Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation

AIM:To investigate large tumor suppressor 1 (LATS1 ) expression, promoter hypermethylation, and microsatellite instability in colorectal cancer (CRC).METHODS:RNA was isolated from tumor tissue of 142 CRC patients and 40 colon mucosal biopsies of healthy controls. After reverse transcription, quantitative polymerase chain reaction (PCR) was performed, and LATS1 expression was normalized to expression of the ACTB and RPL32 housekeeping genes. To analyze hypermethylation, genomic DNA was isolated from 44 tumor CRC biopsies, and methylation-specific PCR was performed. Microsatellite instability (MSI) status was checked with PCR using BAT26, BAT25, and BAT40 markers in the genomic DNA of 84 CRC patients, followed by denaturing gel electrophoresis. RESULTS:Decreased LATS1 expression was found in 127/142 (89.4%) CRC cases with the average ratio of the LATS1 level 10.33 ± 32.64 in CRC patients vs 32.85 ± 33.56 in healthy controls. The lowest expression was found in Dukes' B stage tumors and G1 (welldifferentiated) cells. Hypermethylation of the LATS1 promoter was present in 25/44 (57%) CRC cases analyzed. LATS1 promoter hypermethylation was strongly associated with decreased gene expression; methylated cases showed 162× lower expression of LATS1 than unmethylated cases. Although high-grade MSI (mutation in all three markers) was found in 14/84 (17%) cases and low-grade MSI (mutation in 1-2 markers) was found in 30/84 (36%) cases, we found no association with LATS1 expression. CONCLUSION:Decreased expression of LATS1 in CRC was associated with promoter hypermethylation, but not MSI status. Such reduced expression may promote progression of CRC.

The role of aberrant promoter hypermethylation of DACT1 in bladder urothelial carcinoma

The purpose of this study was to determine the relationship between hypermethylation of DACT1 gene pro-moter and lower mRNA expression in bladder urothelial carcinoma tissue.The methylation status of 29 urothelial carcinoma samples and 29 normal tissue samples were examined by methylation-specific polymerase chain reac-tion（MSP）.The DACT1 mRNA transcript levels and DACT1 protein levels in all samples were then evaluated to define the relationship between the methylation status of the DACT1 promoter and its expression at the transcrip-tional and translational levels.Decreased expression of DACT1 was detected in 89.66% of urothelial carcinomas（26/29;P 〈 0.005）.Promoter hypermethylation was found in 58.62%（17/29） urothelial carcinomas and 25%（7/29） normal tissues,respectively（P 〈 0.05）.DACT1 expression was lower in tissues where the DACT1 gene promoter was hypermethylated than in unmethylated tissues（0.25±0.17 vs 0.69±0.30,P 〈 0.05）.DACT1 gene hyper-methylation was closely related to tumor size,grade and stage（P 〈 0.05）.Our results indicate that silencing and downregulation of DACT1 mRNA may be implicated in carcinogenesis and the progression of bladder urothelial carcinoma,and may be a potential prognostic factor.

HYPERMETHYLATION STATUS OF E-CADHERIN AND p16^(INK4a) IN GASTROINTESTINAL STROMAL TUMOR

Objective： To investigate the methylation status of CpG island in E-cadherin（CDHl）, P16^INK4a（ P16）promoter region ,and to analyze their role in gastrointestinal stromal rumor （GISTs）. Methods： A total of 56 surgically resected GISTs were obtained from January 2003 to December 2005. The routine H＆E-stained sections and CD117, CD34-immunoreactions were reviewed to verify the morphologic diagnosis. Methylation status of the CDH1, P16^INK4a promoter region was analyzed by methylation specific polymerase chain reaction （MSP） from chemically modified DNA after Na-bisulfite treatment. Results： The frequency of CDHlgene methylation was 32% （18 of 56） in GISTs. The rate was 9% （1 of 11）, 21% （4 of 19）, 41.6% （5 of 12）, and 57% （8 of 14） for very low risk, low risk, intermediate risk, and high risk GISTs; P16^INK4a methylation was found in 19 of 56（34%） cases. The rate was 0% （0 of 11）, 16% （3 of 19）, 50% （6 of 12）, and 71% （10 of 14） for very low risk, low risk, intermediate risk, and high risk GISTs. Statistical analysis indicated that of the 56 cases, there was significant association of CDH^INK4a and/or P16^INK4a methylation status with tumor malignant behavior （methylation rate 23/56, 41%, P〈0.01） and site （P〈0.05）. Conclusion： E-cadherin （CDHI） and/or P16^INK4a promoter hypermethylation is strongly associated with risk grade, may be a useful biomarker for GISTs risk assessment, and may shed light on new therapeutic options to treat GISTs

Promoter hypermethylation and loss of CD133 gene expression in colorectal cancers

AIM: To understand CD133 promoter hypermethyl-ation and expression in 32 colorectal cancer cell lines. METHODS: Nucleic acid was isolated from 32 colorectal cancer cell lines and CD133 expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Promoter methylation status of the CD133 gene was analyzed with a methylation-specific PCR after sodium-bisulfi te modification and by clonal sequencing analysis. The correlation between expression and promoter methylation of CD133 gene was confirmed with treatment of 5-aza-2’-deoxycytidine. RESULTS: We measured CD133 expression levels in 32 colorectal cancer cell lines. RT-PCR analysis showed undetectable or low levels of CD133 expression in 34.4%of cell lines. To verify the relation between CD133 expression and methylation status of the CD133 gene promoter in colorectal carcinogenesis, CD133 gene promoter hypermethylation was analyzed in 32 cancer cell lines. Promoter hypermethylation was detected in 13 (40.6%) of the cell lines using methylation specificPCR and confirmed by bisulfite sequencing analysis. Treatment of 11 of the cell lines with the demethylation agent 5-aza-2’-deoxycytidine recovered CD133 expression in most of them. CONCLUSION: Transcriptional repression of CD133 is caused by promoter hypermethylation of the CD133 CpG islands in some of colorectal cancer cell lines. The study may contribute to the understanding of the role of CD133 inactivation in the progression of colorectal cancers.

Hypermethylation of tumor suppressor and tumor-related genes in neoplastic and non-neoplastic gastric epithelia

A number of tumor suppressor and tumor-related genes exhibit promoter hypermethylation with resultant gene silencing in human cancers.The frequencies of methylation differ among genes and genomic regions within CpG islands in different tissue types.Hypermethylation initially occurs at the edge of CpG islands and spreads to the transcription start site before ultimately shutting down gene expression.When the degree of methylation was quantitatively evaluated in neoplastic and non-neoplastic gastric epithelia using DNA microarray analysis,highlevel methylation around the transcription start site appeared to be a tumor-specific phenomenon,although multiple tumor suppressor genes became increasingly methylated with patient age in non-neoplastic gastric epithelia.Quantitative analysis of DNA methylation is a promising method for both cancer diagnosis and risk assessment.

Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, tissues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 （34.8 %） laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades （χ^2= 3. 130, P=0. 077） or in samples from patients with different TNM status （χ^2= 3. 957, P=0. 138）. No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

The role of XPC protein deficiency in tobacco smoke-induced DNA hypermethylation of tumor suppressor genes

DNA hypermethylation of tumor suppressor genes has been frequently observed in cancer patients, and therefore, may provide a valuable biomarker for cancer prevention and treatment. DNA hypermethylation may also provide an important mechanism in cancer progression. Lung cancer is strongly associated with exposure to environmental carcinogens, especially tobacco smoke. DNA damage generated by tobacco smoke is believed to play an important role in lung cancer development. XPC is a DNA damage recognition protein required for DNA repair and other DNA damage responses and attenuated XPC protein levels have been detected in many lung cancer patients. We studied the role of XPC protein deficiency in tobacco smoke-caused DNA hypermethylation of important tumor suppressor genes. Using both normal human fibroblasts (NF) and XPC-deficient hu man fibroblasts (XPC), our DNA methylation studies demonstrated that the XPC deficiency caused elevated levels of DNA hypermethylation in both Brca1 and Mlh1 tumor suppressor genes following exposure to tobacco smoke condensate (TSC). The results of our ChIP studies revealed that the XPC deficiency led to an increased binding of DNA methyltransferase 3A (DNMT3A) at the promoter region CpG island-containing sequences of these genes under the TSC treatment;however, this increase was partially diminished with prior treatment with caffeine. The results of our immuno-precipitation (IP) studies demonstrated a protein-protein interaction of the ATR with DNMT3A. Our western blots revealed that the XPC deficiency caused an increase in TSC-induced ATR phosphorylation at serine 428, an indicator of ATR activation. All these results suggest that XPC deficiency causes an accelerated DNA hypermethylation in important tumor suppressor genes under tobacco smoke exposure and activation of the ATR signaling pathway is involved in this DNA hypermethylation process.

Epigenetic Aberrant Hypermethylation of DNA in Endometrial Cancer: Application as a Biomarker

Endometrial cancer is the seventh most common cancer worldwide among females and accounts for about 40% of cancers of the uterus in Japan. An increase in incidence and a reduction in onset age of this disease are also likely, which makes it important to define the pathogenesis and develop effective treatment. However, the mechanism of canceration in the endometrium is unclear and development of endometrial cancer cannot be explained only by mutations of cancer-related genes. In contrast, epigenetic analyses have shown the importance of aberrant DNA hypermethylation in the canceration mechanism. In development of type 1 endometrial cancer, breakdown of the DNA mismatch repair system plays a large role, with changes in the human mutL homologue 1 (hMLH1) gene being of most importance. Studies to detect aberrant DNA hypermethylation of cancer cells present in microscopic amounts in vivo and to apply these data to diagnosis of cancer have been started. Epigenetic changes have also been examined as a marker of sensitivity to anticancer drugs. Aberrant hypermethylation of checkpoint with forkhead-associated and ring finger (CHFR), a mitotic phase checkpoint gene, is correlated with sensitivity to treatment with microtubule inhibitors and may be a marker for the response of endometrial cancer to anticancer drugs. Epigenetic aberrant DNA methylation of other genes may also be useful as clinical biomarkers for diagnosis and treatment of endometrial cancer.

No Association between p53 Immunohistochemical Staining and RASSF1 or DAPK1 Hypermethylation in Non-Small Cell Lung Cancer

p53 mutations have been linked with shortened survival rates in non-small cell lung cancer (NSCLC). Hypermethylation of RASSF1 and DAPK1 genes, which are downstream targets of p53, has also been linked to a poor prognosis in lung cancer patients. We investigated whether p53 mutations, assessed as p53 stabilization by immunohistochemistry (IHC), were independent of DAPK1 and RASSF1 promoter hypermethylation. We examined 103 resected NSCLC tumors for which we had p53 IHC and RASSF1 and DAPK1 methylation data. p53 protein expression was determined by IHC and graded using a semi-quantitative scoring method. DAPK1 and RASSF1 methylations were determined on tumor blocks by MethyLight real-time PCR assays represented as the percent of methylated reference DNA (PMR). Our primary results found no evidence for an association between the p53 IHC score and RASSF1 and DAPK1 PMR values, P = 0.46 and P = 0.68, respectively.

Promoter hypermethylation of tissue specific tumor supressor genes and point mutation in K-ras, c-myc proto-oncogenes in urinary (transitional cell) bladder carcinoma

In a total of 83 UN specimens were investigated for proto-oncogene mutations, tumor supressor genes promoter methylation status and c-myc and Ki-67 expression. Point mutations in c-myc were detected in cases with high grade and proliferation index. Mutated K-ras proto-onco- gene profiles were detected in 17 (21%) tumoral spiecemens that examined. Tumor specimens were also showed hypermethylated promoter domain for the SFRP2, MGMT tumor supressor genes. These findings showed the combine effect of mutated c-myc and K-ras oncogene and epigenetic inactivation of tissue specific tumor supressor genes (TS) play a crucial role in tumor progression and recurrence in UN carcinogenesis.

Aberrant p16 promoter hypermethylation in bronchial mucosae as a biomarker for the early detection of lung cancer

Lung cancer is the leading cause of cancer related death in the world and its mortality could be greatly reduced by diagnosis and treatment in its early stages. Effective tools for the early detection of lung cancer and its high risk factors remain a major challenge. Biomarkers that detect lung cancer in its early stages or identify its pretumour lesions, enabling early therapeutic intervention, would be invaluable to improve its dismal prognosis.

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients

Background Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine- DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas （pGBMs） and nine secondary glioblastomas （sGBMs） with the expectation to identify valuable prognostic and therapeutic targets. Methods We evaluated the methylation status of testis derived transcript （TES） gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines （U87 and U251） with 5-aza-2-deoxycytidines （5-aza-dC） and detected cell biological behaviors. Results Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs （P 〈0.05）. Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival （P 〈0.05）. This abnormality is also confirmed in glioblastoma cell lines （U87 and U251）. Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth （P=0.013）. Conclusions Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for gliobtastoma.

Realgar(α-As_(4)S_(4))Treats Myelodysplastic Syndromes through Reducing DNA Hypermethylation

DNA hypermethylation is an epigenetic modification that plays a critical role in the oncogenesis of myelodysplastic syndromes(MDS).Aberrant DNA methylation represses the transcription of promotors of tumor suppressor genes,inducing gene silencing.Realgar(α-As_(4)S_(4))is a traditional medicine used for the treatment of various diseases in the ancient time.Realgar was reported to have efficacy for acute promyelocytic leukemia(APL).It has been demonstrated that realgar could efficiently reduce DNA hypermethylation of MDS.This review discusses the mechanisms of realgar on inhibiting DNA hypermethylation of MDS,as well as the species and metabolisms of arsenic in vivo.

Hypermethylation of HIC1 and aberrant expression of HIC1 / SIRT1 contribute to papillary thyroid carcinoma

Objective To explore promoter methylation of HIC1gene and the expression of HIC1/SIRT1 related to the occurrence,development,and metastasis of papillary thyroid carcinoma.Methods Using Bisulfite sequencing PCR to analyze the promoter methylation of HIC1 gene.Using quantitative real-time PCR and Western blot to analyze expression differences of HIC1 and SIRT1 genes

Revolutionizing Non-Invasive Biomarker Discoveries: The Power of Methylation Screening Analysis in Cell-Free DNA Liquid Biopsy

Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.

Role of RECK methylation in gastric cancer and its clinical significance

AIM:To investigate the relation between RECK methylation and clinicopathological characteristics of gastric cancer patients and evaluate the role of RECK methylation in peritoneal metastasis of gastric cancer.METHODS:Methylation of RECK gene in 40 paired samples of gastric cancer and its corresponding adjacent normal mucosa,lymph nodes and peritoneal irrigation fluid was detected by methylation-specific polymerase chain reaction.RESULTS:Aberrant methylation of RECK gene was detected in 27.5%(11/40)of the adjacent normal mucosa samples,in 47.5%(19/40)of gastric cancer samples,in 57.1%(12/21)of the lymph node samples,and in 35%(14/40)of peritoneal irrigation fluid samples,respectively,with a significant difference between the adjacent normal mucosa and lymph node samples(P=0.023).Presence of RECK methylation in the primary tumor samples was significantly correlated with tumor invasion(P=0.023).The accuracy of RECK methylation in peritoneal lavage fluid samples for the diagnosis of peritoneal metastasis of gastric cancer was 72.5%(26/40),with a sensitivity of 66.7%(6/9) and a specificity of 74.2%(23/31).CONCLUSION:Aberrant methylation of RECK gene may provide useful information for the early diagnosis and treatment of peritoneal metastasis of gastric cancer.

Cdx2 expression and its promoter methylation during metaplasia-dysplasia-carcinoma sequence in Barrett's esophagus

AIM:To examine how the expression of caudal type homebox transcription factor 2(Cdx2) is regulated in the development of malignancy in Barrett's esophagus.METHODS:Cdx2,mucin(MUC) series(MUC2,MUC5AC and MUC6),p53 and E-cadherin expression in Barrett's esophagus and adenocarcinoma specimens were examined by immunostaining.Isolated clusters of cells from(1) MUC2 and Cdx2-positive intestinal metaplastic mucosa;(2) MUC5AC and MUC6-positive,and MUC2 and Cdx2-negative high-grade dysplasia(HD),or intramucosal adenocarcinoma(IMC);and(3) MUC5AC,MUC6 and Cdx2-positive poorly-differentiated invasive adenocarcinoma(PDA) were analyzed by methylationspecific polymerase chain reaction using sets of primers for detecting methylation status of the Cdx2 gene.RESULTS:Most of the non-neoplastic Barrett's esophageal mucosa showing intestinal-type metaplasia with or without low-grade dysplasia was positive for E-cadherin,MUC series and Cdx2,but negative for p53.A portion of the low-grade to HD was positive for E-cadherin,MUC5AC,MUC6 and p53,but negative for MUC2 and Cdx2.The definite IMC area was strongly positive for MUC5AC,MUC6 and p53,but negative for MUC2 and Cdx2.Methylation of the Cdx2 promoter was not observed in intestinal metaplasia,while hypermethylation of part of its promoter was observed in hot dipped and IMC.Hypermethylation of a large fraction of the Cdx2 promoter was observed in PDA.CONCLUSION:Cdx2 expression is restored irrespective of the methylation status of its promoter.Apparent positive immunohistochemical results can be a molecular mark for gene silencing memory.

Aberrant methylation of secreted protein acidic and rich in cysteine gene and its significance in gastric cancer

BACKGROUND Aberrant methylation in DNA regulatory regions could downregulate tumor suppressor genes without changing the sequences.However,our knowledge of secreted protein acidic and rich in cysteine(SPARC)and its aberrant methylation in gastric cancer(GC)is still inadequate.In the present research,we performed fundamental research to clarify the precise function of methylation on SPARC and its significance in GC.AIM To investigate promoter methylation and the effects of the SPARC gene in GC cells and tissues and to evaluate its clinical significance.METHODS Plasmids that overexpressed the SPARC gene were transfected into human GC BGC-823 cells;non-transfected cells were used as a control group(NC group).Quantitative real-time polymerase chain reaction and western blotting(WB)were then used to detect the expression of SPARC.Methylation-specific polymerase chain reaction was executed to analyze the gene promoter methylation status.Cell viability was measured by the cell counting kit-8 assay.The migration and invasion ability of cells were detected by scratch assays and transwell chamber assays,respectively.Cell cycle events and apoptosis were observed with a flow cytometer.RESULTS The expression of SPARC mRNA in GC tissues and cells was significantly lower and showed differing degrees of hypermethylation,respectively,than that in normal adjacent tissues and control cells.Treatment with 5-Aza-2’-deoxycytidine(5-Aza-Cdr)was able to restore the expression of SPARC and reverse promoter hypermethylation.Overexpression of the SPARC gene significantly inhibited proliferation,migration,and invasion of GC cells,while also causing cell cycle arrest and apoptosis;the NC group exhibited the opposite effects.CONCLUSION This study demonstrated that SPARC could function as a tumor suppressor and might be silenced by promoter hypermethylation.Furthermore,in GC cells,SPARC inhibited migration,invasion,and proliferation,caused cell cycle arrest at the G0/G1 phase,and promoted apoptosis.

MTNR1B polymorphisms with CDKN2A and MGMT methylation status are associated with poor prognosis of colorectal cancer in Taiwan

BACKGROUND Identifying novel colorectal cancer(CRC)prognostic biomarkers is crucial to helping clinicians make appropriate therapy decisions.Melatonin plays a major role in managing the circadian rhythm and exerts oncostatic effects on different kinds of tumours.AIM To explore the relationship between MTNR1B single-nucleotide polymorphism(SNPs)combined with gene hypermethylation and CRC prognosis.METHODS A total of 94 CRC tumour tissues were investigated.Genotyping for the four MTNR1B SNPs(rs1387153,rs2166706,rs10830963,and rs1447352)was performed using multiplex polymerase chain reaction.The relationships between the MTNR1B SNPs and CRC 5-year overall survival(OS)was assessed by calculating hazard ratios with 95%CIs.RESULTS All SNPs(rs1387153,rs2166706,rs10830963,and rs1447352)were correlated with decreased 5-year OS.In stratified analysis,rs1387153,rs10830963,and rs1447352 risk genotype combined with CDKN2A and MGMT methylation status were associated with 5-year OS.A strong cumulative effect of the four polymorphisms on CRC prognosis was observed.Four haplotypes of MTNR1B SNPs were also associated with the 5-year OS.MTNR1B SNPs combined with CDKN2A and MGMT gene methylation status could be used to predict shorter CRC survival.CONCLUSION The novel genetic biomarkers combined with epigenetic biomarkers may be predictive tool for CRC prognosis and thus could be used to individualise treatment for patients with CRC.

Evidence for Tumour Suppressor Function of DOK7 in Human Breast Cancer

Introduction: Downstream of tyrosine kinase 7 (DOK-7) is a member of the DOK family, which has been associated with the development and progression of various humancancers. Previously, identification of CpG hypermethylation in DOK-7 promoter was identified in breast cancer. Method: DOK-7 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples and normal background breast tissue. Transcript levels of expression were analyzed against TNM stage, tumour grade and clinical outcome over a 10-year follow-up period. Results: Levels of DOK-7 expression decreased significantly with increasing TNM stage. Higher DOK-7 expression was correlated with longer disease free and overall survival times. Conclusion: To our knowledge, this is the first study to investigate DOK-7 expression in human breast cancer. We identify a potential DOK-7 tumour suppressor role. DOK-7 as a prognostic biomarker in human breast cancer should be included in future validation studies.