Lower overall survival in male patients with advanced disease undergoing allogeneic hematopoietic stem cell transplantation is associated with CYP1B1 Leu432Val polymorphism

Abstract

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic key enzyme involved in estrogen metabolism, steroid synthesis, and pro-carcinogen activation. In a single-center retrospective study, 382 patients who underwent allogeneic hematopoetic stem cell transplantation and their donors were genotyped for CYP1B1 C432G polymorphism by reverse transcription polymerase chain reaction. One hundred and sixty-nine patients (44%) were homozygous wild-type (wt) gene CC, 157 (41%) heterozygous CG and 56 (15%) homozygous gene mutated GG. Of interest, mutated CYP1B1 was more common in male (62%) than in female patients (48%) P=0.006, unlike in donors. Five-year estimate for overall survival (OS) was 58±4% (CC) versus 48±3% (CG and GG), P=0.048. Surprisingly, this difference was only evident in males (P=0.024): OS 58±6% versus 42±4%, whereas it was virtually absent in females. Importantly, this difference was only evident in male patients with advanced disease (AD) (n=118, P=0.002): OS 44±8% (CC) versus 32±6% (CG) versus 6±6% (GG), whereas it was virtually absent in male patients with early disease. One-year non-relapse mortality in male patients with AD was 8±4% (CC) versus 21±5% (CG) versus 50±12% (GG), P=0.002. Three-year relapse rate in male patients with AD was 31±7% (wt) versus 42±6% (mut), P=0.04. Multivariate analysis for OS in male patients with AD revealed CYP1B1 polymorphism as the only prognostic factor: RR 1.78, P=0.001. In conclusion, these results suggest that male patients with AD and mutant CYP1B1 polymorphism have lower OS after allogeneic hematopoetic stem cell transplantation due to a higher non-relapse mortality and a higher relapse rate.

Introduction

Hematopoietic stem cell transplantation (HSCT) offers the only cure for many hematological neoplasms; however, the mortality rate remains high. Complications after allogeneic HSCT include relapse, graft-versus-host disease (GVHD), graft rejection, organ damage, and infection. The outcome of HSCT is influenced both by clinical and genetic factors. Human leukocyte antigen (HLA) compatibility is a well-known limiting factor for the success of allogeneic HSCT.¹ In addition, genes other than those of the HLA system (both minor HLA and non-HLA antigens including KIR), in particular those that are also polymorphic, have been shown as potential factors affecting the success of HSCT.² The polymorphic expression of drug-metabolizing enzymes is one of the major factors which causes interindividual variability in drug metabolism, and thereby in pharmacologic and toxicologic responses. Single nucleotide polymorphisms (SNP) within non-HLA genes that are involved with an individual’s capability to mount an immune response to infectious pathogens, residual leukemia, alloantigens or genes involved in drug metabolism, have been studied for their association with acute GVHD,³ host organ function, cancer cells, and immune cells, and thus HSCT outcome.⁴ These include chemokines and cytokines,⁵ and other predictive biomarkers like CYP2C19,⁶ TLR9, NOD2 and IL23R,⁷ TGFB1,⁸ and NOD2/CARD15.^9,10

Human cytochrome P450 1B1 (CYP1B1) is an extrahepatic key enzyme overexpressed in many tumors. It is involved in the production of reactive metabolites, and in the bioactivation of environmental carcinogens.^11-14 CYP1B1 is also involved in sex hormone and estrogen metabolism, and steroid and lipid synthesis. It specifically catalyzes the 4-hydroxylation of 17β-estradiol into 4-hydroxyestradiol, which enables the formation of free radicals that cause DNA damage through redox cycling from compounds such as hydroquinones.¹⁵ The enzyme is primarily found in the endoplasmatic reticulum, and its gene is located on the human chromosome 2p,^16,17 whereas the genes for the HLA system are located on chromosome 6p. The CYP1B1 gene is transcriptionally activated by polycyclic aromatic hydrocarbons, which act via the dioxin inducible Ah receptor complex.¹⁸

CYP1B1 is strongly overexpressed in multiple human malignancies^18,19 and has been used as a target for cancer chemotherapy^20,21 and immunotherapy.^22,23 Several anticancer agents interact with CYP1B1,^20,24 and CYP1B1 inhibitors have been investigated for their anticancer effects.²⁵ CYP1B1 is expressed in human lymphocytes, variable and highly inducible,²⁶ and also in monocytes and macrophages.²⁷ It plays a role in hematopoietic stem and progenitor cell (HSPC) expansion.²⁸ CYP1B1 is also overexpressed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), lymphoma, and myeloma.^29,30 Methylation of CYP1B1 was associated with worse overall survival (OS) and disease-free survival in young adults with ALL and correlated with decreased CYP1B1 expression.³¹ Several polymorphisms were identified in the CYP1B1 gene causing interindividual variability^32,33 and being associated with a variety of cancers.^13,34,35 The CYP1B1 polymorphism C432G = Leu432Val also called rs1056836 causes an amino acid exchange at codon 432, where the C-allele codes for Leu432 (wild-type [wt]), and the G-allele codes for Val432 (variant). Amongst others, this affects the conversion of estradiol to the carcinogenic metabolite 4-hydroxyestradiol.^32,33 Mutant (mut) CYP1B1 Val432Leu is possibly linked with myeloid leukemia.³⁶ We hypothesized that altered polymorphic drug metabolism by CYP1B1 in patients who underwent allogeneic HSCT, might influence host organ function, cancer cells and donor immune cells, and thus transplant outcome. The aim of our study was to evaluate whether the Leu432Val (or C432G) polymorphism of CYP1B1 enzyme in patients influences the outcome of allogeneic HSCT, in particular: OS, non-relapse mortality (NRM), relapse of disease, and incidence of GVHD.

Methods

Patients

We included a total of 382 patients (and donors) for the CYP1B1 gene polymorphism analysis, who were transplanted at the University Hospital of Essen, Department of Bone amended Declaration of Helsinki. All aspects of this study were approved by the Institutional Review Board on Medical Ethics at the University Hospital of Essen (BO 06-3057), and all patients and donors gave their written informed consent.

Marrow Transplantation. Patient characteristics are outlined in Table 1.

Selection of patients was performed purely on the basis that genetic material (peripheral blood) from both the recipient and the donor were available. Genotyping was performed without knowledge of clinical data, or outcome of the patients analyzed. This study was conducted in accordance with the

Table 1.Patient demographics, disease and transplant characteristics.

Early disease versus advanced disease

Early disease (ED): patients with acute leukemia (AL) in first complete remission (CR), chronic myloid leukemia (CML) in first chronic phase, osteomyelofibrosis, severe aplastic anemia, paroxysmal nocturnal hemoglobinuria, hypereosinophilic syndrome, refractory anemia (RA) and refractory anemia with ring sideroblast (RARS) (both untransformed myelodysplastic syndrome [MDS]).

Advanced disease (AD): patients with AL in partial remission (PR) or second CR, secondary AML, relapsed AL, CML with accelerated phase or blast crisis, refractory anemia with excess blasts (RAEB), chronic myelomonocytic leukemia, transformed MDS, multiple myeloma, non-Hodgkin lymphoma, chronic lymphocytic leukemia stage ≥3.

Conditioning regimen and clinical study endpoints

All protocols and procedures are described in detail in the Online Supplementary Appendix.

Isolation of genomic DNA

DNA was prepared from peripheral blood mononuclear cells obtained from the donor and patient before the transplant, using the phenol/chloroform method.⁴³

Genotyping for CYP1B1 codon 432 polymorphism

CYP1B1 Leu432Val polymorphism (rs1056836) was analyzed by a LightCycler (Roche Diagnostics; Mannheim, Germany) protocol using the polymerase chain reaction (PCR) primers and hybridization probes published by Brüning et al.⁴⁴ The primers for the PCR (and the hybridization probes) were synthesized by MWG Biotech (Ebersberg, Germany). Polymorphisms of CYP1B1 at codon 432 were determined by use of the hybridization probe format. The following genotypes were determined by reverse transcription (rt)-PCR: CYP1B*1/*1 = CC (wt), CYP1B*1/*2 = CG, CYP1B*2/*2 = GG (homozygous mut). PCR and subsequent melting curve analysis were performed using the LightCycler device and software (Roche Applied Science; Mannheim, Germany). Control samples confirmed by sequencing were included in each run.

Statistical analysis

Probabilities of OS were calculated using the Kaplan-Meier method.⁴⁵ Differences between time-to-event distribution functions were compared by a log-rank test (SPSS).⁴⁶ Cumulative incidence was used to estimate the endpoints of NRM, relapse and GVHD to accommodate for competing risks (R statistic).⁴⁷ Relapse and NRM were analyzed as competing risks.⁴⁸ Combined data are shown as mean ± standard error. In order to study acute and chronic GVHD, we considered relapse and death to be competing events. Comparisons between categorical covariates were conducted using the χ² test where appropriate. P values are always two-sided. A Cox proportional hazards model was used for multivariate regression to evaluate the prognostic significance of different covariates on the endpoints of OS, NRM, and relapse. Covariates analyzed - see the Online Supplementary Appendix. All interactions between patient sex, AD and other covariates were tested. Relative risk (RR) estimates and their 95% confidence intervals (95% CI) were derived from Cox regression after adjustment for all other covariates in the model. Statistics Software used are presented in the Online Supplementary Appendix.

Results

CYP1B1 genetic polymorphism in patients and donors

Among recipients (R) 169 (44%) were genotyped as homozygous wt gene CC, 157 (41%) as heterozygous genotype CG, and 56 (15%) had the homozygous gene mutation GG. As shown in Table 2, regarding demographic and transplant characteristics, there was no difference between the three CYP1B1 C432G polymorphism groups CC, CG and GG in male patients with AD. With exception to age group >50 years, where there appeared to be a higher percentage of wt R SNP. The same holds true when looking at all male patients and the patient cohort overall (data not shown). Interestingly, male R were more likely to have the mut SNP compared to female R (62% vs. 48%), whereas the wt SNP was more common in females (52% vs. 38%), P=0.006 (x²), see Table 3A. This difference was even more pronounced in AD: the prevalence of mut SNP in male R was 67% versus 50% in female R.

Table 2.Male patients with advanced disease - demographic and transplant characteristics (N=118).

Among donors (D) 167 (44%) were genotyped as homozygous wt gene CC, 162 (42%) as heterozygous genotype CG, and 53 (14%) had the homozygous gene mutation GG. Calculated genotype frequencies were comparable to the R group. Within the donor group however, there was no sex difference in distribution between wt and mut SNP, as shown in Table 3B, in contrast to the R group. Our data with donor CYP1B1 polymorphism or D SNP yielded interesting results. However, D SNP data did not influence the outcome results presented in this paper. Due to clarity and space constraints, they are not included here.

Across all patients with wt R SNP (n=169), 52% of women, and 51% of men had AD (P=0.843). Whereas across all patients with mut R SNP (n=213), 56% of women, and 62% of men had AD (P=0.351). Of all male patients (n=204), mut R SNP was 56% in ED, and 67% in AD (P=0.105). Whereas of all female patients (n=178), mut R SNP was 46% in ED, and 50% in AD (P=0.626).

Overall survival

Across all patients (n=382) the 5-year estimate for OS was 58±4% for the CC group, 50±4% for the CG, and 46±7% for the GG group (P=0.141), whereas it was 58±4% for wt R SNP, and 48±3% for the mut R SNP (CG and GG combined) (P=0.048). Multivariate analysis was performed. In all patients the following factors available at transplant influenced the OS: AD (RR 2.70; 95% CI: 1.94-3.77; P<0.001), mismatch donor (RR 1.64; 95% CI: 1.19-2.27; P=0.003), and age >40 years (RR 1.54; 95% CI: 1.16-2.06; P=0.003), and R SNP (RR 1.22; 95% CI: 1.01-1.49; P=0.044).

Importantly, this difference was evident especially in male patients (n=204) where the group with CYP1B1 mutations did significantly worse: 5-year OS was 58±6% versus 44±5% versus 37±9% (P=0.062) (Figure 1A), whereas it was 58±6% for wt R SNP versus 42±4% for mut R SNP (P=0.024). In sharp contrast, OS of female patients was almost identical between the three groups (5-year OS: 58±5% vs. 58±6% vs. 58±10%) (Figure 1B). Multivariate analysis was performed. In male patients only the following factors available at transplant influenced OS: AD (RR 2.52; 95% CI: 1.59-4.00; P<0.001), R SNP (RR 1.46; 95% CI: 1.12-1.91; P=0.005), and age >40 years (RR 1.50; 95% CI: 1.03-2.19; P=0.034).

Interestingly, this difference in survival rate was observed primarily in male patients with AD (n=118), where the group with CYP1B1 mutations did significantly worse: their 5-year OS was 44±8% versus 32±6% versus 6±6% (P=0.002) (Figure 2A). In contrast, regarding male patients with ED, OS was almost identical between the three groups (5-year OS: 74±7% vs. 69±8% vs. 69±12%) 95% CI: 0.16-0.89; P=0.026) with PBSC being associated with a lower relapse rate.

Table 3.CYP1B1 polymorphism (Leu432Val) of recipients (A) and donors (B), N=382.

(Figure 2B). Multivariate analysis was performed (Table 4). In male patients with AD, only the following factor available at transplant influenced OS: R SNP with RR 1.78; 95% CI: 1.25-2.52; P=0.001.

Non-relapse mortality

One-year estimate for NRM in male patients with AD was 8±4% for wt/wt versus 21±5% for mut/wt versus 50±12% for mut/mut CYP1B1 R SNP (P=0.002) (Figure 3A). In contrast, there was no difference in the outcome concerning male patients with ED (NRM 13% vs. 13% vs. 19%) (Figure 3B). Multivariate analysis was performed (Table 4). In male patients with AD, only the following factor influenced NRM: CYP1B1 R SNP (RR 1.98; 95% CI: 1.21-3.25; P=0.007). Causes of death (n=15) in the GG group (n=16) of male patients with AD were: relapse n=6, sepsis n=4, invasive fungal infection n=3, and veno-occlusive disease n=2 patients. When looking at the R SNP group GG in male patients with AD, there was no pattern with regards to clinical features (see Table 2), other outcomes or causes of death compared to group CG and group CC. Relapse and infections were a common cause of death.

Occurrence of relapse

When looking at relapse, CYP1B1 genetic polymorphism in patients (R SNP) at first did not seem to make a difference in either sex. However, in male patients with AD (n=118), mut R SNP was associated with more relapses at 3 years than wt R SNP: 31±7% versus 43±6% versus 38±12% (P=0.08) (Figure 4A). When combining wt/mut and mut/ mut the numbers were 31±7% versus 42±6% (P=0.04). In contrast, male patients with ED did not show this phenomenon (relapse 24% vs. 16% vs. 19%) (Figure 4B). This was confirmed by multivariate analysis in male patients with AD (Table 4) where R SNP was the strongest predictor for relapse (RR 1.92; 95% CI: 1.21-3.07; P=0.006), with mut R SNP performing worse, and graft type (RR 0.38;

Figure 1.Overall survival estimate by CYP1B1 polymorphism. (A) Male patients (N=204): patients with mutant (mut) recipient single nucleotide polymorphism (R SNP) (wt/mut and mut/mut) had lower overall survival (OS) than patients with wild-type (wt) R SNP (wt/wt). (B) Female patients (N=178) displaying no difference (not significant [NS]). R SNP here CYP1B1 C432G polymorphism CC, CG and GG. HSCT: hematopoietic stem cell transplantation.

Figure 2.Overall survival estimate in male patients by CYP1B1 polymorphism. (A) Male patients with advanced disease (N=118):

Figure 3.Non-relapse mortality estimate in male patients by CYP1B1 polymorphism. (A) Male patients with advanced disease (N=118): male patients with mutant (mut) recipient single nucleotide polymorphism (R SNP) had a higher non-relapse mortality (NRM) than those with wild-type (wt) R SNP. (B) Male patients with early disease (N=86) displaying no difference (not significant [NS]). HSCT: hematopoietic stem cell transplantation.

Figure 4.Relapse estimate in male patients by CYP1B1 polymorphism. (A) Male patients with advanced disease (N=118). Male patients with mutant (mut) recipient single nucleotide polymorphism (R SNP) had a higher relapse rate than those with wild-type (wt) R SNP. (B) Male patients with early disease (N=86) displaying no difference (not significant [NS]). HSCT: hematopoietic stem cell transplantation.

Acute and chronic graft-versus-host disease

In male patients with AD (n=118) the estimated incidence of acute GVHD, grade 3-4 on day 30 post HSCT was 3±3% versus 19±6% versus 40±14% for R SNP (P=0.177). Whereas for male patients with early disease it was 12% versus 13% versus 7% (P=0.633). Although displaying a trend, the numbers were relatively small and not significant. R SNP was also not significant in multivariate analysis for severe acute GVHD. The incidence of chronic GVHD (all stages) was similar in the three R subgroups (R SNP), with 64% in CC, 60% in CG, and 44% in GG (χ², P=0.367) in male patients with AD. R SNP was also not significant in multivariate analysis for all stages of chronic GVHD.

CYP1B1 is not involved in metabolism of immunosuppressants like cyclosporine, and there was no difference observed between the three subgroups of R SNP when comparing cyclosporine or cortisone doses upon discharge.

For the role of sex mismatch on outcome please see the Online Supplementary Appendix.

Engraftment, kidney and liver toxicity

For 198 of 382 patients (52%) additional data was available. This was an unselected group. Male patients with AD, 57 of 198 (29%) displayed no significant difference in engraftment of neutrophils or thrombocyytes between wt and mut R SNP. We also evaluated the maximum creatinine and bilirubin values after transplant. While there was a tendency towards higher mean (not median) creatinine and bilirubin values in patients with mut R SNP, this difference was small and by no means significant.

Discussion

In this retrospective single-center study we evaluated the influence of CYP1B1 polymorphism (Leu432Val) on the clinical outcome after allogeneic HSCT in adult patients. We found that mut SNP is associated with lower OS in male patients with AD due to increased non-relapse mortality, and a higher relapse rate after HSCT. This outcome difference in male patients is not explained by other prognostic factors available at time of transplant. In contrast, this association has not been seen in male patients with ED or in female patients. Of note, the above has been found in a primarily myeloablative setting.

Interestingly, male patients were more likely to have the mut SNP of CYP1B1 compared to female patients (62% vs. 48%). However, in contrast to the R group (which had hematological cancer - therefore, had also been exposed to anticancer drugs and radiation), there was no sex difference in distribution between wt and mut SNP within the donor group. Calculated Leu432Val (or C432G) genotype frequencies of CYP1B1 did not differ much from that reported earlier for Caucasians: CC 37%, CG 46% and GG 17%.^44,50

It could therefore be postulated that men with mut SNP have a higher risk of hematological cancer, or are more likely to relapse post induction and consolidation therapy (and thus become transplant candidates more often) than men with wt SNP. This conclusion is corroborated by Tang et al.⁵¹ who found that among Caucasians 34% of men with prostate cancer were homozygous for the Val432 polymorphism (or GG in other words), while only 12% of matched control subjects had this genotype. Also by Fritsche et al.⁵⁰ who reported that genotypes heterozygous and homozygous for the Val432 polymorphism (i.e., CG and GG or mut SNP) were more frequent in patients with colorectal cancer compared to controls.

Table 4.Results of multivariate analysis for main outcomes after hematopoietic stem cell transplantation - male patients with advanced disease only (N=118).

Of interest, even though wt SNP was more common in female patients, it does not appear to be associated with a better outcome. Whereas mut SNP appears to have a clear negative effect in men with AD. Female patients with mut SNP seem to have a better outcome than their male counterparts. It could be speculated that this is due to the presence of estrogens. Sissung et al.⁵² made a similar observation in male patients with prostate cancer: patients with CYP1B1*3 polymorphic variant (L432V) GG had a decreased OS after docetaxel-based therapies compared with individuals carrying at least one copy of the CYP1B1*1 (allele CC or CG).

NRM was higher in male patients with mut SNP compared to male patients with wt SNP in the AD setting, whereas this difference did not show at all in female patients. An explanation for the NRM increase in male patients with mut SNP could be a higher rate of organ toxicity or more infections due to chemotherapy, radiotherapy or immunotherapy.

Relapse was higher in male patients with AD for mut SNP compared to wt SNP. This could be due to a poor response of cancer cells with mut SNP to chemotherapy, radiotherapy, and immunotherapy, thus leading to a higher relapse rate in patients with AD.

Of note, most patients analyzed received myeloablative transplants in the years 1993-2006, median 2002. The field of transplantation has changed over the years due to advances in donor selection and supportive care, i.e., infection disease and GVHD control, resulting in improved survival.

The sex differences observed in this study comparing wt and mut SNP in recipients were striking. So far, sex differences in outcome after allogeneic HSCT have not been reported with the exception of increased rates of chronic GVHD for female donors and male recipients.⁵³ T cells of female donors specific for minor histocompatibility antigens encoded by genes on chromosome Y may contribute to GVHD, graft rejection, and graft-versus-leukemia effects in hematological malignancies.⁵⁴

In conclusion, our retrospective single-center cohort study provides strong evidence that male patients with mut genetic polymorphism of CYP1B1 have a lower OS in AD after HSCT, and that this is due to a higher NRM and relapse rate.

Genotyping for CYP1B1 C432G polymorphism might therefore help identify patients with higher risk for allogeneic HSCT in the future, and may perhaps also explain some sex differences in outcome. Validation of the role of CYP1B1 C432G polymorphism in a prosppective study or confirmation in an independent cohort are warranted before any clinical conclusions are drawn. We also suggest further in vitro and in vivo studies on hematological cancers and lymphocytes regarding the role of CYP1B1 and its Leu432Val polymorphism.

Footnotes

• Received June 1, 2023
• Accepted September 18, 2023

Correspondence

M. Koldehoff koldehoff@zotzklimas.de

Disclosures

No conflicts of interest to disclose.

Contributions

NS was responsible for the analysis of data, interpretation of results, and literature research. He performed the statistical analyses, created tables and figures, and wrote the manuscript. MK was responsible for the design of the study and conduct of the research. He obtained funds, designed the laboratory protocol, supervised experiments, and extracted and collected both clinical and lab data. He also revised and edited the manuscript. Both authors approved the final version of the manuscript.

Data-sharing statement

The data supporting the findings of this study are available upon request from the corresponding author.

Funding

This work was supported by a grant from Deutsche Kulturstiftung Essen e.V. - research project (05 045).

Acknowledgments

We thank Christiane Schary, Melanie Kroll, Silke Gottwald, and Ines Riepenhoff (all Essen, Germany) for their excellent technical assistance with PCR analyses. We also wish to thank patients and donors who participated in this study, as well as our colleagues, and the entire medical team at the BMT Center in Essen, especially Prof. Dr. Ahmet H. Elmaagacli. The EBMT statistics course in Leiden 2018 was very beneficial for implementing R. As a native English speaker Rachael Mellor assisted with grammar.