ABSTRACT
Background: Non-small cell lung cancer (NSCLC), which accounts for about 80%-85% of lungcancer cases, is a leading cause of cancer-related death worldwide. Lorlatinib is a potent third-generation anaplastic lymphoma kinase (ALK) inhibitor approved for the treatment of patients with advanced, ALK-positive NSCLC previously treated with at least one second-generation ALK tyrosine kinase inhibitor. Objective: The present study assessed the cost-effectiveness of lorlatinib vs pemetrexed with platinum combination of carboplatin or cisplatin (P-ChT) in Greece. Methods: A partitioned survival model with three health states, referring to pre-progression, progressed disease, and death, was locally adapted from a Greek payer perspective over a lifetime horizon. Clinical and safety data and utility values applied in the model were extracted from the literature. A matching-adjusted indirect comparison of lorlatinib and P-ChT was performed. Only direct medical costs () from 2020 were included in the analysis. Primary outcomes were patient life years (LYs), quality-adjusted life years (QALYs), total costs, and incremental cost-effectiveness ratios per QALY and LY gained. All future outcomes were discounted at 3.5% per annum. A probabilistic sensitivity analysis was conducted to account for model uncertainty. Results: The analysis showed that, over a lifetime horizon, the estimated total costs of lorlatinib and P-ChT were 81â¯754 and 12â¯343, respectively. Lorlatinib was more effective than P-ChT with 2.4 and 1.5 more LYs and QALYs gained, respectively. The generated incremental cost-effectiveness ratios of lorlatinib compared with P-ChT were 28â¯613 per LY gained and 46â¯102 per QALY gained. Probabilistic sensitivity analysis confirmed the deterministic results. Conclusion: The present analysis suggests that lorlatinib may be considered as a cost-effective option compared with P-ChT in Greece for the treatment of patients with advanced, ALK-positive NSCLC whose disease has progressed after at least one second-generation ALK tyrosine kinase inhibitor. In addition, this option addresses a significant unmet medical need.
ABSTRACT
BACKGROUND: Neuroblastoma (NB) often presents with metastatic disease and poor survival. The need for new prognostic markers remains invaluable. The FAK-Src-Paxillin protein system is associated with aggressive phenotype in adult malignancies but is largely unexplored in pediatric NB. OBJECTIVE: To assess FAK-Src-Paxillin protein expression in human NB cell lines and clinical cytology material and to delineate its association with survival. DESIGN/METHODS: Western blot and immunohistochemistry were applied for FAK-Src-Paxillin expression in NB cell lines and 23 human cytology specimens, respectively. Protein expression in human clinical samples was correlated with clinicopathological parameters, MYCN amplification and survival. RESULTS: FAK, Src and Paxillin proteins are expressed in human NB cells lines, and can be detected in clinical cytology specimens from NB patients, (59%, 32% and 33% respectively). Simultaneous FAK-Src-Paxillin expression was noted in 30% of NB patients. Children with concomitant positivity FAK, Src, and Paxillin tumors, as well as MYCN amplification, had increased mortality compared to those without. CONCLUSIONS: FAK-Src-Paxillin system is a marker of unfavorable prognosis for human NB patients but also a promising therapeutic target.
Subject(s)
Biomarkers, Tumor/biosynthesis , Focal Adhesion Kinase 1/biosynthesis , Gene Expression Regulation, Neoplastic , Neuroblastoma , Paxillin/biosynthesis , Proto-Oncogene Proteins pp60(c-src)/biosynthesis , Animals , Child , Child, Preschool , Disease-Free Survival , Female , Humans , Infant , K562 Cells , Male , Mice , N-Myc Proto-Oncogene Protein/biosynthesis , NIH 3T3 Cells , Neuroblastoma/metabolism , Neuroblastoma/mortality , Neuroblastoma/pathology , Survival RateABSTRACT
Clinically useful molecular tools to triage gastric cancer patients are not currently available. We aimed to develop a molecular tool to predict gastric cancer risk in endoscopy-driven biopsies obtained from high-risk gastric cancer clinics in low resource settings.We discovered and validated a DNA methylation biomarker panel in endoscopic samples obtained from 362 patients seen between 2004 and 2009 in three high-risk gastric cancer clinics in Lima, Perú, and validated it in 306 samples from the Cancer Genome Atlas project ("TCGA"). Global, epigenome wide and gene-specific DNA methylation analyses were used in a Phase I Biomarker Development Trial to identify a continuous biomarker panel that combines a Global DNA Methylation Index (GDMI) and promoter DNA methylation levels of IRF4, ELMO1, CLIP4 and MSC.We observed an inverse association between the GDMI and histological progression to gastric cancer, when comparing gastritis patients without metaplasia (mean = 5.74, 95% CI, 4.97-6.50), gastritis patients with metaplasia (mean = 4.81, 95% CI, 3.77-5.84), and gastric cancer cases (mean = 3.38, 95% CI, 2.82-3.94), respectively (p < 0.0001). Promoter methylation of IRF4 (p < 0.0001), ELMO1 (p < 0.0001), CLIP4 (p < 0.0001), and MSC (p < 0.0001), is also associated with increasing severity from gastritis with no metaplasia to gastritis with metaplasia and gastric cancer.Our findings suggest that IRF4, ELMO1, CLIP4 and MSC promoter methylation coupled with a GDMI>4 are useful molecular tools for gastric cancer risk stratification in endoscopic biopsies.
Subject(s)
Adenocarcinoma/diagnosis , Biomarkers, Tumor/genetics , Early Detection of Cancer/methods , Stomach Neoplasms/diagnosis , Adaptor Proteins, Signal Transducing/genetics , Adenocarcinoma/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Basic Helix-Loop-Helix Transcription Factors/genetics , Biopsy , Carrier Proteins/genetics , DNA Methylation/genetics , Female , Gastroscopy , Genome-Wide Association Study , Humans , Interferon Regulatory Factors/genetics , Male , Membrane Proteins , Middle Aged , Stomach Neoplasms/genetics , Young AdultABSTRACT
Microbiome studies show altered microbiota in head and neck squamous cell carcinoma (HNSCC), both in terms of taxonomic composition and metabolic capacity. These studies utilized a traditional bioinformatics methodology, which allows for accurate taxonomic assignment down to the genus level, but cannot accurately resolve species level membership. We applied Resphera Insight, a high-resolution methodology for 16S rRNA taxonomic assignment that is able to provide species-level context in its assignments of 16S rRNA next generation sequencing (NGS) data. Resphera Insight applied to saliva samples from HNSCC patients and healthy controls led to the discovery that a subset of HNSCC saliva samples is significantly enriched with commensal species from the vaginal flora, including Lactobacillus gasseri/johnsonii (710x higher in saliva) and Lactobacillus vaginalis (52x higher in saliva). These species were not observed in normal saliva from Johns Hopkins patients, nor in 16S rRNA NGS saliva samples from the Human Microbiome Project (HMP). Interestingly, both species were only observed in saliva from Human Papilloma Virus (HPV) positive and HPV negative oropharyngeal cancer patients. We confirmed the representation of both species in HMP data obtained from mid-vagina (n=128) and vaginal introitus (n=121) samples. Resphera Insight also led to the discovery that Fusobacterium nucleatum, an oral cavity flora commensal bacterium linked to colon cancer, is enriched (600x higher) in saliva from a subset of HNSCC patients with advanced tumors stages. Together, these high-resolution analyses on 583 samples suggest a possible role for bacterial species in the therapeutic outcome of HPV positive and HPV negative HNSCC patients.
ABSTRACT
Systemic inflammatory events and localized disease, mediated by the microbiome, may be measured in saliva as head and neck squamous cell carcinoma (HNSCC) diagnostic and prognostic biomonitors. We used a 16S rRNA V3-V5 marker gene approach to compare the saliva microbiome in DNA isolated from Oropharyngeal (OPSCC), Oral Cavity Squamous Cell Carcinoma (OCSCC) patients and normal epithelium controls, to characterize the HNSCC saliva microbiota and examine their abundance before and after surgical resection.The analyses identified a predominance of Firmicutes, Proteobacteria and Bacteroidetes, with less frequent presence of Actinobacteria and Fusobacteria before surgery. At lower taxonomic levels, the most abundant genera were Streptococcus, Prevotella, Haemophilus, Lactobacillus and Veillonella, with lower numbers of Citrobacter and Neisseraceae genus Kingella. HNSCC patients had a significant loss in richness and diversity of microbiota species (p<0.05) compared to the controls. Overall, the Operational Taxonomic Units network shows that the relative abundance of OTU's within genus Streptococcus, Dialister, and Veillonella can be used to discriminate tumor from control samples (p<0.05). Tumor samples lost Neisseria, Aggregatibacter (Proteobacteria), Haemophillus (Firmicutes) and Leptotrichia (Fusobacteria). Paired taxa within family Enterobacteriaceae, together with genus Oribacterium, distinguish OCSCC samples from OPSCC and normal samples (p<0.05). Similarly, only HPV positive samples have an abundance of genus Gemellaceae and Leuconostoc (p<0.05). Longitudinal analyses of samples taken before and after surgery, revealed a reduction in the alpha diversity measure after surgery, together with an increase of this measure in patients that recurred (p<0.05). These results suggest that microbiota may be used as HNSCC diagnostic and prognostic biomonitors.
Subject(s)
Carcinoma, Squamous Cell , Microbiota/genetics , Mouth Neoplasms , Oral Surgical Procedures , Papillomavirus Infections/genetics , RNA, Ribosomal, 16S/genetics , Aged , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/microbiology , Carcinoma, Squamous Cell/surgery , Carcinoma, Squamous Cell/virology , Cohort Studies , Female , Gene Amplification , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/microbiology , Head and Neck Neoplasms/surgery , Head and Neck Neoplasms/virology , Humans , Longitudinal Studies , Male , Middle Aged , Mouth Neoplasms/genetics , Mouth Neoplasms/microbiology , Mouth Neoplasms/surgery , Mouth Neoplasms/virology , Papillomaviridae/genetics , Papillomavirus Infections/microbiology , Papillomavirus Infections/pathology , Papillomavirus Infections/surgery , Prognosis , Risk Factors , Squamous Cell Carcinoma of Head and NeckABSTRACT
Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor's formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient's age and tumor's clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adenocarcinoma/metabolism , Adenomatous Polyposis Coli Protein/metabolism , Cadherins/metabolism , Colonic Neoplasms/metabolism , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenomatous Polyposis Coli Protein/genetics , Aged , Antigens, CD , Cadherins/genetics , Colon/pathology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA Methylation , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Male , MutL Protein Homolog 1 , Nuclear Proteins/genetics , Polymerase Chain Reaction , Promoter Regions, Genetic , Tumor Suppressor Proteins/geneticsABSTRACT
Using high-throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus-related (HPV+) and HPV- HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF-κB and p53. Immunohistochemical analysis confirmed that HPV- HNSCC is characterized by co-activated STAT3 and NF-κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti-NF-κB and anti-STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF-κB and AP1 in HNSCC. We identified five top-scoring pair biomarkers from STATs, NF-κB and AP1 pathways that distinguish HPV+ from HPV- HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC.
Subject(s)
Carcinoma, Squamous Cell/genetics , Head and Neck Neoplasms/genetics , NF-kappa B/genetics , Papillomavirus Infections/genetics , STAT3 Transcription Factor/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/virology , Cell Line, Tumor , DNA Methylation , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/metabolism , Head and Neck Neoplasms/virology , Humans , NF-kappa B/metabolism , Oligonucleotide Array Sequence Analysis , Papillomavirus Infections/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Squamous Cell Carcinoma of Head and NeckABSTRACT
Exposure to toxicants leads to cumulative molecular changes that overtime increase a subject's risk of developing urothelial carcinoma. To assess the impact of arsenic exposure at a time progressive manner, we developed and characterized a cell culture model and tested a panel of miRNAs in urine samples from arsenic-exposed subjects, urothelial carcinoma patients, and controls. To prepare an in vitro model, we chronically exposed an immortalized normal human bladder cell line (HUC1) to arsenic. Growth of the HUC1 cells was increased in a time-dependent manner after arsenic treatment and cellular morphology was changed. In a soft agar assay, colonies were observed only in arsenic-treated cells, and the number of colonies gradually increased with longer periods of treatment. Similarly, invaded cells in an invasion assay were observed only in arsenic-treated cells. Withdrawal of arsenic treatment for 2.5 months did not reverse the tumorigenic properties of arsenic-treated cells. Western blot analysis demonstrated decreased PTEN and increased AKT and mTOR in arsenic-treated HUC1 cells. Levels of miR-200a, miR-200b, and miR-200c were downregulated in arsenic-exposed HUC1 cells by quantitative RT-PCR. Furthermore, in human urine, miR-200c and miR-205 were inversely associated with arsenic exposure (P = 0.005 and 0.009, respectively). Expression of miR-205 discriminated cancer cases from controls with high sensitivity and specificity (AUC = 0.845). Our study suggests that exposure to arsenic rapidly induces a multifaceted dedifferentiation program and miR-205 has potential to be used as a marker of arsenic exposure as well as a maker of early urothelial carcinoma detection.
Subject(s)
Arsenic/adverse effects , Cell Transformation, Neoplastic/pathology , Epigenesis, Genetic/genetics , Epithelial-Mesenchymal Transition , MicroRNAs/analysis , Urinary Bladder Neoplasms/pathology , Urinary Bladder/pathology , Adult , Aged , Aged, 80 and over , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Case-Control Studies , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic/chemically induced , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Cohort Studies , DNA Methylation , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoenzyme Techniques , Male , MicroRNAs/genetics , Middle Aged , Neoplasm Invasiveness , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Urinary Bladder/drug effects , Urinary Bladder/metabolism , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/genetics , Young AdultABSTRACT
By a candidate gene approach, we analyzed the promoter methylation (PM) of 8 genes genes (ARF, TIMP3, RAR-ß2, NID2, CCNA1, AIM1, CALCA and CCND2) by quantitative methylation specific PCR (QMSP) in DNA of 17 non-recurrent and 19 recurrent noninvasive low grade papillary urothelial cell carcinoma (LGPUCC) archival tissues. Among the genes tested, by establishing an empiric cutoff value, CCND2, CCNA1, NID2, and CALCA showed higher frequency of methylation in recurrent than in non-recurrent LGPUCC: CCND2 10/19 (53%) vs. 2/17 (12%) (p=0.014); CCNA1 11/19 (58%) vs. 4/17 (23.5%) (p=0.048); NID2 13/19 (68%) vs. 3/17 (18%) (p=0.003) and CALCA 10/19 (53%) vs. 4/17 (23.5%) (p=0.097), respectively. We further analyzed PM of CCND2, CCNA1, and CALCA in urine DNA from UCC patients including LGPUCC and controls. The frequency of CCND2, CCNA1 and CALCA was significantly higher (p<0.0001) in urine of UCC cases [ 38/148 (26%), 50/73 (68%) and 94/148 (63.5%) respectively] than controls [0/56 (0%), 10/60 (17%) and 16/56 (28.5%), respectively)]. Most importantly we found any one of the 3 markers methylation positive in 25 out of 30 (83%) cytology negative LGPUCC cases. We also explored the biological function of CCNA1 in UCC. Prospective confirmatory studies are needed to develop a reliable tool for prediction of recurrence using primary LGPUCC tissues and/or urine.
Subject(s)
Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/urine , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/urine , Adult , Aged , Aged, 80 and over , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Biomarkers, Tumor/genetics , Case-Control Studies , Cyclin A1/genetics , Cyclin D2/genetics , DNA Methylation , Decitabine , Epigenesis, Genetic , Female , Genetic Predisposition to Disease , Humans , Hydroxamic Acids/pharmacology , Male , Middle Aged , Neoplasm Grading , Predictive Value of Tests , Promoter Regions, Genetic , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/surgeryABSTRACT
Tumor suppressor genes (TSGs) are commonly inactivated by somatic mutation and/or promoter methylation; yet, recent high-throughput genomic studies have not identified key TSGs inactivated by both mechanisms. We pursued an integrated molecular analysis based on methylation binding domain sequencing (MBD-seq), 450K Methylation arrays, whole exome sequencing, and whole genome gene expression arrays in primary head and neck squamous cell carcinoma (HNSCC) tumors and matched uvulopalatopharyngoplasty tissue samples (UPPPs). We uncovered 186 downregulated genes harboring cancer specific promoter methylation including PAX1 and PAX5 and we identified 10 key tumor suppressor genes (GABRB3, HOXC12, PARP15, SLCO4C1, CDKN2A, PAX1, PIK3AP1, HOXC6, PLCB1, and ZIC4) inactivated by both promoter methylation and/or somatic mutation. Among the novel tumor suppressor genes discovered with dual mechanisms of inactivation, we found a high frequency of genomic and epigenomic alterations in the PAX gene family of transcription factors, which selectively impact canonical NOTCH and TP53 pathways to determine cell fate, cell survival, and genome maintenance. Our results highlight the importance of assessing TSGs at the genomic and epigenomic level to identify key pathways in HNSCC, deregulated by simultaneous promoter methylation and somatic mutations.
Subject(s)
Carcinoma, Squamous Cell/genetics , DNA Methylation , Gene Silencing , Genes, Tumor Suppressor , Head and Neck Neoplasms/genetics , Promoter Regions, Genetic , Carcinoma, Squamous Cell/metabolism , Cell Line, Tumor , Cohort Studies , CpG Islands , Female , Head and Neck Neoplasms/metabolism , Humans , Male , Mutation , PAX5 Transcription Factor/genetics , PAX5 Transcription Factor/metabolism , Paired Box Transcription Factors/genetics , Paired Box Transcription Factors/metabolism , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
The majority of the epigenomic reports in hepatocellular carcinoma have focused on identifying novel differentially methylated drivers or passengers of the oncogenic process. Few reports have considered the technologies in place for clinical translation of newly identified biomarkers. The aim of this study was to identify epigenomic technologies that need only a small number of samples to discriminate HCC from non-HCC tissue, a basic requirement for biomarker development trials. To assess that potential, we used quantitative Methylation Specific PCR, oligonucleotide tiling arrays, and Methylation BeadChip assays. Concurrent global DNA hypomethylation, gene-specific hypermethylation, and chromatin alterations were observed as a hallmark of HCC. A global loss of promoter methylation was observed in HCC with the Illumina BeadChip assays and the Nimblegen oligonucleotide arrays. HCC samples had lower median methylation peak scores and a reduced number of significant promoter-wide methylated probes. Promoter hypermethylation of RASSF1A, SSBP2, and B4GALT1 quantified by qMSP had a sensitivity ranging from 38% to 52%, a specificity of 100%, and an AUC from 0.58 to 0.75. A panel combining these genes with HCC risk factors had a sensitivity of 87%, a specificity of 100%, and an AUC of 0.91.
ABSTRACT
Although specific mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) identify tumors that are responsive to EGFR tyrosine kinase inhibitors (TKI), these genetic alterations are present in only a minority of patients. Patients with tumors expressing wild-type EGFR lack reliable predictive markers of their clinical response to EGFR TKIs. Although epithelial-mesenchymal transition (EMT) has been inversely correlated with the response of cancers to EGFR-targeted therapy, the precise molecular mechanisms underlying this association have not been defined and no specific EMT-associated biomarker of clinical benefit has been identified. Here, we show that during transforming growth factor ß (TGFß)-mediated EMT, inhibition of the microRNAs 200 (miR200) family results in upregulated expression of the mitogen-inducible gene 6 (MIG6), a negative regulator of EGFR. The MIG6-mediated reduction of EGFR occurs concomitantly with a TGFß-induced EMT-associated kinase switch of tumor cells to an AKT-activated EGFR-independent state. In a panel of 25 cancer cell lines of different tissue origins, we find that the ratio of the expression levels of MIG6 and miR200c is highly correlated with EMT and resistance to erlotinib. Analyses of primary tumor xenografts of patient-derived lung and pancreatic cancers carrying wild-type EGFR showed that the tumor MIG6(mRNA)/miR200 ratio was inversely correlated with response to erlotinib in vivo. Our data demonstrate that the TGFß-miR200-MIG6 network orchestrates the EMT-associated kinase switch that induces resistance to EGFR inhibitors, and identify a low ratio of MIG6 to miR200 as a promising predictive biomarker of the response of tumors to EGFR TKIs.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm , Epithelial-Mesenchymal Transition , ErbB Receptors/antagonists & inhibitors , MicroRNAs/genetics , Protein Kinase Inhibitors/pharmacology , Transforming Growth Factor beta/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Line, Tumor , Disease Models, Animal , Drug Resistance, Neoplasm/genetics , Enzyme Activation/drug effects , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , Organ Specificity/genetics , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Phenotype , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Transforming Growth Factor beta/pharmacology , Tumor Suppressor Proteins/genetics , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Securing the negative surgical margin is the first step in surgical cancer treatment. However, tumor recurrence sometimes occurs even with histologically negative surgical margins. To detect minimal residual cancer cells in the deep margin intraoperatively, a time-efficient molecular approach is required. METHODS: We established an innovative rapid quantitative methylation PCR (QMSP) assay, which consists of substantially time-minimized DNA extraction, bisulfite treatment, and QMSP assays. To demonstrate the feasibility of this procedure, 10 serial surgical specimens of primary head and neck squamous cell carcinoma (HNSCC) were evaluated by both rapid and conventional QMSP. Two frequently methylated genes in head and neck cancer, homeobox A9 (HOXA9) and endothelin receptor type B (EDNRB) were analyzed in 10 HNSCCs and surgical margin tissues, as well as normal muscle and oral mucosa samples. RESULTS: The product quality of DNA extraction and bisulfite treatment using the time-saving procedure was comparable to the conventional procedure. In the QMSP assay, target gene methylation and reference gene methylation were equally detected by both the rapid and conventional method. Finally, relative results of rapid and conventional QMSP were quite similar to each other in tumors, margins, and normal tissues. The average total time required for the rapid QMSP procedure was less than 3 h and could be accomplished by a single person. CONCLUSION: From the viewpoint of accuracy, cost, and time consumption, the innovative rapid QMSP maintains highly sensitive methylation detection accomplished within the time frame of a major ablative and reconstructive procedure.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/diagnosis , DNA Methylation , Head and Neck Neoplasms/diagnosis , Neoplasm Recurrence, Local/diagnosis , Neoplasm, Residual/diagnosis , Aged , Aged, 80 and over , Base Sequence , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/surgery , DNA, Neoplasm/genetics , Female , Follow-Up Studies , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/surgery , Homeodomain Proteins/genetics , Humans , Intraoperative Period , Male , Middle Aged , Molecular Sequence Data , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/surgery , Neoplasm Staging , Neoplasm, Residual/genetics , Neoplasm, Residual/surgery , Polymerase Chain Reaction/methods , Prognosis , Promoter Regions, Genetic/genetics , Receptor, Endothelin B , Receptors, Endothelin/geneticsABSTRACT
Cervical cancer is a major health concern among women in Latin America due to its high incidence and mortality. Therefore, the discovery of molecular markers for cervical cancer screening and triage is imperative. The aim of this study was to use a genome wide DNA methylation approach to identify novel methylation biomarkers in cervical cancer. DNA from normal cervical mucosa and cervical cancer tissue samples from Chile was enriched with Methylated DNA Immunoprecipitation (MeDIP), hybridized to oligonucleotide methylation microarrays and analyzed with a stringent bioinformatics pipeline to identify differentially methylated regions (DMRs) as candidate biomarkers. Quantitative Methylation Specific PCR (qMSP) was used to study promoter methylation of candidate DMRs in clinical samples from two independent cohorts. HPV detection and genotyping were performed by Reverse Line Blot analysis. Bioinformatics analysis revealed GGTLA4, FKBP6, ZNF516, SAP130, and INTS1 to be differentially methylated in cancer and normal tissues in the Discovery cohort. In the Validation cohort FKBP6 promoter methylation had 73% sensitivity and 80% specificity (AUC = 0.80). ZNF516 promoter methylation was the best biomarker, with both sensitivity and specificity of 90% (AUC = 0.92), results subsequently corroborated in a Prevalence cohort. Together, ZNF516 and FKBP6 exhibited a sensitivity of 84% and specificity of 81%, when considering both cohorts. Our genome wide DNA methylation assessment approach (MeDIP-chip) successfully identified novel biomarkers that differentiate between cervical cancer and normal samples, after adjusting for age and HPV status. These biomarkers need to be further explored in case-control and prospective cohorts to validate them as cervical cancer biomarkers.
Subject(s)
DNA Methylation , Human papillomavirus 18 , Promoter Regions, Genetic , Tacrolimus Binding Proteins/genetics , Uterine Cervical Neoplasms/genetics , Zinc Fingers , Adult , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Chile , Female , Genome, Human , Humans , Middle Aged , Oligonucleotide Array Sequence Analysis , Tacrolimus Binding Proteins/metabolism , Uterine Cervical Neoplasms/ethnology , Uterine Cervical Neoplasms/virologyABSTRACT
We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (<20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients' plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer.
Subject(s)
DNA Methylation/drug effects , Imidazoline Receptors/genetics , Intracellular Signaling Peptides and Proteins/genetics , Smoking/adverse effects , Adult , Aged , Cohort Studies , Female , Genes, Tumor Suppressor , Humans , Imidazoline Receptors/blood , Intracellular Signaling Peptides and Proteins/blood , Keratinocytes/pathology , Lung Neoplasms/blood , Male , Middle Aged , Promoter Regions, Genetic , Tobacco ProductsABSTRACT
Urothelial cell carcinoma (UCC) is the second most common genitourinary malignant disease in the USA, and tobacco smoking is the major known risk factor for UCC development. Exposure to carcinogens, such as those contained in tobacco smoke, is known to directly or indirectly damage DNA, causing mutations, chromosomal deletion events and epigenetic alterations in UCC. Molecular studies have shown that chromosome 9 alterations and P53, RAS, RB and PTEN mutations are among the most frequent events in UCC. Recent studies suggested that continuous tobacco carcinogen exposure drives and enhances the selection of epigenetically altered cells in UCC, predominantly in the invasive form of the disease. However, the sequence of molecular events that leads to UCC after exposure to tobacco smoke is not well understood. To elucidate molecular events that lead to UCC oncogenesis and progression after tobacco exposure, we developed an in vitro cellular model for smoking-induced UCC. SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. Morphological alterations and increased cell proliferation of non-malignant urothelial cells were observed after 4 months (mo) of treatment with CSE. Anchorage-independent growth assessed by soft agar assay and increase in the migratory and invasive potential was observed in urothelial cells after 6 mo of CSE treatment. By performing a PCR mRNA expression array specific to the PI3K-AKT pathway, we found that 26 genes were upregulated and 22 genes were downregulated after 6 mo of CSE exposure of HUC1 cells. Among the altered genes, PTEN, FOXO1, MAPK1 and PDK1 were downregulated in the transformed cells, while AKT1, AKT2, HRAS, RAC1 were upregulated. Validation by RT-PCR and western blot analysis was then performed. Furthermore, genome-wide methylation analysis revealed MCAM, DCC and HIC1 are hypermethylated in CSE-treated urothelial cells when compared with non-CSE exposed cells. The methylation status of these genes was validated using quantitative methylation-specific PCR (QMSP), confirming an increase in methylation of CSE-treated urothelial cells compared to untreated controls. Therefore, our findings suggest that a tobacco signature could emerge from distinctive patterns of genetic and epigenetic alterations and can be identified using an in vitro cellular model for the development of smoking-induced cancer.
Subject(s)
DNA Methylation , Genome, Human , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Smoking , Carcinoma, Transitional Cell/metabolism , Carcinoma, Transitional Cell/pathology , Cell Cycle Proteins , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , DCC Receptor , Down-Regulation/drug effects , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Humans , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Plant Extracts/pharmacology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Nicotiana/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Up-Regulation/drug effects , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
BACKGROUND: DNA repair is a major defense mechanism, which contributes to the maintenance of genetic sequence, and minimizes cell death, mutation rates, replication errors, DNA damage persistence and genomic instability. Alterations in the expression levels of proteins participating in DNA repair mechanisms have been associated with several aspects of cancer biology. The present study aimed to evaluate the clinical significance of DNA repair proteins MSH2, MLH1 and MGMT in benign and malignant thyroid lesions. MATERIAL/METHODS: MSH2, MLH1 and MGMT protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 90 patients with benign and malignant lesions. RESULTS: The expression levels of MLH1 was significantly upregulated in cases with malignant compared to those with benign thyroid lesions (p = 0.038). The expression levels of MGMT was significantly downregulated in malignant compared to benign thyroid lesions (p = 0.001). Similar associations for both MLH1 and MGMT between cases with papillary carcinoma and hyperplastic nodules were also noted (p = 0.014 and p = 0.026, respectively). In the subgroup of malignant thyroid lesions, MSH2 downregulation was significantly associated with larger tumor size (p = 0.031), while MLH1 upregulation was significantly associated with the presence of lymphatic and vascular invasion (p = 0.006 and p = 0.002, respectively). CONCLUSIONS: Alterations in the mismatch repair proteins MSH2 and MLH1 and the direct repair protein MGMT may result from tumor development and/or progression. Further studies are recommended to draw definite conclusions on the clinical significance of DNA repair proteins in thyroid neoplasia.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , DNA Repair , MutS Homolog 2 Protein/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Female , Humans , Immunohistochemistry , Male , Middle Aged , MutL Protein Homolog 1 , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathologyABSTRACT
Focal Adhesion Kinase (FAK) and Src have been reported to regulate tumor growth, invasion, metastasis and angiogenesis. The present study aimed to evaluate by immunohistochemistry the clinical significance of FAK and Src expression in 108 patients with benign and malignant thyroid lesions. Total FAK expression provided a distinct discrimination between malignant and benign (p = 0.00001), as well as between papillary carcinoma and hyperplastic nodules thyroid lesions (p = 0.00005), being also associated with follicular cells' proliferative capacity (p = 0.0003). In malignant thyroid lesions, total FAK expression was associated with tumor size (p = 0.0455), and presence of capsular (p = 0.0102) and lymphatic (p = 0.0173) invasion. Total Src expression was borderline increased in cases of papillary carcinoma compared to hyperplastic nodules (p = 0.0993), being also correlated with tumor size (p = 0.0169). FAK and Src expression was ascribed to a significant extent to the phosphorylated forms of the enzymes, which provided a better discrimination between malignant and benign thyroid lesions. The current data revealed that FAK and to a lesser extent Src expression could be considered of clinical utility in thyroid neoplasia with potential use as therapeutic targets.
Subject(s)
Focal Adhesion Kinase 1/biosynthesis , Proto-Oncogene Proteins pp60(c-src)/biosynthesis , Thyroid Neoplasms/enzymology , Adult , Aged , Carcinoma, Papillary/enzymology , Diagnosis, Differential , Female , Humans , Hyperplasia/enzymology , Immunohistochemistry , Male , Middle Aged , Statistics, Nonparametric , Thyroid Diseases/enzymology , Young AdultABSTRACT
Thyroid cancer presents a growing tendency during the last decades, particularly in regions affected by radiation exposure. The present review describes expression alterations and gene polymorphisms of DNA repair related molecules, leading to genomic instability and cell death, being associated with thyroid cancer. The referred variations in DNA repair related genes depict that indirect repair mechanisms are mainly correlated with thyroid gland carcinogenesis. Such abnormalities could participate in thyroid tumor development and progression and could be targeted for future prevention and therapy.
Subject(s)
Carcinoma/genetics , Cell Transformation, Neoplastic/genetics , DNA Repair/genetics , Thyroid Neoplasms/genetics , HumansABSTRACT
p16 (INK4a) is a known negative regulator of the cell cycle acting up-stream of Rb to inhibit cellular growth. While the contribution of p16 to the tumorigenic process has been extensively studied, little is known about its role in the cellular differentiation process of normal cells. p16 expression in mammary gland epithelial cells and its possible mediation by DNA methylation was explored. The mammary glands from female mice (mus musculus) at distinct developmental stages (virgin, day 10 of lactation and day 3 of involution) were used. The expression pattern of p16 and the DNA methylases, DNMT1, 3a and 3b was investigated by semi-quantitative RT-PCR, in situ hybridization (ISH) and immunohistochemistry. The p16 methylation status was assesed by methylation-specific PCR (MSP). p16 was differentially expressed during distinct developmental stages and its transcriptional regulation was DNA methylation-independent, which was also corroborated by the expression pattern of the three known DNA methyltransferases (DNA MTase). The p16 expression level was elevated during involution compared to the corresponding expression level during lactation. p16 is differentially expressed during normal mammary gland development, which is not mediated by DNA methylation.