Clonal Cytopenia Of Undetermined Significance Research Articles

Can molecular patterns help to classify overlapping entities in myeloid neoplasms?

Myeloid neoplasms include myeloproliferative and myelodysplastic neoplasms and acute myeloid leukaemia. Historically, these diseases have been diagnosed based on clinicopathological features with sometimes arbitrary thresholds that have persisted even as molecular features were gradually incorporated into their classification. As such, although current diagnostic approaches can classify the majority of myeloid neoplasms accurately using a combination of molecular and clinicopathological features, some areas of overlap persist and occasionally pose diagnostic challenges. These include overlap across BCR::ABL1-negative myeloproliferative neoplasms; between clonal cytopenia of undetermined significance and myelodysplastic neoplasms; myelodysplastic/myeloproliferative neoplasms; and, detection of KIT mutations in myeloid neoplasms other than mastocytosis, raising the prospect of systemic mastocytosis. Molecular testing has become state of the art in the diagnostic work-up of myeloid neoplasms, and molecular patterns can inherently help to classify overlapping entities if considered within a framework of haematological presentations. For future development, molecular testing will likely include whole genome and transcriptome sequencing, and primarily molecular classifications of myeloid neoplasms have already been suggested. As such, genetically defined groups should still constitute the basis for our understanding of disease development from early onset to progression, while clinicopathological features could then be used to describe the stage of the disease rather than the specific type of myeloid neoplasm.

Detection and Interpretation of Clonal Hematopoiesis Variants during Routine Solid Tumor Next-Generation Sequencing: A Single-Institution Experience

Clonal hematopoiesis (CH) and Clonal Cytopenia of Undetermined Significance (CCUS) are recently recognized diagnostic entities that serve as an independent risk factor for cardiovascular disease and myeloid malignancy. CH is an incidental finding and evaluation of the incidence of CH/CCUS associated mutations in solid tumor Next Generation Sequencing (NGS) samples was undertaken to better understand the prevalence of mutations in this population. A retrospective review of clinical sequencing data for solid tumor malignancies diagnosed February 2022-April 2023 on NGS data was performed. Cases were reviewed for variants in genes associated with CH/CCUS. Variant allele frequencies and other factors of the sequencing data were assessed for determining risk of CH/CCUS. 2,479 cases were evaluated during the study period. Of these, 29 cases demonstrated potential CH/CCUS associated mutations with a total of 33 variants identified. These were identified in a variety of tumor types, with gliomas being the most common. Significant cardiac histories were found in over half of cases identified and few cases had abnormal blood counts. Detailed criteria for flagging variants as suspicious for CH and recommend these criteria as future guidelines for reporting are described. These variants are incidental findings which require more extensive follow up or change in therapy management utilizing a single institutional cohort.

Systemic deficits in lipid homeostasis promote aging-associated impairments in B cell progenitor development.

Organismal aging has been associated with diverse metabolic and functional changes across tissues. Within the immune system, key features of physiological hematopoietic cell aging include increased fat deposition in the bone marrow, impaired hematopoietic stem and progenitor cell (HSPC) function, and a propensity towards myeloid differentiation. This shift in lineage bias can lead to pre-malignant bone marrow conditions such as clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenias of undetermined significance (CCUS), frequently setting the stage for subsequent development of age-related cancers in myeloid or lymphoid lineages. At the systemic as well as sub-cellular level, human aging has also been associated with diverse lipid alterations, such as decreased phospholipid membrane fluidity that arises as a result of increased saturated fatty acid (FA) accumulation and a decay in n-3 polyunsaturated fatty acid (PUFA) species by the age of 80 years, however the extent to which impaired FA metabolism contributes to hematopoietic aging is less clear. Here, we performed comprehensive multi-omics analyses and uncovered a role for a key PUFA biosynthesis gene, ELOVL2 , in mouse and human immune cell aging. Whole transcriptome RNA-sequencing studies of bone marrow from aged Elovl2 mutant (enzyme-deficient) mice compared with age-matched controls revealed global down-regulation in lymphoid cell markers and expression of genes involved specifically in B cell development. Flow cytometric analyses of immune cell markers confirmed an aging-associated loss of B cell markers that was exacerbated in the bone marrow of Elovl2 mutant mice and unveiled CD79B, a vital molecular regulator of lymphoid progenitor development from the pro-B to pre-B cell stage, as a putative surface biomarker of accelerated immune aging. Complementary lipidomic studies extended these findings to reveal select alterations in lipid species in aged and Elovl2 mutant mouse bone marrow samples, suggesting significant changes in the biophysical properties of cellular membranes. Furthermore, single cell RNA-seq analysis of human HSPCs across the spectrum of human development and aging uncovered a rare subpopulation (<7%) of CD34 + HSPCs that expresses ELOVL2 in healthy adult bone marrow. This HSPC subset, along with CD79B -expressing lymphoid-committed cells, were almost completely absent in CD34 + cells isolated from elderly (>60 years old) bone marrow samples. Together, these findings uncover new roles for lipid metabolism enzymes in the molecular regulation of cellular aging and immune cell function in mouse and human hematopoiesis. In addition, because systemic loss of ELOVL2 enzymatic activity resulted in down-regulation of B cell genes that are also associated with lymphoproliferative neoplasms, this study sheds light on an intriguing metabolic pathway that could be leveraged in future studies as a novel therapeutic modality to target blood cancers or other age-related conditions involving the B cell lineage.

Inflammation in myelodysplastic syndrome pathogenesis

Inflammation is a key driver of the progression of preleukemic myeloid conditions, such as clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS), to myelodysplastic syndromes (MDS). Inflammation is a critical mediator in the complex interplay of the genetic, epigenetic, and microenvironmental factors contributing to clonal evolution. Under inflammatory conditions, somatic mutations in TET2, DNMT3A, and ASXL1, the most frequently mutated genes in CHIP and CCUS, induce a competitive advantage to hematopoietic stem and progenitor cells, which leads to their clonal expansion in the bone marrow. Chronic inflammation also drives metabolic reprogramming and immune system deregulation, further promoting the expansion of malignant clones. This review underscores the urgent need to fully elucidate the role of inflammation in MDS initiation and highlights the potential of the therapeutical targeting of inflammatory pathways as an early intervention in MDS.

8. Reduced subclone diversity in clonal cytopenia of undetermined significance compared to myelodysplastic syndrome

8. Reduced subclone diversity in clonal cytopenia of undetermined significance compared to myelodysplastic syndrome

Clonal cytopenia of undetermined significance: definitions, risk and therapeutic targets

Clonal cytopenia of undetermined significance: definitions, risk and therapeutic targets

Clonal Hematopoiesis in Patients With Neuroendocrine Tumor Treated With Lutetium-177 and the Risk of Thrombocytopenia: A Prospective Study.

Thrombocytopenia is a relatively common dose-limiting toxicity during peptide receptor radionuclide therapy (PRRT) in patients with NET. Although uncommon, some patients develop persistent cytopenia and eventually therapy-related myeloid neoplasm (t-MN), which has a dismal prognosis. As the indications for PRRT are expanding, it is important to investigate factors that may predict cytopenias during/after PRRT. We prospectively evaluated the prevalence of clonal hematopoiesis (CH) and cytopenia in patients with NET undergoing PRRT. Patients with metastatic NET with plan to receive four cycles of lutetium-177 were enrolled. CH was evaluated before PRRT using a panel of 220 genes with a targeted depth of ≥1,000×. Patients were followed during PRRT and every 3 months thereafter. Of 37 patients enrolled, the median age was 68 years and 51.4% were male. Previous treatment exposures included alkylating agents in 30%, platinum agents in 8%, and external radiation in 13%. CH was detected in 35.1% using a variant allele frequency (VAF) cutoff of ≥2% and 45.9% with a VAF of ≥1%. The most common mutations were in age-related genes (DNMT3A, TET2). CH was not associated with anemia or neutropenia; however, it was associated with lower platelet count at baseline and more time spent in a thrombocytopenic state during/after PRRT. Five patients had bone marrow biopsies (BMBs) because of sustained hematologic dysfunction post-PRRT, and of those, diagnoses included clonal cytopenia of undetermined significance (CCUS) in three and idiopathic cytopenia of undetermined significance (ICUS) in two. CH is present in 35.1% of patients with NET and is associated with thrombocytopenia risk during PRRT. Future studies with long-term follow-up will delineate whether CH might be a predictor for higher risk of t-MN after PRRT.

The impact of cytotoxic therapy on the risk of progression and death in clonal cytopenia(s) of undetermined significance

AbstractClonal cytopenia of undetermined significance (CCUS) is defined by a myeloid driver mutation in the context of otherwise unexplained cytopenia. CCUS has an inherent risk of progressing to myeloid neoplasm. However, it is unknown how exposure to previous cytotoxic therapy may impact the risk of progression and survival. We stratified patients with CCUS by prior exposure to DNA-damaging therapy. Of 151 patients, 46 (30%) had received cytotoxic therapy and were classified as therapy-related CCUS (t-CCUS), whereas 105 (70%) had de novo CCUS. A lower proportion of t-CCUS had hypercellular marrows (17.8% vs 44.8%, P = .002) but had higher median bone marrow blast percentages. After a median follow-up of 2.2 years, t-CCUS had significantly shorter progression-free survival (PFS, 1.8 vs 6.3 years; hazard ratio [HR], 2.1; P = .007) and median overall survival (OS; 3.6 years vs not reached; HR, 2.3; P = .007) compared with CCUS. Univariable and multivariable time-to-event analyses showed that exposure to cytotoxic therapy independently accounted for inferior PFS and OS. Despite the similarities in clinical presentation between CCUS and t-CCUS, we show that exposure to prior cytotoxic therapies was an independent risk factor for inferior outcomes. This suggests that t-CCUS represents a unique clinical entity that needs more stringent monitoring or earlier intervention strategies.

Genetic studies in clonal haematopoiesis, myelodysplastic neoplasms and acute myeloid leukaemia- a practical guide to WHO-HAEM5.

In recent years, technology developments and increase in knowledge have led to profound changes in the diagnostics of haematologic neoplasms, particularly myeloid neoplasms. Therefore an updated, fifth edition of the World Health Organization (WHO) classification of haematolymphoid neoplasms (WHO-HAEM5) will be issued in 2024. In this context, we present a practical guide for analysing the genetic aspects of clonal haematopoiesis of indeterminate potential (CHIP), clonal cytopenia of undetermined significance (CCUS), myelodysplastic neoplasms (MDS), and acute myeloid leukaemia (AML) based on WHO-HAEM5. This guide navigates through the genetic abnormalities underlying myeloid neoplasms which are required to be detected for classification according to WHO-HAEM5 and provides diagnostic algorithms.

Cancer patients with clonal hematopoiesis die from primary malignancy or comorbidities despite higher rates of transformation to myeloid neoplasms.

The occurrence of somatic mutations in patients with no evidence of hematological disorders is called clonal hematopoiesis (CH). CH, whose subtypes include CH of indeterminate potential and clonal cytopenia of undetermined significance, has been associated with both hematologic cancers and systemic comorbidities. However, CH's effect on patients, especially those with concomitant malignancies, is not fully understood. We performed a retrospective evaluation of all patients with CH at a tertiary cancer center. Patient characteristics, mutational data, and outcomes were collected and analyzed. Of 78 individuals included, 59 (76%) had a history of cancer and 60 (77%) had moderate to severe comorbidity burdens. DNMT3A, TET2, TP53, and ASXL1 were the most common mutations. For the entire cohort, the 2-year overall survival rate was 79% (95% CI: 70, 90), while the median survival was not reached. Of 20 observed deaths, most were related to primary malignancies (n = 7, 35%), comorbidities (n = 4, 20%), or myeloid neoplasms (n = 4, 20%). Twelve patients (15%) experienced transformation to a myeloid neoplasm. According to the clonal hematopoiesis risk score, the 3-year transformation rate was 0% in low-risk, 15% in intermediate-risk (p = 0.098), and 28% in high-risk (p = 0.05) patients. By multivariate analysis, transformation was associated with variant allele frequency ≥0.2 and hemoglobin <10 g/dL. In a population including mostly cancer patients, CH was associated with comorbidities and myeloid transformation in patients with higher mutational burdens and anemia. Nevertheless, such patients were less likely to die of their myeloid neoplasm than of primary malignancy or comorbidities.

148 - An odd presentation of abdominal cellulitis in the setting of clonal cytopenia of undetermined significance

148 - An odd presentation of abdominal cellulitis in the setting of clonal cytopenia of undetermined significance

Predicting cytopenias, progression, and survival in patients with clonal cytopenia of undetermined significance: a prospective cohort study

Somatic mutations are frequently reported in individuals with cytopenia but without a confirmed haematological diagnosis (clonal cytopenia of undetermined significance; CCUS). These patients have an increased risk of progression to a myeloid malignancy and worse overall survival than those with no such mutations. To date, studies have been limited by retrospective analysis or small patient numbers. We aimed to establish the natural history of CCUS by prospectively investigating outcome in a large, well defined patient cohort. This prospective cohort study was conducted at the Haematological Malignancy Diagnostic Service, a diagnostic laboratory in Leeds, UK. Patients aged at least 18 years who were referred for investigation of cytopenia were eligible for inclusion; those with a history of myeloid malignancy were not eligible. Targeted sequencing was conducted alongside routine clinical testing. Baseline mutation analysis was then correlated with the main study outcomes: longitudinal blood counts, disease progression to a myeloid malignancy, and overall survival with a median follow-up of 4·54 years (IQR 4·03-5·04). Data were collected manually from hospital records or extracted from laboratory or clinical outcome databases. Bone marrow samples from 2348 patients were received at the Haematological Malignancy Diagnostic Service between July 1, 2014, and July 31, 2016. Of these, 2083 patients (median age 72 years [IQR 63-80, range 18-99]; 854 [41·0%] female and 1229 [59·0%] male) met the inclusion criteria and had samples of sufficient quality for further analysis. 598 (28·7%) patients received a diagnosis on the basis of their biopsy sample, whereas 1485 (71·3%) samples were classified as non-diagnostic; of these, CCUS was confirmed in 400 (26·9%) patients (256 [64·0%] male and 144 [36·0%] female). TET2, SRSF2, and DNMT3A were the most frequently mutated genes in patients with CCUS, with 320 (80%) of 400 patients harbouring a mutation in at least one of these genes. Age (p<0·0001), sex (p=0·0027), and mutations in ASXL1 (p=0·0009), BCOR (p=0·0056), and TP53 (p=0·0055) correlated with a worse overall survival; however, the number of mutations was the strongest predictor for progression to a myeloid malignancy (two mutations, p=0·0024; three or more mutations, p=0·0004). Extended sequencing of samples from a subgroup of patients with sequential samples and no mutations in the initial myeloid gene panel showed recurrent mutations in both DDX41 and UBA1, suggesting that these genes should be included in clinical test panels. Mutation analysis is advised in patients who have undergone bone marrow examination and have an otherwise-unexplained cytopenia. High-risk genetic mutations and increased numbers of mutations are predictive of both survival and progression within 5 years of presentation, warranting clinical surveillance and, when necessary, intervention. MDS Foundation.

Clinical and pathological features of clonal cytopenia of undetermined significance presenting with isolated thrombocytopenia (CCUS-IT).

Clonal cytopenia of undetermined significance (CCUS) is defined as somatic mutations of myeloid malignancy-associated genes in the blood or bone marrow with one or more persistent unexplained cytopenias that do not meet diagnostic criteria for a defined myeloid neoplasm. CCUS with isolated thrombocytopenia (CCUS-IT) is rare. This is a retrospective case series of patients with prolonged isolated thrombocytopenia, a pathogenic mutation on a myeloid molecular panel, and a bone marrow biopsy with morphologic atypia below the WHO-defined diagnostic threshold for dysplasia. Five male patients were identified with a median age at CCUS-IT diagnosis of 61 years (56-74). Median duration of thrombocytopenia prior to CCUS-IT diagnosis was 4 years (3-12), and median platelet count at CCUS-IT diagnosis was 41 × 103 /μL (26-80). All patients had megakaryocytic hyperplasia and megakaryocytes with hyperchromasia and high nuclear-cytoplasmic ratio. Pathogenic SRSF2 mutations were identified in all 5 patients with median variant allele frequency of 36% (28%-50%). Three patients were treated with IVIg and/or steroids with no response; one of three responded to thrombopoietin receptor agonists. Three patients progressed to MDS and one to AML. We describe the clinicopathological features of CCUS-IT which can mimic immune thrombocytopenia.

How to classify risk based on clinical and molecular modeling: integrating molecular markers in the risk assessment of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS), also known as "myelodysplastic neoplasm," is a heterogeneous group of clonal myeloid neoplasms that typically affects older adults. The clinical phenotype, symptoms, and complications relate to the depth of cytopenia and progression to acute myeloid leukemia (AML). The diagnosis of MDS relies on morphologic criteria, such as evidence of dysplasia, disordered maturation, and increasing blast counts, which separate the disease into histologic subtypes with different probabilities for progression to AML. The treatment of MDS is often risk-adapted depending on the prognostic profile of each patient's disease. There has been a coevolution of diagnostic and prognostic systems for MDS developed over the past 40 years, both of which have now incorporated molecular markers. The new International Prognostic Scoring System-Molecular (IPSS-M) improves partitioning of patients compared to prior versions with resultant upgrading of 34% of patients into higher-risk groups due to the presence of mutations. The new IPSS-M also more accurately distinguishes intermediate-risk patients separating them into two tiers. The two new diagnostic classifications include MDS defined by mutations in SF3B1 and TP53, though there are differences in diagnostic criteria. Future efforts to refine MDS prognostication could investigate the interface between MDS and clonal cytopenia of undetermined significance, expand access to genomic testing, obtain results in a less invasive manner, and develop treatment-response predictors and dynamic risk models.

GATA2 Mutation with Bone Marrow Failure. A Review of Ordering Next Generation Sequencing Testing while Triaging Bone Marrows in Patients with Cytopenias – Pathologists Challenge

Abstract Introduction/Objective GATA2 (germline) mutations are a cause of bone marrow (BM) failure in younger patients characterized by pancytopenia and hypoplastic BM with minimal or no dysplasia. These cases may have negative cytogenetic (CG)/FISH (FH) results and are diagnosed by next-generation sequencing (NGS) testing. We present a case where GATA2 testing was performed on a repeat BM resulting in delayed diagnosis. Insurance companies challenge providers and pathologists with frequent claims denials, thus the need to revise pre-authorization (PA) policies for triaging BMs in real time. We also reviewed our data where NGS was performed in patients with cytopenias, negative morphology (MO), flow cytometry (FC) &amp; CG/FH studies. Methods/Case Report We did a retrospective analysis and selected 455 BMs (2018 to 2022) where the clinical indication was cytopenias and had negative MO, FC, CG, and FH. We evaluated MO again with NGS results to ensure diagnostic features were not overlooked. Results (if a Case Study enter NA) The index case was a 22-year-old female failing to show abnormality by MO, FL, CG, or FH. The patient continued to have cytopenias; BM was performed a year later and NGS showed GATA2 mutation. This prompted a retrospective analysis of 455 BMs as described under methods. 101/455 (22%) cases revealed abnormal NGS results with pathogenic mutation resulting in amended reports. 42/455 (9%) cases revealed mutations consistent with clonal hematopoiesis of indeterminate potential (CHIP) or clonal cytopenia of undetermined significance (CCUS). Conclusion 1) PA policy for BM triaging is not practical since a decision for NGS testing is made in real-time; 2) Claim denials lead to underutilization of NGS, thus resulting in delayed patient care; 3) NGS may show pathogenic mutation despite negative MO/FL/CG/FH results; and 4) Insurance companies need to consider reviewing local coverage determination (LCD) policy for hematopoietic disorders as these tests are medically necessary, thus not requiring PA.

Reduced Subclone Diversity in Clonal Cytopenia of Undetermined Significance Compared to Myelodysplastic Syndrome

Introduction: Clonal cytopenias of undetermined significance (CCUS) and myelodysplastic syndromes (MDS) represent a disease continuum that are distinguished by the presence of dysplasia in &amp;gt;10% of blood cells. However, morphologic dysplasia is subject to high inter-observer variability, suggesting that dysplasia may not be ideal to differentiate CCUS from MDS. Defining the clonal architecture of samples from cytopenic patients may provide a more accurate objective measure of disease status than dysplasia. We hypothesize that the number and size of subclones is reduced in patients with CCUS compared to MDS, suggesting that increased subclonal diversity is a hallmark of higher-risk disease. Methods: We performed whole genome sequencing with higher exome coverage (eWGS) on bone marrow (n=58) or peripheral blood (n=4) and paired normal DNA from baseline banked samples from patients with CCUS (n=13), MDS (n=29; IPSS-R very low/low [3], intermediate [5], high/very high [19], no score [2]), and secondary AML (sAML) (n=20), including 32 previously reported patients, to define clonal architecture. Putative somatic variants, including 76 genes that are recurrently mutated in myeloid neoplasms, were validated in hematopoietic and paired normal samples, and available serial samples (n=23), using orthogonal target enrichment sequencing platforms achieving higher coverage (~600-1000x). Clonality of validated somatic mutations was defined using SciClone. Results: The absolute neutrophil count, hemoglobin, and platelet counts trended lower in MDS vs CCUS patients (median, 1.3 vs 2 K/ul [ns]; 9.3 vs 11.35 g/dL [p&amp;lt;0.01]; 63 vs 109.5 K/uL [ns], respectively). The ages and median number of total validated somatic mutations identified by eWGS at baseline was similar for CCUS (n=428), MDS (n=300), and sAML patients (n=420.5). We first defined the founding clone (i.e., the dominant clone with the highest median variant allele frequency [VAF]) and subclones. While the number of total clones per sample was not different between CCUS, MDS, and sAML (median, n=3 for each), the median VAF of all the mutations present in the founding clone (i.e., measure of the maximal molecular disease burden) was significantly lower in CCUS (21.3%) compared to MDS (39.2%, p&amp;lt;0.05) and sAML samples (45.2%, p&amp;lt;0.001), but MDS and sAML were not different (p=0.2) (Figure 1A). In contrast, there was no significant difference in the median VAF of subclones in CCUS and MDS samples (13.4% vs 16.8%), which were both lower than sAML (25.3%, p&amp;lt;= 0.01). However, the proportion of patients with no subclones (i.e., only the founding clone detected) was higher for CCUS (5/13 [38.5%]) compared to MDS and sAML (4/29 [13.8%] and 0/20 [0%], respectively, p 0.0006), suggesting that CCUS samples have reduced subclonal diversity compared to MDS. Consistent with this, genes commonly mutated in subclones occurred less frequently in CCUS vs MDS samples, including activated signaling (0/13 vs 6/29) and transcription factor genes (3/13 vs 9/29, respectively). Nine CCUS patients had serial samples sequenced (median 2 serial samples, range 1-5) at a median follow-up of 539 days (range, 84-4066 days). In CCUS patients with serial samples, there was minimal change in the founding clone (0.16%, range -3.7-23.9%) or the subclone median VAFs (0.2%, range -22.4-22.2%), with only 2 patients having a founding clone or subclone VAF increase of &amp;gt;5%. UPN 529198 had the acquisition of a new SRSF2(P95H)-mutant subclone 4066 days after initial banking but remained with stable cytopenias and no MDS (Figure 1B). One CCUS patient developed MDS 70 days after banking with a blast count that increased from 2% to 15% but had a negligible change in the founding clone VAF (2.8% increase). However, 3 subclones did increase in size (max VAF increase of 6.1%). In summary, while the total number of mutations is similar between CCUS, MDS and sAML samples, the clonal architecture varies across diseases. CCUS has a lower total molecular disease burden and a lower proportion of patients with a subclone compared to MDS patients in our cohort. The data suggest that defining clonal architecture and incorporating subclonal complexity in the evaluation of cytopenic patients could provide an objective measure to characterize and monitor disease burden rather than relying on the presence or absence of dysplasia, especially when considering the diagnosis of CCUS and lower-risk MDS.

1-Year Data Analysis from a Cancer Center Molecular Tumor Board Dedicated to Clonal Hematopoiesis

Background Nearly 25% of cancer patients harbor clonal hematopoiesis of indeterminate potential (CHIP). Detection of clonal hematopoiesis (CH) in routine clinical practice is increasing due to incidental findings during sequencing analysis of circulating cell-free DNA (cfDNA) or exploration of unexplained cytopenia. To improve clinical management of these patients in our institution, we set up a multidisciplinary molecular tumor board (MTB) dedicated to patient cases with CH, CHIP, and clonal cytopenia of undetermined significance (CCUS) in 2022. Methods Between June 2022 and June 2023, our MTB reviewed a total of 345 cases, of which 212 referred by the solid tumor MTB and 133 referred by hematologists. The former 212 cases with CH were identified through cfDNA sequencing (FoundationOne Liquid CDx) within the Gustave Roussy Cancer Profiling study (NCT04932525), which includes some genes recurrently mutated in hematologic malignancies ( ASXL1, DNMT3A, IDH1, IDH2, JAK2, MPL, MYD88, SF3B1, TET2, U2AF1). A variant allele frequency (VAF) of at least 10% for the CH mutation was required to refer cases to the MTB, except for JAK2, MPL, and MYD88 mutations. The latter 133 cases were screened for CH mutations on peripheral blood leukocytes using a next-generation sequencing (NGS) assay targeting 174 genes recurrently mutated in hematologic malignancies. In addition to molecular data, standard clinical and biological parameters were recorded for each individual case. Results Patients referred by the solid tumor MTB In this cohort of 212 patients, median age was 72 years old (range, 48-91), sex ratio (M/F) was 1.4, median prior anticancer treatment lines was 2 (0-9), and time between MTB and cancer diagnosis was 2.3 years (0.2-34). The most frequent tumor types were lung (23%), colorectal (20%), prostate (19%), gynecological (12%) and breast (9%) cancers. The most frequently mutated genes were DNMT3A (59%), TET2 (30%), ASXL1 (24.5%), JAK2 (23%), SF3B1 (7.5%), and MYD88 (6%), with a median VAF of 4.2%, 3.0%, 8.5%, 1.0%, 5.0%, and 0.20%, respectively. Fifty percent of patients harbored more than 1 mutation. Six patients had a known history of myeloproliferative neoplasm (MPN). The proportion of cases with CH, CHIP, and CCUS was 15%, 59%, and 17%, respectively. The CH risk score of myeloid malignancy (CHRS, Weeks et al.) could be calculated in 126 cases but may underestimate risk due to the limited gene list considered, and revealed 69 (55%) low-risk, 52 (41%) intermediate-risk, and 5 (4%) high-risk patients. Our MTB recommended hematologic consultation for additional investigations in 58 patients, of which 22 were actually referred (main reason for not referring patients was advanced cancer disease). Four patients were diagnosed with hematologic malignancy (1 MPN, 1 chronic myeloid leukemia, 2 myelodysplastic syndromes [MDS]). Patients referred by a hematologist In this cohort of 133 patients, median age was 69 years old (53-89), and sex ratio (M/F) was 0.6. Eighty-six percent of patients had a cancer history, median prior anticancer treatment lines was 2 (0-7), time between MTB and cancer diagnosis was 3.5 years (0.2-34). The most frequent tumor types were lymphoid disorders (39%), breast cancer (15%), and gynecological cancer (15%). The proportion of CH, CHIP, and CCUS was 11%, 33%, and 41%, respectively. The most frequently mutated genes were DNMT3A (48,5%), PPM1D (24%), TET2 (22%), TP53 (17%), ASXL1 (6%), JAK2 (4.5%) and CHEK2 (4.5%), with a VAF of 2.5%, 2,3%, 4.0%, 2.0%, 8.0%, 1.0%, and 2.5%, respectively. Sixty percent of patients harbored more than 1 mutation. The CHRS could be calculated in 98 cases and revealed 34% low-risk, 50% intermediate-risk, and 16% high-risk CH patients. No need for a specific follow-up was recommended by the MTB in 33 patients. Among the 85 patients with hematologic follow-up, 3 (for which the CHRS was intermediate in 2 cases and high in 1 at the time of MTB evaluation) rapidly progressed to a myeloid malignancy ( Figure 1) (2 MDS and 1 MDS/MPN). Conclusions Our study highlights the utility of a MTB dedicated to CH, CHIP and CCUS for the identification of high-risk patients, early detection of hematologic malignancies, and appropriate therapeutic management. Long-term follow-up is needed to validate the CHRS but the design of a specific score for patients with cancer may be helpful due to the specific features of this population (high prevalence of PPM1D mutations, post-treatment cytopenia).

Comparison of the 2022 ICC and Who Classification Systems of Myelodysplastic Neoplasms

Introduction: Recently, the World Health Organization (WHO) updated the 5th edition classification of myelodysplastic neoplasms (MDS) and the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemia was published. In this study, we aimed to compare several differences between WHO 2022 and ICC classifications. Methods: Our study consisted of 989 newly-diagnosed primary MDS subjects classified using the WHO 2016 criteria from 08/2016 to 07/2022. CD34 and CD117 immunostaining were used in 30 of 38 subjects with low blasts and BM fibrosis to evaluate a proportion of immature myeloid precursors. All subjects were negative for CD117+ cells. CD34+ BM cells ranged from 0% to 5%. Results: According to WHO 2022 classification, 36 MDS subjects with NPM1 mutation were reclassified as AML and 10 MDS-U subjects were reclassified as clonal cytopenia of undetermined significance (CCUS). Applying to the ICC criteria, 20 MDS subjects with NPM1 mutation were reclassified as AML and 8 MDS-U subjects were reclassified as CCUS. We analyzed the remaining patients. Among the 81 subjects classified as MDS-biTP53 according to WHO 2022, 10 subjects did not meet ICC criteria for MDS or MDS/AML with mutated TP53 because the VAF of TP53 mutation(s) was less than 10%. Subjects not qualifying for MDS or MDS/AML with mutated TP53 had lower Revised International Prognostic Scoring System (IPSS-R) scores, better IPSS-R-defined cytogenetics, a higher frequency of SF3B1 mutations and longer survival compared with subjects with MDS or MDS/AML with mutated TP53 according to ICC (All P-values＜ 0.005, Figure 1 Left). Our data suggested that TP53 mutational burden should also be considered in the diagnostic classification and the cut-off value of 10% seems reasonable. An additional difference between these two classifications is that ≥15% ring sideroblasts may be a surrogate for SF3B1 mutation in WHO 2022. But only those with SF3B1 mutation (VAF≥10%), without 5q-, -7 or complex karyotype and RUNX1 mutation can be diagnosed as MDS with mutated SF3B1(MDS- SF3B1) according to ICC. 23 of 76 subjects (30.3%) with MDS- SF3B1 according to the WHO 2022 classification did not meet the ICC criteria for MDS- SF3B1. Subjects not meeting the ICC for MDS- SF3B1 had lower WBCs ( P = 0.003) ,platelet concentrations ( P = 0.001), higher IPSS-M categories ( P = 0.024) and shorter survival compared with those with MDS- SF3B1 according to ICC( P = 0.003, Figure 1 middle). Therefore, our study shown that SF3B1-unmutated MDS-RS cases have different clinical features and outcome in comparison with SF3B1-mutated subjects and ICC criteria defined a more homogeneous disease entity of MDS- SF3B1. Apart from MDS with defining genetic abnormalities, substantial differences in morphologically defined MDS subtypes between WHO 2022 and ICC also need to be addressed. According to the WHO 2022 classification, only MDS-EB patients were furtherly categorized into MDS-EB with or without BM fibrosis. Here, we explore the impact of BM fibrosis in patients with MDS-LB. Among MDS patients without excess blasts and specific genetic abnormalities, 38 patients had fibrosis. Subjects with low blasts and fibrosis (MDS-LBf) had lower platelet concentrations ( P = 0.002), worse IPSS-R-defined cytogenetics and IPSS-R categories ( P ＜0.05) compared with those without excess blasts and myelofibrosis. MDS-LBf had worse survival compared with those with low blasts and without fibrosis (Figure 1 right, P = 0.007). Conclusion: Both the WHO 2022 and ICC criteria allow more accurate diagnoses and risk stratification. However, the presence of two separate classifications leads to the dilemma in clinical application. When it comes to MDS with defining genetic abnormalities, our analyses suggest ICC defines more homogeneous disease entities of MDS-SF3B1, MDS or MDS/AML with mutated TP53 subjects compared with WHO 2022. Although WHO 2022 places an emphasis on morphologically defined MDS and listed hypoplastic MDS (MDS-h) and MDS with fibrosis (MDS-f) as new entities. Our data shown that fibrosis is a poor prognostic factor in MDS subjects without excess blasts. These subjects tend to have more aggressive clinical manifestations than other MDS subjects with low blasts. Collectively, we propose to integrate these two classifications into one pipeline for diagnosis of MDS (Figure 2), avoiding disadvantages for each one. Bing Liand Zhijian Xiao were co-corresponding authors

Mesenchymal Stromal Cells from Individuals with DNMT3A mut Clonal Hematopoiesis Instruct Young Healthy Hematopoietic Stem/Progenitor Cells Towards a Myeloid-Biased Aging Phenotype Resembling That of Patients with Clonal Cytopenia of Undetermined Significance (CCUS)

Clonal hematopoiesis of indeterminate potential (CHIP) describes the age-related acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPC) leading to clonal blood cell expansion and variable risk of progression to myeloid malignancy. DNMT3A is the most commonly mutated gene and while single mutations do not raise the likelihood of progression, high variant allele frequencies (VAF) and presence of co-mutations do. In myeloid diseases, the bone marrow (BM) microenvironment exhibits alterations promoting malignant transformation. However, it is unknown whether similar alterations exist in the pre-malignant niche. We set out to analyze whether human mesenchymal stromal cells (MSC) from CHIP individuals with a DNMT3A mutation ( DNMT3A mut) are altered and whether they are able to remodel the HSPC compartment in healthy hematopoiesis. From our published cohort of previously sequenced BM samples from hematologically healthy individuals undergoing hip replacement surgery, we selected samples with either an isolated DNMT3A mut at a low VAF (2.47-6.8%, n=4, median age 73.5y, termed low-risk CHIP), or DNMT3A mut samples with a high VAF (17.5-28.5%) and at least one co-mutation (n=5, median age 73y, termed high-risk CHIP) as well as 12 age-matched samples without any mutation (n=12, median age 72y). HSPC were isolated from 3 younger healthy donors (median age 50y). MSC were expanded by plastic adherence and subjected to multilevel characterization. Compared to age-matched healthy controls, MSC from DNMT3A mut BM samples (CHIP-MSC) exhibited higher proliferation rates, lower colony-forming-unit (CFU) fibroblast capacity, as well as reduced adipogenic and increased osteogenic differentiation potential. To understand how altered CHIP-MSC influence the hematopoietic compartment, younger healthy CD34+ HSPC were co-cultured with the different MSC subgroups for 42 hours in a low cytokine-containing medium, followed by bulk RNAseq on subsequently sorted cell populations (MSC: CD45-/CD90+; HSPC: CD45+/CD34+). We found 31 differentially expressed genes (DEG) (logFC±0.5, FDR&amp;lt;0.05) in CHIP-MSC compared to their age-matched counterparts. Functional analyses suggested an activation of communication towards HSPC via extracellular vesicles. Strikingly, differential gene expression of sorted young healthy HSPC after co-culture revealed 375 DEG (322 up-regulated, 53 down-regulated; logFC±0.5, FDR&amp;lt;0.05) in HSPC cultured with CHIP compared to non-CHIP age-matched MSC (Figure panel A). We then used gene set variation analysis (GSVA) to analyze HSPC compartment composition after co-culture. Based on enrichment scores, unsupervised clustering identified two distinctive profiles that correlated with HSPC cultured with either non-CHIP or high-risk CHIP-MSC, while those cultured with low-risk CHIP-MSC showed an intermediate phenotype (Figure panel B). Co-culture of HSPC with high-risk CHIP-MSC resulted in enrichment of hematopoietic stem cells (HSC) and multipotent progenitors (MPP), as well as myeloid skewing represented by higher scores for common-myeloid (CMP) and granulocyte-monocyte (GMP) progenitors. Conversely, in contrast to the control group, megakaryocyte-erythroid progenitors (MEP) were underrepresented. CFU capacity was increased in healthy HSPC cultured with CHIP-MSC and multiparameter flow cytometry analysis showed a trend towards increased CMP/GMP and decreased MEP numbers, consistent with the RNAseq data. Finally, we evaluated the BM HSPC compartment composition of a cohort of 49 CCUS patients with DNMT3A mut (VAF 1-49%, median age 76y) compared to 56 control BM samples using whole transcriptome sequencing data. Intriguingly, we observed the same profile with a myeloid shift in progenitor populations as in our healthy young HSPC exposed to high-risk CHIP-MSC. Taken together, our results indicate that MSC from individuals harboring a DNMT3Amutation are able to instruct young healthy HSPC and influence their fate, thereby promoting a differentiation bias that shifts the hematopoietic compartment toward that associated with an aged pre-malignant state, even in the absence of DNMT3A mut HSPC. These findings suggest that focusing on the interaction between MSC and HSPC is a promising therapeutic strategy to intercept disease progression. Functional analysis of identified deregulated pathways in DNMT3Amut CHIP is ongoing.

Clonal Hematopoiesis Is Associated with Severe Cytokine Release Syndrome in Patients Treated with Chimeric Antigen Receptor T-Cell (CAR-T) Therapy

Background: Among patients receiving CD19 or B-cell maturation antigen (BCMA) CAR-T therapy, inflammation pre- and post-CAR-T infusion is attributed in the development of toxicities such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and prolonged cytopenias. Clonal hematopoiesis (CH), the clonal expansion of hematopoietic stem cells with somatic mutations, has been associated with inflammatory disorders such as rheumatoid arthritis and graft-versus-host disease, as well as clonal cytopenias of undetermined significance. In the setting of CAR-T, others have reported an association between CH and both ICANS and treatment-associated myeloid neoplasms. Herein, we examined the prevalence of pre-CAR-T CH in a predominantly transplant-naïve cohort of recipients with non-Hodgkin lymphoma (NHL) or multiple myeloma (MM), and assessed the relationship between CH and CAR-T related outcomes including CRS, ICANS, prolonged cytopenia, progression-free survival (PFS), and overall survival (OS). Methods: This study included 62 patients with NHL or MM who underwent CD19 or BCMA CAR-T therapy from 2017 to 2022 at City of Hope and had available pre-CAR-T cryopreserved peripheral blood mononuclear cells (PBMCs). DNA was isolated with QIAamp DNA Mini Kit (Qiagen) from PBMC samples (94% collected &amp;lt;30d of CAR-T infusion), on which we performed targeted exome sequencing (108 pre-defined gene panel with 1000x sequencing depth) to determine the presence of CH (variant allele frequency [VAF] ≥2%). Multivariable logistic regression was used to examine the association between CH (Yes/No) and absolute neutrophil count (ANC) recovery at day +60 (Y/N), maximum grade CRS and ICANS, grade &amp;lt;2 vs 2+, and OS and PFS at 1y. Covariables considered were age at CAR-T, baseline ANC, sex, race, CAR-HEMATOTOX (Rejeski et al. 2021), bridging therapy (Y/N), and number of prior lines of therapy. ANC recovery was defined as grade 2 or better for 3 measurements without growth factor support &amp;gt;2 weeks. Those without ANC recovery and who started new treatment before day +60 were excluded from that analysis. Results: Overall, the median age was 64y (range 21y-85y); 37 (60%) had de novo diffuse large B-cell lymphoma, 17 (27%) had transformed follicular lymphoma, 2 (3%) had MM, 2 (3%) had mantle cell lymphoma, and the remaining patients had other transformed indolent lymphomas. Median prior lines of therapy was 3 (1-12), and only 6% had previously undergone autologous hematopoietic cell transplantation. Systemic bridging therapy prior to CAR-T was required in 27 (44%), and 8 (13%) received radiation as bridging. All received fludarabine and cyclophosphamide for lymphodepleting chemotherapy. Axicabtagene ciloleucel was the most common (76%) CAR-T product, followed by tisagenlecleucel in 16% of patients. Overall, 51 (82%) and 29 (47%) patients experienced CRS and ICANS, respectively. had delayed ANC recovery. One-year PFS was 50% and 1y OS was 73%. Clonal hematopoiesis: 15 (24%) patients had at least one pathogenic CH mutation; 4 (6.5%) had ≥2 CH mutations. Of the 28 CH mutations identified, the most common were DNMT3A (18%), CHEK2 (11%), PMS2 (7%) and ASXL1 (7%); 29% of mutations had VAFs &amp;gt;10%. Outcomes after CAR-T: Patients with CH were significantly more likely to develop grade ≥2 CRS (60% vs. 28%, p=0.03) compared to those without CH (odds ratio [OR] 3.9, 95% CI 1.2-13.2; p=0.027). Accounting for baseline ANC (which was unexpectedly higher among the CH cohort and associated with delayed ANC recovery, p=0.02) patients with CH did not have a significantly different rate of delayed ANC recovery compared to those without CH (adjusted OR 0.37, 95% CI 0.09-1.5; p=0.17). There was no association between CH and ICANS, nor with 1y PFS and OS. Conclusion: CH was frequent (24%) in this cohort of CAR-T recipients and was associated with a higher risk of ≥2 CRS after CAR-T. Additional validation studies are currently underway, which may set the stage for consideration of pre-CAR-T CH as a biomarker for risk stratification towards more proactive CRS prophylaxis Acknowledgements: Funding through the CoH Hematological Malignancies Program Pilot Project Award. The authors acknowledge the work provided by CoH Center for Informatics, notably Research Informatics, and the utilization of the POSEIDON platform and the Honest Broker process.