Genetic biomarkers in multiple myeloma
Doctoral thesis
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http://hdl.handle.net/11250/2611114Utgivelsesdato
2019Metadata
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Sammendrag
Multiple myeloma is a cancer of plasma cells in the bone marrow that accounts for 2 % of cancer cases globally. Improved understanding of the molecular basis of this disease has resulted in new treatment options and prolonged survival; however, the disease is still considered incurable. Currently an important biomarker for the diagnosis and management of multiple myeloma is the monoclonal immunoglobulin secreted by tumor cells (M protein). There is however an increasing need for new biomarkers, as treatment options continue to increase, while there are hardly any validated tools to guide treatment choice and intensity in the individual patient.
This thesis focuses on the use of genomic alterations in the cancer cells as biomarkers for management of multiple myeloma patients. The two first studies addressed the role of somatic mutations in the cancer cells. The two last studies addressed the use of tumorspecific DNA sequences for tracking of ‘minimal residual disease’ (MRD), which is a more sensitive biomarker than M protein for evaluation of treatment response.
In study one, we investigated the BRAF V600E mutation as a potential prognostic biomarker and therapeutic target. We found the mutation in a bone marrow biopsy from 11 out of 209 patients (5.3 %) at the time of diagnosis. The mutation was most often only detected in a fraction of cells, suggesting that responses to targeted therapy would be partial at best. Presence of a BRAF V600E mutation was not related to prognosis or clinical phenotype.
In study two, we explored circulating tumor DNA (ctDNA) as a novel biomarker for tumor mass and the tumor genome in multiple myeloma. In our study of 20 patients, we focused on mutations in genes recurrently mutated in myeloma, including KRAS, NRAS and BRAF. We achieved 96 % sensitivity to detect known tumor mutations in serum (34/35). In 11 patients from whom we had available sequential samples, we monitored the concentration of mutations over time and compared them to the concentration of M protein. In 10/11 patients, mutation levels and M protein showed striking co-variation, suggesting that ctDNA is a biomarker of total tumor mass in myeloma. We also found serum mutation levels to increase during terminal and aggressive disease, a situation where M protein does not adequately reflect the disease activity. Measuring the serum concentration of mutations may be a way to monitor patients who do not have measurable M protein, or in other situations where monitoring is challenging. Circulating DNA is also an informative tool for genomics research, allowing easy access to the tumor genome for a wide range of applications.
In study three, we addressed the identification of clonal immunoglobulin V(D)J rearrangement sequences in multiple myeloma for subsequent MRD tracking by next generation sequencing. This approach has proven to be highly sensitive for MRD and has strong prognostic implications. However, previous studies have found variable rates of V(D)J sequence detection at baseline, which could limit tracking. Bone marrow mononuclear cells from 177 myeloma patients underwent V(D)J sequencing by the LymphoTrack assays (Invivoscribe). As a molecular control for tumor cell content, we sequenced the samples using our in-house myeloma panel myTYPE. V(D)J sequence clonality was identified in 81 % of samples overall, as compared with 95 % in samples where tumor-derived DNA was detectable by myTYPE. Clonality was detected more frequently in patients with lambdarestricted disease, mainly because of increased detection of kappa gene rearrangements. Finally, we describe how the tumor cell content of bone marrow aspirates decrease gradually in sequential pulls because of hemodilution: From the initial pull used for aspirate smear, to the final pull that is commonly used for research. In conclusion, baseline clonality detection rates of 95 % or higher are feasible in multiple myeloma. Optimal performance depends on the use of good quality aspirates and/or subsequent tumor cell enrichment.
In study four, we addressed the stability over time of clonal V(D)J sequences for MRD tracking and whether light chain sequences are sufficiently unique to be used for tracking. To accomplish this, we analyzed immunoglobulin gene repertoires from 905 plasma cell tumors and healthy controls. Clonal heavy and/or light chain expression was identified in all patients at baseline, with one or more subclones related to the main clone in 3.2 %. In 45 patients with 101 sequential samples, the dominant clonal V(D)J sequences remained identical over time despite differential clonal evolution by exome sequencing in 49 % of patients. This indicates that V(D)J sequence diversity occurs before the most recent common ancestor in evolutionary history, prior to acquisition of subclonal driver events. Importantly, we show that dominant clonal sequences at baseline are reliable biomarkers over time. Next, we identified somatic hypermutation and non-templated insertions as the most important determinants of light chain clonal uniqueness. Based on these factors we identified a trackable light chain sequence in the majority of patients. Taken together, we provide biological evidence that dominant clonal sequences are in fact reliable biomarkers for longterm tracking of the malignant clone, including both IGH and the majority of light chain clones.
As a result of our work and others’, somatic mutations have been found to be of limited clinical relevance in multiple myeloma. Instead, the most important genomic changes appear to be copy number changes and structural variants. Circulating tumor DNA continues to be an area of active research with several potential applications for genomic characterization of multiple myeloma. The strong prognostic value of MRD testing has been demonstrated in several studies, and MRD negativity appears to overcome traditional risk factors. Clinical trials are on-going to test whether MRD-guided treatment can improve patient outcomes. For example, patients who remain positive for MRD after initial treatment may benefit from further consolidation treatment. MRD testing may turn out to be the first biomarker that provides a real clinical benefit from personalized therapy for multiple myeloma.