Clinical Relevance of Telomere Status and Telomerase Activity in Colorectal Cancer

The role of telomeres and telomerase in colorectal cancer (CRC) is well established as the major driving force in generating chromosomal instability. However, their potential as prognostic markers remains unclear. We investigated the outcome implications of telomeres and telomerase in this tumour type. We considered telomere length (TL), ratio of telomere length in cancer to non-cancer tissue (T/N ratio), telomerase activity and TERT levels; their relation with clinical variables and their role as prognostic markers. We analyzed 132 CRCs and paired non-cancer tissues. Kaplan-Meier curves for disease-free survival were calculated for TL, T/N ratio, telomerase activity and TERT levels. Overall, tumours had shorter telomeres than non-tumour tissues (P < 0.001) and more than 80% of CRCs displayed telomerase activity. Telomere lengths of non-tumour tissues and CRCs were positively correlated (P < 0.001). Considering telomere status and clinical variables, the lowest degree of telomere shortening was shown by tumours located in the rectum (P = 0.021). Regarding prognosis studies, patients with tumours showing a mean TL < 6.35 Kb experienced a significantly better clinical evolution (P < 0.001) and none of them with the highest degree of tumour telomere shortening relapsed during the follow-up period (P = 0.043). The mean TL in CRCs emerged as an independent prognostic factor in the Cox analysis (P = 0.017). Telomerase-positive activity was identified as a marker that confers a trend toward a poor prognosis. In CRC, our results support the use of telomere status as an independent prognostic factor. Telomere status may contribute to explaining the different molecular identities of this tumour type.     

Levels of expression of lipoxygenases and cyclooxygenase-2 in human breast cancer

Lipoxygenases and cyclooxygenase are key mediators of arachidonic acid metabolism. The eicosanoids metabolites from these oxygynases have been shown to regulate the growth and death of cancer cells. This study determined the level of expression of 5-, 12-, 15-lipoxygenase and cyclooxygenase-2 expression in a cohort of breast cancer patients and their correlation with clinical outcomes. Compared with normal breast tissues, tumour tissues exhibited a significantly higher levels of 12-lipoxygenase and cyclooxygenase-2 (P<0.05), and significantly lower level of 15-lipoxygenase (P=0.05). Lobular carcinomas had a higher level of cyclooxygenase-2 and lower level of 15-lipoxygenase than ductal carcinomas. The lowest level of 15-lipoxygenase was seen in TNM3 and TNM4 tumours and from patients who died of breast cancer. Levels of 12- and 5-lipoxygenases were also particularly high in tumours from patients who died of breast cancer. This study shows that human breast tumours aberrantly express lipoxygenases and cyclooxygenase-2 and that decreased level of 15-lipoxygenase and raised level of cyclooxygenase-2 and 12-lipoxygenase has prognostic value in patients with breast cancer.     

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer.

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (> or = 30%) negatively influenced the disease-free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl-2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.     

Serum exosomal lncRNA XIST is a potential non-invasive biomarker to diagnose recurrence of triple-negative breast cancer

Exosomal lncRNAs secreted by cancer cells can serve as potential biomarkers in the diagnosis and prognosis of various tumours. Here, we are committed to explore the diagnostic and prognostic value of serum exosomal XIST secreted by tumour cells to predict recurrence in patients with triple-negative breast cancer (TNBC). Significant increments in XIST and exo-XIST from tumour tissues and blood serum were found in reoccurring TNBC patients by comparison with non-recurrences. Levels of serum exo-XIST were only significantly increased in TNBC recurrence and no association with other clinicopathological parameters. Additionally, serum exo-XIST levels could be served as an assessment of change in the load of triple-negative breast cancer. Expressions of exo-XIST were markedly decreased after resection of the primary breast tumours and obviously elevated at the time of recurrence. Finally, an obvious association was identified between serum exo-XIST levels and a poorer overall survival (OS) in TNBC patients. Levels of serum exo-XIST may serve as a diagnostic and prognostic biomarker to predict the recurrent TNBC-loading status.     

Evaluation of cell-free tumour DNA and RNA in patients with breast cancer and benign breast disease

High levels of DNA and RNA released by apoptotic and necrotic cells circulate in the blood of cancer patients. In the present study we determined the applicability of the quantification of nucleic acids and their genetic alterations as minimally invasive tool for breast cancer screening. The relative concentrations of DNA and RNA were determined in preoperative serum of 102 breast cancer patients, 32 patients with benign breast disease and 53 healthy women. The mean follow-up time of the cancer patients was 6.2 years. Loss of heterozygosity (LOH) at four polymorphic markers (D13S159, D13S280, D13S282 at region 13q31-33 and D10S1765 at PTEN region 10q23.31) was analyzed by PCR-based fluorescence microsatellite analyses using cell-free DNA. The serum levels of DNA (p = 0.016) and RNA (p = 0.001) could differentiate between healthy women and cancer patients, but could not discriminate malignant from benign breast lesions. A significant correlation of serum DNA with RNA levels was observed in all groups (p = 0.018). Increased serum DNA levels (but not RNA levels) in cancer patients were associated with a poorer overall (p = 0.021) and disease-free survival (p = 0.025). The occurrence of LOH at all markers significantly correlated with lymph node status (p = 0.026). In addition, the LOH frequency at D13S280 (p = 0.047) and D13S159 (p = 0.046) associated with overall and disease-free survival, respectively. In conclusion, the quantification of cell-free tumour DNA had diagnostic and prognostic values in breast cancer patients, and DNA loss at the region 13q31-33 may be an indication of lymphatic tumour cell spread.     

Relationship between tumour aromatase activity, tumour characteristics and response to therapy

Aromatase activity has been measured in human breast cancers by incubating tumour minces with [7-³H]testosterone and characterizing purified oestradiol (E₂) fractions by chemical derivative formation. Of 247 primary tumours, 178 showed evidence of oestrogen biosynthesis, levels varying between 0.5 and 12.5 fmol E₂ produced/h/g tissue. These values were quantitatively small but at least comparable with those in other peripheral tissues. There was no correlation between presence or level of aromatase activity and the histopathology of the tumours although oestrogen biosynthesis was more likely to be present in more cellular tumours. Aromatase activity was also unrelated to age, menopausal status, lymph node status and T stage of the patient from which the tumour was derived. In a subgroup of patients presenting without clinical evidence of distant metastatic disease, no significant relation was detected between tumour aromatase and disease-free interval, but tumours without aromatase activity were associated with increased survival at 36 months after primary treatment. A statistically significant correlation was also detected between the presence of tumour aromatase and oestrogen receptors. Furthermore, in small subgroups of patients with “advanced” breast cancer tumour aromatase was related to response to aminoglutethimide but not tamoxifen therapy. Whilst these results do not conclusively define a role for local synthesis of oestrogen in the progression of breast cancer, this possibility still exists and further studies on tumour aromatase are warranted.     

Expression of Erk5 in Early Stage Breast Cancer and Association with Disease Free Survival Identifies this Kinase as a Potential Therapeutic Target

Background

Breast cancer is the most common neoplasia in women. Even though advances in its treatment have improved disease outcome, some patients relapse. Therefore, attempts to better define the molecular determinants that drive breast cancer cell proliferation may help in defining potential therapeutic targets. Mitogen-activated protein kinases (MAPK) play important roles in tumorigenesis. One of them, Erk5, has been linked to the proliferation of breast cancer cells in vitro. Here we have investigated the expression and prognostic value of Erk5 in human breast cancer.

Methodology/Principal Findings

Animal and cellular models were used to study Erk5 expression and function in breast cancer. In 84 human breast tumours the expression of Erk5 was analyzed by immunohistochemistry. Active Erk5 (pErk5) was studied by Western blotting. Correlation of Erk5 with clinicopathological parameters and with disease-free survival in early stage breast cancer patients was analyzed. Expression of Erk5 was detected in most patients, and overexpression was found in 20%. Active Erk5 was present in a substantial number of samples, as well as in tumours from an animal breast cancer model. Overexpression of Erk5 was associated with a decrease in disease-free survival time, which was independent of other clinicopathological parameters of prognosis. Transient transfection of a short hairpin RNA (shRNA) targeting Erk5, and a stable cell line expressing a dominant negative form of Erk5 (Erk5^AEF), were used to investigate the influence of Erk5 on drugs used in the clinic to treat breast tumours. We found that inhibition of Erk5 decreased cancer cell proliferation and also sensitized these cells to the action of anti-HER2 therapies.

Conclusions/Significance

Overexpression of Erk5 is an independent predictor of disease-free survival in breast cancer, and may represent a future therapeutic target.     

Association of the G473A Polymorphism and Expression of Lysyl Oxidase with Breast Cancer Risk and Survival in European Women: A Hospital-Based Case-Control Study

Background

Lysyl oxidase (LOX) is an extracellular enzyme essential for the covalent crosslinking of extracellular matrix proteins and may also have additional functions. LOX expression can be both up- and downregulated in cancer and is associated both with tumour suppression and metastasis progression. The G473A polymorphism (rs1800449) results in the Arg158Gln amino acid substitution in the LOX propeptide, compromises its tumour suppressive activity, and was associated with an increased breast cancer risk in a Chinese Han population. In the first hospital-based case-control study in European women, we aimed at investigating the association of LOX expression and the G473A polymorphism with breast cancer risk and survival in unselected and estrogen receptor (ER) negative patients.

Methodology/Principal Findings

The G473A polymorphism was genotyped in 386 breast cancer patients and 243 female controls. Moreover, LOX mRNA expression was quantified in the tumors of 105 patients by qRT-PCR. We found that the minor A-allele of this polymorphism is associated with a later age at breast cancer onset, a trend towards a decreased disease-free and metastasis-free survival, but not with an increased breast cancer risk. LOX mRNA expression was significantly elevated in tumours of patients older than 55 years, postmenopausal patients, estrogen receptor positive tumours, and p53 negative tumours, but was unaffected by G473A genotype in tumours and breast cancer cell lines. High LOX expression was associated with a poor disease-free and metastasis-free survival in ER negative but not ER positive patients. LOX expression was an independent prognostic parameter in multivariate analysis, whereas G473A genotype was not. A small, distinct subgroup of the ER negative patients was identified which exhibited a considerably elevated LOX expression and a very poor disease-free (p = 0.001) and metastasis-free survival (p = 0.0003).

Conclusions/Significance

This newly identified ER negative/LOX high subgroup may be a suitable collective for future individualized breast cancer diagnosis and therapy.     

Pre-treatment systemic immune-inflammation index is a useful prognostic indicator in patients with breast cancer undergoing neoadjuvant chemotherapy

The systemic immune-inflammation index (SII = N × P/L) based on neutrophil (N), platelet (P) and lymphocyte (L) counts is used to predict the survival of patients with malignant tumours and can fully reflect the balance between host inflammatory and immune status. This study is conducted to explore the potential prognostic significance of SII in patients with breast cancer undergoing neoadjuvant chemotherapy (NACT). A total of 262 patients with breast cancer received NACT were enrolled in this study. According to the receiver operating characteristic curve, the optimal cut-off value of SII was divided into two groups: low SII group (<602 × 10⁹/L) and high SII group (≥602 × 10⁹/L). The associations between breast cancer and clinicopathological variables by SII were determined by chi-squared test or Fisher's exact test. The Kaplan-Meier plots and log-rank test were used to determine clinical outcomes of disease-free survival (DFS) and overall survival (OS). The prognostic value of SII was analysed by univariate and multivariate Cox proportional hazards regression models. The toxicity of NACT was accessed by National Cancer Institute Common Toxicity Criteria (NCICTC). According to univariate and multivariate Cox regression survival analyses, the results showed that the value of SII had prognostic significance for DFS and OS. The patients with low SII value had longer DFS and OS than those with high SII value (31.11 vs 40.76 months, HR: 1.075, 95% CI: 0.718-1.610, P = .006; 44.47 vs 53.68 months, HR: 1.051, 95% CI: 0.707-1.564, P = .005, respectively). The incidence of DFS and OS in breast cancer patients with low SII value was higher than that in those patients with high SII value in 3-, 5- and 10-year rates. The common toxicities after NACT were haematological and gastrointestinal reaction, and there were no differences by SII for the assessment of side effects of neoadjuvant chemotherapy. Meanwhile, the results also proved that breast cancer patients with low SII value and high Miller and Payne grade (MPG) survived longer than those breast cancer with high SII value and low MPG grade. In patients without lymph vessel invasion, these breast cancer patients with low SII value had better prognosis and lower recurrence rates than those with high SII value. Pre-treatment SII with the advantage of reproducible, convenient and non-invasive was a useful prognostic indicator for breast cancer patients undergoing neoadjuvant chemotherapy and is a promising biomarker for breast cancer on treatment strategy decisions.     

A basal membrane-like structure surrounding tumour nodules may prevent intraepithelial leucocyte infiltration in colorectal cancer

Epithelial tumours consist of an epithelial compartment and a stromal compartment, which are sometimes separated by a basal membrane-like structure. We sought to determine whether these factors have prognostic value in 84 curatively resected stage II and III colorectal cancer by immunohistochemically staining tumours for leucocytes (CD45) and extracellular matrix, and to assess the presence of a basal membrane-like structure. Leucocyte infiltration was also assessed in hematoxylin-eosin (HE) stained sections. Most leucocytes were located in the tumour stroma. A relatively high intraepithelial leucocyte infiltration was significantly correlated with a lower level of tumour recurrence (P=0.03) and a longer disease-free survival (P=0.05), whereas leucocytes located in the tumour stroma (P=0.92) or at the advancing margin (p=0.06) were not. Intraepithelial leucocyte infiltration was also significantly correlated with leucocyte infiltration in the tumour stroma (P=0.02) and at the advancing tumour margin (P=0.005), and as assessed in HE-stained tumour sections (P=0.05), but each of these parameters on its own did not have a prognostic value in predicting disease-free survival. Moreover, the presence of a basal membrane-like structure surrounding the tumour epithelium was inversely correlated with the number of intraepithelial leucocytes (P=0.05), suggesting that this membrane-like structure functions as a barrier to intraepithelial leucocyte infiltration. We conclude that leucocytes must be in the direct vicinity of tumour cells to affect tumour growth. The presence of an extracellular matrix barrier seems to prevent this interaction.     

Interleukin 8 in progression of hormone-dependent early breast cancer

The only way to perceive the real clinical course of disease and the prognostic significance of potential biomarkers is follow-up of patients who did not receive any kind of adjuvant therapy. Many studies have confirmed high levels of interleukin 8 (IL8) in HER2-enriched and basal-like (ER–) primary breast tumours, but less is known about the significance of IL8 in hormone-dependent breast cancer. The aim of this study was to evaluate the prognostic significance of IL8 and clinicopathological parameters in hormone-dependent breast cancer, and to examine possible associations between them that might imply possible biological dependence. The study included 91 early-stage breast cancer patients with detectable levels of hormone receptors (ER>0, PR>0). None of the patients received adjuvant therapy according to valid protocol at that time. HER2 status was determined on paraffin-embedded tumour tissue sections by CISH. IL8 levels were determined by ELISA in cytosol tumour extracts of 65 patients with long-term follow-up (144 months). Nonparametric statistical tests were used for data analyses. Patients with low IL8 levels (M<88.8 pg/mg) had significantly longer relapse-free survival (RFS) compared to patients with high IL8 levels (M≥88.82 pg/mg) (Log rank test, p=0.002). Patients with ERhighIL8low phenotype had significantly longer RFS compared to those with ERhighIL8high and ERlowIL8high phenotypes (p=0.04 and p=0.02, respectively); patients with PRlowIL8low phenotype had significantly longer RFS compared to those with PRlowIL8high and PRhighIL8high phenotypes (p=0.003 and p=0.02, respectively); patients with HER2-IL8low phenotype had significantly longer RFS compared to those with HER2-IL8high and HER2+IL8high phenotypes (p=0.01 and p=0.02, respectively). Our results indicate significant contribution of IL8 on survival of hormone-dependent early-stage breast cancer patients and association with established parameters such as ER/PR and HER2.     

Expression of cyclooxygenase-2 in invasive breast carcinomas and its prognostic impact

Representative tumour sections from 468 patients with invasive breast cancer were immunostained for cyclooxygenase-2 (COX-2) and evaluated. The relationships between COX-2 expression, clinical outcome and various clinicopathological variables, including tumour vascularity and disseminated tumour cells (DTC) in the bone marrow were examined. COX-2 expression in invasive breast carcinoma cells was positively associated with oestrogen receptor and/or progesterone receptor positivity (p<0.001). Triple-negative tumours showed no/low COX-2 expression more frequently than other tumour types (p<0.001). Expression of COX-2 was not associated with breast cancer-specific survival (p=0.49, log-rank) or distant disease-free survival (p=0.67, log-rank) for all patients, including lymph node-negative, untreated patients (p>0.14, log-rank). There was also no significant association between COX-2 expression and histological grade, tumour size, nodal status, DTC in bone marrow, p53, HER2, or tumour vascularity. In conclusion, COX-2 expression in this series was associated with the presence of hormone receptors. Low COX-2 expression was observed in triple-negative breast carcinomas.     

The Expression Level and Prognostic Value of Y-Box Binding Protein-1 in Rectal Cancer

The aims of this study were to simultaneously evaluate the expression of Y-box binding protein-1 (YB-1) in non-neoplastic rectal tissue and rectal cancer tissue, and to collect clinical follow-up data for individual patients. Additionally, we aimed to investigate the developmental functions and prognostic value of YB-1 in rectal cancer. We performed immunohistochemical studies to examine YB-1 expression in tissue samples from 80 patients with rectal cancer, 30 patients with rectal tubular adenoma, and 30 patients with rectitis. The mean YB-1 histological scores for rectal cancer, rectal tubular adenoma, and rectitis tissue specimens were 205.5, 164.3, and 137.7, respectively. Shorter disease-free and overall survival times were found in patients with rectal cancer who had higher YB-1 expression than in those with lower expression (38.2 months vs. 52.4 months, P = 0.013; and 44.4 months vs. 57.3 months, P = 0.008, respectively). Our results indicate that YB-1 expression is higher in rectal cancer tissue than in rectal tubular adenoma and rectitis tissue and that it may be an independent prognostic factor for rectal cancer.     

Only Missense Mutations Affecting the DNA Binding Domain of P53 Influence Outcomes in Patients with Breast Carcinoma

The presence of a TP53 gene mutation can influence tumour response to some treatments, especially in breast cancer. In this study, we analysed p53 mRNA expression, LOH at 17p13 and TP53 mutations from exons 2 to 11 in 206 patients with breast carcinoma and correlated the results with disease-free and overall survival. The observed mutations were classified according to their type and location in the three protein domains (transactivation domain, DNA binding domain, oligomerization domain) and correlated with disease-free and overall survival. In our population, neither p53 mRNA expression nor LOH correlated with outcome. Concerning TP53 mutations, 27% of tumours were mutated (53/197) and the presence of a mutation in the TP53 gene was associated with worse overall survival (p = 0.0026) but not with disease-free survival (p = 0.0697), with median survival of 80 months and 78 months, respectively. When alterations were segregated into mutation categories and locations, and related to survival, tumours harbouring mutations other than missense mutations in the DNA binding domain of P53 had the same survival profiles as wild-type tumours. Concerning missense mutations in the DNA binding domain, median disease-free and overall survival was 23 months and 35 months, respectively (p = 0.0021 and p<0.0001, respectively), compared with 78 and 80 months in mutated tumours overall. This work shows that disease-free and overall survival in patients with a frameshift mutation of TP53 or missense mutation in the oligomerization domain are the same as those in wild-type TP53 patients.     

Do poor-prognosis breast tumours express membrane cofactor proteins (CD46)?

CD46 or membrane cofactor protein (MCP) is a complement regulatory protein that has been identified on all nucleated cells and which protects them from attack by autologous complement. Breast carcinomas are reported to consistently express CD46.Aim and methods: Our previous immunohistochemical study showed that in breast carcinomas, loss of CD59 and CD55 correlated with poor survival. This study aimed to investigate the prognostic significance of CD46 on breast tumours using a rabbit polyclonal anti-CD46 antibody with a standard immunohistochemistry method. A total of 510 breast tissues from patients with primary operable breast cancer diagnosed between 1987 and 1992 had previously been included in tissue microarrays. They included patients 70 years of age or less (mean = 54 years) with a long-term follow-up (median = 82 months).Results: Immunohistochemical study revealed that 507/510 (99.4%) of breast tumours expressed CD46. Strong immunoreactivity was exhibited by 136/510 (27%) tumours, while moderate and weak staining was observed in 43% and 29% of tumours, respectively. Intensity of CD46 expression was significantly associated with tumour grade (p<0.05), histological type of tumour (p<0.001) and tumour recurrence (p<0.05). There was no correlation with lymph node stage or the presence of vascular invasion, nor with patient age or menopausal status. Interestingly, as most tumours expressed CD46, it would appear that poor-prognosis tumours that lose CD55 and CD59 still express CD46.Conclusion: Breast tumours express high levels of CD46 that correlates with tumour grade and recurrence. It is therefore likely that loss of CD55 and CD59 could be compensated by expression of CD46. However, loss of CD55 and CD59, even for tumours that still express CD46, is still associated with a poor prognosis. This may suggest that CD46 alone can protect from complement lysis but that loss of CD55 and CD59 are associated with other roles in immune regulation.