Evaluation of circulating transcript analysis (NETest) in small intestinal neuroendocrine neoplasms after surgical resection

PURPOSE: Surgical resection is the only effective curative strategy for small intestinal neuroendocrine neoplasms (SINENs). Nevertheless, the evaluation of residual disease and prediction of disease recurrence/progression remains a problematic issue. METHODS: We evaluated 13 SINENs that underwent surgical resection of the primary tumour and/or mesenteric mass. Patients were divided in three groups: (a) Group 1: SINENs that underwent resection with curative intent, (b) Group 2: SINENs treated with resection in the setting of metastatic disease, which remained stable and (c) Group 3: SINENs treated with resection in the setting of metastatic disease, with evidence of any progression at follow-up. NETest and chromogranin A were measured pre-operatively and post-operatively during a 22-month median follow-up period and compared with imaging studies. NETest score <20% was determined as normal, 20-40% low, 41-79% intermediate and ≥80% high score. RESULTS: NETest score was raised in all (100%) SINENs pre-operatively. Surgery with curative intent resulted in NETest score reduction from 78.25 ± 15.32 to 25.25 ± 1.75 (p < 0.05). Low NETest scores post-operatively were evident in all cases without clinical evidence of residual disease (Group 1). However, the low disease activity score suggested the presence of microscopic residual disease. In three cases (75%) with stable disease (Group 2) the NETest score was low consistent with indolent disease. In the progressive disease group (Group 3), a high NETest score was present in three cases (60%) and an intermediate NETest score in the remainder (40%). CONCLUSIONS: Blood NETest scores accurately identified SINENs and were significantly decreased by curative surgery. Monitoring NETest post-operatively may facilitate management by identifying the presence of residual/progressive disease

Clinicopathological correlations of mesenteric fibrosis and evaluation of a novel biomarker for fibrosis detection in small bowel neuroendocrine neoplasms

PURPOSE: Mesenteric fibrosis (MF) in small intestinal neuroendocrine neoplasms (SINENs) is often associated with significant morbidity and mortality. The detection of MF is usually based on radiological criteria, but no previous studies have attempted a prospective, multidimensional assessment of mesenteric desmoplasia to determine the accuracy of radiological measurements. There is also a lack of non-invasive biomarkers for the detection of image-negative MF. METHODS: A multidimensional assessment of MF incorporating radiological, surgical and histological parameters was performed in a prospective cohort of 34 patients with SINENs who underwent primary resection. Pre-operative blood samples were collected in 20 cases to evaluate a set of five profibrotic circulating transcripts-the "fibrosome"-that is included as an "omic" component of the NETest. RESULTS: There was a significant correlation between radiological and surgical assessments of MF (p < 0.05). However, there were several cases of image-negative MF. The NETest-fibrosome demonstrated an accuracy of 100% for the detection of microscopic MF. CONCLUSIONS: The detection of MF by radiological criteria has limitations. The NETest-fibrosome is a promising biomarker for fibrosis detection and further validation of these results would be needed in larger, multicentre studies

COVID-19 and vertical transmission: assessing the expression of ACE2 / TMPRSS2 in the human fetus and placenta to assess the risk of SARS-CoV-2 infection

Background: While pregnant women have been identified as a potentially at-risk group concerning COVID-19 infection, little is known regarding the susceptibility of the fetus to infection. Co-expression of ACE2 and TMPRSS2 has been identified as a pre-requisite for infection, and expression across different tissues is known to vary between children and adults. However, the expression of these proteins in the fetus is unknown. Methods: We performed a retrospective analysis of single cell data repositories. This data was then validated at both gene and protein level by performing qRT-PCR and two-colour immunohistochemistry on a library of second-trimester human fetal tissues. Findings: TMPRSS2 is present at both gene and protein level in the predominantly epithelial fetal tissues analysed. ACE2 is present at significant levels, only in the fetal intestine and kidney and is not expressed in the fetal lung. The placenta is also negative for the two proteins both during development and at term. Interpretation: This dataset indicates that the lungs are unlikely to be a viable route of SARS-CoV2 fetal infection. The fetal kidney, despite presenting both the proteins required for the infection, is anatomically protected from the exposure to the virus. However, the GI tract is likely to be susceptible to infection due to its high co-expression of both proteins, as well as its exposure to potentially infected amniotic fluid

Transcriptomic Profiling of In Vitro Tumor-Stromal Cell Paracrine Crosstalk Identifies Involvement of the Integrin Signaling Pathway in the Pathogenesis of Mesenteric Fibrosis in Human Small Intestinal Neuroendocrine Neoplasms.

Aim: Analysis of the pathophysiology of mesenteric fibrosis (MF) in small intestinal neuroendocrine tumors (SI-NETs) in an in vitro paracrine model and in human SI-NET tissue samples. Methods: An indirect co-culture model of SI-NET cells KRJ-I and P-STS with stromal cells HEK293 was designed to evaluate the paracrine effects on cell metabolic activity, gene expression by RT2 PCR Profilers to analyse cancer and fibrosis related genes, and RNA sequencing. The integrin signaling pathway, a specific Ingenuity enriched pathway, was further explored in a cohort of human SI-NET tissues by performing protein analysis and immunohistochemistry. Results: RT Profiler array analysis demonstrated several genes to be significantly up- or down-regulated in a cell specific manner as a result of the paracrine effect. This was further confirmed by employing RNA sequencing revealing multiple signaling pathways involved in carcinogenesis and fibrogenesis that were significantly affected in these cell lines. A significant upregulation in the expression of various integrin pathway - related genes was identified in the mesenteric mass of fibrotic SI-NET as confirmed by RT-qPCR and immunohistochemistry. Protein analysis demonstrated downstream activation of the MAPK and mTOR pathways in some patients with fibrotic SI-NETs. Conclusion: This study has provided the first comprehensive analysis of the crosstalk of SI-NET cells with stromal cells. A novel pathway - the integrin pathway - was identified and further validated and confirmed in a cohort of human SI-NET tissue featured by a dual role in fibrogenesis/carcinogenesis within the neoplastic fibrotic microenvironment

Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci

Pathogenic variants in the human m(6)A reader YTHDC2 are associated with primary ovarian insufficiency

Primary ovarian insufficiency (POI) affects 1% of women and carries significant medical and psychosocial sequelae. Approximately 10% of POI has a defined genetic cause, with most implicated genes relating to biological processes involved in early fetal ovary development and function. Recently, Ythdc2, an RNA helicase and N6-methyladenosine reader, has emerged as a regulator of meiosis in mice. Here, we describe homozygous pathogenic variants in YTHDC2 in 3 women with early-onset POI from 2 families: C. 2567C>G, p.P856R in the helicase-associated (HA2) domain and c.1129G>T, p.E377*. We demonstrated that YTHDC2 is expressed in the developing human fetal ovary and is upregulated in meiotic germ cells, together with related meiosisassociated factors. The p.P856R variant resulted in a less flexible protein that likely disrupted downstream conformational kinetics of the HA2 domain, whereas the p.E377*variant truncated the helicase core. Taken together, our results reveal that YTHDC2 is a key regulator of meiosis in humans and pathogenic variants within this gene are associated with POI

Indocyanine Green (ICG) Lymphography Is Superior to Lymphoscintigraphy for Diagnostic Imaging of Early Lymphedema of the Upper Limbs

BACKGROUND: Secondary lymphedema causes swelling in limbs due to lymph retention following lymph node dissection in cancer therapy. Initiation of treatment soon after appearance of edema is very important, but there is no method for early diagnosis of lymphedema. In this study, we compared the utility of four diagnostic imaging methods: magnetic resonance imaging (MRI), computed tomography (CT), lymphoscintigraphy, and Indocyanine Green (ICG) lymphography. PATIENTS AND METHODS: Between April 2010 and November 2011, we examined 21 female patients (42 arms) with unilateral mild upper limb lymphedema using the four methods. The mean age of the patients was 60.4 years old (35-81 years old). Biopsies of skin and collecting lymphatic vessels were performed in 7 patients who underwent lymphaticovenous anastomosis. RESULTS: The specificity was 1 for all four methods. The sensitivity was 1 in ICG lymphography and MRI, 0.62 in lymphoscintigraphy, and 0.33 in CT. These results show that MRI and ICG lymphography are superior to lymphoscintigraphy or CT for diagnosis of lymphedema. In some cases, biopsy findings suggested abnormalities in skin and lymphatic vessels for which lymphoscintigraphy showed no abnormal findings. ICG lymphography showed a dermal backflow pattern in these cases. CONCLUSIONS: Our findings suggest the importance of dual diagnosis by examination of the lymphatic system using ICG lymphography and evaluation of edema in subcutaneous fat tissue using MRI

A case series of Diffuse Glioneuronal Tumours with Oligodendroglioma-like features and Nuclear Clusters (DGONC).

In this study, we report three paediatric cases of Diffuse Glioneuronal Tumours with Oligodendroglioma-like features and Nuclear Clusters (DGONC)