New test for monitoring MRD

[Guest guest]

This abstract describes a new method of monitoring MRD (minimal residual

disease - once patients have reached CCR, that is), using an antibody based

test (immunoassay) to look for the bcr/abl protein instead of the BCR/ABL

gene target of PCR testing. The potential advantage of such a test is that

it's easier to standardize and shows less variability than regular PCR

(though there's a new qPCR test in development at Dana Farber that is more

sensitive and more easily standardized than the old ones). It's not clear to

me that the immunoassay test described here is a sensitive as PCR though.

R

_____________________

[2006] Measurement of Free Circulating Bcr-Abl Fusion Protein and Its

Phosphorylation in Patients with Chronic Myeloid Leukemia. Session Type:

Poster Session 210-II

I. Jilani, H. Kantarjian, H. Faraji, M. Gorre, J. Cortes, O. Ottmann, K.

Bhalla, S. O'Brien, F. Giles, M. Albitar . Hematology, Quest Diagnostics

Nichols Institute, San Capistrano, CA, USA; Leukemia, M.D.

Cancer Ctr. Univ. of Texas, Houston, TX, USA; Leukemia, JW Goethe

Universitat, furt, Germany; Leukemia, H.L. Moffitt Cancer Ctr, Tampa,

FL, USA

Diagnosis and monitoring of therapy in chronic myeloid leukemia (CML) depend

on cytogenetic, FISH, and PCR assays for detecting the fusion bcr-abl gene.

However, the there is an inherent variability and difficulty in

standardizing quantitative PCR-based assays. We assessed the utility of a

simplified immunoassay for measuring levels of Bcr-Abl protein and

phosphorylated Bcr-Abl for diagnosis of CML and detection of minimal

residual disease (MRD). Bcr-Abl protein was immunoprecipitated on beads with

anti-Bcr antibody, and the fusion protein was detected with anti-Abl

antibody. Phosphorylation of the immunoprecipitated Bcr-Abl was measured

using antibodies against phosphorylated Abl protein at tyr245 and thr735.

Because the relatively high turnover of leukemic cells enriches plasma with

leukemic DNA, RNA, and protein, as we previously reported, this assay can

use cell lysate as well as PB plasma. In vitro studies with denatured K562

cells in plasma showed reliable detection at levels as low as 10 cells/mL

plasma. After exposure of cell cultures to 0.5 µM imatinib, K562 cell

lysates exhibited a decreased ratio of phosphorylated Bcr-Abl protein:total

Bcr-Abl protein with both the tyr245 (from 0.53 to 0.11) and the thr735

(from 0.36 to 0.08) antibodies. The assay was validated by demonstrating

negativity (specificity) in 116 normal control subjects (or patients with

other translocations) and positivity in all 380 positive samples, as

confirmed by FISH or cytogenetics. The CV for inter- and intra-assay

variation was <6%. In CML patients receiving imatinib (n=52), the

immunoassay yielded Bcr-Abl measurements parallel to those of cell-based

RT-PCR. Of 32 plasma and PB cell samples collected at 6, 9, and 12 months of

imatinib therapy, 10 were negative by RT-PCR on cells but 4 of these were

positive by the plasma immunoassay. Conversely, 9 of the plasma samples were

negative by immunoassay, 4 of which were positive by RT-PCR on PB cells. At

3 months of therapy, the immunoassay yielded negative results in 5 of 33

available samples, all of which were positive by RT-PCR on PB cells.

Patients with a high level of disease at 3 months of therapy as assessed by

RT- PCR (Bcr-Abl:Abl mRNA ratio >0.05) had significantly more relative

phosphorylated Bcr-Abl protein than those with low disease activity

(P=0.05). In conclusion, these findings demonstrate that Bcr-Abl and

phosphorylated Bcr-Abl are detectable at significant levels in the plasma of

CML patients and that these levels can be used to monitor MRD. This free

circulating Bcr-Abl protein is most likely complexed with other proteins.

Unlike PCR-based assays, our ELISA-like assay can be standardized for use in

various laboratories. More importantly, the CV of the immunoassay (<6%) is

much lower than that typically reported for RT-PCR (up to 30%). We speculate

that the use of plasma rather than cells in this assay allows more accurate

quantitative measurement of the disease in the whole body, without potential

bias due to sampling errors.