Targeted DNA Sequencing for Identifying Prognostic Copy Number Alterations and Mutations Among Patients With CLL

Erin Wiedmeier-Nutor, MD, MPH, Mayo Clinic, Phoenix, Arizona discusses study results comparing an alternative approach for screening prognostic chromosomal abnormalities, a custom DNA sequencing panel designed to detect prognostic copy number alterations and gene mutations, versus the widely used fluorescence in situ hybridization (FISH) panel among a large cohort of untreated patients with chronic lymphocytic leukemia (CLL) and high-count monoclonal B-cell lymphocytosis, a precursor to CLL.

“Our study showed that targeted sequencing can infer prognostic copy number alterations while also providing additional clinically relevant genetic alterations,” concluded Dr Wiedmeier-Nutor.

Transcript:

Hi, my name is Dr. Wiedmeier-Nutor. I'm [an] assistant professor at Mayo Clinic [in] Arizona, and today I will be talking to you about the utility of targeted sequencing compared to FISH for detection of CLL copy number alterations, which was published in Cancers in July of 2024.

Chromosomal copy number changes are a hallmark of many hematologic malignancies, including CLL. Several of these chromosomal alterations, for example, deletion 17p, trisomy 12, deletion 11Q, and deletion 13Q in CLL are evaluated as standard of care because they have important prognostic and predictive implications. Traditionally, chromosomal alterations have been evaluated using FISH. FISH is a useful tool, but it requires prior knowledge of the genetic abnormality.

Next-generation sequencing techniques are now common practice in many hematologic malignancies as the identification of specific mutations also have important prognostic and predictive consequences. Whole genome sequencing is the best sequencing approach for detecting copy number alterations followed by whole exome sequencing, but both methods are still relatively expensive, and the analysis is bioinformatically more intensive compared to gene panels.

Targeted DNA sequencing is an alternative [and] less expensive approach to identifying mutations. 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.

We evaluated the accuracy of using a custom sequencing panel for simultaneously detecting prognostic copy number alterations and gene mutations commonly tested in CLL in a large cohort of untreated patients with CLL and high-count monoclonal B-cell lymphocytosis, which is the precursor to CLL. A total of 509 patients were sequenced with our targeted sequencing panel and had successful copy number alteration calls and were included in the final analysis. Using a method called Pattern (cytomegalovirus) CMV, we found a high specificity sensitivity, positive predictive value, and negative predictive value, comparing targeted next generation sequencing to FISH as the gold standard.

Importantly, targeted sequencing provided mutation results across 59 genes of which FISH and genomic arrays cannot evaluate. Mutation analysis included TP53, which is one of the most clinically important adverse prognostic markers in CLL and many other hematologic malignancies. Patients with any TP53 abnormality including deletion 17p, have shorter progression-free survival and overall survival, and are less responsive to traditional chemoimmunotherapy. Additional genes included and detected in the panel such as SF3B1, NOTCH1, BIRC3, and EGR2 have also been associated with differential prognosis in multiple studies. Targeted sequencing also provided complex karyotype status, which is known to be associated with inferior outcomes, some of which were missed with FISH analysis alone.

In conclusion, our study showed that targeted sequencing can infer prognostic copy number alterations while also providing additional clinically relevant genetic alterations. Thank you for allowing me to speak to you today.

Source:

Wiedmeier-Nutor E, McCabe C, O’Brien D, et al. Utility of targeted sequencing compared to FISH for detection of chronic lymphocytic leukemia copy number alterations. Cancers. Published online July 3, 2024. doi: 10.3390/cancers16132450