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<p>Abstract</p> <p>Background</p> <p>HIV-negative, CD20-negative <it>de novo</it> diffuse large B-cell lymphoma (DLBCL) patients has rarely been reported. To elucidate the nature of this entity, we retrospectively reviewed the data of 1,456 consecutive <it>de novo</it> DLBCL patients who were treated at Sun Yat-Sen University Cancer Center between January 1999 and March 2011.</p> <p>Methods</p> <p>The pathologic characteristics of CD20-negative patients, clinical features, response to initial treatment, and outcomes of 28 patients with available clinical data (n = 21) were reviewed. Then, a matched case-control (1:3) analysis was performed to compare patients with CD20-negative and -positive DLBCL.</p> <p>Results</p> <p>The median age of the 28 CD20-negative DLBCL patients was 48 years, with a male-female ratio of 20:8. Seventeen of 22 (77.3%) CD20-negative DLBCL cases were of the non-germinal centre B-cell (non-GCB) subtype. High Ki67 expression (≥80%), an index of cell proliferation, was demonstrated in 17 of 24 (70.8%) cases. Extranodal involvement (≥ 1 site) was observed in 76.2% of the patients. Following initial therapy, 9 of 21 (42.9%) cases achieved complete remission, 4 (19%) achieved partial remission, 1 (4.8%) had stable disease, and 7 (33.3%) had disease progression. The median overall survival was 23 months. The 3-year progression-free survival (PFS) and overall survival (OS) rates were 30.5% and 35%, respectively. A matched case-control analysis showed that patients with CD20-negative and -positive DLBCL did not exhibit a statistically significant difference with respect to the main clinical characteristics (except extranodal involvement), whereas the patients with CD20-positive DLBCL had a better survival outcome with 3-year PFS (<it>P</it> = 0.008) and OS (<it>P</it> = 0.008) rates of 52% and 74.1%, respectively.</p> <p>Conclusions</p> <p>This study suggests that HIV-negative, CD20-negative <it>de novo</it> DLBCL patients have a higher proportion of non-GCB subtype, a higher proliferation index, more frequent extranodal involvement, a poorer response, and a poorer prognosis to conventional treatment compared to patients with CD20-positive DLBCL. Further studies are warranted to investigate new target and optimal therapy of CD20-negative <it>de novo</it> DLBCL.</p>