The CD30 gene promoter microsatellite binds transcription factor Yin Yang 1 (YY1) and shows genetic instability in anaplastic large cell lymphoma.

CD30 is a member of the TNF receptor family. Our interest lies in understanding the control of CD30 expression, particularly as its over-expression provides a diagnostic marker for a subset of non-Hodgkin's lymphomas, particularly anaplastic large cell lymphoma (ALCL), and because anti-CD30 treatment has been shown to be efficacious. We have identified a number of regulatory regions, including an Sp1 element in the minimal promoter, and a downstream promoter element that is required for start site selection. The discovery of both an activating AP1 site and an upstream microsatellite that represses transcriptional activity of CD30 suggests that this region is involved in dysregulation of CD30 expression. We have now identified the major microsatellite binding activity as transcription factor Yin Yang 1 by both one-hybrid cDNA library screening and peptide mass fingerprinting. Due to the strong repressive effect of the microsatellite, we also investigated whether microsatellite instability may induce changes in CD30 expression and hence explain the over-expression of CD30 in ALCL. Laser capture microdissection of ALCL biopsies and CD30 microsatellite typing indicated that the neoplastic cells show a high degree of variation, but this does not correlate with high CD30 expression seen in ALCL.

Crystalline plasma cell inclusions in helicobacter-associated gastritis.

BACKGROUND: Crystalline cytoplasmic inclusions are well documented in B cell lymphomas but have rarely been described in reactive plasmacytic infiltrates. AIM: Three cases of Lelicobacter-associated gastritis are described in which plasma cells focally contained rhomboid and needle-shaped crystalline inclusions. METHODS: Crystalline inclusions were identified in the gastric biopsy specimens from three patients undergoing routine upper gastrointestinal endoscopy. The cells were characterised immunohistochemically using the following antisera: cytokeratin, leucocyte common antigen, desmin, CD20, CD68, CD79a, CD138, immunoglobulin (Ig)G, IgA and IgM heavy chains, and kappa and lambda Ig light chains. Clinical follow-up data were obtained. RESULTS: All biopsies showed a Lelicobacter-associated active chronic gastritis. Variable numbers of plasma cells with intracytoplasmic crystalline inclusions in the superficial lamina propria were seen. The crystals were not stained with any of the antisera tested, but the cells containing the crystals expressed CD79a and CD138 and, in the two assessable cases, showed IgA and lambda light chain immunoreactivity. The more numerous morphologically normal plasma cells in each patient were polytypic, and there were no histological features to suggest lymphoma. Crystals were not identified in the plasma cells in mucosal biopsy specimens from other sites in any of the patients. CONCLUSIONS: Crystalline inclusions in plasma cells can occur in association with Lelicobacter gastritis. Although light chain restriction was shown in two patients, the overall histological and clinical findings indicated a reactive process. The presence of plasma cell crystals in isolation should not be considered to be diagnostic of lymphoma.

Extraskeletal myxoid chondrosarcoma: a light microscopic, immunohistochemical, ultrastructural and immuno-ultrastructural study indicating neuroendocrine differentiation.

AIMS: Extraskeletal myxoid chondrosarcoma is a rare low-grade soft-tissue sarcoma with locally aggressive and metastasizing potential. Extraskeletal myxoid chondrosarcoma has distinctive clinical, light microscopic, immunophenotypic, cytogenetic and ultrastructural features. Evidence that extraskeletal myxoid chondrosarcoma often shows neuroendocrine features was first provided by Chhieng et al. on the basis of an immunohistochemical and ultrastructural study of seven cases. Our study aims to further confirm by immunohistochemistry and ultrastructural studies, including immunoelectron microscopy, that extraskeletal myxoid chondrosarcoma indeed may show neuroendocrine differentiation. METHODS AND RESULTS: Fifteen cases of extraskeletal myxoid chondrosarcoma and seven control cases of skeletal chondrosarcomas were studied. Extensive immunohistochemical analysis was performed in all cases and ultrastructural studies were done in 11 extraskeletal myxoid chondrosarcomas and three skeletal chondrosarcomas. Immunoelectron microscopy was performed on one case each of extraskeletal myxoid chondrosarcoma and skeletal chondrosarcoma. Extraskeletal myxoid chondrosarcomas expressed neuron-specific enolase (100%), synaptophysin (87%), S100 (50%), PGP 9.5 (40%), and epithelial membrane antigen (25%). Co-expression of synaptophysin and PGP 9.5 was observed in six tumours. Skeletal chondrosarcomas showed expression of S100 protein, vimentin and neuron-specific enolase in all cases. Synaptophysin, chromogranin and PGP 9.5 were not expressed in any skeletal chondrosarcoma case. Ultrastructurally, extraskeletal myxoid chondrosarcoma was characterized by distinct cords of cells immersed in a glycosaminoglycan-rich matrix. The cells were rich in mitochondria, had well-developed Golgi apparatus and there were numerous smooth vesicles. In three cases there were easily found 140-180 nm diameter membrane-bound dense-core granules in cell bodies and in processes, unrelated to the Golgi, compatible with neurosecretory granules. Fewer such granules were present in the remaining extraskeletal myxoid chondrosarcoma cases, three of which also contained intracisternal tubules typical of extraskeletal myxoid chondrosarcoma. The skeletal chondrosarcomas had scalloped cell surfaces, prominent rough endoplasmic reticulum focally distended with secretory product, and lacked neurosecretory granules. Intermediate filaments were prominent in both extraskeletal myxoid chondrosarcoma and skeletal chondrosarcomas. Immunoelectron microscopy showed synaptophysin expression in the extraskeletal myxoid chondrosarcoma but not in the skeletal chondrosarcoma case. CONCLUSIONS: This study confirms that a substantial proportion of extraskeletal myxoid chondrosarcomas show immunophenotypic and/or ultrastructural evidence of neuroendocrine differentiation, and are unlikely to be related to conventional skeletal chondrosarcomas.

The diagnositc significance of Myf-3 hypermethylation in malignant lymphoproliferative disorders.

Deregulated methylation of cytosine in DNA is a frequent finding in malignancy that is reflected by general genomic hypomethylation and regional hypermethylation that includes the myogenic gene Myf-3. In this study of 198 DNA samples from 186 patients with a wide range of lymphoproliferative disorders (LPD), the methylation status of Myf-3 was assessed to evaluate its significance in the diagnosis of malignant LPD. DNA was digested with the restriction endonucleases HpaII and MspI, and using the Southern blot (SB) technique, the size and density of fragments that hybridized with a Myf-3 probe were used to assign the methylation status. None of the samples from 45 patients from a wide age range with benign LPDs had evidence of altered Myf-3 methylation and there was no age-related methylation change. By contrast, 115/123 (93%) of samples from patients with non-Hodgkin lymphoma (NHL) or lymphoid leukemia had increased Myf-3 methylation. There was no methylation alteration in 22/24 (92%) of samples from patients with Hodgkin lymphoma (HL), nor in five of six samples from LPDs that had atypical histopathologic features which were not diagnostic of lymphoma, while the remaining sample of atypical LPD had hypermethylated Myf-3 fragments. There was an association between increasing Myf-3 methylation and higher histopathologic grade of malignancy within specific lymphoma categories. It is concluded that the detection of increased Myf-3 methylation is a sensitive and specific test of malignancy which may complement other molecular methods that are currently used for the assessment of clonality. It may be of particular diagnostic use in natural killer (NK) and null cell malignancies for which other indicators of clonality are lacking. Furthermore, methylation status may prove to be of potential prognostic value.

Pulmonary toxicity of inhaled aerosolized prostacyclin therapy--an observational study.

Large white/landrace piglets (mass 11 to 21 kg) were exposed to aerosolized alkaline glycine diluent (n = 2) or inhaled aerosolized prostacyclin (n = 2) for five to eight hours. Pigs receiving these aerosols developed mild acute sterile tracheitis, involving the superficial layers of the trachea, shown histologically and ultrastructurally. Pigs receiving the diluent aerosol also showed mild inflammatory changes in the bronchioles. These findings suggest caution with the use of high volumes of aerosolized alkaline glycine diluent during inhaled aerosolized prostacyclin therapy.

Vertebral synovial osteochondromatosis with compressive myelopathy.

STUDY DESIGN: A case report of vertebral synovial osteochondromatosis with compressive myelopathy. OBJECTIVES: To describe the clinical, radiologic, and histopathologic features of vertebral facet synovial osteochondromatosis with compressive myelopathy. SUMMARY OF BACKGROUND DATA: There has been only one previously reported case of synovial osteochondromatosis affecting the vertebral facet joint and no previous report of associated compressive myelopathy. METHODS: The case history, radiology, surgical findings, and histopathology are reviewed. RESULTS: Vertebral facet synovial osteochondromatosis is a potential and readily manageable cause of spinal cord compression. CONCLUSIONS: Synovial osteochondromatosis of the vertebral facet joint should be considered as a cause of compressive myelopathy.

Medullary carcinoma of the thyroid: accuracy of diagnosis of fine-needle aspiration cytology.

BACKGROUND: A preoperative diagnosis of medullary carcinoma of the thyroid (MCT) allows for the investigation of associated multiple endocrine neoplasia/ pheochromocytoma, and definitive surgery without the need for frozen section. Criteria for cytodiagnosis are well known but variable patterns of presentation may cause diagnostic difficulty. METHODS: This study examines the accuracy of cytodiagnosis and the value of ancillary tests in 17 patients seen between 1976 and 1997. Nine patients underwent thyroid gland aspirations, five patients underwent fine-needle aspiration (FNA) of the thyroid and cervical lymph nodes, and three patients underwent cervical lymph node aspiration alone. Electron microscopy (EM) of aspirated material was performed in nine cases and immunocytochemistry in two cases. RESULTS: In all cases the diagnosis was suggested by FNA. In four cases, diagnosis and management were based on cytology alone. EM of FNA material was confirmatory in nine cases, two of which also showed positive calcitonin immunocytochemistry. In three cases the diagnosis was not proven until surgical resection, and in one case FNA confirmed lymph node metastasis in known MCT. Frozen section in five patients did not change the level of diagnostic confidence over the FNA diagnosis in any case. In four other thyroid tumors (one Hürthle cell follicular carcinoma, two anaplastic carcinomas, and one hyperplastic nodule) MCT was suspected in the FNA differential diagnosis but later excluded. In the Hürthle cell tumor immunoperoxidase staining was positive for calcitonin and in one anaplastic carcinoma, a neuroendocrine phenotype was suggested. In the latter case, additional EM excluded MCT. CONCLUSIONS: Although correct diagnosis is made by cytology in the majority of instances, other tumors may show cytologic findings similar to MCT. EM of FNA material was found to be the most definitive method of proving or excluding MCT. Immunocytochemistry may be misleading for rarely performed tests.

Granulomatous glomerulonephritis in intravenous drug users: a report of three cases in oxycodone addicts.

Various well-documented renal lesions are associated with intravenous drug use; however, intraglomerular mesangial granulomas have not been previously described. We report three patients who developed an unusual granulomatous glomerulonephritis and interstitial nephritis after intravenous injection of oxycodone, derived from suppositories. Granulomas were seen in an intraglomerular mesangial and also interstitial location. In both sites, the granulomas were associated with filamentous material, presumably derived from a component of the suppositories. This material was periodic acid-Schiff-positive, but negative with Congo red and silver stains. Ultrastructurally, the filamentous material was seen within the mesangial granulomas and also in a subendothelial location, suggesting derivation from the circulation with subsequent transport across the basement membrane and accumulation in the mesangium, where a granulomatous reaction was elicited. All patients developed a degree of renal failure; two of the patients require hemodialysis 20 and 30 months after presentation.

A novel MspI PCR-RFLP in the human cytosine 5-methyltransferase gene: lack of relevance for malignant lymphoproliferative disease and breast cancer.

Two alternate allelic forms of human cytosine 5-methyltransferase, 5-MT I and 5-MT II, which differ by the absence or presence of an intronic MspI recognition sequence, have been recognised. The polymorphic region was localised using a series of subprobes prepared upon MspI digestion of the 2.5-kb cDNA probe (hmt-2.5). A PCR-based method was then developed for rapid 5-MT genotyping. The gene and phenotype frequencies of 5-MT I and 5-MT II were not significantly different in genomic DNA samples from a series of non-Hodgkin's lymphomas and breast cancer cases compared with DNA from normal subjects. Allelism of 5-MT allows new approaches to the assessment of variation in gene copy number of 5-MT in different types of neoplasia.

Atypical cervical polyp with intracytoplasmic inclusions.

Benign polyps containing atypical stromal cells are described at many anatomical sites and some such lesions have been shown to contain intracytoplasmic actin-rich inclusions, believed to represent deranged filament metabolism in proliferating myofibroblastic cells. We present a case of an atypical cervical polyp with intracytoplasmic inclusions, occurring in a 23 year old female, and provide support for the proposal that these inclusions are composed of actin filaments, identical to those initially reported in infantile digital fibromatosis. This report emphasises the need to recognise the benign nature of such stromal proliferations and expands the range of myofibroblastic lesions in which actin inclusions may occur. Characterisation of the inclusions will provide further insight into the complexities of actin metabolism.

Evidence that DNA methylation imbalance is not involved in the development of malignant mesothelioma.

Methylation dysregulation has been a consistent finding in various malignancies, particularly those where the pathogenetic mechanisms are unclear. In order to test the hypothesis that methylation imbalance may not be a feature of cancers where the aetiologic agent or process is known, we studied the methylation status of the myogenic genes Myf-3 and Myf-4 by Southern blotting in malignant mesothelioma, a cancer strongly associated with asbestos exposure. DNA samples obtained from controls and mesothelioma patients did not exhibit hypermethylation of Myf-3 and hypomethylation of Myf-4, as noted in malignant lymphomas. The methylation status of Myf-3 and Myf-4 in malignant mesothelioma was similar to that of non-malignant cells indicating that dysregulation of the DNA methylating machinery may not be involved in mesothelioma development. The present findings do not support the view that methylation imbalance is a consequence of neoplastic transformation, but indicate that it may be one of the early molecular events involved in the genesis of some cancers.

B-cell target DNA quantity is a critical factor in the interpretation of B-cell clonality by PCR.

Criteria for the assessment of clonality by Southern blotting are well established but this is not the case for polymerase chain reaction (PCR)-based assays. Our studies, and infrequent reports in the literature, indicate that B-cell clonality may be erroneously inferred if only small numbers of polyclonal B-cells are present in test samples. In order to establish criteria to minimise the false positive assignment of B-cell clonality, DNA was analysed in a semi-nested PCR to detect rearrangement of the immunoglobulin heavy chain gene using a range (1 microgram-0.1 ng) of target DNA amounts from four tonsils and five lymph nodes showing reactive follicular hyperplasia, and from six B-cell lymphomas. A discrete, narrow band of PCR product of constant size was detected throughout the range of target DNA amounts in most lymphomas indicating the presence of a monoclonal B-cell population. In contrast, from the non-malignant tonsils and lymph nodes, larger target amounts generated a broad band of PCR products indicating populations of polyclonal B-cells, but smaller target amounts generated discrete, narrow PCR product bands of inconstant size indicating oligo- or monoclonal B-cell populations. Results of this study demonstrate that a range of DNA target amounts should be tested when the proportion of B-cells in a sample is unknown, thus preventing the analysis of insufficient target DNA which may lead to the false assignment of clonality.

DNA methylation and developmental genes in lymphomagenesis--more questions than answers?

There is now considerable evidence suggesting that alterations in the DNA methylating machinery play an important role in tumorigenesis and tumour progression. For example, focal hypermethylation and generalised genomic demethylation are features of many different types of neoplasms. It is thought that tumorigenesis and tumour progression may be caused by hypermethylation induced mutational events and silencing of genes which control cellular proliferation and/or demethylation induced reactivation of genes which may only be required during embryological development. Consequently, we have begun to investigate the role of DNA methylation and developmental genes in malignant lymphoproliferative diseases. Previously, in all cases of non-Hodgkins lymphoma and leukemia studied, we have shown that the myogenic developmental gene Myf-3 is abnormally hypermethylated. In this review we discuss the possible significance of these findings since in vitro studies suggest that Myf-3 may play an important role in control of the cell cycle and therefore lymphomagenesis. In vitro and in vivo evidence suggests that PAX genes may also have oncogenic potential. The PAX family of developmental genes are involved in cellular differentiation, proliferation and cell migration. Expression of PAX3 in particular is associated with cellular mobility. Our previous studies have indicated that alternate regional expression of PAX genes may be controlled by DNA methylation. Therefore, we have proposed that abnormal methylation profiles of PAX3 may be associated with neoplastic transformation and/or metastatic potential. Results thus far reveal that the paired box of PAX3 is abnormally hypermethylated and the homeobox abnormally hypomethylated in lymphomas and leukemias. These new findings are consistent with our postulate and support the idea that inappropriate methylation induced activation or inactivation of developmental genes such as Myf-3 and PAX3 play an important role in lymphomagenesis and disease progression and that inspection of the methylation status of other developmental genes is warranted.

Hypermethylation of the Myf-3 gene in human colorectal cancer.

Abnormal methylation of DNA is one of the earliest detectable changes in human tumors. We investigated hypermethylation of the Myf-3 gene, the human homolog of the mouse myogenic gene Myo-D1, in colorectal adenomas and adenocarcinomas. Histologically normal gut mucosa from colorectal cancer patients showed moderately higher levels of methylation than in other normal tissues. Varying degrees of Myf-3 hypermethylation were found in all five adenomas and eight adenocarcinomas examined. Carcinomas arising from within adenomas generally showed a decrease in both the heterogeneity and intensity of Myf-3 methylation. Regional hypermethylation of this gene therefore appears to be an early event in human colorectal neoplasia.

Clonal analysis of colorectal tumors using K-ras and p53 gene mutations as markers.

Mutations to the K-ras oncogene and p53 tumor suppressor gene are two of the most common genetic lesions in human cancers. In the present study we examined the clonality of colorectal tumors with respect to each of these genetic alterations. Screening for mutations was carried out using the polymerase chain reaction-based technique of single-strand conformation polymorphism. Eleven primary colorectal adenocarcinomas and two secondary adenocarcinomas were analyzed at four different sites within the tumor. Involved pericolic lymph nodes were collected from nine of these cases, a metastatic deposit in the liver was obtained in one case, and adjacent adenomatous lesions were collected in two cases. Seven tumors contained mutations in either the K-ras or p53 genes. In all cases, DNA derived from multiple sites within an individual tumor or metastatic deposits arising from that tumor showed the same pattern of gene mutation. Immunohistochemical staining for p53 protein overexpression also showed similar patterns of reactivity within individual tumors and their metastatic deposits. These results suggest that the major clonal expansion of colorectal carcinomas occurs after the acquisition of mutations in these genes. Our results also indicate that sampling errors are unlikely to occur in molecular studies aimed at defining the role of these genes in colorectal cancer progression.

Tumor genes and their proteins in cytologic and surgical specimens: relevance and detection systems.

Oncogenesis is the consequence of a series of genetic alterations that allow unrestrained cellular growth, tissue invasion, and eventual metastases. Tumor-related genes can be classified into functional categories. Proto-oncogenes/oncogenes have a stimulatory role in cell growth, and the inactivation of cancer-suppressor genes/antioncogenes results in the loss of cell cycle regulation. More recently, three other groups of tumor-related genes have been recognized. They include the antiapoptosis genes which protect from programmed cell death, the antimetastasis genes, and multidrug resistance genes. Besides aiding in tumor diagnosis, the detection of such tumor-associated genes and their products allows the identification of individuals with an inherited predisposition to neoplastic growths, and the overexpression of many of these oncogene products has been shown to be a potential marker of tumor behavior and a predictor of treatment outcome and response. The ability to utilize DNA and RNA probes for nucleic acid hybridization and polymerase chain reaction procedures in cell and tissue preparations of solid tumors and lymphoid proliferations expands and complements the information provided by immunohistochemical techniques. These probes allow direct visualization and correlation of specific genes and their protein products with cytomorphologic features, and form a powerful addition to the armamentarium of the cytopathologist and surgical pathologist.

Gastrointestinal autonomic nerve tumors: a clinicopathological, immunohistochemical and ultrastructural study of 10 cases.

Gastrointestinal Autonomic Nerve Tumors (GANTs) are an underrecognized group of gastrointestinal stromal tumors (GISTs) putatively arising from the neural plexuses of the bowel wall. Approximately 24 cases have been previously reported. Their histogenesis, malignant potential, morphology and phenotypic features are not well defined. We present details of 10 GANTs iterating features, predominantly ultrastructural, allowing distinction from other GISTs. Clinical details are: sex-7M, 3F; age range 31-79 yrs, mean 53; symptoms/signs--abdominal pain 3, GI bleeding 3, mass 2, anemia 2. Follow-up ranged from 1-102 mths, mean 29. Seven tumors involved the small intestine and 3 were gastric. Tumor size ranged from 30-160 mm, mean 79. They were solid and cystic, often transmural and usually involved mesentery and retroperitoneum. Spindled and epithelioid cells were "compartmentalized" by a branching microvasculature. Eosinophilic, PAS positive stromal globules were prominent. Paraffin immunostaining results were (number positive/total): vimentin (8/9), NSE (10/10), S100 protein (6/10), neurofilament protein (0/9), synaptophysin (3/9), desmin (2/9, focal), smooth-muscle actin (0/9). Ultrastructural diagnostic features were elaborate, branching cytoplasmic processes containing microtubules, intermediate filaments and varying numbers of neurosecretory granules. Characteristic features were elaborate smooth endoplasmic reticulum enmeshed with intermediate filaments, pleomorphic mitochondria with lamellar cristae, mitochondrial-RER complexes, confronting RER cisternae, and circumscribed collections of stromal "skeinoid" fibres. There were no features of smooth muscle, Schwannian or perineurial differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Southern blot analysis of lymphoproliferative disorders: use and limitations in routine surgical pathology.

Antigen receptor gene rearrangement studies are a sensitive means of determining lineage and clonality in lymphoproliferative disorders (LPDs) which remain difficult to classify after assessment of morphology and immunohistochemistry (IHC). This study investigates the utility of genotyping LPDs in a surgical pathology laboratory servicing a large teaching hospital. Ninety-eight specimens with detailed frozen (FS) and/or paraffin section IHC were studied, including 65 B-cell lymphomas, 14 T-cell lymphomas, 2 biopsies of T-zone dysplasia, one unclassifiable lymphoma, 8 Hodgkin's disease (HD) and 8 reactive nodes. Southern blotting (SB) was performed on tumor and control DNA cleaved with restriction enzymes EcoR1, Hind III and BamH1, using radiolabelled probes for the immunoglobulin heavy chain joining region, constant regions of kappa and lambda light chains, and the constant region of the T-cell receptor beta chain. All reactive nodes and those harbouring HD and DNA in the germline configuration, apart from JH rearrangement in one case each of HD and florid reactive hyperplasia. Of the non-Hodgkin's lymphomas (NHL), 17% did not reveal clonal rearrangements (11% B-NHL; 44% T-NHL). Most of the negative results could be explained by sampling error in partially involved nodes, highly polymorphous infiltrates where the neoplastic population may have been below the 1% threshold detectable by SB, and instances of anaplastic large cell lymphoma. After accounting for these cases, a 5% negative rate of genoclonality remained (3% B-NHL; 13% T-NHL). In the majority of NHL (95%), the diagnosis could be established on the basis of morphology and/or IHC alone.(ABSTRACT TRUNCATED AT 250 WORDS)