Molecular and Genetic Characterization of Fit, Elderly Patients Receiving Oral Fludarabine, Oral Cyclophosphamide and Intravenous Rituximab (OFOCIR) As Initial Treatment of Chronic Lymphocytic Leukemia (CLL)
Authors: Badoux Xavier C , Best Giles , Gabrielli Sara , Hayes Melanie , Zhu Ying , Mulligan Stephen , Kuss Bryone J 
Published: October 2019

Abstract
Background
Cytogenetic characterization by fluorescence in-situ hybridization (FISH) identifies subgroups of patients with chronic lymphocytic leukemia likely to have poor responses or short remission duration following standard frontline chemoimmunotherapy. Next-generation sequencing (NGS) has identified new molecular targets associated with refractory or poorly responsive disease (eg. Notch1, SF3B1 or BIRC3) independent of cytogenetic abnormalities. We have performed genetic and molecular characterization of fit, elderly patients enrolled on the Australasian Leukemia and Lymphoma Group (ALLG) CLL5 randomized clinical trial of oral fludarabine, cyclophosphamide and rituximab (ACTRN12608000404325).
Methods
Pre-treatment peripheral blood and bone marrow aspirate samples were obtained from patients enrolled on a phase II randomized clinical trial investigating oral fludarabine, oral cyclophosphamide and intravenous rituximab (poFCivR) tolerance in previously untreated fit elderly patients with CLL (ALLG CLL5 study). Fitness was defined as Cumulative Illness Rating Scale (CIRS) score of ²6. Bone marrow aspirate samples were analysed for CLL-associated genomic changes with a Vysis CLL FISH probe kit (Abbott, Des Moines, IL) and ranked according to Dohner hierarchical classification. DNA was extracted from peripheral blood lymphocytes and we performed targeted exome sequencing of genes including TP53, ATM, NOTCH1, SF3B1, BIRC3, MYD88 and FBXW7 using a TruSeq Custom Amplicon Design Panel on a MiSeq DNA sequencer as per manufacturerÕs protocol (Illumina, San Diego, CA). Gene mutations were confirmed by Sanger sequencing. Data was analyzed using Illumina proprietary software, annotated using ANNOVAR software and compared to COSMIC and other genomic mutation databases.
Results
Of 116 analyzable patients enrolled on the clinical trial, 78 pts had available FISH results and 76 patients DNA sequencing. The ORR and CR for all patients on study were 96% and 56% respectively. There was no significant difference in ORR between cytogenetic risk groups (Table 1); however, only 1 of 9 patients with ATM deletion achieved CR (11%, p=0.0095). We identified 8 pts with TP53 mutations, only one patient (12.5%) achieved a CR (p=0.0084). CR rate for patients with mutations in ATM (n=9, CR 44%), NOTCH1 (n=10, CR 60%), SF3B1 (n=11, CR 91%), BIRC3 (n=2, CR 0%), XPO1 (n=6, CR 33%), myd88 (n=5, 100%) were not significantly different to patients without the respective mutations. Of 14 pts with normal FISH, 10 pts (71%) had molecular abnormalities identified by NGS (Figure 1). Median follow-up of patients is 20 months, with 91% patients alive at last follow up. At the time of analysis, there was no significant difference in progression free survival (PFS) between different FISH cytogenetic risk groups (Figure 1). Multivariable analysis identified patients with TP53 mutations (HR 4.3, p=0.04) and XPO1 mutations (HR 3.2, p=0.035) as independently associated with shorter PFS. Our analysis was limited by the small subgroups of patients with individual molecular mutations and currently relatively short follow-up of this study.
Conclusions
Molecular characterization by DNA sequencing increases the yield of pre-treatment genetic alterations discovered in CLL patients. In this randomized clinical trial of elderly patients requiring first line therapy of CLL, we identified a high proportion of genomic alterations. Identification of genomic mutations may help further risk stratify CLL patients undergoing chemoimmunotherapy.
Table 1 N CR (n) CR (%) ORR (n) ORR (%) All patients 116 65 56 111 96 FISH 17p deletion 19 10 52 17 89 11q deletion 9 1 11 8 89 Trisomy 12 15 9 60 14 93 13q deletion 33 18 55 32 97 No abnormal 16 13 81 16 100 Not Done 24 14 81 24 100 NGS All Available 76 44 58 72 95 mutations TP53 8 1 13 7 87.5 ATM 9 4 44 9 100 NOTCH1 10 6 60 9 90 SF3B1 11 10 91 10 91 BIRC3 2 0 0 2 100 XPO1 6 2 33 5 83 Myd88 5 5 100 5 100
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
Badoux: Roche: Honoraria. Mulligan:Roche, Abbvie: Consultancy, Honoraria. Kuss:Roche: Research Funding; Sanofi: Research Funding.