Prospective evaluation of 68 Ga-DOTATATE PET/CT in limited disease neuroendocrine tumours and/or elevated serum neuroendocrine biomarkers.

CONTEXT: The 68 Ga-labelled somatostatin analogues (68 Ga-DOTA-SSAs) is becoming popular as an important diagnostic tool in neuroendocrine tumours as evidenced by a growing number of reports detailing institutional experience with various DOTA peptides. However, only few prospective studies have compared 68 Ga-DOTA-SSAs and somatostatin receptor scintigraphy (SRS) in gastroenteropancreatic neuroendocrine tumours (GEP-NETs) and pulmonary neuroendocrine tumours. OBJECTIVE: The aim of our prospective study was to perform head-to-head comparison between 68  Ga-DOTATATE PET/CT and standard imaging work-up (SI) that included multiphasic CT, liver MRI and SRS using single photon emission computed tomography. DESIGN: In this prospective study, the patients were enrolled only if they met any of the following inclusion criteria: (i) initial staging of a NETs without distant metastases on SI or neuroendocrine tumour with unknown primary on SI; (ii) restaging of NETs that could be treated by focused therapeutic interventions; (iii) elevated serum neuroendocrine hormones or peptides. The exclusion criteria was grade 3 GEP-NETs. RESULTS: Thirty-two patients were enrolled in the study. Eleven patients (6 pancreas, 4 ileum, 1 duodenal) were included for initial evaluation and staging of NETs, 8 patients (5 pancreas, 1 ileal, 1 lung, 1 duodenal gastrinoma) for restaging, and 13 patients for elevated serum neuroendocrine biomarkers (5 ectopic Cushing's syndrome, 5 organic hypoglycaemia, 1 patient each with elevated vasoactive inhibitory peptide, chromogranin A and neuron-specific enolase). 68 Ga-DOTATATE PET/CT detected more primary tumours than SRS (15/18 vs 10/18: P = .074). The missed tumours on 68 Ga-DOTATATE PET/CT were located in the lung in two cases and duodenum in one case. For other anatomical regions (nodal and distant metastasis), no statistical difference was observed between imaging modalities using 68 Ga-DOTATATE PET/CT and SRS. Overall, 68 Ga-DOTATATE PET/CT+CT+MRI detected 31/33 of the involved regions (including primaries) (29 and 22 for 68 Ga-DOTATATE and SRS, respectively). CONCLUSION: Our study shows that 68 Ga-DOTATATE PET/CT detected similar number of sites than combination of SRS, liver MRI and thoraco-abdominopelvic CT on region-based analysis. 68 Ga-DOTATATE PET/CT missed half of primary lung carcinoids with ectopic Cushing's syndrome.

Lymph node thyroglobulin in the diagnosis of metastases of thyroid carcinoma with the thyroid in situ: A prospective intraoperative study.

BACKGROUND: The use of thyroglobulin concentration in washout fluid of fine-needle aspiration (FNA-Tg) is a procedure advocated by international guidelines to diagnose metastatic LN in papillary thyroid cancer. With the increasing use of active follow-up or lobectomy alone for low-risk thyroid cancers, the determination of the diagnostic performance of FNA-Tg in the detection of metastatic PTC when the thyroid is in situ is paramount. MATERIALS AND METHODS: Prospective study with measurement of Tg in washout fluid obtained from intraoperative fine needle aspiration (FNA) cytology in order to avoid contamination from thyroid tissue and rigorously isolated punctured nodes. Receiver-operating characteristic (ROC) curve and area under the curve (AUC), optimal threshold to discriminate benign and malignant LN, sensitivity and specificity were provided. RESULTS: a total of 58 lymph nodes from 32 patients were analyzed. ROC analysis defined the optimal cutoff values of FNA-Tg at 60 ng/ml for the diagnosis of malignant LNs in patients with a thyroid in situ. Sensitivity and specificity were 75% (95% confidence interval 57.89-86.75) and 87.5% (95%CI: 69-95.66), respectively. CONCLUSION: Our results support the hypothesis that the Tg-FNA threshold for a safe diagnosis of LN metastasis in PTC is higher in presence of a thyroid gland in situ. The use of lower thresholds could result in false positive results and lead to unnecessary surgery.

Gemcitabine induces the VMP1-mediated autophagy pathway to promote apoptotic death in human pancreatic cancer cells.

BACKGROUND/AIM: Autophagy is a degradation process of cytoplasmic cellular constituents. We have described the vacuole membrane protein-1 (VMP1) whose expression triggers autophagy in mammalian cells. The aim of this study was to analyze the role of autophagy in human pancreatic cancer cell death. METHODS/RESULTS: Here we show that gemcitabine, the standard chemotherapy for pancreatic cancer, induced autophagy in PANC-1 and MIAPaCa-2 cells, as evidenced by the accumulation of acidic vesicular organelles, the recruitment of microtubule-associated protein-1 light chain-3, and electron microscopy. In addition, gemcitabine treatment induced early expression of VMP1 in cancer cells. Gemcitabine also induced apoptosis detected by morphology, annexin V-positive cells, and cleavage of caspase-3. Surprisingly, 3-methyladenine, an autophagy inhibitor, decreased apoptosis in gemcitabine-treated cells, showing that autophagy leads to cancer cell apoptotic death. Finally, VMP1 knockdown decreased autophagy and apoptosis in gemcitabine-treated cancer cells. CONCLUSIONS: The VMP1-autophagy pathway promotes apoptosis in pancreatic cancer cells and mediates gemcitabine-induced cytotoxicity. and IAP.

p8 is a new target of gemcitabine in pancreatic cancer cells.

Gemcitabine is the only available chemotherapeutic treatment of pancreatic cancers. It is, however, moderately effective, showing a tumor response rate of only 12%. The aim of this work was to identify new pathways involved in the resistance of pancreatic cancer cells to gemcitabine, in the hope of developing new adjuvant strategies to enhance its therapeutic efficacy. Comparison of gene expression patterns of five human pancreatic cancer cell lines showing different degrees of resistance to gemcitabine revealed specific overexpression of several genes in the most resistant. One of them encoded the antiapoptotic p8 protein. We found that (a) knocking down p8 expression in gemcitabine-resistant cells promoted cell death and increased caspase-3 activity; (b) forced overexpression of p8 in gemcitabine-sensitive cells increased their resistance to gemcitabine-induced apoptosis; and (c) gemcitabine down-regulated p8 mRNA expression. These results suggest that, in pancreatic cancer cells, a large part of gemcitabine-induced apoptosis results from the inhibition of the constitutive antiapoptotic activity of p8. Hence, targeting the p8-associated pathway could be a new adjuvant therapy improving the response of patients with pancreatic cancer to gemcitabine treatment.

Performance of 18F-FDG PET/CT in the Characterization of Adrenal Masses in Noncancer Patients: A Prospective Study.

Context: Few prospective studies have evaluated the role of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) in the characterization of adrenal masses. Objective: To assess the performance of 18F-FDG PET/CT in the malignancy diagnosis of adrenal masses in noncancer patients. Design: Prospective multicenter study. Material and Methods: The study population consisted of 87 patients (87 adrenal masses) referred to endocrine surgeons: 56 with mass diameter ≥40 mm and 31 with a diameter <40 mm and of indeterminate nature based on unenhanced and washout CT attenuation densities. Fourteen patients had hypercortisolism. Adrenal masses were characterized by 18F-FDG PET/CT. Histology was the gold standard for the diagnosis of malignancy. In the absence of pathological proof (n = 23), the nature of the lesion was based on the 12-month imaging follow-up. Results: Fifteen adrenal masses were classified as malignant (including 11 adrenocortical carcinomas) and 72 as benign. Compared with benign lesions, malignant lesions were larger in size (P = 0.003), had higher unenhanced densities (P = 0.002), lower relative washout values (P = 0.007), and higher 18F-FDG uptake parameters (P < 10-3). The optimal threshold value of (Tumor SUVmax:Liver SUVmax) the ratio for malignancy was >1.5 with sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 86.7%, 86.1%, 56.5%, 96.9%, and 86.2%, respectively. Conclusions: Our results show that 18F-FDG PET/CT complements adrenal washout CT in the evaluation of adrenal masses and should be recommended in the evaluation of large and/or indeterminate adrenal masses.

Quality of life, clinical outcomes and safety of early prophylactic levothyroxine administration in patients with Graves' hyperthyroidism undergoing radioiodine therapy: a randomized controlled study.

OBJECTIVE: While radioiodine therapy is commonly used for treating Graves' disease, a prolonged and clinical hypothyroidism may result in disabling symptoms leading to deterioration of quality of life (QoL) of patients. Introducing levothyroxine (LT4) treatment in the early post-therapeutic period may be an interesting approach to limit this phenomenon. METHODS: A multicenter, prospective, open-label randomized controlled trial enrolled 94 patients with Graves' hyperthyroidism randomly assigned to the experimental group (n=46) (group A: early prophylactic LT4 treatment) or the control group (n=48) (group B: standard follow-up). The primary endpoint was the 6-month QoL. The secondary endpoints were other QoL scores such as Graves' ophthalmopathy (GO) outcomes, thyroid function tests and safety. RESULTS: The primary endpoint at 6 months was achieved: the mental composite score (MCS) of Short Form 36 (SF-36) was significantly higher in group A compared to group B (P=0.009). Four other dimension scores of the SF-36 and four dimension scores of the thyroid-specific patient-reported outcome (ThyPRO) significantly differed between the two groups, indicating better QoL in group A. After adjustment for variables, the early LT4 administration strategy was found as an independent factor for only two scores of SF-36: the MCS and the general health (GH) score. There were no differences in GO, final thyroid status and changes in the anti-TSH receptor antibodies (TRAbs) levels between the two groups. No adverse cardiovascular event was reported. CONCLUSION: Early LT4 administration post-radioactive iodine (RAI) could represent a safe potential benefit for patients with regard to QoL. The optimal strategy taking into account administered RAI activities and LT4 treatment dosage and timing remains to be determined.

Targeting dopamine receptors subtype 2 (D2DR) in pheochromocytomas: head-to-head comparison between in vitro and in vivo findings.

CONTEXT: Dopamine subtype 2 receptors (D2DRs) are overexpressed in pheochromocytomas (PHEOs). D2DR-expressing tumors can be visualized by iodine-123 labeled iodobenzamide (¹²³I-IBZM) single-photon emission computed tomography (SPECT). OBJECTIVE: The hypothesis of this study was that D2DR high expression in PHEOs would allow in vivo visualization through ¹²³I-IBZM SPECT. The present prospective pilot study aims to evaluate the performance of ¹²³I-IBZM SPECT in PHEOs and to correlate the tumor uptake with D2DR expression in tumor samples after surgery. SETTING, MATERIALS, AND METHODS: Ten unrelated patients with PHEOs were evaluated, prior to adrenalectomy, with ¹²³I-IBZM SPECT (whole body scan at 4 and 24 h after the injection; and SPECT centered on the abdomen at 24 h). D2DR mRNA and protein expressions were evaluated in all tumors by quantitative real-time RT-PCR and immunohistochemistry, respectively. MAIN OUTCOME MEASURE: Intensity of tumoral uptake of ¹²³I-IBZM was measured. RESULTS: All PHEOs express D2DR mRNA (ranging from 2.1 to 14.7 copy/copy ß-glucuronidase) and protein (immunostaining score: moderate or strong in 9 of 10 cases). However, none of the patients (0%) showed an increased tumor uptake of ¹²³I-IBZM. CONCLUSIONS: These results suggest that ¹²³I-IBZM is not a useful radiopharmaceutical in the detection and characterization of PHEOs despite D2DR expression. Our findings and data from the related literature may support different hypotheses to explain the failure of D2DR targeting by ¹²³I-IBZM.

An N-terminally truncated mutant of human chemokine CXCL14 has biological activity.

Chemokines are members of the superfamily of cytokines involved in: (i) cell migration to sites of infection; or (ii) cellular stress during an immune response. Human CXCL14/BRAK is a monocyte-selective chemokine expressed in all normal tissues, but is also involved in the development of several cancers. We describe the expression, structural characterization and biological activity of an N-terminal truncated mutant of CXCL14, ΔCXCL14, where the first eleven residues and the two disulphide bridges were removed. We designed this species in order to analyse the biological importance of the disulphide bonds and the flexible N terminus of CXCL14 for its protein folding, stability and function. The mutant ΔCXCL14 is biologically active, as suggested by the in vitro assays with migration of pancreatic cancer cells, but also its structure is not well-fixed, as suggested by fluorescence, CD and NMR. We conclude that the disulphide bridges are important in maintaining the structure of this chemokine, but they are not necessary for the biological activity of CXCL14 species.

The TP53INP2 protein is required for autophagy in mammalian cells.

Using a bioinformatic approach, we identified a TP53INP1-related gene encoding a protein with 30% identity with tumor protein 53-induced nuclear protein 1 (TP53INP1), which was named TP53INP2. TP53INP1 and TP53INP2 sequences were found in several species ranging from Homo sapiens to Drosophila melanogaster, but orthologues were found neither in earlier eukaryotes nor in prokaryotes. To gain insight into the function of the TP53INP2 protein, we carried out a yeast two-hybrid screening that showed that TP53INP2 binds to the LC3-related proteins GABARAP and GABARAP-like2, and then we demonstrated by coimmunoprecipitation that TP53INP2 interacts with these proteins, as well as with LC3 and with the autophagosome transmembrane protein VMP1. TP53INP2 translocates from the nucleus to the autophagosome structures after activation of autophagy by rapamycin or starvation. Also, we showed that TP53INP2 expression is necessary for autophagosome development because its small interfering RNA-mediated knockdown strongly decreases sensitivity of mammalian cells to autophagy. Finally, we found that interactions between TP53INP2 and LC3 or the LC3-related proteins GABARAP and GABARAP-like2 require autophagy and are modulated by wortmannin as judged by bioluminescence resonance energy transfer assays. We suggest that TP53INP2 is a scaffold protein that recruits LC3 and/or LC3-related proteins to the autophagosome membrane by interacting with the transmembrane protein VMP1. It is concluded that TP53INP2 is a novel gene involved in the autophagy of mammalian cells.

The WSB1 gene is involved in pancreatic cancer progression.

BACKGROUND: Pancreatic cancer cells generate metastases because they can survive the stress imposed by the new environment of the host tissue. To mimic this process, pancreatic cancer cells which are not stressed in standard culture conditions are injected into nude mice. Because they develop xenografts, they should have developed adequate stress response. Characterizing that response might provide new strategies to interfere with pancreatic cancer metastasis. METHODOLOGY/PRINCIPAL FINDINGS: In the human pancreatic cancer cell lines Panc-1, Mia-PaCa2, Capan-1, Capan-2 and BxPC3, we used Affymetrix DNA microarrays to compare the expressions of 22.000 genes in vitro and in the corresponding xenografts. We identified 228 genes overexpressed in xenografts and characterized the implication of one of them, WSB1, in the control of apoptosis and cell proliferation. WSB1 generates 3 alternatively spliced transcripts encoding distinct protein isoforms. In xenografts and in human pancreatic tumors, global expression of WSB1 mRNA is modestly increased whereas isoform 3 is strongly overexpressed and isoforms 1 and 2 are down-regulated. Treating Mia-PaCa2 cells with stress-inducing agents induced similar changes. Whereas retrovirus-forced expression of WSB1 isoforms 1 and 2 promoted cell growth and sensitized the cells to gemcitabine- and doxorubicin-induced apoptosis, WSB1 isoform 3 expression reduced cell proliferation and enhanced resistance to apoptosis, showing that stress-induced modulation of WSB1 alternative splicing increases resistance to apoptosis of pancreatic cancer cells. CONCLUSIONS/SIGNIFICANCE: Data on WSB1 regulation support the hypothesis that activation of stress-response mechanisms helps cancer cells establishing metastases and suggest relevance to cancer development of other genes overexpressed in xenografts.

The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells.

Autophagy is a degradation process of cytoplasmic cellular constituents, which serves as a survival mechanism in starving cells, and it is characterized by sequestration of bulk cytoplasm and organelles in double-membrane vesicles called autophagosomes. Autophagy has been linked to a variety of pathological processes such as neurodegenerative diseases and tumorigenesis, which highlights its biological and medical importance. We have previously characterized the vacuole membrane protein 1 (VMP1) gene, which is highly activated in acute pancreatitis, a disease associated with morphological changes resembling autophagy. Here we show that VMP1 expression triggers autophagy in mammalian cells. VMP1 expression induces the formation of ultrastructural features of autophagy and recruitment of the microtubule-associated protein 1 light-chain 3 (LC3), which is inhibited after treatment with the autophagy inhibitor 3-methiladenine. VMP1 is induced by starvation and rapamycin treatments. Its expression is necessary for autophagy, because VMP1 small interfering RNA inhibits autophagosome formation under both autophagic stimuli. VMP1 is a transmembrane protein that co-localizes with LC3, a marker of the autophagosomes. It interacts with Beclin 1, a mammalian autophagy initiator, through the VMP1-Atg domain, which is essential for autophagosome formation. VMP1 endogenous expression co-localizes with LC3 in pancreas tissue undergoing pancreatitis-induced autophagy. Finally, VMP1 stable expression targeted to pancreas acinar cell in transgenic mice induces autophagosome formation. Our results identify VMP1 as a novel autophagy-related membrane protein involved in the initial steps of the mammalian cell autophagic process.