A study using whole exome sequencing in approximately 500 myeloma patients has revealed a range of different mutation types in genes and molecular pathways—some negative and some neutral—that appear to influence patient survival (Blood, doi.org/10.1182/blood-2015-05-644039).
Using the findings, the researchers also used the data to develop a staging system to better identify patients at higher risk of relapse and premature death. The study was funded by the United Kingdom's Institute of Cancer Research at Royal Marsden Hospital's Division of Molecular Pathology, as well as the Fondation Française pour la Recherche contre le Myélome et les Gammapathies, in Paris.
Believed to be the first whole exome analysis exclusively among myeloma patients, the results are published online ahead of print in the Journal of Clinical Oncology ( doi: 10.1200/JCO.2014.59.1503). The team also used the data to develop a staging system to better identify patients at higher risk of relapse and premature death.
The research was led by Gareth J. Morgan, MD, PhD, now Director of the Myeloma Institute for Research and Therapy at the University of Arkansas for Medical Sciences, and previously Professor of Hematology in the Division of Molecular Pathology at the Institute of Cancer Research, where much of the study was conducted. He continues his affiliation with the London-based Institute.
“This is next-generation whole exome sequencing and an entirely different technology,” he told OT. “It was very difficult to get data on almost 500 patients and their outcomes, and that we were able to do this was very surprising to me.”
Whole exome sequencing selectively details exon mutations and variants and their role in different diseases. The expression of exons, part of all genes, steers the final development of mature RNA. Although coding exons represent just one percent of the entire human genome, they are believed to be involved in an estimated 85 percent of mutations in many diseases, Morgan explained.
Whole exon sequencing is much less expensive and takes a significantly shorter time than sequencing the entire genome, he noted.
In all, 15 significant mutations were found, including alterations in the following genes: IRF4, KRAS, NRAS, MAX, HIST1H1E, RB1, EGR1, TP53, TRAF3, FAM46C, DIS3, BRAF, LTB, CYLD, and FGFR3.
Of these, 43 percent were found in RAS pathways and 17 percent in nuclear factor-kB pathways. While the latter were not found to be useful in prognosis, they might be helpful in testing targeted therapeutics, the investigators said.
Mutations in CCND1 and DNA repair pathways (TP53, ATM, ATR, and ZNFHX4 mutations) were associated with an overall negative impact on survival while those in IRF4 and EGR1 were linked to more favorable survival.
Among the mutations were translocations caused by a rearrangement of parts between non-homologous chromosomes—those that are not a member of the same pair. These often correlated with negative outcomes, as did copy number variations and structural abnormalities (CNSAs). Copy number variations are alterations in the number of copies of one or more sections of cells' DNA, and a greater number of such variations have been linked to others cancers.
‘Clearly Driven by...’
Based on this and earlier research, “myeloma is clearly a disease driven by RAS pathway mutations and by MYC translocations,” Morgan said. “With the exception of NRAS and KRAS, other genes had fewer mutations, suggesting that deregulation of these key pathways rather than single-gene mutations are important.”
The ability to deliver an effective apoptotic response to DNA damage provided the most significant prognostic value. By combining the known poor-prognostic marker TP53 with additional mutations in ATM or ATR, the researchers found that 17 percent of patients with these had significantly worse outcomes.
In addition to ATM and ATR, other variants involved in repairing damaged DNA were identified—notably ZFHX4.
Mutations with Positive Impact
On a more positive note, the researchers also identified mutations that seem to have a positive impact on survival, primarily those in IRF4 and EGR1.
“We have shown that there are a limited number of recurrent variants that are seen in a significant proportion of myeloma cases and that they co-segregate with the known recurrent CNSA typical of myeloma,” Morgan said.
“Minimal differences in significantly mutated genes were seen between the major subtypes of myeloma, and it seems likely that, once initiated, the same mutated pathways are pushing the disease forward.”
Previous research by the team showed that integration of in situ stabilization and solidification (ISS) and cytogenetic data (ISS–fluorescent in situ hybridization) can identify high-risk and ultra-high-risk patients.
“Further integration of mutational prognostic data can improve this,” Morgan said. “ISS is the standard for identifying copy number changes and translocations in genes, but being able to sequence the entire exome provides us with more data—and more is always better.
“Adding both ISS and mutations in our score increases precision for early mortality and progression detection. Overall, we identified a set of potential actionable mutations comprising 309 targets applicable to 53 percent of patients. In the years to come, we foresee this to increase to 440 targets applicable to 62 percent of patients.”
Variants associated with a greater risk of relapse and reduced survival pose interesting implications about current therapies, Morgan added. “These data suggest that patients with DNA repair pathway alterations may not benefit from alkylating agents, supporting the prospect that novel agents might be useful.”
He and his colleagues are now analyzing data on 600 patients, the results of which are expected to be published in December at the American Society of Hematology Annual Meeting.
“The next step will be to get investigators at different research facilities together and try to reach a consensus, and we are trying to broker long-term collaborations in this area. I cannot express how important it is to get a consensus so that we are all using the same staging system.”
Suzanne Lentzsch: ‘Big Step Forward’
Asked for her perspective for this article, Suzanne Lentzsch, MD, PhD, Director of the Multiple Myeloma and Amyloidosis Service at Columbia University Medical Center, said: “Knowing how myeloma will progress in patients and which ones might respond to therapeutic interventions, is a big problem—all patients are more or less treated the same.”
But the new study indicates that it might be possible to identify patients who are most at risk and to choose the best treatment approach: “This data appears to address the inherent need to address these versus ‘wait and watch,’ which is now the norm.”
That the researchers were able to use their data to develop a possible staging system is also a big step forward, she continued: “Can you really pick patients using these data to predict outcomes? I think this is still open to debate. But this is the first time that whole exome sequencing has been used, and should help things move forward.”
Nikhil Munshi: Need for International Consensus
Also commenting, Nikhil C. Munshi, MD, Professor of Medicine at Harvard Medical School and Associate Director of the Jerome Lipper Multiple Myeloma Center at Dana-Farber Cancer Institute, said he was very impressed by the findings: “This is outstanding work on many levels and contributes to our own understanding of exon sequencing in myeloma. We have all used exon sequencing for our patients, but the amount of data on specific mutations has not been that large.”
He said that he believes that a staging system like that proposed by the investigators needs to be incorporated into myeloma analysis of patients, but suspects that reaching an international agreement will take some time.
“We are hoping to reach a consensus and are currently working on this. Hopefully one will be reached in the United States sometime soon. I personally do not think we will find many new mutations.”
Munshi also said that the findings represent a “small step” toward development of new therapeutic approaches targeting those mutations that were linked to a higher rate of relapse and poorer outcomes.
“Whole exome sequencing is easy to do today and the technology is not different at all from what is currently available—it is pretty straightforward. The cost of sequencing is around $1,000 and it is possible to perform it using just one cell.
“The big challenge is going to be gathering a large data set and further analysis, but we are all working to solve the problem.”