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Thioredoxin Reductase (TrxR) is a key enzyme in reactive oxygen species (ROS) detoxification and in redox regulation. Because cancer cells produce increased steady-state levels of ROS (i.e., superoxide and hydrogen peroxide), TrxR is viable target in clinical trials using the anti-rheumatic drug, auranofin (AF). To extend these observations to small cell lung cancer (SCLC), AF-mediated TrxR inhibition as well as tolerability and tumor growth inhibition was determined in a xenograft model. AF was administered intraperitoneal, daily or twice daily for 1 to 5 days in mice bearing DMS273 xenografts. AF uptake was determined by mass spectrometry of gold and inhibition of TrxR in the tumor was determined. The optimal dose was 4 mg/kg once daily resulting in 18 μM gold in the plasma and 50% inhibition of TrxR activity in DMS273 SCLC tumors. This regimen given for 14 days provided a trend for prolonged median survival from 17.5 to 22 days (p=0.058, N=20 controls, 19 AF) without causing changes in bodyweight, bone marrow toxicity, blood urea nitrogen or creatinine. These results support the hypothesis that AF is an effective inhibitor of TrxR and suggest that AF could be used as an adjuvant in radio-chemotherapy protocols to enhance therapeutic efficacy.

Background: Histone deacetylase (HDAC) inhibitors have been shown in preclinical studies to upregulate norepinephrine transporters in neuroblastoma and pheochromocytoma, and somatostatin receptors in pulmonary carcinoid, small cell lung cancer, and pancreatic neuroendocrine malignancies. This pilot imaging study in humans focuses on midgut neuroendocrine carcinoma metastatic to the liver, evaluating the effect of pretreatment with the HDAC inhibitor vorinostat on uptake of (123)I-MIBG and (68)Ga-DOTATOC. Materials and Methods: Multiple midgut neuroendocrine liver metastases in clinically stable subjects were imaged with (123)I-MIBG and (68)Ga-DOTATOC before and after a 4-d course of vorinostat. Scans were performed with strict attention to detail and timed about 1 month apart occurring just before monthly long-acting octreotide administrations. Uptake changes in tumor and normal liver parenchyma were assessed on positron emission computed tomography (PET/CT) with standardized uptake values and on single photon emission computed tomography (SPECT) with qualitative ratio images. Results: The experimental units were metastatic liver lesions within patients (n = 50). There was no significant difference in administered activity or uptake time between pairs of scans for either radiotracer. Statistically significant increase in maximum standardized uptake values (SUV(max)) averaged over all lesions was noted on the (68)Ga-DOTATOC PET scans (+11%, p

Peptide receptor radionuclide therapy (PRRT) is an effective treatment for metastatic neuroendocrine tumors. Delivering a sufficient tumor radiation dose remains challenging because of critical-organ dose limitations. Adding 131I-metaiodobenzylguanidine (131I-MIBG) to PRRT may be advantageous in this regard. Methods: A phase 1 clinical trial was initiated for patients with nonoperable progressive neuroendocrine tumors using a combination of 90Y-DOTATOC plus 131I-MIBG. Treatment cohorts were defined by radiation dose limits to the kidneys and the bone marrow. Subject-specific dosimetry was used to determine the administered activity levels. Results: The first cohort treated subjects to a dose limit of 1,900 cGy to the kidneys and 150 cGy to the marrow. No dose-limiting toxicities were observed. Tumor dosimetry estimates demonstrated an expected dose increase of 34%–83% using combination therapy as opposed to 90Y-DOTATOC PRRT alone. Conclusion: These findings demonstrate the feasibility of using organ dose for a phase 1 escalation design and suggest the safety of using 90Y-DOTATOC and 131I-MIBG.

Introduction/Background: Atypical lung carcinoids and lung neuroendocrine carcinomas (NECs) are currently incurable. Most of these cancers express the C-X-C chemokine receptor 4 (CXCR4), making CXCR4 an attractive target for cancer diagnosis and treatment using radioligand therapy (RLT) with nuclides such as alpha emitter Pb212 conjugated to Pentixather (Pent). Alpha particles produce dense ionization tracks and damage cytosolic structures such as mitochondria, leading to the generation of reactive oxygen species such as superoxide (O2 ●−) and hydroperoxides (H2O2 and ROOH) in targeted cancer cells. The FDA approved thioredoxin reductase inhibitor, auranofin (Aur), inhibits peroxiredoxin-mediated hydroperoxide metabolism increasing cell killing in targeted cancer cells. Hypothesis: Inhibition of hydroperoxide metabolism using Aur can enhance therapeutic efficacy of alpha particle emitting 212Pb-Pent in preclinical models of lung carcinoids and NECs. Results: Clinically, IHC revealed greater than 75% of atypical carcinoids and NECs tested had moderate to high CXCR4. qRT-PCR, flow cytometry and immunohistochemistry performed on different lung carcinoid and NEC lung cell lines, determined that the majority moderately or highly express CXCR4. The minimal non-toxic dose of Aur that inhibits 50-70% of thioredoxin reductase activity was 4 mg/kg (intraperitoneal once a day) in DMS273 (small cell lung cancer) xenografts 212Pb-Pent given IV (1 and 3 µCu/g) was effective at increasing the survival of mice with DMS273 xenografts. Mice were then treated with 1.5, 3 or 6 µCu/g 212Pb-Pent w/wo Aur 4 mg/kg/day. A trend test showed that both increasing dose of 212Pb-Pent and the addition of Aur, caused a significant (p Conclusion: The use of 212Pb-Pent therapy to target CXCR4 on NECs showed pre-clinical efficacy in a dose dependent manner that was enhanced by Aur without significant normal tissue toxicity supporting the hypothesis that CXCR4 can be successfully targeted with the radionuclide theragnostic pair, Pb212/Pb203-pentixather. Citation Format: Melissa A. Fath, Dijie Liu, Claudia Robles-Planells, Jordan T. Ewald, Keegan A. Christensen, Spenser S. Johnson, Stephen A. Graves, Douglas R. Spitz, Yusuf Menda, M. Sue O’Dorisio. Targeting CXCR4 and thioredoxin reductase in high grade neuroendocrine tumors and neuroendocrine carcinomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5034.

Simple Summary We compared the safety and efficacy of targeted radionuclide therapy between elderly (79 years old and older) and disease-matched younger patients (between 60 and 70 years of age) with metastatic neuroendocrine tumour (NET). To our knowledge, this is the first paper addressing this important clinical question of the outcome of radionuclide therapy in this particularly vulnerable population. We found that targeted radionuclide therapy did not cause increased side effects in the elderly NET population, while toxicity remains modest and comparable in both groups. We also find that survival (after adjusting for differences in life expectancy) is not inferior for the elderly compared to younger NET patients. Abstract Peptide receptor radionuclide therapy (PRRT) is a well-established treatment in somatostatin receptor-expressing neuroendocrine tumours (NETs). The safety and efficacy of PRRT in >79 years old patients (EP) have not been systematically investigated. All patients with inoperable/metastatic/progressive G1/G2 NET, >79 years (EP), treated with PRRT at the University Hospital of Basel between 2006 and 2018, were enrolled in this retrospective matched cohort study. Each patient was manually matched with ≥1 younger patient (YP = 60–70 years). The primary endpoint was toxicity. Toxicity (subacute, long-term) was graded according to the criteria for adverse events (CTCAE) v5.0. All toxicity grades ≥ 3, or whose delta (Δ) to baseline were ≥2, were considered significant. The odds ratio (OR) for developing toxicity was tested for non-inferiority of EP vs. YP. Clinical response to PRRT and overall survival (OS) were assessed as secondary outcome measures. Forty-eight EP and 68 YP were enrolled. Both cohorts were balanced regarding median time since diagnosis, tumour location, grading, treatment scheme, and baseline biochemical parameters, except for eGFR (EP: 61 ± 16 vs. YP: 78 ± 19; mL/min/1.73 m2). Twenty-two grade ≥ 3 or Δ ≥ 2 subacute hematotoxicities occurred in 10 EP (10.3% of cycles) and 37 in 19 YP (11.6% of cycles; p = NS). Long-term grade ≥ 3 renal toxicity occurred in 7 EP and 2 YP (p = NS). The median OS was 3.4 years (EP) vs. 6.0 years (YP), HR: 1.50 [0.75, 2.98], p = NS. PRRT is a valid therapeutic option in elderly NET patients with similar toxicity and non-inferior survival compared to matched younger patients.

Peptide receptor radionuclide therapy (PRRT) is an effective treatment for metastatic neuroendocrine tumors. Delivering a sufficient tumor radiation dose remains challenging because of critical-organ dose limitations. Adding

Objectives A prospective clinical trial evaluated the effect of Ga-DOTATOC positron emission tomography-computerized axial tomography (PET-CT) on change in management of patients with lung, pancreatic, and small bowel neuroendocrine tumors. The primary eligibility criterion was a histologically proven tumor with positive somatostatin receptor subtype 2A immunohistochemistry. The primary and secondary end points were change in patient management and safety. Methods Referring physicians completed questionnaires pre- and post-Ga-DOTATOC PET-CT, stating current and planned patient management, respectively, with tumor board adjudication of final management decisions. Change in management was categorized as follows: no change; minor change (additional imaging, supportive care); or major change (octreotide/lanreotide therapy, tumor biopsy, surgery, peptide receptor radiotherapy, chemotherapy, biological therapy, liver embolization). Results A major change in management was recommended for 54 (47.37%) of 114 subjects and a minor change for 6 (5.26%) of 114 subjects, with no change for 54 (47.37%) of 114 subjects. Grade 1 adverse events were observed in 26 of 114 subjects (nausea, headache, back pain, diarrhea); one grade 2 (petechiae) and one grade 3 (abdominal pain) adverse event were observed. No grade 2 or 3 adverse events were related to study drug and none required intervention. Conclusions Imaging with Ga-DOTATOC PET-CT has a significant impact on management of patients with neuroendocrine tumors.

To better understand developments in treatment of neuroendocrine tumors of the gastroenteropancreatic system, and the pivotal roles of native somatostatin and its long-acting analogues play in normal peptide regulation and neuropeptide excess associated with neuroendocrine tumors (NETs), this article delineates and defines distinct eras in the history and discovery of gastrointestinal endocrinology. We highlight the collaboration between academia and industry in basic science and the clinical research that advanced Lu-177-DOTATATE to approval as standard of care therapy for low-grade NETs. Examples of new radioisotopes and therapy compounds currently in development for diagnosis and therapy for high-grade NETs are also discussed.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

OBJECTIVE Although personalized dosimetry may be desirable for radionuclide therapy treatments, the multiple time samples required to determine the total integrated activity puts a burden on patients and clinic resources. The aim of this paper is to demonstrate that when some prior knowledge is known about the tracer kinetic parameters, the total integrated activity (and thus radiation dose) can be estimated from a single time sample. METHODS Mathematical derivations have been performed to generate equations for the total integrated activity in terms of a single time sample of activity for monoexponential and biexponential clearance. Simulations were performed using both exponential models where the rate constants and associated parameters were randomly sampled from distributions with a known mean. The actual total integrated activity for each random sample was compared with the estimated total integrated activity using the mean value of the parameters. Retrospective analysis of 90 Y DOTATOC data from a clinical trial provided a comparison of actual kidney dose with the estimated kidney dose using the single time point approach. RESULTS The optimal sampling time for the single point approach was found to be equal to the mean time of the rate constant. The simulation results for the monoexponential and biexpoential models were similar. Regressions comparing the actual and estimated total integrated activity had very high correlations (r2 > 0.95) along with acceptable standard errors of estimate, especially at the optimal sampling point. The retrospective analysis of the 90 Y DOTATOC data also yielded similar results with an r2 = 0.95 and a standard error of estimate of 61 cGy. CONCLUSIONS In situations where there is prior knowledge about the population averages of kinetic parameters, these results suggest that the single time point approach can be used to estimate the total integrated activity and dose with sufficient accuracy to manage radionuclide therapy. This will make personalized dosimetry much easier to perform and more available to the community.

Localization of the site of the unknown primary tumor is critical for surgical treatment of patients presenting with neuroendocrine tumor (NET) with metastases. Methods: Forty patients with metastatic NET and unknown primary site underwent 68Ga-DOTATOC PET/CT in a single-site prospective study. The 68Ga-DOTATOC PET/CT was considered true-positive if the positive primary site was confirmed by histology or follow-up imaging. The scan was considered false-positive if no primary lesion was found corresponding to the 68Ga-DOTATOC-positive site. All negative scans for primary tumor were considered false-negative. A scan was classified unconfirmed if 68Ga-DOTATOC PET/CT suggested a primary, however, no histology was obtained and imaging follow-up was not confirmatory. Results: The true-positive, false-positive, false-negative, and unconfirmed rates for unknown primary tumor were 38%, 7%, 50%, and 5%, respectively. Conclusion:68Ga-DOTATOC PET/CT is an effective modality in the localization of unknown primary in patients with metastatic NET.

Background: Pheochromocytoma and paraganglioma (PHEO/PGL) are rare neuroendocrine tumors which may cause potentially life-threatening complications, with about a third of cases found to harbor specific gene mutations. Thus, early diagnosis, treatment, and meticulous monitoring are of utmost importance. Because of low incidence of succinate dehydrogenase complex subunit A (SDHA)-related metastatic PHEO/PGL, currently there exists insufficient clinical information, especially with regards to its diagnostic and treatment characteristics. Methods: Ten patients with SDHA-related metastatic PHEO/PGL were followed-up prospectively and/or retrospectively between January 2010-July 2018. They underwent biochemical tests (n = 10), 123I-MIBG (n = 9) scintigraphy, and multiple whole-body positron emission tomography/computed tomography (PET/CT) scans with 68Ga-DOTATATE (n = 10), 18F-FDG (n = 10), and 18F-FDOPA (n = 6). Results: Our findings suggest that these tumors can occur early and at extra-adrenal locations, behave aggressively, and have a tendency to develop metastatic disease within a short period of time. None of our patients had a family history of PHEO/PGL, making them appear sporadic. Nine out of 10 patients showed abnormal PHEO/PGL-specific biochemical markers with predominantly noradrenergic and/or dopaminergic phenotype, suggesting their utility in diagnosing and monitoring the disease. Per patient detection rates of 68Ga-DOTATATE (n = 10/10), 18F-FDG (n = 10/10), 18F-FDOPA (n = 5/6) PET/CT, and 123I-MIBG (n = 7/9) scintigraphy were 100, 100, 83.33, and 77.77%, respectively. Five out of 7 123I-MIBG positive patients had minimal 123I-MIBG avidity or detected very few lesions compared to widespread metastatic disease on 18F-FDG PET/CT, implying that diagnosis and treatment with 123/131I-MIBG is not a good option. 68Ga-DOTATATE PET/CT was found to be superior or equal to 18F-FDG PET/CT in 7 out of 10 patients and hence, is recommended for evaluation and follow-up of these patients. All 7 out of 7 patients who received conventional therapies (chemotherapy, somatostatin analog therapy, radiation therapy, 131I-MIBG, peptide receptor radionuclide therapy) in addition to surgery showed disease progression. Conclusion: In our cohort of patients, SDHA-related metastatic PHEO/PGL followed a disease-course similar to that of SDHB-related metastatic PHEO/PGL, showing highly aggressive behavior, similar imaging and biochemical phenotypes, and suboptimal response to conventional therapies. Therefore, we recommend careful surveillance of the affected patients and a search for effective therapies.

68Ga-DOTA-tyr3-Octreotide (68Ga-DOTATOC) PET/CT has been shown to have high accuracy in adults with neuroendocrine tumors, however has not been studied in pediatric patients. This study evaluated the safety and accuracy of 68Ga-DOTATOC PET/CT in children and young adults with solid tumors that express somatostatin receptor type 2. A series of three prospective, IRB approved, clinical trials evaluating safety and efficacy of 68Ga-DOTATOC PET/CT were conducted for subjects aged 6 months to 90 years. This study reports the results for the 26 children and young adults, aged 16 months to 29 years who participated in these trials. The administered activity of 68Ga-DOTATOC was 1.59 MBq/kg with an upper limit of 111 MBq for subjects < 18 years and 148 MBq for young adults. Safety was assessed with laboratory studies and patient/parent report of symptoms before and after the scan. Scans were interpreted in consensus by two board-certified nuclear medicine physicians. Each scan was categorized on a patient basis as true positive, true negative, false negative or false positive against a reference standard that included a combination of histopathology, other imaging modalities and clinical follow-up. Nine Grade I adverse events (AEs) occurred among 26 subjects, none of which were attributable to 68Ga-DOTATOC. Sensitivity of 68Ga-DOTATOC PET/CT was 88% (14 true positive, 2 false negative) and specificity was 100% (10 true negative, 0 false positive). 68Ga-DOTATOC PET/CT is safe and accurate in children and young adults with solid tumors expressing somatostatin receptor type 2.

Small bowel (SBNETs) and pancreatic neuroendocrine tumors (PNETs) often present with liver metastases. Although liver biopsy establishes a neuroendocrine diagnosis, the primary tumor site is frequently unknown without exploratory surgery. Gene expression differences in metastases may distinguish primary SBNETs and PNETs. This study sought to determine expression differences of four genes in neuroendocrine metastases and to create a gene expression algorithm to distinguish the primary site. Nodal and liver metastases from SBNETs and PNETs (n = 136) were collected at surgery under an Institutional Review Board-approved protocol. Quantitative PCR measured expression of bombesin-like receptor-3, opioid receptor kappa-1, oxytocin receptor, and secretin receptor in metastases. Logistic regression models defined an algorithm predicting the primary tumor site. Models were developed on a training set of 21 nodal metastases and performance was validated on an independent set of nodal and liver metastases. Expression of all four genes was significantly different in SBNET compared to PNET metastases. The optimal model employed expression of bombesin-like receptor-3 and opioid receptor kappa-1. When these genes did not amplify, the algorithm used oxytocin receptor and secretin receptor expression, which allowed classification of all 136 metastases with 94.1 % accuracy. In the independent liver metastasis validation set, 52/56 (92.9 %) were correctly classified. Positive predictive values were 92.5 % for SBNETs and 93.8 % for PNETs. This validated algorithm accurately distinguishes SBNET and PNET metastases based on their expression of four genes. High accuracy in liver metastases demonstrates applicability to the clinical setting. Studies assessing this algorithm's utility in prospective clinical decision-making are warranted.

Small bowel and pancreatic neuroendocrine tumors (SBNETs and PNETs) are rare tumors whose incidence is increasing. Drugs targeting the somatostatin receptor are beneficial in these tumors. To identify additional cell-surface targets, we recently found receptors and membrane proteins with gene expression significantly different from adjacent normal tissues in a small number of primary SBNETs and PNETs. We set out to validate these expression differences in a large group of primary neuroendocrine tumors and to determine whether they are present in corresponding liver and lymph node metastases.Primary SBNETs and PNETs, normal tissue, nodal, and liver metastases were collected and mRNA expression of six target genes was determined by quantitative PCR. Expression was normalized to GAPDH and POLR2A internal controls, and differences as compared to normal tissue were assessed by Welch's t test.Gene expression was determined in 45 primary PNETs with 20 nodal and 17 liver metastases, and 51 SBNETs with 50 nodal and 29 liver metastases. Compared to normal tissue, the oxytocin receptor (OXTR) showed significant overexpression in both primary and metastatic SBNETs and PNETs. Significant overexpression was observed for MUC13 and MEP1B in PNET primary tumors, and for GPR113 in primary SBNETs and their metastases. SCTR and ADORA1 were significantly underexpressed in PNETs and their metastases. OXTR protein expression was confirmed by immunohistochemistry.OXTR is significantly overexpressed relative to normal tissue in primary SBNETs and PNETs, and this overexpression is present in their liver and lymph node metastases, making OXTR a promising target for imaging and therapeutic interventions.

Neuroendocrine tumors (NETs) arise from the diffuse neuroendocrine system with approximately 55 % arising in the small bowel, 25 % in lung bronchioles, and 20 % in the pancreas. Less frequent sites include the appendix, cervix, ovaries, prostate, thyroid, breast, and rectum (Fig. 20.1). Pheochromocytoma and paraganglioma will not be considered in this review as excellent evaluations of genetics and imaging in these rare tumors have been published recently [1, 2]. Neuroendocrine tumors are graded according to the mitotic index and/or Ki-67 expression. Grade I tumors demonstrate neuroendocrine differentiation with mitotic index and Ki-67 < 2 %; grade II tumors are also well differentiated with a mitotic index of 3–10 % and Ki-67 index of 2–20 %. Grade III NETs may be either well differentiated or poorly differentiated with mitotic index greater than 10 % and Ki-67 greater than 20 %. Poorly differentiated, grade III tumors are classified as neuroendocrine carcinoma (NEC) and can be either small-cell or large-cell malignancies. However, it is important to recognize that neuroendocrine tumors of any grade can metastasize, most often to the liver, lymph nodes, or bone. The incidence and prevalence of NETs in the USA have been tracked in the Surveillance, Epidemiology, and End Results (SEER) database of the National Institutes of Health since 1973. The most recent comprehensive analysis of SEER data with regard to neuroendocrine tumors was published in 2008 [3]; the estimated rate of new diagnoses was 5.2/100,000 person-years, with a prevalence of approximately 103,000. NETs are primarily a disease of older adults, but may also be diagnosed in childhood. According to the SEER database, the incidence of NETs in the 0–29-year age group is much lower at 0.3/100,000 with a prevalence in the USA of 7724 children and young adults referenced to 1 January 2004 [4]. A rapid increase in the worldwide incidence of NETs has been observed over the past decade as first pointed out for tumors of the lung, small bowel, and pancreas [5]. This increase was confirmed for all NETs in Norway, where the incidence increased from 13.3 to 21.3 per 100,000 person-years from 1993 to 2010 [6] and for gastroenteropancreatic (GEP) NETs in Italy [7]. Much of this increase in incidence is likely due to both the increased sensitivity of newer diagnostic techniques and to the recognition that even NETs formerly classified as “benign” can metastasize.

International audience; Background: Vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating polypeptide (PACAP) are two highly homologous neuropeptides. In vitro and ex vivo experiments repeatedly demonstrate that these peptides exert pronounced immunomodulatory (primarily anti-inflammatory) actions which are mediated by common VPAC1 and VPAC2 G protein-coupled receptors. In agreement, we have shown that mice deficient in PACAP ligand or VPAC2 receptors exhibit exacerbated experimental autoimmune encephalomyelitis (EAE). However, we observed that VIP-deficient mice are unexpectedly resistant to EAE, suggesting a requirement for this peptide at some stage of disease development. Here, we investigated the involvement of VPAC1 in the development of EAE using a VPAC1-deficient mouse model.Methods: EAE was induced in wild-type (WT) and VPAC1 knockout (KO) mice using myelin oligodendrocyte glycoprotein 35–55 (MOG35–55), and clinical scores were assessed continuously over 30 days. Immune responses in the spinal cords were determined by histology, real-time PCR and immunofluorescence, and in the draining lymph nodes by antigen-recall assays. The contribution of VPAC1 expression in the immune system to the development of EAE was evaluated by means of adoptive transfer and bone marrow chimera experiments. In other experiments, VPAC1 receptor analogs were given to WT mice.Results: MOG35–55-induced EAE was ameliorated in VPAC1 KO mice compared to WT mice. The EAE-resistant phenotype of VPAC1 KO mice correlated with reduced central nervous system (CNS) histopathology and cytokine expression in the spinal cord. The immunization phase of EAE appeared to be unimpaired because lymph node cells from EAE-induced VPAC1 KO mice stimulated in vitro with MOG exhibited robust proliferative and Th1/Th17 responses. Moreover, lymph node and spleen cells from KO mice were fully capable of inducing EAE upon transfer to WT recipients. In contrast, WT cells from MOG-immunized mice did not transfer the disease when administered to VPAC1 KO recipients, implicating a defect in the effector phase of the disease. Bone marrow chimera studies suggested that the resistance of VPAC1-deficient mice was only minimally dependent on the expression of this receptor in the immunogenic/hematopoietic compartment. Consistent with this, impaired spinal cord inductions of several chemokine mRNAs were observed in VPAC1 KO mice. Finally, treatment of WT mice with the VPAC1 receptor antagonist PG97-269 before, but not after, EAE induction mimicked the clinical phenotype of VPAC1 KO mice.Conclusions: VPAC1 gene loss impairs the development of EAE in part by preventing an upregulation of CNS chemokines and invasion of inflammatory cells into the CNS. Use of VPAC1 antagonists in WT mice prior to EAE induction also support a critical role for VPAC1 signaling for the development of EAE.

Localization of the site of the unknown primary tumor is critical for surgical treatment of patients presenting with neuroendocrine tumor (NET) with metastases. Methods: Forty patients with metastatic NET and unknown primary site underwent 68Ga-DOTATOC PET/CT in a single-site prospective study. The 68Ga-DOTATOC PET/CT was considered true-positive if the positive primary site was confirmed by histology or follow-up imaging. The scan was considered false-positive if no primary lesion was found corresponding to the 68Ga-DOTATOC–positive site. All negative scans for primary tumor were considered false-negative. A scan was classified unconfirmed if 68Ga-DOTATOC PET/CT suggested a primary, however, no histology was obtained and imaging follow-up was not confirmatory. Results: The true-positive, false-positive, false-negative, and unconfirmed rates for unknown primary tumor were 38%, 7%, 50%, and 5%, respectively. Conclusion: 68Ga-DOTATOC PET/CT is an effective modality in the localization of unknown primary in patients with metastatic NET.

Neuroendocrine tumors (NETs) are rare neoplasms whose incidence has increased five-fold over the past three decades1. These tumors are derived from enterochromaffin and islet cells, and may secrete a variety of polypeptides, which may cause symptoms and can be useful for diagnosis, detection, and treatment of these tumors2. Two of the most common sites of these malignancies involving the digestive system are the small bowel (SBNETs) and pancreas (PNETs), and approximately 58% of patients with SBNETs and 72% of those with PNETs present with metastatic disease3, primarily to the liver. It has been estimated that as many as 10–20% of patients with liver metastases present with an unknown primary site4, and in these cases, being able to determine the primary site has important implications for surgical approach and therapeutic measures. The preferred treatment for SBNETs and PNETs is surgical resection, even in the face of metastatic disease5. Medical therapy for metastatic NETs includes the use of somatostatin analogs, which helps to control hormonal symptoms as well as improve time to progression6. Sandostatin is an 8 amino acid analog of somatostatin which mediates its effects by binding to the type 2 somatostatin receptor (SSTR2, a G-protein coupled receptor [GPCR]) on the cell surface of NETs. Sandostatin has also been useful in localization of tumors when labeled with Indium, but its success in locating primary GI sites of NETs in patients presenting with liver metastases has been limited4. Chemotherapy has also had limited response rates in NETs, with improved survival rates in those with pancreatic tumor sites. Treatment of pancreatic NETs (PNETs) has included combinations of streptozotocin and 5-fluorouracil and/or doxorubicin, with response rates of approximately 40%7. However, in non-pancreatic NETs, the response rate is only 25%7. More recently, Sunitinib and Everolimus have been demonstrated to lead to improved disease free survival for patients with advanced PNETs8, 9. Even patients with advanced disease may have prolonged survival, with median survivals of 56 and 24 months in metastatic SBNETs and PNETs, respectively1. These statistics could potentially be further improved by uncovering new targets, such as additional cell surface receptors (besides SSTR2) which could be exploited for diagnosis, imaging, and therapy. Further understanding of these NETs could come from analysis of their expression patterns, which might then be used to categorize the genetic profiles of different primary sites. This would hold promise for determining the site of origin from biopsies of liver metastases, and to identify targets for future therapy. To date, the limited gene expression studies performed in SBNETs and PNETs have not identified consistent and useful patterns of over and underexpressed genes10–14, and therefore the objective of this study was to examine matched tumor and normal tissues from these sites, then determine whether these could be used to predict the primary sites from liver metastases.

Neuroendocrine tumors, often referred to as carcinoid tumors, are relatively rare within the pediatric and young adult populations. However, when they do occur, the more aggressive tumors can be associated with significant morbidity and even mortality in this younger age group. This article reviews the history of pediatric neuroendocrine tumors, typical clinical presentation, appropriate diagnostic studies, staging, and treatment of this unusual cancer.

This review focuses on neuroendocrine tumors (NETs) that arise in the diffuse neuroendocrine system; these rare tumors can develop in any organ that has dispersed single endocrine cells, for example, the intestine, or in an organ that has clusters of endocrine cells, for example, pancreatic islets. Previously considered benign, NETs are now recognized to recur locally or metastasize to liver and bone if not completely excised early in their course of development. This article summarizes the epidemiology and reviews the diagnostic and therapeutic challenges of NETs in children and youth, noting especially those NETs that are more prevalent in young people than in older adults.

Background The incidence, survival, and prevalence of neuroendocrine tumors (NETs) in children were determined as a first step in improving diagnosis and therapy. Outcomes were compared with neuroblastoma, a pediatric malignancy that shares several biomarkers. Methods Incidence rates, observed survival rates and 31-year limited duration prevalence counts were obtained from SEER*Stat for diagnosis years 1975 to 2006. These rates were compared between and within NETs and neuroblastoma for demographic and tumor-related variables from nine standard SEER registries for ages 0–29 years. Multivariate Cox regression was performed to identify prognostic factors for survival in NETs. Results The number of NETs was 1,073 compared to 1,664 neuroblastomas. The most common NET sites were lung, breast, and appendix. NET 5-year observed survival rates increased from 83% between 1975 and 1979 to 84% for the 2000–2006 period, while analogous neuroblastoma survival rates steadily increased from 45–73%. Five-year observed survival was less than 30% in females with NETs of the cervix and ovary. The estimated 31-year limited duration prevalence for NETs as of January 1, 2006 in the U.S. population was 7,724 compared to 9,960 for neuroblastomas. Age-adjusted multivariate Cox Regression demonstrated small cell histology, primary location in the breast, and distant stage as major predictors of decreased survival. Conclusions While survivorship has significantly increased for neuroblastoma, those diagnosed with NETs have shown no increase in survival during this 31-year period. NETs constitute an unrecognized cancer threat to children and young adults comparable to neuroblastoma in both number of affected persons and disease severity. Pediatr Blood Cancer. 2010;56:50–57. © 2010 Wiley-Liss, Inc.

Pheochromocytomas, intra-adrenal paraganglioma, and extra-adrenal sympathetic and parasympathetic paragangliomas are neuroendocrine tumors derived from adrenal chromaffin cells or similar cells in extra-adrenal sympathetic and parasympathetic paraganglia, respectively. Serious morbidity and mortality rates associated with these tumors are related to the potent effects of catecholamines on various organs, especially those of the cardiovascular system. Before any surgical procedure is done, preoperative blockade is necessary to protect the patient against significant release of catecholamines due to anesthesia and surgical manipulation of the tumor. Treatment options vary with the extent of the disease, with laparoscopic surgery being the preferred treatment for removal of primary tumors. Medullary thyroid cancer (MTC) is a malignancy of the thyroid C cells or parafollicular cells. Thyroid C cells elaborate a number of peptides and hormones, such as calcitonin, carcinoembryonic antigen, and chromogranin A. Some or all of these markers are elevated in patients with MTC and can be used to confirm the diagnosis as well as to follow patients longitudinally for recurrence. Medullary thyroid cancer consists of a spectrum of diseases that ranges from extremely indolent tumors that are stable for many years to aggressive types associated with a high mortality rate. Genetic testing for RET mutations has allowed identification of familial cases and prophylactic thyroidectomy for cure. The only curative treatment is complete surgical resection.

Objective Pancreastatin is a fragment of the chromogranin A (CgA) molecule. Existing pancreastatin assays, which depend on antibodies that cross-react in varying percents with the larger prohormone, may lack sensitivity and specificity to detect small changes in neuroendocrine tumor volume. Methods We developed a highly specific, sensitive pancreastatin assay. The antibody used recognizes the carboxyl terminal of the peptide hormone and was raised against a 17-amino acid porcine pancreastatin fragment with high homology with the carboxy-terminal amino acids 286-301 of the human CgA. Results Our assay measures more than 95% of circulating pancreastatin levels; has little or no cross-reactivity with CgA, even at plasma concentrations of 1000 ng/mL; and can detect pancreastatin levels of 17 pg/mL. Interassay reproducibility for the pancreastatin radioimmunoassay was determined from results of 3 quality control pools in 15 consecutive assays. Coefficients of variation for low, medium, and high pancreastatin levels were less than 20%. The sensitivity of serial pancreastatin assays to detect early liver tumor activity was demonstrated in 2 patients with slowly progressive neuroendocrine tumors and in patients undergoing surgical cytoreduction. Conclusions This highly specific, sensitive pancreastatin assay can detect small changes in liver tumor progression and is up to 100-fold more sensitive and specific than CgA assays in the United States.

Purpose Metastatic carcinoid is an incurable malignancy whose symptoms, such as diarrhea and flushing, can be debilitating and occasionally life-threatening. Although symptom relief is available with octreotide, the disease eventually becomes refractory to octreotide, leaving no proven treatment options. The goal of this study was to evaluate the clinical effect of using 90Y-edotreotide to treat symptomatic patients with carcinoid tumors. Patients and Methods Patients enrolled had metastatic carcinoid, at least one sign/symptom refractory to octreotide, and at least one measurable lesion. Study treatment consisted of three cycles of 4.4 GBq (120 mCi) 90Y-edotreotide each, once every 6 weeks. Results Ninety patients were enrolled in the study. Using Southwest Oncology Group tumor response criteria, 67 (74.%) of 90 patients (95% CI, 65.4% to 83.4%) were objectively stable or responded. A statistically significant linear trend toward improvement was demonstrated across all 12 symptoms assessed. Median progression-free survival was significantly greater (P = .03) for the 38 patients who had durable diarrhea improvement than the 18 patients who did not (18.2 v 7.9 months, respectively). Adverse events (AEs) were reported in 96.7% (87 of 90) of patients. These AEs consisted primarily of reversible GI events (76 of 90), which could be caused in part by concomitant administration of amino acid solution given to reduce radiation exposure to the kidneys. There was one case each of grade 3 oliguria and grade 4 renal failure, each lasting 6 days. Conclusion 90Y-edotreotide treatment improved symptoms associated with malignant carcinoid among subjects with no treatment alternatives. Treatment was well-tolerated and had an acceptable expected AE profile.

Mutations in the RET proto-oncogene cause multiple endocrine neoplasia type 2A (MEN2A), and prophylactic thyroidectomy has generally been recommended before the age of 5 years. Patients with codon 609 mutations develop MTC at a later age and therefore the timing of prophylactic thyroidectomy is less clear. We report a three-generation family with C609Y RET mutation where members having prophylactic or therapeutic thyroidectomy call the current recommendations for age at thyroidectomy into question. Sixteen family members underwent thyroidectomy, for which clinical, laboratory, and pathological data were analyzed. A literature review of RET codon 609 mutations was carried out. Data were collected from 16 patients from this 38-member kindred. None of these affected members had pheochromocytoma, and one had a parathyroid adenoma. Nine of 16 patients had MTC (mean age 44.7 years, range 29–59 years) and elevated basal calcitonin levels; 6 of these 9 had lymph node metastases. Two patients had C-cell hyperplasia (CCH) at ages 18 and 37 years, and five patients had normal thyroid pathology (mean age 16 years, range 5–37 years). In the literature, a family with C609Y mutation was reported, with 15 members having MTC (mean age 42 years, range 21–59 years), and 6 with CCH (mean age 24 years, range 15–37 years). The youngest patient with C609Y RET mutation and MTC was 21 years old, and the youngest patient with CCH was 15 years old at diagnosis. These data suggest that patients with RET C609Y mutations can delay thyroidectomy until 10–15 years of age, with annual calcitonin screening prior to thyroidectomy.

Increased extracellular matrix 1 (ECM1) expression, as determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), has been recently proposed as a novel and independent diagnostic marker for malignant thyroid nodules. However ECM1 protein expression has not been previously evaluated in thyroid tumors. Real-time RT-PCR is not widely available and this has important implications for the widespread use of the expression of this gene for diagnosis. The purpose of the current study is to evaluate and compare ECM1 expression by real-time RT-PCR with immunohistochemistry (IHC) as diagnostic aids in thyroid neoplasms.ECM1 expression was studied in normal thyroid tissues (n = 14) and primary thyroid tumors (19 benign, 30 malignant) using both techniques. The performance characteristics of ECM1 expression were examined with receiver operating characteristic curves and the resulting area under the curve.ECM1 was highly expressed in thyroid carcinomas, compared with benign thyroid tissues, both at the mRNA (P0.01) and protein (P = 0.06) levels. However, ECM1 mRNA expression appeared to be a more sensitive marker of thyroid malignancy than IHC (AUC = 0.77 and 0.65, respectively). ECM1 expression by both methods was useful in identifying malignant follicular, but not Hürthle cell neoplasms.We confirm that ECM1 expression has potential utility as an independent diagnostic marker for thyroid cancer, although, performance characteristics were lower than previously published. Furthermore, IHC was not as sensitive as real-time RT-PCR in identifying malignant lesions. Prospective studies are needed to further clarify the role of ECM1 expression as a diagnostic marker.

Megakaryocytopoiesis is a multistage process that involves differentiation of hematopoietic stem cells through the myeloid lineage, ultimately producing megakaryocytes and platelets. Vasoactive intestinal peptide (VIP) stimulates adenylate cyclase and induces differentiation in multiple cell types; VIP is expressed in hematopoietic stem cells and in megakaryocytes, but its function in these cells has not yet been delineated. The present study was designed to investigate whether the type 1 VIP receptor, VPAC1, mediates VIP effects on megakaryocytopoiesis. The human megakaryoblastic leukemia cell line (CMK) was transfected with VPAC1 and the transgene expression was confirmed by qualitative polymerase chain reaction and immunohistochemistry. The rate of proliferation and the patterns of differentiation were then compared for CMK and CMK/VPAC1 through multiple growth cycles. Upregulation of VPAC1 expression resulted in a decreased proliferation rate (p = 0.0003) and enhanced differentiation with CMK/VPAC1 cells having twice the cell surface area of control CMK cells (p = 0.001), thus increasing potential for proplatelet formation. These results suggest that VIP acts in an autocrine fashion via VPAC1 to inhibit megakaryocyte proliferation and induce proplatelet formation.

Combining anatomical and functional imaging can improve sensitivity and accuracy of tumor diagnosis and surveillance of pediatric malignancies. MRI is the state-of-the-art modality for demonstrating the anatomical location of brain tumors with contrast enhancement adding additional information regarding whether the tumor is neuronal or glial. Addition of SPECT imaging using a peptide that targets the somatostatin receptor (Octreoscan) can now differentiate medulloblastoma from a cerebellar pilocytic astrocytoma. Combined MRI and Octreoscan is now the most sensitive and accurate imaging modality for differentiating recurrent medulloblastoma from scar tissue. CT is the most common imaging modality for demonstrating the anatomical location of tumors in the chest and abdomen. Addition of SPECT imaging with either MIBG or Octreoscan has been shown to add important diagnostic information on the nature of tumors in chest and abdomen and is often more sensitive than CT for identification of metastatic lesions in bone or liver. Combined anatomical and functional imaging is particularly helpful in neuroblastoma and in neuroendocrine tumors such as gastrinoma and carcinoid. Functional imaging with MIBG and Octreoscan is predictive of response to molecularly targeted therapy with 131I-MIBG and 90Y-DOTA-tyr3-Octreotide. Dosimetry using combined anatomical and functional imaging is being developed for patient-specific dosing of targeted radiotherapy and as an extremely sensitive monitor of response to therapy. Both MIBG and Octreotide are now being adapted to PET imaging which will greatly improve the utility of PET in medulloblastoma as well as increase the sensitivity for detection of metastatic lesions in neuroblastoma and neuroendocrine tumors.

In this paper, we review the pediatric oncologic applications of somatostatin receptor–targeted imaging and therapy. Somatostatin receptors are expressed in high densities by embryonal tumors, such as neuroblastoma and medulloblastoma, and neuroendocrine tumors like carcinoids and islet cell tumors. We first review the distribution of these receptors in normal tissues and tumor cells. We then discuss the technique of somatostatin receptor scintigraphy (SRS) in the pediatric population. Next, the specific clinical applications of SRS with regard to the imaging of neuroblastoma, central nervous system tumors, and gastroenteropancreatic neuroendocrine tumors of childhood are discussed. Finally, we discuss the potential role of somatostatin receptor–targeted radiotherapy for improving the duration and quality of life of children with these tumors.

Plasmacytoid dendritic cells (PDC) are considered the main sentinels against viral infections and play a major role in immune tolerance. Vasoactive intestinal peptide (VIP) is a potent immunomodulator, whose role in PDC function is unknown. The present study was designed to investigate whether human PDC express VIP receptors and whether VIP has immunological effects on PDC. Using real-time RT-PCR and immunofluorescence, we demonstrated that VIP receptors VPAC1 and VPAC2 are expressed on PDC. After culturing PDC with VIP and CpG oligodeoxynucleotides for 48 h, expression of surface molecules with significance for PDC-T cell interactions as well as IFN-α secretion were quantified using FACS analysis and ELISA, respectively. For functional assays, CFSE-stained CD4+ T cells were coincubated with differentially treated PDC. T cell proliferation and production of various cytokines were determined by FACS analysis and ELISA. VIP enhanced PDC expression of CD86, MHC II, and CCR7. In contrast, VIP inhibited PDC secretion of IFN-α and expression of Neuropilin-1 and MHC I. The potential of CpG oligodeoxynucleotide-activated PDC to induce proliferation of allogeneic CD4+ T cells was impaired when VIP was present during activation. Furthermore, pretreatment of PDC with VIP resulted in a decrease of the IFN-γ:IL-4 ratio in cocultured T cells, suggesting a modulation of the immune response toward Th2. Taken together, these results strongly suggest that VIP regulates the immunological function of human PDC. VIP may thus be involved in the modulation of immune responses to viral infections as well as in the maintenance of immune tolerance.

BACKGROUND. Allelotype studies have suggested that chromosome 1p is frequently lost in thyroid cancers, thus suggesting that there is an important tumor suppressor at this location. RIZ1 (PRDM2), located on 1p36, is a recently described tumor suppressor gene and is a member of the protein methyltransferase superfamily. RIZ1 expression is lost in a variety of tumors, primarily by means of epigenetic mechanisms that involve promoter hypermethylation. METHODS. RIZ1 expression was examined in a panel of thyroid tumor cell lines and primary thyroid tissues (14 normal, 19 benign, and 31 cancerous) by using real-time polymerase chain reaction (PCR). Methylation status of the RIZ1 promoter was studied using bisulfite sequencing and methylation-specific PCR. RESULTS. The authors demonstrated that RIZ1 expression is lost in thyroid tumor cell lines and is also significantly reduced in thyroid carcinomas, when compared with normal thyroid tissues (P < .0001) and benign tumors (P = .0003). The current study results also showed that loss of RIZ1 is mediated by aberrant cytosine methylation of the RIZ1 promoter. One hundred percent of carcinomas were methylated, compared with 33% of normal thyroid tissues (P = .001). RIZ1 mRNA expression was significantly higher (P = .02) in unmethylated (1.22 ± 1.2, mean ± standard deviation [SD]), compared with methylated tissues (0.37 ± 0.42, mean ± SD). Last, treatment with a DNA methyltransferase inhibitor led to reactivation of RIZ1 expression in cell lines that had negligible RIZ1 expression at baseline. CONCLUSIONS. The current study suggested an important role for RIZ1 expression in thyroid tumorigenesis and identified a potential novel therapeutic target for tumors unresponsive to other therapies. Cancer 2006. © 2006 American Cancer Society.

Intracranial atypical teratoid/rhabdoid tumors (AT/RT) are rare with a poor prognosis. We report one case of a 7-year old girl living over 17 months after the diagnosis of AT/RT in the left frontal lobe. Treatment was partial surgical resection and post-operative radiation therapy. Radiation therapy resulted in complete response with no evidence of residual or recurrent disease more than 17 months after diagnosis. The patient has been maintained on an extensive regimen of alternative therapies since completion of radiation therapy.

Purpose: Although gliomas predominate among central nervous system (CNS) neoplasms in adulthood, embryonal tumors are the most common malignant brain tumors in children. Despite novel treatment approaches, including improved radiotherapy and high-dose chemotherapy, survival rates remain unsatisfactory. The timely diagnosis of residual or recurrent embryonal CNS tumors and thus the earliest possible time point for intervention is often hampered by inaccuracies of conventional imaging techniques. Novel and refined imaging methodologies are urgently needed. Experimental Design: We have previously demonstrated the use of somatostatin receptor imaging (SRI) in the diagnosis of recurrent and residual medulloblastomas. Here, we evaluated somatostatin receptor type 2 (sst2) expression using an antibody in an array of CNS tumors of childhood. Eight high-grade gliomas, 4 atypical teratoid/rhabdoid tumors, 7 supratentorial primitive neuroectodermal tumors (stPNET), 1 medulloepithelioma (ME), and 8 ependymomas were screened. Tumors positive in vitro were additionally analyzed in vivo using SRI. Results: Abundant expression of somatostatin receptor type 2 in stPNET, a ME, and ependymomas warranted in vivo imaging of 7 stPNET, 1 rhabdomyosarcoma, 3 ependymomas, 1 ME, and 1 glioblastoma. Although SRI was positive in 6/7 stPNET, 1 rhabdomyosarcoma, and 1 ME, none of the ependymomas nor the glioblastoma could be imaged using SRI. In selected cases SRI was more sensitive in the detection of relapse than conventional imaging by magnetic resonance imaging and computed tomography. Conclusions: SRI should be considered in the evaluation of residual or recurrent embryonal CNS tumors, especially stPNET. The strengths of SRI lie in the differentiation of reactive tissue changes versus residual or recurrent tumor, the detection of small lesions, and possibly in the distinction of stPNET from gliomas.

Purpose: To report two patients with progressive optic pathway gliomas who had reversible visual loss after treatment with nitrosourea-based chemotherapy. Design: Retrospective case series. Results: Two patients with optic pathway gliomas and progressive visual field loss were treated with nitrosourea-based chemotherapy consisting of 6-thioguanine, procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and vincristine (TPCV). Both patients experienced dramatic and sustained visual improvement after treatment. Conclusion: Patients with progressive optic pathway gliomas may benefit from initial therapy with nitrosourea-based chemotherapy.This treatment might delay or avoid the need for radiation therapy and its associated side effects, especially in younger aged children.

Vasoactive intestinal peptide (VIP) is a neurotransmitter with neurotropic effects. VIP functions through two distinct G-protein-coupled receptor subtypes (VPAC1 and VPAC2). We have demonstrated expression of VPAC1 in pediatric nervous system tumors, including medulloblastoma arising in the cerebellum and neuroblastoma arising in the adrenal medulla. More recently, we have reported the differentiation of neuroblastoma cells by upregulation of VIP type 1 receptor suggesting a role for VPAC1 in neuronal development. To understand the molecular mechanisms regulating VPAC1 expression in both cerebellum and adrenal medulla, we have cloned the human VPAC1 gene and sequenced 2.6-kb of the 5′-flanking sequence. Expression of the luciferase reporter gene under the control of this 2.6-kb human VPAC1 promoter was induced 35-fold in a human medulloblastoma cell line (DAOY) and 36-fold in a human neuroblastoma cell line (SKNSH). Analysis of 5′-unidirectional deletion derivatives of the 2.6-kb fragment demonstrated that a 241-bp sequence immediately upstream of the VPAC1 coding region retains high activity, suggesting that it contains the core promoter region. Quantitative RT-PCR analysis demonstrated that VPAC1 is expressed in mouse cerebellar and adrenal tissues. The VPAC1 promoter also directed expression of a reporter gene in cerebellum and adrenal medulla in transgenic mice. Along with our previous findings, these results suggest that VPAC1 may play a functional role in development of both cerebellum and adrenal medulla.

Several neuropeptides are secreted in high amounts in pediatric tumors such as neuroblastoma and have been used as markers of residual or recurrent disease. Plasma levels of neuropeptides might be expected to change during development, but have not been determined in normal children. We have obtained fresh plasma from cord blood of six full-term infants and from peripheral blood in 41 healthy children, ages 1 month to 21 years. Levels of six neuropeptides, vasoactive intestinal peptide (VIP), somatostatin, gastrin releasing peptide (GRP), substance P, pancreastatin and neuropeptide Y (NPY) were measured by radioimmunoassay along with insulin-like growth factor-1 (IGF-1) whose plasma levels are known to vary during development. A child with neuroblastoma was treated with the somatostatin analogue, octreotide, and the effect on plasma neuropeptides quantified. Octreotide doses of 2–3 μg/kg daily resulted in a 40–60% decrease in plasma levels of IGF-1, pancreastatin and GRP. These results are the first publication of plasma neuropeptide levels in normal children.

Background Peptide receptor radionuclide therapy (PRRT) is an effective form of treatment for patients with metastatic neuroendocrine tumors (NETs). However, delivering sufficient radiation dose to the tumor to result in a high percentage of long-term tumor remissions remains challenging because of the limits imposed on administered activity levels by radiation damage to normal tissues. The goal of this study was to evaluate the dosimetric advantages of adding 131I meta-iodobenzylguanidine (131I-MIBG) to 90Y DOTA Phe1-Tyr3-octreotide (90Y-DOTATOC) in patients with advanced stage midgut NETs. Methods Ten patients were imaged simultaneously with 131I-MIBG and 111In-pentetreotide (as a surrogate for 90Y-DOTATOC) on days 1, 2, and 3 post-administration. Blood samples were obtained at the same time points. Using dosimetry measures from this data and our previously published methodology for calculating optimal combined administered activity levels for therapy, we determined the amount of 131I-MIBG that could be added to 90Y-DOTATOC without exceeding normal organ dose limits (marrow and kidneys) along with the expected increase in associated tumor dose, if any. Results We found that a median value of 34.6 GBq of 131I-MIBG could be safely added to 90Y-DOTATOC (delivered over multiple cycles) by reducing the maximum total deliverable 90Y-DOTATOC by a median value of 24.5%. Taking this treatment approach, we found that there would be a median increase in deliverable tumor dose of 4,046 cGy in six of the ten subjects. Of note, there were a small number of metastases that were positive for only one or the other of these radiopharmaceuticals within the same subject. Conclusions We conclude that approximately half of the patients with midgut NETs that are eligible for PRRT could reasonably be expected to benefit from the addition of 131I-MIBG to 90Y-DOTATOC.

Serum neurokinin A, chromogranin A, serotonin, and pancreastatin reflect tumor burden in neuroendocrine tumors. We sought to determine whether their levels correlate with survival in surgically managed small bowel (SBNETs) and pancreatic neuroendocrine tumors (PNETs). Clinical data were collected with Institutional Review Board approval for patients undergoing surgery at one center. Progression-free (PFS) and overall (OS) survival were from the time of surgery. Event times were estimated by the Kaplan–Meier method. Preoperative and postoperative laboratory values were tested for correlation with outcomes. A multivariate Cox model adjusted for confounders. Included were 98 SBNETs and 78 PNETs. Median follow-up was 3.8 years; 62 % had metastatic disease. SBNETs had lower median PFS than PNETs (2.0 vs. 5.6 years; p

Somatostatin is known to modulate mesangial and tubular cell function and growth, but the somatostatin receptor (sst) subtypes responsible for these effects have not been defined. There are at least five different sst receptor subtypes (sst1-sst5). We used RT-PCR to demonstrate that normal human kidney consistently expresses mRNA for sst1and sst2(9 of 9 donors). Some donors expressed sst4or sst5mRNA, but none expressed sst3mRNA. Expression of sst1and sst2was further assessed by staining serial sections of normal human kidney with sst1and sst2antisera, Arachis hypogaea (AH) lectin (to define distal tubule/collecting duct cells), Phaseolus vulgaris lectin (proximal tubules), and Tamm-Horsfall protein (THP) antiserum (thick ascending limb of the loop of Henle). Specificity of antisera was demonstrated by transfection and absorption studies. Sst2, but not sst1, was expressed in glomeruli. Intense sst1and sst2staining localized exclusively to AH+and THP+tubules. Thus sst1and sst2subtype-selective analogs may be useful to beneficially modulate renal cell function in pathological conditions.

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4+ T cells and CD8+ T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4+ T cells than in CD8+ T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4+ T cells and CD8+ T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4+ T cell subpopulations is highly correlated with T cell activation.

Deletions of 17p have been consistently reported in up to 50% of medulloblastomas (MBs), and the major breakpoint interval has been localized to chromosome segment 17p11.2. Based on several reports linking aberrant DNA methylation and chromosomal disruption, we examined the methylation pattern in this region by employing restriction landmark genomic scanning (RLGS). Several CpG islands located in the major breakpoint cluster region were identified using a bacterial artificial chromosome (BAC) contig of the breakpoint region. A long-range methylation map was established for 20 MBs and 5 supratentorial primitive neuroectodermal tumors (stPNETs). Selected CpG islands were examined using Southern and bisulfite sequencing analysis. Aberrantly hypermethylated CpG islands in 17p11. 2 were found in 33% of MBs. Interestingly, one CpG island was methylated in MBs, but not in any of the examined stPNETs. A BAC clone covering three of the methylated CpG islands was partially sequenced in the search for a potential tumor suppressor gene. None of the expressed sequence tag sequences and full-length mouse/human cDNAs that were associated with aberrant methylation showed a change in expression levels due to methylation. The potential link between chromosomal instability in 17p11.2 and hypermethylation in this region is discussed.

Restriction landmark genome scanning (RLGS) is an effective genome-scanning technique capable of identifying DNA amplification and aberrant DNA methylation. Previously published methods for the cloning of human DNA fragments from RLGS gels have been successful only for high-copy-number fragments (repetitive elements or DNA amplifications). We present here the first technique capable of efficiently cloning single-copy human DNA fragments ("spots") identified in RLGS profiles. This technique takes advantage of a plasmid-based, human genomic DNA, NotI/EcoRV boundary library. The library is arrayed in microtiter plates. When clones from a single plate are pooled and mixed with genomic DNA, the resultant RLGS gel is a normal profile with a defined set of spots showing enhanced intensity for that particular plate. This was performed for a set of 32 plates as well as their pooled rows and columns. Thus, we have mapped individual RLGS spots to exact plate, row, and column addresses in the library and have thereby obtained immediate access to these clones. The feasibility of the technique is demonstrated in examples of cloning methylated DNA fragments identified in human breast tumor and testicular tumor RLGS profiles and in the cloning of an amplified DNA fragment identified in a human medulloblastoma RLGS profile.

Background Compounds targeting somatostatin-receptor-type-2 ( SSTR2 ) are useful for small bowel neuroendocrine tumor (SBNET) and pancreatic neuroendocrine tumor (PNET) imaging and treatment. We recently characterized expression of 13 cell surface receptor genes in SBNETs and PNETs, identifying three drug targets ( GIPR , OXTR , and OPRK1 ). This study set out to characterize expression of this gene panel in the less common neuroendocrine tumors of the stomach and duodenum (gastric and duodenal neuroendocrine tumors [GDNETs]). Methods Primary tumors and adjacent normal tissue were collected at surgery, RNA was extracted, and expression of 13 target genes was determined by quantitative polymerase chain reaction. Expression was normalized to GAPDH and POLR2A internal control genes. Expression relative to normal tissue (ddCT) and absolute expression (dCT) were calculated. Wilcoxon tests compared median expression with false discovery rate correction for multiple comparisons. Results Gene expression was similar in two gastric and seven duodenal tumors, and these were analyzed together. Like SBNETs ( n = 63) and PNETs ( n = 51), GDNETs showed significant overexpression compared with normal tissue of BRS3 , GIPR , GRM1 , GPR113 , OPRK1 , and SSTR2 ( P SSTR2 had the highest absolute expression in GDNETs (median dCT 4.0). Absolute expression of BRS3 , GRM1 , GPR113 , and OPRK1 was significantly lower than SSTR2 in GDNETs ( P GIPR was similar to SSTR2 (median 4.3, P = 0.4). Conclusions As in SBNETs and PNETs, GIPR shows absolute expression close to SSTR2 but has greater overexpression relative to normal tissue (21.1 versus 3.5-fold overexpression). We conclude that GIPR could provide an improved signal-to-noise ratio for imaging versus SSTR2 and represents a promising novel therapeutic target in GDNETs.