Abstract

T cell large granular lymphocytic (LGL) leukemia is a clonal proliferation of cytotoxic T-lymphocytes (CTLs) often resulting in severe cytopenia, including neutropenia and anemia. Clinical observations and laboratory data suggest that LGL evolves in the context of initially normal polyclonal immune responses. Current treatment options favor chronic immunosuppression as high-dose chemotherapy is mostly ineffective. Since 2002 we have diagnosed and treated 75 patients (pts) with LGL leukemia, 45 of whom required therapy. We have identified 8 pts with severe refractory cytopenia despite multiple treatment regimens and who in the course of their care received salvage therapy with subcutaneous alemtuzumab (total dose 60-120 mg in triweekly injections of 10 mg). Previous therapies involved cyclosporine (7/8), cyclophosphamide (2/8), methotrexate (2/8), prednisone (5/8), rituximab (4/8) and high-dose chemotherapy (4/8) including CHOP, ifosfamide, fludarabine, and autologous stem cell transplantation. Analysis of the effects of alemtuzumab therapy revealed remissions with restoration of platelets in 1/1, red blood cell transfusion independence in 3/5 patients and improvement of neutropenia in 1/3 resulting in an overall response rate of 4/8 pts (50%). In the course of analysis we explored cases refractory to alemtuzumab, a monoclonal antibody which recognizes glycosyl phosphatidylinositol (GPI) anchored CD52 antigens ubiquitously expressed on most leukocytes. Previously, the evolution of CD52 deficient T and B cells has been described following alemtuzumab therapy for CLL. Flow cytometric analysis of archived cells collected post therapy demonstrated that clonal CTLs exhibit decreased CD52 expression within the CD8+CD3+CD57+ cell compartment. Vb TCR repertoire analysis by flow also showed that the CD8+ clonal LGL cells expressing a restricted Vb region of the TCR were the predominant CD52 negative cells. Based on this finding, we tested samples collected prior to therapy and unexpectedly found a significant proportion of CD8+CD3+CD57+ cells were CD52 negative before alemtuzumab therapy. In one pt, biclonal LGL was discovered with both clones (one Vb7.1+ and the other Vb3+) lacking expression of CD52 on ~20% of cells. Following alemtuzumab therapy not resulting in a hematologic response, the Vb 7.1+ clone dominated the T cell repertoire, accounting for 92.8% of CD8 cells. Furthermore, 85.7% of these cells lacked CD52 expression, suggesting that alemtuzumab selected for CD52 negative cells. To assess the prevalence of LGL cases with deficient CD52 expression we prospectively analyzed 12 LGL pts. All pts were positive for TCR-g rearrangement and showed expansion of a CD8+CD3+CD57+ Vb restricted CTL population. In 9/12 LGL pts varying amounts of CD52 deficient CTLs were identified while virtually all CD8 cells from healthy controls (n=12) expressed CD52 (p=.006). Further analysis of CD52 deficient cells demonstrated the absence of other GPI linked proteins CD55 and CD59. Previously, GPI-deficient lymphocytes observed after alemtuzumab therapy were shown to contain a wild type PIG-A gene. In the absence of a mutation such as in paroxysmal nocturnal hemoglobinuria, we conclude that deficient expression of GPI-linked proteins is rather due to downmodulation of genes associated with synthesis of a GPI anchor. Reduced mRNA levels of the PIG-A gene have been reported in memory cells suggesting that GPI negativity of LGL cells in some patients may be due to a higher proportion of memory cells within the leukemic cell population. In sum, we conclude that while alemtuzumab may be highly effective in LGL leukemia, prospective monitoring for the presence of CD52 deficient clonal CTLs may be necessary prior to and during therapy.

Disclosures: Off Label Use: Alemtuzumab is a monoclonal antibody approved for the treatment of chronic lymphocytic leukemia..

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