Distribution of clonal hematopoiesis of indeterminate potential (CHIP) is not associated with race in patients with plasma cell neoplasms

Dear Editor, Several studies have recently raised mounting interest regarding clonal hematopoiesis (CH) in the setting of plasma cell neoplasms (PCNs). CH has been shown to occur at an increased frequency among patients with multiple myeloma (MM) undergoing autologous stem cell transplantation and to adversely affect overall survival (OS) and progression-free survival (PFS) in the absence of immunomodulatory drug maintenance [1]. While evidence regarding a role for CH in PCN disease biology is growing, research efforts have largely focused on patients who self-report as non-Hispanic White (NHW). Given the increased risk of MM among Black/AA individuals and the association between CHIP and MM progres- sion, we sought to interrogate CH in a diverse cohort and compare the frequency of this condition in individuals who self- identify as Black/AA vs. NHW. Following Mayo Clinic Institutional Review Board approval and patient informed consent, we performed targeted next-generation sequencing in a cohort of 174 patients with a PCN including MGUS, smoldering MM, MM, amyloidosis and other PCN. Samples were selected from Mayo Clinic patients with available DNA from the diagnostic bone marrow biopsy (subset from the cohort previously described in Baughn et al. [2]). Genomic DNA was extracted from bone marrow aspirates following a 24-h culture period using the QIAmp DNeasy Blood and Tissue Kit (Qiagen, Germantown, Maryland) and subjected to deep sequencing using a custom target bait panel including 30 genes recurrently mutated in CH. Libraries were sequenced on an Illumina HiSeq 4000 (average sequencing depth of ~4000x). Additional details regard- ing bioinformatics analyses and variant curation are available in Supplementary Materials. Allele frequency thresholds for CH were set at 0.01 and 0.02 as per the recognized de ﬁ nition of medullary microenvironment have also been posited [4]. Of distinct interest, an African ancestry-speci ﬁ c germline variant at the locus of an enhancer regulating TET2 expression was associated with an increased risk of CHIP [6]. We thus sought to evaluate whether the frequency of CHIP may be differentially affected by race among patients with PCNs, yet we found similar frequencies of CH occurrence among Black/AA and NHW patients with PCN. Our ﬁ ndings are in accordance with and expand on previous studies offering more modest representation of Black/AA individuals, affording the largest assessment of CH frequency in this population across different PCNs [1, 7 – 9]. Previous reports have revealed variable prognostic implications of CH in MM [1, 7]. In our general cohort, while only a trend for an adverse prognostic effect of CH at a VAF ≥ 0.01 was observed, a statistically signi ﬁ cant deleterious impact on PFS and OS was documented with a VAF ≥ 0.02. PFS was not differentially altered by self-reported race and ethnicity; however, similarly to the recent study by Peres et al. [9], an adverse effect on OS was noted in the setting of CH for Black/AA patients. In summary, our study suggests that the prevalence of CH in the setting of PCNs does not signi ﬁ cantly differ between Black/AA and NHW individuals. While African-speci ﬁ c germline variants predisposing to CH have been uncovered, our ﬁ ndings support that aging and potential factors related to PCN biology prevailingly in ﬂ uence CH frequency. Among Black/AA patients, our results suggest that CH may however be associated with deleterious implications on OS. writing — and editing. AKS: conceptualization, resources. LBB: conceptualiza-tion, data curation, formal analysis, supervision, methodology, writing — review and editing, project administration.


Dear Editor,
Several studies have recently raised mounting interest regarding clonal hematopoiesis (CH) in the setting of plasma cell neoplasms (PCNs). CH has been shown to occur at an increased frequency among patients with multiple myeloma (MM) undergoing autologous stem cell transplantation and to adversely affect overall survival (OS) and progression-free survival (PFS) in the absence of immunomodulatory drug maintenance [1]. While evidence regarding a role for CH in PCN disease biology is growing, research efforts have largely focused on patients who self-report as non-Hispanic White (NHW). Given the increased risk of MM among Black/AA individuals and the association between CHIP and MM progression, we sought to interrogate CH in a diverse cohort and compare the frequency of this condition in individuals who selfidentify as Black/AA vs. NHW.
Following Mayo Clinic Institutional Review Board approval and patient informed consent, we performed targeted next-generation sequencing in a cohort of 174 patients with a PCN including MGUS, smoldering MM, MM, amyloidosis and other PCN. Samples were selected from Mayo Clinic patients with available DNA from the diagnostic bone marrow biopsy (subset from the cohort previously described in Baughn et al. [2]). Genomic DNA was extracted from bone marrow aspirates following a 24-h culture period using the QIAmp DNeasy Blood and Tissue Kit (Qiagen, Germantown, Maryland) and subjected to deep sequencing using a custom target bait panel including 30 genes recurrently mutated in CH. Libraries were sequenced on an Illumina HiSeq 4000 (average sequencing depth of~4000x). Additional details regarding bioinformatics analyses and variant curation are available in Supplementary Materials. Allele frequency thresholds for CH were set at 0.01 and 0.02 as per the recognized definition of CHIP. Considering the overlap in genes mutated in CH and in MM, analyses were restricted to mutations in DNMT3A, TET2 and ASXL1 to ensure unambiguous attribution of mutations to the CH population. Patients were grouped according to self-reported race and ethnicity (Black/AA and NHW) and compared for CH frequency and outcome (OS and PFS). Survival and time-toevent curves were constructed using the Kaplan-Meier method and compared by the log-rank test. Cox proportional-hazards regression models were used for multivariable analysis to determine hazard ratios and associated confidence intervals. Detailed statistical methods and information regarding other selfreported racial groups are provided in Supplementary Materials.
In our full cohort, CH (VAF ≥ 0.01) was detected in 21% (n = 37/ 174) of patients. Median allele frequency was 4% (range: 1-98.9%). CH was detected in 21 patients with MM (23% of 91), 5 with MGUS (17% of 30), 5 with SMM (25% of 20) and 6 with amyloidosis (22% of 27). When analyses were restricted to mutations with a VAF of ≥ 0.02, 13 mutations (7.5%) within DNMT3A, TET2 and ASXL1 were documented (4 with MM, 3 with MGUS, 2 with SMM, 4 with AL amyloidosis). When CH was classified based on VAF ≥ 0.01, patients with CH were significantly older than patients without CH (median age: 71 and 64 years, respectively, p value <0.001). When a VAF threshold of 0.02 was considered, no significant difference in age (median age: 66 vs. 65 years respectively, p = 0.26) was seen. CH, as defined by an VAF threshold ≥ 0.01, occurred at a lower frequency in Black/AA individuals (n = 9/64, 14%) as compared with NHW individuals (n = 24/81, 30%; p value = 0.03). In multivariable analysis, race and ethnicity were not significantly associated with the incidence of CH and age remained the significant predictor of CH frequency. These findings suggest that the lower incidence of CH in Black/AA patients was likely confounded by lower median age in our cohort of Black/AA patients. Among Black/AA individuals with CH, mutations in DNMT3A (n = 7, 11%) and TET2 (n = 6, 9%) were most common. The individual frequencies of DNMT3A, TET2 and ASXL1 mutations did not significantly differ from those of NHW individuals when mutations with VAF ≥ 0.01 were considered. While the limited number of events calls for caution in the interpretation of data, TET2 mutations appeared less prevalent in AA individuals when restricting analyses to mutations with allele frequencies of VAF ≥ 0.02 (0% vs. 9.9% respectively, p value = 0.009).
We next assessed and compared OS and PFS based on CH status and self-reported race and ethnicity. Given the differential  (Fig. 1B). In assessing OS of the patients who were NHW or Black/AA, the median follow-up was 46.9 months (95% CI: 34. 8-64.4). OS was similar between Black/AA vs. NHW patients, even with stratification on PCN type (HR = 1.05, 95% CI: 0.53-1.86; p value = 0.99) (Fig. 1C,  Supplementary Figs. 1 and 5). When evaluating the influence of CH (VAF ≥ 0.01), a tendency toward poorer OS for those with CH in comparison to those without (HR = 1.80, 95% CI: 0.24-3.53; p value = 0.088) was observed, even after adjustment for race group and stratification on PCN type. This association was more substantial when considering CH with a VAF ≥ 0.02 (HR = 3.93, 95% CI: 1.60-9.65; p value = 0.003). Adjusting for age in these models confounded these results, mostly due to the high multicollinearity between age and CH incidence. However, inclusion of CH status (VAF ≥ 0.02) yielded a better predictive model for OS than models with age. Among patients with available data, cause of death did not differ between patients with CH and without CH and was mostly related to PCN progression and infection (p value = 0.8). To explore potential effect modification based on race and ethnicity, we further evaluated OS within Black/AA and NHW patients. In the NHW patients, CH status using VAF ≥ 0.01 did not significantly influence OS when stratifying on PCN type (HR = 1.11, 95% CI: 0.43-2.82; p value = 0.82) (Supplementary Fig. 7). When applying the same model to Black/AA patients with PCN, CH was associated with significantly worse OS when stratifying on PCN type (HR = 4.57, 95% CI: 1.48-14.1; p value = 0.008) (Fig. 1D). The influence of CH was similar in NHW and Black/AA patients at a VAF threshold ≥ 0.02. Black/AA and NHW patient with MM with and without CH did not otherwise differ regarding additional prognostic factors of relevance suggesting that this effect was not attributable to differences in established prognostic features (Table 2).
While genotoxic stress and selective pressure on hematopoietic stem cells with an increased fitness is a well-recognized risk factor for CH [3], the increased frequency of CH does not appear to be restricted to patients with previous exposure to cytotoxic therapy [4,5]. In our cohort, which included untreated patients, similar frequencies of CH were obtained in MM (23%) and in various PCNs (17% of MGUS, 25% of SMM and 22% with amyloidosis). A contribution of treatment-independent factors such as common underlying environmental exposures predisposing to CH and MM, MM-modulated immune dysfunction and alterations in the Correspondence medullary microenvironment have also been posited [4]. Of distinct interest, an African ancestry-specific germline variant at the locus of an enhancer regulating TET2 expression was associated with an increased risk of CHIP [6]. We thus sought to evaluate whether the frequency of CHIP may be differentially affected by race among patients with PCNs, yet we found similar frequencies of CH occurrence among Black/AA and NHW patients with PCN. Our findings are in accordance with and expand on previous studies offering more modest representation of Black/AA individuals, affording the largest assessment of CH frequency in this population across different PCNs [1,[7][8][9].
Previous reports have revealed variable prognostic implications of CH in MM [1,7]. In our general cohort, while only a trend for an adverse prognostic effect of CH at a VAF ≥ 0.01 was observed, a statistically significant deleterious impact on PFS and OS was documented with a VAF ≥ 0.02. PFS was not differentially altered by self-reported race and ethnicity; however, similarly to the recent study by Peres et al. [9], an adverse effect on OS was noted in the setting of CH for Black/AA patients.
In summary, our study suggests that the prevalence of CH in the setting of PCNs does not significantly differ between Black/AA and NHW individuals. While African-specific germline variants predisposing to CH have been uncovered, our findings support that aging and potential factors related to PCN biology prevailingly influence CH frequency. Among Black/AA patients, our results suggest that CH may however be associated with deleterious implications on OS.