Melissa Hoffman (Presenter)
Moffitt Cancer Center/University of South Florida
Bio: Melissa Hoffman, a third year Cancer Biology PhD student at Moffitt Cancer Center/University of South Florida, has dedicated her studies to the application of mass spectrometry in the field of cancer research. She earned her B.S. in Cellular and Molecular Biology with a minor in Biochemistry from California State University, Chico in 2013. Her research focus was on transcriptional regulation by microRNAs in diabetes. After coming to Moffitt Cancer Center in the Fall of 2013, she completed lab rotations in Dr. Jin Cheng’s lab studying the function of a long non-coding RNA in breast and ovarian cancer. Then, she joined Dr. John Koomen’s lab, which focuses on translational proteomics. Since then, she has worked on developing personalized assays to detect tumor burden in Multiple Myeloma patients and profiled kinase expression levels in lung adenocarcinoma tumor samples. She has also served
Authorship: Melissa Hoffman; Luis Saavedra-Roman; Sean Yoder; Wei Guan; Rachid Baz; Kaaron Benson; Aunshka Collins; Jamie Teer; John Koomen
Moffitt Cancer Center
| Short Abstract Multiple Myeloma, an incurable disease with poor patient outcomes, is characterized by clonal expansion of the plasma cells in the bone marrow, which secrete a monoclonal immunoglobulin. This study utilizes a proteogenomics approach to develop individualized, peptide-based mass spectrometry assays to quantify each patient’s disease-specific biomarker. Personalized LC-MRM assays developed for variable region peptides using de novo RNA sequencing have shown a substantial increase in sensitivity compared to clinical methods. The increase in sensitivity improves minimal residual disease detection, which could improve patient outcomes. |
Long Abstract
Introduction
Multiple myeloma (MM) is a devastating disease characterized by clonal expansion of plasma cells in the bone marrow, which secretes a monoclonal immunoglobulin (Ig). Over the past decade, the introduction of new, less toxic therapies has increased median overall survival to six years.[1] Despite great progress in understanding the disease biology and developing new treatment strategies, the disease remains incurable due to the persistence of undetectable amounts of minimum residual disease (MRD). Quantification of the monoclonal Ig using current clinical methods (e.g. serum protein electrophoresis[2]) does not detect MRD sufficiently, and other methods including RNA-seq or PCR amplification,[3] require multiple bone marrow samples, placing a heavy burden on the patient. Currently, clinical treatment decisions are made based on semi-quantitative results from multiple tests. This study aims to change the clinical decision paradigm to the use of a personalized, quantitative biomarker assay that will demystify the state of disease burden in the patient. Previously, the lab has developed liquid chromatography-multiple reaction monitoring (LC-MRM) assays to quantify each immunoglobulin class.[4] To complement this, LC-MRM assays are developed for variable region peptides (VRPs), which improves the limit of detection to at least 100 fold to µg/dl levels in serum, allowing more effective detection of MRD and earlier therapeutic intervention.
Methods
A proteogenomics approach is used to design personalized peptide-based mass spectrometry assays for each patient to monitor their disease progression, which have increased sensitivity and specificity compared with current clinical methods. The monoclonal immunoglobulin’s light and heavy variable regions are determined for each patient using 5’-RACE coupled with RNA-seq (MiSeq, Illumina) on CD138+ plasma cells isolated from bone marrow at diagnosis or recurrence. The protein sequence is generated using translation of de novo RNA sequencing to create a full-length immunoglobulin sequence, which is then validated using the IMGT tools.[5] Using the predicted variable region protein sequence and trypsin digestion of the Ig, LC-MRM assays for VRPs have been developed and characterized. Additionally, Lys-C is utilized to generate longer, more unique VRPs for patients lacking unique tryptic sequences amenable to LC-MRM analysis. These assays are developed using a targeted approach on a hybrid quadrupole-orbital ion trap instrument (Q Exactive Plus, Thermo) to analyze the longer peptides. VRP uniqueness is measured against a cohort of > 80 serum samples. The goal is to use the repertoire of the group of patients to represent the Ig repertoire of the individual patient and screen the most unique sequences. Tumor burden is measured in newly diagnosed patients undergoing treatment and in remission to evaluate the ability to detect MRD and disease progression using excess sera from serial samples collected as part of the standard of care. Results are compared with current clinical measurements. Using this approach, we are generating a database of immunoglobulin variable region RNA and predicted amino acid sequences. The personalized assays are compared between analytical and nanoflow liquid chromatography methods.
Results
Previously developed LC-MRM assays targeting Ig constant region peptides have been compared to current clinical methods (e.g. SPEP, immunofixation electrophoresis, and Ig quantification). These assays are combined with newly developed LC-MRM assays for VRPs designed specifically for each patient. Longitudinal sample collection is ongoing for a growing cohort of 48 patients that represent two groups: newly diagnosed beginning treatment and patients in remission that are monitored for relapse. 5’ RACE is used to amplify the Ig variable region from RNA isolated from CD138+ cells isolated from bone marrow aspirates using class specific primers (from the literature[6] or newly designed). RNA-seq data provides copy number counts that enable identification of the amplified sequence corresponding to the monoclonal Ig. We have shown the 5’ RACE amplification minimizes the cost of sequencing and the time necessary for the bioinformatics analysis. In addition, this method sufficiently amplifies the monoclonal Ig in bone marrow aspirates from patients with MRD as well as the bulk bone marrow cells without CD138+ enrichment.
Based on their uniqueness in the antibody repertoire, VRPs were prioritized for 20 patients to date and selected for assay development and characterization of sensitivity, accuracy, precision, and linearity were assessed. Although these assays will be peptide- and patient-dependent, LC-MRM measurements for VRPs have demonstrated a > 100-fold increase in sensitivity over SPEP and the SFLC assay for monitoring the monoclonal Ig in serum. Additionally, LC-MRM reproducibility is compared to clinical methods.
Conclusions
Personalized LC-MRM assays have been developed using de novo RNA sequencing to define unique candidate biomarkers to quantify Multiple Myeloma patient tumor burden. We have demonstrated a 100-fold increase in sensitivity compared to clinical methods. We are able to detect the diseased Ig at the MRD stage in both bone marrow and serum, which could lead to improved patient outcomes. If this assay can be translated to the clinical lab, it could change the paradigm for patient evaluation and clinical decision-making, increasing the ability of clinicians to continue first line therapy to eliminate more MRD or to intervene at an earlier time point in disease recurrence. We have reduced the cost and time necessary to implement the assay, improving its translational potential. Also, in the event that a patient enters the clinic in with low tumor burden, preliminary results indicate that assays can be developed with whole bone marrow aspirates with MRD. Currently, additional tests must be used to monitor light chain disease and rare MM cases, including IgD and IgE. This personalized assay is a single test that results in quantitative readouts on all immunoglobulin classes of both heavy and light chain, potentially simplifying the analytical process for evaluation of MM tumor burden.
References & Acknowledgements:
References:
1. Kumar et al. (2014) Leukemia. 28, 1122.
2. Katzmann et al. (1997) Electrophoresis. 18, 1775
3. Billadeau (1991) Blood. 78, 3021
4. Wood et al. (2014) Proteomics Clin Appl. 8, 783.
5. Brochet et al. (2008) Nucl. Acids Res. 36, W503.
6. Doenecke et al. (1997) Leukemia. 11, 1787
Acknowledgements:
The Moffitt Genomics Core assisted with the RNA-sequencing.
The Moffitt Proteomics Core synthesized peptide standards for assay development and assisted with equipment maintenance and upkeep.
My PhD mentor, John Koomen, has been immensely helpful in the scientific development of this project and my development as a young investigator.
| Description | Y/N | Source |
| Grants | yes | DeBartolo Personalized Medicine Award |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |