Serum anti‑KIAA0513 antibody as a common biomarker for mortal atherosclerotic and cancerous diseases

Numerous antibody biomarkers have been reported for cancer and atherosclerosis-related diseases. The major complications of atherosclerosis and diabetes mellitus (DM) are acute ischemic stroke (AIS), cardiovascular disease (CVD) and chronic kidney disease (CKD). Cancer development is accompanied by arterial disorders, such as angiogenesis and atherosclerosis, and DM is a risk factor for the development of certain types of cancer. Atherosclerosis-related diseases and cancers are therefore interrelated and could be detected using a common biomarker. In the present study, the initial screening using the protein array method identified KIAA0513 as an antigen recognized by serum IgG antibodies in patients with atherosclerosis. The amplified luminescent proximity homogeneous assay-linked immunosorbent assay revealed significantly higher serum antibody levels against recombinant KIAA0513 protein in patients with AIS, transient ischemic attack (TIA), DM, CVD, obstructive sleep apnea syndrome (OSAS), CKD and solid cancers, such as esophageal, gastric, colon, lung and breast cancers, compared with healthy donors. A receiver operating characteristic (ROC) analysis revealed that the highest areas under the ROC curves of anti-KIAA0513 antibodies were obtained for esophageal cancer, nephrosclerosis-type CKD and DM. Spearman's correlation analysis revealed that serum anti-KIAA0513 antibody levels were associated with maximum intima-media thickness and plaque score, which are indices of atherosclerosis and stenosis. Serum anti-KIAA0513 antibody markers appear to be useful for diagnosing AIS, TIA, DM, CVD, OSAS, CKD and solid cancers, and may reflect common arterial alterations leading to atherosclerotic and cancerous diseases.


Materials and methods
Patients and control sera.The present study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Local Ethical Review Board of the Chiba University, Graduate School of Medicine (Chiba, Japan), as well as by the review boards of the participating hospitals (approval no.2018-320).The Ethics Committee of Toho University, Graduate School of Medicine, Tokyo, Japan (No. A18103_A17052_A16035_A16001_26095_25024_24038_22 047_22047) and Port Square Kashiwado Clinic, Kashiwado Memorial Foundation, China, Japan (approval no.2012-001) also approved the study protocol.Sera were collected from patients who had provided written informed consent.Each serum sample was centrifuged at 3,000 x g for 10 min at 4˚C, and the supernatant was stored at -80˚C until use.
Serum samples collected from patients with AIS, transient ischemic attack (TIA), asymptomatic ischemic stroke (Asympt-CI), chronic-phase cerebral infarction (cCI), and deep and subcortical white matter hyperintensity (DSWMH) were obtained from Chiba Prefectural Sawara Hospital.The stroke subtypes were determined according to the criteria of the Trial of Org 10172 in the Acute Stroke Treatment classification system (46), and large-artery atherosclerosis and small-artery occlusion (lacune) were included as AIS and ischemic stroke.Samples from patients with DM, CVD and OSAS were obtained from the Chiba University Hospital.CVD included acute myocardial infarction (AMI) and unstable angina.The serum samples of patients with AIS, TIA and AMI were obtained within 2 weeks following disease onset.Samples collected from patients with CKD were obtained from the Kumamoto cohort (47,48), whereas those collected from patients with EC, GC, CRC, lung cancer (LC), and breast cancer (BC) were obtained from the Department of Surgery, Toho University Hospital.Serum samples from healthy donors (HDs) were obtained from Chiba University, Port Square Kashiwado Clinic, the National Hospital Organization, Shimoshizu Hospital (Yotsukaido, Japan), and Chiba Prefectural Sawara Hospital (Katori, Japan).For comparisons with TIA and AIS, serum samples from HDs were selected from patients who exhibited no abnormalities in cranial magnetic resonance imaging.
ProtoArray ® screening.The initial screening was performed using ProtoArray ® Human Protein Microarrays v4.0 (Thermo Fisher Scientific, Inc.), which were loaded with 9,480 species of proteins, as previously described (11,14,15).In total, 20 serum samples (10 each from the patients and HDs) were employed to detect antigens specifically recognized by IgG antibodies in the patient sera.The complete results from the ProtoArray ® screening are presented in Table SI.
Nested case-control study.A nested case-cohort study was conducted using the aforementioned AlphaLISA detection antibody levels.The present study was nested within the Japan Public Health Center-based Prospective Study (50-52), which involved ~30,000 Japanese individuals aged 40-69 years at a baseline period from 1990-1994 whose plasma samples were stored.The plasma samples employed were from 202 cases of incidental AIS in the cohort that occurred between baseline and 2008 and from 202 controls whose age (within 2 years), sex, date of blood sampling (within 3 months), time since last meal (within 4 h), and study location (Public Health Center area) were matched with those of the cases.A conditional logistic regression model was used to estimate the odds ratios (ORs) and 95% confidence intervals (95% CIs).The study participants were informed of the objectives and methods of the study, and those who answered the questionnaire and donated blood were regarded as having given informed consent to participate.
Statistical analysis.The Mann-Whitney U test was employed to determine the significant differences between two groups and the Kruskal-Wallis test (with the Bonferroni correction applied) was used to evaluate the differences among ≥3 groups.Correlations were analyzed using Spearman's correlation analysis and logistic regression analysis.All the statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc.).The predictive values of the putative disease markers were assessed via a receiver operating characteristic (ROC) curve analysis and determined the sensitivity and specificity.Patient survival was evaluated using the Kaplan-Meier method and compared using the log-rank test.X-tile 3.6.1 software (Yale University, New Haven, CT, USA) (53) was used to determine the optimal cut-off values for discrimination of the survival rates between antibody positive and negative groups.All tests were two-tailed, and P-values <0.05 were considered to indicate statistically significant differences.

Recognition of KIAA0513 by serum components from patients with atherosclerosis.
The present study employed a ProtoArray loaded with 9,480 protein species to identify the antigens recognized by antibodies in the sera of patients with atherosclerosis.It was found that KIAA0513 isoform c (Accession no.BC030280.1)reacted with 6 of the 10 serum samples from the patients with atherosclerosis, and with only 1 of the 10 samples from the HDs (Table SI).Subsequently, GST-fused full-length KIAA0513 protein was expressed in E. coli and purified by affinity-chromatography.
Presence of autoantibodies against KIAA0513 in the sera of patients with AIS, TIA, DM, EC, or CC.The present study examined the presence of autoantibodies against KIAA0513 in sera using western blot analysis (Fig. 1).GST-KIAA0513 protein reacted with commercial anti-GST and anti-KIAA0513 antibodies, whereas the control, GST, reacted with anti-GST, but not with anti-KIAA0513 antibodies.GST-KIAA0513 protein was also recognized by serum IgG antibodies in the patients with AIS (anonymization nos.#07065 and #070684), TIA (#02337), DM (#22226), EC (#EC-6) and CRC (#Co-58), but not in the HDs (#09101).GST alone exhibited no apparent reaction with any serum from the patients or HDs.
Elevation of s-KIAA0513-Ab levels in the patients with AIS and TIA.The s-KIAA0513-Ab levels were then examined in the patients with AIS or TIA.Sera from HDs, and patients with AIS and TIA were obtained from the Chiba Prefectural Sawara Hospital.The results of AlphaLISA revealed that the s-KIAA0513-Ab levels were significantly higher in the patients with AIS or TIA than in the HDs (Fig. 2A).Using the cut-off values of the average plus two standard deviations (SDs) of the HD values, the s-KIAA0513-Ab positivity rates for the HDs, patients with AIS, and those with TIA were 0.0, 7.6 and 15.6%, respectively (Table SII).ROC analysis revealed that the areas under the ROC curves (AUCs) of s-KIAA0513-Abs were 0.6439 (95% CI, 0.587-0.700)for AIS (Fig. 3A) and 0.6604 (95% CI, 0.563-0.758)for TIA (Fig. 3B).Thus, TIA (which can be a prodromal stage of AIS) and AIS were equally associated with the s-KIAA0513-Ab marker.

Elevation of serum antibody levels against KIAA0513
in patients with DM.The present study then examined the s-KIAA0513-Ab levels in patients with DM.Serum samples from HDs and patients with DM were obtained from Chiba University and Chiba University Hospital.The s-KIAA0513-Ab levels were significantly higher in the samples from the patients with DM than in those from HDs (Fig. 2B).At a cut-off value equivalent to the average plus two SDs of the HD specimen values, the positive rates of s-KIAA0513-Abs in the HDs and patients with DM were 2.5 and 26.5%, respectively (Table SIII).ROC analysis was performed to evaluate the ability of these antibody markers to indicate the presence of DM.The AUC for s-KIAA0513-Abs was 0.736, yielding a sensitivity and specificity of 50.55 and 87.65%, respectively (Fig. 3C).
Association between s-KIAA0513-Ab levels and CVD and OSAS.Subsequently, the antibody levels in serum samples from patients with CVD obtained from Chiba University Hospital were examined.Given that OSAS is related to atherosclerosis and is associated with a high risk of AIS and CVD (36)(37)(38)(39), the present study also examined the sera of patients with OSAS obtained from Chiba University Hospital.Compared with those of the HDs, the s-KIAA0513-Ab levels were significantly higher in the patients with CVD or OSAS (Fig. 2C), although the positive rates in the patients with CVD and those with OSAS were not markedly high (10.3 and 11.6%, respectively) (Table SIV).ROC analysis revealed that the AUCs for CVD and OSAS were 0.649 (95% CI, 0.582-0.716)(Fig. 3D) and 0.646 (95% CI, 0.562-0.731)(Fig. 3E), respectively.Compared with the low P-value (<0.001) of s-KIAA0513-Ab for CVD, the P-value for OSAS was <0.01 (Table SIV), suggesting a weaker association of the s-KIAA0513-Ab marker with OSAS than with CVD.

Elevation of s-KIAA0513-Ab levels in patients with CKD.
The present study then examined the antibody levels in the sera of patients with CKD, which is also closely related to atherosclerosis.CKD was divided into three groups as follows: Type 1, diabetic kidney disease; type 2, nephrosclerosis; and type 3, glomerulonephritis.Samples from patients with CKD were obtained from the Kumamoto cohort, and samples from HDs were obtained from Chiba University.Patients from all three CKD groups had significantly higher s-KIAA0513-Ab levels than the HDs (Fig. 2D).The s-KIAA0513-Ab positivity rates in the HDs and patients with types 1, 2 and 3 CKD were 6.1, 29.0, 37.5 and 20.3%, respectively (Table SV), indicating that the highest positive rate was observed in the patients with type 2 CKD.ROC analysis revealed s-KIAA0513-Ab AUCs as high as 0.7434 (95% CI, 0.678-0.809)for type 1 CKD (Fig. 3F), 0.808 (95% CI, 0.726-0.890)for type 2 CKD (Fig. 3G) and 0.691 (95% CI, 0.618-0.764)for type 3 CKD (Fig. 3H).
s-KIAA0513-Ab levels in solid cancer.Given that atherosclerotic diseases are frequently related to cancer with certain common biomarkers being reported (20), the present study examined the serum samples from patients with EC, GC, CRC, LC and BC obtained from Toho University Hospital.The s-KIAA0513-Ab levels were significantly higher in the samples from all patients with cancer than in those from the HDs (Fig. 2E and Table SVI).The highest average value and positive rate of s-KIAA0513-Ab levels were observed for EC.Similarly, the AUC values were highest for EC (0.830), but lowest for BC among the cancers examined (Fig. 3I-M).
The present study then examined whether the s-KIAA0513-Ab levels are related to the post-operative survival of patients with EC or GC.The s-KIAA0513-Ab levels were divided into the positive and negative groups using the cut-off values obtained using X-tile software (53).The s-KIAA-Ab-positive group presented a more unfavorable prognosis than the negative group in all of EC and CRC (Fig. 4A and B).The X-tile-determined cut-off values are best ones to distinguish the favorable and poor survivals.The cut-off value of EC samples (180,487) was much higher than the average (87,535) (Table SVI), whereas that of CRC (56,879) was lower than the average of CRC (69,308) (Table SVI).Thus, the considerably high levels of s-KIAA-Abs in patients with EC and the moderately high levels in patients with CRC were associated with the prognosis.
The expression levels of KIAA0513 antigenic protein in EC tissues were examined using immunohistochemical staining.A representative example of the staining is illustrated in Fig. 4C.EC tissues were heavily stained by anti-KIAA0513 antibody, whereas surrounding healthy esophageal tissues were not.KIAA0513 protein was localized in the cytoplasm, which is consistent with the findings in a previous study (54).Thus, the high KIAA0513 expression levels may account for some, if not all, of the development of serum KIAA0513-Abs.
Association analysis.An analysis of the association analysis between the s-KIAA0513-Ab levels and participant data was performed using 665 specimens from Chiba Prefectural Sawara Hospital, including 139 specimens from HDs, 225 from patients with AIS, 44 from patients with TIA, 17 from patients with Asympt-CI, 122 from patients with DSWMH, 59 from patients with cCI and 41 from disease controls.The remaining 18 subjects were excluded because they did not have disease information.In this analysis, the Mann-Whitney U test we employed to compare the s-KIAA0513-Ab levels between the male and female participants, with or without DM, hypertension, CVD, dyslipidemia and obesity [body mass index (BMI) ≥25] and with or without smoking and alcohol intake habits.A significant difference in the s-KIAA0513-Ab levels was observed only between the patients with hypertension and those without hypertension (Table I).
Correlation analysis.Spearman's correlation analysis was performed a to determine the correlation between the s-KIAA0513-Ab levels and the continuous variables of participant parameters, including general information such as age, height, weight and BMI; the degree of artery stenosis, such as the maximum intima-media thickness (max-IMT); lifestyle factors such as smoking duration (years) and alcohol intake frequency (times/week); and blood test data.The average values of these parameters are listed in Table SVII.There was a significant correlation between the s-KIAA013-Ab levels and age, max-IMT, alkaline phosphatase, potassium, C-reactive protein (CRP), blood sugar and smoking duration (Table II), and an inverse correlation with height and weight.The correlation with max-IMT suggests that the s-KIAA0513-Ab levels are associated with stenosis and atherosclerosis, which was further confirmed by using other cohorts.Spearman's correlation analysis of the CKD cohort (300 participants) revealed a significant correlation with plaque score, max-IMT (55,56) and cardio-ankle vascular index (CAVI) (right) (57) (Table SVIII), which are indices of atherosclerosis.CRP was also associated with s-KIAA0513-Abs in the CKD cohort, suggesting the involvement of inflammation.By contrast, age, height, weight, BMI and potassium levels exhibited no significant correlation with s-KIAA0513-Abs in the CKD cohort.AIS is closely related to age, which may be indirectly associated with s-KIAA0513-Abs.
Japan Public Health Center (JPHC) cohort analysis.A casecontrol study nested within the Japan Public Health Center-based Prospective Study was then conducted, which involved ~30,000 plasma samples (50)(51)(52).The level of antibodies against the KIAA0513 protein was positively associated with the risk of AIS.The ORs (95% CIs) were 2.11 (1.17-3.81)and 2.23 (1.18-4.21)for those in the third and highest quartiles of antibody levels, respectively, compared with those in the lowest quartile (Table III).These results indicate that the antibody markers against the KIAA0513 protein are useful for predicting the onset of AIS.

Discussion
In the present study, the initial ProtoArray screening identified KIAA0513 as an antigen, as recognized by serum IgG in patients with atherosclerosis, and subsequently recombinant GST-tagged KIAA0513 protein of 301 amino acids was purified.Western blot analysis confirmed the presence of autoantibodies against KIAA0513 (Fig. 1).Using the KIAA0513 protein as an antigen, the serum antibody levels were examined using AlphaLISA.The results revealed significantly higher s-KIAA0513-Ab Table I.Association analysis of antibody levels against KIAA0513 protein with data of subjects in the Sawara Hospital cohort.

Sex
Average The subjects were divided as follows: Sex (male and female); presence (+) or absence (-) of complication of DM, hypertension, CVD, or dyslipidemia, and lifestyle factors (smoking and alcohol intake habits, and obesity).Antibody levels (Alpha counts) were compared using the Mann-Whitney U test.Sample numbers, averages and SDs of counts, as well as P-values are shown.Significant associations (P<0.05) are indicated in bold font.BMI, body mass index; DM, diabetes mellitus; CVD, cardiovascular disease.levels in the patients with AIS, TIA, DM, CVD, OSAS, CKD, EC, GC, CC, LC and MC than in the HDs (Fig. 2A-E and Tables SII-VI).Among these diseases, the highest AUC values were observed for EC, type 2 CKD and DM (Fig. 3A-M).
The close association between s-KIAA0513-Ab levels and hypertension (Table I) could account for the association with OSAS, which is frequently accompanied by hypertension (58).
Table II.Correlation analysis of serum antibody levels against KIAA0513 with data on subjects in the Sawara Hospital cohort.
There are three known splicing variants of KIAA0513: Isoform a (411 amino acids, NP_055547.1),isoform b (301 amino acids, NP_001273495.1)and isoform c (301 amino acids, NP_001284695.1).The full-length 301 amino acids of KIAA0513 isoforms c and b are exactly the same as the first 301 amino acids of KIAA0513 isoform a.The present study also purified GST-fused KIAA0513 isoform a and examined the serum antibodies using sera from HDs and patients with AIS and CVD.Both isoforms a and c of KIAA0513 exhibited higher antibody levels in the sera from patients with AIS or CVD than in the sera from HDs (Fig. S1A and B).The reactivity of KIAA0513 isoform c against serum antibodies was closely associated with that of KIAA0513 isoform a, although the former was higher than the latter (Fig. S1C), implying that the major epitope sites of serum autoantibodies are located in the 301 amino acids of isoform c.
KIAA0513 mRNA expression has been observed predominantly in the neurons and glial cells of the brain, with low-level expression in most human tissues, whereas the KIAA0513 protein was exclusively found in the brain (54).Among brain regions, the highest expression was in the cerebellum, cortex, hippocampus, pons, putamen and amygdala.Using a yeast 2-hybrid analysis of a fetal brain cDNA library, Lauriat et al (54) found that the N-terminal portion of KIAA0513 interacted with KIBRA, HAX1 and INTS4.A coimmunoprecipitation analysis revealed a physical association between KIAA0513 and KIBRA.Given that KIBRA, HAX1 and INTS4 are involved in synaptic and apoptotic signaling, KIAA0513 can also participate in these signaling pathways.
In addition to the KIAA0513-Abs employed in the present study, autoantibodies against ATPase, Ca ++ transporting, plasma membrane 4, bone morphogenetic protein 1, deoxyhypusine synthase, low-density lipoprotein receptor-related protein-associated protein 1 and additional sex combs-like 2, which are markers of atherosclerosis, were also elevated in the sera of patients with EC (13,14,17,18), indicating that arterial abnormalities can also affect the carcinogenic process.In fact, angiogenesis is essential for the development of solid tumors (62), and diabetes and obesity, which induce arteriosclerosis, are risk factors for CRC and EC (63)(64)(65).Given that all tissues and organs require oxygen and nutrition provided by arteries, the alteration of arterial structure and/or function can affect numerous tissues and organs.All tissues and organs present in a body can affect each other to a certain degree (66).In other words, the AIS, CVD and CKD caused by atherosclerosis, the atherosclerosis induced by DM, and the solid cancer caused by arterial lesions can be interrelated with each other via arterial abnormalities.Markers associated with such abnormalities could therefore detect all of the above disorders.
Cancer, heart disease, cerebrovascular disease and renal failure are the first, second, fourth and eighth leading causes of mortality in Japan, respectively (Ministry of Health, Labor and Welfare 2018 vital statistics; https://www.mhlw.go.jp/toukei/saikin/hw/jinkou/kakutei18/dl/10_h6.pdf).The majority of the other causes of death are unavoidable, such as senility and accidents.In other words, the onset and progression of cancer, heart disease, cerebrovascular disease and renal failure (as well as their risk factor DM) can be suppressed by proper health management, such as early diagnosis and intervention.Notably, cancer, heart disease, cerebrovascular disease, renal failure and DM can be detected by the s-KIAA0513-Ab marker, making it applicable for diagnostic purposes and providing appropriate treatment, lifestyle guidance, etc., leading to improved quality of life.
As of 2020, numerous reports have shown that the presence of underlying diseases, such as DM, heart disease, cerebrovascular disease, cancer, OSAS and kidney disease aggravate the coronavirus disease 2019 (COVID-19) (67)(68)(69)(70).The s-KIAA0513-Ab marker is therefore a highly useful tool for detecting patients with COVID-19 who are at a higher risk of mortality.Antibody markers are generally more sensitive than antigen markers.Given that the KIAA0513 protein has particularly high antigenicity, this KIAA0513-Ab marker is extremely sensitive.Given the major life-threatening diseases can be detected by this marker, the KIAA0513-Ab marker could be referred to as a 'supermarker'.
The present study has certain limitations, which should be mentioned.First, although the increase in s-KIAA0513-Ab levels could be attributable to the high KIAA0513 expression levels (Fig. 4C) as suggested above, the association between the expression of the antigen and the antibody has not been completely verified.The antigen levels can be examined by immunohistochemistry, western blot analysis and mass spectrometry.However, accurately quantifying protein amounts across many specimens using these methods is still challenging.The introduction of the AlphaLISA method for the quantification of antigenic proteins may be another approach.Second, the significant differences of the prognosis between the s-KIAA-Ab-positive and -negative groups were observed in EC and CRC (Fig. 4A and B) but not GC, LC, or BC.Further accumulation of the latter specimens may clarify the association between s-KIAA0513-Ab levels and their prognosis.
In conclusion, the serum anti-KIAA0513 antibody marker appears to be useful for diagnosing the progress of atherosclerosis, which can lead to the onset of life-threatening AIS, CVD and cancer.

Figure 2 .
Figure 2. Comparison of serum anti-KIAA0513 antibody (s-KIAA0513-Ab) levels between HDs and patients.The s-KIAA0513-Ab levels of HDs and patients with (A) AIS or TIA, (B) DM, (C) CVD or OSAS, (D) CKD, and (E) EC, GC, CRC, LC, or BC were examined by AlphaLISA using GST-KIAA0513 2-302 protein as the antigen, followed by subtraction of the levels against control GST.Scatter dot plots of the s-KIAA0513-Ab levels are shown.The bars represent the average and average ± SD.P-values were calculated using the Mann-Whitney U test to analyze the differences between two groups, and the Kruskal-Wallis test (with the Bonferroni correction applied) to evaluate the differences among ≥3 groups.** P<0.01 and *** P<0.001 vs. HD specimens.Type-1, type-2, and type-3 CKDs represent diabetic kidney disease, nephrosclerosis, and glomerulonephritis, respectively.The total (male/female) numbers, average ages ± SDs, average antibody levels ± SDs, cut-off values, positive numbers, positive rates (%), and P-values vs. HDs are summarized and shown in Tables SII-VI.HD, healthy donors; AIS, acute ischemic stroke; TIA, transient ischemic attack; DM, diabetes mellitus; EC, esophageal cancer; CVD, cardiovascular disease; OSAS, obstructive sleep apnea syndrome; CKD, chronic kidney disease; GC, gastric cancer; LC, lung cancer; BC, breast cancer; SD, standard deviation.

Figure 4 .
Figure 4. Survival analysis and immunohistochemical staining of cancer specimens.Comparison of overall survivals of the patients with (A) EC and (B) CRC according to s-KIAA0513-Ab-positive (s-KIAA0513-Ab +) and negative (s-KIAA0513-Ab -) groups.Cut-off values of EC and CRC were determined as 180,487 and 56,879, respectively, using X-tile analysis.Statistical analyses were performed using the log-rank test.(C) Surgically resected EC tissue was stained using anti-KIAA0513 antibody (rabbit polyclonal antibodies, Atlas Antibodies).EC and N represent cancerous and normal cells, respectively.EC, esophageal cancer; CRC, colorectal cancer.

Table III .
Odds ratios of incident KIAA0513-Abs vs. AIS.