MLL-AF4 Positive Adult ALL Patients: VH6 Immunoglobulin Gene Rearrangements Predominate While FLT3 Mutations Are Rare.
Authors: Duke Veronique M. , Rai Lena , Mortuza Jeasmin , Saglio Giuseppe , Foa Robert , Macintyre Elisabeth M. , Hoffbrand Victor A. , Kottaridis Panos , Foroni Letizia 
Published: April 2020

Abstract
Among patients with adult acute lymphoblastic leukemia (ALL) the t(4;11) translocation (resulting in the MLL-AF4 fusion gene) is a recurrent chromosomal abnormality occurring in approximately 10–15% of cases. It is associated with poor outcome and in the majority of cases stem cell transplantation is the only possible cure. The MLL-AF4 cases show a more immature phenotype than other subtypes frequently lacking Ig light chain and/or TCR gene rearrangements (Brumpt et al; Blood, 2000; 96:2254). We have previously observed that in adult ALL there is a bias towards the use of JH-proximal VH genes and in particular an over-usage of the VH6 gene segment, the most JH-proximal VH element. This is also common in MLL-AF4 patients while rarer in ALL carrying other chromosomal abnormalities (Mortuza et al, 2001; Blood, 97: 2716). VH6 gene usage is also predominant (8–12%) in adult ALL when compared to the mathematical expected VH6 gene usage (0.8%) or the usage observed in normal PB or in other B-cell malignancies, such as CLL (3.6%) and B cell lymphomas (0%).
Finally, mutations of FLT3 are detected in a large proportion of acute myeloid leukemias and in a small proportion of childhood ALL carrying the MLL-AF4 fusion gene while it has seldom been observed in adult ALL with t(4;11).
The aims of our study were twofold: 1) to expand on the initial observation that the VH6 gene rearrangement may be more frequent in adult patients carrying the t(4;11) translocation; 2). To assess the incidence of FLT3 mutations in an homogenous cohort of adult ALL patients carrying t(4;11) abnormality.
Thirty-two cases of MLL-AF4 fusion positive adult ALL patients (17M/15F) were analysed in addition to the RS4;11 cell line. Pro-B phenotype (n=11)predominated. Age ranged between 22–55 yrs(median: 32 yrs). Patients had cytogenetic or molecular evidence of t(4;11). The WBC count at presentation was high (60–550x109/l; median: 220). Patients were tested for VH gene rearrangement using FRI and consensus-JH primers. Internal tandem duplication and codon 835–836 kinase mutations of the FLT3 were screened by DNA PCR analysis.
Twenty-seven patients (84%) of the 32 tested carried a VH6 gene rearrangement. Of 36 IgH alleles, 27 (75%) were VH6 rearrangements. VH1 in 4 (11%) rearrangements was the second most common rearrangement while 3 were VH3 (8%) and 2 (5.5%) were VH4. Compared to our previous study (Mortuza et al, 2001; Blood,97:2716) the incidence of VH6 IGH rearrangements in MLL positive cases represents a nine-fold increase (75% vs 9.4%) compared to non-MLL + cases. No Internal Tandem Duplication of the FLT3 gene was detected in the 31 cases tested, while one (3.6%) of the 28 cases analysed showed the recurrent mutation of the Asp835Tyr of the FLT3 gene. In conclusion, patients carrying the t(4;11) translocation show a very skewed pattern of IgH gene rearrangement restricted almost exclusively to the VH6 usage, in agreement with both the more immature B cell phenotype and observation in the mouse immune system. However, in t(4;11) adult ALL FLT3 is rarely found mutated and may therefore play a minor role in the poor outcome of this ALL subgroup.