A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5' and 3' rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (approximately 208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a approximately 7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETS) are unusual and rare neoplasms for which prognostic assessment and the diagnosis of malignancy, on the basis of histology alone, represent considerable challenges for the pathologist. To date, many molecular markers have been identified with a view to providing accurate and timely prediction of response to treatment and long-term survival. Proliferation remains a key feature of tumor progression, which has been widely estimated by the immunohistochemical use of the Ki-67 nuclear antigen. Given the continued difficulties inherent in prediction of malignancy in pancreatic neuroendocrine tumors (PETs) in particular, it has become unclear whether Ki-67 is truly a reliable prognostication marker. This review seeks to better establish what the consensus is on the role of the Ki-67 proliferation index as a prognostic indicator of long-term outcome in pancreatic neuroendocrine tumors. We conclude that most studies favor the utility of the Ki-67 proliferation index despite different critical percentages and in concert with other pathological parameters in the routine work-up of PETs.

OBJECTIVE

Somatostatin receptors (SSTR) are known for an overexpression in gastroenteropancreatic neuroendocrine tumours (GEP-NET). The aim of the present study was to find out if the receptor density predicted by the semi-quantitative parameters generated from the static positron emission tomography (PET/CT) correlated with the in vitro immunohistochemistry using a novel rabbit monoclonal anti-SSTR2A antibody (clone UMB-1) for specific SSTR2A immunohistochemistry and polyclonal antibodies for SSTR1 and 3-5.

METHODS

Overall 14 surgical specimens generated from 34 histologically documented GEP-NET patients were correlated with the preoperative (68)Ga-DOTA-NOC PET/CT. Quantitative assessment of the receptor density was done using the immunoreactive score (IRS) of Remmele and Stegner; the additional 4-point IRS classification for immunohistochemistry and standardized uptake values (SUV(max) and SUV(mean)) were used for PET/CT.

RESULTS

The IRS for SSTR2A and SSTR5 correlated highly significant with the SUV(max) on the PET/CT (p < 0.001; p < 0.05) and the IRS for SSTR2A with the SUV(mean) (p < 0.013). The level of SSTR2A score correlated significantly with chromogranin A staining and indirectly to the tumour grading.

CONCLUSIONS

The highly significant correlation between SSTR2A and SSTR5 and the SUV(max) on the (68)Ga-DOTA-NOC PET/CT scans is concordant with the affinity profile of (68)Ga-DOTA-NOC to the SSTR subtypes and demonstrates the excellent qualification of somatostatin analogues in the diagnostics of NET. This study correlating somatostatin receptor imaging using (68)Ga-DOTA-NOC PET/CT with immunohistochemically analysed SSTR also underlines the approval of therapy using somatostatin analogues, follow-up imaging as well as radionuclide therapy.

OBJECTIVE

Monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) comprise heterogeneous disorders with incompletely understood molecular defects and variable clinical features. We performed gene expression profiling (GEP) with microarray data to better dissect the molecular phenotypes, sensitivity to particular chemotherapeutic agents, and prognoses of these diseases.

METHODS

Using gene expression and clinical data from 877 patients ranging from normal plasma cells (NPC) to relapsed MM (RMM), we applied gene expression signatures reflecting deregulation of oncogenic pathways and tumor microenvironment to highlight molecular changes that occur as NPCs transition to MM, create a high-risk MGUS gene signature, and subgroup International Staging System (ISS) stages into more prognostically accurate clusters of patients. Lastly, we used gene signatures to predict sensitivity to conventional cytotoxic chemotherapies among identified clusters of patients.

RESULTS

Myc upregulation and increasing chromosomal instability (CIN) characterized the evolution from NPC to RMM (P < .0001 for both). Studies of MGUS revealed that some samples shared biologic features with RMM, which comprised the basis for a high-risk MGUS signature. Regarding MM, we subclassified ISS stages into clusters based on shared features of tumor biology. These clusters differentiated themselves based on predictions for prognosis and chemotherapy sensitivity (eg, in ISS stage I, one cluster was characterized by increased CIN, cyclophosphamide resistance, and a poor prognosis).

CONCLUSIONS

GEP provides insight into the molecular defects underlying plasma cell dyscrasias that may explain their clinical heterogeneity. GEP also may also refine current prognostic and therapeutic models for MGUS and MM.

Outcome analyses for patients with gastroenteropancreatic neuroendocrine tumors (GEP NET) after peptide receptor radionuclide therapy (PRRT) are still limited, especially with regard to the impact of the Ki-67 index. Using a single-center analysis, we aimed to establish predictors of survival.

METHODS

We retrospectively analyzed a consecutive cohort of 74 patients who had metastatic GEP NET and underwent PRRT with (177)Lu-octreotate (mean activity of 7.9 GBq per cycle, aimed at 4 treatment cycles at standard intervals of 3 mo). Patients (33 with pancreatic NET and 41 with nonpancreatic GEP NET) had unresectable metastatic disease graded as G1 or G2 (G1/G2) and documented morphologic or clinical progression within less than 12 mo or uncontrolled disease under somatostatin analog treatment. Responses were evaluated according to modified Southwest Oncology Group criteria. Potential predictors of survival were analyzed with the Kaplan-Meier curve method (log-rank test) and multivariate analysis (P < 0.05).

RESULTS

The response rates were 36.5% partial response, 17.6% minor response, 35.1% stable disease, and 10.8% progressive disease for the entire cohort; 54.5% partial response, 18.2% minor response, 18.2% stable disease, and 9.1% progressive disease for pancreatic NET; and 22.0% partial response, 17.1% minor response, 48.8% stable disease, and 12.2% progressive disease for nonpancreatic GEP NET. The median progression-free survival and overall survival were 26 mo (95% confidence interval, 18.3-33.7) and 55 mo (95% confidence interval, 48.8-61.2), respectively. Besides the Ki-67 index, a Karnofsky performance score of less than or equal to 70%, a hepatic tumor burden of greater than or equal to 25%, and a baseline plasma level of neuron-specific enolase of greater than 15 ng/mL independently predicted shorter overall survival (hazard ratio, 2.1-3.1). Patients with a Ki-67 index of greater than 10% still had median progression-free survival and overall survival of 19 and 34 mo, respectively.

CONCLUSIONS

The results of this study demonstrated the favorable response and long-term outcome of patients with G1/G2 GEP NET after PRRT. Independent predictors of survival were the Ki-67 index, the patient's performance status (Karnofsky performance scale score), the tumor burden, and the baseline neuron-specific enolase level. Even patients with a Ki-67 index of greater than 10% seemed to benefit from PRRT, with a good response and a notable long-term outcome. We present the first evidence, to our knowledge, that even in patients with metastatic disease the distinction between G1 and G2-in particular, between G1 (Ki-67 index of 1%-2%) and low-range G2 (Ki-67 index of 3%-10%)-provides prognostic stratification.

OBJECTIVE

Epstein-Barr virus-positive (EBV(+)) diffuse large B-cell lymphoma (DLBCL) of the elderly is a variant of DLBCL with worse outcome that occurs most often in East-Asian countries and is uncommon in the Western hemisphere. We studied the largest cohort of EBV(+) DLBCL, independent of age, treated with rituximab combined with CHOP (R-CHOP) in developed Western countries.

METHODS

A large cohort (n = 732) of patients with DLBCL treated with R-CHOP chemotherapy is included from the multicenter consortium. This study group has been studied for expression of different biomarkers by immunohistochemistry, genetic abnormalities by FISH and mutation analysis, genomic information by gene expression profiling (GEP), and gene set enrichment analysis (GSEA).

RESULTS

Twenty-eight patients (4.0%) were positive for EBV with a median age of 60.5 years. No clinical characteristics distinguished patients with EBV(+) DLBCL from patients with EBV-negative (EBV(-)) DLBCL. Genetic aberrations were rarely seen. NF-κB p50, phosphorylated STAT-3, and CD30 were more commonly expressed in EBV(+) DLBCLs (P < 0.05). Significant differences in survival were not observed in patients with EBV(+) DLBCL versus EBV(-) DLBCL. However, CD30 expression combined with EBV conferred an inferior outcome. GEP showed a unique expression signature in EBV(+) DLBCL. GSEA revealed enhanced activity of the NF-κB and JAK/STAT pathways independent of molecular subtype.

CONCLUSIONS

The clinical characteristics of patients with EBV(+) versus EBV(-) DLBCL are similar and EBV infection does not predict a worse outcome. EBV(+) DLBCL, however, has a unique genetic signature. CD30 expression is more common in EBV(+) DLBCL and, consistent CD30 and EBV is associated with an adverse outcome. Clin Cancer Res; 20(9); 2338-49. ©2014 AACR.

Anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-negative (ALCL-ALK-) is a provisional entity in the WHO 2008 Classification that represents 2-3% of NHL and 12% of T-cell NHL. No particular risk factor has been clearly identified for ALCL, but a recent study showed an odds ratio of 18 for ALCL associated with breast implants. Usually, the architecture of involved organs is eroded by solid, cohesive sheets of neoplastic cells, with peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS) and classical Hodgkin lymphoma being the main differential diagnoses. In this regard, staining for PAX5 and CD30 is useful. Translocations involving ALK are absent, TCR genes are clonally rearranged. CGH and GEP studies suggest a tendency of ALCL-ALK- to differ both from PTCL-NOS and from ALCL-ALK+. Patients with ALCL-ALK- are usually adults with a median age of 54-61 years, and a male-to-female ratio of 0.9. At presentation, ALCL-ALK- is often in III-IV stage, with B symptoms, high International Prognostic Index score, high lactate dehydrogenase serum levels, and an aggressive course. ALCL-ALK- presents with lymph node involvement in ∼50% of cases; extranodal spread (20%) is less common. Staging work-up for ALCL-ALK- is similar to that routinely used for nodal NHL. Overall prognosis is poor, with a 5-year OS of 30-49%, which is significantly worse when compared to OS reported in patients with ALCL-ALK+ (5-year: 70-86%). Patients with systemic ALCL exhibit a significantly better survival compared with patients with PTCL-NOS, with a 5-year OS of 51% and 32%, respectively. Age, PIT scoring system, β2-microglobulin, and bone marrow infiltration are the main prognostic factors. The expression of proteins involved in the regulation of apoptosis (caspase 3, Bcl-2, PI9) and of CD56 is related to clinical outcome. ALCL-ALK- is generally responsive to doxorubicin-containing chemotherapy, but relapses are frequent. CHOP is the most commonly used regimen to treat systemic ALCL with complete remission rates of 56%, and a 10-year DFS of 28%. Encouraging results have been reported with more intensive chemotherapy regimens. The addition of etoposide improved outcome. Alemtuxumab-CHOP regimen was associated with excellent remission rate but increased toxicity. The role of high-dose chemotherapy supported by ASCT has not been investigated in a trial of exclusively ALCL patients. When used in first remission, it was associated with a 5-year PFS of 64%. High-dose chemotherapy with ASCT is the standard therapeutic option for patients with relapsed or refractory disease. The role of allogeneic transplantation in patients with relapsed/refractory ALCL remains to be defined but there are data to support the contention that a graft-versus-lymphoma effect does exist. Myeloablative conditioning has been associated with 5-year PFS and OS of 40% and 41%, respectively, but a 5-year TRM of 33% was reported. Allo-SCT can be an option for relapsed/refractory ALCL in younger patients, preferably in the setting of a clinical trial. Pralatrexate, anti-CD30 monoclonal antibodies, brentuximab vedotin (SGN-35) in particular, (131)I-anti-CD45 radioantibody, yttrium-anti-CD25 radioimmunoconjugates, histone deacetylase inhibitors, bortezomib, gemcitabine, vorinostat, lenalidomide, and their combinations represent the most appealing chemotherapy and/or targeted agents to be investigated in future trials.

Somatostatin receptors are expressed in selected human cancers. They are particularly frequently expressed in gastroenteropancreatic neuroendocrine tumors (GEP NET), including both primaries and metastases. The density is often high, the distribution is usually homogeneous. While various somatostatin receptor subtypes can be expressed in these tumors, sst2 is clearly predominant. These receptors represent the molecular basis for a number of clinical applications, including symptomatic therapy with cold octreotide in hormone-secreting GEP NET, in vivo diagnostic with Octreoscan to evaluate the extend of the disease, and 90Y-DOTATOC radiotherapy. GEP NET can, however, express peptide receptors other than somatostatin receptors: insulinomas have more glucagon-like peptide 1 receptors than somatostatin receptors, gut NET (carcinoids) may also express cholecystokinin 2, bombesin or vasoactive intestinal peptide receptors. Often, several of these peptide receptors are expressed simultaneously in GEP NET, providing a molecular basis for in vivo multireceptor targeting of those tumors.

The WHO definition of the 3 classes of well differentiated endocrine tumor, well differentiated endocrine carcinoma and poorly differentiated carcinoma, allows an efficient general classification of GEP NETs fitting European Union (EU) current clinical use. The ENETS grading and TNM staging systems are complementary tools allowing to improve patients' stratification, and their adoption is strongly recommended. However, the prognostic value of these tools is still under investigation and more data are needed to support their usefulness at all gut sites and in perspective series. Similarly, the four classes system adopted by the WHO for the endocrine tumors of the lung allows a prognostically effective stratification of patients. A common grading system is advocated for lung and GEP NETS.

Neuroendocrine tumours (NETs) constitute a heterogeneous group of tumours that are able to express cell membrane neuroamine uptake mechanisms and/or specific receptors, such as somatostatin receptors, which can be of great value in the localization and treatment of these tumours. Scintigraphy with (111)In-pentetreotide has become one of the most important imaging investigations in the initial identification and staging of gastro-enteropancreatic (GEP) tumours, whereas helical computed tomography (CT), magnetic resonance imaging (MRI), endoscopic and/or peri-operative ultrasonography are used for the precise localization of GEPs and in monitoring their response to treatment. Scintigraphy with (123)I-MIBG (meta-iodobenzylguanidine) is sensitive in the identification of chromaffin cell tumours, although scintigraphy with (111)In-pentetreotide may also have a role in the localization of malignant chromaffin cell tumours and medullary thyroid carcinoma; for further localization and monitoring of the response to treatment both CT and MRI are used with high diagnostic accuracy. More recently, positron emission tomography (PET) scanning is being increasingly used for the localization of NETs, particularly when other imaging modalities have failed, although its precise role and utility remain to be defined. Surgery is still the usual initial therapeutic, and only curative, modality of choice; however, the majority of NETs will require further treatment with somatostatin analogues and/or interferon; chemotherapy may be used for progressive and highly aggressive NETs, but its role has not been clearly defined. For those NETs that demonstrate uptake to a diagnostic scan with (123)I-MIBG or (111)In-octreotide, therapy with radionuclides such as (131)I-MIBG or (111)In/(90)Y-octreotide or other isotopes, presents a further evolving therapeutic modality.

The pathobiology of neuroendocrine tumors (NETs) is hampered by the lack of scientific tools that define their mechanisms of secretion, proliferation, and metastasis; and, currently, there are no accurate means to assess tumor behavior and disease prognosis. Molecular biologic techniques and genetic analysis may facilitate the delineation of the molecular pathology of NETs and provide novel insights into their cellular mechanisms. The current status and recent advances in assessment of the molecular basis of tumorigenesis of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) were reviewed (1981-2004). The objectives of this retrospective study were to provide a cohesive overview of the current state of knowledge and to develop a molecular understanding of these rare tumor entities to facilitate the establishment of therapeutic targets and rational management strategies. Multiple differences in chromosomal aberration patterns were noted between gastrointestinal (GI) neuroendocrine and pancreatic endocrine tumors (PETs). Divergence in gene expression patterns in the development of GI carcinoids and PETs was identified, whereas examination of the PET and GI carcinoid data demonstrated only few areas of overlap in the accumulation of genetic aberrations. These data suggest that the recent World Health Organization classification of GEP-NETs may require updating. In addition, previous assumptions of tumor similarity (pancreatic vs. GI) may be unfounded when they are examined at a molecular level. On the basis of the evolution of genetic information, enteric neuroendocrine lesions (carcinoids) and PETs may need to be classified as two distinct entities rather than grouped together as the single entity "GEP-NETs."

BACKGROUND

Tumour marker measurement gives clinicians useful information for the follow-up and management of patients with neuroendocrine tumours (NETs). The currently used tumour markers for NETs are neuron-specific enolase (NSE) and chromogranin A (CgA). The clinical accuracy of these biomarkers depends on histotype and disease extent. CgA is thought to be the optimal marker for most NETs, as it is independent of the biological characteristics of the tumour.

OBJECTIVE

In this study we investigated the value of CgA assessment with respect to the other biomarkers in the diagnosis and follow-up of patients with different types of NETs. PATIENTS AIND METHODS: We measured CgA, NSE, carcinoembryonic antigen (CEA) and urine 5-hydroxy-3-indoleacetic acid (5-HIAA) in 290 patients with 127 gastroenteropancreatic (GEP) tumours, 49 neuroblastomas, 36 lung tumours, 24 medullary thyroid carcinomas (MTCs). 15 pNETs, 12 paragangliomas. 7 Merkel's cell carcinomas (MCCs) and 20 NETs of unknown origin. CgA and 5-HIAA were quantitated by immunoenzymatic assays, while NSE and CEA were determined by radioimmunoassays.

RESULTS

The biomarkers' specificity in GEP tumours was 86% for CgA, 100% for NSE, 91% for CEA and 100% for 5-HIAA. The corresponding sensitivity was 68% for CgA, 33% for NSE, 15.4% for CEA and 35% for 5-HIAA. The sensitivity of CgA largely depends on disease extent or presence of functioning tumours and is highest in metastatic and syndromic patients. CgA determination in GEP tumour monitoring is useful to evaluate the response to therapy and to follow up patients with liver metastases. In neuroblastomas the overall specificity of NSE and CgA was 50%, and 83%, respectively. In these tumours NSE sensitivity was close to 90% in all clinical stages, while the sensitivity of CgA depended on clinical stage (50% for stage I and II, 60% for stage III and 100% for stage IV tumours). Also in this type of tumour changes in CgA levels correlated with objective response. In paragangliomas CgA measurement may provide useful clinical information. Measurement of CgA is of use in the diagnosis of lung carcinoids, while its value in MTCs, pNETs and MCCs is very limited.

CONCLUSIONS

CgA was confirmed to be the best tumour marker currently available for identifying patients suffering from NETs of the GEP system, lung carcinoids and neuroblastomas. CgA evaluation is recommended in the follow-up of patients with such tumours.

The medical treatment of neuroendocrine GEP tumours must be based on the growth properties of the tumour. Medical treatment includes chemotherapy, somatostatin analogues and alpha interferons. Chemotherapy has been particularly active in patients with high proliferating neuroendocrine tumours such as endocrine pancreatic tumours and lung carcinoids. Streptozotocin-based combinations including 5-flourouracil and doxorubicin have generated partial remissions in 40%-60% of the patients giving a median survival of about two years in patients with advanced disease. Cisplatinum plus etoposide have demonstrated significant antitumour effects in anaplastic endocrine pancreatic tumours and lung carcinoids. However, in low proliferating tumours such as classical midgut carcinoids the response rates with the same combinations of cytotoxic agents have only generated short lasting responses in less than 10% of patients. In these patients, biological treatment has been of benefit. Alpha interferon at doses of 3-9 million units three to seven times per week subcutaneously, has given biochemical response rates of 50% and significant tumour reduction in about 15% of patients with long duration, up to three years. Somatostatin analogues have been widely used in the treatment of neuroendocrine gut and pancreatic tumours. The currently available somatostatin analogues particularly bind somatostatin receptor 2 and 5 and with low affinity also receptor subtype 3. Octreotide is registered in most countries for the treatment of patients with carcinoid syndrome and also VIP and glucagon producing tumours. Regular octreotide at standard doses of 100-300 microg/day gives symptomatic responses in a medium of 60% of patients and biochemical responses in up to 70% of patients. Significant tumour responses are rare, less than 5%. Long-acting formulations of somatostatin analogues have been of significant benefit for the patients with similar response rates as for regular formulations. The quality of life has been significantly improved by using the long-acting formulations.

Little is known about the consequences of releasing genetically engineered plants (GEP) into the environment. Using opine-producing GEP, we show that transgenic plants alter their biological environment, more precisely the root-associated bacterial populations. The alterations were both transgene-specific and target population-specific. Therefore, assessment studies on the introduction of a given transgene into a GEP will be valid on the given transgene. Evidence of any transgene-associated biological effect will depend on the determination of the pertinent target populations, the identification of which is a key step of such studies.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are a heterogeneous group of neoplasms that arise from cells of the diffuse neuroendocrine system and are characterized by a wide spectrum of clinical manifestations. All NETs are potentially malignant but differ in their biologic characteristics and the probability of metastatic disease. The pathologic classification of these tumors relies on their proliferation and differentiation. In the past decades, several nomenclatures have been proposed to stratify neuroendocrine tumors, but the World Health Organization classification is the one that is most widely accepted and used. The diagnosis of neuroendocrine tumor relies on clinical manifestation, laboratory parameters, imaging features, and tissue biomarkers in a biopsy specimen. With improved understanding of the natural history and lesion biology, management of GEP-NETs has also evolved. Although surgery remains the only potentially curative therapy for patients with primary GEP-NETs, other available treatments include chemotherapy, interferon, somatostatin analogs, and targeted therapies. Recent improvements in both morphologic and functional imaging methods have contributed immensely to patient care. Morphologic imaging with contrast agent-enhanced multidetector computed tomography and magnetic resonance imaging is most widely used for initial evaluation and staging of disease in these patients, whereas functional imaging techniques are useful both for detection and prognostic evaluation and can change treatment planning.

Gastroenteropancreatic (GEP) endocrine tumors are hypervascular tumors able to synthesize and secrete high amounts of VEGF. We aimed to study the regulation of VEGF production in GEP endocrine tumors and to test whether some of the drugs currently used in their treatment, such as somatostatin analogues and mTOR inhibitors, may interfere with VEGF secretion. We therefore analyzed the effects of the somatostatin analogue octreotide, the mTOR inhibitor rapamycin, the PI3K inhibitor LY294002, the MEK1 inhibitor PD98059 and the p38 inhibitor SB203850 on VEGF secretion, assessed by ELISA and Western blotting, in three murine endocrine cell lines, STC-1, INS-r3 and INS-r9. Octreotide and rapamycin induced a significant decrease in VEGF production by all three cell lines; LY294002 significantly inhibited VEGF production by STC-1 and INS-r3 only. We detected no effect of PD98059 whereas SB203850 significantly inhibited VEGF secretion in INS-r3 and INS-r9 cells only. By Western blotting analysis, we observed decreased intracellular levels of VEGF and HIF-1alpha under octreotide, rapamycin and LY294002. For rapamycin and LY294002, this effect was likely mediated by the inhibition of the mTOR/HIF-1/VEGF pathway. In addition to its well-known anti-secretory effects, octreotide may also act through the inhibition of the PI3K/Akt pathway, as suggested by the decrease in Akt phosphorylation detected in all three cell lines. In conclusion, our study points out to the complex regulation of VEGF synthesis and secretion in neoplastic GEP endocrine cells and suggests that the inhibition of VEGF production by octreotide and rapamycin may contribute to their therapeutic effects.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease.

Chromogranin A (CGA), a protein at first detected in the adrenal medulla, has recently been found also in other organs, e.g. the endocrine pancreas. However, immunohistochemical findings concerning the cellular source of pancreatic CGA were controversial. Therefore, the endocrine pancreas of 10 mammalian species (man, tupaia, mole, cat, dog, pig, guinea pig, rabbit, rat) was investigated immunohistochemically for CGA-like immunoreactivities on serial semithin plastic sections using a high-titer polyclonal antiserum against bovine CGA. The results show that basically all pancreatic endocrine cell types are CGA-immunoreactive; however, every species has its own pattern of CGA-immunoreactive cell types. Other findings of the present studies indicate that the physiological function of CGA in pancreatic endocrine cells is related to the storage mechanisms of peptide hormones. Finally, a methodological approach is given to obtain not only qualitative but also semi-quantitative data during immunohistochemical investigations.

OBJECTIVE

To evaluate the feasibility, safety, and efficacy of transarterial chemoembolization (TACE) of progressive liver metastases from well differentiated gastroenteropancreatic endocrine (GEP) tumors with drug-eluting beads (DEBs).

METHODS

From June 2004 to July 2005, eight men and 12 women aged 34 to 75 years (mean +/- SD, 59 y +/- 12), including 13 patients with bilobar disease and seven with unilobar disease, underwent 34 sessions of TACE with DEBs (500-700 mum) loaded with doxorubicin. Morphologic response was evaluated with computed tomography (CT) at 1 and 3 months according to Response Evaluation Criteria In Solid Tumors. Clinical and laboratory data were also assessed.

RESULTS

The complete dose of 4 mL of DEBs loaded with 100 mg doxorubicin was injected during 22 TACE sessions and 1-3.5 mL of DEBs was injected during 12 TACE sessions. Three months after TACE, 16 of 20 patients (80%) exhibited a partial response, three (15%) had stable disease, and one (5%) had progressive disease. The mean size of the largest metastasis in each patient decreased from 42 mm +/- 24 before treatment (median, 39.5 mm) to 33 mm +/- 23 (median, 29 mm) 1 month after treatment and 30 mm +/- 21 (median, 26.5 mm) 3 months after treatment. After a median follow-up of 15 months (range, 6-24 months), nine patients' disease remained controlled without tumor progression and 10 patients had progressive disease. The median time to progression was 15 months. Postembolization syndrome lasted less than 7 days in 23 sessions (67%) and more than 7 days in seven sessions (22%), and no symptoms at all were observed in four sessions (11%). Peak aspartate aminotransferase, alanine aminotransferase, and bilirubin levels after TACE were 35-490 IU (mean, 125 IU +/- 77; normal, <35 IU), 20-440 IU (mean, 149 IU +/- 155; normal, <45 IU), and 8-90 mol/L (mean, 26 IU +/- 25; normal, <17 IU), respectively, at 2-3 days. In five patients, follow-up CT at 1 month revealed TACE-induced peripheral liver necrosis.

CONCLUSIONS

TACE with DEBs is well tolerated and appears effective. A comparative study with a standard TACE or transarterial embolization regimen is warranted to define the best protocol for transarterial treatment of GEP liver metastases.

Granulin-epithelin precursor (GEP/progranulin) is an autocrine growth factor for ovarian cancer. We examined the production and function of GEP and report that: (1) GEP production is regulated by endothelin (ET-1), lysophosphatidic acid (LPA), and cAMP; (2) cAMP signals GEP production through exchange protein activated by cAMP (EPAC); (3) ET-1 and cAMP/EPAC induce GEP through ERK1/2; and (4) neutralization of GEP results in apoptosis. Exposure of HEY-A8 and OVCAR3 ovarian cancer cells to LPA and ET-1 yielded GEP production and secretion in a dose- and time-dependent fashion; neither stimulated significant concentrations of cAMP directly. Stimulation of cAMP production with pertussis and cholera toxin, or forskolin induced GEP in a PKA-independent fashion. EPAC, an intracellular cAMP receptor, is activated specifically by the cAMP analog, 8-CPT-2'-O-Me-cAMP (8-CPT); 8-CPT treatment stimulated GEP production and secretion. The MEK inhibitor, U0126, abrogated GEP production in response to ET-1 and 8-CPT, confirming involvement of MAPK. A partial inhibition of basal and stimulated GEP production was observed when cells were treated with a internal calcium chelator, BAPTA. Neutralizing anti-GEP antibody reversed basal as well as LPA, ET-1 and 8-CPT-induced ovarian cancer cell growth and induced apoptosis as demonstrated by caspase-3 and PARP cleavage, DNA fragmentation, and nuclear condensation. These results indicate that GEP is a growth and survival factor for ovarian cancer, induced by LPA and ET-1 and cAMP/EPAC through ERK1/2.

The disturbed erythropoiesis in patients with refractory anaemia with ring-sideroblasts (RARS) is characterized by intramedullary apoptosis of erythroid precursors and increased iron accumulation in mitochondria. To gain insight into these pathophysiological mechanisms we compared the gene expression profile (GEP) of erythroid precursors from RARS patients to the GEP of normal erythroid precursors. Three hundred sixty four probe sets were up-, and 253 probe sets downregulated in RARS cells. Interestingly, Growth Differentiation factor 15 (GDF15), a cytokine from the TGFbeta family, was dramatically upregulated in all RARS patients. Measurement of GDF15 in the sera from twenty RARS patients confirmed this finding by showing significantly, 7.2-fold, increased protein levels (3254 +/- 1400 ng/ml vs. 451 +/- 87 ng/ml in normals). In vitro studies demonstrated erythroid-specific production of GDF15 and dependence on erythropoietin. Induction of apoptosis by arsenic trioxide, a drug which acts via reduction of the mitochondrial membrane potential, also stimulated GDF15 production. Downregulation of endogenous GDF15 production in erythoblasts by specific siRNA led to diminished erythroid differentiation. Taken together, our findings demonstrate a new role for GDF15 in normal erythropoiesis as well as in the ineffective erythropoiesis of RARS patients.

Elevated circulating chromogranin A (CgA) levels are found in neuroendocrine tumors (NETs), but the diagnostic usefulness of this marker is still debatable. To assess the role of CgA for the diagnosis of gastroenteropancreatic (GEP) NETs and the identification of metastatic patients, an Italian multicenter observational study has been performed. CgA was evaluated in 202 GEP NET patients by IRMA and ELISA. The cutoffs for diagnosis and presence of metastases were identified by receiver-operating characteristic (ROC) curve. We found good correlation between IRMA and ELISA. The ROC analysis identified a cutoff of 53 ng/ml for IRMA and 16 U/l for ELISA as discriminating between controls and patients with active disease (sensitivity 71.3 and 84%; specificity 71 and 85% respectively). Metastases were present in 123 patients, having significantly higher CgA levels than patients without metastases. ROC analysis identified a cutoff of 146 ng/ml for IRMA and 67.3 U/l for ELISA as discriminating between patients with and without metastases (sensitivity 57 and 63.3%; specificity 55.6 and 71.4% respectively). For pancreatic NETs positive and negative predictive values were 84 and 78% respectively (90% specificity and 68% sensitivity). We found lower CgA levels in patients with extensive metastatic spread than in those with liver metastases only. These data assess the role of CgA evaluation in GEP NETs, and demonstrate that higher CgA levels associate with metastatic disease, confirming that CgA levels can provide a helpful practical biochemical marker for the clinical management of NETs, but with low sensitivity and specificity.

Multiple myeloma is a hematologic malignancy characterized by the proliferation of neoplastic plasma cells in the bone marrow. Although the first-to-market proteasome inhibitor bortezomib (Velcade) has been successfully used to treat patients with myeloma, drug resistance remains an emerging problem. In this study, we identify signatures of bortezomib sensitivity and resistance by gene expression profiling (GEP) using pairs of bortezomib-sensitive (BzS) and bortezomib-resistant (BzR) cell lines created from the Bcl-XL/Myc double-transgenic mouse model of multiple myeloma. Notably, these BzR cell lines show cross-resistance to the next-generation proteasome inhibitors, MLN2238 and carfilzomib (Kyprolis) but not to other antimyeloma drugs. We further characterized the response to bortezomib using the Connectivity Map database, revealing a differential response between these cell lines to histone deacetylase (HDAC) inhibitors. Furthermore, in vivo experiments using the HDAC inhibitor panobinostat confirmed that the predicted responder showed increased sensitivity to HDAC inhibitors in the BzR line. These findings show that GEP may be used to document bortezomib resistance in myeloma cells and predict individual sensitivity to other drug classes. Finally, these data reveal complex heterogeneity within multiple myeloma and suggest that resistance to one drug class reprograms resistant clones for increased sensitivity to a distinct class of drugs. This study represents an important next step in translating pharmacogenomic profiling and may be useful for understanding personalized pharmacotherapy for patients with multiple myeloma.

OBJECTIVE

The development of a genetic signature for the identification of high-risk cutaneous melanoma tumors would provide a valuable prognostic tool with value for stage I and II patients who represent a remarkably heterogeneous group with a 3% to 55% chance of disease progression and death 5 years from diagnosis.

METHODS

A prognostic 28-gene signature was identified by analysis of microarray expression data. Primary cutaneous melanoma tumor tissue was evaluated by RT-PCR for expression of the signature, and radial basis machine (RBM) modeling was performed to predict risk of metastasis.

RESULTS

RBM analysis of cutaneous melanoma tumor gene expression reports low risk (class 1) or high risk (class 2) of metastasis. Metastatic risk was predicted with high accuracy in development (ROC = 0.93) and validation (ROC = 0.91) cohorts of primary cutaneous melanoma tumor tissue. Kaplan-Meier analysis indicated that the 5-year disease-free survival (DFS) rates in the development set were 100% and 38% for predicted classes 1 and 2 cases, respectively (P < 0.0001). DFS rates for the validation set were 97% and 31% for predicted classes 1 and 2 cases, respectively (P < 0.0001). Gene expression profile (GEP), American Joint Committee on Cancer stage, Breslow thickness, ulceration, and age were independent predictors of metastatic risk according to Cox regression analysis.

CONCLUSIONS

The GEP signature accurately predicts metastasis risk in a multicenter cohort of primary cutaneous melanoma tumors. Preliminary Cox regression analysis indicates that the signature is an independent predictor of metastasis risk in the cohort presented.