Strong correlation between N-cadherin and CD133 in breast cancer: role of both markers in metastatic events

Purpose

Epithelial mesenchymal transition is a major mechanism to explain metastatic events in breast cancer. Another important aspect is that cells with stem cell properties are able to become resistant against chemotherapeutics. Our main goal was to investigate the role of the EMT marker, N-cadherin, and of the stem cell marker, CD133, in breast cancer.

Methods

The expressions of N-cadherin and CD133 were assessed by immunohistochemistry in 307 primary tumors from breast cancer patients and for 30 patients, in the related recurrences and/or metastases. We studied the correlation between both markers, their associations with known clinicopathological parameters and their role as predictive markers for survival time. Different expressions of both markers in primary tumor and recurrences or metastases were examined.

Results

N-cadherin and CD133 expressions correlated positively in the 261 primary tumor samples (p = 0.000) and in the 45 primary tumor, recurrence or metastasis samples (p = 0.010). In patients without lymph node metastases, the 10-year survival time was significantly lower when the tumor was N-cadherin-positive (p = 0.042). Expression of N-cadherin was also significantly higher in metastases than in the related primary tumors (p = 0.039).

Conclusion

N-cadherin and CD133 expressions are strongly correlated and N-cadherin appears as a potential metastases marker in a specific patient subpopulation.     

Androgen receptor expression is a predictive marker in chemotherapy-treated patients with endocrine receptor-positive primary breast cancers

Purpose

The androgen receptor (AR) is intensively discussed as a prognostic and/or predictive marker in breast cancer patients.

Methods

We evaluated the value of AR mRNA expression with the Affymetrix HG-U 133A array in 3 different cohorts: a cohort of breast cancer patients who received adjuvant treatment (cohort A; n = 165), a cohort of untreated breast cancer patients (cohort B; n = 200) and a cohort of chemotherapy-treated breast cancer patients with estrogen receptor (ER)-positive tumors (cohort C; n = 223).

Results

AR mRNA expression was associated with lower grading (Grades 1 and 2) as well as ER and progesterone receptor (PgR) positivity in all cohorts. In the treated cohort (cohort A), low androgen receptor expression was associated with shorter event-free survival (OR 2,34, 95 % CI 1.01–5.43, p = 0.047) which was not seen in the untreated cohort B. Subgroup analysis revealed that shorter survival of patients with low AR mRNA expression was observed mainly in the ER-positive subgroup of patients treated with adjuvant chemotherapy. In the validation cohort C we could confirm a benefit of chemotherapy for the group of tumors with high AR mRNA expression (5-year event-free survival (EFS) 74 % versus 57 %, p = 0.013). In this cohort, low AR mRNA expression was associated with shorter event-free survival also in multivariate analysis (OR 2.86, 95 % CI 1.29–6.35, p = 0.010) adjusted for HER2, ki-67, tumor size, age and tumor grade.

Conclusions

We provide evidence that AR expression is associated with chemotherapy responsiveness in ER-positive patients.     

Predictive value of sphingosine kinase 1 expression in neoadjuvant treatment of breast cancer

Purpose

Sphingolipids play important roles in apoptosis and cell proliferation. Sphingosine kinase 1 (SphK1) expression has a prognostic impact in primary breast cancer, but its predictive value is currently unknown.

Methods

A total of 112 breast cancer specimens from a prospective neoadjuvant chemotherapy trial (GeparDuo) were studied. Using tissue microarrays of pre-treatment core cut biopsies, we determined the expression of SphK1 by immunohistochemistry. The upper quartile of the cohort according to an immune reactive score of SphK1 was used as cutoff for high expression.

Results

We observed a larger number of samples with high SphK1 expression among ER-negative cancers (36.8 vs. 20.5 % among ER-positive cancers; Fisher test p = 0.073). Eighteen of the 112 patients demonstrated a pathological complete response. A significant predictive value for pathological complete response was observed for ER negativity (p = 0.003), young age (p = 0.037), and high tumor grade (p = 0.049). An increased pCR rate was observed in tumors with high SphK1 expression within the luminal subtype (26.7 vs. 5.8 %; Fisher test p = 0.040). No significant difference in survival was detected according to SphK1 expression.

Conclusions

Our results suggest that SphK1 may be a predictive factor for pCR after neoadjuvant treatment in luminal type breast cancers and warrants further investigation.     

Receptor change-clinicopathologic analysis of matched pairs of primary and cerebral metastatic breast cancer

Objective

A challenge in the management of breast cancer is development of brain metastases (BM) with limited survival. In primary breast cancer, ER/PR/HER2 are important prognostic markers and are important for making effective treatment decisions. Changes in immunohistochemical markers of metastases are with unclear clinical significance, and mechanisms of resistance to endocrine therapy are an additional challenge. The aim of this retrospective study is to detect changes in immunohistochemical markers of primary and BM and to recognize if receptor change has prognostic impact.

Methods

Twenty-four consecutive primary breast cancer patients who developed BM and got surgical resection of BM were enrolled. Matched pair analyses of primary and BM were done with evaluation by immunostaining (ER/PR/HER2).

Results

A small tumor size, ductal histology and HER2+ tumors were associated with BM. Loss of ER/PR receptor positivity was observed in BM compared to primary (ER: 50.0 %/22.7 %; p = 0.004; PR: 45.8 %/9.1 %; p = n.s), respectively, and almost no change in HER2 status (>80 %; p = 0.012). Patients with ER-/PR-negative or HER2-positive primary had shorter time to recurrence than ER-/PR-positive and HER2-negative patients. Receptor change has negative prognostic impact.

Conclusion

With the observed loss of receptor positivity, therapeutic options are diminished. Identification of patients with a high risk for BM is warranted to evaluate preventive strategies.     

Plasma HER2 amplification in cell-free DNA during neoadjuvant chemotherapy in breast cancer

Purpose

Measurement of human epidermal growth factor receptor 2 (HER2) gene amplification in cell-free DNA (cfDNA) is an evolving technique in breast cancer, enabling liquid biopsies and treatment monitoring. The present study investigated the dynamics of plasma HER2 gene copy number and amplification in cfDNA during neoadjuvant chemotherapy.

Patients and methods

The study included 50 patients from a prospective cohort analyzed during neoadjuvant chemotherapy. Fifty healthy women with no history of cancer served as control group and 15 patients with metastatic breast cancer were used to validate the assay. Total cfDNA and HER2 gene amplification were measured by quantitative real-time polymerase chain reaction.

Results

Plasma HER2 gene copy number (p = 0.794), HER2 gene amplification (p = 0.127) and total cfDNA (p = 0.440) did not differ significantly from the levels in the control group. Eighteen patients (36 %) obtained pathological complete response (pCR). HER2 gene copy number before the operation was significantly higher than the baseline level (p < 0.0001), but there was no difference between patients with and without pCR (p = 0.569). Likewise, there was no difference in plasma HER2 gene amplification between tissue HER2-positive and -negative patients (p = 0.754).

Conclusions

The results indicate that neither total cfDNA nor HER2 gene copy number is elevated in primary breast cancer patients compared to healthy controls. The level of both parameters increased during neoadjuvant chemotherapy, but without any relation to treatment effect. There was no indication of plasma HER2 gene amplification in the HER2-positive patients in the neoadjuvant setting.     

RUNX3 is a prognostic marker and potential therapeutic target in human breast cancer

Purpose

To evaluate the role of RUNX3 in breast cancer pathogenesis, we examined the RUNX3 expression in breast cancer tissues and analyzed the correlation between RUNX3 expression and clinicopathologic variables and patients survival.

Methods

We evaluated the RUNX3 expression by immunohistochemistry using a tissue microarray containing 256 specimens of breast cancer patients. We also studied the role of RUNX3 in cell migration and invasion by performing cell migration and invasion assay. Differential expression of metastasis-related genes after RUNX3 restoration was analyzed using the Human Tumor Metastasis PCR Array.

Results

The RUNX3 expression was significantly correlated with breast cancer histology grade (P = 0.000), and low RUNX3 expression strongly correlated with worse 5-year overall and disease-specific survival rates (P = 0.000 and P = 0.001, respectively). Furthermore, we found that RUNX3 restoration suppressed breast cancer metastasis by controlling cell migration and invasion capacity. Finally, gene expression profiles of RUNX3-549 and Ctrl-549 cells showed matrix metalloproteinase-2 (MMP-2) was the most significant gene among the 84 metastasis-related genes influenced by RUNX3 reintroduction.

Conclusions

Reduced RUNX3 expression is significantly correlated with breast cancer progression and predicts worse survival. RUNX3 regulates breast cancer cell migration and invasion through the MMP-2 pathway.     

Estrogen receptor beta 2 is associated with poor prognosis in estrogen receptor alpha-negative breast carcinoma

Purpose

Our aim was to examine the prognostic significance of ERbeta1 and ERbeta2 expression in ERalpha-negative breast carcinomas.

Materials and methods

We evaluated nuclear and cytoplasmic expression of ERbeta1 and ERbeta2 by immunohistochemistry in a group of 95 patients with long follow-up. ERbeta1 and ERbeta2 status was correlated with clinicopathological parameters and disease outcome. Univariate and multivariate analyses of ERbeta1 and ERbeta2 as independent markers of disease-free survival (DFS) were carried out using the Cox proportional hazards model.

Results

Nuclear ERbeta1 (nERbeta1) and nERbeta2 status was positively correlated (p = 0.01). nERbeta1 positivity was associated with low histological grade (p = 0.01) in all patients and in the nERbeta2-positive subgroup (p = 0.03) but not in the nERbeta2-negative (p = 0.27). nERbeta2 positivity was associated with lymph node involvement and tumor relapse in all cases (p < 0.00 and p < 0.00, respectively) and in the nERbeta1-negative subgroup (p < 0.00 and p < 0.00, respectively) but not in the nERbeta1-positive (p = 0.09 and p = 0.20, respectively). nERbeta2 positivity was associated with poor DFS in all patients (log-rank p <0.00), in the post-menopausal patient subgroup (log-rank p = 0.02) and in the HER2-negative (triple-negative) subgroup (log-rank p = 0.04). Cox multivariate analysis including ERbeta1, ERbeta2 and established clinicopathological variables highlighted ERbeta2 as an independent marker of early disease recurrence (hazard ratio 4.87; 95 % confidence interval 1.07–22.3; p = 0.04).

Conclusion

High nERbeta2 is an independent marker of early relapse in ERalpha-negative breast carcinoma, and in particular, in the nERbeta1-negative, the post-menopausal patient and the triple-negative subgroups. These findings suggest that inhibition of expression and/or function of ERbeta2 could improve disease outcome.     

l-Dopa decarboxylase (DDC) constitutes an emerging biomarker in predicting patients’ survival with stomach adenocarcinomas

Purpose

Stomach adenocarcinoma represents a major health problem and is regarded as the second commonest cause of cancer-associated mortality, universally, since it is still difficult to be perceived at a curable stage. Several lines of evidence have pointed out that the expression of l-Dopa decarboxylase (DDC) gene and/or protein becomes distinctively modulated in several human neuroendocrine neoplasms as well as adenocarcinomas.

Methods

In order to elucidate the clinical role of DDC on primary gastric adenocarcinomas, we determined qualitatively and quantitatively the mRNA levels of the gene with regular PCR and real-time PCR by using the comparative threshold cycle method, correspondingly, and detected the expression of DDC protein by immunoblotting in cancerous and normal stomach tissue specimens.

Results

A statistically significant association was disclosed between DDC expression and gastric intestinal histotype as well as tumor localization at the distal third part of the stomach (p = 0.025 and p = 0.029, respectively). Univariate and multivariate analyses highlighted the powerful prognostic importance of DDC in relation to disease-free survival and overall survival of gastric cancer patients. According to Kaplan–Meier curves, the relative risk of relapse was found to be decreased in DDC-positive (p = 0.031) patients who, also, exhibited higher overall survival rates (p = 0.016) than those with DDC-negative tumors.

Conclusions

This work is the first to shed light on the potential clinical usefulness of DDC, as an efficient tumor biomarker in gastric cancer. The provided evidence underlines the propitious predictive value of DDC expression in the survival of stomach adenocarcinoma patients.     

Metachronous esophageal squamous cell cancer after gastrectomy for gastric cancer

Background

The clinical and biological characteristics of metachronous esophageal squamous cell cancer (ESCC) after gastrectomy for gastric cancer have yet to be sufficiently elucidated. The aim of the present study was to examine carcinogenesis in such patients.

Methods

Subjects comprised 11 patients with metachronous carcinoma in whom ESCC occurred after gastric cancer (metachronous ESCC), 9 patients with simultaneously occurring gastric cancer and ESCC (simultaneous ESCC) and 52 patients with ESCC alone. We investigated the clinicopathological findings and biological properties using p53, p21 and cyclin D1 expression.

Results

The positive rate for the intraepithelial spread of tumor was higher for metachronous ESCC than for simultaneous ESCC (p < 0.05). The number of dysplastic lesions in metachronous ESCC, simultaneous ESCC and ESCC alone was 56, 41 and 44, respectively. The rate of positive p53 expression in dysplasia was significantly higher for metachronous ESCC than for ESCC alone (p = 0.03).

Conclusions

Positive expression of p53 was found in not only the primary tumor, but also intraepithelial neoplasia around the tumor in metachronous ESCC. Chronic gastroesophageal reflux due to gastrectomy may be involved in the process of carcinogenesis in addition to environmental and genetic factors for metachronous ESCC. Further studies of a larger number of patients with metachronous ESCC and a history of gastrectomy are warranted.     

Association of a common genetic variant of the IGF-1 gene with event-free survival in patients with HER2-positive breast cancer

Purpose

Insulin-like growth factor 1 (IGF-1) stimulates mitosis and inhibits apoptosis. High circulating IGF-1 levels are linked with an increased risk of colorectal and breast cancer. Recently, IGF-1 single nucleotide polymorphisms (SNPs), especially variant rs2946834, have been associated with poor clinical outcome in patients with colorectal cancer. In the present study, we aimed to investigate the influence of IGF1 polymorphisms associated with IGF-1 plasma levels on event-free survival in patients with HER2-positive breast cancer.

Methods

The present study included 161 consecutive white patients with HER2-positive breast cancer. Event-free survival was calculated as the time from cancer diagnosis to either relapse or death from any cause. Genomic DNA was extracted from archived formalin-fixed paraffin-embedded tumor tissue samples; five IGF-1 polymorphisms (rs2946834, rs6220, rs1520220, rs5742694, and rs5742678), all associated with IGF-1 levels, were genotyped by SNaPshot assays.

Results

Kaplan–Meier analysis showed a poorer clinical outcome for carriers of the rare allele of SNP rs2946834 (log-rank test, p = 0.020). Concordantly, in univariate Cox regression analyses, the rare allele of SNP rs2946834 was significantly associated with a decreased event-free survival (HR = 3.06 [1.14–8.22]; p = 0.027). Multivariate analysis adjusted for age and tumor stage confirmed this result (HR = 4.02 [1.36–11.90]; p = 0.012). Other investigated polymorphisms of the IGF1 gene were not significantly associated with event-free survival (all p values >0.05).

Conclusions

This study provides first evidence that IGF1 rs2946834 polymorphism is associated with clinical outcome of HER2-positive breast cancer patients. Further studies are warranted to validate these findings.     

PPARγ and Wnt/β-Catenin pathway in human breast cancer: expression pattern, molecular interaction and clinical/prognostic correlations

Purpose

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor expressed in a large number of human cancers and plays important roles in breast cancer cell proliferation. Its association with clinicopathologic features and Wnt/β-Catenin signaling pathway, a crucial factor in embryonic and malignant development, in breast cancer has not been reported systematically. In the present study, expression patterns, interaction and the correlations with clinical/prognostic factors of PPARγ and β-Catenin were investigated among patients with breast cancer.

Methods

Using immunohistochemistry, we performed a study on 70 patient-derived human breast tumors and compared the protein expression levels of PPARγ, β-Catenin and Ki-67. Correlations were then analyzed between IHC-assessed level of these molecules and major clinicopathologic variables and survival. Furthermore, western blot (WB) analysis before and after immunoprecipitation with PPARγ and β-Catenin were performed on breast cancer tissues and cell lines to evaluate their protein level and molecular interaction.

Results

We showed that PPARγ expression was of significant prognostic value in the outcome of breast carcinomas, which positively correlated with ER status (P = 0.012) and inversely associated with histologic grade (P = 0.012), tumor size (P = 0.007), axillary lymph node status (P = 0.044), TNM stage (P = 0.026), Ki-67 (P = 0.006) and abnormal β-Catenin expression (P = 0.023), whereas no correlation was seen between PPARγ and age (P = 0.513), histology (P = 0.764), PR (P = 0.099) or HER-2 status (P = 0.175). Kaplan–Meier survival curves of the study population showed that high expression level of PPARγ significantly correlated with long-term survival. Molecular interaction could also be demonstrated between PPARγ and β-Catenin both in breast cancer cell lines and tissue samples.

Conclusions

On the basis of these results, we suggested that PPARγ might serve as a future target for the development of novel treatments in breast cancer.     

DNA Methylation of NDRG2 in Gastric Cancer and Its Clinical Significance

Background

Gastric cancer is one of the most common digestive malignancies worldwide. N-myc downstream-regulated gene 2 (NDRG2) is a differentiation-related gene that is considered to be a metastasis suppressor gene. In this study, we examined the expression and DNA methylation of NDRG2 in gastric cancer cell lines and tissues, as well as its clinical significance.

Methods

Six gastric cancer cell lines and 42 paired normal and gastric cancer tissue samples were used to assess NDRG2 mRNA expression using RT-PCR. NDRG2 DNA methylation status was evaluated by methylation-specific PCR (MSP) in gastric cancer cell lines and tissues. The suppression of NDRG2 in BGC823 cells by siRNA transfection was utilized to detect the role of NDRG2 in gastric cancer progression.

Results

NDRG2 mRNA was down-regulated in gastric cancer cell lines and tissues, and its expression was just related to lymph node metastasis (p = 0.032). MSP showed methylation of NDRG2 in 54.0 % (47/87) of primary gastric cancer specimens and in 20.0 % (16/80) of corresponding nonmalignant gastric tissues. NDRG2 methylation was related to depth of tumor invasion, Borrmann classification and TNM stage (p < 0.05). Upon treatment with 5-aza-2′-deoxycytidine and trichostatin A, NDRG2 expression was upregulated in HGC27 cells, and demethylation of the highly metastatic cell line, MKN45, inhibited cell invasion. Furthermore, the suppression of NDRG2 by siRNA transfection enhanced BGC823 cells invasion.

Conclusions

Our results suggest that the aberrant methylation of NDRG2 may be mainly responsible for its downregulation in gastric cancer, and may play an important role in the metastasis of gastric cancer.     

Increased Expression of Cellular Repressor of E1A-Stimulated Gene (CREG) in Gastric Cancer Patients: A Mechanism of Proliferation and Metastasis in Cancer

Background

The cellular repressor of E1A-stimulated genes (CREG), a secreted glycoprotein, has been studied with human embryonic carcinoma cells, vascular smooth muscle cells, and NIH3T3 fibroblasts. However, its relationship to tumor cell proliferation and metastasis has not been examined in human gastric cancers (GC) until now.

Aim

To investigate the expression of CREG in GC and its association with GC cell proliferation and metastasis.

Methods

Forty-two cases of GCs, matched normal gastric tissues, and the human gastric cancer cell lines BGC-823, SGC-7901, MKN45, normal gastric mucosa cell line GES, and HUVEC cell line ECV304 were used to analyze CREG expression at the level of mRNA and protein. The expression of CREG was then further examined by immunohistochemistry in 42 GC tissues, and the correlation between the level of CREG and the pathological and clinical data was evaluated. Finally, we down-regulated the expression of CREG in GC cells with specific siRNA, and assessed the role of CREG in the proliferation and metastasis/invasion of the GC cell line.

Results

The level of CREG was found to be higher in malignant GC tissues and cells compared to adjacent normal tissues and normal gastric cells (p < 0.001). Additionally, the expression levels of CREG were positively correlated with tumor clinical stage (p = 0.001), tumor metastasis (p < 0.001), and stages of tumor infiltration (p = 0.019). Furthermore, by using siRNA, we found that the down-regulated expression of CREG inhibited the proliferation of GC cells (p < 0.05), and migration of both GC cells (p = 0.001).

Conclusions

Our data suggest that CREG plays an important role in gastric cancer cell proliferation and metastasis and that CREG may be a potential therapeutic target for GC.     

Positive Expression of LSD1 and Negative Expression of E-cadherin Correlate with Metastasis and Poor Prognosis of Colon Cancer

Background

The first identified lysine-specific demethylase, LSD1, plays an important role in the metastatic progression of several types of cancer.

Aims

The aim of this study was to investigate LSD1, E-cadherin, and N-cadherin expression in colon cancer specimens and their clinical significance.

Methods

The expression of LSD1, E-cadherin, and N-cadherin in colon cancer specimens was determined by immunohistochemistry, and the relationship between the expression of the respective molecules and clinicopathological characteristics was analyzed.

Results

The positive expression rates of LSD1, E-cadherin, and N-cadherin in colon cancer specimens were 66.7 % (72/108), 85.2 % (92/108), and 41.7 % (45/108), respectively. LSD1 was significantly more highly expressed in colon cancer specimens classified as high TNM stage lesions and with distant metastasis (P < 0.05). Further analysis demonstrated that LSD1 expression was positively correlated with lymph node and distant metastases (P < 0.05). However, E-cadherin expression was significantly downregulated in colon cancer specimens classified as high TNM stage lesions and with distant metastasis (P < 0.05), whereas the expression of N-cadherin did not differ significantly according to clinical and pathological characteristics (P > 0.05). Correlation analysis revealed that LSD1 expression was negatively correlated with E-cadherin expression (r _s = ?0.318, P = 0.001), but not evidently correlated with N-cadherin expression (r _s = 0.182, P = 0.06). Colon cancer specimens with positive LSD1 expression and negative E-cadherin expression were correlated with significantly lower overall survival.

Conclusions

LSD1 showed a significantly higher expression, in contrast to the significantly lower expression of E-cadherin, in colon cancer specimens classified as high TNM stage lesions and with distant metastasis. Positive expression of LSD1 and negative expression of E-cadherin may be predictors of a worse colon cancer prognosis.     

p42.3: A promising biomarker for the progression and prognosis of human colorectal cancer

Purpose

As a novel cell cycle-related gene, p42.3 has been shown to play a key role in the cell proliferation and tumorigenicity of gastric cancer. To date, the association between p42.3 and colorectal cancer (CRC) has not been reported. This study investigated the expression of p42.3 and its potential role in human colorectal cancers.

Methods

Real-time polymerase chain reaction and western blotting were used to evaluate p42.3 mRNA and protein expression in 14 pairs of fresh frozen CRC samples, matched with adjacent normal mucosa. The p42.3 protein was evaluated by immunohistochemistry using CRC tissue microarrays, which included 212 CRC specimens and corresponding normal colorectal mucosa. The expression profiles of p42.3 in CRC tissues were analyzed against clinicopathological factors and post-surgical survival status. The expression profiles of p42.3 were also investigated in six human colon carcinoma cell lines.

Results

p42.3 was demonstrated to be over-expressed in colorectal cancer tissues compared with normal mucosa in the 14 tissue pairs (P = 0.011) and was significantly higher in patients with poor tumor differentiation (P = 0.045); patients positive for p42.3 expression had a poorer prognosis than those not expressing this protein (P = 0.033). In a multivariate survival analysis, p42.3 expression was identified as an independent prognostic factor for CRC patients (P = 0.030).

Conclusions

The results indicated that p42.3 might play an important role in the progression of CRC, and it has a great value for assessing CRC patient prognosis after surgery.