This is an abbreviated presentation of the methods of the current study because the details were published prior to undertaking analyses .
Prior studies—background on selection of pharmacogenomic biomarkers for current study
To screen for genomic biomarkers, a Genome Wide Association Study (GWAS) of the PROWESS study  was performed (unpublished data) using DNA from 1446 patients to genotype approximately 1.2 million SNPs (Illumina® Human1 M-Duo BeadChip). These results were taken forward to an independent cohort of patients who had septic shock, some of whom were treated with DrotAA and some of whom were not. This small replication cohort was drawn from St. Paul’s Hospital (SPH) and the Vasopressin and Septic Shock Trial (VASST) .
The replication cohort was used to confirm two IRPs. Two-SNP composite improved response polymorphisms (IRPs), A and B, were constructed. Patients were classified as IRP A+ or − and IRP B+ or − if they had one of both of the responsive genotype. For each IRP, individual patients were considered biomarker positive if they had the responsive genotype for either of the SNPs or for both of the SNPs in the IRP. The individual SNPs in each IRP were associated with a differential DrotAA treatment effect in PROWESS (derivation cohort) and replicated in the replication cohort (unpublished).
The two SNPs comprising IRP A were chosen based first on the alignment of direction and strength of their signals by analyzing the interaction of SNP and treatment effect on mortality in both the PROWESS study and the replication cohort. Secondly, these two SNPs were chosen based on biological plausibility linking the proteins coded by these genes to pathways of sepsis or pathways regarding mechanisms of action of DrotAA. The two SNPs of IRP A are RYR2 (ryanodine receptor 2 gene) rs684923 on chromosome 1 and ACIN1 (apoptotic chromatin condensation inducer 1 gene) rs3751501 on chromosome 14. The SNP of RYR2 could act to enhance efficacy of activated protein C on protection of endothelial permeability via its effects on endothelial protein C receptor and sphingosine-1-phosphate receptor 1 (S1P). When activated protein C (APC) binds to PAR1, this triggers more conversion of sphingosine to S1P, and this could decrease the amount of sphingosine and thus disinhibit the ryanodine receptor. We also suggest that this disinhibition of the ryanodine receptor by the actions of APC varies according to the genotype of the ryanodine receptor.
Phosphorylation of a residue (S422) inACIN1 (Acinus-S variant) by AKT (prosurvival kinase) completely inhibits cleavage of Acinus-S by caspase-3, abrogating the formation of fragment p17 which is essential for chromatin condensation during apoptosis. Apoptosis is increased in some tissues and cells (lymphocytes, dendritic cells, pulmonary and gut epithelial cells) and is decreased in other tissues and cells (neutrophils) in sepsis. This gene modulates apoptosis and activated protein C has anti-apoptotic actions apoptosis so we suggest that there could be an interaction between polymorphisms of ACIN1 and response to DrotAA. More specifically, the genetic variants rs3751501 (AA|AG), associated with increased ARR (absolute risk reduction) and coding for amino acid 478 F in ACIN1, would render ACIN1 constitutively nonphosphorylated at residue 478 F and hence constitutively nonphosphorylated at S422, leading to AKT-independent regulation of chromatin condensation by Acinus-S during apoptosis, because nonphosphorylated acinus-S would be constitutively cleavable by caspase-3.
The two SNPs comprising IRP B were chosen based solely on the strength of their signals in the PROWESS and replication cohorts. These two SNPs are SPATA7 (spermatogenesis associated 7 gene) rs3179969 on chromosome 14 and FLI1 (Friend leukemia virus integration 1 gene) rs640098 on chromosome 11.
For the replication cohort, the ARR was 19.7% for IRP A + patients (95% confidence interval (CI) 2.2–37.1%), whereas for the IRP A − patients the ARR was −8.9% (95% CI −22.6–4.9%)(p = 0.018 unadjusted). The ARR was 21.2% for IRP B+ patients (95% CI 3.2–39.2%), whereas for the IRP B − patients the ARR was −5% (95% CI −18.2–8.2%)(p = 0.04 unadjusted).
The current study—overall design
This was an international, multicenter, retrospective, controlled, outcome-blinded, genotype-blinded, and matched-patients study . Retrospectively accessed DNA and clinical data were analyzed to validate the prespecified IRPs. Prospective aspects of this study were the genotyping of patients with regard to the IRPs and the statistical testing of the prespecified genotype hypothesis. Eight academic centers contributed the data and DNA from 10 cohorts (5 EU, 4 US, 1 Canada).
Study population and treatment groups
Patients included in the current study (the INDICATED population) met prespecified eligibility criteria  and DrotAA-treated patients were matched to DrotAA-free patients. Eligibility criteria (aligned with the approved use of DrotAA in the USA and EU) were used to select the primary study population (INDICATED). Such patients with high risk of death represented common practice for use of DrotAA [10,11,12,13,14]. No patient in this study was part of a prospective randomized trial of DrotAA. Patients were treated according to standard care at their sites, and data and DNA samples collected at that time were retrospectively accessed for this study. All patients were enrolled after the Food and Drug Administration (FDA) or European Medicines Agency (EMA) approval of DrotAA.
Matching DrotAA-treated to control patients
The current study incorporated a robust, well-accepted matching strategy. A propensity score of the estimated probability that a patient would have received DrotAA given their key baseline characteristics was calculated, and patients were selected as matches had to be within a prespecified tolerance on this score. Combining the use of propensity scores with covariate matching is superior to the use of either strategy alone . The intended clinical variables for the calculation of the Mahalanobis distance and the reasons why these variables were chosen were described previously .
Syreon Corporation (a clinical research organization) conducted the study. A two-phase transfer of data from each center was performed to ensure that the selection of matched control patients was blinded and unbiased. Data transfer 1 included variables to confirm eligibility and to conduct the matching. Once the matching was achieved, the matched sets of treated and control patients were “locked” together. Then data transfer 2 (outcomes and genotypic data) was sent to Syreon.
Genotyping for the IRP SNPs was done using a validated Taqman®-based analytical method and the laboratory was blinded to treatment and outcome. A 91-SNP ancestry informative marker (AIM) panel was genotyped by the GoldenGate® analytical method.
As previously noted , this study had 90% power to detect a treatment-by-IRP interaction assuming an absolute mortality reduction of 15% in the DrotAA-treated group compared to control in IRP + patients and with 1–2% difference in mortality between the treated and control groups in IRP- patients.
The primary analysis was done on the Matched-INDICATED population (comparing the effect of treatment in the IRP + and IRP- groups) by testing for the effect of the interaction between IRP and DrotAA treatment on the primary endpoint in a conditional logistic regression model, conditioning on the matching while incorporating the principal component scores from the 91-SNP AIM panel data (as covariates to control for potential population stratification). The primary endpoint was in-hospital mortality through Day 28 (i.e., patients were followed until hospital discharge or Day 28, whichever came first). Each of the primary analyses, one for IRP A and one for IRP B, was done as a two-sided test with α = 2.5% for an overall, Bonferroni-corrected, type I error rate of 5%.