Association of PARP inhibitor treatment on the prevalence and progression of clonal hematopoiesis in patients with advanced prostate cancer.

BACKGROUND: Poly ADP-ribose polymerase (PARP) inhibitors are approved for the treatment of some men with advanced prostate cancer. Rare but serious side effects include myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The impact of PARP inhibitors on clonal hematopoiesis (CH), a potential precursor lesion associated with MDS and AML, is incompletely understood in prostate cancer. We hypothesized that PARP inhibitors would increase CH prevalence and abundance. METHODS: We prospectively enrolled participants with advanced prostate cancer treated with PARP inhibitors. The presence of CH was assessed from leukocytes using an ultra-deep error-corrected dual unique molecular identifiers sequencing method targeting 49 genes most commonly mutated in CH and myeloid malignancies. Variant allele frequencies (VAF) of ≥0.5% were considered clinically significant. Blood samples were collected before and after PARP inhibitor treatment. RESULTS: Ten men were enrolled; mean age of 67 years. Six patients had Gleason 7 disease, and four had Gleason ≥8 disease at diagnosis. Nine had localized disease at diagnosis, and eight had prior treatment with radiation. The mean time between pre- and post-treatment blood samples was 11 months (range 2.6-31 months). Six patients (60%) had CH identified prior to PARP inhibitor treatment, three with multiple clones. Of 11 CH clones identified in follow-up, 5 (45%) appeared or increased after treatment. DNMT3A, TET2, and PPM1D were the most common CH alterations observed. The largest post-treatment increase involved the PPM1D gene. CONCLUSION: CH alterations are frequently found after treatment with PARP inhibitors in patients with prostate cancer and this may be one mechanism by which PARP inhibitors lead to increased risk of MDS/AML.

Impact of diagnostic genetics on remission MRD and transplantation outcomes in older patients with AML.

Older patients with acute myeloid leukemia (AML) have high relapse risk and poor survival after allogeneic hematopoietic cell transplantation (HCT). Younger patients may receive myeloablative conditioning to mitigate relapse risk associated with high-risk genetics or measurable residual disease (MRD), but older adults typically receive reduced-intensity conditioning (RIC) to limit toxicity. To identify factors that drive HCT outcomes in older patients, we performed targeted mutational analysis (variant allele fraction ≥2%) on diagnostic samples from 295 patients with AML aged ≥60 years who underwent HCT in first complete remission, 91% of whom received RIC, and targeted duplex sequencing at remission in a subset comprising 192 patients. In a multivariable model for leukemia-free survival (LFS) including baseline genetic and clinical variables, we defined patients with low (3-year LFS, 85%), intermediate (55%), high (35%), and very high (7%) risk. Before HCT, 79.7% of patients had persistent baseline mutations, including 18.3% with only DNMT3A or TET2 (DT) mutations and 61.4% with other mutations (MRD positive). In univariable analysis, MRD positivity was associated with increased relapse and inferior LFS, compared with DT and MRD-negative mutations. However, in a multivariable model accounting for baseline risk, MRD positivity had no independent impact on LFS, most likely because of its significant association with diagnostic genetic characteristics, including MDS-associated gene mutations, TP53 mutations, and high-risk karyotype. In summary, molecular associations with MRD positivity and transplant outcomes in older patients with AML are driven primarily by baseline genetics, not by mutations present in remission. In this group of patients, where high-intensity conditioning carries substantial risk of toxicity, alternative approaches to mitigating MRD-associated relapse risk are needed.

CCRL2 affects the sensitivity of myelodysplastic syndrome and secondary acute myeloid leukemia cells to azacitidine.

Better understanding of the biology of resistance to DNA methyltransferase (DNMT) inhibitors is required to identify therapies that can improve their efficacy for patients with high-risk myelodysplastic syndrome (MDS). CCRL2 is an atypical chemokine receptor that is upregulated in CD34+ cells from MDS patients and induces proliferation of MDS and secondary acute myeloid leukemia (sAML) cells. In this study, we evaluated any role that CCRL2 may have in the regulation of pathways associated with poor response or resistance to DNMT inhibitors. We found that CCRL2 knockdown in TF-1 cells downregulated DNA methylation and PRC2 activity pathways and increased DNMT suppression by azacitidine in MDS/sAML cell lines (MDS92, MDS-L and TF-1). Consistently, CCRL2 deletion increased the sensitivity of these cells to azacitidine in vitro and the efficacy of azacitidine in an MDS-L xenograft model. Furthermore, CCRL2 overexpression in MDS-L and TF-1 cells decreased their sensitivity to azacitidine. Finally, CCRL2 levels were higher in CD34+ cells from MDS and MDS/myeloproliferative neoplasm patients with poor response to DNMT inhibitors. In conclusion, we demonstrated that CCRL2 modulates epigenetic regulatory pathways, particularly DNMT levels, and affects the sensitivity of MDS/sAML cells to azacitidine. These results support CCRL2 targeting as having therapeutic potential in MDS/sAML.

Haploidentical donor hematopoietic cell transplantation for myelodysplastic/myeloproliferative overlap neoplasms: results from a North American collaboration.

Haploidentical donors offer a potentially readily available donor, especially for non-White patients, for hematopoietic cell transplantation (HCT). In this North American collaboration, we retrospectively analyzed outcomes of first HCT using haploidentical donor and post-transplantation cyclophosphamide (PTCy) in myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap neoplasms (MDS/MPN). We included 120 consecutive patients who underwent HCT using a haploidentical donor for MDS/MPN across 15 centers. Median age was 62.5 years and 38% were of non-White/Caucasian ethnicity. The median follow-up was 2.4 years. Graft failure was reported in seven of 120 (6%) patients. At 3 years, nonrelapse mortality (NRM) was 25% (95% confidence interval [CI]: 17-34), relapse 27% (95% CI: 18-36), grade 3-4 acute graftversus- host disease 12% (95% CI: 6-18), chronic graft-versus-host disease requiring systemic immunosuppression 14% (95% CI: 7-20), progression-free survival (PFS) 48% (95% CI: 39-59), and overall survival (OS) 56% (95% CI: 47-67). On multivariable analysis, NRM was statistically significantly associated with advancing age at HCT (per decade increment, subdistribution hazard ratio [sdHR] =3.28; 95% CI: 1.30-8.25); relapse with the presence of mutation in EZH2/RUNX1/SETBP1 (sdHR=2.61; 95% CI: 1.06-6.44); PFS with advancing age at HCT (per decade increment, HR=1.98, 95% CI: 1.13-3.45); and OS with advancing age at HCT (per decade increment, HR=2.01; 95% CI: 1.11-3.63) and splenomegaly at HCT/prior splenectomy (HR=2.20; 95% CI: 1.04-4.65). Haploidentical donors are a viable option for HCT in MDS/MPN, especially for those disproportionately represented in the unrelated donor registry. Hence, donor mismatch should not preclude HCT for patients with MDS/MPN, an otherwise incurable malignancy. In addition to patient age, disease-related factors including splenomegaly and high-risk mutations dominate outcomes following HCT.

Gender-related differences in the outcomes and genomic landscape of patients with myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes.

Myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndromes show a male predominance and men with MDS/MPN have worse outcomes, but it is unknown if the mutational burden differs between genders. We reviewed 167 patients with MDS/MPN and found that men had worse overall survival [hazard ratio (HR) 2·09, 95% confidence interval (CI) 1·16-3·75; P = 0·013] independent of subtype, Revised International Prognostic Scoring System score and age at diagnosis. We analysed the genomic data of a subset of 100 patients. Men had 0·88 more somatic mutations on average (95% CI 0·20-1·56, P = 0·011) independent of subtype, sample source and blast percentage. More somatic mutations was associated with a higher incidence of transformation to acute myeloid leukaemia (subdistribution HR 1·30, 95% CI 1·01-1·70; P = 0·046). Men had 0·70 more mutations in high-risk genes [additional sex combs like-1 (ASXL1), enhancer of zeste homolog 2 (EZH2), Runt-related transcription factor 1 (RUNX1), SET binding protein 1 (SETBP1), NRAS proto-oncogene, GTPase (NRAS), stromal antigen 2 (STAG2)] on average (95% CI 0·11-1·29, P = 0·021), and 13-times higher odds of harbouring an EZH2 mutation (95% CI 1·64-102·94, P = 0·015). The presence of an EZH2 mutation was associated with worse survival among men (HR 2·98, 95% CI 1·1-8·0; P = 0·031). Our present findings suggest that the worse outcomes in men with MDS/MPN are associated with a higher number of somatic mutations, especially in high-risk genes. These results warrant validation in larger cohorts and investigation of the underlying mechanisms.

Clonal hematopoiesis in the setting of hematopoietic cell transplantation.

Clonal hematopoiesis (CH) in autologous transplant recipients and allogeneic transplant donors has genetic features and clinical associations that are distinct from each other and from non-cancer populations. CH in the setting of autologous transplant is enriched for mutations in DNA damage response pathway genes and is associated with adverse outcomes, including an increased risk of therapy-related myeloid neoplasm and inferior overall survival. Studies of CH in allogeneic transplant donors have yielded conflicting results but have generally shown evidence of potentiated alloimmunity in recipients, with some studies showing an association with favorable recipient outcomes.

Recipient clonal hematopoiesis in allogeneic bone marrow transplantation for lymphoid malignancies.

Allogeneic blood and marrow transplantation (alloBMT) is increasingly being used in older patients with blood cancer. Aging is associated with an increasing incidence of clonal hematopoiesis (CH). Although the effects of donor CH on alloBMT has been reported, the impact of recipient CH on alloBMT outcomes is unknown. In this retrospective study, alloBMT recipients age 60 and older with lymphoid malignancies were included. Among 97 consecutive patients who received alloBMT between 2017 and 2022, CH was detected in 60 (62%; 95% CI 51-72%). CH was found in 45% (95% CI 28-64%) of patients aged 60-64, 64% (95% CI 44-81%) of patients aged 65-69, and 73% (95% CI 59-87%) in those above 70. Pretransplant CH was associated with worse survival after alloBMT: 3-year overall survival (OS) was 78% (95% CI 65-94%) for patients without CH versus 47% (95% CI 35-63%) for those with CH, [unadjusted HR 3.1 (95%CI 1.4-6.8; P<0.001)]. Non-relapse mortality (NRM) was higher in patients with CH; cumulative incidence of NRM at one-year was 11% (95% CI 1-22%) versus 35% (95% CI 23-48%), [HR 3.4 (95% CI 1.4-8.5), p=0.009]. Among CH patients, worse OS and NRM was associated with CH burden and number of mutations. Recipient CH had no effect on relapse. In conclusion, older patients with CH experience worse outcomes after alloBMT, almost exclusively attributable to increased NRM. CH is a strong, independent predictor of outcomes. Novel strategies to ameliorate the adverse impacts of patient CH on transplant outcomes are being evaluated.

Prognostic impact of SNP array karyotyping in myelodysplastic syndromes and related myeloid malignancies.

Single nucleotide polymorphism arrays (SNP-As) have emerged as an important tool in the identification of chromosomal defects undetected by metaphase cytogenetics (MC) in hematologic cancers, offering superior resolution of unbalanced chromosomal defects and acquired copy-neutral loss of heterozygosity. Myelodysplastic syndromes (MDSs) and related cancers share recurrent chromosomal defects and molecular lesions that predict outcomes. We hypothesized that combining SNP-A and MC could improve diagnosis/prognosis and further the molecular characterization of myeloid malignancies. We analyzed MC/SNP-A results from 430 patients (MDS = 250, MDS/myeloproliferative overlap neoplasm = 95, acute myeloid leukemia from MDS = 85). The frequency and clinical significance of genomic aberrations was compared between MC and MC plus SNP-A. Combined MC/SNP-A karyotyping lead to higher diagnostic yield of chromosomal defects (74% vs 44%, P < .0001), compared with MC alone, often through detection of novel lesions in patients with normal/noninformative (54%) and abnormal (62%) MC results. Newly detected SNP-A defects contributed to poorer prognosis for patients stratified by current morphologic and clinical risk schemes. The presence and number of new SNP-A detected lesions are independent predictors of overall and event-free survival. The significant diagnostic and prognostic contributions of SNP-A-detected defects in MDS and related diseases underscore the utility of SNP-A when combined with MC in hematologic malignancies.

The impact of epigenetic modifications on allogeneic hematopoietic stem cell transplantation.

The field of epigenetics studies the complex processes that regulate gene expression without altering the DNA sequence itself. It is well established that epigenetic modifications are crucial to cellular homeostasis and differentiation and play a vital role in hematopoiesis and immunity. Epigenetic marks can be mitotically and/or meiotically heritable upon cell division, forming the basis of cellular memory, and have the potential to be reversed between cellular fate transitions. Hence, over the past decade, there has been increasing interest in the role that epigenetic modifications may have on the outcomes of allogeneic hematopoietic transplantation and growing enthusiasm in the therapeutic potential these pathways may hold. In this brief review, we provide a basic overview of the types of epigenetic modifications and their biological functions, summarizing the current literature with a focus on hematopoiesis and immunity specifically in the context of allogeneic hematopoietic stem cell transplantation.

Persistent IDH mutations are not associated with increased relapse or death in patients with IDH-mutated acute myeloid leukemia undergoing allogeneic hematopoietic cell transplant with post-transplant cyclophosphamide.

The presence of measurable residual disease (MRD) prior to an allogeneic hematopoietic transplant (alloHCT) in Acute Myeloid Leukemia (AML) has been shown to be associated with an increased risk of post-transplant relapse. Since the Isocitrate Dehydrogenase genes (IDH1/2) are mutated in a considerable proportion of patients with AML, we studied if these mutations would serve as useful targets for MRD. Fifty-five IDH-mutated AML patients undergoing non-myeloablative alloHCT with post-transplant cyclophosphamide at a single center were sequenced at baseline using a multi-gene panel followed by targeted testing for persistent IDH mutations at the pre- and post-alloHCT timepoints by digital droplet PCR or error-corrected next generation sequencing. The cohort included patients who had been treated with IDH inhibitors pre- and post-transplant (20% and 17% for IDH1 and 38% and 28% for IDH2). Overall, 55% of patients analyzed had detectable IDH mutations during complete remission prior to alloHCT. However, there were no statistically significant differences in overall survival (OS), relapse-free survival (RFS), and cumulative incidence of relapse (CIR) at 3 years between patients who tested positive or negative for a persistent IDH mutation during remission (OS: IDH1 p=1, IDH2 p=0.87; RFS: IDH1 p=0.71, IDH2 p= 0.78; CIR: IDH1 p=0.92, IDH2 p=0.97). There was also no difference in the prevalence of persistent IDH mutation between patients who did and did not receive an IDH inhibitor (p=0.59). Mutational profiling of available relapse samples showed that 8 out of 9 patients still exhibited the original IDH mutation, indicating that the IDH mutations remained stable through the course of the disease. This study demonstrates that persistent IDH mutations during remission is not associated with inferior clinical outcomes after alloHCT in patients with AML.

Recurrent germline variant in ATM associated with familial myeloproliferative neoplasms.

Genetic predisposition (familial risk) in the myeloproliferative neoplasms (MPNs) is more common than the risk observed in most other cancers, including breast, prostate, and colon. Up to 10% of MPNs are considered to be familial. Recent genome-wide association studies have identified genomic loci associated with an MPN diagnosis. However, the identification of variants with functional contributions to the development of MPN remains limited. In this study, we have included 630 MPN patients and whole genome sequencing was performed in 64 individuals with familial MPN to uncover recurrent germline predisposition variants. Both targeted and unbiased filtering of single nucleotide variants (SNVs) was performed, with a comparison to 218 individuals with MPN unselected for familial status. This approach identified an ATM L2307F SNV occurring in nearly 8% of individuals with familial MPN. Structural protein modeling of this variant suggested stabilization of inactive ATM dimer, and alteration of the endogenous ATM locus in a human myeloid cell line resulted in decreased phosphorylation of the downstream tumor suppressor CHEK2. These results implicate ATM, and the DNA-damage response pathway, in predisposition to MPN.

Cell-free DNA measurable residual disease as a predictor of postallogeneic hematopoietic cell transplant outcomes.

The measurable residual disease (MRD) assessment provides an attractive predictor of allogeneic hematopoietic cell transplnat (alloHCT) outcomes. Cell-free DNA (cfDNA) has been applied to diagnosis, early detection, and disease burden monitoring in various tumors, but its utility as an MRD test in myeloid malignancies has not been systematically evaluated. We sought to determine the differential sensitivity between bone marrow (BM) and cfDNA MRD and to assess the effect of cfDNA MRD on alloHCT outcomes. The technical and clinical validation cohorts, including 82 patients participating in clinical trials (Bone Marrow Transplant Clinical Trials Network-0201 and 0402), were used. Ultradeep error-corrected targeted sequencing was performed on plasma and BM-derived DNA. We demonstrated that 94.6% (range, 93.9-95.3) of cfDNA was derived from hematopoietic tissue. The mutant allele fraction was congruent between BM and cfDNA (rho = 0.8; P < .0001); however, cfDNA seemed to be more sensitive in detecting clones with a variant allele frequency (VAF) of <0.26%. cfDNA-MRD clearance by day 90 after alloHCT (D90) was associated with improved relapse-free survival (RFS, median survival not reached vs 5.5 months; P < .0001) and overall survival (OS, median survival not reached vs 7.3 months; P < .0001) when compared with patients with persistent MRD. Irrespective of pre-alloHCT MRD, D90 cfDNA MRD was associated with inferior 2-year OS (16.7% vs 84.8%; P < .0001) and RFS (16.7% vs 80.7%; P < .0001). cfDNA seems to be an accurate, minimally invasive alternative to BM aspirates in MRD assessment and confers important prognostic implications in patients with myeloid malignancies undergoing alloHCT.

Co-occurring mutations in ASXL1, SRSF2, and SETBP1 define a subset of myelodysplastic/ myeloproliferative neoplasm with neutrophilia.

Identification of genomic signatures with consistent clinicopathological features in myelodysplastic/myeloproliferative neoplasm (MDS/MPN) is critical for improved diagnosis, elucidation of biology, inclusion in clinical trials, and development of therapies. We describe clinical and pathological features with co-existence of mutations in ASXL1 (missense or nonsense), SRSF2, and SKI homologous region of SETBP1, in 18 patients. Median age was 68 years with a male predominance (83%). Leukocytosis and neutrophilia were common at presentation. Marrow features included hypercellularity, granulocytic hyperplasia with megakaryocytic atypia, while the majority had myeloid hyperplasia and/or erythroid hypoplasia, myeloid dysplasia, and aberrant CD7 expression on blasts. Mutations in growth signaling pathways (RAS or JAK2) were noted at diagnosis or acquired during the disease course in 83% of patients. Two patients progressed upon acquisition of FLT3-TKD (acute myeloid leukemia) or KIT (aggressive systemic mastocytosis) mutations. The prognosis is poor with only two long-term survivors, thus far, who underwent blood or marrow transplantation. We propose that the presence of co-occurring ASXL1, SRSF2, and SETBP1 mutations can be diagnostic of a subtype of MDS/MPN with neutrophilia if clinical and morphological findings align. Our report underscores the association between genotype and phenotype within MDS/MPN and that genomic signatures should guide categorization of these entities.

Venetoclax in combination with hypomethylating agent for the treatment of advanced myeloproliferative neoplasms and acute myeloid leukemia with extramedullary disease.

The combination of venetoclax and hypomethylating agent (HMA/venetoclax) has emerged as a treatment option for patients with de novo acute myeloid leukemia (AML) who are unfit to receive intensive chemotherapy. In this single-center retrospective study, we evaluated clinical outcomes following treatment with HMA/venetoclax in 35 patients with advanced myeloproliferative neoplasms, myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes or AML with extramedullary disease. The composite complete remission (CR) rate (including confirmed/presumed complete cytogenetic response, acute leukemia response-complete, CR and CR with incomplete hematologic recovery) was 42.9% with median overall survival (OS) of 9.7 months. Complex karyotype was associated with inferior median OS (3.7 versus 12.2 months; p = 0.0002) and composite CR rate (22% versus 50.0%; p = 0.2444). Although SRSF2 mutations were associated with higher composite CR rate (80.0% versus 28.0%; p = 0.0082), this was not associated with longer median OS (10.9 versus 8.0 months; p = 0.2269). Future studies should include these patient subgroups.

Deleterious heteroplasmic mitochondrial mutations are associated with an increased risk of overall and cancer-specific mortality.

Mitochondria carry their own circular genome and disruption of the mitochondrial genome is associated with various aging-related diseases. Unlike the nuclear genome, mitochondrial DNA (mtDNA) can be present at 1000 s to 10,000 s copies in somatic cells and variants may exist in a state of heteroplasmy, where only a fraction of the DNA molecules harbors a particular variant. We quantify mtDNA heteroplasmy in 194,871 participants in the UK Biobank and find that heteroplasmy is associated with a 1.5-fold increased risk of all-cause mortality. Additionally, we functionally characterize mtDNA single nucleotide variants (SNVs) using a constraint-based score, mitochondrial local constraint score sum (MSS) and find it associated with all-cause mortality, and with the prevalence and incidence of cancer and cancer-related mortality, particularly leukemia. These results indicate that mitochondria may have a functional role in certain cancers, and mitochondrial heteroplasmic SNVs may serve as a prognostic marker for cancer, especially for leukemia.