Expanding the mutational spectrum of LZTR1 in schwannomatosis

Schwannomatosis is characterized by the development of multiple non-vestibular, non-intradermal schwannomas. Constitutional inactivating variants in two genes, SMARCB1 and, very recently, LZTR1, have been reported. We performed exome sequencing of 13 schwannomatosis patients from 11 families without SMARCB1 deleterious variants. We identified four individuals with heterozygous loss-of-function variants in LZTR1. Sequencing of the germline of 60 additional patients identified 18 additional heterozygous variants in LZTR1. We identified LZTR1 variants in 43% and 30% of familial (three of the seven families) and sporadic patients, respectively. In addition, we tested LZTR1 protein immunostaining in 22 tumors from nine unrelated patients with and without LZTR1 deleterious variants. Tumors from individuals with LZTR1 variants lost the protein expression in at least a subset of tumor cells, consistent with a tumor suppressor mechanism. In conclusion, our study demonstrates that molecular analysis of LZTR1 may contribute to the molecular characterization of schwannomatosis patients, in addition to NF2 mutational analysis and the detection of chromosome 22 losses in tumor tissue. It will be especially useful in differentiating schwannomatosis from mosaic Neurofibromatosis type 2 (NF2). However, the role of LZTR1 in the pathogenesis of schwannomatosis needs further elucidation.     

Solution‐Processable Donor‐Acceptor‐Donor Oligomers with Cross‐Linkable Functionality

Electron‐acceptor units, combined with bithiophene substituted with flexible chains end‐functionalized with cross‐linkable moieties, provide soluble donor‐acceptor‐donor (DAD) π‐conjugated oligomer‐type molecules with cross‐linking ability and broad absorption in the visible spectrum. A study on the cross‐linking conditions of the new oligomers to yield insoluble polymer networks is presented, including conditions for obtaining polymer films over poly(3,4‐ethylenedioxythiophene):polystyrene sulfonate‐covered substrates. The combination of the DAD molecular design and cross‐linking functionality opens prospects for applications in solution‐processed small‐molecule solar cells with morphologically‐stable organic layers.

     

Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient

Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.Hepatocellular carcinoma (HCC) is a global health concern with an estimated 750,000 new cases per year (1). In more than 80% of cases, HCC arises in a setting of liver cirrhosis mainly of alcoholic or viral origin (2). The prognosis for HCC patients is poor, with less than 30% qualifying for curative treatments such as tumor resection or liver transplantation (2). Median survival time of patients that cannot be treated surgically is less than 1 y. Sorafenib is the only approved targeted therapy for HCC, prolonging median patient survival by ∼3 mo (3). Sorafenib is a multikinase inhibitor of Raf (B and C), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR) (4), which presumably inhibits not only tumor cells but also endothelial cells responsible for tumor vascularization.Resistance to a targeted cancer drug can be intrinsic or adaptive (5). Sorafenib is largely cytostatic (6), suggesting that intrinsic resistance is more common in tumors, although some reports describe tumor shrinkage upon sorafenib treatment (7). Studies involving HCC cell lines or immunohistochemical staining of tumor sections revealed that sorafenib resistance correlates with the up-regulation of several signaling pathways, including the mammalian target of rapamycin (mTOR) pathway as assayed by S6 S235/236 (8) and Akt S473 phosphorylation (9). Other potential resistance mechanisms involve epithelial-to-mesenchymal transition (EMT) and autophagy (10, 11). However, the molecular mechanisms of sorafenib resistance in patients are largely unknown. Understanding the pathways that confer intrinsic or adaptive resistance would allow precision medicine and increase treatment efficacy.Proteomic analysis allows the identification of drug targets for cancer treatment and biomarkers for cancer classification or recurrence. In particular, MS is a powerful tool for resolving the complexity of cancer signaling pathways. With regard to HCC, qualitative proteomics has been performed on resected tumor material (12), laser-capture microdissected material from tissue sections (13, 14), and primary hepatocytes or serum derived from patients (15, 16). These studies (17, 18) identified HCC biomarkers such as glutamine synthetase and heat shock protein 70 (Hsp70) that are currently in use for diagnosis (19, 20). Quantitative proteomics has been performed on HCC resected tissue and serum (21, 22). Recently, proteomics has been performed on tumor biopsies of renal cell carcinoma patients (23). Several studies also have described phosphoproteomic analyses of resected HCC or other cancer material (24–26), in some cases quantifying up to 8,000 phosphorylated sites (hereafter referred to as “phosphosites”) starting with 2 mg of protein (18, 27–30). However, to our knowledge, quantitative proteomics and phosphoproteomics, hereafter collectively referred to as “(phospho)proteomics,” have yet to be performed on tumor biopsies, possibly because biopsy material is nonrenewable and typically provides only a very small amount of protein. Importantly, quantitative (phospho)proteomics on serial biopsies taken before and during treatment has not been described. We note that although a biopsy procedure generates less material than a resection, it has the important advantage of capturing normally dynamic properties of a tumor, such as the phosphorylation status of signaling pathways. Biopsies are immediately snap-frozen upon removal from the patient and, unlike resected tissue, are obtained without causing ischemia or hypoglycemia in the collected tissue. Needle biopsies are taken routinely to diagnose and stage the disease. Another important consideration is a method to perform quantitative (phospho)proteomics, such as super-SILAC (“SILAC” is an acronym for “stable isotope labeling of amino acids in cell culture”), that allows direct comparison of biopsies obtained at different times or from different patients (31).We describe quantitative (phospho)proteomic analyses of needle biopsies of HCC and matched nontumor tissue from a human patient. These analyses provide a global snapshot of signaling pathways in the biopsy material. Analyzing serial biopsies taken from a patient before and during therapy, we measured differences in signaling pathways between tumor and matched nontumor control tissue and the changes in these signaling pathways upon sorafenib treatment. Our findings provide insight into mechanisms of tumor progression and resistance to cancer therapy.     

A Higher Angiogenin Expression is Associated With a Nonnuclear Maspin Location in Laryngeal Carcinoma

Objectives

In numerous malignancies, angiogenin (ANG) and Maspin are important proangiogenic and antiangiogenic regulators, respectively. The aim of this study was to identify potential relationships between the biological roles of these two proteins in laryngeal squamous cell carcinoma (LSCC).

Methods

Immunohistochemical staining for ANG and Maspin was performed on specimens from 76 consecutive LSCC patients treated with surgery alone, considering the subcellular pattern of Maspin expression. Univariate and multivariate statistical models were used for prognostic purposes.

Results

On univariate analysis, a different level of ANG expression was seen for patients stratified by subcellular Maspin expression pattern: the mean ANG expression was higher in cases with a nonnuclear MASPIN expression than in those with a nuclear pattern (P=0.002). Disease-free survival (DFS; in months) differed significantly when patients were stratified by N stage (P=0.01). Patients whose Maspin expression was nonnuclear (i.e., it was cytoplasmic or there was none) had a significantly higher recurrence rate (P<0.001), and shorter DFS (P=0.01) than those with a nuclear Maspin pattern. The mean ANG expression was significantly higher in cases with loco-regional recurrent disease (P=0.007); and patients with an ANG expression ≥5.0% had a significantly shorter DFS than those with an ANG expression <5.0% (P=0.007). On multivariate analysis, ANG expression ≥5.0% was a significant, independent, negative prognostic factor in terms of DFS (P=0.041).

Conclusion

Our results support the hypothesis that a higher ANG expression is associated with a nonnuclear Maspin expression pattern in patients with LSCC. Further studies are needed to clarify the relationship between the ANG and Maspin pathways, and their potential diagnostic and therapeutic role in LSCC.     

Absence of steroid-dependent, endogenous opioid peptide suppression of pulsatile luteinizing hormone release between diestrus 1 and diestrus 2 in the rat estrous cycle

The objective of this study was to determine whether the negative feedback action of ovarian steroids on pulsatile luteinizing hormone (LH) release in the diestrous 1 (D1)-diestrous 2 (D2) interval of the rat estrous cycle is mediated by endogenous opioid peptides (EOPs), by examining the pulsatile LH release response to naloxone infusions in the presence or absence of D1-D2 levels of estradiol (E2) and progesterone (P). As plasma E2 and P levels increased between D1 and D2, mean blood LH levels decreased due solely to a decrease in LH pulse amplitude as frequency remained stable. However, ovariectomy increased both parameters of pulsatile LH release, indicating the effect of loss of ovarian steroid-negative feedback in this interval. Replacement of D1-D2 plasma levels of E2 and P restored D2 values for both parameters of pulsatile LH release, and E2 + P did not alter in vivo pituitary responsiveness to LH-releasing hormone (LHRH). In ovariectomized rats lacking the negative feedback provided by E2 + P in this cycle interval, continuous infusion of naloxone caused a further dose-dependent augmentation in both LH pulse amplitude and frequency. This stimulatory action of naloxone was prevented by simultaneous infusion with morphine, and was not associated with any change in in vivo pituitary responsiveness to LHRH, indicating that this was an action exerted through centrally located EOP receptors. Naloxone also increased both parameters of pulsatile LH release in E2 + P-treated rats. However, the magnitudes of the naloxone-induced increments in LH pulse amplitude and frequency in ovariectomized, steroid-treated rats were not greater than those seen in ovariectomized, nonsteroid-treated rats given naloxone versus saline. In addition, mean values for both parameters of pulsatile LH secretion during EOP receptor blockade in steroid-treated rats were reduced when compared to values in ovariectomized, nonsteroid-treated rats infused with naloxone. Thus the stimulatory effect of naloxone on pulsatile LH release was similar in the presence or absence of the negative feedback action of D1-D2 plasma levels of E2 + P. This indicates that the negative feedback effect of E2 + P on pulsatile LH release in this interval is not mediated by EOPs whose actions are blocked by naloxone.     

Korean red ginseng (Panax ginseng) improves glucose and insulin regulation in well-controlled, type 2 diabetes: results of a randomized, double-blind, placebo-controlled study of efficacy and safety

Background and aimTo address the paucity of randomized clinical studies assessing ginseng on long-term outcomes in type 2 diabetes, we assessed the clinical antidiabetic efficacy and safety of 12 weeks of supplementation with a Korean red ginseng (KRG) preparation, dose, and mode of administration, selected from an acute, clinical, screening model.Methods and resultsNineteen participants with well-controlled type 2 diabetes (sex: 11 M:8 F, age: 64 ± 2 years, BMI: 28.9 ± 1.4 kg/m², HbA_1c: 6.5%) completed the study. Using a double-blind, randomized, crossover design, each participant received the selected KRG preparation (rootlets) and placebo at the selected dose (2 g/meal = 6 g/day) and mode of administration (preprandial oral agent [−40 min]) for 12 weeks as an adjunct to their usual anti-diabetic therapy (diet and/or medications). Outcomes included measures of efficacy (HbA_1c and fasting- and 75-g oral glucose tolerance test [OGTT]-plasma glucose [PG], plasma insulin [PI], and insulin sensitivity index [ISI] indices); safety (liver, kidney, haemostatic, and blood-pressure function); and compliance (returned capsules, diet-records, and body-weight).There was no change in the primary endpoint, HbA_1c. The participants, however, remained well-controlled (HbA_1c = 6.5%) throughout. The selected KRG treatment also decreased 75g-OGTT-PG indices by 8–11% and fasting-PI and 75g-OGTT-PI indices by 33–38% and increased fasting-ISI (homeostasis model assessment [HOMA]) and 75g-OGTT-ISI by 33%, compared with placebo (P < 0.05). Safety and compliance outcomes remained unchanged.ConclusionsAlthough clinical efficacy, as assessed by HbA_1c, was not demonstrated, 12 weeks of supplementation with the selected KRG treatment maintained good glycemic control and improved PG and PI regulation safely beyond usual therapy in people with well-controlled type 2 diabetes. Further investigation with similarly selected KRG treatments may yield clinical efficacy.     

A comparison of the performance characteristics of classification criteria for the diagnosis of psoriatic arthritis

OBJECTIVE: To compare the accuracy of published classification criteria for the diagnosis of psoriatic arthritis (PsA) and to see whether data-derived classification criteria would be more accurate. METHODS: Data were abstracted from case-note review and radiographic review of patients identified with PsA or rheumatoid arthritis (RA) from 2 clinical disease registers. Each patient was classified according to 7 criteria sets. The test performance characteristics were compared using conditional logistic regression analysis. In an attempt to overcome the problems of the diagnostic gold standard, latent class analysis also was used to calculate test-performance characteristics. Classification and regression-tree methodology was used to derive new criteria and to indicate the diagnostic importance of particular data items, especially rheumatoid factor (RF). RESULTS: Four hundred ninety-nine patients were identified with RA (n=156) or PsA (n=343). Excluding the criteria of Fournie, which could not be applied in 24% of subjects, 446 cases could be classified by all of the other 6 methods. The most sensitive criteria for the diagnosis of PsA were those of Vasey and Espinoza, McGonagle, and Gladman (99%), whereas the others were significantly less sensitive (between 56% and 94%). The specificity of the criteria was high and statistically similar (between 93% and 99%). The Fournie criteria were the most difficult to use, whereas the Vasey and Espinoza and Moll and Wright criteria were the easiest (98% of subjects were able to be classified). A 2-latent class model found very similar test-performance characteristics. Logistic regression and classification and regression-tree models suggested that negative RF was not necessary for diagnosis in the presence of other characteristic features of PsA. CONCLUSIONS: Apart from the Bennett and European Spondyloarthropathy Study Group criteria, which have inadequate sensitivity, the published classification criteria for PsA have similar test-performance characteristics. These data suggest that the criteria proposed by Vasey and Espinoza, Gladman, or McGonagle are the most accurate and feasible in distinguishing between PsA and RA. Relevance International agreement about classification criteria for PsA will assist the interpretation of clinical and epidemiologic research. However, further prospective studies on unselected patients with and without PsA, including controls with non-rheumatoid inflammatory arthritis, are required to confirm these findings.     

The corticotrophin-releasing hormone test is the most reliable noninvasive method to differentiate pituitary from ectopic ACTH secretion in Cushing's syndrome

OBJECTIVE: It has been reported previously that the paired interpretation of the corticotrophin-releasing hormone (CRH) test and the 8-mg dexamethasone suppression test (HDDST) could have higher diagnostic power than any single test in the differential diagnosis of ACTH-dependent Cushing's syndrome. This finding has not been confirmed thereafter in large series. The aim of the present study has been to assess the operating characteristics of either the CRH test or the overnight HDDST and also to evaluate the potential utility of combining the interpretation of both tests in the differential diagnosis of ACTH-dependent Cushing's syndrome. DESIGN AND PATIENTS: We have reviewed the medical records of 59 consecutive cases with ACTH-dependent Cushing's syndrome: 49 patients with proven Cushing's disease (CD) and 10 patients with proven ectopic ACTH syndrome (EAS). Univariate curves of the receiver operating characteristics (ROC) have been performed to define the best cut-off values, the sensitivity and the specificity for CRH and overnight HDDST. A comparison between the areas under the ROC curves has also been performed. RESULTS: For the CRH test, the point on the ROC curve closest to 1 corresponded to a value of ACTH percentage increment of 50%[sensitivity 86% (72.6-94.8) and specificity 90% (55.5-98.3)]. The best threshold for cortisol percentage (30%) increment gave inferior results [sensitivity 61% (45.5-75.6) and specificity 70% (34.8-93.0)]. For the HDDST, the point on the ROC curve closest to 1 corresponded to a value of cortisol decrease from the baseline of 50%[sensitivity 77% (62.7-88.5), specificity 60% (26.4-87.6)]. The area under the ROC curve of the ACTH percentage increment after CRH was significantly greater than the area under the diagonal [0.9 (0.7-1.0), P= 0.0001]. Conversely, the area under the cortisol percentage decrement after dexamethasone was not different from that obtained by chance [0.7 (0.5-0.9), P= ns]. The area under the ROC curve of CRH is significantly greater than that of overnight HDDST (P = 0.03). A correct diagnosis has been achieved by the CRH test in 86.5% of cases and by the HDDST in 73% (P = 0.06). The combination of both tests has given a correct diagnosis in a significantly lower percentage of cases than the CRH test alone (69%, P= 0.04). The bilateral inferior petrosal sinus sampling (BIPSS) has been performed in 29 patients (24 CD, five EAS) who had negative imaging and/or discordant results of the noninvasive tests. Considering the criterion of a central to peripheral ACTH ratio > 3 after CRH stimulation, a correct diagnosis was achieved in all cases. CONCLUSIONS: The present data suggest that the CRH is likely to be the most reliable noninvasive diagnostic procedure for the differential diagnosis of the ACTH-dependent Cushing's syndrome. The criterion for a diagnosis of EAS is an ACTH percentage increment lower than 50%. The use of a combination of tests is not recommended because it does not add valuable information and may even impair the outcome of the CRH test. Cases with discordant results in pituitary imaging and CRH test should undergo BIPSS. The validity of this approach, which is straightforward and easily applicable in clinical practice, should be verified in larger series.     

Acute left ventricular failure after transcatheter closure of a secundum atrial septal defect in a patient with coronary artery disease: a critical reappraisal.

We report a case of acute left ventricular failure after transcatheter closure of a single secundum atrial septal defect in a 68-year-old man with coronary artery disease. Just before the procedure, two coronary lesions had been treated with direct stenting. Transcatheter closure of atrial septal defects should always be deferred in ischemic heart disease patients who need percutaneous myocardial revascularization.     

Novel markers of cell kinetics to evaluate progression from cirrhosis to hepatocellular carcinoma.

BACKGROUND: We investigated cell cycle kinetics of nodular lesions in cirrhosis to differentiate hepatocellular carcinoma (HCC) from its precursor lesions. METHODS: Twelve small HCC, 10 regenerative (RN), six large regenerative (LRN), and five dysplastic nodules (DN), identified in explant cirrhotic livers of five consecutive patients transplanted at Royal Free Hospital in 2002. Immunoperoxidase for MCM2, geminin and Ki67 was performed and the percentage of positive cells counted. RESULTS: The proportion of cells expressing MCM2 was more than those expressing Ki67, which in turn was more than those expressing geminin (overall median=16%, 2% and 0.5%, respectively, P<0.001). There was a statistically significant trend of increasing Ki67 expression (P=0.006), from RN to HCC; this trend was not statistically significant for geminin (P=0.18) or MCM2 (P=0.51). The median percentage of cells expressing Ki67 was 1% in RN, 0.5% in LRN, 2.2% in DN and 5.4% in HCC. The combination of these markers identified four different cell kinetics patterns: 'resting' (G0 cells: MCM2 -ve, Ki67 -ve, geminin -ve); 'licensed' (MCM2 +ve, Ki67 -ve, geminin -ve); 'slowly growing' (G1 phase arrest, MCM2 +ve, Ki67 +ve, low (0.4%) geminin) and expanding (MCM2 +ve, Ki67 +ve, geminin +ve) nodules. CONCLUSIONS: The combination of MCM2, geminin and Ki67 could represent a valuable tool in the understanding of HCC progression in cirrhosis.