Sage Journals: Discover world-class research

Abstract

Refractory acute myeloid leukemia (AML), defined as failure of two cycles of induction therapy at diagnosis or of one cycle at relapse, represents a subgroup with poor outcomes. Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (fludarabine + cytosine + granulocyte colony-stimulating factor ± idarubicin or mitoxantrone + etoposide) followed by 1-week rest and then reduced-intensity transplant with fludarabine + melphalan. We used the same backbone for this trial (CTRI/2019/02/017505) with the addition of CD56-positive cells from a family donor infused 1 day after the completion of chemotherapy. CD56-positive selection was done using a CliniMACS Prodigy system (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by overnight incubation in autologous plasma with 2 micromolar arsenic trioxide and 500 U/mL of interleukin-2. From February 2019, 14 patients with a median age of 29 years (interquartile range [IQR]: 16.5–38.5) were enrolled in this trial. Six were females. Six had primary refractory AML while eight had relapsed refractory AML. The median CD56-cell dose infused was 46.16 × 106/kg (IQR: 25.06–70.36). One patient withdrew consent after NK cell infusion. Of the 13 patients who proceeded to transplant, five died of immediate post-transplant complications while two did not engraft but were in morphologic leukemia-free state (both subsequently died of infective complications after the second transplant). Of the remaining six patients who engrafted and survived beyond 1 month of the transplant, two developed disease relapse and died. The remaining four patients are alive and relapse free at the last follow-up (mean follow-up duration of surviving patients is 24 months). The 2-year estimated overall survival for the cohort was 28.6% ± 12.1% while the treatment-related mortality (TRM) with this approach was 38.5% ± 13.5%. Haploidentical NK cell therapy as an adjunct to transplant is safe and needs further exploration in patients with AML. For refractory AML, post-transplant NK infusion and strategies to reduce TRM while using pre-transplant NK infusion merit exploration.

Keywords

NK cell therapy sequential transplant primary refractory relapsed refractory acute myeloid leukemia

Introduction

Refractory acute myeloid leukemia (AML) is associated with dismal long-term outcomes (<10% long-term survival) with allogeneic hematopoietic cell transplant (allo-HCT) being the current standard of care¹. However, even among patients with refractory AML who undergo a myeloablative allo-HCT, the outcomes are poor (19% long-term survival) with pre-transplant factors like first complete remission duration, circulating blasts, donor type, Karnofsky or Lansky score, and cytogenetic risk being predictive of survival². Due to concerns of high transplant-related mortality (TRM) rates with a myeloablative approach, a sequential reduced-intensity transplant approach (peak cytopenia transplant) has been tried in primary refractory AML; however, the 2-year relapse incidence with this approach was very high (54%)³. Historically, at our center, patients with refractory AML have been treated with a sequential transplant, that is, cytoreductive therapy (FLAG [fludarabine, cytosine, granulocyte colony-stimulating factor (GCSF)] ± idarubicin regimen⁴ or mitoxantrone + etoposide,⁵ followed by 1-week rest, and then a reduced-intensity transplant with fludarabine + melphalan conditioning while in peak cytopenia, with which we have reported long-term survival of 16% in this cohort)⁶. Hence, further strategies are needed to reduce the relapse risk in patients with refractory AML undergoing stem cell transplantation.

In order to reduce relapse risk, haploidentical natural killer cell (NK) therapy is a strategy that is being explored in high-risk AML wherein NK cells have been given either before or after transplant and with or without ex-vivo expansion^7–10.

Pre-transplant minimal residual disease seems to be a strong predictor of the post-transplant relapse¹¹. Hence, devising strategies to target pre-transplant disease in order to reduce post-transplant relapse risk seems intuitive. Our previously reported in-vitro and animal model data suggest that exposure to arsenic trioxide (ATO) results in enhanced NK cytotoxicity against acute promyelocytic leukemia (APL). This effect is mediated by upregulation of activating NK ligands on APL cells along with upregulation of both activating (NKG2D, NKp30, and KIR2DS4) and inhibitory receptors (NKG2A) on NK cells. This effect was seen at concentrations not affecting the cell viability and proliferation of NK cells¹². Hence, we decided to evaluate the feasibility of ATO- and interleukin-2 (IL-2)-exposed CD56-positive cells from haploidentical family donors given as an adjunct to sequential reduced-intensity stem cell transplantation patients with refractory AML. Our hypothesis was that haploidentical NK cells given before transplant would potentially reduce the pre-transplant disease burden and result in improved outcomes after transplant in patients with refractory AML.

Methods

From February 2019, we initiated a phase II single-arm clinical trial (CTRI/2019/02/017505) enrolling patients of any age with primary/relapsed refractory AML planned for a sequential reduced-intensity stem cell transplant.

Patient Eligibility

The key inclusion criteria were (1) patients with refractory AML defined as failure of two cycles of induction therapy at diagnosis or of one cycle at relapse; (2) patients planned for a sequential reduced-intensity stem cell transplant; (3) any age; (4) an Eastern Cooperative Oncology Group performance score < 2 at screening.

The key exclusion criteria were (1) a severe illness or organ dysfunction involving the heart, kidneys, liver, or other organ systems, for example, active infection, clinically relevant impairment of cardiac function, QTc prolongation, unstable angina pectoris or history of recent myocardial infarction, total bilirubin or liver enzymes greater than three times the upper limit of normal, estimated creatinine clearance <60 ml/min by the Cockcroft–Gault formula, active hepatitis B or hepatitis C, or laboratory evidence for a chronic infection, such as HIV infection; (2) second malignancy currently requiring an active therapy (except for hormonal/anti-hormonal treatment, eg, in prostate or breast cancer); (3) any significant concurrent psychiatric disorder or social situation that, according to the investigator’s judgment, would compromise patient’s safety or compliance, interfere with consent, study participation, or interpretation of study results; (4) known or suspected active alcohol or drug abuse.

Treatment Schema

After a written informed consent, patients received cytoreductive chemotherapy which was primarily FLAG chemotherapy consisting of priming GCSF of 300 µg per m² body surface area (BSA) along with fludarabine 30 mg per m² BSA and cytosine 2 g per m² BSA for 5 days. Patients who had received and not attained remission with high-dose cytosine before enrollment were offered mitoxantrone 10 mg per m² BSA and etoposide 100 mg per m² BSA for 3–5 days¹³ or a GCLAC (GCSF, clofarabine, cytosine) regimen consisting of priming GCSF of 300 µg per m² BSA along with clofarabine 30 mg per m² BSA and cytosine 2 g per m² BSA for 5 days¹⁴ as the cytoreductive chemotherapy before infusion of CD56-positive cells (Fig. 1).

Figure 1.

Clinical trial schema. GCSF: granulocyte colony-stimulating factor; Flu: fludarabine; AraC: cytosine; MRD: measurable residual disease testing; Mel: melphalan; TBI: total body irradiation; GVHD: graft versus host disease; NK: natural killer cell.

Donor Selection for NK Cells

Along with the patient’s sample, samples from all potential donors within the patient’s family were sent for high-resolution Human Leukocyte Antigen (HLA) and killer immunoglobulin-like receptor (KIR) typing. A flowcytometry crossmatch for donor-specific antibodies was performed for all HLA haploidentical donors. An HLA haploidentical family donor (other than the stem cell donor, wherever feasible) was selected for NK cell donation. The NK cell donor preference strategy included presence of KIR ligand mismatch¹⁵, greater number of KIR B motifs (or the B score), lower donor age, and negative donor-specific antibodies tested using flowcytometry crossmatch (Supplemental Table 1).

Clinical-Grade NK Separation, Release Criteria, and Characterization

The donor underwent unstimulated leukapheresis along with a plasma collection on a Spectra Optia apheresis system (Terumo BCT Inc, Lakewood, CO, USA) 1 day after completion of the patient’s cytoreductive chemotherapy. The apheresed product was processed in a class 10,000 (International Standards Organization Class 7) clean-room good manufacturing practices (GMP) facility using the CliniMACS Prodigy^® system (Miltenyi Biotec, Bergisch Gladbach, Germany). CD56-positive selection was done using the CliniMACS CD56 reagent and CliniMACS—TS310—Prodigy Tubing set and phosphate buffered saline/ethylenediaminetetraacetic acid buffer (Miltenyi Biotec). This was followed by overnight incubation of the CD56-positive cells in autologous plasma with 2 micromolar ATO (from Intas Pharmaceuticals, Thaltej, India) and 500 U/mL of MACS^® GMP recombinant IL-2 (from Miltenyi Biotec)¹⁶. Product release criteria were as shown in Supplemental Table 2. Flowcytometry characterization of the product including expression of CD16, PD1, CXCR4, CD94, and CD57 was done before and after the overnight incubation using a panel of antibodies as shown in Supplemental Table 3.

NK Cell Infusion and Monitoring

The CD56-positive cells after overnight incubation were infused to the patient 1 day after the completion of cytoreductive chemotherapy. Flowcytometry-based measurable residual disease (MRD) testing was done prior to the infusion and then again 6 days later coinciding with the start of conditioning for the transplant.

Transplantation Procedure

Patients underwent a reduced-intensity stem cell transplant with fludarabine 30 mg/m²/day for 4 days + melphalan 140 mg/m² for 1-day (with single-fraction total body irradiation 200 cGy on day 1 for haploidentical transplants) conditioning. The best available stem cell donor was taken (matched sibling > matched related > matched unrelated > haploidentical). The graft versus host disease (GVHD) prophylaxis used was intravenous cyclosporine 2.5 mg/kg Q12H along with a short course of intravenous methotrexate on days 1 (10 mg/m²), 3 (7 mg/m²), and 6 (7 mg/m²) for HLA-matched and HLA-mismatched donor transplantation while it was post-transplant intravenous cyclophosphamide at 50 mg/kg on days 3 and 4 along with oral tacrolimus at 0.03 mg/kg BD and oral mycophenolate mofetil at 15 mg/kg TID from day 5 for haploidentical donor transplantation. Bone marrow assessment after transplant at day 28 was performed along with flowcytometry-based MRD testing.

Statistical Analysis

Descriptive statistics were used to describe the demographic, clinical, and laboratory data. The primary outcome variable was 1-year relapse-free survival. A survival analysis was performed using the Kaplan–Meier method to estimate the 1-year relapse-free survival and 1-year event-free survival (event defined as death or relapse) of the current clinical trial cohort who underwent transplant and compared using the log-rank test with the survival of an historical cohort of patients with refractory AML as defined in the current trial (failure of two lines of therapies at diagnosis and failure of one therapy at relapse). All analyses were performed using SPSS version 25 (IBM SPSS Inc., Chicago, IL, USA)

Results

Patient Characteristics

From February 2019, 14 patients with a median age of 28 years (IQR: 15.75–31.5) were enrolled in this trial. Six were females. Six had primary refractory AML while eight had relapsed refractory AML. The median Duval/Center for International Blood and Marrow Transplant Research (CIBMTR) score² was 3 (IQR: 1.25–4). The cytoreductive chemotherapy was FLAG ± idarubicin (n = 7), mitoxantrone + etoposide (n = 6), and GCLAC (n = 1). Baseline characteristics of the patients is summarized in Table 1 while the NK cell donor characteristics and characterization of the CD56-enriched product before and after incubation with ATO and IL-2 are summarized in Supplemental Tables 4 and 5.

Table 1.

Patients and Disease Characteristics (n = 14).

Patient no.	Age (years)	Sex	Primary or relapsed refractory AML	Treatment details	Peripheral blood blasts% at enrollment	Bone marrow blasts% at enrollment	Duval/CIBMTR score	Karyotype at enrollment	FLT3 ITD/NPM1 status at enrollment	Pre-NK cytoreductive chemotherapy
1	9	Female	Primary	AML with myelodysplasia-related changes with persistent disease and hypocellular marrow after BFM 98 induction	1	5	3	46, XX[20]	FLT3 ITD mutated	FLAG
2	27	Female	Relapsed	7/3 followed by 3 HIDAC consolidation; relapsed after 11 months; treated with FLAG-IDA, HIDAC	90	NA	5	45~46, X,-X,t(9;11)(p21; q23)[13]/46, XX,t(2;17)(p13; q25)[1cell]/46,XX[6]	Not done	Mitoxantrone-etoposide × 3 days
3	5	Female	Primary	ADE followed by 2 HIDAC	0	66	3	46, XX,t(6;11)(q27; q23)[13]/46,XX[7].	Negative	FLAG-IDA
4	48	Female	Primary	Therapy-related AML; 3 cycles of azacitidine, 1 cycle of decitabine–lenalidomide and 2 cycles of decitabine–venetoclax	78	NA	1	46, XX,t(11;19)(q23; p13.3)[18]/46,XX[2]	Negative	FLAG
5	14	Male	Relapsed	7/3 × 2 followed by 3 HIDAC consolidation; relapsed after 5 months; treated with FLAG, mitoxantrone–etoposide	20	35	4	46, XY, del(9)(q21q32)[17]/46, XY[3]	Negative	GCLAC
6	3	Male	Relapsed	7/3 followed by 3 HIDAC consolidation; relapsed after 3 months; treated with FLAG-IDA, CLAG	25	96	5	“41~46, XY,add(1)(p36), del(4)(q31.1), der(7), t(9;11)(p21; q21), add(10)(q22), add(13)(q34),-15, der(16),-17, der(19)t(1;19)(q24; p13.1),+mar,+mar[cp19]/82~92, XXYY, add(1)(p36), del(4)(q31.1)×2, t(9;11)(p21; q21)×2, add(10)(q22)×2, add(13)(q34)×2,-15,-15, der(16)×2,-17,-17, der(19)t(1;19)(q24; p13.1)×2,+mar×2,+mar×2[cp2]/46,XY[1cell]”	Negative	FLAG
7	29	Female	Relapsed	Systemic mastocytosis with AML; 7/3, FLAG-IDA followed by allotransplant; relapsed in 13 months; treated with mitoxantrone–etoposide	16	NA	2	46, XX[17]//46, XY[3]	Not done	FLAG
8	40	Female	Primary	7/3, FLAG and HIDAC	60	32	4	45, X,-X[6]/46, XX[14]	Negative	Mitoxantrone–etoposide × 5 days
9	34	Male	Relapsed	7/3, FLAG-IDA followed by 3 HIDAC consolidation; relapsed after 3 years; treated with FLAG, 1 cycle of azacitidine	0	23	2	47, XY,+mar[8]/46, XY[12]	Not done	Mitoxantrone–etoposide × 5 days
10	22	Male	Primary	7/3, FLAG	10	42	1	46, XY,t(1;4)(p32; q26)[2]/46, XY,t(12;14)(q21; q21)[1cell]/46,XY[17]	FLT3 ITD mutated	Mitoxantrone–etoposide × 5 days
11	24	Male	Primary	7/3, 2 HIDAC	0	17	1	46, XY,t(11;17)(q23.3; q21)[15]/47, idem,+8[3]/46,XY[2]	Not done	FLAG-IDA
12	35	Male	Relapsed	7/3, 5/2 followed by 3 HIDAC consolidation; relapsed after 2 years; treated with FLAG, azacitidine–midostaurin	0	20	1	45, XY,del(5)(q23),-7, der(10)[9]/46,XY[11]	Negative	Mitoxantrone–etoposide × 3 days
13	29	Male	Relapsed	5/2, 7/3 followed by 3 HIDAC; relapsed after 3 months; treated with FLAG	70	95	4	47, XY,+8[17]/47, idem,t(1;3)(p22; p25)[3]	NPM1 and FLT3 ITD mutate	Mitoxantrone–etoposide × 3 days
14	30	Male	Relapsed	7/3 followed by 3 HIDAC consolidation; relapsed after 3 months; treated with 3 cycles of azacitidine	0	31	3	46, XY[20]	Negative	FLAG-IDA

7/3, 5/2: cytosine and daunorubicin; AML: acute myeloid leukemia; NK: natural killer cell; FLAG: fludarabine, cytosine, GCSF; HIDAC: high-dose cytosine; FLAG-IDA: FLAG with idarubicin; NA: not available; GCLAC: clofarabine, cytosine, GCSF; CLAG: cladribine, cytosine, GCSF; GCSF: granulocyte colony-stimulating factor, CIBMTR: Center for International Blood and Marrow Transplant Research; BFM: Berlin-Frankfurt-Munster; FLT3 ITD: FMS-like tyrosine kinase 3 internal tandem duplication; NPM1: Nucleophosmin 1, ADE: cytosine, daunorubicin and etoposide.

In summary, the median B score for the NK cell donors was 2 (IQR: 1–3). The median age of the NK cell donor was 43 years (IQR: 36–49.5). KIR ligand mismatch with the patient was noted in two donors. The median CD56-cell dose infused was 46.16 × 10⁶/kg (IQR: 25.06–70.36). Pre-defined release criteria (Table 2), including sterile bacterial cultures, were met in all cases. There was no infusion-related toxicity. Figs. 2 and 3 show the representative dot plot of flowcytometry characterization of the CD56-enriched product before and after overnight incubation with ATO and IL-2. Supplemental Table 5 shows the mean fluorescence intensity (MFI) for PD-1, CD25, CXCR4, CD94, and CD57 before and after incubation with ATO and IL-2. There was a significant increase in the MFI of CXCR4 and CD94 after incubation with ATO and IL-2 while there was a reduction in the MFI of CD25 and CD57 on the NK cells.

Table 2.

Flowcytometry-Based MRD Testing Done Before and After the NK Cell Infusion Along With the Day-28 Post-Transplant MRD.

Patient no.	Bone marrow blast at enrollment	Flowcytometry MRD after cytoreductive chemotherapy and before NK infusion	Flowcytometry MRD after NK infusion and before transplant	Flowcytometry MRD at day 28 after transplant
1	5%	Negative	Not done	NA
2	NA (90% in PB)	90%	75.05%	NA
3	66%	5%	Events inadequate	Negative
4	NA (78% in PB)	Events inadequate	Events inadequate	Negative
5	35%	Events inadequate	Negative	Negative
6	96%	72%	89%	NA
7	NA (16% in PB)	Events inadequate	Events inadequate	NA
8	32%	72.78%	Not done	NA
9	23%	15.87%	5.95%	Negative
10	42%	28.65%	38.48%	0.13%
11	17%	8.92%	6.23%	Negative
12	20%	9.24%	17.61%	NA
13	95%	36.30%	1.63%	Engraftment failure, events inadequate
14	31%	11.06%	Not done	Engraftment failure, events inadequate
Median	32%	15.87%	11.92%	0%

MRD: measurable residual disease; NK: natural killer cell; NA: not applicable; PB: peripheral blood.

Figure 2.

Representative dot plot of flowcytometry expression of CD16, PD-1, CXCR4, CD94, and CD57 on the NK and NKT cells before overnight incubation with 2 µM arsenic trioxide with 500 IU/mL of interleukin-2. NK: natural killer cell, NKT: Natural Killer T cell, SSC-A: side scatter parameter.

Figure 3.

Representative dot plot of flowcytometry expression of CD16, PD-1, CXCR4, CD94, and CD57 on the NK and NKT cells after overnight incubation with 2 µM arsenic trioxide with 500 IU/mL of interleukin-2. NK: natural killer cell.

Flowcytometry-Based MRD Testing Before and After NK Cell Infusion

The median blast percentage on flowcytometry MRD testing prior to NK infusion was 15.9% (IQR: 9.1%–54.5%) (n = 11). The median blast percentage on flowcytometry MRD testing done 6 days following NK cell infusion was 11.9% (IQR: 4.9%–47.6%) (n = 8) (on comparison with the MRD prior to NK infusion, P = 0.29). Of the 10 patients for whom MRD was unequivocally detectable prior to NK infusion, eight patients had MRD assessment done after NK infusion as well. Among these eight, three patients (patient no. 6, 10, and 12) had increased MRD after NK infusion, and all these patients died (two due to immediate post-transplant complications, and one was MRD-positive after transplant and subsequently died due to a post-transplant relapse). Of the remaining five patients (patient no. 2, 3, 9, 11, and 13) who showed reduction in MRD after NK infusion, one died of immediate post-transplant complications, three were MRD-negative after transplant and in remission at the last follow-up, and one had morphologic leukemia-free state at day 28 after transplant. There were no significant differences in the NK cell dose and flowcytometry MFI of PD1, CD25, CXCR4, CD94, and CD57 on NK cells in the patients who had an increase in MRD following NK cell infusion (n = 3, patient no. 6, 10, and 12) versus the remaining evaluable patients (see Table 2).

Transplant Characteristics

One patient withdrew consent after NK cell infusion and did not undergo transplant. For the 13 patients who underwent stem cell transplant, the stem cell donor was HLA-matched for four patients, one had 9/10 HLA-matched related donor, while the remaining eight had haplo-matched donors. The median age of the stem cell donor was 35 years (IQR: 25–50). The median CD34 dose infused was 10 × 10⁶/kg (IQR: 7.51–11.6 × 10⁶/kg). Five (38.5%) patients died of immediate post-transplant complications, sepsis (n = 3) on days 1, 2, and 28; cerebral venous sinus thrombosis (n = 1) on day 1; and sinusoidal obstruction syndrome (n = 1) on day 15. All deaths were unrelated to the NK cell infusion and attributed to anticipated complications after stem cell transplantation like sepsis and sinusoidal obstruction syndrome. Sinusoidal obstruction syndrome contributing to death occurred in a patient who had relapsed after a previous allogeneic transplant and was undergoing a second transplant. The patient who developed cerebral venous sinus thrombosis had received hormonal contraceptives in the immediate pre-transplant period for menorrhagia, and this was considered as a likely contributory factor for the event (see Table 3).

Table 3.

Transplant Characteristics and Clinical Outcomes.

Patient no.	Stem cell donor						Conditioning	CD34 cell dose (× 10⁶/kg)	GVHD prophylaxis	Day of engraftment		Acute GVHD	AGVHD grade	Chronic GVHD	Limited/extensive	Relapse	Death	Cause of death	Status at last follow-up
Patient no.	Relation	Age	Donor-specific antibodies	HLA	B score	KIR ligand mismatch	Conditioning	CD34 cell dose (× 10⁶/kg)	GVHD prophylaxis	Neutrophil	Platelet	Acute GVHD	AGVHD grade	Chronic GVHD	Limited/extensive	Relapse	Death	Cause of death	Status at last follow-up
1	Mother	31	Neg	7/10	3	HVG direction	Flu-Mel-TBI	10	PTCy-Tac-MMF	21	NA	NA	NA	NA	NA	NA	Yes	Sepsis, transplant-associated microangiopathy	Died on day 28 post-transplant due to sepsis
2	Mother	54	Neg	5/10	3	GVH direction	Flu-Mel-TBI	16	PTCy-Tac-MMF	NA	NA	NA	NA	NA	NA	NA	Yes	Cerebral venous thrombosis	Died on day 2 post-transplant due to cerebral venous sinus thrombosis
3	Mother	35	Neg	8/10	1	Neg	Flu-Mel-TBI	20	CSA-MTX	16	17	No	NA	Yes	Extensive	No	No	NA	Post-transplant MRD negative; alive and relapse-free at day 1061 post-transplant; chronic GVHD
4	Brother	51	NA	8/8		Neg	Flu-Mel	10	CSA-MTX	14	16	No	NA	Yes	Extensive	Yes	Yes	Sepsis	Post-transplant MRD negative; however, relapsed 7 months post-transplant and died during treatment for relapse
5	Cousin brother	20	Neg	5/10	1	Neg	Flu-Mel-TBI	6.27	PTCy-Tac-MMF	20	106	No	NA	Yes	Extensive	No	No	NA	Post-transplant MRD negative; alive and relapse-free at day 923 post-transplant; chronic GVHD
6	Mother	29	Neg	5/10	0	GVH direction	Flu-Mel	22	NA	NA	NA	NA	NA	NA	NA	NA	Yes	Sepsis	Died on day 1 post-transplant due to sepsis
7	Sister	35	Neg	10/10	2	Neg	Flu-Mel	11.67	CSA-MTX	10	NA	No	NA	NA	NA	NA	Yes	Veno-occlusive disease, invasive aspergillosis	Died on day 15 post-transplant due to veno-occlusive disease
8	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	NA	Withdrew consent after NK infusion; did not undergo transplant
9	Mother	58	Neg	8/10	2	Neg	Flu-Bu-TBI	2.53	PTCy-Tac-MMF	11	15	Yes	II	Yes	Extensive	No	No	NA	Post-transplant MRD negative, acute GVHD—resolved, alive and relapse-free at day 481 post-transplant
10	Mother	36	NA	12/12	1	Neg	Flu-Mel	9.08	CSA-MTX	16	22	Yes	IV	No	NA	Yes	Yes	Pseudomonas sepsis with shock	Post-transplant MRD 0.13%; relapsed at day 54 and died
11	Brother	13	NA	11/12	0	Neg	Flu-Mel	10	CSA-MTX	13	20	Yes	III	Yes	Extensive	No	No	NA	Post-transplant MRD negative, acute GVHD—resolving, alive and relapse-free at day 447 post-transplant
12	MUD	22	NA	10/10		Neg	Flu-Mel	6.45	CSA-MTX	NA	NA	No	NA	NA	NA	NA	Yes	Sepsis	Died on day 2 post-transplant due to sepsis
13	Mother	50	Neg	6/10	4	Neg	Flu-Mel-TBI	7.51	PTCy-Tac-MMF	NA	NA	NA	NA	NA	NA	NA	Yes	Bilateral pneumonia	Did not engraft, morphologic leukemia-free state post 1st transplant, died of bilateral pneumonia following engraftment after 2nd transplant
14	Brother	25	Neg	7/10	1	Neg	Flu-TBI	9.16	PTCy-Tac-MMF	NA	NA	NA	NA	NA	NA	NA	Yes	Bilateral pneumonia	Did not engraft, morphologic leukemia-free state post 1st transplant, died of bilateral pneumonia following engraftment after 2nd transplant

GVHD: graft versus host disease; AGVHD: acute graft versus host disease; HLA: Human Leukocyte Antigen; KIR: killer immunoglobulin-like receptor; HVG: host versus graft; TBI: total body irradiation; PTCy: post-transplant cyclophosphamide; Tac: tacrolimus; MMF: mycophenolate mofetil; NA: not applicable; CSA: cyclosporine; MTX: methotrexate; MRD: measurable residual disease testing; MUD: matched unrelated donor; Neg: negative; Flu: fludarabine; Mel: melphalan; Bu: busulfan; NK: natural killer cell.

Post-Transplant Day-28 Bone Marrow Assessment

Of the remaining eight patients who were alive at day 28 after transplant, six were in morphologic remission (of which five were MRD negative and one had detectable MRD [0.13%]) while two were in morphologic leukemia-free state with graft rejection (MRD events were inadequate).

Clinical Outcomes Following Transplantation

Two patients (15.4%) who failed to engraft after a haploidentical stem cell transplant (marrow showed morphologic leukemia-free state at rejection) engrafted following a second transplant and died of an immediate infective post-transplant complication (bilateral pneumonia). One patient received a CD34 cell boost on day 96 (cell dose: 8.55 × 10⁶/kg) for poor graft function. On follow-up, two (15.4%) patients developed disease relapse (on days 54 and 218, respectively) and died. The remaining four (30.8%) patients were alive and relapse-free at the last follow-up (for data cutoff date of February 28, 2022, the follow-up time of the four surviving patients was 35, 30, 16, and 15 months, respectively). There were no statistically significant differences between the patients who survived (n = 4) versus those who died (n = 9) following the transplant in terms of the CD34 cell dose (in × 10⁶/kg) (median 8.14 vs 10, P = 0.65), pre-transplant MRD (median 5.95% vs 38.5%, P = 0.12), and the NK cell dose infused (in × 10⁶/kg) (median 33.96 vs 25.06, P = 0.69).

For all the enrolled patients (n = 14), the 2-year estimated overall survival was 28.6% ± 12.1%, event-free survival was 28.6% ± 12.1%, while the TRM with the approach was 38.5% ± 13.5% (Fig. 4). Acute GVHD was noted in three patients (grade II, III, and IV in one patient each) (50%; out of six evaluable patients), and chronic GVHD was noted in five patients (all had extensive chronic GVHD, 84%; out of the six evaluable patients).

Figure 4.

Kaplan–Meier curves showing the estimated 2-year overall survival, event-free survival, and the treatment-related mortality. For all the enrolled patients (n = 14), the 2-year estimated overall survival was 28.6% ± 12.1%, event-free survival was 28.6% ± 12.1%, while the treatment-related mortality with the approach was 38.5% ± 13.5%.

Discussion

Delayed NK cell recovery after transplant has been shown to be associated with risk of relapse and non-relapse mortality in transplants using post-transplant cyclophosphamide¹⁷. In addition, NK cell dysfunction due to higher levels of TGF-β1 has been reported in AML¹⁸. Post-transplant NK cell therapy in the setting of haploidentical transplants has been shown to reduce relapse risk^9,19 and also GVHD⁸. Other approaches have utilized cytokine-induced memory like NK cells after transplant to reduce the relapse risk in AML¹⁰ or for treatment of post-transplant relapse²⁰, and the use of CTLA4-Ig primed donor lymphocyte infusions for augmenting NK cell function to reduce relapse risk²¹. There have been limited approaches testing NK cell therapy before transplant in refractory AML. Haploidentical NK cell therapy has been shown to be feasible in the setting of a matched donor transplant wherein NK cells given before transplant did not affect engraftment⁷.

Currently there are no approved therapies to target MRD in AML before transplant. We hypothesized that NK cell therapy would potentially reduce the pre-transplant disease burden in patients with refractory AML undergoing a sequential transplantation and hence improve the post-transplant outcomes.

Our results show the safety of haploidentical NK cell therapy given as an adjunct to a sequential transplantation approach in refractory AML. We could not unequivocally demonstrate efficacy due to the limited sample size and high non-relapse mortality in this cohort. However, we noted that the median blast percentage reduced from 15.87% to 11.92% following the NK infusion, although this did not reach statistical significance. Patients who showed reduction in flowcytometry MRD following the NK infusion seemed to have better disease control following the transplant in our study. Although not unequivocal, this is a signal toward efficacy, and hence, the strategy of using haploidentical NK cell therapy to target pre-transplant diseases in AML merits further evaluation in other cohorts like high-risk AML and AML with pre-transplant MRD positivity.

The high TRM rate (38.5%) seen with the current protocol combining NK cell infusion before the transplant was largely due to infective complications during the neutropenic period, which was prolonged in the current sequential transplant approach. The way forward would be to use hypomethylating agent venetoclax-based regimens ± targeted therapies wherever applicable ± gemtuzumab ozogamicin. The objective will be to reduce the off-target severe side effects that we see with intensive salvage regimens, especially prolonged neutropenia and mucosal and epithelial toxicities, which subsequently compromise the clinical outcome after transplant. Another alternative is to move away from the sequential (peak cytopenia) approach altogether and consider post-transplant infusion of ex-vivo expanded and stem cell donor-derived NK cells.

We did not find any favorable effect of ATO and IL-2 overnight exposure on the NK cells except for increased CXCR4 expression which seems to be a non-specific phenomenon unrelated to the drug exposure²². The way forward can be pharmacologic modulation of the leukemic cells to enhance NK cytotoxicity like treatment with agents like poly-ADP ribose polymerase (PARP) inhibitors²³ or ATO¹² to upregulate the NKG2DL on the leukemic cells before giving NK cell therapy.

In conclusion, our study shows the safety of infusion of CD56-positive cells from haploidentical family donors given as an adjunct to a sequential reduced-intensity stem cell transplant for refractory AML with no immediate or long-term safety concerns. For assessment of efficacy, this strategy merits further evaluation in larger cohorts of high-risk AML and AML with pre-transplant MRD positivity. For relapsed refractory AML, post-transplant NK infusion and strategies to reduce TRM while using pre-transplant NK infusion merit further exploration.

Supplemental Material

sj-docx-1-cll-10.1177_09636897231198178 – Supplemental material for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia

Supplemental material, sj-docx-1-cll-10.1177_09636897231198178 for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia by Uday Kulkarni, Arun Kumar Arunachalam, Hamenth Kumar Palani, Reeshma Radhakrishnan Nair, Nithya Balasundaram, Arvind Venkatraman, Anu Korula, Sushil Selvarajan, Sharon Lionel, Poonkuzhali Balasubramanian, Madhavi Maddali, Aby Abraham, Biju George and Vikram Mathews in Cell Transplantation

Supplemental Material

sj-jpg-2-cll-10.1177_09636897231198178 – Supplemental material for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia

Supplemental material, sj-jpg-2-cll-10.1177_09636897231198178 for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia by Uday Kulkarni, Arun Kumar Arunachalam, Hamenth Kumar Palani, Reeshma Radhakrishnan Nair, Nithya Balasundaram, Arvind Venkatraman, Anu Korula, Sushil Selvarajan, Sharon Lionel, Poonkuzhali Balasubramanian, Madhavi Maddali, Aby Abraham, Biju George and Vikram Mathews in Cell Transplantation

Supplemental Material

sj-jpg-3-cll-10.1177_09636897231198178 – Supplemental material for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia

Supplemental material, sj-jpg-3-cll-10.1177_09636897231198178 for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia by Uday Kulkarni, Arun Kumar Arunachalam, Hamenth Kumar Palani, Reeshma Radhakrishnan Nair, Nithya Balasundaram, Arvind Venkatraman, Anu Korula, Sushil Selvarajan, Sharon Lionel, Poonkuzhali Balasubramanian, Madhavi Maddali, Aby Abraham, Biju George and Vikram Mathews in Cell Transplantation

Supplemental Material

sj-jpg-4-cll-10.1177_09636897231198178 – Supplemental material for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia

Supplemental material, sj-jpg-4-cll-10.1177_09636897231198178 for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia by Uday Kulkarni, Arun Kumar Arunachalam, Hamenth Kumar Palani, Reeshma Radhakrishnan Nair, Nithya Balasundaram, Arvind Venkatraman, Anu Korula, Sushil Selvarajan, Sharon Lionel, Poonkuzhali Balasubramanian, Madhavi Maddali, Aby Abraham, Biju George and Vikram Mathews in Cell Transplantation

Supplemental Material

sj-jpg-5-cll-10.1177_09636897231198178 – Supplemental material for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia

Supplemental material, sj-jpg-5-cll-10.1177_09636897231198178 for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia by Uday Kulkarni, Arun Kumar Arunachalam, Hamenth Kumar Palani, Reeshma Radhakrishnan Nair, Nithya Balasundaram, Arvind Venkatraman, Anu Korula, Sushil Selvarajan, Sharon Lionel, Poonkuzhali Balasubramanian, Madhavi Maddali, Aby Abraham, Biju George and Vikram Mathews in Cell Transplantation

Supplemental Material

sj-jpg-6-cll-10.1177_09636897231198178 – Supplemental material for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia

Supplemental material, sj-jpg-6-cll-10.1177_09636897231198178 for Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia by Uday Kulkarni, Arun Kumar Arunachalam, Hamenth Kumar Palani, Reeshma Radhakrishnan Nair, Nithya Balasundaram, Arvind Venkatraman, Anu Korula, Sushil Selvarajan, Sharon Lionel, Poonkuzhali Balasubramanian, Madhavi Maddali, Aby Abraham, Biju George and Vikram Mathews in Cell Transplantation

Footnotes

Acknowledgements

The content of this article has been presented in part at the Annual Conference of the American Society of Hematology 2021, Kulkarni U et al.; Haploidentical Natural Killer Cell Therapy As an Adjunct to Stem Cell Transplantation for Refractory Acute Myeloid Leukemia. Blood 2021; 138 (Supplement 1): 3827. doi: .

Availability of Data and Material

The data sets generated and analyzed in the current study are available from the corresponding author upon reasonable request.

Author Contributions

U.K.: Designed study, performed research, analyzed data, provided clinical care to patients enrolled, wrote the paper.

A.K.A.: Performed research (Clinical grade cell processing and flowcytometry), analyzed data.

H.K.P.: Performed research (Clinical grade cell processing), analyzed data.

R.R.N.: Performed research, analyzed data.

N.B.: Performed research, analyzed data.

A.V.: Performed research, analyzed data.

A.K.: Performed research, analyzed data, provided clinical care to patients enrolled.

S.S.: Performed research, analyzed data, provided clinical care to patients enrolled.

S.L.: Performed research, analyzed data, provided clinical care to patients enrolled.

P.B.: Performed research, analyzed data.

M.M.: Performed research, analyzed data.

A.A.: Performed research, analyzed data, provided clinical care to patients enrolled.

B.G.: Performed research, analyzed data, provided clinical care to patients enrolled.

V.M.: Designed study, performed research, analyzed data, provided clinical care to patients enrolled, wrote the paper.

Ethical Approval

This study was approved by the institutional ethics committee (IRB:10982 [INTERVEN] dated 22.11.2017).

Statement of Human and Animal Rights

The study procedures followed were in accordance with the institutional ethics committee (IRB:10982 [INTERVEN] dated 22.11.2017) approved protocol.

Statement of Informed Consent

All participants were enrolled after providing a written informed consent.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study is supported by a DBT Wellcome Trust India Alliance research grant (IA/CPHE/17/1/503351). V.M. is supported by the senior fellowship program of the DBT Wellcome Trust India Alliance (IA/CPHS/18/1/503930), New Delhi, India. P.B. is supported by the senior fellowship program of the DBT Wellcome Trust India Alliance (IA/S/15/1/501842) New Delhi, India. U.K. is supported by an early career fellowship program of DBT Wellcome Trust India Alliance (IA/CPHE/17/1/503351), New Delhi, India.

ORCID iDs

Uday Kulkarni

Sushil Selvarajan

Supplemental Material

Supplemental material for this article is available online.

References

Ferguson

Hills

Grech

Betteridge

Kjeldsen

Dennis

Vyas

Goldstone

Milligan

Clark

Russell

, et al. An operational definition of primary refractory acute myeloid leukemia allowing early identification of patients who may benefit from allogeneic stem cell transplantation. Haematologica. 2016;101(11):1351–58. doi:10.3324/haematol.2016.148825.

Duval

Klein

Cahn

Cairo

Camitta

Kamble

Copelan

de Lima

Gupta

Keating

, et al. Hematopoietic stem-cell transplantation for acute leukemia in relapse or primary induction failure. J Clin Oncol. 2010;28(23):3730–38. doi:10.1200/JCO.2010.28.8852.

Mohty

Malard

Blaise

Milpied

Socié

Huynh

Reman

Yakoub-Agha

Furst

Guillaume

Tabrizi

, et al. Sequential regimen of clofarabine, cytosine arabinoside and reduced-intensity conditioned transplantation for primary refractory acute myeloid leukemia. Haematologica. 2017;102(1):184–91. doi:10.3324/haematol.2016.150326.

Pastore

Specchia

Carluccio

Liso

Mestice

Rizzi

Greco

Buquicchio

Liso

. FLAG-IDA in the treatment of refractory/relapsed acute myeloid leukemia: single-center experience. Ann Hematol. 2003;82(4):231–35. doi:10.1007/s00277-003-0624-2.

Trifilio

Rademaker

Newman

Coyle

Carlson-Leuer

Mehta

Altman

Frankfurt

Tallman

. Mitoxantrone and etoposide with or without intermediate dose cytarabine for the treatment of primary induction failure or relapsed acute myeloid leukemia. Leuk Res. 2012;36(4):394–96. doi:10.1016/j.leukres.2011.10.027.

Ganapule

Nemani

Korula

Lakshmi

Abraham

Srivastava

Balasubramanian

George

Mathews

. Allogeneic stem cell transplant for acute myeloid leukemia: evolution of an effective strategy in India. J Glob Oncol. 2017;3(6):773–81. doi:10.1200/JGO.2016.006650.

Lee

Denman

Rondon

Woodworth

Chen

Fisher

Kaur

Fernandez-Vina

Cao

Ciurea

Shpall

, et al. Haploidentical natural killer cells infused before allogeneic stem cell transplantation for myeloid malignancies: a phase I trial. Biol Blood Marrow Transplant. 2016;22(7):1290–98. doi:10.1016/j.bbmt.2016.04.009.

Jaiswal

Zaman

Nedunchezhian

Chakrabarti

Bhakuni

Ahmed

Sharma

Rawat

O’Donnell

Chakrabarti

. CD56-enriched donor cell infusion after post-transplantation cyclophosphamide for haploidentical transplantation of advanced myeloid malignancies is associated with prompt reconstitution of mature natural killer cells and regulatory T cells with reduced incidence of acute graft versus host disease: a pilot study. Cytotherapy. 2017;19(4):531–42. doi:10.1016/j.jcyt.2016.12.006.

Ciurea

Kongtim

Soebbing

Trikha

Behbehani

Rondon

Olson

Bashir

Gulbis

Indreshpal

Rezvani

, et al. Decrease post-transplant relapse using donor-derived expanded NK-cells. Leukemia. 2022;36(1):155–64. doi:10.1038/s41375-021-01349-4.

10.

Berrien-Elliott

Foltz

Russler-Germain

Neal

Tran

Gang

Wong

Fisk

Cubitt

Marin

Zhou

, et al. Hematopoietic cell transplantation donor-derived memory-like NK cells functionally persist after transfer into patients with leukemia. Sci Transl Med. 2022;14(633):eabm1375. doi:10.1126/scitranslmed.abm1375.

11.

Thol

Gabdoulline

Liebich

Klement

Schiller

Kandziora

Hambach

Stadler

Koenecke

Flintrop

Pankratz

, et al. Measurable residual disease monitoring by NGS before allogeneic hematopoietic cell transplantation in AML. Blood. 2018;132(16):1703–13. doi:10.1182/blood-2018-02-829911.

12.

Alex

Ganesan

Palani

Balasundaram

David

Lakshmi

Kulkarni

Nisham

Korula

Devasia

Janet

, et al. Arsenic trioxide enhances the NK cell cytotoxicity against acute promyelocytic leukemia while simultaneously inhibiting its bio-genesis. Front Immunol. 2018;9:1357. doi:10.3389/fimmu.2018.01357.

13.

Lipp

Ehninger

Illiger

Meyer

Freund

Hunstein

. Combination of mitoxantrone and etoposide in refractory acute myelogenous leukemia—an active and well-tolerated regimen. J Clin Oncol. 1988;6(2):213–17. doi:10.1200/JCO.1988.6.2.213.

14.

Becker

Medeiros

Stein

Othus

Appelbaum

Forman

Scott

Hendrie

Gardner

Pagel

Walter

, et al. G-CSF priming, clofarabine, and high dose cytarabine (GCLAC) for upfront treatment of acute myeloid leukemia, advanced myelodysplastic syndrome or advanced myeloproliferative neoplasm. Am J Hematol. 2015;90(4):295–300. doi:10.1002/ajh.23927.

15.

Robinson

Mistry

McWilliam

Lopez

Marsh

SGE

. IPD—the immuno polymorphism database. Nucleic Acids Res. 2010;38:D863–69.

16.

Carlens

Gilljam

Chambers

Aschan

Guven

Ljunggren

Christensson

Dilber

. A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells. Hum Immunol. 2001;62(10):1092–98. doi:10.1016/s0198-8859(01)00313-5.

17.

McCurdy

Radojcic

Tsai

Vulic

Thompson

Ivcevic

Kanakry

Powell

Lohman

Adom

Paczesny

, et al. Signatures of GVHD and relapse after posttransplant cyclophosphamide revealed by immune profiling and machine learning. Blood. 2022;139(4):608–23. doi:10.1182/blood.2021013054.

18.

Wang

Sun

Zhu

Zheng

Zhou

Yan

Wang

Liu

Jin

Zhu

, et al. GARP-mediated active TGF-β1 induces bone marrow NK cell dysfunction in AML patients with early relapse post-allo-HSCT. Blood. 2022;140(26):2788–804. doi:10.1182/blood.2022015474.

19.

Chang

Zhao

Huang

. Strategies for enhancing and preserving anti-leukemia effects without aggravating graft-versus-host disease. Front Immunol. 2018;9:3041. doi:10.3389/fimmu.2018.03041.

20.

Shapiro

Birch

Vergara Cadavid

Nikiforow

Baginska

Ali

Tarannum

Sheffer

Abdulhamid

Rambaldi

, et al. Expansion, persistence, and efficacy of donor memory-like NK cells infused for post-transplant relapse. J Clin Invest. 2022;132:e154334. doi:10.1172/JCI154334.

21.

Jaiswal

Chakraborty

Lakhchaura

Shashi

Mehta

Soni

Chakrabarti

. Early and sustained expansion of adaptive natural killer cells following haploidentical transplantation and CTLA4Ig-primed donor lymphocyte infusions dissociate graft-versus-leukemia and graft-versus-host effects. Transplant Cell Ther. 2021;27(2):144–51. doi:10.1016/j.jtct.2020.10.005.

22.

Shalekoff

Tiemessen

. Duration of sample storage dramatically alters expression of the human immunodeficiency virus coreceptors CXCR4 and CCR5. Clin Diagn Lab Immunol. 2001;8(2):432–36. doi:10.1128/CDLI.8.2.432-436.2001.

23.

Paczulla

Rothfelder

Raffel

Konantz

Steinbacher

Wang

Tandler

Mbarga

Schaefer

Falcone

Nievergall

, et al. Absence of NKG2D ligands defines leukaemia stem cells and mediates their immune evasion. Nature. 2019;572(7768):254–59. doi:10.1038/s41586-019-1410-1.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB

0.05 MB

0.08 MB

0.03 MB

0.37 MB

0.32 MB