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

Jun 15, 2021
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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.

Figure 1. Treatment schema*

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*

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

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

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*

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)

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

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*

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)

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 

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. 

  1. Muñiz P, Andrés-Zayas C, Carbonell D, et al. Association of gene polymorphisms in cyclophosphamide metabolism pathway with complications after haploidentical hematopoietic stem cell transplantation. Oral abstract #OS9-8. 47th Annual Meeting of the EBMT; Mar 14, 2021; Virtual.

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