Choline phosphate cytidylyltransferase‐α is a novel antigen detected by the anti‐ERCC1 antibody 8F1 with biomarker value in patients with lung and head and neck squamous cell carcinomas

Abstract

BACKGROUND Determination of in situ protein levels of ERCC1 with the antibody (8F1) is prognostic of survival in non-small-cell lung cancer (NSCLC). We previously demonstrated that 8F1 recognizes a second nuclear antigen. We identified this antigen and analyzed its value as a biomarker of clinical outcomes. METHODS The second antigen was identified by mass spectrometry. Protein identity and antibody specificity were confirmed through knockdown and overexpression experiments. Immunohistochemistry (IHC) of 187 early stage NSCLC samples and 60 head and neck squamous cell carcinomas (HNSCC) was used to examine the influence of the second antigen on 8F1 immunoreactivity and association with patient outcomes. RESULTS Cholinephosphate citidylyl transferase–α (CCTα, a.k.a. phosphate citidylyl transferase 1 choline alpha (PCYT1A), a phospholipid synthesis enzyme regulated by RAS, is the second antigen of 8F1. In NSCLC, CCTα contributed (rho = 0.38) to 8F1 immunoreactivity. In squamous cell carcinoma of the lung, CCTα was the dominant determinant of 8F1 immunoreactivity, while its contribution in other subtypes of lung cancer was negligible. High expression of CCTα, but not ERCC1, was prognostic of longer disease-free (log-rank p = 0.002), and overall survival (log-rank p = 0.056). Similarly, in HNSCC, CCTα contributed strongly to 8F1 immunoreactivity (rho = 0.74), and high CCTα expression was prognostic of survival (log-rank p = 0.022 for DFS and p = 0.027 for OS). CONCLUSIONS CCTα is the second antigen detected by 8F1. High CCTα expression is prognostic of survival in NSCLC treated by surgery alone and HNSCC. CCTα is a promising biomarker of patient survival and deserves further study.

The prognostic and predictive value of excision repair cross-complementation group 1 (ERCC1) protein in 1288 patients with head and neck squamous cell carcinoma treated with platinum-based therapy: a meta-analysis

“…It has been also reported that the most frequently used antibody 8F1 has not been specific for ERCC1 [42]. It also tags a spurious 45 kDa band on immunoblotting, a cross reaction that results in ERCC1-XPF deficient cells being falsely characterized as having ERCC1-XPF expression [43], and the unrelated nuclear membrane protein choline phosphate cytidylyltransefrase- (PCYT1 ) [44][45]. Fiboulet et al [46] reported that the major limitation to ERCC1 employment in guiding the treatment decision was the lack of ERCC1 antibodies that would detect functionally different ERCC1 isoforms.…”

CTP:phosphocholine cytidylyltransferase: Function, regulation, and structure of an amphitropic enzyme required for membrane biogenesis

“…Furthermore, studies on CCT expression in tumors have incompletely assessed CCT regulatory parameters and/or did not differentiate between isoforms [252]. An exception to this is a recent study that analyzed CCTa expression in non-small cell lung cancer and head and neck squamous cell carcinomas [255]. In this study, a previously identified monoclonal antibody against a nuclear antigen associated with increased cancer survival was shown to primarily detect CCTa.…”

“…In this study, a previously identified monoclonal antibody against a nuclear antigen associated with increased cancer survival was shown to primarily detect CCTa. The level of expression of nuclear CCTa detected with this monoclonal was prognostic for disease-free survival of patients with squamous cell carcinomas of the lung, head, and neck [255].…”

A phase 2 cooperative group adjuvant trial using a biomarker‐based decision algorithm in patients with stage I non‐small cell lung cancer (SWOG‐0720, NCT00792701)

“…First, ERCC1 has multiple isoforms that cannot be specifically distinguished by the available reagents, and only 1 isoform appears to be involved in platinum‐induced DNA damage repair . Second, the monoclonal antibody 8F1, which is consistently used for ERCC1 protein expression analysis, detects a second and unrelated protein that shares a common epitope with ERCC1 . This observation may account for the highly batch‐dependent performance of this antibody, which may explain the significantly lower ERCC1 values in the current study compared with prior results .…”

“…Results from the first trial (NCT00499109) demonstrated no improvement in patient survival; however, the authors raised the possibility of a false‐negative result because of an inexplicably divergent survival in an internal control group . The second trial (NCT00801736) and third trial (NCT00775385) were terminated early after the discovery of ERCC1 isoforms and specificity problems with the 8F1 antibody . The fourth trial is using ERCC1 and tumor thymidylate synthase mRNA expression levels for treatment assignment compared with a cisplatin‐based control treatment with OS as the primary endpoint and a planned accrual of 700 patients.…”