SCG3 transcript in peripheral blood is a prognostic biomarker for REST-deficient small cell lung cancer.

PURPOSE: Specific markers of circulating tumor cells may be informative in managing lung cancer. Because the RE-1 silencing transcription factor (REST/NRSF) is a transcriptional repressor that is inactivated in neuroendocrine lung cancer, we identified REST-regulated transcripts (CHGA, CHGB, SCG3, VGF, and PCSK1) for evaluation as biomarkers in peripheral blood. EXPERIMENTAL DESIGN: Transcripts were screened across lung cancer and normal cell lines. Candidates were assessed by reverse transcription-PCR and hybridization of RNA extracted from the peripheral blood of 111 lung cancer patients obtained at clinical presentation and from 27 cancer-free individuals. RESULTS: Expression profiling revealed multiple chromogranin transcripts were readily induced on REST depletion, most notably SCG3 was induced >500-fold. The SCG3 transcript was also overexpressed by 12,000-fold in neuroendocrine compared with nonneuroendocrine lung cancer cells. In peripheral blood of lung cancer patients and cancer-free individuals, we found that SCG3 was more tumor-specific and more sensitive than other chromogranin transcripts as a biomarker of circulating tumor cells. Overall, 36% of small cell lung cancer (SCLC) and 16% of non-SCLC patients scored positively for normalized SCG3 transcript. This correlated with worse survival among SCLC patients with limited disease (n = 33; P = 0.022) but not extensive disease (n = 29; P = 0.459). Interestingly, the subcohort of 6 SCLC patients with resistance to platinum/etoposide chemotherapy all scored positively for peripheral blood SCG3 transcript (P = 0.022). CONCLUSIONS: SCG3 mRNA, a component of the REST-dependent neurosecretory transcriptional profile, provides a sensitive prognostic biomarker for noninvasive monitoring of neuroendocrine lung cancer.

CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I-restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.

Epstein-Barr virus nuclear antigen 1 (EBNA1) induced cytotoxicity in epithelial cells is associated with EBNA1 degradation and processing.

Epstein-Barr virus nuclear antigen 1 (EBNA1) has a central role in the maintenance and segregation of the Epstein-Barr virus (EBV) episome and by virtue of a glycine-alanine repeat domain is prevented from being endogenously processed for recognition by HLA class I restricted cytotoxic T lymphocytes (CTLs). We found that EBNA1 expression resulted in growth inhibition and a G2/M arrest in human squamous epithelial cell lines (SCC12F, SVK) but not epithelial cell lines of glandular origin (Hela, Ad/AH). The cytotoxicity of EBNA1 was associated with EBNA1 degradation and both these effects were blocked in SCC12F cells expressing either the anti-apoptotic bcl-2 protein or the EBV homolog of bcl-2, BHRF1. The endogenous degradation of EBNA1 in SVK epithelial cells was associated with specific CTL recognition, an effect not evident in EBNA1-expressing Hela cells. Consistent with the inability of SVK cells to tolerate EBNA1 expression, studies with a recombinant EBV demonstrated that SVK cells are unable to maintain stable virus infection, whereas Hela cells are able to efficiently establish latent EBV infection. These data have important implications for both the cellular requirements necessary to sustain a stable EBV infection and for the possible role of CTL responses in controlling EBV infection of epithelial cells.

B cells immortalized by a mini-Epstein-Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells.

Lymphoblastoid cell lines (LCLs) are human B cells latently infected and immortalized by Epstein-Barr virus (EBV). Presenting viral antigens, they efficiently induce EBV-specific T-cell responses in vitro. Analogous ways to generate T-cell cultures specific for other antigens of interest are highly desirable. Previously, we constructed a mini-EBV plasmid that consists of less than half the EBV genome, is unable to cause virus production, but still immortalizes B cells in vitro. Mini-EBV-immortalized B-cell lines (mini-LCLs) are efficiently produced by infection of B cells with viruslike particles carrying only mini-EBV DNA. Mini-EBV plasmids can be engineered to express an additional gene in immortalized B cells. Here we present a mini-EBV coding for a potent CD8(+) T-cell antigen, the matrix phosphoprotein pp65 of human cytomegalovirus (CMV). By means of this pp65 mini-EBV, pp65-expressing mini-LCLs could be readily established from healthy donors in a one-step procedure. We used these pp65 mini-LCLs to reactivate and expand effector T cells from autologous peripheral blood cells in vitro. When generated from cytomegalovirus (CMV)-seropositive donors, these effector T-cell cultures displayed strong pp65-specific HLA-restricted cytotoxicity. A large fraction of CD8(+) T cells with pp65 epitope specificity was present in such cultures, as demonstrated by direct staining with HLA/peptide tetramers. We conclude that the pp65 mini-EBV is an attractive tool for CMV-specific adoptive immunotherapy. Mini-EBVs could also facilitate the generation of T cells specific for various other antigens of interest.