Monoclonal B-cell Lymphocytosis Research Articles

Genomic instability and genetic heterogeneity in aging: insights from clonal hematopoiesis (CHIP), monoclonal gammopathy (MGUS), and monoclonal B-cell lymphocytosis (MBL).

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

A rare case of IgG4-related aortitis in the thoracic aorta mimicking an intramural hematoma: navigating the diagnostic labyrinth

A 54-year-old female presented with recurrent abdominal pain and new onset chest pain. Chest computed-tomography angiogram detected a thoracic aortic aneurysm with suspected Type A intramural hematoma (IMH) versus aortitis. Initially, conservative management was pursued while awaiting a definitive diagnosis. Differential workup was negative, while additional imaging modalities favored IMH, prompting expedited surgical intervention. During ascending aortic and hemiarch replacement, severe aortitis was unexpectedly discovered without evidence of IMH. Histopathological examination of the aortic specimens identified transmural aortic inflammation with lymphoplasmacytic infiltrate and irregular fibrosis. Numerous IgG4-positive plasma cells were present with IgG4/IgG ratio of 40–50% suggesting IgG4-related disease (IgG4-RD). Subsequent analysis revealed B cells positive for clonal IgH gene rearrangement, and bone marrow biopsy then revealed the same clonal B cells. She was ultimately diagnosed with CLL, the most common phenotype of monoclonal B-cell lymphocytosis, thought to account for the IgG4-predominant plasma cells causing aortitis. Although rare, this case highlights the importance of considering IgG4-related disease (IgG4-RD) as a cause of aortitis when assessing symptomatic patients with aortic pathologies, emphasizing the complexities involved in diagnosing due to a variety of imaging presentation, differentiating, and managing large-vessel vasculitides. Moreover, it underscores the importance of Multidisciplinary Aortic Team care and the use of multiple diagnostic modalities in evaluating ambiguous aortic pathologies.

Incidence, risk factors, and outcomes of patients with monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia who develop venous thromboembolism

BackgroundThe incidence, risk factors, and outcomes of venous thromboembolism (VTE) in patients with chronic lymphocytic leukemia (CLL) and monoclonal B-cell lymphocytosis (MBL) are not well described. ObjectivesWe aimed to determine the clinical characteristics, risk factors, and outcomes of incident VTE in patients with newly diagnosed MBL/CLL and compare the incidence to the age- and sex-matched general population. MethodsUsing the Mayo Clinic CLL Database, we identified 946 patients with newly diagnosed MBL/CLL between 1998 and 2021. Incidence of VTE was identified by querying the electronic health record for VTE-specific International Classification of Diseases-9 and -10 codes and reviewing results of radiographic studies. ResultsEighty patients developed VTE. The incidence of VTE in patients with newly diagnosed MBL/CLL was ∼1% per year. In multivariable analyses, prior history of VTE (hazard ratio [HR]: 5.33; 95% CI: 1.93-14.68, P = .001) and high/very high-risk CLL-International Prognostic Index score (HR: 2.63; 95% CI: 1.31-5.26; P = .006) were associated with an increased risk of VTE; receipt of CLL treatment or occurrence of nonhematologic malignancy was not. Development of VTE was associated with shorter overall survival (HR: 1.82, 95% CI: 1.30-2.55) after adjusting for age, sex, prior history of VTE, and Rai stage. The age- and sex-adjusted VTE incidence rate for patients with MBL/CLL and no prior history of VTE (n = 904) was 1254 per 100 000 person-years compared with 204 per 100 000 person-years in the general population, reflecting a 5.9-fold increase. ConclusionOur study demonstrates a 6-fold increased risk of VTE in patients with MBL/CLL compared with the age- and sex-matched general population.

Utility of Targeted Sequencing Compared to FISH for Detection of Chronic Lymphocytic Leukemia Copy Number Alterations.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

Molecular Subtypes and the Role of TP53 in Diffuse Large B-Cell Lymphoma and Richter Syndrome.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy and a heterogeneous entity comprised of several biologically distinct subtypes. Recently, novel genetic classifications of DLBCL have been resolved based on common mutational patterns indicative of distinct pathways of transformation. However, the complicated and costly nature of the novel classifiers has precluded their inclusion into routine practice. In view of this, the status of the TP53 gene, which is mutated or deleted in 20-30% of the cases, has emerged as an important prognostic factor for DLBCL patients, setting itself apart from other predictors. TP53 genetic lesions are particularly enriched in a genetic subtype of DLBCL that shares genomic features with Richter Syndrome, highlighting the possibility of a subset of DLBCL arising from the transformation of an occult chronic lymphocytic leukemia-like malignancy, such as monoclonal B-cell lymphocytosis. Patients with TP53-mutated DLBCL, including those with Richter Syndrome, have a particularly poor prognosis and display inferior responses to standard chemoimmunotherapy regimens. The data presented in this manuscript argue for the need for improved and more practical risk-stratification models for patients with DLBCL and show the potential for the use of TP53 mutational status for prognostication and, in prospect, treatment stratification in DLBCL.

Clinical Risks for Chronic Lymphocytic Leukemia.

Our understanding of risk factors for the development of chronic lymphocytic leukemia (CLL) is still incomplete and includes genetic and environmental factors. CLL is one of the most familial of all cancers, yet common high-penetrance risk alleles have not been identified. Genome-wide association studies have identified many common variants with low relative risks, whereas exome-wide rare variant analysis has implicated ATM in CLL causation. Environmental factors have also been challenging to identify given the limited understanding of the relevant time period of exposure relative to diagnosis, and the inability to quantify past exposures. Agent Orange and glyphosate herbicides have perhaps the most data to support their role. CLL is preceded by a precursor condition called monoclonal B-cell lymphocytosis (MBL), which could therefore be considered a risk factor, but which itself is likely caused by the same risk factors that ultimately give rise to CLL. Although virtually all people with CLL have a preceding MBL phase, most people with MBL will not develop CLL.

Progressive multifocal leukoencephalopathy as the presenting feature in a patient with occult low-count monoclonal B-cell lymphocytosis.

Low Count Monoclonal B-Cell Lymphocytosis (LC-MBL) is a relatively poorly understood entity which has been suggested to be very common in asymptomatic adults and possibly related to infectious complications despite not progressing to CLL. We describe the first case of Progressive Multifocal Leukoencephalopathy (PML) presenting in a 72-year-old man with LC-MBL but no other immunocompromising conditions. A diagnosis of PML was confirmed with classic MRI findings in association with a high CSF John Cunningham polyomavirus (JCV) viral load (4.09' 105 copies/mL). An extensive search for underlying immunocompromising conditions only demonstrated LC-MBL representing approximately 4% of total leukocytes (0.2' 109/L). This is the first report of PML in association with LC-MBL. Careful review of peripheral blood flow cytometry results is necessary to identify this disorder. Further study of the epidemiology and infectious complications of LC-MBL are warranted.

CD20+ T cells in monoclonal B cell lymphocytosis and chronic lymphocytic leukemia: frequency, phenotype and association with disease progression.

In monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), the expansion of malignant B cells disrupts the normal homeostasis and interactions between B cells and T cells, leading to immune dysregulation. CD20+ T cells are a subpopulation of T cells that appear to be involved in autoimmune diseases and cancer. Here, we quantified and phenotypically characterized CD20+ T cells from MBL subjects and CLL patients using flow cytometry and correlated our findings with the B-cell receptor mutational status and other features of the disease. CD20+ T cells were more represented within the CD8+ T cell compartment and they showed a predominant memory Tc1 phenotype. CD20+ T cells were less represented in MBL and CLL patients vs healthy controls, particularly among those with unmutated IGVH gene. The expansion of malignant B cells was accompanied by phenotypic and functional changes in CD20+ T cells, including an increase in follicular helper CD4+ CD20+ T cells and CD20+ Tc1 cells, in addition to the expansion of the TCR Vβ 5.1 in CD4+ CD20+ T cells in CLL.

Abstract 6152: Genetic associations with hematologic malignancies: An evaluation of CLL polygenic risk score and MBL status in the Mayo Clinic Biobank Cohort

Abstract Chronic Lymphocytic Leukemia (CLL) exhibits a substantial familial risk that extends beyond CLL to other B-cell lymphomas (including follicular lymphoma and diffuse large B-cell lymphoma). Monoclonal B-cell Lymphocytosis (MBL), a precursor to CLL, is also associated with a significant risk for hematologic malignancies overall and for lymphoid malignancies, specifically. Moreover, previous research has shown an association between the CLL polygenic risk score (CLL-PRS) and common lymphoma subtypes. This study aims to further evaluate CLL-PRS associations with hematological cancer overall and by lineage while also considering MBL status a strong risk factor for lymphoid malignancies. Utilizing the Mayo Clinic Biobank, we selected individuals aged 40 years or older, residing in proximal counties, who had no prior hematologic malignancies, and with available biospecimens. MBL screening was done using 8-color flow cytometry, and MBL with CLL (CD5+/CD20dim) or atypical CLL (CD5+/CD20+) immunophenotype were included. CLL-PRS was calculated from 41 known susceptibility variants, with weights being the log of the odds ratios from the largest CLL genome-wide association study. Incident hematologic malignancies were ascertained through ICD codes and confirmed via medical record review. Hazard ratios (HR) and 95% confidence intervals (CIs) for associations with hematologic malignancies were estimated through Cox regression. Of 7,332 participants, 1,150 (16%) screened positive for MBL; median age was 67 years (range 40-101), and 37% male. Median follow-up was 2.9 years (range 0-13), and 56 hematologic cancers were identified. After adjusting for age and sex, the CLL-PRS was associated with increased risk of developing hematologic (HR=1.49, 95%CI=1.12-1.98, P=0.006) which was all attributable to lymphoid cancers (HR=1.79, 95%CI=1.26-2.56, P=0.001). When adjusting for baseline MBL status in the models, the CLL-PRS associations remained significant but were attenuated for hematological (HR=1.33, 95%CI=0.99-1.79, P=0.058) and lymphoid (HR=1.56, 95%CI=1.07-2.26, P=0.020) malignancies, while MBL status was also significantly associated with risk of hematologic (HR=2.76, 95%CI=1.55-4.91, P=0.001) and lymphoid (HR=3.61, 95%CI=1.78-3.32, P<0.001) malignancies. In this prospective cohort study of individuals screened for MBL, CLL-PRS was associated with an elevated risk of hematologic malignancies overall and within the lymphoid lineage, underscoring a shared genetic etiology across many B-cell lymphomas. However, the associations between CLL-PRS and hematologic malignancies were attenuated when accounting for MBL status or when analyzing among MBL-positive individuals, highlighting the role of MBL status in these associations. Further research with expanded cohorts is needed to unravel these complex genetic and clinical interactions. Citation Format: Raphael Mwangi, Dennis P. Robinson, Sara J. Achenbach, Geffen Kleinstern, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie Griffin, Nicholas J. Boddicker, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, James R. Cerhan, Tait D. Shanafelt, Susan L. Slager. Genetic associations with hematologic malignancies: An evaluation of CLL polygenic risk score and MBL status in the Mayo Clinic Biobank Cohort [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 6152.

Abstract 7450: The impact of prior history of non-hematologic malignancies on time to first treatment and overall survival among individuals with CLL or MBL

Abstract Background: The impact of a prior history of non-hematologic malignancy (NHM) on clinical outcomes among individuals with newly diagnosed chronic lymphocytic leukemia (CLL) or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, is not well known. Methods: In this prospective study, we identified individuals with CLL/MBL who were seen in the Division of Hematology at the Mayo Clinic, Rochester. Participants provided consent within nine months of diagnosis, had no prior history of hematological malignancy, and completed a self-reported risk-factor questionnaire, including a prior history of NHM. Time to first treatment (TTFT) was defined as the time between the date of diagnosis and the earliest date of the first CLL treatment, death, or the last known treatment-free follow-up date. Overall survival (OS) was defined as the time from diagnosis to death or the last follow-up. We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations between a prior history of a NHM with OS and TTFT. We adjusted for sex and the CLL International Prognostic Index (CLL-IPI), a score comprised of five key CLL prognostic factors, including age and Rai stage. Results: Among the 1,090 CLL/MBL individuals, 833 (76%) had CLL. The median age at diagnosis was 64 years (range 24-89), with 65% being men, and 230 (21%) reported a prior history of NHM. The most common tumor was non-melanoma skin cancer (NMSC, 8.9%, 96 individuals), followed by prostate, testicular, and penile tumors (7.3%, 51 individuals), and melanoma (1.9%, 20 individuals). CLL/MBL patients with a prior history of a NHM were more likely to be over 65 years old (29%) than under 65 years old (14%, p < 0.0001). After a median follow-up of 2.9 years (range 0-21), 470 CLL/MBL patients progressed, requiring CLL treatment. CLL/MBL individuals with a prior history of NHM had a longer TTFT compared to those without (HR=0.69; 95% CI: 0.53-0.90; p=0.006). Individuals with a prior history of NHM who had either intermediate or high-very high CLL-IPI score had significantly longer TTFT (HR= 0.55; 95% CI: 0.33-0.92; p=0.02, and HR=0.65; 95% CI: 0.43-0.97; p=0.03, respectively) compared to those without NHM. After a median follow-up of 8.6 years (range 0-21), 359 individuals died. CLL/MBL patients with a prior history of NHM also experienced reduced OS compared to those without (HR=1.46; 95% CI: 1.08-1.97, p=0.013), particularly among those with low (HR=1.70; 95% CI: 0.96-3.00, p=0.07) or intermediate-risk (HR=2.08; 95% CI: 1.24-3.51, p=0.006), according to CLL-IPI. After excluding NMSC, results remained constant. Conclusions: At the time of diagnosis, one out of five CLL/MBL individuals had a prior history of a NHM. These individuals had a longer time to first CLL directed treatment and reduced OS, even after accounting for other important CLL prognostic factors, which deserves additional studies. Citation Format: Bryan A. Vallejo, Cristine Allmer, Kari G. Rabe, Dennis P. Robinson, Yucai Wang, Paul J. Hampel, Esteban D. Braggio, Neil E. Kay, James R. Cerhan, Sameer A. Parikh, Susan L. Slager. The impact of prior history of non-hematologic malignancies on time to first treatment and overall survival among individuals with CLL or MBL [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 7450.

Genetic alterations in chronic lymphocytic leukemia and plasma cell neoplasms- a practical guide to WHO HAEM5.

The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) provides a revised classification of lymphoid malignancies including chronic lymphocytic leukemia (CLL) and plasma cell myeloma/multiple myeloma (PCM/MM). For both diseases the descriptions of precursor states such as monoclonal B-cell lymphocytosis and monoclonal gammopathy of uncertain significance (MGUS) have been updated including a better risk stratification model. New insights on mutational landscapes and branching evolutionary pattern were embedded as diagnostic and prognostic factors, accompanied by a revised structure for the chapter of plasma cell neoplasms. Thus, the WHO-HAEM5 leads to practical improvements of biological and clinical relevance for pathologists, clinicians, geneticists and scientists in the field of lymphoid malignancies. The present review gives an overview on the landscape of genetic alterations in CLL and plasma cell neoplasms with a focus on their impact on classification and treatment.

Tumor mutational load is prognostic for progression to therapy among high-count monoclonal B-cell lymphocytosis

AbstractHigh-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.

Pseudolymphomatous primary breast epithelioid angiosarcoma: a case report with presentation as a monoclonal B-cell lymphocytosis

Pseudolymphomatous primary breast epithelioid angiosarcoma: a case report with presentation as a monoclonal B-cell lymphocytosis

Mosaic Chromosomal Alterations and Monoclonal B-Cell Lymphocytosis: Relationship and Risk of Hematologic Malignancies

Background: Monoclonal B-cell lymphocytosis (MBL) is a precursor condition to chronic lymphocytic leukemia (CLL) characterized by the presence of circulating monoclonal B-cell populations in the peripheral blood of otherwise healthy individuals. MBL increases risk of several adverse clinical outcomes, including development of hematological malignancies. Mosaic chromosomal alterations (mCAs) are a form of clonal hematopoiesis which affect large segments of DNA and include gains, losses, and copy number neutral loss of heterozygosity events. mCAs can be detected from SNP-array genotyping. Prior work has suggested mCAs as a potent risk factor for hematological malignancies. However, the nature of the relationship between mCAs and MBL is unknown. We sought to determine 1) the association between mCAs and MBL and 2) their combined effect on risk of hematological malignancies, in a large, prospectively collected, and well-annotated cohort of individuals with known MBL, mCA, and clinical outcomes. Methods: We examined the prevalence of mCA in a cohort of individuals from the Mayo Clinic Biobank who were screened for MBL using a sensitive eight-color flow cytometry assay. We classified MBL by the size of the clone relative to total B-cells, with high count (HC) defined as those individuals who had a percent clonal B-cell count ≥85% out of total B-cell count and low-count (LC) otherwise. Autosomal mCAs were detected from whole blood DNA collected at the time of MBL screening using a SNP-array-based assay. The MoChA pipeline was used to identify mCA events. Logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CIs). To examine incident hematological cancers, we identified and validated all hematological cancers through medical record abstraction using the International Statistical Classification of Diseases codes. Cox regression was used to calculate hazard ratios (HR) and 95% CIs, stratified by MBL status or autosomal mCA status. All models were adjusted for age and sex. Results: We evaluated the prevalence of autosomal mCA in 3,764 individuals (792 individuals with MBL of whom 64 had HC-MBL and 2,972 without MBL). The prevalence of autosomal mCA was 7.7% ( n=288) with a median mCA cell fraction of 0.09 (range 0.01-1). Among those with and without MBL overall, the prevalence of mCA was 15.5% ( n=123) and 5.6% ( n=165), respectively (Figure 1). Compared to those without an autosomal mCA, individuals with an autosomal mCA were 2.4-fold more likely to have MBL (95% CI 1.9-3.2; P =2.0x10 -11; Figure 1). CLL-associated mCA (i.e., those commonly altered in CLL including trisomy 12 and deletion of 6q, 11q, 13q, and 17p) were present in 42 individuals, of whom 38 (90.5%) had MBL overall and 30 (71.4%) individuals had HC-MBL. Compared to those without a CLL-associated mCA, individuals with a CLL-associated mCA were 539.39-fold more likely to have HC-MBL (95% CI 190.3-1975.9; P <2.0x10 -16) and 6.7-fold more likely to have LC-MBL (95% CI 2.0-26.5; P =0.0031; Figure 1). Next, we evaluated the relationship between MBL and mCA with risk of developing a hematologic malignancy in 3,009 individuals with follow-up data (median 10 years). Forty-one individuals developed an incident hematological malignancy (68.3% lymphoid). When we evaluated the effect of autosomal mCA stratified by MBL status we observed elevated but non-significant risk of hematological malignancies (Figure 2). When we evaluated the effect of MBL stratified by autosomal mCA status we observed elevated and significant increased risk of developing a hematological malignancy (Figure 2). Conclusions: We found that the presence of an autosomal mCA is highly associated with having MBL. In particular, >90% of CLL-associated mCA carriers had MBL with ~7- and ~540-fold increased association for LC- and HC-MBL, respectively. These results suggest that almost all individuals in large biobanks with CLL-associated mCAs have undiagnosed MBL. However, not all individuals with MBL have an mCA. When evaluating risk of hematological malignancies, we found autosomal mCAs had >2-fold elevated but non-significant effects when stratified by MBL status while MBL remained a significant risk factor regardless of autosomal mCA status. Collectively these results support that MBL remains an important risk factor and suggests that mCAs hold potential to identify a subgroup of individuals at greater risk of developing a hematological malignancy.

Not All Lymphocytosis Is Chronic Lymphocytic Leukemia (CLL): A Retrospective Review of Patients Referred to an Academic Hematology Oncology Center for CLL

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults, especially in elderly patients. Most patients with CLL are asymptomatic at presentation and have routine blood work that prompts further investigation. In patients with CLL, their complete blood cell count with differential typically demonstrates lymphocytosis due to the increased number of malignant B-lymphocytes. Although lymphocytosis can be suggestive of CLL, diagnostic workup can sometimes reveal an alternative diagnosis. Here we describe a retrospective review of 36 patients referred to our hematology oncology clinic for the suspected diagnosis of CLL. Out of the 36 patients referred for CLL, 8 patients (22%) had diagnostic workup confirming an alternative diagnosis including non-Hodgkin lymphoma (NHL) (5 patients), hairy cell leukemia (1 patient), and monoclonal B-cell lymphocytosis (2 patients). Only 28/36 (78%) of total patients had flow cytometry confirming the diagnosis of CLL. Out of the 36 total patients, 34 patients had lab work revealing lymphocytosis (absolute lymphocyte count greater or equal to 5.0 10 3/mL) prompting referral. At presentation, total patients (34/36) with lymphocytosis had an absolute lymphocyte count range of 5-593.0 (10 3/mL), median of 17.6 (10 3/mL), and average of 50.8 (10 3/mL). 27/34 (79%) of patients with lymphocytosis were diagnosed with CLL, while 7/34 (21%) of patients with lymphocytosis initially suggestive of CLL were diagnosed with alternative diagnoses (follicular lymphoma (1), marginal zone lymphoma (2), mantle lymphoma (1), hairy cell leukemia (1), and monoclonal B-cell lymphocytosis (2)). The patients with lymphocytosis not diagnosed with CLL (7/34 patients) had an absolute lymphocyte count range of 5-105.6 (10 3/mL), median of 18.27 (10 3/mL), and average of 45.2 (10 3/mL). Their average age at presentation was 63 years old (range of 53-80). Most common symptoms at presentation were asymptomatic (3), B-symptoms plus lymphadenopathy (2), lymphadenopathy (1), and gastrointestinal symptoms (1). The asymptomatic patients were monoclonal B-cell lymphocytosis (2) and mantle lymphoma (1). Hairy cell leukemia, marginal zone lymphoma, and follicular lymphoma had significantly elevated absolute lymphocyte count (86.8, 105.6, 89.45 (10 3/mL) respectively) and associated anemia. Only one patient referred for CLL was diagnosed with follicular lymphoma had anemia and thrombocytopenia without lymphocytosis. The patients with lymphocytosis confirmed to have CLL (27/34 patients) had an absolute lymphocyte count range of 5-593 (10 3/mL), median of 17.6 (10 3/mL), and average of 50.8 (10 3/mL). Their average age at presentation was 70 years old (range 54-93). Most common symptoms at presentation were asymptomatic (16), B-symptoms plus lymphadenopathy (5), lymphadenopathy only (2), B-symptoms only (2), and neck/back pain (2). 59% of patients were asymptomatic with abnormal lab work including lymphocytosis only (14), anemia (1), and abnormal M-spike on serum protein electrophoresis (1). We wish to emphasize the role of flow cytometry and its value in confirming diagnosis of CLL. Even if clinical suspicion for CLL is high, it is important to not solely rely on complete blood cell count and peripheral lymphocyte count. NHL, hairy cell leukemia, and monoclonal B-cell lymphocytosis can have similar lab work and initial presenting symptoms to patients with CLL. A few patients in this review were receiving chemotherapy for CLL initially and were later found to have a different diagnosis. It is important to keep clinical suspicion high for alternative diagnoses for lymphocytosis to guide appropriate management.

Aberrant Heterochromatin Silences Immune Response Genes in Chronic Lymphocytic Leukemia

Introduction: Recent studies have shown that clonal B cells derived from patients with chronic lymphocytic leukemia (CLL) have altered histone modification landscapes compared to B cells from healthy controls. We have also shown that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells, implicating the interaction between oncogenic signaling and epigenetic machinery in CLL (Wang et al., Blood Cancer J. 2022; Wang et al., Cancer Res (2023) 83 (7_Supplement): 4725). However, the majority of the chromatin studies in CLL concentrate on the active enhancers associated with oncogene expression; there is a very limited understanding of lost enhancers in CLL: 1) What is the function of lost enhancers; 2) How are these lost enhancers maintained in CLL; 3) Can these lost enhancers be detected at the precursor state of CLL, monoclonal B-cell lymphocytosis (MBL); 4) if this aberrant chromatin signature affected by CLL treatment. Methods: We systematically mapped the genome-wide histone modification profile (histone marks: H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, and H3K36me3) in 22 previously untreated CLL samples, 6 high count MBL samples, and peripheral blood B cells from 6 healthy donors by Cleavage Under Targets and Tagmentation (CUT&Tag). We also analyzed B cells from 8 patients before and after one year of continuous Bruton tyrosine kinase inhibitor (BTKi) ibrutinib treatment. The genomic regions with a decrease in both H3K4me1 and H3K27ac enrichment (analyzed by Deseq2, fold change > 1, p < 0.01) were named lost enhancers. We used CRISPR-mediated epigenetic editing to study the role of gained H3K9me3 on EBF1 gene expression. Results: Compared to normal B cells, the lost enhancers (genomic regions defined by decreased enrichment of H3K4me1 and H3K27ac) in CLL B cells showed increased heterochromatin marks (H3K9me3 and H3K27me3) enrichment. Importantly, these enhancers are associated with genes downregulated in CLL and involved in the immune response, indicating the source of epigenetic impact on impaired immunity in CLL. We further found that this aberrant chromatin structure also exits in clonal B cells from the precursor stage of MBL, implicating the importance of epigenetic silencing in CLL evolution. In particular, we found the lost enhancer signature across the gene locus EBF1, an important transcription factor that defines B cell lineage and regulates normal B cell activation. Using CRISPR-mediated epigenetic editing, we demonstrated that the gain of H3K9me3 is sufficient to silence the EBF1 enhancer and its gene expression. Finally, we found that while BTKi ibrutinib treatment partially inhibits the CLL-gained enhancers in some patients, it failed to restore the lost enhancers in CLL B cells. Conclusion: In summary, we have elucidated an epigenetic silencing mechanism that may contribute to the loss of physiological function of normal B cells, which places the clonal B cells at a selection advantage during CLL evolution and increases the risk of serious infections in CLL and MBL ( Fig 1).

A Snapshot of the Management of Chronic Lymphocytic Leukemia in Italy. Preliminary Analysis on over 3000 Patients Enrolled in the Gimema CLL2121 Trial

In recent years the management of patients (pts) with chronic lymphocytic leukemia (CLL) has benefited from a deeper knowledge of the mechanisms underlying the disease and from the development of novel therapeutic approaches. That notwithstanding, local and national accessibility to drugs and tests may lead to distinct “real-world” practices in terms of management of pts that are worth of being recorded and compared to understand the degree of reproducibility and applicability of international guidelines. This could also be relevant for the design of future clinical trials, more tailored to the true patient's needs. To this end, within the GIMEMA cooperative study group the observational retrospective and prospective CLL2121 study (NCT04867915) has been designed with the objective of evaluating the diagnosis and management of CLL in all hematological centers in Italy through the assessment of: 1) the methods and the actual diagnostic/prognostic work-up capacity; 2) the algorithms applied to define disease progression and treatment requirements with respect to national and international guidelines; 3) the clinical and biological variables not strictly associated with CLL, but capable of influencing the clinical course and overall survival; 4) the true incidence of some rarer complications associated with CLL. The study consists of a collection of clinico-biological data from all pts with newly diagnosed CLL, small lymphocytic lymphoma (SLL) or CLL-like monoclonal B-cell lymphocytosis (MBL), according to the iwCLL 2018 criteria in Italy. The retrospective part aims at including all cases followed at the participating centers with a diagnosis between January 2010 and September 2021, while the prospective part will include all pts with a documented diagnosis of CLL, SLL or MBL between September 2021 and September 2025. In this pilot analysis of the study, we examined pts' demographics, diagnosis, treatment line and type. Data were collected using the REDCap electronic data capture platform, analyzed using the SAS software v.9.4 and reported as numbers and frequencies. Between 2 November 2021 and 28 June 2023, 3294 eligible pts were enrolled in 75 hematology centers (out of the total of 110 centers who will be activated during the study) across the entire Italian territory. At the time of the present report, 3033 pts had clinical data available for our preliminary analysis. The vast majority of pts registered (N=2599, 85.7%) belonged to the retrospective cohort while only a minority (N=434, 14.3%) to the prospective cohort. Pts had a median age of 68 years ranging from 29 to 97; 60% of pts were males. 2630 pts (86.8%) had a diagnosis of CLL, 187 (6.2%) of SLL and 214 (7.0%) of MBL (2 missing information). Among those with available results 112 pts (12.6%) were TP53 mutated, 526 (45.6%) del(13q) positive, 174 (14.9%) del(11q) positive, 142 (11.9%) del(17p) positive and 226 (20.4%) presented a trisomy 12. The majority of pts (57.6%) were untreated, while 42.4% have been treated. Within the latter subset, 67.3% of pts have received one line of therapy, 21.4% 2 lines of therapy, 11.3% ≥3 lines of therapy. The most common therapeutic regimens were the combination of chemotherapy with an anti-CD20 antibody (39.3%), mainly rituximab, and those based on BTK inhibitors (33.3%), mainly ibrutinib. Chemotherapy alone was used in 12% of pts. Only 5% of pts was treated with the BCL2 inhibitor venetoclax. In the remaining 10% of pts, other approaches were used. This is the initial report of a nation-based real-world data collection aimed at describing the biological and clinical features of pts diagnosed with CLL in virtually all Italian hematology centers starting from 2010. The pattern of treatments highlighted in our preliminary analysis, with a wide use of the watch & wait policy and of chemoimmunotherapy, will help understand how the introduction of novel therapies impacted treatment habits also in light of the timing of the local reimbursement policy. The different time of drug access in Italy, typically delayed after the EU approval, may also have affected the limited use of venetoclax-based treatment. The continuous accrual of pts in this study will allow to obtain a close-to-registry vision of CLL management in Italy over time, in terms of coverage of the entire country but enriched with the granularity of the data and flexibility of the collection typical of a real-world study.

Whole Genome Classification of Pre-Malignant B-Cells Disorder of the Oxplored Study

Background. Chronic lymphocytic leukaemia (CLL) is always preceded by preclinical clonal B-cell disorders such as monoclonal B-cell lymphocytosis (MBL). Pre-malignant clonal B-cells are identifiable in asymptomatic individuals, a minority of whom will progress to symptomatic CLL. While some people remain at pre-cancerous or asymptomatic early stage CLL, others experience clinically aggressive disease. There is currently no method to effectively stratify individuals at higher risk of developing symptomatic CLL. Despite recent advances in treatment options available, the disease remains incurable. Therefore, we need to expand our knowledge and improve current classification methods. Here, we propose a molecular classifier using whole genome sequencing (WGS). This classifier was obtained by performing genomic characterization in pre-malignancy / early stages of the disease and comparing the genomic subgroups with those of symptomatic CLL. Methods. Individuals with newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic chronic lymphocytic leukaemia were enrolled in the OXPLORED clinical trial (Oxford Pre-cancerous Lymphoproliferative Disorders: Analysis and Interception study). We performed WGS in 400 samples from 200 individuals (sorted CD19+ B-cells as tumour, and matched salivary DNA as germline) to detect somatic alterations. We derived immunoglobulin heavy chain variable (IGHV) mutational status, stereotype and an additional 186 genomic features as previously described (Robbe*, Ridout* et al. Nature Genetics 2022) including known and recently discovered candidate drivers of CLL, recurrent structural variants, mutational signatures, genomic complexity measures, mutational burden, and pathway alterations. All the data was compiled into a matrix to extract meaningful sets of features to cluster patients' genomes using non-negative matrix factorization (NMF). Genomic findings were compared to the largest CLL whole genome cohort of symptomatic patients requiring frontline treatment (n=443). Results. The IGHV mutational status was found unmutated in 31% of individuals and hypermutated in 69% (excluding missing data for 6 genomes). Next, we considered mutations and CNAs in 58 common CLL drivers and 34 regions with recurrent CNAs. Although numbers were lower than in frontline CLL, most individuals presented at least one driver (86%) or more (57%) (by comparison, the CLL frontline cohort presented at least one CLL driver in 98% of patients). Genomic complexity, defined as 4 or more CNAs was detected in 12%, and complex genome defined as the presence of both CN gains and CN losses in 22%, indicating that newly diagnosed monoclonal B-cell lymphocytosis and early asymptomatic CLL present a relatively high degree of genomic complexity similar to what was observed in frontline CLL. The most common driver was del13q (60%), including as a sole driver in a third of these genomes. Other recurrent alterations were IGLL5 mutations (20%), trisomy 12 (9%), and mutations in MYD88 (8%), CREBBP (5%), NOTCH1 (5%), and SPEN (5%). Noncoding elements were also found mutated, including 30 promoters (including BIRC3 in 7%), five UTRs and eight enhancers, including BCL6 and PAX5 enhancers in 18% and 3.4% of individuals, respectively. When comparing this cohort with CLL genomes, we found that alterations in the main cancer pathways were depleted in OXPLORED, including known drivers such as ATM, NOTCH1, SF3B1. Finally, moving away from single-alteration grouping, we clustered the 643 genomes (443 CLL + 200 OXPLORED) using 186 genomic features by applying NMF. Importantly, we found distinct genomic profiles in the early disease cohort, including a subgroup like symptomatic CLL at frontline and another distinct subgroup, hinting towards identifying a genomic subgroup at higher risk of progressing to CLL. Conclusion: The comprehensive characterisation of genomic events that determine progression to malignancy are essential to understand the mechanisms of disease progression. Our study opens new horizons towards identifying individuals at higher risk and might pinpoint to individuals who need early intervention with curative intent. Therefore, we propose to analyse newly generated whole-genome sequencing (WGS) data to establish genomic subgroups in early CLL / pre-malignancy stages.

LGL in MGUS or MGUS in LGL? Lessons of Nature and Their Clinical Implications

Associations between seemingly unrelated entities often reveal important lessons of nature, instructive for the understanding of mutual pathogenic roots. While investigating the clinical features of T-cell large granular lymphocytic leukemia (LGL), we noted the frequent presence of plasma cell dyscrasias/monoclonal gammopathies (MGs), chiefly MGUS. There are several potential explanations for this co-occurrence: 1) similar demographics (both conditions mostly presenting in the elderly); 2) the possibility that the two precursors originate from an initial polyclonal immune response to a common target; and 3) as alternative, that monoclonal plasma cells or the Ig product may be the targets of cytotoxic T-LGL cells in a context of tumor surveillance reaction. For instance, clonal TCR may recognize the M protein idiotype or vice versacausing aberrant activations. In addition, while MGUS should not be associated with cytopenias, responses to anti-myeloma therapies have been observed in LGL with MGUS refractory to traditional immunosuppressive therapies. We hypothesized that the study of clinical and epidemiologic features of MGs and LGL may be helpful to elucidate some of these fundamental questions. To address this, a dual approach was applied: 1) MG-forward strategy: screening of LGL in 2348 individuals with documented diagnosis of MGs and available bone marrow exam; 2) LGL-forward strategy: search of MGs in 235 LGL cases with M protein assessment. Overall, among 2348 cases with MGs, we identified 42 cases (1.8%) with concurrent evidence of LGL. We then selected an exploratory cohort of 787 MG patients with clinical annotations for further phenotypic dissection and comparative analyses. Within 533 patients with non-active MGs (i.e., MGUS or smoldering myeloma -SM), 144 (27%) had cytopenias of any type and grade, which could not be attributable to plasma cell infiltration. Screening for comorbid conditions explaining cytopenias left 32 cases (22%) presumed to be idiopathic. LGL was overrepresented in MGUS-SM with cytopenias (6 vs 0.3% in cases without counts alterations, p< .001). This frequency was higher when considering only cases with idiopathic cytopenias (22 vs 1.8% in cases with cytopenias of other causes, p< .001) ( FigA). Reverse analysis of the LGL cohort demonstrated that 71 (30%) LGL cases had M proteins. Overall, the MG/LGL dyad was more frequently associated with concurrent autoimmune conditions (54%), as well as monoclonal B-cell lymphocytosis and other B-cell lymphoid neoplasms (19%). Next, we compared cases with MG/LGL vs those with MGs (N = 775) or LGL (N = 157) exclusively. When compared to the MGs cohort, MG/LGL cases presented a younger age (median 64 vs 70 years, p < .001), had higher frequency of M protein of the IgM type (21 vs 10%, p= .002), and oftentimes exhibited small/transient paraproteinemia (42 vs 2%, p< .001). No specific features were enriched in the MG/LGL cases when compared to the LGL cohort, except for a higher proportion of proliferations of NK-cell subtype (12 vs 4%, p= .02). Finally, to confirm the observed association, we focused on the subgroup of non-active MGs by excluding cases with initial diagnosis of MM or lymphoma. Multivariable analysis (adjusting by sex, age and MG risk) revealed a lower risk of immunoparesis (aOR: 0.4, 95%CI: 0.2 - 0.7, p= .002) in MG/LGL as opposed to MG without LGL, whereas no effect was found in progression to MM/lymphoma (aHR: 0.9, 95%CI: 0.4 - 2.1, p= .81 in a competitive-risk setting). Moreover, when opposed to its MG and LGL counterparts, MG/LGL more frequently presented with severe cytopenias (aOR vsMGs: 28.9, 95%CI: 14.0 - 59.0, p< .001; aOR vsLGL: 2.7, 95%CI: 1.7 - 5.6, p= .008), and with immune-mediated cytopenias (aOR vsMGs: 10.5, 95%CI: 4.9 - 22.2, p< .001; aOR vsLGL: 2.8, 95%CI: 1.0 - 4.8, p= .04) ( FigB). In conclusion, our study reveals that: 1) the association between MGUS and LGL is more than an age-related epidemiological coincidence; 2) distinctive nosologic features for the MG/LGL dyad; and 3) that even “non-active” MGs associated with LGL might be of hematological clinical significance through humoral or cellular immune mechanisms. These findings point towards alternative treatment strategies of cytopenias in MGUS and LGL (immunosuppression vs anti-myeloma therapies, respectively), and provide impetus for the investigation of tumor surveillance in MGUS vs common etiologic factors intertwining these clonopathies.

Other malignancies in the history of CLL: an international multicenter study conducted by ERIC, the European Research Initiative on CLL, in HARMONY

Patients with chronic lymphocytic leukemia (CLL) have a higher risk of developing other malignancies (OMs) compared to the general population. However, the impact of CLL-related risk factors and CLL-directed treatment is still unclear and represents the focus of this work. We conducted a retrospective international multicenter study to assess the incidence of OMs and detect potential risk factors in 19,705 patients with CLL, small lymphocytic lymphoma, or high-count CLL-like monoclonal B-cell lymphocytosis, diagnosed between 2000 and 2016. Data collection took place between October 2020 and March2022. In 129,254 years of follow-up after CLL diagnosis, 3513 OMs were diagnosed (27.2 OMs/1000 person-years). The most common hematological OMs were Richter transformation, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Non-melanoma skin (NMSC) and prostate cancers were the most common solid tumors (STs).The only predictor for MDS and AML development was treatment with fludarabine and cyclophosphamide with/without rituximab (FC±R) (OR=3.7; 95% CI=2.79-4.91; p<0.001). STs were more frequent in males and patients with unmutated immunoglobulin heavy variable genes (OR=1.77; 95% CI=1.49-2.11; p<0.001/OR=1.89; 95% CI=1.6-2.24; p<0.001).CLL-directed treatment was associated with non-melanoma skin and prostate cancers (OR=1.8; 95% CI=1.36-2.41; p<0.001/OR=2.11; 95% CI=1.12-3.97; p=0.021). In contrast, breast cancers were more frequent in untreated patients (OR=0.17; 95% CI=0.08-0.33; p<0.001).Patients with CLL and an OM had inferior overall survival (OS) than those without. AML and MDS conferred the worst OS (p<0.001). OMs in CLL impact on OS. Treatment for CLL increased the risk for AML/MDS, prostate cancer, and NMSC. FCR was associated with increased risk for AML/MDS. AbbVie, and EU/EFPIAInnovative Medicines Initiative Joint Undertaking HARMONY grant n° 116026.