Asbtract Introduction This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Methods A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. Results We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p= 0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. Conclusion The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

ABSTRACT Background: Acute myeloid leukemia presenting the MYST3-CREBBP fusion gene is a rare subgroup associated with hemophagocytosis in early infancy and monocytic differentiation. The aim of this study was to define the relevant molecular cytogenetic characteristics of a unique series of early infancy acute myeloid leukemia cases (≤24 months old), based on the presence of hemophagocytosis by blast cells at diagnosis. Methods: A series of 266 infant cases of acute myeloid leukemia was the reference cohort for the present analysis. Acute myeloid leukemia cases with hemophagocytosis by blast cells were reviewed to investigate the presence of the MYST3-CREBBP fusion gene by fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction. Results: Eleven cases with hemophagocytosis were identified with hemophagocytic lymphohistiocytosis being ruled out. Six cases were classified as myelomonocytic leukemia, three as AML-M7 and two as AML-M2. In five cases, the presence of the MYST3-CREBBP fusion gene identified by molecular cytogenetics was confirmed by fluorescence in situ hybridization. All patients received treatment according to the Berlin-Frankfürt-Münster acute myeloid leukemia protocols and only one out of the five patients with the MYST3-CREBBP fusion gene is still alive. Conclusions: Our findings demonstrate that the presence of hemophagocytosis in acute myeloid leukemia was not exclusively associated to the MYST3-CREBBP fusion gene. Improvements in molecular cytogenetics may help to elucidate more complex chromosomal rearrangements in infants with acute myeloid leukemia and hemophagocytosis.