Premalignant State Research Articles

Oncogenic HRAS Induces Metformin Resistance in Head and Neck Cancer by Promoting Glycolytic Metabolism.

Metformin administration has recently emerged as a candidate strategy for the prevention of head and neck squamous cell carcinoma (HNSCC). However, the intricate relationship between genetic alterations in HNSCC and metformin sensitivity is still poorly understood, which prevents the stratification of patients, harboring oral premalignant lesions that may benefit from the chemopreventive activity of metformin. In this study, we investigate the impact of prevalent mutations in HNSCC on response to metformin. Notably, we found that the expression of oncogenic HRAS mutants confers resistance to metformin in isogenic HNSCC cell systems, and that HNSCC cells harboring endogenous HRAS mutations display limited sensitivity to metformin. Remarkably, we found that metformin fails to reduce activation of the mTOR pathway in HRAS oncogene-expressing HNSCC cells in vitro and in vivo, correlating with reduced tumor suppressive activity. Mechanistically, we found that this process depends on the ability of HRAS to enhance glycolytic metabolism, thereby suppressing the requirement for oxidative phosphorylation to maintain the cellular energetic balance. Overall, our study revealed that HNSCC cells with oncogenic HRAS mutations exhibit diminished metformin sensitivity, thus shedding light on a potential mechanism of treatment resistance. This finding may also help explain the limited clinical responses to metformin in cancers with RAS mutations. Ultimately, our study underscores the importance of understanding the impact of the genetic landscape in tailoring precision cancer-preventive approaches in the context of HNSCC and other cancers that are characterized by the presence of a defined premalignant state, and therefore, are amenable to cancer interception strategies. Prevention Relevance: Our findings highlight the challenges of using metformin for cancer prevention in RAS-mutant cancers, where elevated glycolysis may reduce drug efficacy. This underscores the need to explore metformin's potential in early, premalignant stages, before metabolic shifts render it less effective.

Machine learning and traditional analysis of the interaction between cardiovascular diseases and hematological malignancies

Abstract Background Cardiovascular diseases and clonal hematopoiesis of indetermine potential (CHIP), the premalignant state of hematological cancers, exhibit reciprocal interactions. While the presence of CHIP confer a risk for heart failure, it is unclear if cardiovascular diseases are associated with elevated risk hematological cancers. Purpose To determine the association between cardiovascular diseases (CVD) and cardiovascular risk, and hematological cancer. Methods We evaluated 27,231 adults, free of CVD and cancer, who participated in preventive healthcare program between 2000-2018. Each participant underwent a complete physical evaluation, blood test and exercise stress test at baseline. CVD was defines as ischemic heart disease, stroke, atrial fibrillation or hypertension. Then, we used machine learning and traditional survival analysis to evaluate the association between CVD and hematological cancer. For machine learning, we used the random forest method using Curat package in r, and incorporated all continuous variables gathered into the model (70% training, 30% test). For survival analysis, we first matches each CVD patient to a control patient by propensity scores to balance for age, sex, smoking, diabeles mellitus, renal function, body mass index and length of follow-up. Then, we compared the CVD and non-CVD patients using cox proportional hazard regression. Results Using machine learning, we found that the strongest predictors for hematological cancer in our cohort are low eGFR, age, high atherosclerosis cardiovascular disease score (ASCVD) and BMI. Of note, systolic and diastolic blood pressure were also strong predictors of hematological cancer (Figure A). Then, we matched 5500 CVD patients to controls with median follow-up of 9.2 years (Interquartile range 5-15 years). During follow-up, 1380 developed cancer. Of them, 186 developed hematological cancer. We used cox proportional hazard regression and Kaplan Mayer method to determine the risk and hazard ratio of developing hematological cancer (Figure B-C). Compared with subjected without CVD, patients with CVD were 91% more likely to develop cancer during follow-up, and 62% more likely to develop hematological cancer (Figure C, HR=1.61. 95%CI 1.21-2.16). Moreover, the 10-year risk to develop hematological cancer was 1.9% for patients with CVD and 1.0% for individuals without CVD (Figure D). Notably, the increased incidence of hematological cancers included 38% increase in cases of leukemia but only 11% increase in cases of lymphoma. Conclusions Using machine learning and traditional analysis we show that CVD risk and CVD were associated with increased incidence of hematological cancer. Understanding this relationship could lead to advances in cardiovascular and cancer treatment and could ultimately help to improve patient outcomes.

Early Detection of Malignant and Premalignant Peripheral Nerve Tumors Using Cell-Free DNA Fragmentomics.

Early detection of neurofibromatosis type 1 (NF1)-associated peripheral nerve sheath tumors (PNST) informs clinical decision-making, enabling early definitive treatment and potentially averting deadly outcomes. In this study, we describe a cell-free DNA (cfDNA) fragmentomic approach that distinguishes nonmalignant, premalignant, and malignant forms of PNST in the cancer predisposition syndrome, NF1. cfDNA was isolated from plasma samples of a novel cohort of 101 patients with NF1 and 21 healthy controls and underwent whole-genome sequencing. We investigated diagnosis-specific signatures of copy-number alterations with in silico size selection as well as fragment profiles. Fragmentomics were analyzed using complementary feature types: bin-wise fragment size ratios, end motifs, and fragment non-negative matrix factorization signatures. The novel cohort of patients with NF1 validated that our previous cfDNA copy-number alteration-based approach identifies malignant PNST (MPNST) but cannot distinguish between benign and premalignant states. Fragmentomic methods were able to differentiate premalignant states including atypical neurofibromas (AN). Fragmentomics also adjudicated AN cases suspicious for MPNST, correctly diagnosing samples noninvasively, which could have informed clinical management. Novel cfDNA fragmentomic signatures distinguish AN from benign plexiform neurofibromas and MPNST, enabling more precise clinical diagnosis and management. This study pioneers the early detection of malignant and premalignant PNST in NF1 and provides a blueprint for decentralizing noninvasive cancer surveillance in hereditary cancer predisposition syndromes.

Exploring serum trace element shifts: Implications for cervical intraepithelial neoplasia

BackgroundCervical intraepithelial neoplasia (CIN) represents a premalignant state presumably related to perturbations in circulating levels of trace elements. Materials and methodsEmploying inductively coupled plasma mass spectrometry (ICP-MS), we quantified essential and toxic trace elements in the sera of 60 women diagnosed with CIN and 60 age-matched healthy counterparts. ResultsOur investigation revealed a noteworthy higher levels in serum of Mn, Zn, and Pb, as well as lower levels in Ni, Se, Rb, and Mo levels within the CIN cohort. Levels of Mn, Zn, and Pb were higher by approximately 5.5-fold, 3.0-fold, and 7.5-fold, respectively, while Mo levels exhibited an approximate 4.5-fold reduction in CIN sera compared to the control group. While the study provided valuable insights into trace element variations, it’s important to note that the adult Serbian population is considered Zn-deficient, so the Zn data should be interpreted with caution. Age stratification (30–40 vs. 40–50 vs. 50–60 years), smoking status (smokers vs. nonsmokers), and CIN severity (CIN 2 vs. CIN 3) yielded no significant disparities in elemental profiles. Among the 10 proposed ratios, 5 demonstrated a significant surge in CIN sera relative to controls: Mn/Se, Mn/Mo, Zn/Se, Zn/Mo, and Se/Mo. Correlation analysis of trace element levels revealed a predominantly consistent pattern between CIN cases and healthy subjects, except for Zn and its negative correlations (antagonistic interactions) with other analyzed trace elements. ConclusionOur findings underscore differences in serum levels of specific trace elements in CIN cases versus controls, implicating their potential involvement in the underlying pathophysiological cascades culminating in cervical neoplasms.

CRISPR Dependency Screens in Primary Hematopoietic Stem Cells Identify KDM3B as a Genotype-specific Vulnerability in IDH2- and TET2-mutant Cells.

Clonal hematopoiesis (CH) is a common premalignant state in the blood and confers an increased risk of blood cancers and all-cause mortality. Identification of therapeutic targets in CH has been hindered by the lack of an ex vivo platform amenable for studying primary hematopoietic stem and progenitor cells (HSPCs). Here, we utilize an ex vivo co-culture system of HSPCs with bone marrow endothelial cells to perform CRISPR/Cas9 screens in mutant HSPCs. Our data reveal that loss of the histone demethylase family members Kdm3b and Jmjd1c specifically reduces the fitness of Idh2- and Tet2-mutant HSPCs. Kdm3b loss in mutant cells leads to decreased expression of critical cytokine receptors including Mpl, rendering mutant HSPCs preferentially susceptible to inhibition of downstream JAK2 signaling. Our study nominates an epigenetic regulator and an epigenetically regulated receptor signaling pathway as genotype-specific therapeutic targets and provides a scalable platform to identify genetic dependencies in mutant HSPCs. Significance: Given the broad prevalence, comorbidities, and risk of malignant transformation associated with CH, there is an unmet need to identify therapeutic targets. We develop an ex vivo platform to perform CRISPR/Cas9 screens in primary HSPCs. We identify KDM3B and downstream signaling components as genotype-specific dependencies in CH and myeloid malignancies. See related commentary by Khabusheva and Goodell, p. 1768.

"Myeloid" Mutations in ALL Are Not Uncommon: Implications for Etiology and Therapies.

In Blood Cancer Discovery, Saygin and colleagues report that somatic variants that are recurrent in myeloid malignancies can also occur with high frequency (16%) in adult acute lymphoblastic leukemia (ALL) where they correlate with older age, diagnosis following genotoxic therapy for a prior malignancy and worse outcome to chemotherapy. Mutations in these "myeloid" genes can precede ALL diagnosis and arise in hematopoietic stem or progenitor cells that clonally expand and differentiate into both lymphoblasts and nonmalignant myeloid cells, supporting a role for clonal hematopoiesis as premalignant state outside the context of myeloid malignancies and providing implications for both ALL etiology and therapeutic intervention. See related article by Saygin et al., p. 164 (4).

Abstract 5635: TPM4 overexpression promotes colon epithelial cell tumorigenesis in vitro and correlates with human colon tumor progression in vivo

Abstract Because colon cancer still exerts a heavy burden on human health, we are interested in identifying very early events in colon epithelial cell tumorigenesis in order to develop strategies to treat and possibly prevent colon carcinogenesis. We hypothesize that aberrant expression of genes involved in crypt-luminal colon epithelial cell migration, needed for proliferative, undifferentiated stem cells at the crypt bottom to mature into growth arrested, mature effector cells near the lumen, may disrupt normal differentiation and initiate events that can trigger the transition from a normal to premalignant state in colon epithelial cells. Forced upregulation of tropomyosin 4 (TPM4), an actin-binding protein that exerts many effects on contractility and migration, prevents certain aspects of Caco2 maturation induced by contact inhibition of growth, including increased expression of the digestive enzyme sucrose isomaltase and glutathione S-transferase alpha 1. Furthermore, TPM4.2-overexpressing cells exhibit numerous morphological abnormalities. Compared to untransfected Caco2 cells and empty-vector stable transfectant controls, TPM4.2-overexpressing Caco2 cell clones elaborate aberrant thin membrane protrusions and expanded membrane ruffles. Particularly striking are defective maintenance of dome structures at middle (day 14) and late (day 21) time points of induced differentiation in TPM4.2-overeexpressing clones. We further hypothesize that TPM4 is overexpressed in human colon tumors, particularly at early stages of tumorigenesis. In order to test this hypothesis, we examined TPM4 RNA expression in datasets obtained from human colon tissue at premalignant as well as tumorigenic stages. TPM4 trends toward overexpression in premalignant, relative to normal, human colon tissue (uninvolved mucosa from patients with familial adenomatous polyposis, or FAP) and is significantly overexpressed in adenocarcinoma tissue relative to normal controls. Interestingly, TPM4 overexpression may be more pronounced at earlier, rather than later, stages of adenocarcinoma. Our results suggest that aberrant TPM4 overexpression in colon epithelial cells can promote adoption of a tumorigenic state. Certain aspects of Caco2 cell maturation are disrupted by forced TPM4 overexpression during induced differentiation, and TPM4 overexpression correlates with early stages of colon tumorigenesis. Misregulation of TPM4 gene expression may therefore represent a critical molecular event that can block normal differentiation and induce early events in colon epithelial cell tumorigenesis. TPM4 expression level may therefore prove to be a useful biomarker that can, along with other diagnostics, help to determine tumorigenic progression in human colon tissue and to develop novel strategies to prevent colon cancer and treat it at early stages. Citation Format: Giulio Ferrero, Barbara Pardini, Alessio Naccarati, Michael Papetti. TPM4 overexpression promotes colon epithelial cell tumorigenesis in vitro and correlates with human colon tumor progression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5635.

Bone marrow adipocytes provide early sign for progression from MGUS to multiple myeloma.

Multiple Myeloma (MM) is the second most common hematological malignancy and is characterized by clonal expansion of malignant plasma cells in the bone marrow. In spite of recent advances in the field of MM, the disease has remained incurable. MM is preceded by a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS), with a risk of progression to MM of 1% per year. Establishing a scalable approach that refines the identification of MGUS patients at high risk of progression to MM can transform the clinical management of the disease, improve the patient's quality of life, and will have significant socioeconomic implications. Here, we provide evidence that changes in the bone marrow adipose tissue (BMAT) provide an early sign for progression from MGUS to MM. We employed AI-assisted histological analysis of unstained bone marrow biopsies from MGUS subjects with or without progression to MM within 10 years (n = 24, n = 17 respectively). Although the BMAT fraction was not different between the two groups, bone marrow adipocyte (BMAd) density was decreased in MGUS patients who developed MM, compared to non-progressing MGUS patients. Importantly, the distribution profile for BMAd size and roundness was significantly different between the two groups, indicating a shift toward increased BMAd size and roundness in MGUS patients who developed MM. These early changes in the BMAT could serve as valuable early indicators for the transition from MGUS to MM, potentially enabling timely interventions and personalized treatment strategies. Finally, the AI-based approach for histological characterization of unstained bone marrow biopsies is cost-effective and fast, rendering its clinical implementation feasible.

Can Vaccines Stop Cancer Before It Starts? Assessing the Promise of Prophylactic Immunization Against High-Risk Preneoplastic Lesions

Background: Cancer remains a leading cause of mortality with modest declines, highlighting the need for more efficacious prevention strategies like early immunological intervention against premalignant disease. Main body of abstract: Oncogenic viruses demonstrate prophylactic vaccines can successfully reduce malignancy by blocking precipitating infections. However, most cancers lack viral etiology, requiring novel approaches targeting sporadic precancerous states to enable early immunoprevention. Preneoplastic tissues exhibit biological changes making them appealing targets for stimulating immune surveillance before additional mutations cause unconstrained proliferation. High-risk precancers also provide sources of dysregulated self-antigens. Yet challenges exist in lesion identification, overcoming tolerance, and avoiding inflammation potentially worsening progression. Multidisciplinary insights into precancer immunology, predictive biomarkers, antigen discovery, and combinatorial vaccination strategies are illuminating rational vaccine design. Despite obstacles, prophylactic immunization against early dysplastic changes holds disruptive potential if key steps advance this approach. Elucidating preneoplasia immunobiology and progression risk modeling will be critical to guide productive immune targeting while mitigating immunotherapy hazards. Thoughtful translation could eventually shift paradigms by priming immunosurveillance against peak vulnerability lesions. Short Conclusion: Advancements in precancer vaccines may profoundly expand prevention horizons. Cautious immune targeting of premalignant states could intercept progression toward widely disseminated malignancies. This warrants methodical efforts to unravel the promise of thwarting lethal cancers before they start.

Exploring Aging Dependent Adaptations of Splicing Factor Mutations in Clonal Hematopoiesis

Introduction: It has been shown that leukemia-associated mutations can be detected in the blood of healthy individuals and their clonal expansion in hematopoietic stem and progenitor cell (HSPC) populations is referred to as Clonal Hematopoiesis (CH). In one study, a population analysis of a cohort of 17,182 patients performed by Bick et al. [PMID7944936] selected without prior screening for hematological malignancies, indicated that advancing age is directly associated with an increase in CH. When the mutant clone accounts for 4% of the nucleated blood cells, it is then referred to as Clonal Hematopoiesis of Indeterminant Potential (CHIP). CHIP has been classified by the World Health Organization as a premalignant state and risk criteria for neoplastic development. CHIP is highly associated with adverse outcomes including auto-immune or auto-inflammatory conditions, cardiovascular disease, cytopenia, cancers in general, and other hematological neoplasms. The associated hematopoietic neoplasms include myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD), and acute myeloid leukemia (AML). Population studies reveal splicing factor mutations to be among the most common mutations in CHIP and are highly correlated with age and show a high hazard ratio for risk of developing secondary neoplasms including AML. RNA splicing factors U2AF1, SRSF2, and SF3B1 make up core components of the spliceosome and are critical to prevent aberrant splicing. Mutations in these factors have widespread effects implicating a multitude of cellular processes. There are several established mutant splicing factor mouse models that can be utilized; however, many employ non-specific or leaky Cre drivers. Methods: To avoid non-specific and leaky Cre activation during aging, we used a Cre/lox-mediated U2af1(Q157R) conditional knock-in mouse model that does not contain an internal Cre driver. This mouse has a floxed wild-type U2af1 cDNA insert that expresses wild-type U2AF1 under normal circumstances. However, upon the exogenous addition of recombinant Cre the U2af1 cDNA is excised and the U2af1(Q157R) splicing factor mutation in exon 6 is expressed instead. This mouse model also possesses lox-stop-lox-YFP, leading to expression of YFP along with mutant U2AF1(Q157R) when Cre is introduced. We then isolated bone marrow from 6 week (young) and 6 months (aged) floxed Q157R/YFP mice and treated lineage-depleted bone marrow with recombinant Cre protein for two hours, followed by transplantation into mildly conditioned 6 week (young) and 18 month (old) wild-type mice. The percentage of YFP in the peripheral blood is quantified every 3 weeks. Full analyses, including RNA-seq, of the bone marrow progenitor populations after 3 months is in progress. Results and Discussion: Using the previously mentioned bone marrow transplant model our work shows that when a U2AF1 mutation is introduced into young and old recipient mice, there is significant preferential outgrowth when the donor mice are aged (Figure 1). This is indicated by an increase in the ratio of YFP, which represents the U2AF1(Q157R) mutant, to CD45.1 events, which represent an internal engraftment control. This would suggest that the expansion of U2AF1(Q157R) splicing mutant cells seen in clinical data may be the result of intrinsic factors from the aging cell population. Comprehensive analysis of the effects of alternative splicing in this model is ongoing, however, these data provide support for further investigation of the intrinsic effects of splicing factor mutations in an aged context.

Identification, Monitoring and Reversibility of PARP Inhibitor Associated Clonal Haematopoiesis and Myelodysplastic Syndrome: A Case Series

Background: Poly ADP ribose polymerase inhibitors (PARPi) are widely used as maintenance treatment for ovarian cancer (OC) after response to platinum chemotherapy in the first line setting and recurrent disease. Their use has significantly improved outcomes, particularly in patients with BRCA mutations in whom first-line PARPi maintenance therapy increases progression-free survival from 13.8 to 56.0 months (Hazard Ratio, 0.33; 95% CI, 0.25-0.43). However, a recent meta-analysis of clinical trial adverse events and pharmacovigilance data showed that PARPi therapy significantly increases the risk of therapy related myeloid neoplasms (tMN), myelodysplastic syndrome (MDS) and acute myeloid leukaemia with poor outcomes. Platinum based chemotherapy is the mainstay of OC treatment and is associated with clonal haematopoiesis (CH) with mutations in DNA damage response genes. CH is recognised as a premalignant myeloid state but real world data on clonal dynamics during PARPi therapy are lacking. We present a case series of OC patients receiving PARPi-therapy referred for haematological review for beyond-expected HT or unexplained full blood count (FBC) abnormalities with serial FBC and myeloid next generation sequencing (mNGS) clonal monitoring in relation to PARPi therapy. Methods: Seven patients with stage III/IV high-grade OC were referred to haematology at the discretion of the treating oncologist between February 2017 and May 2022 (mean age (SD): 68 (7.0) years, median PARPi duration: 18 months (range: 4-86)). All patients had received prior platinum-based chemotherapy and were screened with mNGS in peripheral blood (PB) or bone marrow aspirate and trephine (BMAT) at initial assessment. Results: Reasons for referral are detailed in Table 1 and include two patients with isolated elevated nucleated red blood cell (NRBC) counts. Six patients had clonal pathology (CP), five of whom had single-nucleotide variants typical of platinum-exposure defined by the involved gene or platinum-associated mutational signatures. Clinical details including BRCA-status, haematological diagnosis and mNGS results are detailed in Table 1. PARPi-therapy was discontinued in four patients with CP, with resolution or improvement of FBC and m-NGS abnormalities (variant allele frequencies (VAFs) and/or copy number variations (CNVs)) in three patients. These findings were most marked in Patient 1, referred with grade 3 thrombocytopenia and transfusion dependence and in whom PARPi cessation resulted in transfusion independence and resolution of morphological and cytogenetic abnormalities (clinical course including BMAT results and haematological parameters illustrated in Figure 1), and Patient 3, who demonstrated selective regression of a TP53 mutated clone on PARPi cessation. Two patients with germline BRCA mutations continued PARPi therapy with 3 monthly PB mNGS monitoring after discussion at multidisciplinary team meetings. Conclusions: We show using sequential molecular and haematological monitoring before and after PARPi cessation that morphological, FBC and clonal abnormalities can be reversed on PARPi discontinuation, potentially mitigating the risk of life-threatening tMNs in some patients. We also highlight that, in addition to cytopenias, persistently elevated NRBCs may be an indicator of clinically relevant CP not well described in pharmacovigilance data based on common terminology criteria for adverse events criteria. Large scale prospective data on clonal dynamics in PARPi therapy are needed to inform mNGS screening and tMN risk stratification as scoring systems based on age associated clonal haematopoiesis of indeterminate potential may not be applicable. Until these data are available we demonstrate the value in MDT discussion and serial PB mNGS monitoring in formulating case-by-case therapeutic decisions regarding CP and PARPi therapy, particularly patients with high risk BRCA mutated OC.

High Throughput Risk-Adapted Monoclonal Gammopathy Community Monitoring Program

Introduction Monoclonal gammopathy (MG) is present in 4.5% of adults older than 40 years. Given an annual 1% rate of progression to multiple myeloma (MM), regular MG monitoring has the potential to improve MM outcomes through earlier diagnosis. To this end, a variety of surveillance systems have been implemented in the NHS, including hospital, primary care, and nurse-led surveillance. However, as the vast majority of new MG patients are low-risk and unlikely to progress, most services are inefficient and unsustainable to support population-based monitoring. There is a need for a robust high-throughput system that can monitor most patients in the community, whilst streamlining high-risk MG for review in secondary care. Here, we describe a risk-adapted MG monitoring program (OxCOM), in which patients with incidental MG in Thames Valley were prospectively stratified and monitored. Methods Patients with incidental MG were captured over a 24-month period (between 4 th March 2021 and 3 rd March 2023) in an Immunology Laboratory covering a population of 2.5 million people. An attending immunologist risk-stratified patients daily into high-risk [IgG >30g/L or IgA/IgM >10g/L; or free light chain (FLC) ratio outside 0.1-7.0; or CRAB criteria], intermediate-risk [IgG 15-30g/L or IgA/IgM 5-10g/L; or FLC ratio outside 0.3-3.0; or monoclonal IgD/E or FLC; or urinary band] or low-risk [IgG <15g/L or IgA/IgM <5g/L] MG. Patients were excluded from analysis if out-of-area, aged 90 years or older with low-risk MG, under regular haematology review or had malignancy diagnosed as an inpatient following MG detection. The laboratory advised the requesting clinician to refer low-risk patients for 4-12-monthly remote blood monitoring by primary care, supervised by an OxCOM administrator. Intermediate and high-risk patients were advised for routine or urgent clinical assessment under haematology, respectively, with blood monitoring in secondary care. Intent for monitoring was assessed by patients receiving subsequent surveillance within 4 months (IgA or IgM) or 12 months (IgG), with a 2-month grace period. Follow-up rates were compared to a reference cohort of new MG detected in 2019 (prior to setup of OxCOM, in which monitoring was managed in the community) that were risk stratified retrospectively. Results 1,290 patients had MG detected over a 24-month period [962 low-risk (75%), 200 intermediate-risk (16%) and 128 high-risk (9%)] (Figure 1), of which 50% were female and median age was 75.4 years. Testing was requested equally between primary and secondary care (50% each); the most common indications were anemia (13%), bone pain (13%) and fracture (13%), all of which were over-represented in intermediate and high-risk MG [ p=0.0002]. Of 25% patients referred to secondary care [5% low-risk, 77% intermediate-risk and 91% high-risk], resources were concentrated towards higher-risk patients. Patients with high-risk MG were reviewed more quickly than those with intermediate-risk MG (median 14 vs. 54 days since test) [ p<0.0001], more likely to be assessed F2F (98% vs. 55%) [ p<0.0001] and receive investigations including CT/MRI imaging (86% vs. 44%) and bone marrow biopsy (59% vs. 17%) [ p<0.0001]. OxCOM risk stratification criteria distinguished risk of haematological malignancy diagnosis between low-risk (1%, n=10), intermediate-risk (14%, n=18) and high-risk (57%, n=57) MG at last known follow-up [ p<0.0001] (Figure 2). OxCOM enabled risk reclassification based on clinical judgement; of 253 patients initially assessed by secondary care, 65 (26%) had been transferred to remote primary care monitoring after a median of 3 clinical reviews and 7.3 months follow-up. There was a higher fidelity of subsequent monitoring at an appropriate interval under OxCOM (89%) than in the pre-OxCOM system (47%), including in those with low-risk (90% vs. 35%) and high-risk (94% vs. 76%) MG [ p<0.0001]. Conclusions We demonstrate the feasibility of a risk-adapted high-throughput clinical monitoring service for incidental MG. Immunology Laboratory led central oversight by stratifying patients for either remote blood surveillance under primary care, or for clinical assessment in secondary care. OxCOM therefore establishes a model to enable tailored monitoring of a premalignant state, which distributes healthcare resource towards higher-risk patients, whilst enabling sustainable monitoring for those with lower-risk.

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<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<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.

Transcriptomic Analysis of Clonal Hematopoiesis in Solid Tumors Reveals Driver Mutation-Specific Patterns of Immune Dysregulation

Introduction: Clonal hematopoiesis (CH) is a pre-malignant state wherein somatic mutations confer a proliferative advantage to hematopoietic stem cells, driving inflammatory changes that increase the risk of age-related diseases and mortality. CH is common in individuals with solid cancers and associated with poorer overall survival due to increased tumor progression, raising the possibility that CH alters the tumor-immune microenvironment (TIME). This study defines the inflammatory program of tumor-infiltrating leukocytes to help elucidate the role of CH in the TIME. Methods: Somatic mutations compatible with CH were detected in the whole exome sequences of 6,537 cancer patients with 16 major cancer types from The Cancer Genome Atlas (TCGA) project using Mutect2. CH variant curation followed a pre-defined list of putative variants in 63 genes known to drive hematologic clonality. Calls were filtered to remove sequencing artifacts, germline variants, and small clones with variant allele fraction (VAF) < 2%. Bulk RNA-seq analysis was conducted on data from the colon adenocarcinoma (n=240) and lung adenocarcinoma (n=402) groups. Sequencing reads were pseudo-aligned and quantified with kallisto, then differential expression analysis was performed with DESeq2, controlled for cancer type, tumor biopsy site, and patient sex. Differentially expressed genes were subject to gene set enrichment analysis (GSEA) of gene ontology (GO) terms using clusterProfiler. Results: The prevalence of CH in this cohort was 8.8% (n=576/6,537) and its presence was strongly associated with age (p=1.0x10 -25). The highest rate of CH was found in cutaneous melanoma (23.5%, n=110/468) and the lowest rate was in esophageal carcinoma (2.4%, n=3/126). Most patients with CH had just 1 variant (82.8%, n=477/576), but some patients exhibited 2 variants (10.1%, n=58/576), and a small group of patients carried 3 or more variants. A total of 772 CH variants were detected in the cohort, and as expected their distribution across the CH driver genes was not uniform: DNMT3A mutations were most common (n=193), followed by TET2 (n=59), and ASXL1 (n=47; Fig 1). KMT2C mutations were also present in this cohort (n=41) at far higher rates than reported in the general population, providing possible evidence that KMT2C-mutant CH is associated with cancer or its treatments. Across the group of colon and lung cancer patients subject to RNA-seq analysis, a total of 220 genes (111 upregulated, 109 downregulated) were found to be differentially expressed by CH status at p adj < 0.05. GSEA of GO terms revealed significant activation of gene sets involved in innate and adaptive immune processes with upregulation of both positive and negative regulatory systems, suggesting more intricate mechanisms of immune dysfunction as opposed to generalized hyper-inflammatory activity in the TIME (Fig. 2). GSEA also revealed enrichment of non-immune pathways tied to tumorigenesis. Among the significantly dysregulated genes are the neutrophil activators PPBP and CXCL6, monocyte/macrophage markers LILRA2, CPM, CHI3L1, and CD163L1, and the natural killer cell receptors KLRC2/3. Strikingly, these inflammatory changes were absent in patients with DNMT3A, TET2, or ASXL1 (DTA) CH, and were instead driven by non-DTA CH mutations in a VAF-dependent manner. Investigation of tumor transcriptomes within each cancer type revealed that CH in colon cancer patients has reciprocal effects on inflammation and immunity - DTA CH in this cancer demonstrated suppression of immune-associated gene sets while non-DTA CH was more consistent with the general trend of immune enrichment and activation. Conclusions: This work demonstrates that while general inflammatory changes are evident at the tumor level with CH, there is a critical need to recognize the nuances of CH-associated immune mechanisms in cancer, which appear to vary by cancer type and CH driver. Ongoing efforts will continue to characterize the TIME of the other TCGA cancers through transcriptomic analysis and in situ histological imaging. This study also provides some early clinical evidence for the selection of KMT2C variants in cancer patients, though more work is needed to understand their clonal dynamics and clinical implications. In light of these findings, future studies will pave the way for CH as a biomarker in cancer medicine and a cornerstone of precision immuno-oncology.

IPSS-M - Use for Predicting Survival and Progression in Patients with Ccus - a Retrospective Multi-Institutional Study

Background: Clonal cytopenia of undetermined significance (CCUS) is characterized by the presence of a clone with somatic mutations in genes associated with myeloid neoplasms (MN) or abnormal cytogenetics, along with unexplained and persistent cytopenias in the absence of significant marrow dysplasia. CCUS is considered a premalignant state with a risk of progression to MN. The clinical outcomes of patients (pts) with high risk CCUS (Weeks et al, NEJM Evidence 2023) may be similar that of some patients with myelodysplastic syndromes (MDS). This study aims to predict the outcomes of CCUS patients using the molecular international prognostic scoring system (IPSS-M) developed for MDS. Methods: A retrospective analysis was conducted in patients diagnosed with CCUS between 2015 and 2023, we included patients harboring pathogenic mutations (MUT) identified through next-generation sequencing (NGS) testing at two institutions: Mayo Clinic and the University of Texas Southwestern Medical Center, Dallas, TX, USA were included. Progression was considered present if patients with CCUS progressed into myeloid neoplasm at follow up. Statistical analysis was performed using JMP® 17.1.0 Software. IPSS-M risk scores were calculated via the IPSS-M Risk Calculator (mds-risk-model.com). Results: A total of 103 patients were included in the study. The median age was 72 years, the majority were male patients (73%), and the median bone marrow blast % at the time of NGS was 1%. Forty-five % had an isolated mutation in a single gene, and 55% had co-mutations in ≥1 gene. The median number of mutations was 2 (range 1 - 5), occurring across 43 different genes. The most common mutations were in TET2 (N=28; 27%), ASXL1 (N=21; 20%), SRSF2 (N=21; 20%), U2AF1 (N=18; 17%), DNMT3A (N=16; 16%), and TP53 (N=12; 12%). Additionally, 25% of patients had an abnormal karyotype. Hematologic and genetic characteristics are summarized in (Table 1A). The median IPSS-M score was -0.9 (-3 to +2.34) and the median IPSS- Revised (R) score was 2 (0 - 6). The majority of patients had low (L) IPSS-M risk category (53%), followed by moderate low (ML) (19%) and very low (VL) (17%). Per IPSS-R, the majority had low-risk category (47%), followed by VL (38%) and intermediate (14%). Overall, 26 (25%) pt died after a median follow-up of 34 months and 21% progressed to MN (MDS 14 & CMML 8), and 1 patient with MDS and 1 with CMML progressed to secondary AML (2%). The median overall survival (mOS) was not reached, with a 2-year estimated (YE) OS and leukemia-free survival (LFS) of 80%. Predictive Power: When utilizing the IPSS-M risk category ( L, M, H) * by combining the risk categories into L *(L+VL), M * (ML+MH), and H *(H+VH) to overcome the low sample size limitation, it significantly predicted OS (p < .0001) ( Figure 1A) and median MN-free survival (FS) (p < .0001) ( Figure 1B). The 2YE-OS rates by IPSS-M risk category were as follows: VL - 93%, L - 87%, ML - 75%, high (H) - 25%, very high (VH) - 0% and the 2YE-OS rates by IPSS-M ( L, M, H) are summarized in Table 1B. IPSS-M hazard ratio (HR) for OS was 1.6 for ( M*) compared to 25 for ( H *) risk category (p < .0001). The ( L *) category, being the largest, was used as the reference level. Conclusion: This study provides the first real-world experience of using the IPSS-M and a modified version of the IPSS-M ( L, M, H) * risk category model to predict survival and progression into MN in CCUS (MUT) pt. The IPSS-M risk category was found to be a significant predictor of OS and MN-free survival. We propose that larger cohorts are needed to validate our findings.

One-Carbon Metabolites Promote Systemic Inflammation, Gut Dysbiosis, and a Myeloid Lineage Differentiation Bias

TET methylcytosine dioxygenase 2 ( TET2) loss-of-function mutations induce a pre-malignant state known as clonal hematopoiesis of indeterminate potential (CHIP). CHIP occurs in approximately 10% of people over 65 years of age and confers a 10-fold greater risk of developing hematological malignancy. Several environmental factors, including radiation, sleep deprivation, atherosclerosis, and diet, have been associated with the expansion of pre-malignant clones in CHIP patients. It has previously been shown that hematopoietic Tet2 deficiency in mice triggers a pro-inflammatory state with increased intestinal permeability, gut bacterial translocation, and accelerated clonal expansion. Gut microbes themselves can exert an influence on myeloid leukemia progression through synthesis of compounds including short-chain fatty acids (SCFAs), which promote intestinal barrier integrity. Dietary levels of one-carbon metabolites and cofactors have been found to alter gut microbial composition, affecting SCFA production and intestinal permeability, in disease-free adults. Given the connection between diet, SCFAs and gut permeability, we sought to determine the impact of dietary one-carbon metabolites on gut microbial composition and function in CHIP progression. We performed competitive bone marrow transplantation assays with Tet2+/- cells in mice supplemented with altered one-carbon metabolites: a control diet, high or low methionine diets, high or low folate diets, and high or low vitamin B12 supplementation. Mice were treated for a total of 7 months before timed sacrifice. Altered supplementation of the one carbon metabolites tested did not influence the competitiveness of Tet2-deficient cells in peripheral blood, spleen or bone marrow, however, high vitamin B12 levels promoted a myeloid differentiation bias in the spleen and bone marrow. Plasma cytokine analysis also showed that high vitamin B12 supplementation caused increased circulating inflammatory cytokines (IL-1b, IL-23, IL-27, CCL3/4, CXCL2). Based on this strong inflammatory phenotype, we performed scRNA-sequencing of splenic Tet2+/- CD11b+ cells. These data confirmed a neutrophilic cell expansion in the spleens of vitamin B12-treated mice along with increased expression of innate inflammatory genes S100a8/9 and Lyz2 as well as IL-1 b and Cxcl2 in the neutrophil clusters. Gut microbial-dependent inflammation is known to drive TET2-dependent myeloproliferation, thus we collected fecal samples before timed sacrifice and performed shotgun sequencing. Vitamin B12 supplementation decreased alpha diversity and the composition of genera that produce butyrate, an SCFA known to promote intestinal barrier integrity. Metagenomic analysis revealed increased amino acid metabolism and fatty acid biosynthesis in the gut microbiome of vitamin B12-supplemented mice, along with decreased butanoate metabolism, suggesting an alteration in microbial function that could influence SCFA content. To test whether these alterations in SCFA-producers led to increased gut permeability, we performed fluorescence in situ hybridization (FISH) against bacterial ribosomal 16S in livers from our mouse cohorts and observed increased levels of bacterial dissemination in mice with vitamin B12 supplementation. Our findings suggest that vitamin B12 supplementation in mice exacerbates the myeloid lineage differentiation bias of Tet2+/- hematopoiesis, possibly due to decreased gut barrier integrity which contributes to a heightened innate inflammatory response. This study highlights the potential for micronutrients of one-carbon metabolism to influence CHIP progression through maintenance of proper gut microbial homeostasis.

Bacterial dysbiosis and epithelial status of the California sea lion (Zalophus californianus) in the Gulf of California.

Despite the high incidence of urogenital carcinoma (UGC) in California sea lions stranded along California, no UGC has been reported in other areas of their distribution; however, cell morphologies typical of premalignant states have been found. Risk factors for UGC include high of organochlorines and infection with a gammaherpesvirus, OtHV-1, but the importance of the bacteriome for epithelial status remains unknown. We characterized the genital bacteriome of adult female California sea lions along their distribution in the Gulf of California and examined whether the diversity and abundance of the bacteriome varied spatially, whether there were detectable differences in the bacteriome between healthy and altered epithelia, and whether the bacteriome was different in California sea lions infected with OtHV-1 or papillomavirus. We detected 2270 ASVs in the genital samples, of which 35 met the criteria for inclusion in the core bacteriome. Fusobacteriia and Clostridia were present in all samples, at high abundances, and Actinobacteria, Alphaproteobacteria, and Campylobacteria were also well-represented. Alpha diversity and abundance of the California sea lion genital bacteriome varied geographically. The abundance of bacterial ASVs varied depending on the genital epithelial status and inflammation, with differences driven by classes Fusobacteriia, Clostridia, Campylobacteria and Alphaproteobacteria. Alpha diversity and abundance were lowest in samples in which OtHV-1 was detected, and highest those with papillomavirus. Our study is the first investigation of how the bacteriome is related to epithelial status in a wild marine species prone to developing cancer.

Progressive disruption of hematopoietic architecture from clonal hematopoiesis to MDS

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. Although CHIP mutations drive myeloid malignancies like myelodysplastic syndromes (MDS) it is unknown if clonal expansion is attributable to changes in cell type kinetics, or involves reorganization of the hematopoietic hierarchy. Using computational modeling we analyzed differentiation and proliferation kinetics of cultured hematopoietic stem cells (HSC) from 8 healthy individuals, 7 CHIP, and 10 MDS patients. While the standard hematopoietic hierarchy explained HSPC kinetics in healthy samples, 57% of CHIP and 70% of MDS samples were best described with alternative hierarchies. Deregulated kinetics were found at various HSPC compartments with high inter-individual heterogeneity in CHIP and MDS, while altered HSC rates were most relevant in MDS. Quantifying kinetic heterogeneity in detail, we show that reorganization of the HSPC compartment is already detectable in the premalignant CHIP state.

Somatic mutational landscape of hereditary hematopoietic malignancies caused by germline variants in RUNX1, GATA2, and DDX41

AbstractIndividuals with germ line variants associated with hereditary hematopoietic malignancies (HHMs) have a highly variable risk for leukemogenesis. Gaps in our understanding of premalignant states in HHMs have hampered efforts to design effective clinical surveillance programs, provide personalized preemptive treatments, and inform appropriate counseling for patients. We used the largest known comparative international cohort of germline RUNX1, GATA2, or DDX41 variant carriers without and with hematopoietic malignancies (HMs) to identify patterns of genetic drivers that are unique to each HHM syndrome before and after leukemogenesis. These patterns included striking heterogeneity in rates of early-onset clonal hematopoiesis (CH), with a high prevalence of CH in RUNX1 and GATA2 variant carriers who did not have malignancies (carriers-without HM). We observed a paucity of CH in DDX41 carriers-without HM. In RUNX1 carriers-without HM with CH, we detected variants in TET2, PHF6, and, most frequently, BCOR. These genes were recurrently mutated in RUNX1-driven malignancies, suggesting CH is a direct precursor to malignancy in RUNX1-driven HHMs. Leukemogenesis in RUNX1 and DDX41 carriers was often driven by second hits in RUNX1 and DDX41, respectively. This study may inform the development of HHM-specific clinical trials and gene-specific approaches to clinical monitoring. For example, trials investigating the potential benefits of monitoring DDX41 carriers-without HM for low-frequency second hits in DDX41 may now be beneficial. Similarly, trials monitoring carriers-without HM with RUNX1 germ line variants for the acquisition of somatic variants in BCOR, PHF6, and TET2 and second hits in RUNX1 are warranted.

A digital score of peri-epithelial lymphocytic activity predicts malignant transformation in oral epithelial dysplasia.

Oral squamous cell carcinoma (OSCC) is amongst the most common cancers, with more than 377,000 new cases worldwide each year. OSCC prognosis remains poor, related to cancer presentation at a late stage, indicating the need for early detection to improve patient prognosis. OSCC is often preceded by a premalignant state known as oral epithelial dysplasia (OED), which is diagnosed and graded using subjective histological criteria leading to variability and prognostic unreliability. In this work, we propose a deep learning approach for the development of prognostic models for malignant transformation and their association with clinical outcomes in histology whole slide images (WSIs) of OED tissue sections. We train a weakly supervised method on OED cases (n = 137) with malignant transformation (n = 50) and mean malignant transformation time of 6.51 years (±5.35 SD). Stratified five-fold cross-validation achieved an average area under the receiver-operator characteristic curve (AUROC) of 0.78 for predicting malignant transformation in OED. Hotspot analysis revealed various features of nuclei in the epithelium and peri-epithelial tissue to be significant prognostic factors for malignant transformation, including the count of peri-epithelial lymphocytes (PELs) (p < 0.05), epithelial layer nuclei count (NC) (p < 0.05), and basal layer NC (p < 0.05). Progression-free survival (PFS) using the epithelial layer NC (p < 0.05, C-index = 0.73), basal layer NC (p < 0.05, C-index = 0.70), and PELs count (p < 0.05, C-index = 0.73) all showed association of these features with a high risk of malignant transformation in our univariate analysis. Our work shows the application of deep learning for the prognostication and prediction of PFS of OED for the first time and offers potential to aid patient management. Further evaluation and testing on multi-centre data is required for validation and translation to clinical practice. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.