T-cell immune profile in blood of systemic mastocytosis: Association with disease features.

BACKGROUND: Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated mast cells (MC). KIT-mutated MC display activated features and release MC mediators that might act on the tumour microenvironment and other immune cells. Here, we investigated the distribution of lymphocyte subsets in blood of patients with distinct subtypes of SM and determined its association with other disease features. METHODS: We studied the distribution of TCD4+ and TCD4- cytotoxic cells and their subsets, as well as total NK- and B cells, in blood of 115 SM patients-38 bone marrow mastocytosis (BMM), 67 indolent SM (ISM), 10 aggressive SM (ASM)- and 83 age-matched healthy donors (HD), using spectral flow cytometry and the EuroFlow Immunomonitoring panel, and correlated it with multilineage KITD816V , the alpha-tryptasemia genotype (HαT) and the clinical manifestations of the disease. RESULTS: SM patients showed decreased counts (vs. HD) of TCD4- cytotoxic cells, NK cells and several functional subsets of TCD4+ cells (total Th1, Th2-effector memory, Th22-terminal effector and Th1-like Tregs), together with increased T-follicular-helper and Th1/Th17-like Treg counts, associated with different immune profiles per diagnostic subtype of SM, in multilineal versus MC-restricted KITD816V and in cases with a HαT+ versus HαT- genotype. Unique immune profiles were found among BMM and ISM patients with MC-restricted KITD816V who displayed HαT, anaphylaxis, hymenoptera venom allergy, bone disease, pruritus, flushing and GI symptoms. CONCLUSION: Our results reveal altered T- and NK-cell immune profiles in blood of SM, which vary per disease subtype, the pattern of involvement of haematopoiesis by KITD816V , the HαT genotype and specific clinical manifestations of the disease.

Improved Sézary cell detection and novel insights into immunophenotypic and molecular heterogeneity in Sézary syndrome.

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring.

GlcNAc is a mast-cell chromatin-remodeling oncometabolite that promotes systemic mastocytosis aggressiveness.

Systemic mastocytosis (SM) is a KIT-driven hematopoietic neoplasm characterized by the excessive accumulation of neoplastic mast cells (MCs) in various organs and, mainly, the bone marrow (BM). Multiple genetic and epigenetic mechanisms contribute to the onset and severity of SM. However, little is known to date about the metabolic underpinnings underlying SM aggressiveness, which has thus far impeded the development of strategies to leverage metabolic dependencies when existing KIT-targeted treatments fail. Here, we show that plasma metabolomic profiles were able to discriminate indolent from advanced forms of the disease. We identified N-acetyl-d-glucosamine (GlcNAc) as the most predictive metabolite of SM severity. High plasma levels of GlcNAc in patients with advanced SM correlated with the activation of the GlcNAc-fed hexosamine biosynthesis pathway in patients BM aspirates and purified BM MCs. At the functional level, GlcNAc enhanced human neoplastic MCs proliferation and promoted rapid health deterioration in a humanized mouse model of SM. In addition, in the presence of GlcNAc, immunoglobulin E-stimulated MCs triggered enhanced release of proinflammatory cytokines and a stronger acute response in a mouse model of passive cutaneous anaphylaxis. Mechanistically, elevated GlcNAc levels promoted the transcriptional accessibility of chromatin regions that contain genes encoding mediators of receptor tyrosine kinases cascades and inflammatory responses, thus leading to a more aggressive phenotype. Therefore, GlcNAc is an oncometabolite driver of SM aggressiveness. This study suggests the therapeutic potential for targeting metabolic pathways in MC-related diseases to manipulate MCs effector functions.

Population matched (pm) germline allelic variants of immunoglobulin (IG) loci: Relevance in infectious diseases and vaccination studies in human populations.

Immunoglobulin (IG) loci harbor inter-individual allelic variants in many different germline IG variable, diversity and joining genes of the IG heavy (IGH), kappa (IGK) and lambda (IGL) loci, which together form the genetic basis of the highly diverse antigen-specific B-cell receptors. These allelic variants can be shared between or be specific to human populations. The current immunogenetics resources gather the germline alleles, however, lack the population specificity of the alleles which poses limitations for disease-association studies related to immune responses in different human populations. Therefore, we systematically identified germline alleles from 26 different human populations around the world, profiled by "1000 Genomes" data. We identified 409 IGHV, 179 IGKV, and 199 IGLV germline alleles supported by at least seven haplotypes. The diversity of germline alleles is the highest in Africans. Remarkably, the variants in the identified novel alleles show strikingly conserved patterns, the same as found in other IG databases, suggesting over-time evolutionary selection processes. We could relate the genetic variants to population-specific immune responses, e.g. IGHV1-69 for flu in Africans. The population matched IG (pmIG) resource will enhance our understanding of the SHM-related B-cell receptor selection processes in (infectious) diseases and vaccination within and between different human populations.

Proteomics for Low Cell Numbers: How to Optimize the Sample Preparation Workflow for Mass Spectrometry Analysis.

Nowadays, massive genomics and transcriptomics data can be generated at the single-cell level. However, proteomics in this setting is still a big challenge. Despite the great improvements in sensitivity and performance of mass spectrometry instruments and the better knowledge on sample preparation processing, it is widely acknowledged that multistep proteomics workflows may lead to substantial sample loss, especially when working with paucicellular samples. Still, in clinical fields, frequently limited sample amounts are available for downstream analysis, thereby hampering comprehensive characterization at protein level. To aim at better protein and peptide recoveries, we compare existing and novel approaches in the multistep sample preparation protocols for mass spectrometry studies, from sample collection, cell lysis, protein quantification, and electrophoresis/staining to protein digestion, peptide recovery, and LC-MS/MS instruments. From this critical evaluation, we conclude that the recent innovations and technologies, together with high quality management of samples, make proteomics on paucicellular samples possible, which will have immediate impact for the proteomics community.

The presence of CLL-associated stereotypic B cell receptors in the normal BCR repertoire from healthy individuals increases with age.

BACKGROUND: Aging is known to induce immunosenescence, resulting in alterations in both the innate and adaptive immune system. Here we evaluated the effects of aging on B cell subsets in peripheral blood of 155 immunologically healthy individuals in four age categories (range 20-95y) via multi-parameter flow cytometry. Furthermore, we studied the naive and antigen-experienced B cell receptor (BCR) repertoire of different age groups and compared it to the clonal BCR repertoire of chronic lymphocytic leukemia (CLL), a disease typically presenting in elderly individuals. RESULTS: Total numbers and relative frequencies of B cells were found to decline upon aging, with reductions in transitional B cells, memory cell types, and plasma blasts in the 70 + y group. The BCR repertoire of naive mature B cells and antigen-experienced B cells did not clearly alter until age 70y. Clear changes in IGHV gene usage were observed in naive mature B cells of 70 + y individuals, with a transitional pattern in the 50-70y group. IGHV gene usage of naive mature B cells of the 50-70y, but not the 70 + y, age group resembled that of both younger (50-70y) and older (70 + y) CLL patients. Additionally, CLL-associated stereotypic BCR were found as part of the healthy control BCR repertoire, with an age-associated increase in frequency of several stereotypic BCR (particularly subsets #2 and #5). CONCLUSION: Composition of the peripheral B cell compartment changes with ageing, with clear reductions in non-switched and CD27 + IgG+ switched memory B cells and plasma blasts in especially the 70 + y group. The BCR repertoire is relatively stable until 70y, whereafter differences in IGHV gene usage are seen. Upon ageing, an increasing trend in the occurrence of particular CLL-associated stereotypic BCR is observed.

Bone Marrow Mast Cell Antibody-Targetable Cell Surface Protein Expression Profiles in Systemic Mastocytosis.

Despite recent therapeutic advances, systemic mastocytosis (SM) remains an incurable disease due to limited complete remission (CR) rates even after novel therapies. To date, no study has evaluated the expression on SM bone marrow mast cells (BMMC) of large panel of cell surface suitable for antibody-targeted therapy. In this study, we analyzed the expression profile of six cell-surface proteins for which antibody-based therapies are available, on BMMC from 166 SM patients vs. 40 controls. Overall, variable patterns of expression for the markers evaluated were observed among SM BMMC. Thus, CD22, CD30, and CD123, while expressed on BMMC from patients within every subtype of SM, showed highly variable patterns with a significant fraction of negative cases among advanced SM (aggressive SM (ASM), ASM with an associated clonal non-MC lineage disease (ASM-AHN) and MC leukemia (MCL)), 36%, 46%, and 39%, respectively. In turn, CD25 and FcεRI were found to be expressed in most cases (89% and 92%) in virtually all BMMC (median: 92% and 95%) from both indolent and advanced SM, but with lower/absent levels in a significant fraction of MC leukemia (MCL) and both in MCL and well-differentiated SM (WDSM) patients, respectively. In contrast, CD33 was the only marker expressed on all BMMC from every SM patient. Thus, CD33 emerges as the best potentially targetable cell-surface membrane marker in SM, particularly in advanced SM.

Flow cytometric assessment of leukocyte kinetics for the monitoring of tissue damage.

Leukocyte populations quickly respond to tissue damage, but most leukocyte kinetic studies are not based on multiparameter flow cytometry. We systematically investigated several blood leukocyte populations after controlled tissue damage. 48 patients were assigned to either an anterior or posterolateral total hip arthroplasty. Peripheral blood was collected pre-operatively and at 2 h, 24 h, 48 h, 2 and 6 weeks postoperatively and assessed by flow cytometry for absolute counts of multiple leukocyte populations using standardized EuroFlow protocols. Absolute counts of leukocyte subsets differed significantly between consecutive time points. Neutrophils increased instantly after surgery, while most leukocyte subsets initially decreased, followed by increasing cell counts until 48 h. At two weeks all leukocyte counts were restored to pre-operative counts. Immune cell kinetics upon acute tissue damage exhibit reproducible patterns, which differ between the leukocyte subsets and with "opposite kinetics" among monocyte subsets. Flow cytometric leukocyte monitoring can be used to minimally invasively monitor tissue damage.

KIT D816V-mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression.

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome.

Low-count monoclonal B-cell lymphocytosis persists after seven years of follow up and is associated with a poorer outcome.

Low-count monoclonal B-cell lymphocytosis is defined by the presence of very low numbers of circulating clonal B cells, usually phenotypically similar to chronic lymphocytic leukemia cells, whose biological and clinical significance remains elusive. Herein, we re-evaluated 65/91 low-count monoclonal B-cell lymphocytosis cases (54 chronic lymphocytic leukemia-like and 11 non-chronic lymphocytic leukemia-like) followed-up for a median of seven years, using high-sensitivity flow cytometry and interphase fluorescence in situ hybridization. Overall, the clone size significantly increased in 69% of low-count monoclonal B-cell lymphocytosis cases, but only one subject progressed to high-count monoclonal B-cell lymphocytosis. In parallel, the frequency of cytogenetic alterations increased over time (32% vs 61% of cases, respectively). The absolute number of the major T-cell and natural killer cell populations also increased, but only among chronic lymphocytic leukemia-like cases with increased clone size vs age- and sex-matched controls. Although progression to chronic lymphocytic leukemia was not observed, the overall survival of low-count monoclonal B-cell lymphocytosis individuals was significantly reduced vs non-monoclonal B-cell lymphocytosis controls (P=0.03) plus the general population from the same region (P≤0.001), particularly among females (P=0.01); infection and cancer were the main causes of death in low-count monoclonal B-cell lymphocytosis. In summary, despite the fact that mid-term progression from low-count monoclonal B-cell lymphocytosis to high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia appears to be unlikely, these clones persist at increased numbers, usually carrying more genetic alterations, and might thus be a marker of an impaired immune system indirectly associated with a poorer outcome, particularly among females.

Maturation-associated gene expression profiles along normal human bone marrow monopoiesis.

Human monopoiesis is a tightly coordinated process which starts in the bone marrow (BM) haematopoietic stem cell (HSC) compartment and leads to the production of circulating blood mature monocytes. Although mature monocytes/macrophages have been extensively studied in both normal or inflammatory conditions, monopoiesis has only been assessed in vitro and in vivo animal models, due to low frequency of the monocytic precursors in the normal human BM. Here we investigated the transcriptional profile along normal human BM monopoiesis. Five distinct maturation-associated stages of monocytic precursors were identified and isolated from (fresh) normal human BM through fluorescence-activated cell sorting, and the gene expression profile (GEP) of each monocytic precursor subset was analysed by DNA-oligonucleotide microarrays. Overall, >6000 genes (18% of the genes investigated) were expressed in ≥1 stage of BM monopoiesis at stable or variable amounts, showing early decrease in cell proliferation with increased levels of expression of genes linked with cell differentiation. The here-defined GEP of normal human BM monopoiesis might contribute to better understand monocytic differentiation and the identification of novel monocytic candidate markers, while also providing a frame of reference for the study of monocytic maturation in both neoplastic and non-neoplastic disease conditions involving monocytic precursor cells.

Comprehensive and Systematic Analysis of the Immunocompatibility of Polyelectrolyte Capsules.

The immunocompability of polyelectrolyte capsules synthesized by layer-by-layer deposition has been investigated. Capsules of different architecture and composed of either non-degradable or biodegradable polymers, with either positively or negatively charged outer surface, and with micrometer size, have been used, and the capsule uptake by different cell lines has been studied and quantified. Immunocompatibility studies were performed with peripheral blood mononuclear cells (PBMCs). Data demonstrate that incubation with capsules, at concentrations relevant for practical applications, did not result in a reduced viability of cells, as it did not show an increased apoptosis. Presence of capsules also did not result in an increased expression of TNF-α, as detected with antibody staining, as well as at mRNA level. It also did not result in increased expression of IL-6, as detected at mRNA level. These results indicate that the polyelectrolyte capsules used in this study are immunocompatible.

Clinical, immunophenotypic, and molecular characteristics of well-differentiated systemic mastocytosis.

BACKGROUND: Well-differentiated systemic mastocytosis (WDSM) is a rare variant of systemic mastocytosis (SM) characterized by bone marrow (BM) infiltration by mature-appearing mast cells (MCs) often lacking exon 17 KIT mutations. Because of its rarity, the clinical and biological features of WDSM remain poorly defined. OBJECTIVE: We sought to determine the clinical, biological, and molecular features of a cohort of 33 patients with mastocytosis in the skin in association with BM infiltration by well-differentiated MCs and to establish potential diagnostic criteria for WDSM. METHODS: Thirty-three patients with mastocytosis in the skin plus BM aggregates of round, fully granulated MCs lacking strong CD25 and CD2 expression in association with clonal MC features were studied. RESULTS: Our cohort of patients showed female predominance (female/male ratio, 4:1) and childhood onset of the disease (91%) with frequent familial aggregation (39%). Skin involvement was heterogeneous, including maculopapular (82%), nodular (6%), and diffuse cutaneous (12%) mastocytosis. KIT mutations were detected in only 10 (30%) of 33 patients, including the KIT D816V (n = 5), K509I (n = 3), N819Y (n = 1), and I817V (n = 1) mutations. BM MCs displayed a unique immunophenotypic pattern consisting of increased light scatter features, overexpression of cytoplasmic carboxypeptidase, and aberrant expression of CD30, together with absent (79%) or low (21%) positivity for CD25, CD2, or both. Despite only 9 (27%) of 33 patients fulfilling the World Health Organization criteria for SM, our findings allowed us to establish the systemic nature of the disease, which fit with the definition of WDSM. CONCLUSIONS: WDSM represents a rare clinically and molecularly heterogeneous variant of SM that requires unique diagnostic criteria to avoid a misdiagnosis of cutaneous mastocytosis per current World Health Organization criteria.

Diagnosis and classification of mastocytosis in non-specialized versus reference centres: a Spanish Network on Mastocytosis (REMA) study on 122 patients.

The diagnosis of 'rare diseases', such as mastocytosis, remains a challenge. Despite this, the precise benefits of referral of mastocytosis patients to highly specialized reference centres are poorly defined and whether patients should be managed at non-specialized versus reference centres remains a matter of debate. To evaluate the quality and efficiency of diagnostic procedures performed at the reference centres for mastocytosis in Spain (REMA) versus other non-reference centres, we retrospectively analysed a series of 122 patients, for the overall degree of agreement obtained for the World Health Organization (WHO) diagnostic and classification criteria betwen the referring and REMA centres. Our results showed that not all WHO diagnostic criteria were frequently investigated at the referring centres. Among the five WHO diagnostic criteria, the highest degree of agreement was obtained for serum tryptase levels [median 90% (95% confidence interval 84-96%)]; in turn, the overall agreement was significantly lower for the major histopathological criterion [80% (72-89%)], and the other three minor criteria: cytomorphology [68% (56-80%)] immunophenotyping of BM mast cells [75% (62-87%)] and detection of the KIT mutation [34% (8-60%)]. Referral of patients with diagnostic suspicion of mastocytosis to a multidisciplinary reference centre improves diagnostic efficiency and quality.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

The protein expression profile of meningioma cells is associated with distinct cytogenetic tumour subgroups.

AIMS: Limited information exists about the impact of cytogenetic alterations on the protein expression profiles of individual meningioma cells and their association with the clinicohistopathological characteristics of the disease. The aim of this study is to investigate the potential association between the immunophenotypic profile of single meningioma cells and the most relevant features of the tumour. METHODS: Multiparameter flow cytometry (MFC) was used to evaluate the immunophenotypic profile of tumour cells (n = 51 patients) and the Affymetrix U133A chip was applied for the analysis of the gene expression profile (n = 40) of meningioma samples, cytogenetically characterized by interphase fluorescence in situ hybridization. RESULTS: Overall, a close association between the pattern of protein expression and the cytogenetic profile of tumour cells was found. Thus, diploid tumours displayed higher levels of expression of the CD55 complement regulatory protein, tumours carrying isolated monosomy 22/del(22q) showed greater levels of bcl2 and PDGFRß and meningiomas carrying complex karyotypes displayed a greater proliferation index and decreased expression of the CD13 ectoenzyme, the CD9 and CD81 tetraspanins, and the Her2/neu growth factor receptor. From the clinical point of view, higher expression of CD53 and CD44 was associated with a poorer outcome. CONCLUSIONS: Here we show that the protein expression profile of individual meningioma cells is closely associated with tumour cytogenetics, which may reflect the involvement of different signalling pathways in the distinct cytogenetic subgroups of meningiomas, with specific immunophenotypic profiles also translating into a different tumour clinical behaviour.

Nonaggressive systemic mastocytosis (SM) without skin lesions associated with insect-induced anaphylaxis shows unique features versus other indolent SM.

BACKGROUND: Indolent systemic mastocytosis (ISM) without skin lesions (ISMs(-)) shows a higher prevalence in males, lower serum baseline tryptase levels, and KIT mutation more frequently restricted to bone marrow (BM) mast cells (MCs) than ISM with skin lesions (ISMs(+)). Interestingly, in almost one-half of ISMs(-) patients, MC-mediator release episodes are triggered exclusively by insects. OBJECTIVE: We aimed to determine the clinical and laboratory features of ISMs(-) associated with insect-induced anaphylaxis (insectISMs(-)) versus other patients with ISM. METHODS: A total of 335 patients presenting with MC activation syndrome, including 143 insectISMs(-), 72 ISMs(-) triggered by other factors (otherISMs(-)), 56 ISMs(+), and 64 nonclonal MC activation syndrome, were studied. RESULTS: Compared with otherISMs(-) and ISMs(+) patients, insectISMs(-) cases showed marked male predominance (78% vs 53% and 46%; P < .001), a distinct pattern of MC-related symptoms, and significantly lower median serum baseline tryptase levels (22.4 vs 28.7 and 45.8 µg/L; P ≤ .009). Moreover, insectISMs(-) less frequently presented BM MC aggregates (46% vs 70% and 81%; P ≤ .001), and they systematically showed MC-restricted KIT mutation. CONCLUSIONS: ISMs(-) patients with anaphylaxis triggered exclusively by insects display clinical and laboratory features that are significantly different from other ISM cases, including other ISMs(-) and ISMs(+) patients, suggesting that they represent a unique subgroup of ISM with a particularly low BM MC burden in the absence of adverse prognostic factors.