Chromogranin A: its role in endocrine function and as an endocrine and neuroendocrine tumor marker.

CgA is a 49 kilodalton protein that is present in the secretory granules of most endocrine and many neuroendocrine cells. Detection of CgA in cells by immunocytochemistry and measurement of CgA in serum by immunoassay can serve as tissue and serum markers for CgA-producing tumors. CgA is of diagnostic value in classical endocrine tumors, in hormone-negative tumors, and in endocrine tumors in which other diagnostic procedures have their limitations. Although the biological function of CgA is yet unknown, it may serve as a precursor molecule, like POMC, for a family of biologically active peptides. CgA is an important new tool for the endocrinologist in the diagnosis and management of patients with endocrine and neuroendocrine tumors.

Sperm protein 17 is expressed in human nervous system tumours.

BACKGROUND: Human sperm protein 17 (Sp17) is a highly conserved protein that was originally isolated from a rabbit epididymal sperm membrane and testis membrane pellet. It has recently been included in the cancer/testis (CT) antigen family, and shown to be expressed in multiple myeloma and ovarian cancer. We investigated its immunolocalisation in specimens of nervous system (NS) malignancies, in order to establish its usefulness as a target for tumour-vaccine strategies. METHODS: The expression of Sp17 was assessed by means of a standardised immunohistochemical procedure [(mAb/antigen) MF1/Sp17] in formalin-fixed and paraffin embedded surgical specimens of NS malignancies, including 28 neuroectodermal primary tumours (6 astrocytomas, 16 glioblastoma multiforme, 5 oligodendrogliomas, and 1 ependymoma), 25 meningeal tumours, and five peripheral nerve sheath tumours (4 schwannomas, and 1 neurofibroma). RESULTS: A number of neuroectodermal (21%) and meningeal tumours (4%) were found heterogeneously immunopositive for Sp17. None of the peripheral nerve sheath tumours was immunopositive for Sp17. The expression pattern was heterogeneous in all of the positive samples, and did not correlate with the degree of malignancy. CONCLUSION: The frequency of expression and non-uniform cell distribution of Sp17 suggest that it cannot be used as a unique immunotherapeutic target in NS cancer. However, our results do show the immunolocalisation of Sp17 in a proportion of NS tumour cells, but not in their non-pathological counterparts. The emerging complex function of Sp17 makes further studies necessary to clarify the link between it and immunopositive cells.

Expression of CRF1 and CRF2 receptors in human cancers.

Overexpressed peptide receptors in human tumors represent clinically relevant targets for cancer diagnosis and therapy. Corticotropin-releasing factor (CRF) and its receptors have not been known to be involved in human cancer. The aim of the present study was to investigate such possibility by evaluating the expression of CRF(1) and CRF(2) receptors using in vitro autoradiography with subtype-selective CRF analogs in more than 200 primary human cancer samples. We show that a majority of pituitary adenomas express CRF receptors, often in high amounts. Whereas ACTH-producing adenomas preferentially express CRF(1) receptors, nonfunctioning adenomas (gonadotropin-producing and null-cell adenomas) and GH- and TSH-producing adenomas express CRF(2) receptors. Furthermore, several central and peripheral nervous system tumors express CRF receptors: medulloblastomas, paragangliomas, neuroblastomas, and some meningiomas express CRF(1) receptors, but ependymomas or Ewing sarcomas do not. Insulinomas can also express CRF receptors, whereas ductal pancreatic cancers or prostatic, colorectal, and non-small cell lung cancers lack CRF receptors. In all receptor-positive tumors, the receptors were located on tumor cells. The high incidence of CRF(1) or CRF(2) receptors in selected human tumors suggests that unlabeled CRF agonists may be evaluated as inhibitors of tumor cell proliferation in cancer therapy, and radiolabeled CRF analogs may be used for cancer diagnosis and/or radiotherapy.

Localization of endogenous sugar-binding proteins (lectins) in tumors of the central and peripheral nervous system by biotinylated neoglycoproteins.

The carbohydrate part of cellular glycoconjugates - glycoproteins, glycoproteins, glycolipids and proteoglycans - and specific endogenous sugar receptors, i.e. lectins, can establish a system of biological recognition based on protein-sugar interactions on the cellular and subcellular levels. To gain insight into the role of proteins in this type of interaction, sections of surgically removed tumor specimens of central and peripheral nervous tissue were analyzed glycohistochemically, using biotinylated neoglycoproteins with different sugar part. A specific staining with this type of probe, exposing different sugar moieties as ligands, indicated the presence of sugar receptors in different types of meningiomas, glioblastomas, gangliocytomas, anaplastic and well-differentiated oligodendrogliomas and ependymomas as well as in neurinomas and neurofibromas of peripheral nerves. In comparison to the well-differentiated ependymomas, the anaplastic form of this tumor exhibited a generally higher capacity to specifically bind the neoglycoproteins, containing alpha- or beta-glucosides. Inverse intensity of the glycohistochemical reaction was observed with galactose-6-phosphate-, galactose-beta(1.3)-N-acetylglucosamine-N-acetyl-D-glucosamine- and mannose- (BSA- biotin), respectively, when anaplastic and differentiated oligodendrogliomas were compared with each other. Tumorously dedifferentiated neurons, i.e. in gangliocytomas, showed a changed spectrum of endogenous sugar receptors in comparison to neurons of normal cerebral cortex. Qualitative and quantitative differences of sugar receptors were observed among the distinct subtypes of meningiomas. Receptors for N-acetyl-D-galactosamine were present only in the anaplastic form, while glucuronic acid-specific receptors were only found in the meningotheliomatous meningiomas. Distinctions in binding spectrum of neoglycoproteins suggest the presence of a possible additional subtype of meningiomas, called submalignant meningioma. Analysis of the spectrum of endogenous sugar receptors can serve to distinguish between different cell populations composing a given tumor, as shown in neurofibromas in the cases of Schwann cells and fibroblastoid cells stained with N-acetyl-D-glucosamine-(BSA-biotin). The analysis of expression of endogenous sugar receptors, as part of an intercellular information code system, may represent a further way of studying the mechanism of tumor differentiation and propagation.

AgNOR content and PCNA expression in transplanted malignant neurinoma in rats.

Malignant neurinomas can be induced in BD IX rats by transplacental application of ethylnitroso-urea during pregnancy. Tumors develop in the offspring in trigeminal and spinal nerves and can be easily transplanted upon rats of the same strain. During the first passages a considerable shortening of subsequent induction periods takes place. Concurrently, silver stained Nucleolar Organizer Regions (AgNORs) increase in number and other measured AgNOR parameters change in a similar way. The number of cells that express the proliferation marker PCNA equally becomes more frequent during the first subcutaneously transplanted generations. There is high correlation between AgNOR parameters, number of PCNA expressing cells, induction times and passage. It is concluded that the first generations of the transplantation model of these tumors can be used to test the validity of proliferation indicators. Our results show further that AgNORs in fact belong to the group of markers of proliferation.

Immunohistochemistry of synapsin I and synaptophysin in human nervous system and neuroendocrine tumors. Applications in diagnostic neuro-oncology.

Synapsin I is a phosphoprotein localized to the cytoplasmic surface of synaptic vesicles and is one of the best characterized neuron-specific proteins. Synaptophysin is an integral membrane glycoprotein, also located on presynaptic vesicles, which has been shown to be a useful immunohistochemical marker for neuroendocrine/neuronal differentiation in tumor diagnosis. The sensitivity and specificity of immunohistochemical staining for these two proteins in formalin-fixed, paraffin-embedded tissues was studied in a series of 67 neuroectodermal, neuroendocrine, and non-neural tumors. Intense immunoreactivity for both synapsin I and synaptophysin was observed in tumors containing well-differentiated neurons (gangliocytoma, ganglioglioma, neurocytoma). In these tumors, immunostaining was primarily concentrated along the outer surface of the cell membrane of the neuronal cells. Primitive neuroectodermal tumors (PNETs) (cerebral PNET, medulloblastoma, neuroblastoma) and most neuroendocrine tumors generally showed less intense and more variable immunoreactivity for these proteins. In most cases, immunostaining for synapsin I was sharper and often more intense than for synaptophysin. Some PNETs and neuroendocrine tumors that were immunoreactive for synapsin I did not stain for synaptophysin. We conclude that synapsin I is a reliable, sensitive immunohistochemical marker for neuronal/neuroendocrine differentiation in human neoplasms and may offer some advantages over synaptophysin when applied to formalin-fixed, paraffin-embedded tissues, particularly in the evaluation of primitive neuroectodermal tumors and neuroendocrine tumors.

An immunohistochemical study of peripheral neuroblastoma, ganglioneuroblastoma, anaplastic ganglioglioma, schwannoma and neurofibroma in cattle.

Immunohistochemical analysis of five paraffin wax-embedded neoplasms was performed to elucidate the characteristics of bovine nervous-tissue tumours. In case 1 (peripheral neuroblastoma), the neoplastic tissue was characterized by the formation of true and Homer-Wright rosettes and the existence of neuron-specific enolase. The neoplastic cells were possibly more immature than those of common neuroblastomas, because similar features are observed in human malignant neuroepitheliomas. The neoplastic cells in case 2 (ganglioneuroblastoma) ranged from large cells with abundant neurofilaments to immature small cells, rarely with neurofilaments or glial fibrillary acidic protein (GFAP). Such expression suggests the presence of pluripotential cells. The neoplastic tissue in case 3 (anaplastic ganglioglioma) was strikingly polymorphous, and had five elements; neuronal, astrocytic, oligodendrocytic, spindle cell and small oval cell. The neoplastic neurocytes and astrocytes were, respectively, characterized by neurofilament and GFAP positivity. The neoplastic oligodendrocytes made a honeycomb appearance, and the neoplastic spindle cells and small oval cells were considered to be less differentiated. The tumours of cases 2 and 3, which contained poorly differentiated cells and revealed both neuronal and glial differentiation, may be specific to calves. In case 4 (schwannoma), almost all the neoplastic cells were positive for S100 protein, while S100-negative fibroblasts were present in many areas of case 5 (neurofibroma). These two tumours were readily distinguished histologically and immunohistochemically.

Morphological and cytoskeletal alterations of nervous system tumor cells with different culturing methods.

Cell culture is one of the most important methods of research in molecular and cellular biology, and various culture systems have been developed, including two-dimensional (2D), three-dimensional (3D) and floating culture systems. In the present study, we examined morphological changes and different expression patterns of cytoskeletal proteins in three different types of nervous system tumor cells grown in 2D, 3D and floating cell cultures. A172, KG-1-C and IMR-32 cells showed marked morphological changes, depending on the cell culture methods. F-actin expression was clearly observed at the level of the cells nearest the plate surface in 2D and 3D cultures. On the other hand, expression of F-actin was weak in the floating culture system. α-tubulin was detected in the cytoplasm of cells in 2D culture, but in floating and 3D cultures, α-tubulin was expressed in the peripheral regions of spheres and spheroids. In conclusion, this study demonstrated that nervous system tumor cells showed different alterations in morphology, and different cytoskeletal protein expression patterns, depending on the culture methods.

The role of immunohistochemistry in predicting behavior of astrocytic tumors.

The purpose of this study was to analyze the significance of p53, bcl-2 and EGFR expression in the grading and biological behavior of astrocytic tumors, especially in the Indian population. A total of 117 cases of astrocytomas graded using the WHO grading system published in 2007 were immunolabeled using p53, EGFR and bcl-2 monoclonal antibodies and analyzed with respect to grade and other relevant parameters. The 117 cases included 16 cases of pilocytic astrocytomas and 25, 15 and 61 cases of diffuse fibrillary astrocytomas WHO grade II, anaplastic astrocytomas WHO grade III and glioblastomas (GBM), respectively. Our results showed that p53 alterations is an early event in astrocytic gliomagenesis, but is not significant in the evolution of pilocytic astrocytomas. Bcl-2 expression did not correlate with grade and no statistical correlation was seen with p53 expression. EGFR protein expression correlated with the severity of tumor grade. Of the GBM cases, 47.5% were p53 positive only, 18% were EGFR positive only, 16.5% were negative for both and 18% were positive for both. The mean age in the dual positive category was significantly higher when compared to the others. EGFR and p53 alterations are not mutually exclusive and might act synergistically to promote progression. We also noted a significantly higher p53 expression in females in GBMs. Though most of our findings correlated with those of previous studies, some differences were noted, especially in the pattern of immunoexpression in GBMs, perhaps because of ethnicity.

Focal cortical dysplasia type II (malformations of cortical development) aberrantly expresses apoptotic proteins.

bcl-XL, bax, bcl-2, and p53 are apoptotic proteins essential to normal neural development. Aberrant expression of these proteins has been observed in several central nervous system neoplasms. Immunoexpression of these markers is studied in 21 patients with focal cortical dysplasia type II (Taylor-type cortical dysplasia; malformations of cortical development) who had undergone lesionectomy for treatment of pharmacoresistant epilepsy. Paraffin immunohistochemistry using standard methodology was performed on representative sections using antibodies to bcl-XL, bax, bcl-2, and p53. Aberrant expression of bcl-XL, bax, bcl-2, and p53 was observed in the majority of cases, with dysmorphic neurons staining positively for bcl-XL, bax, and bcl-2 in 71%, 76%, and 24% of cases, respectively, and balloon cells staining positively for bcl-XL, bax, and bcl-2 in 89%, 78%, and 17% of cases, respectively. Most cases (86%) showed some expression of p53, with the majority showing expression of p53 most prominently in balloon cells. Previous work has shown gangliogliomas and dysembryoplastic neuroepithelial tumors, both dysplasia-associated neoplasms, to demonstrate aberrant expression of apoptotic markers, suggesting a possible common mechanism of development for these 2 processes in patients in whom they coexist.

Immunohistochemical demonstration of chromogranin A, chromogranin B, and secretogranin II in extra-adrenal paragangliomas.

Twelve sympathetic and 14 parasympathetic extra-adrenal paragangliomas were investigated immunohistochemically with antibodies against chromogranin A, chromogranin B, and secretogranin II. In sympathetic paragangliomas chromogranin A was found in 12/12 and chromogranin B in 11/12 tumors in almost all chief cells (the remaining tumor was focally chromogranin B positive), whereas secretogranin II was immunolocalized in the majority of chief cells in 5/12, in a focal distribution in 3/12, and only in a few scattered tumor cells in 3/12 cases. One case showed no secretogranin II immunoreactivity. In parasympathetic paragangliomas both chromogranin B and secretogranin II immunoreactivity was demonstrated in the majority of chief cells of all 14 tumors investigated. Chromogranin A showed a strong immunostaining in 2/14 cases; in 12 tumors chromogranin A was found in only a few chief cells or was completely absent. It is concluded that sympathetic and parasympathetic paragangliomas show a divergent expression of chromogranins/secretogranins that apparently reflects the different histogenetic origins of these tumors.

Immunocytochemistry of paragangliomas--value of staining for S-100 protein and glial fibrillary acid protein in diagnosis and prognosis.

Surgical specimens of 65 adrenal and 27 extra-adrenal paragangliomas, the latter comprising 11 carotid body, five jugulotympanic, one aorticopulmonary, eight aorticosympathetic and two visceral autonomic tumours, were examined immunocytochemically for the presence of glial fibrillary acid protein (GFAP) and S-100 protein. Six adrenal and four extra-adrenal (one parasympathetic and three sympathetic) neoplasms pursued a malignant clinical course. S-100 staining of sustentacular (type 2) cells was seen in both adrenal (48/65) and extra-adrenal (23/27) lesions, the 10 malignant tumours being entirely devoid of S-100 protein positive cells. GFAP positivity of type 2 cells was seen in only 16 of the extra-adrenal tumours, all of these lesions belonging to the group of benign parasympathetic paragangliomas. The presence of S-100 positive type 2 cells may thus help to exclude malignancy in individual paraganglioma cases, while GFAP positivity of such cells renders possible the correct typing of benign parasympathetic paragangliomas.

Expression of CD15 in tumours of the nervous system.

The expression of the CD15 epitope was investigated by immunohistochemistry, western blotting and immuno-thin-layer chromatography on a large series of human nervous system tumours and ethylnitrosourea-induced rat gliomas. Our results show that CD15 is expressed as a glycoprotein- or glycolipid-associated epitope in normal human and rat brain. In contrast, immunoreactivity for CD15 was absent in tumour cells of experimental rat gliomas. In human tumours we found a more complex expression pattern. While intra- and perivascular granulocytes as well as macrophages in necrotic areas of anaplastic tumours were always strongly CD15-positive, immunoreactive tumour cells were detectable only in a fraction of low-grade gliomas. Anaplastic gliomas and glioblastomas consistently did not express the epitope on their tumour cells. In addition to individual low-grade gliomas, we found CD15-positive cases among metastatic carcinomas, craniopharyngeomas, meningiomas, germinomas and malignant melanomas. Our results suggest that immunohistochemistry for CD15 is potentially useful in diagnostic neuropathology as a marker for granulocytes in paraffin sections, as a supplementary tool for the histopathological grading of gliomas, and as an aid for differentiation between anaplastic glioma cells and non-neoplastic glia. Furthermore, it can be speculated that the lack of CD15 expression on anaplastic glioma cells may potentially be responsible for some of their characteristics--such as altered cellular interaction and loss of contact inhibition.

Relationship between skeinoid fibers and stromal matrix in gastrointestinal stromal tumors: morphometric analysis with quick-freezing and deep-etching method.

Our previous study of a gastrointestinal autonomic nerve tumor with skeinoid fibers (SF) using the quick-freezing and deep-etching method, suggested that the distance between one radix and a neighboring radix (DRNR) in pre-existing meshwork structures around the tumor cells is consistent with the periodicity of the SF. Therefore, measurement of the DRNR in the meshwork could clarify the significance of the pericellular matrix for SF development. In the present study, we analyzed the meshwork in three cases of gastrointestinal stromal tumor (GIST), which showed different immunohistochemical stainings, but confirmed to have smooth muscle differentiation (SMD) by immunohistochemistry and/or electron microscopy. The DRNR from the three cases of GIST showed similar histogram patterns (a peak of 20-30 nm, mean values of 28.02, 25.74 and 26.45 nm), which were significantly shorter than the periodicity of SF (a peak of 40-45 nm, mean value of 42.14). Although we need further studies with additional GIST cases, we speculate that the pericellular matrix of GIST with SMD is not suitable for SF development.

Gastrointestinal autonomic nerve tumor: a common type of gastrointestinal stromal neoplasm.

Seven of 15 gastrointestinal stromal tumors from the author's file were classified as plexosarcomas when examined by electron microscopy and immunohistochemistry. Electron microscopy was essential for the diagnosis.

Jejunal stromal tumor with skeinoid fibers or myenteric plexoma: a case report.

Small intestinal stromal tumors with 'skeinoid fibers' are uncommon stromal tumors with an associated controversial histogenesis. Although their microscopic appearance is suggestive of a smooth muscle nature, they lack specific smooth muscle features, as evident by electron microscopy and immunohistochemistry. They also appear to lack features of neurogenic origin because they fall to react with neural/neuroendocrine markers such as S-100 protein, neuron-specific enolase and chromogranin. It is interesting, nonetheless, to note that the ultrastructural examination of these tumors may show structures reminiscent of neural differentiation, such as cytoplasmic projections, containing occasional membrane-bound, dense-core, neurosecretory-type granules, which mimick the long cytoplasmic processes seen in tumors of neural origin. Moreover, the association of these tumors with Von Recklinghausen's neurofibromatosis, as well as the presence of 'skeinoid fibers' in proven neurogenic spindle cell neoplasms such as gastrointestinal autonomic nerve tumors and schwannomas, suggests that these tumors might also be neurogenic in origin and enhances the diagnostic value of 'skeinoid fibers' as a possible ultrastructural marker of neural differentiation. Thus, light microscopic evaluation is clearly insufficient to accurately diagnose these tumors and to determine their histogenesis, electron microscopic and immunohistochemical studies being necessary. In this article the histogenesis of small intestinal stromal tumors with 'skeinoid fibers', regarding a jejunal neoplasm in a 63-year-old patient, is reviewed. The light microscopic, immunohistochemical and ultrastructural features are described and compared with findings usually seen in all those stromal tumors which may raise a differential diagnosis, such as smooth muscle stromal tumors, gastrointestinal autonomic nerve tumors, schwannomas, paragangliomas and fibrosarcomas.

Characterization of rat NCA/CD9 cell surface antigen and its expression by normal and malignant neural cells.

As part of investigations on ethylnitrosourea (EtNU)-induced neuro-oncogenesis in the rat, we have produced monoclonal antibodies (Mabs) specific for neural cell surface antigens (NCAs) by immunization with cells of the clonal tumorigenic neural rat cell line BT4Ca. Mabs designated as anti-NCA (alpha NCA1, alpha NCA2, alpha NCA3, alpha NCA4, and alpha NCA5) recognize proteins of 25 kDa and 23 kDa, as shown by immunoprecipitation and Western blot. The predominant 25-kDa protein was purified from BT4Ca cells by immunoaffinity chromatography with immobilized Mab alpha NCA1 and identified by N-terminal sequencing as the rat homologue of the CD9 antigen. Identification of proline as N-terminal amino acid of the purified protein suggests post-translational modification of CD9 in the rat central nervous system. The NCA/CD9 protein was localized in distinct regions of fetal and adult rat brain by immunofluorescence staining of frozen sections. Flow cytometric analyses of isolated fetal rat brain cells (FBC) showed that the proportion and number of NCA/CD9-expressing cells increased during prenatal development. Immunoreactivity of approximately 40% of brain cells isolated 13 days post conception (p.c.) indicated that NCA/CD9 is expressed by neuronal precursors at this stage of development. In primary cultures of rat FBC isolated 18 days p.c., the NCA/CD9 antigen was expressed by all premature and mature astrocytes, oligodendrocytes, ependymal cells, and microglial cells, but not by E-N-CAM-expressing neuronal progenitor cells and neurons. Furthermore, eight out of ten EtNU-induced malignant neural rat cell lines as well as EtNU-induced tumors of the central and peripheral nervous system exhibited intermediate or strong immunoreactivity with Mab alpha NCA1. Expression of the NCA/CD9 protein is, therefore, characteristic of both normal glial precursor cells and their malignant counterparts in the rat.

Metastatic multicentric neurofibrosarcoma of the lumbosacral plexus in a cow.

A metastatic multicentric neurofibrosarcoma of the lumbosacral plexus in an adult cow is described. The left lumbosacral plexus was obliterated by a mass which extended through the intervertebral foramen into the spinal canal and between the dorsal arches of the fifth and sixth lumbar vertebrae. A closely associated (possibly contiguous) mass extended into and separated the left sacroiliac joint. Multiple similar masses involved peripheral nerves and skeletal muscles of the pelvis, pelvic limbs, and abdominal wall. Metastatic lesions were scattered throughout the lungs. The lumbosacral lesion and all other masses consisted of interwoven bundles of loosely cohesive, elongated cells separated by variable collagenous matrix. Many neoplastic cells were positive for S-100 protein. Ultrastructurally, fibroblastic cells were mixed with scattered cells possessing schwannian characteristics.