Clonal Hematopoiesis and Evolution to Hematopoietic Malignancies

Bolouri H., Farrar J.E., Triche T., Ries R.E., Lim E.L., Alonzo T.A., Ma Y., Moore R., Mungall A.J., Marra M.A., Zhang J., Ma X., Liu Y., Liu Y., Auvil J.M., et. al.

A comprehensive molecular analysis of almost 1,000 pediatric subjects with acute myeloid leukemia (AML) uncovers widespread differences in pediatric AML as compared to adult AML, including a higher frequency of structural variants and different mutational patterns and epigenetic signatures. Future studies are needed to characterize the functional relevance of these alterations and to explore age-tailored therapies to improve disease control in younger patients. We present the molecular landscape of pediatric acute myeloid leukemia (AML) and characterize nearly 1,000 participants in Children's Oncology Group (COG) AML trials. The COG–National Cancer Institute (NCI) TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and microRNA sequencing and CpG methylation profiling. Validated DNA variants corresponded to diverse, infrequent mutations, with fewer than 40 genes mutated in >2% of cases. In contrast, somatic structural variants, including new gene fusions and focal deletions of MBNL1, ZEB2 and ELF1, were disproportionately prevalent in young individuals as compared to adults. Conversely, mutations in DNMT3A and TP53, which were common in adults, were conspicuously absent from virtually all pediatric cases. New mutations in GATA2, FLT3 and CBL and recurrent mutations in MYC-ITD, NRAS, KRAS and WT1 were frequent in pediatric AML. Deletions, mutations and promoter DNA hypermethylation convergently impacted Wnt signaling, Polycomb repression, innate immune cell interactions and a cluster of zinc finger–encoding genes associated with KMT2A rearrangements. These results highlight the need for and facilitate the development of age-tailored targeted therapies for the treatment of pediatric AML.

Kastenhuber E.R., Lowe S.W.

TP53 is the most frequently mutated gene in human cancer. Functionally, p53 is activated by a host of stress stimuli and, in turn, governs an exquisitely complex anti-proliferative transcriptional program that touches upon a bewildering array of biological responses. Despite the many unveiled facets of the p53 network, a clear appreciation of how and in what contexts p53 exerts its diverse effects remains unclear. How can we interpret p53's disparate activities and the consequences of its dysfunction to understand how cell type, mutation profile, and epigenetic cell state dictate outcomes, and how might we restore its tumor-suppressive activities in cancer?

Coombs C.C., Zehir A., Devlin S.M., Kishtagari A., Syed A., Jonsson P., Hyman D.M., Solit D.B., Robson M.E., Baselga J., Arcila M.E., Ladanyi M., Tallman M.S., Levine R.L., Berger M.F.

Clonal hematopoiesis (CH), as evidenced by recurrent somatic mutations in leukemia-associated genes, commonly occurs among aging human hematopoietic stem cells. We analyzed deep-coverage, targeted, next-generation sequencing (NGS) data of paired tumor and blood samples from 8,810 individuals to assess the frequency and clinical relevance of CH in patients with non-hematologic malignancies. We identified CH in 25% of cancer patients, with 4.5% harboring presumptive leukemia driver mutations (CH-PD). CH was associated with increased age, prior radiation therapy, and tobacco use. PPM1D and TP53 mutations were associated with prior exposure to chemotherapy. CH and CH-PD led to an increased incidence of subsequent hematologic cancers, and CH-PD was associated with shorter patient survival. These data suggest that CH occurs in an age-dependent manner and that specific perturbations can enhance fitness of clonal hematopoietic stem cells, which can impact outcome through progression to hematologic malignancies and through cell-non-autonomous effects on solid tumor biology.

Cimmino L., Dolgalev I., Wang Y., Yoshimi A., Martin G.H., Wang J., Ng V., Xia B., Witkowski M.T., Mitchell-Flack M., Grillo I., Bakogianni S., Ndiaye-Lobry D., Martín M.T., Guillamot M., et. al.

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and α-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer. PAPERCLIP.

Agathocleous M., Meacham C.E., Burgess R.J., Piskounova E., Zhao Z., Crane G.M., Cowin B.L., Bruner E., Murphy M.M., Chen W., Spangrude G.J., Hu Z., DeBerardinis R.J., Morrison S.J.

Stem-cell fate can be influenced by metabolite levels in culture, but it is not known whether physiological variations in metabolite levels in normal tissues regulate stem-cell function in vivo. Here we describe a metabolomics method for the analysis of rare cell populations isolated directly from tissues and use it to compare mouse haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which decreased with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, in part by reducing the function of Tet2, a dioxygenase tumour suppressor. Ascorbate depletion cooperated with Flt3 internal tandem duplication (Flt3ITD) leukaemic mutations to accelerate leukaemogenesis, through cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate therefore accumulates within HSCs to promote Tet activity in vivo, limiting HSC frequency and suppressing leukaemogenesis. Ascorbate depletion in mice increased haematopoietic stem-cell frequency and promoted leukaemogenesis, partly by reducing the function of the Tet2 tumour suppressor enzyme. The effect of changes in metabolite levels on stem-cell fate in vivo has been unclear. Sean Morrison and colleagues survey the metabolites of haematopoietic stem cells (HSCs) and progenitors. They show that each type of blood cell has a specific signature and that human and mouse HSCs have high levels of ascorbate, which drop during differentiation. Depletion of ascorbate in mice increases the number and function of HSCs and cooperates with a mutation associated with leukaemia to accelerate tumorigenesis. Analysis of the phenotypes indicates that ascorbate can act in a non-cell-autonomous fashion, partly by modulating the function of the tumour suppressor Tet2.

Pan W., Zhu S., Qu K., Meeth K., Cheng J., He K., Ma H., Liao Y., Wen X., Roden C., Tobiasova Z., Wei Z., Zhao J., Liu J., Zheng J., et. al.

Ten-Eleven-Translocation-2 (Tet2) is a DNA methylcytosine dioxygenase that functions as a tumor suppressor in hematopoietic malignancies. We examined the role of Tet2 in tumor-tissue myeloid cells and found that Tet2 sustains the immunosuppressive function of these cells. We found that Tet2 expression is increased in intratumoral myeloid cells both in mouse models of melanoma and in melanoma patients and that this increased expression is dependent on an IL-1R-MyD88 pathway. Ablation of Tet2 in myeloid cells suppressed melanoma growth in vivo and shifted the immunosuppressive gene expression program in tumor-associated macrophages to a proinflammatory one, with a concomitant reduction of the immunosuppressive function. This resulted in increased numbers of effector T cells in the tumor, and T cell depletion abolished the reduced tumor growth observed upon myeloid-specific deletion of Tet2. Our findings reveal a non-cell-intrinsic, tumor-promoting function for Tet2 and suggest that Tet2 may present a therapeutic target for the treatment of non-hematologic malignancies.

Zink F., Stacey S.N., Norddahl G.L., Frigge M.L., Magnusson O.T., Jonsdottir I., Thorgeirsson T.E., Sigurdsson A., Gudjonsson S.A., Gudmundsson J., Jonasson J.G., Tryggvadottir L., Jonsson T., Helgason A., Gylfason A., et. al.

Key Points Whole-genome sequencing of 11 262 Icelanders reveals that clonal hematopoiesis is very common in the elderly. Somatic mutation of some genes is strongly associated with clonal hematopoiesis, but in most cases, no driver mutations were evident.

Buscarlet M., Provost S., Zada Y.F., Barhdadi A., Bourgoin V., Lépine G., Mollica L., Szuber N., Dubé M., Busque L.

Key Points Somatic mutations driving clonal hematopoiesis occur mainly in DNMT3A and TET2 and have no significant impact on hematological phenotypes. There is a familial predisposition to acquire TET2 mutation.

Malcovati L., Gallì A., Travaglino E., Ambaglio I., Rizzo E., Molteni E., Elena C., Ferretti V.V., Catricalà S., Bono E., Todisco G., Bianchessi A., Rumi E., Zibellini S., Pietra D., et. al.

Publisher's Note: There is an Inside Blood Commentary on this article in this issue.

Jaiswal S., Natarajan P., Silver A.J., Gibson C.J., Bick A.G., Shvartz E., McConkey M., Gupta N., Gabriel S., Ardissino D., Baber U., Mehran R., Fuster V., Danesh J., Frossard P., et. al.

Clonal hematopoiesis of indeterminate potential (CHIP), which is defined as the presence of an expanded somatic blood-cell clone in persons without other hematologic abnormalities, is common among older persons and is associated with an increased risk of hematologic cancer. We previously found preliminary evidence for an association between CHIP and atherosclerotic cardiovascular disease, but the nature of this association was unclear.We used whole-exome sequencing to detect the presence of CHIP in peripheral-blood cells and associated such presence with coronary heart disease using samples from four case-control studies that together enrolled 4726 participants with coronary heart disease and 3529 controls. To assess causality, we perturbed the function of Tet2, the second most commonly mutated gene linked to clonal hematopoiesis, in the hematopoietic cells of atherosclerosis-prone mice.In nested case-control analyses from two prospective cohorts, carriers of CHIP had a risk of coronary heart disease that was 1.9 times as great as in noncarriers (95% confidence interval [CI], 1.4 to 2.7). In two retrospective case-control cohorts for the evaluation of early-onset myocardial infarction, participants with CHIP had a risk of myocardial infarction that was 4.0 times as great as in noncarriers (95% CI, 2.4 to 6.7). Mutations in DNMT3A, TET2, ASXL1, and JAK2 were each individually associated with coronary heart disease. CHIP carriers with these mutations also had increased coronary-artery calcification, a marker of coronary atherosclerosis burden. Hypercholesterolemia-prone mice that were engrafted with bone marrow obtained from homozygous or heterozygous Tet2 knockout mice had larger atherosclerotic lesions in the aortic root and aorta than did mice that had received control bone marrow. Analyses of macrophages from Tet2 knockout mice showed elevated expression of several chemokine and cytokine genes that contribute to atherosclerosis.The presence of CHIP in peripheral-blood cells was associated with nearly a doubling in the risk of coronary heart disease in humans and with accelerated atherosclerosis in mice. (Funded by the National Institutes of Health and others.).

Bejar R.

Clonal hematopoiesis can be identified by the presence of somatic mutations in blood or bone marrow even in individuals without a myeloid malignancy. Advances in DNA sequencing have led to the discovery that clonal hematopoiesis is remarkably common and occurs in a wide variety of settings, each often described by unique acronym. These distinctions can be useful as the implications of clonal hematopoiesis depend almost entirely on the clinical context in which it is identified. However, some generalizations can be made. The prevalence of clonal hematopoiesis increases with age, particularly after the fifth decade of life. Clonal hematopoiesis in normal individuals with very small clones is typically benign, while patients with clinically abnormal hematopoiesis, larger clones and more driver gene mutations appear to be at much greater risk. Understanding the significance of clonal hematopoiesis in the various contexts in which it occurs can influence how physicians assess risk, select therapies and counsel their patients. This concise review examines the implications of clonal hematopoiesis in several settings, including normal aging, aplastic anemia, unexplained cytopenias and patients receiving cytotoxic chemotherapy.

Dai Y., Wang Y., Huang J., Xia L., Shi X., Xu J., Lu J., Su X., Yang Y., Zhang W., Wang P., Wu S., Huang T., Mi J., Han Z., et. al.

Significance DNMT3A is a critical epigenetic modifier and tumor suppressor in the hematopoietic system. This gene is frequently mutated in hematopoietic malignancies, including acute myeloid leukemia (AML), with Dnmt3a R878H being the most common mutant. By using a conditional knockin approach, this study shows that Dnmt3a R878H is sufficient to initiate AML and recapitulate human leukemic features in mice. The leukemia-initiating cells are enriched in hematopoietic stem/progenitor cells. Through gene expression profiling, DNA methylation and histone modification analysis, and functional tests on important regulators for cell proliferation and differentiation in an animal model, this study has not only discovered mTOR pathway activation as a key player in the disease mechanism but also revealed the potential therapeutic effects of mTOR inhibition on DNMT3A mutation-related leukemia.

Gibson C.J., Lindsley R.C., Tchekmedyian V., Mar B.G., Shi J., Jaiswal S., Bosworth A., Francisco L., He J., Bansal A., Morgan E.A., Lacasce A.S., Freedman A.S., Fisher D.C., Jacobsen E., et. al.

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.

McConnell M.J., Moran J.V., Abyzov A., Akbarian S., Bae T., Cortes-Ciriano I., Erwin J.A., Fasching L., Flasch D.A., Freed D., Ganz J., Jaffe A.E., Kwan K.Y., Kwon M., Lodato M.A., et. al.

Single-cell diversity in the brain The cells that make up an organism may all start from one genome, but somatic mutations mean that somewhere along the line of development, an organism's individual cellular genomes diverge. McConnell et al. review the implications and causes of single-cell genomic diversity for brain function. Somatic mutations caused by mobile genetic elements or errors in DNA repair may underlie certain neuropsychiatric disorders. Science , this issue p. eaal1641

Pan F., Wingo T.S., Zhao Z., Gao R., Makishima H., Qu G., Lin L., Yu M., Ortega J.R., Wang J., Nazha A., Chen L., Yao B., Liu C., Chen S., et. al.

TET2 is a dioxygenase that catalyses multiple steps of 5-methylcytosine oxidation. Although TET2 mutations frequently occur in various types of haematological malignancies, the mechanism by which they increase risk for these cancers remains poorly understood. Here we show that Tet2−/− mice develop spontaneous myeloid, T- and B-cell malignancies after long latencies. Exome sequencing of Tet2−/− tumours reveals accumulation of numerous mutations, including Apc, Nf1, Flt3, Cbl, Notch1 and Mll2, which are recurrently deleted/mutated in human haematological malignancies. Single-cell-targeted sequencing of wild-type and premalignant Tet2−/− Lin−c-Kit+ cells shows higher mutation frequencies in Tet2−/− cells. We further show that the increased mutational burden is particularly high at genomic sites that gained 5-hydroxymethylcytosine, where TET2 normally binds. Furthermore, TET2-mutated myeloid malignancy patients have significantly more mutational events than patients with wild-type TET2. Thus, Tet2 loss leads to hypermutagenicity in haematopoietic stem/progenitor cells, suggesting a novel TET2 loss-mediated mechanism of haematological malignancy pathogenesis. TET2 catalyses DNA demethylation and is mutated in various blood cancers; in particularTet2null mice develop haematological neoplasms. Here the authors show that this effect could be due to the increased frequency of mutation associated with TET2 loss in haematopoietic stem/progenitor cells.

Zhang N., Tian X., Sun D., Tse G., Xie B., Zhao Z., Liu T.

Wang X., Wang K., Zhang W., Tang Z., Zhang H., Cheng Y., Zhou D., Zhang C., Zhong W., Ma Q., Xu J., Hu Z.

Hawking Z.L., Allan J.M.

ABSTRACTBackgroundThe ten–eleven translocation (TET) enzyme family is a key regulator of DNA methylation, responsible for the conversion of 5‐methylcytosine to 5‐hydroxymethylcytosine to promote locus‐specific demethylation. Thus, it is not surprising that loss or attenuation of TET enzymes is implicated in genomic hypermethylation and transcriptional reprogramming that drives cancer development. Somatic mutations in TET2 are observed in the bone marrow of 5%–10% of healthy adults over 65 years of age, imparting a hematopoietic stem cell advantage and subsequent clonal hematopoiesis of indeterminate potential (CHIP), a condition which is associated with increased risk of myeloid malignancy. Somatic TET2 mutations are frequently reported in myeloid disorders, including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Evidence suggests that TET2 mutations also affect prognosis in myeloid leukemia and other hematopoietic malignancies. However, there is a paucity of collated data on the frequency of TET2 mutations in solid human cancers.ObjectivesWe review the published literature on TET2 mutation in human solid cancers and explore their frequency and impact on patient outcomes.Results & ConclusionsSomatic TET2 mutations are reported in numerous solid human cancers, including those arising in the skin, lung and prostate. Many of the somatic TET2 mutations reported in solid cancers are recurrent, suggesting functionality. There is also evidence to suggest that somatic TET2 mutations affect prognosis in solid human cancers.

Kunevich E.O., Mikhaleva M.A., Krysyuk O.B., Bogdanov A.N., Zhernyakova A.A., Voloshin S.V.

Qiu H., Zhang C., Ma X., Li Y.

Totani H., Matsumura T., Yokomori R., Umemoto T., Takihara Y., Yang C., Chua L.H., Watanabe A., Sanda T., Suda T.

Latorre-Crespo E., Robertson N.A., Kosebent G., Macgillivray L., Murphy L., Uddin M., Honigberg M., Whitsel E.A., Bick A.G., Reiner A.P., Orru V., Marongiu M., Cucca F., Fiorillo E., Deary I., et. al.

AbstractClonal hematopoiesis (CH) is characterized by expanding blood cell clones carrying somatic mutations in healthy aged individuals and is associated with various age-related diseases and all-cause mortality. While CH mutations affect diverse genes associated with myeloid malignancies, their mechanisms of expansion and disease associations remain poorly understood. We investigate the relationship between clonal fitness and clinical outcomes by integrating data from three longitudinal aging cohorts (n=713, observations=2,341). We demonstrate pathway-specific fitness advantage and clonal composition influence clonal dynamics. Further, the timing of mutation acquisition is necessary to determine the extent of clonal expansion reached during the host individual’s lifetime. We introduce MACS120, a metric combining mutation context, timing, and variant fitness to predict future clonal growth, outperforming traditional variant allele frequency measurements in predicting clinical outcomes. Our unified analytical framework enables standardized clonal dynamics inference across cohorts, advancing our ability to predict and potentially intervene in CH-related pathologies.

Iyama S., Chi S., Idogawa M., Ikezoe T., Fukushima K., Utsu Y., Kanda J., Yoshimoto G., Kobayashi T., Hosono N., Yamauchi T., Kondo T., Nakamura Y., Kojima K., Yoshida C., et. al.

Ten-eleven translocation-2 (TET2) gene mutations are observed in 12–20% of adult patients with acute myeloid leukemia (AML). The prognostic impact of TET2 mutations in patients with AML and myelodysplastic syndromes has been reported in several studies; however, their results remain controversial. Therefore, we aimed to analyze the prevalence and significance of TET2 mutations in patients with AML. Data were obtained from 331 patients with AML according to the Hematologic Malignancies-SCREEN-Japan 01 and 02 studies, which were prospective multicenter genomic profiling analyses. We found a distinct type of TET2 mutations, with a particularly detrimental prognosis in the patients. Thirty-five patients with TET2 ‘significant’ mutations were identified (26 with frameshift mutations and nine with nonsense mutations). The proportion of patients with TET2 mutations was 31.7% (10.6% and 21.1% in the TET2 significant and non-significant mutation groups). The TET2 significant mutation group had a shorter OS than the TET2 non-significant mutation or wild-type TET2 group (median: 15.9 vs. 35.0 vs. 25.9 months). Regarding the response to chemotherapy according to TET2 status, the complete response (CR) or CR with incomplete count recovery rate was 37.1% in the TET2 significant mutation group and 46.6% in the non-significant mutation or wild-type group. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) improved patient prognosis in the TET2 non-significant mutation or wild-type TET2 group; however, allo-HSCT did not affect prognosis in the TET2 significant mutation group. This study indicates that certain TET2 mutations in patients with AML may have detrimental effects. 

Jin G., Rong S., Yin D., Deng Z., Ding X., Sheng M., Gao H., Kohli R.M., Xu G., Zhou D.

Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (mC) in DNA, contributing to the regulation of gene transcription. Diverse mutations of TET2 are frequently found in various blood cancers, yet the full scope of their functional consequences has been unexplored. Here, we report that a subset of TET2 mutations identified in leukemia patients alter the substrate specificity of TET2 from acting on mC to thymine. This neomorphic activity results from substitutions at key residues involved in the interactions with the mC base, including Asn1387 and His1904. Recombinant human TET2 proteins harboring the mutation of these residues can catalyze the oxidation of thymine to 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU). Exogenous expression of the mutant TET2 Asn1387Thr (N1387T) in HEK293T cells leads to hmU accumulation, with levels further increased in cells lacking the glycosylase SMUG1. Endogenous knock-in of N1300T, the murine equivalent of N1387T, in mouse embryonic stem cells induces hmU production, causing DNA lesions and transcriptional activation of DNA damage response genes. N1300T cells accumulate more additional mutations with extended culture and exhibit heightened sensitivity to ATR inhibition compared to Tet2 knockout cells. Our study reveals that certain patient-derived TET2 mutations can acquire unexpected gain-of-function activities beyond impairing mC oxidation, offering a fresh perspective on the diverse molecular etiology of mutant TET2-related leukemogenesis.

Martínez-Vila C., Teixido C., Aya F., Martín R., González-Navarro E.A., Alos L., Castrejon N., Arance A.

The treatment landscape for advanced melanoma has transformed significantly with the advent of BRAF and MEK inhibitors (BRAF/MEKi) targeting BRAFV600 mutations, as well as immune checkpoint inhibitors (ICI) like anti-PD-1 monotherapy or its combinations with anti-CTLA-4 or anti-LAG-3. Despite that, many patients still do not benefit from these treatments at all or develop resistance mechanisms. Therefore, prognostic and predictive biomarkers are needed to identify patients who should switch or escalate their treatment strategies or initiate an intensive follow-up. In melanoma, liquid biopsy has shown promising results, with a potential role in predicting relapse in resected high-risk patients or in disease monitoring during the treatment of advanced disease. Several components in peripheral blood have been analyzed, such as circulating tumor cells (CTCs), cell-free DNA (cfDNA), and circulant tumoral DNA (ctDNA), which have turned out to be particularly promising. To analyze ctDNA in blood, different techniques have proven to be useful, including digital droplet polymerase chain reaction (ddPCR) to detect specific mutations and, more recently, next-generation sequencing (NGS) techniques, which allow analyzing a broader repertoire of the mutation landscape of each patient. In this review, our goal is to update the current understanding of liquid biopsy, focusing on the use of ctDNA as a biological material in the daily clinical management of melanoma patients, in particular those with advanced disease treated with ICI.

Watt S.M., Roubelakis M.G.

Human hematopoietic stem cells (HSCs) have traditionally been viewed as self-renewing, multipotent cells with enormous potential in sustaining essential steady state blood and immune cell production throughout life. Indeed, around 86% (1011–1012) of new cells generated daily in a healthy young human adult are of hematopoietic origin. Therapeutically, human HSCs have contributed to over 1.5 million hematopoietic cell transplants (HCTs) globally, making this the most successful regenerative therapy to date. We will commence this review by briefly highlighting selected key achievements (from 1868 to the end of the 20th century) that have contributed to this accomplishment. Much of our knowledge of hematopoiesis is based on small animal models that, despite their enormous importance, do not always recapitulate human hematopoiesis. Given this, we will critically review the progress and challenges faced in identifying adult human HSCs and tracing their lineage differentiation trajectories, referring to murine studies as needed. Moving forward and given that human hematopoiesis is dynamic and can readily adjust to a variety of stressors, we will then discuss recent research advances contributing to understanding (i) which HSPCs maintain daily steady state human hematopoiesis, (ii) where these are located, and (iii) which mechanisms come into play when homeostatic hematopoiesis switches to stress-induced or emergency hematopoiesis.

Wang N., Pachai M.R., Li D., Lee C.J., Warda S., Khudoynazarova M.N., Cho W.H., Xie G., Shah S.R., Yao L., Qian C., Wong E.W., Yan J., Tomas F.V., Hu W., et. al.

Abstract Members of the KMT2C/D–KDM6A complex are recurrently mutated in urothelial carcinoma and in histologically normal urothelium. Here, using genetically engineered mouse models, we demonstrate that Kmt2c/d knockout in the urothelium led to impaired differentiation, augmented responses to growth and inflammatory stimuli and sensitization to oncogenic transformation by carcinogen and oncogenes. Mechanistically, KMT2D localized to active enhancers and CpG-poor promoters that preferentially regulate the urothelial lineage program and Kmt2c/d knockout led to diminished H3K4me1, H3K27ac and nascent RNA transcription at these sites, which leads to impaired differentiation. Kmt2c/d knockout further led to KMT2A–menin redistribution from KMT2D localized enhancers to CpG-high and bivalent promoters, resulting in derepression of signal-induced immediate early genes. Therapeutically, Kmt2c / d knockout upregulated epidermal growth factor receptor signaling and conferred vulnerability to epidermal growth factor receptor inhibitors. Together, our data posit that functional loss of Kmt2c/d licenses a molecular ‘field effect’ priming histologically normal urothelium for oncogenic transformation and presents therapeutic vulnerabilities.

Zhang Q., Yim R., Lee P., Chin L., Li V., Gill H.

Clonal hematopoiesis (CH) is associated with an increased risk of developing myeloid neoplasms (MNs) such as myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML). In general, CH comprises clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). It is an age-related phenomenon characterized by the presence of somatic mutations in hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) that acquire a fitness advantage under selection pressure. Individuals with CHIP have an absolute risk of 0.5–1.0% per year for progressing to MDS or AML. Inflammation, smoking, cytotoxic therapy, and radiation can promote the process of clonal expansion and leukemic transformation. Of note, exposure to chemotherapy or radiation for patients with solid tumors or lymphomas can increase the risk of therapy-related MN. Beyond hematological malignancies, CH also serves as an independent risk factor for heart disease, stroke, chronic obstructive pulmonary disease, and chronic kidney disease. Prognostic models such as the CH risk score and MN-prediction models can provide a framework for risk stratification and clinical management of CHIP/CCUS and identify high-risk individuals who may benefit from close surveillance. For CH or related disorders, therapeutic strategies targeting specific CH-associated mutations and specific selection pressure may have a potential role in the future.

Villegas-Valverde C.A., Bencomo-Hernandez A.A., Castillo-Aleman Y.M., Ventura-Carmenate Y., Casado-Hernandez I., Rivero-Jimenez R.A.

Hematopoietic stem cell transplantation (HSCT) is a widely used therapy, but its success largely depends on the number and quality of stem cells collected. Current evidence shows the complexity of the hematopoietic system, which implies that, in the quality assurance of the apheresis product, the hematopoietic stem cells are adequately characterized and quantified, in which mass cytometry (MC) can provide its advantages in high-dimensional analysis. This research aimed to characterize and enumerate CD45dim/CD34+ stem cells using the MC in apheresis product yields from patients with chronic lymphoid malignant diseases undergoing autologous transplantation at the Abu Dhabi Stem Cells Center. An analytical and cross-sectional study was performed on 31 apheresis products from 15 patients diagnosed with multiple myeloma (n = 9) and non-Hodgkin lymphomas (n = 6) eligible for HSCT. The MC was employed using the MaxPar Kit for stem cell immunophenotyping. The analysis was performed manually in the Kaluza and unsupervised by machine learning in Cytobank Premium. An excellent agreement was found between mass and flow cytometry for the relative and absolute counts of CD45dim/CD34+ cells (Bland-Altman bias: -0.029 and -64, respectively), seven subpopulations were phenotyped and no lineage bias was detected for any of the methods used in the pool of collected cells. A CD34+/CD38+/CD138+ population was seen in the analyses performed on four patients with multiple myeloma. The MC helps to characterize subpopulations of stem cells in apheresis products. It also allows cell quantification by double platform. Unsupervised analysis allows results completion and validation of the manual strategy. The proposed methodology can be extended to apheresis products for purposes other than HSCT.

Singh S., Sarkar T., Gudmundsson K.O., Jakubison B.L., Morris H.M., Burkett S., Baktiar K., Pauly G.T., Sigano D.M., Schneider J.P., Keller J.R.

ABSTRACTHematopoietic malignancies emerge through the gradual acquisition of genetic mutations within hematopoietic stem and progenitor cells (HSPCs). Mutations that occur early in disease progression impart a selective growth advantage to HSPCs, which allows them to expand and contribute to a substantial percentage of mature blood cells. This increased expansion is termed clonal hematopoiesis (CH) and is a preleukemic phase associated with an increased risk of developing leukemia. Inhibitor of DNA binding 1 (ID1) protein is a transcriptional regulator of proliferation/differentiation of neuronal, muscle, hematopoietic and other cells, and is frequently overexpressed in cancer.Id1is expressed at low levels in normal HSCs and is induced by growth factors and other mediators of inflammatory stress and promotes HSPC proliferationin vitro and in vivo.Since chronic inflammation is associated with the progression of hematopoietic malignancies, reducingId1expression during CH may be therapeutic. Mutations inTET2are frequently observed in patients with CH, andTet2−/−mice develop CH that progress to hematopoietic malignancies.Id1is upregulated in murineTet2−/−HSPCs and in AML, CMML and MDS patient samples withTET2mutations. Genetic ablation ofId1inTet2−/−HSPCs reduces HSPC expansion/self-renewal/CH, extramedullary hematopoiesis, myeloid skewing, genetic instability and delays the onset of disease. Mechanistically, p16 expression, senescence and apoptosis were increased and proliferation decreased inTet2−/−; Id1−/−HSPCs. Thus, ID1 may represent a potential therapeutic target to reduce CH, hematopoietic hyperplasia, and delay the onset of disease.One Sentence SummaryGenetic ablation ofId1inTet2−/−mice rescues clonal hematopoiesis by increasing CDKI expression, apoptosis, senescence, and differentiation, and reducing cell growth.