Biological tumor markers associated with local control after primary radiotherapy in laryngeal cancer:A systematic review

Background The choice of treatment in laryngeal cancer is mainly based on tumor stage, post-treatment morbidity and quality of life. Biological tumor markers might also be of potential clinical relevance. Objective of the review The aim was to systematically review the value of published biological tumor markers to predict local control in laryngeal cancer patients treated with definitive radiotherapy. Type of Review Systematic review. Search strategy PubMed, Embase, Cochrane Library. Evaluation Method A literature search was performed using multiple terms for laryngeal cancer, radiotherapy, biological markers, detection methods and local control or survival. Studies regarding the relation between biological tumor markers and local control or survival in laryngeal cancer patients primarily treated with radiotherapy were included. Markers were clustered on biological function. Quality of all studies was assessed. Study selection, data extraction and quality assessment was performed by two independent reviewers. Results A total of 52 studies out of 618 manuscripts, concerning 118 markers, were included. EGFR and P53 showed consistent evidence for not being predictive of local control after primary radiotherapy, whereas proliferation markers (ie high Ki-67 expression) showed some, but no consistent, evidence for being predictive of better local control. Other clusters of markers (markers involved in angiogenesis and hypoxia, apoptosis markers, cell cycle, COX-2 and DNA characteristics) showed no consistent evidence towards being predictors of local control after primary radiotherapy. Conclusions Cell proliferation could be of potential interest for predicting local control after primary radiotherapy in laryngeal cancer patients, whereas EGFR and p53 are not predictive in contrast to some previous analyses. Large diversity in research methods is found between studies, which results in contradictory outcomes. Future studies need to be more standardised and well described according to the REMARK criteria in order to have better insight into which biomarkers can be used as predictors of local control after primary radiotherapy

Standardised Ki-67 proliferation index assessment in early-stage laryngeal squamous cell carcinoma in relation to local control and survival after primary radiotherapy

ObjectivesAmbiguous results have been reported on the predictive value of the Ki-67 proliferation index (Ki-67 PI) regarding local control (LC) and survival after primary radiotherapy (RT) in early-stage laryngeal squamous cell cancer (LSCC). Small study size, heterogenic inclusion, variations in immunostaining and cut-off values are attributing factors. Our aim was to elucidate the predictive value of the Ki-67 PI for LC and disease-specific survival (DSS) using a well-defined series of T1-T2 LSCC, standardised automatic immunostaining and digital image analysis (DIA). MethodsA consecutive and well-defined cohort of 208 patients with T1-T2 LSCC treated with primary RT was selected. The Ki-67 PI was determined using DIA. Mann-Whitney U-tests, logistic and Cox regression analyses were performed to assess associations between Ki-67 PI, clinicopathological variables, LC and DSS. ResultsIn multivariate Cox regression analysis, poor tumour differentiation (HR 2.20; 95% CI 1.06-4.59, P = .04) and alcohol use (HR 2.84, 95% CI 1.20-6.71; P = .02) were independent predictors for LC. Lymph node positivity was an independent predictor for DSS (HR 3.16, 95% CI 1.16-8.64; P = .03). Ki-67 PI was not associated with LC (HR 1.59; 95% CI 0.89-2.81; P = .11) or DSS (HR 0.98; 95% CI 0.57-1.66; P = .97). In addition, continuous Ki-67 PI was not associated with LC (HR 2.03; 95% CI 0.37-11.14, P = .42) or DSS (HR 0.62; 95% CI 0.05-8.28; P = .72). ConclusionThe Ki-67 PI was not found to be a predictor for LC or DSS and therefore should not be incorporated in treatment-related decision-making for LSCC

The role of ATM and 53BP1 as predictive markers in cervical cancer

Treatment of advanced-stage cervical cancers with (chemo)radiation causes cytotoxicity through induction of high levels of DNA damage. Tumour cells respond to DNA damage by activation of the DNA damage response (DDR), which induces DNA repair and may counteract chemoradiation efficacy. Here, we investigated DDR components as potential therapeutic targets and verified the predictive and prognostic value of DDR activation in patients with cervical cancer treated with (chemo)radiation. In a panel of cervical cancer cell lines, inactivation of ataxia telangiectasia mutated (ATM) or its substrate p53-binding protein-1 (53BP1) clearly gave rise to cell cycle defects in response to irradiation. Concordantly, clonogenic survival analysis revealed that ATM inhibition, but not 53BP1 depletion, strongly radiosensitised cervical cancer cells. In contrast, ATM inhibition did not radiosensitise non-transformed epithelial cells or non-transformed BJ fibroblasts. Interestingly, high levels of active ATM prior to irradiation were related with increased radioresistance. To test whether active ATM in tumours prior to treatment also resulted in resistance to therapy, immunohistochemistry was performed on tumour material of patients with advanced-stage cervical cancer (n = 375) treated with (chemo)radiation. High levels of phosphorylated (p-)ATM [p = 0.006, hazard ratio (HR) = 1.817] were related to poor locoregional disease-free survival. Furthermore, high levels of p-ATM predicted shorter disease-specific survival (p = 0.038, HR = 1.418). The presence of phosphorylated 53BP1 was associated with p-ATM (p = 0.001, odds ratio = 2.206) but was not related to any clinicopathological features or survival. In conclusion, both our in vitro and patient-related findings indicate a protective role for ATM in response to (chemo)radiation in cervical cancer and point at ATM inhibition as a possible means to improve the efficacy of (chemo)radiation

Clinical and public health research using methylated DNA immunoprecipitation (MeDIP) A comparison of commercially available kits to examine differential DNA methylation across the genome

The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp (TM) Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies

Immunoblotting analysis of phospo-AKT , total-AKT, phospo-PI3K, total- NF-κB and β-actin.

<p>In <b>A.</b> SiHa and CaSki cell lines 48 hours after transient transfection of OGDHL and empty vector; and <b>B.</b> HeLa and ME180 cell lines after OGDHL siRNA and scramble siRNA transfection; OGDHL inhibition by OGDHL siRNA has dramatic effect on total AKT and phospho-AKT level <b>C.</b> Immunoblotting analysis of phospo-NF-κB and total-NF-κB of SiHa cell lines 48 hours after OGDHL and empty vector transfection; β-actin and lamin was used as a loading control for cytosolic and nuclear fraction respectively; NF-κB translocation from cytoplasm towards the nucleus was decreased after OGDHL overexpression in SiHa cells; <b>D.</b> NF-κB gel shift assay in SiHa and HeLa cell lines; OGDHL overexpression in SiHa cells decreased the DNA binding activity of NF-κB compared to that of empty vector control; OGDHL siRNA increase the binding activity of NF-κB in HeLa cell; <b>E.</b> NF-κB luciferase assay in SiHa (upper panel) and CaSki (lower panel) cell lines after transient over-expression of OGDHL with or without AKT1 over-expression. It is evident that OGDHL suppresses AKT activity and lead to inhibition of NF-κB-dependent gene transcription (* p<0.001). <b>F.</b> Immunoblotting analysis of phospho- NF-κB, total- NF-κB, β-actin and lamin in nuclear and cytosolic fractions of SiHa cell lines after OGDHL over-expression with or without AKT1 overexpression. The data showed that co-transfection of OGDHL and AKT1 partially increased the translocation of NF-κB from the cytoplasm to the nucleus.</p

Relationship among OGDHL expression, ROS production and alterations of AKT pathway genes.

<p><b>A.</b> Immunoblotting analysis of total-AKT (T-AKT), total NF-κB (T -NF-κB), pro and cleaved caspase 3, cleaved PARP1/2 in SiHa cell line 48 hours after OGDHL over-expression in the presence or absence of ROS (N-acetyl l-cysteine or NAC, 2.5 mM), Lipid-peroxidation (butylated hydroxytoluene or BHT, 0.2 mM) and caspase 3 (DEVD-FMK, 25 µM) inhibitor. β-actin was used as a loading control. All the inhibitors protect AKT from getting cleaved. ROS and lipid peroxidation inhibitors have protective effect on caspase 3 activation; <b>B.</b> MTT assay of SiHa cell line 48 hours after OGDHL or AKT1 over-expression in the presence or absence of NAC (ROS inhibitor), BHT (Lipid peroxidation inhibitor) or DEVD-FMK (caspase 3 inhibitor); NAC, BHT and DEVD-FMK significantly prevented cell death induced by OGDHL; <b>C.</b> MTT assay of HeLa cell line after OGDHL knockdown with or without AKT1 knockdown. AKT1 down-regulation by AKT1 siRNA decreased HeLa cell survival that was induced by knockdown of OGDHL (*<i>P</i><0.05).</p

Phenotypic alterations of cells after forceful expression of OGDHL.

<p><b>A....... </b><b>MTT assay</b>: Significantly decreased numbers of SiHa and CaSki cells were observed after forceful expression of OGDHL in a time dependent manner (after 0, 24, 48 and 72 h of transfection). Cell growth rate is expressed as absorbance at 570 to 650 nm (<b>*</b><b><i>P</i></b><b><0.001</b>).; <b>B....... </b><b>BrdU assay</b>: OGDHL inhibit cell proliferation of SiHa and Caski cells as detected by DNA synthesis (*<i>P</i><0.001 and 0.001 for SiHa and CaSki cells respectively); <b>C....... </b><b>Tunel assay</b>: Forty-eight hours after transfection of OGDHL, the ratio of apoptotic versus total number of cells was evaluated by TUNEL assay. Significant differences were observed between OGDHL and mock transfected cells (*<i>P</i><0.001 and 0.001 for SiHa and CaSki cell lines respectively); <b>D....... </b><b>Caspase 3 assay</b>: Caspase-3 activity was measured using the Caspase colorimetric assay kit in SiHa and CaSki cells 48 hours after mock or OGDHL-transfection. (<i>*P</i><0.001,<i>**P</i><0.001 respectively); <b>E....... </b><b>Immunoblot analysis</b>: Cleaved Caspase 3 and PARP1/2 were observed in SiHa and CaSki cell lines after overexpression of OGDHL; <b>F....... </b><b>Colony formation assay</b>: The effect of exogenous OGDHL expression in colony formation of SiHa cell line. The SiHa cell line was transfected with constructs encoding OGDHL or the empty vector. Cells were harvested 24 h after transfection, and equal cell numbers were seeded in 100 mm dishes and grown under selection in G418 for 21 days. The representative photograph after 21 days (left) and the quantification of the number of G418 selected SiHa cell colonies counted in 3 plates (right). The vector control was set at 100%. The data represent the mean ±SD of three independent experiments, each done in triplicate. <b>G....... </b><b>Invasive assay</b>: SiHa cells 48 hours after transient transfection of OGDHL and empty vector. Cells that invaded the polycarbonate membrane of transwell chamber (Left). The number of cells that invaded the polycarbonate membrane of transwell chamber (right) (*<i>P</i><0.001). The data represent the mean ±SD of three independent experiments, each done in triplicate.</p

Phenotypic alterations of cells after siRNA mediated down-regulatin of OGDHL.

<p><b>A..... </b><b>MTT assay:</b> MTT assay 48 hours after transient transfection of OGDHL siRNA in HeLa and ME180 cell lines; Cell growth rate is expressed as absorbance at 570 to 650 nm <b>B..... </b><b>BrdU assay:</b> OGDHL siRNA increased cell proliferation of HeLa and ME180 cells as detected by DNA synthesis (*<i>P</i><0.001 and 0.001 for HeLa and ME180 cells respectively); <b>C..... </b><b>Caspase 3 assay:</b> Caspase-3 activities in HeLa and ME180 cells were measured in mock or 48 hours after OGDHL siRNA-transfected cells using the Caspase colorimetric assay kit. *<i>P</i><0.001. <b>D..... </b><b>Immunoblot analysis</b>: Immunoblotting analysis of Caspase 3 and PARP1/2 in HeLa and ME180 cell lines after siRNA mediated knockdown of OGDHL and <b>E..... </b><b>Invasive assay</b>: Invasive assay with HeLa cells 48 hours after transient down-regulation of OGDHL by siRNA and scramble siRNA (control). Representative photograph of HeLa cells that invaded the polycarbonate membrane of transwell chamber (Left). The number of HeLa cells that invaded the polycarbonate membrane of transwell chamber were significantly more when OGDHL expression were inhibited by siRNA (* <i>P</i><0.001) (right). The data represent the mean ±SD of three independent experiments, each done in triplicate.</p

Tumor suppressive properties of OGDHL using stable clone.

<p><b>A.</b> RT-PCR analysis of SiHa cells stably transfected with the mammalian expression vector pcDNA3 with full-length OGDHL cDNA and empty pcDNA3 vector (mock). All the three OGDHL clones (clone number at the bottom) containing full-length OGDHL cDNA expressed OGDHL at various levels. There is a very low level endogenous OGDHL expression in mock-transfected clones. <b>B.</b> Immunoblot analysis of Flag, OGDHL and β-actin. All the three OGDHL over-expressing clones showing Flag tagged with OGDHL and OGDHL expression while no Flag-OGDHL and OGDHL expression in empty vector stable clones (clone number at the bottom). <b>C.</b> Soft agar assay: number of soft agar colonies was significantly lower in the OGDHL over-expressed cells compared with empty vector-transfected cells (<i>*P</i><0.001) (Bottom). Magnification, ×100 in representative photograph (Top). <b>D.</b> Invasion assay: Compared with empty vector, stable OGDHL over-expressing SiHa cell clone showed significantly less number of invading cells (*<i>P</i><0.001) (right). Magnification, ×100 in representative photograph (left. Each experiment was repeated twice. In general all the findings are consistent with the transient transfection data; <b>E.</b> Immunoblotting analysis of phospho-AKT, total-AKT, phospho-PI3K, total-NF-κB and β-actin in empty vector (left lane) and OGDHL over-expressing stable SiHa cell clone (right lane) and the findings are similar to transient transfection. <b>F.</b> NF-κB gel shift assay in SiHa cell lines stably transfected with empty vector or vector containing OGDHL-Flag indicate that OGDHL inhibited the binding of NF-κB to the nuclear DNA. <b>G.</b> Inhibition of OGDHL mediated ROS generation by over-expression of Mn-SOD increases the viable cells determined by MTT assay (*<i>P</i><0.05) (left panel) and this increase number of cells is partially due to decrease ROS production as shown in right panel (*<i>P</i><0.05).</p