How Fast Can Acute Promyelocytic Leukemia Be Diagnosed? - Evaluation of PML Immunofluorescence, Flowcytometric Immunophenotypic Analysis and RT-PCR for PML/RARa.
Authors: Varma Neelam , Agarwal Chetna , Varma Subhash , Malhotra Pankaj 
Published: October 2019

Abstract
Abstract 4674

Introduction
Acute promyelocytic leukemia (APL) constitutes nearly 5-8% of all leukemias, however its frequency is higher in some populations. It is essential to diagnose APL rapidly and accurately as it often presents as a devastating coagulopathy and shows unique sensitivity to all-trans retinoic acid (ATRA). A great deal of morphological, immunophenotypic and cytogenetic heterogeneity of APL adversely affects efforts for providing an accurate and rapid diagnosis. APL is associated with t(15;17)(q22;q12) with generation of a novel PML/RARa fusion protein in 95% cases. Cases positive for t(15;17)/ PML/RARa are ATRA sensitive, while some of those without it [eg t(11;17)(q23:q21)] are not. Several techniques such as karyotyping, fluorescent in situ hybridization (FISH), and reverse –transcriptase polymerase chain reaction (RT-PCR), used for its detection are time consuming, laborious, costly and require specialized laboratories. Lately, immunnostaining methods have been described using PML antibodies for faster diagnosis of APL. The distinction between APL (AML-M3) and non AML-M3 AML is based upon microgranular versus speckled pattern observed in the nuclei of leukemic cells. Only few studies have described use of PML monoclonal antibody (Moab) – PG-M3. Typical flowcytometric (FCM) immunophenotype of APL cells reveals positivity for CD33 and CD13, with negativity for HLA-DR and CD34. RT-PCR (gold standard method) is used to detect classical APL genetic abnormality PML/RARa hybrid transcripts resulting from t(15;17)(q22;q21) and also the alternate translocations associated with APL. We undertook this study to assess the role of PML immunoflourescence (IF), flowcytometry and RT-PCR for quick diagnosis of APL.

Patients and methods
During last 17 months, peripheral blood and/or bone marrow samples from 93 consecutive acute non-lymphocytic leukemia (ANLL) cases were obtained after informed consent. All the cases were classified using standard morphological criteria (FAB classification). RT-PCR for PML/RARa, FCM immunophenotyping (IP) (Moab panel comprising of CD13, CD33, CD34, HLA-DR, CD56, CD2, CD19, CD14 and CD64) and PML IF (using anti-PML Moab PG-M3 clone) were performed. In the PML IF study, staining pattern of leukemic cells was noted within 2 hours of staining, using Leica DM LB2 epifluorescence microscope equipped with chilled digital color camera and Leica FW 4000 software. In our experience PML-IF could be completed in 2-4 hrs, FCM-IP in 3-5 hrs and RT-PCR in 12-24 hrs.

Results
27/93 (29%) cases belonged to AML-M3 category and rest to different categories of AML (M0:1, M1:9, M2: 39, M4:10, M5:6, M6:1). RT–PCR for PML/RARa was positive in 24/27 (88.88%) cases with morphological diagnosis of AML-M3. bcr1 transcripts were detected in 2 (8.33%), bcr2 transcripts in 8 (33.33%) and bcr3 transcripts in 14 (58.33%) among 24 cases. 22/24 cases positive for PML/RARa by RT-PCR showed typical microgranular pattern of nuclear staining on PML IF (91.66% concordance). PML IF did not give any signal in one case and another one showed speckled pattern. In non AML-M3 cases, RT-PCR for PML-RARa was negative in all and PML immunoflourescence staining revealed speckled pattern in 59/66 (89.39%) cases. Typical FCM signature for APL was seen in 86.66% cases positive for PML/RARa by RT-PCR. Two cases were HLA DR positive and one out of these was positive for PML-RARa RT-PCR.

Conclusions
Immunostaining with PML-antibody was found to be a rapid, simple, cost effective & less time consuming technique to detect PML-RARA with high rate of concordance with the gold standard RT-PCR. This could be routinely applied as an upfront investigation to pick up the APL cases.

Disclosures:
No relevant conflicts of interest to declare.