The interest of actin immunocytochemistry in diagnostic histopathology

The immunohistochemical detection of different isoforms of actin provides useful data in the histopathological diagnosis of human tumours. The marked resistance of this protein to fixation and embedding procedures makes it a practical alternative to the ultrastructural search of microfilaments in routine tissue sections. Staining with monoclonal antibodies against alpha smooth muscle actin is helpful in the diagnosis of benign and malignant smooth muscle tumours and allows the differentiation of various types of spindle cell sarcomas. These antibodies are also useful in tracing the myoepithelial cells in normal and various pathological conditions of the breast and salivary glands. Skeletal muscle actin is a reliable marker in the diagnosis of rhabdomyosarcomas. Its use should be combined and complemented with other markers (i.e. desmin; foetal, slow and fast myosins; myoglobin) to monitor the degree of rhabdomyoblastic differentiation of the neoplastic cells in single cases, a parameter of potential prognostic value.     

Immunohistochemical and morphological features of a small bowel leiomyoma in a black crested macaque (Macaca nigra)

ABSTRACT: BACKGROUND: Spontaneous gastrointestinal neoplasms in non-human primates are commonly seen in aged individuals. Due to genetic similarities between human and non-human primates, scientists have shown increasing interest in terms of comparative oncology studies. CASE PRESENTATION: The present study is related to a case of an intestinal leiomyoma in a black crested macaque (Macaca nigra), kept on captivity by Matecana Zoo, Pereira City, Colombia. The animal had abdominal distension, anorexia, vomiting, diarrhea and behavioral changes. Clinical examination showed an increased volume in the upper right abdominal quadrant caused by a neoplastic mass. The patient died during the surgical procedure. Necropsy revealed several small nodules in the peritoneum with adhesion to different portions of the small and large intestines, liver, stomach and diaphragm. Tissue samples were collected, routinely processed and stained by H&E. Microscopic examination revealed a mesenchymal tumor limited to tunica muscularis, resembling normal smooth muscle cells. Neoplastic cells were positive for alpha-smooth muscle actin and vimentin, and negative for cytokeratin AE1/AE3 by immunohistochemistry. Those morphological and immunohistochemical findings allowed to diagnose the intestinal leiomyoma referred above. CONCLUSION: Neoplastic diseases in primates have multifaceted causes. Their manifestations are understudied, leading to a greater difficulty in detection and measurement of the real impact provides by this disease.     

Pleomorphic vulvar leiomyoma with local invasive behavior

A case of pleomorphic leiomyoma in Bartholin gland's area in a 26-year-old woman is reported. After diagnostic treatment, primary excision was done. A large, solid tumor 10 x 7.5 cm was extirpated. The tumor showed locally invasive behavior, which suggested a malignant tumor of Bartholin gland, because of it's localization and outlook. Pathohistological examination and immunohistochemical reactions proved that it was a mesenchymal tumor of smooth muscle origin with marked polymorphism, without mitosis, with a myxoid stroma and with biological aggressivity, and the possibility of local recurrence. Thus, a second more radical surgical procedure, was performed. In the excised tissue, no residual tumor was found and all lymph nodes were negative.     

Fine needle aspiration biopsy and immunostaining findings in an aggressive inflammatory myofibroblastic tumor of the lung: a case report

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) can vary from benign pseudosarcomatous tumors to low grade sarcomas. To date, fine needle aspiration (FNA) findings of lung IMTs, especially in the aggressive form, have not been fully described. Here we present FNA biopsy findings in conjunction with immunohistochemical studies in a case of primary and recurrent pulmonary IMT. CASE: A 22-year-old man first presented with a left lung mass and 4.5 years later with a recurrent mass. Preoperative computed tomography-guided FNA was performed on both tumors. FNA cytologic smears of both specimens consisted of scant, distorted spindle cells suggestive of a spindle cell lesion but were insufficient for further classification. Needle core biopsies as well as touch imprints were performed during the FNA procedures. The imprints revealed abundant, well-preserved spindle cells with mild to moderate atypia and intermixed lymphocytes and plasma cells. The spindle cells in both specimens were immunoreactive for vimentin and smooth muscle actin and were negative for pancytokeratin, desmin, CD34 and c-kit. Thirty percent of the tumor cells were positive for p53. The findings were compatible with those of IMT. Histologic examination of the surgically resected initial and recurrent masses confirmed the diagnosis of lMT. CONCLUSION: The cytologic findings of pulmonary IMT in FNA specimens are suggestive of, although not specific for, IMT. Immunohistochemical studies can assist in the diagnosis by excluding other spindle cell lesions. Cytologic atypia and p53 immunoreactivity may be indicators of aggressive IMTs.     

Contractile proteins in pericytes at the blood-brain and blood-retinal barriers

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (α-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that α-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to α-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.     

Kit-like immunopositive cells in sheep mesenteric lymphatic vessels

Recent electrophysiological studies have suggested that there is a subpopulation of cells in lymphatic vessels which act as pacemakers controlling the characteristic spontaneous contractile activity in this tissue. In this study, electron microscopy and immunohistochemical techniques were used on sheep mesenteric lymphatic vessels to investigate the morphology of the cells comprising the lymphatic wall. The smooth muscle cells were not orientated in circular and longitudinal layers as is seen in the gastrointestinal tract, but were arranged in bundles which interlock and cross over in a basket-weave fashion. Antibodies to Kit and vimentin, which are widely used to label specialised pacemaking cells in the gastrointestinal tract (known as interstitial cells of Cajal), demonstrated the existence of an axially orientated subpopulation of cells lying between the endothelium and the bulk of the smooth muscle. Examination of this area using electron microscopy showed cells which were electron dense compared to the underlying smooth muscle and contained caveolae, Golgi complexes, mitochondria, 10-nm filaments, a well-developed endoplasmic reticulum and a basal lamina. The smooth muscle cells typically contained caveolae, dense bodies, mitochondria, abundant filaments, sER and basal laminae. Cells dispersed for patch-clamp studies were also stained for vimentin and myosin. Myosin-staining cells had the typical spindle appearance of smooth muscle cells whereas the vimentin-positive cells could either be branched or more closely resemble the smooth muscle cells. The present study provides the first morphological evidence that specialised cells exist within the vascular system which have the ultrastructural characteristics of pacemaker cells in other tissues and are vimentin and Kit positive.     

Localization of myosin and actin in ocular nonmuscle cells

Summary Myosin and actin were localized by indirect immunofluorescence microscopy using specific antibodies prepared in rabbits against highly purified gizzard myosin and actin. A strong fluorescence staining with both antibodies was observed in rat corneal epithelial cells, anterior lens epithelial cells, rod inner segments, and in rat and frog pigment epithelial cells. The immunohistochemical localization of myosin in corneal epithelial cells was further supported by the electrophoretic and immunological identification of smooth muscle type myosin heavy chain in pure corneal epithelial abrasions. Electron-microscopic observations revealed a clear correlation between staining with actin antibodies and the presence of numerous thin cytoplasmic filaments (50–80 Å in diameter). The functional and biochemical nature of 90–110 Å filaments occurring in corneal and lens epithelial cells, as well as the ultrastructural localization of myosin in ocular nonmuscle cells under study remains obscure.     

Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels

Myosin, tropomyosin, and actin were localized in the epithelial cells of rat intestine by means of specific antibodies to chicken gizzard smooth muscle myosin, tropomyosin, and actin by immunohistochemical studies at both the light and electron microscope levels (unlabeled antibody enzyme technique). The pattern of antibody staining was the following (a) Anti-actin was associated with the microfilament bundles of the microvilli in their entire length, as well as with the microfilament network in the terminal web. (b) Anti-myosin was concentrated along the rootlets of the microvillar microfilament bundles and within the filamentous feltwork forming the terminal web. (c) Anti-tropomyosin showed a distribution similar to that of anti- myosin. In addition, the three antibodies also labeled the subplasmalemmal web underneath the cell membrane bordering on the basal lamina. Utilizing the above ultrastructural findings, we wish to propose a functional model of microvillar contraction.     

The Eker rat: establishing a genetic paradigm linking renal cell carcinoma and uterine leiomyoma

Renal Cell Carcinoma (RCC) and uterine leiomyoma (often referred to as fibroids) are tumors arising from tubular epithelium and myometrial compartments of the kidney and uterus, respectively. These tumors have a very different clinical presentation, with RCC being one of the less common cancers, having a very poor prognosis, and occurring predominantly in men, whereas uterine leiomyoma are the most common tumor of women and are benign. Although they are distinct histologically, with RCC arising from epithelial cells and leiomyoma arising from smooth muscle cells, they share a common embryological origin. Renal tubular epithelial cells arise during nephrogenesis as a result of the mesenchymal-epithelial transition of condensed mesenchyme induced by the developing ureteric bud, and have a shared mesenchymal lineage with smooth muscle cells of the uterus. In addition to a common embryological origin, RCC and leiomyoma have been demonstrated to share a common genetic etiology. The Eker rat model was the first demonstration of a specific genetic linkage between RCC and uterine leiomyoma. Eker rats carry a germline defect in the rat homologue of the tuberous sclerosis complex 2 (TSC-2) tumor suppressor gene and develop spontaneous RCC and uterine leiomyoma with a high frequency. TSC patients are also at risk for RCC, and sporadic human uterine leiomyomas exhibit loss of function of the TSC-2 gene product, tuberin. Individuals with the inherited cancer syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC) that have germline defects in the fumarate hydratase (FH) gene develop papillary RCC and uterine and skin leiomyomas. Benign cutaneous lesions and uterine leiomyoma also arise in German Shepherd dogs with germline mutations in the Birt-Hogg-Dube (BHD) gene, and these animals develop RCC and uterine leiomyoma with a high frequency. Identification of the tumor suppressor genes involved in these diseases, TSC, FH and BHD, and the elucidation of the function of their protein products, tuberin, fumarate hydratase and folliculin, respectively, opens new avenues for understanding the pathogenesis of both RCC and uterine leiomyoma.     

Lacrimal gland myoepithelioma in a rhesus macaque (Macaca mulatta)

An 18-year-old rhesus macaque (Macaca mulatta) developed ptosis of the left upper eyelid due to a mass that had first been observed 10 years previously. The 11 x 7 x 7-mm mass was surgically excised, and the ptosis resolved after 5 days. Histologic examination of the mass revealed two confluent cell populations. Most cells were spindle-shaped and were arranged in loose fascicles. Smaller numbers of cells had squamous differentiation. The spindle-shaped cells expressed smooth muscle actin. Cells with squamous differentiation did not express smooth muscle actin, but did, along with around half of the spindle-shaped cells, express pan-cytokeratin. On the basis of histologic and immunohistochemical findings, the mass was diagnosed as myoepithelioma. The neoplasm most likely originated from the palpebral lobe of the lacrimal gland, although accessory lacrimal gland origin could not be excluded. Recurrence of the neoplasm has not been observed 6 months after surgery.     

Fine needle aspiration cytology of a dedifferentiated liposarcoma: report of a case with histologic and immunohistochemical follow-up

BACKGROUND: Dedifferentiation is a histologic progression of a neoplasm from low grade to high grade histology. It occurs in tumors of the retroperitoneum and in those undergoing treatment. This usually occurs in the setting of radiation or chemotherapy or as a spontaneous process over a long period. The features of dedifferentiation can be toward any mesenchymal element of the underlying neoplastic process. CASE: We report the cytologic features of a dedifferentiated liposarcoma arising in a 76-year-old man who had a history of well-differentiated liposarcoma. Papanicolaou- and Diff-Quik-stained smears from a radiologically guided fine needle aspiration biopsy showed a hypercellular sample. The smears showed a mixed population of cells. There were multinucleated, pleomorphic giant cells with abundant cytoplasm, smaller clusters of cells with a high nuclear/cytoplasmic ratio and cells with spindled and elongated nuclear features. The follow-up surgical resection specimen showed a dedifferentiated liposarcoma with strong and diffuse immunoreactivity to vimentin, desmin and CD68 in the large, pleomorphic cells; focal and weak immunoreactivity to smooth muscle actin and S-100 in these cells; and strong and focal immunoreactivity to desmin, smooth muscle actin and muscle-specific actin in the spindle cells. This supports the dedifferentiated components of this tumor to be of fibrohistiocytic and leiomyosarcomatous differentiation. CONCLUSION: Dedifferentiation of a well-differentiated liposarcoma should be entertained in the setting of a mass lesion in the retroperitoneum in patients with prior histories of well-differentiated liposarcoma. The radiologic features of a particular neoplastic process can be very helpful in determining the nature of this process.     

Morphological examination of the termination pattern of substance P-immunoreactive nerve fibers in human antral mucosa

The termination pattern of substance P (SP)-containing axons in human antral mucosa was examined using immunohistochemical techniques at the light and electron microscopic level. SP-immunoreactive (IR) axons were found to extend towards the pit region of the glands, where intraepithelial axons were observed. Electron microscopy showed immunostained axon profiles in close contact with the basement membrane of surface mucous cells. Membrane-to-membrane contacts between labeled axons and myofibroblast-like cells were identified, and SP-IR axons that were apposed to the epithelium were also in contact with subjacent myofibroblast-like cells. The anatomical relationship between SP-IR axons and the cells of the muscularis mucosae was investigated by light microscopy. Immunoreactivity for alpha-smooth muscle actin (alpha-sma) was used to visualize the smooth muscle cells, and the alpha-sma-IR cells were found to create a network that surrounded the gastric glands. Immunostained varicose axons ran alongside and in close apposition to the labeled muscle strands. Ultrastructural examination showed close contacts between SP-IR axon profiles and smooth muscle-like cells. In conclusion, SP-containing neurons may be important for sensory and secretomotor functions in the human antral mucosa.     

Dual secretory and myoepithelial differentiation in the transplantable R3230AC rat mammary carcinoma

The hormone-responsive R3230AC mammary carcinoma, serially transplantable in Fisher rats, shows striking functional and morphological similarities to the normal mammary gland. We have studied its cellular composition by both light and electron microscopy, employing markers of myoepithelial and epithelial cells. We identified two cell types: the major cellular component corresponded to epithelial milk-protein secreting cells, while a second component showed immunocytochemical and ultrastructural characteristics of the myoepithelial cells. These cells were positive with a monoclonal antibody detecting alpha smooth muscle actin. The dual differentiation which normally occurs in breast ducts is therefore reproduced in a malignant experimental tumor. The coexistence of neoplastic cell populations, divergent in morphology and function, that persist in a tumor despite many transplant generations, leads to reconsideration of the relationship between cellular differentiation and malignant transformation.     

Malignant epithelioid gastrointestinal stromal tumors: report of a case with cytologic and immunohistochemical studies

BACKGROUND: Gastrointestinal stromal tumors (GISTs) may exhibit a fusiform, epithelioid or mixed pattern of growth. Only rare articles report the cytologic and immunohistochemical features of malignant epithelioid tumors. CASE: A 57-year-old woman presented with a tumor mass in the small intestine omentum measuring 8 x 7 cm; it was surgically removed. Five years later 2 mesenteric relapses were studied by fine needle aspiration biopsy and later surgically excised. Cytologically the smears contained small clusters of epithelioid and plasmacytoid cells with round nuclei. The presence of nucleoli and occasional nuclear grooves were prominent. Focally the background was myxoid. Histologically the tumor showed an epithelioid pattern with moderate pleomorphism and mitoses in 6 of 50 high-power fields. Immunohistochemical study showed positivity for c-kit (CD117), vimentin, smooth muscle actin and caldesmon, and focally for desmin and cytokeratin. CONCLUSION: This case illustrates the difficulty in making a reliable diagnosis of the epithelioid variant of GIST by cytology alone. The immunohistochemical panel (apart from c-kit) should include smooth muscle markers and cytokeratins because they are more likely to be reactive. A complete cytoimmunohistochemical evaluation is mandatory to make an accurate diagnosis.     

Myxoid leiomyosarcoma of the uterus. Report of a case with cytologic findings

BACKGROUND: Myxoid leiomyosarcoma is a rare variant of uterine sarcoma, exhibiting malignant biologic behavior despite the absence of cytologic atypia and of significant mitotic activity. CASE: A 20-year-old female was referred with a cystic pelvic mass. At laparotomy, the tumor, weighed 2,200 g and originating in the left lateral uterine wall, was removed. Microscopic examination revealed well-differentiated smooth muscle cells without atypia and with a few mitotic figures in the copious myxoid matrix, suggesting myxoid leiomyosarcoma. Three years following laparotomy, an irregular mass around the uterus was noted on sonographic examination, suggesting local recurrence. Two years and six months later, the second operation was performed, and a locally recurrent, multicystic tumor weighing 3,500 g was excised. The histopathology was similar to that of the primary tumor. Cytologic findings on imprint material from the tumor revealed a few isolated or sheet like small cells consisting of spindle and polygonal cells with round and oval nuclei. Cytologic atypia was also minimal. CONCLUSION: Myxoid leiomyosarcoma should be included in the differential diagnosis of smooth muscle neoplasia.     

Immunohistochemical and ultrastructural characteristics of interstitial cells of Cajal in the rabbit duodenum. Presence of a single cilium

Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duodenum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce perinuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular contacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium showing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-Kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single cilium. Some populations of ICCs in the rabbit duodenum present certain immunohistochemical and ultrastructural characteristics that often are present in progenitor cells.     

Telocytes: novel interstitial cells present in the testis parenchyma of the Chinese soft‐shelled turtle Pelodiscus sinensis

Telocytes (TCs) are novel interstitial cells that have been found in various organs, but the existence of TCs in the testes has not yet been reported. The present ultrastructural and immunohistochemical study revealed the existence of TCs and differentiate these cells from the peritubular cells (Pc) in contact with the surrounding structures in the testes. Firstly, our results confirmed the existence of two cell types surrounding seminiferous tubules; these were Pc (smooth muscle like characteristics) and TCs (as an outer layer around Pc). Telocytes and their long thin prolongations called telopodes (Tps) were detected as alternations of thin segments (podomers) and thick bead‐like portions (podoms), the latter of which accommodate the mitochondria and vesicles. The spindle and irregularly shaped cell bodies were observed with small amounts of cytoplasm around them. In contrast, the processes of Pc contained abundant actin filaments with focal densities, irregular spine‐like outgrowths and nuclei that exhibited irregularities similar to those of smooth muscle cells. The TCs connected with each other via homocellular and heterocellular junctions with Pc, Leydig cells and blood vessels. The Tps of the vascular TCs had bands and shed more vesicles than the other TCs. Immunohistochemistry (CD34) revealed strong positive expression within the TC cell bodies and Tps. Our data confirmed the existence and the contact of TCs with their surroundings in the testes of the Chinese soft‐shelled turtle Pelodiscus sinensis, which may offer new insights for understanding the function of the testes and preventing and treating testicular disorders.