Study design and patients
The design of the RESPONSE trial (
ClinicalTrials.gov identifier, NCT01243944) has been reported in detail elsewhere [
4]. Briefly, patients with PV and spleen volume ≥450 cm
3 who had not undergone prior JAK inhibitor therapy and were resistant to or intolerant of hydroxyurea were randomized 1:1 to ruxolitinib (initial dosage, 10 mg twice daily) or BAT; dose modification was permitted. BAT options included hydroxyurea (at a dose that did not cause unacceptable side effects), interferon (IFN) or pegylated (PEG) IFN, pipobroman, anagrelide, immunomodulators (e.g., lenalidomide, thalidomide), phlebotomy, or no medication. Phosphorus-32, busulfan, and chlorambucil were prohibited. In cases of lack of response or toxicity requiring drug discontinuation, a change of BAT was permitted. Patients also received low-dose aspirin unless its use was medically contraindicated. At week 32, crossover from BAT to ruxolitinib was permitted if the primary end point had not been met; crossover was also permitted after week 32 in cases of disease progression.
The primary end point of the study was the proportion of patients who had both Hct control and a reduction in spleen volume of ≥35% from baseline at week 32. Hct control was defined as phlebotomy ineligibility from weeks 8 to 32 and ≤1 instance of phlebotomy eligibility between randomization and week 8. Patients were considered eligible for phlebotomy if they had a confirmed Hct >45% that was ≥3 percentage points higher than their baseline Hct level or a confirmed Hct >48%, whichever was lower (confirmed 2–14 days after the initial observation).
The study was conducted in accordance with the Declaration of Helsinki. Each participating site’s institutional review board reviewed and approved the study, and all patients provided written informed consent before inclusion in the study.
Assessment of allele burden
Evaluating changes in
JAK2 p.V617F allele burden was a predefined exploratory objective of the RESPONSE trial. Blood samples for
JAK2 p.V617F allele burden quantitation were drawn from each patient at baseline; at weeks 32, 56, 80, 112, 144, 176, and 208; at the crossover visit, if applicable; and at the end of treatment visit. Genomic DNA samples were isolated from peripheral blood using previously validated methods [
6].
This post hoc analysis measured the
JAK2 p.V617F allele burden in peripheral blood with a limit of detection of 1%
JAK2 p.V617F and a lower limit of quantitation (LLOQ) of 2%
JAK2 p.V617F [
6].
JAK2 p.V617F allele burden was defined as the percentage of mutant allele present relative to the total (i.e., wild-type plus mutant). Changes from baseline in
JAK2 p.V617F allele burden among patients randomized to ruxolitinib and those who crossed over from BAT to ruxolitinib were reported up to week 208; for the crossover cohort, baseline allele burden was defined as the last observation before receiving ruxolitinib. Changes from baseline in
JAK2 p.V617F allele burden were also evaluated in subgroups of patients who had and who did not have IFN treatment before study enrollment and in patients randomized to IFN as BAT who crossed over to ruxolitinib.
The JAK2 p.V617F mutation was detected by real-time PCR or high-resolution melting analysis. Deep sequencing based on the Ion Torrent™ Personal Genome Machine™ (PGM) System (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to analyze mutations in 22 genes associated with the JAK/STAT pathway (JAK1, JAK2 [other than JAK2 p.V617F], JAK3, EZH2, ASXL1, TET2, IDH1, IDH2, CBL, SRSF2, DNMT3A, NFE2, SOCS1, SOCS2, SOCS3, SH2B3, STAT1, STAT3, STAT5A, STAT5B, SF3B1, and U2AF1). Ion Torrent PGM data were aligned against the human genome (version 19) using NextGENe® software 2.3.1 (SoftGenetics, LLC, State College, PA, USA). Mutations were confirmed using conventional sequencing methods. Mutations in CALR were analyzed using high-resolution capillary electrophoresis and confirmed with Sanger sequencing.
Complete molecular response (CMR) and partial molecular response (PMR) were defined using the International Working Group–Myeloproliferative Neoplasms Research and Treatment/European LeukemiaNet consensus criteria [
7]. CMR was defined as reduction of the
JAK2 p.V617F allele burden to below the LLOQ for patients who had an allele burden above the LLOQ at baseline (i.e., a 100% reduction in allele burden from baseline). PMR was defined as achieving ≥50% reduction in
JAK2 p.V617F allele burden for patients who had ≥20% allele burden at baseline.
Potential associations with clinical outcomes were evaluated by analyzing reductions in the JAK2 p.V617F allele burden and relationships to changes in spleen volume, Hct levels, white blood cell counts, and platelet counts at last observation. These evaluations were also conducted in subgroups of patients who experienced a JAK2 p.V617F allele burden reduction <20 versus ≥20%.