Association of HMGB1 Polymorphisms with Outcome after Allogeneic Hematopoietic Cell Transplantation

Article Outline

• Abstract
• Introduction
• Materials and Methods
  • Patients
  • Genotyping
  • Statistics
• Results
  • Genetic Variation in the HMGB1 Gene
  • Transplantation Outcome
  • Association of Patient HMGB1–1377delA Genotype with Relapse
  • Association of Patient HMGB1 3814C > G, 1177 G > C and 2351insT Genotype with OS, PFS, and aGVHD
  • Association of Donor HMGB1 3814C > G, 1177G > C, and 2351insT Genotype with cGVHD
• Discussion
• Aknowledgments
• References
• Copyright

Several studies have demonstrated that genetic variation in cytokine genes can modulate the immune reactions after allogeneic hematopoietic cell transplantation (HCT). High mobility group box 1 protein (HMBG1) is a pleiotropic cytokine that functions as a pro-inflammatory signal, important for the activation of antigen presenting cells (APCs) and propagation of inflammation. HMGB1 is implicated in the pathophysiology of a variety of inflammatory diseases, and we have recently found the variation in the HMGB1 gene to be associated with mortality in patients with systemic inflammatory response syndrome. To assess the impact of the genetic variation in HMGB1 on outcome after allogeneic HCT, we genotyped 276 and 146 patient/donor pairs treated with allogeneic HCT for hematologic malignancies following myeloablative (MA) or nonmyeloablative (NMA) conditioning. Associations between genotypes and outcome were only observed in the cohort treated with MA conditioning. Patient homozygosity or heterozygosity for the–1377delA minor allele was associated with increased risk of relapse (hazard ratio [HR] 2.11, P = .02) and increased relapse related mortality (RRM) (P = .03). Furthermore, patient homozygosity for the 3814C > G minor allele was associated with increased overall survival (OS; HR 0.13, P = .04), progression free survival (PFS; HR 0.30, P = .05) and decreased probability of RRM (P = .03). Patient carriage of the 2351insT minor allele reduced the risk of grade II to IV acute graft-versus-host disease (aGVHD) (HR 0.60, P = .01), whereas donor homozygosity was associated with chronic GVHD (cGVHD) (HR 1.54, P = .01). Our findings suggest that the inherited variation in HMGB1 is associated with outcome after allogeneic HCT following MA conditioning. None of the polymorphisms were associated with treatment-related mortality (TRM).

Key Words: Genetic polymorphism, High mobility group box 1 protein, HMGB1, Allogeneic hematopoietic cell transplantation

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Introduction 

An essential feature of allogeneic hematopoietic cell transplantation (HCT) is the interaction between antigen-presenting cells (APCs) and donor T lymphocytes. In the context of acute graft-versus-host disease (aGVHD) following allogeneic HCT with myeloablative (MA) conditioning the pathophysiology of the initial immunoreactions have been conceptualized into a 3-phase model where tissue damage and release of pro-inflammatory cytokines, caused by total body irradiation (TBI) and/or chemotherapy of the conditioning regimen, activates APCs leading to subsequent proliferation of T lymphocytes enabling them to damage target cells [1].

The origin of the APCs is thought to be important in relation to their ability to induce GVHD or the graft-versus-tumor (GVT) effect. Patient APCs are critical for the development of aGVHD and the GVT effect 2, 3, 4, whereas donor APCs are important for the development and perpetuation of chronic GVHD (cGVHD) 5, 6. In allogeneic HCT after nonmyeloablative (NMA) conditioning, the mostly immunosuppressive conditioning regimen is associated with lower peritransplant toxicity compared to MA conditioning 7, 8. In NMA conditioning, tumor eradication mainly depends on the immunologic GVT effect, and lower incidence of disease progression has been associated with the development of cGVHD, independently of aGVHD [9].

High mobility group box 1 protein (HMGB1) is an ubiquitously expressed [10], highly conserved [11], 25-kDa DNA binding protein [12] that has been identified as an endogenous damage-associated molecular pattern (DAMP), whih could be detected in serum hours after an initial septic insult [13]. HMGB1 has been implicated in the pathology of a wide variety of infectious 13, 14, 15, 16, noninfectious 17, 18, and autoimmune diseases, such as primary Sjögren's disease and systemic sclerosis 19, 20. In cancer, HMGB1 has been observed to influence immunogenicity of antigens presented by APCs [21], and overexpression of HMGB1 has been associated with proliferation of cancer cells, metastasis, and a generally worse prognosis 22, 23, 24.

HMGB1 diffuses freely from necrotic cells and is tightly sequestered in the nucleus of apoptotic cells, providing an endogenous danger signal for the organism to distinguish between programmed and nonprogrammed cell death [25]. Extracellular HMGB1 exhibits inflammatory cytokine-like activity and acts as a potent mediator of APC activation 26, 27, 28 and proliferation of T cells 26, 29. As HMGB1 is secreted by activated immune cells in response to pro-inflammatory cytokines, it has an ability to self-amplify and prolong inflammation 13, 30. Other functions of extracellular HMGB1 are abrogation of the epithelial barrier function 31, 32, 33 and promotion of tissue repair and regeneration 34, 35, 36

The human HMGB1 gene consists of 5 exons, and is located on the short arm of chromosome 13 [37]. We have identified several polymorphic loci throughout the gene [38], and, in a recent study of patients with systemic inflammatory response syndrome (SIRS) admitted to an intensive care unit, we showed that the–1377delA and 982C > T polymorphisms were significant risk factors for late and early mortality, respectively [39].

Genetic variation in cytokine genes can modulate the immune reactions after allogeneic HCT and hereby influence the outcome [40]. Because of the pro-inflammatory nature of HMGB1, its central role in the activation of APCs and our observation that HMGB1 polymorphisms were associated with outcome in SIRS patients, we hypothesized that the HMGB1 genotype of either recipients or donors could influence the risk of GVHD, relapse, and death in patients treated with allogeneic HCT. The objective of the current study was, therefore, to analyze the association between HMGB1 genotype and outcome in patients treated with allogeneic HCT after 2 different conditioning regimens, namely, MA conditioning, characterized by a high inflammatory milieu posttransplant, and NMA conditioning, where the immediate period posttransplant is characterized by a lower state of inflammation.

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Materials and Methods 

Patients 

The study population consisted of 2 independent cohorts undergoing allogeneic HCT. One of 276 patients treated with MA conditioning between January 1990 and November 2007, and a second of 146 patients treated with NMA conditioning between March 2000 and July 2007. Both cohorts were treated for malignant hematologic disease at the Bone Marrow Transplantation Unit at Rigshospitalet, Copenhagen, Denmark. The inclusion criteria were the availability of pretransplantation blood samples from either recipients or donors. Patient demographics are listed in Table 1.

Table 1. Pretransplantation Demographics

Variable	Myeloablative Cohort (N = 276)	Nonmyeloablative Cohort (N = 146)
Patient age, median years (range)	28 (0.7-57)	53 (19-69)
< 16 years, no. (%)	78 (28)	0
16–40 years, no. (%)	118 (43)	21 (14)
> 40 years, no. (%)	80 (29)	125 (86)
Donor age, median years (range)	35 (0.7-62)	44 (19-68)
< 16 years, no. (%)	33 (12)	0
16–40 years, no. (%)	148 (54)	50 (34)
> 40 years, no. (%)	89 (32)	91 (63)
Not available	6 (2)	5 (3)
Type of donor
Matched related, no. (%)	138 (50)	86 (59)
Mismatched related, no. (%)	11 (4)	0
Matched unrelated, no. (%)	97 (35)	49 (34)
Mismatched unrelated, no. (%)	30 (11)	11 (8)
Sex of patient / donor
Male / female, no. (%)	50 (18)	34 (23)
Other combinations, no. (%)	226 (82)	112 (77)
Underlying disease
Low risk, no. (%)	0	30 (21)
Standard risk, no. (%)	95 (34)	71 (48)
High risk, no. (%)	181 (66)	45 (31)
Conditioning regimen
Cyclophosphamide and 12Gy TBI, no. (%)	155 (56)	0
Etoposide and 12Gy TBI, no. (%)	51 (18)	0
Busulfan and cyclophosphamide, no. (%)	49 (18)	0
Other conditioning regimens, no. (%) (20 included cyclophosphamide)	21 (8)	0
Fludarabine and 2Gy TBI, no. (%)	0	143 (98)∗
2Gy TBI, no. (%)	0	3 (2)
Use of ATG in conditioning
No ATG, no. (%)	165 (60)	146
ATG, no. (%)	111 (40)	0
Stem cell source
Peripheral blood, no. (%)	186 (67)	146
Bone marrow, no. (%)	90 (33)	0
Immuno suppression
Cyclosporine alone, no. (%)	41 (15)	—
Methotrexate, and cyclosporine, no. (%)	234 (85)	—
Mycophenolate mofetil and cyclosporine or tacrolimus, no. (%)	—	146
None	1	—
CMV serostatus of patient / donor
CMV-negative / CMV-negative	69 (25)	31 (21)
Other combinations	203 (74)	115 (79)
Inconclusive CMV serology	4 (1)	0
Pre-transplantation Karnofsky score
100 - 80, no. (%)	260 (94)	68 (47)
< 80, no. (%)	16 (5)	3 (2)
Not available	0	75 (51)

TBI indicates total body irradiation; CMV, cytomegalovirus; ATG, antithymocyte globulin.

In the cohort treated with myeloablative conditioning, standard risk was defined as acute leukemia in first remission and chronic leukemia in first chronic phase, whereas all other diseases and stages were considered high risk. The diseases in recipients treated with nonmyeloablative conditioning were classified as low, standard, or high risk according to Kahl et al. [67].

In the MA conditioning cohort, DNA was available from 275 patients and 274 donors, whereas DNA from all patients and donors was available in the NMA conditioning cohort. With improving HLA-typing techniques the definition of matched unrelated donors has changed from 1990 to 2007. Patient-donor pairs from the MA conditioning cohort that were HLA-typed early in the study period were typed by a combination of low-resolution serological typing for HLA-A and HLA-B and high-resolution genotyping for DRB1, whereas patient donor pairs HLA-typed later in the study period were high-resolution genotyped for HLA-A, -B, -C, and DRB1 and DQB1. All patients and donors in the NMA conditioning cohort were matched with high-resolution HLA-typing for HLA-A, -B, -C, DRB1, and DQB1, and only a single HLA allele disparity was allowed. For the purpose of the current study, a matched unrelated donor was defined, as a donor fully HLA matched using the typing methods available at the time of transplantation, realizing that the group of patient donor pairs partially matched with serological techniques could include high-resolution mismatches.

The diagnoses in the MA conditioning cohort included acute lymphoblastic leukemia (ALL; n = 92), acute myelogenous leukemia (AML; n = 93), chronic myelogenous leukemia (CML; n = 65), and other hematologic malignancies (n = 26). Acute leukemias in first remission and CML in first chronic phase (CP1) were classified as standard risk disease, whereas all other diagnoses or disease stages as high risk. The diagnoses in the NMA conditioning cohort included AML/myelodysplastic syndrome (MDS; n = 62), non-Hodgkin lymphoma (NHL; n = 30) follicular lymphoma, (FL; n = 19); diffuse large B-cell lymphoma, (DLBCL; n = 4); mantle cell lymphoma, (n = 4); peripheral T-cell lymphoma (n = 3), chroni lymphocytic leukemia (CLL; n = 21), multiple myeloma (MM; n = 15), Hodgkin disease (HD; n = 15), and CML (n = 3). In the MA conditioning cohort, 7 patients transplanted with a related donor received additional immunosuppression with antithymocyte globulin (ATG), whereas all, except 23, transplanted with an unrelated donor received ATG. ATG was administered in the form of ATGAM (20 mg/kg/day) or Thymoglobulin (2.5 mg/kg/day) for 3 days (day–5, −4, and–3 before transplantation). One patient in the MA conditioning cohort received an ex vivo T lymphocyte-depleted bone marrow (BM) from a fully HLA-matched related donor. The conditioning regimen and prophylactic antibiotics employed in the NMA conditioning cohort has been described previously [41]. aGVHD and cGVHD were diagnosed according to standard criteria 42, 43.

Informed consent was obtained from all patients and the local ethics committee approved the study.

Genotyping 

Reference single nucleotide polymorphism numbers (rs) are provided for all polymorphisms. The –1615A > G (rs1412125), −1377delA (rs41369348), 1747delT (rs55946320), 1888insT (rs41497949), and 2351insT (rs41376448) polymorphisms were genotyped by direct sequencing as previously described [39]. The 3814C > G (rs2249825), 982C > T (rs1060348), and 1177 G > C (rs3742305) polymorphisms were genotyped using a 12-plex format GenomeLab SNPstream Genotyping system (Beckman Coulter Inc., Fullerton, CA). For each of the polymorphism genotyped by the SNPstream system, the genotypes, in 10 to 20 samples, were validated by direct sequencing as previously described [38]. Figure 1 is a schematic drawing of the HMGB1 gene locus, showing the location of the genetic variants in relation to each and their exons. For each genetic variant, the allele with the lowest frequency will be referred to as the minor allele, whereas the allele with the highest frequency will be referred to as the major allele (Table 2).

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• Figure 1 

  Schematic illustration of the high mobility group box 1 protein gene locus with exon I to V, marked as solid boxes, and the approximate location of the genetic variants. The most common inferred haplotypes (frequency >3%), called H1 to H4, in patients and donors are shown. Modified from Ferrari et al. [37].

Table 2. Distribution of Genotypes

Rs number indicates reference single nucleotide polymorphism number.

Observed frequencies of HMGB1 genotypes in patients and donors according to type of conditioning. There were no significant differences in genotype frequencies between patients and donors in their respective cohorts (all P > .05). The minor alleles were defined as the alleles with the lowest frequencies, whereas the major alleles were defined as the alleles with the highest frequency.

Statistics 

The inferred haplotypes and linkage disequilibrium (LD), expressed as D'Lewontin's coefficient and squared correlation (R²) coefficient quantified between all pairs of biallelic loci, were estimated using SNPAlyze version 4.0 (Dynacom, Yokohoama, Japan). The Hardy-Weinberg equilibrium (HWE) was analyzed using gene frequencies obtained by simple gene counting and the chi-squared test with Yates' correction. Fisher's exact test was used to compare frequencies. To maximize power, inferred haplotypes, LD and HWE, were analyzed separately for patient and donor groups, with each group consisting of patients or donors from the MA conditioning and NMA conditioning cohorts. As the 982C > T polymorphism has been previously associated with mortality [39], association analyses were performed despite a low minor allele frequency, whereas the 1888insT and 1747delT polymorphisms were excluded from further analyzes because of minor allele frequencies <5 % (Table 2). Because of sporadic failed genotypes during genotyping lowering the number of possible full haplotypes (Table 2), association analyzes between haplotypes and transplantation outcome were not performed. Cox regression was used to estimate the association between HMGB1 genotypes and overall survival (OS), progression free survival (PFS), relapse incidence (RI), relapse-related mortality (RRM), treatment-related mortality (TRM), grade II to IV aGVHD, grade III to IV aGVHD, limited or extensive cGVHD, and extensive cGVHD. OS was measured from the time of transplantation until death from any cause. Patients still alive at the time of analysis were censored at the date of last follow-up. PFS was calculated from date of transplantation to date of first relapse or death. Patients who were alive and in remission where censored at date of last follow-up. TRM was defined as death in complete remission or death related to transplantation where it was not possible to assess disease status before death. RRM was defined as death during relapsed or progressive disease. Probability of OS and PFS were estimated by the Kaplan-Meier method, and comparisons were made with the log-rank test. In the calculation of cumulative incidences for RI, RRM, TRM, and GVHD, death before relapse, death with or without relapse, death without GVHD, and retransplantation were handled as competing events, where appropriate in these analyzes [44]. Comparisons of cumulative incidences were performed using Gray's k-sample test [45]. For the purpose of Cox regression, competing events were censored. Multivariate Cox regression models were used to evaluate the risk associated with HMGB1 polymorphisms on transplantation outcome. Each covariate listed in Table 1 and grade 2-4 aGVHD and limited or extensive cGVHD as posttransplant time-dependent covariates, were entered 1 by 1 in a pair wise model together with HMGB1 genotype. The covariates were kept in the final model if they remained significant (P ≤ .05) or altered the association with the HMGB1 variant by more than 10%. All P-values were 2-tailed, and P ≤ .05 was considered significant.

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Results 

Genetic Variation in the HMGB1 Gene 

The HWE, LD, and haplotype inference was analyzed for recipients and donors separately in a combined MA conditioning and NMA conditioning cohort. All polymorphisms adhered to the HWE (P > .05), except for 1747delT (P = .03) because of 1 individual homozygous for the minor allele, which was confirmed by both forward and reverse sequencing. In the pairwise investigation of LD between the 8 polymorphic loci the strongest squared correlation coefficients were between 3814C > G, 1177 G > C, and 2351insT (R² 0.7869- 0.9339) in both recipients and donors (data not shown). Of 55 and 42 possible inferred haplotypes, the 4 most common accounted for 90% of the chromosomes in both donors and recipients (Figure 1). There were no significant differences in the distribution of genotype frequencies between recipients and donors in the separate MA conditioning and NMA conditioning cohorts (Table 2).

Transplantation Outcome 

In the cohort of patients who underwent MA conditioning HCT, the median follow-up time was 1190 days (range: 10-3653 days). Five-year OS, PFS, day 100 probability of grade II to IV, and 3 years probability of cGVHD were 68%, 58%, 40%, and 40%, respectively. In the NMA conditioning HCT cohort, the median follow-up was 887 days (range: 30-3229), and the 5 years OS, PFS, 1-year probability of grade II to IV aGVHD, and 3 years probability of cGVHD were 48%, 46%, 66%, and 54%, respectively.

As no significant associations were observed in the in the NMA conditioning cohort, only genotype association data from the MA conditioning cohort will be presented. However, results from the NMA conditioning cohort corresponding to the main findings in the MA conditioning cohort are shown in parallel in Table 3, Table 5, Table 6. No data concerning the −1615A > G and 982C > T genotypes will be presented, as no significant associations with transplant outcome were observed.

Association of Patient HMGB1–1377delA Genotype with Relapse 

Patient heterozygosity or homozygosity for the–1377delA minor allele was an independent risk factor significantly associated with increased RI both in the unadjusted (P = .02) (Table 3) and adjusted Cox regression analyzes (P = .02) (Table 4), whereas only a trend toward increased RRM was observed (P = .05) (Table 3). The incidence rates of RI (P = .01) and RRM (P = .03) stratified according to the–1377delA genotype were significantly higher in patients carrying the–1377delA minor allele (Figure 2). There were no significant associations between patient or donor genotype and any other transplantation outcome (Table 3).

Table 3. Univariate Cox Regression Analyzes According to the Patient–1377delA Genotype

HR indicates hazard ratio; CI, confidence interval.

Table 4. Multivariate Cox Regression Analyzes of the Association of HMGB1 Genotype with Transplantation Outcome in the Cohort Treated with Myeloablative Conditioning

HR indicates hazard ratio; CI, confidence interval; Ref, reference group; CsA, cyclosporine; MTX, methotrexate; GVHD, graft-versus-host disease; Cy, cyclophosphamide; TBI, total body irradiation; Eto, etoposide; Bu, busulfan.

The covariates in the table have only been included in the final models, if they changed the estimate of the polymorphisms of interest by at least 10% or were significantly associated with outcome in pair wise analyzes.

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• Figure 2 

  Cumulative incidence of relapse incidence and relapse related mortality according to patient −1377delA genotype in the transplantation cohort treated with myeloablative conditioning. Gray's k-test between patients with A/A genotype versus patients with A/− or −/− genotypes.

Association of Patient HMGB1 3814C > G, 1177 G > C and 2351insT Genotype with OS, PFS, and aGVHD 

In general, the 3 polymorphisms, 3814C > G, 1177 G > C, and 2351insT tended to have the same influence on transplantation outcome, because of a moderate to strong LD (R² = .7869-0.9339) between the 3 loci. Patient homozygosity for the minor allele of 3814C > G was the strongest independent protective factor associated with increased OS and PFS, both in the unadjusted (OS: P = .04; PFS: P = .02) (Table 5) and adjusted (OS: P = .04; PFS: P = .05) Cox regression analyses (Table 4). Furthermore, patient homozygosity for the minor allele of 3814C > G showed a trend toward lower RI in the unadjusted Cox regression analysis (P = .06) (Table 5). The probabilities of OS, PFS, RI, and RRM, were significantly superior in patients homozygous for the 3814C > G minor allele, with no observed deaths because of relapse (Figure 3).

Table 5. Univariate Cox Regression Analyses of Association between Patient HMGB1 Genotype and Transplantation Outcome

HR indicates hazard ratio; CI, confidence interval; Ref, reference group; ; GVHD, graft-versus-host disease.

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• Figure 3 

  Cumulative incidence of OS, PFS, RI, and RRM according to patient 3814C > G genotype in the transplantation cohort treated with myeloablative conditioning. Gray's k-test between patients with G/G genotype versus patients with C/G or C/C genotype.

Although, all 3 polymorphisms (3814C > G, 1177 G > C, and 2351insT) showed the same tendency toward patient heterozygosity decreasing the risk of grade II to IV aGVHD, only the 2351insT polymorphism was significant at the .05 level (Table 5). As the probability of developing grade II to IV aGVHD was similar in patients carrying the 2351insT −/T and T/T genotypes (2351insT: −/− 47%, −/T 32%, and T/T 36%), they were analyzed together in both unadjusted (data not shown) and adjusted Cox regression models, which showed that carriage of the 2351insT minor allele was an independent factor reducing the risk of grade II to IV aGVHD (P = .01) (Table 4).

Association of Donor HMGB1 3814C > G, 1177G > C, and 2351insT Genotype with cGVHD 

The cumulative incidence of developing limited or extensive cGVHD showed a successive increase with the donor carrying 0, 1, or 2 minor alleles of the 3814C > G, 1177 G > C, and 2351insT polymorphisms, implying a gene dosage effect (Figure 4, only 2351insT shown). Table 6 shows the results of the univariate Cox regression analyzes, where the 3814C > G, 1177 G > C, and 2351insT polymorphisms were analyzed as quantitative variables (0, 1, and 2 minor alleles). For all 3 polymorphisms, the HR, denoting the risk of developing limited or extensive cGVHD per unit of time per minor allele, was significantly associated with the risk of limited or extensive cGVHD. The 2351insT polymorphism was also evaluated as a quantitative variable in a multivariate Cox regression analysis, where donor carriage of the minor alleles successively increased the risk of developing limited or extensive cGVHD significantly (P = .01) (Table 4).

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• Figure 4 

  Cumulative incidence of limited or extensive cGVHD, according to donor 2351insT genotype in the transplantation cohort treated with myeloablative conditioning.

Table 6. Cumulative Incidence and Univariate Cox Regression Analyses of Limited and Extensive Chronic Graft-versus-Host Disease, According to Donor 3814C > G, 1177 G > C, and 2351insT Genotype

CI indicates confidence interval; HR, hazard ratio.

In the Cox regression analysis donor 3814C > G, 1177 G > C, and 2351insT genotypes were ascribed the value 0, 1, and 2, with respect to the carriage of 0, 1 or 2 minor alleles. The HR denotes the risk per minor allele.

No other significant associations with transplant outcome were observed (data not shown).

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Discussion 

In the present study we have investigated the association between HMGB1 genotype and outcome after allogeneic HCT. There were no differences in the distribution of HMGB1 genotypes between patients and donors, indicating that the HMGB1 polymorphisms as such do not play a role in the susceptibility to diseases treated with allogeneic HCT in the present study. The frequencies of the inferred haplotypes H1 to H4 showed similar distributions compared to the SIRS cohort and normal population 38, 39.

We observed that presence of the minor allele of the –1377delA polymorphism in patients treated with allogeneic HCT after MA conditioning, was an independent risk factor for RI increasing the probability of RRM significantly. In a previous study of patients with SIRS admitted to an intensive care unit, we observed that carriage of the–1377delA minor allele also was independently associated with mortality [39]. Although SIRS and allogeneic HCT are different entities, the confirmative association of this polymorphic locus with mortality in 2 independent studies suggests that it is of pathophysiological importance [39].

The 3 polymorphisms 3814C > G, 1777 G > C, and 2351insT tended to have the same effect on transplantation outcome in the MA conditioning cohort, because of a moderate to strong LD between loci. Patient homozygosity for the minor allele of 3814C > G was the strongest independent factor associated with increased OS and PFS, and also associated with lower probabilities of RI and RRM. Patient carriage of the 2351insT minor allele was an independent factor associated with decreased risk of grade II to IV aGVHD, whereas donor carriage of the minor allele displayed a gene dosage effect, with a successive increase in risk of developing limited or extensive cGVHD per minor allele carried. Neither patient nor donor 2351insT genotype was associated with TRM, which is consistent with the genotype not being independently associated with grade III to IV aGVHD and extensive cGVHD.

Only associations between HMGB1 genotype and outcome after allogeneic HCT following MA conditioning but not following NMA conditioning were observed, suggesting a differential effect of HMGB1 depending on the intensity of the conditioning regimen. It is generally accepted that the biology of the immunoreactions following MA conditioning and NMA conditioning, in part, differ in respect to the degree of tissue damage and secretion of inflammatory cytokines, with MA conditioning being more pro-inflammatory and tissue damaging 46, 47, 48, whereas NMA conditioning mostly being immunosuppressive. In the highly pro-inflammatory milieu of MA conditioning 46, 47, 48, HMGB1 can be passively released by cells damaged by TBI and/or exposure to cyclophosphamide (Cy) 21, 49 and actively secreted as in response to LPS and tumor necrosis factor (TNF)-α [13].

Apart from the conditioning regimen, other differences between the MA conditioning and NMA conditioning cohorts are present. These pertain to the patient demographics and are mainly: age at transplantation, type of malignant hematologic disease, stem cell source, and the HLA-match resolution level, as all NMA conditioning treated patients had high-resolution HLA-matching performed. Whether this heterogeneity also can explain the different impact, in the 2 allogeneic HCT cohorts, of the HMGB1 polymorphisms cannot be excluded.

Collectively, there was a tendency toward patient HMGB1 genotypes being associated with outcomes dependent on primarily patient APCs, such as RI, RRM, and aGVHD 2, 3, 4, and that donor genotypes were associated with, in part, a donor APC-dependent outcome, namely, cGVHD 5, 6, suggesting that the polymorphisms in HMGB1 influence the transcription of HMGB1 in APCs induced by the pro-inflammatory milieu after MA conditioning, rather than the passively released from damaged cells, although these 2 mechanism are not mutually exclusive.

Although we do not yet know the molecular mechanisms responsible for the polymorphisms influence on outcome after allogeneic HCT following MA conditioning, these observations suggest that the–1377delA, 3814C > G, 1777 G > C, and 2351insT polymorphisms are important for the function of HMGB1. As the role of HMGB1 in the setting of allogeneic HCT also is unknown, a hypothesis unifying all the current observations would be premature. The seemingly close relation between HMGB1 genotype and relapse-related outcome measures was somewhat surprising, as associations with aGVHD and TRM would be expected because of its pro-inflammatory properties. Nevertheless, the strongest and most consistent observations in the study were associations with RI, RRM, and cGVHD. In a previous study, scanning of the genomic sequence for pattern matches against known transcription factor binding motifs revealed that the minor allele of the 3814C > G polymorphism created a potential binding site for the transcriptional enhancer v-Myb 38, 50, 51, suggesting that the 3 polymorphisms 3814C > G, 1777 G > C, and 2351insT, because of LD, could be associated with increased function of HMGB1. An association between increased function of HMGB1 and decreased RI and RRM is supported by recent experiments by Apetoh et al. [21] and Curtin et al. [52], where tumor antigen-specific T lymphocyte responses were dependent on HMGB1, as neutralization of HMGB1 abolished immunogenicity of cancer cells in vivo. Furthermore, in a murine tumor model, tumor growth was abrogated by using HMGB1 competent fibroblasts as immunoadjuvants, whereas unabated tumor growth was observed when fibroblasts unable to release HMGB1 were used [53]. The association of the 2351insT minor allele with cGVHD is also in coherence with increased function of HMGB1, as increased extracellular expression of HMGB1 is a feature of some autoimmune diseases that share clinical features with cGVHD 19, 54, 55. In line with these data, the association of the–1377delA minor allele with increased RI and RRM, should be associated with decreased function of HMGB1. Albeit no measurements or HMBG1 in plasma or serum were performed in the present study, no association between the–1377delA, 3814C > G, 1177 G > C, and 2351insT and serum levels were observed in a previous study of SIRS patients [39]. The posttranslational modification of HMGB1 varies depending on whether it is actively secreted from APCs or passively released by necrotic cells [56], and it has been suggested that the biological activity of HMGB1 could vary according to these differences [57]. If the genetic variations in the HMGB1 gene influence the active secretion from APCs, the effect on outcome could be explained by changes in the ratio between different subsets of HMGB1. The presented interpretation of the current data implies that the polymorphisms influence the HMGB1-dependent antigen presentation by APCs. However, the association between the minor allele of the 2351insT polymorphism and decreased aGVHD is in contradiction and cannot readily be explained by the current model.

Although only the 3814C > G polymorphism colocated with a potential transcription factor [38], an influence of the remaining polymorphisms cannot be ruled out. Genetic polymorphisms can influence gene expression levels by several mechanisms. Polymorphisms found in the regulatory regions can alter the structure of transcription factor binding sites, hereby affecting the affinity of the transcriptional apparatus. In exons and in the exon-intron boundaries polymorphisms can alter the structure and function of proteins, by inducing amino acid changes, stop codons, or altering mRNA splicing and stability. The 2351insT located in the 3′-UTR could interfere with binding of micro-RNA and hereby influence mRNA stability. Because of strong LD or haplotype effects the genotyped polymorphisms could also represent surrogate markers for other distant or yet-unknown polymorphic loci that influence the gene expression of HMGB1. The 3814C > G and 1177 G > C polymorphisms, which have been genotyped in the International HapMap Project [58], are in strong LD with rs1360485 and rs1045411 also located in the 3-UTR, although outside the genetic regions sequenced in our study.

Several non-HLA gene polymorphisms have been assessed in allogeneic HCT with the most thoroughly assessed being in TNF-α [40], IL-10 59, 60, 61, IL-6 [62], NOD2/CARD15 63, 64, and mannose binding lectin 65, 66. Although they have been analyzed in multiple trials confusion concerning their roles still exists for most. The impact of non-HLA genetics in allogeneic HCT after NMA conditioning has recently been assessed for mannose binding lectin, where no association was found between genotype and outcome [66]. The inconsistent and sometimes contradictory data can to some extent be explained by the multigeneic regulation of immune responses, where the pleiotropic effects of cytokines differ depending on the activating stimuli and timing of activation. This is further complicated by the heterogeneity in patient demographics across the different study cohorts introducing “noise” both genetic and immunobiological that can mask influence of the polymorphisms.

With the presented data and proposed model in mind, an obvious limitation of the current study is the risk of false-positive associations because of the numerous comparisons. Although, the significance level of the discussed results was below .05, the statistical power of the study was too low to allow most results to withstand formal Bonferroni correction, by multiplying all P-values by 6. As no corrections for multiple testing were applied in the current study, cautious interpretation of the obtained P-values and rigorous validation of the results in independent cohorts is warranted.

In conclusion, this is the first report of implications of the genetic variation in the human HMGB1 gene in a population of patients treated with allogeneic HCT following MA conditioning. The–1377delA polymorphism, which previously has been associated with increased mortality in patients with SIRS was observed to increase the risk of RI and probability of RRM. Moreover, 3 polymorphisms in moderate to strong LD also influenced the risk of relapse related events and GVHD. Currently, the functional aspects of the HMGB1 polymorphisms and the role of HMGB1 in the setting of allogeneic HCT are unknown. However, HMGB1 is likely to play a role in the development of GVHD, GVT effect, and possibly engraftment, because of its central placement in the activation of APCs and tissue regeneration. Although, the current study is purely descriptive, our findings suggest that the inherited variation in the HMBG1 gene locus could affect outcome after allogeneic HCT following MA conditioning. Further studies, both clinical, in independent cohorts, and experimental with in vitro analysis of the functional relevance of the different polymorphisms, are needed to confirm these findings and explain their molecular background.

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Aknowledgments 

Financial disclosure: The authors wish to thank Anne Bjørlig and Ulla Lang for excellent technical assistance, and the staff of the Department of hematology at Rigshospitalet for invaluable help and support. The work was supported by grants from the Danish Medical Research council, the Novo Nordisk Research Foundation, The Benzon Foundation, The Danish Rheumatism Association, The Danish Cancer Society, Rigshospitalet, and The Lundbeck Foundation. MD, PhD-student Brian Kornblit was supported by a grant from Rigshospitalet

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