For clinical follow-up, features of SDHB mutation-associated disease include a later age of onset, extraadrenal (abdominal or thoracic) tumors, and a higher rate of malignancy. In contrast, SDHD mutation carriers, in addition to head and neck paragangliomas, should be observed for multifocal tumors, infrequent malignancy, and the possibility of extraadrenal pheochromocytoma.

Pheochromocytomas are neural crest–derived tumors that arise from inherited or sporadic mutations in at least six independent genes. The proteins encoded by these multiple genes regulate distinct functions. We show here a functional link between tumors with VHL mutations and those with disruption of the genes encoding for succinate dehydrogenase (SDH) subunits B (SDHB) and D (SDHD). A transcription profile of reduced oxidoreductase is detected in all three of these tumor types, together with an angiogenesis/hypoxia profile typical of VHL dysfunction. The oxidoreductase defect, not previously detected in VHL-null tumors, is explained by suppression of the SDHB protein, a component of mitochondrial complex II. The decrease in SDHB is also noted in tumors with SDHD mutations. Gain-of-function and loss-of-function analyses show that the link between hypoxia signals (via VHL) and mitochondrial signals (via SDH) is mediated by HIF1α. These findings explain the shared features of pheochromocytomas with VHL and SDH mutations and suggest an additional mechanism for increased HIF1α activity in tumors.

Background: Hyperparathyroidism is a common endocrinopathy characterised by the formation of parathyroid tumours. In this study, we determine the role of the recently identified gene, HRPT2, in parathyroid tumorigenesis. Methods: Mutation analysis of HRPT2 was undertaken in 60 parathyroid tumours: five HPT-JT, three FIHP, three MEN 1, one MEN 2A, 25 sporadic adenomas, 17 hyperplastic glands, two lithium associated tumours, and four sporadic carcinomas. Loss of heterozygosity at 1q24-32 was performed on a subset of these tumours. Results: HRPT2 somatic mutations were detected in four of four sporadic parathyroid carcinoma samples, and germline mutations were found in five of five HPT-JT parathyroid tumours (two families) and two parathyroid tumours from one FIHP family. One HPT-JT tumour with germline mutation also harboured a somatic mutation. In total, seven novel and one previously reported mutation were identified. ''Two-hits'' (double mutations or one mutation and loss of heterozygosity at 1q24-32) affecting HRPT2 were found in two sporadic carcinomas, two HPT-JT-related and two FIHP related tumours. Conclusions:The results in this study support the role of HRPT2 as a tumour suppressor gene in sporadic parathyroid carcinoma, and provide further evidence for HRPT2 as the causative gene in HPT-JT, and a subset of FIHP. In light of the strong association between mutations of HRPT2 and sporadic parathyroid carcinoma demonstrated in this study, it is hypothesised that HRPT2 mutation is an early event that may lead to parathyroid malignancy and suggest intragenic mutation of HRPT2 as a marker of malignant potential in both familial and sporadic parathyroid tumours.

Parathyroid carcinoma is notoriously difficult to diagnose with confidence in borderline cases. Commonly there is a long lag time between diagnosis and clinical evidence of malignant behavior even in histopathologically straightforward lesions. There is therefore a need for a novel adjunctive marker to assist in the diagnosis of carcinoma. Parafibromin is the protein encoded by the putative tumor suppressor gene HRPT2. Mutations predicted to inactivate parafibromin were first detected in the germline of patients with hyperparathyroidism-jaw tumor (HPT-JT) syndrome. Subsequently, somatic mutations have been identified in the majority of sporadic carcinomas. We performed immunohistochemistry for parafibromin on 115 parathyroid tissues comprising 4 HPT-JT-related tumors (3 adenomas and 1 carcinoma), 11 sporadic parathyroid carcinomas, 79 sporadic adenomas, 3 multiple endocrine neoplasia 2A-related adenomas, 2 sporadic primary hyperplasias, 2 multiple endocrine neoplasia (MEN)-1-related hyperplasias, 6 secondary hyperplasias, 4 tertiary hyperplasias, and 4 normal parathyroid glands. There was complete absence of nuclear staining in 3 of 4 (75%) HPT-JT-related tumors and 8 of 11 (73%) sporadic parathyroid carcinomas and focal weak staining in 1 of 4 HPT-JT tumors and 2 of 11 sporadic parathyroid carcinomas. Only 1 parathyroid carcinoma exhibited diffuse strong nuclear expression of parafibromin. In contrast, 98 of 100 non-HPT-JT-related benign parathyroids showed diffuse strong nuclear positivity and 2 of 100 showed weak positive staining. We conclude that, in the correct clinical and pathologic context, complete absence of nuclear staining for parafibromin is diagnostic of parathyroid carcinoma or an HPT-JT-related tumor.

The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as ‘Low’, ‘Intermediate’ or ‘High’ for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.

microRNAs have emerged as powerful regulators of many biological processes, and their expression in many cancer tissues has been shown to correlate with clinical parameters such as cancer type and prognosis. Present in a variety of biological fluids, microRNAs have been described as a ‘gold mine’ of potential noninvasive biomarkers. Release of microRNA content of blood cells upon hemolysis dramatically alters the microRNA profile in blood, potentially affecting levels of a significant number of proposed biomarker microRNAs and, consequently, accuracy of serum or plasma-based tests. Several methods to detect low levels of hemolysis have been proposed; however, a direct comparison assessing their sensitivities is currently lacking. In this study, we evaluated the sensitivities of four methods to detect hemolysis in serum (listed in the order of sensitivity): measurement of hemoglobin using a Coulter® AcT diff™ Analyzer, visual inspection, the absorbance of hemoglobin measured by spectrophotometry at 414 nm and the ratio of red blood cell-enriched miR-451a to the reference microRNA miR-23a-3p. The miR ratio detected hemolysis down to approximately 0.001%, whereas the Coulter® AcT diff™ Analyzer was unable to detect hemolysis lower than 1%. The spectrophotometric method could detect down to 0.004% hemolysis, and correlated with the miR ratio. Analysis of hemolysis in a cohort of 86 serum samples from cancer patients and healthy controls showed that 31 of 86 (36%) were predicted by the miR ratio to be hemolyzed, whereas only 8 of these samples (9%) showed visible pink discoloration. Using receiver operator characteristic (ROC) analyses, we identified absorbance cutoffs of 0.072 and 0.3 that could identify samples with low and high levels of hemolysis, respectively. Overall, this study will assist researchers in the selection of appropriate methodologies to test for hemolysis in serum samples prior to quantifying expression of microRNAs.

BackgroundThere is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC). MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers.MethodsTo identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28) and age-matched healthy donors (n = 28). Serum microRNA levels were assessed by quantitative RT-PCR following preamplification.ResultsmicroRNA (miR)-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48) were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years) for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P < 0.05; 0.05; 0.0005 respectively) and in combination, miR-200b + miR-200c normalized to serum volume and miR-103 was the best predictive classifier of SEOC (ROC-AUC = 0.784). This predictive model (miR-200b + miR-200c) was further confirmed by leave one out cross validation (AUC = 0.784).ConclusionsWe identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

Ubiquitination has traditionally been viewed in the context of polyubiquitination that is essential for marking proteins for degradation via the proteasome. Recent discoveries have shed light on key cellular roles for monoubiquitination, including as a post-translational modification (PTM) of histones such as histone H2B. Monoubiquitination plays a significant role as one of the largest histone PTMs, alongside smaller, better-studied modifications such as methylation, acetylation and phosphorylation. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) has been shown to have key roles in transcription, the DNA damage response and stem cell differentiation. The H2Bub1 enzymatic cascade involves E3 RING finger ubiquitin ligases, with the main E3 generally accepted to be the RNF20-RNF40 complex, and deubiquitinases including ubiquitin-specific protease 7 (USP7), USP22 and USP44. H2Bub1 has been shown to physically disrupt chromatin strands, fostering a more open chromatin structure accessible to transcription factors and DNA repair proteins. It also acts as a recruiting signal, actively attracting proteins with roles in transcription and DNA damage. H2Bub1 also appears to play central roles in histone cross-talk, influencing methylation events on histone H3, including H3K4 and H3K79. Most significantly, global levels of H2Bub1 are low to absent in advanced cancers including breast, colorectal, lung and parathyroid, marking H2Bub1 and the enzymes that regulate it as key molecules of interest as possible new therapeutic targets for the treatment of cancer. This review offers an overview of current knowledge regarding H2Bub1 and highlights links between dysregulation of H2Bub1-associated enzymes, stem cells and malignancy.