Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size

OBJECTIVE: Recent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown. DESIGN: An international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS). RESULTS: ATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p\u3c0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p\u3c0.001 and p\u3c0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p\u3c0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%). CONCLUSIONS: ATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary

Monitoring Protein Kinases in Cellular Media with Highly Selective Chimeric Reporters

Protein kinases are important regulators of cellular function, and the dynamics of their activities are critical indicators of the health or pathology of living systems.[1, 2] In particular, extracellular-signal regulated kinases 1 and 2 (ERK1/2) play a pivotal role in the mitogen-activated protein kinase (MAPK) signaling pathway responsible for regulated cell survival and proliferation.[3] The centrality of these enzymes in normal and diseased cell states underscores the need for high throughput, selective, and sensitive methods that accurately and directly diagnose kinase activities. The benchmark phosphorylation assays for ERK1/2 rely on transfer of radioactive γ-phosphate of [γ-32P]ATP to peptide or protein substrates.[4] While broadly employed, this approach has limitations, including the discontinuous nature of the radioactive assay and the non-native ATP concentrations that are utilized. Alternatively, for cellular imaging, genetically-encoded sensors that rely on phosphorylation-based changes in fluorescence resonance energy transfer (FRET) between fluorescent protein pairs[5, 6] have been constructed for several kinases, including ERK1/2.[7-10] These sensors are powerful because they can be expressed in cells, however, they cannot be used for high throuput screening of recombinant enzymes and unfractionated cell lysates due to the very limited fluorescence changes that accompany phosphorylation. As a complementary approach, probes based on small, organic fluorophores with direct readouts[6, 11] can give sensitive and robust signals under physiogical conditions and are thus amenable to high throughput applications. For example, we have incorporated a sulfonamido-oxine (Sox) chromophore into peptides[12, 13] to report phosphorylation via chelation-enhanced fluorescence (CHEF) (Figure 1a). The weak binding affinity of the unphosphorylated substrate for Mg2+ increases significantly upon phosphorylation, resulting in robust (2- to 12-fold) fluorescence enhancements. This versatile peptide-based sensor design has been applied to monitor the activity of numerous Ser/Thr and Tyr kinases both in vitro[13] and in cell lysates.National Institutes of Health (U.S.) (NIH Cell Migration Consortium (GM064346)

Strategies for Site-Specific Radiolabeling of Peptides and Proteins

Although anatomical imaging modalities (X-ray, computed tomography (CT), magnetic resonance imaging (MRI)) still have a higher spatial resolution (0.1–1 mm) than molecular imaging modalities (single-photon emission computed tomography (SPECT), positron emission tomography (PET), optical imaging (OI)), the advantage of molecular imaging is that it can detect molecular and cellular changes at the onset of a disease before it leads to morphological tissue changes, which can be detected by anatomical imaging. During the last decades, noninvasive diagnostic imaging has encountered a rapid growth due to the development of dedicated imaging equipment for preclinical animal studies. In addition, the introduction of multimodality imaging (PET/CT, SPECT/CT, PET/MRI) which combines high-resolution conventional anatomical imaging with high sensitivity of tracer-based molecular imaging techniques has led to successful accomplishments in this exciting field. In this book chapter, we will focus on chemical synthesis techniques for site-specific incorporation of radionuclide chelators. Subsequently, radiolabeling based on complexation of a radionuclide with a chelator will be discussed, with focus on: diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-triacetic acid (NOTA), hexa-histidine (His-tag), and 6-hydrazinonicotinic acid (HYNIC) that allow the production of peptides labeled with 18F, 68Ga, 99mTc, and 111In – the currently most widely used isotopes

Clear cell chondrosarcoma in Von Hippel-Lindau disease

A diagnosis of clear cell chondrosarcoma of the ulna was made in a patient with Von Hippel-Lindau disease (VHL). After surgery, genetic analysis of the tumor tissue showed loss of heterozygosity at the VHL gene locus. Immunohistochemical analysis confirmed loss of expression of the VHL protein in the tumor cells. In addition, abundant Cyclin D1 expression in the tumor was observed. Chondrosarcoma has been described before in a VHL patient and VHL protein expression has been correlated to tumor grade in a series of sporadic chondrosarcomas. In this report, we show that clear cell chondrosarcoma may be a rare but canonical VHL manifestation through a cell-autonomous mechanism involving somatic loss-of-heterozygosity of the VHL tumor suppressor gene. We discuss the relevance of this observation with regard to the pathogenesis of clear cell chondrosarcoma in the context of VHL