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Researchers evaluated whether targeted next-generation sequencing was comparable to fluorescence in situ hybridization for detecting genetic alterations in CLL.
A team of researchers at the 2024 ASCO Annual Meeting investigated the use of AI and machine learning for diagnosing and classifying chronic lymphocytic leukemia (CLL). The investigators reported that their study underscored the heterogeneity in CLL, as well as the potential for clinicians to use genetic markers to guide prognoses and treatment decision-making.
Similarly, in a recent study not presented at the annual meeting, researchers from the Mayo Clinic evaluated whether targeted next generation sequencing (NGS) was comparable to fluorescence in situ hybridization (FISH), the current gold standard, for identifying genetic mutations in CLL. The findings were published in Cancers.
“FISH panel assays, which typically detect 4 to 6 common recurring genetic defects, are relatively quick and have high specificity, but they require prior knowledge of the genetic abnormality to be screened and can detect only a limited number of alterations per assay,” wrote J. Erin Wiedmeier-Nutor, MD, MPH, and coauthors. “NGS has recently become more readily available for clinical applications and has the ability to screen the entire genome or multiple genes of interest in hematologic malignancies.”
The researchers added that whole-genome sequencing is the best approach for detecting copy number alterations, followed by whole exome sequencing. However, these methods are expensive and require clinicians to perform more intensive analyses than if they were to use gene panels.
“Although targeted sequencing is not an established method for clinical copy number alteration screening, it would be ideal to have a single clinical assay that could evaluate multiple relevant copy number alterations and mutations in a cost-effective manner. Hence, we evaluated the accuracy of using a custom sequencing panel for simultaneously detecting prognostic copy number alterations and gene mutations commonly assessed in CLL in a large cohort of untreated patients with CLL and high-count monoclonal B-cell lymphocytosis (HC-MBL), the precursor to CLL.”
Targeted NGS vs FISH
The study included 522 patients diagnosed with CLL or HC-MBL between 2001 and 2019. The patients provided a blood sample within two years of diagnosis for NGS and had available data from FISH assays performed within three months of submitting their blood specimen.
The researchers designed a DNA targeted sequencing panel that covered 59 recurrently mutated genes in CLL, as well as amplicons typically affected by clinically relevant copy number alterations. The investigators also used an algorithm called PatternCNV to detect common abnormalities in FISH assays.
The final analysis involved 509 patients, including 379 with CLL and 130 with HC-MBL, with successful copy number alteration calls. Patients averaged 62 years of age and were mostly male (70.3%). Half of the participants (49.8%, n=242) had IGHV mutations, and most patients had low- or intermediate-risk disease.
Targeted sequencing showed high concordance with FISH, especially for del(17p) and del(11q) mutations.
FISH demonstrated the following prevalence rates:
- 3% for del(17p)
- 0% for del(11q)
- 5% for trisomy 12
- 2% for del(13q)
Targeted sequencing showed similar rates:
- 1% for del(17p)
- 4% for del(11q)
- 7% for trisomy 12
- 7% for del(13q)
There were 77 discordant cases between FISH and targeted sequencing. For del(17p), targeted sequencing produced four false negatives and three false positives; manual review corrected three false negatives, and chromosomal microarray (CMA) confirmed one false positive as true. For del(11q), targeted sequencing showed five false negatives and two false positives, with CMA confirming one false negative. For trisomy 12, there were ten false negatives and one false positive, with CMA confirming one false negative.
Del(13q) had the highest discordance with 52 cases (40 false negatives, 12 false positives); manual review corrected nine false negatives, and CMA confirmed five false positives.
Targeted sequencing also identified other chromosomal aberrations, such as del(6q) (3.5%), del(14q) (2.1%), gain 8q (3.1%), and gain 2p (5.1%). Additionally, targeted sequencing detected complex karyotypes (3 or more chromosomal abnormalities) in 26 samples. More than half (66.4%) of the participants had at least one mutation in recurrently mutated genes in CLL, and 34.8% had two or more mutations. Targeted sequencing detected TP53 mutations in 11.4% of patients, with 4.9% having both TP53 mutations and del(17p).
Limitations & Future Directions in CLL
Dr. Wiedmeier-Nutor and colleagues concluded that a single targeted sequencing assay could infer copy number alterations relevant to CLL’s prognosis and provide information about additional clinically relevant genetic alterations.
The researchers noted, however, that their study “supports the need for manual visual inspection of negative copy number variation (CNV) calls that are slightly below the threshold of CNV calling to identify additional CNVs.”
Dr. Wiedmeier-Nutor and colleagues also called for further research to validate their findings.
“It is important to note that we are not proposing targeted NGS as the ideal method for the detection of adverse mutations and CNAs in every circumstance (for example, turn-around time for targeted sequencing may take one to two weeks, whereas FISH may be available in a few days),” Dr. Wiedmeier-Nutor and the coauthors wrote. “Rather, it is a cost-effective NGS tool that can provide additional prognostic information.”
The team added that future investigations will compare targeted NGS and FISH for relapsed/refractory CLL since these patients’ samples will have more molecular aberrations that traditional FISH assays may not detect.
