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The degree of somatic hypermutation, determined as percent deviation of immunoglobulin heavy chain gene variable region sequence from the germline (IGHV%), is an important prognostic factor in chronic lymphocytic leukaemia (CLL). Currently, a cut-off of 2% deviation or 98% sequence identity to germline in IGHV sequence is routinely used to dichotomize CLL patients into mutated and unmutated groups. Because dissimilar IGHV% cut-offs of 1-5% were identified in different studies, we wondered whether no cut-off should be applied and IGHV% treated as a continuous variable. We analysed the significance of IGHV% in 203 CLL patients enrolled on the original frontline fludarabine, cyclophosphamide and rituximab (FCR) trial with a median of 10 years follow-up. Using the Cox Proportional Hazard model, IGHV% was identified as a continuous variable that is significantly associated with progression-free (PFS) and overall survival (OS) (P < 0·001). Furthermore, we validated this finding in 323 patients treated with FCR off-protocol and in the total cohort (n = 535). Multivariate analysis revealed a continuous trend. Higher IGHV% levels were incrementally associated with favorable PFS and OS in both FCR-treated cohorts (P < 0·001, both cohorts). Taken together, our data suggest that IGHV% is a continuous variable in CLL patients treated with FCR.

Zeta-associated protein-70 (ZAP70) expression measured by flow cytometry has been proposed as a surrogate marker of the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes in chronic lymphocytic leukemia. However, attempts to implement this approach in clinical flow cytometry laboratories have been problematic; many commercially available antibodies give unreliable results. Assessment of ZAP70 protein expression by immunohistochemistry in chronic lymphocytic leukemia tissue sections is an easy, alternative approach, although lack of quantitation and subjective interpretation of results are potential pitfalls. In this study, we correlated ZAP70 protein expression, assessed by immunohistochemistry, with ZAP70 messenger RNA (mRNA) transcript expression, assessed by semi-quantitative real-time reverse transcriptase-polymerase chain reaction assay, with the somatic mutation status of the IGHV genes in previously untreated patients with chronic lymphocytic leukemia. Expression of ZAP70 protein and mRNA transcripts correlated strongly (P=8.238 × 10(-12)). Expression of ZAP70 protein and mRNA transcripts also correlated strongly with the somatic mutation status of the IGHV genes (P=0.000071 and P=0.00076, respectively). Further, ZAP70 positivity by immunohistochemistry was associated with an increased risk of progression to therapy requirement (3-year risk 83% vs 31% for ZAP70 negative by immunohistochemistry, P=0.03). These results show that ZAP70 expression assessed by immunohistochemistry is a reliable surrogate marker of the somatic mutation status of the IGHV genes, and predicts time to progression.

Rituximab is a human/mouse chimeric monoclonal antibody that binds to the CD20 antigen and is expressed at all stages of B-cell development. Rituximab has demonstrated efficacy as monotherapy and in combination with chemotherapy in the treatment of both indolent and aggressive non-Hodgkin's lymphoma (NHL). Rituximab treatment results in rapid depletion of B-cells and this has led to the consideration of other B-cell disorders as candidates for rituximab therapy. Recent studies have demonstrated the efficacy of rituximab in a variety of such disorders, including chronic lymphocytic leukemia (CLL), post-transplant lymphoprollferative disorder (PTLD),Waidenstrom's macroglobulinemia (WM), multiple myeloma (MM), idiopathic thrombocytopenic purpura (ITP), hairy-cell leukemia (HCL) and cold agglutinin disease (CAD). In patients with CLL, increasing the dose and/or frequency of rituximab treatment has given improved response rates compared with the standard dose schedule used in NHL, and combination immunochemotherapy has yielded an overall response rate of 92% (with a 60% complete response rate). Clinical trials have also demonstrated evidence of efficacy for rituximab in PTLD, WIVI and relapsed or refractory ITP. Efficacy of rituximab in CAD and relapsed or refractory HCL has also been demonstrated in small studies and case reports. Available data thus indicate that rituximab can be an effective therapy in a wide range of CD20(+) lymphoid disorders. [(C) 2002 Lippincott Williams Wilkins.]