FLT3-ITD As a Target for Minimal Residual Disease Monitoring in Early T-Cell Precursor (ETP) Acute Lymphoblastic Leukemia
Authors: Andriano Nellina , Buldini Barbara , Silvestri Daniela , Villa Tiziana , Locatelli Franco , Parasole Rosanna , Barisone Elena , Testi Anna Maria , Biondi Andrea , Valsecchi Maria Grazia , Conter Valentino , Basso Giuseppe , Lo Nigro Luca , Cazzaniga Giovanni 
Published: October 2019

Abstract
Background. The ‘early T-cell precursor’ (ETP) subtype of T-ALL comprises up to 15% of T-ALL and has been reported to be associated with high risk of relapse. In addition to properties of T cell development, gene expression profile and immunophenotype of ETP-ALL show stem cell and early myeloid features. Consistently, this leukemia subgroup shows lower frequencies of prototypical T-ALL lesions and a higher prevalence of mutations typically associated with AML, including RAS and FLT3 mutations. In particular, FLT3-ITD was identified in up to 35% of adult ETP-ALL but data on its prevalence in pediatric ETP-ALL are lacking.
In agreement with its stem-cell signature, ETPs frequently lack Immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements, the most used and sensitive targets for MRD monitoring. As a consequence, alternative markers are required to extend the application of molecular MRD to most ETP-ALL patients.
Aim. We explored the prevalence of FLT3-ITD mutation in a large series of pediatric ETP-ALL enrolled in two consecutive protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP), and we evaluated the potential use of FLT3-ITD as an alternative DNA marker for MRD monitoring.
Methods. Out of 439 T-ALL patients enrolled in Italy into the AIEOP-BFM ALL2000 and AIEOP ALLR2006 consecutive protocols, 145/168 Early-T ALL (TI/II) patients were screened for FLT3-ITD occurrence. Among Early-T patients, 34 were defined as ETP according to immunophenotype, and 31 of them were screened for FLT3-ITD. Twenty-two ETP patients enrolled in Italy into the ongoing AIEOP-BFM ALL2009 were also screened, together with T-ALL cases without IG/TR molecular markers only for the technical validation of the method. PCR screening and RQ-PCR for FLT3-ITD were performed as previously reported (Nakao, Leukemia 1996;10:1911; Beretta, Leukemia 2004;18:1441). Parallel MRD analysis for IG/TR on the same samples, and flow cytometry-MRD were performed by standard procedures. EuroMRD guidelines were applied for performance and interpretation of RQ-PCR.
Results. Among ALL2000/R2006 and ALL2009 ETP cases, 4/31 (12.9%) and 3/22 (13.6%) were FLT3-ITD positive, respectively; 5/7 were PPR, and all 7 were stratified as high risk. For ALL2000/R2006 patients, IG/TR MRD monitoring was feasible in 2 cases, and both were MRD-HR; 3/4 cases are alive in CCR, and one died after HSCT. Overall, the FLT3-ITD marker was detected in 12 T-ALL cases; only 4 of them had valuable IG/TR markers, while 8/12 (66%) did not present a suitable IG/TR MRD marker. FLT3-ITD MRD monitoring was performed on 11/12 FLT3-ITD positive T-ALL cases. Mean length of the ITD was 44 nucleotides (nts) (range 24-71), with a mean of 7 randomly inserted nts (range 1-26). Standard curves performed by 10-fold dilutions in DNA from PB Healthy Donor, showed a quantitative range of at least 5.0E-04 in all cases and 1.0E-04 in 5/11. Sensitivity of the assay was at least 1.0E-04 in all tested cases, and 1.0E-05 in 7/11.
A comparison between IG/TR and FLT3-ITD was feasible in 3 out of 4 cases (1 is ongoing); all 3 cases were monitored by 2 IG/TR markers. At day15 and day33 of the Induction therapy, when MRD was very high (10-1 to 10-3 range), the IG/TR and FLT3-ITD were fully comparable, with less than 2 times difference. At day78 (after IB Induction block) 1 case was fully negative for both markers, 1 was slightly positive by FLT3-ITD (although not quantifiable and at the limit of the sensitivity) and negative for both IG/TR. The latter case was highly positive for both IG/TR (5.0E-03) but low positive (<1.0E-04) by FLT3-ITD. In this case, the IG/TR and FLT3-ITD were concordant and finally both were negative at subsequent time points.
Conclusions. This is the first report on FLT3-ITD prevalence in a consecutive series of children with ETP-ALL, which resulted to be 14.8% (4/31), a value lower than that reported in adult ETP-ALL. The limited number of cases does not allow to draw conclusions on the prognostic impact of FLT3-ITD in ETP-ALL, although 3 out of 4 patients are alive in CCR. Although available in a limited subset, FLT3-ITD can be used as a marker for sensitive molecular MRD monitoring in ETP-ALL, when IG/TR markers are not available (about 2/3 of cases). The results of MRD monitoring in a limited set of cases suggests that ETP patients might respond well to IB Induction therapy. As already known for AML, caution for false negative results is required when only FLT3-ITD is monitored.

Disclosures
No relevant conflicts of interest to declare.