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.

BACKGROUND: The determination of in situ protein levels of ERCC1 with the 8F1 monoclonal antibody is prognostic of survival in patients with non-small cell lung cancer (NSCLC). The authors previously demonstrated that 8F1 recognizes a second nuclear antigen. This antigen was identified and its value as a biomarker of clinical outcomes analyzed. METHODS: The second antigen was identified by mass spectrometry. Protein identity and antibody specificity were confirmed through knockdown and overexpression experiments. Immunohistochemistry of 187 early-stage NSCLC samples and 60 head and neck squamous cell carcinomas (HNSCCs) was used to examine the influence of the second antigen on 8F1 immunoreactivity and its association with patient outcomes. RESULTS: Choline phosphate cytidylyltransferase-α (CCTα, also known as phosphate cytidylyltransferase 1 choline alpha [PCYT1A], a phospholipid synthesis enzyme regulated by RAS) is the second antigen recognized by 8F1. In NSCLC samples, CCTα contributed (rho, 0.38) to 8F1 immunoreactivity. In samples of squamous cell carcinomas of the lung, CCTα was found to be the dominant determinant of 8F1 immunoreactivity, whereas its contribution in other subtypes of lung cancer was negligible. High expression of CCTα, but not ERCC1, was found to be prognostic of longer disease-free survival (log-rank P = .002) and overall survival (log-rank P = .056). Similarly, in patients with HNSCC, CCTα contributed strongly to 8F1 immunoreactivity (rho, 0.74), and high CCTα expression was found to be prognostic of survival (log-rank P = .022 for disease-free survival and P = .027 for overall survival). CONCLUSIONS: CCTα is the second antigen detected by 8F1. High CCTα expression appears to be prognostic of survival in patients with NSCLC who are treated by surgery alone and patients with HNSCC. CCTα is a promising biomarker of patient survival and deserves further study.

Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology.

BACKGROUND: An antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein. RESULTS: By using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application. CONCLUSION: In summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.

CTP:phosphocholine cytidylyltransferase alpha is required for B-cell proliferation and class switch recombination.

CTP:phosphocholine cytidylyltransferase (CCT) is a key rate-controlling enzyme in the biosynthetic pathway leading to the principle membrane phospholipid, phosphatidylcholine. CCTalpha is the predominant isoform expressed in mammalian cells. To investigate the role of CCTalpha in the development and function of B-lymphocytes, mice with B-lymphocytes that selectively lacked CCTalpha were derived using the CD19-driven Cre/loxP system. When challenged with a T-cell-dependent antigen, the animals harboring CCTalpha-deficient B-cells exhibited a hyper-IgM secretion phenotype coupled with a lack of IgG production. The inability of CCTalpha-/- B-cells to undergo class switch recombination correlated with a proliferation defect in vivo and in vitro in response to antigenic and mitogenic stimuli. Lipopolysaccharide stimulation of CCTalpha-/- B-cells resulted in an early trigger of the unfolded protein response-mediated splicing of Xbp-1 mRNA, and this was accompanied by accelerated kinetics of IgM secretion and higher incidence of IgM-secreting cells. Thus, the inability of stimulated B-cells to produce enough phosphatidylcholine prevents proliferation and class switch recombination but leads to unfolded protein response activation and a hyper-IgM secretion phenotype.