Putting Mutation Testing in Myeloid Malignancies to the Test

In 2014, the World Health Organization (WHO) convened a clinical advisory committee to begin updating and revising the existing classification of myeloid neoplasms (MPNs) and acute myeloid leukemia (AML).1 In the 6 years since the prior classification was published in 2008, investigators had uncovered numerous unique biomarkers associated with these diseases, thanks to the wider availability of gene-expression analysis and next-generation sequencing (NGS). In 2016, the WHO published the update, incorporating novel data on genetic characterizations of genes associated with MPNs, myelodysplastic syndromes (MDS), and AML.

The availability of NGS and gene-expression data on these malignancies has led to improvements in diagnosing MPNs, predicting patient outcomes, and identifying gene mutations as potential therapeutic targets.

“For myelofibrosis, a subtype of MPN, prior to the use of clinical genomics, there were patients whose disease progressed despite treatment and, based on their clinical laboratory results and pathologic parameters, we really didn’t understand why,” Ann Mullally, MD, Associate Professor of Medicine at Harvard Medical School and a medical oncologist at the Dana-Farber Cancer Institute, told ASH Clinical News, describing one of the “success stories” of using mutation testing in myeloid malignancies.

In her lab, which studies the genetics and biology of MPNs, “we’ve come to understand the importance of a molecular genetics characterization in these patients,” Dr. Mullally said. “With the power of genetic tests, we are now able to identify a subset of patients with myelofibrosis [MF] and high-risk molecular features, for example. This allows us to more closely monitor these patients and consider more aggressive treatments such as allogeneic hematopoietic cell transplant.”

ASH Clinical News spoke with Dr. Mullally and other experts in the field of genetics and myeloid malignancies about the potential value of mutation testing for myeloid malignancies, which are increasingly being used in the clinic, as well as the challenges yet to overcome.

Mutation Testing in the Clinic

The wider availability of NGS methods – in the form of targeted gene panels and polymerase chain reaction (PCR)–based diagnostic tests – means that clinical diagnostic laboratories are incorporating these tools into their daily practices. These genetic tests identify mutations associated with MPNs in patients who have already been diagnosed with a myeloid malignancy or in those who are suspected of having a myeloid malignancy.2

“At diagnosis, a targeted NGS panel tailored for myeloid neoplasms as well as select PCR-based diagnostics are now standard because clinicians need that information up front to make management decisions, including whether to augment induction chemotherapy for patients with AML,” said Jay Patel, MD, MBA, Medical Director of Molecular Oncology and Hematopathology at ARUP Laboratories and Associate Professor at the University of Utah School of Medicine. Clinicians can use commercial gene panels, as well as gene panels made in-house at academic cancer centers’ clinical testing laboratories.

“We now sequence all of our patients with AML, most patients with MDS, and certain patients with cytopenia using our in-house 40-gene panel called MyeloSeq,” added Eric Duncavage, MD, Associate Professor of Pathology and Immunology and Section Head of Hematopathology at Washington University School of Medicine in St. Louis. “Results from MyeloSeq are most often used for risk stratification and making decisions about bone marrow transplant but can also be used to guide initial therapy in certain patients.”

At Dana-Farber, “every patient who has any kind of blood cancer or any blood abnormality that we’re trying to understand gets an in-house NGS test that comprises about 100 genes,” Dr. Mullally said of her institution’s practices. “For MPNs, we can identify the causative mutation using the gene panel, which is typically in one of three genes – JAK2, CALR or MPL, the so-called MPN phenotypic driver mutations.”

In addition to these mutations, the gene panels developed by Dana-Farber and other labs can also identify concomitant mutations present in myelofibrosis (MF), including ASXL1, SRSF2, EZH2, IDH1/2, and U2AF1-Q157, which can inform a patient’s prognosis. “This is important to know because we might change how intensively we monitor a patient based on his or her molecular risk,” she added.

“Most patients in the U.S. are likely getting a diagnostic panel, as are many MDS patients, though fewer compared with the AML population because of the wide spectrum of clinical presentations in MDS,” noted Dr. Duncavage.3 “Also, in AML, there are mutations that can be targeted with FDA-approved treatments.”

NGS gene panels allow laboratory scientists and clinicians to detect mutations in up to 100 genes, providing information used in three categories: diagnosis, prognostic risk stratification, and eligibility for targeted therapy.4 See the FIGURE for examples of mutations in genes used for these purposes.

At ARUP Laboratories, patient samples are assessed with a 64-gene panel. According to Dr. Patel, the turnaround time for the panel is 7 to 10 days, while the turnaround time for the single- or multiple-gene PCR diagnostic tests is 3 to 5 days. But the team at ARUP and other labs plan to decrease the turnaround time for the NGS gene panels to 7 days or fewer get the vital information to treating clinicians more quickly –through automation, standardization, and other workflow improvements.

“A practical solution we can implement now is to identify patients who are strong candidates for targeted therapy through close communication with treating hematologists and expedite their test results,” says Dr. Patel.

Targeted Drugs to Treat Myeloid Malignancies

While there are fewer molecular targeted therapies for myeloid malignancies than for solid tumors, the FDA has approved several therapies based on a patient’s mutational profile. In AML, mutations of the FLT3 gene occur in approximately 30% of cases, with the most common FLT3 mutation, an internal tandem duplication (ITD), representing about 25% of all FLT3 mutations. The FLT3-ITD mutation is a common driver mutation in AML and confers a poor prognosis.5 Several FLT3 inhibitors have been approved by the FDA in recent years. In 2017, the agency approved midostaurin for newly diagnosed AML patients with a FLT3 mutation in combination with standard induction chemotherapy. The following year, the agency approved the FLT3 inhibitor gilteritinib to treat patients with relapsed or refractory AML. Several additional FLT3 inhibitors are being evaluated in clinical trials.

The IDH1 and IDH2 genes, which together occur in about 20% of patients with AML, also are targets of approved AML therapies: ivosidenib, an oral drug that targets IDH1 mutations, and enasidenib, an oral drug that targets IDH2 mutations.6,7

Ruxolitinib, an oral JAK1/2 inhibitor, was first approved by the FDA in 2011 for the treatment of patients with intermediate- and high-risk, primary, post-polycythemia vera and post-essential thrombocythemia MF – the first therapy to be approved for MF in the U.S. Of note, ruxolitinib is approved for patients with intermediate- or high-risk MF, regardless of whether they harbor a JAK2 mutation. In 2019, a second JAK2 inhibitor, fedratinib, also was approved for MF and additional JAK2 inhibitors are being evaluated in late-phase clinical trials. However, the presence of other concomitant mutations in MF can influence the effectiveness of the JAK2 inhibitor therapy, Dr. Mullally explained, noting that “patients with myelofibrosis with three or more concomitant mutations are likely to have a shorter response to JAK2 inhibitor therapy.”

The Caveats of Mutation Testing

The advent of routine mutational testing has provided clinicians with information to help guide treatment decisions, but there are several obstacles on the way to their widespread adoption in the clinic. First, as with any new technology, reimbursement of NGS gene panel testing can still be a challenge, according to Dr. Duncavage. One emerging use for genetic panels is the detection and monitoring of measurable residual disease (MRD), but insurance companies often refuse to pay for MRD testing using sequencing panels.

“Many payers will pay for the mutation testing only once,” he said. “They see it as a genetic test for inherited disorders, like for the BRCA gene that increases the risk of developing breast cancer, where a person needs to be tested only once in a lifetime because the information is static. Of course, that is not the correct way of looking at MRD testing, but the payers have been slow to accept the modern utility of these testing tools.”

Another issue is standardization of NGS panel results. Panels have varying ranges of sensitivity of detection for the different sets of genes included in the panel. “A good example of a standardized genetic assay is the quantitative BCR-ABL1 RT-PCR test for chronic myeloid leukemia,” said Dr. Duncavage. “Results from a test from one lab are directly comparable to those from a test performed at another lab. We are not there yet with these myeloid neoplasm genetic tests.”

However, Dr. Patel predicted that, in the near future, “we’re going to continue to see hematopathologists and hematologists collaborate to develop consensus guidelines for appropriate biomarker testing in hematologic malignancies as technology continues to advance and the potential for clinical impact expands.”

Variability in how the results of genetic panels are interpreted – for example, determining whether a detected aberration is a potentially clinically meaningful mutation or a polymorphism – represents another challenge. There are no standardized cutoff values or recommended variant annotations for mutations, although the Association for Molecular Pathology and College of American Pathologists are working toward developing such a resource,” Dr. Duncavage noted. “We need harmonization, but it takes a long time to get everyone together and to agree on a mutual way to create such a resource.”

“Interpretation of test results is a shared responsibility between pathologists, clinical scientists, and treating hematologists,” added Dr. Patel. ARUP developed its first NGS gene panel for myeloid malignancies in 2014. “There was a steep learning curve with how to interpret the data, and we’ve learned quite a lot in the past several years,” he said. “The field is still evolving on how to classify sequence variants as pathogenic, variants of unknown significance, or benign.”

Dr. Duncavage sees clinical NGS methods continuing to expand to include whole-exome and whole-genome sequencing. “We can do so much with whole-genome sequencing to detect structural rearrangements, copy number alterations, and translocations that are currently detected by cytogenetics,” he said. “The ultimate goal is to create a single, rapid diagnostic assay capable of identifying the full spectrum of DNA mutations in cancer.”

Dr. Patel agreed, noting that whole-genome sequencing and whole-exome sequencing are being explored in research studies but are not yet ready for the clinic.

On the research side, scientists are working with large amounts of gene panel data from patients to identify genetic predictors of outcomes and other trends. “We are prospectively acquiring data that we can then analyze to link genetics with clinical outcomes, asking questions such as ‘Which patients progressed rapidly, which ones suffered thrombosis, and what are the molecular predictors of these outcomes that we can find?’” Dr. Mullally explained. “Having high-resolution, comprehensive genomic data acquired prospectively over many years will allow us to use the data to inform our clinical practice in the future. This highlights the power of genomic data and the critical importance of research – we must learn from our current patients to do better for them and for those who follow.”  —By Anna Azvolinsky

References

  1. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127:2391-2406.
  2. Tefferi A, Lasho TL, Patnaik MM, et al. Targeted next-generation sequencing in myelodysplastic syndromes and prognostic interaction between mutations and IPSS-R. Am J Hematol. 2017;92:1311-1317.
  3. Döhner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373:1136-1152.
  4. Haferlach T, Nagata Y, Grossmann V, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia. 2014;28:241-247.
  5. Daver N, Schlenk RF, Russell NH, Levis MJ, et al. Targeting FLT3 mutations in AML: review and current knowledge and evidence. Leukemia. 2019;33:299-312.
  6. Chou WC, Lei WC, Ko BS, et al. The prognostic impact and stability of IDH2 mutation in adult patients with AML. Leukemia. 2011;25:246-253.
  7. Chou WC et al. Distinct clinical and biological characteristics in adult AML bearing IDH1 mutation. Blood. 2010;115:2749-2754.