We enrolled 51 patients (M/F 16/35; median age 70 years, IQR 16.5). Nineteen patients (41.3%) suffered from NSCLC, 15 (32.6%) from head and neck cancer, 5 (10.8%) from melanoma, 4 (8.7%) from urothelial, 6 (13%) from kidney, 1 (1.9%) from squamous cell carcinoma and 1 (1.9%) from Merkel cell carcinoma. Moving on treatment, 28 (54.9%) patients were treated with pembrolizumab, 19 (37.2%) with nivolumab, 2 (3.9%) with cemiplimab, 1 (1.9%) with avelumab and 1 (1.9%) with durvalumab.

A complete serological evaluation was available for 46 patients (90.2%). Sixteen patients (34.8%) were ANA positive with a titer of at least 1:80; specifically, according to ICAP classification, 13 were characterized by a homogeneous pattern (AC-1), 1 by centromeric pattern (AC-3), 1 by speckled pattern (AC-2), and 1 by both homogenous and speckled (AC-1 + AC-2). Moving to the ANA titer, 7 patients had an ANA title —1:80; 4— patients  1:160; 4 patients 1:320; 1 patient 1:640.

Furthermore, three (6.5%) were ENA positive and all of them showed a high titer positivity for anti-SSA antibodies; two were (4.3%) Ratest positive (one for IgM RF, IgA RF) and one (2.1%) showed ACPA positivity (Fig. 1a).

a Prevalence of autoantibodies in the analyzed cohort before immune-checkpoint inhibitors treatment, b Kaplan–Meier analysis of the occurrence of immune-related Adverse Events in the overall patients and by dividing ANA + and ANA − patients (Log-rank Mantel-cox test, (62.5% versus 31.0%; p = 0.029). c Prevalence of immune-related Adverse Events (irAEs) developed during follow-up

Patients were followed up for a median period of 21 months (IQR 38.75). The median overall survival (OS) was 18 months (IQR 18) and the median progression-free survival (PFS) was 6 months (IQR 11).

During this period, 19 patients (41.3%) developed irAEs after a median interval equal 6 weeks (IQR 16; mean 13.05 weeks, SD 14.0). The Kaplan–Meier analysis is reported in Fig. 1b.

In detail, 6 patients (13.3%) experienced arthritis, 4 patients (8.8%) thyroiditis, 3 (6.6%) dermatological reactions, 2 (4.4%) neurotoxicity, 2 patients (4.4%) nephritis, 1 patient (2.2%) severe neutropenia, and 1 (2.2%) pneumonia (Fig. 1c). During observation, any adverse effects not directly attributable to immunotherapy were recorded but not considered as ICIs induced, including nausea, fatigue, low-grade anemia, peripheral neuropathic damage, hearing loss, and severe hyponatremia.

No differences were found in the irAEs prevalence according to administered drug.

Considering the high prevalence of ANA antibodies, we performed a comparison between ANA + and ANA − patients. Indeed, the incidence of irAEs was significantly higher in ANA + patients [10/16 patients (62.5%) versus 9/30 (31%), p = 0.03; Fig. 2]; furthermore, the relative risk (RR) of developing any irAEs for ANA-positive patients was 2.01 (95% CI 1.03–3.92; p = 0.04). Indeed, we stratified patients according to ANA titer: seven patients (43.7%) showed 1:80 titer, the remaining 9 showed a titer of ≥ 1:160. No differences were found according to ANA titer in the occurrence of irAEs (p = 0.14).

Incidence of immune-related Adverse Events (irAEs) in ANA + and ANA −  patients

No differences were found between ANA + and ANA −  patients in terms of treatment response and PD-L1 expression (Supplementary Table 1-S1). Similarly, no differences were identified when comparing ANA + and ANA −  patients in terms of PFS [9.5 months (IQR 19) versus 5 months (IQR 9.5); p = 0.08) and OS [18.5 months (13.75) versus 18 months (IQR 20); p = 0.39]. When stratifying patients by the occurrence of irAEs, the PFS was found to be significantly longer in patients developing adverse events compared to those who did not experience these events [10 months (IQR 16.5) versus 4 months (IQR 9.5); p = 0.007].

Indeed, arthritis was found to be the most frequent "irAEs" and developed in six patients (30% of total irAEs). Three of them were treated with nivolumab and three with pembrolizumab. The serological features of these six patients are reported in Table 1: of note, 5 out of 6 (83.3%) were ANA positive, associated with other autoantibodies positivity in 4 patients. Of note, one patient positive for ANA, Ratest, and anti-SSA, developed lower limb purpura, xerostomia and lymphopenia associated with arthritis. Accordingly, a diagnosis of Sjögren Syndrome (SS) was made (patient 2).

All the patients were treated with a median glucocorticoids (GCs) dose of 10 mg (IQR 5) with fast tapering. Treatment with cDMARDs was added in three patients; specifically, hydroxychloroquine (HCQ) was added in the patient diagnosed with SS and in a second patient due to the persistence of arthralgia at GCs tapering (patient 4). Finally, one patient received methotrexate (10 mg weekly) due to persistent arthritis despite GCs treatment; this patient was diagnosed with undifferentiated arthritis (patient 6).