scholarly journals Diagnostic Utility of Flow Cytometric Immunophenotyping in Chronic Myelomonocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5526-5526
Author(s):  
Leonor Arenillas ◽  
Ivonne Parraga ◽  
Lourdes Florensa ◽  
Sara Montesdeoca Romero ◽  
Anna Puiggros ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Chronic Myelomonocytic Leukemia ◽  
Nonspecific Esterase ◽  
Age Related ◽  
Hla Dr ◽  
Myelomonocytic Leukemia ◽  
Classical Monocytes

Abstract INTRODUCTION The diagnosis of chronic myelomonocytic leukemia (CMML) according to WHO 2017 requires the presence of ≥1x109/L and ≥10% of monocytes in peripheral blood (PB). Establish an accurate diagnostic is difficult since many clinical situations present persistent monocytosis. The presence of dysplasia, mainly dysgranulopoiesis, is frequent but not always present in CMML. Cytogenetic aberrations are infrequent in this disease (20-25% of cases). Although 85-90% of CMML patients present at least one mutation in TET2, SRSF2 or ASXL1 genes, the use of NGS panels is not widespread. Furthermore, mutations in these genes are among the most frequently observed in age-related clonal hematopoiesis. Therefore, complementary techniques are required to support the diagnosis of this entity. The study of the peripheral monocyte subsets by flow cytometry (FC) has gained special interest due to a high sensitivity and specificity for the diagnosis of CMML (S = 90.6%, E = 95.1%, Selimoglu-Buet et al., Blood, 2015). An increase in the fraction of classical monocytes (Mo1) to >94% of total monocytes is an event frequently observed in CMML. There are no specific bone marrow (BM) FC panels for the diagnosis of CMML and very few have been validated for the diagnosis of MDS. "Ogata score", the only multicenter validated score in MDS, has not been applied in CMML. The aim of our study was to evaluate the usefulness of FC in PB and BM for the diagnosis of CMML. METHODS Twenty-two CMML were prospectively studied from 02/2016 to 04/2018. Patients' characteristics are summarized in Table 1. Diagnostic procedure consisted of morphological, cytochemical (Perls, myeloperoxidase, nonspecific esterase), cytogenetic and FC studies in BM, and morphological and FC studies in PB. "Ogata Score" was applied in BM samples (Table 2). Aberrant coexpression of CD2, CD7 and CD56 in BM monocytes was assessed. Immunophenotypic maturation profile of the monocytic elements in BM distinguishes: promonocytes (CD34-/CD117-/CD64++/CD14- or dim/CD45+/HLA-DR+++), mature monocytes (CD34-/CD117-/CD64++/CD14++/CD45++/HLA-DR++) and mature monocytes in terminal stage (CD300e+). In PB, the monocytes FC subsets (Mo1, Mo2 and Mo3) were studied, as well as the aberrant coexpression of CD2, CD7 and CD56 (Table 3). RESULTSThe presence of ≥2 aberrations in Ogata Score predicted properly the diagnosis of CMML in all patients analyzed (100% sensitivity). Due to the study design, we could not obtain results about specificity.An increase in Mo1 (classical monocytes) > 94% was detected in 18/20 patients (Table 3). This method predicted the diagnosis of CMML with a sensitivity of 91%, a result almost identical to the original study (Selimoglu-Buet et al., Blood, 2015).A good positive correlation was established between the percentage of BM promonocytes detected by morphology and by FC (Rho Spearman 0.61, P = 0.003).A negative correlation was found between the percentage of promonocytes by FC in MO and the expression of CD56 (Rho Spearman -0.612, P = 0.002). Similarly, CD56+ CMML presented a percentage of promonocytes by FC significantly lower than the CD56- CMML group (median: 24.5% (14-40) vs. 41% (23-71), P = 0.005). The expression of CD56 seems to be related to a more mature immunophenotypic profile of the monocytic population. On the other hand, the correlation between the percentage of CD56+ monocytes in BM and PB was almost perfect (Rho Spearman 0.928, P <0.001). CONCLUSION Our findings support the usefulness of flow cytometry in bone marrow and peripheral blood for the diagnosis of CMML. Disclosures No relevant conflicts of interest to declare.

MIR150 Is Involved in the Monocyte Subset Differentiation and Its Down-Regulation Leads to Classical Monocyte Accumulation in Chronic Myelomonocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1133-1133
Author(s):  
Dorothee Selimoglu-Buet ◽  
Julie Riviere ◽  
Margot Morabito ◽  
Catherine Lacout ◽  
Aurelie Chauveau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Human Monocyte ◽  
Chronic Myelomonocytic Leukemia ◽  
Monocyte Subset ◽  
Monocyte Subsets ◽  
Down Regulation ◽  
Cd34 Cells ◽  
Myelomonocytic Leukemia ◽  
Classical Monocytes

Abstract Background. Monocytes are a heterogeneous population of peripheral blood leukocytes. The expression of CD14 and CD16 distinguishes CD14+/CD16- classical from CD14+/CD16+ intermediate and CD14low/CD16+ non-classical monocytes. We have shown (Selimoglu-Buet D et al, Blood 2015) that monocytes that accumulate in the peripheral blood of patients with chronic myelomonocytic leukemia (CMML) are predominantly CD14+/CD16- classical monocytes that typically represent more than 94% of total blood monocytes. Strikingly, this phenotypic signature efficiently distinguishes CMML from a reactive monocytosis. Importantly, the CMML-associated increase in classical monocyte fraction disappears in patients who respond to hypomethylating drugs. Whereas in the mouse, the transcription factor Nr4a1 is required for the development of the Ly6Clowmonocytes, the molecular mechanisms that regulate the formation of the three human monocyte populations remain poorly understood. Analysis of the classical monocytes accumulation in CMML may provide insights into the regulation of monocyte subset differentiation. Methods. A microarray screen of miRNA expression was performed in monocytes sorted from 33 CMML and 5 healthy donor blood samples. Validation was performed by qRT-PCR, in monocytes of a cohort of 160 CMML patients and 20 controls, and in CD34+ cells from 44 CMML patients and 19 controls. A mouse model of MIR150-knock-out (Mir150-/-) was used to examine the consequences of the miRNA down-regulation. Multi-color flow cytometry assays were designed to explore mouse and human monocyte subsets. Results. Microarray analyses and validation experiments identified a decreased expression of miR150 in monocytes and CD34+cells from CMML patients compared to controls. Mir150-/- mouse model does not generate monocytosis even in ageing animals. However, an increase in Ly6Chigh inflammatory monocyte fraction at the expense of Ly6Clowpatrolling monocytes was observed in the bone marrow and peripheral blood, leading to further explore the link between MIR150 and monocyte populations. The abnormal repartition of monocyte populations in Mir150-/- mice is a cell-autonomous phenotype as wild-type (WT) mice receiving bone marrow from Mir150-/-mice demonstrated a reduced fraction of Ly6Clow monocytes. This phenotype was rescued by re-expression of MIR150 in LIN- cells of Mir150-/-mice before engraftment. The number of myeloid progenitors was normal in Mir150-/-mice, and the remaining Ly6Clow monocytes did not demonstrate an increased sensitivity to apoptosis. Competitive reconstitution experiments combining WT and Mir150-/-LIN- cells did not identify any significant fitness advantage to Mir150-/-cells, but Mir150-/-donor cells developed reduced numbers of Ly6Clow monocytes than cells from WT donors. These data suggest that MIR150 is involved during late stages of monocyte development and has a key role in the generation of Ly6Clowmonocytes. Finally, TET2 is the main gene mutated in CMML, and Tet2-/- animals develop a monocytosis. Mir150-/- crossed with Tet2-/-mice developed a CMML-like phenotype. In human, the expression of MIR150 decreases along myeloid differentiation and is low in classical compared to intermediate and non-classical monocytes. Depletion or overexpression of MIR150 in human CD34+ cells alters the repartition of CD14+/CD16- and CD14+/CD16+ cells generated in culture. In CMML patients who respond to hypomethylating agents, the normalization of monocyte subset repartition correlates with an increased expression of MIR150, suggesting an epigenetic regulation. MIR150 has several promoters. By combining ChIP-Seq and DNA methylation analyses, a differentially methylated region was detected in one of the MIR150 promoters in CMML patients compared to controls. Using monocyte differentiation conditions, RNA Sequencing performed in CD34+cells overexpressing MIR150, identified ID1 gene as a potential MIR150 target. Conclusion: We demonstrate a role for MIR150 in the generation of intermediate and non-classical monocyte subsets, and its down-regulation in CMML accounts for the characteristic accumulation of classical monocytes. Disclosures Fenaux: Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding.

scholarly journals Disappearance of Slan-Positive Non-Classical Monocytes As a New Parameter for the Diagnosis of Chronic Myelomonocytic Leukemia with Associated Inflammatory State

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4376-4376
Author(s):  
Sihem Tarfi ◽  
Bouchra Badaoui ◽  
Nicolas Freynet ◽  
Margot Morabito ◽  
Jeffie Lafosse ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Chronic Myelomonocytic Leukemia ◽  
Blood Samples ◽  
Healthy Donors ◽  
Inflammatory State ◽  
Whole Blood Samples ◽  
Myelomonocytic Leukemia ◽  
Classical Monocytes

Abstract Introduction: Even though the diagnosis criteria of chronic myelomonocytic leukemia (CMML) have been recently revised by the World Health Organization (WHO), recognition of this disease may be challenging. This myelodysplastic/myeloproliferative neoplasm can also be diagnosed by a relative accumulation of classical monocytes (cMO, CD14++CD16-) ≥94% of total peripheral blood monocytes and a decrease of intermediate (iMO, CD14++CD16+) and non-classical monocytes (ncMO, CD14lowCD16+) percentages measured by flow cytometry (Selimoglu-Buet, 2015; Talati, 2017; Patnaik, 2017; Hudson, 2018). However, inflammatory diseases concomitant to CMML or inflammatory state in CMML patients can provoke an increase of the iMO percentage leading to a decrease of the relative cMO percentage below the 94% threshold (Selimoglu-Buet, 2017). In these cases, the decrease of the relative ncMO percentage persists, hence it might be a useful diagnostic criterion relevant for CMML diagnostic. Since accurate delineation of the iMO and ncMO populations remains debated, the use of a ncMO specific marker, such as slan (6-sulfo LacNac), could be of interest. Objective:We aimed to assess the clinical utility of the slan marker in peripheral monocytosis exploration and CMML diagnosis, especially in inflammatory state. Methods: From November 2017 to July 2018, whole blood samples collected on EDTA or peripheral blood mononuclear cells (PBMC) were stained with the following antibodies as previously described: anti-CD45, CD2, CD56, CD24, CD14, CD16 (purchased either from Beckman-Coulter or Becton-Dickinson) and anti-slan (Miltenyi Biotec). Sample analysis was performed either with a Navios (BC) or a Fortessa (BD) cytometer. Fifty-four controls (19 young healthy blood donors and 35 age-matched healthy donors), 13 patients with reactive monocytosis and 37 patients newly diagnosed with a CMML were enrolled in this study. Results: Firstly, we analyzed the expression of the slan marker in the different circulating mature populations from control whole blood samples. We found that slan is only expressed in monocytes and not in neutrophils or lymphocytes (Figure 1A), especially not in NK cells. Among the three monocyte subpopulations, the expression of slan is restricted to ncMO with 98.9%±0.7% of slan-positive cells gathering within this subpopulation (Figure 1B). However, only 49.1%±12.3% of the ncMO are slan positive, corresponding to ncMo cells with the weakest expression of CD14 (Figure 1C, compare red population to blue one). Yet, both slan-positive and slan-negative ncMO subpopulations displayed similar morphological features after cell-sorting and MGG staining (Figure 1D). Next, we assessed slan expression within the ncMO subpopulation in comparison with relative cMO percentage in healthy donors, patients presenting a reactive monocytosis or a CMML. Thirty-two out of the 37 CMML patients displayed cMO percentage above 94% as expected (Figure 1E). A significant decrease of slan-positive ncMO percentage was observed in CMML patients compared to healthy donors and patients with reactive monocytosis (Figure 1G). All the five patients whose cMO percentage was below the threshold (Figure 1E, blue triangles amongst red ones) displayed the well-recognized "bulbous" aspect (Figure 1F), with an increase of the iMO leading to the decrease of the relative cMO percentage. Interestingly, these patients that couldn't be diagnosed as CMML using the relative accumulation of cMO displayed a low percentage of slan-positive ncMO (Figure 1G, blue triangles amongst red ones). Eventually, we established a Receiver Operator Curve (ROC) and obtained a 1.4% cut-off value of slan-positive ncMo with an area under the ROC curve (AUC) of 0.999 (Figure 1H). The use of the relative slan-positive ncMo percentage led to an improvement of the sensitivity of the CMML flow cytometry assay compared to the relative cMO% (100% vs 86%), as all the false negatives were retrieved. Conclusion: Here, we describe a new parameter for CMML diagnosis, namely the decrease of the relative slan-positive ncMo percentage below 1.4%. This criterion, associated to the relative cMO quantification, may be useful, especially when CMML patient displays an inflammatory state. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

Morphologic and Phenotypic Features of Concurrent Clonal T-Cell Large Granular Lymphocytic Expansion and Myelodysplastic Syndrome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2803-2803
Author(s):  
Xiaohui Zhang ◽  
Lynn Moscinski ◽  
John M. Bennett ◽  
Reza Setoodeh ◽  
Deniz Peker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cell ◽  
Peripheral Blood ◽  
Low Grade ◽  
Gene Rearrangements ◽  
High Grade ◽  
Bone Marrow Cellularity ◽  
Hla Dr ◽  
Marrow Cellularity

Abstract Abstract 2803 Myelodysplastic syndrome (MDS) and T-cell large granular (T-LGL) leukemia are both bone marrow failure disorders. It has been reported in a small number of cases that clonal T-LGL proliferation or leukemia can coincidentally occur with MDS. Also, clonal CD8+/CD57+ effector T cells expansion was detected in as many as 50% of MDS bone marrows [Epling-Burnette, 2007]. How clonal LGL cells that reside in the bone marrow interfere with hematopoiesis remains unclear, particularly in the setting of MDS. We analyzed the clinicopathological features of concomitant MDS and T-LGL, and evaluated bone marrow status for lineage or pan-hypoplasia in these patients. Design: Clinical and pathologic data from patients with a diagnosis of MDS and flow cytometry performed on the peripheral blood between 1/2005 and 12/2009 were reviewed. The concurrent bone marrow biopsies from each patient at the time of flow cytometric analysis were reviewed by two hematopathologists. Bone marrow cellularity, lineage hypoplasia (M:E >5: 1 or <1:2) were documented. Peripheral lymphocyte count and CD3+/CD57+ and CD8+/CD57+ populations by flow cytometry were calculated and T cell gene receptor (TCR) rearrangements were correlated. Results: We performed LGL flow cytometry panel on 76 MDS patients (high grade MDS, n=23; low grade, n=54), as well as TCR gene rearrangements, and identified clonal T-LGL cells in peripheral blood of 37 patients (48.7%), including 15 high grade MDS (40.5%, RAEB-I and RAEB-II), and 22 low grade MDS (59.4%), including RCMD(13), RA(1), RS(1), RCMD-RA(1), RCMD-RS (2), 5q- MDS(1), and MDS unclassifiable(3). The immunophenotype of the T-LGL cells was typically CD3+/CD57+/CD7 dim+/CD5 dim+/CD8+ with variable CD11b,CD11c, CD16, CD56 and HLA-DR. A frequent variant in these MDS patients was CD11b-,CD11c -, CD16+/−, CD56+/−, HLA-DR- and CD62L+.The TCRβ or/and TCRγ gene rearrangements were positive in 35 of the 38 cases (92.1%). The peripheral blood lymphocyte counts were 300–3820 cells/μL (1199±799 cells/μL); the CD3+/CD8+/CD57+ T-LGL cell counts were 30–624 cells/μL (229±154 cells/μL). In comparison, the remaining 39 patients with non-clonal T-LGL included 11 high grade MDS cases, and 28 low grade MDS cases. The peripheral blood lymphocyte counts were 308–2210 cells/μL (1030±461 cells/μL). CD3+/CD57+ cells were 1–425 cells/μL (105±98 cells/μL). There was no identifiable phenotypic features suggestive of clonal T-LGL cells such as dim CD5 and/or dim CD7 with aforementioned aberrant expressions on T-cells, although 7 of the 39 cases had TCRβ or/and TCRγ gene rearrangements. Thirty healthy donors were included for controls with absolute lymphocyte counts of 2136±661 cells/μL and baseline CD3+/CD57+ cells of 162±109 cells/μL. All showed no clonal LGL phenotype and negative TCR gene rearrangements. Since the presence of T-LGL cells may impair bone marrow hematopoiesis, we examined if there are bone marrow status differences between these two groups. All the bone marrows were obtained at diagnosis or not on chemotherapy. The bone marrow cellularity of the MDS patients with clonal T-LGL ranged from <3% to almost 100%, averaging 56%, with 8 cases with dramatic hypocellularity (<3%-20%), while the bone marrow cellularity of the MDS patients without clonal T-LGL ranged from 20% to 90%, averaging 62%, with only 2 cases with mild hypocellularity (20% in 73- and 65-year-old). In addition, among MDS patients with clonal T-LGL cells, 14 of 37 (37.8%; 5 high grade, and 9 low grade) bone marrows had certain lineage hypoplasia, including 3 cases of trilineal hypoplasia, 9 cases of erythroid hypoplasia, and 2 cases of myeloid hypoplasia. In contrast, among 39 MDS patients without T-LGL, there were only 1 bone marrow with trilineal hypoplasia and 3 others with erythroid hypoplasia (10.2%). The difference between the two groups was statistically significant (p=0.004, chi square test). In conclusion, our studies indicate that clonal T-LGL cells expansion is a fairly common finding in high grade as well as low grade MDS. The clonal T-LGL cells have more than one variant immunophenotypes and are typically positive for TCR gene rearrangements. Additionally, we observed that the clonal LGL cells present in MDS bone marrows could be associated with lineage hypoplasia, which, in this respect, might impact clinical treatment. Disclosures: No relevant conflicts of interest to declare.

Clinical Experience with Azacitidine In Chronic Myelomonocytic Leukemia (CMML) in Spain

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5040-5040
Author(s):  
Pablo Gonzalez Navarro ◽  
Regina García Delgado ◽  
Alicia Bailén Garcia ◽  
Juan Antonio Múñoz Múñoz
Keyword(s):  
Bone Marrow ◽  
Overall Survival ◽  
Clinical Trials ◽  
Clinical Experience ◽  
Peripheral Blood ◽  
Progression Free Survival ◽  
Complete Response ◽  
Chronic Myelomonocytic Leukemia ◽  
Free Survival ◽  
Myelomonocytic Leukemia

Abstract Abstract 5040 Clinical Experience with Azacitidine In Chronic myelomonocytic leukemia (CMML) in Spain Pablo González Navarro 1*, Regina García Delgado 2*, Alicia Bailén Garcia 3*, Juan Antonio Muñoz Muñoz 4* 1MD, PhD. Hospital San Cecilio, 18014 Granada, Spain, Teléfono: 958023600 [email protected]; 2Hospital Virgen De La Victoria, Málaga, Spain; 3Hospital Carlos Haya, Málaga, Spain; 4MD, PhD. Hospital Universitario Puerta del Mar, Cádiz, Spain Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal disorder of hematopoietic stem cells often occurring in elderly patients. In the new WHO classification, CMML has been reclassified as a myelodysplastic/myeloproliferative disease. CMML has been subdivided in two subclasses: CMML-1:<5% blasts in peripheral blood and 5–9% blasts in bone marrow, and CMML-2: <10% blasts in peripheral blood and 10–19% blasts in bone marrow (Greco et al. Mediterr J Hematol Infect Dis.2011). Azacitidine (AZA) is an hypomethylating agent approved in Europe for the treatment of myelodysplastic syndromes, with an intermediate to high risk of progressing to AML or death; chronic myelomonocytic leukemia (CMML) and AML that has developed from a myelodysplastic syndrome (prescribing information EMEA 2011). Until its approval in May 2009, AZA was used in Spain under compassionate use in clinical trials. AZA produce a direct decrease of DNA methyltransferase activity, reverting aberrant DNA methylation and increasing the expression of silenced genes, leading to celular differentiation and/or apoptosis (Greco et al. Mediterr J Hematol Infect Dis. 2011). Materials and Methods: We report the results of a retrospective, longitudinal, multicenter Spanish study of 27 patients to assess the effectiveness of AZA to treat CMML. We present results of: Response, Overall Response, Overall Survival and Progression Free Survival. Results: Eighteen of the patients (69.23%) had Chronic Myelomonocytic Leukemia (CMML) type 1 and nine (30.77%) CMML type 2. Median age at diagnosis was 69 years. Male/female ratio: 19/8. ECOG performance status score 1–2 was 78%, twenty patients (74%) received an initial dose of 75 mg/m2 of AZA, whereas three patients (11%) received 50mg/ m2. The mean number of cycles received was 8.32, 95%IC (5.91; 10.73). Overall response to treatment was 53% (CR+PR+HI+mCR): 14.81% complete response, 7.4% partial response, 3,7% Medular complete response and 29,62% Hematological Improvement. In addition, 18,51% had stable disease. Thirty-six percent of patients were alive at the end of treatment with AZA. Median Overall Survival and Progression Free Survival were 17.47 months (95%CI 9.33, upper limit not reached) and 10.97 (95%IC 3.97, 17.47) respectively (Figure 1, 2). Conclusion: Our results show that AZA is an active drug in the treatment of patients with CMML, with similar response rates in the published literature. More data from this study and further investigation with different clinical trials are needed to confirm these outcomes as well as safety and effectiveness of this treatment. Disclosures: García Delgado: Celgene and Novartis: Speakers Bureau.

scholarly journals Deubiquitnase BAP1 Downregulation in Myeloid Malignances: A New Pathogenic Mechanism, Dominant in Chronic Myelomonocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5594-5594
Author(s):  
Ana Maria Hurtado ◽  
Eva Caparrós ◽  
Jose Miguel Torregrosa ◽  
Ginés Luengo ◽  
Mara Toderici ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Chronic Myelomonocytic Leukemia ◽  
Common Mechanism ◽  
Regulation Of Transcription ◽  
Brca1 Protein ◽  
Protein Levels ◽  
Increased Risk ◽  
Myelomonocytic Leukemia

Abstract Background: De-ubiquitinating enzyme BAP1, a fundamental deubiquitinase in the epigenetic regulation of transcription factors and functionally related to ASXL1, is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. In a recent murine study, absolute BAP1 depletion generated specimens with similar characteristics to myelodysplastic / myeloproliferative syndromes in humans (ineffective hematopoiesis and myeloproliferation), mainly to chronic myelomonocytic leukemia (CMML) (Dey, et al. Science 2012). Aim: The aim of this study was to quantify BAP1 gene expression in patients diagnosed with a variety of myeloid neoplasms, and compared it with healthy donors. We furthermore explored the possible association of BAP1 low expression level and the presence of ASXL1 mutations or BRCA1 protein levels. In addition, a regression analysis to determine the possible correlation of peripheral blood and bone marrow expression levels was performed. Methods: We included patients diagnosed between 2008-2014 of CMML, myelodysplastic sydrome (MDS) chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), of whom bone marrow DNA and RNA were available at diagnosis. As controls, 6 healthy bone marrow donors were used. BAP1 and BRCA1 expressions levels were quantified by RT-qPCR, using the same healthy bone marrow donor sample as an inter-assay normalizing- calibrator. The study of somatic ASXL1 mutations was carried out by the Sanger method. For statistical studies, the T-Student, Pearson correlation and/or U Mann-Whitney test, were used when needed. For survival analysis COX regression and the ROC curves were used. A two-side P<0.05 was used as statistical significance threshold. Results: Samples of 116 patients were included in the study: CMML=26; MDS=15; AML=50; CML=25 and 6 controls. This study shows that levels of BAP1 expressions are decreased when compared to controls along the spectrum of myeloid diseases. In the comparison among entities, CMML shows the lowest values (percentage respect to the calibrator), significantly lower than the other groups, except for CML patients: CMML vs MDS, p=0.001; CMML vs AML, p<0.001; CMML vs Controls, p<0.001; LMMC vs CML, p=0.346. No differences were found between CMML patients with dysplastic and myeloproliferative variant, WHO types I and II or according to the presence of ASXL1 mutations (33% CMML patients were mutated). Of potential clinical interest, BAP1 expression in bone marrow and peripheral blood showed a direct and significant correlation ( r=0.884, p= 0.001). BRCA1 expression were decreased uniformly through the different myeloid diseases, suggesting that the heterogeneous BAP1 expression could be responsible for different BRCA1 protein levels by posttranslational regulation. Conclusion:In summary; this study shows that BAP1 decreased expression is a common mechanism among the myeloid malignances, being CMML mainly affected. This mechanism is independent of the presence of ASXL1 mutations, and it could constitute a new therapeutic target in chronic myelomonocytic leukemia. Disclosures No relevant conflicts of interest to declare.

Myeloid-Derived Suppressor Cells in Patients with Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3662-3662
Author(s):  
Nunziatina Parrinello ◽  
Piera La Cava ◽  
Daniele Tibullo ◽  
Cesarina Giallongo ◽  
Orazio Di Bartolo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Suppressor Cells ◽  
Cell Number ◽  
Myeloid Derived Suppressor Cells ◽  
Peripheral Blood Mononuclear ◽  
Hla Dr

Abstract Abstract 3662 Poster Board III-598 Background Immune suppression and angiogenesis are mechanisms key to tumour growth and progression. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin and include immature macrophages, dendritic cells (DC) and other myeloid cells. In mice are phenotypically characterized as CD11b+Gr-1+ cells, while in human they have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC reduce activated T-cell number and inhibit their function through different mechanisms including: L-arginine metabolism, nitric oxide (NO), up-regulation of reactive oxygen species (ROS), and secretion of immunosuppressive cytokines. MDSC also promote tumor-dependent angiogenesis as well as tumor metastasis. Their accumulation has been described in patients affected by some solid tumors but information on haematological neoplasms are lacking. Our study investigated by flow cytometry the presence of MSDC in the peripheral blood of patients affected by Hodgkin Lymphoma (HL). Methods We studied 14 patients with HL at diagnosis and 10 age-matched healthy controls (HC). Peripheral blood mononuclear cells were stained with the following monoclonal antibodies:CD11b, CD13, CD14, CD34, CD45, for 20 minutes at room temperature. After lysing red cells, cells were analyzed by flow cytometry. Results we observed a increased number of MDSC (CD11b+,CD13+,CD34+,CD14-, CD45+) in the peripheral blood of patients with HL compared to HC (13,37 ± 17,77 ×109/l vs 1,45± 0,98 ×109/l, p=0,0007). We also found that patients with advanced-stage Hodgkin disease (III and IV) have higher number of MDSC, compared to patients stage I and II (p= 0,04). Conclusion These data suggest a role for myeloid-derived suppressor cells in promoting tumor cell proliferation in hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.

CML Shows Characteristic Pattern by Flow Cytometry Analysis of Peripheral Blood

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4430-4430
Author(s):  
Kalley Scott ◽  
Wojciech Gorczyca
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Characteristic Pattern ◽  
Differential Count ◽  
Aberrant Expression ◽  
Chromosomal Changes ◽  
Wbc Count

Abstract Abstract 4430 A total of 75 chronic myeloid leukemia (CML) cases from peripheral blood (40 cases) and bone marrow (BM, 35 cases) with adequate flow cytometry (FC) data, smear, blood cell count, and the presence of t(9;22)/BCR-ABL by fluorescence in-situ hybridization (FISH) studies were analyzed for immunophenotypic pattern. The FC pattern in CML was compared with benign (healthy) controls (blood, 20 cases; BM, 20 cases), blood with reactive neutrophilia (15 cases), myelodysplastic syndrome (BM, 15 cases), and blood with eosinophilia (15 cases). CML showed a characteristic pattern by FC in blood, which can be easily differentiated from reactive neutrophilia or eosinophilia, regardless of WBC count. The identification of distinct population of blasts, basophilia, lack of CD10, CD11b, CD13 and/or CD16 on subset of granulocytes, decreased granularity, and/or aberrant expression of CD56 on granulocytes and monocytes, can be easily identified by routine FC analysis. We suggest using FC analysis of blood as a screening tool for patients with leukocytosis (neutrophilia) with follow-up FISH studies in cases with the phenotypic features suggestive of CML. Patients with confirmed CML diagnosis by FISH will undergo marrow biopsy for differential count including blast and basophil enumeration (to exclude accelerated phase or blast crisis), degree of reticulin fibrosis and cytogenetic studies (for additional chromosomal changes present at diagnosis). This approach, in our opinion, allows to diagnose early (unsuspected) CML and eliminates the need for unnecessary cytogenetic/FISH testing, and especially bone marrow biopsy in patients with reactive leukocytosis or eosinophilia. Disclosures: No relevant conflicts of interest to declare.

PD-1 and PD-L1 Are Overexpressed in the "Intermediate CD14+CD16+" and "Non Classical CD14lowCD16+" but Not in the "Classical CD14+CD16-" Monocytes in the Peripheral Blood of Chronic Myelomonocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1694-1694
Author(s):  
Hae Tha Mya ◽  
Maria Diez Campelo ◽  
Sandra Valle Herrero ◽  
Agustin Díaz-Alvarez ◽  
Domingo Bustos ◽  
...  
Keyword(s):  
Internal Control ◽  
Peripheral Blood ◽  
Research Funding ◽  
Chronic Myelomonocytic Leukemia ◽  
Programmed Death ◽  
Cd16 Expression ◽  
Myelomonocytic Leukemia ◽  
Classical Monocytes ◽  
Intermediate Monocytes ◽  
Normal Controls

Abstract Chronic myelomonocytic Leukemia (CMML) is a heterogeneous clonal disorder highly resistant to the few therapies that are available nowadays. There is increasing evidence to suggest that Programmed Death-1 (PD-1), and its major ligand Programmed Death Ligand-1 (PD-L1), are involved in immune suppression and disease progression, are highly expressed in many hematological malignancies, and can be involved in MDS pathogenesis and resistance mechanisms to hypomethylating agents. However, the expression of PD-1 and PD-L1 is not widely explored in CMML. Different types of monocytes based on CD14 and CD16 expression show different genetic profiles and functions, having different distribution in several conditions, including malignancy. In our study, we studied the expression of PD-1 and PD-L1 in the peripheral blood (PB) monocytic compartment of patients with CMML using flow cytometry , to better understand their potential role in the pathogenesis of the disease, and as a basis for the evaluation of this pathway for the development of future immunotherapy strategies. Peripheral blood samples from 16 CMML, and age matched normal (n=10) and reactive (n=9) monocytosis (>1 x109 /L) were studied. Two hundred µl of each PB sample were stained with an 8-color panel of monoclonal antibodies (CD16-FITC, CD64-PE, PD1-PCP5.5, PDL1-PC7, CD300-APC, CD14-APCH7, HLADR-V450 and CD45-OC515). A minimum of 1x 106 events were acquired by FACSCanto II (BD Biosciences, San Jose, USA) and the data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytic population was selected first on the automated population separator plot and confirmed by the expression of CD64 and HLADR expression. Lymphocytes were used as the internal control. Three types of monocytes were defined based on the CD14 and CD16 expression, as previously described. As expected, CMML type 1 patients had higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (0.001). The distribution (median) of the monocytic subpopulations based on CD14 and CD16 expression among the monocytic compartment in PB of CMML, reactive and normal cases, respectively, was as follows: "classical"(CD14+CD16-) were of 98%, 90% and 85% (p<0.000); "intermediate" (CD14+CD16+) 1.4%, 3.7% and 2.6% (p=0.01); and "non-classical" (CD14lowCD16+) monocytes 1%, 5% and 12% (p<0.001). The expression of PD-1 in the major population ("classical" monocyte) was similar among CMML (Median MFI 370), reactive (Median MFI 403), and normal cases (Median MFI 265). However, in the "intermediate CD14+CD16+" and in the "non-classical CD14lowCD16+" monocytes, PD-1 was overexpressed in CMML and reactive cases, compared to normal controls. Reactive cases had even a higher overexpression of PD1 in both "intermediate" and "non-classical" monocytes compared to CMML (Median MFI of 312, 529, and 398 for "intermediate" and Median MFI of 185, 465, and 271 for "non- classical" in normal, reactive and CMML cases, respectively-p=0.01, and p<0.01). For PDL1, we did not find differences in their expression in "classical" nor "intermediate" monocytes among CMML, reactive and normal cases (Median MFI in "classical" monocytes of 2415 vs 2086 vs 2003 for CMML, reactive and normal cases -p>0.05-; and Median MFI in "intermediate" monocytes of 3803 vs 2737 vs 3200 for CMML, reactive and normal cases -p>0.05-). However, in the "non-classical" monocytic population, PD-L1 was clearly overexpressed in CMML (Median MFI of 1782) compared to normal controls (Median MFI of 699), and this was also significantly higher than in reactive cases (Median MFI of 1040) (p=0.002). We found that PD-1 and PD-L1 were overexpressed in CMML, but not in the main "classical" monocyte population of the PB, but in the less represented "intermediate" and "non classical" monocytic compartment. Interestingly, the CD16+ monocytes (intermediate and non-classical) were proposed to have a more important role in inflammation and immunomodulation. Therefore, these populations could have an important function in the pathogenesis of the CMML, and the overexpression of PD-1 and PD-L1 could be investigated as a target for immunotherapy in the development of new therapeutical strategies to improve the adverse prognosis of the CMML. Disclosures Diez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Puig:The Binding Site: Consultancy; Janssen: Consultancy.

Diagnostic Approach To Oligomonocytic Chronic Myelomonocytic Leukemia

2020 ◽  
Vol 154 (Supplement_1) ◽  
pp. S112-S112
Author(s):  
J Muldoon ◽  
M Czader
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Molecular Genetic ◽  
Chronic Myelomonocytic Leukemia ◽  
Differential Count ◽  
Normocytic Anemia ◽  
Molecular Testing ◽  
Monocyte Count ◽  
Diagnostic Threshold ◽  
Myelomonocytic Leukemia

Abstract Casestudy: Pathologic diagnosis of chronic myelomonocytic leukemia (CMML) is typically straightforward with the majority of patients presenting with persistent monocytosis (&gt;1x109/L, &gt;=10%) and bone marrow dysplasia. The diagnosis may be challenging in patients with unusual features such as lack pf peripheral blood monocytosis and non-diagnostic bone marrow morphology. In this abstract, we present a 67-year-old female with a 5-year history of anemia of unclear etiology. At the time of initial presentation, the laboratory work-up of normocytic anemia was non- contributory, the bone marrow was reported as normocellular with maturing trilineage hematopoiesis and no significant dysplasia. Over the course of the disease, the peripheral blood monocyte count fluctuated from 11% to 18% with absolute monocyte count ranging from 0.4 to 0.7x109/L. The most recent bone marrow was markedly hypercellular with increased trilineage hematopoiesis with left shift, dysgranulopoiesis and dysmegakaryopoiesis. Blasts (including promonocytes) constituted 10% of the differential count and were immunophenotypically abnormal with uniform expression of CD117, dim to negative CD13, and partial CD15. Monocytes were elevated at 12% and were strongly positive for CD64 and partially for CD14. They were negative for CD16 consistent with classical monocytes, and showed partial loss of CD13. The karyotype was normal. Molecular testing revealed TET2, RELN and SRSF2 mutations at high allelic frequencies. This case illustrates a value of flow cytometric immunophenotyping and molecular genetic studies in diagnosing challenging cases of CMML. While the patient’s absolute monocyte count remained below the diagnostic threshold of 1x109/L throughout the course of the disease, peripheral blood and bone marrow monocytes showed skewed classical immunophenotype, immunophenotypic abnormalities of myeloid series and high allelic frequency mutations. These findings should raise a differential diagnosis of oligomonocytic CMML, even when morphologic abnormalities and monocyte count threshold are not diagnostic.

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