SUMMARY

INTRODUCTION

Allogeneic hematopoietic cell transplantation (HCT) is an effective therapy for patients with acute leukemia because of the anti-leukemia effect driven by the donor graft, known as the graft-versus-leukemia effect.¹ The 3-year probabilities of matched unrelated donor post-HCT survival for adults with acute myeloid leukemia are 53%, 50% and 27% for patients in first, second remission and not in remission, respectively.² Despite recent improvements in HCT modalities, the risks of transplant-related complications and non-relapse mortality (NRM) remain high, with deaths primarily caused by infections, graft-versus-host disease (GvHD), and organ failure. Together, these causes account for approximately 60% and 40% of deaths occurring within and beyond the first 100 days post-HCT, respectively.²

The interferon lambda 4 gene (IFNL4) encodes IFN-λ4, a recently discovered member of the type III interferon gene family.³ Protein expression of IFN-λ4 is controlled by a dinucleotide genetic variant (rs368234815-TT/dG) located in the first exon of IFNL4. The rs368234815-dG allele supports the production of IFN-λ4 protein, while the TT allele introduces a protein-abrogating frameshift. Thus, IFNL4 genotype can be categorized into IFNL4-positive (those producing 1 or 2 copies of IFN-λ4 protein), and IFNL4-Null (those not producing IFN-λ4 protein).³ Another IFNL4 polymorphism (rs12979860) is located within the first intron and can be used as a proxy when rs368234815 is not available. Individuals with IFNL4-positive genotype can be further genotyped for rs117648444 (a missense variant P70S in exon 2 of IFNL4) and categorized into those who can only produce functionally weak IFN-λ4-S70 protein and those who carry at least one haplotype for producing functionally strong IFN-λ4-P70 protein (reviewed in⁴; details are presented Appendix p10). IFNL4-Null individuals are more likely to clear hepatitis C virus (HCV) infection and less likely to reactivate cytomegalovirus (CMV) infection after solid organ transplantation.^,5 IFN-λ4 activates the JAK/STAT pathway,³ and induces expression of negative regulators of cytokine and interferon signaling,⁶ which may have a broad impact on both the adaptive and innate immune responses, and explain diverse and generally negative clinical outcomes associated with its activity.⁷

We hypothesized that IFN-λ4 might be important for outcomes of HCT by affecting infection clearance and/or alloreactivity. In this study, we evaluated the association between the recipient and donor IFNL4 genetic polymorphisms with post-HCT survival outcomes in patients with acute leukemia.

METHODS

Study Design and Participants:

The Center for International Blood and Marrow Transplant Research (CIBMTR) maintains one of the world’s largest databases and biorepositories serving HCT and cellular therapy-related studies. CIBMTR collects detailed information on almost all allogeneic HCT performed in the United States. The research sample biorepository primarily includes samples collected in the context of unrelated donor transplants facilitated by the National Marrow Donor Program (NMDP/ Be The Match®). Data collected by the CIBMTR is thoroughly reviewed and audited to ensure high quality (details at https://www.cibmtr.org/Data/Available/Pages/index.aspx).

Eligibility criteria for this study included: patients with acute myeloid leukemia (AML) or acute lymphocytic leukemia (ALL), who received HCT at any age from an unrelated 10/10 HLA-matched donor, with myeloablative conditioning regimen, between January 1, 2000 - December 31, 2008, and had available pre-HCT recipient and/or donor blood sample.

The discovery cohort (NCI set) used an available set (N=446) that was part of a previously published study to test this hypothesis.⁸ Patients in the original study were in first or second complete remission and had an available donor pre-HCT blood sample (since the original study focused on donor telomere length) in the CIBMTR biorepository. Of those, 410 recipient-donor pairs met study eligibility criteria; genotyping was completed for 404 donors and 319 recipients with available DNA.

A validation cohort was sought after promising results were obtained in the first set. We used genomic and outcome data from the DISCOVeRY-BMT study (DBMT; a genome-wide association study aiming to determine the influence of susceptibility variants on one-year mortality after HCT).⁹ The DBMT included patients with AML, ALL, and myelodysplastic syndrome (MDS) selected from the CIBMTR database and biorepository. Of the 1593 HCT recipients meeting study eligibility criteria, we excluded 348 patients who were already included in the NCI set. This left 1245 unique patients for an independent validation analysis. We also performed a combined analysis using the full DBMT set of 1593 eligible patients (independent validation + overlapping patients in the NCI set) to improve statistical power. Figure 1 presents the study flow chart.

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Figure 1:

Study flow chart.

The study was approved by the NMDP Institutional Review Board. All study participants provided written informed consent for participation in the CIBMTR repository and database.

RESULTS

Table 1 summarizes the patient characteristics by study sets. The discovery and validation sets (NCI; N=404 and DBMT; N=1245; all 10/10 HLA matched) were similar in the demographics of recipients and donors, disease frequency, and predominance of tacrolimus-based regimens for GvHD prophylaxis. Notable differences between the two sets included: graft source (bone marrow grafts in 129 patients (31.9%) of 404 vs. 596 (47.9%) of 1245, for the NCI and DBMT, respectively), and disease status at HCT (404 patients (100%) vs. 802 (64.2%) of 1245 were in remission, respectively). The distributions of recipient and donor IFNL4 genotypes were similar in both sets, such as 156 (49.4%) of 316 and 550 (44.9%) of 1225 of the NCI and DBMT recipients; and 177 (43.8%) of 404 and 556 (44.7%) of 1245 of the donors, respectively were IFNL4-Null (Table 1).

Results from both the discovery and validation sets showed no statistically significant association between recipient IFNL4 genotype and post-transplant NRM (HR=1.12, 95% CI=0.84–1.51, p=0.44, and HR=1.11, 95% CI=0.95–1.28, p=0.19, respectively) or OS (HR=1.01, 95% CI=0.81–1.26, p=0.96, and HR=1.07, 95 % CI=0.97–1.19, p=0.17). Analysis of the full DBMT set (combined set) showed similar results (Appendix p 6).

On the other hand, donor IFNL4-positive genotype was statistically significantly associated with an excess risk of NRM in both cohorts (HR=1.60, 95% CI=1.23–2.10, p=0.0005, and HR=1.22, 95% CI=1.05–1.41, p=0.007, in the discovery and validation sets, respectively) (Table 2). Similar result was noted in the combined set (HR =1.27, 95% CI=1.12–1.45, p=0.0001) (Table 2 & Figure 2A).

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Figure 2:

IFNL4 genotype and hematopoietic cell transplant: Donor IFNL4 genotype and the probability of post-transplant non-relapse mortality (A); Overall survival (B) in the combined DBMT set.

The observed higher risk of NRM in patients receiving HCT from donors with IFNL4-positive genotype resulted in statistically significant inferior OS in the discovery and combined sets (HR=1.24, 95% CI=1.02–1.51, p=0.03, and HR =1.11, 95% CI=1.02–1.22, p=0.02, respectively) (Table 2 & Figure 2B). The association did not reach statistical significance in the validation cohort (HR=1.10, 95% CI=0.98–1.20, p=0.10) (Table 2).

The associations between donor IFNL4 genotypes and one-year post-HCT causes of death are summarized in Table 3. The data showed statistically significant associations between donor IFNL4-positive genotype and risk of death from GvHD in all study sets. A significant relationship with infection-related death was only noted in the validation, and combined sets. No associations were noted with death from organ failure (HR-combined=1.05, 95% CI=0.78–1.40, p=0.74) or leukemia relapse (HR-combined=0.96, 95% CI=0.82–1.12, p=0.60).

We then used the combined set and complete the following secondary analyses: 1) restricted to patients who received HCT while in remission, this showed similar results to that observed in all patients (HR for NRM =1.27, p=0.001, and for OS=1.12, p=0.04); 2) stratified on graft source, the HR for NRM=1.36, p=0.0003; and HR=1.18, p=0.11, for peripheral blood vs. bone marrow stem cell transplants respectively, p-interaction=0.56); 3) stratified on donor-recipient pre-HCT CMV serostatus as a proxy for risk of post-HCT CMV viremia or use of anti-viral prophylaxis, and found no risk differences in NRM or OS in relation to donor IFNL4 genotype by donor-recipient CMV matching or donor seropositivity (Appendix p.8).

In further analysis, compared with IFNL4-Null donors, a higher risk of NRM was observed in patients who received HCT from donors with IFN-λ4-Strong, but not from donors with IFN-λ4-Weak protein; similar results were noted in all study sets. Results for OS followed the same trend, but did not reach statistical significance in the validation cohort or the combined analysis (Table 2 & Appendix p.12).

To explain the association between IFNL4 genotype and outcomes of HCT, we hypothesized that the transplanted graft is the source of IFNL4-expressing cells. We analyzed mRNA expression of all human interferons - IFNL4 and 21 other transcripts in bone marrow samples of three healthy donors. We showed that the IFNL4 was the main interferon transcript expressed in bone marrow of all three individuals tested, with expression detected in 0.67% of all sequenced cells (range of expression of other interferons=0–0.07%), and in 83.1% of all interferon-expressing cells (range for other interferons=0–8.8%). Based on the IFNL4 proxy variant rs73930703 within the transcribed region, all three individuals are likely to be IFNL4-Null (Appendix p 7&13).

Using risk stratification tools to guide donor selection strategy based on IFNL4 genotype testing, we found that selecting IFNL4-Null donors was associated with an absolute risk reduction of post-HCT NRM of 6.4% (95% CI=1.2–11.6%) versus 11.0% (95% CI=2.7–19.4%) if rejecting donors homozygous for the IFNL4-positive genotype. The Mean Risk Stratification (MRS) provided by selecting HCT donors with the common IFNL4-Null status (~50% frequency) changed the average risk of NRM by MRS=3.2% (95% CI=0.67% to 5.74%). Our estimate for the corresponding MRS for 8/8 vs. 7/8 HLA-matching is 3.5% (95% CI=1.83%−5.23%). In contrast, the MRS of the rare homozygous IFNL4-positive genotype (frequency=12.1%) was only 2.4% (95% CI=0.56% to 4.15%), despite contributing the highest AR difference (11.0%). These results suggest that the strongest impact on population-average risk of NRM would be achieved by attempting to use donors with IFNL4-Null status rather than merely avoiding homozygous IFNL4-positive donors. For OS, the MRS for selecting donors with the IFNL4-Null status was 1.4% (95% CI= −1.3% to 4.1%), which is less than that for HLA matching (2.1%; 95% CI= 0.53% to 3.7%). See Supplemental Results for MRS and AUC comparison. (Appendix p. 19–24)

DISCUSSION

In this study of acute leukemia HCT recipients, we showed that donor IFNL4 genotype is associated with patient risk of NRM. The observed risk reduction when selecting IFNL4-Null donors was beyond that already provided by the HLA matching since all patients in this study received an HCT from a 10/10 HLA-matched donor. Our findings suggest potential clinical utility for prioritizing 10/10 HLA-matched donors with IFNL4-Null genotype to further reduce patient risk of NRM and improve OS.

Donor selection is key for HCT success, with HLA matching being the most important factor. A CIBMTR study of 3,857 patients who received myeloablative regimens for malignant conditions showed an associated mortality risk of 1.25 with every single HLA mismatch.¹³ Growing evidence suggests a role for additional donor genetic factors in post-HCT survival. For example, longer donor telomere length was associated with an improved OS after unrelated donor HCT in patients with SAA.^14–16 Yet, results for the possible post-HCT benefit of longer donor telomere length in acute leukemia are still inconclusive.¹⁷ Other examples include polymorphisms in Vitamin D receptor gene, cytokine genes, and HLA-DP permissiveness and expression.^18–20 None of these markers made it to the donor selection clinical practice with the exception of considering HLA-DP.

Consistent with the unfavorable effect of IFNL4-positive genotype detected in our study, a large study of 840 solid organ transplant recipients showed a high incidence of CMV reactivation in patients with the rs368234815-dG/dG genotype (HR=1.46, p=0.05), while no association was noted in patients receiving antiviral prophylaxis.⁵ Two small studies evaluated the possible effect of IFNL4 genotype on CMV viremia after HCT with inconclusive results.^21,22 The use of infection prophylaxis after HCT is a common practice. In 2009, a guideline for preventing infection-related complications after HCT was published in an effort to mainstream recommendations and practices.²³ In recent years, new and more effective drugs to prevent and treat infections- particularly CMV and invasive fungal disease- became available,^24,25 leading to decrease in post-HCT infections and improving patient survival. In our current analysis, donor IFNL4 genotype was associated with increased risk of death from infections, but detailed information on infections or infection prophylaxis was not available to further explore this question. We found no risk differences in NRM or OS in relation to donor IFNL4 genotype by donor-recipient CMV matching or donor seropositivity, as a proxy for CMV risk of infection or need for prophylaxis. The use of actual detailed data is necessary to answer the question since strategies of infection prophylaxis may differ between transplant centers.

The impact of donor IFNL4 genotype on HCT survival was less pronounced in the validation cohort where bone marrow grafts were more frequent than in the discovery set. Our evaluation suggested a possible higher risk of NRM associated with donor IFNL4-positive genotype in patients receiving peripheral blood than bone marrow stem cell source, although the difference was not statistically significant. This needs further evaluation since unrelated donor bone marrow transplants are associated with lower risk of NRM and improved OS, and may explain the observed difference in the impact of IFLN4 genotype between study sets.

The donor IFNL4 genotype was also associated with a higher risk of death due to GvHD (HR=1.54, p=0.003). This association may reflect a higher risk of developing severe forms of GvHD or a lower response to GvHD immunosuppressive therapies. We evaluated the risk of severe acute GvHD (grade 3–4) and chronic GvHD in relation to donor IFNL4 genotype in the combined DBMT set. No statistically significant association was noted with acute GvHD grade 3–4 (HR=1.11, p=0.16), but a suggestive association with the risk of chronic GvHD existed (HR=1.12, p=0.03). Type III interferons, including IFN-λ4, signal through a distinct receptor complex of IFNLR1 and IL-10R2, to activate the expression of IFN-stimulated genes (ISG) via the JAK/STAT signaling pathway (reviewed in²⁶). Donor IL10R2 genotype and JAK/STAT signaling have been associated with risk of GvHD,²⁷ which was reduced by JAK inhibitors,²⁸ thus providing a plausible link between GvHD and type III interferon signaling.

It is intriguing that death from both immune complications of HCT – inefficient ability to clear infection and overreaction in GvHD - are associated with the genetically determined ability to produce IFN-λ4. It is possible that this observation is attributed to ISG expression; furthermore, IFN-λ4 expression was associated with enhanced negative regulator of IFN signaling upon exposure to viral infection,⁶ and interferon-related DNA damage resistance signature in prostate cancer.²⁹ Notably, IFNL4 was the predominant interferon transcript detected by scRNA-seq in the bone marrow of healthy blood donors. It is possible that IFN-λ4 produced in bone marrow in individuals with IFNL4-positive genotype can reprogram progenitor cells that later contribute to multiple negative phenotypes, especially when placed into an immunosuppressed environment of transplant recipients. The results suggest a novel functional role of IFN-λ4 in hematopoietic stem cells in normal and disease conditions; our scRNA-seq analysis was limited by the small sample size warranting future investigation.

IFN-λ4-P70S status only affects IFN-λ4 and not other IFN-λs encoded in the same genomic region,⁴ and therefore can provide evidence of a direct relationship with IFNL4. Patients receiving HCT from donors with IFN-λ4-Weak protein had NRM risk comparable to those with IFNL4-Null donors, while high NRM risk was observed in patients who received HCT from donors with IFN-λ4-Strong protein. This is in line with increased interferon gene signature and impaired HCV clearance in patients with the IFN-λ4-Strong compared to patients with IFN-λ4-Weak protein and IFNL4-Null genotype. ³⁰

Our calculated absolute risk difference (RD) and mean risk stratification (MRS) for NRM showed a potential benefit for selecting donors based on their IFNL4 genotype in the current era of optimal HLA recipient-donor matching. Our data showed an additional 6.4% AR reduction in NRM in donors with IFNL4-Null genotype and 10/10 HLA matching. The effect of selecting donors based on their IFNL4 genotype on the reduction of NRM risk was comparable to that of selecting 8/8 vs. 7/8 HLA matched donors (3.2% vs. 3.5%, respectively), but a lower MRS for risk of OS was noted (1.4% vs. 2.1%, respectively). Thus, our findings reinforce the primary importance of optimal HLA matching, while suggesting that using donors with IFNL4-Null genotype may further reduce NRM risk. A comprehensive study is needed to definitively decide which of the two strategies (merely avoiding donors homozygous for IFNL4-positive vs. actively selecting those with IFNL4-Null genotype) is more effective for clinical practice.

The strengths of this study include the availability of comprehensive clinical and outcome data for the study population, together with pre-HCT samples from both recipients and donors for a relatively large number of patients. Yet, the study was limited by the unavailability of information on infection prophylaxis regimens or episodes of post-HCT infections. Our results may not be generalizable beyond the target study population and need to be studied further for other HCT indications.

In conclusion, our study showed that donor IFNL4-positive genotype is associated with an increased risk of NRM and inferior OS in patients with acute leukemia who received a 10/10 HLA-matched unrelated donor myeloablative HCT. If confirmed, prioritizing donors with the IFNL4-Null genotype may improve HCT survival.

Supplementary Material

ACKNOWLEDGMENTS:

Footnotes

Contributor Information

Shahinaz M. Gadalla, Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.

Youjin Wang, Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.

Tao Wang, Division of Biostatistics, Medical College of Wisconsin, Milwaukee, WI. Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI.

Olusegun O. Onabajo, Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.

A. Rouf Banday, Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.

Adeola Obajemu, Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.

Ezgi Karaesman, College of Pharmacy, Ohio State University Columbus, OH.

Lara Sucheston-Campbell, College of Pharmacy, Ohio State University Columbus, OH.

Theresa Hahn, Roswell Park Comprehensive Cancer Center, Buffalo, NY.

Jennifer A. Sees, Center for International Blood and Marrow Transplant Research, Minneapolis, MN.

Stephen R. Spellman, Center for International Blood and Marrow Transplant Research, Minneapolis, MN.

Stephanie J. Lee, Center for International Blood and Marrow Transplant Research, Medical College of Wisconsin, Milwaukee, WI. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

Hormuzd A. Katki, Biostatistics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.

Ludmila Prokunina-Olsson, Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD.

REFERENCES