Tang et al. recently conducted a systematic review of microarray-based expression profiling studies in human sepsis [19
]. One conclusion from this review is that the transcriptomic response is highly variable in human sepsis. Our previous data are consistent with this conclusion by demonstrating the potential existence of 3 subclasses of children with septic shock, as defined by variable gene expression. We have now prospectively validated that a 100-gene expression signature, depicted using visually intuitive mosaics, can be used to allocate patients with septic shock into subclasses having clinically relevant phenotypic differences and biologically relevant differential gene expression. While the subclassification strategy needs to be further refined, and one would expect that computer-based subclassification would be superior to that of clinician-based subclassification, we have nonetheless demonstrated that clinically relevant subclasses of children with septic shock can be identified via gene expression profiling.
The assertion that the subclasses have clinically relevant phenotypes is based on a higher level of illness severity in the subclass A patients, as measured by mortality, maximal number of organ failures, PRISM scores, and PICU free days. The profound negative impact of multiple organ failure on outcomes in critical illness is well established [20
], and is therefore a clinically relevant measure. The reliance on PRISM scores could lead one to conclude that it would be more straightforward to calculate PRISM scores as a means of stratification. However, illness severity scores such as PRISM and APACHE are intended for population-based predictions, rather than for individual patient stratification, and do not provide biological information [23
Patients in validation cohort subclass A had a trend toward higher mortality, which did not reach statistical significance. However, when we combined both the derivation and validation cohorts, the subclass A patients have a significantly higher mortality rate. Notably, the mortality rates of the patients in subclasses B and C are consistent with current estimates for the U.S. [24
], whereas the mortality rate in subclass A patients is 3-fold higher.
The assertion that the subclasses have biologically relevant differences in gene expression is based on the functional significance of the 100 class-defining genes, which are enriched for genes corresponding to adaptive immunity [6
]. The majority of these genes corresponding to adaptive immunity are repressed in subclass A patients, relative to subclass B and C patients [6
]. It is unlikely that lymphopenia accounts for this observation because the absolute lymphocyte counts were not significantly different between the 3 subclasses. Recent literature indicates that enhancement of adaptive immune function may be a rational therapeutic strategy in sepsis [28
]. Optimization of such a strategy, however, will need to take into account the potential existence of subclass A patients, characterized by a higher illness severity and repression of adaptive immunity-related genes.
The 100 class-defining genes are also enriched for genes corresponding to glucocorticoid receptor signaling [6
], and these genes are also repressed in subclass A patients, relative to subclass B and C patients [6
]. Glucocorticoid replacement therapy and the concept of relative adrenal insufficiency in septic shock are highly controversial topics in critical care medicine [34
]. The potential existence of a subclass of patients with septic shock (i.e. subclass A) having a higher illness severity and repression of genes corresponding glucocorticoid receptor signaling, may have important implications for future clinical trials and may have been confounders in previous clinical trials.
Subclass A patients were significantly younger than subclass B patients in both our previous study [6
] and the current study. This observation is consistent with pediatric septic shock epidemiology, which has identified young age as a risk factor for increased illness severity [24
]. However, subclass C patients are of a similar age to subclass A patients, but have a lower illness severity in both studies. Thus, while younger age is a risk factor for illness severity, the current data indicate that the expression pattern of the 100 class-defining genes also impacts illness severity, independent of age.
Our subclassification strategy is focused on a single time point and therefore does not take into account potential temporal shifts in patient status from one subclass to another. However, the major goal of the strategy is to allocate patients into subclasses at a time point that affords earlier clinical management decisions or early stratification for clinical trials [4
In conclusion, we have addressed the challenges of septic shock heterogeneity and stratification by prospectively validating the existence of septic shock subclasses based on a 100-gene expression signature. The subclasses can be identified at a clinically relevant time point and have relevant clinical differences. Finally, the expression patterns of the 100 class defining-genes may therapeutic implications.