Quantitative Proteomic Analysis in Metastatic Renal Cell Carcinoma Reveals a Unique Set of Proteins with Potential Prognostic Significance

Metastatic renal cell carcinoma (RCC) is one of the most treatment-resistant malignancies, and patients have a dismal prognosis, with a <10% five-year survival rate. The identification of markers that can predict the potential for metastases will have a great effect in improving patient outcomes. In this study, we used differential proteomics with isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify proteins that are differentially expressed in metastatic and primary RCC. We identified 1256 non-redundant proteins, and 456 of these were quantified. Further analysis identified 29 proteins that were differentially expressed (12 overexpressed and 17 underexpressed) in metastatic and primary RCC. Dysregulated protein expressions of profilin-1 (Pfn1), 14–3-3 zeta/delta (14–3-3ζ), and galectin-1 (Gal-1) were verified on two independent sets of tissues by means of Western blot and immunohistochemical analysis. Hierarchical clustering analysis showed that the protein expression profile specific for metastatic RCC can distinguish between aggressive and non-aggressive RCC. Pathway analysis showed that dysregulated proteins are involved in cellular processes related to tumor progression and metastasis. Furthermore, preliminary analysis using a small set of tumors showed that increased expression of Pfn1 is associated with poor outcome and is a potential prognostic marker in RCC. In addition, 14–3-3ζ and Gal-1 also showed higher expression in tumors with poor prognosis than in those with good prognosis. Dysregulated proteins in metastatic RCC represent potential prognostic markers for kidney cancer patients, and a greater understanding of their involved biological pathways can serve as the foundation of the development of novel targeted therapies for metastatic RCC.Renal cell carcinoma (RCC)¹ is the most common neoplasm of the adult kidney. Worldwide incidence and mortality rates of RCC are rising each decade (1). Seventy-five percent of kidney tumors are of the clear cell (ccRCC) subtype (2). Although modern imaging techniques for abdominal screening have led to increased incidental detection of renal tumors (3), unfortunately ∼25% to 30% of patients still have metastases at presentation.The prognosis with RCC is quite variable. The greatest risk of recurrence following nephrectomy is within the first 3 to 5 years (4). The ability to predict which tumors will metastasize would have a significant effect on patient outcomes, because the likelihood of a favorable response to treatment is greater when the metastatic burden is limited, and surgical resection of a single or limited number of metastases can result in longer survival (5). Furthermore, ∼3% of patients will develop a second primary renal tumor, either synchronous or metachronous. Currently, patient prognosis is assessed based on histological parameters and a multivariate analysis developed at Memorial Sloan Kettering (6), but neither is sufficiently accurate. A more accurate assessment of prognosis is urgently needed to better guide patient management.Although surgery can be curative for localized disease, many patients eventually relapse. Metastatic RCC is one of the most treatment-resistant malignancies, with chemotherapy and radiotherapy having limited effect. The five-year survival rate for metastatic RCC is ≤10% (7). Although there has been much progress in RCC treatment with the new era of antiangiogenic therapy, the majority of patients ultimately suffer a relapse and die from progression of the cancer. A more in-depth understanding of the pathogenesis of metastasis will be a cornerstone in the development of new targeted therapies. A number of prognostic markers have previously been identified based on comparative analysis of primary and metastatic tumors, including C-reactive protein, tetraspanin 7, hypoxia-inducible factor 1 α, phos-S6, U3 small nucleolar ribonucleoprotein protein, carbonic anhydrase IX, and microvascular density (8–14). However, no biomarker has yet had an established clinical role independent of stage (15). Differential protein expression between primary RCC and normal tissues was previously studied (16–18). Also, differential expression between primary and metastatic kidney disease has been investigated at the microRNA level (19, 20). Molecular analyses hold the promise of providing a better understanding of the pathogenesis of kidney cancer (21).In this study, we aimed to elucidate the pathogenesis of RCC metastasis through proteomic analysis and to identify potential prognostic markers for kidney cancer. We performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS to identify proteins that were dysregulated in metastatic RCC relative to primary RCC. Differential expressions of selected biologically interesting proteins—profilin-1 (Pfn1), 14–3-3 zeta/delta (14–3-3ζ), and galectin-1 (Gal-1)—were validated on two independent sets of tumors by means of western blot (WB) analysis and immunohistochemistry (IHC). Hierarchical clustering analysis showed that differential protein expression can distinguish between aggressive and non-aggressive tumors. In order to explore the role of these dysregulated proteins in tumor progression, we performed Gene Ontology (GO) and pathway analyses. In addition, we carried out a preliminary analysis to assess the potential of Pfn1, 14–3-3ζ, and Gal-1 as prognostic markers in RCC.     

Difference in Ki67 and Thymidylate Synthase Expression in Primary Tumour Compared with Metastatic Nodes in Breast Cancer Patients

Breast cancer is a heterogeneous disease, so therapeutic predictive biological markers need to be identified. To date an accurate evaluation of predictive markers is mainly done at the primary site; however, the main goal of adjuvant therapy for breast cancer is the control of micrometastases. The aim of this study is to assess as therapeutic and/or prognostic marker, the proliferation status of primary tumors and involved nodes as measured by Ki67 and thymidylate synthase (TS) expression, in 30 breast cancer node positive patients. TS is the main target of 5-fluorouracil (5-FU) activity, and its overexpression is one of the mechanisms of 5-FU drug resistance; however, in some studies its absence is responsible for a worse response to 5-FU. Our results show that malignant cells of involved nodes were in a post mitotic phase of the cell cycle, and show a low proliferation index and TS expression, while the primary tumours and controls, were strongly positive. On these basis we can hypothesize that these cells could be less sensitive to 5-FU. Further studies are necessary to identify other mechanisms responsible for their metastasing capability and/or for their aggressiveness.     

Immunohistochemical LRIG3 Expression in Cervical Intraepithelial Neoplasia and Invasive Squamous Cell Cervical Cancer: Association with Expression of Tumor Markers,Hormones, High-Risk HPV-Infection,Smoking and Patient Outcome

The novel biomarker LRIG3 is a member of the LRIG family (LRIG1-3). While LRIG1 has been associated with favorable prognosis and LRIG2 with poor prognosis in invasive cervical cancer, little is known about the role of LRIG3. The aim of this study was to investigate the expression of LRIG3 in invasive cancer and cervical intraepithelial neoplasia (CIN) for possible correlation with other tumor markers, to hormones and smoking, as a diagnostic adjunct in CIN, and prognostic value in invasive cancer. Cervical biopsies from 129 patients with invasive squamous cell carcinoma and 170 biopsies showing low grade and high grade CIN, or normal epithelium were stained for LRIG3 and 17 additional tumor markers. Among other variables the following were included: smoking habits, hormonal contraceptive use, serum progesterone, serum estradiol, high-risk HPV-infection, menopausal status and ten-year survival. In CIN, high expression of the tumor suppressors retinoblastoma protein, p53, and p16, and Ecadherin (cell-cell interaction), or low expression of CK10, correlated to LRIG3 expression. In addition, progestogenic contraceptive use correlated to high expression of LRIG3. In invasive cancer there was a correlation between expression of the major tumor promoter c-myc and high LRIG3 expression. High LRIG3 expression correlated significantly to presence of high-risk HPV infection in patients with normal epithelium and CIN. There was no correlation between LRIG3 expression and 10-year survival in patients with invasive cell cervical cancer. LRIG3 expression is associated with a number of molecular events in CIN. Expression also correlates to hormonal contraceptive use. The results on expression of other tumor markers suggest that LRIG3 is influenced by or influences a pattern of tumor markers in cancer and precancerous cells. Further studies are needed to elucidate if LRIG3 expression might be clinically useful.Key words: LRIG3, cervical cancer, cervical intraepithelial neoplasia, biological markers, human papillomavirus, hormonal contraceptives, smoking     

Prognostic Relevance of Pretherapeutic Gamma-Glutamyltransferase in Patients with Primary Metastatic Breast Cancer

Background

Gamma-glutamyltransferase (GGT) is a known marker for apoptotic balance and cell detoxification. Recently, an association of baseline GGT levels and breast cancer incidence, tumor progression and chemotherapy resistance was shown. The purpose of this study was to evaluate the association of pre-therapeutic GGT levels, clinical-pathological parameters and survival in patients with primary metastatic breast cancer (PMBC).

Methods

In this multicenter analysis, pre-therapeutic GGT levels and clinical-pathological parameters of 114 patients diagnosed with PMBC between 1996 and 2012 were evaluated. The association between GGT levels and clinical-pathological parameters were analysed. Patients were stratified into four GGT risk-groups (GGT < 18.00 U/L: normal low, 18.00 to 35.99 U/L: normal high, 36.00 to 71.99 U/L: elevated and ≥ 72.00 U/L: highly elevated) and survival analyses were performed.

Findings

Patients in the high risk GGT group had a poorer overall survival, when compared to the low risk group with five-year overall survival rates of 39.5% and 53.7% (p = 0.04), respectively. Patients with larger breast tumors had a trend towards higher GGT levels (p = 0.053). Pre-therapeutic GGT levels were not associated with indicators of aggressive tumor biology such as HER2-status, triple negative histology, or poorly differentiated cancers.

Conclusion

Pre-therapeutic GGT serum level might serve as a novel prognostic factor for overall-survival in patients with PMBC.     

Correlation of Hypertension and Proteinuria with Outcome in Elderly Bevacizumab-Treated Patients with Metastatic Colorectal Cancer

Background

Studies suggest a relationship between hypertension and outcome in bevacizumab-treated patients with metastatic colorectal cancer (mCRC). We performed a retrospective analysis of two phase II studies (BECA and BECOX) to determine if hypertension and proteinuria predict outcome in elderly patients with mCRC treated with bevacizumab.

Patients and Methods

Patients ≥70 years of age received either capecitabine 1250 mg/m² bid days 1–14 + bevacizumab 7.5 mg/kg day 1 every 21 days (BECA study) or capecitabine 1000 mg/m² bid days 1–14 with bevacizumab 7.5 mg/kg and oxaliplatin 130 mg/m² day 1 (BECOX study). The primary objective was to correlate hypertension and proteinuria with overall response rate (ORR), time to progression (TTP) and overall survival (OS). Secondary objectives included identification of risk factors associated with the development of hypertension and proteinuria and determining whether development of hypertension or proteinuria in the first 2 cycles was related to ORR, disease-control rate (DCR), TTP or OS.

Results

In total, 127 patients (median age 75.5 years) were included in the study. Hypertension correlated with DCR and OS; proteinuria correlated with ORR and DCR. Proteinuria or hypertension in the first 2 cycles did not correlate with efficacy. Risk factors for hypertension were female gender (odds ratio [OR] 0.241; P = 0.011) and more bevacizumab cycles (OR 1.112; P = 0.002); risk factors for proteinuria were diabetes (OR 3.869; P = 0.006) and more bevacizumab cycles (OR 1.181; P<0.0001). Multivariate analysis identified as having prognostic value: baseline lactate dehydrogenase, haemoglobin, number of metastatic lesions and DCR.

Conclusion

This analysis of two phase II studies suggests that hypertension is significantly correlated with OS but not with ORR and TTP, whereas proteinuria is correlated with ORR but not with OS and TTP. Both hypertension and proteinuria are associated with the duration of bevacizumab treatment and do not represent an independent prognostic factor.     

Detection of Long Non-Coding RNA in Archival Tissue: Correlation with Polycomb Protein Expression in Primary and Metastatic Breast Carcinoma

A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.     

Identification of Tissue microRNAs Predictive of Sunitinib Activity in Patients with Metastatic Renal Cell Carcinoma

Purpose

To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell-carcinoma (MRCC) and to evaluate in vitro their mechanism of action in sunitinib resistance.

Methods

We screened 673 microRNAs using TaqMan Low-density-Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n = 41). The most relevant differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenotypes selected from an independent cohort (n = 101). In vitro experiments were conducted to study the role of miR-942 in sunitinib resistance.

Results

TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p = 0.0074). High expression of miR-942, miR-628-5p, miR-133a, and miR-484 was significantly associated with decreased time to progression and overall survival. These microRNAs were also overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive cell line. MiR-942 overexpression in Caki-2 up-regulates MMP-9 and VEGF secretion which, in turn, promote HBMEC endothelial migration and sunitinib resistance.

Conclusions

We identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity or resistance to sunitinib. MiR-942 was the best predictor of efficacy. We describe a novel paracrine mechanism through which high miR-942 levels in MRCC cells up-regulates MMP-9 and VEGF secretion to enhance endothelial migration and sunitinib resistance. Our results support further validation of these miRNA in clinical confirmatory studies.     

Gene Expression Profiles Accurately Predict Outcome Following Liver Resection in Patients with Metastatic Colorectal Cancer

Purpose

The aim of this study was to build a molecular prognostic model based on gene signatures for patients with completely resected hepatic metastases from colorectal cancer (MCRC).

Methods

Using the Illumina HumanHT-12 gene chip, RNA samples from the liver metastases of 96 patients who underwent R0 liver resection were analyzed. Patients were randomly assigned to a training (n = 60) and test (n = 36) set. The genes associated with disease-specific survival (DSS) and liver-recurrence-free survival (LRFS) were identified by Cox-regression and selected to construct a molecular risk score (MRS) using the supervised principle component method on the training set. The MRS was then evaluated in the independent test set.

Results

Nineteen and 115 genes were selected to construct the MRS for DSS and LRFS, respectively. Each MRS was validated in the test set; 3-year DSS/LRFS rates were 42/32% and 79/80% for patients with high and low MRS, respectively (p = 0.007 for DSS and p = 0.046 for LRFS). In a multivariate model controlling for a previously validated clinical risk score (CRS), the MRS remained a significant predictor of DSS (p = 0.001) and LRFS (p = 0.03). When CRS and MRS were combined, the patients were discriminated better with 3-year DSS/LRFS rates of 90/89% in the low risk group (both risk scores low) vs 42/26% in the high risk group (both risk scores high), respectively (p = 0.002/0.004 for DSS/LRFS).

Conclusion

MRS based on gene expression profiling has high prognostic value and is independent of CRS. This finding provides a potential strategy for better risk-stratification of patients with liver MCRC.     

ERCC1 and TS Expression as Prognostic and Predictive Biomarkers in Metastatic Colon Cancer

In patients with metastatic colon cancer, response to first line chemotherapy is a strong predictor of overall survival (OS). Currently, oncologists lack diagnostic tests to determine which chemotherapy regimen offers the greatest chance for response in an individual patient. Here we present the results of gene expression analysis for two genes, ERCC1 and TS, measured with the commercially available ResponseDX: Colon assay (Response Genetics, Los Angeles, CA) in 41 patients with de novo metastatic colon cancer diagnosed between July 2008 and August 2013 at the University of California, San Diego. In addition ERCC1 and TS expression levels as determined by RNAseq and survival data for patients in TCGA were downloaded from the TCGA data portal. We found that patients with low expression of ERCC1 (n = 33) had significantly longer median OS (36.0 vs. 10.1 mo, HR 0.29, 95% CI .095 to .84, log-rank p = 9.0x10^-6) and median time to treatment to failure (TTF) following first line chemotherapy (14.1 vs. 2.4 mo, HR 0.17, 95% CI 0.048 to 0.58, log-rank p = 5.3x10^-4) relative to those with high expression (n = 4). After accounting for the covariates age, sex, tumor grade and ECOG performance status in a Cox proportional hazard model the association of low ERCC1 with longer OS (HR 0.18, 95% CI 0.14 to 0.26, p = 0.0448) and TTF (HR 0.16, 95% CI 0.14 to 0.21, p = 0.0053) remained significant. Patients with low TS expression (n = 29) had significantly longer median OS (36.0 vs. 14.8 mo, HR 0.25, 95% CI 0.074 to 0.82, log-rank p = 0.022) relative to those with high expression (n = 12). The combined low expression of ERCC1/TS was predictive of response in patients treated with FOLFOX (40% vs. 91%, RR 2.3, Fisher’s exact test p = 0.03, n = 27), but not with FOLFIRI (71% vs. 71%, RR 1.0, Fisher’s exact test p = 1, n = 14). Overall, these findings suggest that measurement of ERCC1 and TS expression has potential clinical utility in managing patients with metastatic colorectal cancer.     

Functional Analysis of Prognostic Gene Expression Network Genes in Metastatic Breast Cancer Models

Identification of conserved co-expression networks is a useful tool for clustering groups of genes enriched for common molecular or cellular functions [1]. The relative importance of genes within networks can frequently be inferred by the degree of connectivity, with those displaying high connectivity being significantly more likely to be associated with specific molecular functions [2]. Previously we utilized cross-species network analysis to identify two network modules that were significantly associated with distant metastasis free survival in breast cancer. Here, we validate one of the highly connected genes as a metastasis associated gene. Tpx2, the most highly connected gene within a proliferation network specifically prognostic for estrogen receptor positive (ER+) breast cancers, enhances metastatic disease, but in a tumor autonomous, proliferation-independent manner. Histologic analysis suggests instead that variation of TPX2 levels within disseminated tumor cells may influence the transition between dormant to actively proliferating cells in the secondary site. These results support the co-expression network approach for identification of new metastasis-associated genes to provide new information regarding the etiology of breast cancer progression and metastatic disease.     

Circulating Endothelial Cells and Microparticles as Prognostic Markers in Advanced Non-Small Cell Lung Cancer

Background

Circulating endothelial cells and microparticles have prognostic value in cancer, and might be predictors of response to chemotherapy and antiangiogenic treatments. We have investigated the prognostic value of circulating endothelial cells and microparticles in patients treated for advanced non-small cell lung cancer.

Methodology/Principal Findings

Peripheral blood samples were obtained from 60 patients before first line, platinum-based chemotherapy +/− bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Circulating endothelial cells were measured by an immunomagnetic technique and immunofluorescence microscopy. Phosphatidylserine-positive microparticles were evaluated by flow cytometry. Microparticle-mediated procoagulant activity was measured by the endogen thrombin generation assay. Results: pre- and posttreatment levels of markers were higher in patients than in controls (p<0.0001). Elevated levels of microparticles were associated with longer survival. Elevated pretreatment levels of circulating endothelial cells were associated with shorter survival.

Conclusions/Significance

Circulating levels of microparticles and circulating endothelial cells correlate with prognosis, and could be useful as prognostic markers in patients with advanced non-small cell lung cancer.     

Survivin在人非小细胞肺癌中的表达及其与VEGF表达的相关性

目的:探讨Survivin在人非小细胞肺癌组织中表达、临床意义及其与VEGF表达的相关性。方法:采用免疫组织化学(Envision二步法)检测Survivin和VEGF在116例肺鳞癌、腺癌组织及15例肺良性病变组织中的表达情况。结果:Survivin、VEGF的阳性率分别为62.1%(72/116)、72.4%(84/116),显著高于肺良性病变组织的0(0/15)、13.3%(2/15);Survivin表达与非小细胞肺癌的低分化(P<0.05)、淋巴结转移呈正相关(P<0.05),与患者预后负相关(P<0.05);VEGF的表达与肺癌组织的分化程度、TNM分期及淋巴结转移均有关(P<0.05);并且Survivin表达与VEGF表达呈正相关(P<0.05)。结论:Survivin有望成为肺癌诊断和基因治疗的新靶点。     

Survivin在人非小细胞肺癌中的表达及其与VEGF表达的相关性

目的：探讨Survivin在人非小细胞肺癌组织中表达、临床意义及其与VEGF表达的相关性。方法：采用免疫组织化学（Envision二步法）检测Survivin和VEGF在116例肺鳞癌、腺癌组织及15例肺良性病变组织中的表达情况。结果：Survivin、VEGF的阳性率分别为62.1%（72/116）、72.4%（84/116）,显著高于肺良性病变组织的0（0/15）、13.3%（2/15）;Survivin表达与非小细胞肺癌的低分化（P〈0.05）、淋巴结转移呈正相关（P〈0.05）,与患者预后负相关（P〈0.05）;VEGF的表达与肺癌组织的分化程度、TNM分期及淋巴结转移均有关（P〈0.05）;并且Survivin表达与VEGF表达呈正相关（P〈0.05）。结论：Survivin有望成为肺癌诊断和基因治疗的新靶点。     

Impact of miR-21, miR-126 and miR-221 as Prognostic Factors of Clear Cell Renal Cell Carcinoma with Tumor Thrombus of the Inferior Vena Cava

Clear cell renal cell carcinoma (ccRCC) characterized by a tumor thrombus (TT) extending into the inferior vena cava (IVC) generally indicates poor prognosis. Nevertheless, the risk for tumor recurrence after nephrectomy and thrombectomy varies. An applicable and accurate prediction system to select ccRCC patients with TT of the IVC (ccRCC/TT) at high risk after nephrectomy is urgently needed, but has not been established up to now. To our knowledge, a possible role of microRNAs (miRs) for the development of ccRCC/TT or their impact as prognostic markers in ccRCC/TT has not been explored yet. Therefore, we analyzed the expression of the previously described onco-miRs miR-200c, miR-210, miR-126, miR-221, let-7b, miR-21, miR-143 and miR-141 in a study collective of 74 ccRCC patients. Using the expression profiles of these eight miRs we developed classification systems that accurately differentiate ccRCC from non-cancerous renal tissue and ccRCC/TT from tumors without TT. In the subgroup of 37 ccRCC/TT cases we found that miR-21, miR-126, and miR-221 predicted cancer related death (CRD) accurately and independently from other clinico-pathological features. Furthermore, a combined risk score based on the expression of miR-21, miR-126 and miR-221 was developed and showed high sensitivity and specificity to predict cancer specific survival (CSS) in ccRCC/TT. Using the combined risk score we were able to classify ccRCC/TT patients correctly into high and low risk cases. The risk stratification by the combined risk score (CRS) will benefit from further cohort validation and might have potential for clinical application as a molecular prediction system to identify high- risk ccRCC/TT patients.     

Analysis of Death Receptor 5 and Caspase-8 Expression in Primary and Metastatic Head and Neck Squamous Cell Carcinoma and Their Prognostic Impact

Death receptor 5 (DR5) and caspase-8 are major components in the extrinsic apoptotic pathway. The alterations of the expression of these proteins during the metastasis of head and neck squamous cell carcinoma (HNSCC) and their prognostic impact have not been reported. The present study analyzes the expression of DR5 and caspase-8 by immunohistochemistry (IHC) in primary and metastatic HNSCCs and their impact on patient survival. Tumor samples in this study included 100 primary HNSCC with no evidence of metastasis, 100 primary HNSCC with lymph node metastasis (LNM) and 100 matching LNM. IHC analysis revealed a significant loss or downregulation of DR5 expression in primary tumors with metastasis and their matching LNM compared to primary tumors with no evidence of metastasis. A similar trend was observed in caspase-8 expression although it was not statistically significant. Downregulation of caspase-8 and DR5 expression was significantly correlated with poorly differentiated tumors compared to moderately and well differentiated tumors. Univariate analysis indicates that, in HNSCC with no metastasis, higher expression of caspase-8 significantly correlated with better disease-free survival and overall survival. However, in HNSCC with LNM, higher caspase-8 expression significantly correlated with poorer disease-free survival and overall survival. Similar results were also generated when we combined both DR5 and caspase-8. Taken together, we suggest that both DR5 and caspase-8 are involved in regulation of HNSCC metastasis. Our findings warrant further investigation on the dual role of caspase-8 in cancer development.     

The Role of Cancer-Testis Antigens as Predictive and Prognostic Markers in Non-Small Cell Lung Cancer

Background

Cancer-Testis Antigens (CTAs) are immunogenic proteins that are poor prognostic markers in non-small cell lung cancer (NSCLC). We investigated expression of CTAs in NSCLC and their association with response to chemotherapy, genetic mutations and survival.

Methods

We studied 199 patients with pathological N2 NSCLC treated with neoadjuvant chemotherapy (NAC; n = 94), post-operative observation (n = 49), adjuvant chemotherapy (n = 47) or unknown (n = 9). Immunohistochemistry for NY-ESO-1, MAGE-A and MAGE-C1 was performed. Clinicopathological features, response to neoadjuvant treatment and overall survival were correlated. DNA mutations were characterized using the Sequenom Oncocarta panel v1.0. Affymetrix data from the JBR.10 adjuvant chemotherapy study were obtained from a public repository, normalised and mapped for CTAs.

Results

NY-ESO-1 was expressed in 50/199 (25%) samples. Expression of NY-ESO-1 in the NAC cohort was associated with significantly increased response rates (P = 0.03), but not overall survival. In the post-operative cohort, multivariate analyses identified NY-ESO-1 as an independent poor prognostic marker for those not treated with chemotherapy (HR 2.61, 95% CI 1.28–5.33; P = 0.008), whereas treatment with chemotherapy and expression of NY-ESO-1 was an independent predictor of improved survival (HR 0.267, 95% CI 0.07–0.980; P = 0.046). Similar findings for MAGE-A were seen, but did not meet statistical significance. Independent gene expression data from the JBR.10 dataset support these findings but were underpowered to demonstrate significant differences. There was no association between oncogenic mutations and CTA expression.

Conclusions

NY-ESO-1 was predictive of increased response to neoadjuvant chemotherapy and benefit from adjuvant chemotherapy. Further studies investigating the relationship between these findings and immune mechanisms are warranted.     

Expression of Estrogen-Related Gene Markers in Breast Cancer Tissue Predicts Aromatase Inhibitor Responsiveness

Aromatase inhibitors (AIs) are the most effective class of drugs in the endocrine treatment of breast cancer, with an approximate 50% treatment response rate. Our objective was to determine whether intratumoral expression levels of estrogen-related genes are predictive of AI responsiveness in postmenopausal women with breast cancer. Primary breast carcinomas were obtained from 112 women who received AI therapy after failing adjuvant tamoxifen therapy and developing recurrent breast cancer. Tumor ERα and PR protein expression were analyzed by immunohistochemistry (IHC). Messenger RNA (mRNA) levels of 5 estrogen-related genes–AKR1C3, aromatase, ERα, and 2 estradiol/ERα target genes, BRCA1 and PR–were measured by real-time PCR. Tumor protein and mRNA levels were compared with breast cancer progression rates to determine predictive accuracy. Responsiveness to AI therapy–defined as the combined complete response, partial response, and stable disease rates for at least 6 months–was 51%; rates were 56% in ERα-IHC-positive and 14% in ERα-IHC-negative tumors. Levels of ERα, PR, or BRCA1 mRNA were independently predictive for responsiveness to AI. In cross-validated analyses, a combined measurement of tumor ERα and PR mRNA levels yielded a more superior specificity (36%) and identical sensitivity (96%) to the current clinical practice (ERα/PR-IHC). In patients with ERα/PR-IHC-negative tumors, analysis of mRNA expression revealed either non-significant trends or statistically significant positive predictive values for AI responsiveness. In conclusion, expression levels of estrogen-related mRNAs are predictive for AI responsiveness in postmenopausal women with breast cancer, and mRNA expression analysis may improve patient selection.     

Differential Matrix Rigidity Response in Breast Cancer Cell Lines Correlates with the Tissue Tropism

Metastasis to a variety of distant organs, such as lung, brain, bone, and liver, is a leading cause of mortality in the breast cancer patients. The tissue tropism of breast cancer metastasis has been recognized and studied extensively, but the cellular processes underlying this phenomenon, remain elusive. Modern technologies have enabled the discovery of a number of the genetic factors determining tissue tropism of malignant cells. However, the effect of these genetic differences on the cell motility and invasiveness is poorly understood. Here, we report that cellular responses to the mechanical rigidity of the extracellular matrix correlate with the rigidity of the target tissue. We tested a series of single cell populations isolated from MDA-MB-231 breast cancer cell line in a variety of assays where the extracellular matrix rigidity was varied to mimic the environment that these cells might encounter in vivo. There was increased proliferation and migration through the matrices of rigidities corresponding to the native rigidities of the organs where metastasis was observed. We were able to abolish the differential matrix rigidity response by knocking down Fyn kinase, which was previously identified as a critical component of the FN rigidity response pathway in healthy cells. This result suggests possible molecular mechanisms of the rigidity response in the malignant cells, indicating potential candidates for therapeutic interventions.