Intrapatient variability in fetal hemoglobin measurements over time in sickle cell patients not on fetal hemoglobin inducing agents
Authors: Esther Knapp, Hillel Cohen, Abdullah Kutlar, Richard Ghalie, Deepa Manwani
Journal: American Journal of Hematology (2015)

To the Editor: Sickle cell disease (SCD) is a condition that causes significant morbidity and mortality worldwide, yet a limited array of therapies exists. Fetal hemoglobin inducing agents are a mainstay of therapy. Several studies have confirmed the beneficial effects of higher fetal hemoglobin on sickle cell-associated morbidity and mortality. 1-4 Serial fetal hemoglobin (Hb F) monitoring is used as a marker of response to fetal hemoglobin inducing agents. In addition, Hb F levels are often used as an approximate gauge of medication compliance, especially when followed over time. We previously reported in this journal results of the placebo-controlled phase II study of the short-chain fatty acid derivative 2,2-dimethylbutyrate (HQK-1001) in inducing Hb F in 76 patients with SCD. 5 The primary endpoint was a comparison of Hb F levels in the treatment versus the placebo arms. Week 24 interim analyses revealed no statistically significant difference in change in Hb F levels between the two groups. We now report an examination of the placebo arm alone to assess untreated, intra-patient variability of Hb F%. Patients with SCD between 12 and 60 years of age were eligible for enrollment if they had hemoglobin SS or S/β0 thalassemia, symptomatic disease (defined as at least one acute SCD-related complication or leg ulcers in the 12 months prior to enrollment), and no current treatment with hydroxyurea (i.e., within 3 months prior to enrollment). This was a multinational study, conducted at various sites worldwide. A more detailed description of the methods can be found in Reid et al, 2014. 5 Hemoglobin separation and quantification was performed by cation exchange high-performance liquid chromatography, once during the screening period, prior to starting study drug, and then every 4 weeks. Study visits occurred every 4 weeks, including 4 weeks after completion of therapy. Only Hb F values performed by HPLC at the central reference laboratory (Georgia Health Sciences University) were included. Any Hb F values determined within a 3-month period after a blood transfusion were excluded. Baseline levels and at least one subsequent Hb F level were available in 37 of the 38 patients randomized to the placebo arm, so these 37 patients were included in the analysis. Serial determinations were performed at 4 week intervals, resulting in a total of 348 determinations and a median of nine values per patient (range 2–15). Mean and standard deviation of repeat measures of Hb F% in each individual patient were calculated. A mean ± SD of the individual coefficients of variation (SD/mean of repeat measures for each individual) was calculated. To address potential regression to the mean, we calculated peak-to-trough (maximum–minimum) ranges of repeated measures, both as an absolute difference and also as a percent of peak value per individual, and determined quartiles for the group. Absolute peak-to-trough values are probably the most clinically relevant representation of variability. Considerable intrapatient variability in Hb F levels was noted over time and is clinically meaningful, at least in the top quartile. The mean coefficient of variation was 13%. Peak-to-trough differences ranged from 0.1 to 9.8, with a median of 1.8 and a mean of 2.36. The median variability in Hb F% over time (range) expressed as a percent of peak value per individual was 26.1% (7.2–80) and is depicted in Table 1. Table 2 shows the peak-to-trough differences for the top quartile of subjects. The variability in this group has potential clinical relevance; the four subjects with the greatest variability all had peak-to-trough differences of greater than 5.0. The baseline Hb F percentage value had a statistically significant inverse correlation with percentage variability (Spearman rho −0.34, P = 0.04). There was also a statistically significant difference in peak to trough values between black and nonblack patients, with black patients showing less intra-patient variability (mean peak to trough of 1.7 versus 3.1, P = 0.01). There were 20 black patients, and 17 nonblack patients (15 white patients, one patient of multiple races, and one patient who identified as Native American/Alaskan, and Hispanic). Age and gender were not found to be significantly associated with peak to trough values (both P > 0.78). Genotype was also not significantly associated with peak to trough values between patients with Hb SS (n = 29) versus Hb S/β0 thalassemia (n = 8) (P = 0.39). There was no apparent association between genotype and race (P = 0.69). We have identified substantial variability in Hb F among patients who are not on any fetal hemoglobin inducing medication. We have also found an interesting correlation between race and Hb F variability. To our knowledge, these observations have not been previously reported. If this variability can be extrapolated to a broader population, these findings could inform estimates of the standard deviation, standard error, and power of any comparative analysis in future clinical trials of Hb F modulating agents, and larger sample sizes should be considered to detect differences between experimental and control groups. There may also be utility in obtaining several measurements and using a mean or median of these measurements (for instance, at baseline), in order to reduce intrapatient variability. Further, it may be meaningful to correlate changes in Hb F with changes in other relevant lab parameters. Limitations of the study include that this is a secondary analysis and potentially confounding variables such as change in reticulocyte count, renal function, and bone infarctions were not measured. Strengths of the study are that this was a multicenter, international cohort of patients of diverse race and country of origin, and that all Hb F analyses were performed at a single reference laboratory. In conclusion, we report a notable baseline variability in fetal hemoglobin among patients not on any Hb F inducing medications, and we believe these findings may have important implications for future studies relying on Hb F values as a measure of drug efficacy. Esther Knapp1, Hillel Cohen2, Abdullah Kutlar3, Richard Ghalie4, Deepa Manwani5* 1Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, Tennessee; 2Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, New York; 3Division of Hematology/Oncology and Stem Cell Transplantation, Department of Medicine, Sickle Cell Center, Georgia Regents University, Augusta, Georgia; 4HemaQuest Pharmaceuticals/Viracta Therapeutics, San Diego, California; 5Division of Pediatric Hematology, Oncology, and Marrow and Blood Cell Transplantation, Albert Einstein College of Medicine, Bronx, New York