Immunophenotypic stability of T‐cell large granular lymphocytic leukaemia by flow cytometry

T-cell large granular lymphocytic leukaemia (T-LGLL) is a persistent, clonal expansion of large granular lymphocytes (LGLs) with a typically indolent clinical course, although severe neutropenia and/or anaemia can be frequent causes of morbidity and mortality (Chan et al, 2008; Lamy & Loughran, 2003). Autoimmune diseases and concomitant malignancies have also been reported in association with T-LGLL (Lamy & Loughran, 2003). Increased LGLs can also be seen in reactive conditions, which makes diagnosing T-LGLL challenging; however, flow cytometry (FC) has proven useful in demonstrating immunophenotypic aberrancies, commonly seen in virtually all T-LGLL, similar to other T-cell neoplasms (Lundell et al, 2005; Morice et al, 2003). Clonality can be demonstrated in T-LGLL by polymerase chain reaction (PCR) and Southern blot analysis, or indirectly by T-cell receptor (TCR) variable beta (Vb) chain repertoire and killer immunoglobulin-like receptor (KIR) analysis by FC (Lundell et al, 2005; Morice et al, 2004). While clonality alone does not always equate to malignancy, the combination of immunophenotypic aberrancies and evidence of clonality provides strong evidence of neoplasia (Kroft, 2004). Increasingly, clinicians are relying on FC for tracking disease burden and response to therapy (Mohan et al, 2009). This utility depends upon the assumption that the unique immunophenotypic fingerprint being followed remains constant. It is conceivable that a false negative study could occur in the setting of a narrowly targeted antibody panel, in combination with loss of immunophenotypic aberrancies present at diagnosis. The ability of haematological malignancies to undergo immunophenotypic shifts has been well established, particularly in B-cell and myeloid neoplasms (Chen et al, 2007). However, data regarding immunophenotypic stability in T-LGLL is limited. To address this question, we compared diagnostic and follow-up specimens from 10 T-LGLL patients by FC for changes from the baseline immunophenotype over time. Patients were diagnosed according to World Health Organization 2008 criteria (Chan et al, 2008), including: (i) persistent (>6 months) increase of peripheral blood LGLs, without a clearly identified cause; (ii) presence of cytopenias; (iii) aberrant T-cell antigen expression by FC; (iv) establishment of clonality by TCR gene rearrangement studies and/or Vb receptor analysis; and (v) demonstration of bone marrow involvement by LGLs with an intrasinusoidal distribution. We examined 67 sequential peripheral blood specimens (February 2006 to April 2010) by 4-colour FC, using antibodies to CD2, CD3, CD4, CD5, CD7, CD8, CD16, CD56, CD57, TCRab/cd, CD94, CD158a, CD158b, CD158e, according to previously reported methods (Olteanu et al, 2010). For Vb analysis, the Beta Mark TCR Repertoire kit (Beckman Coulter, Miami, FL, USA), was used according to the manufacturer’s recommendations. FC data was analysed using Paint-A-Gate Pro (Becton Dickinson Biosciences, San Jose, CA, USA). Neoplastic T-cell populations were identified based on aberrant antigen expression, relative to internal normal populations, as previously described (Jamal et al, 2001). Antigen overor under-expression was defined as >0Æ25 log difference in intensity between aberrant and normal populations. A change in immunophenotype was recorded for each antigen as either loss (i.e., reversion to normal level of expression) or gain of aberrancy. Clinical and laboratory data were obtained from review of medical records. Statistical analysis was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). The study was approved by the Medical College of Wisconsin Institutional Review Board. Our study included five men and five women, with a median age of 55 years (range 47–72). At presentation, cytopenias were present in 10/10 patients, including anaemia (8/10), thrombocytopenia (2/10), and neutropenia <1Æ5 · 10/l (7/10). The median absolute number of aberrant T-cells at diagnosis was 3Æ36 · 10/l (range 1Æ76–6Æ24 · 10/l). Clonal rearrangement of TRG@ was detected by PCR in 7/7 T-LGLLs tested. The remaining three patients had restricted Vb antigen expression, and one patient had evidence of clonality by both PCR and Vb analysis. Bone marrow involvement was documented in 9/9 cases examined. Five patients received chemotherapy. Table I summarizes the FC findings at diagnosis. All T-LGLLs showed aberrant expression of at least two T-cell antigens, including CD3, CD5, CD7, and CD8. An example is shown in Fig 1. One case was CD4(+)/CD8(+), while the remaining were CD4())/CD8(+). Restricted KIR expression pattern was seen in 4/4 cases tested. Similar to previous studies, CD16 and CD56 expression were predominantly mutually exclusive, and most cases were CD57(+) (Lundell et al, 2005; Morice et al, 2003). A total of 47 aberrancies were present at diagnosis (median 4Æ5/case; range 2–7). The number of aberrancies/case was comparable to that from previous studies, even with different antibody combinations and data analysis techniques used, thus demonstrating the validity of this analytic approach (Lundell et al, 2005; Morice et al, 2003). Median follow-up was 27 months (range 12–44), with 3–24 analyses per patient (total = 57 analyses). All cases retained multiple aberrancies at follow-up (total = 239: median 5/ analysis; range 2–6). A change in immunophenotype was Correspondence