In Acute Promyelocytic Leukemia (APL) with Multiple Relapses Additional Prognostic Information Is Provided by FLT3 and Telomerase (hTERT) Quantitative Real-Time PCR.
Authors: Bernasconi Paolo , Calatroni Silvia , Rocca Barbara , Cavigliano Paola Maria , Castagnola Carlo , Boni Marina , Giardini Ilaria , Zappatore Rita , Quarna Jessica , Alessandrino Emilio Paolo , Astori Cesare , Lazzarino Mario 
Published: April 2020

Abstract
The present study, carried out in two APL with multiple relapses, was aimed at determining 1) which correlation does exist between FLT3/hTERT expression levels and PML-RARA results; 2) whether high FLT3 and hTERT expression levels might be predictive of relapse; 3) whether FLT3 expression is better than FLT3 Internal Tandem Duplication (ITD) for evaluating disease outcome. Relative quantifications of FLT3/hTERT transcripts were performed by real-time PCR using SybrGreen I. For FLT3 calibration total RNA from a normal subject was used, for hTERT total RNA from K562 cells. In both cases the ΔΔCt method was used for quantification. On clinical diagnosis one patient with a WBC of 124.0x109/L and a PML-RARA fusion at PML BCR1 presented the FLT3/hTERT genes highly expressed. On qualitative PCR the patient also showed the ITD of FLT3. He was treated with the AIDA protocol and succeeded in achieving a haematological but not molecular remission. During CR FLT3/hTERT expression remained high and the ITD was never detected. Fourteen months later when on first clinical relapse FLT3 expression abruptly increased, the ITD reappeared, hTERT levels were still high. A re-induction chemotherapy induced a second haematological but not molecular remission lasting five months. FLT3 as well as hTERT expression levels became similar to those of the control, and FLT3 ITD disappeared. A progressive increase of FLT3 expression and an abrupt increase of hTERT expression preceded the second relapse which was accompanied by the reappearance of the ITD. After re-induction chemotherapy FLT3/hTERT expression dropped down to values of the control. A third CR was obtained but the patient remained PML/RARA and Flt3 ITD positive and soon after died of a CNS relapse.
The other patient was treated in another Centre and came to our observation in haematological CR. At that time he was PML-RARA negative with high FLT3/hTERT expression. Eight months later he was still in clinical but not molecular CR having a PML-RARA fusion at BCR3, high FLT3/hTERT expression levels and presenting FLT3 ITD. One month later when clinical relapse occurred FLT3 expression levels were unchanged, hTERT expression dropped down to normal values and FLT3 ITD was still present. A re-induction chemotherapy induced a second CR with alternatively positive and negative PML-RARA results, high FLT3 and low hTERT expression levels. The patient underwent an allogeneic bone marrow transplant from an unrelated donor but five months later he relapsed for the second time with an abrupt rise of hTERT expression that preceded a quick increase of FLT3 expression. A third clinical but not molecular CR was achieved after chemotherapy, but the patient remained PML-RARA positive with a normal FLT3/hTERT expression. Two months later a rapid increase of hTERT expression preceded that of FLT3 and the occurrence of the third relapse.
In conclusion i) increased FLT3 and hTERT levels during CR are associated with alternative positive/negative PML-RARA results on nested RT PCR and are always predictive of pending relapse; ii) on disease recurrence a marked elevation of hTERT expression often preceded that of FLT3; iii) quantitative real-time PCR of the FLT3 gene was more effective in predicting disease outcome than the ITD, this last being discovered only when FLT3 expression was already high.