Utility and Impact Of Early t(15;17) Identification By Fluorescence In Situ Hybridization (FISH) In Clinical Decision Making For Patients With Acute Promyelocytic Leukemia (APL)
Authors: Mangaonkar Abhishek , Kota Vamsi , Rojiani Amyn , Jillella Anand , Kolhe Ravindra B. 
Published: October 2019

Abstract

Background
Acute Promyelocytic Leukemia (APL) is a highly curable malignancy with most large co-operative group studies showing greater than 90% long term survival. Despite this, recent studies looking at survival in population-based studies suggest that approximately 30% of patients with APL die during induction. This has been confirmed in large population-based studies in Sweden and the US. A recent analysis of Surveillance, Epidemiology & End Results (SEER) data from 13 population-based cancer registries with 1400 APL patients in the US showed that 17% of all patients and 24% of patients > 55 years of age die within one month of diagnosis. Swedish registry data also showed that 29% of patients died within 30 days of diagnosis with most of them dying in the first two weeks. Early initiation of treatment is critical in improving outcomes. Early demonstration of t(15;17) will lead to more accurate decision making regarding treatment especially in the setting when patients are referred from rural areas or treated in academic institutions where confirmation is essential for research protocol purposes. The aim of this project was to validate earlier time frames for the Abbott Molecular Vysis LSI PML/RARA FISH probe (ASR 6-16 hrs).

Materials and Methods
15 patients (AML-M0, M1, leukocytosis, NHL, pregnancy loss & APL) were selected for validating various hybridization (hybe) times for FISH probe. After preparing the slides, they were chemically aged by treatment of 2XSSC at 73°C for 2 minutes. 2uL of probe (Abbott Molecular Vysis LSI PML/RARA) was applied to each sample after dehydrating with an ethanol series (70%, 85% & 90%) for 1 minute each. The slides were then placed in the Thermobrite for 2, 4, 6 hours and overnight to denature and hybridize. After each hybe the slides were treated with 0.4xSSC/0.3%NP-40 for 2 minutes at 73°C and later immersed in 0.2xSSC/0.3% NP-40 for 1 minute at room temp. 2uL of DAPI Antifade SS was added with a micropipette onto each hybridized area, and a coverslip was applied. 200 cells with robust signals were identified and calculated. Hybe efficiency was graded as acceptable (good), intermediate (okay) and unacceptable (poor). Expected normal signal pattern was two red and two green signals (2R2G), and the expected most common abnormal signal pattern was two fusion (yellow) signals, one red and one green (2F1R1G) and/or one fusion, one red and one green (1F1R1G).

Results
The specificity of the probe ranged from 84% at two hours, 86% at four hours, 84% at 6 hours, and 87% for overnight hybe. The sensitivity increased from 79% at two hours, 80% at four hours, 81% at 6 hours, to 87% for overnight hybe, which was expected.

Conclusion and Discussion
Majority of the APL patients who survive the induction phase are cured with a low chance of relapse. Early mortality continues to be a significant problem with deaths attributable to bleeding, differentiation syndrome and infection. Coagulopathy abnormalities are a hallmark of this disorder and can be rapidly corrected with early institution of treatment. Also, in patients with co-morbid conditions that would exclude the use of anthracyclines, arsenic is the treatment of choice and starting of arsenic is not recommended unless cytogenetic diagnosis is confirmed. The utility of early t(15;17) detection by FISH helps in initiating treatment early and may lead to improved outcomes. This can be easily achieved by shortening the timeframe for FISH results. Based on the validation studies the current recommendations are to read FISH results at the 4 hour incubation mark for a preliminary diagnosis and confirmation with overnight hybridization.

Disclosures:
Kota: Teva: Speakers Bureau; Ariad: Advisory board, Advisory board Other.