Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes

Prevalence of Clonal Hematopoiesis in Patients with Monoclonal Gammopathy of Undetermined Significance

Relationship and Susceptibility to Serious Infections Among Monoclonal B-Cell Lymphocytosis (MBL), Monoclonal Gammopathy of Undetermined Significance (MGUS), and Clonal Hematopoiesis (CH) Premalignant Conditions

Aging is a multifaceted process characterized by a gradual decline in physiological function and increased susceptibility to a range of chronic diseases. Among the molecular and cellular mechanisms driving aging, genomic instability is a fundamental hallmark, contributing to increased mutation load and genetic heterogeneity within cellular populations. This review explores the role of genomic instability and genetic heterogeneity in aging in the hematopoietic system, with a particular focus on clonal hematopoiesis of indeterminate potential (CHIP), monoclonal gammopathy of undetermined significance (MGUS), and monoclonal B-cell lymphocytosis (MBL) as biomarkers. CHIP involves the clonal expansion of hematopoietic stem cells with somatic mutations. In contrast, MGUS is characterized by the presence of clonal plasma cells producing monoclonal immunoglobulins, while MBL is characterized by clonal proliferation of B cells. These conditions are prevalent in the aging population and serve as measurable indicators of underlying genomic instability. Studying these entities offers valuable insights into the mechanisms by which somatic mutations accumulate and drive clonal evolution in the hematopoietic system, providing a deeper understanding of how aging impacts cellular and tissue homeostasis. In summary, the hematopoietic system serves as a powerful model for investigating the interplay between genomic instability and aging. Incorporating age-related hematological conditions into aging research, alongside other biomarkers such as epigenetic clocks, can enhance the precision and predictive power of biological age assessments. These biomarkers provide a comprehensive view of the aging process, facilitating the early detection of age-related diseases and hopefully enabling personalized healthcare strategies.

This document represents an update of the British Society of Haematology guideline published in 2014 due to advances in understanding the biology and therapy of the myelodysplastic syndromes (MDS).1 The objective of these guidelines is to provide healthcare professionals with clear guidance on the diagnosis and evaluation of prognosis of adult patients with MDS. A separate BSH guideline covers the Management of Adult MDS which is published alongside this guideline. A separate good practice paper detailing the management of patients with chronic myelomonocytic leukaemia (CMML) will follow and is not considered in these guidelines. These guidelines were compiled according to the BSH process https://b-s-h.org.uk/media/16732/bsh-guidance-development-process-dec-5-18.pdf. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) nomenclature was used to evaluate levels of evidence and to assess the strength of recommendations. The GRADE criteria can be found at http://www.gradeworkinggroup.org. The guideline group was selected to be representative of UK medical experts and the manuscript was reviewed by the UK MDS Patient Support Group. Recommendations are based on a review of the literature using Medline/Pubmed searches. Search terms included: Myelodysplasia, MDS, myelodysplastic, refractory an(a)emia, refractory cytopenia, deletion 5q, del(5q), idiopathic cytopenia of undetermined significance (ICUS), clonal cytopenia of undetermined significance (CCUS), clonal haematopoiesis of indeterminate potential (CHIP), diagnosis, diagnostic, investigation, cytogenetic, molecular, mutation, bone marrow, flow cytometry risk, prognosis. Only English-language publications from January 2012 to December 2020 were included in the literature search. Additional searches and subsection heading terms were conducted by members of the writing committee at the time of final submission to the British Journal of Haematology. Titles and/or abstracts of publications obtained from the database searches described were curated and manually reviewed by members of the writing committee. Review of the manuscript was performed by the BSH Guidelines Committee Haemato-oncology Task Force, the BSH Guidelines Committee and the haemato-oncology sounding board of the BSH. It was also posted on the members section of the BSH website for comment. This guideline has also been reviewed by patient representatives from the MDS UK Patient Support Group (mdspatientsupport.org.uk). These organisations do not necessarily endorse the contents. The myelodysplastic syndromes (MDS) are a group of clonal bone marrow neoplasms characterised by ineffective haematopoiesis and manifested by morphological dysplasia in haematopoietic cells and by peripheral cytopenia(s).2 They have a variable predilection for the development of acute myeloid leukaemia (AML). The incidence of MDS in the UK is 3·72/100,000 population/year; it is predominantly a disease of the elderly (median age at diagnosis 75·7 years) and more common in men (approximately 2:1).3 Patients with suspected MDS should be assessed by a haematologist with a specialist interest in the disease. They should be referred for a second opinion to a regional or national centre when required by the clinician, or requested by the patient. All patients with a diagnosis of MDS must be discussed at a multidisciplinary team meeting (MDT), which should include allogeneic stem cell transplantation representation. All patients diagnosed with MDS should be reported to the National Cancer Registry, via the MDT, and to MDS-specific registries if appropriate. Myelodysplastic syndrome is defined by a combination of cytopenias and morphological bone marrow dysplasia. Myelodysplastic syndromes should be considered in all patients with otherwise unexplained cytopenia(s). World Health Organisation (WHO) thresholds for cytopenias are haemoglobin <100 g/l, absolute neutrophil count <1·8 × 109/l and platelets <100 × 109/l.2 However, higher values (as defined by local laboratory ranges) do not exclude the diagnosis if definitive morphological and/or cytogenetic abnormalities are present. A diagnostic algorithm for suitable patients is outlined in Fig. 1. Table I shows the minimum clinical assessment and laboratory investigation of a patient with possible MDS. Selected patients may require further investigations (Table II). Alternative causes of marrow dysplasia should also be considered. In the context of persistent and otherwise unexplained cytopenias, a WHO-defined diagnosis of MDS requires either (i) morphological dysplasia (involving ≥10% of bone marrow cells in ≥1 lineage); (ii) increased myeloblasts (≥5%, but <20%); or (iii) evidence of clonality with a typical MDS-associated cytogenetic abnormality.2, 4 Dysplasia is not restricted to MDS patients and can occur following a toxic insult, in reactive conditions or secondary to haematinic deficiencies. Furthermore, dysplasia has been reported in healthy individuals.5, 6 Identifying MDS can therefore be challenging and caution is required when the diagnosis is based solely on morphology, particularly in borderline cases or those with unilineage dysplasia. Other causes of morphological dysplasia should be excluded and a period of observation followed by repeat sampling may be warranted. New technologies, in particular genomic testing, may help in challenging cases by providing additional markers of clonality. Although the presence of clonal markers should not be considered in isolation of other diagnostic modalities, there are strong associations between particular genetic lesions (for example mutations in SF3B1 or isolated deletion of chromosome 5q) with WHO-defined MDS subtypes. In patients with <10% marrow dysplasia and lacking a clonal abnormality, the term 'idiopathic cytopenia of undetermined significance' (ICUS) may be used where cytopenias are sustained (>6 months) and there is no other identifiable cause.7 Such patients should be observed (with repeat investigation if necessary) for subsequent development of overt MDS. Chronic myelomonocytic leukaemia (CMML) has been reclassified to the WHO subgroup of myelodysplastic/myeloproliferative neoplasms (MDS/MPN)2 and is not considered further in this guideline. In confirmed cases of MDS, family history and clinical features should be reviewed to identify those with germline predisposition, which may have implications for prognosis, genetic counselling and management. Both blood film and bone marrow examination by a haematologist or haematopathologist with experience in diagnosing MDS, looking for characteristic morphological features of dysplasia, are necessary for diagnosis, classification and prognostic evaluation of MDS. Blood films should be assessed for dysplasia in erythroid, platelet and white-cell lineages.2, 8 Bone marrow examination of May–Grünwald–Giemsa (or equivalent)-stained smears should routinely comment on myeloid, megakaryocyte and erythroid maturation, and report dysplasia if present. Blast percentage should be enumerated. Optimal differential count should evaluate 500 or more nucleated cells, including 30 or more megakaryocytes. Good quality smears and stains are essential for accurate diagnosis. Fresh specimens should be processed within 2 hours, where possible, and excess of ethylenediamine tetra-acetic acid (EDTA) should be strictly avoided. Stains should be well controlled and checked by examining non-MDS films. Prussian Blue or Perls' stain should be performed on all marrow aspirates to assess iron stores and to quantitate ring sideroblasts. In the revised WHO classification,2 the presence of an SF3B1 mutation reduces the ring sideroblast percentage threshold required for a diagnosis of MDS with ring sideroblasts (MDS-RS) from 15% to 5%.2 A trephine biopsy (decalcified, paraffin or plastic-embedded) should be taken from all patients and sectioned for analysis alongside the aspirate. Whilst dysplasia can be harder to assess, the histology of the trephine section provides supportive information for diagnosis, including architectural disruption (e.g. disruption of erythroid islands; abnormal localisation of immature precursors), cellularity and fibrosis (with reticulin staining). Trephine section histology is especially helpful for the diagnosis of hypocellular MDS and MDS/myeloproliferative neoplasms (MPN) overlap syndromes.9 Patients with MDS/MPN overlap including CMML are now considered a distinct entity by the WHO when features of both MDS and MPN are present. This includes MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) which may evolve from MDS-RS. Around 10–20% of patients with MDS have decreased marrow cellularity.10 The WHO classification of myeloid neoplasm terms this hypoplastic MDS (h-MDS), although it does not give it a distinct category.2 Hypocellularity in MDS can present diagnostic difficulties with other bone marrow failure (BMF) syndromes especially aplastic anaemia. A study integrating cytohistological and genetic features in adult patients with hypocellular bone marrows has led to proposed criteria to define h-MDS.10 This separates patients into two distinct groups, one with features highly consistent with myeloid neoplasm and one more consistent with a non-malignant BMF. The two groups have significantly different risk of blast progression and overall survival (OS). Flow cytometry should be performed for paroxysmal nocturnal haemoglobinuria in patients with h-MDS. Enumeration of blast percentage should be undertaken by morphological assessment of the bone marrow aspirate. This is considered the gold standard. However, if the aspirate smear is suboptimal, then the bone marrow trephine section may be used to quantitate blasts using immunohistochemistry. There is no specific immunophenotypic finding diagnostic of MDS, and flow cytometry is therefore not mandatory. Aberrant flow cytometric profiles may support the diagnosis of MDS but should be interpreted with morphological and cytogenetic or molecular findings. Common findings are aberrant antigen expression on myeloid progenitors, maturing myeloid, monocytic and erythroid lineages, reduced numbers of B-cell progenitors,11 and increased CD34+ cells. Many cases also show lineage infidelity antigen expression. Flow cytometry can be useful to enumerate myeloid progenitor cells (CD34+ cells) which may in turn be a proxy for morphological blast percentage but these do not always correlate precisely, for example due to haemodilution of the aspirate or the progenitor cell phenotype lacking CD34 expression. Recommendations for standardisation of flow cytometric methodology, including consensus recommendations for cell sampling, handling and processing have been published;12-16 validation is ongoing. Chromosomal abnormalities evidencing a clonal disorder are detected by cytogenetic analyses in approximately 50% of MDS patients. Some recurrent abnormalities [most commonly, −5, del(5q), −7, del(7q), i(17q)] are considered MDS-defining in a cytopenic patient, even without morphological dysplasia (a comprehensive list is shown in Fig 1 and Table III).2, 17 G-banding or metaphase cytogenetic analysis should be performed on all suspected MDS cases to aid diagnosis, prognosis and inform management. When no abnormality is found in a diagnostic sample, a minimum of 20 metaphases should be examined and reported using International System for Human Cytogenetic Nomenclature Recommendations.18 Cytogenetic assessment is essential for international prognostic scoring systems.17 Furthermore, specific cytogenetic abnormalities may provide a marker for assessing response to therapy and evaluating residual disease. Since both the type and number of karyotypic abnormalities may have prognostic significance, adherence to International Working Group on MDS Cytogenetics consensus guidelines in the enumeration of abnormalities is recommended.19 <15% / <5%b b If SF3B1 mutation is present. BM <5%, PB <1%, No Auer rods <15% / <5%b b If SF3B1 mutation is present. BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods BM 5-9% or PB 2-4%, BM <10% and PB <5%, No Auer rods BM 10-19% or PB 5-19%, Or Auer rods BM and PB <20% BM <5%, PB <1% c c 1% PB blasts must be recorded on ≥2 separate occasions. , No Auer rods BM <5%, PB <1%, No Auer rods BM <5%, PB <1%, No Auer rods In cases where G-banding analysis is not possible or fails, fluorescence in situ hybridisation (FISH) analysis of marrow aspirate or peripheral blood smears for selected common cytogenetic anomalies (e.g. −7, del(5q), +8) may be performed, to detect key abnormalities of prognostic significance or provide confirmation of clonality in borderline diagnostic cases. Where available, single nucleotide polymorphisms array analysis (SNP-A) can provide a more precise, genome-wide analysis which is independent of metaphases.20-22 Although not currently mandated in diagnostic work-up, this can provide useful additional information. In particular, where conventional cytogenetics fails SNP-A array can provide a full karyotype, and should be strongly considered in such cases. SNP-A may also detect karyotypic abnormalities in ~16–30% additional cases where they were not detected by metaphase cytogenetics (MC).20-22 Importantly, copy number abnormalities detected by SNP-A in cases where none were found by MC, are prognostic;23 thus prognostic equivalence can be reasonably assumed for larger structural abnormalities detected by this approach, and should be reported as such. This, however, cannot currently be assumed for smaller abnormalities below the detection resolution of conventional cytogenetics. SNP-A reports should state clearly those lesions considered detectable by MC and which should (and should not) be considered when calculating the cytogenetic risk score for current prognostic systems (e.g. Revised International Prognostic Scoring System [IPSS-R]). Furthermore, SNP-A have limited capacity for detecting translocations which are confined to those with associated microdeletions or uniparental disomy.24 Next-generation sequencing (NGS) has identified recurrent gene mutations in DNA from haematopoietic cells of ~90% of MDS patients, some of which may have independent prognostic significance.25-27 Molecular testing using targeted mutation panels is now widely available, increasingly affordable and should be considered in all patients (unless clearly not appropriate) for its potential to inform on diagnosis, prognosis and management. Sensitivity is highest on bone marrow, but can usefully be performed on peripheral blood in situations in which bone marrow biopsy is impractical or undesirable (provided that circulating myeloid cells are present). Patients should be counselled and at least verbal consent taken prior to genetic testing to explain the possible results including the implications of identifying a germline mutation. Detection of certain MDS-associated mutations can be used to establish subtypes with prognostic relevance. For example, SF3B1 mutations are found in >95% of MDS cases with ring sideroblasts, and are associated with a relatively favourable prognosis28 compared with SF3B1 wild-type MDS-RS cases.29 Due to its characteristic features SF3B1-mutated MDS has been proposed by The International Working Group as a distinct MDS subtype, although this is not yet formally incorporated into the WHO classification.30 TP53 mutations in MDS with isolated del(5q) helps identify early clonal evolution and predict disease progression and poorer prognosis in this generally favourable subgroup.31 In MDS more broadly, combinations of mutation, deletion and/or loss of heterozygosity events, resulting in 'double-hit' biallelic loss of TP53, are strongly associated with complex (typically monosomal) karyotype and exceptionally poor survival outcomes.32 In contrast, patients with single-hit, monoallelic TP53 mutations often lack associated chromosomal aneuploidies and display similar therapy response and outcomes to MDS patients without mutated TP53.32, 33 Mutations in genes such as ASXL1, EZH2 and RUNX1 confer adverse prognosis in univariate analysis but their prognostic significance in multivariate analysis has not yet been consistently reproduced in independent series.28, 34 Mutation status will likely inform prognosis in future models (e.g. IPSS-Molecular; in development) and guide eligibility for clinical trials of emerging targeted therapies (e.g. IDH1/IDH2 inhibitors; spliceosome inhibitors). In view of potential challenges of morphological diagnosis of MDS, mutation analysis can provide objective evidence of clonal disease. However, somatic mutations can be identified in healthy individuals and detection of mutations alone is not considered diagnostic.2 Notably, MDS patients tend to have a higher allele fraction and greater number of mutations than healthy, older individuals.35, 36 In an attempt to standardise testing, NHS England has created the NHS Genomic Medicine service, comprised of a national Genomic Laboratory Hub (GLH) network. A National Genomic Test Directory specifies genomic tests commissioned by the NHS in England and patients who are eligible for testing. Each GLH will provide cytogenetics and DNA sequencing with analysis and expert interpretation. Currently, those with suspected or confirmed MDS are eligible for a targeted NGS panel. Classification of MDS remains largely based upon morphological examination.2 The latest WHO revision has updated nomenclature and removed the focus on specific lineages of cytopenia (Table III and Fig 2).2 A WHO classification subtype should be recorded for every patient in the bone marrow report. In adult patients with at least 20% blasts the disease is classified as AML, although cases with 20–30% blasts were included in derivation of the IPSS. Myelodysplastic syndrome secondary to prior cytotoxic therapy is classified separately, under therapy-related myeloid neoplasms. Clonal haematopoiesis can be detected in the healthy population, typically with increasing age.37-40 This is frequently characterised by acquisition of MDS-associated mutations, but without other clinicopathological features of MDS. This has been termed 'clonal haematopoiesis of indeterminate potential' (CHIP) or 'age-related clonal haematopoiesis' (ARCH), and can be found in >10% of healthy individuals over 70 years of age.38 The most commonly identified mutations are in genes involved in epigenetic regulation (DNMT3A, TET2, ASXL1). These are commonly mutations in single genes only, at low allele frequency (<10%). Risk of transformation to haematological malignancy is low (<1% per year). Annual monitoring of blood counts in individuals found to have CHIP may, therefore, be appropriate. Factors that might increase risk of progression to myeloid malignancy include higher variant allele frequency, presence of multiple CHIP mutations or particular high-risk mutations (e.g. TP53, IDH2).35 A new nomenclature has emerged for conditions related to MDS but not fulfilling the formal diagnostic criteria (Table IV). These are increasingly used to describe observed states bearing isolated molecular, cytopenic or morphological features associated with MDS, and which might predispose to haematological malignancy. Clonal haematopoiesis of indeterminate potential Age-related clonal haematopoiesis ICUS carries approximately 9% risk of developing myeloid malignancy at 10 years.41 Evidence-based recommendations on monitoring cannot yet be made and decisions should be guided by the overall clinical picture and context; the possibility of non-MDS-related causes for the cytopenia should be reviewed during follow-up. In contrast, close monitoring of patients with CCUS is recommended, given emerging evidence that these patients carry a high — possibly universal — risk of progression to frank haematological malignancy.41 Beyond securing a diagnosis, identification of a germline condition underlying MDS can have important implications for treatment planning; for example, when selecting sibling donors for allogeneic stem cell transplantation. A three-generational family history should be taken. Table V outlines individuals in whom the possibility of a myeloid neoplasm with germline predisposition should be considered. Some germline mutations, such as those in TP53, RUNX1 and GATA2, may also be detected by NGS platforms aimed at detecting somatic mutations. Germline variants may be suggested by a variant allele frequency around 50%, although this can be the case too for dominant, deeply established somatic clones, so cannot alone be routinely taken as presumptive evidence. Early contact with a centre having clinical experience of constitutional marrow failure syndromes and a clinical genetics department is indicated in cases of suspected germline conditions. Patients and family members should ideally be offered genetic counselling before genetic screening if there is a high clinical suspicion.42 Since its publication in 1997, the IPSS has been an important tool for assessing the outcome of patients with untreated, primary adult MDS.43 Additional prognostic variables have been identified, the most important of which are newer cytogenetic groupings (Table VI) that give more accurate prognostic information.17 The IPSS-R described the relative importance of defined clinical factors to prognosis by multivariate analysis of 7012 primary, adult MDS patients not treated with disease-modifying therapies. Using the same parameters as the IPSS (cytogenetic groups, marrow blast percentage and cytopenias), it provided extended categorisation of cytogenetic subgroups, refinement of blast counts <5% and depth of cytopenias (Table VII).44 The IPSS-R stratifies into 5 risk categories and has improved the prognostic ability to determine survival and AML evolution in untreated adult patients with primary MDS (Table VIII). A tool to the IPSS-R can be via the UK MDS website In some the IPSS-R has both the IPSS and prognostic at least for some and is currently the scoring for prognosis. However, as as for is based on IPSS risk, that clinical in the Mutation do not currently inform prognostic scoring in MDS. is currently under should be given to a review of prognosis for MDS patients. For example, loss of response to or is associated with a in overall In contrast, IPSS or IPSS-R that for MDS, the the patient remains low risk, the the overall prognosis compared with the prognosis at In patients eligible for allogeneic stem cell should be given to bone marrow testing. Although of of transplantation was based on a to AML transformation in MDS, expert opinion transplantation following identification of of such as increased bone marrow blast clonal evolution or increasing fibrosis in subtypes such as del(5q) Such should be in with the All the to the writing of these guidelines. The writing committee to the team of MDS experts at the MDS UK Patient Support Group for their review of the manuscript on of the MDS UK Patient Support for help in the literature also the BSH Haemato-oncology Task Force, the BSH sounding board and the BSH Guidelines Committee for their support in this guideline. All and the MDS UK Patient Support Group have made a of to the BSH and Task which may be on of the writing group will inform the writing group if new evidence that the strength of the recommendations made in this document or it The document will be reviewed by the Task and the literature will be every years to for new evidence that may have been The document will be and removed from the BSH current guidelines website if it If new recommendations are made an will be published on the BSH guidelines the and information in this guidance is to be and accurate at the time of to the the BSH the for the of this

INTRODUCTION: Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the expansion of progeny derived from hematopoietic stem cells that have acquired somatic mutations at a VAF greater than 2%. CHIP manifests in 10% of patients older than 65 and is associated with an increased risk of progression to malignancies such as the MDS or AML. Although the risk factors for developing CHIP remain incompletely defined, they include prior exposure to chemotherapy and a history of smoking. Monoclonal gammopathy of undetermined significance (MGUS) is characterized by the abnormal growth of clonal plasma cells in the bone marrow and carries a risk of 1% for progression to multiple myeloma (MM) per year. Like CHIP, it becomes more prevalent with age and is associated with smoking. Additionally, patients with MM have demonstrated an increased risk for malignancies. Thus, an examination for a correlation between CHIP and MGUS promises to reveal a link between these two pre-malignant conditions. A recent study did not demonstrate such an association, but this study was performed in a very elderly population and may not be applicable to younger patients. In this study, we aim to assess the relationship between CHIP and MGUS in a population-based cohort of MGUS patients seen at UT Southwestern Medical Center. METHODS: To evaluate an association between CHIP and MGUS, we collected bone marrow samples from 37 patients diagnosed with MGUS. We employed a hybridization capture-based next generation sequencing assay in order to detect CHIP. We identified 24 genes known to cause CHIP in adults. We also evaluated patients risk for developing MM after having been diagnosed with CHIP. RESULTS: The mean age was 68, (range 26-92). 22 patients were white, 8 were black and 3 Hispanic/Latino. 17 patients had IgG, 7 had IgA, 3 had IgM, 3 had biclonal gammopathy and 7 light-chain MGUS. We identified 18 mutations to validate the presence of CHIP in 10 (27%) patients, with the most frequent being DNMT3A (7 patients) and TET2 (5 patients). Other common mutations noted were PPM1D (2), GND1 (1), SF3B1 (1), ASXL1 in (1), and NRAS in (1). 3 out of the 10 patients harbored 2 mutations and 1 harbored 4 mutations. History of chemotherapy (n=6) and smoking (n=14) was taken into consideration to determine the relative risk of patients with MGUS developing CHIP. We found that those who had a prior history of smoking and chemotherapy displayed a higher risk of CHIP. CONCLUSION: There was no significant association between CHIP and MM progression. Our analysis showed 1 patient with CHIP progression and 2 without CHIP progression. Because the rates of CHIP and MGUS are positively correlated with characteristics like aging and a history of smoking, we expected to see high rates of CHIP in patients within our cohort. However, our data suggests that CHIP is frequent (27%) in MGUS patients, but larger future cohorts need to be evaluated to validate this association. Citation Format: Vianey Quaney, Benjamin Kroger, Aishwarya Sannareddy, Umar Khan, Fatma Kalkan, Robert H. Collins, Yazan F. Madanat, Madhuri Vusirikala, Yi Huang, Farrukh T. Awan, Praveen Ramakrishnan, Aimaz Afrough, Larry D. Anderson, Stephen S. Chung, Gurbakhash Kaur. Prevalence of clonal hematopoiesis in patients with monoclonal gammopathy of undetermined significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5925.

Prevalence of Clonal Hematopoiesis of Indeterminate Potential in Patients with Monoclonal B-Cell Lymphocytosis Compared to Untreated Chronic Lymphocytic Leukemia

Next-Generation Sequencing of Matched Normal Blood Identifies Clonal Hematopoiesis in a Significant Subset of Solid Tumor Patients without Hematologic Malignancies

Novel and Significant Impact of Germline Variants Predisposed to Pathogenic Somatic Mutations and Loss of Heterozygosity (LOH) in Myelodysplastic Syndromes (MDS) and Clonal Hematopoiesis of Indeterminate Potential (CHIP)

MGUS and Chip: Two Faces, but Not of the Same Medal

Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the age-dependent accumulation of somatic leukemia-associated driver mutations in hematopoietic stem cells, in individuals with normal blood counts and with absence of an underlying myeloid neoplasm (MN).1, 2 CHIP is associated with an increased risk of developing MN and an increased all-cause mortality, largely due to cardiovascular disease.3 The presence of CHIP prior to receiving chemotherapy and radiation has been associated with therapy related MN (T-MN), such as myelodysplastic syndromes (MDS) and acute myeloid leukemia.4 Autologous stem cell transplantation (ASCT) is an effective treatment modality for managing higher-risk patients with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM). In a seminal NHL study, 30% of patients were found to have CHIP at the time of ASCT, with the presence of CHIP being associated with an increased rate of T-MN (10-year cumulative incidence of 14.1% vs 4.3%) and an inferior overall survival (10 year OS 30.4% vs 60.9%).5 In MM, targeted sequencing of 629 patients prior to ASCT detected CHIP in 21.6% of patients, with the presence of CHIP strongly associating with inferior OS (HR 1.34, p = .02) and an inferior progression-free survival (PFS, HR 1.45, p < .001). Interestingly, in this study, adverse CHIP-associations were apparently overcome by lenalidomide maintenance therapy.6 Unlike in NHL, CHIP in MM was not associated with T-MN; while lenalidomide maintenance therapy, independent of the presence or absence of CHIP, was associated with T-MN (p = .047) and second primary malignancies (SPM).6 We carried out this study to assess the prevalence and prognostic impact of CHIP in a relatively uniform cohort of MM patients at the time of ASCT, with all patients going on to receive lenalidomide maintenance therapy. Successive MM patients who consented to have their pre-ASCT bone marrow (BM) sample collected and who underwent first ASCT at Mayo Clinic, followed by lenalidomide maintenance therapy, were included in the study. The BM mononuclear cell DNA from pre-ASCT samples was extracted after excluding CD38/CD138+ (negative selection) plasma cells and then subjected to targeted NGS testing (42-myeloid related genes) by previously described methods.7 All patients were closely followed for the development of T-MN as defined by the 2016 WHO criteria, arterial and venous thromboembolism (VTE) and SPM.8, 9 Response to therapy was assessed using the international myeloma working group (IMWG) consensus criteria 2016.10 Statistical methods are highlighted in the supplemental material. Clonal hematopoiesis was detectable in 23 (23%) of 101 MM patients assessed in the study (Table S1, Figure S1). Clinical characteristics, MM risk stratification, median number of prior therapies, response to therapy, ASCT conditioning regimens, engraftment data, day +100 post ASCT outcomes and median duration of lenalidomide maintenance are outlined in Table 1. Except for a higher median age at MM diagnosis in MM patient with CHIP (p = .002), there were no other significant differences between the two groups (Figure S2). Ten (43.5%) patients in the MM CHIP group and 30 (38.0%) in the MM no CHIP group, received alkylatingagent-based induction therapy prior to ASCT (p = .66). Melphalan 200 mg/m2 conditioning was used in 87.1% of patients (95.7% in the CHIP vs 84.6% in the no CHIP group, p = .45), while the remainder received melphalan 140 mg/m2 conditioning. The median duration of lenalidomide maintenance therapy was 21 months (10–36); 16 months in MM patients with CHIP and 22 months in MM patients without CHIP (p = .76), with the median lenalidomide dose being 15 mg (range 10–15 mg; 10 mg in the CHIP group and 15 mg in the no CHIP group, p = .08). The most frequent CHIP mutations encountered included DNMT3A [52%; median variant allele frequency (VAF) 9.0%, range 2.0%–29.2%], TET2 (26%; median VAF 3.0%, range 2.0%–4.8%), followed by TP53, PPM1D and BRAF (10.0% each), respectively (Figures 1(A) and S1). Sixteen patients (69.6%) had one mutation, while seven (30.4%) had >1 mutation and two patients had four mutations each (Figure 1(B)). Eight (66.6%) of 12 patients with DNMT3A mutations had nonsynonymous missense mutations, while four had deletion variants: with no patient harboring the commonly mutated DNMT3A R882 hot spot. There were no statistically significant differences in CHIP mutation distribution, including TP53 and PPM1D mutations, between MM CHIP patients that received alkylating-agent based induction therapy prior to ASCT, vs MM CHIP patients that did not (Figure 1(C)). At last follow up, 70 (69.3%) relapses after ASCT and 41 (40.6%) deaths were documented. Twenty-nine patients (28.7%) were on salvage therapy whereas 13 (12.9%) were on lenalidomide maintenance and 14 (13.8%) were on observation alone, with no statistically significant differences between MM patients with CHIP vs MM without CHIP. Rates of VTE were similar between MM patients with CHIP (30%) and those without CHIP (24%), with similar rates of provoked thromboses (33% vs 29%, p = .4). Thromboses were diagnosed in typical locations in individuals with CHIP including seven lower extremity deep vein thromboses (DVT) and two pulmonary emboli (PE). In contrast, a variety of VTE locations were diagnosed in those without CHIP, including 10 lower extremity DVT, three PE, one DVT with PE, two upper extremity DVT and one portal vein thrombosis. There was a distinction in VTE timing with regards to lenalidomide use between MM patients with CHIP and those without CHIP. For MM patients with CHIP, 2/9 (22.2%) VTE occurred while on lenalidomide, 2/9 (22.2%) occurred prior to lenalidomide and 4/9 (44.4%) occurred at least 3 months after discontinuation of lenalidomide therapy. For MM patients without CHIP, majority (13/17, 76.5%) of VTE occurred while on lenalidomide, with a minority (11%) occurring either before or at least 3 months after discontinuation of lenalidomide. While lenalidomide is a known risk factor for thrombosis in MM, the fact that 44% of VTE in MM with CHIP occurred >3 months after discontinuing lenalidomide, suggests that CHIP might increase VTE risk in this setting (P = .04). Median OS from the time of diagnosis of the entire cohort was 124.6 months (95%CI 97.5-N/A months) with a corresponding 5 year OS of 82.0% (95% CI 74.8%–89.9%). There was no difference in median OS between MM with CHIP vs those without CHIP (100.2 months; 95%CI 76.2-NA months vs 135.6 months; 95%CI 106.3-N/A months, p = .27) (Figure 1(D)), including assessments with individual CHIP-mutations. The median EFS after ASCT was 36.4 months (95%CI 30.5–48.5 months), with there being no difference in median PFS between MM CHIP (36.4 months, 95%CI 24.1–58.5 months) patients vs MM patients without CHIP (36.4 months, 95%CI 29.9–52.4 months) (p = .34) (Figure 1(E)), including TP53 mutations (Figure S3). There were also no differences between the two groups with regards to non-relapse mortality (Figure S4), cumulative incidence of relapse after ASCT (Figure S5) and time to next treatment. Nineteen (18.8%) SPM were documented, 7 (30.4%) in MM CHIP group vs 12 (15.3%) in the MM no CHIP group (Figure 1(F), p = .13), with corresponding 5-year cumulative incidence rates of 22% and 13%, respectively. These SPM included five (4.9%) hematological malignancies (two in MM CHIP vs three in MM no CHIP), seven (6.9%) skin cancers (three in MM CHIP vs four in MM no CHIP) and seven visceral malignancies (two in MM CHIP vs five in MM no CHIP) (Table S2, Figure S6). The two MM CHIP patients who developed T-MN/MDS had TP53 and PPM1D mutations, respectively. In the MM no CHIP group, there was one patient with B-acute lymphoblastic leukemia and two patients with T-MDS with monosomal karyotypes. In summary, we define the CHIP landscape in MM patients at the time of ASCT, with mutations in epigenetic regulator genes being most common (57%), followed by tumor suppressor genes (17%). Unlike in NHL, presence of CHIP at time of ASCT in MM did not impact OS, PFS and incidence of T-MN; a finding potentially attributable to immunomodulatory properties of lenalidomide, or to the use of maintenance therapy in general.6, 11 While the presence of CHIP and lenalidomide therapy have individually been associated with increased risk of thromboses,1-3, 9 we did not see synergy in MM patients with CHIP, although the timing and patterns of thromboses suggest that CHIP might negatively influence thrombotic risk. While SPM have been well described with lenalidomide maintenance therapy,9 we did not see any differences in SPM and hematological malignancies between the two groups. The findings of this study independently confirm a prior observation on the potential ability of lenalidomide maintenance to mitigate the expected adverse effects of CHIP on OS and PFS in MM patients' post-ASCT6; an important consideration given that approximately 13 000 MM patients undergo ASCT in the US annually, with lenalidomide maintenance considered as standard of care.12, 13 Given the smaller sample size and the inherent flaws of a retrospective analysis, future clinical trials evaluating therapies in MM patients' post-ASCT should consider accounting for the presence of CHIP and its impact on outcomes. The authors would like to acknowledge the “Henry Predolin Leukemia Foundation”, Mayo Clinic, Rochester, MN, USA. Mrinal Patnaik has served on the advisory board of Kura Oncology. A Keith Stewart has served on the advisory board for Celgene. Rafael Fonseca has the following disclosures: Consulting: Amgen, BMS, Celgene, Takeda, Bayer, Janssen, Novartis, Pharmacyclics, Sanofi, Karyopharm, Merck, Juno, Kite, Aduro, OncoTracker, Oncopeptides, GSK, AbbVie. Scientific Advisory Board: Adaptive Biotechnologies, Caris Life Sciences and OncoTracker. Gene mutations annotated in the study have been provided in the supplementary material. Raw sequencing data can be made available on request. Appendix S1 Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Suspicious, Non-MDS-Diagnostic Bone Marrows Have a High Incidence of Clonal Hematopoiesis (CHIP), with MDS-like Clone Size but Restricted Mutation Burden

Monoclonal B-cell lymphocytosis (MBL) and clonal hematopoiesis of indeterminate potential (CHIP) are prevalent clonal precursors associated with increased risk of lymphoid malignancies. However, the relationship between MBL and CHIP and their combined impact on lymphoid malignancy risk remains poorly understood. We screened participants from the Mayo Clinic Biobank to identify MBL using eight-color flow cytometry; CHIP was detected using whole-exome sequencing of whole-blood DNA in 291 genes related to myeloid or lymphoid malignancies. Incident myeloid or lymphoid hematological malignancies were identified using ICD codes and confirmed via medical record review. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI). Cox regression was used to estimate hazard ratios (HR). Analyses were adjusted for age and sex. In 10,067 participants, 15% had MBL, and 9% had CHIP. No evidence of an association between MBL and CHIP (OR = 1.00; 95% CI: 0.82–1.20) was observed. With a median follow-up of 5.4 years, 138 participants developed hematological malignancies (94 lymphoid). MBL (HR = 3.48; 95% CI: 2.27–5.34; P < 0.001) and CHIP (HR = 1.89; 95% CI: 1.10–3.27; P = 0.022) were each associated with incident lymphoid malignancy. Compared to individuals with no precursors, the combined presence of MBL and CHIP significantly amplified lymphoma risk (HR = 7.18; 95% CI: 3.33–15.47; P < 0.001), more than doubling the risk among individuals with MBL alone (HR = 3.30; 95% CI: 2.06–5.30; P < 0.001). In contrast, the risk associated with CHIP alone was attenuated and no longer statistically significant (HR = 1.63; 95% CI: 0.77–3.47; P = 0.20). MBL and CHIP are independent hematological precursor conditions. While their combined presence amplifies the risk of lymphoid malignancy, CHIP alone may not be a strong independent risk factor.

Myelodysplastic syndromes: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†☆

Clonotype-Naïve Detection of Clonality in Patients Suspected of Having Multiple Myeloma or Monoclonal Gammopathy Using Peripheral Blood Cell-Free RNA (cfRNA)

Monoclonal gammopathy of undetermined significance (MGUS) is a precursor to multiple myeloma (MM). Clonal hematopoiesis of indeterminate potential (CHIP) is a precursor to either myeloid or lymphoid malignancy, depending on whether the mutation is in a gene associated with lymphoid (L-CHIP) or myeloid (M-CHIP) outcome. Little is known about the association between CHIP and MGUS and their combined effect on risk of MM. We examined this question within Mayo Clinic Biobank cohort of participants aged 50 years or older with no prior history of hematological malignancies. Using whole blood, individuals were screened for MGUS using a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry assay. M-CHIP and L-CHIP were defined based on mutations in 56 genes associated with myeloid malignancies or mutations in 235 genes associated with lymphoid malignancies, respectively, using whole-exome-sequencing. Incident MM was identified using International Classification of Diseases (ICD) codes and confirmed via medical record review. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals for the association of CHIP and MGUS. Cox proportional hazards regression was used to estimate hazard ratios (HR), with time defined as date between blood sample and the first incident MM, date of death, or 12/31/2023. All models were adjusted for age and sex. Of the 16, 479 individuals (median age 65 years, 45% male) screened, 12.3% were positive for MGUS, 9.8% were positive for M-CHIP, and 1.5% were positive for L-CHIP. There was no evidence of an association between M-CHIP and MGUS (OR=0.97, 95% CI:0.83-1.12) nor L-CHIP and MGUS (OR=1.12, 95% CI:0.78-1.57), including with any MGUS isotype (IgA, IgG, or IgM; P-values&amp;gt;0.22). Next, we investigated the association of these precursors on risk of MM. Median follow-up was 11.7 years (range 0.1-15.1), and 46 individuals developed incident MM. Both MGUS (HR=29.7, 95% CI: 14.1-62.4) and L-CHIP (HR=4.1, 95% CI: 1.3-13.2) were significantly associated with incident MM, but not M-CHIP (HR-1.0, 95% CI: 0.4-2.5). Investigating the combined effect of MGUS and L-CHIP, we found those with MGUS alone had a 27.5-fold (95% CI: 13.0-58.2) increased risk of incident MM compared to those with no precursors. No individuals with L-CHIP only developed MM, but those with both L-CHIP and MGUS had a 129.4-fold (95% CI: 34.4-486.4) increased risk of incident MM. In the largest cohort with screened MGUS and CHIP, we found no evidence of an association between either definition of CHIP with MGUS. However, individuals with both L-CHIP and MGUS were at the highest risk of developing MM. Citation Format: Nicholas J. Boddicker, Cristine Allmer, Danelle H. Moonen, Aaron D. Norman, Angelica Macauda, Alyssa Clay-Gilmour, Janet E. Olson, Mrinal S. Patnaik, Vincent Rajkumar, Esteban Braggio, David L. Murray, Shaji Kumar, Susan L. Slager, Celine M. Vachon. The association between MGUS and CHIP and their impact on multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4917.