Discovery of B Cell Monoclonal Lymphocytosis (BCML). Our work led to the discovery of BCML at CBER FDA several years ago. The goal that led to this discovery was the early, pre-clinical detection and characterization of BCML in unaffected first-degree relatives in familial CLL. This has been accomplished using less sophisticated 2 or 3-color analysis. Using present technology, 0.14% of blood donors in a large mid-western blood bank have BCML as well as 0.7% of individuals living near Superfund toxic waste sites. This work uses CLL as a model for the early detection of disease and the application of the technology of multi-parameter flow cytometry. High-resolution flow cytometry combined with digital signal processing and multi-color analysis will allow further characterization of the precursor state in this common, hematological malignancy. This will also permit the detection of changes in B cell subsets in autoimmune disease, post bone marrow transplantation and in environmental exposure involving agents of bioterrorism. Single Cell PCR. These experiments combine high-speed cell sorting, B cell purification (enrichment) and flow cytometric multi-color analysis with single cell PCR. The goal is to define the abnormal B cell repertoire in unaffected first-degree relatives in kindreds of familial CLL. The accomplishment to date has been the identification and confirmation of B cell abnormalities in 3 of 4 unaffected siblings in a single family. We believe that these initial findings suggest a whole new area of investigation of the human B cell repertoire and will permit us to detect the molecular lesions associated with familial CLL. The application of this combined technology to normal B cell subsets will be important in both health and disease. Morphometric Image Analysis. Using CLL as a model, the goal is to be able to differentiate typical CLL from atypical CLL by morphological sub-classification. The accomplishment to date has been to standardize the method of slide preparation. Image analysis is relevant to CBER as a patient screening procedure for protocol eligibility, remote consultation via remote digital microscopic teleconferencing and is becoming a standard in medical care. It is also used for product evaluation and macroscopic confocal image analysis is at the heart of the microarray technology. The relationship between morphology, immunophenotype and molecular cytogenetics is sought in this work. Aneuploidy Analysis. We have developed and used the New Zealand Black (NZB) as a murine model for B-CLL/B-NHL. Using CLL and the NZB mouse as a model, the goal of this project is to resolve normal diploid cells from hyperdiploid, aneuploid cells. The conditions for resolution of these two populations of cells have been defined in both the NZB and CLL. Aneuploidy is relatively easy to detect in the NZB mouse and should permit the separation of clonal diploid cells from aneuploid cells. This would allow analysis of the molecular changes prior to karyotypic change. In a pilot experiment using a flow cytometer developed for NASA, we have been able to detect aneuploidy in CLL samples. Genetic Linkage Analysis in Murine Model. Historically the NZB has been used as a model for auto-immune disease and lymphoproliferative disease (LPD). In fact, the NZB has been said to represent the murine model of human CLL. However, our histopathological analysis of both the NZB parent and an F1 backcross shows that the predominant LPD is a marginal zone lymphoma. Although the cell of origin in human CLL remains unknown, there is one hypothesis that it arises from a marginal zone cell. A PCR based, linkage analysis of the F1 backcross using short sequence length polymorphisms (SSLP) was conducted on mice with histological and flow cytometric evidence of lymphoid neoplasia. Four loci of interest were found in the initial study and the analysis of a replication study is pending. BCML Nomenclature. The existence and international recognition of BCML (discovered at CBER) will allow the definition of a precursor cell and will serve as a surrogate disease marker for CLL in appropriate pedigrees. The goal of the NCI International Consortium for Familial CLL is to redefine BCML as """"""""Essential B Cell Monoclonal Lymphocytosis."""""""" CBER participation in this classification brings the perspective of genetic testing and the development of IVD [in-vitro diagnostic(s)] testing reagents. I have been asked to the second meeting of the NCI Familial CLL Consortium in conjuction with the IWCLL meeting Oct 2003 in Italy where I will deliver the key note talk on this matter.
