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Association between recipient TNF rs361525 and acute GVHD: results from analysis of BMT CTN-0201 samples

To the Editor,

Acute graft-versus-host disease (aGVHD) complicates up to half of all allogeneic hematopoietic cell transplants (allo-HCT) [1]. Given our limited progress in the treatment of aGVHD [2], predictive methods that allow for early intervention are desirable. In a recent meta-analysis of 6 studies (627 patients), we demonstrated a borderline association between the A allele of recipient tumor necrosis factor (TNF) single nucleotide polymorphism (SNP) rs361525 and higher risks of grade II-IV aGVHD (risk ratio: 1.29, 95% confidence interval: 0.99–1.69, P = 0.06) [3]. rs361525G > A is an intronic upstream variant (2KB) TNF SNP with a global minor allele frequency of 6%. Our rationale for focusing on this SNP was the following: (i) rs361525G > A is a functional promoter variant associated with reduced TNFα production [4]; (ii) Increased TNFα expression by tissue macrophages and higher serum TNFα levels after conditioning have been associated with severe aGVHD [5,6,7]; (iii) SNPs increasing TNFα production by gut-resident macrophages could potentially increase donor T-cell reactivity and thus the risk of subsequent aGVHD [8]; and (iv) TNFα is uniformly and highly specifically expressed in gut biopsies of patients with aGVHD [9].

Considering the known association between the A allele of rs361525 and lower TNFα production, the observation of increased rates of aGVHD in patients with this allele was unexpected. Our conclusions from the meta-analysis was that validation on a large independent cohort was needed to permit a more definitive conclusion. Here we report our results from this validation.

We analyzed samples from BMT CTN-0201, a large randomized trial that compared unrelated donor HCT using peripheral blood (PB) vs. bone marrow (BM) [10]. The specific hypothesis we tested was: Recipient TNF rs361525G > A is associated with increased risk of grade II-IV aGVHD by Day + 180. Patients with 1 or more HLA locus mismatch (HLA-A, B, C, or DRB1; n = 142), active disease at the time of HCT (n = 24), no remaining research aliquots (n = 176), or only 1 remaining research aliquot (n = 22, reserved for future studies) were excluded. Genotyping was performed at the University of Minnesota Genomics Core by multiplexed PCR of genomic DNA extracted from pre-HCT recipient peripheral blood mononuclear cells provided by the BMT CTN. An iPLEX Gold method was used for genotyping. Multiplexed PCR was performed in 5-µl reactions on a 384-well plate containing 10 ng of genomic DNA. Reactions contained 0.5 U HotStar Taq polymerase (QIAGEN), 100 nM primers, 1.25 × HotStar Taq buffer, 1.625 mM MgCl2, and 500 µM dNTPs. Following enzyme activation at 94 °C for 15 min, DNA was amplified with 45 cycles of 94 °C x 20 s, 56 °C x 30 s, 72 *C x 1 min, followed by a 3-min extension at 72 °C. Unincorporated dNTPs were removed using shrimp alkaline phosphatase (0.3 U) (Agena, San Diego). Single-base extension was carried out by addition of single base primer extension (SBE) primers at concentrations from 0.625 µM (low MW primers) to 1.25 µM (high MW primers) using iPLEX enzyme and buffers (Agena, San Diego) in 9-µl reactions. Reactions were desalted and SBE products measured using the MassARRAY system, and mass spectra analyzed using TYPER software (Agena, San Diego), in order to generate genotype calls and allele frequencies.

The unadjusted cumulative incidence method was used for the analysis of aGVHD using death without aGVHD as a competing risk. Fine and Gray multiple regression models using SNP, graft source (PB vs. BM), and conditioning intensity (reduced intensity [RI] vs. myeloablative [MA]), with pre-specified interaction terms (SNP x graft source) and (SNP x conditioning intensity) were built. A total of 187 patients (median [range] age: 45 [31–56] years; graft source: 94 BM, 93 PB) were included with a median follow up for survivors of 3 years. PB and BM groups were matched in all baseline characteristics as expected from the randomized design of BMT CTN-0201. Call rate was 100% and genotype distribution was in Hardy–Weinberg equilibrium (G/G: 92.5%, G/A: 7.5%; P = 0.99). We found no association between recipient TNF rs361525 and grade II-IV aGVHD in any of the tested models (Table 1, Fig. 1).

Table 1 Association between TNF rs361525 and grade II-IV aGVHD
Fig. 1
figure1

Association between TNF rs361525 and grade II-IV acute graft-versus-host disease (aGVHD). No association was found (hazard ratio: 0.79, 95% confidence interval: 0.34–1.80, P = 0.57)

In conclusion, the analysis of samples from a large randomized clinical trial did not reproduce the suggestive SNP/aGVHD association demonstrated in a recent meta-analysis of 6 studies. Our study is limited by research sample availability. Additionally, while excluding patients with active disease and those with HLA mismatch increased our population homogeneity, it further decreased the sample size. The G/A genotype was infrequent in the present study, resulting in a wide confidence interval for the hazard ratio. Non-reproducibility of our previous meta-analytic observation is consistent with studies demonstrating validation failure of most SNP associations with allo-HCT outcomes [11, 12]. Reasons for spurious genetic associations include, but are not limited to, small sample size, population stratification (especially ethnic differences between cases and controls), biased or inaccurate measurements of genetic variants, assessment of clinical phenotypes by individuals not blinded to the genetic information, and failure to adjust for multiple comparisons [13]. Unbiased genome-wide association analyses, clearly defined hypothesis-driven questions, confirmation in independent study populations, and incorporating mechanistic approaches are critical for the success of SNP studies associating variant alleles with complex clinical outcomes.

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Acknowledgements

Support for the original trial was provided to the Blood and Marrow Transplant Clinical Trials Network by grant #U10HL069294 from the National Heart, Lung, and Blood Institute and the National Cancer Institute. The Department of the Navy, Office of Naval Research, and the National Marrow Donor Program also supported this study. Enrollment support was provided by DKMS Germany. Any views, opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not reflect the views or the official policy or position of the above mentioned parties.

Funding

This study was funded by a University of Minnesota Marrow on the Move Award to Armin Rashidi, and in part by NIH P30 CA77598 utilizing the Biospecimen Repository in the Translational Therapy Laboratory Shared Resource of the Masonic Cancer Center, University of Minnesota.

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Rashidi, A., Shanley, R., Yohe, S.L. et al. Association between recipient TNF rs361525 and acute GVHD: results from analysis of BMT CTN-0201 samples. Bone Marrow Transplant 53, 1069–1071 (2018). https://doi.org/10.1038/s41409-018-0127-3

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