NEW ORLEANS—New research provides insights into the genomic architecture of multiple myeloma and will help identify molecular alterations associated with progression of the disease and the development of drug resistance, according to a presentation here at the American Society of Hematology Annual Meeting (Abstract 399).
“Multiple myeloma shows dynamic evolution, both spontaneous and after treatment, and next-generation sequencing can, at diagnosis, identify clones likely to be resistant to a given treatment,” lead author Niccolo Bolli, MD, PhD, Academic Clinical Lecturer in Hematology at the University of Cambridge, said in an interview.
“Post-treatment sequencing can allow assessment of response at the subclonal level and identify new genomic events that may influence treatment decisions.”
The pathogenesis of multiple myeloma is only partially understood. To investigate genomic events underlying the disease's pathogenesis and evolution, he and his colleagues used whole exome sequencing, copy number profiling, and cytogenetics in 67 patients and 84 samples. For 15 patients, two or three serial samples taken a median of 299 days apart were available.
The researchers used genome-wide single nucleotide polymorphism array or exome reads to estimate the allele-specific copy number of the tumor. The team used mutation burden, corrected for copy number, and normal cell contamination to cluster variants and estimate the clonal architecture of each sample and its evolution over time.
Analysis of the clonal structure of the tumors showed at least one subclone in 94 percent of the patients at diagnosis, suggesting that myeloma is a heterogeneous disease at presentation, Bolli said.
“Interestingly, many mutations of known multiple myeloma driver genes—KRAS, NRAS, BRAF, TP53, and FAM46C—were subclonal at diagnosis. Mitogen-activated protein kinase signaling pathway variants are frequent, can be subclonal, and overlapping.”
In five of the 67 patients, BRAF and KRAS/NRAS mutations coexisted in the same sample, raising the therapeutic implications, he said, “given the paradoxical ERK-activating effect of BRAF inhibitors in RAS-mutated cells.” Only three of 10 BRAF variants were V600E, the current target of most inhibitors.
This suggests that a small minority of patients with BRAF-mutated myeloma have the potential to be treated with vemurafenib, and that perhaps oncologists should look to other genes as targets, he said.
“Most myeloma samples show a complex clonal architecture. Every sample is a complex mixture of subclones. Tumors are composed of different groups of cells that behave differently. Some subgroups are more resistant to treatment, while some are more prone to progression and aggressive clinical features.”
Altogether, only the five previously known genes were significantly enriched in the cohort, highlighting marked heterogeneity of the spectrum of candidate driver gene mutations across multiple myeloma patients.
“Nevertheless, we identified several new recurrent gene lesions,” he said. These included inactivating mutations of SP140, a gene previously linked to germline susceptibility to chronic lymphocytic leukemia, and in ROBO1, a gene recently implicated in pancreatic cancer; clustered missense substitutions in EGR1, a gene previously implicated in plasma cell apoptosis; and clustered truncating mutations in LTB, a tumor-necrosis factor-family protein implicated in lymphoid development.
Genomic Evolution at Relapse
The subclonal structure of the sample changed over time in 72 percent of paired samples, highlighting genomic evolution at relapse. “We described four different scenarios with striking concordance between mutations and chromosomal copy number changes: (1) no change; (2) linear evolution (a new clone appears in the later sample); (3) differential clonal response (the relative proportions of the subclones change over time); and (4) branching evolution (new clones emerge, while others decline in frequency or disappear).”
All subclonal variants in known driver myeloma genes increased their clonal fraction at the later time-point, which was consistent with the expected positive selection for the subclones harboring them, he said.
“Subclonal structure evolves over time, and the type of genomic evolution correlates with clinical features. Upon treatment, the subclonal structure changes in at least two-thirds of patients. Some changes are minimal and subtle, while other changes are dramatic and correlate with resistance to treatment.”
Next, the researchers analyzed the mutational signatures to investigate the mutational processes responsible for the generation of the mutational repertoire in multiple myeloma. Two signatures were found: The most represented one, Bolli reported, is enriched for spontaneous deamination of methylated cytosines, a common process in cancer and aged cells.
The second signature was more represented in samples showing extremely high numbers of variants, sometimes clustered in small regions. The team hypothesized that this results from aberrant activity of the APOBEC family of cytosine deaminases, which have recently been described in breast cancer.
“Interestingly, cases of intramedullary relapse were always associated with branching evolution and showed increased contribution from this APOBEC signature,” he said.
“There are different scenarios for driver variants at relapse. Disease relapse can be driven by clonal variants not responsive to treatment, subclonal variants increasing their clonal fraction, and new variants acquired after treatment.”
In conclusion, Bolli said, “in our cohort of multiple myeloma samples we show evidence of tumor heterogeneity at the time of diagnosis, discernable genetic changes, and shifts in the clonal structure of disease at the time of progression, different mutational processes responsible for an heterogeneous mutational repertoire across patients and over time in the same patient, and a comprehensive list of recurrent variants, many of which are previously unreported.
“Multiple myeloma is a multifocal disease, and this is the tip of the iceberg about clonality. If we go deeper, we may find something more. We can eradicate a fraction of the cells in each clone. We speculate that this predicts for stable clinical features. But the numbers are low, so this is pure speculation at this point.”
In the question-and-answer session, in response to a question about the collection method for bone marrow samples, he said bone marrow aspirate was collected wherever the needle falls for each sample, but that in the future, the researchers will look at multiple samples at multiple time points.
“Multiple myeloma is more treatable now than ever before, but still is not curable for the majority of patients. We have provided old and new targets for future studies aiming at better disease control.”
Asked for her opinion, Janis L. Abkowitz, MD, Hematology Division Head and Clement A. Finch Professor of Medicine and Adjunct Professor of Genome Sciences at the University of Washington, called the research an example of precision medicine, locating where patient-specific, molecular targets can hit in a disease. The information helps distinguish between understanding targets and what this means to patients, she said.
“The study of whole exome sequencing of multiple myeloma shows that myeloma can exist as an expanded clone.
“This study shows how to use genomics to distinguish disease progression in individual patients. The researchers have found many more genes, and identified patients with recurrent signatures of myeloma cancer cells that look like breast cancer cells and aging cells. We thought myeloma was a simple disease. Now we have more clues about how the cancer develops and how cell aging happens, and the connections between these processes.
“This is a time for change in hematology. Genomics are entering our space. What we do for the patient will include thinking through genetics.”