Lymphoma Cell Detection and Follow-Up in the Rituximab Era: Concordance between Flow Cytometry, Qualitative/Quantitative PCR and FISH in the Marrow and Blood from 647 Patients.
Authors: Mancuso Patrizia , Muratori Elisabetta , Rabascio Cristina , Calleri Angelica , Corsini Chiara , Bertolini Francesco 
Published: April 2020

Abstract
The anti-CD20 monoclonal antibody Rituximab is now widely used in B-cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL) patients. Its administration is associated with a profound reduction of the number of normal and malignant CD20+ B cells, so that the procedures used so far for NHL and CLL diagnosis, detection of minimal residual disease (MRD) and follow-up (F-UP) should be revised. We have retrospectively evaluated our consecutive 4-year record of 647 NHL and CLL patients. Our aims were: 1) to compare sensitivity and concordance between 4-colour flow cytometry, qualitative and quantitative PCR and FISH. 2) to compare concordance between bone marrow (BM) and peripheral blood (PB). Sensitivity of the procedures (evaluated by serial dilution of NHL cell lines in healthy donors’ BM and PB) were found to be 10−5, 10−6, 10−4/5 and 10−3 for 4-colour flow cytometry, qualitative PCR, quantitative PCR and FISH, respectively. A total of 955 paired BM and PB samples were investigated. Most frequent diagnoses were follicular NHL (29%), diffuse large B-cell NHL (22%), marginal zone NHL (11%), CLL (10%), and mantle cell NHL (8%). In 236/955 cases (25%), 4-color flow cytometry identified a monoclonal or a phenotypically aberrant cell population. Concordance between BM and PB was 87% (p&lt;0.001). Most frequent diagnosis of discordant cases was lymphoplasmocytoid-Waldenstrom NHL (36% of discordant cases). Qualitative and quantitative PCR were used to investigate clonality (Fr2A/VLJH and Fr2A/VLJH genes) and the presence of translocations involving BCL-1 or BCL-2 genes (in mantle cell or follicular NHL, respectively). A total of 91/333 samples (27%) were positive. Concordance between BM and PB was 91% (p&lt;0.001). Discordance was found in 11% of mantle cells NHL and 8% of follicular NHL cases. When 118 patients were evaluated 3-month after Rituximab (alone or in combination with chemotherapy), qualitative PCR still detected malignant cells in 95% of previously PCR-positive patients, flow cytometry in 72% of previously cytometry-positive patients. On the other hand, at 7 month after Rituximab, in these patients malignant cells were found by qualitative PCR in 14/72 (19%) cases, by flow-cytometry in 22/118 (19%) cases. These procedures identified disease persistence or recurrence significantly earlier than quantitative PCR (evaluated in 98 cases, p&lt;0.01) and FISH (evaluated in 11 cases, p&lt;0.001). We conclude that 1) BM and PB examination by means of 4-color flow cytometry and qualitative PCR offer nearly-overlapping results. In Rituximab-treated patients, differences seen at 3-month F-UP, probably due to the higher sensitivity of qualitative PCR, are no more observed at 7-month F-UP. 2) Four-color flow cytometry and qualitative PCR have higher sensitivity and detect MRD and relapses significantly earlier than quantitative PCR and FISH. 3) With the exception of lymphoplasmocytoid-Waldenstrom NHL, PB examination may be considered a sensitive and specific alternative to BM examination in NHL and CLL patients, including those treated with Rituximab.