The clinical heterogeneity of Hu-PNS remains immunologically unexplained. Hu-antibodies target the neuronal Embryonic Lethal Abnormal Vision-Like (nELAVL) protein family and often coexist with antibodies against other neural proteins.

To evaluate Hu-antibodies epitope reactivity and new candidate autoantigens in samples of patients with Hu-associated paraneoplastic neurological syndromes (Hu-PNS), correlating the findings with their clinical presentation.

Using a proteome-wide PhIP-seq library, we analyzed the serum (n=203) and CSF (n=84) from 210 Hu-PNS patients. Z-scores were generated using serum from 100 healthy controls as a background. nELAVL fragment sequences were aligned to map Hu-Abs epitopes. Proteins with Z-scores ≥10 in ≥10% of Hu-PNS samples, compared to ≤2% of control samples, were considered positive hits.

Among 210 Hu-PNS patients, 159 (76%) had samples with Z-scores ≥10 for nELAVL-derived fragments. Most serum (162/203, 80%) and CSF (56/84, 67%) samples enriched fragments spanning nELAVL RNA recognition motifs 2 and 3. Only 19/76 (25%) patients with paired samples targeted the same nELAVL epitope in both serum and CSF. These 19 patients had central nervous system involvement and never exhibited isolated peripheral nervous system involvement (0/19, 0% vs. 24/57, 42%; P=0.003) compared to patients with different serum and CSF nELAVL epitope reactivity. We identified 364 positive hits other than nELAVL proteins: 70 in serum, 80 in CSF, and 214 in both. Forty-one positive hits were significantly associated with specific clinical phenotypes: 2 with limbic involvement, 18 with brainstem/cerebellar, 12 with sensory neuropathy and 10 with myenteric involvement.

Our study reveals significant variability in antigens and epitope reactivity between serum and CSF samples, varying with clinical presentation. This highlights the need to study CSF to understand the immune mechanisms underlying neurological presentations in Hu-PNS. Future longitudinal studies could clarify the roles of epitope reactivity and other autoantibodies in disease progression and patient stratification.