Gene polymorphisms in the cyclophosphamide metabolism pathway correlate with complications after haplo-HSCT

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is used as curative treatment for patients with hematologic malignancies who do not have a full-matched donor. Posttransplant cyclophosphamide is used as a prophylactic treatment to prevent graft-versus-host disease (GvHD) after haplo-HSCT. Cyclophosphamide is an inactive prodrug that is metabolized by polymorphic enzymes in the liver to produce phosphoramide mustard, a DNA alkylating agent with antineoplastic and immunosuppressive activities.¹

During the 47th Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT), Paula Muñiz, Gregorio Marañón General University Hospital, Madrid, ES, presented a retrospective study aimed at identifying polymorphisms in genes of the cyclophosphamide metabolism pathway that correlate with post-HSCT complications, including GvHD, transplant-related mortality (TRM), sinusoidal obstruction syndrome (SOS), and hemorrhagic cystitis (HC).¹

Study design and patient characteristics

182 consecutive patients that had received a haplo-HSCT with posttransplant cyclophosphamide (50mg/kg on Days 3 and 4) at the Gregorio Marañón General University Hospital between 2007–2019 were included in the analysis. Treatment schema for this patient population can be seen in Figure 1. Patients received either myeloablative conditioning or reduced intensity conditioning prior to haplo-HSCT.

GvHD, graft-versus-host disease; Haplo-HSCT, haploidentical hematopoietic stem cell transplantation; IV, intravenous; MMF, mycophenolate mofetil.

*Adapted from Paula Muñiz et al., 2021¹

 A custom panel of 11 genes relating to cyclophosphamide metabolism were analyzed by genotyping in a MiSeq platform.

• Variations located in the coding and splicing sites were analyzed.
• Polymorphisms that corresponded to a read depth ≥30X in the canonical isoform with an allele frequency greater than 40%, and represented in at least 5% of the cohort, were selected.

Patient characteristics can be seen in Table 1.

• Approximately half of the patients had acute myeloid leukemia or non-Hodgkin lymphoma, and in total, 57% were in complete remission pretransplant.
• Posttransplant complications were: 41% of patients developed Grade II-IV acute GvHD (aGvHD), 35% developed global chronic GvHD (cGvHD), 9% developed SOS, and 25% developed HC.

Table 1. Patient characteristics*

aGvHD, acute graft-versus-host disease; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; cGvHD, chronic graft-versus-host disease; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; HC, hemorrhagic cystitis; HL, Hodgkin lymphoma; MDS, myelodysplastic syndrome; MM, multiple myeloma; NHL, non-Hodgkin lymphoma; SOS, sinusoidal obstruction syndrome. *Adapted from Paula Muñiz et al., 20211
Characteristic	N = 182
Median age of recipient, years (range)	48 (16–67)
Median age of donor, years (range)	40 (14–74)
Sex of recipient, male/female %	67.03/32.97
Sex of donor, male/female %	54.95/45.05
Diagnosis, %
AML	35.71
NHL	13.74
ALL	10.44
MDS	9.89
MM	2.75
HL	11.54
Other (aplastic anemia, CLL, CML)	15.93
Pretransplant status, %
Active disease/partial response	43.41
Complete remission	56.6
Conditioning regimen, %
Myeloablative	45.05
Reduced intensity	54.95
Prior transplant, %	35.16
Serum ferritin levels >1,648 (µg/mL), %	50.54
Busulfan >1.5 days, %	80.76
aGvHD, %
Grade II–IV	41.20
Grade III–IV	13.19
cGvHD, %
Global	35.16
Extensive	18.68
SOS, %	9.34
HC, %	24.73

Results¹

In total, 35 polymorphisms across nine genes were found. The 19 single nucleotide polymorphisms (SNPs) that demonstrated a correlation with post-haplo-HSCT complications by univariate analysis are shown in Table 2.

• SNPs associated with a decrease in activity of cyclophosphamide activation enzymes (cytochrome P450) were associated with a higher incidence of GvHD, TRM, and SOS.
• SNPs associated with a decrease in activity of detoxification enzymes (glutathione S-transferases) were associated with a higher incidence of severe GvHD, TRM, and SOS.

Table 2. Univariate analysis of SNPs associated with posttransplant complications^*

aGvHD, acute graft-versus-host disease; cGvHD, chronic graft-versus-host disease; HC, hemorrhagic cystitis; Mod-sev, moderate to severe; OR, odds ratio; SNP, single nucleotide polymorphism; SOS, sinusoidal obstruction syndrome; TRM, transplant-related mortality. *Adapted from Paula Muñiz et al., 20211
Gene		SNP	Modulation of enzyme activity	Variant effect	Posttransplant complication, OR (p value)
Activation	CYP2A6	rs4986892	Unknown	Synonymous	cGVHD 0.38 (0.02) SOS 5.17 (0.03)
rs1801272	Loss of function	Missense	II–IV aGvHD 2.74 (0.03) cGvHD 3.86 (0.04) Mod-sev cGvHD 7.28 (0.0002)
rs143731390	Decrease	Missense	TRM 3.1 (0.01)
CYP2B6	rs3745274	Decrease	Missense	Mod-sev cGvHD 0.37 (0.01)
rs3211371	Decrease	Missense	II–IV aGvHD 2.46 (0.01)
rs2279341	Unknown	Synonymous	cGvHD 2.11 (0.03)
rs2279343	Unknown	Missense	HC 3.16 (0.03)
rs3745274(wt)	—	—	SOS 1.6 (0.002)
CYP2C8	rs10509681	Decrease	Missense	II–IV aGvHD 1.59 (0.01) III–IV aGvHD 4.07 (0.02)
rs11572080	Decrease	Missense	II–IV aGvHD 2.41 (0.04) III–IV aGvHD 4.12 (0.03)
CYP2C9	rs1799853	Decrease	Missense	II–IV aGvHD 1.67 (0.03) III–IV aGvHD 4.35 (0.02)
CYP2C19	rs4244285	Decrease	Synonymous	TRM 2.45 (0.01)
rs3758580	Unknown	Synonymous	TRM 2.02 (0.04)
Detoxification	GSTA1	rs1051775	Unknown	Synonymous	III–IV aGvHD 0.46 (0.003) TRM 1.86 (0.04)
GSTA1*B	Decrease	Missense	III–IV aGvHD 2.53 (0.01) TRM 1.43 (0.03)
GSTM1	rs1065411	Unknown	Missense	TRM 1.94 (0.03)
GTSM1*0	Loss of function	Null allele	SOS 2.3 (0.04)
GSTP1	rs1659	Decrease	Missense	III–IV aGvHD 2.77 (0.04)
GSTT1	GSTT1*0	Loss of function	Null allele	III–IV aGvHD 2.62 (0.01)

Multivariate analysis revealed that eight polymorphisms in five genes were associated with posttransplant complications (Table 3).

• SNPs associated with a decrease in activity of activation enzymes, were associated with a higher incidence of GvHD.
• SNPs that were associated with a decrease in activity of detoxification enzymes were associated with a higher incidence of severe GvHD, TRM, and SOS.

Table 3. Multivariate analysis of SNPs associated with posttransplant complications^*

aGvHD, acute graft-versus-host disease; cGvHD, chronic graft-versus-host disease; HS, hemorrhagic cystitis; Mod-sev, moderate to severe; SHR, subdistribution hazard ratio; SNP, single nucleotide polymorphisms; SOS, sinusoidal obstruction syndrome; TRM, transplant-related mortality. *Adapted from Paula Muñiz et al., 20211
Gene		SNP	Modulation of enzyme activity	Variant effect	Posttransplant complication, SHR (p value)
Activation	CYP2A6	rs143731390	Decrease	Missense	Mod-sev cGvHD 3.44 (0.003)
	CYP2B6	rs3745274	Decrease	Missense	Mod-sev cGvHD 0.38 (0.02)
		rs3211371	Decrease	Missense	II–IV aGvHD 2.02 (0.008)
Detoxification	GSTA1	rs1051775	Unknown	Synonymous	III–IV aGvHD 0.42 (0.042) TRM 2.56 (0.004)
		GSTA1*B	Decrease	Missense	TRM 2.32 (0.036)
	GSTM1	rs1065411	Unknown	Missense	TRM 2.13 (0.01)
		GTSM1*0	Loss of function	Null allele	SOS 1.36 (0.032)
	GSTT1	GSTT1*0	Loss of function	Null allele	III–IV aGvHD 3.29 (0.005)

Conclusions

Genetic variation in genes involved in the cyclophosphamide metabolism pathway correlated with several post-haplo-HSCT complications, most notably GvHD, but also TRM, and SOS. Only one SNP correlated with HC in the univariate analysis. Therefore, the analysis of these variants before haplo-HSCT transplant could facilitate personalized risk approaches and clinical management of patients with hematologic malignancies. However, these results need to be validated in other patient cohorts.