Protein profiles distinguish stable and progressive chronic lymphocytic leukemia.

Patients with a stable chronic lymphocytic leukemia (CLL) double their blood lymphocyte count in >5 years, but may develop progressive disease with lymphocytes doubling in <12 months. To identify a protein signature for progressive CLL, whole cell extracts of peripheral blood mononuclear cells from patients with CLL (n=27) were screened using iTRAQ (isobaric tags for relative and absolute quantification) analysis. A total of 84 differentially abundant proteins were identified from patients with stable and progressive CLL. Subsequently, 32 of these proteins were quantified by SRM (selected reaction monitoring) using extracts of purified CD19+ CLL cells from patients (n=50). Hierarchical clustering of these protein profiles showed two clusters of patients that correlated with progressive and stable CLL, providing signatures that should be useful for triaging patients. Some of the proteins in the progressive cluster have not been linked with CLL, for example, glutamate dehydrogenase 1 and transcription intermediary factor 1-beta.

Cell surface phenotype profiles distinguish stable and progressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20-30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging system, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 overexpression, cytogenetic abnormalities (13q-, + 12, 11q-, 17p-) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all cases of CLL at risk. In a recent review, we summarized those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan(™) CLL antibody microarray (Version 3; 182 CD antibodies), and with refined analysis of purified CD19 + B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage patients with CLL according to probability of clinical progression and potential earlier requirement for treatment.

Profiles of surface mosaics on chronic lymphocytic leukemias distinguish stable and progressive subtypes.

PURPOSE: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, some patients may survive for many years, while 20-30% of patients progress and may die within several years. Currently, there is not a single procedure that enables accurate prognosis and triaging of those patients who need immediate and aggressive treatment. All CLL cells are characterised by the expression of the B-cell antigens CD19, CD20, CD21, CD22 and CD23, with aberrant expression of the T-cell antigen CD5. METHODS: We have developed a CD antibody microarray (DotScan) containing 182 immobilised CD antibodies that has been used to obtain extensive surface profiles of CLL cells obtained from 96 patients. RESULTS: Of these 182 antigens, 27 were significantly differentially expressed between stable, stable-progressive and progressive CLL. Some of these antigens are not expressed on normal B-cells and may be targets for therapeutic antibodies against CLL. Unsupervised hierarchical clustering of the surface profiles from 96 patients showed that those with progressive CLL could be distinguished based solely upon this 'disease signature'. The sensitivity (proportion of actual positives correctly identified) was 67.9%, the specificity (proportion of negatives correctly identified) was 77.5%, and the accuracy was 71.9%. CONCLUSIONS: Considerable effort by a number of research groups has resulted in identification of individual markers for progressive CLL, but their collective use is yet to provide a test that identifies CLL patients at risk. Data presented here provide a basis for development of a simple test using an antibody microarray.

Surface profiles for subclassification of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Some patients have stable disease while others progress and require treatment. Levels of several cluster of differentiation (CD) antigens are known to correlate with prognosis and may be used to stratify patients according to risk. In this review, we summarize current information on surface CD antigens found on CLL, their pathological significance and their detection using CD antibody microarrays. The use of extensive immunophenotypic patterns or surface profiles as disease signatures for CLL subclassification, prognosis and patient management is discussed with a focus on triaging patients with CLL with progressive disease.

Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing.

A procedure is described for the disaggregation of colorectal cancers (CRC) and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognising 122 different surface antigens (DotScan CRC microarray). Cell binding patterns recorded by optical scanning of microarrays provide a surface profile of antigens on the cells. Sub-populations of cells bound on the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with Quantum Dots or other fluorescent dyes. Surface profiles are presented for 6 CRC cell lines (T84, LIM1215, SW480, HT29, CaCo and SW620) and surgical samples from 40 CRC patients. Statistical analysis revealed significant differences between profiles for CRC samples and mucosal controls. Hierarchical clustering of CRC data identified several disease clusters that showed some correlation with clinico-pathological stage as determined by conventional histopathological analysis. Fluorescence multiplexing using Phycoerythrin- or Alexa Fluor 647-conjugated antibodies was more effective than multiplexing with antibodies labelled with Quantum Dots. This relatively simple method yields a large amount of information for each patient sample and, with further application, should provide disease signatures and enable the identification of patients with good or poor prognosis.