Myofibroblast activation in colorectal cancer lymph node metastases

Background:

Myofibroblasts have an important role in regulating the normal colorectal stem cell niche. While the activation of myofibroblasts in primary colorectal cancers has been previously described, myofibroblast activation in lymph node metastases has not been described before.

Methods:

Paraffin-embedded lymph node sections from patients with macrometastases, micrometastases and isolated tumour cells were stained to identify myofibroblasts and to characterise the distribution of different cell types in tumour-containing lymph nodes. The extent of myofibroblast presence was quantified and compared with the size of the metastasis and degree of proliferation and differentiation of the cancer cells.

Results:

We show substantial activation of myofibroblasts in the presence of colorectal metastases in lymph nodes, which is intimately associated with glandular structures, both in micro- and macrometastases. The degree of activation is positively associated with the size of the metastases and the proportion of Ki67+ve cancer cells, and negatively associated with the degree of enterocyte differentiation as measured by CK20 expression.

Conclusion:

The substantial activation of myofibroblasts in tumour-containing lymph nodes strongly suggests that these metastatic cancer cells are still significantly dependent on their microenvironment. Further understanding of these epithelial–mesenchymal interactions could lead to the development of new therapies in metastatic disease.     

P4H9-detected molecule expression on spindle-shaped fibroblasts indicates malignant phenotype of colorectal cancer

Background:

Our previous study using a mammary fat pad mouse model showed that P4H9, produced by the β2 integrin epitope, detected a molecule on fibroblasts in response to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1)-expressing cancer cells. P4H9-detected molecule (PDM) expression appeared to be associated with myofibroblast differentiation. In this study, we investigated whether PDM is expressed on fibroblasts and cancer cells in clinical tissue samples, and whether the presence of PDM-expressing colorectal cancer cells is correlated with clinicopathological features of patients.

Methods:

Immunohistochemistry was conducted to detect P4H9 on clinical tissue samples from 156 patients with colorectal cancer. Risk factors for metastases and survival were calculated for clinical implication of PDM-expressing spindle-shaped fibroblasts.

Results:

Multivariate analysis showed that PDM-expressing spindle-shaped fibroblasts were an independent risk factor for lymph node metastasis, hematogenous metastasis, and poor survival. A Kaplan–Meier survival curve indicated that PDM-expressing spindle-shaped fibroblasts were associated with shorter survival time (P<0.0001). Immunofluorescence showed PDM expression on CCD-18Co fibroblasts and two colorectal cancer cell lines (HCT116 and HCT-15).

Conclusions:

PDM-expressing spindle-shaped fibroblasts are associated with metastasis and shorter survival in colorectal cancer patients. PDM-expressing spindle-shaped fibroblasts may have a role in eliciting the malignant phenotype of colorectal cancer.     

Tumour-site-dependent expression profile of angiogenic factors in tumour-associated stroma of primary colorectal cancer and metastases

Background:

Tumour-associated stroma has a critical role in tumour proliferation. Our aim was to determine a specific protein expression profile of stromal angiogenic cytokines and matrix metalloproteinases (MMPs) to identify potential biomarkers or new therapy targets.

Methods:

Frozen tissue of primary colorectal cancer (n=25), liver (n=25) and lung metastases (n=23) was laser-microdissected to obtain tumour epithelial cells and adjacent tumour-associated stroma. Protein expression of nine angiogenic cytokines and eight MMPs was analysed using a multiplex-based protein assay.

Results:

We found a differential expression of several MMPs and angiogenic cytokines in tumour cells compared with adjacent tumour stroma. Cluster analysis displayed a tumour-site-dependent stromal expression of MMPs and angiogenic cytokines. Univariate analysis identified stromal MMP-2 and MMP-3 in primary colorectal cancer, stromal MMP-1, -2, -3 and Angiopoietin-2 in lung metastases and stromal MMP-12 and VEGF in liver metastases as prognostic markers (P>0.05, respectively). Furthermore, stroma-derived Angiopoietin-2 proved to be an independent prognostic marker in colorectal lung metastases.

Conclusion:

Expression of MMPs and angiogenic cytokines in tumour cells and adjacent tumour stroma is dependent on the tumour site. Stroma-derived MMPs and angiogenic cytokines may be useful prognostic biomarkers. These data can be helpful to identify new agents for a targeted therapy in patients with colorectal cancer.     

Maspin is a marker for early recurrence in primary stage III and IV colorectal cancer

Background:

Little is known about the factors that drive metastasis formation in colorectal cancer (CRC). Here, we set out to identify genes and proteins in patients with colorectal liver metastases that correlate with early disease recurrence. Such factors may predict a propensity for metastasis in earlier stages of CRC.

Methods:

Gene expression profiling and proteomics were used to identify differentially expressed genes/proteins in resected liver metastases that recurred within 6 months following liver surgery vs those that did not recur for >24 months. Expression of the identified genes/proteins in stage II (n=243) and III (n=176) tumours was analysed by immunohistochemistry on tissue microarrays. Correlation of protein levels with stage-specific outcome was assessed by uni- and multivariable analyses.

Results:

Both gene expression profiling and proteomics identified Maspin to be differentially expressed in colorectal liver metastases with early (<6 months) and prolonged (>24 months) time to recurrence. Immunohistochemical analysis of Maspin expression on tumour sections revealed that it was an independent predictor of time to recurrence (log-rank P=0.004) and CRC-specific survival (P=0.000) in stage III CRC. High Maspin expression was also correlated with mucinous differentiation. In stage II CRC patients, high Maspin expression did not correlate with survival but was correlated with a right-sided tumour location.

Conclusion:

High Maspin expression correlates with poor outcome in CRC after spread to the local lymph nodes. Therefore, Maspin may have a stage-specific function possibly related to tumour cell dissemination and/or metastatic outgrowth.     

Potential role of a navigator gene NAV3 in colorectal cancer

Background:

The recently described navigator proteins have a multifaceted role in cytoskeletal dynamics. We report here on the relevance of one of them, navigator 3 (NAV3), in colorectal cancer (CRC).

Methods:

We analysed changes in chromosome 12 and NAV3 copy number in CRC/adenoma samples of 59 patients and in 6 CRC cell lines, using fluorescence in situ hybridisation, loss of heterozygosity, and array-CGH. NAV3 target genes were identified by siRNA depletion, expression arrays, and immunohistochemistry.

Results:

NAV3 deletion and chromosome 12 polysomy were detected in 30 and 70% of microsatellite stability (MSS) carcinomas, in 23 and 30% of adenomas and in four of six CRC cell lines. NAV3 amplification was found in 25% of MSS samples. NAV3 alterations correlated with lymph node metastasis. In normal colon cells, NAV3 silencing induced upregulation of interleukin 23 receptor (IL23R) and gonadotropin releasing hormone receptor. In MSS and microsatellite instability tumours, IL23R immunoreactivity correlated with Dukes'' staging and lymph node metastases, whereas nuclear beta-catenin correlated with lymph node metastases only.

Conclusion:

NAV3 copy number changes are frequent in CRC and in adenomas, and upregulation of IL23R, following NAV3 silencing, strongly correlates with Dukes'' staging and lymph node metastases. This suggests that NAV3 has a role in linking tissue inflammation to cancer development in the colon.     

Risks of myeloid malignancies in patients with autoimmune conditions

Background:

We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential.

Methods:

Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases.

Results:

Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT–PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse.

Conclusion:

Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.     

KRAS mutation analysis: a comparison between primary tumours and matched liver metastases in 305 colorectal cancer patients

Background:

KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor antibody in metastatic colorectal cancer (CRC). KRAS mutation analysis is usually performed on primary tumour tissue because metastatic tissue is often not available. However, controversial data are available on the concordance of test results between primary tumours and corresponding metastases. We assessed the concordance of KRAS mutation status in a study of 305 primary colorectal tumours and their corresponding liver metastases.

Methods:

Patients with histologically confirmed CRC who underwent surgical resection of the primary tumour and biopsy or surgical resection of the corresponding liver metastasis were included. KRAS mutation analysis was performed for codons 12 and 13.

Results:

KRAS mutation was detected in 108 out of 305 primary tumours (35.4%). In 11 cases (3.6%), we found a discordance between primary tumour and metastasis: 5 primary tumours had a KRAS mutation with a wild-type metastasis, 1 primary tumour was wild type with a KRAS mutation in the metastasis, and in 5 cases the primary tumour and the metastasis had a different KRAS mutation.

Conclusion:

We observed a high concordance of KRAS mutation status of 96.4% (95% CI 93.6–98.2%) between primary colorectal tumours and their corresponding liver metastases. In only six patients (2.0% 95% CI 0.7–4.2%), the discordance was clinically relevant. In this largest and most homogenous study to date, we conclude that both primary tumours and liver metastases can be used for KRAS mutation analysis.     

HER2/neu testing in primary colorectal carcinoma

Background:

Anti-HER2/neu therapy is well-established in breast and gastric carcinoma. The increased understanding of this pathway led to the identification of new promising drugs in addition to trastuzumab, offering further perspectives. The role of HER2/neu in colorectal carcinoma is controversially discussed, as discrepant data has been reported.

Methods:

Here, we retrospectively assessed the prevalence of HER2/neu positivity in a large series of colorectal carcinoma, testing HER2/neu status according to current recommendations. We correlated the results to clinico-pathological data and patient survival.

Results:

Overall, in 1645 primary colorectal carcinoma cases, 1.6% of the cases were HER2/neu positive. HER2/neu positivity significantly correlated with higher UICC stages (P=0.017) and lymph node metastases (P=0.029). In the subgroup of sigmoideal and rectal carcinomas, positive HER2/neu status was associated with T-category (P=0.041) and higher UICC stages (P=0.022). Although statistically not significant, HER2/neu-positive colorectal carcinomas displayed a tendency to poorer overall survival.

Conclusions:

These results illustrate the importance of testing HER2/neu by approved diagnostic techniques and scoring systems. We assume that although the prevalence of HER2/neu positivity in colorectal carcinoma is low, HER2/neu testing in advanced, nodal-positive colorectal carcinoma is reasonable, offering a potential target in high risk colorectal carcinoma.     

Expression of young HERV-H loci in the course of colorectal carcinoma and correlation with molecular subtypes

Background

Expression of the human endogenous retrovirus (HERV)-H family has been associated with colorectal carcinomas (CRC), yet no individual HERV-H locus expression has been thoroughly correlated with clinical data.Here, we characterized HERV-H reactivations in clinical CRC samples by integrating expression profiles, molecular patterns and clinical data. Expression of relevant HERV-H sequences was analyzed by qRT-PCR on two well-defined clinical cohorts (n = 139 pairs of tumor and adjacent normal colon tissue) including samples from adenomas (n = 21) and liver metastases (n = 16). Correlations with clinical and molecular data were assessed.

Results

CRC specific HERV-H sequences were validated and found expressed throughout CRC disease progression. Correlations between HERV-H expression and lymph node invasion of tumor cells (p = 0.0006) as well as microsatellite instable tumors (p < 0.0001) were established. No association with regard to age, tumor localization, grading or common mutations became apparent. Interestingly, CRC expressed elements belonged to specific young HERV-H subfamilies and their 5′ LTR often presented active histone marks.

Conclusion

These results suggest a functional role of HERV-H sequences in colorectal carcinogenesis. The pronounced connection with microsatellite instability warrants a more detailed investigation. Thus, HERV-H sequences in addition to tumor specific mutations may represent clinically relevant, truly CRC specific markers for diagnostic, prognostic and therapeutic purposes.     

Establishing the link between hepatitis B virus infection and colorectal adenoma

Background

Chronic hepatitis B (CHB) infection has been associated with malignancy, most notably hepatocellular carcinoma. Previous research has shown that hepatitis C is associated with increased colorectal adenomas and neoplasia. Currently, there are no studies on the association of CHB and colorectal adenomas. We aimed to identify a possible link between CHB and colorectal adenoma.

Methods

A retrospective chart review was performed on 588 consecutive patients undergoing screening or diagnostic colonoscopy that were previously screened or diagnosed with hepatitis B. Comparisons between categorical variables were analyzed with Chi Square test and t-test for continuous variables. Unconditional logistic regression was used to generate age-, gender-and race-adjusted odds ratios and their 95% confidence intervals (CI) comparing medication users with non-users. Statistical analyses were performed with SAS 9.3 software.

Results

A total of 487 patients were analyzed in the control group vs. 71 in the hepatitis B group. The adenoma detection rate was 23.9% in hepatitis B vs. 15.9% in the non-hepatitis B group for all cause colonoscopy; however this did not reach statistical significance. There was a significantly higher number of adenomas present in the distal colon compared to control (OR =2.16; 95% CI, 1.06-4.43; P=0.04). There were no significant findings between hepatitis B infection with size, multiplicity or presence of proximal adenomas. There was a significant difference noted in regards to smoking history, BMI and age between two groups.

Conclusions

Although the adenoma detection rate was higher in hepatitis B population vs. the non-hepatitis B group this did not reach statistical significance. However, we did find an association between CHB infection and the presence of distal colorectal adenomas. Larger prospective studies are needed to strengthen our findings along with future studies examining hepatitis B virus (HBV) and mechanisms inducing colorectal carcinogenesis.     

Tumour-suppressive function of SIRT4 in human colorectal cancer

Background:

SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers.

Methods:

We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry.

Results:

SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis.

Conclusions:

SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.     

Diagnostic value of preoperative serum carcinoembryonic antigen and carbohydrate antigen 19-9 in colorectal cancer

Background

Since the first introduction of tumour markers, their usefulness for diagnosis has been a challenging question. The aim of the present prospective study was to investigate, in colorectal cancer patients, the relationship between preoperative tumour marker concentrations and various clinical variables.

Methods

The study prospectively enrolled 131 consecutive patients with a confirmed diagnosis of colorectal carcinoma and 131 age- and sex-matched control subjects with no malignancy. The relationships of the tumour markers carcinoembryonic antigen (cea) and carbohydrate antigen (ca) 19-9 with disease stage, tumour differentiation (grade), mucus production, liver function tests, T stage, N stage, M stage were investigated.

Results

Serum concentrations of cea were significantly higher in the patient group than in the control group (p = 0.001); they were also significantly higher in stage iii (p = 0.018) and iv disease (p = 0.001) than in stage i. Serum concentrations of cea were significantly elevated in the presence of spread to lymph nodes (p = 0.005) in the patient group. Levels of both tumour markers were significantly elevated in the presence of distant metastasis in the patient group (p = 0.005 for cea; p = 0.004 for ca 19-9).

Conclusions

Preoperative levels of cea and ca 19-9 might provide an estimate of lymph node invasion and distant metastasis in colorectal cancer patients.     

Comparison of the epidermal growth factor receptor protein expression between primary non-small cell lung cancer and paired lymph node metastases: implications for targeted nuclide radiotherapy

Background

The knowledge of Epidermal growth factor receptor (EGFR) expression in metastases of NSCLC was limited. In receptor-mediated targeted nuclide radiotherapy, tumor cells are killed with delivered radiation and therapeutic efficiency is mainly dependent on the receptor expression. Thus, the level and stability of receptor expression in both primary tumors and corresponding metastases is crucial in the assessment of a receptor as target. The goal of this study was to evaluate whether EGFR is suitable as target for clinical therapy.

Methods

Expression of EGFR was investigated immunohistochemically in paired samples of lymph node metastases and corresponding NSCLC primary lesions (n = 51). EGFR expression was scored as 0, 1+, 2+ or 3+.

Results

Positive (1+, 2+ or 3+) EGFR immunostaining was evident in 36 of 47 (76.6%) analysed NSCLC primary tumors, and in 78.7% of the corresponding lymph node metastases. When EGFR expression is classified as positive or negative, discordance between the primary tumors and the corresponding metastases was observed in 5 cases (10.6%). EGFR overexpression (2+ or 3+) was found in 53.2% (25/47) of the NSCLC primary tumors and 59.6% of the corresponding metastases. Nine out of the 47 paired samples (19.2%) were discordant: Only three patients who had EGFR overexpression in the primary tumors showed EGFR downregulation (0 or 1+) in lymph node metastases, while six patients changed the other way around.

Conclusions

The EGFR expression in the primary tumor and the corresponding metastasis is discordant in about 10% of the patients. When overexpression is considered, the discordance is observed in about 20% of the cases. However, concerning EGFR overexpression in the primary tumors, similar expression in the metastases could be predicted with a reasonably high probability, which is encouraging for testing of EGFR targeted nuclide radiotherapy.     

The incidence of pelvic and para-aortic lymph node metastasis in uterine papillary serous and clear cell carcinoma according to the SEER registry

Objective

In this study we utilized the Surveillance, Epidemiology and End-Results (SEER) registry to identify risk factors for lymphatic spread and determine the incidence of pelvic and para-aortic lymph node metastases in patients with uterine papillary serous carcinoma (UPSC) and uterine clear cell carcinoma (UCCC) who underwent complete surgical staging and lymph node dissection.

Methods

Nine hundred seventy-two eligible patients diagnosed between 1998 to 2009 with International Federation of Gynecology and Obstetrics (FIGO) 1988 stage IA-IVA UPSC (n=685) or UCCC (n=287) were identified for analysis. Binomial logistic regression was used to determine risk factors for lymph node metastasis, with the incidence of pelvic and para-aortic lymph node metastases reported for each FIGO primary tumor stage. The Cox proportional hazards regression model was used to determine factors associated with overall survival.

Results

FIGO primary tumor stage was the only independent risk factor for lymph node metastasis (p<0.01). The incidence of pelvis-only and para-aortic lymph node involvement according to the FIGO primary tumor stage were as follows: IA (2.3%/3.8%), IB (7.5%/5.2%), IC (22.5%/16.9%), IIA (20.8%/13.2%), IIB (25.7%/14.9%), and III/IV (25.7%/24.3%). Prognostic factors for overall survival included lymph node involvement (hazard ratio [HR], 1.42; 95% confidence interval [CI], 1.09 to 1.85; p<0.01), patient age >60 years (HR, 1.70; 95% CI, 1.21 to 2.41; p<0.01), and advanced FIGO primary tumor stage (p<0.01). Tumor grade, histologic subtype, and patient race did not predict for either lymph node metastasis or overall survival.

Conclusion

There is a high incidence of both pelvic and para-aortic lymph node metastases for FIGO stages IC and above uterine papillary serous and clear cell carcinomas, suggesting a potential role for lymph node-directed therapy for these patients.     

Intratumoural budding (ITB) in preoperative biopsies predicts the presence of lymph node and distant metastases in colon and rectal cancer patients

Background:

In colorectal cancer (CRC), tumour budding at the invasion front is associated with lymph node (LN) and distant metastasis. Interestingly, tumour budding can also be detected in biopsies (intratumoural budding; ITB) and may have similar clinical importance. Here we investigate whether ITB in preoperative CRC biopsies can be translated into daily diagnostic practice.

Methods:

Preoperative biopsies from 133 CRC patients (no neoadjuvant therapy) underwent immunohistochemistry for pan-cytokeratin marker AE1/AE3. Across all biopsies for each patient, the densest region of buds at × 40 (high-power field; HPF) was identified and buds were counted.

Results:

A greater number of tumour buds in the biopsy was associated with pT stage (P=0.0143), LN metastasis (P=0.0007), lymphatic (P=0.0065) and venous vessel invasion (P=0.0318) and distant metastasis (cM1) (P=0.0013). Using logistic regression, a ‘scale'' was developed to estimate the probability of LN and distant metastasis using the number of tumour buds (e.g. 10 buds per HPF: 64% chance of LN metastasis; 30 buds per HPF: 86% chance). Inter-observer agreement for ITB was excellent (intraclass correlation coefficient: 0.813).

Conclusion:

Tumour budding can be assessed in the preoperative biopsy of CRC patients. It is practical, reproducible and predictive of LN and distant metastasis. Intratumoural budding qualifies for further investigation in the prospective setting.     

Impact of the coxsackievirus and adenovirus receptor on the adenoma-carcinoma sequence of colon cancer

Background:

Coxsackie and adenovirus receptor (CAR) has been suggested to function as a tumour suppressor. Its impact on the adenoma–carcinoma sequence of the colon, however, is unclear.

Methods:

Coxsackie and adenovirus receptor was analysed in non-cancerous and neoplastic colon samples using immunohistochemistry and quantitative RT–PCR. The function of CAR in colon cancer cell lines was determined following application of CAR siRNA or ectopic expression of a human full-length CAR cDNA.

Results:

Compared with healthy mucosa, increased CAR-mRNA expression was found in adenomas, whereas primary cancers and metastases displayed a marked decline. At the plasma membrane, CAR was present in normal mucosa samples (93%), adenomas, and metastases (100% ea.), whereas in colon cancers, it was found less frequently (49%, P<0.0001). Cytoplasmic CAR immunopositivity increased from normal mucosa (22%), to adenomas (73%, P=0.0006), primary cancers (83%, P<0.0001), and metastases (67%, P=0.0019). In cancer cell lines, CAR inhibition resulted in increased proliferation, whereas enforced ectopic CAR expression led to opposite results. Blocking the extracellular portion of CAR increased cell invasion in vitro. In mice, xenotransplants of colon cancer cells with enforced CAR expression formed significantly smaller tumours, whereas CAR inhibition increased the formation of liver metastases.

Conclusion:

We conclude that CAR facilitates complex effects during colon carcinogenesis, potentially mediated by its stage-dependent subcellular distribution; high CAR expression potentially prevents apoptosis in adenomas, loss of CAR at the plasma membrane promotes growth, and dissemination of primary cancers, and high membranous CAR presence may support the establishment of distant metastases.