Failure to Prevent Severe Graft-Versus-Host Disease in Haploidentical Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide in Chronic Granulomatous Disease

Abstract

Purpose

Haploidentical related donor (HRD) transplantation was performed in 7 recipients with chronic granulomatous disease (CGD) who had no matched-related or unrelated donor.

Methods

Peripheral blood cell (PBC) products were used with a conditioning regimen consisting of low-dose cyclophosphamide, fludarabine, total body irradiation, and busulfan. Graft-versus-host disease (GVHD) prophylaxis consisted of high-dose post-transplant cyclophosphamide and sirolimus. Recipients were ages 14–26 years, and 3 had severe infections active at transplant.

Results

All 7 recipients achieved full engraftment with complete donor chimerism early in the post-transplant period. Acute GVHD occurred in all cases and was grade 3 or steroid refractory in 3. Two patients with steroid-refractory GVHD died. Three patients with severe infectious complications active at transplant, 1 Nocardia pneumonia and 2 extensive invasive fungal infections), survived and were cured of their infection at last follow-up. Bacterial disease occurred post-transplant in all recipients, and viral infections/reactivation were common, including 4 cases of BK virus–associated hemorrhagic cystitis.

Conclusions

Seven patients with CGD achieved rapid and full-donor engraftment from HRDs utilizing PBCs and a conditioning regimen with PTCy and sirolimus GVHD prophylaxis. However, the incidence of grade 3 and steroid-refractory GVHD was high and led to 2 deaths. Patients with active infections at transplant had successful transplant courses and were cured of their disease. Although there was an initial success with this regimen, the cumulative experience does not support its use in CGD due to an unacceptable rate of severe GVHD.

Chronic granulomatous disease (CGD) is a phenotypically complex primary immunodeficiency. Immunologically first characterized by neutrophil dysfunction mediated by genetic changes in the phox reactive oxygen intermediate generating system, CGD is associated with an increased risk of infection but also non-infectious inflammatory disease, often involving the gastrointestinal tract; hematopoietic cell transplantation can be curative [1].

The severe and life-threatening manifestations of CGD can be cured by hematopoietic cell transplant (HCT). Multiple protocols now exist to achieve engraftment and avoid graft-versus-host disease (GVHD). Since there is no association with an antileukemic benefit in this non-malignant disease, avoiding GVHD is a priority. With increasing acceptance of HCT, the requirements for suitable donors are a barrier that can now be overcome by the use of “non-traditional” donors, especially haploidentical donors (HRDs). The predictable risks of rejection and GVHD from HRDs can be attenuated by manipulation of the graft (T cell depletion), or more generally applicable, the use of post-transplant cyclophosphamide (PTCy), as pioneered in hematologic malignancy transplantation at the Johns Hopkins Medical Institutions [2]. Our institution began HRD HCT using PTCy in 2016 in patients with GATA2 and DOCK8 deficiency [3, 4].

We previously reported on our first experience in CGD with HRD HCT and PTCy in a single patient with a severe, refractory fungal infection as the indication for transplantation [5]. We continued to transplant for refractory infection and inflammatory bowel disease, and here report the results of seven patients, with an unacceptable incidence of severe/steroid-refractory gastrointestinal (GI) GVHD that has led us to modify this approach in HCT for CGD.

Methods and Patients

Data Collection

This study was designed to determine the efficacy and safety of HRD HCT using PTCy, and was approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases. This study was independently monitored for safety and data accuracy (Clinical Trials. gov. number, NCT02282904).

The following pretransplant data were collected: age at transplant, bone marrow biopsy, CGD genetic characteristics, and disease phenotype. The transplant data collected consisted of donor source with either peripheral blood cells (PBC) or bone marrow (BM), HLA match, busulfan area under the curve (AUC) based on a test dose of busulfan, total busulfan dose/kg weight of recipient, CD34+ and CD3+ donor cells infused per kg recipient weight, myeloid and CD3+ cell chimerism at day 100, time after transplant in months, GVHD prophylaxis, incidence of acute and chronic GVHD (aGVHD and cGVHD), and current status including the presence or absence of continued immunosuppression.

Methods

A busulfan test dose was performed to inform the conditioning regimen. HCT was performed with peripheral blood stem cells and conditioning with fludarabine days − 6 through − 2, (30 mg/m² × 5), intravenous once daily busulfan on days − 4, − 3, and − 2, with levels on day − 4 to provide a measured AUC to inform changes in the day − 2 dose to achieve total AUC 4000–6000 min × μMol/L, cyclophosphamide 14.5 mg/kg × 2, days − 6 and − 5, and total body irradiation (200 cGy, day − 1) conditioning with post-transplant cyclophosphamide (50 mg/kg days 3 and 4) and sirolimus GVHD prophylaxis.

Hemorrhagic cystitis prevention was in accordance with institutional BMT consortium “Supportive Care Guidelines: Hemorrhagic Cystitis Prevention with High-Dose Cyclophosphamide” at http://intranet.cc.nih.gov/bmt/education/supportive-care.shtml.

Haploidentical donors had HLA compatibility representing a minimum match of 5/10 loci. If more than one haploidentical donor was available, each donor was evaluated for overall health, age, ABO match, cytomegalovirus (CMV) serostatus, etc., to select the best donor.

Patients

The study was designed to enroll 10 patients with no HLA matched-related or unrelated donor and disease of sufficient severity to justify the risk of HCT. Patients ages 2–65 were eligible.

HLA antibody screens were performed, with ABO typing and KIR typing (at the investigator’s discretion), and cytomegalovirus (CMV) and Epstein-Barr virus (EBV) serologies for donor and recipient were performed.

Supportive Care

Standard guidelines for supportive care established at the National Institutes of Health Clinical Center for patients undergoing allogeneic HCT were used. These guidelines agree with international guidelines for preventing infectious complications among HCT recipients.

Results

Patients and Transplant Outcomes

Seven patients, ages 14–26 years, were transplanted between November 2014 and December 2016 (Table 1). All patients had a history of mixed infectious and inflammatory conditions. All patients but one (patient 2, Table 1) had inflammatory bowel disease (IBD) requiring various intensities and durations of immunosuppression. The indications for transplantation were extensive active infection in 3 (one Nocardia, one Aspergillus nidulans, one Scedosporium apiospermum), and refractory inflammatory disease, especially colitis, in 4. One patient had protein-losing enteropathy (PLE), one extensive dermatitis, and one discoid lupus. Patient 7 had undergone an experimental expanded cord blood transplant as a child which failed.

Table 1.

Recipients, indications for transplantation, conditioning, and cell dose

Age/sex	CGD type	Clinical disease	Manifestations of CGD present at transplant/pretransplant CRP (mg/L) and immunosuppression	Busulfan AUC min × microm/L	TNC (109)	Cd 34+ cells/kg (106)	CD3+ cells/kg (107)
14/M	XL (p91-phox)	Infection/protein-losing enteropathy, colitis	Protein-losing enteropathy and Nocardia pneumonia/2.90, prednisone 7.5 mg daily and mesalamine DR 400 mg every 8 h	2461	31.5	7.98	16.89
26/F	P67 (NCF-2)	Infection	A. nidulans pneumonia and osteomyelitis/17.10, none	3610	92.4	9.24	9.31
24/M	P47 (NCF-1)	Infection, colitis, and dermatitis	Colitis and dermatitis/5.00, prednisone 20 mg daily, and mesalamine DR, 1200 mg twice daily	3828	50.3	9.84	21.5
16/M	XL (p91-phox)	Infection, colitis, cystitis, recurrent fever, and headaches	Colitis, bladder disease/12.60, prednisolone 5 mg twice daily	3619	91.0	15.9	20.5
14/M	XL (p91-phox)	Infection and colitis	S. apiospermum pneumonia and pericarditis, Colitis/5.90, prednisone 5 mg daily, and sulfasalazine 500 mg daily	3008	27.6	12	14.9
22/F	XL (p91-phox) carrier	Infection (Nocardia), colitis	Colitis and discoid lupus/5.40, azathioprine 150 mg daily, mesalamine 1.2 g every 12 h, prednisone 15 mg daily, and hydroxychloroquine 200 mg daily	3349	105	10.5	14.1
19/M	XL (p91-phox)	Infection and colitis	Colitis/6.60, mesalamine DR 1200 mg twice daily, and prednisone 7.5 mg twice daily	4250	55.2	9.85	12.32

CGD chronic granulomatous disease, CRP high-sensitivity comprehensive C-reactive protein, AUC area under the curve, TNC total nucleated cell dose, XL X-linked, DR delayed release: A. nidulans, Aspergillus nidulans; S. apiospermum, and Scedosporium apiospermum

All products were peripheral blood cells (PBC). The median busulfan AUC was 3446 min × microm/L (IQR 3008–3828). Cell products contained total nucleated cells median 64.7 × 10⁹ (IQR 31.5–92.4,), CD34+ cells median 10.8 × 10⁶/kg, (IQR 9.24–12), and CD3 15.6 × 10⁷/kg (IQR 12.32–20.5). Patients with active infections at transplant received 2–5 granulocyte transfusions starting at neutropenia through the evidence of neutrophil recovery.

Pretransplant inflammatory disease was characterized by a high-sensitivity C-reactive protein within the week prior to transplantation and immunosuppression related to inflammatory bowel disease (Table 1).

All patients engrafted, with persistent evidence of full chimerism throughout the measured period. Neutrophil engraftment occurred at median day 19 (IQR 15–22) and platelets at median day 34 (IQR 17–31).

Five patients survived with median follow-up of more than 3 years (Table 2). All patients had grade 2 or higher acute GVHD; one patient had Grade 3 aGVHD of the skin. Two of these 5 also developed mild cGVHD.

Table 2.

Transplant donor, engraftment, complications, and outcome

Age/sex	Donor (age), HLA match, and ABO compatible	Engraftment day/granulocyte transfusion days	Myeloid/CD3+ cell chimerism	aGVHD grades/cGVHD severity	Viral reactivation/intervention	Post-transplant infections	Follow-up or cause of death/interval
14/M	Father (33), 8/10, yes	PMN 15, platelets 14/5 and 11	100/≥ 97% from first assessment	Grade 2 acute GI/no	EBV/none Grade 2 HC with BK viruria	C. difficile-associated diarrhea	Resolved Nocardia pneumonia 18 months, persistent PLE, cure/> 3 years
26/F	Mother (45), 10/10, yes	PMN 21, platelets 23/9, 10, 12, 13, and 14	100/≥ 98% from first assessment	Grade 2 acute GI, grade 1 skin/no	CMV/preemption EBV/none	S. aureus sternal wound infection	Aspergillus infection resolved 11 months, Cure/> 3 years
24/M	Brother (18), 8/10, yes	PMN 17, platelets 31	100/100% at all points	Grade 2 acute skin and grade 1 GI/yes, mild-limited	EBV/none Grade 1 HC with BK viruria	C. difficile-associated diarrhea	Cure > 3 years
16/M	Father (43), 5/10, yes	PMN 12, platelets 19	100/100% at all points	Grade 3 skin, GI/ mild	CMV/preemption EBV/none Grade III HC with BK viruria	A. fumigatus pneumonia E. coli bacteremia B. fragilis bacteremia	Cure > 2 years
14/M	Father (34), 6/10, yes	PMN 24, platelets 17/9, 21	100/100% at all points	Grade 2 GI	EBV/none CMV colitis/treatment	S. mitis bacteremia	Resolved fungal pneumonia/pericarditis 12 months, cure > 4 years
22/F	Father (53), 5/10, yes	PMN 20, Platelets 29	96–100%, 98–100%	Steroid-refractory GI	EBV/none Disseminated adenovirus	K. pneumoniae/E.coli pneumonia	Death day 131
19/M	Father (52), 3/6, yes	PMN 22, platelets 107	100/100% At all points	Liver steroid- refractory GI	EBV PTLD/rituximab CMV/preemption Grade III HC with BK viruria	E. faecium bacteremia Nocardia/Enterobacter pneumonia Rhizopus sp. pneumonia	Death day 316

HLA human leukocyte antigen, PMN polymorphonuclear leukocytes, aGVHD acute graft-versus-host disease, cGVHD chronic graft-versus-host disease, GI gastrointestinal, EBV Epstein-Barr virus, CMV cytomegalovirus, PTLD post-transplant lymphoproliferative disease, HC hemorrhagic cystitis,C. difficile Clostridioides difficile, S. aureus Staphylococcus aureus, A. fumigatus, Aspergillus fumigatus, E. coli Escherichia coli, B. fragilis Bacteroides fragilis, S. mitis Streptococcus mitis, K. pneumoniae Klebsiella pneumoniae, E. faecium Enterococcus faecium, PLE protein-losing enteropathy

Two patients died, both as a result of steroid-refractory GVHD, one complicated by disseminated adenovirus disease. Both of these patients had severe and refractory IBD prior to transplantation.

Transplant Complications

Viral reactivation occurred in all cases. One case of EBV reactivation led to preemptive therapy with rituximab without evidence of disease. Preemptive therapy for CMV was used in 2 cases. One patient had CMV colitis with concomitant lower GI GVHD and was treated with a prolonged course of antiviral therapy with resolution. Hemorrhagic cystitis associated with BK viruria was present in 4 cases, and in 2 cases was Grade 3. Disseminated adenovirus disease with pneumonia led to death in one patient as noted above with steroid-refractory GVHD after combined anti-CD25 (basiliximab) and anti-tumor necrosis factor (infliximab) antibody treatment.

Bacterial infections complicated all cases, including Clostridioides difficile–associated colitis in 2 cases and bacteremias with enteric/mucosal organisms in 4 cases. All were successfully treated without apparent influence on clinical outcome. Patient 1, who had Nocardia pneumonia as the immediate indication for transplantation, had no pulmonary complication or progression of disease during transplantation.

Invasive molds (A. nidulans and S. apiospermum) refractory to standard therapies were the indications for transplantation in 2 cases and in neither case was there progression of disease during the transplant. One patient with severe GVHD had multiple new pulmonary nodules during the course of treatment of GVHD, with culture of bronchoalveolar lavage fluid that yielded Aspergillus fumigatus, which was successfully treated. One patient with steroid-refractory GVHD had a culture from BAL that grew a Rhizopus species, without clear association with a radiographic lesion, and there was no discernible progression of pulmonary disease.

Discussion

The need for alternative donors in CGD parallels the progression of disease to the degree that HCT becomes a necessary therapy in patients with complications that do not respond to standard therapies for infection and inflammation. We adapted a transplant protocol utilizing haploidentical donors and a conditioning regimen similar to that pioneered at the Johns Hopkins Medical Institutions and adapted to other congenital immunodeficiencies (DOCK8 deficiency and GATA2 deficiency) at the Clinical Center of the NIH.

The results of this transplantation protocol were unsatisfactory. Although engraftment was essentially complete in the CD3+ and myeloid compartments early after transplant, all patients experienced acute GVHD, severe in 3/7 cases, and steroid refractory with subsequent death in 2. This degree of GVHD varies from that reported in our previous experience using a similar regimen in DOCK8 and GATA2 deficiency [3, 4]. Whether these results are due to transplantation in CGD, to changes in the regimen (the use of PBCs or sirolimus GVHD prophylaxis), or to a combination of these and other factors is difficult to ascertain. The most severe outcomes, steroid-refractory GVHD and death, occurred in patients who had the most severe CGD-associated IBD.

The small sample size and the possible relationship between the severity of pretransplant IBD and outcomes complicate this analysis. Prior to this experience, we did not have a formal severity scale related to IBD that would allow us to provide an adequate quantitative rather than qualitative assessment. It is clear from the series that the patients transplanted for active infection (patients 1, 2, and 5 in Table 1) had qualitatively less severe or even absent IBD, and that the two patients who died with steroid-refractory GVHD had chronic and severe disease requiring multiple immunosuppressive regimens over the course of many years. IBD has been associated with high-grade GVHD, and the scale of responses here, with death from steroid-refractory GI GVHD in the two patients with the most chronic and severe manifestations prior to transplant, would support the association [6]. To approach this with appropriate caution, we have added several evaluations, including consultation with a gastroenterologist, the Crohn Disease Activity Index, corticosteroid and other immunosuppressant doses, and endoscopic evaluation of colitis (simplified endoscopic score of Crohn Disease), to provide a composite severity evaluation of IBD pretransplant and will exclude patients with severe disease from the initial stages of an HRD protocol.

The first patient transplanted has been previously reported, and the justification for that alternative donor transplant was a fungal infection in an anatomic distribution that threatened the patient’s life (pericarditis). Both this patient, one with extensive Nocardia pneumonia and a third with chronic, destructive A. nidulans pneumonia and osteomyelitis had successful engraftment. Their GVHD and immunosuppressive therapy did not impede the cure of these underlying infections. The ability to transplant through medically refractory infectious diseases is a notable result and has been replicated in our conventional donor experience (manuscript in preparation).

With this discordance between early and complete engraftment and unacceptable outcomes related to GVHD, the need for an alternative method using mismatched donors remains. Post-transplant cyclophosphamide is an established agent to facilitate haploidentical donor transplantation and has marked advantages in terms of its availability. An increased incidence of infectious complications (especially viral infections) has been noted, but as in our experience, these have not produced unacceptable outcomes clearly attributable to viral reactivation [7]. In our series, only disseminated adenovirus disease after T cell- and TNF-depleting therapy contributed to a fatal outcome.

This is the largest published experience with HRD transplant in CGD, and should serve as a note of caution to single patient case reports, our own as well as others [8, 9]. HCT with PTCy led to complete engraftment in patients with CGD, but unacceptable levels of GVHD with 2 deaths out of 7 recipients. We are modifying our transplantation approach for haploidentical donors by initially excluding recipients with severe inflammatory bowel disease. Our interpretation of these outcomes is that the intensity of the regimen and rapid full engraftment may have contributed to the unacceptable rates of GVHD. The conditioning regimen will be modified to eliminate pretransplant cyclophosphamide and fludarabine and replace those agents with what we believe to be a lower-toxicity regimen based on scheduled moderate dose distal and proximal low-dose alemtuzumab, in order to decrease inflammatory disease that may contribute to an environment hostile to engraftment and to provide improved GVHD prophylaxis along with the continued use of post-transplant cyclophosphamide. This approach will allow us to assess the comparative intensity related to engraftment and the probable inverse relationship with GVHD, reverting to a standard consideration in transplantation for non-malignant disease in which failure of engraftment is preferable to GVHD, certainly with the mortality demonstrated with the regimen described here. As transplantation for CGD becomes a preferred treatment, the need for alternative donors continues to increase, justifying these attempts to achieve the standard goal of successful engraftment without severe complications and mortality.