Pathogenic and likely pathogenic germline variation in patients with myeloid malignancies and their unrelated HLA-matched hematopoietic stem cell donors

INTRODUCTION

Germline studies have demonstrated that inherited variants contribute to familial myeloid leukemia risk in adults, even in the absence of a known family history of the disease^[1-3]. With the growing availability of clinical genetic testing, there is an increasing appreciation for several inherited predisposition syndromes and germline causal variants, which may underlie apparent de novo presentations of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS)^[1]. Large genomic studies of these cancer predisposition genes in leukemia populations have the potential to help inform medical management , donor selection for blood and marrow transplant (BMT), and genetic counseling practice around medical management for patients and their families.

Evaluation for an underlying familial syndrome in a patient with acute leukemia or MDS should involve a medical and family screening history, focused physical examination, and diagnostic genetic testing^[4-6]. The World Health Organization (WHO) and National Comprehensive Cancer Network (NCCN) have recently made such recommendations for both AML and MDS^[7]. However, there is no consensus on the optimal management of individuals diagnosed with a hereditary hematologic malignancy predisposition syndrome, with or without a personal history of acute leukemia or MDS, so management must be individualized^[6]. Genetic counseling of leukemia patients is challenging because of the inability to prevent or even attenuate marrow failure in at-risk individuals, the need for skin punch biopsy (versus blood) which leads to delayed test turnaround time, the ill-defined prevention strategies for at-risk relatives, and undefined medical management due to lack of a complete picture of related diseases, disease spectrum, penetrance, and age of onset^[8]. Genetic counseling in the context of BMT is especially nuanced, given the role relatives can play in patients’ curative therapy as blood or marrow donors, and there is burgeoning evidence that donor genetics may play a role in survival after transplant^[9-11]. Additionally, the optimal delivery model including when to perform genetic counseling and germline testing for a patient and donor is not well described.

To identify pathogenic variations in hematologic and solid tumor predisposition genes and determine their impact on survival after transplant, we use exome chip data on two cohorts of almost 2,000 AML and MDS patients and their HLA-matched unrelated healthy donors representing over a decade (2000-2011) of data reported to the Center for International Blood and Marrow Transplant (CIBMTR)^[12]. Here, we report the existence of LPV/PV in AML and MDS patients and donors and that donor variation impacts patient survival after BMT. These data further highlight the need for continued genomic science and genetic counseling research around the characterization of germline variation in susceptibility to hematologic malignancies and to survival after curative therapy for these diseases.

METHODS

Study design and population

Data in our study are derived from the Determining the Influence of Susceptibility-COnveying Variants Related to 1-Year Mortality after Blood and Marrow Transplant (DISCOVeRY-BMT) cohorts designed to find common and rare germline genetic variation associated with survival after an unrelated donor BMT. DISCOVeRY-BMT consists of two cohorts (termed cohorts 1 and 2) of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS) patients and their human leukocyte antigen (HLA)-matched unrelated healthy donors reported to the CIBMTR from 2000-2008 (Cohort 1) and 2009-2011 (Cohort 2)^[10-12]. Criteria for unrelated donors include at least 18 years of age and passing a medical examination, including family history, to determine if the donor is healthy. We selected AML and MDS patients and used all available unrelated donors for both Cohort 1 and Cohort 2 for analyses. Study design is shown in Figure 1.

Figure 1. Study schema for frequency and survival analysis of LPV/PV in DISCOVeRY-BMT.

RESULTS

Following quality control, exome, clinical and demographic data were available on 1,990 donor-recipient pairs with an additional 256 donor genomes available with corresponding clinical and demographic data on the matched recipient but no recipient exome data [Table 1]. Most of the 1,523 AML cases were de novo and approximately 9% of patients had therapy-related AML (t-AML). The most frequently reported previous cancers in patients with t-AML were breast, non-Hodgkin lymphoma (NHL), and Hodgkin lymphoma (HL). Therapy-related MDS (t-MDS) comprised 18% of the 467 MDS patients and the most frequently reported antecedent cancers in MDS patients were NHL, breast, ALL, HL, and AML [Table 1].

The literature review and MESH search yielded 122 articles; after removing clinical reviews or uninformative articles for building a gene list, 32 articles were identified. When combined with information from OMIM, this resulted in 667 genes, including genes in commercially available clinical genetic tests for hereditary hematologic malignancies. Gene information from ClinVar on pathogenicity is provided in Supplementary Table 1. For frequency analyses, we first identified variants and genes in ClinVar that were available on the HumanExome and then determined the frequency in DISCOVeRY-BMT. For survival analyses, we analyzed the most frequent LPV/PV in donors and patients to determine if the identified susceptibility variants correlated with survival.

DISCUSSION

Autosomal dominant acting LPV/PV cancer variants were identified in both pre-transplant AML and MDS patients and donors. We identified 18 patients and 1 donor with LPV/PV in the known myeloid leukemia gene DDX41. Recently, a large-scale study on DDX41 mutations in 5,609 multi-ethnic patients with AML or MDS showed that germline or somatic mutation in DDX41 was seen in 202 of the 5,609 patients (3.6%)^[34]. These data also show that if a DDX41 variant is present in a patient’s NGS marrow biopsy, there is a 78% chance of a germline mutation; in contrast, without a somatic DDX41 mutation, the probability of a germline DDX41 variant is around two percent. When considering a history of cytopenia, family history of cancer, or personal history of another cancer, it is almost certain the patient will have a germline DDX41 pathogenic variant if a somatic mutation is present^[35]. These studies provide genetics professionals with concrete numbers to discuss the risks of having a germline PV in DDX41 before and after NGS testing, irrespective of family or personal history.

While there was no distinction made for those undergoing transplant in Makishima et al., the 1% DDX41 mutation frequency in DISCOVeRY-BMT is attributable to only two mutations^[34]. Variant coverage on the exome chip represents a fraction of the data available with sequencing, so unsurprisingly, this fraction is lower in our study. Our sex finding, showing a higher DDX41 LPV/PV frequency in males versus females, and age results, that patients with DDX41 mutations were significantly older than AML and MDS patients without the mutation, are in line with previously published literature and Makishima et al.’s findings^[34,36]. In the future, sex-specific information could be used to further refine estimates of mutation likelihood following bone marrow biopsy results for counseling patients with AML and MDS.

We identified several AR inherited PVs in donors and patients, including those responsible for Fanconi Anemia in a homozygous state and solid tumors in a heterozygous state^{[21,24,25,37,38]}. FA mutation carriers do not exhibit the AR disorder; however, there is evidence that heterozygous Fanconi Anemia gene carriers may be at increased risk of developing MDS and AML^[38]. The identification of the donor FA association with recipient survival and our previous publications on exome and genome-wide significant associations with survival in DISCOVeRY-BMT provide strong evidence that non-HLA genetics is relevant to patient survival and replication of these findings in additional cohorts could have implications for patient clinical care and for counseling patients around outcomes following transplant^[10,11,39]. At present, there are no NCCN or WHO guidelines on germline testing for survival outcomes following transplant. New genes and PVs with biological relevance will likely continue to be identified and validated and may need to be considered in the context of donor selection, strategies for transplant timing and/or pre-transplant conditioning intensity. While a larger and more diverse cohort with sequence data and follow-up time greater than 1 year will be necessary to definitively answer these questions, this is an arena where genetic counselors could play a role in patient care.

Given the presence of both AD and AR pathogenic variants in our AML and MDS patients, it is also worth considering questions that arise around the return of results, which have direct implications for related family as individuals and as potential donors to their affected relatives. First, hematologic malignancy prevention strategies for unaffected, mutation-positive at-risk relatives are not well defined^[6]. Medical management often utilized in the care of families affected by a predisposition to solid tumors, such as prophylactic surgeries, is not feasible in mitigating leukemia risk. For example, a prophylactic BMT in an unaffected individual is not an acceptable medical prevention for AML or MDS due to the high morbidity and mortality after BMT and the increased risk of t-AML or t-MDS following transplant. A second issue centers around family members serving as blood or marrow donors for related or haplo-identical transplants^[9]. While unrelated donors must be 18 years of age, this is not true of related donors; a sibling, child, or related donor who is a minor may further add to families’ psychological and financial difficulties at the time of diagnosis. A systematic review of 16 studies across 1,023 related hematopoietic stem cell donors on experiences around information and psychosocial support found few studies addressed donors’ unmet needs. Importantly, genetic testing in the context of cancer predisposition contribution to psychosocial stressors has not been measured or evaluated in published literature^[40]. For mutations associated with adult age at onset of disease, such as DDX41, this could translate to testing for a mutation with average onset decades later and why the National Society of Genetic Counselors (NSGC) does not encourage genetic testing for adult-onset diseases in individuals < 18. This, and more broadly the issue of testing coercion that could arise due to lack of matched unrelated donors, may disproportionately impact individuals of African, Asian, Native American, Pacific Islander, and mixed genomic ancestries given the likelihood of finding an unrelated match is substantially reduced due to a smaller donor pool coupled with more human leukocyte antigen (HLA) diversity^[41]. In January of 2023, the NMDP initiated a policy that allows donors to receive results about genetic variation detected in a recipient following transplant, but this information is not made available at donation^[42].

Previous DISCOVeRY-BMT variant and gene-based analyses were performed irrespective of variant pathogenicity and were the first to identify rare and common variations related to one-year survival following transplant^[10-12]. The analyses herein focus on LPV/PV variants in a select list of genes related to cancer and demonstrate that germline mutations in known cancer susceptibility genes with medical management recommendations are present at low frequency in unrelated donors. While broad unrelated donor germline screening is not standard practice, it is imperative that research on the psychosocial implications of germline testing for hereditary hematologic malignancies keep pace with genomic discovery and the framework for donor disclosure is continually developed and refined^[43]; it is only in this way that we will be able to effectively incorporate appropriate genomic testing.

Counseling BMT patients and their families can be further complicated by the type of genomic variation identified. While a novel germline DNMT3A mutation was recently identified in a mother-son pair with leukemia^[44], the two DNMT3A variants we identified in AML patients are likely somatic. Those in the five donors are likely CHIP. CHIP is associated with an increased risk of hematologic cancer, an increase in all-cause mortality, decreased overall survival, and susceptibility to heart disease, which makes the finding of these mutations in our young donors of interest^[31,45,46]. At present, opinions are divided in the field regarding screening donors for CHIP^[47,48]. To understand the impact of donor or recipient CHIP on transplant outcomes, and subsequently make clinical recommendations for donor selection and genetic counseling, larger population studies and clinical trials will be necessary^[49,50]. From a patient care and genetic counseling perspective, CHIP highlights unique considerations of caring for transplant patients and their donors. While the identification and clinical implications of CHIP in high-risk populations have recently been explored, there is a paucity of articles on counseling patients with CHIP^[51]. An extensive MESH search identified one article written in the last year focusing on counseling older adults with cancer^[52].

This study is not without limitations. The patient and donor population in DISCOVeRY-BMT, while representative of HLA-matched unrelated transplants in the United States, is not representative of the racial and/or ethnic diversity of AML or MDS patients. Larger more diverse studies with follow-up time longer than one year are imperative. In addition, patient samples are blood, and while taken pre-transplant from patients in complete remission (CR) helps distinguish between somatic germline, the exclusion of mutations that are only somatic was not possible. The selection of genes with known function-related cancer susceptibility can be viewed as a strength; however, the assumption that genes associated with susceptibility are also associated with one-year survival following treatment is an independent hypothesis. Lastly, while some work has been performed for validation and functional purposes^[10,53,54], additional cohorts with full sequencing data and further functional studies are warranted.

We identified LPV/PV across cancer genes in AML and MDS patients and their HLA-matched unrelated blood or marrow donors. These data are the first to demonstrate that collectively, donor PV across Fanconi Anemia genes significantly impacts recipient survival in the first year following transplant. Using a decade of data from CIBMTR on AML and MDS patients and donors, these results highlight the need for genomic science and genetic counseling research around the characterization of susceptibility and survival-correlated germline variation in patients receiving a BMT, donor selection, and the conveyance of this information to the patient and their family.