US 2017.0029891A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0029891 A1 SHARP et al. (43) Pub. Date: Feb. 2, 2017 (54) BOMARKERS FOR DAGNOSING (60) Provisional application No. 61/584.232, filed on Jan. SCHEMA 7, 2012. (71) Applicant: The Regents of the University of California, Oakland, CA (US) Publication Classification (51) Int. C. (72) Inventors: Frank SHARP, Davis, CA (US); Glen CI2O I/68 (2006.01) C. JICKLING, Sacramento, CA (US) (52) U.S. C. (73) Assignee: The Regents of the University of CPC ....... CI2O 1/6883 (2013.01); C12O 2600/158 California, Oakland, CA (US) (2013.01); C12O 2600/118 (2013.01) (21) Appl. No.: 15/226,844 (57) ABSTRACT (22) Filed: Aug. 2, 2016 The present invention provides methods and compositions for diagnosing and predicting the occurrence of ischemia. Related U.S. Application Data For example, the present invention provides methods and (63) Continuation of application No. 14/370.709, filed on compositions for diagnosing and predicting the risk and Jul. 3, 2014, now Pat. No. 9,410,204, filed as appli cause of transient neurological events (TNE) as ischemic or cation No. PCT/US2013/020240 on Jan. 4, 2013. non-ischemic. Patent Application Publication Feb. 2, 2017. Sheet 1 of 3 US 2017/0029891 A1 TA (n=263 WS schemic Stroke (n=94) VRF Contro ra.) vs WRF Control (n=44) Fig. 1 A. Derivation Cross-Walidation 26 Gene 10-fold leave-one-out DA Prediction Model TA-DW+f Minor Stroke (n=17) Fia. 1 B. Validation g Nonischemic TNE {n=13) Fig. 1 C. Prediction Unclear TNE (n=14) Fig. 1A-C Patent Application Publication Feb. 2, 2017. Sheet 2 of 3 US 2017/0029891 A1 lschemic Stroke TA CD4 2% Monocytes 22% Granulocytes 64% Granulocytes 38% Fig. 2 Patent Application Publication Feb. 2, 2017. Sheet 3 of 3 US 2017/0029891 A1 --Control Probability -Crischemia Probability s s s 2. 25 so Fig. 3B ar ischemic "“” Stroke 3.25v.au :---------------------------------------------------------------------------------------------c. goo &x3xxxx8 s 2. 3. a. ESO Fig. 3C 1.00 is 0.7s 3 Control Q.50 : - .2: US 2017/0029891 A1 Feb. 2, 2017 BIOMARKERS FOR DIAGNOSING (Rothwell, et al., Lancet. (2007) 370:1432-1442). Addition ISCHEMA ally, identifying nonischemic TNE (which have a very low risk of stroke) will improve risk stratification and use of CROSS-REFERENCE TO RELATED health care resources. Thus, improved methods to distin APPLICATIONS guish ischemic from nonischemic causes of TNE are needed. 0001. This application is a continuation of U.S. applica 0006 Previous studies have demonstrated an immune tion Ser. No. 14/370,709, filed on Jul. 3, 2014, issued as U.S. response in patients with stroke by evaluating leukocyte Pat. No. 9,410,204 on Aug. 9, 2016, which is a U.S. national RNA expression using whole genome microarrays (Barr, et phase under 35 U.S.C. S371 of International Application No. al., Neurology. (2010) 75:1009-1014; Jickling, et al. Ann PCT/US2013/020240, filed on Jan. 4, 2013, which claims Neurol. (2010) 68:681-692; and Stamova, et al., Stroke. the benefit under 35 U.S.C. S 119(e) of U.S. Provisional (2010) 41:2171-2177). However, the distinction between Application No. 61/584.232, filed on Jan. 7, 2012, which are immune response to cerebral ischemia and cerebral infarc hereby incorporated herein by reference in their entireties tion was unclear. We report herein the pathways associated for all purposes. with the peripheral immune response to cerebral ischemia, which has clinical utility to identify ischemic causes of TNE. STATEMENT OF GOVERNMENTAL SUPPORT SUMMARY OF THE INVENTION 0002 This invention was made with government support under Grant No. NS056302 awarded by the National Insti 0007. The present invention provides biomarkers useful tutes of Health. The government has certain rights in the for diagnosing the occurrence or risk of ischemia, particu invention. larly transient cerebral ischemia, and for differentiating a transient neurological event as ischemic or non-ischemic. FIELD OF THE INVENTION Accordingly, in one aspect, the invention provides methods for diagnosing transient cerebral ischemia or a predisposi 0003. The present invention provides methods and com tion for experiencing transient cerebral ischemia. In some positions for diagnosing and predicting the occurrence of embodiments, the methods comprise: determining a level of ischemia. For example, the present invention provides meth expression of a plurality of ischemia-associated biomarkers ods and compositions for diagnosing and predicting the risk in a biological sample from a patient, wherein an increase or and cause of transient neurological events (TNE) as isch decrease of the level of expression compared to a control is emic or non-ischemic. correlative with or indicates that the patient has suffered or is at risk of experiencing transient cerebral ischemia, BACKGROUND OF THE INVENTION wherein the plurality of ischemia-associated biomarkers is 0004 Systemic inflammation is linked to ischemic stroke selected from the biomarkers set forth in Tables 3 and 5, and transient ischemic attack (TIA). Cerebral ischemia pro thereby diagnosing transient cerebral ischemia or a predis duces many endogenous ligands and cytokines that elicit an position for experiencing transient cerebral ischemia. immune response (Arumugam, et al., Shock. (2009) 32:4- 10008. In another aspect, the invention provides methods 16). Leukocytes, including neutrophils and monocytes, are for diagnosing and/or differentiating a transient neurological activated and recruited to initiate processes of containment, event (TNE) as ischemic or non-ischemic. In some embodi removal, and repair. We have previously demonstrated in a ments, the methods comprise: determining a level of expres rat model of TIA that a peripheral immune response occurs Sion of a plurality of ischemia-associated biomarkers in a to transient brain ischemia with similarities to that observed biological sample from a patient, wherein an increase or in experimental brain infarction (Zhan, et al., J. Cereb Blood decrease of the level of expression compared to a control is Flow Metab. (2010) 30:110-118; Zhan, et al., Brain Res. correlative with or indicates that the patient has suffered or (2008) 1234:183-197). This suggests that aspects of the is at risk of experiencing transient cerebral ischemia, immune response to cerebral ischemia in TIA and stroke are wherein the plurality of ischemia-associated biomarkers is common and may be useful in identifying ischemic events. selected from the biomarkers set forth in Tables 3 and 5, 0005. In clinical practice, deciphering whether a transient thereby diagnosing a transient neurological event (TNE) as neurological event (TNE) is of ischemic or nonischemic ischemic or non-ischemic. etiology is often difficult. Many common neurological con 0009. In a related aspect, the invention provides methods ditions mimic the symptoms of TIA, including migraine, for identifying the occurrence or a predisposition for expe seizure, and syncope. The transient nature of TIA adds to the riencing ischemia. In some embodiments, the methods com diagnostic challenge as objective deficits generally resolve prise: determining a level of expression of a plurality of by the time of presentation and assessment of symptoms is ischemia-associated biomarkers in a biological sample from reliant on patient recall and physician interpretation. Current a patient, wherein an increase or decrease of the level of diagnostic tests including neuroimaging, electrocardiogram, expression compared to a control is correlative with or and electroencephalogram are frequently unremarkable, indicates that the patient has suffered or is at risk of leaving the cause of a TNE unclear. As a result, TIA is experiencing ischemia, wherein the plurality of ischemia estimated to be incorrectly diagnosed in as many as 50% of associated biomarkers is selected from the biomarkers set cases (Ferro, et al., Stroke. (1996) 27:2225-2229; Kraai forth in Tables 3 and 5, thereby identifying the occurrence or jeveld, et al., Stroke. (1984) 15:723-725; Castle, et al., a predisposition for experiencing ischemia. In some embodi Stroke. (2010) 41: 1367-1370; Bos, et al., JAMA. (2007) ments, the ischemia is selected from the group consisting of 298:2877-2885; and Johnston S. C.JAMA. (2007)298:2912 cerebral ischemia, myocardial ischemia and pulmonary isch 2913). However, correctly identifying ischemic TNE is emia. critical as the risk for stroke is high in TIA and can be 10010 With respect to embodiments of the methods, in reduced by early initiation of stroke prevention therapy Some embodiments, an increased expression level of one or US 2017/0029891 A1 Feb. 2, 2017 more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all) decreased expression level of one or more (e.g., 2, 3, 4, 5, ischemia-associated biomarkers selected from the group 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or all) consisting of UBE2J1, ELAVL3, FCGR2B, BLVRA, ischemia-associated biomarkers selected from the group JMJD6, DDAH2, PTRH2, CARD16, CAV 1, ZNF608, consisting of CARD16, IRF7, TLR6, NMU, C13orf16, NDUFB3, SLC22A4, PCMT1, CACNA1A, CASP1 and TAPBP. BTC, ZBP1, HSPA6, TWIST1, PLSCR1, LOC100129105 indicates that the patient has suffered or is SAMD9L, OSTCL, C90rf66, GYPA, ADM, ANKRD22, at risk of experiencing ischemia. In some embodiments, an SHOX, ZNF354A, SRGAP1, GRM5, BAGE, XRCC4, increased expression level of one or more (e.g., 2, 3, 4, 5, 6, SLC37A3, OVOL2, LIFR, RASAL2, hCG 1749898, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or all) IQGAP3, HS3ST3A1, NPR3, SIX3 and HCN1 indicates ischemia-associated biomarkers selected from the group that the patient has not suffered or is not at risk of experi consisting of AIM2, C14orf101, DNAH17, UBE2J1, encing ischemia. In some embodiments, a decreased expres LOC203274 PGS1, ZEB2.