Investigations on Novel Gene Variants Associated with Longterm Response to Tyrosine Kinase Inhibitors (TKIs) in Chronic Myeloid Leukemia: Implication in TKI-Cessation Clinical Trails
Authors: Iqbal Zafar , Absar Muhammad , Jameel Abid , Akhtar Tanveer , Basit Sulman , Mahmood Aamer , Aleem Aamir , Alanazi Nawaf , Anhar Ullah , Khalid Naila Batool , Afzal Sibtain , Hashmi Jamil Amjad , Akram Afia M , Ramzan Khushnooda , Rasool Mahmood , Aziz Muhammad Hasaan , Sabar Muhammad Farooq , Iqbal Mudassar , Khalid Ahmad Mukhtar 
Published: November 2019

Introduction:
Tyrosine kinase inhibitor (TKIs) have revolutionized CML treatment causing more than 80% patients to achieve durable cytogenetic and molecular remissions1. Initial CML treatment guidelines were to keep CML patients on TKIs for the life time2. Nevertheless, cost issues related to TKI-based treatment have given rise to thinking if TKI treatment can de safely discontinued in a subset of long-term treatment CML responders2. This led to carry out STOP-TKI trials in long-term TKI responders in CML globally1-3. Such efforts can culminate into significant reduction of costs related to very expensive TKI treatment that can spare billions of US dollars annually for treatment and research related to other hematological disorders1.
One of the hindrance for successful application of STOP-TKI efforts is lack of any molecular and other biological biomarkers in CML patients with long-term response as potential candidates for treatment discontinuation3.As next generation sequencing (NGS) technology has proved to be very useful in elucidating novel biomarkers in hematological and other disorders, this study was designed to find novel genetic variants associated with long-term imatinib response in our CML patients.
Materials and Methods:
Selection of Study Subjects (N=123):
1.Chronic phase treatment-naïve CML patients (Control 1)
2.Chronic phase CML long-term TKI responders (at least 3 continuous years of MMR)2 (Experimental group)
3.CML patients with resistant to TKIs (Control 2)
4.CML patients in accelerated and blast crisis phases (Control 3)
5.Healthy controls
All patients were recruited from Hayatabad Medical Complex, Peshawar, Pakistan.
Sample Collection and DNA extraction:10 ml peripheral blood was collected from all study subjects. DNA was extracted at HOPES, University of the Punjab, Lahore, Pakistan. Patient follow-up was carried out during course of this study (2013-8).
Whole Exome Sequencing (WES): WES was carried out using Illumina NGS instrument (HiSeq). bcl files were converted to fastq files by using bcl2fastqtool 4. Raw reads were aligned to genome using BWA tools while whole exome variants were annotated using Illumina Variant Studio 4. R package was employed to align specific gene mutants to disease phenotypes 5. Variants detected by WES were confirmed using Sanger sequencing.
Results & Discussion:
Novel genes/variants associated exclusively with long-term CML responders to imatinib and absent in all control groups are provided in Table 1. One gene of particular interest is
RA1 gene that had multiple frameshift mutations, active throughout the body and controls functions of many genes involved in daily rhythms6.
Our studies are supported by some previous studies that reported CML patients with some genotypes (rs460089-GC in SLC22A4 gene) showing stable major molecular response 7.
Clinical Significance:
Variants found in this study can serve as biomarkers of long term response that can help identify candidates of cessation of TKIs which is one of the major focus of many ongoing CML STOP-TKI trials.
References:
1.Saussele S,et al.Lancet Oncol. 2018 Jun;19(6):747-757.
2.Etienne G,et al. J Clin Oncol. 2017 Jan 20;35(3):298-305.
3.McMullan RR, McConville C, McMullin MF. Ulster Med J. 2019 May;88(2):105-110.
4.Hashmi JA, et al. 2018 Jan;58(1):10-15.
5.R Core Team (2012). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0, URL http://www.R-project.org/
6.https://www.genecards.org
7.Jaruskova M. et al. J Exp Clin Cancer Res. 2017; 36: 55.

Disclosures
No relevant conflicts of interest to declare.