The Spatial Distribution of LGR5+ Cells Correlates With Gastric Cancer Progression

In this study we tested the prevalence, histoanatomical distribution and tumour biological significance of the Wnt target protein and cancer stem cell marker LGR5 in tumours of the human gastrointestinal tract. Differential expression of LGR5 was studied on transcriptional (real-time polymerase chain reaction) and translational level (immunohistochemistry) in malignant and corresponding non-malignant tissues of 127 patients comprising six different primary tumour sites, i.e. oesophagus, stomach, liver, pancreas, colon and rectum. The clinico-pathological significance of LGR5 expression was studied in 100 patients with gastric carcinoma (GC). Non-neoplastic tissue usually harboured only very few scattered LGR5+ cells. The corresponding carcinomas of the oesophagus, stomach, liver, pancreas, colon and rectum showed significantly more LGR5+ cells as well as significantly higher levels of LGR5-mRNA compared with the corresponding non-neoplastic tissue. Double staining experiments revealed a coexpression of LGR5 with the putative stem cell markers CD44, Musashi-1 and ADAM17. Next we tested the hypothesis that the sequential changes of gastric carcinogenesis, i.e. chronic atrophic gastritis, intestinal metaplasia and invasive carcinoma, are associated with a reallocation of the LGR5+ cells. Interestingly, the spatial distribution of LGR5 changed: in non-neoplastic stomach mucosa, LGR5+ cells were found predominantly in the mucous neck region; in intestinal metaplasia LGR5+ cells were localized at the crypt base, and in GC LGR5+ cells were present at the luminal surface, the tumour centre and the invasion front. The expression of LGR5 in the tumour centre and invasion front of GC correlated significantly with the local tumour growth (T-category) and the nodal spread (N-category). Furthermore, patients with LGR5+ GCs had a shorter median survival (28.0±8.6 months) than patients with LGR5− GCs (54.5±6.3 months). Our results show that LGR5 is differentially expressed in gastrointestinal cancers and that the spatial histoanatomical distribution of LGR5+ cells has to be considered when their tumour biological significance is sought

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Synchronous metastatic gastric cancer-molecular background and clinical implications with special attention to mismatch repair deficiency

Background: Current guidelines recommend that metastatic gastric cancer should not be treated with surgery unless this is required for symptom control. We hypothesized that patients with mismatch repair deficiency (MMRd) gastric cancer and metastatic disease detected at the timepoint of surgical resection would have superior survival compared to patients with MMRd cancers in the same setting. Methods: Clinicopathological details and survival data were collected from prospective databases at two large European centers on patients who had undergone surgery and were diagnosed with synchronous stage IV gastric cancer (distant lymph nodes, positive peritoneal cytology, peritoneal, and distant metastases) at the timepoint of surgery. Resection specimens were tested for the presence of microsatellite instability using a standard 5 mononucleotide repeat panel. Results: One hundred and seventy six patients with resected stage IV gastric cancer were identified. 14/176 (8.0%) had MSI-H (high) disease. There was no significant difference between the clinical and pathological characteristics of MSI and microsatellite stable (MSS) patients. No differences in the type of metastases were observed between MSI and MSS groups. Patients who were MSI-H had superior OS compared to MSS patients (median OS 15.9 vs. 8 months, p = 0.023). However, in Cox regression multivariate analysis only liver and peritoneal metastases were independent predictors of survival. Conclusions: Surgically treated patients with MSI-H stage IV gastric cancer have a better survival than patients with MSS gastric cancer. Further analysis of the role of surgery in MSI stage IV GC is required

Cytoplasmic TRAIL-R1 is a positive prognostic marker in PDAC

Abstract Background The death receptors TRAIL-R1 and TRAIL-R2 are frequently overexpressed in cancer and there is an emerging evidence for their important role in malignant progression, also in the case of pancreatic ductal adenocarcinoma (PDAC). In their canonical localization at the plasma membrane, TRAIL-R1/−R2 may induce cell death and/or pro-inflammatory signaling leading to cell migration, invasion and metastasis. Although, they have repeatedly been found intracellular, in the cytoplasm and in the nucleus, their functions in intracellular locations are still not well understood. Likewise, studies dealing with the prognostic relevance of TRAIL-Rs located in particular cellular compartments are very rare. For PDAC, the correlation of nuclear TRAIL-R2 with worse patients’ prognosis has been shown recently. Corresponding data on TRAIL-R1 are not available so far. Methods In the present study we analyzed the expression of TRAIL-R1 in 106 PDACs and 28 adjacent, peritumoral non-malignant pancreatic ducts with special emphasis on its cytoplasmic and nuclear localization and correlated the immunohistochemical findings with clinico-pathological patient characteristics. Results TRAIL-R1 was found in 93.4% of all PDAC samples. Cytoplasmic staining was present with very similar intensity in tumor and normal tissue. In contrast, nuclear TRAIL-R1 staining was significantly stronger in tumor compared to normal tissue (p = 0.006). Interestingly, we found that the number of cells with cytoplasmic TRAIL-R1 staining negatively correlates with tumor grading (p = 0.043). No such correlation could be detected for nuclear TRAIL-R1. Neither, cytoplasmic nor nuclear TRAIL-R1 staining showed a correlation with other clinico-pathological parameter such as pTNM categories. However, Kaplan-Meier analyses revealed significantly prolonged median survival of patients with positive cytoplasmic TRAIL-R1 expression in more than 80% of tumor cells compared to patients with tumors containing a smaller quantity of cells positively stained for cytoplasmic TRAIL-R1 (20 vs. 8 months; p = 0.004). Conclusion Cytoplasmic TRAIL-R1 is a positive prognostic marker for patients with PDAC. Our findings indicate that loss of cytoplasmic TRAIL-R1 results in recurrent disease with more malignant phenotype thus suggesting anti-tumor activities of cytoplasmic TRAIL-R1 in PDAC

Assessment of LGR5 expression in whole mount tissue sections of intestinal type gastric carcinomas.

<p>Correlation of the spatial distribution of LGR5⁺ cancer cells at the luminal surface, the tumour centre, and the invasion front with clinico-pathological patient characteristics.</p>*<p>Fisher's exact test.</p>§<p>Kendall's tau.</p>£<p>log-rank test (Mantel-Cox) with a 95% confidence interval (CI). Number (n) and percentage (%) of LGR5⁺ cases. SD = standard deviation.</p

LGR5-immunoreactivity in hepato-gastrointestinal tissues.

<p>Expression of LGR5 in normal oesophageal mucosa (A) compared with an adenocarcinoma (B) and a squamous cell carcinoma (C). LGR5 expression in the normal liver (D) compared with hepatocellular carcinoma (E), normal (F) and malignant (G) epithelium of the bile duct, as well as non-neoplastic (H) and neoplastic (I) pancreatic tissue. Original magnifications ×400.</p

Immunofluorescence staining with anti-LGR5-antibody.

<p>Immunofluorescence staining of HEK293 EBNA cells with LGR5 specific antibody (green) and anti-myc tag antibody (red). Cells were counterstained with DAPI to show the cell nucleus (blue). Cells transfected with the myc-tagged LGR5 cDNA (first panel) compared to control cells, transfected with the empty vector (control empty vector); leaving untransfected (control untransfected); or incubated without the primary antibodies , respectively. Original magnifications ×400.</p

LGR5 expression in hepato-gastrointestinal tissues measured by Real-time RT-PCR.

<p>Boxplots depicting overall distribution of LGR5 comparing malignant versus adjacent non-malignant tissue in (A) Barrett's adenocarcinoma and (B) squamous cell carcinoma of the oesophagus, (C) intestinal type gastric cancer, (D) diffuse type gastric cancer, (E) hepatocellular carcinoma, (F) cholangiocarcinoma, (G) colon and (H) rectal carcinoma.</p

Validation of the anti-LGR5-antibody by western blotting.

<p>Coomassie blue staining (Coomassie) depicts the quality of loaded total protein lysates. In western blot analysis the anti-LGR5-antibody (1∶20,000) detects a single band in protein lysates of stably transfected MKN74 cells, overexpressing LGR5 (MKN74+LGR5), a weaker band of cells transfected with the control empty vector (MKN74 + empty vector), and no band in lysates of human non-neoplastic stomach mucosa, respectively. On a parallel blot no target bands are visible when the anti-LGR5-antibody was pre-incubated with its immunizing blocking peptide (peptide blocking). The top bands (100 kDa, arrow) display the LGR5 protein, whereas the bottom bands (∼43 kDa, arrow) depict β-actin, used as a loading control.</p