Minimal Residual Disease By Patient-Specific Taqman Real Time PCR in Newly Diagnosed Hairy Cell Leukemia Patients Randomized to Initial Vs Delayed Rituximab in Combination with Cladribine

Background: Patients with newly diagnosed hairy cell leukemia (HCL) have high complete remission (CR) rates to purine analogs including cladribine, but most patients appear to relapse within 5-16 years depending on follow-up testing, suggesting that most patients, young, will require treatment later for relapsed disease. Minimal residual disease (MRD) is often present after CR to cladribine, and its later growth may lead to relapse. Standard tests of HCL MRD include bone marrow biopsy (BMBx) immunohistochemistry (IHC) and flow cytometry of the blood or bone marrow aspirate (BMA), the latter being most sensitive. PCR to amplify the patient-specific HCL immunoglobulin heavy chain gene rearrangement (IgH) is less sensitive than flow cytometry because of the presence of IgH from normal B-cells. For improved sensitivity, HCL IgH sequences are used to create patient-specific real-time PCR (RQ-PCR) TaqMan assays utilizing sequence-specific primers and probes, and are capable of detecting 1 HCL in 10⁶normal cells.

Methods: To determine the value of rituximab added to cladribine for initial treatment of HCL, using MRD as a surrogate endpoint, 68 newly diagnosed patients in a phase 2 trial were randomized 1:1 between cladribine alone at 0.15 mg/Kg/day on days 1-5, vs cladribine with 8 weekly doses of rituximab 375 mg/m²begun on day 1, to optimize synergy with cladribine. Patients with MRD in blood at least 6 months after cladribine received delayed rituximab at the same dose as initial rituximab. Bone marrow was assessed 1, 6, 18, and 30 months after cladribine, then every 2 years, and blood for RNA and flow cytometry was obtained at 1, 3, 6, 9, 12, 18, 24, and 30 months after cladribine, then yearly. The same schedule was used after delayed rituximab except for the 1 month bone marrow.

Results: All 68 randomized patients were evaluable for response. At 1-6 months after cladribine + initial rituximab, 100% of 34 patients achieved CR, compared to 28 (90%) of 31 evaluable patients after cladribine alone (p=0.1). By standard tests of MRD 6 months after cladribine, 31 (97%) of 32 with vs 10 (32%) of 31 without rituximab were MRD-negative (p<0.0001), and the one sample of MRD+ HCL after cladribine plus immediate rituximab contained variant hairy cells not observed before treatment. About half of the patients were evaluable for molecular MRD assessment, which required IgH sequencing and RQ-PCR TaqMan assay. The blood RQ-PCR assay was negative in 100% of 15 with vs 4 (40%) of 10 without immediate rituximab (p=0.001). No molecular relapses occurred with vs 2 of 4 relapses without rituximab (p=0.035). Bone Marrow RQ-PCR was negative in 100% of 14 with vs 3 (38%) of 8 without immediate rituximab (p=0.002). Of 16 patients receiving delayed rituximab after cladribine alone, 12 (75%) cleared MRD by standard tests, and of these 12, 5 were tested by RQ-PCR TaqMan, which was negative in 5 (100%) by blood and 3 (60%) by bone marrow. By standard studies at 6-60 (median 30) months of follow-up, 31 (97%) of 32 with vs 20 (65%) of 31 without initial rituximab remain MRD-free (p=0.001) and all continue both standard and molecular testing.

Conclusions: Rituximab was highly effective in clearing MRD, both by standard and patient-specific methods, begun immediately with cladribine for newly diagnosed HCL. RQ-PCR TaqMan analysis was slightly more sensitive than standard methods in detecting HCL MRD. Since MRD could be cleared in a minority of patients each by cladribine alone or by delayed rituximab, long term follow-up of these patients will be needed to determine the full value of immediate vs delayed rituximab in newly diagnosed HCL. Randomization is continuing in separate stratifications with patients having either once-relapsed HCL or HCL variant.

This work is supported in part by the Intramural Program, National Cancer Institute, Genentech, and the Hairy Cell Leukemia Research Foundation.