Page 220 - Haematologica May 2020

J. Edelmann et al.
Introduction
Advanced understanding of the pathophysiology of chronic lymphocytic leukemia (CLL) has led to targeted therapy approaches such as inhibition of B-cell receptor signaling by BTK inhibitors or PI3K inhibitors and antago- nism of BCL-2.1,2 These treatment strategies clearly improved the clinical outcome of high-risk CLL,1,2 although inadequate responses have been observed in as yet insuffi- ciently characterized subgroups of patients.3-5 In the era of chemo(immuno)therapy, high-risk CLL was defined by TP53 deletion/mutation or refractoriness to purine analog- based treatment (no response or progression-free survival <6 months).6 For chemotherapy-free regimens, the prog- nostic value of TP53 alterations is less clear, but the pres- ence of a complex karyotype, which often occurs together with TP53 deletion/mutation,7 has been identified as an independent risk factor for early progression during vene- toclax or ibrutinib treatment.8,9 However, a more recent study has shown that CLL with a complex karyotype is a heterogeneous group with variable clinical behaviors.10
To better understand treatment failure in CLL, a com- prehensive characterization of the genomic architecture in high-risk CLL is vital. With 5-10% high-risk cases included in large-scale studies on DNA copy number changes and gene mutations, these cases were underrep- resented for systematic analyses restricted to this sub- group.11-14
Available results from single nucleotide polymorphism (SNP)-array profiling of high risk CLL support the notion of increased genomic complexity in the majority of these cases.11-13 However, it should be noted that TP53 dysfunc- tion and defects in other DNA damage response systems such as ATM cause chromosomal instability with random secondary events not necessarily associated with adverse prognosis.15 This constitutes a challenge, to identify those alterations contributing to a high-risk form of disease.
In order to get a more thorough understanding of the pivotal genomic alterations contributing to high-risk CLL biology, we performed high-resolution SNP-array profil- ing and targeted sequencing on 75 relapsed/refractory CLL cases including 18 cases without TP53 alterations. We extended our cohort by including 71 treatment-naïve, TP53-deficient, primary high-risk cases. All patients’ sam- ples were derived from prospective clinical trials of the French/German CLL study groups (FCLLSG/GCLLSG).
To identify DNA copy number alterations (CNA) occur- ring more often than would be expected by chance, we applied the Genomic Identification of Significant Targets in Cancer algorithm 2.0 (GISTIC2.0).16 In relapsed/refrac- tory CLL, in which tumor cell clones underwent selective pressure imposed by therapy, CNA with significance assigned by GISTIC2.0 harbored genes with key roles in cell-cycle control. Furthermore, we identified NOTCH1 as a central pathway frequently affected by genomic alterations enhancing its signaling strength.
Methods
Patients and samples
The study included peripheral blood mononuclear cells (PBMC) from 146 high-risk cases (TP53 aberration or refractori- ness to purine analogs) enrolled on prospective trials of the GCLLSG/FCLLSG (CLL2O trial, clinicaltrials.gov identifier:
NCT01392079; CLL8 trial, NCT00281918; CLL11 trial, NCT01010061). Written informed consent from all patients and ethics committee approval were obtained in accordance with the Declaration of Helsinki.
Selection of cases was guided by sample availability and included 110 of 135 cases from the CLL2O trial,17 27 of 51 cases with 17p deletion from the CLL8 trial18 and nine of 52 cases with 17p deletion from the CLL11 trial.19 All samples were taken at trial enrollment and tumor cells were enriched via CD19 immunomagnetic beads (MACS, Miltenyi Biotec®, Bergisch Gladbach, Germany). CD19 negative PBMC fractions with a tumor cell load <5% were available for paired analysis in 91 cases. Cases lacking matched normal material were analyzed against a pool of ten gender-matched reference samples.
IGHV mutational analysis, fluorescence in situ hybridization (FISH) studies for 11q22.3, 13q14, 12p11.1-q11, 17p13.1, t(11;14)(q13;q23) and TP53 mutational analysis were performed at trial enrollment. Cases positive for t(11;14)(q13;q23) were excluded from the study. Telomere length was determined as described previously.20
Single nucleotide polymorphism array and gene enrichment analysis
Analysis for CNA, including copy neutral losses of heterozy- gosity, was done using 6.0 SNP arrays (Affymetrix®, Santa Clara, CA, USA). CNA positions and gene locations were determined with the UCSC Genome Browser, assembly March 2006, NCBI36/hg18. CNA frequencies were compared to those observed in treatment-naïve, standard-risk cases (n=304, no TP53 deletion/mutation).13 Microarray raw data were made pub- licly available at Gene Expression Omnibus (GEO accession number: GSE131114).
GISTIC2.0 was applied on manually curated DNA copy num- ber data.16 According to default settings, CNA with a q value <0.25 were defined as significant. CNA that reached high confi- dence levels for being significantly enriched (q value <0.01) were manually curated for minimally affected regions. Genes located within these minimally affected regions were assigned to WikiPathways21,22 and analyzed for pathway enrichments using PathVisio, version 3.2.3.23,24
Next-generation sequencing
Amplicon-based, targeted next-generation sequencing (tNGS) was performed on TP53 exons 2-11, NOTCH1 exon 34, and SF3B1 exons 13-16. In 17 cases TP53, NOTCH1 and SF3B1 mutational status was determined as previously described.25 All coding regions of MGA, SPEN, RBPJ, and SNW1 (in 108 cases each), and CDKN2A and MYC (in 93 cases each) were screened by tNGS.
Quantitative gene expression analysis
Gene expression of CCAT1, MGA, RBPJ, SNW1, HES1, DTX1, MYC, CDKN2A, p14ARF, and p15INK4b was analyzed by quan- titative reverse transcription (qRT) polymerase chain reaction (PCR) (TaqMan® Gene Expression Assays; Applied Biosystems®, Foster City, CA, USA). Sample selection was based on highly clonal presence of respective CNA/gene mutations (log2 ratio <- 0.8 for deletions and >0.75 for gains; variant allele frequency >0.3 for mutations). Promoter DNA methylation of CDKN2A/B transcripts was assessed by bisulfite PCR followed by Sanger sequencing.
Statistical analysis
Associations between genomic alterations were tested by Fisher exact tests; differences between datasets by Mann
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