Phosphorylated p-70S6K predicts tamoxifen resistance in postmenopausal breast cancer patients randomized between adjuvant tamoxifen versus no systemic treatment

Introduction

Activation of the phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways results in anti-estrogen resistance in vitro, but a biomarker with clinical validity to predict intrinsic resistance has not been identified. In metastatic breast cancer patients with previous exposure to endocrine therapy, the addition of a mammalian target of rapamycine (mTOR) inhibitor has been shown to be beneficial. Whether or not patients on adjuvant endocrine treatment might benefit from these drugs is currently unclear. A biomarker that predicts intrinsic resistance could potentially be used as companion diagnostic in this setting. We tested the clinical validity of different downstream-activated proteins in the PI3K and/or MAPK pathways to predict intrinsic tamoxifen resistance in postmenopausal primary breast cancer patients.

Methods

We recollected primary tumor tissue from patients who participated in a randomized trial of adjuvant tamoxifen (1–3 years) versus observation. After constructing a tissue micro-array, cores from 563 estrogen receptor α positive were immunostained for p-AKT(Thr308), p-AKT(Ser473), p-mTOR, p-p706SK and p-ERK1/2. Cox proportional hazard models for recurrence free interval were used to assess hazard ratios and interactions between these markers and tamoxifen treatment efficacy.

Results

Interactions were identified between tamoxifen and p-AKT(Thr308), p-mTOR, p-p70S6K and p-ERK1/2. Applying a conservative level of significance, p-p70S6K remained significantly associated with tamoxifen resistance. Patients with p-p70S6K negative tumors derived significant benefit from tamoxifen (HR 0.24, P < 0.0001), while patients whose tumor did express p-p70S6K did not (HR = 1.02, P =0.95), P for interaction 0.004. In systemically untreated breast cancer patients, p-p70S6K was associated with a decreased risk for recurrence.

Conclusions

Patients whose tumor expresses p-p70S6K, as a marker of downstream PI3K and/or MAPK pathway activation, have a favorable prognosis, but do not benefit from adjuvant tamoxifen. A potential benefit from inhibitors of the PI3K/Akt/mTOR pathway in these patients needs to be further explored.     

Novel therapeutic strategy targeting the Hedgehog signalling and mTOR pathways in biliary tract cancer

Background:

Activation of the PI3K/mTOR and Hedgehog (Hh) signalling pathways occurs frequently in biliary tract cancer (BTC). Crosstalk between these pathways occurs in other gastrointestinal cancers. The respective signalling inhibitors rapamycin and vismodegib may inhibit BTC synergistically and suppress cancer stem cells (CSCs).

Methods:

Gene expression profiling for p70S6k and Gli1 was performed with BTC cell lines. Tumour and pathway inhibitory effects of rapamycin and vismodegib were investigated in BTC preclinical models and CSCs.

Results:

Rapamycin and vismodegib synergistically reduced BTC cell viability and proliferation. This drug combination arrested BTC Mz-ChA-1 cells in the G1 phase but had no significant effect on the cell cycle of BTC Sk-ChA-1 cells. Combined treatment inhibited the proliferation of CSCs and ALDH-positive cells. Nanog and Oct-4 expression in CSCs was decreased by the combination treatment. Western blotting results showed the p-p70S6K, p-Gli1, p-mTOR, and p-AKT protein expression were inhibited by the combination treatment in BTC cells. In an Mz-ChA-1 xenograft model, combination treatment resulted in 80% inhibition of tumour growth and prolonged tumour doubling time. In 4 of 10 human BTC specimens, tumour p-p70S6K and Gli1 protein expression levels were decreased with the combination treatment.

Conclusions:

Targeted inhibition of the PI3K/mTOR and Hhpathways indicates a new avenue for BTC treatment with combination therapy.     

Aberrant activation of the mTOR pathway and anti-tumour effect of everolimus on oesophageal squamous cell carcinoma

Background:

The mammalian target of rapamycin (mTOR) protein is important for cellular growth and homeostasis. The presence and prognostic significance of inappropriate mTOR activation have been reported for several cancers. Mammalian target of rapamycin inhibitors, such as everolimus (RAD001), are in development and show promise as anti-cancer drugs; however, the therapeutic effect of everolimus on oesophageal squamous cell carcinoma (OSCC) remains unknown.

Methods:

Phosphorylation of mTOR (p-mTOR) was evaluated in 167 resected OSCC tumours and 5 OSCC cell lines. The effects of everolimus on the OSCC cell lines TE4 and TE11 in vitro and alone or in combination with cisplatin on tumour growth in vivo were evaluated.

Results:

Mammalian target of rapamycin phosphorylation was detected in 116 tumours (69.5%) and all the 5 OSCC cell lines. Everolimus suppressed p-mTOR downstream pathways, inhibited proliferation and invasion, and induced apoptosis in both TE4 and TE11 cells. In a mouse xenograft model established with TE4 and TE11 cells, everolimus alone or in combination with cisplatin inhibited tumour growth.

Conclusion:

The mTOR pathway was aberrantly activated in most OSCC tumours. Everolimus had a therapeutic effect both as a single agent and in combination with cisplatin. Everolimus could be a useful anti-cancer drug for patients with OSCC.     

Neuroendocrine and epithelial phenotypes in small-cell lung cancer: implications for metastasis and survival in patients

Background:

Small-cell lung cancer (SCLC) has a very aggressive clinical course with early metastasis. This study investigated how the distinctive neuroendocrine characteristics contribute to disease progression and invasion in human SCLC.

Methods:

The neuroendocrine phenotype (pro-opiomelanocortin (POMC)) was quantified by ELISA in blood samples from 43 SCLC patients. The neuroendocrine (POMC, chromogranin A, neuron-specific enolase, NCAM) and epithelial (cytokeratin and E-cadherin) phenotypes were investigated, using ELISA and immunocytochemistry/immunohistochemistry.

Results:

In SCLC patients, 16% had elevated circulating POMC, which was associated with significantly worse survival (P=0.02) and liver metastases (P=0.004). In addition, POMC correlated with epithelial-positive circulating tumour cells (P=0.0002). In a panel of SCLC cell lines, all POMC-secreting cell lines expressed cytokeratin (40% of total). Even after cloning, DMS 79 cells expressed both neuroendocrine and epithelial markers. DMS 79 xenografts secreted POMC into the blood, which mirrored the tumour volume. These xenografts expressed both neuroendocrine and epithelial phenotypes in all tumours, with both phenotypes prevalent in cells invading the surrounding tissue.

Conclusion:

Both neuroendocrine and epithelial phenotypes coexist in human SCLC tumours in vitro and in vivo and this persists in invading tumour cells. In patients, POMC secretion predicts poor survival and liver metastases, suggesting a crucial role of the neuroendocrine phenotype.     

Prognostic effect of activated EGFR expression in human colon carcinomas: comparison with EGFR status

Background:

Evidence suggests that epidermal growth factor receptor (EGFR)-activation status may better predict the clinical behaviour of colon cancers than does EGFR expression. However, the prognostic effect of phospho-EGFR in primary colon cancer remains undefined.

Methods:

Phospho-EGFR (Tyr-1173) and EGFR expression were analysed by immunohistochemistry (IHC) in tissue microarrays of TNM stage II and III colon cancers from completed adjuvant therapy trials (n=388). Staining intensity was scored and correlated with clinicopathological variables, DNA mismatch repair (MMR) status, rates of cell proliferation (Ki-67), apoptosis (caspase-3), and patient survival.

Results:

Phospho-EGFR expression was detected in 157 of 388 (40%) tumours, whereas EGFR was found in 214 of 361 (59%). Although phospho-EGFR was unrelated to clinicopathological variables, strong EGFR intensity was associated with higher tumour stage (P=0.03). Tumours overexpressing EGFR (P=0.0002) or phospho-EGFR (P=0.015) showed increased Ki-67, but not caspase-3 expression. Phospho-EGFR was not prognostic. EGFR intensity was associated with worse disease-free survival (DFS) (hazard ratio (HR): 1.21 (1.03, 1.41); P=0.019) and overall survival (OS) (HR: 1.19 (1.02, 1.39); P=0.028). Tumours expressing both EGFR and phospho-EGFR had similar survival as EGFR alone. Stage and lymph node number were prognostic for DFS and OS, and histological grade for OS. EGFR was an independent predictor of DFS (P=0.042) after adjustment for stage, histological grade, age, and MMR status.

Conclusion:

Phospho-EGFR and EGFR expression were associated with tumour cell hyperproliferation. Phospho-EGFR was not prognostic, whereas increased EGFR intensity was independently associated with poor DFS.     

The prognostic impact of the neutrophil-to-lymphocyte ratio in patients with small-cell lung cancer

Background:

The neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are prognostic factors for various types of cancer. In this study, we assessed the association of NLR and PLR with the prognosis of small-cell lung cancer (SCLC) in patients who received the standard treatment.

Methods:

We retrospectively reviewed patients who were diagnosed with SCLC and treated with platinum-based chemotherapy between July 2006 and October 2013 in Gyeongsang National University Hospital Regional Cancer Center and Changwon Samsung Hospital.

Results:

In total, 187 patients were evaluated. Compared with low NLR (<4), high NLR (⩾4) at diagnosis was associated with poor performance status, advanced stage, and lower response rate. Median overall survival (OS) and progression-free survival (PFS) were worse in the high-NLR group (high vs low, 11.17 vs 9.20 months, P=0.019 and 6.90 vs 5.49 months, P=0.005, respectively). In contrast, PLR at diagnosis was not associated with OS or PFS (P=0.467 and P=0.205, respectively). In multivariate analysis, stage, lactate dehydrogenase, and NLR at diagnosis were independent prognostic factors for OS and PFS.

Conclusions:

NLR is easily measurable and reflects the SCLC prognosis. A future prospective study is warranted to confirm our results.     

Activation of the EGFR/ERK pathway in high-grade mucoepidermoid carcinomas of the salivary glands

Background:

Mucoepidermoid carcinoma (MEC) shows differences in biological behaviour depending mainly on its histological grade. High-grade tumours usually have an aggressive biological course and they require additional oncological treatment after surgery.

Methods:

In a series of 43 MECs of the salivary glands, we studied the epidermal growth factor receptor (EGFR) gene by using dual-colour chromogenic in situ hybridisation (CISH). Moreover, we assessed the protein expressions of the EGFR and the activated extracellular signal-regulated kinases (pERK1/2) by using immunohistochemistry. These results were correlated with the histological grade of the tumours and the outcome of the patients.

Results:

The CISH study demonstrated a high-EGFR gene copy number, with balanced chromosome 7 polysomy, in 8 out of 11 high-grade MECs (72.7%), whereas 27 low-grade and 15 intermediate-grade tumours had a normal EGFR gene copy number (P<0.001). The EGFR gene gains correlated with disease-free interval (P=0.003) and overall survival of the patients (P=0.019). The EGFR protein expression had a significant correlation with the histological grade of the tumours but not with the outcome of the patients. The pERK1/2 expression correlated with histological grade of tumours (P<0.001), disease-free interval (P=0.004) and overall survival (P=0.001).

Conclusions:

The EGFR/ERK pathway is activated in high-grade MECs with aggressive behaviour. Patients with these tumours who require oncological treatment in addition to surgery could benefit from EGFR and mitogen-activated protein kinase pathway inhibitors.     

MET phosphorylation predicts poor outcome in small cell lung carcinoma and its inhibition blocks HGF-induced effects in MET mutant cell lines

Background:

Small cell lung carcinoma (SCLC) has poor prognosis and remains orphan from targeted therapy. MET is activated in several tumour types and may be a promising therapeutic target.

Methods:

To evaluate the role of MET in SCLC, MET gene status and protein expression were evaluated in a panel of SCLC cell lines. The MET inhibitor PHA-665752 was used to study effects of pathway inhibition in basal and hepatocyte growth factor (HGF)-stimulated conditions. Immunohistochemistry for MET and p-MET was performed in human SCLC samples and association with outcome was assessed.

Results:

In MET mutant SCLC cells, HGF induced MET phosphorylation, increased proliferation, invasiveness and clonogenic growth. PHA-665752 blocked MET phosphorylation and counteracted HGF-induced effects. In clinical samples, total MET and p-MET overexpression were detected in 54% and 43% SCLC tumours (n=77), respectively. MET phosphorylation was associated with poor median overall survival (132 days) vs p-MET negative cases (287 days)(P<0.001). Phospho-MET retained its prognostic value in a multivariate analysis.

Conclusions:

MET activation resulted in a more aggressive phenotype in MET mutant SCLC cells and its inhibition by PHA-665752 reversed this phenotype. In patients with SCLC, MET activation was associated with worse prognosis, suggesting a role in the adverse clinical behaviour in this disease.     

Analysis of circulating angiogenic biomarkers from patients in two phase III trials in lung cancer of chemotherapy alone or chemotherapy and thalidomide

Background:

Thalidomide has potent anti-inflammatory and anti-angiogenic properties. It was evaluated in combination with chemotherapy in two randomised placebo-controlled trials in patients with small cell lung cancer (SCLC, n=724) and advanced non-small cell lung cancer (NSCLC, n=722). Neither study demonstrated an improvement in overall survival with the addition of thalidomide to chemotherapy. This study investigated circulating angiogenic biomarkers in a subset of these patients.

Methods:

Serial plasma samples were collected in a cohort of patients enrolled in these two trials (n=95). Vascular endothelial growth factor (VEGF), soluble truncated form of VEGF receptor-2 (sVEGFR-2), interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), basic fibroblast growth factor (bFGF) and soluble intercellular adhesion molecule-1 (sICAM-1) levels were measured by enzyme-linked immunosorbent assays. Results were correlated with patient clinical data including stage, response rate and progression-free survival (PFS).

Results:

Baseline biomarker levels were not significantly different between SCLC and NSCLC. For pooled treatment groups, limited stage SCLC was associated with lower baseline VEGF (P=0.046), sICAM-1 (P=0.008) and IL-8 (P=0.070) than extensive stage disease. Low baseline IL-8 was associated with a significantly improved PFS in both SCLC and NSCLC (P=0.028), and a greater reduction in IL-8 was associated with a significantly improved tumour response (P=0.035). Baseline angiogenic factor levels, however, did not predict response to thalidomide.

Conclusion:

Circulating angiogenic biomarkers did not identify patients who benefited from thalidomide treatment.     

RAD21 cohesin overexpression is a prognostic and predictive marker exacerbating poor prognosis in KRAS mutant colorectal carcinomas

Background:

RAD21 is a component of the cohesion complex and is integral to chromosome segregation and error-free DNA repair. RAD21 is functionally important in tumour progression but its role in colorectal carcinoma (CRC) is unclear. We therefore assessed its clinicopathological and prognostic significance in CRC, as well as its effect on chemosensitivity.

Methods:

A retrospective observation study examined RAD21 expression in 652 CRCs using a tissue microarray approach. Correlation with clinicopathological factors including gender, tumour grade, mucinous subtype, TNM stage, disease-specific survival (DSS), BRAF and KRAS mutation status, tumour p53 immunostaining, tumour microsatellite instability and tumour CpG island methylator phenotype was performed. Colorectal cancer cell clones with stable RAD21 knockdown were generated and tested for cellular sensitivity to conventional chemotherapeutic drugs.

Results:

RAD21 expression was significantly correlated with male gender (56.7% vs 43.3%, P=0.02), well-differentiated histology (14.4% vs 4.0%, P=0.0001), higher T-stage (36.1% vs 27.0%, P=0.01), presence of metastasis (18.8% vs 12.6%, P=0.03), and shorter DSS (hazard ratio (HR) 1.4, 95% CI 1.1 to 1.9, P=0.01) in both univariate and multivariate analysis. RAD21 expression was associated with shorter DSS in patients with KRAS mutant tumours (HR:2.6, 95% CI:1.4–4.3, P=0.001) and in patients receiving adjuvant chemoradiotherapy (HR:1.9, 95% CI:1.2–3.0, P=0.008). Colorectal cancer cells with RAD21 knockdown exhibited enhanced sensitivity to 5-fluorouracil, either alone or in combination with oxaliplatin.

Conclusions:

RAD21 expression in CRC is associated with aggressive disease especially in KRAS mutant tumours and resistance to chemoradiotherapy. RAD21 may be an important novel therapeutic target.     

Multipoint targeting of the PI3K/mTOR pathway in mesothelioma

Background:

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis.

Methods:

Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations.

Results:

We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation.

Conclusions:

These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.     

CXCR7 expression is associated with disease-free and disease-specific survival in cervical cancer patients

Background:

The CXC chemokine receptor (CXCR)7 is involved in tumour development and metastases formation. The aim of the present study was to determine protein expression of CXCR7, its putative co-receptors epidermal growth factor receptor (EGFR) and CXCR4, its predominant ligand CXCL12, their co-dependency and their association with survival in cervical cancer patients.

Methods:

CXC chemokine receptor 7, EGFR, CXCR4 and CXCL12 expression were determined immunohistochemically in 103 paraffin-embedded, cervical cancers. Subsequently, associations with patient characteristics were assessed and survival analyses were performed.

Results:

CXC chemokine receptor 7 was expressed by 43% of tumour specimens, in a large majority of cases together with either EGFR or CXCR4 (double positive), or both (triple positive). The CXCR7 expression was associated with tumour size (P=0.013), lymph node metastasis (P=0.001) and EGFR expression (P=0.009). CXC chemokine receptor 7 was independently associated with disease-free survival (hazard ratio (HR)=4.3, 95% confidence intervals (CI) 1.7–11.0, P=0.002), and strongly associated with disease-specific survival (HR=3.9, 95% CI 1.5–10.2, P=0.005).

Conclusion:

CXC chemokine receptor 7 expression predicts poor disease-free and disease-specific survival in cervical cancer patients, and might be a promising new therapeutic marker. In a large majority of cases, CXCR7 is co-expressed with CXCR4 and/or EGFR, supporting the hypothesis that these receptors assist in CXCR7 signal transduction.     

Tetraspanins CD9 and CD151, epidermal growth factor receptor and cyclooxygenase-2 expression predict malignant progression in oral epithelial dysplasia

Background:

Prognostic biomarkers aim to improve on the current inadequate method of histological assessment to identify patients with oral epithelial dysplasia at greatest risk of malignant transformation. We aimed to assess the prognostic ability of six protein biomarkers linked to the epidermal growth factor receptor (EGFR) pathway, including three tetraspanins, in a large multicentre oral dysplasia cohort.

Methods:

One hundred and forty-eight cases with varying degrees of epithelial dysplasia underwent immunohistochemical assessment for CD9, CD151, CD82, EGFR, Her-2, and COX-2. Scoring was performed independently by two observers. Univariate analyses using both logistic and Cox regression models and a multivariate regression were performed.

Results:

Malignant progression was significantly greater in those cases with decreased expression of CD9 (P=0.02), and increased expression of CD151 (P=0.02), EGFR (P=0.04), and COX-2 (P=0.003). Histological grade (P=0.0002) and morphology (P=0.03) were also prognostic, whereas smoking and alcohol were not. The optimal combination by backward-variable selection was of histological grade (hazard ratio (HR) 1.64; 95% CI 1.12, 2.40), COX-2 overexpression (HR 1.12; 1.02, 1.24) and CD9 underexpression (HR 0.88; 0.80, 0.97). CD82 and Her-2 demonstrated no prognostic ability.

Conclusion:

This is the first study of the expression and prognostic potential of the tetraspanins in oral dysplasia. A combination of certain biomarkers with clinical factors appeared to improve the accuracy of determining the risk of malignancy in individuals with oral dysplasia. These findings may also offer potential new therapeutic approaches for this condition.     

Clinical impact of mutation fraction in epidermal growth factor receptor mutation positive NSCLC patients

Background:

We examined clinical outcomes in a population-based cohort of EGFR mutant advanced NSCLC patients, exploring the potential role of factors including tumour EGFR mutation fraction and cellularity in predicting outcomes.

Methods:

A cohort of patients with EGFR mutant advanced NSCLC was identified (N=293); clinical outcomes, pathologic and treatment details were collected. Tumour response was determined from radiology and clinical notes. Association between demographic and pathologic variables EGFR TKI response, time to treatment failure (TTF) and overall survival (OS) was examined using logistic regression and proportional hazards regression. EGFR TKI response rates were summarised by percent mutation fraction to explore their association.

Results:

Higher mutation fraction was associated with greater EGFR TKI response rate (odds ratio 1.58, 95% CI=1.21–2.07, P=0.0008), longer TTF (hazard ratio 0.80, 95% CI=0.68–0.92, P=0.003) and better OS (hazard ratio 0.81, 95% CI=0.67–0.99, P=0.04). However, even in patients with ⩽5% mutation fraction, response rate was 34%. Females had longer TTF (P=0.02).

Conclusions:

EGFR mutation fraction in tumour samples was significantly associated with response, TTF and OS. Despite this, no lower level of mutation fraction was detected for which EGFR TKI should be withheld in those with activating EGFR mutations.     

Epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are prognostic biomarkers for patients with resected colorectal cancer liver metastases

Background:

Resection of colorectal cancer liver metastasis (CRCLM) with curative intent has long-term benefit in ∼40% of cases. Prognostic biomarkers are needed to improve clinical management and reduce futile surgeries. Expression of epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase-2) has been associated with carcinogenesis and survival. We investigated the prognostic value of EGFR and PTGS2 expression in patients with resected CRCLM.

Methods:

Formalin-fixed paraffin-embedded CRCLM tissue and corresponding primary tumour specimens from a multi-institutional cohort of patients who underwent liver resection between 1990 and 2010 were incorporated into tissue microarrays (TMAs). TMAs were stained for EGFR and PTGS2 by immunohistochemistry. The hazard rate ratio (HRR) for the association between expression in CRCLM and overall survival was calculated using a 500-fold cross-validation procedure.

Results:

EGFR and PTGS2 expression could be evaluated in 323 and 351 patients, respectively. EGFR expression in CRCLM was associated with poor prognosis (HRR 1.54; P<0.01) with a cross-validated HRR of 1.47 (P=0.03). PTGS2 expression was also associated with poor prognosis (HRR 1.60; P<0.01) with a cross-validated HRR of 1.63 (P<0.01). Expression of EGFR and PTGS2 remained prognostic after multivariate analysis with standard clinicopathological variables (cross-validated HRR 1.51; P=0.02 and cross-validated HRR 1.59; P=0.01, respectively). Stratification for the commonly applied systemic therapy regimens demonstrated prognostic value for EGFR and PTGS2 only in the subgroup of patients who were not treated with systemic therapy (HRR 1.78; P<0.01 and HRR 1.64; P=0.04, respectively), with worst prognosis when both EGFR and PTGS2 were highly expressed (HRR 3.08; P<0.01). Expression of PTGS2 in CRCLM was correlated to expression in patient-matched primary tumours (P=0.02, 69.2% concordance).

Conclusions:

EGFR and PTGS2 expressions are prognostic molecular biomarkers with added value to standard clinicopathological variables for patients with resectable CRCLM.     

Bcl-2 and β1-integrin predict survival in a tissue microarray of small cell lung cancer

Introduction:

Survival in small cell lung cancer (SCLC) is limited by the development of chemoresistance. Factors associated with chemoresistance in vitro have been difficult to validate in vivo. Both Bcl-2 and β₁-integrin have been identified as in vitro chemoresistance factors in SCLC but their importance in patients remains uncertain. Tissue microarrays (TMAs) are useful to validate biomarkers but no large TMA exists for SCLC. We designed an SCLC TMA to study potential biomarkers of prognosis and then used it to clarify the role of both Bcl-2 and β₁-integrin in SCLC.

Methods:

A TMA was constructed consisting of 184 cases of SCLC and stained for expression of Bcl-2 and β₁-integrin. The slides were scored and the role of the proteins in survival was determined using Cox regression analysis. A meta-analysis of the role of Bcl-2 expression in SCLC prognosis was performed based on published results.

Results:

Both proteins were expressed at high levels in the SCLC cases. For Bcl-2 (n=140), the hazard ratio for death if the staining was weak in intensity was 0.55 (0.33–0.94, P=0.03) and for β₁-integrin (n=151) was 0.60 (0.39–0.92, P=0.02). The meta-analysis showed an overall hazard ratio for low expression of Bcl-2 of 0.91(0.74–1.09).

Conclusions:

Both Bcl-2 and β₁-integrin are independent prognostic factors in SCLC in this cohort although further validation is required to confirm their importance. A TMA of SCLC cases is feasible but challenging and an important tool for biomarker validation.     

Telomere length is a novel predictive biomarker of sensitivity to anti-EGFR therapy in metastatic colorectal cancer

Background:

Telomeres are TTAGGG tandem repeats capping chromosomal ends and partially controlled by the telomerase enzyme. The EGFR pathway putatively regulates telomerase function, prompting an investigation of telomere length (TL) and its association with anti-epidermal growth factor receptor (EGFR) therapy in metastatic colorectal cancer (mCRC).

Methods:

Colorectal cancer cell lines were treated with multiple drugs and sensitivity determined. Clinical information was gathered from 75 patients who had received anti-EGFR drugs. Telomere length was measured using a validated qRT–PCR technique.

Results:

In CRC cell lines, TL independently predicted cetuximab sensitivity. Cells with shorter TL had growth inhibition of 18.6±3.41% as compared with 41.39±8.58% in longer TL (P=0.02). These in vitro findings were validated clinically, in a robust multivariate model. Among patients with KRas WT tumours, those with longer TL had a superior median progression-free survival (PFS) of 24.9 weeks than those with shorter TL; median 11.1 weeks, HR 0.31; P=0.048.

Conclusion:

Telomere length could be a potential unique biomarker predictive of clinical benefit (PFS) of mCRC patients treated with anti-EGFR therapy. This is the novel demonstration of a complex hitherto undescribed interaction, placing anti-EGFR therapy, EGFR pathway, and the telomerase complex within a clinical context.     

Prognostic significance of overexpression of c-Met oncoprotein in cholangiocarcinoma

Background:

Cholangiocarcinoma (CC) is a highly malignant carcinoma. We attempted to clarify the prognostic significance of c-Met overexpression and its association with clinicopathological factors in patients with CC.

Patients and methods:

One hundred and eleven patients with intrahepatic CC (IHCC) and 136 patients with extrahepatic CC (EHCC) who had undergone curative surgery were divided immunohistologically into c-Met^high and c-Met^low groups. Clinicopathological factors and outcomes were compared between the groups. c-Met and epidermal growth factor receptor (EGFR) expression was also examined in 10 CC cell lines.

Results:

The positivity of c-Met was 45.0% in IHCC and 68.4% in EHCC; c-Met^high expression was demonstrated in 11.7% of IHCC and 16.2% of EHCC. c-Met^high expression was significantly correlated with the 5-year survival rate for CC overall (P=0.0046) and for IHCC (P=0.0013), histopathological classification in EHCC, and for EGFR overexpression in both IHCC and EHCC. Coexpression and coactivation of c-Met and EGFR were also observed in CC cell lines. Multivariate analysis revealed that c-Met^high expression was an independent predictor of poor overall and disease-free survival in patients with IHCC.

Conclusions:

c-Met overexpression is associated with EGFR expression and is a poor prognostic factor in CC.     

VeriStrat? has prognostic value in advanced stage NSCLC patients treated with erlotinib and sorafenib

Background:

The serum proteomic test VeriStrat has been shown to be able to classify advanced non-small cell lung cancer (NSCLC) patients for overall survival (OS) after treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). In this study, VeriStrat was evaluated as a pre-treatment stratification tool in patients with advanced stage NSCLC for treatment with the combination of erlotinib and sorafenib, considering both OS and progression-free survival (PFS) as end points.

Methods:

Serum samples from 50 patients treated within the context of a phase II trial of first-line erlotinib and sorafenib were analysed with VeriStrat, a fully locked mass spectrometry-based test that identifies patients likely to have good or poor outcome on EGFR therapy based on eight distinct features in mass spectra. Analysis was performed fully blinded to all clinical data, and then the outcome data were analysed with respect to the obtained serum classifications.

Results:

VeriStrat classified pre-treatment samples into two groups, VeriStrat Good and VeriStrat Poor, which were significantly different in OS (hazard ratio (HR) 0.30, log-rank P=0.009) and in PFS (HR 0.40, log-rank P=0.035).

Conclusion:

VeriStrat has shown its potential for stratification of unselected, advanced stage NSCLC patients treated in first line with a combination of erlotinib and sorafenib.     

Topo2α protein expression predicts response to anthracycline combination neo-adjuvant chemotherapy in locally advanced primary breast cancer

Background:

This study aimed to identify predictors of response to anthracycline-based chemotherapy (5-fluoro-uracil, epirubicin, cyclophosphamide (FEC)) in locally advanced primary breast cancer (LAPC).

Methods:

A total of 91 LAPC patients were treated with six cycles of FEC before surgery. Protein expression of nine biomarkers (topoisomerase2α (Topo2α), ER, PR, HER2, Ki67, p53, EGFR, CK5/6 and CK14) was assessed in pre-chemotherapy core biopsies using immunohistochemistry (IHC) and results correlated with clinical and pathological response.

Results:

Clinical (cCR) and pathological (pCR) complete response were seen in 34.1% (n=31) and 20% (n=18), respectively. Pathological complete response was concordant with cCR in 14/31 cases; in four cases of cPR with palpable residual breast tumours, histology showed fibrous tissue only (pCR). On univariate analysis, pre-chemotherapy high expression of Topo2α protein (P=0.031), and negativity for ER and EGFR (P=0.001 and P=0.005, respectively) correlated with pCR. Positivity for p53 also showed significance (P=0.015), whereas basal phenotype, HER2, and all the clinicopathological variables of LAPC included in this study did not show significant correlation with response. On multivariate analysis, Topo2α expression had the strongest correlation with pCR (P=0.021) followed by EGFR (P=0.044).

Conclusion:

The study suggests that pre-chemotherapy Topo2α protein expression measured by IHC strongly correlates with pathological CR to neo-adjuvant anthracyclines in this group of LAPC studied.