Vimentin as a Novel Prognostic Factor for Aggressive CLL

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[707] Proteomic Analysis of Chronic Lymphocytic Leukemia Cells

Identifies Vimentin as a Novel Prognostic Factor for Aggressive

Disease. Session Type: Oral Session

Grzegorz S. Nowakowski, Yean K. Lee, D. Bone, G.

Morice, Barnidge, Diane F. Jelinek, Tschumper, Tait D.

Shanafelt, Clive S. Zent, Z. Rassenti, J. Kipps, Neil E.

Kay Division of Hematology, Mayo Clinic; Immunology; Hematopathology,

Mayo Clinic, Rochester, MN, USA; Division of Hematology/Oncology, CLL

Research Consortium, UCSD, LaJolla, CA, USA

The IgVH mutation status is an independent predictor of time to

treatment and survival in CLL. However, the biological basis of this

difference is unknown. Clinical use of the IgVH mutation status as a

prognostic marker is limited due to the technical difficulties of

assessing this feature. As such, we probed for differences in protein

expression by CLL cells with unmutated (UM) vs mutated (M) IgVH using

phage peptide display libraries (PDPL).

Methods: PDPL consists of phages engineered to express random

peptides (7-mer) on the coat protein. From approximately 2x1011

phages displaying all possible permutations of 7-mer peptide, we

selected phages binding to UM cells by incubation with 106 UM cells

and elution of bound phages. Then, from numerous phages initially

binding to UM cells, we subtracted phages binding to M cells by

multiple incubations with M cells (106). Thus, the phages that bound

only to UM cells were selected. The procedure was repeated multiple

times to increase selection specificity. The peptide sequences

displayed by selected phages were evaluated. We identified phage

targets on UM cells by immunoprecipitation (IP) of CLL protein with

specific phage and by sequencing of precipitated protein. The

expression of target protein in CLL cells was confirmed by

immunobloting and flow cytometry.

Results: Sequencing of phages from the 3rd round of selection (UM v.

M) revealed several peptides, with the most predominant being

FPSAHFL. IP identified the cellular target for FPSAHFL as vimentin,

an intermediate filament protein involved in cell motility and

activation. The level of vimentin expressed by CLL cells varied

between patient samples (n=15). UM CLL cases were found to express

higher levels of vimentin (the mean MFI was 1002; SD 381), than cases

of M CLL (mean MFI 547; SD 109), p=0.0128.

To evaluate the prognostic significance of vimentin, we assessed

vimentin expression by flow cytometry in a separate subgroup of 40

CLL patients. Archived cell samples collected between 6/2000 – 6/2001

were used. The median follow up was 81 months (40-233). Vimentin

expression (MFI) as a continuous variable was a risk factor for a

shortened time to treatment (TTT) with a proportional hazard ratio of

4.152 (95% CI 1.33-15.220, p=0.0317). Using MFI closest to the median

(and rounded to 2 decimal places) as a cutoff, we stratified patients

in high (n=21) and low (n=19) vimentin expression groups. The median

times to treatment (TTT) in patients with high vs low level

expression of vimentin were 2.8 and 12.9 years respectively, p=0.0025

(Fig.).

Conclusion: Using novel proteomic technology, we found that CLL cells

with unmutated IgVH expressed higher levels of vimentin than CLL

cells with mutated IgVH. A high-level of vimentin expression defined

by flow cytometry appears to be associated with high-risk disease.

Validation of these findings could allow for the vimentin level to be

used as a surrogate marker for expression of unmutated IgVH in CLL.