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Monitoring measurable residual disease has been postulated to be a surrogate marker of progression free survival in chronic lymphocytic leukemia patients after treatment with immunochemotherapy regimens. In this study, we have analyzed the outcome of 84 patients at 3 years of follow up after first line treatment with fludarabine, cyclophosphamide and rituximab induction followed by 36 months of rituximab maintenance. Measurable residual disease was assessed by a quantitative four-color flow cytometry panel with a sensitivity level of 10-4. Eighty out of 84 evaluable patients (95.2%) achieved at least a partial response or better at the end of induction. After clinical evaluation, 74 patients went into rituximab maintenance and the primary endpoint was assessed in the final analysis at 3-years of follow up. Bone marrow measurable residual disease analysis was performed after the last planned induction course and every 6 months in cases with detectable residual disease during the 36-month of maintenance. Thirty-seven cases (44%) had undetectable bone marrow residual disease prior to maintenance. Interestingly, 29 patients with detectable bone marrow residual disease after induction, had undetectable bone marrow residual disease following maintenance. After a median follow-up of 6.30 years, median overall survival and progression free survival were not reached in patients with either undetectable or detectable bone marrow residual disease, who had achieved a complete response at the time of starting maintenance. Interestingly, univariate analysis showed that after rituximab maintenance overall survival was not affected by IGHV status (mutated vs unmutated overall survival: 85.7% alive at 7.2 years vs 79.6 % alive at 7.3 years, respectively). As per protocol, 15 patients (17.8%), who achieved a complete response and undetectable peripheral blood and bone marrow residual disease after 4 courses of induction, were allowed to stop fludarabine and cyclophosphamide and complete 2 additional courses of rituximab and continue with maintenance for 18 cycles. Surprisingly, the outcome in this population was similar to that observed in patients who received the full 6-cycles induction regimen. These data show that fludarabine, cyclophosphamide and rituximab followed by rituximab maintenance produce high-quality responses with less relapse and improved overall survival compared to historic controls, showing a favorable tolerability. Furthermore, attaining an early undetectable residual disease status could reduce the length of chemoimmunotherapy, reducing toxicities and preventing long-term side effects. The analysis of bone marrow measurable residual disease after fludarabine-based induction could be a powerful predictor of post-maintenance outcomes in patients with chronic lymphocytic leukemia undergoing rituximab maintenance and could be a valuable tool to identify patients at high risk of relapse, influencing further treatment strategies. This trial is registered with EudraCT No.: 2007-002733-36 and ClinicalTrials.gov Identifier: NCT00545714.

The Effects of Different Echinostoma caproni Miracidial Doses on the Lutein and β-Carotene Content of Biomphalaria Glabrata Snails The effects of 5, 25, and 40 Echinostoma caproni miracidia on the lipophilic pigment content of Biomphalaria glabrata snails was studied using reverse phase high performance thin-layer chromatography-densitometry. Analysis was done on the whole body at two and four weeks post exposure. All extraction and analysis steps were performed in minimal light in order to prevent pigment degradation. The pigments were extracted with acetone, evaporated to dryness, reconstituted using heptane, and analyzed on reverse phase silica gel HPTLC plates with a concentrating zone using a petroleum ether (37.8-53.5oC)-acetonitrile-methanol (1:1:2) mobile phase. The analysis yielded significant differences between the lutein and β-carotene content of the snails at two and four weeks post exposure and between the different miracidial doses.