ABSTRACTdeveloped algorithm to describe clonal hierarchy and migration patterns more thoroughly. Methods Patients, histology, and immunohistochemistryThis study comprised three patients with synchronous LN and BM infiltration by FL at presentation. Biopsies were performed during the diagnostic and staging procedures. The selection criteria were the diagnosis of FL according to the fourth edition of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues.1 Clinical information was obtained from the patients' medical records. Material was collected from patients after their informed consent in accordance with the Declaration of Helsinki. The study was approved by the responsible institutional ethic boards. Further details are provided in the Online Supplementary Methods. Sequence analysis of IgV H genes and definition of mutational patternsDNA from the IgV H gene segments was extracted and amplified as described elsewhere. 23,24 Cloning, sequencing, and the mutational analysis of the obtained segments are described in detail in the Online Supplementary Methods. Delineation of tumor cell evolution by construction of pedigreesFor each patient and each compartment (LN and BM separately), the mutational patterns of IgV H gene segments were arranged in an ascending order of mutations to illustrate the mutational hierarchy of intraclonal sequence heterogeneity. Consequently, mutational patterns of early clones with few mutations had to be included in successor clones. When direct transition of one mutation pattern into that of successor clones with higher mutation loads was not observable, hypothetical predecessor clones (HPC) were introduced to retrace the evolution of sequenced clones back to the determined initial V H DJ H gene rearrangement (wild-type sequence). Accordingly, compartment-specific pedigrees were constructed. Thereafter, a third "summary-pedigree" comprising all sequenced clones was constructed, to evaluate the possibility of inter-compartmental exchange between LN and BM. Generation of hypothetical predecessor clones and delineation of migration probabilityFor each sequenced FL population (i.e. LN, BM, and LN and BM together) the pool of possible HPC was derived from mutations shared by at least two sequenced clones. To select the most appropriate predecessor clones from the abundance of generated HPC, the probability measurement was introduced (Figure 1). Only HPC with the highest probability measurement values were introduced until the evolution of sequenced clones could be retraced to the "wild-type sequence". Already established clone groups could not be disrupted by HPC with lower probability measurement values. These calculations resulted in a LN, a BM and an inter-compartment pedigree. If HPC of the inter-compartment pedigree displayed a higher probability measurement value than the corresponding LN or BM counterparts, inter-compartment migration was considered. The LN or BM allocation of these inter-compartment HPC was directed by the LN or BM affiliation of the majority of ev...

Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to membrane microdomains. Here, we show that concomitant antifungal treatment with itraconazole impairs the rituximab antilymphoma effect both in vitro and in vivo. At the molecular level, recruitment of CD20 to lipid rafts is inhibited in the presence of itraconazole. Furthermore, calcium influx, which is crucial for rituximab-mediated cell death, was nearly completely abolished by itraconazole treatment. In contrast, the antifungal drug caspofungin did not inhibit CD20 recruitment to lipid rafts, nor did it affect calcium influx or the cytotoxic effect of rituximab. The finding that itraconazole also abolished the cytotoxic effects of other therapeutic antibodies directed against lipid raft-associated molecules (i.e., CD20 and CD52) but not those against the non-raftassociated molecule CD33 further supported our proposed mechanism of action. Our results argue that concomitant medications must be adjusted carefully to achieve optimal antitumor effects with monoclonal antibodies. Cancer Res; 70(11); 4292-6. ©2010 AACR.

The monoclonal antibody rituximab directed against the cell surface molecule CD20 of mature B cells has been proven to be successful in the treatment in a variety of B cell malignancies. However, resistance against rituximab occurs and there is no prognostic marker to predict individual response. Rituximab has been shown to induce cell killing via antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and the induction of apoptosis. However, the mechanism that renders rituximab so effective in vivo remains elusive as does the reason for treatment failure in a subgroup of patients. On the molecular level the function of rituximab has been associated with the partitioning of CD20 molecules to lipid microdomains. We now show that the extent of CD20 recruitment to lipid rafts correlates with response to rituximab. In addition, analyzing 11 different Non Hodgkin’s lymphoma cell lines we demonstrate that the expression of the raft associated sphingolipid GM1 on lymphoma cells is associated with the susceptibility to rituximab. Of note, lymphoma cells treated with the sphingolipid synthesis inhibitor PDMP exhibited reduced GM1 expression levels and showed impaired susceptibility to rituximab as compared to non treated lymphoma cells. Furthermore, analyzing the GM1 expression on lymphoma cells of 217 patients with various types of indolent Non-Hodgkin’s lymphoma, we demonstrate significantly different GM1 expression in various Non-Hodgkin’s lymphoma subtypes. Whereas chronic lymphocytic leukemia (CLL) cells have a low GM1 expression, marginal zone lymphoma (MZL) cells exhibit much higher levels. Differences were not only detected between various lymphoma subgroups but also within one lymphoma subtype. Interestingly, whereas CLL cells from patients with high GM1 expression responded to rituximab, patients with low GM1 expressing CLL cells did not. Similar results were observed analyzing lymphoma cells from patients with mantle cell lymphoma (MCL) suggesting that GM1 expression correlates with responsiveness to rituximab in further types of Non-Hodgkin’s lymphoma. Collectively, these data demonstrate the importance of membrane microdomains for the effect of rituximab and may offer a predictive factor for the responsiveness of lymphoma cells to rituximab.