Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for FGFR1 amplification by FISH, and positive cases were confirmed with a microarray platform (OncoscanTM). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the FGFR1 status. Results: Tissue-based analyses identified FGFR1 amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% (n = 12) showed concordance for FGFR1 status between tissue and cfDNA. In one case, we were able to detect a high-level FGFR1 amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Conclusions: Screening for FGFR1 amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials.
Our preliminary findings have lead us to propose bone marrow adipocyte secretions as new contributors to bone loss. Indeed, using a coculture model based on human bone marrow stromal cells, we previously showed that soluble factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. In this study, microarray gene expression profiling showed profound transcriptomic changes in osteoblasts following coculture and confirmed the enrichment of the adipocyte gene signature. Double immunofluorescence microscopic analyses demonstrated the coexpression of adipogenic and osteoblastic specific markers in individual cells, providing evidence for a transdifferentiation event. At the molecular level, this conversion was associated with upregulated expression levels of reprogramming genes and a decrease in the DNA methylation level. In line with these in vitro results, preliminary immunohistochemical analysis of bone sections revealed adipogenic marker expression in osteoblasts from elderly subjects. Altogether, these data suggest that osteoblast transdifferentiation could contribute to decreased bone mass upon ageing.
Objectives. We hypothesize that chondrocytes from the deepest articular cartilage layer are pivotal in maintaining cartilage integrity and that the modification of their prehypertrophic phenotype to a hypertrophic phenotype will drive cartilage degradation in osteoarthritis.Design. Murine immature articular chondrocytes (iMACs) were successively cultured into three different culture media to induce a progressive hypertrophic differentiation. Chondrocyte were phenotypically characterized by whole-genome microarray analysis. The expression of IL-34 and its receptors PTPRZ1 and CSF1R in chondrocytes and in human osteoarthritis tissues was assessed by RT-qPCR, ELISA and immunohistochemistry. The expression of bone remodeling and angiogenesis factors and the cell response to IL-1β and IL-34 were investigated by RT-qPCR and ELISA. Results. Whole-genome microarray analysis showed that iMACs, prehypertrophic and hypertrophic chondrocytes each displayed a specific phenotype. IL-1β induced a stronger catabolic effect in prehypertrophic chondrocytes than in iMACs. Hypertrophic differentiation of prehypertrophic chondrocytes increased Bmp-2 (95%CI [0.78;1.98]), Bmp-4 (95%CI [0.89;1.59]), Cxcl12 (95%CI [2.19;5.41]), CCL2 (95%CI [3.59;11.86]), Mmp3 (95%CI [10.29;32.14]) and Vegf mRNA expression (95%CI [0.20;1.74]). Microarray analysis identified IL-34, PTPRZ1 and CSFR1 as being strongly overexpressed in hypertrophic chondrocytes. IL-34 was released by human osteoarthritis cartilage; its receptors were expressed in human osteoarthritis tissues. IL-34 stimulated CCL2 and MMP13 in osteoblasts and hypertrophic chondrocytes but not in iMACs or prehypertrophic chondrocytes. Conclusion.Our results identify prehypertrophic chondrocytes as being potentially pivotal in the control of cartilage and subchondral bone integrity. Their differentiation into hypertrophic chondrocytes initiates a remodeling program in which IL-34 may be involved.
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