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Background: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB., Methods: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available., Results: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB., Conclusions: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples., (© 2021 International Clinical Cytometry Society.)

Background: Multidimensional flow cytometry (MFC) is routinely used for the diagnosis and follow-up of hematolymphoid neoplasms but its contribution to the identification of non-hematolymphoid malignant tumors is limited., Methods: The presence of non-hematolymphoid cells in clinical samples obtained via minimally invasive methods was ascertained by using a panel of monoclonal antibodies previously developed in our laboratory comprising a mixture of antibodies: CD9-PacB/CD45-OC515/CD57-FITC/CD56-PE/CD3-PerCP-Cy5.5/CD117-PE-Cy7/CD326-APC/CD81-APC-C750. Histopathological studies were performed using standard techniques., Results: 164 specimens of different origins were included. Malignancy was finally confirmed in 142 (86.5%), while 22 non neoplastic samples were identified. The most frequent diagnosis was small cell lung carcinoma (SCLC) (50%). High sensitivity (S = 98.6%) was reached combining MFC and conventional pathology. Individual markers differed according to the cellular origin of the neoplasm, with neuroendocrine tumors showing a unique immunophenotypic profile (CD56+ CD326+ CD117-/+ and variable tetraspanins expression). Principal component analysis efficiently distinguished SCLC from other tumor samples. In immune cell populations, differences between reactive and malignant biopsies were found in different cell compartments, especially in B cells and Plasma cells. Differences also emerged in the percentage of CD4+ CD8- T cells, CD4-CD8+ T cells and NK cells and these were dependent on the origin of the tumor cells., Conclusions: These results support the use of MFC as a rapid and valuable technique to detect non-hematolymphoid tumoral cells in clinical specimens, providing an initial orientation to complement hystopathological studies and allow a more precise diagnosis, especially in neuroendocrine neoplasms. The impact of different immune cell patterns warrants further research., (© 2022 International Clinical Cytometry Society.)

The exquisite coupling between herpesvirus and human beings is the result of millions of years of relationship, coexistence, adaptation, and divergence. It is probably based on the ability to generate a latency that keeps viral activity at a very low level, thereby apparently minimising harm to its host. However, this evolutionary success disappears in immunosuppressed patients, especially in haematological patients. The relevance of infection and reactivation in haematological patients has been a matter of interest, although one fundamentally focused on reactivation in the post-allogeneic stem cell transplant (SCT) patient cohort. Newer transplant modalities have been progressively introduced in clinical settings, with successively more drugs being used to manipulate graft composition and functionality. In addition, new antiviral drugs are available to treat CMV infection. We review the immunological architecture that is key to a favourable outcome in this subset of patients. Less is known about the effects of herpesvirus in terms of mortality or disease progression in patients with other malignant haematological diseases who are treated with immuno-chemotherapy or new molecules, or in patients who receive autologous SCT. The absence of serious consequences in these groups has probably limited the motivation to deepen our knowledge of this aspect. However, the introduction of new therapeutic agents for haematological malignancies has led to a better understanding of how natural killer (NK) cells, CD4+ and CD8+ T lymphocytes, and B lymphocytes interact, and of the role of CMV infection in the context of recently introduced drugs such as Bruton tyrosine kinase (BTK) inhibitors, phosphoinosytol-3-kinase inhibitors, anti-BCL2 drugs, and even CAR-T cells. We analyse the immunological basis and recommendations regarding these scenarios., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alonso-Álvarez, Colado, Moro-García and Alonso-Arias.)

The role of decentralized assessment of measurable residual disease (MRD) for risk stratification in acute myeloid leukemia (AML) remains largely unknown, and so it does which methodological aspects are critical to empower the evaluation of MRD with prognostic significance, particularly if using multiparameter flow cytometry (MFC). We analyzed 1076 AML patients in first remission after induction chemotherapy, in whom MRD was evaluated by MFC in local laboratories of 60 Hospitals participating in the PETHEMA registry. We also conducted a survey on technical aspects of MRD testing to determine the impact of methodological heterogeneity in the prognostic value of MFC. Our results confirmed the recommended cutoff of 0.1% to discriminate patients with significantly different cumulative-incidence of relapse (-CIR- HR:0.71, P < 0.001) and overall survival (HR: 0.73, P = 0.001), but uncovered the limited prognostic value of MFC based MRD in multivariate and recursive partitioning models including other clinical, genetic and treatment related factors. Virtually all aspects related with methodological, interpretation, and reporting of MFC based MRD testing impacted in its ability to discriminate patients with different CIR. Thus, this study demonstrated that "real-world" assessment of MRD using MFC is prognostic in patients at first remission, and urges greater standardization for improved risk-stratification toward clinical decisions in AML., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

B7-H6, a ligand for the NK activating receptor NKp30, has been identified as a biomarker of poor prognosis in several solid cancers. However, little is known about the role of B7-H6 and the mechanisms that control its expression in acute myeloid leukemia (AML). Epigenome modulation, including epigenomic reader dysregulation, is one of the hallmarks of AML. Bromodomain-containing protein 4 (BRD4), the best-known member of the BET family of epigenetic readers, is overexpressed in AML cells and regulates the transcription of genes involved in the pathogenesis of AML, as MYC oncogene. Here, we analyze the role of BRD4 in regulating B7-H6 in AML cells. Results demonstrated that the specific inhibition of BRD4 drastically reduces the expression of B7-H6 in AML cells. Histone acetylation mediated by CBP30/P300 facilitates the binding of BRD4 to the B7-H6 promoter, which recruits the P -TEFb elongation factor that phosphorylates RNA polymerase II, thereby activating B7-H6 transcription. BRD4 also co-bounded with JMJD6 at the distal enhancer of the B7-H6 gene. Metabolic modulation with metformin modifies the acetylation pattern in the B7-H6 promoter, impairing BRD4 binding, thereby inhibiting B7-H6 expression. B7-H6 knockdown induces the apoptosis in HEL-R cell line. Moreover, a high level of B7-H6 expression in AML patients is related to increased BRD4 levels, myelodysplastic-derived AML, and del5q, the two latter being associated with poor prognosis. Our data show that BRD4 is a positive regulator of the pro-tumorigenic molecule B7-H6 and that the blockage of the B7-H6 is a potential therapeutic target for the treatment of AML., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Several risk stratification models have been proposed in recent years for systemic mastocytosis but have not been directly compared. Here we designed and validated a risk stratification model for progression-free survival (PFS) and overall survival (OS) in systemic mastocytosis on the basis of all currently available prognostic factors, and compared its predictive capacity for patient outcome with that of other risk scores., Methods: We did a retrospective prognostic modelling study based on patients diagnosed with systemic mastocytosis between March 1, 1983, and Oct 11, 2019. In a discovery cohort of 422 patients from centres of the Spanish Network on Mastocytosis (REMA), we evaluated previously identified, independent prognostic features for prognostic effect on PFS and OS by multivariable analysis, and designed a global prognostic score for mastocytosis (GPSM) aimed at predicting PFS (GPSM-PFS) and OS (GPSM-OS) by including only those variables that showed independent prognostic value (p<0·05). The GPSM scores were validated in an independent cohort of 853 patients from centres in Europe and the USA, and compared with pre-existing risk models in the total patient series (n=1275), with use of Harrells' concordance index (C-index) as a readout of the ability of each model to risk-stratify patients according to survival outcomes., Findings: Our GPSM-PFS and GPSM-OS models were based on unique combinations of independent prognostic factors for PFS (platelet count ≤100 × 10 9 cells per L, serum β2-microglobulin ≥2·5 μg/mL, and serum baseline tryptase ≥125 μg/L) and OS (haemoglobin ≤110 g/L, serum alkaline phosphatase ≥140 IU/L, and at least one mutation in SRSF2, ASXL1, RUNX1, or DNMT3A). The models showed clear discrimination between low-risk and high-risk patients in terms of worse PFS and OS prognoses in the discovery and validation cohorts, and further discrimination of intermediate-risk patients. The GPSM-PFS score was an accurate predictor of PFS in systemic mastocytosis (C-index 0·90 [95% CI 0·87-0·93], vs values ranging from 0·85 to 0·88 for pre-existing models), particularly in non-advanced systemic mastocytosis (C-index 0·85 [0·76-0·92], within the range for pre-existing models of 0·80 to 0·93). Additionally, the GPSM-OS score was able to accurately predict OS in the entire cohort (C-index 0·92 [0·89-0·94], vs 0·67 to 0·90 for pre-existing models), and showed some capacity to predict OS in advanced systemic mastocytosis (C-index 0·72 [0·66-0·78], vs 0·64 to 0·73 for pre-existing models)., Interpretation: All evaluated risk classifications predicted survival outcomes in systemic mastocytosis. The REMA-PFS and GPSM-PFS models for PFS, and the International Prognostic Scoring System for advanced systemic mastocytosis and GPSM-OS model for OS emerged as the most accurate models, indicating that robust prognostication might be prospectively achieved on the basis of biomarkers that are accessible in diagnostic laboratories worldwide., Funding: Carlos III Health Institute, European Regional Development Fund, Spanish Association of Mastocytosis and Related Diseases, Rare Diseases Strategy of the Spanish National Health System, Junta of Castile and León, Charles and Ann Johnson Foundation, Stanford Cancer Institute Innovation Fund, Austrian Science Fund., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Diagnosis of hematolymphoid neoplasm (HLN) requires different technologies which are performed on a patient basis instead of per protocol. We hypothesize that integration of hematimetric and cytological analysis along with multiparametric flow cytometry (MFC) provides a framework to evaluate peripheral blood (PB) samples from Primary Care., Methods: Samples from patients with persistent (>3 months) lymphocytosis (>5 × 10 9 /L) and/or monocytosis (>10 9 /L) or the presence of atypical and/or blast cells upon the smear review were analyzed by MFC concurrent to cytological analysis. MFC studies were carried out following standardized procedures., Results: In a 3-year period, smear review and MFC were performed simultaneously in 350 samples, demonstrating HLN in 194 cases (55.4%). In 156 cases, reactive cell populations were found. The combination of age, absolute lymphocyte count (ALC), hemoglobin and platelets provided the best correlation with MFC for the presence of a chronic lymphoproliferative disorder (CLPD) in lymphocytosis [area under the curve (AUC) 0.891, p < 0.05]. A model evaluating the probability of CLPD has been proposed and validated in an independent cohort., Conclusions: A strategy to perform MFC studies following standardized procedures has proven to be useful to evaluate samples from patients in Primary Care centers for HLN diagnosis or reactive conditions, providing a sensitive and rapid clinical orientation and avoiding unnecessary consultations in routine clinical practice. The probability for the presence of CLPD in PB can be calculated and help guide decision-making regarding further testing., (©2020 Walter de Gruyter GmbH, Berlin/Boston.)

Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF ( KITLG , KIT , PTPN11 , GNB1 , KRAS and NRAS ) were mutated in 12 patients (2.6%), and genes downstream of BRAF ( MAPK2K1 , MAPK2K2 , and MAPK1 ) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P =0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited., (Copyright© 2019 Ferrata Storti Foundation.)

Small lymphocytic lymphoma (SLL) is considered as the non-leukemic form of presentation of chronic lymphocytic leukemia (CLL). We have compared the features, genomic alterations, and outcome of 890 patients with CLL and SLL. One hundred and thirteen patients presented as SLL and more frequently had unmutated-IGHV, CD38 high , ZAP-70 high , CD49d high , +12, alterations in genes of NOTCH1, cell cycle, RNA metabolism, and NFkB pathways than CLL. During the follow-up, 46% of SLL patients developed CLL. Time to first treatment (TTFT) was shorter in SLL (10-year: 75% vs 62%; p = .006). Binet stage, SLL, and IGHV were independent predictive factors for TTFT. Transformation to diffuse large B-cell lymphoma was higher (10-year: 12% vs 6%; p = .003), and overall survival was shorter in SLL (10-year: 55% vs 66%; p = .004). When A0 CLL patients were excluded, only CD38 and CD49d expression, +12, and 10-year TTFT remained different between the SLL and CLL patients. In summary, SLL showed only minor clinicobiological differences when compared with CLL in similar clinical stages.