Brenton G. Mar

Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes

BACKGROUND The incidence of hematologic cancers increases with age. These cancers are associated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders. METHODS We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide variants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events. RESULTS Detectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respectively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8). CONCLUSIONS Age-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the latter possibly due to an increased risk of cardiovascular disease. (Funded by the National Institutes of Health and others.).

Acute myeloid leukemia ontogeny is defined by distinct somatic mutations

Acute myeloid leukemia (AML) can develop after an antecedent myeloid malignancy (secondary AML [s-AML]), after leukemogenic therapy (therapy-related AML [t-AML]), or without an identifiable prodrome or known exposure (de novo AML). The genetic basis of these distinct pathways of AML development has not been determined. We performed targeted mutational analysis of 194 patients with rigorously defined s-AML or t-AML and 105 unselected AML patients. The presence of a mutation in SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 was >95% specific for the diagnosis of s-AML. Analysis of serial samples from individual patients revealed that these mutations occur early in leukemogenesis and often persist in clonal remissions. In t-AML and elderly de novo AML populations, these alterations define a distinct genetic subtype that shares clinicopathologic properties with clinically confirmed s-AML and highlights a subset of patients with worse clinical outcomes, including a lower complete remission rate, more frequent reinduction, and decreased event-free survival. This trial was registered at www.clinicaltrials.gov as #NCT00715637.

Somatic Mutations Predict Poor Outcome in Patients With Myelodysplastic Syndrome After Hematopoietic Stem-Cell Transplantation

PURPOSE Recurrently mutated genes in myelodysplastic syndrome (MDS) are pathogenic drivers and powerfully associated with clinical phenotype and prognosis. Whether these types of mutations predict outcome after allogeneic hematopoietic stem-cell transplantation (HSCT) in patients with MDS is not known. PATIENTS AND METHODS We used massively parallel sequencing to examine tumor samples collected from 87 patients with MDS before HSCT for coding mutations in 40 recurrently mutated MDS genes. RESULTS Mutations were identified in 92% of patients, most frequently in the ASXL1 (29%), TP53 (21%), DNMT3A (18%), and RUNX1 (16%) genes. In univariable analyses, only TP53 mutations were associated with shorter overall (OS; hazard ratio [HR], 3.74; P < .001) and progression-free survival (HR, 3.97; P < .001). After adjustment for clinical variables associated with these end points, mutations in TP53 (HR, 2.30; P = .027), TET2 (HR, 2.40; P = .033), and DNMT3A (HR, 2.08; P = .049) were associated with decreased OS. In multivariable analysis including clinical variables, complex karyotype status, and candidate genes, mutations in TP53 (HR, 4.22; P ≤ .001) and TET2 (HR, 1.68; P = .037) were each independently associated with shorter OS. Nearly one half of patients (46%) carried a mutation in TP53, DNMT3A, or TET2 and accounted for 64% of deaths. Three-year OS in patients without these mutations was 59% (95% CI, 43% to 72%), versus 19% (95% CI, 9% to 33%) in patients with these mutations. CONCLUSION Mutations in TP53, TET2, or DNMT3A identify patients with MDS with shorter OS after HSCT.

Prognostic Mutations in Myelodysplastic Syndrome after Stem-Cell Transplantation

BACKGROUND Genetic mutations drive the pathogenesis of the myelodysplastic syndrome (MDS) and are closely associated with clinical phenotype. Therefore, genetic mutations may predict clinical outcomes after allogeneic hematopoietic stem‐cell transplantation. METHODS We performed targeted mutational analysis on samples obtained before transplantation from 1514 patients with MDS who were enrolled in the Center for International Blood and Marrow Transplant Research Repository between 2005 and 2014. We evaluated the association of mutations with transplantation outcomes, including overall survival, relapse, and death without relapse. RESULTS TP53 mutations were present in 19% of the patients and were associated with shorter survival and a shorter time to relapse than was the absence of TP53 mutations, after adjustment for significant clinical variables (P<0.001 for both comparisons). Among patients 40 years of age or older who did not have TP53 mutations, the presence of RAS pathway mutations was associated with shorter survival than was the absence of RAS pathway mutations (P=0.004), owing to a high risk of relapse, and the presence of JAK2 mutations was associated with shorter survival than was the absence of JAK2 mutations (P=0.001), owing to a high risk of death without relapse. The adverse prognostic effect of TP53 mutations was similar in patients who received reduced‐intensity conditioning regimens and those who received myeloablative conditioning regimens. By contrast, the adverse effect of RAS pathway mutations on the risk of relapse, as compared with the absence of RAS pathway mutations, was evident only with reduced‐intensity conditioning (P<0.001). In young adults, 4% of the patients had compound heterozygous mutations in the Shwachman–Diamond syndrome–associated SBDS gene with concurrent TP53 mutations and a poor prognosis. Mutations in the p53 regulator PPM1D were more common among patients with therapy‐related MDS than those with primary MDS (15% vs. 3%, P<0.001). CONCLUSIONS Genetic profiling revealed that molecular subgroups of patients undergoing allogeneic hematopoietic stem‐cell transplantation for MDS may inform prognostic stratification and the selection of conditioning regimen. (Funded by the Edward P. Evans Foundation and others.)

Mutations in epigenetic regulators including SETD2 are gained during relapse in paediatric acute lymphoblastic leukaemia

Relapsed pediatric acute lymphoblastic leukemia (ALL) has high rates of treatment failure. Epigenetic regulators have been proposed as modulators of chemoresistance, here we sequence genes encoding epigenetic regulators in matched diagnosis-remission-relapse ALL samples. We find significant enrichment of mutations in epigenetic regulators at relapse with recurrent somatic mutations in SETD2, CREBBP, MSH6, KDM6A and MLL2, mutations in signaling factors are not enriched. Somatic alterations in SETD2, including frameshift and nonsense mutations, are present at 12% in a large de novo ALL patient cohort. We conclude that the enrichment of mutations in epigenetic regulators at relapse is consistent with a role in mediating therapy resistance.

Sequencing histone-modifying enzymes identifies UTX mutations in acute lymphoblastic leukemia

Sequencing histone-modifying enzymes identifies UTX mutations in acute lymphoblastic leukemia

Clonal Hematopoiesis Associated With Adverse Outcomes After Autologous Stem-Cell Transplantation for Lymphoma

Purpose Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition characterized by somatic mutations in the blood of otherwise healthy adults. We hypothesized that in patients undergoing autologous stem-cell transplantation (ASCT) for lymphoma, CHIP at the time of ASCT would be associated with an increased risk of myelodysplastic syndrome and acute myeloid leukemia, collectively termed therapy-related myeloid neoplasm (TMN), and other adverse outcomes. Methods We performed whole-exome sequencing on pre- and post-ASCT samples from 12 patients who developed TMN after autologous transplantation for Hodgkin lymphoma or non-Hodgkin lymphoma and targeted sequencing on cryopreserved aliquots of autologous stem-cell products from 401 patients who underwent ASCT for non-Hodgkin lymphoma between 2003 and 2010. We assessed the effect of CHIP at the time of ASCT on subsequent outcomes, including TMN, cause-specific mortality, and overall survival. Results For six of 12 patients in the exome sequencing cohort, mutations found in the TMN specimen were also detectable in the pre-ASCT specimen. In the targeted sequencing cohort, 120 patients (29.9%) had CHIP at the time of ASCT, which was associated with an increased rate of TMN (10-year cumulative incidence, 14.1% v 4.3% for those with and without CHIP, respectively; P = .002). Patients with CHIP had significantly inferior overall survival compared with those without CHIP (10-year overall survival, 30.4% v 60.9%, respectively; P < .001), including increased risk of death from TMN and cardiovascular disease. Conclusion In patients undergoing ASCT for lymphoma, CHIP at the time of transplantation is associated with inferior survival and increased risk of TMN.

Self-renewal related signaling in myeloid leukemia stem cells

A key characteristic of hematopoietic stem cells (HSC) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSC and potentially also in leukemic stem cells. Besides pathways such as Wnt signaling, Hedgehog signaling and Notch signaling, transcription factors (FoxOs) and cell fate determinants may also play a role in stem cells. While some of these pathways seem to be dispensable for maintenance of adult HSC, there may be a distinct requirement in leukemia stem cells for leukemic self-renewal. Here we will focus on self-renewal related signaling in myeloid leukemia stem cells and its therapeutic relevance.

Deletion of ribosomal protein genes is a common vulnerability in human cancer, especially in concert with TP53 mutations

Heterozygous inactivating mutations in ribosomal protein genes (RPGs) are associated with hematopoietic and developmental abnormalities, activation of p53, and altered risk of cancer in humans and model organisms. Here we performed a large‐scale analysis of cancer genome data to examine the frequency and selective pressure of RPG lesions across human cancers. We found that hemizygous RPG deletions are common, occurring in about 43% of 10,744 cancer specimens and cell lines. Consistent with p53‐dependent negative selection, such lesions are underrepresented in TP53‐intact tumors (P ≪ 10−10), and shRNA‐mediated knockdown of RPGs activated p53 in TP53‐wild‐type cells. In contrast, we did not see negative selection of RPG deletions in TP53‐mutant tumors. RPGs are conserved with respect to homozygous deletions, and shRNA screening data from 174 cell lines demonstrate that further suppression of hemizygously deleted RPGs inhibits cell growth. Our results establish RPG haploinsufficiency as a strikingly common vulnerability of human cancers that associates with TP53 mutations and could be targetable therapeutically.

SETD2 alterations impair DNA damage recognition and lead to resistance to chemotherapy in leukemia

Mutations in SETD2, encoding the histone 3 lysine 36 trimethyltransferase, are enriched in relapsed acute lymphoblastic leukemia and MLL-rearranged acute leukemia. We investigated the impact of SETD2 mutations on chemotherapy sensitivity in isogenic leukemia cell lines and in murine leukemia generated from a conditional knockout of Setd2. SETD2 mutations led to resistance to DNA-damaging agents, cytarabine, 6-thioguanine, doxorubicin, and etoposide, but not to a non-DNA damaging agent, l-asparaginase. H3K36me3 localizes components of the DNA damage response (DDR) pathway and SETD2 mutation impaired DDR, blunting apoptosis induced by cytotoxic chemotherapy. Consistent with local recruitment of DDR, genomic regions with higher H3K36me3 had a lower mutation rate, which was increased with SETD2 mutation. Heterozygous conditional inactivation of Setd2 in a murine model decreased the latency of MLL-AF9-induced leukemia and caused resistance to cytarabine treatment in vivo, whereas homozygous loss delayed leukemia formation. Treatment with JIB-04, an inhibitor of the H3K9/36me3 demethylase KDM4A, restored H3K36me3 levels and sensitivity to cytarabine. These findings establish SETD2 alteration as a mechanism of resistance to DNA-damaging chemotherapy, consistent with a local loss of DDR, and identify a potential therapeutic strategy to target SETD2-mutant leukemias.