BACKGROUND

Proteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841.

RESULTS

Similarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP), a twin-arginine translocase (TAT) secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP) were generated, but only mutations affecting the PrsDE (Type I) and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation.

CONCLUSIONS

None of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V) is required for establishing the symbiosis with legumes. The PrsDE (Type I) system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to the genes encoding the secretion system itself.

Little is known about the molecular pathogenesis of neuroendocrine tumors (NET) of the gastro-entero-pancreatic (GEP) system. We analyzed genetic and epigenetic alterations as well as the CpG island methylator phenotype (CIMP). The study comprised 118 well-differentiated fore- and mid-gut GEP-NET from 71 patients. In addition to loss of heterozygosity (LOH), microsatellite instability (MSI) and the methylation status of various tumor associated genes were examined. The expression profile of p16, APC and MENIN was investigated by immunohistochemistry. None of the tumors was highly microsatellite unstable, LOH was found in 22.2%. Significant differences in promoter hypermethylation were identified in the RUNX3 and the O(6)-MGMT genes. We found a significant loss of p16 expression in insulinomas (p = 0.05) and functional NET (p = 0.01), respectively. APC was expressed less in gastrinomas (p = 0.01) and functional GEP-NET (p = 0.05) vs. nonfunctional tumors. MENIN expression was reduced in pancreatic vs. extrapancreatic NET (p = 0.008) and in insulinomas vs. nonfunctional GEP-NET (p = 0.019) and NET associated with the carcinoid syndrome (p = 0.029). Further CIMP and a Ki-67 index >10% showed a close correlation. Outcome analysis of 19 patients showed a better survival for CIMP-negative patients. The analyses identified significant genetic and epigenetic alterations in well-differentiated fore- and mid-gut NET. CIMP, similar to Ki-67, might turn out to be of prognostic relevance.

Microarray analysis with 40 000 cDNA gene chip arrays determined differential gene expression profiles (GEPs) in CD34(+) marrow cells from myelodysplastic syndrome (MDS) patients compared with healthy persons. Using focused bioinformatics analyses, we found 1175 genes significantly differentially expressed by MDS versus normal, requiring a minimum of 39 genes to separately classify these patients. Major GEP differences were demonstrated between healthy and MDS patients and between several MDS subgroups: (1) those whose disease remained stable and those who subsequently transformed (tMDS) to acute myeloid leukemia; (2) between del(5q) and other MDS patients. A 6-gene "poor risk" signature was defined, which was associated with acute myeloid leukemia transformation and provided additive prognostic information for International Prognostic Scoring System Intermediate-1 patients. Overexpression of genes generating ribosomal proteins and for other signaling pathways was demonstrated in the tMDS patients. Comparison of del(5q) with the remaining MDS patients showed 1924 differentially expressed genes, with underexpression of 1014 genes, 11 of which were within the 5q31-32 commonly deleted region. These data demonstrated (1) GEPs distinguishing MDS patients from healthy and between those with differing clinical outcomes (tMDS vs those whose disease remained stable) and cytogenetics [eg, del(5q)]; and (2) molecular criteria refining prognostic categorization and associated biologic processes in MDS.

OBJECTIVE

Foregut carcinoid tumours have a different embryological origin than other gastroenteropancreatic neuroendocrine tumours (GEP NETs). In the total group of GEP NETs (n = 131), treatment with (177)Lu-octreotate resulted in tumour remission in 47% of patients, with a median time to progression (TTP) of >36 months. As patients with foregut carcinoids may respond differently, we here present the effects of this treatment in a subgroup of patients with foregut carcinoids of bronchial, gastric or thymic origin.

METHODS

Nine patients with bronchial, five with gastric and two with thymic carcinoids were treated. All patients had metastasised disease. The intended cumulative dose of (177)Lu-octreotate was 22.2-29.6 GBq. Southwest Oncology Group criteria were used for response evaluation.

RESULTS

Bronchial carcinoids: Five patients had partial remission, one had minor response (MR, tumour size reduction:>> or =25%, <50%), two had stable disease (SD) and one had progressive disease (PD). Median TTP was 31 months. Gastric carcinoids: One patient had complete remission, one had MR and two had SD, including one with PD at baseline. One patient developed PD. Thymic carcinoids: One patient had SD. In the other patient, disease remained progressive. All patients: Overall remission rate was 50%, including MR.

CONCLUSIONS

(177)Lu-octreotate treatment can be effective in patients with bronchial and gastric carcinoids. Its role in thymic carcinoids cannot be determined yet because of the limited number of patients. The overall remission rate of 50% in patients with the studied foregut carcinoids is comparable to that in the total group of GEP NETs.

Oncolytic viruses obliterate tumor cells in tissue culture but not against the same tumors in vivo. We report that macrophages can induce a powerfully protective antiviral state in ovarian and breast tumors, rendering them resistant to oncolytic virotherapy. These tumors have activated JAK/STAT pathways and expression of interferon-stimulated genes (ISGs) is upregulated. Gene expression profiling (GEP) of human primary ovarian and breast tumors confirmed constitutive activation of ISGs. The tumors were heavily infiltrated with CD68+ macrophages. Exposure of OV-susceptible tumor cell lines to conditioned media from RAW264.7 or primary macrophages activated antiviral ISGs, JAK/STAT signaling and an antiviral state. Anti-IFN antibodies and shRNA knockdown studies show that this effect is mediated by an extremely low concentration of macrophage-derived IFNβ. JAK inhibitors reversed the macrophage-induced antiviral state. This study points to a new role for tumor-associated macrophages in the induction of a constitutive antiviral state that shields tumors from viral attack.

Neuroendocrine neoplasms (NENs) are rare, heterogeneous and ubiquitous tumors commonly localized in the gastrointestinal tract, lung, and pancreas. The clinical behavior of NEN is highly unpredictable; in fact, low-grade cases can unexpectedly be associated with metastases. Currently, the 2010 WHO NEN classification employs histological differentiation and the proliferation index for grading tumors but fails to provide reliable prognostic and therapeutic indications. Therefore, there is an urgent need for a better characterization of G2/G3 NENs. Similar to several other tumors, NENs possess immune-escape mechanisms, but very little has yet been done to characterize this crucial aspect. There are no available data describing PD-L1 expression in these tumors. Here we provide, for the first time, evidence of PD-L1 tissue expression in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). PD-L1 expression was significantly associated with a high-grade WHO classification (G3) (P<0.001) but not with gender, primary site, or lymph node status. The PD-L1 positivity rate and signal intensity are directly correlated (P<0.001) with a grade increase from G1 to G3. In particular in G3 cases, we observed a dichotomy between the morphology (WD- and PD-NENs) and Ki67. Moreover, our study demonstrated a significant association with the grade and PD-L1 expression levels in immune-infiltrating cells (P<0.001). In particular, G3 tumors are characterized by strong PD-L1 expression in both the tumor and infiltrating immune cells (P<0.001), reflecting an unfavorable environment for T-cell-mediated tumor aggression. These findings suggest that NENs might acquire resistance to immune surveillance by upregulating PD-L1 and inhibiting peritumoral and intratumoral infiltrating lymphocytes. Here we demonstrate that PD-L1 is currently the best-known biomarker for G3 NENs, becoming the new gold standard for G3 NEN discrimination. Furthermore, pharmacological approaches using anti-PD-1 antibodies may become the logical choice for the treatment of G3 cases with a poor prognosis.

The term general secretory pathway (GSP) has been usurped and misused in the literature over the past few years. The concept of GSP is discussed from an historical perspective, and the definitions of the general export pathway (GEP), the main terminal branch (MTB) of the GSP, the unified GSP nomenclature and the type II, IV and V secretion pathways are also described to show how they have fuelled the confusion. By putting the record straight and using novel findings within the field of bacterial protein secretion, we hope to bring clarity to this area of science and prevent further promulgation of incorrect terminologies.

Nuclear medicine plays a pivotal role in the imaging and treatment of neuroendocrine tumours (NETs). Somatostatin receptor scintigraphy (SRS) with [(111)In-DTPA(0)]octreotide has proven its role in the diagnosis and staging of gastroenteropancreatic NETs (GEP-NETs). New techniques in somatostatin receptor imaging include the use of different radiolabelled somatostatin analogues with higher affinity and different affinity profiles to the somatostatin receptor subtypes. Most of these analogues can also be labelled with positron-emitting radionuclides that are being used in positron emission tomography imaging. The latter imaging modality, especially in the combination with computed tomography, is of interest because of encouraging results in terms of improved imaging quality and detection capabilities. Considerable advances have been made in the imaging of NETs, but to find the ideal imaging method with increased sensitivity and better topographic localisation of the primary and metastatic disease remains the ultimate goal of research. This review provides an overview of the currently used imaging modalities and ongoing developments in the imaging of NETs, with the emphasis on nuclear medicine and puts them in perspective of clinical practice. The advantage of SRS over other imaging modalities in GEP-NETs is that it can be used to select patients with sufficient uptake for treatment with radiolabelled somatostatin analogues. Peptide receptor radionuclide therapy (PRRT) is a promising new tool in the management of patients with inoperable or metastasised NETs as it can induce symptomatic improvement with all Indium-111, Yttrium-90 or Lutetium-177-labelled somatostatin analogues. The results that were obtained with [(90)Y-DOTA(0),Tyr(3)]octreotide and [(177)Lu-DOTA(0),Tyr(3)]octreotate are even more encouraging in terms of objective tumour responses with tumour regression and documented prolonged time to progression. In the largest group of patients receiving PRRT, treated with [(177)Lu-DOTA(0),Tyr(3)]octreotate, a survival benefit of several years compared with historical controls has been reported.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are relatively rare and heterogenous malignancies. Recent advances in histopathological classification according to the anatomical site of origin, proliferation rate, and extend of the disease have created a valid and powerful tool for the prognostic stratification of GEP-NETs. Chromogranin A is still the best available marker used for the biochemical confirmation of these tumors, but new more sensitive markers are urgently required. Although scintigraphy with (111)In-octreotide has widely been applied for the localization and staging of GEP-NETs, newer imaging modalities based on the functional characteristics of these tumors are evolving aiming not only to facilitate the diagnosis but also prognosis and evaluation of treatment. Somatostatin receptors are the primary therapeutic targets through somatostatin analogs and peptide receptor radionuclide therapy (PRRT) producing symptomatic, biochemical and to a lesser extent antiproliferative effects. Due to the relatively limited and erratic response to chemotherapy, new molecular targeted therapies exploiting some of the biological properties of GEP-NETs such as increased vascularity and inhibition of pathways involved in downstream signal transduction have evolved. Some of these therapies, the mTOR inhibitor everolimus and the tyrosine kinase inhibitor sunitinib, have been recently validated in phase III studies producing practice changing outcomes. In addition, two oral chemotherapeutic agents temozolomide and capecitabine, show promising effects and may replace streptozotocin-based regimens whereas combination therapies with the angiogenesis inhibitor bevacizumab are being investigated. Although progression free survival is used as a feasible primary end point due to the long survival of patients even in the presence of extensive disease prolongation of overall survival following the introduction of new therapies needs to be established.

Indium-111-pentetreotide, a radiolabeled somatostatin analog, has been proposed for imaging tumors bearing somatostatin receptors. This study evaluates the safety, efficacy and impact on patient management of this scintigraphic agent in patients with gastroenteropancreatic (GEP) neuroendocrine tumors.

METHODS

We studied 47 consecutive patients with a proven or clinically suspected GEP neuroendocrine tumor who were imaged 4 and 24 hr after injection of 111In-pentetreotide. The patients were monitored for adverse reactions and changes in vital signs or clinical chemistry over 24 hr. The scintigraphic findings were compared with results from conventional imaging methods. The patients were followed over a minimal 6-mo period during which further localization procedures were performed to confirm or refute the additional tumor sites found at scintigraphy.

RESULTS

No adverse reactions or clinically relevant changes in clinical chemistry were noted after injection of the radiopharmaceutical. The final diagnosis of a GEP neuroendocrine tumor was retained in 38 patients. Somatostatin receptor-positive lesions were found in 33 of these patients, whereas conventional methods were positive in 31 patients. Of the 54 sites seen by conventional procedures, 50 sites were also detected scintigraphically.

CONCLUSIONS

Indium-111-pentetreotide is a safe, sensitive imaging agent in the detection of GEP neuroendocrine tumor sites. Indium-111-pentetreotide also provides information on the somatostatin receptor status of the tumor and may therefore aid in therapeutic decisions.

Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa GTPases that are inactive in the GDP-bound form, depends on guanine nucleotide-exchange proteins (GEPs) to accelerate GTP binding. A novel ARF GEP, designated cytohesin-4, was cloned from a human brain cDNA library. Deduced amino acid sequence of the 47-kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an approximately 200-amino acid Sec7 domain with an approximately 100-residue pleckstrin homology domain near the C terminus. The Sec7 domain sequence is 77% identical to those of other cytohesins. Structures of the cytohesin-4 and cytohesin-1 genes were remarkably similar, except for an extra 3-base pair (GAG) exon present in cytohesin-1. Two mRNAs with and without the 3-base pair sequence were found in brain in different ratios for cytohesin-1, -2, and -3 but not cytohesin-4. Recombinant cytohesin-4 stimulated guanosine 5'-3-O-(thio)triphosphate binding by human ARF1 and ARF5 but not ARF6. Like other cytohesins and unlike the approximately 200-kDa ARF GEPs, it was not inhibited by brefeldin A. A cytohesin-4 mRNA of approximately 3.7 kilobases, abundant in leukocytes, was not detected in most tissues. Among separated populations of blood cells, approximately 90% of CD33(+) (monocytes), 80% of CD2(+) (NK/T), and 10-20% of CD19(+) (B) cells contained cytohesin-4 mRNA by in situ hybridization. Thus, in gene structure and brefeldin A-insensitive GEP activity, cytohesin-4 resembles other cytohesins, but its tissue distribution differs considerably, consistent with a different specific function.

Gene expression profiling (GEP) has identified genes whose expression levels predict patient survival in diffuse large-B-cell lymphoma (DLBCL). Such discovery techniques generally require frozen samples unavailable for most patients. We developed a quantitative nuclease protection assay to measure expression levels of prognostic DLBCL genes using formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE tissue was sectioned, permeabilized, denatured in the presence of specific probes, and hybridized to mRNA in situ. Nuclease subsequently destroyed non-hybridized probe. Alkaline hydrolysis freed mRNA-bound probes from tissue, which were transferred to ArrayPlates for probe capture and chemiluminescent quantification. We validated assay performance using frozen, fresh, and FFPE DLBCL samples, then used 39 archived DLBCL, previously microarray analyzed, to correlate GEP and ArrayPlate results. We compared old (>18 years) with new (<2 months) paraffin blocks made from previously frozen tissue from the original biopsy. ArrayPlate gene expression results were confirmed with immunohistochemistry for BCL2, BCL6, and HLA-DR, showing agreement between mRNA species and the proteins they encode. Assay performance was linear to approximately 1 mg sample/well. RNase and DNase treatments demonstrated assay specificity for RNA detection, both fixed and soluble RNA detection. Comparisons were excellent for lysate vs snap-frozen vs FFPE (R(2)>0.98 for all comparisons). Coefficients of variation for quadruplicates on FFPE were generally <20%. Correlation between new and old paraffin blocks from the same biopsy was good (R(2)=0.71). Comparison of ArrayPlate to Affymetrix and cDNA microarrays showed reasonable correlations. Insufficient power from small sample size prevented successfully correlating results with patient survival, although hazard ratios trended the expected directions. We developed an assay to quantify expression levels of survival prediction genes in DLBCL using FFPE, fresh, or frozen tissue. While this technique cannot replace GEP for discovery, it indicates that expression differences identified by GEP can be replicated on a platform applicable to archived FFPE samples.

BACKGROUND

Gastroenteropancreatic neuroendocrine neoplasm (GEP-NEN) is the most common type of neuroendocrine tumors accounting for 65-75% of neuroendocrine neoplasms (NENs). Given the fact that there are few studies on GEP-NENs among Chinese patients, we performed a retrospective study in South China.

METHODS

Totally 178 patients with GEP-NENs treated at the First Affiliated Hospital of Sun Yat-sen University between January 1995 and May 2012 were analyzed retrospectively.

RESULTS

Pancreas was found the most common site of involvement (34.8%). 149 patients (83.7%) presented as non-functional tumors with non-specific symptoms such as abdominal pain (33.7%); carcinoid syndrome was not found in this study. Several methods are useful for localization of GEP-NENs, yielding varied detection rates from 77.8% to 98.7%. Positive rates of chromogranin A (CgA) and synaptophysin (Syn) immunhistochemically were 69.1% and 90.2%, respectively. 87 patients (51.5%) had G1 tumors, 31(18.3%) G2 tumors and 51 (30.2%) G3 tumors. Neuroendocrine tumor (NET), neuroendocrine carcinoma (NEC) and mixed adenoendocrine carcinoma (MANEC) were 69.8%, 27.2% and 3.0%, respectively. 28.1% of patients presented with distant disease. Surgery was performed in 152 (85.4%) patients, and overall 5-year survival rate was 54.5%. Functionality, G1 grading and NET classification were associated with favorable prognosis in univariate analysis. Distant metastasis contributed to unfavorable prognosis of these tumors.

CONCLUSIONS

Nonfunctional tumors with non-specific symptoms account for the majority of GEP-NENs. Diagnosis depends on pathological classification. Multidisciplinary treatments could help improve the outcome.

Patients with multiple myeloma have variable survival and require reliable prognostic and predictive scoring systems. Currently, clinical and biological risk markers are used independently. Here, International Staging System (ISS), fluorescence in situ hybridization (FISH) markers, and gene expression (GEP) classifiers were combined to identify novel risk classifications in a discovery/validation setting. We used the datasets of the Dutch-Belgium Hemato-Oncology Group and German-speaking Myeloma Multicenter Group (HO65/GMMG-HD4), University of Arkansas for Medical Sciences-TT2 (UAMS-TT2), UAMS-TT3, Medical Research Council-IX, Assessment of Proteasome Inhibition for Extending Remissions, and Intergroupe Francophone du Myelome (IFM-G) (total number of patients: 4750). Twenty risk markers were evaluated, including t(4;14) and deletion of 17p (FISH), EMC92, and UAMS70 (GEP classifiers), and ISS. The novel risk classifications demonstrated that ISS is a valuable partner to GEP classifiers and FISH. Ranking all novel and existing risk classifications showed that the EMC92-ISS combination is the strongest predictor for overall survival, resulting in a 4-group risk classification. The median survival was 24 months for the highest risk group, 47 and 61 months for the intermediate risk groups, and the median was not reached after 96 months for the lowest risk group. The EMC92-ISS classification is a novel prognostic tool, based on biological and clinical parameters, which is superior to current markers and offers a robust, clinically relevant 4-group model.

OBJECTIVE

The role of growth factors in ovarian cancer development and progression is complex and multifactorial. We hypothesized that new growth factors may be identified through the molecular analysis of ovarian tumors as they exist in their native environment.

METHODS

RNA extracted from microdissected serous low malignant potential (LMP) and invasive ovarian tumors was used to construct cDNA libraries. A total of 7300 transcripts were randomly chosen for sequencing, and those transcripts were statistically evaluated. Reverse transcription-PCR and immunohistochemistry were used to validate the findings in tumor tissue samples. Ovarian cancer cell lines were used to test gene effects on monolayer growth, proliferative capacity, and density-independent growth.

RESULTS

Analysis of the pooled library transcripts revealed 26 genes differentially expressed between LMP and invasive ovarian cancers. The granulin-epithelin precursor [GEP/PC-cell derived growth factor (PCDGF)] was expressed only in the invasive ovarian cancer libraries (P < 0.028) and was absent in the LMP libraries (0 of 2872 clones). All of the invasive tumor epithelia, 20% of the LMP tumor epithelia, and all of the stroma from both subsets expressed GEP by reverse transcription-PCR. Immunohistochemical staining for GEP was diffuse and cytosolic in invasive ovarian cancer tumor cells compared with occasional, punctate, and apical staining in LMP tumor epithelia. Antisense transfection of GEP into ovarian cancer cell lines resulted in down-regulation of GEP production, reduction in cell growth (P < 0.002), decrease in the S-phase fraction (P < 0.04), and loss of density-independent growth potential (P < 0.01).

CONCLUSIONS

cDNA library preparation from microdissected tumor epithelium provided a selective advantage for the identification of growth factors for epithelial ovarian cancer. Differential granulin expression in tumor samples and the antiproliferative effects of its antisense down-regulation suggest that GEP may be a new autocrine growth factor and molecular target for epithelial ovarian cancer.

Hepatocellular carcinoma (HCC) has a poor prognosis owing to aggressive phenotype. Gα12 gep oncogene product couples to G-protein-coupled receptors, whose ligand levels are frequently increased in tumor microenvironments. Here, we report Gα12 overexpression in human HCC and the resultant induction of zinc-finger E-box-binding homeobox 1 (ZEB1) as mediated by microRNA deregulation. Gα12 expression was higher in HCC than surrounding non-tumorous tissue. Transfection of Huh7 cell with an activated mutant of Gα12 (Gα12QL) deregulated microRNA (miRNA or miR)-200b/a/429, -194-2/192 and -194-1/215 clusters in the miRNome. cDNA microarray analyses disclosed the targets affected by Gα12 gene knockout. An integrative network of miRNAs and mRNA changes enabled us to predict ZEB1 as a key molecule governed by Gα12. Decreases of miR-200a/b, -192 and -215 by Gα12 caused ZEB1 induction. The ability of Gα12 to decrease p53 levels, as a result of activating protein-1 (AP-1)/c-Jun-mediated mouse double minute 2 homolog induction, contributed to transcriptional deregulation of the miRNAs. Gα12QL induced ZEB1 and other epithelial-mesenchymal transition markers with fibroblastoid phenotype change. Consistently, transfection with miR-200b, -192 or -215 mimic prevented the ability of Gα12QL to increase tumor cell migration/invasion. In xenograft studies, sustained knockdown of Gα12 decreased the overall growth rate and average volume of tumors derived from SK-Hep1 cell (mesenchymal-typed). In HCC patients, miR-192, -215 and/or -200a were deregulated with microvascular invasion or growth advantage. In the HCC samples with higher Gα12 level, a correlation existed in the comparison of relative changes of Gα12 and ZEB1. In conclusion, Gα12 overexpressed in HCC causes ZEB1 induction by deregulating p53-responsive miRNAs, which may facilitate epithelial-mesenchymal transition and growth of liver tumor. These findings highlight the significance of Gα12 upregulation in liver tumor progression, implicating Gα12 as an attractive therapeutic target.

BACKGROUND

Gene expression profiling (GEP) and expanded immunohistochemistry (IHC) tests aim to improve decision-making relating to adjuvant chemotherapy for women with early breast cancer.

OBJECTIVE

The aim of this report is to assess the clinical effectiveness and cost-effectiveness of nine GEP and expanded IHC tests compared with current prognostic tools in guiding the use of adjuvant chemotherapy in patients with early breast cancer in England and Wales. The nine tests are BluePrint, Breast Cancer Index (BCI), IHC4, MammaPrint, Mammostrat, NPI plus (NPI+), OncotypeDX, PAM50 and Randox Breast Cancer Array.

METHODS

Databases searched included MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE and The Cochrane Library. Databases were searched from January 2009 to May 2011 for the OncotypeDX and MammaPrint tests and from January 2002 to May 2011 for the other tests.

METHODS

A systematic review of the evidence on clinical effectiveness (analytical validity, clinical validity and clinical utility) and cost-effectiveness was conducted. An economic model was developed to evaluate the cost-effectiveness of adjuvant chemotherapy treatment guided by four of the nine test (OncotypeDX, IHC4, MammaPrint and Mammostrat) compared with current clinical practice in England and Wales, using clinicopathological parameters, in women with oestrogen receptor-positive (ER+), lymph node-negative (LN-), human epidermal growth factor receptor type 2-negative (HER2-) early breast cancer.

RESULTS

The literature searches for clinical effectiveness identified 5993 citations, of which 32 full-text papers or abstracts (30 studies) satisfied the criteria for the effectiveness review. A narrative synthesis was performed. Evidence for OncotypeDX supported the prognostic capability of the test. There was some evidence on the impact of the test on decision-making and to support the case that OncotypeDX predicts chemotherapy benefit; however, few studies were UK based and limitations in relation to study design were identified. Evidence for MammaPrint demonstrated that the test score was a strong independent prognostic factor, but the evidence is non-UK based and is based on small sample sizes. Evidence on the Mammostrat test showed that the test was an independent prognostic tool for women with ER+, tamoxifen-treated breast cancer. The three studies appeared to be of reasonable quality and provided data from a UK setting (one study). One large study reported on clinical validity of the IHC4 test, with IHC4 score a highly significant predictor of distant recurrence. This study included data from a UK setting and appeared to be of reasonable quality. Evidence for the remaining five tests (PAM50, NPI+, BCI, BluePrint and Randox) was limited. The economic analysis suggests that treatment guided using IHC4 has the greatest potential to be cost-effective at a £20,000 threshold, given the low cost of the test; however, further research is needed on the analytical validity and clinical utility of IHC4, and the exact cost of the test needs to be confirmed. Current limitations in the evidence base produce significant uncertainty in the results. OncotypeDX has a more robust evidence base, but further evidence on its impact on decision-making in the UK and the predictive ability of the test in an ER+, LN-, HER- population receiving current drug regimens is needed. For MammaPrint and Mammostrat there were significant gaps in the available evidence and the estimates of cost-effectiveness produced were not considered to be robust by the External Assessment Group.

CONCLUSIONS

Methodological weaknesses in the clinical evidence base relate to heterogeneity of patient cohorts and issues arising from the retrospective nature of the evidence. Further evidence is required on the clinical utility of all of the tests and on UK-based populations. A key area of uncertainty relates to whether the tests provide prognostic or predictive ability.

CONCLUSIONS

The clinical evidence base for OncotypeDX is considered to be the most robust. The economic analysis suggested that treatment guided using IHC4 has the most potential to be cost-effective at a threshold of £20,000; however, the evidence base to support IHC4 needs significant further research.

BACKGROUND

PROSPERO 2011:CRD42011001361, available from www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42011001361.

Rab3A, a member of the Rab3 small G protein family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M(r) of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.

OBJECTIVE

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) constitute a diverse group of neoplasms arising from the diffuse neuroendocrine cell system. During the last 2 years a new classification system, the WHO 2010, has come into clinical practice together with Tumor Nodes Metastases (TNM) staging and grading systems, developed by the European Neuroendocrine Tumor Society/American Joint Cancer Committee. At the same time new targeted agents have been developed for treatment of GEP-NETs and it is important discuss these new agents in relation to the classification and staging system.

RESULTS

The current article is reviewing the most important clinical trials of targeting agents within the field of neuroendocrine tumors. Tyrosine kinase inhibitors as well as PI3 kinase mTOR inhibitors have been applied in the treatment of neuroendocrine tumors.

CONCLUSIONS

Sunitinib and everolimus have recently been registered for treatment of pancreatic neuroendocrine tumors worldwide. The role of these new targeted agents in the treatment algorithm of neuroendocrine tumors will be discussed. A large number of phase I and phase II trials have been performed in GEP-NETs with rather limited results and no significant impact on the clinical management of patients with GEP-NETs. However, there are two phase III trials that have completely changed the treatment landscape for pancreatic neuroendocrine tumors, e.g., sunitinib and everolimus demonstrating an increased progression free survival of 11 vs. 5 months for the placebo group.

Due to the limited efficacy and considerable toxicity of conventional chemotherapy, novel cytotoxic agents and innovative noncytotoxic approaches to cancer treatment are being developed. Amongst the various hormonal agents, increasing attention is being directed to somatostatin analogs. This is largely due to the demonstration of antineoplastic activity of these compounds in a variety of experimental models in vitro and in vivo and to the elucidation of some aspects of the molecular mechanisms underlying their antineoplastic activity. On the other hand, clinical experience with somatostatin analogs in the treatment of conditions like acromegaly and GEP tumors has shown that they are well tolerated compared to other antineoplastic therapies currently in use. As a consequence, there is much ongoing clinical research to determine whether or not results from experimental studies will translate into clinically useful antineoplastic activity. Besides being used in cancer treatment and palliation, radiolabelled somatostatin analogs are employed for the localization of primary and metastatic tumors expressing somatostatin receptors. The so-called 'somatostatin receptor scintigraphy' is indeed the most important clinical diagnostic investigation for patients with suspected neuroendocrine tumors. Targeted radiotherapy, which is being evaluated in clinical trials, represents an obvious extension of somatostatin scintigraphy. Since the short half-life of native somatostatin makes continuous intravenous infusion mandatory, several long-acting analogs have been synthesized. Amongst the hundreds of peptides synthesized, octreotide (which binds mainly to SSTR-2 and SSTR-5 receptor subtypes) has been the most extensively investigated. A thorough analysis of the pharmacological activities and therapeutic efficacy of the native somatostatin and the synthetic analogs (octreotide, lanreotide and vapreotide) reveals that the biological actions of these peptides are not always identical. These differences appear to be related to the different affinities of the natural hormone and synthetic derivatives for the different receptor subtypes. For all the three peptides long-lasting formulations have been developed to provide patients with the convenience of once or twice a month administration and to ensure stable drug serum concentrations between injections. Radiolabelled derivatives of octreotide, lanreotide and vapreotide have been synthesized and used as radiopharmaceuticals for somatostatin receptor scintigraphy and somatostatin receptor-targeted radiotherapy. The safety profile of synthetic somatostatin analogs is well established. Most adverse reactions to these peptides are merely a consequence of their pharmacological activity and consist mainly of gastrointestinal complaints, cholelithiasis and effects on glucose metabolism. They are often of little clinical relevance, thus making somatostatin analogs safe drugs for long-term use. While immediate release preparations are the drugs of choice in the short term, long-acting formulations are better indicated, on an outpatient basis, for the long-term management of chronic conditions. New 'receptor-selective' and 'universal' somatostatin analogs are being developed and combinations of currently available derivatives with other (cytotoxic and/or hormonal) agents are being explored in the search for an efficacious and well-tolerated treatment of the various malignancies. Somatostatin receptor-targeted chemotherapy (with conjugates of somatostatin peptides with cytotoxic drugs) and gene therapy (e.g. transferring the SSTR-2 gene into neoplastic cells), which have been successfully tested in experimental studies, should be applied to human beings in a not too distant future.

BACKGROUND

A gene expression profile (GEP) test able to accurately identify risk of metastasis for patients with cutaneous melanoma has been clinically validated.

OBJECTIVE

We aimed for assessment of the prognostic accuracy of GEP and sentinel lymph node biopsy (SLNB) tests, independently and in combination, in a multicenter cohort of 217 patients.

METHODS

Reverse transcription polymerase chain reaction (RT-PCR) was performed to assess the expression of 31 genes from primary melanoma tumors, and SLNB outcome was determined from clinical data. Prognostic accuracy of each test was determined using Kaplan-Meier and Cox regression analysis of disease-free, distant metastasis-free, and overall survivals.

RESULTS

GEP outcome was a more significant and better predictor of each end point in univariate and multivariate regression analysis, compared with SLNB (P < .0001 for all). In combination with SLNB, GEP improved prognostication. For patients with a GEP high-risk outcome and a negative SLNB result, Kaplan-Meier 5-year disease-free, distant metastasis-free, and overall survivals were 35%, 49%, and 54%, respectively.

CONCLUSIONS

Within the SLNB-negative cohort of patients, overall risk of metastatic events was higher (∼30%) than commonly found in the general population of patients with melanoma.

CONCLUSIONS

In this study cohort, GEP was an objective tool that accurately predicted metastatic risk in SLNB-eligible patients.

BACKGROUND

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are uncommon but clinically challenging and fascinating tumors. GEP-NETs present as either functional or as nonfunctional tumors. Functional tumors are commonly associated with a specific hormonal syndrome directly related to a hormone secreted by the tumor, like gastrinomas with a Zollinger-Ellison syndrome or carcinoid syndrome in patients with neuroendocrine tumors (NET) of the ileum. Nonfunctional tumors do not secrete a hormone resulting in a clinical syndrome.

METHODS

The natural course of GEP-NETs is highly variable. Small, benign neoplasms such as 90% of all insulinomas or gastric endocrine tumors type 1 are readily curable by surgical resection; however, most other GEP-NETs have a much less favorable prognosis. Patients with completely resected tumors generally have a good prognosis, and an aggressive surgical approach in patients with advanced disease may also prolong survival.

CONCLUSIONS

This review focuses on the current standards of surgical treatment of gastric endocrine tumors, NETs of the pancreas (PNET) and NETs of the ileum. Although the evidence level is low in many instances due to the lack of randomized controlled trials, important treatment recommendations can be given.

Synaptic scaffolding molecule (S-SCAM) has six PDZ domains through which it interacts with N-methyl-d-aspartate receptors and neuroligin at synaptic junctions. We isolated here a novel S-SCAM-binding protein. This protein has one PDZ, one Ras association, one Ras GDP/GTP exchange protein (Ras GEP) domain, and one C-terminal consensus motif for binding to PDZ domains. We named it nRap GEP (neural Rap GEP). nRap GEP moreover has an incomplete cyclic AMP (cAMP)-binding (CAB) domain. The domain organization of nRap GEP is similar to that of Epac/cAMP-guanine nucleotide exchange factor (GEF) I, except that Epac/cAMP-GEFI has complete CAB and Ras GEP domains but lacks the other two domains and the C-terminal motif. nRap GEP showed GEP activity for Rap1 but did not bind cAMP. nRap GEP was specifically expressed in rat brain. Immunohistochemical analysis revealed that nRap GEP and S-SCAM were localized at synaptic areas of the cerebellum. These results suggest that nRap GEP is a novel neural Rap1-specific GEP which is associated with S-SCAM.

Gastroenteropancreatic (GEP) neoplasms originate from any of the various cell types belonging to the neuroendocrine system. A general characteristic of GEP endocrine tumours is that the vast majority produce and secrete a multitude of peptide hormones and amines. Many patients with malignant metastasising tumours present clinical symptoms related to hormone hyperproduction. These include the so-called carcinoid syndrome, characterised by flushing, diarrhoea, wheezing and right heart disease, which is predominantly associated with the serotonin- and tachykinins-producing carcinoids of the midgut. Several types of syndrome associated with GEP endocrine tumors are caused by overproduction of a specific hormone. For instance, the well-known Zollinger-Ellison syndrome is gastrin-mediated. The so-called 'insulinoma syndrome' depends on excessive production of insulin and proinsulin, resulting in hypoglycemia. The 'glucagonoma syndrome' is characterised by necrolytic migratory erythema, diabetes and diarrhoea. The Verner-Morrison syndrome, which is brought about by high circulating levels of vasointestinal peptide (VIP). produces severe secretory diarrhoea. Finally the 'somatostatinoma syndrome' involves gallbladder dysfunction and gallstones, diarrhoea with or without steatorrhea, and impaired glucose tolerance. The biochemical diagnosis of endocrine digestive tumors is based on general and specific markers. The best general markers are chromogranin A (CgA) and pancreatic polypeptide (PP). Specific markers for endocrine tumors include insulin, gastrin, glucagon, vaso intestinal polypeptide (VIP), somatostatin and the primary cathabolic product of serotonin, 5-hydroxyndoleacetic acid (5-HIAA). Localisation procedures commonly applied, in the diagnosis of endocrine tumours include ultrasound (US), computed tomography (CT) and somatostatin receptor scintigraphy (SRS).