Clonal Evolution Pattern of Leukemia Progression and Richter Transformation in Ibrutinib-Relapsed CLL Patients
Authors: Kadri Sabah , Lee Jimmy , Fitzpatrick Carrie , Galanina Natalie , Sukhanova Madina , Venkataraman Girish , Sharma Shruti , Long Bradley , Theissen Megan , Ming Mei , Kobzev Yuri , Wenjun Kang , Guo Ailin , Wang Weige V , Niu Nifang , Weiner Howie Lawrence , Thirman Michael J. , Smith Sonali M. , Segal Jeremy P. , Lu Pin , Wang Y. Lynn 
Published: October 2019

Abstract
Introduction
Ibrutinib (ibr), a first-in-class BTK inhibitor, has high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL) independent of high-risk FISH abnormalities (NEJM. 2015; 373:2425-37, NEJM. 2013; 369:32-42). However, about 25% of patients discontinue ibr therapy at a median of 20 months treatment and ~40% patients stop ibr due to disease progression (JAMA Oncol 2015; 1:80-7, Blood 2015; 125:2062-67). Among progressed patients, at least half developed Richter's transformation (RT). Treatment options following progression are limited, with mortality rates exceeding 75% and a short median overall survival of 3 months. As the use of ibr becomes more prevalent in CLL and other types of non-Hodgkin lymphoma (NHL), more patients are at risk to develop resistance (BJH 2015; 170:445-56). Strategies to prevent and treat ibr-relapsed patients by understanding mechanisms of resistance are critically needed and will support rational drug development and therapeutic approaches. Recent studies including ours have provided some insights into ibr-resistance. Both BTK and PLCG2 mutations have been shown to confer ibr-resistance (NEJM 2014; 370:2352-54, NEJM 2014; 370:2286-94). Additionally, TRAIL-R has also been associated with the drug resistance in several ibr-relapsed patients (Nat Comm 2016). However, there is still limited understanding in the setting of disease progression during ibr treatment, such as what risk factors predispose patients to relapse, whether other molecular or cytogenetic lesions are associated with disease progression, and how RT is related to CLL tumor cells in the blood.
Materials & Methods
To address these questions, we analyzed 9 CLL patients treated with ibr and relapsed at the University of Chicago between 2008-2016. Among 9 patients, 6 developed RT at progression. The median age was 66.3 yrs (range, 52-88) and the median number of therapies prior to ibr initiation was 2 (range, 1- 4). The median duration of response to ibr was 16 months (range, 2-30). Eight patients discontinued ibr therapy due to CLL progression or RT. Longitudinal samples including peripheral blood (PB), bone marrow (BM) and tissue were collected from each patient at time points prior to ibr initiation (pre-ibr), post-relapse and at the time of RT. When possible, samples were also collected during the responding phase. Samples were analyzed using both UCM-OncoPlus, a hybrid capture 1,212 cancer-associated Next generation sequencing (NGS) gene panel and Affymetrix SNP arrays (CytoScan and OncoScan) to assess mutations, indels, copy number variations (CNVs) and loss of heterozygosity. A custom algorithm was developed to calculate mutant clonal frequencies (MCFs) using an integrative approach combining allelic frequencies, tumor purity and CNV data. K-means clustering was performed to identify gene mutations belonging to the same clonal populations. An amplicon-based 17-gene CLL panel was further used to sequence BTK in greater depth (~10,000x) to confirm the presence of minor clones (1-5%) in some samples.
Results & Conclusions
To determine the risk factors associated with relapse, we compared all pre-ibr samples for recurrent molecular and cytogenetic abnormalities. Eight of nine patients were found to have TP53 mutations and/or del (17p), several other genetic lesions also seem to be enriched in our cohort compared to larger populations of treated or untreated CLL patients. The mutation profiles of matched pre-ibr and relapsed CLL PB/BM for each patient were then compared to identify relapse-specific alterations that might drive CLL progression. As expected, BTK mutations were found in 5 of 9 patients including previously reported C481S/R as well as a structurally novel T316A located in the SH2 domain. The findings confirm that mutated BTK is the most common mechanism responsible for disease progression on ibr. Furthermore, the emergence of minor BTK clones was detected in progressed patients. Analyses are ongoing to determine the clonal relationship between CLL leukemia in PB/BM and large cell transformation at the tissue site. So far, IGH gene rearrangement assay on three of five patients demonstrated that the CLL and RT are of the same B-cell origin. We are also in the process of performing cluster analyses to understand mutations that travel in the same malignant clones or sub-clones in each patient and updated results will be presented.

Disclosures
Smith: Celgene: Consultancy; Amgen: Other: Educational lecture to sales force; Genentech: Consultancy, Other: on a DSMB for two trials ; AbbVie: Consultancy; TGTX: Consultancy; Gilead: Consultancy; Portola: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy. Wang:Portola Pharmaceuticals: Honoraria, Research Funding.