Cellular localization of the Trk neurotrophin receptor family in human non-neuronal tissues.

We investigated the distribution of the high-affinity neurotrophin receptors TrkA, TrkB, and TrkC in a wide range of normal non-neuronal tissues of adult human by immunohistochemical methods. Trk immunoreactivity (IR) was detected at various levels in all tissues examined, except for the heart and liver. The gastric parietal cells showed strong TrkA and TrkC IR and all of the Trks had IR for the putative intestinal neuroendocrine cells. In the pancreas, TrkA and TrkC IR was detected in the sub-intralobular ducts, whereas TrkB IR was found specifically in the alpha-islet cells. The lymph node and spleen exhibited TrkB IR in monocytes/macrophages. The adrenal cortex showed selective TrkA IR with TrkC IR in the medulla. In the reproductive system, TrkA IR was detected in the prostatic epithelial cells, TrkC in the ovarian theca and granulosa cells, TrkA and TrkC in the secretory-phase endometrium, and TrkA in the mammary ducts. The kidney showed strong TrkA and TrkC IR in it tubules, but no Trk receptors were present in the glomeruli. In the skin, TrkA and TrkB/TrkC were present in the basal and granular layers of the epidermis, respectively.     

Neurotrophins and neurotrophin receptors in human lung cancer.

The expression of neurotrophins (NTs) and related high- and low-affinity receptors was studied in surgical samples of histologically diagnosed human tumors of the lower respiratory tract. The experiment was conducted with 30 non-small cell lung cancer specimens and in eight small cell lung cancer specimens by Western blot analysis and immunohistochemistry to assess expression and distribution of NT and NT receptor proteins in tissues examined. Immunoblots of homogenates from human tumors displayed binding of anti-nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and NT-3 antibodies as well as of anti-tyrosine-specific protein kinase (Trk) A, TrkB, and TrkC receptor antibodies, with similar migration characteristics than those displayed by human beta-NGF and proteins from rat brain. A specific immunoreactivity for NTs and NT receptors was demonstrated in vessel walls, stromal fibroblasts, immune cells, and sometimes within neoplastic cell bodies. Approximately 33% of bronchioloalveolar carcinomas exhibited a strong membrane NGF and TrkA immunoreactivity, whereas 46% adenocarcinomas expressed an intense TrkA immunoreactivity but a weak immunostaining for NGF within tumor cells. Moreover, squamous cell carcinomas developed an intense TrkA immunoreactivity only within stroma surrounding neoplastic cells. A faint BDNF and TrkB immunoreactivity was documented in adenocarcinomas, squamous cell carcinomas, and small cell lung cancers. NT-3 and its corresponding TrkC receptor were found in a small number of squamous cell carcinomas within large-size tumor cells. No expression of low-affinity p75 receptor protein was found in tumor cells. The detection of NTs and NT receptor proteins in tumors of the lower respiratory tract suggests that NTs may be involved in controlling growth and differentiation of human lung cancer and/or influencing tumor behavior.     

The teleost kidney expresses Trk Neurotrophin Receptor-Like Proteins

Neurotrophins are involved in the development and maintenance of some neuronal populations, acting through tyrosine protein kinase (Trk) receptors, TrkA, TrkB and TrkC. In addition, recent studies have demonstrated the occurrence of Trk receptors in a wide variety of adult and embryonic non-neuronal tissues in vertebrates, including kidney. Nevertheless no data are available on Trk proteins distribution in teleost kidney. The present study, by using an immunoperoxidase technique, analyses Trk receptor protein distribution in the kidney of three species of freshwater and saltwater teleost. TrkA-like immunoreactivity was the only one detected in all examined species, while TrkB-like immunoreactivity was completely absent and TrkC-like immunoreactivity was detected only in goldfish. The TrkA immunoreactive cells were mainly localised in the collecting duct system, as this system was the only one to also display TrkC. Such data could provide new clues to Trk in fish and aid assessment of the role of Trk protein receptors during vertebrate evolution. Accepted: 3 November 1999     

The teleost kidney expresses Trk neurotrophin receptor-like proteins

Neurotrophins are involved in the development and maintenance of some neuronal populations, acting through tyrosine protein kinase (Trk) receptors, TrkA, TrkB and TrkC. In addition, recent studies have demonstrated the occurrence of Trk receptors in a wide variety of adult and embryonic non-neuronal tissues in vertebrates, including kidney. Nevertheless no data are available on Trk proteins distribution in teleost kidney. The present study, by using an immunoperoxidase technique, analyses Trk receptor protein distribution in the kidney of three species of freshwater and saltwater teleost. TrkA-like immunoreactivity was the only one detected in all examined species, while TrkB-like immunoreactivity was completely absent and TrkC-like immunoreactivity was detected only in goldfish. The TrkA immunoreactive cells were mainly localised in the collecting duct system, as this system was the only one to also display TrkC. Such data could provide new clues to Trk in fish and aid assessment of the role of Trk protein receptors during vertebrate evolution.     

Differential expression of estrogen, progesterone, and epidermal growth factor receptors in normal, benign, and malignant human breast tissues using dual staining immunohistochemistry.

Distribution of estrogen (ER), progesterone (PR) receptors, and epidermal growth factor (EGF) receptors was assayed by dual staining immunohistochemistry on 28 selected cytosolic ER-positive breast carcinomas and 22 nonmalignant breast tissues. ER-positive tumor cells were detected in 26 (93%) and EGF receptor positive tumor cells were detected in 7 (25%) carcinomas. In five tumors both ER and EGF receptors were detected but localized in distinct tumor cells. Only in one case of ductal carcinoma in situ co-expression was observed in a subset of tumor cells. In contrast, simultaneous expression of ER/PR and EGF receptors was observed in non-neoplastic ductal remnants in the majority of the carcinomas and the fibroadenomas. In addition, double-positive cells were occasionally detected in luminal epithelial cells of normal breast tissue and mastopathies. This study shows that ER/PR and EGF receptors in breast tumor cells are inversely related at the single cell level. However, demonstration of ER/PR and EGF receptors in individual normal luminal cells shows that expression is not mutually exclusive.     

Expression of tyrosine kinase receptors in cultured dorsal root ganglion neurons in the presence of monosialoganglioside and skeletal muscle cells

The neurotrophic factor-like activity of monosialoganglioside (GM1) has been shown to activate tyrosine kinase receptors (Trk). Targets of neuronal innervation play a vital role in regulating the survival and differentiation of innervating neurotrophin-responsive neurons. Both GM1 and target skeletal muscle (SKM) cells are essential for the maintenance of the function of neurons. However, much less is known about the effects of GM1 or/and target SKM cells on the expression of Trk receptors in dorsal root ganglion (DRG) neurons. Here we have tested what extent to the expression of TrkA, TrkB, and TrkC receptors in primary cultured of DRG neurons in absence or presence of GM1 or/and SKM cells. In this experiment, we found that: (1) GM1 promoted expression of TrkA and TrkB but not TrkC in primary cultured DRG neurons; (2) target SKM cells promoted expression of TrkC but not TrkA and TrkB in neuromuscular cocultures without GM1 treatment; and (3) GM1 and target SKM cells had additional effects on expression of these three Trk receptors. The results of the present study offered new clues for a better understanding of the association of GM1 and target SKM on the expression of Trk receptors.     

Enhanced B-Raf protein expression is independent of V600E mutant status in thyroid carcinomas

BRAF (7q24) encodes a serine/threonine protein kinase, and its expression level varies in different tissues. Although a high prevalence of BRAF mutation has been suggested as an important event in thyroid tumorigenesis, little is known about the expression pattern of B-Raf in the thyroid. Thus, we examined the expression of B-Raf in various neoplastic and nonneoplastic thyroid tissues and compared it with BRAF mutational status. Normal and hyperplastic thyroid tissues showed focal and faint immunoreactivity for B-Raf, especially in cuboidal follicular cells of small follicles. In contrast, diffuse expression of B-Raf was observed in follicular adenomas and well-differentiated carcinomas. The missense point mutation BRAF(V600E) was identified in 42% (13/31 cases) of papillary carcinomas and 33% (5/15 cases) of undifferentiated carcinomas but not in normal thyroid tissues, nodular hyperplasia, follicular adenomas, or follicular carcinomas. The immunohistochemical expression of B-Raf did not correlate with BRAF mutational status. Moreover, Western blotting revealed that B-Raf expression in thyroid carcinoma cell lines was also independent of BRAF mutation. Serum or fibroblast growth factor-1 stimulation further activates ERK1/2 in heterozygous BRAF(V600E)-positive carcinoma cells as well as BRAF(V600E) mutation-negative carcinoma cells. In conclusion, heterogeneous focal expression of wild-type B-Raf in nonneoplastic tissues may play a role in the growth or functional activity of thyroid follicular cells. In contrast, diffuse expression of wild-type and/or mutant B-Raf may be involved in the tumorigenic process resulting in activation of the mitogen-activated protein kinase signaling pathway in cooperation with other genetic abnormalities and activation of ligand-receptor signaling pathways.     

Syndecan-1 expression is induced in the stroma of infiltrating breast carcinoma.

Loss of expression of the heparan sulfate proteoglycan syndecan-1 leads to reduced cell adhesion, increased invasive potential, and dysregulated growth of mammary epithelial cells in vitro. We compared syndecan-1 expression in malignant and nonmalignant breast tissues using immunohisto-chemistry with monoclonal antibody B-B4. Staining for syndecan-1 is greatly diminished on malignant cells within infiltrating ductal carcinomas (n = 20) as compared with ductal epithelium of both normal breast (n = 14) and stromal-epithelial neoplasms (n = 10), which exhibit extensive basolateral epithelial staining. Surprisingly, comparison of malignant and nonmalignant breast tissue also reveals a striking difference in expression of syndecan-1 within the stromal compartment. In infiltrating ductal carcinomas, strong staining for syndecan-1 is present both within the connective tissue and on stromal cell surfaces, whereas syndecan-1 expression is absent in the stroma of both normal breast and stromal-epithelial neoplasms. Because syndecan-1 interacts with heparin-binding growth factors such as FGF-2, accumulation of syndecan-1 within the tumor stroma may contribute to the extensive angiogenesis and stromal proliferation characteristic of infiltrating breast carcinoma. Moreover, the induction of syndecan-1 within the stroma, coupled with the loss of syndecan-1 on malignant cells, suggests that changes in syndecan-1 expression are critical in promoting the metastatic phenotype of infiltrating ductal carcinoma of the breast.     

The p75 neurotrophin receptor is widely expressed in conventional papillary thyroid carcinoma

Papillary thyroid carcinomas (PTCs) are associated with alterations in several proto-oncogenes related with nervous system development and function, such as TrkA and RET, which are commonly rearranged in these carcinomas. The other oncogenic event recently identified in PTC is the BRAF V600E mutation. Because the role of TrkA was not completely elucidated in thyroid cancer ethiopathogenesis, we decided to study the expression of active, phosphorylated TrkA and of its coreceptor p75 neurotrophin receptor (p75 NTR) in a series of 92 PTC (37 lesions of conventional PTC, 28 of follicular variant of PTC [FVPTC], and 27 of other variants of PTC) as well as in 21 samples of normal thyroid and nonneoplastic thyroid lesions used as a controls. We observed neoexpression of p75 NTR in PTC, particularly in conventional PTC and in other variants of PTC displaying a papillary growth pattern, rather than in FVPTC. No immunoexpression of p75 NTR was observed in normal thyroid nor in nonneoplastic thyroid lesions. The cellular localization of p75 NTR immunoexpression was also significantly associated with the growth pattern of PTC, being much more frequently detected in an apical localization in PTC with papillary architecture than in PTC with a follicular or solid growth pattern. This apical localization of p75 NTR was significantly associated with the presence of BRAF V600E. No significant differences were detected between normal thyroid, nonneoplastic lesions, and PTC (or any PTC variant) regarding expression/activation of TrkA, thus suggesting that by itself and in contrast to p75 NTR, TrkA is not altered during PTC development.     

Altered expression of the RON receptor tyrosine kinase in various epithelial cancers and its contribution to tumourigenic phenotypes in thyroid cancer cells

Aberrant expression of the RON receptor tyrosine kinase has been implicated in the pathogenesis of epithelial tumours. The aim of this study was to determine RON expression in various normal epithelial cells and their corresponding tumours by immunohistochemistry. The role of RON in regulating tumourigenic phenotypes was also studied using thyroid cancer cells as a model. RON was almost exclusively expressed at variable levels in normal epithelial cells from the digestive track, lung, kidney, pancreas, liver, breast, bladder, skin, and others. Among 15 types of cancer studied, RON was overexpressed in significant numbers in cancers derived from breast (56%), colon (51%), lung (48), thyroid (42%), skin (37%), bladder (36%), and pancreas (33%). In contrast, limited RON overexpression was observed in cancers from stomach, kidney, brain, liver, ovary, and prostate. Detailed analysis of thyroid tissues showed that RON was hardly detected in normal thyroid cells, moderately expressed in adenoma samples, but overexpressed in about half of papillary and follicular cancer specimens. Overexpression correlated with advanced clinical stage and was associated with lymph node metastasis. In cultured thyroid cancer cells, RON was highly expressed, with constitutive phosphorylation. Activation of RON increased cell growth and migration via the MAP kinase and AKT pathways. Silencing RON expression significantly prevented cell growth and increased cell apoptotic death. These findings show that RON overexpression occurs in a particular group of epithelial cancers. The requirement for RON in sustaining tumourigenic phenotypes suggests that it is a potential target for therapeutic intervention.     

Stromal elastosis in papillary thyroid carcinomas

Stromal elastosis, defined as dense aggregations of elastic fibers, is found in some neoplastic tissues especially in malignant tumors of the breast and lung. Although also found in thyroid tissue, stromal elastosis in thyroid neoplasms have received little attention. To clarify the histopathological significance of stromal elastosis in the thyroid, we examined neoplastic (n = 223) and hyperplastic (n = 82) thyroid tissues in conjunction with cancer tissues (n = 193) of various other organs. Stromal elastosis was observed as deposits of pale homogeneous material in hematoxylin and eosin stain, and distinctively highlighted by elastic-van Gieson's stain. On immunohistochemical examination, elastin and tropoelastin were confirmed in these deposits. Stromal elastosis was found in 66% of papillary thyroid carcinomas (PTCs), although it was not identified in other histological types of thyroid neoplasms. In PTCs, deposits of elastic fibers varied in size and shape, and were more frequently distributed in the periphery of the tumor tissue. The histological subtypes of PTC varied in prevalence of elastosis with the follicular variant's (9%) prevalence being significantly lower than that of the classical type (72%). The frequency of stromal elastosis in PTCs was very similar to the frequencies in breast and lung adenocarcinomas, and higher than the frequencies in carcinomas of other organs. In conclusion, our results suggest that stromal elastosis is a characteristic histological finding of PTCs, presumably associated with their growth pattern and/or histological architecture. It is, therefore, reasonable to propose that stromal elastosis is an ancillary feature in the histopathological diagnosis of PTCs.     

Expression of alpha-methylacyl-CoA racemase (P504s) in various malignant neoplasms and normal tissues: astudy of 761 cases

Alpha-methylacyl CoA racemase (AMACR), also known as P504S, plays an important role in peroxisomal beta-oxidation of branched-chain fatty acids. It has recently been shown that AMACR is highly expressed in prostate cancer and that it may be an important diagnostic marker for prostate carcinoma. However, little is known about expression of AMACR in normal tissues and other malignant tumors. In this study, we investigated expression of AMACR in 539 malignant tumors and 222 normal human tissues of various types by immunohistochemical analysis. mRNA levels of AMACR in normal organs and in selected tumors were assessed by real time PCR. In normal tissue, high expression of AMACR mRNA was identified in liver, kidney and salivary gland, while AMACR protein was detected in liver (hepatocytes), kidney (tubular epithelial cells), lung (only bronchial epithelial cells), and gallbladder (only mucosal epithelial cells). High expression of AMACR mRNA was found in prostate, liver, and kidney cancers but rarely in stomach and bladder cancers. A high percent of adenocarcinomas arising from these organs express AMACR, including 17 of 21 (81%) of hepatocellular carcinomas and 18 of 24 (75%) of renal cell carcinomas. In addition, carcinomas arising from tissues normally not expressing AMACR were also positive for the antigen, including 17 of 18 (94%) prostate carcinomas, 9 of 29 (31%) of urothelial carcinomas, and 4 of 15 (27%) of gastric adenocarcinomas. Two hundred and fifty cases of adenocarcinomas from lung, breast, pancreas, bile duct, adrenal gland, salivary gland, ovary, thyroid and endometrium were negative or rarely positive for AMACR. Neuroendocrine carcinomas rarely expressed AMACR. Melanomas, squamous cell carcinomas, basal cell carcinomas, soft tissue tumors (including epithelioid sarcomas and synovial sarcoma), thymomas, and germ cell tumors were negative for AMACR. Our data provide important baseline information for using AMACR in clinical practice and also are valuable in furthering understanding of the pathogenic role of AMACR in malignant neoplasms.     

neu oncogene protein and epidermal growth factor receptor are independently expressed in benign and malignant breast tissues

The neu oncogene protein, p185, and epidermal growth factor receptor (EGFR) were localized immunohistochemically in benign and malignant human breast tissues using monoclonal antibodies. Both benign and malignant epithelial cells were positive for these oncogene proteins in acetone-postfixed frozen sections. Stromal cells were negative for p185, but occasionally positive for EGFR. Myoepithelial cells were consistently positive for EGFR, and p185 was localized predominantly in duct-lining cells, where the basolateral plasma membrane was the normal expression site of both substances. Paraformaldehyde-prefixed frozen sections were less sensitive for antigen demonstration. Based on the intensity of immunoreactivity, 11 of 37 acetone-postfixed breast carcinomas (30%) were judged neu overexpressors, while none of 24 benign tissues overexpressed neu. Epidermal growth factor receptor was demonstrated in 18 of 36 acetone-postfixed cancer tissues (50%) and was overexpressed in three (8%). At the cellular level, heterogenous expression of p185 and EGFR was occasionally observed in both benign and malignant tissues, and a single case of cancer overexpressing both neu and EGFR showed reciprocal patterns of staining, indicating their independent expression. In some carcinomas, EGFR was localized only in stromal cells. Our findings confirmed mutually independent expression of the two closely related protooncogenes in benign and malignant breast tissues.     

Expression of epidermal growth factor receptor in benign and malignant primary tumours of the breast

Summary Using the monoclonal antibody EGFR1, normal mammary gland and a series of 213 unselected primary breast tumours were investigated immunohistochemically for expression of epidermal growth factor receptor (EGFR). In normal breast EGFR was expressed in variable patterns in lobular, ductal, and myoepithelial cells. In fibroadenoma, EGFR was detectable in variable numbers of ductal and myoepithelial cells and in stromal fibroblasts. The myoepithelial compartment of 2 cystosarcomas phyllodes also expressed EGFR. Among the 197 carcinomas tested only 20.3% contained EGFR expressing tumour cells which represented a minority in 12.2%, the majority in 2.1%, and the entire neoplastic population in 6.1% of the cases. Again, non-neoplastic ductal remnants often contained EGFR positive myoepithelial and ductal cells whereas stromal fibro-blasts expressed EGFR only occasionally. We conclude that in contrast to the normal state, EGFR-expression is a rather rare phenomenon in breast carcinoma cells, positively correlated with a declining grade of differentiation (p<0.025) and at least occasionally associated with squamous metaplasia within the tumour, that EGFR expression is not exclusively restricted to cells of the epithelial lineage, and that EGFR may have other functions not related to proliferation, since it is commonly detectable in myoepithelial cells.