Peripheral Blood Disease Monitoring in Marginal Zone Lymphoma Using Flow Cytometry

The use of sensitive flow cytometric techniques for the primary diagnosis and monitoring of patients with B-CLL is now well established. The aim of this study is to examine the feasibility of extending this approach to other indolent B-cell lymphoproliferative disorders. Such a technique, particularly if applicable to peripheral blood samples, would have significant benefits in allowing minimally invasive sequential monitoring of treatment responsiveness and early detection of relapse. Using a similar statistical approach to that used in the design of the CLL minimal disease flow cytometry panels we identified LAIR1, CCR6, CXCR5 and CD10 as markers that allowed discrimination between normal and neoplastic B-cells. These markers were incorporated into a 2 tube 8 colour assay together with CD19, CD5, CD20, CD22, CD45, kappa and lambda. Leucocytes were prepared using ammonium chloride lysis, incubated with these antibodies and washed before acquisition of 500,000 events on a FacsCanto flow cytometer (BD BioSciences, Oxford). As part of the evaluation of the technique we investigated peripheral blood samples from a total of 28 patients (14 male, median age 69.1 years and 14 female, median age 73.4 years), 22 with Marginal Zone Lymphoma/Waldenstrom’s Macroglobulinaemia (MZL) and a further 6 with Extranodal Marginal Zone Lymphoma (EMZL). Of the 22 MZL patients, 8 had active disease, 5 had stable disease that was untreated, 1 was on treatment and 8 were in remission. Of the extranodal MZL patients 1 had stable untreated disease, with 2 on treatment and 3 in remission. 77% (10/13) of MZL patients with active or stable untreated disease had detectable circulating tumour cells. Abnormal B-cells represented a median 2% of leucocytes (range 0.08 – 39%). The median absolute abnormal B-cell count was 0.34 ×10⁹/l (range 0.003–9.6 ×10⁹/l). The phenotypes of the cells detected showed a high level of heterogeneity. Abnormal cells were detectable in 22% (2/9) of cases on treatment or in remission, with a median % abnormal B-cells of 0.88% (range 0.35–5.37%) and a median absolute B-cell count of 0.09×10⁹/l (range 0.02–0.386×10⁹/l). None of the EMZL patients had abnormal cells detectable. Circulating tumour cells were detectable down to a level of 0.003×10⁹/l and were found in a proportion of patients on treatment or considered to be in clinical remission. Methods used to monitor patients with indolent lymphoproliferative disorder need to take into account the phenotypic diversity found in marginal zone lymphoma compared to CLL. However the results demonstrate the feasibility of extending the use of flow cytometric residual disease assays to marginal zone lymphoma.