The laboratory is interested in exploring the use of molecular methods to monitor residual disease in several clinical settings: 1. Serial monitoring of BCR-ABL expression in patients with CML undergoing allo-transplantation. Quantitative RT-qPCR monitoring for BCR-ABL mRNA is a standard for monitoring residual disease during standard tyrosine kinase inhibitor (TKI) treatment of CML. Although TKIs are highly effective in most patients, a subset ultimately develop PTK-resistant, accelerated phase disease requiring allotransplantation. Using a variety of approaches to enhance graft versus tumor effect, Dr. Daniel Fowler in the NCI and John Barrett in the NHLBI have active programs using allogeneic stem cell transplantation to treat TKI-refractory CML. In recent experience, virtually all these patients experience complete hematologic response, but many develop low grade recurrent disease within several months detectable by serial BCR-ABL monitoring. Relying heavily on the BCR-ABL assay to direct their therapy, investigators have been able using a combination of additional immunologic and pharmacologic manipulations to prevent clinical relapse in most patients. Using different approaches, both groups are able to maintain complete or near complete molecular remission in most post transplant patients with extended patient survival. Clinical studies continue for both groups with the goal of further improving the cure rate in this population. 2. Assays designed to detect short tandem repeat (STR) microsatellite polymorphisms are used to monitor engraftment and donor chimerism in patients undergoing stem cell allotransplantation at the NIH. The same assays can also detect the late reappearance of (recipient-derived) leukemia, lymphoma, and myelodysplasia in the blood or bone marrow of transplanted patients. Though limited in sensitivity/specificity, chimerism assays can be a valuable tool for disease monitoring particularly in hematologic malignancies as yet lacking an alternative sensitive biomarker. To improve the sensitivity of our chimerism assays for detection of low-level residual disease, in the past year we have adopted the Osiris software package developed by NCBI for technical analysis of STR capillary electrophoresis tracings. This program, designed originally for STR-based forensic identification of victims after a mass-casualty disaster, uses a highly sophisticated mathematical analysis of peak size and shape to distinguish true STR peaks from noise, stutter, or multichannel compensation errors. Using this improved analytic software has improved our ability to confidently identify potential early recurrent disease in settings such as MDS where other indicators of early recurrence may not be available. 3. The laboratory is interested in testing the usefulness of plasma/serum cell free DNA (cfDNA) used in conjunction with quantitative-PCR assays targeting tumor-specific immunoglobulin variable region (VDJ) DNA rearrangements to monitor residual disease in patients with multiple myeloma. While routine serum and urine electrophoresis are adequate for monitoring most disease responses, the newer highly active agents can sometimes reduce tumor mass below the limits of detectability of these methods and more sensitive techniques will clearly be needed. PCR-based assays by their nature can be designed for high sensitivity. Equally important, a DNA-based approach can be used to provide other genomic information that will in the future provide additional information critical in understanding the mutational causation of malignancy, and clonal evolution with therapy. The most sensitive standard method for monitoring of residual disease currently in use (bone marrow aspirate/biopsy/flow cytometry) is invasive, and can be limited by sampling error and the difficulty in mobilizing intact plasma cells from marrow stroma for analysis. Cell free DNA detected in the circulation as a consequence of plasma cell turnover/apoptosis could be a useful alternative tools for monitoring low level residual disease with time and in tracking genomic changes in tumor with time. In pilot study of 17 patients with multiple myeloma or smoldering myeloma beginning chemotherapy under a protocol initiated by Ola Landgren (NCI), we attempted to detect tumor specific cf VDJ DNA in DNA extracted from 0.5-1.0 ml of plasma or serum taken during staging immediately before therapy with a highly active mutiagent regimen (carfiilzomib, revlimid and dexamethasone). At least trace levels of tumor specific VDJ could be detected in all but 2 patients before treatment. Levels were quite low however, often below the level for accurate quantitation using a conventional q PCR assay. Of greater concern, circulating cf DNA levels did not correlate well with other measures of myeloma cell load, and levels typically dropped below detectability within 1-2 months even in patients with residual disease measureable by other methods. These studies suggest sensitivity could be substantially improved by increasing sample concentration (i.e. extracting cf DNA from larger volumes of blood) and by using more quantitative and sensitive digital PCR methods or deep NGS in lieu of conventional PCR for tumor VDJ detection/quantitation.