Cytotoxic T-cell precursor frequencies to HER-2 (369 – 377) in patients with HER-2/neu-positive epithelial tumours

HER-2/neu oncoprotein contains several major histocompatibility complex class I-restricted epitopes, which are recognised by cytotoxic T lymphocyte (CTL) on autologous tumours and therefore can be used in immune-based cancer therapies. Of these, the most extensively studied is HER-2(9(369)). In the present report, we used dendritic cells pulsed with HER-2(9(369)) to stimulate, in the presence of IL-7 and IL-12, the production of IFN-gamma by patients' CTL detected by the enzyme-linked immunosorbent spot-assay. Frequencies of peptide-specific precursors were estimated in HLA-A2, HLA-A3 and HLA-A26 patients with HER-2/neu-positive (+) breast, ovarian, lung, colorectal and prostate cancers and healthy individuals. We found increased percentages of such precursors in HLA-A2 (25%) and HLA-A26 (30%) patients, which were significantly higher (60%) in HLA-A3 patients. Our results demonstrate for the first time that pre-existing immunity to HER-2(9(369)) occurs in patients with colorectal, lung and prostate cancer. They also suggest that HER-2(9(369)) can be recognised by CTL, besides HLA-A2, also in the context of HLA-A3 and HLA-A26, thus increasing the applicability of HER-2(9(369))-based vaccinations in a considerably broader patients' population.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

The expression of the CEACAM19 gene, a novel member of the CEA family, is associated with breast cancer progression

Breast cancer (BC) continues to affect the lives of millions of women worldwide. Several members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subfamily are involved in tumor progression. Notably, the CEACAM subfamily harbors the already established cancer biomarker CEA, as well as other potential molecular markers. CEACAM19, a recently identified gene belonging to CEACAM subfamily, was discovered and cloned by members of our research group. The present study analyzes, quantitatively, the expression of CEACAM19 and evaluates its clinical relevance in BC. Total RNA was extracted from 143 cancerous and 89 normal adjacent breast tissue specimens. Following reverse transcription, quantitative analysis of CEACAM19 mRNA expression levels was performed via real-time PCR and the comparative Ct (2 -ΔΔCt) method. CEACAM19 expression and detailed clinicopathological data were used for extensive biostatistical analyses. CEACAM19 was found to be overexpressed in breast cancer tissue specimens compared to normal tissue counterparts (p=0.013). CEACAM19 mRNA expression status was also associated with clinicopathological features indicative of aggressive behavior and poor prognosis in BC, such as high tumor grade (p=0.031) and high Ki67 proliferative index (p=0.038). A significant negative association was documented between CEACAM19 expression and tumor ER status (p=0.018) as well as patients' menopausal state (p=0.016). Our results suggest that CEACAM19 mRNA expression represents a promising, novel and clinically useful tissue biomarker for breast cancer management

BCL2L12: A promising molecular prognostic biomarker in breast cancer

Objectives: BCL2-like 12 ( BCL2L12) is a new member of the BCL2 gene family that was discovered and cloned by members of our group and found to be expressed in the mammary gland. Many genes of the BCL2 family were found to be implicated in breast carcinogenesis and to serve as possible prognostic markers. The aim of the present study was the quantification of BCL2L12 mRNA expression in order to assess its value as a prognostic tissue biomarker in breast cancer (BC). Design and methods: BCL2L12 mRNA levels were determined in a statistically significant sample size of cancerous (N=108) and adjacent non-cancerous (N=71) breast tissues using a highly sensitive quantitative real-time polymerase chain reaction (qRT-PCR) method. Relative quantification analysis was conducted using the comparative CT (2-δδCT) method, whereas the association between BCL2L12 expression and clinopathological data, disease-free survival (DFS) and overall survival (OS) were estimated by statistical analysis. Results: BCL2L12 mRNA expression was decreased in malignant samples compared to the histologically normal counterparts ( p= 0.012). Significant relationships between BCL2L12 expression and TNM stages ( p= 0.009), metastatic potential ( p= 0.012), tumor size ( p= 0.04) and age ( p= 0.024) were observed. Moreover, Kaplan-Meier and Cox univariate analyses indicated that BCL2L12 expression is associated with longer DFS, whereas multivariate analysis pointed out the independent favorable prognostic value of BCL2L12. Conclusions: According to our results, BCL2L12 mRNA expression is a favorable prognostic marker of DFS for BC patients, suggesting its possible application as a novel prognostic indicator of this malignancy. © 2014

Kallikrein-related peptidase 6 (KLK6) expression differentiates tumor subtypes and predicts clinical outcome in breast cancer patients

Novel molecular markers that address the heterogeneity of breast cancer (BC) and provide meaningful prognostic information for BC patients are needed. Kallikrein-related peptidase 6 (KLK6) is aberrantly expressed and functionally implicated in BC and, like other members of the KLK family, may prove a useful molecular tool for clinical management. Our objective was to assess, for the first time, the clinical relevance of KLK6 mRNA expression in BC. Total RNA was isolated from 165 breast tumors, as well as 100 adjacent non-cancerous tumor specimens. After cDNA synthesis, and following quality control, quantitative real-time PCR for KLK6 expression analysis took place. Receiver operating characteristic curves were constructed in order to assess the ability of KLK6 mRNA expression levels to differentiate between molecular BC subtypes. Survival analyses, using DFS as endpoint, were performed at the univariate and multivariate levels. Publicly available BC databases and online survival analysis tools were used to validate our findings. A significant downregulation of KLK6 mRNA expression was observed in BC tissue sections compared to the non-cancerous component (P < 0.001). The expression of KLK6 is positively associated with tumor grade (P = 0.038) and is overexpressed in TNBC and HER2-positive tumors (P < 0.001). Aberrant KLK6 expression predicts the clinical outcome of BC patients in terms of DFS, independently of currently used prognostic markers (HR = 7.11, 95% CI = 1.19–42.45). The differential expression of KLK6 and its association with unfavorable outcome in BC patients was validated via in silico analyses. Although an independent external cohort is necessary to confirm our findings, we proved for the first time that KLK6 can provide independent prognostic information for BC patients. © 2018, Springer International Publishing AG, part of Springer Nature

Natural CD8+ T-cell responses against MHC class I epitopes of the HER-2/neu oncoprotein in patients with epithelial tumors

HER-2/neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/neu-positive (+) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2-binding nona-peptide 369-377 (HER-2(9369)). In the present study, we examined patients with HER-2/neu+ breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/neu-derived and HLA-A2-restricted "cytotoxic" peptides and to a novel one spanning amino acids 777-785 also with HLA-A2-binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an Interferon γ (IFN-γ) ELISpot assay detecting T cells at the single cell level secreting IFN-γ. CTLp were denned as peptide-specific precursors per 106 peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/ neu-negative ( -) tumors and healthy individuals. Of the HER-2/neu+ patients examined, 31% had increased CTLp to HER-2(9952), 19% to HER-2(9665), 16% to HER-2(9689), and 12.5% HER-2(9 435), whereas only 2 of 32 patients (6%) responded to HER-2(9 777). The CTLp recognizing HER-2(9952) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu- patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN-γ production, preexisting CTL immunity to all five HER-2/neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9435), HER-2(9952), HER-2(9689), HER-2(9665), and HER-2(9777) is present in patients with HER-2/neu+ tumors of distinct histology, (2) HER-2(9777) is a naturally processed peptide expressed on the surface of HER-2/neu + tumors, as are the other four peptides, and (3) HER-2/neu + prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/neu-based immunotherapy

Identification and characterization of a BER-2/neu epitope as a potential target for cancer immunotherapy

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828-836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu+ tumor cell lines. HER-2/neu(828-836), [HER-2(9 828)], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9828) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8+ T-lymphocytes specifically recognizing HER-2(9828) in 8 out of 20 HLA-A*0201+ HER-2/neu+ breast cancer patients. Moreover, HER-2(9828)-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9828) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9828) as a promising candidate for peptide-based cancer vaccines. © 2009 Springer-Verlag

Identification of a novel immunogenic HLA-A*0201-binding epitope of HER-2/neu with potent antitumor properties

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with aggressive disease. Immunogenic HER-2/neu CTL epitopes have been used as vaccines for the treatment of HER-2/neu positive malignancies with limited success. By applying prediction algorithms for A1HC class I ligands and proteosomal cleavages, in this study, we describe the identification of HER-2/new decamer LIAHNQVRQV spanning residues 85-94 (HER-2(1085)). HER-2(1085) proved to bind with high affinity to HLA-A2.1 and was stable for 4 h in an off-kinetics assay. This peptide was immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded to tumor cell lines of various origin coexpressing human HER-2/neu and HLA-A2.1. This demonstrates that HER-2(1085) is naturally processed from endogenous HER-2/neu. Five of sixteen HER-2/neu+ HLA-A2.1+ breast cancer patients analyzed had HER-2(1085)-reactive T cells ranging from 0.35-0.70% of CD8+ T cells. Depletion of T regulatory cells from PBMC enabled the-rapid expansion of HLA-A2.1/HER-2(10 85)pentamer+/CD8+ cells (PENT +/CD8+), whereas significantly lower numbers of CTL could be generated from unfractionated PBMC. HER-2(1085)-specific human CTL recognized the HER-2/neu+ HLA-A2.1+ tumor cell line SKBR3.A2, as determined by IFN-γ intracellular staining and in the high sensitivity CD107α degranulation assay. Finally, HER-2(1085) significantly prolonged the survival of HHD mice inoculated with the transplantable ALC.A2.1.HER tumor both in prophylactic and therapeutic settings. These data demonstrate that HER-2(1085) is an immunogenic peptide, capable of eliciting CD8-mediated responses in vitro and in vivo, providing the platform for further exploitation of HER-2(1085) as a possible target for anticancer immunotherapy. Copyright © 2008 by The American Association of Immunologists, Inc