Abstract A40: CD44 expression in T-cell acute lymphoblastic leukemia and acute myeloid leukemia associated with RAS mutations
Authors: Marques Luisa V. C. , Noronha Elda P. , Andrade Franciane G. , Pina Eugenia T. G. , Pombo-de-Oliveira Maria S. 
Published: May 2019

CD44 is an adhesion glycoprotein which helps with the homing of hematopoietic precursor cells. In acute myeloid leukemia (AML), CD44 has been investigated as a stem cell marker, and expression of its variant proteins has been associated with poor prognosis. Only in murine T-cell acute lymphoblastic leukemia (T-ALL) models, CD44 expression was associated with tumor progression, organ infiltration, and influencing survival. In early T-cell precursor-ALL (ETP-ALL, a T-ALL subset) several similarities were observed with AML genomic aberrations. Taking the CD44 gene that is a target of the RAS pathway, which promotes its alternative splicing, throughout a positive feedback loop, we have investigated whether the cellular expression of CD44 in different maturational subtypes of pediatric T-ALL and AML would predict RAS mutations. We have tested if the cellular CD44 status would be associated with patient's clinical characteristics.

Methods: A series of 193 T-ALL and 28 AML patients (≤ 21 years) was tested by multiparameter flow cytometry with a panel of monoclonal antibodies combined as CD4FITC/CD7PE/CD45PercPCy5.5/CD8Pe-Cy7/CD44APC (T-ALL), and CD117PE/ CD45PercPCy5.5/HLA-DR PE-Cy7/CD44APC (AML). CD44 expression was evaluated by median fluorescence intensity (MFI) in blast cells and the value of the MFI in the 75th percentile was used as a cutoff to discriminate between high or low expression of CD44. Sanger sequencing was used to detect mutation in codons 12 and 13 of N/KRAS genes, after DNA extraction, amplification through polymerase chain reaction and product purification. Fisher´s exact test or chi-square test was used to evaluate the distribution of categorical variables, whereas Mann-Whitney (two groups) or Kruskal Wallis (more than two groups) tests were used to evaluate the distribution of non-parametric continuous variables; t-test and one-way ANOVA were used for parametric variables; p values of &lt; 0.05 were considered statistically significant.

Results: Only two cases were negative for CD44 (&lt;20% positive blasts). There was no association between high expression of CD44 and organomegaly in T-ALL while for chloroma in AML the evaluation was impaired due to small series. There was no association between the expression of CD44 and T-ALL subtypes. AML patients have a higher cellular expression of CD44 (MFI: 18890 [1019-44720]) than T-ALL (MFI: 1211 [68-8325]) (p&lt;0,0001). The CD44 expression in ETP-ALL were lower than in AML (MFI: 1504 [855-3296], p&lt;0,0001). The frequency of N/KRAS mutations in T-ALL cases was 11,7% (12/102), whereas in AML cases it was 16.7%. There was no significant difference in CD44 expression between cases with N/KRAS mutations (MFI: 1715 [365-8325]) and without mutation (MFI: 1179 [68-5544]) in T-ALL, whereas in AML, N/KRAS mutated cases had a lower CD44 expression (MFI: 10979 [7650-18810]) than the cases without mutation (MFI: 21720 [1019-44720]) (p=0,032).

Conclusions: CD44 cellular status was not relevant for T-ALL tumoral profile and its expression was not associated with T-ALL subtype. CD44 is underexpressed in T-ALL and ETP-ALL when compared with AML. N/KRAS mutation does not seems be associated with different expression of CD44 in pediatric T-ALL whereas further investigation is required in AML subsets.

Citation Format: Luisa V. C. Marques, Elda P. Noronha, Franciane G. Andrade, Eugenia T. G. Pina, Maria S. Pombo-de-Oliveira. CD44 expression in T-cell acute lymphoblastic leukemia and acute myeloid leukemia associated with RAS mutations [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A40.