Non-neuroendocrine Tumors Research Articles

Lung Cancer Histologic and Immunohistochemical Heterogeneity in the Era of Molecular Therapies

On the basis of seminal studies in the 1980s, appreciable histologic heterogeneity, ranging from 45% to 70% of cases, may be encountered in lung cancer. However, the epidemiologic and histologic landscape of lung cancer in the last 3 decades has radically changed. In this study, 172 consecutive surgically resected primary lung carcinomas evaluated from 2010 to 2012 were entirely sampled and examined according to current histologic classifications. In 129 cases, a positive preoperative biopsy was also available. Major histologic heterogeneity (a single tumor showing at least 2 different histologic types) and minor histologic heterogeneity (a single tumor showing just 1 histologic type but at least 2 different growth patterns) were evaluated in all cases. Immunohistochemical heterogeneity (ie, "aberrant" staining) was also assessed using a panel of markers of adenocarcinoma (TTF-1, napsin, and CK7), squamous cell carcinoma (p63, CK5/6), and neuroendocrine differentiation (chromogranin and synaptophysin), both on positive biopsies and surgical specimens. Overall, major and minor histologic heterogeneity on resections were disclosed in 4% (7 cases) and 50.6% (87 cases), respectively, whereas just 1 case of minor heterogeneity (pleomorphic carcinoma) was observed on biopsies. Minor heterogeneity was limited to adenocarcinomas (82.6%, 81/98 cases) and sarcomatoid carcinomas (6 pleomorphic types among 8 sarcomatoid carcinomas). Immunohistochemical heterogeneity was recorded in 22.6% of the cases, with expression of p63 and CK5/6 in a subset of adenocarcinomas (25 cases, 25.5%), CK7 in 17.4% of squamous cell carcinomas, and synaptophysin in 6 cases of non-neuroendocrine tumors (4%, 6/155). The high rate of adenocarcinomas, accounting for 57% (98 cases) of 172 consecutively resected lung cancers, reflects the new scenario of thoracic oncology and may explain the significant lower rate of major histologic heterogeneity (4%) and the higher frequency of different architectural patterns (minor heterogeneity) that we found in lung cancer compared with previous studies.

Long-term results of hepatic resection for non-colorectal, non-neuroendocrine liver metastasis.

The significance of surgical resection for non-colorectal non-neuroendocrine tumor liver metastasis (NCNNLM) remains controversial. The present study sought to clarify the long-term outcomes of surgical resection for NCNNLM and prognostic factors after hepatectomy in a single institution. From 1993 to 2009, 145 patients underwent hepatectomy for NCNNLM. The primary sites of the hepatic tumors were gastrointestinal carcinoma in 80 cases, breast in 30, genitourinary in 12, gastrointestinal stromal tumor in 11, and miscellaneous in 12. The cumulative 1-, 3-, and 5-year overall survival rates of those who underwent hepatectomy for NCNNLM were 83.9, 55.4, and 41.0%, respectively, with median overall survival times of 41.8 months. Multivariate analysis revealed that postoperative complication was the only independent poor prognostic factor impacting on survival. Postoperative morbidity and mortality rate were 17.9% and 1.4%. There are 38 cases survived more than 5 years including 21 patients without remnant tumors due to the repeat hepatic and/or pulmonary resection for recurrence. A total of 32 patients survived without tumor and without any kinds of chemotherapy in the latest condition. Hepatectomy for NCNNLM may be beneficial and might relieve patients from excursive chemotherapy in selected patients. Meticulous surgery avoiding complication may enhance the outcome.

Evaluation of 177Lu-DOTA-sst2 Antagonist Versus 177Lu-DOTA-sst2 Agonist Binding in Human Cancers In Vitro

Somatostatin receptor targeting of neuroendocrine tumors using radiolabeled somatostatin agonists is today an established method to image and treat cancer patients. However, in a study using an animal tumor model, somatostatin receptor antagonists were shown to label sst(2)- and sst(3)-expressing tumors in vivo better than agonists, with comparable affinity even though they are not internalized into the tumor cell. In the present study, we evaluated the in vitro binding of the antagonist (177)Lu-DOTA-pNO(2)-Phe-c (DCys-Tyr-DTrp-Lys-Thr-Cys) DTyrNH(2) ((177)Lu-DOTA-BASS) or the (177)Lu-DOTATATE agonist to sst(2)-expressing human tumor samples. Forty-eight sst(2)-positive human tumor tissue samples (9 ileal carcinoids, 10 pheochromocytomas, 7 breast carcinomas, 10 renal cell carcinomas, and 12 non-Hodgkin lymphomas) were analyzed by in vitro receptor autoradiography for the expression of sst(2), comparing the binding capacity of (177)Lu-DOTA-BASS and (177)Lu-DOTATATE in successive tissue sections. The autoradiograms were quantitated using an electronic autoradiography detection system. In all cases, the radiolabeled antagonist bound to more receptor sites than did the agonist. The mean ratios of the antagonist (177)Lu-DOTA-BASS to the agonist (177)Lu-DOTATATE were 4.2 ± 0.5 in the 9 ileal carcinoids, 12 ± 3 in the 10 pheochromocytomas, 11 ± 4 in the 7 breast carcinomas, 5.1 ± 0.6 in the 10 renal cell carcinomas, and 4.8 ± 0.7 in the 12 non-Hodgkin lymphomas. The present in vitro human data, together with previous in vivo animal tumor data, are strong arguments indicating that sst(2) antagonists may be worth testing in vivo in patients in a wide range of tumors including nonneuroendocrine tumors.

Chromogranin A Restricts Drug Penetration and Limits the Ability of NGR-TNF to Enhance Chemotherapeutic Efficacy

NGR-TNF is a derivative of TNF-α that targets tumor blood vessels and enhances penetration of chemotherapeutic drugs. Because of this property, NGR-TNF is being tested in combination with chemotherapy in various phase II and III clinical trials. Here we report that chromogranin A (CgA), a protein present in variable amounts in the blood of normal subjects and cancer patients, inhibits the synergism of NGR-TNF with doxorubicin and melphalan in mouse models of lymphoma and melanoma. Pathophysiologically relevant levels of circulating CgA blocked NGR-TNF-induced drug penetration by enhancing endothelial barrier function and reducing drug extravasation in tumors. Mechanistic investigations done in endothelial cell monolayers in vitro showed that CgA inhibited phosphorylation of p38 MAP kinase, disassembly of VE-cadherin-dependent adherence junctions, paracellular macromolecule transport, and NGR-TNF-induced drug permeability. In this system, the N-terminal fragment of CgA known as vasostatin-1 also inhibited drug penetration and NGR-TNF synergism. Together, our results suggest that increased levels of circulating CgA and its fragments, as it may occur in certain cancer patients with nonneuroendocrine tumors, may reduce drug delivery to tumor cells particularly as induced by NGR-TNF. Measuring CgA and its fragments may assist the selection of patients that can respond better to NGR-TNF/chemotherapy combinations in clinical trials.

Liver resection for noncolorectal and nonneuroendocrine metastases: Results of a study on 56 patients at a single institution

The usefulness of surgical treatment for hepatic metastases of noncolorectal nonneuroendocrine (NCRNNE) tumors is not yet clear due to the natural history of these tumors, their frequent systemic dissemination and their histological heterogeneity. The aim of this study was to evaluate the long-term outcome of patients who underwent liver resection for NCRNNE metastases. For this purpose we retrospectively analyzed 202 patients who underwent liver resection for metastasis between January 1989 and December 2006 at the Department of Surgery of the University Hospital of Udine. Fifty-six patients underwent liver resection because of NCRNNE metastases. The preoperative assessment was based on hepatic ultrasonography and CT scan; PET was used in a few patients. All patients had intraoperative liver ultrasonography to evaluate the lesions and to define the resection. Gender, age, primary tumor site (gastrointestinal or nongastrointestinal), synchronous or metachronous metastasis, unilobar or bilobar localization, number and diameter of the lesion(s), type of resection, margin status, positive lymph nodes in the hepatoduodenal ligament, and time between surgery and diagnosis of liver metastases were evaluated as possible prognostic factors for survival. Univariate analysis showed that the location of the primary tumor and the disease-free interval since the treatment of the primary tumor were positive predictive factors for longer survival. Multivariate analysis showed that the only independent significant factor was gastrointestinal versus nongastrointestinal origin. Demographic data, the synchronous or metachronous appearance of metastases, their unilobar or bilobar location, number and size, the type of resection, the resection margin status and the involvement of lymph nodes did not prove to be prognostic factors.

The potential role of somatostatin analogues in the treatment of gastrointestinal (GI) cancers.

150 Background: Somatostatin receptors (sstr) are expressed in various normal and malignant tissues, including non-neuroendocrine GI solid tumors. Gastric cancer (GC) cells mainly express sstr2 and sstr5, colorectal cancer (CRC) cells predominantly express sstr1 followed by sstr5 and sstr2, and hepatocellular carcinoma (HCC) cells express mainly sstr5 as well as sstr1,2,3. Somatostatin analogues (SAs) may have direct and indirect antitumor activity. Octreotide and lanreotide primarily target sstr2, whereas the new agent pasireotide has high affinity for sstr1,2,3 and sstr5. Methods: Published data evaluating the antitumor properties of SAs in GI tumors were reviewed. Results: The table lists the results of clinical studies in which SAs were evaluated as antitumor therapies in GI cancers. Conclusions: Although octreotide and lanreotide are efficacious in the treatment of symptoms of neuroendocrine tumors, these agents have demonstrated mixed results in the antitumor treatment of non-neuroendocrine GI tumors. Data suggesting potential favorable outcomes with SAs alone or in combination with other agents have been seen in solid tumors with predominant sstr2 expression such as GC. Novel SAs such as pasireotide may have improved antitumor effects in non-neuroendocrine solid tumors expressing multiple sstr compared with octreotide and lanreotide. [Table: see text] [Table: see text]

Chromogranin A and the tumor microenvironment.

Chromogranin A (CgA) is an acidic glycoprotein belonging to a family of regulated secretory proteins stored in the dense core granules of the adrenal medulla and of many other neuroendocrine cells and neurons. This protein is frequently used as a diagnostic and prognostic serum marker for a range of neuroendocrine tumors. Circulating CgA is also increased in patients with other diseases, including subpopulations of patients with non-neuroendocrine tumors, with important prognostic implications. A growing body of evidence suggests that CgA is more than a diagnostic/prognostic marker for cancer patients. Indeed, results of in vitro experiments and in vivo studies in animal models suggest that this protein and its fragments can affect several elements of the tumor microenvironment, including fibroblasts and endothelial cells. In this article, recent findings implicating CgA as a modulator of the tumor microenvironment and suggesting that abnormal secretion of CgA could play important roles in tumor progression and response to therapy in cancer patients are reviewed and discussed.

Pasireotide (SOM230) as a Potential Treatment for Endocrine and Non- Endocrine Tumors

Neuroendocrine tumors are usually characterized by the overexpression of somatostatin receptors, the presence of secretory granules and the ability to secrete amines or hormones. Somatostatin receptor expression profiles vary by tumor type and may even vary over the course of the disease. Furthermore, multiple somatostatin receptor subtypes are also expressed in a number of non-neuroendocrine tumors. Pasireotide is a multi-receptor targeted somatostatin analogue with high binding affinity for four of the five somatostatin receptor subtypes sst1,2,3 and sst5. Prolonged inhibition of hormone secretion by pasireotide in animal models and expression of multiple somatostatin receptors in neuroendocrine tumors suggest that pasireotide may have clinical advantages over the sst2-preferential somatostatin analogues, octreotide and lanreotide. Clinical trials demonstrated pasireotide to be a promising treatment for patients with acromegaly, Cushings disease and GI-NETs, and results from ongoing Phase III clinical studies will determine the role of pasireotide in these indications. Somatostatin analogues may have direct antitumor effects through receptor-mediated signal transduction pathways and indirect antitumor effects via inhibition of angiogenesis, suppression of growth factors and growth-promoting hormone synthesis/secretion or immunomodulatory effects. As such, pasireotide is under preclinical and clinical investigation as an antiproliferative therapy in several malignancies, both endocrine and non-endocrine. Keywords: Pasireotide, somatostatin, sst, somatostatin analogue, neuroendocrine tumor, solid tumor, Neuroendocrine tumors, somatostatin receptors, hormones, analogue, octreotide, lanreotide, Cushing's disease, antiproliferative, malignancies, oncological disorders, prolactin, alcitonin, adrenocorticotrophin hormone, sst5 regulates, G-protein, corticosteroids, antisecretory effect, carcinoid syndrome, pituitary adenoma, bronchoalveolar, bleomycin, endocrine islets, hypopituitarism, corticotroph tumor cells, dexamethasone treatment, Acromegaly, transsphenoidal surgery, cynomologus, prolactinomas, gonadotroph adenomas, Gastroenteropancreatic, gastrinomas, insulinomas, androgen- independent growth, tamoxifen

Resection of metastases from pancreatic and periampullary nonneuroendocrine tumors.

e14683 Background: The survival benefit from resection of hepatic metastases from colorectal and neuroendocrine tumors has been well documented in the literature. There are limited studies examining the role of metastasectomy in patients with nonneuroendocrine pancreatic or periampullary primary tumors. Methods: We performed a retrospective chart review of 983 patients who underwent resections for pancreatic and periampullary tumors at our institution between 1991 and 2009. Survival was defined in two ways: overall survival was defined as time from primary resection to death or last follow-up, while metastasectomy-survival was defined as time from metastasectomy to death or last follow-up. Results: We identified 15 patients who underwent metastasectomy for nonneuroendocrine tumors, including adenocarcinoma (n = 14) and acinar cell carcinoma (n = 1). The majority of patients underwent resection of hepatic metastases (n = 9), followed by lung (n = 3) and ovarian (n = 3) metastases. Four patients had periamp...

Extensive Expression of Collapsin Response Mediator Protein 5 (CRMP5) is a Specific Marker of High-grade Lung Neuroendocrine Carcinoma

The diagnosis of high-grade neuroendocrine tumors has strong clinical relevance because it identifies patients at higher risk of an unfavorable outcome who should receive multimodal treatment. However, these tumors can be mistaken for poorly differentiated nonsmall cell carcinoma or carcinoid lung tumors. In fact, no immunohistochemical marker can currently distinguish between histologic lung subtypes. Because the collapsin response mediator protein (CRMP) family is involved in an autoimmune disease associated with small cell lung carcinoma, we explored the relationship between CRMP5 expression and lung tumor behavior. Using World Health Organization morphologic criteria, 123 lung neuroendocrine tumors and 41 randomly selected non-neuroendocrine tumors were classified. CRMP5 protein expression in tumors, metastases, and healthy lung tissue was assessed using immunostaining method. Strong and extensive CRMP5 expression was seen in 98.6% of high-grade neuroendocrine lung tumors, including small cell lung carcinoma and large cell lung neuroendocrine carcinoma, but not in any of the squamous cell carcinomas or lung adenocarcinomas in our series. In contrast, the majority of low-grade neuroendocrine lung tumors were negative for CRMP5 staining, although weak CRMP5 expression was seen in some, with 2 different staining patterns of either scattered positive cells or small foci of positive cells. Our findings point at CRMP5 as a novel marker for routine pathologic evaluation of lung tumors surgical samples in distinguishing between highly aggressive neuroendocrine carcinoma and the other lung cancers.

Genetics of a combined lung small cell carcinoma and large cell neuroendocrine carcinoma with adenocarcinoma

Combined nonneuroendocrine-neuroendocrine lung tumors are relatively infrequent and little is known as for their genetic basis. Here, we report the case of a 69-year-old male with a solitary neoplasm in the upper lobe of the right lung. At histological examination, the tumor showed two components. The main part was an adenocarcinoma of the acinar type. The second part showed morphological and immunohistochemical phenotype of a neuroendocrine carcinoma composed of a small cell lung carcinoma and a large cell neuroendocrine carcinoma. The aim of our study was to investigate the genetic relationship between neuroendocrine and nonneuroendocrine tumor components. To this purpose, we performed a loss of heterozygosity (LOH) analysis with 40 chromosomal microsatellite markers. Microallelotyping revealed a common genetic profile in the different tumor areas. In 9 of 30 informative regions analyzed, LOH involved the same allele in all components, regardless of their histological type and grade. These findings support the true combined nature of this exocrine-neuroendocrine carcinoma of the lung and suggest a common monoclonal origin from a pluripotent epithelial (alveolar or bronchial) precursor cell for the two different tumor components.

Unresectable Chemorefractory Liver Metastases: Radioembolization with90Y Microspheres—Safety, Efficacy, and Survival

To prospectively evaluate the safety, efficacy, and survival of patients with chemorefractory liver metastases who have been treated with yttrium 90 ((90)Y) glass microspheres. Institutional review boards from two institutions approved the HIPAA-compliant study; all patients provided informed consent. One hundred thirty-seven patients underwent 225 administrations of (90)Y microspheres by using intraarterial infusion. Primary sites (origins) included colon, breast, neuroendocrine, pancreas, lung, cholangiocarcinoma, melanoma, renal, esophageal, ovary, adenocarcinoma of unknown primary, lymphoma, gastric, duodenal, bladder, angiosarcoma, squamous cell carcinoma, thyroid, adrenal, and parotid. Patients underwent evaluation of baseline and follow-up liver function and tumor markers and computed tomographic or magnetic resonance imaging. Patients were observed for survival from first treatment. Median survival (in days) and corresponding 95% confidence intervals were computed by using the Kaplan-Meier method. The log-rank statistic was used for statistical significance testing of survival distributions between various subgroups of patients. There were 66 men and 71 women. All patients were treated on an outpatient basis. Median age was 61 years. The mean number of treatments was 1.6. The median activity and dose infused were 1.83 GBq and 112.8 Gy, respectively. Clinical toxicities included fatigue (56%), vague abdominal pain (26%), and nausea (23%). At follow-up imaging, according to World Health Organization criteria, there was a 42.8% response rate (2.1% complete response, 40.7% partial response). There was a biologic tumor response (any decrease in tumor size) of 87%. Overall median survival was 300 days. One-year survival was 47.8%, and 2-year survival was 30.9%. Median survival was 457 days for patients with colorectal tumors, 776 days for those with neuroendocrine tumors, and 207 days for those with noncolorectal, nonneuroendocrine tumors. (90)Y hepatic treatments are well tolerated with acceptable toxicities; tumor response and median survival are promising.

Elevated Cocaine- and Amphetamine-Regulated Transcript Immunoreactivity in the Circulation of Patients with Neuroendocrine Malignancy

Cocaine- and amphetamine-regulated transcript (CART) codes for a peptide widely distributed in nervous and endocrine tissues. CART immunoreactivity (CART-LI) has been detected in human insulinomas. The objective of the study was to investigate the measurement of plasma CART-LI as a tumor marker of neuroendocrine malignancy. Plasma CART-LI levels were measured in 401 patients with a range of diagnoses: neuroendocrine malignancy (n = 131), after removal of neuroendocrine malignancy (n = 27), without any form of tumor or renal impairment (n = 192), with renal impairment (n = 17) and with nonneuroendocrine tumors (n = 34). Chromatography methods were used to investigate CART-LI circulating in human plasma. The upper limit of normal calculated for CART-LI was 150 pmol/liter. Mean circulating plasma CART-LI among neuroendocrine tumor patients was 440 pmol/liter, 56% of subjects having levels greater than 150 pmol/liter. Measuring CART-LI in addition to chromogranin (Cg)-A improved the sensitivity for neuroendocrine malignancy from 85 to 91%, whereas combined use of CgA and CgB had a joint sensitivity of 89%. Of 38 patients with pancreatic neuroendocrine tumors, 71% had plasma CART-LI levels greater than 150 pmol/liter, increasing to 95% in those classified with progressive disease (n = 20, mean CART-LI 625 pmol/liter), compared with 80% for CgA. Chromatographic analysis suggests that circulating CART-LI is present as one major form, which may correspond to CART (62-102) or another unknown form. We demonstrate CART-LI as a specific tumor marker in patients with a range of neuroendocrine tumors. Used in combination with CgA, CART-LI measurement has the potential to improve sensitivity in diagnosis and follow-up of neuroendocrine tumors, in particular progressive pancreatic neuroendocrine tumors.

Octreotide in Palliative Treatment of Hepatic Metastases: Is it Effective for Clinical Stabilization?

Octreotide in Palliative Treatment of Hepatic Metastases: Is it Effective for Clinical Stabilization?

Octreotide in Palliative Treatment of Unresectable Cholangiocarcinoma: Is it Effective for Disease Stabilization?

also reported a positive experience in liver metastases from non-neuroendocrine tumors with the use of octreotide LAR.A possible antitumor mechanism of octreotide is a stimulatory effect on Kupffer cells, induction of apoptosis or other antiproliferative actions, inhibition of proliferation, which have been suggested but not proved.

Methods in Neuroendocrine Histopathology, A Methodological Overview

Light microscopy is still the main tool in diagnostic histopathology, though it does not always lead to a definitive diagnosis. It has therefore been a constant ambition to develop methods which can add further information to the diagnosis.In endocrine pathology, a major problem has been to distinguish between neuroendocrine and non-neuroendocrine tumours. The silver stains, such as the Bodian, Grimelius and Sevier-Munger methods, were the first useful “general neuroendocrine” markers. Electron microscopy can also be useful for identifying neuroendocrine tumours.A further step forward was the introduction of histochemical fluorescence methods, as these could identify biogenic amines. With the introduction of immunohistochemical techniques, tumours could be characterized in a more specific way regarding peptide hormones and biogenic amines content, proliferation factors, hormone receptors, etc. Another method, DNA cytometry, has been used mainly in predicting the prognosis. In situ hybridization can be a useful complement to the histopathological diagnosis when other methods have failed to demonstrate the neuroendocrine nature of the tumour. Some endocrine tumours, especially the well-differentiated ones, still cause diagnostic problems in predicting tumour behaviour, why further complementary methods would be of great value.

Somatostatin, cortistatin and their receptors in tumours

Somatostatin (SS) and its synthetic analogs have a role in the treatment of neuroendocrine tumours both in terms of symptoms control and antiproliferative activities. These effects are mediated by five SS receptors, widely expressed in both human neuroendocrine and non-neuroendocrine tumours, which were demonstrated to be diagnostically and therapeutically valuable targets. Cortistatin (CST), a brain cortex peptide, partially homologous to SS and having similar functions is also expressed in peripheral tissues and tumours. CST binds all SS receptors, and, differently from SS, also the ghrelin receptor GHSR1a and the CST specific receptor MrgX2. The expression profile of CST is mostly restricted to neuroendocrine tumours (gastrointestinal, pancreas, lung, parathyroid, thyroid, adrenal). In these tumours, CST probably acts via the SS or ghrelin receptor, the MrgX2 receptor being absent. Thus, in comparison to SS analogs, CST synthetic analogs may represent additional diagnostic/therapeutic tools in those tumours expressing the receptors for SS, for ghrelin or for both peptides.

Future clinical prospects in somatostatin/cortistatin/somatostatin receptor field

Somatostatin receptors (sst), somatostatin (SS) and cortistatin (CST) are widely expressed in the various systems in the human and rodent organisms and are "responsible" for maintaining homeostasis, which is essential for survival. Because of their broad expression pattern sst, SS and CST interactions may play regulatory roles in both physiology and pathophysiology in mammalian organisms. SS analogue treatment strategies as well as the use of SS analogues for diagnostic purposes have been established in diseases of different origins. This review focuses on the currently determined role for SS analogues in today's clinical practice and the potential clinical prospects for SS, CST and sst interactions in the future, with a focus on neuroendocrine and non-neuroendocrine tumours and immune-mediated diseases. Moreover, the role of new SS analogues and new insights in sst physiology will be discussed.

Localization of immunoreactive HIF‐1α and HIF‐2α in neuroendocrine cells of both benign and malignant prostate glands

Hypoxia induces increased tumor growth by promoting angiogenic and glycolytic pathways. Tumors expressing hypoxia-inducible factor-1alpha (HIF-1alpha), an important transcriptional activator of oxygen-regulated genes, are resistant to chemotherapy and radiotherapy. The major challenge in prostate cancer therapy today is to gain a better understanding of the development of hormone-refractory tumors, which is often characterized by neuroendocrine differentiation. Here we studied the expression of HIF-1alpha and HIF-2alpha in neuroendocrine cells of the benign prostate and in prostate cancer. Tissue sections from 30 patients who underwent radical prostatectomy and from 21 patients operated by transurethral resection of the prostate were selected for immunohistochemical analysis for expression of HIF-1alpha, HIF-2alpha, androgen receptor (AR), neuroendocrine markers (chromogranin A, synaptophysin), and two gene products downstream of HIF-1alpha: VEGF and GAPDH. Immunoreactive HIF-1alpha was detected in a subpopulation of AR-negative neuroendocrine cells in benign and malignant prostate tissue. Analysis of serial sections showed that the levels of expression of GAPDH and VEGF proteins are increased in AR-negative malignant neuroendocrine cells expressing HIF-1alpha. In situ-hybridization indicated that HIF-1alpha mRNA levels are not higher in neuroendocrine prostate cancer cells relative to corresponding non-neuroendocrine tumor cells. We also demonstrated induced stabilization of nuclear HIF-1alpha in LNCaP cells by hypoxia and long-term stimulation with interleukin-6. Focal HIF-2 expression was detected in benign neuroendocrine-like cells and in malignant prostatic cells. The expression of HIF-1alpha and HIF-2alpha in prostate cancer has been confirmed, but we also identified immunoreactive HIF-1alpha and downstream gene products in benign and malignant prostate neuroendocrine cells.

Sequence, "subtle" alternative splicing and expression of the CYYR1 (cysteine/tyrosine-rich 1) mRNA in human neuroendocrine tumors

BackgroundCYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors.MethodsThe CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG- isoform, the first described mRNA for CYYR1 locus) or the presence (CAG+ isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species.ResultsThe CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG- or CAG+ isoform expression compared to control tissues. CYYR1 CAG- isoform was significantly more expressed than CAG+ isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms.ConclusionA new "subtle" splicing isoform (CAG+) of CYYR1 mRNA, the sequence and the expression of this gene were defined in a large series of NE tumors.