Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia.

Jelena V Jovanovic,Paresh Vyas,Kristian Rennie,Justin Harrison,Andrew Peniket,Dominic Culligan,David Grimwade,Anne Lennard

doi:10.3389/fonc.2011.00035

Abstract

Molecular detection of minimal residual disease (MRD) has become established to assess remission status and guide therapy in patients with ProMyelocytic Leukemia–RARA+ acute promyelocytic leukemia (APL). However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF–RARA and STAT5b–RARA. Despite their rarity (<1% of APL) we identified 6 cases (PLZF–RARA, n = 5; STAT5b–RARA, n = 1), established the respective breakpoint junction regions and designed reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17) – associated APL, affording assay sensitivities of ∼1 in 104−105. Serial samples were available from two PLZF–RARA APL patients. One showed persistent polymerase chain reaction positivity, predicting subsequent relapse, and remains in CR2, ∼11 years post-autograft. The other, achieved molecular remission (CRm) with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b–RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RT-qPCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly defined subsets of acute leukemia.

Highlights

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the gene encoding retinoic acid receptor alpha (RARα), which is most commonly fused to the ProMyelocytic Leukemia (PML) gene consequent upon the t(15;17)(q22;21)
DEVELOPMENT OF reverse transcription-quantitative real-time PCR (RT-qPCR) ASSAYS FOR APL FUSION TRANSCRIPTS The assay designs to amplify PLZF–RARA and STAT5b–RARA fusion transcripts were adapted from the standardized PML– RARA assay developed in the EAC program (Gabert et al, 2003), using the EAC probe and reverse primer located in RARA exon 3 in conjunction with newly designed forward primers located within PLZF and STAT5b, respectively (Figure 2; Table 2)
Application of molecular monitoring by RT-qPCR to establish remission status and identify patients needing additional therapy to achieve disease cure is firmly established as a key component of the management of patients with PML–RARA+ APL (Sanz et al, 2009)

Summary

Introduction

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the gene encoding retinoic acid receptor alpha (RARα), which is most commonly fused to the ProMyelocytic Leukemia (PML) gene consequent upon the t(15;17)(q22;21) (reviewed Mistry et al, 2003). The majority of these cases lacking the classic t(15;17) still harbor an underlying PML–RARA fusion gene, while in ∼1–2% of cases presenting with APL an alternative fusion partner is involved (Grimwade et al, 2000) These include PLZF (ZBTB16), NPM1, NuMA, FIP1L1, and BCOR, formed as a result of the t(11;17)(q23;q21), t(5;17)(q35;q21), t(11;17)(q13;q21), t(4;17)(q12;q21), and t(X;17)(p11;q21), respectively; while the PRKAR1A, and STAT5b genes are fused to RARA following rearrangements involving 17q (Chen et al, 1993; Redner et al, 1996; Wells et al, 1997; Arnould et al, 1999; Catalano et al, 2007; Kondo et al, 2008; Yamamoto et al, 2010). Sensitivity to arsenic has only been demonstrated in PML–RARα positive APL, reflecting the capacity of ATO to bind directly to the PML moiety of the fusion www.frontiersin.org

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