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Novel DNA/RNA Genomic Profiling Assay Identifies Broad Range of Genomic Alterations in Hematologic Malignancies

December 30, 2021

Researchers have developed a novel, next-generation sequencing-based assay that successfully characterized genomic alterations in 93 percent of patients with hematologic malignancies. This genomic-profiling approach has the potential of increasing the ability to identify clinically and therapeutically relevant alterations, according to Jie He, PhD, from Foundation Medicine in Cambridge, Massachusetts, and colleagues, whose findings were published in Blood.

"Cytogenetic studies have identified recurrent mutations and amplifications/deletions in a spectrum of hematologic malignancies, creating a pressing need to develop comprehensive genomic assays to identify somatic alterations," Dr. He and co-authors wrote. "However, current diagnostic assays, including FISH and real-time PCR, are designed ad hoc to identify specific genomic alterations, and in some cases there are no assays that can reliably identify specific rearrangements."

The newly developed assay uses targeted next-generation DNA and RNA sequencing to identify single-nucleotide substitutions, insertions and deletions, copy number alterations (CNAs), and rearrangements.

To test the accuracy of this assay, Dr. He and researchers first extracted DNA and RNA from each of the 3,696 patients, categorized them into barcoded libraries through separate workflow streams, and then pooled and sequenced the samples. The investigators then performed blinded comparisons between the novel assay and DNA-only diagnostic assays, including real-time PCR, FISH, and PRC fragment analysis, for a large number of clinical samples. The concordance between the two sets of results was 99.4 percent, "[demonstrating] that the current DNA profiling platform can accurately detect DNA sequence variants and CNAs, as established for the reference assay," the authors reported.

The novel assay was also evaluated for its ability to detect genomic rearrangements. Sensitivity and specificity (determined by comparing the genomic rearrangements detected in the pooled samples relative to the constituent cell-lines) were high (at 100% and 98%, respectively), and very few false-positive responses were observed, with a positive predictive value of ≥98 percent (n=245/248). Results from the novel assay were reproducible between batches, as well.

The new assay has been used to perform genomic profiling in 3,696 patient samples, and 3,433 (93%) were successfully characterized via DNA and RNA, including:

  • 27% formalin-fixed paraffin-embedded samples
  • 21% bone marrow aspirates
  • 18% blood samples
  • 34% samples from pre-extracted nucleic acids from relevant tumor tissues

The specimens submitted came from patients with the following diagnoses:

  • 39% multiple myeloma
  • 22% non-Hodgkin lymphoma
  • 41% diffuse large B-cell lymphoma
  • 13% follicular lymphoma
  • 8% B-cell lymphoma not otherwise specified
  • 8% mantle cell lymphoma
  • 17% acute myeloid leukemia
  • 13% myelodysplastic syndromes/myeloproliferative neoplasms

"We showed that a single, integrated assay can be used to find the spectrum of clinically relevant disease alleles, including mutations, translocations, and gains and losses," Ross Levine, MD, a co-author of the study, told ASH Clinical News. "In many cases, having this information is of value with respect to clinical care."

Extracted DNA was sequenced to an average depth of 500x and RNA to an average of 6.9 million unique pairs. Seven percent of the specimen (n=263/3,433) did not attain quality control specifications, including failures in DNA or RNA preparation, or blood samples older than acceptable criteria with no evidence of disease, or no somatic driver mutations reported under the study's qualifying conditions.

"Genomic profiling of hematologic malignancies demonstrates that only a relatively small number of genes are commonly mutated while many specimens harbor a wide range of rarer events, including base substitutions, indels, copy number amplification/losses, and rearrangements," the authors reported.

At least one driver mutation was identified in 95 percent of tumor specimens (n=3,246/3,433), 77 percent (n=2,650) of which harbored at least one alteration linked to an approved targeted therapy or one in clinical trials, including:

  • NRAS (14%)
  • KRAS (13%)
  • DNMT3A (7%)
  • CDKN2A (7%)
  • IDH1/2 (5%)
  • BRAF (4%)
  • FLT3 (4%)

In 61 percent of cases, at least one alteration with known prognostic relevance in a particular tumor type was identified, including:

  • TP53 (19%)
  • ASXL1 (9%)
  • TET2 (8%)
  • CDKN2B (5%)
  • CREBBP (5%)
  • MLL (4%)
  • NPM1 (2%)

A total of 1,524 genomic rearrangements were identified in 37 percent (n=1,256/3,433) of tumor specimens, involving genes implicated in:

  • Chromatin/histone remodeling (20%)
  • Transcriptional regulation (16%)
  • Kinase/oncogene activation (15%)
  • Apoptosis regulation (13%)
  • Cell cycle regulation (13%)
  • Truncation of tumor suppressors (5%), including novel in-frame fusions in kinase drug targets in ALK, BRAF, FLT3, JAK2, and ROS1

Dr. He and researchers were also able to identify genomic alterations in different patients that activated specific subsets of oncogenes, such as somatic alterations involving the FLT3 locus that have been shown to be of prognostic importance in acute leukemia. Eighty-nine alterations involving FLT3 in 84 specimens were found, as well as other base substitutions (n=14), in-frame deletions (n=1), CNAs (n=6), and fusions involving FLT3 (n=1), "indicating that the sequencing-based assay can robustly detect a wide range of alterations in driver genes that are not fully evaluated using conventional methods."

"The assay is rapid, comprehensive, accurate, and able to find lesions in DNA and RNA," Dr. Levine told ASH Clinical News. "This allows us to offer genomic testing to a broader set of patients with blood cancers."

The study was limited by RNA preparation failure that occurred in 132 cases, thus indicating a challenge of processing and sequencing RNA samples.

Dr. He, the study's first author is an employee of Foundation Medicine, a company that develops, manufactures, and sells genomic analysis diagnostics.


He J, Abdel-Wahab O, Naha MK, et al. Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting. Blood. 2016 March 10. [Epub ahead of print]


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