Michael Tachezy

Low levels of cell-free circulating miR-361-3p and miR-625* as blood-based markers for discriminating malignant from benign lung tumors.

The high mortality rate of lung cancer patients is mainly due to the late stage at which lung cancer is diagnosed. For effective cancer prevention programs and early diagnosis, better blood-based markers are needed. Hence, blood-based microarray profiling of microRNA (miR) expression was performed in preoperative serum of 21 non-small cell lung cancer (NSCLC) patients and 11 healthy individuals by microfluid biochips containing 1158 different miRs. Two out of the 30 most dysregulated miRs were further validated in serum of 97 NSCLC patients, 20 patients with benign lung diseases and 30 healthy individuals by TaqMan MicroRNA Assays. Microarray profiling showed that miR-361-3p and miR-625* were significantly down-regulated in serum of lung cancer patients. Their further evaluation by quantitative RT-PCR showed that the levels of miR-361-3p and miR-625* were lower in NSCLC than in benign disease (p = 0.0001) and healthy individuals (p = 0.0001, p = 0.0005, respectively). Moreover, the levels of miR-625* were significantly lower in patients with large cell lung cancer (LCLC, p = 0.014) and smoking patients (p = 0.030) than in patients with adenocarcinoma and non-smoking patients, respectively. A rise in the levels of both miRs was observed in the postoperative samples compared with the preoperative levels (p = 0.0001). Functional analyses showed that Smad2 and TGFß1 are not dysregulated by miR-361-3p and miR-625* in the lung cell line A549, respectively. Our present pilot study suggests that miR-361-3p and miR-625* might have a protective influence on the development of NSCLC, and the quantitative assessment of these miRs in blood serum might have diagnostic potential to detect NSCLC, in particular in smokers.

Prognostic impact of CXCR4 and CXCR7 expression in pancreatic adenocarcinoma.

Chemokines and their receptors are known to play important roles in the tumorigenesis of many malignancies. The aim of this study was to evaluate the prognostic impact of the expression of the chemokine receptors CXCR4 and CXCR7 in patients with pancreatic adenocarcinoma (PAC).

Activated leukocyte cell adhesion molecule (CD166)--its prognostic power for colorectal cancer patients.

BACKGROUND The activated leukocyte cell adhesion molecule (ALCAM, CD166) has been reported to be involved in tumorigenesis of colorectal cancer (CRC) and to function as a cancer stem cell marker. Controversial data exist regarding the prognostic power of ALCAM expression in CRC. Here, we evaluate the expression of ALCAM in a cohort of CRC patients and its usage as a prognostic marker for survival. MATERIALS AND METHODS Tissue specimens from 299 patients with CRC treated between 1993 and 2006 were analyzed via ALCAM immunohistochemistry (clone MOG/07) using a tissue microarray. Results were correlated with clinical, histopathological, and patient survival data (Chi-square test, Kaplan-Meier analysis, and log-rank test, respectively). Multivariate analysis also was performed (Cox regression). RESULTS ALCAM is expressed in most primary (76%) and secondary (62%) CRC lesions (P = 0.014). Immunohistochemistry revealed an inverse association with tumor grading (P = 0.002) but not with any other clinical or histopathological data. Kaplan-Meier survival analysis revealed a significant overall survival benefit in the group of ALCAM-positive patients (P = 0.019). Multivariate analysis showed that ALCAM is an independent positive prognostic marker for overall survival (P = 0.023). CONCLUSIONS ALCAM expression is a positive prognostic marker for overall survival of CRC patients, and its detection might help to optimize the existing prognostic staging system. Elevated expression in higher differentiated tumors might indicate a potential role in the early steps of tumorigenesis, and its loss might be associated with reduced cellular adhesion, resulting in a higher metastatic potential of the tumor. Further studies must be conducted investigating these hypotheses.

ALCAM (CD166) expression and serum levels are markers for poor survival of esophageal cancer patients

The expression of the activated leukocyte cell adhesion molecule (ALCAM and CD166) is increased in various types of cancer. We aimed to evaluate its role as a prognostic marker for esophageal cancer (EC). We retrospectively analyzed ALCAM expression in 299 primary lesions, 147 lymph node and 46 distant metastases from EC patients, on a tissue microarray using immunohistochemistry. Bone marrow samples from representative cancer patients (n = 16), taken before primary surgery, were stained by double‐immunofluorescence for ALCAM and cytokeratins (CK). Blood serum samples from 236 cancer patients and 127 controls were analyzed for serum ALCAM (s‐ALCAM) by ELISA. The immunohistochemical analysis showed increased ALCAM expression in the majority of lesions (primary tumor 71%, lymph node 76% and distant metastases 80%). ALCAM expression was not associated with histopathological parameters except for tumor grading (p = 0.015). ALCAM‐positive patients had significantly worse recurrence‐free and overall survival (OS; p = 0.002). Disseminated tumor cells (DTC) in bone marrow showed two phenotypes, ALCAM+/CK+ (36%) and ALCAM‐/CK+ (64%). Multivariate analysis revealed that ALCAM expression and elevated s‐ALCAM serum values are powerful prognostic variables for OS in patients with EC (hazard ratio [HR] 3.987, 95% confidence interval [95%CI] 1.906–8.340, p < 0.001 and HR 1.915, 95%CI 1.021–3.592, p = 0.043). The results of our study provide preliminary evidence for the potential clinical utility of ALCAM as a prognostic biomarker for EC, which might be a basis for future clinical application. In addition, ALCAM expression in a subset of DTC of the bone marrow indicates a potential function in the metastatic cascade of EC.

Detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer

ABSTRACT Pancreatic cancer presents with a dismal mortality rate and is in urgent need of methods for early detection with potential for timely intervention. All living cells, including cancer cells, generate exosomes. We previously discovered double stranded genomic DNA in exosomes derived from the circulation of pancreatic cancer patients, which enabled the detection of prevalent mutations associated with the disease. Here, we report a proof-of-concept study that demonstrates the potential clinical utility of circulating exosomal DNA for identification of KRASG12D and TP53R273H mutations in patients with pancreas-associated pathologies, including pancreatic ductal adenocarcinoma (PDAC), chronic pancreatitis (CP) and intraductal papillary mucinous neoplasm (IPMN), and in healthy human subjects. In 48 clinically annotated serum samples from PDAC patients, digital PCR analyses of exosomal DNA identified KRASG12D mutation in 39.6% of cases, and TP53R273H mutation in 4.2% of cases. KRASG12D and TP53R273H mutations were also detected in exosomal DNA from IPMN patients (2 out of 7 with KRASG12D, one of which also co-presented with TP53R273H mutation). Circulating exosomal DNA in 5 out of 9 CP patients enabled the detection of KRASG12D mutation. In 114 healthy subject-derived circulating exosomal DNA, 2.6% presented with KRASG12D mutation and none with TP53R273H mutation. This study highlights the value of circulating exosomal DNA for a rapid, low-cost identification of cancer driving mutations. The identification of mutations in IPMN patients and healthy subjects suggests that liquid biopsies may allow potential assessment of cancer risk but with a cautionary note that detection of clinical cancer cannot be assumed.

Low activated leukocyte cell adhesion molecule expression is associated with advanced tumor stage and early prostate-specific antigen relapse in prostate cancer

Activated leukocyte cell adhesion molecule (CD166) is a member of the immunoglobulin superfamily and is aberrantly expressed in different tumors, including prostate cancer. To learn more on the prevalence and clinical significance of activated leukocyte cell adhesion molecule expression in prostate cancer, a tissue microarray containing 3261 primary prostate cancers treated by radical prostatectomy was used. A total of 2390 different prostate cancers were analyzed by immunohistochemistry in a tissue microarray format. Activated leukocyte cell adhesion molecule immunostaining in cancers was compared with clinical follow-up, which was available for 1746 patients. Membranous activated leukocyte cell adhesion molecule immunostaining was recorded in 1663 (69.6%) of cases. High activated leukocyte cell adhesion molecule expression levels were significantly associated with favorable tumor features (pT: P = .0015; pN: P = .0008; preoperative prostate-specific antigen: P = .0057) and a lower risk of a biochemical recurrence (P = .0067). Cytoplasmatic activated leukocyte cell adhesion molecule staining was usually associated with membranous staining. The small number of cancers with pure cytoplasmatic staining did not reveal any particularities with respect to clinical outcome or tumor phenotype. It is concluded that activated leukocyte cell adhesion molecule protein is almost always expressed in prostate cancer and that decreased levels of activated leukocyte cell adhesion molecule expression may lead to an aggressive behavior of tumor cells. The abundant presence of activated leukocyte cell adhesion molecule and its membranous localization in prostate cancer epithelium make activated leukocyte cell adhesion molecule a potentially attractive structure for targeted therapy.

Activated Leukocyte Cell Adhesion Molecule (CD166): An “Inert” Cancer Stem Cell Marker for Non-Small Cell Lung Cancer?

Recently, the activated leukocyte cell adhesion molecule (CD166) was identified as an “inert” cancer stem cell (CSC) marker for non‐small cell lung cancer (NSCLC). Few data exist regarding the clinical relevance of CD166 expression in NSCLC. We evaluated the expression of CD166 using immunohistochemistry in a large cohort of NSCLC patients (n = 1,910) on a tissue microarray basis. Expression was inversely associated with tumor size and lymph node status. Grading slightly failed to be significantly inversely associated, and survival analysis revealed no significant survival benefit of CD166‐positive patients. Due to the results of this study, the theory of CD166 as a CSC marker for NSCLC must be questioned. The association of CD166 with smaller tumors and no nodal metastases does not make it a typical CSC marker. Further studies are required to investigate the functional role of CD166 in NSCLC. Stem Cells 2014;32:1429–1436

CXCR7 expression in esophageal cancer

BackgroundThe chemokine CXCL12 and its receptor CXCR4 play a major role in tumor invasion, proliferation and metastasis in different malignant diseases, including esophageal carcinoma, amongst others. CXCR7 was recently identified as a novel alternate receptor for CXCL12. The aim of this study was to evaluate the prognostic impact of expression of chemokine receptor CXCR7 in patients with esophageal carcinoma (EC).MethodsExpression of CXCR7 in primary tumors, lymph nodes and distant metastases of 299 patients with EC was evaluated by immunohistochemistry on a tissue microarray and compared with clinical and histopathological data.ResultsIn esophageal cancer sections, CXCR7-specific reactivity was apparent in 45% of the squamous cell carcinomas (ESCC), but only occasionally in adenocarcinomas. No correlation between CXCR4 and CXCR7 expression was evident. We correlated expression with clinical and histopathological characteristics, but could not find any association.ConclusionsContrary to the other known CXCL12 receptor, CXCR4, CXCR7 is expressed in ESCC only, underlining the divergent mechanisms and backgrounds of EAC and ESCC. The results of the study do not indicate a significant functional role for CXCR7 in EAC or ESCC of the esophagus. However, its variable expression in the main two main types of EC needs to be further investigated.

Disseminated tumor cells in pancreatic cancer-an independent prognosticator of disease progression and survival.

Pancreatic cancer is one of the most devastating cancers with a 6‐month median survival and a 5‐year survival rate of 3–5%. Still important aspects of its aggressive biology remain elusive and advanced therapeutic regimens have not been substantially successful. We investigated the prognostic role of disseminated tumor cells (DTC) in bone marrow, a reservoir for early DTC potentially contributing to metastatic progression, of pancreatic cancer patients. After exclusion of patients with different postsurgery diagnosis or missing DTC status (n = 40) a total of 175 patients remained for final analyses. One‐hundred and nineteen patients were male and 96 female with a median age of 67 years, 96 patients underwent complete resection. Bone marrow aspirates taken at primary surgery were analyzed for DTC by an immunocytochemical cytokeratin assay and correlated to survival data. Overall 13.7% of patient samples (24/175) harbored DTC in their bone marrow. Histopathological parameters did not correlate significantly. Univariate survival analysis revealed a borderline significant correlation between DTC and decreased progression‐free survival (p = 0.069), and was significant for overall survival (p = 0.036). Regarding patients with resected tumors, the respective p‐values were 0.058 for progression‐free and 0.016 for overall survival. Importantly, the prognostic influence was independent from other risk factors as shown by multivariate analyses for progression‐free (p = 0.030, HR: 2.057; CI (95%): 1.073–3.943) and overall survival (p = 0.006, HR: 2.283; CI (95%): 1.260–4.135). The presence of DTC in bone marrow is a strong and independent prognostic factor of survival in patients with pancreatic cancer. Thus, bone‐targeting may be a new future therapeutic option for DTC‐positive patients.

Circulating levels of cell adhesion molecule L1 as a prognostic marker in gastrointestinal stromal tumor patients

BackgroundL1 cell adhesion molecule (CD171) is expressed in many malignant tumors and its expression correlates with unfavourable outcome. It thus represents a target for tumor diagnosis and therapy. An earlier study conducted by our group identified L1 expression levels in primary gastrointestinal stromal tumors (GIST) as a prognostic marker. The aim of the current study was to compare L1 serum levels of GIST patients with those of healthy controls and to determine whether levels of soluble L1 in sera could serve as a prognostic marker.MethodsUsing a sensitive enzyme-linked immunosorbent assay (ELISA), soluble L1 was measured in sera of 93 GIST patients und 151 healthy controls. Soluble L1 levels were then correlated with clinicopathological data.ResultsMedian levels of soluble L1 were significantly higher (p < 0.001; Mann-Whitney U test) in sera of GIST patients compared to healthy individuals. Median soluble L1 levels were particularly elevated in patients with recurrence and relapse (p < 0.05; Mann Whitney U test).ConclusionThese results suggest that high soluble L1 levels predict poor prognosis and may thus be a promising tumor marker that can contribute to individualise therapy.