Page 10 - 2021_03-Haematologica-web

EDITORIALS
Validating genomic tools for precision medicine in CLL: ERIC leads the way
Adrian Wiestner
National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA E-mail: ADRIAN WIESTNER - wiestnera@mail.nih.gov
doi:10.3324/haematol.2020.270652
In this issue of Haematologica, Sutton and colleagues report the comparative analysis of targeted next-genera- tion sequencing (NGS) panels for the detection of gene mutations in chronic lymphocytic leukemia (CLL).1 This important contribution to the standardization of mutation profiling for clinical practice was made possible by a multi- center study conducted by the European Research Initiative on CLL (ERIC).
CLL is characterized by the clonal expansion of mature B cells in blood, bone marrow, and lymph nodes.2, 3 CLL has a highly variable clinical course. While some patients may never develop symptomatic disease and do not require treat- ment, others can progress rapidly and may need repeat lines of therapy. Relapsing disease often carries additional genetic abnormalities that contribute to progression and/or treat- ment resistance. Determination of the mutational status of the rearranged immunoglobulin heavy chain variable (IGHV) genes contributes greatly to the dissection of disease hetero- geneity. IGHV sequences with ≥98% homology to germline are considered unmutated. Compared to CLL with mutated IGHV genes, IGHV unmutated CLL is associated with more rapidly progressive disease, early relapse, and more frequent acquisition of additional genetic lesions. IGHV mutation sta- tus is therefore one of the most important prognostic factors in CLL. Fluorescence in situ hybridization (FISH) cytogenetics detect additional well-validated prognostic markers. Deletion of chromosomal arms 17p (del17p) or 11q (del11q) are associated with shorter overall survival and early progres- sion after treatment with chemoimmunotherapy. Dependencies of the clonal cells on B-cell receptor signaling and BCL2 are now specifically targeted by kinase inhibitors and venetoclax, respectively. Having demonstrated superior disease control compared to chemoimmunotherapy in ran- domized trials, these targeted agents have become preferred treatment options for patients with CLL.3
Over the last years, genome-wide analyses using NGS con- tributed a large number of putative driver mutations as well as mutations associated with treatment resistance. Aggregating data from two large NGS studies covering approximately 1,000 patient samples, Lazarian et al. counted 75 significantly mutated genes.4 Thirty-five were found at a frequency >1% in the combined data set, and 28 were common to both studies. However, most gene mutations were present in <5% of patients. The most commonly mutated genes function in DNA damage response and cell cycle control (ATM, TP53, POT1), RNA and ribosomal processing (SF3B1, XPO1, RPS15), NOTCH1 signaling (NOTCH1), and immunoreceptor signal- ing (BIRC3, MYD88, EGR2).2,4 Several of the somatic muta- tions have been validated as independent prognostic markers. In most patients, the tumor cells carry more than one putative driver mutation often in combination with one or more chro- mosomal aberration detected by FISH. Integrating somatic mutations and FISH cytogenetics improves the separation of risk groups regarding overall survival.5
©2021 NIH (National Institutes of Health)
The increasing number of mutations and the complexity and heterogeneity of the tumor genome in CLL pose a need for techniques that can concurrently screen multiple genes. Targeted amplicon-based gene panels are widely pursued as an option that can meet the demands while being practical for routine testing. A number of technical options exist. The comparative performance of these different technologies in the hands of clinical laboratories is the focus of the study reported here.1 The coordinating ERIC site distributed tumor DNA from 48 CLL patients to six participating European cen- ters. Three different amplicon-based targeted assays were used in the study: HaloPlex Target Enrichment System (Agilent Technologies), Illumina TruSeq Custom Amplicon (Illumina) and Multiplicom CLL Multiplex MASTR Plus (Agilent). Each assay was used by two centers allowing for a comparison of the performance of the individual assays. Paired-end sequencing was performed on Illumina MiSeq instruments. All sequencing data were centrally analyzed using a custom bioinformatics pipeline. Across all six centers, overall reproducibility of targeted NGS was assessed by look- ing at the inter-laboratory variation in detecting mutations. 107 of 115 mutations were detected in all six centers for a remarkable concordance of 93%. A variant allele frequency (VAF) of 5% had to be reached in at least one center for the variant to be included in the concordance counts. Noteworthy, eight variants were detected in five of the six centers and missed in one. Six of theses eight variants con- cerned minor subclonal mutations present at low frequency. The full report is rich in detail on the performance of the indi- vidual assays and the reasons for false negatives and the rare false positives having VAF ≥5%. Suffice it to say that all the assays performed well with high concordance between sister centers utilizing the same methodology.1
The concordance of 93% for inter-laboratory performance is based on mutations with VAF ≥5%. VAF cutoffs of 10%, the approximate detection limit of Sanger sequencing, are widely used in clinical routine. Despite advantages of NGS, reliable detection of low-level variants may be difficult. Pushing the limit of detection lower requires additional tech- nical adaptions. In the ERIC study, the investigators explored high sensitivity assays containing unique molecular identi- fiers to detect low-level variants.1 While successful in princi- ple, this method adds to complexity and will require addi- tional efforts to implement in routine diagnostics.
Given the technical difficulties, do we really need to know about low frequency mutations? Yes, because, at least for some genes, low-level variants may be clinically relevant. In patients treated with chemoimmunotherapy, minor clones harboring TP53 mutations have been shown to expand by the time of relapse.6 The strong selective advantage conferred by TP53 mutations may explain observations that even minor clones harboring TP53 mutations (VAF <10%) confer poor prognosis.7,8 Nadeu and colleagues screened for known driver mutations in 28 genes. They identified some as having a prog-
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haematologica | 2021; 106(3)