Ralph M. Wirtz

Prognostic significance of Dicer expression in ovarian cancer - link to global microRNA changes and oestrogen receptor expression

MicroRNA (miRNA) deregulation is a hallmark of human cancer. However, the mechanisms underlying miRNA alteration and the specific role of proteins involved in miRNA processing remains to be elucidated. Dicer is a key enzyme in the miRNA processing pathway that is essential for the production of mature miRNAs from their precursors. We tested the hypothesis that Dicer has biological and clinical relevance in ovarian cancer, using a range of methods including in vitro manipulation of Dicer expression. We observed down‐regulation of Dicer in a subgroup of ovarian carcinomas, and found that decreased Dicer expression correlates significantly with reduced patient survival in serous cancers and advanced disease stages. Moreover, microarray and functional analysis suggest that reduced Dicer expression is connected with a global down‐regulation of the microRNAome and with gene expression changes, particularly reduced expression of oestrogen receptor (ER) mRNA and protein in tumour tissue and in cell culture. Our data suggest a common mechanism for miRNAs changes by alterations in the basic machinery controlling miRNA biogenesis, of which Dicer is a central enzyme. These alterations of miRNA processing are of prognostic value and may play a role in the molecular pathogenesis of ovarian carcinoma and, possibly, other tumours. Knowledge of these molecular pathways may help toward new targeted therapeutic approaches for ovarian cancer. Copyright

Evaluation of the prognostic and predictive value of HER family mRNA expression in high-risk early breast cancer: A Hellenic Cooperative Oncology Group (HeCOG) study

The aim of the study was to evaluate the prognostic ability of the transcriptional profiling of the HER family genes in early breast cancer, as well as to investigate the predictive value of HER2 mRNA expression for adjuvant treatment with paclitaxel. RNA was extracted from 268 formalin-fixed paraffin-embedded (FFPE) tumour tissue samples of high-risk breast cancer patients enrolled in the randomised HE10/97 trial, evaluating the effect of dose-dense anthracycline-based sequential adjuvant chemotherapy with or without paclitaxel. The mRNA expression of all four HER family members was assessed by kinetic reverse transcription-polymerase chain reaction (kRT–PCR). The overall concordance between kRT–PCR and IHC/FISH for HER2 status determination was 74%. At a median follow-up of 8 years, multivariate analysis showed that EGFR and HER2 mRNA expression was associated with reduced overall survival (OS). HER3 and HER4 mRNA level had a favourable prognostic value in terms of OS and disease-free survival (DFS), respectively. Adjusting for HER2 mRNA expression, OS and DFS did not differ between treatment groups. These data indicate that EGFR as well as HER2 are prognostic factors of worse clinical outcomes, whereas HER3 and HER4 gene transcription is associated with better prognosis in high-risk early breast cancer. However, HER2 mRNA expression did not predict clinical benefit from paclitaxel. Kinetic RT–PCR represents an alternative method for evaluating the expression of HER family members in FFPE breast carcinomas.

The prognostic impact of epidermal growth factor receptor in patients with metastatic gastric cancer

BackgroundThe epidermal growth factor receptor (EGFR) is a potential target of anticancer therapy in gastric cancer. However, its prognostic role in metastatic gastric or gastroesophageal junction (GE) cancer has not been established yet.MethodsEGFR status was analyzed by immunohistochemistry (IHC) in paraffin-embedded samples from 357 patients who received chemotherapy in 4 first-line trials. Automated RNA extraction from paraffin and RT-quantitative PCR were additionally used to evaluate EGFR mRNA expression in 130 patients.ResultsEGFR protein expression (any grade) and overexpression (3+) were observed in 43% and 11% of patients, respectively. EGFR positivity correlated with intestinal type histology (p = 0.05), but not with other clinicopathologic characteristics. Median follow-up was 18.2 months. Median overall survival (OS) was similar in patients with EGFR positive vs. those with EGFR negative tumors, regardless whether positivity was defined as ≥1+ (10.6 vs. 10.9 months, p = 0.463) or as 3+ (8.6 vs. 10.8 months, p = 0.377). The multivariate analysis indicated that EGFR status is not an independent prognostic factor (hazard ratio 0.85, 0.56 to 1.12, p = 0.247). There were also no significant differences in overall survival when patients were categorized according to median (p = 0.116) or quartile (p = 0.767) distribution of EGFR mRNA gene expression. Similar distributions of progression-free survival according to EGFR status were observed.ConclusionsUnlike different cancer types where EGFR-positive disease is associated with an adverse prognostic value, EGFR positivity is not prognostic of patient outcome in metastatic gastric or GE cancer.

Prognostic significance of UBE2C mRNA expression in high-risk early breast cancer. A Hellenic Cooperative Oncology Group (HeCOG) Study

BACKGROUND The ubiquitin-proteasome system (UPS) plays a pivotal role in tumorigenesis. Components of the UPS have recently been implicated in breast cancer progression. In the present study, we sought to explore the prognostic and/or predictive significance of UBE2C messenger RNA (mRNA) expression on disease-free survival (DFS) and overall survival (OS) in high-risk operable breast cancer patients. METHODS Five hundred and ninety-five high-risk breast cancer patients were treated in a two-arm trial evaluating postoperative, dose-dense sequential chemotherapy with epirubicin followed by CMF (cyclophosphamide, methotrexate and 5-fluorouracil) with or without paclitaxel (Taxol). RNA was extracted from 313 formalin-fixed primary tumor tissue samples followed by one-step quantitative RT-PCR for assessment of mRNA expression of UBE2C. RESULTS High UBE2C mRNA expression was associated with poor DFS (Walds P = 0.003) and OS (Walds P = 0.005). High tumor grade, as well as high Ki67 protein expression, was more frequent in the high-expression group of UBE2C. Results of the Cox multivariate regression analysis revealed that high UBE2C mRNA expression remained an independent adverse prognostic factor for relapse (P = 0.037) and death (P = 0.05). CONCLUSIONS High UBE2C mRNA expression was found to be of adverse prognostic significance in high-risk breast cancer patients. These findings need to be validated in larger cohorts.

Human papillomavirus DNA load and p16INK4a expression predict for local control in patients with anal squamous cell carcinoma treated with chemoradiotherapy.

As the detection rate of HPV‐DNA in anal carcinoma commonly exceeds 90%, a comparison between sole HPV‐positive and HPV‐negative cancers with respect to treatment response following chemoradiotherapy (CRT) and long‐term oncological outcome is challenging. Against this background, we aimed to assess HPV types and HPV DNA load in formalin‐fixed paraffin‐embedded tissue (FFPE) of 95 patients treated with standard CRT for anal cancer to correlate viral load (≤/> median) with local failure, distant metastases, cancer‐specific (CSS) and overall survival (OS) rates. Various clinicopathologic parameters and the immunohistochemical marker p16INK4a were evaluated for any correlation with HPV16 DNA load and were included in uni‐ and multivariate analyses. The overall prevalence of HPV DNA was 95.8% with HPV16 monoinfection being the most commonly encountered HPV type (78.9%), followed by HPV16 and 31, 35, 39, 44, 58, 66 and 81 dual infection in 9 patients (9.5%). HPV16 DNA load was significantly associated with p16INK4a expression (p = 0.001). Patients with HPV16 DNA load ≤ median and low p16INK4a expression showed significantly worse local control (HPV16 DNA load: univariate p = 0.023, multivariate p = 0.042; p16INK4a: univariate p = 0.021), and OS (HPV16 DNA load: univariate p = 0.02, multivariate p = 0.03). Moreover, a combined HPV16 DNA load and p16INK4a variable revealed a significant correlation to decreased local failure, and increased CSS and OS (p = 0.019, p = 0.04 and p = 0.031). In conclusion, these data indicate that HPV16 DNA load and p16INK4a expression are significant prognostic factors for local tumor control and overall survival of patients with anal SCC following CRT.

Validity of the proliferation markers Ki67, TOP2A, and RacGAP1 in molecular subgroups of breast cancer

High proliferation rates are characteristic of cancer, and proliferation markers make up the majority of genes included in RNA-based prognostic gene signatures applied for breast cancer patients. Based on prior data on differences in molecular subgroups of breast cancer, we hypothesized that the significance of single proliferation markers might differ in luminal, Her2-positive and triple-negative subtypes. Therefore, we compared mRNA expression data of Ki67, TOP2A, and RacGAP1 using a pool of 562 Affymetrix U133A microarrays from breast cancer samples. “Luminal,” “triple-negative,” and “Her2-positive” subcohorts were defined by ESR1 and ERBB2 mRNA expression using pre-defined cut-offs. The analysis of the three potential proliferation markers revealed subtype-specific differences: in luminal carcinomas, expression of all three markers was a significant indictor of early recurrence in univariate and multivariate analysis, but RacGAP1 was superior to Ki67 and TOP2A in significance. In triple-negative tumors, only Ki67 was a significant and independent marker, whereas none of the markers showed a significant prognostic impact in Her2-positive cases. Within the group of luminal carcinomas, the proliferation markers had different impact depending on the treatment of patients: in untreated patients, Ki67, TOP2A, and RacGAP1 were significant and independent prognostic markers. In chemotherapy-treated patients, overexpression of all three markers was predictive for early recurrence, but only RacGAP1 retained significance in multivariate analysis. In contrast, RacGAP1 was the only predictive proliferation marker in the endocrine treatment group. These data point to subtype-specific differences in the relevance of proliferation-associated genes, and RacGAP1 might be a strong prognostic and predictive marker in the luminal subgroup.

Prognostic relevance of glycosylation-associated genes in breast cancer

Glycosylation of cellular proteins has important impact on their stability and functional properties, and glycan structures strongly influence cell adhesion. Many enzymes are involved in glycoconjugate synthesis and degradation, but there is only limited information about their role in breast cancer progression. Therefore, we retrieved RNA expression data of 202 glycosylation genes generated by microarray analysis (Affymetrix HG-U133A) in a cohort of 194 mammary carcinomas with long-term follow-up information. After univariate and multivariate Cox regression analysis, genes with independent prognostic value were identified. These were further analysed by Kaplan–Meier analysis and log-rank tests, and their prognostic value was validated in a second cohort of 200 tumour samples from patients without systemic therapy. In our first cohort, we identified 24 genes with independent prognostic value, coding for sixteen anabolic and eight catabolic enzymes. Functionally, these genes are involved in all important glycosylation pathways, namely O-glycosylation, N-glycosylation, O-fucosylation, synthesis of glycosaminoglycans and glycolipids. Eighteen genes also showed prognostic significance in chemotherapy-treated patients. In the second cohort, six of the 24 relevant genes were of prognostic significance (FUT1, FUCA1, POFUT1, MAN1A1, RPN1 and DPM1), whereas a trend was observed for three additional probesets (GCNT4, ST3GAL6 and UGCG). In a stratified analysis of molecular subtypes combining both cohorts, great differences appeared suggesting a predominant role of N-glycosylation in luminal cancers and O-glycosylation in triple-negative ones. Correlations of gene expression with metastases of various localizations point to a role of glycan structures in organ-specific metastatic spread. Our results indicate that various glycosylation reactions influence progression and metastasis of breast cancer and might thus represent potential therapeutic targets.

Improved outcome of high-risk early HER2 positive breast cancer with high CXCL13-CXCR5 messenger RNA expression.

UNLABELLED The CXCL13-CXCR5 is a chemokine axis that is activated in some breast cancers. A total of 321 tissue blocks from a group of patients who received adjuvant, dose-dense chemotherapy for high-risk early breast cancer were examined. Activation of this axis was found to be associated with determinants of poor prognosis but also with improved outcome in the human epidermal growth factor receptor 2 overexpressing subpopulation. BACKGROUND Chemokines are important in cell migration and are thought to play a key role in metastasis. We explored the prognostic significance of C-X-C ligand-motif (CXCL) 12, CXCL13, and receptor (CXCR) 5 on disease-free survival (DFS) and overall survival (OS) in early breast cancer. METHODS A total of 595 patients with high risk, [corrected] early breast cancer were treated in a 2-arm trial (HE10/97) with dose-dense sequential epirubicin followed by cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) with or without paclitaxel. RNA was extracted from 321 formalin-fixed paraffin-embedded primary tumor tissue samples and quantitative reverse-transcriptase polymerase chain reaction was used to assess messenger RNA (mRNA) expression of CXCL12, CXCL13, and CXCR5; estrogen receptor; progesterone receptor (PgR); microtubule-associated protein tau and human epidermal growth factor receptor 2 (HER2). RESULTS CXCL13 and CXCR5 were found to be negatively associated with estrogen receptor and microtubule-associated protein tau mRNA expression and with dense lymphocytic infiltration, and were positively associated with nuclear grade. Only CXCL13 was positively associated with HER2. Multivariate analysis revealed an association between high CXCL13 mRNA expression and improved DFS (hazard ratio [HR] 0.48 [95% CI, 0.25-0.90]; Wald, P = .023) but not OS; whereas high CXCL12 expression was significantly associated with increased OS (HR 0.53 [95% CI, 0.33-0.85]; Wald, P = .009). In the HER2 mRNA overexpressing subgroup, high CXCL13 mRNA expression was associated with improved DFS (P < .001), whereas high CXCR5 was associated with increased DFS and OS (P = .004 and P = .049, respectively). CONCLUSIONS The CXCL13-CXCR5 axis is associated with classic determinants of poor prognosis, such as high grade, hormone receptor negativity, and axillary node involvement. Interestingly, this chemokine axis seems to be strongly associated with improved outcome in patients with HER2(+) disease.

HER2 and TOP2A in high-risk early breast cancer patients treated with adjuvant epirubicin-based dose-dense sequential chemotherapy.

BackgroundHER2 and TOP2A parameters (gene status, mRNA and protein expression) have individually been associated with the outcome of patients treated with anthracyclines. The aim of this study was to comprehensively evaluate the prognostic/predictive significance of the above parameters in early, high-risk breast cancer patients treated with epirubicin-based, dose-dense sequential adjuvant chemotherapy.MethodsIn a series of 352 breast carcinoma tissues from patients that had been post-operatively treated with epirubicin-CMF with or without paclitaxel, we assessed HER2 and TOP2A gene status (chromogenic in situ hybridization), mRNA expression (quantitative reverse transcription PCR), as well as HER2 and TopoIIa protein expression (immunohistochemistry).ResultsHER2 and TOP2A amplification did not share the same effects on their downstream molecules, with consistent patterns observed in HER2 mRNA and protein expression according to HER2 amplification (all parameters strongly inter-related, p values < 0.001), but inconsistent patterns in the case of TOP2A. TOP2A gene amplification (7% of all cases) was not related to TOP2A mRNA and TopoIIa protein expression, while TOP2A mRNA and TopoIIa protein were strongly related to each other (p < 0.001). Hence, TOP2A amplified tumors did not correspond to tumors with high TOP2A mRNA or TopoIIa protein expression, while the latter were characterized by high Ki67 scores (p = 0.003 and p < 0.001, respectively). Multivariate analysis adjusted for nodal involvement, hormone receptor status, Ki67 score and HER2/TOP2A parameters revealed HER2/TOP2A co-amplification (21.2% of HER2 amplified tumors) as an independent favorable prognostic factor for DFS (HR = 0.13, 95% CI: 0.02-0.96, p = 0.046); in contrast, increased HER2/TOP2A mRNA co-expression was identified as an independent adverse prognostic factor for both DFS (HR = 2.41, 95% CI: 1.31-4.42, p = 0.005) and OS (HR = 2.83, 95% CI: 1.42-5.63, p = 0.003), while high TOP2A mRNA expression was an independent adverse prognostic factor for OS (HR = 2.06, 95% CI: 1.23-3.46, p = 0.006). None of the parameters tested was associated with response to paclitaxel.ConclusionsThis study confirms the favorable prognostic value of HER2/TOP2A co-amplification and the adverse prognostic value of high TOP2A mRNA expression extending it to the adjuvant treatment setting in early high-risk breast cancer. The strong adverse prognostic impact of high HER2/TOP2A mRNA co-expression needs further validation in studies designed to evaluate markers predictive for anthracyclines.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12611000506998.

MMP9 but Not EGFR, MET, ERCC1, P16, and P-53 Is Associated with Response to Concomitant Radiotherapy, Cetuximab, and Weekly Cisplatin in Patients with Locally Advanced Head and Neck Cancer

Concomitant administration of radiotherapy with cisplatin or radiotherapy with cetuximab appear to be the treatment of choice for patients with locally advanced head and neck cancer. In the present retrospective analysis, we investigated the predictive role of several biomarkers in an unselected cohort of patients treated with concomitant radiotherapy, weekly cisplatin, and cetuximab (CCRT). We identified 37 patients treated with this approach, of which 13 (35%) achieved a complete response and 10 (27%) achieved a partial response. Severe side effects were mainly leucopenia, dysphagia, rash, and anemia. Tumor EGFR, MET, ERCC1, and p-53 protein and/or gene expression were not associated with treatment response. In contrast, high MMP9 mRNA expression was found to be significantly associated with objective response. In conclusion, CCRT is feasible and active. MMP9 was the only biomarker tested that appears to be of predictive value in cetuximab treated patients. However, this is a hypothesis generating study and the results should not be viewed as definitive evidence until they are validated in a larger cohort.