Purpose In contrast to the classic form, variant hairy cell leukemia (HCLv) responds poorly to single-agent purine analogs, expresses unmutated BRAF, has shorter overall survival, and lacks effective standard therapy. No treatment has achieved a high complete remission rate even in small series, and of 39 reported cases from 6 studies, overall response rate after cladribine was 44% with 8% complete remissions. Rituximab has been found to increase the sensitivity of malignant cells to cladribine, suggesting that combination with cladribine might improve response in HCLv. To test this hypothesis, HCLv patients were treated with simultaneous cladribine and rituximab. Experimental design HCLv patients with 0-1 prior courses of cladribine received cladribine 0.15 mg/Kg days 1–5, with 8 weekly doses of rituximab 375 mg/m2 beginning day 1. Restaging was performed, and minimal residual disease (MRD) in blood and marrow was quantified using PCR, immunohistochemistry, and flow cytometry. Results By 6 months, 9 (90%) of 10 patients achieved complete remission (CR), compared to 3 (8%) of 39 reported cases treated with cladribine alone (p<0.0001). Of the 9 CRs, 8 remain free of MRD at 12–48 (median 27) months of follow-up. No dose-limiting toxicities were observed when beginning cladribine and rituximab on the same day, although most patients required short-term steroids to prevent and treat rituximab infusion reactions. Cytopenias in CRs resolved in 7–211 (median 34) days without major infections. Conclusion Although cladribine alone lacks effectiveness for early or relapsed HCLv, cladribine with immediate rituximab achieves CRs without MRD and is feasible to administer.

To compare hairy cell leukemia (HCL) with chronic lymphocytic leukemia (CLL) and normal B cells with respect to their B-cell receptors, somatic hypermutation (SHM) features in HCL were examined in a series of 130 immunoglobulin gene heavy chain rearrangements, including 102 from 100 classic (HCLc) and 28 from 26 variant (HCLv) patients. The frequency of unmutated rearrangements in HCLc was much lower than that in HCLv (17% vs 54%, P < .001) or historically in CLL (17% vs 46%, P < .001), but HCLv and CLL were similar (P ‫؍‬ .45). As previously reported for CLL, evidence of canonical SHM was observed in HCLc rearrangements, including: (1) a higher ratio of replacement to silent mutations in the complementarity determining regions than in the framework regions (2.83 vs 1.41, P < .001), (2) higher transition to transversion ratio than would be expected if mutations were random (1.49 vs 0.5, P < .001), and (3) higher than expected concentration of mutations within RGYW hot spots (13.92% vs 3.33%, P < .001). HCLv met these 3 criteria of canonical SHM to a lesser extent. These data suggest that, whereas

2503^ Background: The anti-CD22 recombinant immunotoxin moxetumomab pasudotox, also known as HA22 or CAT-8015, was recently reported in phase I testing to achieve complete remissions (CRs) in 13 (46%) of 28 patients with relapsed/refractory hairy cell leukemia (HCL); 3 of 13 patients have relapsed. Methods: To complete this trial, 20 additional patients received the highest dose level (50 µg/Kg every other day x 3 doses); none of the 48 HCL patients had dose-limiting toxicity (DLT). Results: Of the first 42 patients with >6 mo of follow up off-treatment, 23 (55%) had CRs, with an overall response rate of 88%. Of the 23 CRs, 21 were evaluable for minimal residual disease (MRD) using flow cytometry of blood and immunohistochemistry of the bone marrow biopsy, and 17 (81%) were negative. Of these 17 patients, 11 (65%) were negative by bone marrow aspirate (BMA) flow cytometry. PCR using consensus primers for the heavy chain immunoglobulin (IgH) rearrangement was less specific than flow cytometry of blood, since IgH rearrangements of normal B cells, which recovered rapidly after immunotoxin treatment, were also amplified. For better MRD detection in blood, patient IgH sequences were cloned and sequence specific primers and probes designed for real-time quantitative PCR (RQ-PCR). RQ-PCR of blood was negative in 6 (100%) of 6 patients achieving flow-negativity in both blood and BMA and positive in 3 (100%) of 3 patients flow-negative in blood but not BMA (p=0.01). No relapses from CR have been observed in 10 patients who became RQ-PCR-negative in blood or flow-negative in BMA, with 5-38 (median 11) mo of follow-up. Conclusions: We conclude that clone-specific RQ-PCR is the most sensitive blood test for MRD in our HCL patients after moxetumomab pasudotox, and could be used to assess the possibility of long-term molecular remissions. We believe these results, including durable CRs without DLT, support a pivotal trial in which moxetumomab pasudotox is compared with alternative therapy. Note: this summary contains investigator reported data. This study was funded by MedImmune, LLC, and supported by NCI’s Intramural Research Program and the Hairy Cell Leukemia Research Foundation.