Polyclonal nature of diffuse proliferation of interstitial cells of Cajal in patients with familial and multiple gastrointestinal stromal tumours.

BACKGROUND: Diffuse proliferation of interstitial cells of Cajal (ICCs) in the myenteric plexus layer of the intestine has been described in patients with familial and multiple gastrointestinal stromal tumours (GISTs). However, it is not fully understood whether proliferation is polyclonal or monoclonal. AIMS: To evaluate the clonal nature of diffuse ICC proliferation in familial and multiple GIST cases, we carried out clonal analysis using inactivation at the human androgen receptor (HUMARA) locus. MATERIALS AND METHODS: Diffuse ICC proliferation tissues from three female patients were microdissected using a laser capture microdissection (LCM) system. Normal intestinal mucosal tissues were also microdissected for polyclonal controls and GIST tissues for monoclonal controls from the same patients, and genomic DNA was extracted. After digestion by restriction enzyme HhaI, the HUMARA locus was amplified by a fluorescent polymerase chain reaction (PCR) procedure and the PCR products were analysed. RESULTS: One case was uninformative because it was homozygous at the HUMARA locus. In the two other cases, PCR products from the diffuse ICC proliferation showed two alleles as well as those from normal intestinal mucosal tissues, indicating that ICC proliferation was polyclonal. In contrast, PCR products from associated GIST tissues showed only one allele, indicating that GISTs were monoclonal. CONCLUSION: The results suggested that diffuse ICC proliferation in familial and multiple GIST cases was non-neoplastic hyperplasia.

The importance of dedifferentiation in recurrent acinic cell carcinoma.

The biological activity of acinic cell carcinoma is uncertain. Histological dedifferentiation is one possible reason for recurrent disease, and this study was undertaken to assess its importance in acinic cell carcinoma. The initial and recurrent specimens from five patients with acinic cell carcinoma were assessed histologically and using flow cytometry, AgNOR estimation and morphometric analysis for evidence of dedifferentiation. No objective evidence of a change in biological aggressiveness in recurrent acinic cell carcinoma was identified. In this limited series of a rare salivary gland tumour, it would appear that factors other than dedifferentiation, such as close/involved margins, histological type and stage have a more meaningful effect on the likelihood of recurrence and prognosis.

Submandibular gland adenocarcinoma of intercalated duct origin in Smgb-Tag mice.

A line of transgenic mice that develops submandibular gland adenocarcinoma of intercalated duct origin was established. In these mice, the oncogene SV40 T antigen (Tag) is expressed from the neonatal submandibular gland secretory protein b (Smgb) gene promoter. This hybrid gene directs expression of the oncoprotein to neonatal submandibular gland proacinar and terminal tubule cells and to intercalated ducts of the adult gland. Transgene expression resulted in duct luminal cell hyperplasia as early as 20 to 30 days postnatally, which progressed to dysplasia by 3 to 4 months of age. Marked dysplasia and in situ carcinoma were evident at 4 to 6 months of age. All histologic changes were more pronounced in males. Submandibular gland adenocarcinoma developed stochastically in more than half of the adult male mice by 12 months of age (average age: 10.8 months, range: 6 to 13.5 months). Tag expression persisted in in situ carcinoma and all tumors. Using a combination of immunocytochemical and ultrastructural criteria, submandibular gland dysplasia and tumors were found to originate from intercalated ducts. The dysplastic ducts and adenocarcinoma in Smgb-Tag mice were morphologically similar to previously reported Tag-induced dysplasias of striated ducts and granular convoluted tubules and a Tag-induced adenocarcinoma of striated duct origin. These findings demonstrate that salivary gland dysplasias and tumors of similar histologic appearance can arise from distinct differentiated cell types. Analysis of the molecular changes accompanying tumor formation in Smgb-Tag mice could increase knowledge of human salivary gland tumorigenesis.

Intraosseous salivary tissue: jawbone examples of choristomas, hamartomas, embryonic rests, and inflammatory entrapment: another histogenetic source for intraosseous adenocarcinoma.

PROBLEM: Hundreds of primary salivary neoplasms have been found to be completely enclosed within the marrow spaces of the maxilla and mandible, yet nonneoplastic salivary tissue has never been convincingly identified within marrow, either separately or adjacent to such neoplasms. This situation has forced the acceptance of an inherently awkward odontogenic origin for all intramedullary salivary carcinomas and adenomas. OBJECTIVE: The purpose of this study was to microscopically evaluate a large number of maxillofacial marrow samples for the presence of intramedullary salivary tissue. STUDY DESIGN: We microscopically reviewed 5034 maxillofacial bone samples from the Latvala Inflammatory Bone Registry for evidence of heterotopic salivary inclusions within the marrow tissues. Contributing surgeons were contacted for each identified case of intraosseous salivary tissue to assure that all submitted tissue was removed from within the marrow spaces rather than from overlying soft tissue. RESULTS: Thirteen of 5034 marrow samples (0.3%) contained heterotopic acinic hamartomas, salivary choristomas, embryonic salivary rests, or entrapped surface glands. Four additional hamartomas of the condyle are described. We report also the chance finding of incipient odontogenic epithelial neoplasms (n = 6) and odontogenic epithelial rests (n = 84) within the fatty marrow and outside the periodontal ligament spaces, confirming that not all odontogenic neoplasms are necessarily of periodontal ligament origin. CONCLUSION: The frequency rate for salivary choristomas, hamartomas, embryonic rests, and displaced surface glands within alveolar bone is no less than 2.6 of 1000 biopsied marrow samples. This provides an additional and quite logical histogenetic explanation for the presence of intraosseous salivary neoplasms.

Adenoid cystic carcinoma: use of cell proliferation, BCL-2 expression, histologic grade, and clinical stage as predictors of clinical outcome.

BACKGROUND: Although the three basic histologic growth patterns of adenoid cystic carcinomas (tubular, cribriform, and solid) provide some indication of clinical outcome, additional, perhaps superior, predictors of biologic activity are needed for patient management. METHODS: This series is composed of 31 adenoid cystic carcinomas that presented in Linköping between 1982 and 1997. The tumors were clinically staged and histologically graded. For each case, after immunohistochemical identification, the proportion of tumor cells expressing the cell cycle markers MIB-1 and bcl-2 (as an indicator of proliferation and apoptosis, respectively) were quantified. Statistical correlation was sought between tumor stage and grade and the two cell cycle markers. RESULTS: The proportions of cycling tumor cells in adenoid cystic carcinomas ranged from 0.3% to 55%. For patients with no evidence of disease and a follow-up of at least 5 years, the mean percent MIB-1 value was significantly lower than for those patients who were alive with local recurrence and/or metastasis or who had died from their adenoid cystic carcinoma (p =. 024). MIB-1 tumor cell positivity also correlated strongly with tumor grade (p =.053), but not with stage (p =.22). Neither clinical stage nor histologic grade correlated with the degree of bcl-2 tumor cell positivity (p =.97 and p =.49, respectively). CONCLUSIONS: Staging and grading continue to play a vital role in the management of patients with adenoid cystic carcinoma. Furthermore, in this series of patients with adenoid cystic carcinoma, a cycling tumor cell population as measured by the MIB-1 antibody greater than 10% indicates this group as biologically more aggressive and at an increased risk for a fatal course.

Basal cell adenocarcinoma of the salivary gland: an ultrastructural and immunohistochemical study.

OBJECTIVE: The purpose of this study was to examine the ultrastructural and immunohistochemical characteristics of basal cell adenocarcinoma. STUDY DESIGN: Three cases of basal cell adenocarcinoma of the salivary glands were studied by means of light microscopy, electron microscopy, and immunohistochemistry. RESULTS: Some of the architectural tumor patterns encountered were solid, some were trabecular, and some were mixed. Ultrastructurally, solid areas were composed of nonluminal cells, some of which contained tonofilaments and well-formed desmosomes; tubulo-trabecular areas differentiated into both luminal and nonluminal cells. Both growth patterns were associated with the formation of excess basal lamina, marginally and between nonluminal cells. Myofilaments were infrequent in nonluminal cells of solid or trabecular areas. Cytokeratin (AE1/AE3) stained all 3 tumors, more peripherally in the solid pattern and usually centrally in the trabecular areas; vimentin stained all 3 tumors diffusely; smooth muscle actin (IA4) stained all 3 tumors but was mainly confined to peripheral tumor cells in both the solid and the trabecular growth patterns; epithelial membrane antigen and carcinoembryonic antigen stained 1 of the 3 tumors, predominantly in the luminal cells; p53 oncoprotein was focally positive in 2 of the 3 tumors; Ki-67 stained less than 5% of the tumor cells in all cases; and c-erb-B2 was uniformly negative in all cases. Staining patterns of cytokeratin and actin varied with the architecture of the tumor. CONCLUSIONS: Neither ultrastructural characteristics nor immunohistochemistry findings appear to distinguish basal cell adenocarcinoma from basal cell adenoma.

Multiple familial gastrointestinal autonomic nerve tumors and small intestinal neuronal dysplasia.

Multiple small intestinal stromal tumors were removed from mother and natural daughter within 15 months of each other. Both had long histories of recurrent iron deficiency anemia and upper gastrointestinal bleeding. Light microscopy revealed that the tumors had arisen in conjunction with diffuse hyperplasia/ dysplasia of Auerbach's myenteric plexus. Immunohistochemistry generally did not show myogenic or paraganglionic phenotypes; CD34 was positive in most tumors. Electron microscopy confirmed the association with the abnormal Auerbach's plexus and showed the structure of gastrointestinal autonomic nerve tumors (GANTs). These findings provide information as to the origin and evolution of GANTs, and also have implications for the clinical management of these tumors which appear to occur more frequently than previously thought.

Differentiation and the cytomorphology of salivary gland tumors with specific reference to oncocytic metaplasia.

OBJECTIVE: The different cell types and many growth patterns found in salivary gland tumors provide ample reason for the diagnostic problems caused by these tumors. To improve criteria for differential diagnosis, the potential range of cytologic features possible in salivary gland tumor cells must be better appreciated. STUDY DESIGN: From our respective pathology archives, normal salivary tissue and salivary gland tumours--other than Warthin's tumor and oncocytoma--with oncocytic differentiation were identified and studied by means of light and electron microscopy. RESULTS: In this article, we cite a number of different salivary gland tumors, including basal cell adenoma, pleomorphic adenoma, myoepithelioma, polymorphous low-grade adenocarcinoma, and mucoepidermoid carcinoma, showing varying degrees of oncocytic differentiation. CONCLUSIONS: Variable cellular differentiation is probably the basis for foci of tumor cells unexpected for a particular salivary gland neoplasm, further compounding differential diagnosis. Illustration of oncocytic differentiation serves 2 purposes. First, it can alert pathologists to this potential in otherwise typical salivary gland tumors; an awareness of this and other possible variations in cellular differential patterns can help prevent misdiagnosis. Second, these particular tumors illustrate the role of the cellular differentiation that is responsible for the range of histologic features within any one subtype of salivary gland tumors.

Mounting evidence against current histogenetic concepts for salivary gland tumorigenesis.

Various morphological observations of salivary gland tumors are frequently used to support histogenetic concepts, such as the semipluripotential bicellular reserve cell hypothesis, for these tumors. Singularly, evidence in support of this hypothesis remains unavailable. Indeed, physiological evidence from studies of salivary glands have long existed proving the semipluripotential bicellular reserve cell hypothesis was incorrect. More recent studies reconfirm this and show that all cell types in these complex glands are quite capable of replication and, therefore, of being involved in tumorigenic processes. The demise of the bicellular reserve cell hypothesis is long overdue.

A model for spontaneous B-lineage lymphomas in IgHmu-HOX11 transgenic mice.

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR alpha/delta regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHmu-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin's lymphoma of peripheral mature B cell origin.

Hybrid tumors or salivary gland tumors sharing common differentiation pathways? Reexamining adenoid cystic and epithelial-myoepithelial carcinomas.

Three adenoid cystic carcinomas and two epithelial-myoepithelial carcinomas, which focally shared common histologic features, were studied to examine the common differentiation pathways manifested by these tumors and to discuss criteria for hybrid salivary gland tumors. Regions of the adenoid cystic carcinomas had cellular features ranging from simple clear cell change of basal/myoepithelial cells to combined clear cells and prominent ductal structures mimicking epithelial-myoepithelial carcinoma. Conversely, two epithelial-myoepithelial carcinomas had adenoid cystic carcinoma-like regions caused by the formation of "pseudocysts"; this resulted in a focal cribriform pattern. Electron microscopy of two additional but typical epithelial-myoepithelial carcinomas revealed both excess basal lamina at the margins of cellular nests and widened intercellular spaces containing reduplicated basal lamina and accumulations of glycosaminoglycans; these ultrastructural features were identical to those seen in adenoid cystic carcinomas. The five current cases are not examples of hybrid tumors, but they demonstrate the effects of gene expression and the resulting differentiation of synthetic products and tumor cells that are generally restricted to one or the other of these two tumor types by as-yet-unknown means. To avoid misdiagnosis and its prognostic implications, adenoid cystic carcinoma-like regions in epithelial-myoepithelial carcinoma and epithelial-myoepithelial-like regions in adenoid cystic carcinoma should be recognized simply as anomalous differentiation.

Cell population changes during atrophy and regeneration of rat parotid gland.

Limited data exist regarding the changes in number and location of myoepithelial cells during salivary gland atrophy and regeneration. Through the use of double immunohistochemical labeling for muscle-specific actin and amylase coupled with morphometric analysis, this study investigated the changes in distribution and proportion of cell types during salivary gland atrophy/regeneration phases in a model previously used to study proliferation in rat parotid gland. The double immunohistochemical labeling clearly showed the changes in proportion of cell types in the atrophying and regenerating glands. The morphometric analysis showed that the relative myoepithelial area increased (as did the intercalated duct and striated duct areas) as the gland atrophied. Myoepithelial cells occupied 19.0% of the total epithelial area by day 7 of atrophy, up from 2.7% in the resting gland. Regeneration of acinar cells was obvious 1 day after duct release. The myoepithelial cell area decreased to 4.3% of the total epithelial area by day 14 of regeneration; this value was higher than the percentage of area in the resting gland (p = 0.02). The relative areas of acinar, striated duct, and intercalated duct cells returned to resting levels after 14 days of regeneration. The morphometric and histologic results of this study show that the parotid gland is capable of regenerating to essentially normal anatomic condition after 7 days of gland atrophy and then 14 days of regeneration. Each type of cell, however, responded to the atrophy and regeneration differently. Atrophy of salivary glands from radiation therapy. Sjögren's syndrome, or sialadenitis is an important clinical problem. Study of the salivary gland response to atrophy and regeneration may provide a framework for designing strategies for the radioprotection of salivary glands or methods by which to treat or reverse the effects of gland atrophy.

Quantitation and localization of cycling tumor cells in pleomorphic adenomas and myoepitheliomas: an immunohistochemical analysis.

Previous investigations have suggested that in certain salivary gland tumors only nonluminal (myoepithelial-like) tumor cells proliferate and that this may have histogenetic implications; however, no quantitative assessment of the cycling component is available. We decided, therefore, to enumerate the cycling luminal and nonluminal cells in 15 pleomorphic adenomas and six myoepitheliomas by using an antibody to proliferating cell nuclear antigen. The mean percentages of cycling luminal and nonluminal cells in pleomorphic adenomas were 2.3 +/- 1.4 and 2.4 +/- 2.3, respectively, and 2.1 +/- 0.7 of the tumor cells in myoepitheliomas. Even though duct-like structures in pleomorphic adenomas are considerably separated by more numerous nonluminal cells and, therefore, cycling cells may seem fewer, both cell types proliferate at similar rates. The results indicate that nonluminal cells are not the sole proliferative component in salivary gland tumors.

Squamous cell carcinomas of the head and neck cultured in floating collagen gels: 1. The maintenance of stromal and epithelial elements in vitro without fibroblast overgrowth.

The study of the molecular biology of head and neck squamous cell carcinomas has been heavily reliant on the analysis of cell lines. This is largely because the maintenance of primary cell cultures is difficult. However, being monoclonal, cell lines are not representative of the primary tumor because of the loss of tumor cell heterogeneity. We report a technique for primary culture of squamous cell carcinomas with maintenance of epithelial and stromal cell components without overgrowth of the fibroblast cells. Phenotypic markers for fibroblasts and squamous cells were present up to 45 days after initiation of culture, and expression of epidermal growth factor receptor and involucrin in cultures paralleled that in the primary tumor. In vivo, tumor stromal elements are thought to play an important role in the support of epithelial cell growth. In the collagen gel system the preservation of the stromal cell component likely improves culture viability and growth. More importantly, this culture system allows the in vitro tumor to more accurately reflect the tumor from which it was derived, and it permits the study of primary squamous cell carcinomas under in vitro conditions.

Myoepithelial cells actively proliferate during atrophy of rat parotid gland.

Despite limited supporting evidence, salivary gland myoepithelial cells are said to be differentiated cells with little or no capacity to replicate; they presumably develop from stem cells. This study investigated the proliferative potential of myoepithelial cells with an antibody to proliferating cell nuclear antigen and a rat model. This model involved clamping of the parotid duct causing atrophy of the gland and then releasing the duct followed by gland regeneration. Rats were sacrificed at time points during atrophy and regeneration phases and the number and location of cycling myoepithelial cells assessed. Cycling myoepithelial cells were identified with double immunohistochemical staining, cycling cells with proliferating cell nuclear antigen-positive nuclei within muscle-specific actin-positive cytoplasm (the latter identified with antibody HHF35). The results show that baseline proliferative rates of myoepithelial cells in both the resting and fully regenerated gland ranged from 0.3% to 2%, similar to rates for other major cell types in the normal rat gland. A peak myoepithelial cell proliferative rate of 23% occurred at day 5 during the atrophy phase. Rates during the regenerative phase were not significantly different than the baseline levels. Similarities of rat and human parotid gland and the definite proliferative capacity of myoepithelial cells indicates that these specialized cells must be considered one of the potential progenitor cells for human salivary gland tumors.

Differentiating myoepithelial and acinar cells in rat neonatal parotid gland and histogenetic concepts for salivary gland tumors.

Histogenetic concepts for salivary gland tumors are predicated on the presence of reserve or undifferentiated cells in normal glands, presumably the source for cell renewal and induction of tumors. Developing rat parotid gland, which remains fetal-like at birth, provides the opportunity to study differentiation and observe whether cytologically undifferentiated cells do or do not have functional indicators of specific differentiation pathways. Immunohistochemistry and immuno-electron microscopy, when applied to parotid gland at birth, at 12 days of age and in the adult gland, indicate that commitment to myoepithelial cell differentiation occurs prior to development of structural changes characteristic of these cells. Conversely, secretory granules are evident in differentiating acinar cells prior to synthesis of amylase. The results suggest that an appearance of undifferentiation does not confer reserve cell status either in the normal salivary gland or their tumors.

Immunoelectron microscopy for chromogranin A in small cell neuroendocrine carcinoma of lung.

To see if immunoelectron microscopy can improve localization of neurosecretory granules, postembedding immunolabelling for chromogranin A was performed on 15 examples of small cell anaplastic (neuroendocrine) carcinomas primary in lung, five cases of bronchopulmonary carcinoids, and two cases of pheochromcytoma; both the carcinoids and pheochromocytomas had neurosecretory granule-rich cytoplasm by routine electron microscopy and some neurosecretory granules were identified in each of the small cell carcinomas. Immunolabeling for chromogranin A resulted in many colloidal gold particles over cytoplasmic secretory granules in both pheochromcytomas and four of the carcinoids. One carcinoid that was focally positive by immunoperoxidase staining was negative by immunoelectron microscopy. None of the 15 cases of small cell carcinoma stained for chromogranin A using immunoperoxidase techniques, but three had a small number of secretory granules weakly labeled by the anti-chromogranin A/colloidal gold complex. Immunoelectron microscopy, at least using standard glutaraldehyde fixation and epoxy resin embedding, does not increase the sensitivity of neurosecretory granule identification in small cell neuroendocrine carcinomas of the lung. Results of other studies of this neoplasm suggest that despite transcription of chromogranin genes, synthesis of the specific protein may occur to a limited extent or not at all.