Premalignant State Research Articles

Association between excess body mass index trajectory and change in monoclonal protein level in patients diagnosed with monoclonal gammopathy of undetermined significance.

10538 Background: Obesity is a known risk factor for the development of multiple myeloma (MM) and for progression of its asymptomatic premalignant state, monoclonal gammopathy of undetermined significance (MGUS) to MM. Prior studies have demonstrated that monoclonal protein (M-spike) at MGUS diagnosis as well as high M-spike velocity within a year following MGUS diagnosis predict progression of MGUS to MM, making M-spike a useful, readily available, and less invasive (compared to bone marrow biopsy) marker to assess progression risk. However, no studies have evaluated to what extent exposure to high body mass index (BMI) over time influences M-spike trajectory. Methods: Patients diagnosed with MGUS from 1999-2021 in the Veterans Health Administration were identified. We used a natural language processing-based algorithm to confirm MGUS diagnosis and progression to smoldering MM or MM. MGUS patients without IgM or IgD subtype or Diabetes Mellitus and were black or white were included in the analysis. Other exclusion criteria were progression within 5 years of MGUS diagnosis, patients without M-spike level within a year before or after MGUS diagnosis, and patients without BMI data 3 years or more after MGUS diagnosis. The exposure was the trajectory of excess BMI (defined as BMI > 25 kg/m2), defined by an area under the curve (AUC) of excess BMI during the 5 years following MGUS diagnosis. The outcome was continuous M-spike trajectory post-MGUS diagnosis since a prior study demonstrated that this was a reliable marker for progression risk A multivariable mixed model was used to estimate the association of excess BMI trajectory AUC with M-spike trajectory to control for correlation between M-spike values overtime. The covariates included BMI at MGUS diagnosis, M-spike level ( > = 1.5g/dL) at MGUS diagnosis, age at MGUS diagnosis, sex, race, MGUS heavy chain subtype, and light-chain MGUS. Results: After applying the inclusion and exclusion criteria, the analytic cohort consisted of 6757 MGUS patients. Our multivariable analysis demonstrated a positive association between excess BMI trajectory and increasing M-spike trajectory (β = 0.005, p = 0.007). Conclusions: In patients diagnosed with MGUS, increasing BMI over time following MGUS diagnosis was associated with increasing M-spike level. This further supports the relationship between obesity and weight gain in the pathway of MGUS to MM progression.

Alterations of miRNA Expression in Diffuse Hyperplastic Perilobar Nephroblastomatosis: Mapping the Way to Understanding Wilms' Tumor Development and Differential Diagnosis.

Wilms' tumor (WT) is the most common renal malignancy in children. In diffuse hyperplastic perilobar nephroblastomatosis (DHPLN), nephrogenic rests result in a bulky enlargement of the kidney, a condition considered as a premalignant state before WT. Despite relevant clinical differences between WT and DHPLN, they are often challenging to distinguish based on histology. Molecular markers would improve differential diagnosis, but none are available at present. In our study, we investigated the potential of microRNAs (miRNAs) as such biomarkers, also aiming to shed light on the chronological order of expression changes. Formalin-fixed, paraffin-embedded (FFPE) samples from four DHPLN cases and adjacent healthy tissues were tested using a PCR array containing primers for 84 miRNAs implicated in genitourinary cancer. Expression in DHPLN was compared to WT data available in dbDEMC. Let-7, miR-135, miR-146a-5p, miR-182-5p, miR-183-5p, miR-20b-3p, miR-29b-3p, miR-195-5p and miR-17-5p showed potential to be used as biomarkers to distinguish WT and DHPLN in cases when traditional differential diagnosis is inconclusive. Our study also revealed miRNAs which may play a role in the initial steps of the pathogenesis (at a precancerous stage) and ones which become deregulated later in WT. More experiments are needed to confirm our observations and find new candidate markers.

SOHO State of the Art Updates and Next Questions | Infections in Chronic Lymphocytic Leukemia Patients: Risks and Management.

Although chronic lymphocytic leukemia (CLL) is a malignancy characterized by accumulation of tumor cells in the blood, bone marrow, lymph nodes and secondary lymphoid tissues, the hallmark of the disease and the major cause of death for patients with CLL is actually immune dysfunction and associated infections. Despite improvement in treatment based on combination chemoimmunotherapy and targeted treatment with BTK and BCL-2 inhibitors leading to longer overall survival for patients with CLL, the mortality due to infections have not improved over the last 4 decades. Thus, infections are now the main cause of death for patients with CLL, posing threats to the patient whether during the premalignant state of monoclonal B lymphocytosis (MBL), during the watch & wait phase for treatment naïve patients, or upon treatment in terms of chemoimmunotherapy or targeted treatment. To test whether the natural history of immune dysfunction and infections in CLL can be changed, we have developed the machine learning based algorithm CLL-TIM.org to identify these patients. The CLL-TIM algorithm is currently being used for selection of patients for the clinical trial PreVent-ACaLL (NCT03868722), testing whether short-term treatment with the BTK inhibitor acalabrutinib and the BCL-2 inhibitor venetoclax can improve immune function and decrease the risk of infections for this high-risk patient population. We here review the background for and management of infectious risks in CLL.

Abstract A44: The influence of dietary one-carbon metabolites on gut dysbiosis during pre-malignant hematopoiesis

Abstract 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. Tet2-deficiency in mice has also been shown to trigger a pro-inflammatory state with increased intestinal permeability and accelerated myeloid expansion. Gut microbes exert an influence on host disease progression through the synthesis of many compounds including short-chain fatty acids (SCFAs), which promote gut barrier integrity. Dietary levels of one-carbon metabolites and cofactors, including methionine and folate, have been found to alter gut microbial composition in disease-free adults and influence SCFA production in the gut in murine models. 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 a murine model of pre-malignant hematopoiesis. We performed competitive bone marrow transplantation assays in mice fed diets with altered one-carbon metabolite supplementation, such as high and low folate or methionine. We found that differential supplementation with these one-carbon metabolites did not influence the competitiveness of Tet2-deficient hematopoietic cells, however, increased dietary methionine promoted a myeloid lineage differentiation bias and an elevation in circulating pro-inflammatory cytokines. After 8 months of dietary treatment, fecal samples were collected, and shotgun sequencing was performed to examine the role of one-carbon metabolite levels on gut microbial diversity. Alterations in dietary methionine and folate caused significant changes to gut microbial composition in Tet2-deficient mice. High folate or methionine supplementation led to a decrease in the ratio of Firmicutes to Bacteroidetes and high folate supplementation was also associated with decreased alpha diversity and a decrease in SCFA-producing bacteria in the gut. These studies highlight the potential influence of dietary one-carbon metabolites on the microbiome and inflammatory microenvironment of pre-malignant hematopoiesis. Citation Format: Peter Lyon, Praveen Singh, Byron Fang, Victoria Strippoli, Sabita Roy, Luisa Cimmino. The influence of dietary one-carbon metabolites on gut dysbiosis during pre-malignant hematopoiesis [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A44.

Monoclonal B-cell lymphocytosis with a non-CLL immunophenotype - Review of 34 cases.

The monoclonal B-cell lymphocytosis (MBL) introduced as new entities in the 2008 WHO classification, are defined by circulating B-cell clone <5.109/L without organomegaly and previous and/or simultaneous lymphoproliferative disorders. The MBL were subclassified in MBL CLL type (the most frequent), MBL atypical CLL type and MBL non-CLL type (rarely reported in literature). Here the clinic, cytologic, immunologic and genetic features of MBL non-CLL type were described from a series of 34 cases. As previously reported, present cases presented immunologic and genetic similarities to MZL and could be associated to the new proposed entity CBL-MZ (clonal B-cell lymphocytosis of marginal zone origin). In addition, few cases presented similarities to splenic diffuse red pulp lymphoma (SDRPL). In conclusion, according to the literature, MBL with non-CLL type (assimilated to CBL-MZ) may be a premalignant state of MZL and/or SDRPL.

Abstract PR004: The role of NOTCH1 in bronchial pre-malignant lesions

Abstract Continuous carcinogen exposure through smoking creates a field of injury along the airway and resulting in mutations which promote remodeling of the bronchial epithelium. Over time lesions can form and exhibit increasing abnormalities progressing from goblet cell hyperplasia (GCH) which is the earliest detectable premalignant state through hyperplasia and squamous metaplasia to incremental stages of dysplasia, carcinoma in situ (CIS) and eventually to frank carcinoma. Unfortunately, more than half of all lung cancer patients are diagnosed with metastatic disease with a dismal 5-year survival rate of only 5.2%. However, when diagnosed at an early stage, the 5-year survival rate is more than 10 times as high. Our group has identified NOTCH1 as the most frequently mutated gene in progressive lung premalignant lesions (PMLs). However, these mutations are significantly less common in advanced lung squamous cell carcinoma (LUSC), indicating a role for NOTCH1 in the pre-tumorigenic environment rather than contributing to the actual tumor mass. Transcriptomic profiling of NOTCH1 mutated lesions identified up regulation of peri-goblet cells (PGC), a newly identified bronchial epithelial cell type that is associated with smoking, and a down regulation of immune components. Using highly multiplex Imaging Mass Cytometry (IMC) to analyze the epithelium of NOTCH1 mutated PMLs and its interplay with the immune system in situ. We identified a shift in epithelial composition with an increase in un- and de-differentiated cells. Interestingly, PGC appear to be an intermediate cell type on a previously undescribed path to goblet cell differentiation. In a parallel approach we are modeling NOTCH1 driven dysplasia in vitro to gain insight into the molecular mechanisms of lesion progression. Primary human bronchial epithelial cells (hBECs) grown at the air-liquid interface (ALI) into mature epithelia are exposed to cigarette smoke condensate (CSC) or IL-13 to model the early stages of bronchial airway dysplasia. Continuous exposure of these differentiating cultures to either stimulus increases the abundance of goblet cells and PGCs in a dose dependent manner with IL-13 additionally leading to the loss of baso-apical organization of the epithelium. After successfully modeling the first stage of bronchial epithelial dysplasia we are now advancing our model by co-culturing primary hBECs with NOTCH1 mutated cells and to disentangle the pathway of basal-PGC-goblet cell differentiation. Understanding driving forces in early lesion progression will further our ability to discern progressive lesions from those that regress naturally and facilitate interventions that force lesions into regression. Citation Format: Roxana Michaela Pfefferkorn, Darren Jianjhih Chiu, Divya Lakshmi Venkatraman, Jennifer Ellen Beane, Sarah Anne Mazzilli, Joshua David Campbell. The role of NOTCH1 in bronchial pre-malignant lesions. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR004.

Targeting TGF-β signaling in the multiple myeloma microenvironment: Steering CARs and T cells in the right direction.

Multiple myeloma (MM) remains a lethal hematologic cancer characterized by the expansion of transformed plasma cells within the permissive bone marrow (BM) milieu. The emergence of relapsed and/or refractory MM (RRMM) is provoked through clonal evolution of malignant plasma cells that harbor genomic, metabolic and proteomic perturbations. For most patients, relapsed disease remains a major cause of overall mortality. Transforming growth factors (TGFs) have pleiotropic effects that regulate myelomagenesis as well as the emergence of drug resistance. Moreover, TGF-β modulates numerous cell types present with the tumor microenvironment, including many immune cell types. While numerous agents have been FDA-approved over the past 2 decades and significantly expanded the treatment options available for MM patients, the molecular mechanisms responsible for drug resistance remain elusive. Multiple myeloma is uniformly preceded by a premalignant state, monoclonal gammopathy of unknown significance, and both conditions are associated with progressive deregulation in host immunity characterized by reduced T cell, natural killer (NK) cell and antigen-presenting dendritic cell (DC) activity. TGF-β promotes myelomagenesis as well as intrinsic drug resistance by repressing anti-myeloma immunity to promote tolerance, drug resistance and disease progression. Hence, repression of TGF-β signaling is a prerequisite to enhance the efficacy of current and future immunotherapeutics. Novel strategies that incorporate T cells that have been modified to express chimeric antigen receptor (CARs), T cell receptors (TCRs) and bispecific T cell engagers (BiTEs) offer promise to block TGF-β signaling, overcome chemoresistance and enhance anti-myeloma immunity. Here, we describe the effects of TGF-β signaling on immune cell effectors in the bone marrow and emerging strategies to overcome TGF-β-mediated myeloma growth, drug resistance and survival.

Monoclonal gammopathy of undetermined significance: evaluation, risk assessment, management, and beyond.

Monoclonal gammopathy of undetermined significance (MGUS) is a premalignant state for a spectrum of lymphoplasmacytic malignancies. The risk of progression of MGUS to a symptomatic therapy requiring plasma cell dyscrasia is about 1% per year. Studies carried out over the previous 10 years have improved risk stratification of MGUS based on serologic and genomic evaluations, which has led to better management of patients. In this review, we address the epidemiology, diagnosis, and pathogenesis of MGUS and discuss risk-adapted best practice approaches to monitor patients.

Genotyping of Multiple Genomic Loci with Chromatin Accessibility Profiling in Single Cells Links Clonal Hierarchy with Epigenetic Variation in Acute Myeloid Leukemia

Acquisition of multiple somatic mutations and epigenetic changes can result in malignant transformation, intra-tumoral heterogeneity, therapy resistance and disease relapse. However, our current knowledge of how cooperating driver genetic and epigenetic events shape gene regulation and chromatin landscapes in patient samples is still limited. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), a novel method for high efficiency capture of open chromatin with parallel amplification of multiple genomic targets. This provides genetic clonal identity and epigenetic state at single-cell resolution (Fig. 1). We first tested this method in leukemia cell lines. GTAC successfully detected 8 genomic loci encoding leukemic variants in 96-100% of single cells. Low allelic dropout rates (1.5% - 11%) allowed high-confidence mutation calling for heterozygous loci in 90-99% of cells. Corresponding scATAC-seq libraries were of high quality, independent of the number of genotyped loci, with >105 unique fragments/cell on average and unperturbed signal-to-noise ratio. 93.75% of cells had simultaneous detection of all 8 genomic targets and efficient scATAC-seq library generation. Finally, a mixing experiment on three cell lines showed that 97%, 96% and 99% of cells had correct mutation calls for the ASXL1, RUNX1 and NRAS loci, respectively. We next applied GTAC to 3520 single cells from a primary human acute myeloid leukemia (AML) sample harbouring mutations in TET2, ASXL1, RUNX1, and two BCOR loci (Fig. 2). Single-cell genotyping successfully assigned clonal state for 96.8% of cells. This showed that TET2, ASXL1, and RUNX1 mutations were acquired sequentially, followed by branched acquisition of two independent clones with BCOR loss-of-function mutations. Integration of clonal structure and chromatin accessibility revealed that wild type cells were mainly found in T/NK cell clusters, while TET2 and TET2/ASXL1 mutant cells were predominant in the hemopoietic stem and multipotent progenitor clusters, which were most likely preleukemic. In contrast, on acquisition of the RUNX1 mutation, cells were entirely distributed within lympho-myeloid progenitor-like compartments, establishing the importance of the RUNX1 mutation in transformation from preleukemia to leukemia. Global transcription factor binding motif analysis confirmed profound chromatin landscape changes between clones. RUNX1 mutant cells were strongly enriched in motifs of pro-proliferative transcription factors and those central to lympho-myeloid differentiation, such as SPI1, CEBPA, BCL11A/B and FOS/JUN. Further clonal evolution occurred within leukemic cells, resulting in a significant enrichment of BCOR mutant clones within the lymphoid-primed multipotent progenitor (LMPP)-like differentiation state (P<0.0001). Interestingly, this cluster was characterized by higher accessibility of stemness-related HOXA9, MECOM, HLF, FOXO, ERG and ETV6 binding motifs coupled with higher CD34 and lower CD38 accessibility. LMPP-like cells also displayed higher accessibility of leukemia stem cell (LSC)-associated genes (Gentles et al., JAMA, 2010). Moreover, BCOR mutant cells were enriched in MEF2 family motifs linked to AML therapy resistance. This, together with our previous data showing LSC function within LMPP-like cells, suggests a bias towards an LSC-like chromatin state upon BCOR loss. Our dataset uniquely allowed us to examine the impact that BCOR loss-of-function exerts on chromatin accessibility specifically within LMPP-like cells by comparing ATAC profiles of BCOR-mutant and wild type cells within this compartment. BCOR mutant LMPP-like cells had increased accessibility of motifs bound by HOXA/B, NANOG, and NF-kB transcription factors, and a more open INPP4B gene, recently associated to LSC maintenance (Woolley et al., BioRxiv, 2022). This suggests that aberrant PRC1.1 activity, caused by BCOR gene loss, may further confer a fitness advantage to BCOR mutant cells in the TET2/ASXL1/RUNX1-mutant context. In summary, GTAC enables detailed single-cell analysis of chromatin landscapes in normal, pre-malignant and fully transformed subclones within complex clonal hierarchies in primary patient cells, revealing how gene regulation is corrupted as clones evolve. GTAC is a widely applicable tool to study malignant and pre-malignant states. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Somatic Mutational Landscape of Hereditary Hematopoietic Malignancies Associated with Germline Variants in RUNX1, GATA2 and DDX41

BACKGROUND: Germline variants in RUNX1, GATA2 and DDX41 may confer a predisposition to hereditary haematopoietic malignancies (HHMs) such as MDS and AML yet have distinct age ranges of malignancy diagnosis and a highly variable overall risk for leukemogenesis. The increased awareness and identification of carriers of these germline variants, particularly before development of malignancy, has changed the way in which individuals and families need to be managed in the clinic. Individuals need lifelong monitoring and may also need modification to treatments when malignancy does develop, compared to sporadic counterparts. Gaps in understanding pre-malignant states in HHM syndromes have hampered efforts to design effective clinical surveillance regimes, provide personalized pre-emptive treatments, and appropriate counselling to patients. METHODS: We have addressed some of these knowledge gaps using the largest known international comparative cohort of germline RUNX1, GATA2, or DDX41 variant carriers, which includes carriers both without a malignancy diagnosis "unaffected-carriers" and those with a malignancy "affected-carriers". Our cohort included 245 patient samples evenly distributed between unaffected-carrier (n=122) and affected-carrier (n=123) samples. We utilized a uniform bioinformatics approach, to identify somatic variants from next generation sequencing data provided by our network of investigators. RESULTS: We identified clonal hematopoiesis (CH) in 34% and 38% of RUNX1 and GATA2 unaffected-carriers, respectively (Figure 1A top), with overlap in the ages of observation CH and malignancy in different individuals observed (Figure 1A bottom). In contrast CH was seen in only 4% of DDX41 unaffected-carriers, significantly less frequently than RUNX1 or GATA2 (p=0.002). RUNX1- and GATA2 variant carriers were at an increased risk of early onset CH (0-29 years) present in 19% of RUNX1 carriers and 25% of GATA2 variant carriers compared to 0.2% of the general population (PMID: 25426837; PMID: 25426838). RUNX1 unaffected-carriers had an increased prevalence of CH at all ages compared to population controls, with BCOR being the most frequently mutated gene in RUNX1 carriers, present across the entire age spectrum from as young as 16 years to 76 years (Figure 1B). In RUNX1-driven CH specifically we detected "high-risk" variants in TET2, PHF6 and most frequently in BCOR, all of which were also recurrently mutated in RUNX1-driven malignancies, suggesting CH is a direct precursor to malignancy. Consistent with a multi-step process towards leukaemia, we found both an increase in clone size (variant allele frequency, VAF) and the number of variants present per sample, as the age of carriers increased (Figure 1B). Tumor profiling similarly highlighted differences in the pathways to myeloid malignancy development for the three syndromes. Leukemogenesis in both RUNX1 and DDX41 carriers was often driven by second-hit variants in RUNX1 and DDX41 respectively. Somatic RUNX1 variants were not seen in germline RUNX1 unaffected-carriers, only in tumors, therefore this likely represents a "late" leukemogenic event. This was not able to be inferred for somatic DDX41 variants in DDX41 related predisposition where mutations before malignancy were rarely seen. In contrast, GATA2 germline driven malignancy was not associated with somatic mutation of GATA2, instead this process favored mutations in ASXL1. Both germline RUNX1 and DDX41 cohorts presented with a sex bias for HM development, but in opposite directions (AML 1:2.5 (RUNX1) and HM 3:1 (DDX41), Male:Female). Disruption of RUNX1 regulation of estrogen signalling, and DDX41 mediated exaggeration of the known sex difference in innate immunity, are postulated as mechanisms worth investigation to explain these observed sex biases (PMID: 25479752; PMID:28223406). CONCLUSIONS: This study lays the foundation for developing novel-preventative therapies and the implementation of gene-specific clinical monitoring requirements for carriers of germline variants in RUNX1, DDX41 and GATA2. Individuals with RUNX1 variants will require regular monitoring for somatic variants in several genes throughout their lifetime, while DDX41 carriers are likely to benefit from monitoring throughout adulthood for driver DDX41 variants using sensitive technology to detect low-frequency initiating events. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Natural History of Patients with Clonal Hematopoiesis Seen in Hematology Clinics

Background: The International Consensus Classification (ICC) recently recognized clonal hematopoiesis of indeterminate potential (CHIP) as a clinically defined pre-malignant state. A subgroup of patients with clonal hematopoiesis (CH), herein termed progressors, ultimately develop to a frank clinical diagnosis, while others, herein termed non-progressors, may have CH indefinitely with variable risk of progression. Although eight years have passed since the large-scale descriptions of CHIP as an age-related phenomenon, the risk factors for progression and management guidelines are still not well-defined. Moreover, while many quaternary care centers have developed CHIP databases, little is known about the epidemiology of CHIP from tertiary care centers that draw patients from the community setting. Methods: Patients with CH were identified via concurrent morphologic, cytogenetic, and molecular diagnostics of bone marrow aspirates reviewed by UMass Hematopathology between 2013 and 2022. CH was defined here as per the 2022 ICC: presence of a somatic mutation with variant allele frequency (VAF) ≥ 2% on molecular diagnostics or non-myelodysplastic syndrome (MDS)-defining clonal cytogenetic aberration in a patient without morphologic evidence of myeloid neoplasm. Patients with germline mutations were excluded. Independent review of all bone marrow aspirates was performed by at least two pathologists. This study included a pre-selected group of patients who had an indication for bone marrow evaluation; reasons for performing bone marrow evaluation included presentation with pancytopenia (41.7%), anemia (14.6%), leukocytosis (12.5%), thrombocytopenia (6.3%), leukopenia (4.2%), bicytopenia (4.2%), staging workup for a lymphoma (4.2%), erythrocytosis (4.2%), thrombocytosis (4.2%), abnormal radiographic marrow signal (2.1%), eosinophilia (2.1%). A total of 48 patients with CH were identified. Results: A total of 102 aberrations were found among 48 patients with CH. The mean, median, mode, and range for CH aberrations were 2.15, 1.5, 1, and 5, respectively. The mean age at detection of CH for non-progressors was 69.2 ± 1.8 years vs. 63.1 ± 3.5 years for progressors (p = 0.072). The most prevalent somatic variants in CH were: TET2 (29.1%), DNMT3A (14.5%), and gross structural abnormalities (7.7%) (Panel A, outer circle). Progressors had a distinct profile (Panel A, inner circle). Zero of 48 patients with CH carried TP53, IDH1, or EZH2 aberrations. The most prevalent genomic disruptions were missense mutations (45.6%), nonsense mutations (15.6%), and frameshift deletions (13.3%). We assessed the clinical trajectory of patients with CH (Panel B). Nine (18.8%) of 48 patients progressed to neoplasm (confirmed by bone marrow evaluation). Median overall survival (OS) at the time of analysis was 6.85 years, with longest follow up of 9.58 years. For non-progressors, median OS was not reached. For progressors, median OS was 6.85 years (from time of identification of CH). Median time from identification of CH to frank neoplasm was 1.05 years. For progressors, median time from diagnosis of frank neoplasm to death was 1.76 years. Of the 14 patients harboring DNMT3A, TET2, or ASXL1 (DTA) mutations alone, without mutations in other biological clusters, none (0%) progressed (Panel B,blue bars). Conclusion: In contrast to the original reports of CH, which studied unselected patients from multiple consortia, the patients in our study were pre-selected for suspicion of an underlying hematologic condition, reflecting real-world data. CH spans multiple functional clusters that are recurrently mutated in MDS and/or acute myeloid leukemia (AML), with the notable exception of TP53, IDH1, and EZH2. This may be due to more rapid progression to malignancy for those mutations, with only a transient, if any, CH state. All patients who progressed with DTA mutations did so only in the presence of additional mutations - at least a second mutation was required for DTA cases to progress. There was a trend toward significance for younger mean age at CH detection for progressors vs. non-progressors. Lead-time bias may skew OS results for patients found to have CH at an early point; and CH detected at a younger age may be more likely progress, leading to shorter OS. Systematic studies are needed to more comprehensively profile CH and develop formalized risk-adapted management strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonal hematopoiesis and solid neoplasms

Clonal hematopoiesis (CH) is a premalignant state of the hematopoietic system that frequently occurs in old age and is associated with an elevated cardiovascular risk and higher overall mortality. The prevalence and clinical implications of CH in patients with solid neoplasms were examined. A review, summary and discussion of the recent literature was carried out. CH occurs in20-30% of patients with solid neoplasms. In the molecular diagnostics of tumor or cell-free DNA from plasma, CH mutations can be falsely interpreted as tumor mutations. CH and in particular mutations in the genes of the DNA damage repair machinery are associated with ahigher risk of therapy-associated myeloid neoplasms (t-MN) following chemotherapy, radiotherapy and poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor therapy. CH is afrequent phenomenon in patients with solid neoplasms. It has high clinical relevance due to the associated risk of t‑MN. More research is needed for a better understanding of the role of CH in this patient collective and to derive evidence-based recommendations for action.

Importance of clonal hematopoiesis for hematologic neoplasms

Clonal hematopoiesis of indeterminate potential (CHIP) is afairly newly described phenomenon characterized by myeloid cancer-associated somatic mutations detectable in the peripheral blood of individuals without evidence of hematologic disease. Individuals with CHIP have asignificantly increased risk of developing ahematologic malignancy, although the overall rate of transformation is low. We review the current state of knowledge on causes of clonal expansion of blood cells as well as identifiable risk factors for progression to overt hematologic malignancy. CHIP is considered apremalignant state and predisposes to the development of hematologic malignancy. Because the overall rate of transformation is low, clear identification and subsequent monitoring of those CHIP individuals at a higher risk is of paramount importance. In the future, prospective studies evaluating preventive and/or preemptive therapeutic strategies may aid in avoiding progression to blood cancer in individuals with CHIP.

Calorimetric Markers for Detection and Monitoring of Multiple Myeloma.

Simple SummaryThis review highlights the potential of differential scanning calorimetry for multiple myeloma diagnosis and monitoring of the treatment outcome. The thermodynamic signatures of blood sera from patients with multiple myeloma are strongly dependent on the concentration and isotype of the secreted monoclonal immunoglobulins. Mathematical methods developed to analyze the biocalorimetry data and distinguish “diseased” from “healthy” thermogram to stratify plasma calorimetric profiles and determine specific interrelations between calorimetric and biochemical/clinical data are discussed.This review summarizes data obtained thus far on the application of differential scanning calorimetry (DSC) for the analysis of blood sera from patients diagnosed with multiple myeloma (MM) with the secretion of the most common isotypes of monoclonal proteins (M-proteins), free light chains (FLC) and non-secretory MM, as well as Waldenström macroglobulinemia and the premalignant state monoclonal gammopathy of undetermined significance. The heterogeneous nature of MM is reflected in the thermal stability profiles of the blood serum proteome of MM patients found to depend on both the level and the isotype of the secreted M-proteins or FLC. Common calorimetric markers feature the vast majority of the different myeloma types, i.e., stabilization of the major serum proteins and decrease in the albumin/globulin heat capacity ratio. A unique calorimetric fingerprint of FLC molecules forming amorphous aggregates is the low-temperature transition centered at 57 °C for a calorimetric set of FLC MM and at 46–47 °C for a single FLC MM case for which larger aggregates were formed. The calorimetric assay proved particularly advantageous for non-secretory MM and is thus a suitable tool for monitoring such patients during treatment courses. Thus, DSC provides a promising blood-based approach as a complementary tool for MM detection and monitoring.

Hematopoiesis of Indeterminate Potential and Atherothrombotic Risk.

Hematopoiesis is the process of blood production, essential for the continued supply of immune cells and red blood cells. However, the proliferative nature of hematopoietic stem cells (HSCs) renders them susceptible to developing somatic mutations. HSCs carrying a mutation can gain a selective advantage over normal HSCs and result in hematological disorders. One such disorder is termed clonal hematopoiesis of indeterminate potential (CHIP), a premalignant state associated with aging, where the mutant HSCs are responsible for producing a small portion of mature immune cells in the circulation and subsequently in tissues. People with CHIP have been shown to have an increased risk of mortality due to cardiovascular disease (CVD). Why this occurs is under rigorous investigation, but the majority of the studies to date have suggested that increased atherosclerosis is due to heightened inflammatory cytokine release from mutant lesional macrophages. However, given CHIP is driven by several mutations, other hematopoietic lineages can be altered to promote CVD. In this review we explore the relationship between mutations in genes causing CHIP and atherothrombotic disorders, along with potential mechanisms of enhanced clonal outgrowth and potential therapies and strategies to slow CHIP progression.

Mutant PPM1D- and TP53-Driven Hematopoiesis Populates the Hematopoietic Compartment in Response to Peptide Receptor Radionuclide Therapy.

Hematologic toxic effects of peptide receptor radionuclide therapy (PRRT) can be permanent. Patients with underlying clonal hematopoiesis (CH) may be more inclined to develop hematologic toxicity after PRRT. However, this association remains understudied. We evaluated pre- and post-PRRT blood samples of patients with neuroendocrine tumors. After initial screening, 13 cases of interest were selected. Serial blood samples were obtained on 4 of 13 patients. Genomic DNA was analyzed using a 100-gene panel. A variant allele frequency cutoff of 1% was used to call CH. Sixty-two percent of patients had CH at baseline. Persistent cytopenias were noted in 64% (7 of 11) of the patients. Serial sample analysis demonstrated that PRRT exposure resulted in clonal expansion of mutant DNA damage response genes (TP53, CHEK2, and PPM1D) and accompanying cytopenias in 75% (3 of 4) of the patients. One patient who had a normal baseline hemogram and developed persistent cytopenias after PRRT exposure showed expansion of mutant PPM1D (variant allele frequency increased to 20% after exposure from < 1% at baseline). In the other two patients, expansion of mutant TP53, CHEK2, and PPM1D clones was also noted along with cytopenia development. The shifts in hematopoietic clonal dynamics in our study were accompanied by emergence and persistence of cytopenias. These cytopenias likely represent premalignant state, as PPM1D-, CHEK2-, and TP53-mutant clones by themselves carry a high risk for transformation to therapy-related myeloid neoplasms. Future studies should consider CH screening and longitudinal monitoring as a key risk mitigation strategy for patients with neuroendocrine tumors receiving PRRT.

A large-area organic transistor with 3D-printed sensing gate for noninvasive single-molecule detection of pancreatic mucinous cyst markers

Early diagnosis in a premalignant (or pre-invasive) state represents the only chance for cure in neoplastic diseases such as pancreatic-biliary cancer, which are otherwise detected at later stages and can only be treated using palliative approaches, with no hope for a cure. Screening methods for the purpose of secondary prevention are not yet available for these cancers. Current diagnostic methods mostly rely on imaging techniques and conventional cytopathology, but they do not display adequate sensitivity to allow valid early diagnosis. Next-generation sequencing can be used to detect DNA markers down to the physical limit; however, this assay requires labeling and is time-consuming. The additional determination of a protein marker that is a predictor of aggressive behavior is a promising innovative approach, which holds the potential to improve diagnostic accuracy. Moreover, the possibility to detect biomarkers in blood serum offers the advantage of a noninvasive diagnosis. In this study, both the DNA and protein markers of pancreatic mucinous cysts were analyzed in human blood serum down to the single-molecule limit using the SiMoT (single-molecule assay with a large transistor) platform. The SiMoT device proposed herein, which exploits an inkjet-printed organic semiconductor on plastic foil, comprises an innovative 3D-printed sensing gate module, consisting of a truncated cone that protrudes from a plastic substrate and is compatible with standard ELISA wells. This 3D gate concept adds tremendous control over the biosensing system stability, along with minimal consumption of the capturing molecules and body fluid samples. The 3D sensing gate modules were extensively characterized from both a material and electrical perspective, successfully proving their suitability as detection interfaces for biosensing applications. KRAS and MUC1 target molecules were successfully analyzed in diluted human blood serum with the 3D sensing gate functionalized with b-KRAS and anti-MUC1, achieving a limit of detection of 10 zM and 40 zM, respectively. These limits of detection correspond to (1 ± 1) KRAS and (2 ± 1) MUC1 molecules in the 100 μL serum sample volume. This study provides a promising application of the 3D SiMoT platform, potentially facilitating the timely, noninvasive, and reliable identification of pancreatic cancer precursor cysts.Graphical abstract

Biological Characterization and Clinical Relevance of Circulating Tumor Cells: Opening the Pandora's Box of Multiple Myeloma.

Simple SummaryBone marrow (BM) aspirates are mandatory for diagnosis and follow-up of patients with multiple myeloma (MM). However, they present two important caveats: Their invasiveness and limited scope to capture the broad tumor heterogeneity. Conversely, circulating tumor cells (CTCs) are detectable in the peripheral blood of patients with precursor and malignant disease states and have strong prognostic value. Moreover, the high genetic and transcriptomic overlap between both plasma cell compartments suggests that CTCs might reflect with notable precision the medullar clone. Furthermore, the study of CTCs could be used as a model to identify mechanisms favoring BM egression and disease spreading. Here, we summarize the state of the art on MM CTCs and provide insights on what they may offer in research and clinical scenarios.Bone marrow (BM) aspirates are the gold standard for patient prognostication and genetic characterization in multiple myeloma (MM). However, they represent an important limitation for periodic disease monitoring because they entail an aggressive procedure. Moreover, recent findings show that a single BM aspirate is unable to reflect the complex MM heterogeneity. Recent advances in flow cytometry, microfluidics, and “omics” technologies have opened Pandora’s box of MM: The detection and isolation of circulating tumor cells (CTCs) offer a promising and minimally invasive alternative for tumor assessment and metastasis study. CTCs are detectable in premalignant and active MM states, and their enumeration has strong prognostic value, to the extent that it is challenging current stratification systems. In addition, CTCs reflect with high precision both intra- and extra-medullary disease at the phenotypic, genomic, and transcriptomic levels. Despite this high resemblance between tumor clones in distinct locations, some subtle (not random) differences might shed some light on the metastatic process. Thus, it has been suggested that a hypoxic and pro-inflammatory microenvironment could induce an arrest in proliferation forcing tumor cells to recirculate. Herein, we summarize data on the characterization of MM CTCs as well as their clinical and research potential.

Abstract P3-09-07: R-loop forming sequences determine early response genes and tumorigenic pathways driven in BRCA1-deficient carriers

Abstract Transcriptional R-loops are triple-stranded RNA:DNA hybrid genome structures which are defined in more than 75% of genes of the human genome, functionally versatile in cells, and mechanistically involved in tumorigenesis. In cancer cells, excessive/aberrant R-loop structures promote mutagenesis and genome instability due to their key regulatory roles in transcription and genotoxic stress. Thus, a balance in the regulation of R-loop initiation, stabilization, and suppression is required for proper function. Recent studies have shown that R-loop formation increases in luminal but not in basal-like cells of the tumor-free mammary epithelium of BRCA1 mutation carriers. R-loop forming sequences (RLFSs) are strand-specific G-rich regions of ssDNA that initiate and stabilize the formation of R-loops. The roles of the RLFSs in functional R-loops associated with premalignant states of BRCA1-mc have not been studied. Using our QmRLFS model and R-loopDB (http://rloop.bii.a-star.edu.sg/), we identified RLFS-associated (RLFS(+)) and non-associated (RLFS(-)) RNA:DNA hybrids detected by published DRIP-seq data in gene regions in luminal precursors, mature luminal, and basal-like epithelial cell type populations, extracted from BRCA1 wild-type and mutated non-cancer breast tissue samples (GSE96672). We found that genome regions of RLFS(+) RNA:DNA hybrid signals are strongly associated with G4-quadruplex, ssDNA regions, histone markers (acetylation, demethylation), and other open chromatin markers while the RLFS(-) RNA:DNA hybrid signal regions were not significantly associated with the positions of these regulatory markers. Our statistical modeling and cell-type-specific gene analysis of normal and BRCA1-deficient non-cancer epithelial cells samples shown that RLFS(+) R-loop frequency in luminal cells was mostly increased; in contrast, RLFS(+) R-loops were essentially decreased in basal-like breast cells. Analyzing published gene expression profiles of mammary epithelial cells obtained from disease-free prophylactic mastectomy tissues of BRCA1-mutation carriers and reduction mammoplasty tissues from non-mutation carriers (GSE25835 &amp; GSE19383), we found the differential expressed genes (DEGs) associated with RLFS(+) RNA-DNA hybrid (DRIP-seq) signals. Integrating these findings with our RLFS RNA-DNA hybrid characterizations, we dichotomized the DEGs referring to the luminal and basal-like cell types. According to pathway analysis, a majority of BRCA1-deficient altered transcripts referring to luminal cell’s genes are involved metabolism and cell cycle while basal-like cells involved immune functions, cell signaling, and epithelial-mesenchymal transition pathways. We also found that the genes associated with RLFS clusters present on the sense strand are more associated with nucleic acid binding and transcription regulation, while the genes with an RLFS present on the anti-sense strand are more associated with immune system functions depleted in BRCA1 deficient non-cancer carriers. Thus, for the first time, we demonstrate the roles of RLFS mediated R-looping in the luminal and basal-like breast cells from BRCA1-mutation carrier tissues that specify aberrantly transcribed genes involving in tumorigenesis and immune function pathways. This study suggests novel regulatory mechanics of BRCA1-driven pre-malignant state development and prospective biomarker discovery. Citation Format: Andre Grageda, Vladimir A Kuznetsov. R-loop forming sequences determine early response genes and tumorigenic pathways driven in BRCA1-deficient carriers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-07.

Macrophage migration inhibitory factor is overproduced through EGR1 in TET2low resting monocytes

Somatic mutation in TET2 gene is one of the most common clonal genetic events detected in age-related clonal hematopoiesis as well as in chronic myelomonocytic leukemia (CMML). In addition to being a pre-malignant state, TET2 mutated clones are associated with an increased risk of death from cardiovascular disease, which could involve cytokine/chemokine overproduction by monocytic cells. Here, we show in mice and in human cells that, in the absence of any inflammatory challenge, TET2 downregulation promotes the production of MIF (macrophage migration inhibitory factor), a pivotal mediator of atherosclerotic lesion formation. In healthy monocytes, TET2 is recruited to MIF promoter and interacts with the transcription factor EGR1 and histone deacetylases. Disruption of these interactions as a consequence of TET2-decreased expression favors EGR1-driven transcription of MIF gene and its secretion. MIF favors monocytic differentiation of myeloid progenitors. These results designate MIF as a chronically overproduced chemokine and a potential therapeutic target in patients with clonal TET2 downregulation in myeloid cells.