Multipotential adult mesenchymal stem cells (MSCs) are able to differentiate along several known lineages, and lineage commitment is tightly regulated through specific cellular mediators and interactions. Recent observations of a low/high bone-mass phenotype in patients expressing a loss-/gain-of-function mutation in LRP5, a coreceptor of the Wnt family of signaling molecules, suggest the importance of Wnt signaling in bone formation, possibly involving MSCs. To analyze the role of Wnt signaling in mesenchymal osteogenesis, we have profiled the expression of WNTs and their receptors, FRIZZLEDs (FZDs), and several secreted Wnt inhibitors, such as SFRPs, and examined the effect of Wnt 3a, as a representative canonical Wnt member, during MSC osteogenesis in vitro. WNT11, FZD6, SFRP2, and SFRP3 are upregulated during MSC osteogenesis, while WNT9A and FZD7 are downregulated. MSCs also respond to exogenous Wnt 3a, based on increased beta-catenin nuclearization and activation of a Wnt-responsive promoter, and the magnitude of this response depends on the MSC differentiation state. Wnt 3a exposure inhibits MSC osteogenic differentiation, with decreased matrix mineralization and reduced alkaline phosphatase mRNA and activity. Wnt 3a treatment of fully osteogenically differentiated MSCs also suppresses osteoblastic marker gene expression. The Wnt 3a effect is accompanied by increased cell number, resulting from both increased proliferation and decreased apoptosis, particularly during expansion of undifferentiated MSCs. The osteo-suppressive effects of Wnt 3a are fully reversible, i.e., treatment prior to osteogenic induction does not compromise subsequent MSC osteogenesis. The results also showed that sFRP3 treatment attenuates some of the observed Wnt 3a effects on MSCs, and that inhibition of canonical Wnt signaling using a dominant negative TCF1 enhances MSC osteogenesis. Interestingly, expression of Wnt 5a, a non-canonical Wnt member, appeared to promote osteogenesis. Taken together, these findings suggest that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor MSC population, whereas non-canonical Wnts facilitate osteogenic differentiation. Release from canonical Wnt regulation is a prerequisite for MSC differentiation. Thus, loss-/gain-of-function mutations of LRP5 would perturb Wnt signaling and depress/promote bone formation by affecting the progenitor cell pool. Elucidating Wnt regulation of MSC differentiation is important for their potential application in tissue regeneration.

While the incidence of gastric cancer has decreased worldwide in recent decades, the incidence of signet-ring cell carcinoma (SRCC) is rising. SRCC has a specific epidemiology and oncogenesis and has two forms: early gastric cancer, which can be resected endoscopically in some cases and which has a better outcome than non-SRCC, and advanced gastric cancer, which is generally thought to have a worse prognosis and lower chemosensitivity than non-SRCC. However, the prognosis of SRCC and its chemosensitivity with specific regimens are still controversial as SRCC is not specifically identified in most studies and its poor prognosis may be due to its more advanced stage. It therefore remains unclear if a specific therapeutic strategy is justified, as the benefit of perioperative chemotherapy and the value of taxane-based chemotherapy are unclear. In this review we analyze recent data on the epidemiology, oncogenesis, prognosis and specific therapeutic strategies in both early and advanced SRCC of the stomach and in hereditary diffuse gastric cancer.

Markers of chemotherapy efficacy in metastatic colorectal cancer (mCRC) are essential for optimization of treatment strategies. We evaluated the applicability of early changes in circulating tumor DNA (ctDNA) as a marker of therapeutic efficacy. This prospective study enrolled consecutive patients with mCRC receiving a first- or second-line chemotherapy. CtDNA was assessed in plasma collected before the first (C), second (C) and/or third (C) chemotherapy cycle, using picodroplet-digital PCR assays based either on detection of gene mutation () or hypermethylation (). CT scans were centrally assessed using RECIST v1.1 criteria. Multivariate analyses were adjusted on age, gender, ECOG performance status (PS), metastatic synchronicity, and treatment line. Eighty-two patients with mCRC treated in first- (82.9%) or second- (17.1%) line chemotherapy were included. Patients with a high (>10 ng/mL) versus low (≤0.1 ng/mL) ctDNA concentration at C had a shorter overall survival (OS; 6.8 vs. 33.4 months: adjusted HR, 5.64; 95% CI, 2.5-12.6; < 0.0001). By analyzing the evolution of the ctDNA concentration between C and C or C (C), we classified the patients in two groups (named "good" or "bad ctDNA responders"). In multivariate analysis, patients belonging to the group called "good ctDNA responder" ( = 58) versus "bad ctDNA responder" () had a better objective response rate ( < 0.001), and a longer median progression-free survival (8.5 vs. 2.4 months: HR, 0.19; 95% CI, 0.09-0.40; < 0.0001) and OS (27.1 vs. 11.2 months: HR, 0.25; 95% CI, 0.11-0.57; < 0.001). This study suggests that early change in ctDNA concentration is a marker of therapeutic efficacy in patients with mCRC. .

In the adult human, mesenchymal stem cells (hMSCs) resident in the bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. Glucocorticoids (GCs) are required for chondrogenic differentiation of hMSCs in vitro; however, the exact role of GCs in this process is not known. In this study, we examined the effects of dexamethasone (DEX) on chondrogenic differentiation of hMSCs in the presence or absence of DEX, transforming growth factor-␤ (TGF-␤), or DEX plus TGF-␤. GC treatment upregulated gene expression of cartilage matrix components aggrecan, dermatopontin, and collagen type XI; enhanced TGF-␤-mediated upregulation of collagen type II and cartilage oligomeric matrix protein; and increased aggrecan and collagen type II production as well as cartilage matrixsulfated proteoglycans as assessed by immunohistochemistry and alcian blue staining. Inclusion of an antagonist of GCs inhibited expression of chondrogenic differentiation markers, suggesting that the GC effects during chondrogenesis are mediated by the GC receptor (GR). Steady levels of the major active form of GR, GR␣, were detected in both undifferentiated and differentiating hMSCs, whereas the dominant-negative isoform GR␤, present at low levels in undifferentiated hMSCs, was downregulated during chondrogenesis. In the presence of DEX and TGF-␤, expression of a collagen type II gene promoter luciferase reporter construct in hMSCs was upregulated. However, coexpression of GR␤ dramatically inhibited promoter activity, suggesting that GR␣ is required for GC-mediated modulation of chondrogenesis and that GCs may play an important role in the maintenance of cartilage homeostasis.

The Drosophila drifter (dfr) gene, previously referred to as Cfla, encodes a POU-domain DNA-binding protein implicated as a neuron-specific regulator in the developing central nervous system (CNS). We have isolated full-length dfr cDNA clones that encode a 46-kD protein containing the conserved POU-domain DNA-binding domain. The use of alternate polyadenylation sites produces two dfr mRNA transcripts that are first expressed in stage 10 embryos at 5-to 6-hr of development. A specific anti-dfr polyclonal antiserum generated against a dfr-glutathione S-transferase fusion protein recognizes a 46-kD protein on Western blots and has been used to analyze the cell-specific distribution of dfr protein during embryonic development, dfr protein is distributed in a complex expression pattern including the tracheal system, the middle pair of midline glia, and selected CNS neurons. We have carried out a genetic characterization of the dfr locus, previously localized to region 65D of the third chromosome, by generating a series of overlapping deficiencies between 65A and 65E1 that were used to isolate dfr rs2, an EMS-induced lethal allele. Analysis of dfr ~sz mutant embryos shows a disruption of the developing tracheal tree as well as commissural defects in the developing CNS. Based on an examination of a cell-specific marker for tracheal cells and midline glia, these defects appear to be caused by a failure of these cells to follow their characteristic routes of migration. The dfr r82 tracheal phenotype is rescued by a dfr minigene present as a P-element transposon expressing wild-type dfr protein in tracheal cells. These results suggest that the dfr protein plays a fundamental role in the differentiation of tracheal cells and midline glia possibly by regulating the expression of essential cell-surface proteins required for cell-cell interactions involved in directed cell migrations.

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5–1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical ‘hot-spot’ mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.

BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

No abstract