A Study to Evaluate the Sensitivity, Specificity, and Overall Accuracy of an Amyotrophic Lateral Sclerosis Diagnostic Test Developed by Iron Horse Diagnostics

ALS, also known as Lou Gehrig's disease, is a rapidly progressive, degenerative disease of motor neurons in the brain and spinal cord that leads to muscle atrophy and spasticity in limb and bulbar muscles. Clinical presentations of this fatal disease include weakness, loss of ambulation, oropharyngeal dysfunction, weight loss, and ultimately respiratory failure. Average survival is approximately 3 years after symptom onset. The initial symptoms are similar to other neuropathies (e.g., motor neuropathy, progressive muscle atrophy, etc.) and include limb weakness leading to partial or total paralysis, and increasing difficulties in speech and breathing. ALS clinical diagnosis typically takes 12 months from symptom onset and relies on exclusion of other potential causes of the clinical symptoms. To date there are no FDA-approved diagnostic tests for ALS. Diagnostic tools for accurate and early diagnosis are under investigation. These tools would not only permit early intervention, but also would improve clinical trial design for new drug therapies.

Axonal degeneration and inflammation are among proposed pathogenic mechanisms for ALS; therefore, proteins that function within these pathways are being evaluated as potential biomarkers. Iron Horse Diagnostics, Inc. (Iron Horse) has focused its ALS diagnostic efforts on studying the levels of cytoskeletal and inflammatory proteins in the cerebrospinal fluid (CSF) of ALS patients.

Cytoskeletal proteins, including neurofilament proteins and tau, have been shown to be elevated in the CSF of patients with various neurodegenerative diseases. Interestingly, levels of phosphorylated neurofilament heavy subunit (pNfH) have been found to be significantly increased in the CSF of ALS patients as compared with healthy subjects, Alzheimer's disease patients or disease mimics Numerous inflammatory proteins, including cytokines and complement proteins, have been shown to be altered in ALS; complement proteins, including complement c3 (C3), have been found to be increased in the CSF of ALS patients.

Iron Horse has evaluated CSF samples from 106 subjects (45 ALS patients, 25 disease controls with a range of neurodegenerative conditions, and 36 healthy controls.CSF levels of pNfH, tau, C3, and C reactive protein (CRP) were measured. pNfH was significantly elevated in CSF from ALS subjects as compared with healthy and disease controls; there were no significant differences for CRP or tau. To further distinguish between ALS subjects and disease controls, data from cytoskeletal and inflammatory pathways were combined. A ratio of CSF levels of pNfH and C3 showed significant differences between ALS and both the disease and healthy control groups. Results were verified on a separate test set of CSF samples. Overall, the predictive pNfH/C3 ratio identified ALS with 87.3 % sensitivity and 94.6% specificity in a total of 71 ALS subjects, 52 disease control subjects, and 40 healthy subjects.

In the same study, it was observed that plasma levels of pNfH were significantly elevated in ALS subjects as compared with healthy controls; however there was no significant difference between ALS and disease controls. Comparison of pNfH levels between the CSF and plasma for each study subject revealed a weak correlation between plasma and CSF pNfH levels.

The accuracy of pNfH and pNfH/C3 ratio as ALS predictors has also been tested by Iron Horse in a recent prospective, blinded study in collaboration with the Northeast ALS Consortium (NEALS) [unpublished data]. CSF (n=126) and plasma (n=220) samples were collected from subjects from 30 medical centers across the United States (US) who were undergoing diagnostic assessment for neurodegenerative conditions or who were recently diagnosed with ALS. Accuracy for predicting ALS diagnosis was 93% for CSF pNfH/C3 ratio and 82% for pNfH plasma levels.

The prospective study proposed in this concept will test the sensitivity, specificity, and overall accuracy of pNfH and pNfH/C3 ratio as ALS predictors on a separate sample set. CSF and matching plasma samples will be collected at up to 6 neurodegenerative clinics from up to 300 subjects who were recently diagnosed with ALS or another similar neurologic disease or who are currently undergoing diagnostic procedures.