Page 74 - 2019_04-Haematologica-web

M. Kenealy et al.
and no prior demethylating agent or immunomodulatory drug. They were to have a performance status of ECOG 0-2 and ade- quate organ function (Online Supplementary Appendix). GCSF was only used for short term management of severe neutropenic infec- tions with no response assessment performed within 21 days of use. Patients on a stable dose of EPO prior to study entry were allowed to continue unchanged while on study.
Statistical plan
Analyses were carried out using the SAS (Statistical Analysis System, Version 9.3, SAS Institute, North Carolina, USA) software and graphs were produced in R version 3.2.3 software (R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org). All comparisons were by intention to treat. The close-out date for this analysis was 12th March 2016.
A sample size of 160 patients (80 per arm) would provide 90% power, assuming a two-sided type I error of 5%, to detect an improvement of at least 25% in clinical benefit at 12 months, where the expected rate of clinical benefit at 12 months in the con- trol arm of 50%, given a median time to progressive disease, relapse or death in the AZA001 study of 14.1 months,9 and an expected rate of clinical benefit in the combination arm being 75%.
Toxicity
Adverse event rates are based on the worst grade reported dur- ing study treatment for those patients who commenced treat- ment. Fisher’s exact test was used to compare adverse event rates between the randomized arms. All events and grades are based on the CTCAE v4.0 unless otherwise specified. Emerging grade 3+ haematologic toxicity applied to patients who did not have a haematologic toxicity at baseline but developed whilst on treat- ment. Results are based on absolute value from the screening aver- aged haematology counts. A grade 3+ toxicity for neutrophil and platelet data was defined as a reduction of more than 50% from baseline but for haemoglobin Grade 3+ was defined as Hb <80g/L.
Efficacy
All patients who were randomized (intention to treat group) were considered in efficacy analysis. 2006 IWG criteria were used for all responses.27 The primary endpoint was “clinical benefit at 12 months”, defined as the patient being alive and progression/relapse free at 12 months (+/- 1 month) post com- mencement of treatment, and so included those patients with sta- ble disease at 12 months as achieving clinical benefit. Best response was determined using all assessments performed at the commencement of each cycle from C3 until treatment discontin- uation, with bone marrow biopsies performed after C2, C4, C8 and C12. The overall response rate (ORR) included all patients achieving improvement (HI), marrow CR, PR and CR as best response.
Univariable logistic regression models were used to assess the impact of the following pre-defined variables on response (mar- row CR or better): treatment arm, IPSS-R, IPSS, cytogenetic risk group, WHO diagnosis (MDS vs. AML vs. CMML).
Progression-free survival (PFS) was measured from the com- mencement of treatment to disease progression or death from any cause. Overall survival (OS) was measured from the commence- ment of treatment to death from any cause. OS and PFS duration was censored at the study close-out date for patients who were still being followed up without having experienced the relevant event by the close-out date, or at the date of last contact for patients who were lost to follow up before the study close-out date. The Kaplan-Meier (product-limit) method was used to esti- mate PFS and OS and median follow-up time (using the censoring
distribution). The logrank test was used to compare survival between the treatment arms and IPSS-R subgroups.
Quality of life (QoL)
The EORTC QLQ C30 was utilized to describe differences in QoL parameters. These analyses were performed on five function- al scales (physical, role, emotional, social and cognitive), three symptom scales (fatigue, nausea & vomiting and pain) and a glob- al health status/QoL scale and six single items. Regression meth- ods accounting for repeated measures (i.e., generalized estimating equations (GEE) with an exchangeable correlation structure) were used to estimate the difference between the treatment arms adjusted for baseline QoL score and weeks on trial. Differences between arms are expressed such that positive differences favour the LEN+AZA arm and negative differences favour the AZA arm.
Molecular and Biomarkers
Next generation sequencing (NGS). Sequence analysis of targeted regions within 26 genes involved in myeloid malignancy (Peter MacCallum Cancer Centre Myeloid Amplicon Panel (v5.4)) was performed in duplicate using Access Array methodology (Fluidigm, South San Francisco, CA, USA) to prepare amplicon- based, indexed libraries that were sequenced to a depth of ∼1000 reads per amplicon on a MiSeq instrument using v2 chemistry (Illumina, San Diego, CA, USA). Alignment, variant calling and annotation were performed using a custom pipeline. Variants were evaluated using multiple functional and quality filters to identify likely pathogenic variants.
SNP-Array testing. DNA (200 ng) was hybridized to CytoSNP-12 BeadChip arrays (Illumina, San Diego, CA) according to the man- ufacturer’s instructions. Analysis was performed using Karyostudio v1.4 software (Illumina). Karyotypes were reported according to the International System for Cytogenetic Nomenclature (ISCN 2013).
Exploratory analyses include changes in promoter DNA methy- lation during cycle 1 and at additional time points on treatment, immunophenotyping of MDS population, T-cell subsets, NK-cell function and cytokine profile as predictors of response to treat- ment and will be reported separately.
Results
Baseline demographics and disease features (Table 1)
One hundred and sixty patients with a median age of 70.7 years (range 42.5-87.2) were enrolled on study between August 2010 and August 2012; 159 received study drug. The median time from diagnosis to treatment was 1.0 year (0.0-13.2). Twenty-two patients (14%) had CMML, 19 (12%) AML and the remaining 74% MDS were mostly RCMD or RAEB 1/2 subtypes. Overall IPSS was Low-Int 1 in 61%; by IPSS-R Very Low/Low/Intermed in 63% with no difference in prog- nostic or cytogenetic subgroups between arms. Fifty- seven percent of patients were transfusion dependent at study entry. The 5q- cytogenetic abnormality was present as an isolated abnormality in only 3 patients ( IPSS Int-1 in 2 patients, Int-2 in 1).
Molecular characteristics at baseline
Targeted amplicon sequencing and SNP-A testing was successfully performed in 66 cases. Targeted amplicon sequencing detected pathogenic mutations in one or more genes in 94% (62/66) of patients and SNP-array detected
702
haematologica | 2019; 104(4)