Peripheral Blood Analysis By Flow Cytometry for the Diagnosis of Type I Chronic Myelomonocytic Leukemia
Authors: Mya Hae Tha , Diez Campelo Maria , Valle Herrero Sandra , Díaz-Alvarez Agustin , Bustos Domingo , Vaquero Luis M , Pérez José J , González Verónica , Bastida Jose M , Garcia-Coca Alfonso , de Cabo Erik , Gonzalez Tomas , Puig Noemi , Martin-Sancho Alejandro , FernÃ°ndez Patricia , Davila Julio , Africa Martín López Ana , González Marcos , Del Cañizo Consuelo , Vidriales Maria Belen 
Published: November 2019

Abstract
Chronic myelomonocytic leukemia (CMML) is a clonal disorder with a diagnostic challenge encountered frequently in routine hematology consultations, especially when few dysplastic features in bone marrow and/or no cytogenetics or molecular alterations are detected. Thus, easily applicable parameters in peripheral blood analysis are warranted to aid diagnosis. We studied the utility of flow cytometry analysis of peripheral blood (PB) for the diagnosis of CMML type I.
PB samples from 16 CMML type I, age- matched normal (n=10) and reactive monocytosis (>1 x109/L) cases (n=9) were studied. A large panel of monoclonal antibodies was used ( FITC:CD36, CD35, CD15 and CD4, CD16,PCP5.5:CD34, CD163, CD13, PC7: CD117, CD38, CD2,CD7, APC: CD300, CD33, CD11B , CD11C ,CD56 , APCH7: CD14 , HLADR V450, CD64 PE and CD45 OC515) in an 8-color combination, including a backbone of 4 colors in all tubes. At least 1x 10(6) events were acquired by FACSCanto II (BD Biosciences, San Jose, USA). The data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytes were selected on the automated population separator plot and confirmed by the expression of CD64 and HLADR. Lymphocytes were used as the internal control.
As expected, CMML type 1 patients showed higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (p=0.001).
CD64bright and CD163 expression were equally valid to identify monocytic population in all three groups (correlation r2 = 1). The majority of the monocytic population belonged to the late stages of maturation, being CD64+CD35+CD14+CD300+ in CMML as well as reactive and normal cases (p=0.14). CMML and reactive cases showed significantly lower levels of early stages of monocytic maturation (CD64+CD35-/+ CD14-CD300-) than normal controls ( p=0.04).
The majority of type I CMML (69%) had more than 95% "classical" monocytes (CD14+CD16-) in the monocytic compartment, whereas only 22% of reactive cases and none of the normal cases had such condition (p=0.000). Statistically significant differences among the three groups were also found for "intermediate" (CD14+CD16+) and "non-classical" (CD14lowCD16+) monocytic populations, with the normal cases having higher percentage of "non classical" monocytes, and reactive cases higher "intermediate" (CD14+CD16+) monocytes (12% vs. 5% vs. 1%; -p <0.000-, and 2.6% vs. 3.7% vs. 1.4%; -p=0.01- for "non classical" and "intermediate" monocytes in normal, reactive and CMML cases, respectively).
As for the aberrant antigenic expression, CD56 was most frequently expressed in monocytes of CMML (75% CMML with >20% CD56 positivity) followed by CD2 (31%) and CD7 (6%). CD56 could also be detected in reactive (22%) and normal (7%) cases, whereas CD2 and CD7 were always negative. Though useful, CD56 expression alone could overestimate the diagnosis of CMML type I whereas the utility of CD7 expression was doubtful due to its low frequency in CMMLs.
When the pattern of distribution of "classical", "intermediate" and "non classical" monocytes together with the aberrant expression of CD56 and CD2 were considered, only CMML cases met both criteria (>95% of "classical" monocytes among the monocytic compartment in PB, and having an aberrant expression of CD56 or CD2 in >20% of monocytic cells), excluding all the reactive monocytosis and normal cases, although not all CMML (62%) met both criteria.
Concerning the antigens analyzed on the monocytes, only CD163 and CD11b had a higher expression in CMML than in reactive or normal cases (p=0.01 & p=0.05). CD64, CD15, CD4, and CD45 were similarly expressed in CMML and reactive, but higher than in normal cases (p<0.03). CD35 was highest (p=0.01) and HLA-DR (p=0.01) and CD33 (p=0.02) lowest in reactive cases, compared to CMML or normal cases. No significant difference was found for the remaining antigens. In spite of these findings, in our opinion, its prospective utility to diagnose CMML or to distinguish it from reactive monocitosis was doubtful.
Flow cytometry analysis can be useful to diagnose CMML type I in the routine laboratory by employing a few easily applicable parameters in PB (the pattern of distribution of "classical", "intermediate" and "non classical" monocytes together with the aberrant expression of CD56 or CD2), distinguishing it from reactive monocytosis, and helping to identify patients in whom other studies, as bone marrow assessment, should be performed.

Disclosures
Diez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Puig:The Binding Site: Consultancy; Janssen: Consultancy.