelan OR, Sanikommu SR, Trivedi JS, et al. Higher incidence of hemorrhagic cystitis following haploidentical related donor transplantation

55.

56.

57.

58.

59.

13

compared with matched related donor transplantation. Biol Blood Marrow Transplant. 2019;25:785–790. Gungor T, Teira P, Slatter M, et al. Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicentre study. Lancet. 2014;383:436–448. Duarte RF, Labopin M, Bader P, et al. Indications for haematopoietic stem cell transplantation for haematological diseases, solid tumours and immune disorders: current practice in Europe. Bone Marrow Transplant. 2019. https://doi.org/10.1038/s41409-019-0516-2. [e-pub ahead of print]. Dvorak CC, Long-Boyle J, Dara J, et al. Low exposure busulfan conditioning to achieve sufficient multilineage chimerism in patients with severe combined immunodeficiency. Biol Blood Marrow Transplant. 2019;25: 1355–1362. Weisdorf D, Ruiz-Arguelles GJ, Srivastava A, Gomez-Almaguer D, Szer J. Economic challenges in hematopoietic cell transplantation: how will new and established programs face the growing costs? Biol Blood Marrow Transplant. 2017;23:1815–1816. Klein OR, Chen AR, Gamper C, et al. Alternative-donor hematopoietic stem cell transplantation with post-transplantation cyclophosphamide for nonmalignant disorders. Biol Blood Marrow Transplant. 2016;22:895–901.