Otitis media/interna and encephalitozoonosis are the most common causes of head tilt in pet rabbits in the UK: 73 cases (2009‒2020).

BACKGROUND: There are limited studies that identify diseases associated with head tilt in pet rabbits. METHODS: This was an observational, retrospective, single-centre study of rabbits with head tilt presented between 2009 and 2020. Descriptive statistics were performed for all cases, whereas univariate and multivariate analyses were only performed for the 36 cases with a final diagnosis. RESULTS: Seventy-three rabbits met the inclusion criteria. The final diagnoses included Encephalitozoon cuniculi meningoencephalomyelitis (EC) (15/36; 41.7%), otitis media/interna (OMI) (8/36; 22.2%) and concurrent EC and OMI (13/38; 36.1%). Subacute-to-chronic onset was more common in rabbits with OMI than in those with EC (p = 0.018). Previous middle ear surgery (p = 0.046) and a diagnosis of otitis externa (p = 0.004) significantly increased the risk of OMI. Meloxicam was associated with improvement of clinical signs (p = 0.007). Upright ears (p = 0.013), recumbency (p = 0.037) and impaired mentation (p = 0.001) were associated with a higher risk of death/euthanasia. The proportions of residual head tilt (66.7%) and relapse of vestibular signs (42.1%) were high. LIMITATIONS: This was a retrospective study with cases varying in their investigation and conclusive final diagnoses. CONCLUSION: OMI and EC were the most common aetiologies of head tilt in pet rabbits in the UK. Meloxicam might be associated with a favourable outcome in affected rabbits. Paired EC serology and a CT scan of the head should be the baseline investigation for head tilt in rabbits.

Clinical signs, causes, and outcome of central cord syndrome in 22 cats.

OBJECTIVE: To describe the signalment, clinical findings, presumptive or definitive diagnosis, and outcome in cats with central cord syndrome (CCS). ANIMALS: 22 cats. CLINICAL PRESENTATION: Cats evaluated for CCS at 7 referral hospitals between 2017 and 2021 were included. Information retrieved from medical records included signalment, physical and neurological examination findings, diagnostic investigations, definitive or presumptive diagnosis, treatment, and follow-up. RESULTS: Median age at presentation was 9 years. Two neuroanatomical localizations were associated with CCS: C1-C5 spinal cord segments in 17 (77.3%) cats and C6-T2 spinal cord segments in 5 (22.7%) cats. Neuroanatomical localization did not correlate with lesion location on MRI in 8 (36.3%) cats. The most common lesion location within the vertebral column was over the C2 and C4 vertebral bodies in 6 (27.2%) and 5 (22.7%) cats, respectively. Peracute clinical signs were observed in 11 (50%) cats, acute in 1 (4.5%), subacute in 4 (18%), and chronic and progressive signs were seen in 6 (40.9%) cats. The most common peracute condition was ischemic myelopathy in 8 (36.3%) cats, whereas neoplasia was the most frequently identified chronic etiology occurring in 5 (22.7%) cats. Outcome was poor in 13 (59%) cats, consisting of 4 of 11 (36.6%) of the peracute cases, 3 of 4 (75%) of the subacute cases, and 6 of 6 of the chronic cases. CLINICAL RELEVANCE: Central cord syndrome can occur in cats with lesions in the C1-C5 and C6-T2 spinal cord segments. Multiple etiologies can cause CCS, most commonly, ischemic myelopathy and neoplasia. Prognosis depends on the etiology and onset of clinical signs.

Early-Stage Breast Cancer Detection in Breast Milk.

Breast cancer occurring during pregnancy (PrBC) and postpartum (PPBC) is usually diagnosed at more advanced stages compared with other breast cancer, worsening its prognosis. PPBC is particularly aggressive, with increased metastatic risk and mortality. Thus, effective screening methods to detect early PrBC and PPBC are needed. We report for the first time that cell-free tumor DNA (ctDNA) is present in breast milk (BM) collected from patients with breast cancer. Analysis of ctDNA from BM detects tumor variants in 87% of the cases by droplet digital PCR, while variants remain undetected in 92% of matched plasma samples. Retrospective next-generation sequencing analysis in BM ctDNA recapitulates tumor variants, with an overall clinical sensitivity of 71.4% and specificity of 100%. In two cases, ctDNA was detectable in BM collected 18 and 6 months prior to standard diagnosis. Our results open up the potential use of BM as a new source for liquid biopsy for PPBC detection. SIGNIFICANCE: For the first time, we show that BM obtained from patients with breast cancer carries ctDNA, surpassing plasma-based liquid biopsy for detection and molecular profiling of early-stage breast cancer, even prior to diagnosis by image. See related commentary by Cunningham and Turner, p. 2125. This article is featured in Selected Articles from This Issue, p. 2109.

Circulating tumor DNA reveals complex biological features with clinical relevance in metastatic breast cancer.

Liquid biopsy has proven valuable in identifying individual genetic alterations; however, the ability of plasma ctDNA to capture complex tumor phenotypes with clinical value is unknown. To address this question, we have performed 0.5X shallow whole-genome sequencing in plasma from 459 patients with metastatic breast cancer, including 245 patients treated with endocrine therapy and a CDK4/6 inhibitor (ET + CDK4/6i) from 2 independent cohorts. We demonstrate that machine learning multi-gene signatures, obtained from ctDNA, identify complex biological features, including measures of tumor proliferation and estrogen receptor signaling, similar to what is accomplished using direct tumor tissue DNA or RNA profiling. More importantly, 4 DNA-based subtypes, and a ctDNA-based genomic signature tracking retinoblastoma loss-of-heterozygosity, are significantly associated with poor response and survival outcome following ET + CDK4/6i, independently of plasma tumor fraction. Our approach opens opportunities for the discovery of additional multi-feature genomic predictors coming from ctDNA in breast cancer and other cancer-types.

Early Diagnosis of Oral Cancer and Lesions in Fanconi Anemia Patients: A Prospective and Longitudinal Study Using Saliva and Plasma.

Fanconi anemia (FA) patients display an exacerbated risk of oral squamous cell carcinoma (OSCC) and oral potentially malignant lesions (OPMLs) at early ages. As patients have defects in their DNA repair mechanisms, standard-of-care treatments for OSCC such as radiotherapy and chemotherapy, give rise to severe toxicities. New methods for early diagnosis are urgently needed to allow for treatment in early disease stages and achieve better clinical outcomes. We conducted a prospective, longitudinal study wherein liquid biopsies from sixteen patients with no clinical diagnoses of OPML and/or OSCC were analyzed for the presence of mutations in cancer genes. The DNA from saliva and plasma were sequentially collected and deep-sequenced, and the clinical evaluation followed over a median time of approximately 2 years. In 9/16 FA patients, we detected mutations in cancer genes (mainly TP53) with minor allele frequencies (MAF) of down to 0.07%. Importantly, all patients that had mutations and clinical follow-up data after mutation detection (n = 6) developed oral precursor lesions or OSCC. The lead-time between mutation detection and tumor diagnosis ranged from 23 to 630 days. Strikingly, FA patients without mutations displayed a significantly lower risk of developing precursor lesions or OSCCs. Therefore, our diagnostic approach could help to stratify FA patients into risk groups, which would allow for closer surveillance for OSCCs or precursor lesions.

Development of a mouse model for spontaneous oral squamous cell carcinoma in Fanconi anemia.

Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3-4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research on new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA.

Circulating SOD2 Is a Candidate Response Biomarker for Neoadjuvant Therapy in Breast Cancer.

There is a great need for non-invasive tools that inform of an early molecular response to cancer therapeutic treatment. Here, we tested the hypothesis that proteolytically resistant proteins could be candidate circulating tumor biomarkers for cancer therapy. Proteins resistant to proteolysis are drastically under-sampled by current proteomic workflows. These proteins could be reliable sensors for the response to therapy since they are likely to stay longer in circulation. We selected manganese superoxide dismutase (SOD2), a mitochondrial redox enzyme, from a screening of proteolytic resistant proteins in breast cancer (BC). First, we confirmed the robustness of SOD2 and determined that its proteolytic resistance is mediated by its quaternary protein structure. We also proved that the release of SOD2 upon chemotherapy treatment correlates with cell death in BC cells. Then, after confirming that SOD2 is very stable in human serum, we sought to measure its circulating levels in a cohort of BC patients undergoing neoadjuvant therapy. The results showed that circulating levels of SOD2 increased when patients responded to the treatment according to the tumor shrinkage during neoadjuvant chemotherapy. Therefore, the measurement of SOD2 levels in plasma could improve the non-invasive monitoring of the therapeutic treatment in breast cancer patients. The identification of circulating biomarkers linked to the tumor cell death induced by treatment could be useful for monitoring the action of the large number of cancer drugs currently used in clinics. We envision that our approach could help uncover candidate tumor biomarkers to measure a tumor's response to cancer therapy in real time by sampling the tumor throughout the course of treatment.

Clinical and Magnetic Resonance Imaging (MRI) Features, Tumour Localisation, and Survival of Dogs with Presumptive Brain Gliomas.

Brain gliomas are common tumours diagnosed in dogs. However, limited information is available on the clinical features and overall survival time (OS) in dogs receiving palliative treatment. The aim of this study was to evaluate possible associations between presenting complaint, tumour localisation, Magnetic Resonance Imaging (MRI) features, survival times, and reason for the death of dogs with suspected intracranial glioma treated palliatively. Sixty dogs from a single institution were retrospectively included (from September 2017 to December 2021). Dogs were included if a presumptive diagnosis of brain glioma was obtained based on an MRI scan and medical history. French Bulldogs were overrepresented (40/60); 46 out of 60 dogs (77%) presented due to epileptic seizures (ES) and in 25/60 dogs (42%), cluster seizures or status epilepticus were the first manifestation of the disease. Dogs with suspected gliomas located in the piriform lobe showed a higher probability of presenting due to epilepsy compared to dogs with glioma in other regions, and more frequently died or were euthanised because of increased ES. Magnetic Resonance Imaging (MRI) features differed between localisations. Fronto-olfactory tumours were more frequently, whereas piriform tumours were less frequently, classified as suspected high-grade glioma. The median survival time was 61 days. Dogs with contrast-enhancing suspected gliomas had significantly shorter OS. This study provides additional information on the clinical features and survival of dogs with suspected brain gliomas treated palliatively.

Prognostic value of ctDNA detection in patients with early breast cancer undergoing neoadjuvant therapy: A systematic review and meta-analysis.

Circulating tumor DNA (ctDNA) is increasingly being used as a biomarker in early breast cancer (EBC). We performed a systematic review and meta-analysis to investigate the prognostic value of ctDNA in patients with EBC treated with neoadjuvant therapy (NAT). We searched Medline, Web of Science and Embase for observational or interventional studies that included patients with EBC undergoing NAT, reported outcomes related to the predefined endpoints, and had full text articles available. Study selection followed the PRISMA guidelines and quality assessment the REMARK tool for biomarker studies. Primary endpoint was impact of ctDNA detection in different time points (baseline, on-treatment, and after NAT) on relapse-free survival (RFS) and overall survival (OS). Secondary endpoints included the association of ctDNA detection with pathologic complete response (pCR), and the positive and negative predictive value of ctDNA detection in predicting residual disease after NAT. From the 2908 studies initially identified, 11 met the eligibility criteria and were included in the meta-analysis. Detection of ctDNA, both at baseline and after completion of NAT, significantly associated to worse RFS (HR 4.22, 95% CI: 1.29-13.82 and HR 5.67, 95% CI: 2.73-11.75, respectively) and worse OS (HR 19.1, 95% CI: 6.9-53.04 and HR 4.00, 95% CI: 1.90-8.42, respectively). In contrast, detection of ctDNA did not associate with the probability of achieving a pCR. Our results suggest that ctDNA assessment during NAT for EBC merits further evaluation as a stratification risk factor in prospective trials, in order to better individualize patient's treatment.

The clinical utility of neostigmine administration in the diagnosis of acquired myasthenia gravis.

OBJECTIVE: To assess the clinical utility of neostigmine methylsulfate administration in the diagnosis of suspected acquired myasthenia gravis (MG) in dogs and cats. DESIGN: Retrospective study (2017-2019). SETTING: Five university teaching hospitals and 2 private referral hospitals. ANIMALS: Twenty-two dogs and 3 cats. Criteria for inclusion were clinical signs consistent with acquired MG, performance of a neostigmine challenge and acetylcholine receptor antibody titers. INTERVENTIONS: None. MEASUREMENTS & MAIN RESULTS: The route of neostigmine administration was recorded. Response to neostigmine challenge was determined via sequential evaluation of muscle strength and ambulation following administration of neostigmine methylsulfate. Response to neostigmine challenge was compared to acetylcholine receptor antibody titers, which were used as the biochemical gold standard in this study. Sixteen out of 22 dogs were diagnosed with acquired MG. Thirteen of 16 had a strong positive response to neostigmine challenge whereas 3 of 16 had no response. Two out of 3 dogs with polymyositis also had a strong positive response to neostigmine challenge. Weak positive results were seen with intracranial neoplasia (n = 1) and a dog with dilated cardiomyopathy and coxofemoral joint disease (n = 1). One cat was diagnosed with acquired MG and had a positive response to neostigmine challenge. Two cats had no response to neostigmine challenge and were diagnosed with alternate conditions. Two cats were premedicated with glycopyrrolate, one of which had a mild adverse response to neostigmine challenge (sialorrhea and mild transient tremors). Three out of 22 dogs had minimal adverse effects (sialorrhea and 1 dog with muscle tremors). CONCLUSIONS: The neostigmine challenge appears to be safe and viable alternative to the previously utilized edrophonium challenge, particularly when weak positive responses are considered negative for acquired MG. Polymyositis cases may have a false positive response to neostigmine challenge.

Clinical features, diagnosis, and survival analysis of dogs with glioma.

BACKGROUND: Gliomas in dogs remain poorly understood. OBJECTIVES: To characterize the clinicopathologic findings, diagnostic imaging features and survival of a large sample of dogs with glioma using the Comparative Brain Tumor Consortium diagnostic classification. ANIMALS: Ninety-one dogs with histopathological diagnosis of glioma. METHODS: Multicentric retrospective case series. Signalment, clinicopathologic findings, diagnostic imaging characteristics, treatment, and outcome were used. Tumors were reclassified according to the new canine glioma diagnostic scheme. RESULTS: No associations were found between clinicopathologic findings or survival and tumor type or grade. However, definitive treatments provided significantly (P = .03) improved median survival time (84 days; 95% confidence interval [CI], 45-190) compared to palliative treatment (26 days; 95% CI, 11-54). On magnetic resonance imaging (MRI), oligodendrogliomas were associated with smooth margins and T1-weighted hypointensity compared to astrocytomas (odds ratio [OR], 42.5; 95% CI, 2.42-744.97; P = .04; OR, 45.5; 95% CI, 5.78-333.33; P < .001, respectively) and undefined gliomas (OR, 84; 95% CI, 3.43-999.99; P = .02; OR, 32.3; 95% CI, 2.51-500.00; P = .008, respectively) and were more commonly in contact with the ventricles than astrocytomas (OR, 7.47; 95% CI, 1.03-53.95; P = .049). Tumor spread to neighboring brain structures was associated with high-grade glioma (OR, 6.02; 95% CI, 1.06-34.48; P = .04). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with gliomas have poor outcomes, but risk factors identified in survival analysis inform prognosis and the newly identified MRI characteristics could refine diagnosis of tumor type and grade.

Cerebrospinal fluid analysis in dogs: Main patterns and prevalence of albuminocytological dissociation.

BACKGROUND: Albuminocytological dissociation (ACD) of the cerebrospinal fluid (CSF) is defined as an increased total protein concentration with normal total nucleated cell count. It is suspected to occur in diseases that alter the blood-brain barrier, increase the production of protein or obstruct the flow of CSF. The purposes of this study were to review the CSF analysis results of a large cohort of dogs with neurological conditions, to analyse the total prevalence of ACD and to describe which diseases have a higher prevalence of ACD. STUDY DESIGN AND METHODS: Medical records were retrospectively searched for dogs whom CSF was sampled from 2012-2019. Data collected included signalment, body weight, site of collection of the CSF, CSF analysis results, and final diagnosis. RESULTS: A total of 497 dogs met the inclusion criteria. ACD was identified in 16.5% (82/497) of dogs. The diseases with higher proportion of ACD were cranial nerve neuropathy (6/10; 60.0%), brain tumour (10/24; 41.7%), idiopathic vestibular disease (7/17; 41.2%) and brain vascular disease (4/13; 30.8%). CLINICAL SIGNIFICANCE: This study describes the CSF patterns of the most common neurological conditions in dogs, also characterizing, for the first time in dogs, the prevalence and causes of ACD, which was identified in 16.5% of the samples. The diseases with highest proportions of ACD were cranial nerve neuropathy, brain tumour, idiopathic vestibular disease and brain vascular disease.

MRI of the optic nerve sheath and globe in cats with and without presumed intracranial hypertension.

OBJECTIVES: The study aimed to: (1) test MRI repeatability of measurements of optic nerve sheath diameter (ONSD), optic nerve diameter (OND) and eye globe transverse diameter (ETD); (2) investigate the associations between the OND, ONSD and ETD; (3) assess whether these measurements are affected by age or body weight; and (4) test the association between ONSD, OND, ETD and ONSD:ETD ratio with presumed intracranial pressure (ICP) status. METHODS: This was a retrospective and blinded study where patients were allocated to presumed normal or intracranial hypertension groups based on MRI findings. The ONSD and ETD were measured and recorded. Interclass correlation coefficient (ICC) was calculated to investigate interobserver agreement. Data were analysed using the Pearson correlation coefficient, two-sample t-test and general linear model ANOVA. RESULTS: Seventy-seven cats were included, 62 with presumed normal ICP and 15 with presumed intracranial hypertension. The ICC showed moderate-to-good reliability for all measurements. Positive correlations were identified for: (1) ETD and weight; (2) ONSD and age; (3) OND and age; (4) ONSD and ETD; (5) ONSD:ETD ratio and presumed ICP status; and (6) ONSD and presumed ICP status. No difference was detected between the presumed normal and intracranial hypertension groups and ONSD, as well as ONSD:ETD ratio and presumed ICP status when patient age was considered. CONCLUSIONS AND RELEVANCE: The measurement of the ONSD and the ONSD:ETD ratio on T2-weighted MRI might not be reliable as non-invasive tests for diagnosing intracranial hypertension in cats.

Feline meningioma with extensive nasal involvement.

CASE SUMMARY: A 9-year-old male neutered domestic longhair cat was presented with a 3 week history of lethargy and pain of unknown origin. A large extra-axial mass was demonstrated on MRI of the head, with cribriform plate destruction, extensive nasal invasion and intracranial expansion, producing a severe mass effect. The mass was isointense on T1-weighted imaging, predominantly hypointense with some hyperintense areas on T2-weighted imaging and fluid attenuation inversion recovery, markedly contrast enhancing, and caused transtentorial and cerebellar herniation. Histopathological evaluation confirmed a transitional (mixed) meningioma. RELEVANCE AND NOVEL INFORMATION: To our knowledge this is the first report of a meningioma with extensive nasal involvement in a cat. Based on this case, meningioma should be considered as a differential diagnosis for tumours involving the nasal cavity and frontal lobe with cribriform plate destruction.

Traumatic L7 articular processes fracture and spondylolisthesis following dorsal lumbosacral laminectomy in a cat.

CASE SUMMARY: A 12-year-old neutered male domestic shorthair cat was presented to our referral hospital with a chronic history of tenesmus and lumbosacral pain. A diagnosis of degenerative lumbosacral stenosis (DLSS) was made and a standard dorsal L7-S1 laminectomy was performed uneventfully, with complete recovery within 1 month. The cat was brought back 4 months later for investigation of lumbosacral pain after having suffered a minor traumatic event. Neurological examination identified a low tail carriage, weakness, exercise intolerance, left pelvic limb lameness and diminished withdrawal reflexes in both pelvic limbs with severe sacrocaudal pain. A traumatic facet fracture of the L7 articular processes and subsequent spondylolisthesis was diagnosed. A second surgery was performed to stabilise the region. The cat was normal on neurological examination 1 month later and no further clinical signs were noted. RELEVANCE AND NOVEL INFORMATION: This is the first description of a fracture and spondylolisthesis as a possible postoperative complication after L7-S1 dorsal laminectomy in a cat. The case highlights the importance of postoperative changes in the supportive structures of the lumbosacral spine in cats after surgical treatment of DLSS.

Long-term follow-up of surgical resection alone for primary intracranial rostrotentorial tumors in dogs: 29 cases (2002-2013).

Intracranial neoplasia is frequently encountered in dogs. After a presumptive diagnosis of intracranial neoplasia is established based on history, clinical signs and advanced imaging characteristics, the decision to treat and which treatment to choose must be considered. The objective of this study is to report survival times (ST) for dogs with intracranial meningiomas and gliomas treated with surgical resection alone (SRA), to identify potential prognostic factors affecting survival, and to compare the results with the available literature. Medical records of 29 dogs with histopathologic confirmation of intracranial meningiomas and gliomas treated with SRA were retrospectively reviewed. For each dog, signalment, clinical signs, imaging findings, type of surgery, treatment, histological evaluation, and ST were obtained. Twenty-nine dogs with a histological diagnosis who survived >7 days after surgery were included. There were 15 (52%) meningiomas and 14 (48%) gliomas. All tumors had a rostrotentorial location. At the time of the statistical analysis, only two dogs were alive. Median ST for meningiomas was 422 days (mean, 731 days; range, 10-2735 days). Median ST for gliomas was 66 days (mean, 117 days; range, 10-730 days). Kaplan-Meier analysis indicated that ST was significantly longer for meningiomas than for gliomas (P<0.05). A negative correlation between the presence of a midline shift and ST (P=0.037) and ventricular compression and ST (P=0.038) was observed for meningiomas. For gliomas, there were no significant associations between ST and any of the variables evaluated. In conclusion, the results of this study suggest that, for dogs that survived >7 days postoperatively, SRA might be an appropriate treatment, particularly for meningiomas, when radiation therapy is not readily available. Also, the presence of midline shift and ventricular compression might be negative prognostic factors for dogs with meningiomas.

Biological signatures of asymptomatic extra- and intracranial atherosclerosis: the Barcelona-AsIA (Asymptomatic Intracranial Atherosclerosis) study.

BACKGROUND AND PURPOSE: Intracranial atherosclerotic disease (ICAD) remains a challenge for stroke primary and secondary prevention. Molecular pathways involved in the development of ICAD from its asymptomatic stages are largely unknown. In our population-based study, we aimed to compare the risk factor and biomarker profiles associated with intracranial and extracranial asymptomatic cerebral atherosclerosis. METHODS: The Asymptomatic Intracranial Atherosclerosis (AsIA) study cohort includes a random sample population of 933 white subjects >50 years with a moderate to high vascular risk (based on REGICOR score) and without a history of stroke (64% males; mean age, 66 years). Carotid and intracranial atherosclerosis were screened by cervical and transcranial color-coded Duplex ultrasound, being moderate to severe stenoses confirmed by MR angiography. We registered clinical and anthropometric data and created a biobank with blood samples at baseline. A panel of biomarkers involved in atherothrombogenesis was determined: C-reactive protein, asymmetric-dimethylarginine, resistin, and plasminogen activator inhibitor-1. Insulin resistance was quantified by Homeostasis Model Assessment index. RESULTS: After multinomial regression analyses, male sex, hypertension, smoking, and alcoholic habits were independent risk factors of isolated extracranial atherosclerotic disease. Diabetes and metabolic syndrome conferred a higher risk for ICAD than for extracranial atherosclerotic disease. Moreover, metabolic syndrome and insulin resistance were independent risk factors of moderate to severe ICAD but were not risk factors of moderate to severe extracranial atherosclerotic disease. Regarding biomarkers, asymmetric-dimethylarginine was independently associated with isolated ICAD and resistin with combined ICAD-extracranial atherosclerotic disease. CONCLUSIONS: Our findings show distinct clinical and biological profiles in subclinical ICAD and extracranial atherosclerotic disease. Insulin resistance emerged as an important molecular pathway involved in the development of ICAD from its asymptomatic stage.