Fibroblast activation protein-α expression in fibroblasts is common in the tumor microenvironment of colorectal cancer and may serve as a therapeutic target.

Background: Colorectal cancer (CRC) is still one of the leading causes of cancer death worldwide, emphasizing the need for further diagnostic and therapeutic approaches. Cancer invasion and metastasis are affected by the tumor microenvironment (TME), with cancer-associated fibroblasts (CAF) being the predominant cellular component. An important marker for CAF is fibroblast activation protein-α (FAP) which has been evaluated as therapeutic target for, e.g., radioligand therapy. The aim of this study was to examine CRC regarding the FAP expression as a candidate for targeted therapy. Methods: 67 CRC, 24 adenomas, 18 tissue samples of inflammation sites and 28 non-neoplastic, non-inflammatory tissue samples of colonic mucosa were evaluated for immunohistochemical FAP expression of CAF in tissue microarrays. The results were correlated with clinicopathological data, tumor biology and concurrent expression of additional immunohistochemical parameters. Results: 53/67 (79%) CRC and 6/18 (33%) inflammatory tissue specimens showed expression of FAP. However, FAP was only present in 1/24 (4%) adenomas and absent in normal mucosa (0/28). Thus, FAP expression in CRC was significantly higher than in the other investigated groups. Within the CRC cohort, expression of FAP did not correlate with tumor stage, grading or the MSI status. However, it was observed that tumors exhibiting high immunohistochemical expression of Ki-67, CD3, p53, and ß-Catenin showed a significantly higher incidence of FAP expression. Conclusion: In the crosstalk between tumor cells and TME, CAF play a key role in carcinogenesis and metastatic spread. Expression of FAP was detectable in the majority of CRC but nearly absent in precursor lesions and non-neoplastic, non-inflammatory tissue. This finding indicates that FAP has the potential to emerge as a target for new diagnostic and therapeutic concepts in CRC. Additionally, the association between FAP expression and other immunohistochemical parameters displays the interaction between different components of the TME and demands further investigation.

[Lung transplantation : Histomorphological diagnosis and clinical aspects]. / Lungentransplantation : Histomorphologische Diagnostik und klinische Aspekte.

The function of pulmonary allografts is regularly impaired by alloimmune reactions with quite variable clinical outcomes, different involved effector cells and molecules, as well as affected anatomical compartments. Acute rejection of grafts after lung transplantation (LuTx) is not only associated with the subsequent development of acute graft dysfunction, but can also contribute - among other immunological and nonimmunological factors - to the development of chronic lung allograft dysfunction (CLAD), which is the main reason for the limited long-term survival after LuTx. In addition to ACR and analogous to other solid organ transplants, the importance of antibody-mediated (humoral) rejection (AMR) in LuTx has also been recognized. There are currently no specific laboratory, radiological, or clinical tests available for either ACR or AMR. Only by the synoptic examination of histopathological changes and interpretation against the background of microbiological, virological, serological, and functional findings, can adequate sensitivity and specificity be achieved in the diagnostics of rejection. In this article, the current criteria for histopathological diagnostics of rejection following LuTx are summarized and the most important differential diagnoses are discussed.

Prediction of Microvascular Tumor Invasion in Liver Transplant Candidates With Hepatocellular Carcinoma: A Feasible Concept or a Misleading Illusion?

INTRODUCTION: Hepatocellular carcinoma (HCC) in cirrhosis is a widely accepted indication for liver transplantation (LT). Many scoring systems have been proposed intending to an extension of the established Milan criteria. Bridging treatments are systematically applied in order to maintain or to downstage such patients to the listing criteria. The objective of our study was to estimate the feasibility of the prediction of microvascular tumor invasion in transplant candidates. PATIENTS AND METHODS: Data corresponding to transplanted HCC patients were reviewed for the purposes of this study. All tumor slices were blindly re-evaluated by a single pathologist in order to score for tumor necrosis and microvascular invasion. Recipients of pediatric or split LT were excluded. RESULTS: Eighty patients (30 women and 50 men) were included in the study. Tumor necrosis was absent in 29 of 80 liver explants (36.25%). In the majority of instances (63.75%) tumor necrosis was evident in proportions between 5% and 100%. In 58 liver explants showing 0%-60% tumor necrosis and 22 liver explants showing > 60% tumor necrosis, microvascular tumor invasion was detectable in 11 and 0 cases, respectively (P = .0385). CONCLUSION: In about one-fourth of the cases (27.5%) microvascular tumor invasion could not be detected due to extended areas of tumor necrosis. Preoperative detection of microvascular invasion is misleading.

Methylation of L1RE1, RARB, and RASSF1 function as possible biomarkers for the differential diagnosis of lung cancer.

BACKGROUND: Lung cancer is the major cause of cancer-related deaths worldwide. Differential diagnosis can be difficult, especially when only small samples are available. Epigenetic changes are frequently tissue-specific events in carcinogenesis and hence may serve as diagnostic biomarkers. MATERIAL AND METHODS: 138 representative formalin-fixed, paraffin-embedded (FFPE) tissues (116 lung cancer cases and 22 benign controls) were used for targeted DNA methylation analysis via pyrosequencing of ten literature-derived methylation markers (APC, CDH1, CDKN2A, EFEMP1, FHIT, L1RE1, MGMT, PTEN, RARB, and RASSF1). Methylation levels were analyzed with the Classification and Regression Tree Algorithm (CART), Conditional Interference Trees (ctree) and ROC. Validation was performed with additional 27 lung cancer cases and 38 benign controls. TCGA data for 282 lung cancer cases was included in the analysis. RESULTS: CART and ctree analysis identified the combination of L1RE1 and RARB as well as L1RE1 and RASSF1 as independent methylation markers with high discriminative power between tumor and benign tissue (for each combination, 91% specificity and 100% sensitivity). L1RE1 methylation associated significantly with tumor type and grade (p<0.001) with highest methylation in the control group. The opposite was found for RARB (p<0.001). RASSF1 methylation increased with tumor type and grade (p<0.001) with strongest methylation in neuroendocrine tumors (NET). CONCLUSION: Hypomethylation of L1RE1 is frequent in tumors compared to benign controls and associates with higher grade, whereas increasing methylation of RARB is an independent marker for tumors and higher grade. RASSF1 hypermethylation was frequent in tumors and most prominent in NET making it an auxiliary marker for separation of NSCLC and NET. L1RE1 in combination with either RARB or RASSF1 could function as biomarkers for separating lung cancer and non-cancerous tissue and could be useful for samples of limited size such as biopsies.

MDM2 is an important prognostic and predictive factor for platin-pemetrexed therapy in malignant pleural mesotheliomas and deregulation of P14/ARF (encoded by CDKN2A) seems to contribute to an MDM2-driven inactivation of P53.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive tumour that is first-line treated with a combination of cisplatin and pemetrexed. Until now, predictive and prognostic biomarkers are lacking, making it a non-tailored therapy regimen with unknown outcome. P53 is frequently inactivated in MPM, but mutations are extremely rare. MDM2 and P14/ARF are upstream regulators of P53 that may contribute to P53 inactivation. METHODS: A total of 72 MPM patients were investigated. MDM2 immunoexpression was assessed in 65 patients. MDM2 and P14/ARF mRNA expression was analysed in 48 patients of the overall collective. The expression results were correlated to overall survival (OS) and progression-free survival (PFS). RESULTS: OS and PFS correlated highly significantly with MDM2 mRNA and protein expression, showing a dismal prognosis for patients with elevated MDM2 expression (for OS: Score (logrank) test: P⩽0.002, and for PFS: Score (logrank) test; P<0.007). MDM2 was identified as robust prognostic and predictive biomarker for MPM on the mRNA and protein level. P14/ARF mRNA expression reached no statistical significance, but Kaplan-Meier curves distinguished patients with low P14/ARF expression and hence shorter survival from patients with higher expression and prolonged survival. CONCLUSIONS: MDM2 is a prognostic and predictive marker for a platin-pemetrexed therapy of patients with MPMs. Downregulation of P14/ARF expression seems to contribute to MDM2-overexpression-mediated P53 inactivation in MPM patients.

Alveolar and intraparenchymal proteasome in sarcoidosis.

BACKGROUND: In sarcoidosis, an antigen specific immune response is a putative mechanism, resulting in granulomatous inflammation. Since the proteasome is involved in antigen presentation, alterations in the alveolar and parenchymal proteasomal system may be a feature of sarcoidosis. OBJECTIVES: To explore the role of proteasomes and immunoproteasomes in sarcoidosis. METHODS: Total proteasome concentration and activity was assessed in bronchoalveolar lavage (BAL) supernatant obtained from sarcoidosis patients (n = 67) and healthy controls (n = 18) using ELISA and cleavage of specific fluorogenic substrates (±epoxomicin), respectively. Immunohistochemistry of lung tissue sections and immunocytochemistry of BAL macrophages for immunoproteasome was performed in sarcoidosis patients and controls. RESULTS: Proteasome was present in BAL supernatants of all sarcoidosis patients. In sarcoidosis, abundant immunoproteasome staining was seen in pneumocytes type II and granulomas. Total proteasome concentration was greater in active sarcoidosis, stages II (101 ng/ml ± 79; p = 0.009) and III (119 ng/ml ± 66; p = 0.012), than in inactive sarcoidosis or in healthy controls (35 ng/ml ± 34). In the absence of epoxomicin, all fluorogenic substrates were hydrolyzed by BAL supernatant of sarcoidosis patients and controls. CONCLUSIONS: Patients with active sarcoidosis but not healthy controls demonstrate immunoproteasome in the lung tissue and in granulomas. Thus, the putative immune response in sarcoidosis may be mediated or sustained by the proteasomal system.

Assessment of SHOX2 methylation in EBUS-TBNA specimen improves accuracy in lung cancer staging.

BACKGROUND: Endobronchial ultrasound with transbronchial needle aspiration (EBUS-TBNA) is a well-established method to assess mediastinal lymph nodes for lung cancer. However, a proportion of patients require further investigation, due to the low negative predictive value (NPV). The objective of this study was to determine whether the assessment of short stature homeobox 2 (SHOX2) DNA methylation level in lymph node tissue obtained by EBUS-TBNA improves the accuracy of mediastinal staging. PATIENTS AND METHODS: EBUS-TBNA was carried out for suspicious lymph nodes of 154 patients. Negative or ambiguous histological results were confirmed by surgical means and clinical follow-up over 6 months. EBUS-TBNA was assessed on 80 positive and 85 negative classified lymph nodes and compared with the result of the SHOX2 DNA methylation real-time PCR analysis. Relative methylation measured by delta-delta cycle threshold (ΔΔCt) was used to classify the samples. Clinical performance of the EBUS-TBNA procedure with and without the additional SHOX2 assessment was calculated against the final classification according to the gold standard. RESULTS: Based on data from 105 patients, an average 80-fold increase in the SHOX2 methylation level was measured for positive compared with negative lymph nodes. SHOX2 results with a ΔΔCt value of <6.5 indicate positive lymph nodes. Applying this molecular analysis to EBUS-TBNA cases, not diagnosed by pathologic assessment, the sensitivity of staging was improved by 17%-99%. The NPV increased from 80% to 99%. CONCLUSIONS: The combination of EBUS-TBNA and SHOX2 methylation level strongly improves the assessment of the nodal status by identifying additional malignant lesions and confirming benign nodes and therefore avoiding invasive follow-up procedures.

Controlled oxygenated rewarming of cold stored liver grafts by thermally graduated machine perfusion prior to reperfusion.

The quality of cold-stored livers declines with the extension of ischemic time, increasing the risk of primary dys or nonfunction. A new concept to rescue preserved marginal liver grafts by gentle oxygenated warming-up prior to blood reperfusion was investigated. Porcine livers were preserved by cold storage (CS) in modified HTK-solution for 18 h. Some grafts were subsequently subjected to 90 min of controlled oxygenated rewarming (COR) by machine perfusion with gradual increase of perfusate temperature up to 20°C or simple oxygenated machine perfusion in hypothermia (HMP) or subnormothermia (SNP). Graft viability was assessed thereafter by 4 h of normothermic blood reperfusion ex vivo. Endischemic tissue energetics were significantly improved by COR or SNP and to a notably lesser extent by HMP. COR significantly reduced cellular enzyme loss, gene expression and perfusate activities of TNF-alpha, radical mediated lipid peroxidation (LPO) and increase of portal vascular perfusion resistance upon reperfusion, while HMP or SNP were less protective. Only COR resulted in significantly more bile production than after CS. Histological injury score and caspase 3-activation were significantly lower after COR than after CS. Oxygenated rewarming prior to reperfusion seems to be a promising technique to improve subsequent organ recovery upon reperfusion of long preserved liver grafts.

Loss of Survivin influences liver regeneration and is associated with impaired Aurora B function.

The chromosomal passenger complex (CPC) acts as a key regulator of mitosis, preventing asymmetric segregation of chromosomal material into daughter cells. The CPC is composed of three non-enzymatic components termed Survivin, the inner centromere protein (INCENP) and Borealin, and an enzymatic component, Aurora B kinase. Survivin is necessary for the appropriate separation of sister chromatids during mitosis and is involved in liver regeneration, but its role in regenerative processes is incompletely elucidated. Whether Survivin, which is classified as an inhibitor of apoptosis protein (IAP) based on domain composition, also has a role in apoptosis is controversial. The present study examined the in vivo effects of Survivin ablation in the liver and during liver regeneration after 70% hepatectomy in a hepatocyte-specific knockout mouse model. The absence of Survivin caused a reduction in the number of hepatocytes in the liver, together with an increase in cell volume, macronucleation and polyploidy, but no changes in apoptosis. During liver regeneration, mitosis of hepatocytes was associated with mislocalization of the members of the CPC, which were no longer detectable at the centromere despite an unchanged protein amount. Furthermore, the loss of survivin in regenerating hepatocytes was associated with reduced levels of phosphorylated Histone H3 at serine 28 and abolished phosphorylation of CENP-A and Hec1 at serine 55, which is a consequence of decreased Aurora B kinase activity. These data indicate that Survivin expression determines hepatocyte number during liver development and liver regeneration. Lack of Survivin causes mislocalization of the CPC members in combination with reduced Aurora B activity, leading to impaired phosphorylation of its centromeric target proteins and inappropriate cytokinesis.

Accelerated hyperfractionated radiotherapy within trimodality therapy concepts for stage IIIA/B non-small cell lung cancer: Markedly higher rate of pathologic complete remissions than with conventional fractionation.

BACKGROUND: Radiation dose escalation within definitive radiochemotherapy (RTx/CTx) was not successful for stage III non-small cell lung cancer (NSCLC) using conventional fractionation (CF). Accelerated-hyperfractionation (AHF) counteracts tumour cell repopulation. In this observational study, the effects of neoadjuvant RTx/CTx using AHF or CF were studied by histopathology and using the survival end-point. METHODS: Data from all consecutive lung cancer patients treated with neoadjuvant RTx/CTx and thoracotomy between 08/2000 and 06/2012 were analysed. Patients received induction chemotherapy (cisplatin-doublets) followed by concurrent RTx/CTx using AHF (45 Gy/1.5 Gy bid) or CF-RTx (46 Gy/2 Gy qd). For estimating the AHF versus CF treatment effects, multivariate analysis (MA), propensity score weighting (PS), and instrumental variable analysis (IV) were used. FINDINGS: 239 patients were treated, median age 58 (34-78)years, stage II/IIIA/B: 19/88/132, squamous cell/adenocarcinomas/other: 98/107/34; AHF/CF-RTx 112/127 patients. No significant differences between both groups, in tumour related factors (age, gender, Charlson comorbiditiy score, lactate dehydrogenase (LDH), haemoglobin, stage, histopathology and grading), existed. Crude rates of pathologic complete responses (pCR) in AHF and CF groups were 37% and 24% respectively. The dose fractionation effect on pCR was significant (p ⩽ 0.006, PS and IV analyses). There was a significant dependence of pCR on biologically effective dose. pCR also depended on treatment time (MA, p = 0.04; PS, p = 0.0004). Median treatment time was 22 d or 31 d using AHF or CF (p<0.0001), respectively. Adenocarcinomas had lower pCR rates in comparison to other histologies. Five-year survival of patients with pCR was 65%, independent of the fractionation. INTERPRETATION: This large monoinstitutional analysis demonstrates an increased effect of AHF on pCR of lung cancer which modifies overall survival.

ßV-tubulin expression is associated with outcome following taxane-based chemotherapy in non-small cell lung cancer.

BACKGROUND: Tubulin-binding agents (TBAs) are effective in non-small cell lung cancer (NSCLC) treatment. Both ßIII- and ßV-tubulins are expressed by cancer cells and may lead to resistance against TBAs. METHODS: Pre-treatment samples from 65 locally advanced or oligometastatic NSCLC patients, who underwent uniform induction chemotherapy with paclitaxel and platinum followed by radiochemotherapy with vinorelbine and platinum were retrospectively analysed by immunohistochemistry. Protein expression of ßIII- and ßV-tubulin was morphometrically quantified. RESULTS: Median pre-treatment H-score for ßIII-tubulin was 110 (range: 0-290), and 160 for ßV-tubulin (range: 0-290). Low ßIII-tubulin expression was associated with improved overall survival (OS) (P=0.0127, hazard ratio (HR): 0.328). An association between high ßV-tubulin expression and prolonged progression-free survival (PFS, median 19.2 vs 9.4 months in high vs low expressors; P=0.0315, HR: 1.899) was found. Further, high ßV-tubulin expression was associated with objective response (median H-score 172.5 for CR+PR vs 120 for SD+PD patients, P=0.0104) or disease control following induction chemotherapy (170 for CR+PR+SD vs 100 for PD patients, P=0.0081), but not radiochemotherapy. CONCLUSION: Expression of ßV-tubulin was associated with treatment response and PFS following paclitaxel-based chemotherapy of locally advanced and oligometastatic NSCLC patients. Prolonged OS was associated with low levels of ßIII-tubulin. Prospective evaluation of ßIII/ßV-tubulin expression in NSCLC is warranted.

Cyclo-oxygenase-2 overexpression is a feature of early and well-differentiated hepatocellular carcinoma with a favourable prognosis.

AIMS: To determine the prognostic relevance of cyclo-oxygenase-2 (COX-2) expression in hepatocellular carcinoma (HCC) and its relationship to important clinicopathological parameters. METHODS: A series of 196 patients with HCCs treated either by surgical resection (n = 106) or liver transplantation (n = 90) was investigated. Immunohistochemically confirmed COX-2 expression was correlated with a series of clinicopathologically relevant parameters as well as proliferative activity and apoptosis. RESULTS: Overexpression of COX-2 correlated statistically with high histological tumour differentiation (p<0.001) and early TNM stage (p = 0.003). COX-2 overexpression was associated with lower apoptotic rates (p = 0.001), whereas proliferation activity did not differ significantly. In addition, COX-2 overexpression showed a significant correlation with favourable overall survival (p<0.001). In multivariate survival analysis, COX-2 expression qualified as an independent prognostic parameter (p = 0.030). CONCLUSIONS: Overexpression of COX-2 in HCC indicates early-stage cancer with less aggressive tumour behaviour and constitutes an independent prognostic factor.

White matter changes in HIV-1 infected brains: a combined gross anatomical and ultrastructural morphometric investigation of the corpus callosum.

OBJECTIVE: The HIV-1 associated cognitive/motor complex is characterized by cognitive, motor and behavioral disturbances. Besides a significant loss of neurons in the cerebral cortex and subcortical nuclei, a possible morphological substrate of this complex is also given by changes of the white matter as seen in HIV-1 leucoencephalopathy (HIVL), which is characterized by widespread diffuse pallor of myelin and the presence of gliomesenchymal nodules with multinucleated giant cells. METHODS: The corpus callosum as a sensitive marker for damage of the cerebral white matter was investigated by morphometry both at the macroscopic and electronmicroscopic level. RESULTS: In HIV-1 infected brains, a significant decrease of the profile area of the whole corpus callosum as well as of its different parts was noted. The absolute number of nerve fibers was significantly decreased, in particular in the frontal and occipital parts of the corpus callosum. Moreover, several morphometric parameters for nerve fibers, axons and myelin sheaths indicate in some areas a reduction of nerve fibers and axons, as well as a diminished myelin sheath thickness, whereas, in other regions, swelling of axons and myelin sheaths was observed. CONCLUSIONS: The observed changes are considered to represent subtle changes affecting nerve fibers before histological evidence of HIVL, and might represent one aspect of the morphological substrates preceeding the development of the HIV-1 related cognitive/motor complex.

Role of osteopontin and CD44s expression for patients with hepatocellular carcinoma undergoing liver transplantation or resection.

BACKGROUND: Detection of new biomarkers for hepatocellular carcinoma (HCC) is needed to estimate prognosis after liver transplantation (OLT) or hepatic resection. Osteopontin (OPN) is a secreted, calcium-binding, phosphorylated, acidic glycoprotein that is overexpressed in various cancers. Cluster differentiation 44 standard isoform (CD44s) is one of the primary receptors of OPN; it may contribute to metastatic tumor spread. MATERIALS AND METHODS: Tumor tissue and surrounding hepatic parenchyma were obtained from 53 HCC patients who underwent liver resection. Their RNA was extracted from nitrogen-frozen tissues, and OPN mRNA levels were estimated by quantitative reverse transcription-polymerase chain reactions. Formalin-fixed, paraffin-embedded tissues were obtained from the same patients, and additionally from 60 OLT HCC patients to perform expression analysis for OPN and CD44s by standard avidin-biotin immunostaining methods. RESULTS: Expression of OPN and CD44s was significantly higher among HCC compared with adjacent nontumor tissue. The OPN mRNA expression and protein abundance correlated positively; OPN overexpression was associated with high tumor grade. A positive correlation existed between OPN and CD44s expression; both proteins were significantly overexpressed in HCC lesions with positive lymph nodes. No significant correlation existed between patient survival and OPN and CD44s expression. CONCLUSION: Expression of both OPN and CD44s in HCC is associated with advanced tumor stage, thus possibly contributing prognostic information when evaluated together with classical clinicopathological parameters.

Five-year survival after monotherapy for hepatocellular carcinoma in the setting of cirrhosis.

The purpose of this study was to evaluate the long-term results with monotherapy for hepatocellular carcinoma (HCC) in the setting of cirrhosis. We reviewed data of 14 patients who survived for at least 5 years after performance of liver resection (n = 1), transarterial chemoembolization (TACE, n = 3), or liver transplantation (OLT, n = 19). Eight patients were within the Milan criteria, whereas the remaining 6 were beyond the criteria. Tumor stages according to the UICC were I (n = 8), II (n = 5), and IIIA (n = 1). Vascular invasion was not detected in any patient. The HCCs recurred in 2 patients, at 81 and 48 months' posttransplant. Sites of recurrence were the intrathoracic lymph nodes in the first case, and lungs in the second case. Treatment of recurrence included chemotherapy in the first case and local resection in the second case. Both patients died at 98 and 64 months postoperation (ie, 17 and 16 months, respectively, after the diagnosis of the recurrence). A third patient died of nontumor-related causes at 69 months after his first TACE. Currently, 11 patients are alive with a median survival of 70 months (range, 63-144 months). The alpha-fetoprotein level was demonstrated to be prognostic of recurrence by discriminant function analysis. In conclusion, OLT provided the best long-term results as monotherapy for HCC in the setting of cirrhosis.

AKT and ERK1/2 signaling in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinomas (ICC) are neoplasms that originate from cholangiocytes and can occur at any level of the biliary tree. Surgical resection is the current therapy of choice for this highly aggressive cancer. However, the 5-year survival still is poor, with high recurrence rates. Due to the intrahepatic growth a significant proportion of patients present with advanced disease and are not candidates for curative surgery or transplantation. The existing palliative options are of limited benefit and there is a great necessity for novel therapeutic options. In this article we review the role of the phosphoinositide 3-kinase(PI3K)/AKT and extracellular regulated kinase (ERK) signaling pathways in ICC and present new data on the prognostic value of these protein kinases. Finally, we discuss future upcoming therapeutic options based on targeting these signaling pathways.

Activation of extracellular regulated kinases (ERK1/2) but not AKT predicts poor prognosis in colorectal carcinoma and is associated with k-ras mutations.

Signal transduction and modulation represent central mechanisms in cellular processes such as cell-cycle regulation, oncogenesis, and apoptosis. The aim of this study was to determine the prognostic relevance of two kinases important in the regulation of cell proliferation and apoptosis in 135 colorectal cancer cases: AKT and extracellular regulated kinases (ERK1/2). We investigated the relationship of phospho-ERK1/2 (pERK1/2) and phospho-AKT (pAKT) with associated parameters (EGFR, COX-2, cyclin-D1), proliferative activity (Ki-67), and apoptosis (TUNEL) using immunohistochemistry. Additionally, the k-ras gene was screened for mutations to determine its putative association with ERK1/2 activation. Activation of ERK1/2 but not AKT correlated statistically with the presence of k-ras mutations (P = 0.015). Survival analysis of phospho-ERK1/2 immunoexpression showed a significant correlation with decreased overall survival (OS). The multivariate Cox regression analysis identified pERK1/2 as an independent prognostic parameter (P = 0.005). Activation of ERK1/2 in colorectal cancer may indicate aggressive tumor behavior and may constitute an independent prognostic factor. Furthermore, our data suggest that mutations of the k-ras oncogene may induce activation of ERK1/2. We propose immunohistochemical determination of pERK1/2 status as a promising candidate for the identification of high-risk patients who would benefit from new anticancer drugs targeting the ERK pathway.

Overexpression of cyclo-oxygenase-2 is an independent predictor of unfavourable outcome in node-negative breast cancer, but is not associated with protein kinase B (Akt) and mitogen-activated protein kinase (ERK1/2, p38) activation or with Her-2/neu signalling pathways.

BACKGROUND AND AIM: The production of prostaglandins is regulated by cyclo-oxygenases (COXs), which also have a role in tumour development and progression in various malignancies, including breast cancer. The mechanisms by which COX-2 contributes to unfavourable prognosis are still poorly understood. The association between expression of COX-2 and possible linked signalling pathways-namely, Akt, extracellular regulated kinases (ERK1/2), the stress-activated kinase p38 or Her-2/neu-is assessed in a series of 113 node-negative breast cancers. RESULTS: COX-2 was identified as an independent prognostic factor (p = 0.034) in node-negative breast cancer by survival analysis. The lack of a relationship between COX-2 expression and activated Akt, Erk1/2, p38 and Her-2/neu was indicated by statistical analysis. CONCLUSIONS: The prognostic effect of COX-2 expression on lymph node-negative breast cancer is confirmed-COX-2 is probably not regulated by HER-2, Akt, Erk1/2 or p38. Further studies are necessary for the elucidation of the signalling pathways responsible for the modification of COX-2 expression and the increased aggressiveness of breast cancers overexpressing COX-2.