Augmented whole-exome sequencing

Details regarding DNA sequencing are available in the supplemental Methods on the Blood website, and the overall workflow is illustrated in Figure 1. DNA was extracted from peripheral blood samples stored at −80°C, using a QIAcube robotic workstation (Qiagen, Hilden, Germany). Whole-exome sequencing (WES) was augmented with custom-designed spike-in probes covering noncoding regions known to contain inherited risk alleles, including the following: the 5′ untranslated region (UTR) of ANKRD26 (NM_014915), the 5′ UTR of DKC1 (NM_001363), intron 31 of FANCI (NM_001113378), intron 4 of GATA2 (NM_032638), and the entire RNA gene TERC (NR_001566). Sequencing was performed at the Yale Center for Genome Analysis at 40× coverage. Variant calling and annotation were performed with an in-house custom pipeline (https://github.com/LucyGodley/Pipeline/tree/main/Variant_Calling/WES/hg).

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Figure 1.

Work flow diagram for the identification of P/LP germ line variants. DNA was extracted from thawed peripheral blood from 404 patients with MDS and was sequenced using an augmented whole-exome sequencing platform. SNVs and CNVs were called in 233 genes, using custom bioinformatic pipelines. A total of 21,661 SNVs and 334 CNVs were manually curated according to the ACMG/AMP and ClinGen SVI-WG guidelines. P/LP germ line variants were validated by Sanger sequencing, subcloning, and/or RT-qPCR. ACMG, American College of Medical Genetics; AMP, Association for Molecular Pathology; ClinGen SVI-WG, Clinical Genome Sequence Variant Interpretation Working Group; CNV, copy number variant; LP, likely pathogenic; MDS, myelodysplastic syndrome; P, pathogenic; RT-qPCR, quantitative real-time PCR; SNV, single-nucleotide variant; WES, whole-exome sequencing.

Variants in 233 genes associated with inherited hematopoietic malignancies, BMF syndromes, TBD, DNA repair deficiency, immunodeficiency, RASopathies, cancer predisposition syndromes, and congenital cytopenias were analyzed (supplemental Table 1). DNA from 9 of the patients was unavailable for augmented WES; however, sequencing data from a next-generation sequencing panel including 77 of these 233 genes (supplemental Table 1) were available for our use.²⁹ Somatic variants in all 404 recipients and 25 donors sharing a germ line variant were assessed in an additional 167 genes known to be somatically mutated in MDS based on large MDS data sets from cBioPortal³⁰ and the Catalogue of Somatic Mutations in Cancer³¹ (supplemental Table 2).

Variant interpretation

Details regarding variant calling, interpretation, validation, and the multiple-criteria decision analysis are available in the supplemental Methods. Briefly, germ line single-nucleotide variants (SNVs) and copy number variants (CNVs) were analyzed manually according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP), and Clinical Genome Sequence Variant Interpretation Working Group guidelines.^{32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48} P/LP germ line variants were validated by custom-designed polymerase chain reaction (PCR) amplification and Sanger sequencing. For diseases with recessive inheritance, subcloning of PCR amplicons/sequencing was done to confirm the variants were in trans. Validation of germ line CNVs was performed by quantitative real-time PCR.

Germ line status was determined by the presence of the identical P/LP variant within both the patients with MDS and their related donors, and/or variants with a variant allele frequency (VAF) of 40% to 60% seen previously only as germ line alleles,⁴⁹ including DDX41 (NM_016222.4) p.Asp140Glyfs2, and p.Arg53Alafs16 (Figure 2). Presumed germ line status was determined for P/LP variants with a VAF within germ line range (0.4-0.6 or 1.0) that were not shared in the donor sample, with no other potential P/LP variants within germ line range in the same recipient, and a combination of the following criteria based on a multiple-criteria decision analysis: (1) presence and frequency of somatic variants in the gene of question; (2) previous report of the identical variant in the somatic setting; (3) additional somatic variants and their VAF; (4) reported predisposing condition; (5) MDS subtype with higher bone marrow blast count (>5%) or advanced disease; and (6) high pretest probability as has been described for genes, such as BRCA1/2, DDX41, GATA2, RUNX1, and others (supplemental Table 3).^{49, 50, 51, 52} The interpretation of somatic variants was conducted using the ACMG/AMP and American Society of Clinical Oncology guidelines.⁵³

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Figure 2.

Variant subgroups based on donor status. This schematic identifies subgroups of patients with MDS/HSCT recipients based on the presence or absence of a predisposing variant, the manner of inheritance, whether the variant is shared by the donor and would be disease-causing, as well as subgroups chosen for statistical analyses of outcome parameters. ^†Healthy carriers refer to recipients with a P/LP variant who received donor stem cells lacking an AD P/LP variant or cells with a heterozygous P/LP allele with an AR mode of inheritance. AD, autosomal dominant; AR, autosomal recessive; het, heterozygous; HSCT, hematopoietic stem cell transplantation; LP, likely pathogenic; MDS, myelodysplastic syndrome; mut, mutated; P, pathogenic; wt, wild-type.

Yield and variant spectrum of confirmed P/LP germ line variants

We identified P/LP germ line variants in 28 of 404 patients with MDS (7%), present in all age deciles from ages 11 to 71 years (Table 2; supplemental Tables 6 and 7). The yield was highest in the 11- to 20-year age group at 33%, followed by 8% in the age deciles 21 to 30, 31 to 40, and 71 to 80 years; 7% in the age decile 51 to 60 years; and 6% in the age deciles 41 to 50 and 61 to 70 years (Figure 3). Twenty-three of the patients were identified with a germ line predisposition syndrome with autosomal dominant (AD) inheritance, and of those, variants were shared with the related donor in 20 cases (Figure 2; Table 2). Three patients with MDS with truncating DDX41 variants (p.Asp140Glyfs2 [Nos. 12R and 13R] and p.Arg53Alafs16 [No. 15R]) did not share the variant with their donors (Table 2). However, these variants were considered germ line based on the VAF within germ line range (0.4-0.6) and the historical absence of truncating DDX41 variants in tumor tissue (Figure 2).⁴⁹ In the 5 patients with autosomal recessive (AR) conditions, 3 were homozygous for a P/LP variant in ERCC6L2 (No. 16R), FANCA (No. 17R), and FANCG (No. 18R), respectively, and 2 individuals (Nos. 24R and 25R) were confirmed to have 2 different SBDS variants in the compound heterozygous state (Table 2). The related donors of these 5 patients with AR disease were heterozygotes for 1 of the P/LP variants (Figure 2). One patient with MDS (No. 3R) had a shared LP BLM variant as well as a second LP BLM variant with a VAF in germ line range that was not shared (Table 2), but the phase of these alleles could not be determined due to technical limitations. If both variants were present in the compound heterozygous state, the patient would be diagnosed with AR Bloom syndrome.

Table 2.

Overview of all P/LP confirmed germ line variants detected in this cohort of patients with MDS

ID	Shared	Gene	Inheri-tance	Variant type	cDNA	Protein	Transcript	VAF	ACMG/AMP classifi-cation	Age at diagnosis (y)	MDS WHO 2008 classification	Somatic driver variants	Outcome
1R	Yes	BARD1	AD	Frameshift	c.1935_1954dup	p.Glu652Valfs69	NM_000465.4	0.61	LP	64	RAEB-2	—	Alive
2R	Yes	BLM	AD	Frameshift	c.2506_2507del	p.Arg836Glyfs18	NM_000057.4	0.52	LP	71	CMML	MPL p.Ser204Pro TET2 p.Arg544Ter TET2 p.Gln1053Ter	Relapse, death from infection
3R	Yes	BLM	AD	Nonsense	c.1642C>T	p.Gln548	NM_000057.4	0.43	LP	57	MDS, unclassifiable†	—	Alive
4R	Yes	BRCA2	AD	Frameshift	c.5946delT	p.Ser1982Argfs22	NM_000059.4	0.51	P	52	RAEB-1†	RAD50 p.Asn934Lysfs TP53 p.Lys132Glu	Relapse, death from primary disease
5R	Yes	BRCA2	AD	Frameshift	c.5946delT	p.Ser1982Argfs22	NM_000059.4	0.58	P	59	RCMD†	—	Relapse, death from primary disease
6R	Yes	BRCA2	AD	Nonsense	c.5217_5223del	p.Tyr1739	NM_000059.4	0.61	P	56	RAEB-2	TP53 c.783-1G>A PPM1D p.Val465Aspfs SMC1A p.Lys752Argfs	Relapse, death from primary disease
7R	Yes	BRIP1	AD	Frameshift	c.1236delA	p.Val413Phefs10	NM_032043.3	0.53	LP	63	RAEB-2	—	Relapse, death from infection
8R	Yes	BRIP1	AD	Frameshift	c.3196delT	p.Ser1066Hisfs12	NM_032043.3	0.45	LP	64	MDS, unclassifiable†	CBL p.Cys404Tyr DNMT3A p.Arg882His NRAS p.Gly12Asp SETBP1 p.Asp868Asn ASXL1 p.Tyr591Ter	Relapse, death from primary disease
9R	Yes	BRIP1	AD	Frameshift	c.2392C>T	p.Arg798	NM_032043.3	0.69	LP	60	RAEB-2	NF1 c.3198-1G>T	Relapse, death from primary disease
10R	Yes	CHEK2	AD	Frameshift	c.1229del	p.Thr410Metfs15	NM_001005735.2	0.39	P	49	MDS, unclassifiable	—	Relapse, alive
11R	Yes	CHEK2	AD	Frameshift	c.1392delT	p.Ser465Valfs15	NM_001005735.2	0.53	LP	54	MDS associated with isolated del(5q)	NFE2 p.Arg323Alafs CSNK1A1 p.Asp140Ala	Alive
12R	No	DDX41	AD	Nonsense	c.415_418dup	p.Asp140Glyfs2	NM_016222.4	0.43	P	66	MDS, unclassifiable	ASXL1 p.Gly646Trpfs	Alive
13R	No	DDX41	AD	Nonsense	c.415_418dup	p.Asp140Glyfs2	NM_016222.4	0.49	P	57	RAEB-2	—	Alive
14R	Yes	DDX41	AD	Nonsense	c.415_418dup	p.Asp140Glyfs2	NM_016222.4	0.42	P	49	RAEB-2	—	Alive
15R	No	DDX41	AD	Frameshift	c.155dupA	p.Arg53Alafs16	NM_016222.4	0.49	P	69	RAEB-2	—	Relapse, death from primary disease
16R	Yes	ERCC6L2	AR	Nonsense	c.19C>T	p.Gln7	NM_001010895.4	1	LP	34	RAEB-1	SF3B1 p.Arg238Cys TP53 p.Arg175His	Relapse, death from primary disease
17R	Yes	FANCA	AR	Indel	c.3791_3793del	p.Ser1264del	NM_000135.4	1	P	11	RAEB-2	—	Nonrelapse mortality, cause of death not specified
18R	Yes	FANCG	AR	Frameshift	c.907_908dup	p.Glu304Trpfs4	NM_004629.2	0.93	LP	27	RA	ASXL1 p.Arg693Ter	Alive
19R	Yes	MRE11	AD	Nonsense	c.1516G>T	p.Glu506	NM_005591.4	0.54	LP	54	RCMD†	—	Death from infection
20R	Yes	MSH6	AD	Frameshift	c.1634_1635del	p.Lys545Argfs17	NM_000179.3	0.46	P	49	RA†	—	Nonrelapse mortality, death from secondary malignancy
21R	Yes	NBN	AD	Splicing	c.2071-1G>A	na	NM_002485.5	0.62	P	55	CMML	TET2 p.His667Ilefs GATA2 p.Met388_Lys389del RUNX1 p.Leu161_Arg162insSer CBL p.Leu380Pro SRSF2 p.Pro95Leu TET2 p.Lys875Ter	Primary graft failure/rejection, alive
22R	Yes	PALB2	AD	Copy number variant	na‡	na	NC_000016.10	0.52	LP	65	RAEB-2	—	Relapse, death from primary disease
23R	Yes	PMS2	AD	Nonsense	c.11C>G	p.Ser4	NM_001322015.2	0.41	LP	60	RCMD	—	Nonrelapse mortality, death from GVHD
24R	Yes/no§	SBDS	AR	Nonsense/splicing	c.184A>T/c.258+2T>C	p.Lys62/na	NM_016038.4	0.38/0.43	P/P	18	RAEB-2	—	Relapse, death from primary disease
25R	Yes/no§	SBDS	AR	Splicing/splicing	c.258+1G>C/c.258+2T>C	na/na	NM_016038.4	0.7/0.26	P/P	19	MDS associated with isolated del(5q)	TP53 c.559+1G>A	Alive
26R	Yes	TERT	AD	Nonsense	c.951G>A	p.Trp317	NM_198253.3	0.43	LP	51	RAEB-2	—	Alive
27R	Yes	TERT	AD	Nonsynonymous	c.193C>G	p.Pro65Ala	NM_198253.3	0.63	LP	64	RAEB-2	TP53 p.Arg248Gln TP53 p.Thr125Thr	Relapse, death from primary disease
28R	Yes	TNFRSF13B	AD	Frameshift	c.204dupA	p.Leu69Thrfs12	NM_012452.3	0.46	P	66	MDS, unclassifiable	—	Relapse, alive

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ACMG, American College of Medical Genetics and Genomics; AD, autosomal dominant; AMP, Association for Molecular Pathology; AR, autosomal recessive; GVHD, graft-versus-host disease; indel, insertion/deletion; na, not applicable; VAF, variant allele frequency; WHO, World Health Organization. Other abbreviations are explained in Table 1.

Donor shares AD germ line variant.

^†Therapy related.

^‡NC_000016.10:g.(?_23607316)_(23641590_?)del.

^§Donor only shares 1 of the variants in the heterozygous state.

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Figure 3.

Age distribution of pathogenicknownand presumed germ line variants in patients with MDS. The number of patients with MDS (left y-axis, red) with a confirmed germ line variant (red, solid line), a presumed germ line variant (red, dashed line), or wild-type (ie, without a germ line variant; peach, solid line with shading) is plotted per age decile (x-axis). The percentage (right y-axis, blue) of confirmed germ line variants (blue, solid line) and presumed germ line variants (blue, dashed line) is plotted per age decile. Presumed germ line variants based on a multiple-criteria decision analysis.

Among the 28 P/LP variants, 27 were SNVs. One CNV, a PALB2 deletion including exons 1 to 12, was found. The vast majority of variants were truncating variants (n = 26), followed by 1 small in-frame deletion and 1 missense variant (Table 2). P/LP variants affected genes involved in BMF syndromes [ERCC6L2 (n = 1), FANCA (n = 1), FANCG (n = 1), SBDS (n = 2)], TBD [TERT (n = 2)], general tumor predisposition syndromes [BARD1 (n = 1), BLM (n = 2), BRCA2 (n = 3), BRIP1 (n = 3), CHEK2 (n = 2), MRE11 (n = 1), MSH6 (n = 1), NBN (n = 1), PALB2 (n = 1), PMS2 (n = 1)], and DDX41 (n = 4). One P/LP variant was found in an immunodeficiency-related gene, TNFRSF13B (Figure 4A-B).⁵⁶ The subgroup of patients with BMF syndromes was younger at the time of diagnosis (median age, 20 years) compared with patients with TBD and general tumor predisposition syndromes, who were diagnosed at a median age of 58 and 59 years, respectively. Patients with DDX41 variants presented with the oldest median age at disease onset, 65 years (Figure 4C), consistent with prior literature.^{16, 17, 18} This pattern is also upheld when comparing the age of confirmed and presumed germ line variant carriers in our cohort and other larger cross-sectional studies on germ line variants in patients with MDS using either panel-based or WES covering most genes of interest. Deleterious variants in DDX41 were associated with an age at onset greater than 40 years, whereas those in GATA2 were associated with an age at onset less than 40 years (Figure 4D).^{5, 57, 6, 7, 8,12,15,57}

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Figure 4.

Spectrum and distribution of deleterious germ line variants among subgroups and association with age at diagnosis. (A) Bar chart depicting the number of confirmed P/LP germ line variants per gene identified in 28 patients with MDS. Compound heterozygous variants in the AR gene SBDS were counted as 1 variant per patient. Color schematic: purple, DDX41; green, general tumor predisposition genes; blue, telomere biology disorders; red, bone marrow failure genes; gray, others. (B) Nested pie chart showing the distribution of gene subgroups as well as biological functions. (C) Violin plots of the age at diagnosis of the 4 most common groups of predisposition syndromes identified in this study. (D) Association between the affected gene in the germ line and the age at MDS diagnosis. The data are derived from our study (confirmed/presumed germ line, n = 44) and other larger cross-sectional studies on germ line variants in patients with MDS using either panel-based or WES covering most genes of interest.^{5, 57, 6, 7, 8,12,15,57} Each circle represents a gene, and the circle size corresponds to the frequency of variants within this gene in the total cohort. Genes in the upper left quadrant were associated with an age at diagnosis of less than 40 years, whereas genes in the upper right quadrant were associated with an age at diagnosis greater than 40 years, based on calculated odds ratio. Color schematic: purple, DDX41; green, general tumor predisposition genes; blue, telomere biology disorders; red, bone marrow failure genes; gray, others. DSB, double-strand break repair; MMR, mismatch repair; RM, ribosome maturation.

In total, 20 of the 28 donors (71%) shared the P/LP AD germ line variant with the recipients. Five of the 28 donors were heterozygous carriers for a P/LP germ line variant with AR inheritance (Nos. 16D, 17D, 18D, 24D, and 25D) (Table 2; supplemental Table 7A). One donor was found to have a LP germ line DDX41 variant (NM_016222.4: c.3G>A, p.?) that was found only in the donor and not the patient with MDS, although it was confirmed that these 2 individuals were related based on the presence of shared variants (No. 29D) (supplemental Tables 7A and 8). This donor was aged 54 years, which is lower than the average age at onset for DDX41-related malignancies (65-69 years), for which the penetrance is known to be incomplete.^{16, 17, 18} Of the 404 related donors, 21 (5%) were heterozygous for a LP/P variant in a gene with AD inheritance with a VAF within the germ line range (supplemental Figure 1; supplemental Table 9).

Patients with MDS with P/LP germ line variants were more likely to have a reported predisposing condition, compared with those patients without such disorders (11% vs 4%, respectively; P < .04) (supplemental Table 5). Moreover, patients with MDS with P/LP germ line variants were more likely to have higher-grade MDS than those without such variants (43% vs 25%, respectively; P = .04) (Table 1). There was no difference in the age at onset, sex, ethnicity, cytogenetic abnormalities, IPSS-R classification, bone marrow cellularity, graft source, or donor type between patients with or without a P/LP germ line variant (Table 1; supplemental Table 5). The power to detect differences in cytogenetic abnormalities or IPSS-R classification was limited because of missing data.

Germ line variants of unknown significance (VUSs) were detected frequently in this study. Fifty-six shared germ line VUSs were found, and among these, 6 (10%) were deemed highly suspicious by calculating posterior probabilities in a Bayesian framework³⁸ based on ACMG/AMP criteria (posterior probabilities between 67.5% and 90%) (supplemental Table 10). These highly suspicious VUSs were identified in MLH1 (n = 2), SRC (n = 1), and TERT (n = 3) (supplemental Tables 7B and 10). The median age of the patients with MDS with these variants was 53 years (range, 45-69 years). If these highly suspicious VUSs are considered P/LP, it would raise the total yield of germ line variants to 12%.

Yield and variant spectrum of presumed P/LP germ line variants

Two hundred fifty-one other P/LP variants were identified among the 404 patients with MDS, but could not be confirmed as germ line variants because they were not shared between the patient and the related donor (supplemental Table 7A). By performing a multiple-criteria decision analysis, we determined that 16 variants were most likely germ line, including 5 GATA2 variants, 3 RUNX1 variants, 2 DDX41 variants, 2 TP53 variants, and single variants in ACD, CSF3R, NBN, and TUBB1, respectively (supplemental Figure 2; supplemental Tables 3 and 11). The penetrance of recognized phenotypes, such as the presence of cytopenias, in patients with P/LP germ line GATA2 or RUNX1 variants, would be expected to result in rejection of related donors who share the germ line variant in most cases, explaining why these variants were not identified as shared in our study.

Somatic variants

Because our methodology used sequencing of peripheral blood samples from patients with MDS, we were able to use the data to determine the somatic variants in the malignant cells and to test whether particular somatic variants were associated with specific predisposition disorders. However, sequencing was performed at an average depth of 40× for germ line variant calling, giving us the ability to identify relatively large somatic clones with a VAF greater than 10%. Smaller clone sizes were rarely detected. Variants were only considered somatic variants when: (1) they occurred below a population frequency of 0.5% in the general Genome Aggregation Database population or continental subpopulations; (2) they were not shared between donor and recipient; and (3) they were not presumed germ line based on the multiple-criteria decision analysis.

Among all patients with MDS and 167 genes analyzed, we identified 652 driver variants. Forty-seven of those 167 genes were also analyzed for germ line variants (supplemental Table 2), with 58 presumed somatic variants detected within a germ line VAF range (8.9%). The top 10 genes most commonly somatically mutated were as follows: ASXL1 (19.1%), TP53 (11.9%), DNMT3A (11.6%), TET2 (10.4%), SRSF2 (9.7%), RUNX1 (9.2%), U2AF1 (7.2%), SF3B1 (5.7%), SETBP1 (4.7%), and STAG2 (3.7%) (Figure 5A). Variants were found mostly in genes affecting DNA methylation (27.7%), chromatin modification (25.5%), splicing (24.5%), transcription (24%), checkpoint/cell cycle regulation (13.1%), and Ras signaling (11.1%) (Figure 5B; supplemental Table 12A). The somatic variants in individuals with confirmed/presumed germ line variants were grouped by broad gene ontology and biological pathways and are shown in Figure 5C, together with the age at diagnosis, MDS subgroup, cytogenetics, and outcomes. We found no differences in the pattern of somatic variants in the group with confirmed/presumed germ line variants (n = 44) against the group with no germ line predisposition (n = 360), either gene-wise or by affected gene groups, suggesting that the pathways leading to MDS were similar (supplemental Table 13). Two of 25 donors sharing a germ line variant had clonal hematopoiesis of indeterminate potential, with 1 variant in ARID1A (No. 20D at a VAF of 23%) and another variant in U2AF1 (No. 26D at a VAF of 19%) (supplemental Table 12B).

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Figure 5.

Spectrum and distribution of germ line variants, co-occurring somatic variants, and clinical data. (A) Bar chart displaying the top 20 most commonly affected genes for somatic variants (dark blue, wild-type; light blue, confirmed or presumed germ line variant). Percentages are given at the end of each bar. (B) Bar chart showing the top 10 most commonly affected gene groups (by broad gene ontology and biological pathways) (dark red, wild-type; light red, confirmed or presumed germ line variant). Percentages are given at the end of each bar. (C) Mutational grid with all 28 patients with MDS with confirmed germ line P/LP variants (left) and the 16 patients with MDS with presumed germ line variants (right). Patients are grouped by age at diagnosis in both panels. (Top) Clinical data such as age, MDS subtype, cytogenetic analysis, and outcome parameters, including death, relapse and nonrelapse mortality, and primary graft failure/rejection as well as co-occurring somatic variants (grouped by broad gene ontology and biological pathways) are included. (Bottom) The legend indicates the colors used for age deciles, MDS subgroups, cytogenetic analysis, variant status, and missing data. CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndrome; RA, refractory anemia; RAEB, refractory anemia with excess blasts; RARS, refractory anemia with ring sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; RCMD-RS, refractory cytopenia with multilineage dysplasia and ring sideroblasts.

The authors thank the patients and their donors for participating in research through the CIBMTR. The authors acknowledge all participating centers and researchers from the CIBMTR and thank the members of the Chronic Leukemia Writing Committee, especially for their critical review of this work and their helpful suggestions: Mahmoud Aljurf, Ulrike Bacher, Amer Beitinjaneh, Jean-Yves Cahn, Jan Cerny, Edward Copelan, Miguel A. Diaz, Shahinaz Gadalla, Robert Peter Gale, Siddhartha Ganguly, Michael Grunwald, Nasheed M. Hossain, Mohamed A. Kharfan-Dabaja, Tania Jain, Mark Juckett, Hillard M Lazarus, Mark Litzow, Sunita Nathan, Richard F. Olsson, Attaphol Pawarode, David A. Rizzieri, Lynn Savoie, Kirk R. Schultz, Sachiko Seo, Melhem Solh, Celalettin Ustun, David Valcárcel, and Baldeep Wirk.

Funding for this work was provided by the Department of Defense (W81XWH-19-1-0241) (L.A.G.). S.F. receives funding from the Olympia Morata Program of the Medical Faculty Heidelberg. A.M.T. was supported by generous donors from the QEII Health Sciences Centre Foundation. M.W.D. was supported by the Damon Runyon Cancer Research Foundation, the Edward P. Evans Foundation, and the National Institutes of Health Paul Calabresi K12 Program in Oncology.

The CIBMTR is supported primarily by Public Health Service U24CA076518 from the National Cancer Institute (NCI), the National Heart, Lung, and Blood Institute (NHLBI), and the National Institute of Allergy and Infectious Diseases (NIAID); HHSH250201700006C from the Health Resources and Services Administration (HRSA); and N00014 to 20-1-2705 and N00014 to 20-1-2832 from the Office of Naval Research. Support is also provided by Be The Match Foundation, the Medical College of Wisconsin, the National Marrow Donor Program, and the following commercial entities: AbbVie; Accenture; Actinium Pharmaceuticals, Inc; Adaptive Biotechnologies Corporation; Adienne SA; Allovir, Inc; Amgen, Inc; Astellas Pharma US; bluebird bio, Inc; Bristol Myers Squibb Co; CareDx; CSL Behring; CytoSen Therapeutics, Inc; Daiichi Sankyo Co, Ltd; Eurofins Viracor, DBA Eurofins Transplant Diagnostics; Fate Therapeutics; Gamida-Cell, Ltd; Gilead; GlaxoSmithKline; HistoGenetics; Incyte Corporation; Iovance; Janssen Research & Development, LLC; Janssen/Johnson & Johnson; Jasper Therapeutics; Jazz Pharmaceuticals, Inc; Kadmon; Karius; Karyopharm Therapeutics; Kiadis Pharma; Kite Pharma Inc; Kite, a Gilead Company; Kyowa Kirin International plc; Kyowa Kirin; Legend Biotech; Magenta Therapeutics; Medac GmbH; Medexus; Merck & Co; Millennium, the Takeda Oncology Co; Miltenyi Biotec, Inc; MorphoSys; Novartis Pharmaceuticals Corporation; Omeros Corporation; OncoImmune,Inc; Oncopeptides, Inc; OptumHealth; Orca Biosystems, Inc; Ossium Health, Inc; Pfizer, Inc; Pharmacyclics, LLC; Priothera; Sanofi Genzyme; Seagen, Inc; Stemcyte; Takeda Pharmaceuticals; Talaris Therapeutics; Terumo Blood and Cell Technologies; TG Therapeutics; Tscan; Vertex; Vor Biopharma; Xenikos BV.