Novel Assay Identifies Mutations, Fusions, Exon Skipping in Myeloid and Lymphoid Malignancies

November 20, 2020
Nichole Tucker
Nichole Tucker

In an interview with Targeted Oncology during the Association for Molecular Pathology 2020 Annual Meeting, Rachel Sparks, MD, discussed the findings observed in hematologic malignancies with the use of a new RNA-based NGS assay.

In modern practice of hematologic malignancy management, clinicians utilize various assays to help identify gene fusions and driver mutations to diagnosis and classify patients as well as select the appropriate targeted therapy to treat their disease. The majority of assays only have the ability to pinpoint either fusions, mutations, or exon skipping.

With the assistance of using anchored multiplex polymerase chain reaction technology, oncologic pathologists at the University of Iowa have curated a new next-generation sequencing (NGS) assay with the ability to identify a minimum of 76 alterations, including hotspot mutations, fusions, and exon skipping mutations.

To evaluate the ability of the assay, 63 patient cases were assessed. The patients had a wide range of hematologic malignancies, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), Ph-like acute lymphoblastic leukemia (Ph-ALL), Philadelphia chromosome positive B-cell lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, acute promyelocytic leukemia (APL), and myeloproliferative neoplasm (MPN) with hypereosinophilia.

Results of the assay evaluation showed that 55 alterations were identified, so that were expected with an NGS platform. Notably, however, there were some unexpected findings.

In an interview with Targeted Oncology during the Association for Molecular Pathology (AMP) 2020 Annual Meeting, Rachel Sparks, MD, fellow, Molecular Pathology, Carver College of Medicine, University of Iowa, discussed the findings observed in hematologic malignancies with the use of a new RNA-based NGS assay.

TARGETED ONCOLOGY: What is the current of next-generation sequencing in myeloid and lymphoid neoplasms?

Sparks: NGS in hematopoetic and lymphoid malignancies is increasingly complex, but it's used

routinely for diagnosis. Some of the subtypes are based on the genetic mutations that are identified and NGS is also used for identification of prognostic factors and other findings that would influence therapy.

TARGETED ONCOLOGY: In the past five years, what advances have we seen in terms of identification of driver mutations in these diseases?

Sparks: With widespread use of NGS in this field, we’ve identified at least 76 genes that are driven mutation. If you look at just AML alone, you can see that there's at least 1 driver mutation that's identified in about 96% of cases. Some cases have 2 driver mutations at the time of diagnosis.

In the past 5 years, we’ve also identified mutation that are early like DNMT3A, ASXL1, and IDH1/2. Some of these genes are also associated with age-related clonal hematopoiesis. From all of this information, we can identify changes that are occurring later, such as changes in the RAS pathway genes or NPM1 mutations that are secondary events.

We’re starting to get a better feel of driver mutations in general as well as the early versus later drivers.

TARGETED ONCOLOGY: Can you provide background on the analysis you presented during AMP 2020?

Sparks: We custom-designed an assay that is an RNA-based NGS. Our assay was primarily designed to identify Ph-like ALL cases. We also included targets for single nucleotide variants (SNVs), fusions and exon skipping so that we could identify mutation that would indicate resistance to tyrosine kinase inhibitors. We also included the tyrosine kinase receptor genes, like PDGFRA, PDGFR, and FGFR, that would be identified in myeloid and lymphoid neoplasms with hypereosinophilia which have those specific abnormalities.

TARGETED ONCOLOGY: What were the results of the analysis?

Sparks: I presented cases that we looked at. Some of the cases were from our initial validation sample and the rest of the cases are from the patient sample that we conducted in our clinical practice.

Using our assay, we were able to identify about 87% of cases that had genomic alterations. These cases were either fusions, exon skipping mutations, or SNVs.

If we look at it based on disease, for example in CML and Ph-positive ALL, we were able to identify the BCR/ABL fusions. This was expected, but we also identified some cases at the head like IKZF1 exon skipping, which is a prognostic indicator that is no seen in all of the cases. If we just look at our AML cases, we identified a genomic alteration in over 90% of our cases. The majority of those were fusions either in KMT2A or RUNX1. These are fusions that would provide prognostic information for the clinicians and their patients. We also had a cohort of Ph-like ALL cases where again, we identified IKZF1 exon skipping, which is also associated with prognosis.

Then, we found specific fusions that were strongly associated with individual disease. For APL, we discovered a PML/RARA fusion, which is diagnostic. We also had 1 case of MPN with hypereosinophilia where we found a PDGFRA fusion.

TARGETED ONCOLOGY: What are the implications of these results?

Sparks: Our assay is able to simplify the workflow through its ability to identify fusions. It is also cost-effective. Our results are efficient and therefore able to guide clinical management for our patients.

TARGETED ONCOLOGY: How would you discuss these findings with an oncologist?

Sparks: I would mention this assay is helpful both from a diagnostic standpoint as well as from its ability to stratify patients prognostically and to guide therapeutic interventions.