Cell Enrichment for FISH: A Novel Diagnostic Tool in Challenging Cases of Low Bone Marrow Involvement By Mature B-Cell Neoplasms

Cytogenetic studies are useful tools which can provide diagnostic, prognostic and management information for mature B-cell neoplasms. Mature B-cells do not grow well in cytogenetic cultures. Therefore, detection, characterization and differentiation of scant mature B-cell neoplasms or minimal residual disease can be difficult in bone marrow or peripheral blood specimens. FISH provides more sensitive information than G-band chromosome analysis. However, in cases with low-level involvement of the marrow, abnormal FISH results may be missed or not reported when abnormalities do not exceed experimentally determined cut-off values.

To increase the sensitivity of abnormal mature B-cell detection, we developed an enrichment method utilizing pan B-cell antibodies. This method separates mature B-cells from the remaining bone marrow and/or peripheral blood cells.

We selected 59 bone marrow and peripheral blood specimens from patients referred for mature B-cell neoplasms (except for myeloma) for use in the validation. These specimens had 1% to 10% monoclonal mature B-cells detected by flow cytometry and were enriched using cell-separating technologies. Each specimen was divided into four equal portions, with three of four portions undergoing enrichment using different pan B-cell antibodies. The fourth portion was reserved for standard non-enriched testing and was used for comparison to results obtained in the enriched portions. A variety of corresponding FISH analyses were performed in each of the four portions, based upon the disease state. FISH results were obtained by two independent scoring technologists.

Enrichment with B-cell antibodies improved detection of FISH abnormalities that may not have otherwise been observed in the patient specimens. 42% (25/59) of samples had abnormalities detected within the enriched portion that were not detected in the standard non-enriched portion. Of these, 64% (16/25) had a FISH abnormality that was a critical finding for the final diagnosis, prognosis and/or management of the patient. Enrichment also increased the sensitivity of FISH abnormality detection. 29% (17/59) of samples had abnormalities that were detected in both the enriched and non-enriched portions. However, detection was on average 15-fold more sensitive. The average detection rate of FISH abnormalities in the non-enriched portion was 3%, which is at or near the experimentally determined cut-off value for most FISH probes. In contrast, the average detection rate of FISH abnormalities in the enriched portion was 56%. In 5% (3/59) of cases, detection of FISH aberrations in enriched specimens

helped to distinguish two separate neoplastic processes in the bone marrow. These results demonstrate the increased opportunity for detecting FISH aberrations in enriched versus non-enriched specimens.

Mature B-cell enrichment and subsequent FISH testing in cases of scant mature B-cell neoplastic involvement of the bone marrow and/or peripheral blood is a novel and powerful cytogenetic technique. This technique enriches bone marrow and/or peripheral blood specimens for targeted abnormal cells and increases the number of those cells analyzed by FISH testing, thus allowing for a higher detection rate of genetic abnormalities.