In this study we demonstrate that a gene expression profile can distinguish patients with lacunar stroke from non-lacunar stroke. Further, when this gene expression profile is applied to patients with SDI of unclear cause (SDI>15mm and SDI<15mm with potential embolic source), both lacunar and non-lacunar causes are predicted. Given the difficulty in distinguishing lacunar from non-lacunar causes of SDI and the importance of this distinction in management, developing a reliable marker for lacunar etiology may be clinically useful.
The profile to distinguish lacunar from non-lacunar stroke has potential applications in the diagnosis of stroke cause, particularly in SDI where cause of stroke is unclear. Patients presenting with acute stroke can be classified based on their profile of gene expression in blood as either of high or low probability of being lacunar stroke. This in conjunction with clinical symptoms and imaging suggesting lacunar stroke increases a physician’s confidence in making a diagnosis of small vessel lacunar stroke as the cause of SDI. Further studies are required to determine the extent to which a gene profile can improve diagnosis, though based on the sensitivity and specificity observed in this study there is promise that a profile of differentially expressed RNA in blood can add to the ascertainment of SDI cause.
Arterial Small Deep Infarcts
Patients with arterial stenosis >50% ipsilateral to an SDI are often classified as a non-lacunar infarction 29, 30
. However, whether the arterial disease is the actual cause of the SDI or a coincidental disease occurring in a patient with symptomatic small vessel disease remains unclear 31
. Symptomatic carotid stenosis derives greater benefit from vascular intervention compared to asymptomatic carotid stenosis. Thus ascertaining whether the SDI is of lacunar or arterial etiology is of clinical significance. Furthermore, correct classification of stroke cause is important in stroke research and therapeutic development.
Carotid endarterectomy in SDI patients with carotid stenosis does improve outcomes, supporting the argument that arterial disease is a cause of some SDI 32, 33
. Other studies also suggest that arterial disease is the cause some SDI, with the degree of vascular stenosis, intima medial thickness and arterial stiffness all having been reported as predictors of non-lacunar stroke 7, 18, 29, 30, 34–42
. Additionally, Tejada et al.
reported a 7% absolute increase in ipsilateral compared to contralateral carotid stenosis in patients with SDI, suggesting carotid disease contributes to some SDI 43
. However, this finding has not been demonstrated by others 44
. Our study supports the notion that the presence of arterial disease is associated with non-lacunar infarction. Among the 32 patients with SDI of unclear cause, those predicted to have non-lacunar infarction were over five times more likely to have ipsilateral arterial disease.
Not all SDI with arterial disease were predicted to be of non-lacunar etiology. In 4 out of the 12 SDI with arterial disease, a lacunar etiology was predicted. This suggests that some patients with SDI have asymptomatic arterial disease, coincidental to infarction. There were no clinical features recorded that were significantly different between SDI with arterial disease of predicted lacunar etiology compared to those of predicted non-lacunar etiology. Further evaluation regarding the clinical significance of SDI with arterial disease predicted to be of lacunar etiology is required.
Cardioembolic Small Deep Infarcts
The presence of a cardiac source has also been suggested as a marker of non-lacunar SDI 20, 35, 38, 45–50
. In our study, there was a trend for a cardiac source to be more common in SDI of predicted non-lacunar etiology, though statistically significance was not achieved. There were two subjects with cardioembolic source predicted to have lacunar stroke, one with atrial fibrillation and the other with cardiomyopathy. This suggests that some cardiac sources are coincidental to SDI, and some are probably causal. No clinical features were significantly different between SDI with a potential cardiac source of predicted lacunar versus non-lacunar etiology, though sample size was small. Further study in larger cohorts is required.
Lacunar Small Deep Infarcts
The diagnosis of lacunar stroke was made using clinical symptoms, imaging and ancillary investigations to rule out other potential etiologies. Such features have been shown to make lacunar small vessel disease the most likely cause of a small deep infarct. This is indeed true in our study, where 22 out of 30 lacunar strokes were classified as lacunar on cross-validation analysis. However, there were 8 patients who met the criteria for lacunar stroke who were predicted to have a non-lacunar etiology based on their pattern of gene expression. Of interest none of these 8 patients had evidence of microhemorrhage on gradient echo recall MRI, whereas 6 of the 22 lacunar strokes of predicted lacunar etiology did (p=0.09). Though sample size in our study was small, the suggestion that microhemorrhages may be an important marker of lacunar stroke has previously been reported 51–53
. In future studies, more detailed analysis of small vessel disease markers including microhemorrhage, retinal imaging, blood brain barrier permeability and blood endothelial markers may provide better insight into features characteristic of lacunar stroke.
The identified differences in blood reflect immune differences between lacunar and embolic stroke, including differences in immune response to vascular risk factors. The genes identified as differentially expressed in lacunar stroke were over represented in canonical pathways involving innate and adaptive immune cell communication, TREM1 signaling, T-helper cell differentiation and immune cell signaling (). Over represented functional pathways included growth, activation and recruitment of leukocytes and myeloid cells, endothelial adhesion and angiogenesis. Specific inflammatory and/or genetic factors may predispose to endothelial damage. Indeed, others have identified markers of inflammation and endothelial dysfunction to be associated with lacunar strokes 54–56
. Further study of identified pathways as well as specific immune cell responses may provide insight into lacunar stroke pathophysiology.
This study has its limitations. Derivation of predictors from a large number of variables, as in the case of microarray analysis, increases the risk of false discoveries. A method to estimate this risk is to evaluate the predictors using cross-validation analysis and in a second test cohort, as performed in this study. The best test, however, remains validation of predictors in a second, completely independent cohort. There were differences between the groups analyzed, with the lacunar group being slightly younger, having a lower NIHSS on admission and having more non-Caucasian subjects. These are not unexpected findings of a lacunar stroke population. Though ethnicity does affect gene expression, in this study genes differentially expressed in Caucasian compared to non-Caucasian were not present in the 41 gene list reported, and the identified predictors performed equally well in the prediction of Caucasian and non-Caucasian strokes. However, further study in larger populations is required. Sample size also limited the ability to identify clinical features associated with SDI’s of predicted lacunar and non-lacunar etiology. A definitive diagnosis of lacunar stroke is challenging because it requires neuropathologic examination. Clinical diagnosis of lacunar stroke, as performed in this study, relies on probability assumptions based on clinical features, neuroimaging and ancillary investigations. However the features that characterize lacunar small vessel disease likely remain incompletely delineated. In future studies, evaluation of additional measures of vascular disease may further refine the lacunar stroke group. Retinal imaging, carotid intima media thickness, prolonged cardiac monitoring, brain imaging parameters and serum markers of endothelial function may be important to consider.
In conclusion, small deep infarcts were predicted to be of both lacunar and non-lacunar etiology. This suggests that a comprehensive workup of patients with SDI is required to identify potential cardioembolic and arterial causes. Though clinical and imaging features may distinguish most lacunar strokes, there remains a group of SDI with non-lacunar etiologies that may require different management. Whether a gene expression profile can guide diagnostic testing and treatment specific to SDI etiology requires further study, though they do show promise as a potential method to infer SDI etiology.