Christos K. Kontos

Phosphatidylinositol 3′-Kinase Catalytic Subunit α Gene Amplification Contributes to the Pathogenesis of Mantle Cell Lymphoma

Purpose: Activation of phosphatidylinositol 3′-kinase pathway is implicated in the pathogenesis of mantle cell lymphoma (MCL). The genetic change in phosphatidylinositol 3′-kinase catalytic subunit α (PIK3CA) in MCL has not been identified. Experimental Design: Thirty-five primary MCL cases and 2 MCL cell lines (GRANTA-519 and Rec-1) were used to investigate somatic mutation and gene copy number of PIK3CA. Gene copy number was determined using quantitative real-time PCR and fluorescence in situ hybridization. We used quantitative real-time reverse transcription-PCR to measure PIK3CA transcription levels. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and phoshorylated AKT protein levels were analyzed using Western blotting and immunohistochemistry. Flow cytometry was used to assess apoptosis after treatment of MCL cell lines and one control cell line with LY294002, a specific inhibitor of PI3KCA. Results: Fifteen of 22 (68%) MCL cases and the MCL cell lines harbored a gain (≥3) of PIK3CA gene copy number. In addition, cases with increased PIK3CA gene copy number had elevated PIK3CA mRNA levels. Furthermore, amplification of PIK3CA correlated with the status of AKT phosphorylation in 7 of 12 (58%) primary MCL cases. Inhibition of PIK3CA induced increased apoptosis in the MCL cell lines. PTEN protein expression was present in all 14 primary MCL cases and cell lines by Western blotting, whereas 5 of 33 (15%) cases tested by immunohistochemistry had loss of PTEN expression. Conclusions: We conclude that a gain of gene copy number of PIK3CA is frequent genetic alteration that contributes to MCL progression. PIK3CA is a promising therapeutic target in MCL. (Clin Cancer Res 2009;15(18):5724–32)

Kallikrein-related peptidases (KLKs): a gene family of novel cancer biomarkers

Abstract Early diagnosis of cancer and early detection of relapse following surgery are critical for the effective treatment of the disease and for a positive clinical outcome. Identification of novel diagnostic, prognostic and predictive biomarkers will contribute utmost to clinical decision-making. The human tissue kallikrein and kallikrein-related peptidases (KLKs), encoded by the largest contiguous cluster of protease genes in the human genome, are secreted serine proteases with diverse expression patterns and physiological roles. The aberrant expression of KLKs in various malignancies as well as their involvement in many cancer-related processes, such as cell growth regulation, angiogenesis, invasion, and metastasis, has prompted scientists to investigate their potential as cancer biomarkers. Expression of distinct KLKs is associated with clinicopathological parameters of cancer patients. Moreover, several KLKs possess significant favorable or unfavorable prognostic value in various malignancies, with prostate-specific antigen (PSA) being the most widely used biomarker in clinical practice, today. KLKs are also considered as very promising biomarkers for cancer personalized medicine, especially for prediction and monitoring of patients’ response to chemotherapy, therefore opening up new horizons towards effective patient monitoring post-treatment. This review describes the current status of KLKs as tumor biomarkers.

Apoptosis-related BCL2-family Members: Key Players in Chemotherapy

Classical chemotherapeutic agents such as mitotic inhibitors (spindle poisons), alkylating agents, antimetabolites, topoisomerase inhibitors, and anthracenediones (anthracyclines) inhibit DNA synthesis and mitosis, thereby killing or impeding the proliferation of rapidly dividing cells. During the last decade, targeted therapy has gained advantage over conventional treatment regimens, as it is more effective against cancer and also much less harmful to normal cells, thus minimizing the side-effects of chemotherapy. This type of treatment blocks the proliferation of cancer cells by inhibiting the function of specific targeted molecules needed for tumor growth and metastasis. Targeted therapy agents include monoclonal antibodies and small-molecule inhibitors, which most commonly target receptor and/or non-receptor tyrosine kinases. Most members of the BCL2 apoptosis-related family regulate cellular fate as a response to antineoplastic agents. Modulations at the mRNA and protein levels of these genes are usually associated with sensitivity or resistance of various types of cancer cells to chemotherapeutic drugs. Moreover, alterations in expression of BCL2-family members, induced by anticancer drug treatment, can trigger or simply facilitate apoptosis. In this review, we summarize information about changes in apoptosis-related gene expression caused directly or indirectly by antineoplastic agents, as well as about the impact of BCL2-family members on the chemosensitivity or chemoresistance of cancer cells.

Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma

Purpose: Programmed death-ligand 1 (PD-L1; also known as CD274 or B7-H1) expression represents a mechanism of immune escape for cancer. Our purpose was to characterize tumor PD-L1 expression and associated T-cell infiltration in primary laryngeal squamous cell carcinomas (SCC). Experimental Design: A well-annotated cohort of 260 operable primary laryngeal SCCs [formalin-fixed paraffin-embedded (FFPE) specimens] was morphologically characterized for stromal tumor-infiltrating lymphocytes (TIL), on hematoxylin/eosin-stained whole sections and for PD-L1 mRNA expression by qRT-PCR in FFPE specimens. For PD-L1 protein expression, automated quantitative protein analysis (AQUA) was applied on tissue microarrays consisting of two cores from these tumors. In addition, PD-L1 mRNA expression in fresh-frozen tumors and normal adjacent tissue specimens was assessed in a second independent cohort of 89 patients with primary laryngeal SCC. Results: PD-L1 mRNA levels were upregulated in tumors compared with surrounding normal tissue (P = 0.009). TILs density correlated with tumor PD-L1 AQUA levels (P = 0.021). Both high TILs density and high PD-L1 AQUA levels were significantly associated with superior disease-free survival (DFS; TILs: P = 0.009 and PD-L1: P = 0.044) and overall survival (OS; TILs: P = 0.015 and PD-L1: P = 0.059) of the patients and retained significance in multivariate analysis. Conclusions: Increased TILs density and PD-L1 levels are associated with better outcome in laryngeal squamous cell cancer. Assessment of TILs and PD-L1 expression could be useful to predict response to immune checkpoint inhibitors. Clin Cancer Res; 22(3); 704–13. ©2015 AACR.

The Novel Member of the BCL2 Gene Family, BCL2L12, Is Substantially Elevated in Chronic Lymphocytic Leukemia Patients, Supporting Its Value As a Significant Biomarker

BCL2L12 is a recently identified gene belonging to the BCL2 family, members of which are implicated in hematologic malignancies, including chronic lymphocytic leukemia (CLL). The aim of this study was to analyze the mRNA expression of the novel apoptosis-related gene BCL2L12 in patients with CLL and to examine its prognostic and predictive value and potential clinical application as a novel molecular biomarker for CLL. For this purpose, total RNA was isolated from peripheral blood of 65 CLL patients and 23 healthy donors. An ultrasensitive quantitative real-time polymerase chain reaction methodology for BCL2L12 and BCL2 mRNA quantification was developed using SYBR Green chemistry. After preparing cDNA by reverse transcription, relative quantification analysis was performed using the comparative C(T) (2(-ΔΔCT)) method. Furthermore, analysis of IGHV mutational status, CD38 expression, and detection of early apoptosis by double staining with Annexin V-FITC and propidium iodide were performed. According to our findings, BCL2L12 mRNA expression is significantly higher in CLL patients than in healthy donors. Receiver operating characteristic analysis demonstrated that BCL2L12 expression had significant discriminatory value, distinguishing very efficiently CLL patients from the non-leukemic population. Moreover, BCL2L12 expression predicts the presence of CLL, as demonstrated by both univariate and multivariate logistic regression analyses. Finally, high BCL2L12 mRNA levels are associated with advanced clinical stage and predict shorter overall survival in CLL patients.

BCL2L12 is a Novel Biomarker for the Prediction of Short-Term Relapse in Nasopharyngeal Carcinoma

BCL2-like 12 (BCL2L12) is a new member of the apoptosis-related BCL2 gene family, members of which are implicated in various malignancies. Nasopharyngeal carcinoma is a highly metastatic, malignant epithelial tumor, with a high prevalence in South-east Asia and North Africa. The purpose of the current study was to quantify and investigate the expression levels of the BCL2L12 gene in nasopharyngeal carcinoma biopsies and to assess its prognostic value. Total RNA was isolated from 89 malignant and hyperplastic nasopharyngeal biopsies from Tunisian patients. After testing the quality of the extracted RNA, cDNA was prepared by reverse transcription. A highly sensitive real-time polymerase chain reaction (PCR) method for BCL2L12 mRNA quantification was developed using SYBR® Green chemistry. GAPDH served as a reference gene. Relative quantification analysis was performed using the comparative CT (2−ΔΔCT) method. Higher BCL2L12 mRNA levels were detected in undifferentiated carcinomas of the nasopharynx, rather than in nonkeratinizing nasopharyngeal tumors (P = 0.045). BCL2L12 expression status was also found to be positively associated with the presence of distant metastases (P = 0.014). Kaplan-Meier survival analysis demonstrated that patients with BCL2L12-positive nasopharyngeal tumors have significantly shorter disease-free survival (P = 0.020). Cox regression analysis showed BCL2L12 expression to be an unfavorable and independent prognostic indicator of short-term relapse in nasopharyngeal carcinoma (P = 0.042). Our results suggest that mRNA expression of BCL2L12 may constitute a novel biomarker for the prediction of short-term relapse in nasopharyngeal carcinoma.

Quantitative expression analysis and prognostic significance of the novel apoptosis-related gene BCL2L12 in colon cancer.

Abstract Apoptosis is a tightly regulated process that plays a critical role in many biological events. Members of the BCL2 (Bcl-2) family of apoptosis-related genes have been found to be differentially expressed in various malignancies and have been proposed as prognostic tumor biomarkers. We have recently discovered and cloned a new member of the BCL2 gene family, BCL2L12, expressed in colon tissues. Here we have investigated expression of the BCL2L12 gene in colon cancer tissues and assessed its prognostic value. Total RNA was isolated from 96 specimens of malignant colon tissue. After testing the RNA quality, cDNA was prepared by reverse transcription. A highly sensitive real-time PCR method for BCL2L12 mRNA quantification was developed using SYBR® Green chemistry. GAPDH served as a housekeeping gene. Relative quantification analysis was performed using the comparative CT method (2-ΔΔCT). High BCL2L12 expression levels were found in smaller (≤5 cm, p=0.027) and well-differentiated tumors (p=0.034), as well as in early-stage tumors (p=0.039). Survival analysis demonstrated that patients with BCL2L12-positive colon tumors have significantly longer disease-free survival and overall survival (p=0.015 and p=0.027, respectively). Our results suggest that BCL2L12 gene expression may represent a potential new biomarker for colon cancer.

Enhanced miR-182 transcription is a predictor of poor overall survival in colorectal adenocarcinoma patients.

Abstract Background: Colorectal cancer is the second most frequent cause of cancer-related death in the developed world. Recent studies have tried to associate colorectal cancer with the aberrant expression of several microRNAs. The aim of the present study was the development of a highly sensitive quantitative real-time PCR which can be used to evaluate the miR-182 expression levels in colorectal adenocarcinoma and adjacent non-cancerous tissue specimens and associate them with several clinicopathological characteristics, aiming to examine the prognostic potential of miR-182. Methods: Total RNA was isolated from 116 malignant colorectal adenocarcinoma specimens and 60 paired non-cancerous tissues. Then, polyadenylation of 2 μg total RNA by poly(A) polymerase and reverse transcription with suitable oligo-dT-adapter followed. miR-182 levels were quantified by real-time PCR based on SYBR Green chemistry. The results were analyzed by the comparative quantification cycle method and by extensive biostatistical analysis. Results: miR-182 was found to be significantly upregulated in colorectal adenocarcinoma specimens compared to their non-cancerous counterparts (p<0.001). miR-182 expression increases as the histological grade increases (p=0.013). miR-182 overexpression is associated with high depth of tumor invasion, positive regional lymph node status, and advanced TNM stage of patients. Therefore, miR-182 is an unfavorable prognostic marker in colorectal adenocarcinoma, predicting poor overall survival (p=0.007). Most importantly, miR-182 expression retained its unfavorable prognostic significance among patients with well- or moderately differentiated colorectal adenocarcinoma (p=0.006) and among metastasis-free patients (p=0.025). Conclusions: The increased levels of the oncogene-like miR-182 increase the risk for disease progression and predict poor overall survival for colorectal adenocarcinoma patients.

Expression analysis of mir-17-5p, mir-20a and let-7a microRNAs and their target proteins in CD34+ bone marrow cells of patients with myelodysplastic syndromes.

Mir-17-5p and mir-20a, members of the mir-17-92 family, down-regulate E2F1, which is over-expressed in myelodysplastic syndromes (MDS). Moreover, let-7a down-regulates KRAS, which is aberrantly expressed in MDS. We evaluated the expression of the aforementioned microRNAs in CD34+ cells of 43 MDS patients using real-time PCR and their target proteins (E2F1, MYC, BCL2, CCND1, and KRAS) by Western blot. Mir-17-5p and mir-20a were under expressed in high risk MDS patients, compared to low risk MDS patients. Similarly, let-7a was under expressed in patients with intermediate or high-risk karyotype. Interestingly, there was an inverse correlation between microRNA and the expression levels of their targets. Importantly, mir-17-5p and mir-20a constitute favorable prognostic factors in MDS, since their expression was associated with increased overall survival of MDS patients.

Molecular cloning of novel alternatively spliced variants of BCL2L12, a new member of the BCL2 gene family, and their expression analysis in cancer cells

In the past, we identified and cloned the BCL2-like 12 (BCL2L12) gene, a novel member of the BCL2 family, which is implicated in various malignancies. The classical BCL2L12 protein isoform contains a highly conserved BH2 domain, a BH3-like motif, and a proline-rich region, and is involved in apoptosis. Most members of this apoptosis-related family are subjected to alternative splicing, thus generating multiple protein isoforms with distinct properties, and sometimes even with opposite function (pro- vs. anti-apoptotic). In the current study, we report the identification, molecular cloning, and expression pattern of novel splice variants of the human BCL2L12 gene in cancer cell lines. EST clones displaying high sequence identity (≥90%) with the classical BCL2L12 transcript were aligned, in order to identify those containing at least one novel splice junction. EST database mining led to the identification of three previously unknown splice variants of this apoptotic gene. In our effort to experimentally validate these novel transcripts, we also cloned seven more, previously unidentified, BCL2L12 alternatively spliced variants. Expression analysis of all BCL2L12 splice variants in human cancer cell lines and embryonic kidney cells revealed remarkable differences between their BCL2L12 expression profiles. Interestingly, 7 out of 10 novel splice variants of BCL2L12 are predicted to encode new protein isoforms, some of which are BH3-only proteins, in contrast to the classical BCL2L12 isoform, which also contains a functional BH2 domain. The remaining three novel splice variants of BCL2L12 are nonsense-mediated mRNA decay (NMD) candidates.