Identification of Genes Associated with Resistance and Response in Vivo to Therapy with Rituximab, Fludarabine and Cyclophosphamide in Patients with Chronic Lymphocytic Leukemia.

Fludarabine and cyclophosphamide are the backbone of therapy for patients with CLL. The addition of rituximab leads to further improvement of response rates and progression free survival (results from the randomized CLL8 study of the GCLLSG). However, a considerable number of patients have insufficient or short responses to FC or RFC and there is a need to identify factors influencing response or resistance to therapy. The aims of this study are

• to identify gene expression associated with response or resistance before the start of therapy,

• to investigate changes in the expression of specific genes or pathways associated with response or resistance during the first cycle of FC and RFC,

• to provide a rationale for the additional use of novel drugs to improve remission and overcome resistance.

We investigated peripheral blood samples from 20 patients receiving FC (n=10) or RFC (n=10) by gene expression profiling, flow cytometry, RT-PCR and western blotting before and during therapy. Sixteen patients received FC or RFC as first line (8 within the CLL8 study) and 4 as second line treatment. All patients were in stage Binet B or C. Gene expression was analyzed and correlated to good (CR or PR) or poor clinical response (SD or PD) at the end of therapy based on NCI-WG/IWCLL criteria. CD19⁺ cells were harvested by cell sorting before therapy, 24 hours after FC (FC arm), 24 hours after rituximab, and 24 hours after FC (RFC arm). Microarray analysis was performed using Affymetrix U133A gene chips. Genes with a consistent pattern of expression (high or low) in the majority of samples in the good or poor response group were further analyzed. Overall, 9 patients responded adequately to therapy (3 CR, 7 PR), while 11 did not (7 SD, 3PD). Unmutated IgVH status and poor risk cytogenetics were more frequent in poor responders. Gene expression signature before treatment showed that overexpression of 39 genes strongly correlated with response, while overexpression of 20 genes (including HSPA1B, IFI6, APP, CEACAM1, CD9, GAB1, INPP5F) was associated with resistance. Changes in expression after initiation of treatment was also analyzed. Seven genes (including CENTD1, HBA2, COL9A2 and APRIN) were significantly upregulated after rituximab in non-responders. Upregulation of 13 genes (including PMAIP1, SFRS11, CLK1, EFHC1, MRPL39, TUG1, TBRG1, CD49d, PTPRC) after R-FC and 7 genes (including ITPKB, LOC641298, CD44, TAF5) after FC was associated with poor response (resistance) to RFC and FC respectively. Many of these genes are involved in regulation of apoptosis, cell cycle, integrin and PI3-K signaling Therapeutic antibodies or inhibitors against some of these targets are already available. RT-PCR analysis demonstrated a significant downregulation of Akt1 mRNA 24 hours after rituximab infusion in RFC group but no significant changes were observed in patients receiving FC alone. In vitro exposure to rituximab confirmed its in vivo effect and resulted in a significant downregulation of Akt1 and PI3-K-p85 mRNA expression. FACS analysis demonstrated a decrease in the percentage and mean fluorescence intensity (MFI) of surface CD20 after rituximab infusion. This effect was associated with a significant change in total amount and phosphorylation state of CD20 in the RFC group. There was also a decrease in the MFI of CD44 and CD23 after rituximab in the majority of patients in the RFC group but this effect was not consistent in the FC group. In conclusion, we have identified a set of markers associated with good or poor response to FC or RFC before therapy and during the first cycle of treatment. The data provide a rationale for targeted drug combinations to overcome resistance and improve response to therapy in CLL.