Evoked potentials are widely used in clinical medicine for objective evaluation of sensory disturbances. However, gustatory evoked potentials (GEPs) have not been extensively studied due to lack of agreement among investigators regarding the waveforms. In this study GEPs and gustatory magnetic fields (GEMfs) were simultaneously recorded from five subjects in response to 0.3 M NaCl in an attempt to establish GEP recording as an objective gustatometer. Each subject received a total of 240 stimulus presentations over six sessions. Three GEP components (P1, N1 and P2) were observed and correlated with their corresponding equivalent current dipoles (ECD1, ECD2 and ECD3, respectively). ECD1 was localized to area G in all subjects, P1 being the indicator of intact gustatory projection to area G. No significant GEP activity was detected during the time preceding P1, which suggests that there was no activity in cortical gyri other than that detected by magnetoencephalography. ECD2 and ECD3 were localized to various cortical structures, including the inferior insula and the superior temporal sulcus, indicating that N1 and P2 reflect higher order gustatory functions. The present results indicate that measurement of GEPs may be useful for objective evaluation of gustatory disturbance.

Inflammation as a systemic process plays a central role in atherosclerotic plaque progression (PP). Here we investigated other systemic correlates of PP by global gene expression profiling (GEP) in peripheral blood. From a database of 45,727 coronary angiograms, we identified two patient groups with good risk factor control, but different clinical evolution: First, 16 patients had significant PP leading to repeated coronary interventions, and second, 16 patients had angiographically documented stable courses. GEP revealed 93 differentially expressed genes, of which 23 have unknown function. Among the remaining 70 genes, 10 were associated with progenitor and pluripotent cells, but only three genes with atherosclerosis. We developed a risk prediction gene signature by a multivariable statistical model integrating comprehensive laboratory and clinical patient data. This signature identified PP with high sensitivity and specificity for new patients, as estimated by resampling techniques. GEP results were validated by qPCR for ANK2 and GSTT1.

BACKGROUND

Gene expression profiling (GEP) in cells obtained from peripheral blood has shown that this is a very useful approach for biomarker discovery and for studying molecular pathogenesis of prevalent diseases. While there is limited literature available on gene expression markers associated with Chronic Obstructive Pulmonary Disease (COPD), the transcriptomic picture associated with critical respiratory illness in this disease is not known at the present moment.

RESULTS

By using Agilent microarray chips, we have profiled gene expression signatures in the whole blood of 28 COPD patients hospitalized with different degrees of respiratory compromise.12 of them needed of admission to the ICU, whilst 16 were admitted to the Respiratory Medicine Service. GeneSpring GX 11.0 software was used for performing statistical comparisons of transcript levels between ICU and non-ICU patients. Ingenuity pathway analysis 8.5 (IPA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to select, annotate and visualize genes by function and pathway (gene ontology). T-test showed evidence of 1501 genes differentially expressed between ICU and non-ICU patients. IPA and KEGG analysis of the most representative biological functions revealed that ICU patients had increased levels of neutrophil gene transcripts, being [cathepsin G (CTSG)], [elastase, neutrophil expressed (ELANE)], [proteinase 3 (PRTN3)], [myeloperoxidase (MPO)], [cathepsin D (CTSD)], [defensin, alpha 3, neutrophil-specific (DEFA3)], azurocidin 1 (AZU1)], and [bactericidal/permeability-increasing protein (BPI)] the most representative ones. Proteins codified by these genes form part of the azurophilic granules of neutrophils and are involved in both antimicrobial defence and tissue damage. This "neutrophil signature" was paralleled by the necessity of advanced respiratory and vital support, and the presence of bacterial infection.

CONCLUSIONS

Study of transcriptomic signatures in blood suggests an essential role of neutrophil proteases in COPD patients with critical respiratory illness. Measurement and modulation of the expression of these genes could present an option for clinical monitoring and treatment of severe COPD exacerbations.

Autophagy plays an important role in multiple myeloma (MM) for homeostasis, survival and drug resistance, but which genes participate in this process is unclear. We identified several cytoskeleton genes upregulated in MM patients by gene expression profiling (GEP) datasets; in particular, patients with high profilin 1 (PFN1) expression had poor prognosis in MM. In vitro, overexpressed PFN1 promotes proliferation and bortezomib (BTZ) resistance in MM cells. Further study indicated overexpression of PFN1 significantly promoted the process of autophagy and induced BTZ resistance in MM. Otherwise, knockdown of PFN1 blocked autophagy and sensitized MM to BTZ. Co-immunoprecipitation in MM cells indicated that PFN1 could bind Beclin1 complex and promote the initiation of autophagy. Inhibition of autophagy by blocking the formation of Beclin1 complex could reverse the phenotype of BTZ resistance in MM. Our findings suggested that PFN1 could promote autophagy through taking part in Beclin1 complex and contribute to BTZ resistance, which may become a novel molecular target in the therapy of MM.

Cancer immunotherapy has evolved major breakthroughs in the last years. The cell-surface receptor programmed death-1 (PD-1) and its ligand, programmed death ligand-1 (PD-L1), have been detected in various cancer types. However, the analysis on gastroenteropancreatic neoplasia (GEP-NENs) is limited. Therefore, the aim of this study was to characterize GEP-NENs with regard to PD-1/PD-L1 pathway and tumor-infiltrating lymphocytes (TILs). On protein level, we examined TILs, PD-1 and PD-L1 expression in tumor tissue of 244 GEP-NENs using immunohistochemistry. Expression levels were correlated with clinicopathological parameters including long-term survival in an observational study. In total, 244 patients could be included. Most of the patients had a NEN of the small intestine (52.5%) or the pancreas (29.5%). All tumors could be graded by their morphology and Ki67 index, with 57.8% G1, 34% G2 and 8.2% G3 tumors. High TILs (19.6%) and high PD-1 (16.1%) expression showed a significant correlation with shorter patient survival (P < 0.05) and with a higher grading. Furthermore, expression of PD-L1 (8.7%) showed a trend to shorter patient survival. High TILs and PD-1 expression are significantly associated with shorter patient survival and higher grading in GEP-NENs. PD-L1 expression showed a trend to shorter patient survival. Immunotherapy might be a promising therapeutic approach in GEP-NENs especially in tumors with high TILs.

OBJECTIVE

To evaluate the effects of positive end expiratory pressure and physiotherapy intervention during Phase I of cardiac rehabilitation on the behavior of pulmonary function and inspiratory muscle strength in postoperative cardiac surgery.

METHODS

A prospective randomized study, in which 24 patients were divided in 2 groups: a group that performed respiratory exercises with positive airway expiratory pressure associated with physiotherapy intervention (GEP, n = 8) and a group that received only the physiotherapy intervention (GPI, n = 16). Pulmonary function was evaluated by spirometry on the preoperative and on the fifth postoperative days; inspiratory muscle strength was measured by maximal inspiratory pressure on the same days.

RESULTS

Spirometric variables were significantly reduced from the preoperative to the fifth postoperative day for the GPI, while the GEP had a significant reduction only for vital capacity (P < .05). When the treatments were compared, smaller values were observed in the GPI for peak flow on the fifth postoperative day. Significant reductions of maximal inspiratory pressure from preoperative to the first postoperative day were found in both groups. However, the reduction in maximal inspiratory pressure from the preoperative to the fifth postoperative day was significant only in the GPI (P < .05).

CONCLUSIONS

These data suggest that cardiac surgery produces a reduction in inspiratory muscle strength, pulmonary volume, and flow. The association of positive expiratory pressure with physiotherapy intervention was more efficient in minimizing these changes, in comparison to the physiotherapy intervention alone. However, in both groups, the pulmonary volumes were not completely reestablished by the fifth postoperative day, and it was necessary to continue the treatment after hospital convalescence.

UNASSIGNED

Neuroendocrine tumors (NETs) are rare neoplasms, with an estimated annual incidence of ~ 6.9/100,000. NETs arise throughout the body from cells of the diffuse endocrine system. More than half originate from endocrine cells of the gastrointestinal tract and the pancreas, thus being referred to as gastroenteropancreatic NETs (GEP NETs). The only treatment that offers a cure is surgery, however most patients are diagnosed with metastatic disease, and curative surgery is usually not an option.Since the majority of patients are not candidate for curative surgery, they can be offered long-term systemic treatment, for both symptomatic relief and tumor growth suppression. Evidence based treatment options include somatostatin analogues, everolimus (an mTOR inhibitor), sunitinib (a tyrosine kinase inhibitor), peptide receptor radionuclide therapy (PRRT), chemotherapy, etc., alone or combined with cytoreductive procedures (surgery or liver directed procedures). However, there is an increasing need for novel therapies. Other treatment options being investigated are immunotherapy and epigenetic assessment that may lead to more personalized interventions. Following first line therapy with somatostatin analogues, there is no clear information to date indicating a preferred treatment sequence, and therefore the treatment approach should be individualized based on each NET patient characteristics.

UNASSIGNED

NET patients are increasingly diagnosed throughout the world, usually with metastatic disease and requiring systemic therapy. We believe that each patient should be therefore thoroughly evaluated and individually discussed by a multidisciplinary and dedicated NET-expert team, updated with all treatment options including ongoing clinical trials, and before selecting the proper treatment sequence.

UNASSIGNED

Radiotherapy after breast conservation has become the standard of care. Prior meta-analyses on effects of radiotherapy predated availability of gene expression profiling (GEP) to assess recurrence risk and/or did not include all relevant outcomes. This analysis used GEP information with pooled individual-level data to evaluate the impact of omitting radiotherapy on recurrence and mortality.

UNASSIGNED

We considered trials that evaluated or administered radiotherapy after lumpectomy in women with low-risk breast cancer. Women included had undergone lumpectomy and were treated with hormonal therapy for stage I, ER+ and/or PR+, HER2- breast cancer with Oncotype scores no greater than 18. Recurrence-free interval (RFI), type of RFI (locoregional or distant), and breast cancer-specific and overall survival were compared between no radiotherapy and radiotherapy using adjusted Cox models. All statistical tests were two-sided.

UNASSIGNED

The final sample included 1778 women from seven trials. Omission of radiotherapy was associated with an overall adjusted hazard ratio of 2.59 (95% confidence interval [CI] = 1.38 to 4.89, P = .003) for RFI. There was a statistically significant increase in any first locoregional recurrence (P = .001), but not distant recurrence events (P = .90), or breast cancer-specific (P = .85) or overall survival (P = .61). Five-year RFI rate was high (93.5% for no radiotherapy vs 97.9% for radiotherapy; absolute reduction = 4.4%, 95% CI = 0.7% to 8.1%, P = .03). The effects of radiotherapy varied across subgroups, with lower RFI rates for those with Oncotype scores of less than 11 (vs 11-18), older (vs younger), and ER+/PR+ status (vs other).

UNASSIGNED

Omission of radiotherapy in hormone-sensitive patients with low recurrence risk may lead to a modest increase in locoregional recurrence event rates, but does not appear to increase the rate of distant recurrence or death.

BACKGROUND

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) constitute a rare and heterogeneous group of tumors with varied biology.

OBJECTIVE

The aim of this study was to establish the clinical characteristics of patients with GEP-NEN and identify factors influencing their 5-year survival.

METHODS

The study included 122 patients living in Kraków or its administrative region, who were diagnosed with GEP-NEN between 2002 and 2011.

RESULTS

The mean follow-up period was 4.9 ±2.8 years. The most frequent primary site of the tumor was the small intestine (n = 25; 20%), followed by pancreas (n = 23; 19%), rectum (n = 23; 19%), stomach (n = 21; 17%), appendix (n = 19; 16%), and colon (n = 11; 9%). There were 84 tumors classified as NEN G1; 31, as NEN G2; 5, as neuroendocrine carcinoma; and 1, as mixed adenoneuroendocrine carcinoma. Most well-differentiated GEP-NENs (n = 57; 57%) were diagnosed at stage I according to the American Joint Committee on Cancer / Union for International Cancer Control (AJCC/UICC) classification; 77% of NEN G1 (n = 64) were diagnosed at stage I, but the majority of NEN G2—at stage IV (n = 18; 58%). Metastases at diagnosis were found in 38 patients (34%). In 90% of the cases (n = 101), tumors were hormonally nonfunctional. The overall 5-year survival was 85%. In the univariate analysis, NEN G2 (P = 0.003), higher stage according to the AJCC/UICC classification (P <0.001), and metastases at diagnosis (P <0.001) were associated with poorer prognosis. In standardized multivariate models, higher stage (P = 0.02) and metastases at diagnosis (P = 0.02) were independent risk factors for death.

CONCLUSIONS

The most important factors affecting survival of patients with GEP-NENs are tumor stage and the presence of metastases at diagnosis. The analysis of single-center data improves identification of patients with poorer prognosis requiring a more aggressive approach.

There is limited data on the clinical, cellular and molecular changes in relapsed acute promyeloytic leukemia (RAPL) in comparison with newly diagnosed cases (NAPL). We undertook a prospective study to compare NAPL and RAPL patients treated with arsenic trioxide (ATO) based regimens. 98 NAPL and 28 RAPL were enrolled in this study. RAPL patients had a significantly lower WBC count and higher platelet count at diagnosis. IC bleeds was significantly lower in RAPL cases (P=0.022). The ability of malignant promyelocytes to concentrate ATO intracellularly and their in-vitro IC50 to ATO was not significantly different between the two groups. Targeted NGS revealed PML B2 domain mutations in 4 (15.38%) of the RAPL subset and none were associated with secondary resistance to ATO. A microarray GEP revealed 1744 genes were 2 fold and above differentially expressed between the two groups. The most prominent differentially regulated pathways were cell adhesion (n=92), cell survival (n=50), immune regulation (n=74) and stem cell regulation (n=51). Consistent with the GEP data, immunophenotyping revealed significantly increased CD34 expression (P=0.001) in RAPL cases and there was in-vitro evidence of significant microenvironment mediated innate resistance (EM-DR) to ATO. Resistance and relapse following treatment with ATO is probably multi-factorial, mutations in PML B2 domain while seen only in RAPL may not be the major clinically relevant cause of subsequent relapses. In RAPL additional factors such as expansion of the leukemia initiating compartment along with EM-DR may contribute significantly to relapse following treatment with ATO based regimens.

OBJECTIVE

To assess the impact of C-reactive protein (CRP) kinetics, the effect of dynamic changes of CRP concentration on the survival of patients with locally advanced or metastatic urothelial carcinoma (UC) treated by single chemotherapeutic regimen including cisplatin was examined.

METHODS

Eighty patients with advanced UC, who failed treatment of advanced UC with the first-line chemotherapy or who received perioperative treatment of neoadjuvant or adjuvant settings, were treated with gemcitabine, etoposide and cisplatin (GEP) as second-line chemotherapy. Patients were divided into three groups according to CRP kinetics based on baseline and nadir CRP concentrations. Patients whose baseline CRP levels were <5 mg/L, patients whose baseline CRP levels were ≥5 mg/L and normalized (<5 mg/L), and patients whose baseline CRP levels were ≥5 mg/L and never normalized were assigned to non-elevated, normalized and non-normalized CRP groups, respectively. The prognostic impact of CRP kinetics and the correlation between normalized CRP period and overall survival period were determined.

RESULTS

In 46 (57%) of the 80 patients, CRP levels were elevated at the diagnosis of advanced UC. During treatment, after a median follow-up period of 12 months CRP levels were normalized in 24 (71%) of 34 patients, whereas CRP levels remained elevated in the remaining 10 patients. Overall survival rates were significantly different between the non-elevated, normalized, and non-normalized CRP groups (P < 0.001), with 1-year survival rates of 72, 51 and 14%, respectively. On multivariate analysis including Eastern Cooperative Oncology Group performance status, visceral metastasis, number of metastatic sites, previous definitive surgery, anaemia, baseline and nadir CRP levels (mg/L), and CRP kinetics status, CRP kinetics was an independent and significant factor for overall survival. The normalized CRP period was significantly correlated with the overall survival period in 52 patients who died.

CONCLUSIONS

CRP kinetics is significantly associated with the prognosis and survival period of patients with advanced UC treated by chemotherapy. Although larger confirmatory studies are warranted to validate our results, CRP can potentially be a useful biomarker for patients with advanced UC.

The Oncotype DX® recurrence score (RS) predictor has been clinically utilized to appropriately select adjuvant chemotherapy for patients with estrogen receptor (ER)-positive early breast cancer. However, the selection of chemotherapy for patients with intermediate RSs remains controversial. We assessed the prognostic value of a 70-gene signature (70GS) among patients with ER-positive breast cancer and intermediate RSs. In addition, we sought to identify genes associated with poor 70GS scores based on gene expression profiling (GEP). GEP was performed using gene expression data from 186 patients with ER-positive breast cancer. The RS and 70GS score were calculated on the basis of GEP. Among 186 patients, 82 ER-positive patients with intermediate RSs were identified. These patients were stratified by 70GS, overall survival (OS) significantly differed according to 70GS (p=0.013). In a supervised hierarchical analysis according to 70GS, the expression of several representative genes for cell proliferation was significantly higher in the poor 70GS cluster than in the good 70GS cluster. Furthermore, among these patients, FOXM1, AURKA, AURKB, and BIRC5 displayed prognostic significance for OS. In conclusion, 70GS can help to discriminate survival differences among ER-positive patients with intermediate RSs. FOXM1, AURKA, AURKB, and BIRC5, are associated with poor 70GS scores.

Multiple myeloma (MM) is a malignant hematological neoplasmand constitutes 10% of blood cancers. Recently, it has become evident that almost all cases of MM are preceded by gammopathy of undetermined significance (MGUS). MM is diagnosed by the presence of paraprotein in serum or urine, detected by serum electrophoresis and immunofixation, infiltration of malignant plasma cells in the bone marrow and related organ, or tissue damage. Cytogenetic status, International Staging System (ISS) and the quality of response to therapy are the most important prognostic factors. In the last decade the availability of new effective drugs, such as thalidomide, lenalidomide and bortezomib have provided a new therapeutic scenario expected to impact favorably on the outcome of MM patients. Today, for younger patients a triple-agent induction regimen incorporating novel agents followed by autologous stem cell transplantation (ASCT) is the best approach. In this setting ongoing clinical trials are evaluating the efficacy of consolidation and maintenance therapy based on the use of new drugs. For patients noteligible for ASCT the best initial regimens are combination therapy with melphalan and prednisone plus either thalidomide or bortezomibor the combination of lenalidomide and dexamethasone. Supportive therapy includes the use of erythropoietin and bisphosphonates according to the updated guidelines. In the future the identification of candidate therapeutic targets through gene expression profiling (GEP) and single-nucleotide polymorphism (SNP) analysis, as well as the availability of the targeted investigative agents will lead to a substantial progress in the development and implementation of personalized medicine in MM.

OBJECTIVE

Human papilloma virus (HPV) positive and HPV negative head and neck squamous cell cancer (HNSCC) are biologically distinct with a prognostic advantage for HPV positive patients compared to HPV negative cases. DNA promoter methylation is central to human diseases such as cancer, including HNSCC, with reported genome-wide hypomethylaton and promoter hypermethylation in HPV positive HNSCC tumors. The goal of this study was to identify differentially methylated genes in HPV positive versus HPV negative primary HNSCC genomes with clues to signaling networks.

METHODS

Laboratory-based study.

METHODS

Primary care academic health care system.

METHODS

DNA from 4 HPV positive and 4 HPV negative freshly frozen primary HNSCC were subject to comprehensive genome-wide methylation profiling. Differentially methylated gene lists were examined using the Signal Transduction Pathways (canonical) filter in the Genomatix Pathway System (GePS).

RESULTS

Twofold methylation differences were observed between HPV positive and HPV negative cases for 1168 genes. Pathway analysis applied to investigate the biological role of the 1168 differentially methylated genes revealed 8 signal transduction pathways forming a network of 66 genes, of which 62% are hypermethylated.

CONCLUSIONS

Our study reveals a predominant hypermethylation profile for genes in signal transduction pathways of HPV positive HNSCC tumor genomes. Because signaling events in the cell play a critical role in the execution of key biological functions, insights into how complex cellular signaling cascades and networks may be programmed in HNSCC are likely to be critical in the development of new biological agents designed to hit multiple targets.

Gastroenteropancreatic endocrine tumours (GEP ETs) represent a relatively rare and heterogeneous group of neoplasms whose therapy can be challenging. The poorly differentiated, fast-growing cases are treated with chemotherapy. In the slow-growing ones, biotherapy is usually performed. Several categories of targeted therapies have been studied for their treatment in vitro and in vivo. A critical review of molecular alterations suggests a rationale for targeting angiogenesis, and the phosphatidylinositol 3 kinase (PI(3)K)/AKT/mammalian target of rapamycin (mTOR) pathway. Accordingly, antiangiogenic agents and mTOR inhibitors are presently the most tested agents in phase II and III studies. Bevacizumab, some multitarget inhibitors, and mTOR inhibitors showed promising results in patients with advanced GEP ETs. A limited activity has been reported for imatinib and epidermal growth factor receptor (EGFR) inhibitors. Combinations of molecular targeted therapies with different sites of action, and somatostatin analogues may be relevant to avoid molecular escape pathways. Future trials should include more homogeneous groups of patients and pay more attention to the subgroup with progressive disease.

A characteristic of human gastroenteropancreatic neuroendocrine tumors (GEP-NET) is a minute unobtrusive primary tumor which often cannot be detected by common physical examinations. It therefore remains unidentified until the tumor has spread and space-occupying metastases cause clinical symptoms leading to diagnosis. Cases in which the primary cannot be located are referred to as NET with CUP-syndrome (cancer of unknown primary syndrome). With the help of array-CGH (comparative genomic hybridization, Agilent 105K) and gene expression analysis (Agilent 44K), microdissected primaries and their metastases were compared to identify up- and down-regulated genes which can be used as a marker for tumor progression. In a next analysis step, a hierarchical clustering of 41.078 genes revealed three genes [C-type lectin domain family 13 member A (CD302), peptidylprolyl isomerase containing WD40 repeat (PPWD1) and abhydrolase domain containing 14B (ABHD14B)] which expression levels can categorize the metastases into three groups depending on the localization of their primary. Because cancer therapy is dependent on the localization of the primary, the gene expression level of these three genes are promising markers to unravel the CUP syndrome in NET.

ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that are regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (approximately 180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP, of course). Cholera toxin was the first to be recognized, hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking, ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms, which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells, it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain, a sequence of approximately 200 amino acids that resembles a region in the yeast Sec7 gene product, which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains, among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity, recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4, 5, or 6.

Thirty-four gastroenteropancreatic (GEP) neuroendocrine tumors were evaluated for expression of epithelial (keratin, carcinoembryonic antigen [CEA] and neuroendocrine (neuron-specific enolase, chromogranin, bombesin) markers, and results were correlated with histologic patterns and histochemical staining. Tumors of mixed pattern (insular or trabecular with glandular areas) predominated. CEA localization corresponded to staining for mucin, with polarized apical or lumenal staining in glandular areas. Four trabecular midgut carcinoids, however, revealed diffuse cytoplasmic staining for CEA. Staining for keratin proteins was present in 68% of tumors. Bombesin immunoreactivity was demonstrated in 60% of GEP neuroendocrine tumors, indicating that bombesin positive metastatic tumors may not be predominantly of pulmonary origin, as previously suggested. Chromogranin was a sensitive marker for identifying normal gastrointestinal neuroendocrine cells that were not demonstrated by staining for neuron-specific enolase. Chromogranin was present in most neuroendocrine tumors, but was absent from three of five rectal carcinoids in keeping with the distinctive profile of hormonal and silver staining in these tumors. All GEP neuroendocrine neoplasms expressed both neuroendocrine and epithelial markers, supporting their derivation from endodermal epithelium.

To investigate somatostatin receptor scintigraphy (SRS) usefulness compared to conventional imaging procedures (CIP) in non-functioning gastroenteropatic (GEP) neuroendocrine tumors, we studied 40 patients, 30 in follow-up (FU) after previous operation for primary tumor (27 cases) or non-operable for disseminated metastases (3 cases), and 10 in the phase of initial diagnosis and staging (IDS). All patients were asymptomatic for hormone overexpression, with slightly high serum chromogranine A in 6 FU. The definitive diagnosis was obtained by surgery, laparotomy or percutaneous CT/US guided biopsy. Within 1 month before scintigraphy, all patients had undergone at least 2 of 3 CIP (CT, MRI and US); 4 and 24 h after 250 MBq 111In-pentetreotide i.v. injection, whole body scanning, planar and SPECT over the abdomen or other suspect regions were performed. Globally, 135 neoplastic lesions (84 hepatic, 34 abdominal extra-hepatic and 17 extra-abdominal) were ascertained in 32/40 patients; SPECT was positive in 29 cases, CIP in 28 and planar in 20, while all 3 procedures were true negative in the remaining 8 cases. Per patient sensitivity and accuracy were significantly higher at SPECT and CIP than planar. SPECT showed a significantly higher per lesion sensitivity (89.6%) than CIP (72.6%) and planar (53.3%) while CIP sensitivity was significantly higher than planar. SPECT correctly modified CIP patient classification and thus management in 18.7% of patients, while it downstaged the disease in 9.4% in respect of CIP; planar was incorrect in 51.3% of cases. SPECT and CIP combined use achieved 100% accuracy and correctly classified all patients. 111In-pentetreotide SRS, particularly SPECT, is a useful diagnostic tool in the detection of non-functioning GEP tumors, contributing to correct patient classification and appropriate therapeutic strategy. SPECT proved more sensitive than CIP, but their combined use achieved the highest accuracy values and gave the most accurate disease staging.

Genomic profiles of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are still insufficiently understood, and the genetic alterations associated with drug responses have not been studied. Here, we performed whole exome sequencing of 12 GEP-NETs from patients enrolled in a nonrandomized, open-labeled, single-center phase II study for pazopanib, and integrated our results with previously published results on pancreas (n = 12) and small intestine NETs (n = 50). The mean numbers of somatic mutations in each case varied widely from 20 to 4682. Among 12 GEP-NETs, eight showed mutations of more than one cancer-related gene, including TP53, CNBD1, RB1, APC, BCOR, BRAF, CTNNB1, EGFR, EP300, ERBB3, KDM6A, KRAS, MGA, MLL3, PTEN, RASA1, SMARCB1, SPEN, TBC1D12, and VHL. TP53 was recurrently mutated in three cases, whereas CNBD1 and RB1 mutations were identified in two cases. Three GEP-NET patients with TP53 mutations demonstrated a durable response and one small intestinal grade (G) 1 NET patient with BRAF V600E mutation showed progression after pazopanib treatment. We found BRAF V600E (G1 NET from rectum and two G3 NETs from colon) and BRAF G593S (G2 NET from pancreas) missense mutations (9.1%) in an independent cohort of 44 GEP-NETs from the rectum (n = 26), colon (n = 7), pancreas (n = 4), small intestine (n = 3), stomach (n = 3) and appendix (n = 1) by Sanger sequencing. All tumor specimens were obtained before chemotherapy. In conclusion, BRAF V600E mutation is likely to result in resistance to pazopanib but may be a potentianally actionable mutation in metastatic GEP-NETs patients.

The NCCN Guidelines for Occult Primary tumors provide recommendations for the evaluation, workup, management, and follow-up of patients with occult primary tumors (cancers of unknown primary). These NCCN Guidelines Insights summarize major discussion points of the 2014 NCCN Occult Primary panel meeting. The panel discussed gene expression profiling (GEP) for the identification of the tissue of origin and concluded that, although GEP has a diagnostic benefit, a clinical benefit has not been demonstrated. The panel recommends against GEP as standard management, although 20% of the panel believes the diagnostic benefit of GEP warrants its routine use. In addition, the panel discussed testing for actionable mutations (eg, ALK) to help guide choice of therapy, but declined to add this recommendation.

Huntington's disease is an inherited disorder caused by expanded stretch of consecutive trinucleotides (cytosine-adenosine-guanine, CAG) within the first exon of the huntingtin (HTT) gene on chromosome 4 (p16.3). The mutated huntingtin (mHTT) gains toxic function, probably through mechanisms that involve aberrant interactions in several pathways, causing cytotoxicity. Pathophysiology of disease involves several tissues; indeed it has been shown that there is a broad toxic effect of mHTT in the peripheral tissue of patients with HD, not only in the central nervous system. In this study we compared gene expression profiles (GEP) of HD fibroblasts and matched controls using microarray technology. We used RT-PCR to test the consistency of the microarray data and we found four genes up-regulated in HD patients with respect to control individuals. The genes appear to be involved in different pathways that have been shown to be perturbed even in HD models and patients. Although our study is preliminary and has to be extended to a larger cohort of HD patients and controls, nevertheless it shows that gene expression profiles seem to be altered in the fibroblasts of HD patients. Validation of the differential expressions at the protein level is required to ascertain if this cell type can be considered a suitable model for the identification of HD biomarkers.

We report the results of the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) Collaborative Exercise 2002-2003 on mitochondrial DNA (mtDNA) analysis. Six different samples were submitted to the participating laboratories: four blood stains (M1-M2-M3-M4), one mixture blood sample (M5), and two hair shaft fragments (M6). Most of the labs reported consensus results for the blood stains, slightly improving the results of previous collaborative exercises. Although hair shaft analysis is still carried out by a small number of laboratories, this analysis yielded a high rate of success. On the contrary, the analysis of the mixture blood stain (M5) yielded a lower rate of success; in spite of this, the whole results on M5 typing demonstrated the suitability of mtDNA analysis in mixture samples. We have found that edition errors are among the most common mistakes reported by the different labs. In addition, we have detected contamination events as well as other minor problems, i.e. lack of standarization in nomenclature for punctual and length heteroplasmies, and indels. In the present edition of the GEP-ISFG exercise we have paid special attention to the visual phylogenetic inspection for detecting common sequencing errors.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2-3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls.