Quantitative Ki-67 Proliferative Index Assessment of Human B-Cell Lymphomas By Flow Cytometry

Abstract

Background: The Ki-67 proliferative index is used as a diagnostic and prognostic marker in the classification of B-cell non-Hodgkin lymphomas (NHL). Despite its semiquantitative nature, immunohistochemistry (IHC) evaluation of Ki-67 in histologic specimens is the gold standard. The aim of this study was to determine the correlation of quantitative Ki-67 expression by flow cytometry (FCM) with standard IHC in samples of B-cell NHL. Methods: We performed a retrospective observational study of bone marrow and tissue biopsies evaluated for routine lymphoma screening between January 2021 and December 2022. FCM analysis was performed according to Euroflow standard LST screening. Positive samples were further characterized, and Ki-67 expression was integrated using a PerCP-Cy5.5 fluorochrome-conjugated monoclonal antibody after cell fixation and permeabilization. The Ki-67 proliferative index was calculated as all positive events among the neoplastic population (%). Ki-67 proliferative index by flow cytometry was compared with Ki-67 proliferative index by IHC. Clinical data were retrieved from the electronic database. This study was approved by the institutional IRB and clinical ethics committee. Results: A total of 51 samples from 50 patients were evaluated: 4 bone marrow aspirates, 8 fine-needle aspirations (FNA) and 39 tissue biopsies. Median age at diagnosis was 67 years (interquartile range [IRQ], 60-79 years), and 45% were female. A low-grade lymphoma was diagnosed in 27 cases (52.9%; 20/27 follicular lymphoma), a high-grade lymphoma case in 23 cases (45.1%; 20/23 diffuse large B-cell lymphoma), and mantle cell lymphoma in 1 case. Median Ki-67 proliferative index by FCM was 41.4% (range, 1 - 100%) and 54.8% (range, 5 - 100%) by IHC. Overall, the Ki-67 proliferation index by FCM showed a significant correlation coefficient of 0.925 with IHC (95% IC 0.870 - 0.957, p < 0.0001). There was a negative constant bias of -13.35% (-46.87 to 20.16), which was greater in the range of 30-80% of Ki-67 expression by IHC. Indolent lymphomas displayed a lower proliferative index by FCM compared with IHC (5 vs 20%, p<0.001). No difference was seen among aggressive lymphomas. Fourteen cases had a difference in the proliferative index >20%, 10/14 were follicular lymphoma, 2/14 were indolent lymphoma transformations and 2 were large B-cell NHL. In all cases FCM displayed lower results compared with IHC. Conclusions: Our data suggest that the Ki-67 proliferative index can be accurately assessed by flow cytometry. A correlation bias seems clinically meaningful in intermediate values of Ki-67. The quantitative nature of FCM-based proliferative index may improve the prognostic potential of Ki-67, especially for indolent or borderline histologies. Further validation studies are warranted.