Frequencies and expression levels of programmed death ligand 1 (PD-L1) in circulating tumor RNA (ctRNA) in various cancer types.

BACKGROUND: Precision medicine and prediction of therapeutic response requires monitoring potential biomarkers before and after treatment. Liquid biopsies provide noninvasive prognostic markers such as circulating tumor DNA and RNA. Circulating tumor RNA (ctRNA) in blood is also used to identify mutations in genes of interest, but additionally, provides information about relative expression levels of important genes. In this study, we analyzed PD-L1 expression in ctRNA isolated from various cancer types. Tumors inhibit antitumor response by modulating the immune checkpoint proteins programmed death ligand 1 (PD-L1) and its cognate receptor PD1. The expression of these genes has been implicated in evasion of immune response and resistance to targeted therapies. METHODS: Blood samples were collected from gastric (GC), colorectal (CRC), lung (NSCLC), breast (BC), prostate cancer (PC) patients, and a healthy control group. ctRNA was purified from fractionated plasma, and following reverse transcription, levels of PD-L1 expression were analyzed using qPCR. RESULTS: PD-L1 expression was detected in the plasma ctRNA of all cancer types at varying frequencies but no PD-L1 mRNA was detected in cancer-free individuals. The frequencies of PD-L1 expression were significantly different among the various cancer types but the median relative PD-L1 expression values were not significantly different. In 12 cases where plasma and tumor tissue were available from the same patients, there was a high degree of concordance between expression of PD-L1 protein in tumor tissues and PD-L1 gene expression in plasma, and both methods were equally predictive of response to nivolumab. CONCLUSIONS: PD-L1 mRNA can be detected and quantitated in ctRNA of cancer patients. These results pave the way for further studies aimed at determining whether monitoring the levels of PD-L1 mRNA in blood can identify patients who are most likely to benefit from the conventional treatment.

Folates as adjuvants to anticancer agents: Chemical rationale and mechanism of action.

Folates have been used with cytotoxic agents for decades and today they are used in hundreds of thousands of patients annually. Folate metabolism is complex. In the treatment of cancer with 5-fluorouracil, the administration of folates mechanistically leads to the formation of [6R]-5,10-methylene-tetrahydrofolate, and the increased concentration of this molecule leads to stabilization of the ternary complex comprising thymidylate synthase, 2'-deoxy-uridine-5'-monophosphate, and [6R]-5,10-methylene-tetrahydrofolate. The latter is the only natural folate that can bind directly in the ternary complex, with other folates requiring metabolic activation. Modulation of thymidylate synthase activity became central in the study of folate/cytotoxic combinations and, despite wide use, research into the folate component was neglected, leaving important questions unanswered. This article revisits the mechanisms of action of folates and evaluates commercially available folate derivatives in the light of current research. Better genomic insight and availability of new analytical techniques and stable folate compounds may open new avenues of research and therapy, ultimately bringing increased clinical benefit to patients.

Standing the test of time: targeting thymidylate biosynthesis in cancer therapy.

Over the past 60 years, chemotherapeutic agents that target thymidylate biosynthesis and the enzyme thymidylate synthase (TS) have remained among the most-successful drugs used in the treatment of cancer. Fluoropyrimidines, such as 5-fluorouracil and capecitabine, and antifolates, such as methotrexate and pemetrexed, induce a state of thymidylate deficiency and imbalances in the nucleotide pool that impair DNA replication and repair. TS-targeted agents are used to treat numerous solid and haematological malignancies, either alone or as foundational therapeutics in combination treatment regimens. We overview the pivotal discoveries that led to the rational development of thymidylate biosynthesis as a chemotherapeutic target, and highlight the crucial contribution of these advances to driving and accelerating drug development in the earliest era of cancer chemotherapy. The function of TS as well as the mechanisms and consequences of inhibition of this enzyme by structurally diverse classes of drugs with distinct mechanisms of action are also discussed. In addition, breakthroughs relating to TS-targeted therapies that transformed the clinical landscape in some of the most-difficult-to-treat cancers, such as pancreatic, colorectal and non-small-cell lung cancer, are highlighted. Finally, new therapeutic agents and novel mechanism-based strategies that promise to further exploit the vulnerabilities and target resistance mechanisms within the thymidylate biosynthesis pathway are reviewed.

Regulation of MLH1 mRNA and protein expression by promoter methylation in primary colorectal cancer: a descriptive and prognostic cancer marker study.

BACKGROUND: In colorectal cancer MLH1 deficiency causes microsatellite instability, which is relevant for the patient's prognosis and treatment, and its putative heredity. Dysfunction of MLH1 is caused by sporadic gene promoter hypermethylation or by hereditary mutations as seen in Lynch Syndrome. The aim of this study was to determine in detail how DNA methylation regulates MLH1 expression and impacts clinical management. METHODS: Colorectal cancer samples were collected from 210 patients. The laboratory methods used to study these samples included methylation specific multiplex ligation-dependent probe amplification (MS-MLPA), real-time quantitative PCR (qPCR), and immunohistochemistry (IHC). RESULTS: We found that the MLH1 mRNA and protein expression levels were highly related. MS-MLPA was successful in tumors from 195 patients. In these tumors, hypermethylation was observed in promoter regions A (n = 57), B (n = 30), C (n = 28), and D (n = 47), and in intron 1 (n = 25). The promoter region C and intron 1 methylation levels were found to be excellently suited for discriminating between low and high gene expression levels, whereas those of promoter regions A, B and D were less specific. Hypermethylation in any region (n = 77) served as an independent prognostic factor (hazard ratio 0.56, 95 % confidence interval 0.36-0.89, p = 0.01). CONCLUSIONS: MLH1 inactivation through hypermethylation was found to be related to improved survival. Hypermethylation in promoter region C and intron 1 served as the most specific markers for this inactivation.

EGFR, FLT1 and heparanase as markers identifying patients at risk of short survival in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma remains to be a tumor with very few treatment choices and limited prognosis. In this study, we sought to determine the prognostic role of fms-related tyrosine kinase 1/vascular endothelial growth factor receptor 1 (FLT1/VEGFR1), heparanase (HPSE) and epidermal growth factor receptor (EGFR) gene expression in patients with resected CCC. METHODS: 47 formalin-fixed paraffin embedded FFPE tumor samples from patients with resected CCC were analyzed. FFPE tissues were dissected using laser-captured microdissection and analyzed for FLT1, FLT4, HPSE, Hif1a, VEGFA/C, HB-EGF, PDGFA, PDGF-RA and EGFR mRNA expression using a quantitative real-time RT-PCR method. Gene expression values (relative mRNA levels) are expressed as ratios between the target gene and internal reference genes (beta-actin, b2mg, rplp2, sdha). RESULTS: EGFR, FLT1 and HPSE expression levels were significantly associated with overall survival (OS). FLT1 showed the strongest significant independent association with overall survival in a multivariate cox regression analysis when compared to the other genes and clinicopathological factors with a nearly 5 times higher relative risk (4.74) of dying earlier when expressed in low levels (p = 0.04). ROC Curve Analysis revealed that measuring EGFR potentially identifies patients at risk of a worsened outcome with a sensitivity of 80% and a specificity of 75% (p = 0.01). CONCLUSIONS: EGFR and FLT1 seem to be potential markers to identify those patients at high risk of dying from cholangiocarcinoma. Therefore these markers may help to identify patient subgroups in need for a more aggressive approach in a disease that is in desperate need for new approaches.

Glutathione S-transferase PI (GST-PI) mRNA expression and DNA methylation is involved in the pathogenesis and prognosis of NSCLC.

INTRODUCTION: The aim of this study was to investigate the relevance of mRNA expression and DNA methylation of GST-PI in tumor and non-tumor lung tissue from NSCLC patients in terms of prognostic and pathogenetic value of this biomarker. METHOD: Quantitative real-time PCR was used to measure mRNA expression and DNA methylation of GST-PI in paired tumor (T) and non-tumor (N) lung tissue of 91 NSCLC patients. Of all 91 patients 49% were stage I, 21% stage II and 30% stage IIIA. Forty-seven percent of the patients had squamous cell carcinoma, 36% adenocarcinoma and 17% large cell carcinoma. All patients were R0 resected. RESULTS: GST-PI mRNA expression could be measured in 100% in both (T and N) tissues; GST-PI DNA methylation was detected in 14% (N) and 14% (T). The median GST-PI mRNA expression in N was 7.83 (range: 0.01-19.43) and in T 13.15 (range: 0.01-116.8; p≤0.001). The median GST-PI methylation was not significantly different between T and N. No associations were seen between the mRNA expression or DNA methylation levels and clinical or histopathologic parameters such as gender, age, TNM stage, tumor histology and grading. The median survival of the investigated patients was 59.7 years (the median follow-up was 85.9 months). High GST-PI DNA methylation was significantly associated with a worse prognosis (p=0.041, log rank test). No correlation was found between the GST-PI DNA methylation levels and the correlating mRNA expression levels. CONCLUSION: GST-PI mRNA expression seems to be involved in the pathogenesis of NSCLC. High levels of GST-PI DNA methylation in tumor tissue of NSCLC patients have a potential as a biomarker identifying subpopulations with a more aggressive tumor biology. Quantitation of GST-PI DNA methylation may be a useful method to identify patients with a poor prognosis after curative resection and who will benefit from intensive adjuvant therapy.

Update of prognostic and predictive biomarkers in oropharyngeal squamous cell carcinoma: a review.

Oropharyngeal squamous cell carcinomas (OSCC) constitute about 5% of all cancers in the western world and the incidence and mortality rates of this tumor have shown little improvement over the last 30 years. Molecular targeted therapy, a promising strategy for the treatment of OSCC and other cancers, requires the understanding of specific molecular events of carcinogenesis and the different pathological, partly interrelated pathways. Extended knowledge of the prognostic or predictive value of molecular biomarkers in oropharyngeal cancer is necessary to allow a better characterization and classification of the tumor, improve the appraisal of clinical outcome and help to specify individual multimodal therapy with increased efficiency. This work affords an updated summary regarding recent data about tissue biomarkers in patients with OSCC, based on the six essential hallmarks of cancer described by Hanahan and Weinberg (Cell 100(1):57-70, 2000) providing the characterization of a malignant cell.

Association of epidermal growth factor receptor activating mutations with low ERCC1 gene expression in non-small cell lung cancer.

INTRODUCTION: Patients with non-small cell lung cancer (NSCLC) with cancers harboring activating mutations in the epidermal growth factor receptor (EGFR) show improved efficacy from EGFR tyrosine kinase inhibitors. Some clinical studies also suggest enhanced efficacy of platinum-based chemotherapy in patients with EGFR-mutant cancers. We investigated the relationship of EGFR mutation status and DNA repair capacity, as exemplified by excision repair cross-complementing 1 (ERCC1) gene expression, as a potential explanation for this observation. METHODS: Microdissected formalin-fixed paraffin-embedded tumors from 1207 patients with NSCLC were analyzed by real-time polymerase chain reaction for mRNA expression levels of ERCC1 and for EGFR mutation status by an allele-specific polymerase chain reaction assay. RESULTS: NSCLC subtype was adenocarcinoma (AC) in 712 patients, squamous in 175, and not otherwise specified or other in 320. EGFR activating mutations were detected in 183/1207 patients (15.2%). Median ERCC1 expression overall was 1.82 (range, 0.22-27.31) and was histology related: AC, median = 1.68 (0.22-11.33) and squamous, median = 2.42 (0.51-14.28) (p < 0.001). Using a previously defined reference level of <1.7, ERCC1 expression was categorized as low in 556 of 1207 patients (46.1%). The presence of EGFR mutations was highly associated with ERCC1 expression (p < 0.001). This association was retained when adjusting for AC histologic subtype (p = 0.001). CONCLUSIONS: NSCLC specimens harboring EGFR activating mutations are more likely to express low ERCC1 mRNA levels. Whether these findings translate into enhanced clinical efficacy of EGFR-mutant cancers to platinum-based chemotherapy remains to be determined.

Low thymidylate synthase, thymidine phosphorylase, and dihydropyrimidine dehydrogenase mRNA expression correlate with prolonged survival in resected non-small-cell lung cancer.

BACKGROUND: Thymidylate synthase (TS), thymidine phosphorylase (TP), and dihydropyrimidine dehydrogenase (DPD) are key enzymes in the 5-fluorouracil (5-FU) pathway. The aim of this study was to investigate the mRNA expression of TS, TP, and DPD in tumor and nontumor lung tissue of patients with NSCLC and to determine the potential of these genes as molecular biomarkers. MATERIALS AND METHODS: The TS, TP, and DPD mRNA expression was analyzed in tumor and nontumor tissue of 91 patients with NSCLC by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) with ß-actin as the internal control. All tumors were R0 resected. The median follow-up was 85.9 months. RESULTS: The mRNA expression of TS, TP, and DPD was detectable in both tumor and nontumor tissue. Tumor TP (tTP) seems to correlate with tumor TS (tTS) and tumor DPD (tDPD) mRNA expression, but no correlation in the mRNA expression of tTS and tDPD was found. The TS and TP mRNA expression levels were significantly associated with patient prognosis. The 5-year survival probability was 58.7% (TS), and 59.6% (TP) for patients with a low TS and TP mRNA expression and 33.4% (TS), and 31.8% (TP) for patients with a high mRNA expression (P = .04 [TS]; P = .03 [TP]; log-rank). The probability of survival was significantly different among patients with no and any 1 highly expressed gene compared with patients with any 2 or more of the 3 investigated genes highly expressed (P = .012). CONCLUSION: High TS, TP, and DPD mRNA expression are biomarkers for a more severe malign NSCLC biology. Quantitation of the mRNA expression of these genes seems to be helpful in differing patients with unequal malign tumor entities and therefore possibly helpful in selecting tailored additional therapies to control the disease.

MDR1 and ERCC1 expression predict outcome of patients with locally advanced bladder cancer receiving adjuvant chemotherapy.

PURPOSE: The role of adjuvant chemotherapy in patients with locally advanced bladder cancer still remains to be defined. We hypothesized that assessing the gene expression of the chemotherapy response modifiers multidrug resistance gene 1 (MDR1) and excision repair cross-complementing 1 (ERCC1) may help identify the group of patients benefiting from cisplatin-based adjuvant chemotherapy. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tumor samples from 108 patients with locally advanced bladder cancer, who had been enrolled in AUO-AB05/95, a phase 3 trial randomizing a maximum of three courses of adjuvant cisplatin and methotrexate (CM) versus methotrexate, vinblastine, epirubicin, and cisplatin (M-VEC), were included in the study. Tumor cells were retrieved by laser-captured microdissection and analyzed for MDR1 and ERCC1 expression using a quantitative real-time reverse transcription-polymerase chain reaction assay. Gene expression levels were correlated with clinical outcomes by multivariate Cox proportional hazards regression analysis. RESULTS: Expressions of MDR1 and ERCC1 were independently associated with overall progression-free survival (P = .001, relative risk = 2.9 and P = .01, relative risk = 2.24, respectively). The correlation of high MDR1 expression with inferior outcome was stronger in patients receiving M-VEC, whereas ERCC1 analysis performed equally in the CM and M-VEC groups. CONCLUSIONS: High MDR1 and ERCC1 gene expressions are associated with inferior outcome after cisplatin-based adjuvant chemotherapy for locally advanced bladder cancer. Prospective studies are warranted to define a role for MDR1 and ERCC1 analysis in individualizing multimodality treatment in locally advanced bladder cancer.

The relationship between proangiogenic gene expression levels in prostate cancer and their prognostic value for clinical outcomes.

BACKGROUND: Androgens stimulate the expression of vascular endothelial growth factor (VEGF) through activation of hypoxia inducible factor (HIF). These genes play a major role in cancer angiogenesis. This study assesses the relationship among expression levels for the androgen receptor (AR), HIF1a, VEGF-A, and VEGF-C genes in human prostate cancer tissue and their impact on prostate cancer outcomes. It also examines the impact of pre-operative androgen deprivation therapy (ADT) on the expression of these genes. METHODS: Radical prostatectomy specimens were obtained from 138 patients with D1 prostate cancer from the University of Southern California prostatectomy database; 30% received pre-operative and 23% received post-operative ADT. Gene expression levels were determined by quantitative real-time PCR. Specimens were stratified into three groups for each gene based on expression levels, and groups were compared for clinical outcomes (PSA and clinical recurrence, overall survival). RESULTS: There was a significant correlation in expression levels amongst all genes. Patients treated with pre-operative ADT had significantly lower HIF1a expression, mean 2.64 (CI 2.34-2.94) than patients not treated, mean 3.25 (CI 2.97-3.53, P = 0.006), adjusting for age, PSA, Gleason score, and stage. Higher VEGF-A expression was significantly associated with better overall survival (HR 0.49, P = 0.015). The risk of developing clinical recurrence was significantly lower with higher VEGF-C expression (HR 0.4, P = 0.014). CONCLUSIONS: Significant correlation was noted among AR, HIF1a, VEGF-A, and VEGF-C. This study shows that ADT is associated with lower HIF1a gene expression in human prostate cancer tissue and documents prognostic value for VEGF-A and VEGF-C expression levels.

Ornithine decarboxylase mRNA expression in curatively resected non-small-cell lung cancer.

BACKGROUND: The effect of ornithine decarboxylase (ODC) on the pathogenesis of non-small-cell lung cancer (NSCLC) remains poorly investigated. Hence, the aim of this study was to explore the potential role of ODC mRNA expression as a prognostic biomarker in patients with curatively resected NSCLC. PATIENTS AND METHODS: A total of 91 tumor and matching nontumorous lung tissue samples from patients with NSCLC were analyzed using a quantitative real-time reverse-transcriptase polymerase chain reaction method. The relative ODC mRNA expression was measured in tumorous and nontumorous lung tissue using beta-actin as a reference gene. Squamous cell carcinoma was found in 43 patients (47%), adenocarcinoma in 33 (36%), and large-cell carcinoma in 15 of the patients (17%). All patients' disease was R0 resected. RESULTS: Ornithine decarboxylase was detected in all 91 tumor and nontumorous lung tissue samples. The median tumorous expression of 9.11 (range, 0.92-155.35) was significantly elevated compared with the median ODC expression of 7.89 (range, 0.0-45.8) in nontumorous lung tissue. Ornithine decarboxylase expression levels were not associated with any clinicopathologic parameters. Using an ODC/beta-actin ratio of 10 as a cutoff, tumorous ODC (tODC) expression is a significant prognostic factor in NSCLC. The ODC ratio between tumorous and nontumorous expression was even more prognostic. Moreover, Cox proportional hazards model analysis showed ODC expression to be an independent prognostic factor. CONCLUSION: In this study, ODC is shown to have a prognostic potential in NSCLC. Low levels of tODC expression are associated with a more aggressive tumor biology. Also, an increase of ODC mRNA expression during carcinogenesis seems to have a favorable prognostic effect. Measuring the ODC expression in patients with NSCLC could aid in further chemotherapy decisions. Our results suggest that further investigation of ODC mRNA expression in NSCLC may be warranted.

The prognostic role of Bcl-2 mRNA expression in curatively resected non-small cell lung cancer (NSCLC).

BACKGROUND: The effect of the apoptosis related gene Bcl-2 in the pathogenesis in NSCLC remains poorly investigated. Hence the aim of this study was to explore the potential role of Bcl-2 mRNA expression as a prognostic biomarker in patients with curatively resected NSCLC. METHODS: 91 tumor and matching normal tissue samples from patients with NSCLC were analyzed using a quantitative real-time RT-PCR method. The relative Bcl-2 mRNA expression was measured using beta-actin as a reference gene. 45 of the 91 patients had stage I tumors (49%), 19 had stage II (21%) and 27 had stage IIIa (30%). Squamous cell carcinoma was found in 43 patients (47%), adenocarcinoma in 33 (36%) and in large cell carcinoma in 15 (17%) of the patients. RESULTS: Bcl-2 mRNA expression was detected in 83 (91%) of the investigated tumor samples and in 74 (81%) of the normal lung tissue. The median gene expression was 0.147 in tumor tissue and 0.144 in matching normal lung tissue (p=n.s., Wilcoxon Test). No associations were seen between the tumorous Bcl-2 mRNA expression levels and clinical or histopathologic parameters such as gender, tumor size, TNM stadium and grading, but with tumor histology and smoking. With a follow-up of 85.9 months, the median survival time was 59.7 months. Bcl-2 mRNA expression was significantly associated with patients prognosis (p=0.013, log-rank test). Multivariate regression analysis revealed Bcl-2 expression status and tumor stage as independent prognostic factor. CONCLUSIONS: Bcl-2 expression in NSCLC is not associated with the pathogenesis of this disease. Our data suggests that Bcl-2 mRNA expression plays a crucial role in the biological behavior of NSCLCs. Quantitation of Bcl-2 expression improves estimation of prognosis and appears to identify patients who will benefit from intensive adjuvant therapy.

Epidermal growth factor receptor (EGFR) mRNA levels and protein expression levels in primary colorectal cancer and corresponding liver metastases.

PURPOSE: High expression levels of EGFR mRNA are reported to be associated with a higher response probability in epidermal growth factor receptor (EGFR) targeted drugs. Our aim was to determine how EGFR gene expression levels in primary colorectal cancer (CRC) were related to those in liver metastases. METHODS: 31 pairs of primary CRC and corresponding liver metastases were analyzed. Gene expression level was measured using real-time RT-PCR. RESULTS: No significant difference was observed between median mRNA expression levels of EGFR in primary cancer and those in corresponding liver metastases (P = 0.99). When matched tissue sets were compared on an individual basis, there was a significant correlation for EGFR mRNA expression between primary cancer and corresponding liver metastases (rs = 0.78, P < 0.0001). CONCLUSIONS: A good prediction of EGFR mRNA levels in liver metastases can be obtained by measuring those in the primary CRC.

Prognostic Significance and Clinicopathological Associations of COX-2 SNP in Patients with Nonsmall Cell Lung Cancer.

Background. To further improve the screening, diagnosis, and therapy of patients with nonsmall cell lung cancer (NSCLC) additional diagnostic tools are urgently needed. Gene expression of Cyclooxygenase-2 (COX-2) has been linked to prognosis in patients with NSCLC. The role of the COX-2 926G>C Single Nucleotide Polymorphism (SNP) in patients with NSCLC remains unclear. The aim of this study was to investigate the potential of the COX-2 926G>C SNP as a molecular marker in this disease. Methods. COX-2 926G>C SNP was analyzed in surgically resected tumor tissue of 85 patients with NSCLC using a PCR-based RFLP technique. Results. The COX-2 926G>C SNP genotypes were detected with the following frequencies: GG n = 62 (73%), GC n = 20 (23%), CC n = 3 (4%). There were no associations between COX-2 SNP genotype and histology, grading or gender detectable. COX-2 SNP was significantly associated with tumor stage (P = .032) and lymph node status (P = .016, Chi-square test). With a median followup of 85.9 months, the median survival was 59.7 months. There were no associations seen between the COX-2 SNP genotype and patients prognosis. Conclusions. The COX-2 926G>C SNP is detectable at a high frequency in patients with NSCLC. The COX-2 926G>C SNP genotype is not a prognostic molecular marker in this disease. However, patients with the GC or CC genotype seem more susceptible to lymph node metastases and higher tumor stage than patients with the GG genotype. The results suggest COX-2 926G>C SNP as a molecular marker for lymph node involvement in this disease.

A three-gene signature for outcome in soft tissue sarcoma.

PURPOSE: Finding markers or gene sets that would further classify patients into different risk categories and thus allow more individually adapted multimodality treatment regimens in soft tissue sarcomas is necessary. In this study, we investigated the prognostic values of hypoxia-inducible factor 1a (HIF1a), heparin-binding epidermal growth factor-like growth factor (HB-EGF), vascular endothelial growth factor (VEGF), and other angiogenesis-related gene expressions, as well as their interrelationships. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded tissue samples were obtained from 45 patients with soft tissue sarcoma (median age 57 years, range 16-85 years). After laser capture microdissection direct quantitative real-time reverse transcription-PCR (TaqMan) assays were done in triplicates to determine HIF1a, HB-EGF, VEGF, and other gene expression levels. RESULTS: Multivariate Cox [corrected] regression analysis revealed significant independent associations of HB-EGF, HIF1a, and VEGF-C gene expression to the overall survival (P < 0.0001). A combined factor of these three genes showed a relative risk for shorter survival of 5.5, more than twice higher as in an increasing International Union against Cancer Stage. Receiver operating characteristic curve analysis showed a significant sensitivity of 73% and specificity of 82% of this factor for the diagnosis of short (<3 years) versus long (3-9 years) survival (P = 0.0002). VEGF-A showed significant gender differences in the association to survival. CONCLUSIONS: Measuring HIF1a, HB-EGF, and VEGF-C expression may contribute to a better understanding of the prognosis of patients with soft tissue sarcoma and may even play a crucial role for the distribution of patients to multimodal therapeutic regimens. Prospective studies investigating the response to different adjuvant or palliative therapies seem to be warranted.

Loss of heterozygosity at thymidylate synthase locus in Barrett's metaplasia, dysplasia, and carcinoma sequences.

BACKGROUND: Thymidylate synthase (TS) is known to have a unique 28 bp tandemly repeated sequence in the promoter region, and the majorities of subjects have a heterozygous double repeat/triple repeat genotype in their non-cancerous tissue. Loss of heterozygosity (LOH) at the TS locus is known to occur in cancer patients, but there is no evidence that it is present in precancerous tissue. The aim of this study was to analyze the frequency and timing of LOH at the TS locus in Barrett-associated adenocarcinoma (BA) and its precursory lesions, such as intestinal metaplasia (IM) and dysplasia. METHODS: One hundred twenty-three samples (including 37 with gastroesophageal reflux disease (GERD), 29 with IM, 13 with dysplasia, and 44 with BA) were obtained from 100 patients. Biopsies were obtained from the lower esophageal mucosa/IM/dysplasia/BA, when available. Normal squamous tissue from the upper esophagus was taken as a control. All tissues were analyzed for the TS genotype and TS mRNA expression using the real-time reverse-transcription polymerase chain reaction (RT-PCR) method after laser-capture microdissection. RESULTS: Among the patients with informative heterozygous genotype in their control samples, no sample with LOH at the TS locus was observed in the lower esophageal mucosa in GERD patients (0/22 samples). However, 6 out of 21 samples (28.6%) had LOH in IM, 2 of 7 (28.6%) in dysplasia, and 10 of 25 (40.0%) in BA. No significant difference in TS mRNA expression levels was observed between TS genotypes. CONCLUSION: Our results demonstrate that LOH is a relatively frequent and early event in the IM-BA sequence.

Messenger RNA expression of COX-2 and angiogenic factors in primary colorectal cancer and corresponding liver metastasis.

Several new drugs that are targeted towards various angiogenic factors have shown considerable potential for controlling tumor proliferation and metastases. Expression levels of the targeted genes in primary tumors and metastases should be understood to maximize the use of such drugs. The present study aimed to clarify associations between mRNA levels of cyclooxygenase 2 (COX-2) and angiogenic factors [vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8)] in primary colorectal cancer and in corresponding liver metastasis. We also compared these gene expressions of primary colorectal cancer between patients with and without liver metastasis. In 31 pairs of formalin-fixed and paraffin-embedded primary and metastatic liver tumors as well as 27 specimens of consecutive stage II patients without recurrence, mRNA was quantified by real-time reverse transcription-polymerase chain reaction following the laser capture microdissection. We found a significantly positive correlation in IL-8 between primary tumors and matched liver metastases (p=0.034, rs=0.39) and in VEGF (p=0.0083, rs=0.48), but not in COX-2, which was associated with both VEGF (p=0.044, rs=0.37) and IL-8 (p=0.0004, rs=0.64) in primary colorectal cancers. Multiple regression analysis revealed that COX-2 was independently associated with IL-8 (p<0.0001). There were no differences in mRNA levels between patients with and without liver metastasis. The mRNA levels of VEGF and IL-8 in liver metastasis can be predicted from those in primary colorectal cancer. COX-2 might exert angiogenic activity more through the IL-8, than the VEGF pathway. These angiogenic factors were sufficiently up-regulated before hematogenous metastasis. These preliminary data merit further validation studies.

Alterations of p16 and p14ARF genes and their 9p21 locus in oral squamous cell carcinoma.

The p16 gene encodes a 16-kDa cyclin kinase inhibitor, and the p14ARF gene a 14-kDa protein, which acts as a cell cycle regulator or tumor suppressor in human cancer cells. Both genes are mapped on chromosome 9p21. Previous studies have suggested that the p16 gene has important roles in head and neck squamous cell carcinoma. To clarify carcinogenesis in oral squamous cell carcinoma (OSCC), we examined 44 primary OSCCs for alterations of p16 and p14ARF mRNA expression, the methylation status of the p16 gene promoter, the loss of heterozygosity (LOH) at the 9p21 locus, and p16 and p14ARF gene mutations. Alterations of p16 and p14ARF mRNA expression were seen in 27 (61.4%) of 44 and 10 (22.7%) of 44 of OSCC samples, respectively. Methylation of the p16 gene promoter region was detected in 28 (63.6%) of 44 samples, and LOH at 9p21 locus was found in 30 (68.2%) of 44. p16 and p14ARF gene mutations were observed in 4 (9.0%) of 44 and 2 (4.5%) of 44 samples, respectively. Suspected homozygous deletion (HD) was seen in 9 (20.5%) of 44. All cases except one (97.7%) showed alterations in p16, p14ARF, and their locus. These data indicate that the status of p16 and p14ARF genes in OSCC is frequently influenced by methylation, gene mutation, and allelic deletions. Furthermore, these genes and their 9p21 locus have various roles in the pathogenesis of OSCC.

Gene profiling and pathway analysis of neuroendocrine transdifferentiated prostate cancer cells.

BACKGROUND: Neuroendocrine (NE) cells are present in both normal prostate and prostate cancer. In addition, NE differentiation can be induced by various factors, such as IL-6, in vitro and in vivo. However, the mechanism of this differentiation and the role of NE cells in prostate cancer are not well understood. In this study, we evaluated the gene expression and analyzed the pathways in prostate cancer cells exposed to various NE differentiation inducing factors in vitro. METHODS: Gene expression signatures between control LNCaP cells and each treatment induced NE cell line were compared using Affymetrix GeneChip with network and pathway analysis. RESULTS: All treatments were able to transdifferentiate LNCaP cells into NE phenotype as shown by morphology changes and NE marker measurements. Of the 54,675 oligonucleotide-based probe sets in microarray, 44,975 were mapped into the Ingenuity Pathway Analysis database and were filtered according to the t-test P value. At P < 0.002, the number of genes that were differentially expressed included 302 of the IL-6 treated cells, 201 of genistein, 233 of epinephrine, and 191 of the charcoal stripped serum ones. A pooled data approach also showed 346 differentially expressed genes at the same P value. Gene ontology analysis showed that cancer-related function had the highest significance. CONCLUSIONS: Despite some overlap, each NE transdifferentiation inducing treatment was associated with a changed expression of a unique set of genes, and such gene profiling may help to elucidate the molecular mechanisms involved in NE transdifferentiation of prostate cancer cells.