Genomic and Transcriptomic Characterization of Papillary Microcarcinomas With Lateral Neck Lymph Node Metastases.
ABSTRACT: CONTEXT:Most papillary microcarcinomas (PMCs) are indolent and subclinical. However, as many as 10% can present with clinically significant nodal metastases. OBJECTIVE AND DESIGN:Characterization of the genomic and transcriptomic landscape of PMCs presenting with or without clinically important lymph node metastases. SUBJECTS AND SAMPLES:Formalin-fixed paraffin-embedded PMC samples from 40 patients with lateral neck nodal metastases (pN1b) and 71 patients with PMC with documented absence of nodal disease (pN0). OUTCOME MEASURES:To interrogate DNA alterations in 410 genes commonly mutated in cancer and test for differential gene expression using a custom NanoString panel of 248 genes selected primarily based on their association with tumor size and nodal disease in the papillary thyroid cancer TCGA project. RESULTS:The genomic landscapes of PMC with or without pN1b were similar. Mutations in TERT promoter (3%) and TP53 (1%) were exclusive to N1b cases. Transcriptomic analysis revealed differential expression of 43 genes in PMCs with pN1b compared with pN0. A random forest machine learning-based molecular classifier developed to predict regional lymph node metastasis demonstrated a negative predictive value of 0.98 and a positive predictive value of 0.72 at a prevalence of 10% pN1b disease. CONCLUSIONS:The genomic landscape of tumors with pN1b and pN0 disease was similar, whereas 43 genes selected primarily by mining the TCGA RNAseq data were differentially expressed. This bioinformatics-driven approach to the development of a custom transcriptomic assay provides a basis for a molecular classifier for pN1b risk stratification in PMC.
Project description:PURPOSE OF REVIEW: The global incidence of small papillary thyroid carcinoma (PTC) is increasing remarkably, mostly due to the increased use of imaging studies worldwide. The issue of how to manage low-risk small PTC has become urgent. In this review, we focus on how to treat low-risk papillary thyroid microcarcinomas (PMCs; i.e., PTCs measuring ?10?mm). RECENT FINDINGS: Studies of large numbers of patients with low-risk PMC clarified that most of the PMCs did not grow or grew very slowly and were harmless. Active observations of these patients discriminated rare progressive cases from the majority. Surgery performed after the detection of progression signs was not too late, and surgery immediately after the detection and diagnosis of low-risk PMC may be overtreatment for most patients. Interestingly, low-risk PMCs in elderly patients were most unlikely to progress, in sharp contrast to clinical PTC. The reason for this phenomenon remains unknown. SUMMARY: Active observation without immediate surgery can be a leading alternative to the classical surgical treatment in the majority of the patients with low-risk PMC. It is not too late to perform surgery after the detection of progression signs for these patients. VIDEO ABSTRACT: http://links.lww.com/COON/A10
Project description:In recent years management practices in relation to low-risk papillary microcarcinoma (PMC) have been evolving with increased awareness of the potential overdiagnosis and overtreatment of PMCs, and guidelines recommendations for non-surgical management options such as active surveillance. This study aimed to develop an in-depth understanding of patients' experiences of the communication of their PMC diagnosis, their treatment preferences and decision making.Semi-structured qualitative interviews with 25 patients diagnosed pre-operatively with PMC < 1 year since their diagnosis and treatment. Interviews were conducted between September 2015 and July 2016 and were audio-recorded and transcribed verbatim. Framework analysis method was used to analyse the data.The diagnosis and treatment experience of PMC patients varied widely. The majority of patients were asymptomatic, and their PMC was initially detected via an imaging test requested for a reason unrelated to a thyroid disorder or symptom. Clinicians generally described PMC to patients as being a "small" or "slow-growing" cancer, and there was little evidence that clinicians had discussions about the possibility of overdiagnosis or overtreatment. Overall, surgery was the only option discussed and offered to patients. Patients preference for treatment was largely based on eliminating the possibility of the cancer spreading (thyroidectomy) or not wanting to be on thyroid replacement medication for the rest of their life (hemi-thyroidectomy). Many patients reported emotional and physical side-effects associated with their diagnosis and treatment, however patients generally indicated that active surveillance is not something they would have been interested in if it was offered to them.Evidence continues to emerge that many patients with PMCs may be overdiagnosed, and management guidelines are recommending more conservative management options for these patients. As a result, shared decision making around treatment options is vital so that patients are fully aware of the meaning of their diagnosis and their management options including active surveillance. Importantly, interventions to reduce unnecessary diagnoses of PMC are critically needed.
Project description:The presence of lymph node metastases remains the most reliable prognostic predictor and the gold indicator for adjuvant treatment in colon cancer (CC). In spite of a potentially curative resection, 20 to 30% of CC patients testing negative for lymph node metastases (i.e. pN0) will subsequently develop locoregional and/or systemic metastases within 5 years. The presence of occult nodal isolated tumor cells (ITCs) and/or micrometastases (MMs) at the time of resection predisposes CC patients to high risk for disease recurrence. These pN0(micro+) patients harbouring occult micrometastases may benefit from adjuvant treatment. The purpose of the present study is to delineate the subset of pN0 patients with micrometastases (pN0(micro+)) and evaluate the benefits from adjuvant chemotherapy in pN0(micro+) CC patients.EnRoute+ is an open label, multicenter, randomized controlled clinical trial. All CC patients (age above 18 years) without synchronous locoregional lymph node and/or systemic metastases (clinical stage I-II disease) and operated upon with curative intent are eligible for inclusion. All resected specimens of patients are subject to an ex vivo sentinel lymph node mapping procedure (SLNM) following curative resection. The investigation for micrometastases in pN0 patients is done by extended serial sectioning and immunohistochemistry for pan-cytokeratin in sentinel lymph nodes which are tumour negative upon standard pathological examination. Patients with ITC/MM-positive sentinel lymph nodes (pN0(micro+)) are randomized for adjuvant chemotherapy following the CAPOX treatment scheme or observation. The primary endpoint is 3-year disease free survival (DFS).The EnRoute+ study is designed to improve prognosis in high-risk stage I/II pN0(micro+) CC patients by reducing disease recurrence by adjuvant chemotherapy.ClinicalTrials.gov: NCT01097265.
Project description:The origin of the myofibroblast in fibrotic lung disease is uncertain, and no effective medical therapy for fibrosis exists. We have previously demonstrated that transforming growth factor-?1 (TGF-?1) induces pleural mesothelial cell (PMC) transformation into myofibroblasts and haptotactic migration in vitro. Whether PMC differentiation and migration occurs in vivo, and whether this response can be modulated for therapeutic benefit, is unknown. Here, using mice recombinant for green fluorescent protein (GFP) driven by the Wilms tumor-1 (WT-1) promoter, we demonstrate PMC trafficking into the lung and differentiation into myofibroblasts. Carbon monoxide or the induction of heme oxygenase-1 (HO-1) inhibited the expression of myofibroblast markers, contractility, and haptotaxis in PMCs treated with TGF-?1. Intrapleural HO-1 induction inhibited PMC migration after intratracheal fibrogenic injury. PMCs from patients with idiopathic pulmonary fibrosis (IPF) exhibited increased expression of myofibroblast markers and enhanced contractility and haptotaxis, compared with normal PMCs. Carbon monoxide reversed this IPF PMC profibrotic phenotype. WT-1-expressing cells were present within fibrotic regions of the lungs in IPF subjects, supporting a role for PMC differentiation and trafficking as contributors to the myofibroblast population in lung fibrosis. Our findings also support a potential role for pleural-based therapies to modulate pleural mesothelial activation and parenchymal fibrosis progression.
Project description:BACKGROUND:The developmental gene regulatory network (GRN) that underlies skeletogenesis in sea urchins and other echinoderms is a paradigm of GRN structure, function, and evolution. This transcriptional network is deployed selectively in skeleton-forming primary mesenchyme cells (PMCs) of the early embryo. To advance our understanding of this model developmental GRN, we used genome-wide chromatin accessibility profiling to identify and characterize PMC cis-regulatory modules (CRMs). RESULTS:ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) analysis of purified PMCs provided a global picture of chromatin accessibility in these cells. We used both ATAC-seq and DNase-seq (DNase I hypersensitive site sequencing) to identify >?3000 sites that exhibited increased accessibility in PMCs relative to other embryonic cell lineages, and provide both computational and experimental evidence that a large fraction of these sites represent bona fide skeletogenic CRMs. Putative PMC CRMs were preferentially located near genes differentially expressed by PMCs and consensus binding sites for two key transcription factors in the PMC GRN, Alx1 and Ets1, were enriched in these CRMs. Moreover, a high proportion of candidate CRMs drove reporter gene expression specifically in PMCs in transgenic embryos. Surprisingly, we found that PMC CRMs were partially open in other embryonic lineages and exhibited hyperaccessibility as early as the 128-cell stage. CONCLUSIONS:Our work provides a comprehensive picture of chromatin accessibility in an early embryonic cell lineage. By identifying thousands of candidate PMC CRMs, we significantly enhance the utility of the sea urchin skeletogenic network as a general model of GRN architecture and evolution. Our work also shows that differential chromatin accessibility, which has been used for the high-throughput identification of enhancers in differentiated cell types, is a powerful approach for the identification of CRMs in early embryonic cells. Lastly, we conclude that in the sea urchin embryo, CRMs that control the cell type-specific expression of effector genes are hyperaccessible several hours in advance of gene activation.
Project description:In metazoans, the epithelial-mesenchymal transition (EMT) is a crucial process for placing the mesoderm beneath the ectoderm. Primary mesenchyme cells (PMCs) at the vegetal pole of the sea urchin embryo ingress into the floor of the blastocoele from the blastula epithelium and later become the skeletogenic mesenchyme. This ingression movement is a classic EMT during which the PMCs penetrate the basal lamina, lose adherens junctions and migrate into the blastocoele. Later, secondary mesenchyme cells (SMCs) also enter the blastocoele via an EMT, but they accompany the invagination of the archenteron initially, in much the same way vertebrate mesenchyme enters the embryo along with endoderm. Here we identify a sea urchin ortholog of the Snail transcription factor, and focus on its roles regulating EMT during PMC ingression. Functional knockdown analyses of Snail in whole embryos and chimeras demonstrate that Snail is required in micromeres for PMC ingression. Snail represses the transcription of cadherin, a repression that appears evolutionarily conserved throughout the animal kingdom. Furthermore, Snail expression is required for endocytosis of cadherin, a cellular activity that accompanies PMC ingression. Perturbation studies position Snail in the sea urchin micromere-PMC gene regulatory network (GRN), downstream of Pmar1 and Alx1, and upstream of several PMC-expressed proteins. Taken together, our findings indicate that Snail plays an essential role in PMCs to control the EMT process, in part through its repression of cadherin expression during PMC ingression, and in part through its role in the endocytosis that helps convert an epithelial cell to a mesenchyme cell.
Project description:Pleural mesothelial cells (PMCs), which are derived from the mesoderm, exhibit an extraordinary capacity to undergo phenotypic changes during development and disease. PMC transformation and trafficking has a newly defined role in idiopathic pulmonary fibrosis (IPF); however, the contribution of Wilms' tumor 1 (Wt1)-positive PMCs to the generation of pathognomonic myofibroblasts remains unclear. PMCs were obtained from IPF lung explants and healthy donor lungs that were not used for transplantation. Short hairpin Wt1-knockdown PMCs (sh Wt1) were generated with Wt1 shRNA, and morphologic and functional assays were performed in vitro. Loss of Wt1 abrogated the PMC phenotype and showed evidence of mesothelial-to-mesenchymal transition (MMT), with a reduced expression of E-cadherin and an increase in the profibrotic markers ?-smooth muscle actin (?-SMA) and fibronectin, along with increased migration and contractility, compared with that of the control. Migration of PMCs in response to active transforming growth factor (TGF)-?1 was assessed by live-cell imaging with 2-photon microscopy and 3D imaging, of Wt1-EGFP transgenic mice. Lineage-tracing experiments to map the fate of Wt1(+) PMCs in mouse lung in response to TGF-?1 were also performed by using a Cre-loxP system. Our results, for the first time, demonstrate that Wt1 is necessary for the morphologic integrity of pleural membrane and that loss of Wt1 contributes to IPF via MMT of PMCs into a myofibroblast phenotype.
Project description:Nodal metastases and breast cancer subtypes (BCS) are both well-recognized prognostic indicators. However, the association between nodal metastases and BCS, and the prognostic value of nodal metastases in different BCS are still remains unclear. Our aim was to investigate the association between nodal metastases and BCS, and the prognostic value of nodal metastases in the different BCS.We found that the breast cancer subtype was closely associated with the pN stage. pN stage and breast cancer subtype were significantly associated with disease-free survival. The subgroup analysis showed that the patients in higher pN stage had a poor outcome than patients in lower pN stage in each breast cancer subtype. Furthermore, when the analysis was stratified by breast cancer subtype, we found that even in the same pN stage (pN0-pN2), there was significant survival difference among patients in different BCS, and Luminal A breast cancer patients had the best survival outcome. However, there were no significant survival difference between Luminal A patients and other breast cancer subtype when patients in pN3 stage. Thus, our study suggested that both lymph node status and molecular subtype played important roles in the outcome of breast cancer patients and they cannot replace each other.
Project description:Early development requires well-organized temporal and spatial regulation of transcription factors that are assembled into gene regulatory networks (GRNs). In the sea urchin, an endomesoderm GRN model explains much of the specification in the endoderm and mesoderm prior to gastrulation, yet some GRN connections remain incomplete. Here, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN state. Expression of foxN2/3 mRNA begins in micromeres at the hatched blastula stage and then is lost from micromeres at the mesenchyme blastula stage. foxN2/3 expression then shifts to the non-skeletogenic mesoderm and, later, to the endoderm. Here, we show that Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in micromeres. The later endomesoderm expression of foxN2/3 is independent of the earlier expression of foxN2/3 in micromeres and is independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of the larval skeleton. Early expression of genes for the skeletal matrix is dependent on FoxN2/3, but only until the mesenchyme blastula stage as foxN2/3 mRNA disappears from PMCs at that time and we assume that the protein is not abnormally long-lived. Knockdown of FoxN2/3 inhibits normal PMC ingression and foxN2/3 morphant PMCs do not organize in the blastocoel and fail to join the PMC syncytium. In addition, without FoxN2/3, the PMCs fail to repress the transfating of other mesodermal cells into the skeletogenic lineage. Thus, FoxN2/3 is necessary for normal ingression, for expression of several skeletal matrix genes, for preventing transfating and for fusion of the PMC syncytium.
Project description:Recent work on the sea urchin endomesoderm gene regulatory network (GRN) offers many opportunities to study the specification and differentiation of each cell type during early development at a mechanistic level. The mesoderm lineages consist of two cell populations, primary and secondary mesenchyme cells (PMCs and SMCs). The micromere-PMC GRN governs the development of the larval skeleton, which is the exclusive fate of PMCs, and SMCs diverge into four lineages, each with its own GRN state. Here we identify a sea urchin ortholog of the Twist transcription factor, and show that it plays an essential role in the PMC GRN and later is involved in SMC formation. Perturbations of Twist either by morpholino knockdown or by overexpression result in defects in progressive phases of PMC development, including specification, ingression/EMT, differentiation and skeletogenesis. Evidence is presented that Twist expression is required for the maintenance of the PMC specification state, and a reciprocal regulation between Alx1 and Twist offers stability for the subsequent processes, such as PMC differentiation and skeletogenesis. These data illustrate the significance of regulatory state maintenance and continuous progression during cell specification, and the dynamics of the sequential events that depend on those earlier regulatory states.