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. 2022 Oct 31:12:968570.
doi: 10.3389/fonc.2022.968570. eCollection 2022.

Development of a salivary autoantibody biomarker panel for diagnosis of oral cavity squamous cell carcinoma

Pei-Chun Hsueh  1   2 , Kai-Ping Chang  3   4 , Hao-Ping Liu  5 , Wei-Fan Chiang  6 , Xiu-Ya Chan  7   8 , Chu-Mi Hung  7   8 , Lichieh Julie Chu  4   8 , Chih-Ching Wu  3   4   7   8   9
Affiliations

Development of a salivary autoantibody biomarker panel for diagnosis of oral cavity squamous cell carcinoma

Pei-Chun Hsueh et al. Front Oncol. .

Abstract

Oral cavity squamous cell carcinoma (OSCC) is a destructive disease with increasing incidence. OSCC is usually diagnosed at an advanced stage, which leads to poor outcomes of OSCC patients. Currently, there is a lack of biomarkers with sufficient effectiveness in early diagnosis of OSCC. To ameliorate OSCC screening, we evaluated the performances of salivary autoantibodies (auto-Abs) to nine proteins (ANXA2, CA2, ISG15, KNG1, MMP1, MMP3, PRDX2, SPARC, and HSPA5) as OSCC biomarkers. A multiplexed immunoassay using a fluorescence bead-based suspension array system was established for simultaneous assessment of the salivary levels of the above nine auto-Abs and a known OSCC-associated auto-Ab, anti-p53. Compared to healthy individuals (n = 140), the salivary levels of nine auto-Abs were significantly elevated in OSCC patients (n = 160). Notably, the salivary levels of the 10 auto-Abs in the early-stage OSCC patients (n = 102) were higher than that in the healthy group. Most importantly, utilizing a marker panel consisting of anti-MMP3, anti-PRDX2, anti-SPARC, and anti-HSPA5 for detection of early-stage OSCC achieved a sensitivity of 63.8% with a specificity of 90%. Collectively, herein we established a multiplex auto-Ab platform for OSCC screening, and demonstrated a four-auto-Ab panel which shows clinical applicability for early diagnosis of OSCC.

Keywords: autoantibody; biomarker; cancer screening; oral cancer; saliva.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Workflow for establishment of a bead-based suspension immunoassay for auto-Ab detection in saliva samples. Auto-Ab detection was conducted with a multiplexed bead-based suspension array system (Bio-Plex). To generate the beads used in the system, individual His-tagged recombinant protein were covalently conjugated to the COOH beads with an unique fluorescent identity. The resulting recombinant protein-conjugated beads can capture the salivary IgA specifically responsive to the recombinant proteins. By addition of a biotin-labeled anti-human IgA and streptavidin-phycoerythrin (SA-PE), the levels and identities of salivary IgA auto-Abs can be simultaneously investigated in the Bio-Plex system, in which the “red” laser is applied to interrogate the bead identity which identifies the auto-Ab type, and the “green” laser is used to assess the level of the identified auto-Abs. An anti-His Ab is used to evaluate the protein-coupling efficiency, the cross-reactivity of the beads, and the precision of the multiplex assay.
Figure 2
Figure 2
Evaluation of the range and efficiency of auto-Ab detection with the established bead-based suspension immunoassay. The protein-coupling efficiency of individual beads and their effectiveness in detection of corresponding auto-Abs were verified by using Abs specific to individual proteins, including p53, ANXA2, CA2, ISG15, KNG1, and MMP1, respectively. An anti-His Ab was applied for verification of the beads coupled with His-tagged MMP3, PRDX2, SPARC, and HSPA5, respectively. Initial concentrations of the Abs specific to p53, ANXA2, CA2, ISG15, KNG1, MMP1, and His tag are 0.03, 4, 0.2, 0.2, 20, 0.4, and 20 μg/mL, respectively. Data are acquired as the median fluorescence intensity (MFI) and shown as the mean ± SD of MFI.
Figure 3
Figure 3
Assessment of the cross-reactivity and inter-assay precision of the multiplexed auto-Ab immunoassay. (A) To evaluate the cross-reactivity of the multiplexed immunoassay, six pooled salivary samples, each of which was pooled from saliva samples of six OSCC patients and six healthy controls, were subjected to detection of the 10 auto-Abs using individual protein-conjugated beads (single-plex) and using equally mixed beads respectively for 10 auto-Abs (10-plex) in parallel. Results are presented as a ratio of the auto-Ab level acquired in single-plex to that in 10-plex for each of the six pooled samples (open circles), and the mean auto-Ab ratio of the six pooled samples is indicated with a red thick line. (B) To investigate the inter-assay precision of the 10-plex immunoassay, measures of auto-Ab levels in five pooled saliva samples were taken at three different time points. Results are presented as the coefficient of variation (CV) of auto-Ab levels acquired from the three batches of individual pooled samples (open circles), and the mean CV of the auto-Ab in the five samples is indicated with a red thick line.
Figure 4
Figure 4
Elevated levels of salivary auto-Abs in the OSCC patients. The levels of auto-Abs were detected in saliva samples respectively collected from healthy controls (HC; n = 70), and OSCC patients (n = 80) with the multiplexed bead-based system. Salivary levels of the auto-Abs are shown with the median fluorescence intensity (MFI). Data are presented as the upper and lower quartiles (box), the median value (the horizontal line), and the middle 90% distribution (the whisker) of MFI. *p < 0.05, **p < 0.01 and ***p < 0.001.
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