High frequency of p53 protein expression in thymic carcinoma but not in thymoma

Thymic epithelial tumours are broadly classified into thymomas and thymic carcinomas. Although both tumours occasionally show invasive growth, they exhibit different clinical and biological findings. The oncogene and anti-oncogene in thymic epithelial tumours have not been evaluated fully. We investigated the expression of p53 protein by immunohistochemical analysis using the anti-p53 polyclonal antibody (CM-1) in 17 thymomas and 19 thymic carcinomas. We also examined p53 gene (exon 5-8) mutation in 18 thymic carcinomas by using polymerase chain reaction-single-strand conformation polymorphism methods and direct sequencing. Of the thymoma cases, only one invasive thymoma showed focal nuclear staining. Fourteen of the 19 thymic carcinomas (74%) showed nuclear staining. Point mutations of the p53 gene were recognized in only 2 of the 18 thymic carcinomas (11%). One was the mutation C to T transition in the first letter of codon 222 in exon 6, which results in the amino acid substitution from proline to serine. Another was a silent mutation. p53 protein accumulation is highly frequent in thymic carcinomas but not in thymomas, and gene mutation is uncommon in thymic carcinomas.     

Effect of the mycotoxins penitrem, paxilline and lolitrem B on the electromyographic activity of skeletal and gastrointestinal smooth muscle of sheep

Myasthenia gravis (MG) is mediated by autoantibodies against the acetylcholine receptor at the muscle endplate. Some MG patients have in addition antibodies (Ab) to the skeletal muscle proteins ryanodine receptor (RyR) and titin. We have examined GM and KM allotypes, RyR and titin Ab in 44 MG patients (37 thymoma patients and 7 non-thymoma, late-onset patients) and 292 non-MG controls to see if GM/KM allotypes associate with differences in autoantibody production. All patients had titin Ab, and 15 thymoma patients had also RyR Ab. The phenotype GM 1, 2, 3 23 5, 21 was significantly increased in the patients with titin Ab compared with the non-MG controls (chi2 = 4.93, p < 0.05). Thymoma patients with RyR Ab had a higher frequency of the GM 3 23 5 phenotype compared with RyR Ab negative patients and controls (chi2 = 7.1, p < 0.05). KM allotypes did not differ between RyR Ab positive or titin Ab positive patients and controls. GM phenotypes may thus be associated with an autoimmune response against the muscle proteins titin and RyR in MG patients.     

Apoptosis, bcl-2 protein, and Fas antigen in thymic epithelial tumors

We examined 35 thymic epithelial tumors by immunohistochemical techniques to investigate the extent of apoptosis detected by in situ end-labeling of fragmented DNA and the expression of bcl-2 and Fas. There were 22 thymomas (4 medullary, 9 mixed, 3 predominantly cortical, 6 cortical), 3 well-differentiated thymic carcinomas (WDTCs), and 10 high-grade thymic carcinomas (HGTCs), according to the Müller-Hermelink histologic classification. The HGTC showed the highest apoptotic index of the tumor cells. The apoptotic indices of the thymomas and WDTCs were significantly lower than those of the HGTCs (P < .001), but there was no significant difference among each type of thymoma and the WDTCs. bcl-2 protein was expressed in the tumor cells of the medullary-type thymomas and of the HGTCs but not in the other types of thymoma or WDTC. Fas antigen was negative in the HGTCs but was predominantly expressed in thymomas and WDTCs with advanced clinical stages (P < .05). From the immunostaining pattern, WDTC might be more likely to belong to the class of thymoma than to the category of HGTC.     

Lymphocytes in host defense against Pneumocystis carinii

To determine the neuroendocrine (NE) features of thymic epithelial tumor, immunohistochemistry and electron microscopy studies were performed on eight NE tumors (thymic carcinoids) and 26 non-NE tumors (nine thymic carcinomas, five atypical thymomas, and 12 thymomas other than lymphocytic thymoma). Immunohistochemical studies were performed with antibodies against general markers for NE cells (synaptophysin, alpha subunit of a guanine nucleotide-binding protein, Go, and small-cell lung carcinoma cluster 1 antigen), and a broad panel of antibodies for hormonal substances. Thymic carcinoid showed synchronous diffuse immunoreactivity for the three NE markers and contained cells that were positive for a variety of hormonal products: human chorionic gonadotropin (hCG) alpha-subunit (eight of eight), hCG beta-subunit (three of eight), adrenocorticotropic hormone (ACTH) (three of eight), calcitonin (two of eight), calcitonin gene-related peptide (two of eight), and serotonin (one of eight). Conversely, although positivity for NE markers was neither synchronous nor diffuse in non-NE tumors, seven of nine thymic carcinomas, three of five atypical thymomas (focal or dispersed distribution), and none of the five thymomas were positive for at least two of these NE markers. A small number of neoplastic cells were positive for hCGalpha-subunit or ACTH in three thymic carcinomas and one atypical thymoma. Ultrastructurally, dense core granules (DCG) were much more frequent in thymic carcinoid, but several DCG-like granules were identified in 12 of 13 non-NE tumors with or without immunoexpression of NE markers. The presence of focal or dispersed NE cells in thymic carcinoma and atypical thymoma may reflect multidirectional differentiation within the tumor, which, like cytological atypia, epithelial CD5 expression, and lack of immature T cell infiltration, may be another feature of this group at thymic tumors.     

Immunohistochemical staining for bcl-2 and mcl-1 in intrathyroidal epithelial thymoma (ITET)/carcinoma showing thymus-like differentiation (CASTLE) and cervical thymic carcinoma

Intrathyroidal epithelial thymoma (ITET)/carcinoma showing thymus-like differentiation (CASTLE), a rare thyroid neoplasm, was recently shown to be immunoreactive for CD5, providing immunophenotypic evidence of previously postulated thymic differentiation. To assess whether ectopic malignant neoplasms with thymic differentiation display other markers associated with thymic carcinoma, we studied five cases of ITET/CASTLE, two cases of cervical thymic carcinoma, and one case of cervical thymoma for bcl-2 and mcl-1 immunoreactivity. Both of these antiapoptosis proto-oncogenes have been reported to be expressed by the majority of true thymic carcinomas but only a minority of thymomas. All of the five cases of ITET/CASTLE, both CD5-positive cervical thymic carcinomas, and one CD5-negative cervical thymoma were immunoreactive for bcl-2, as were 10 (91%) of 11 thymic carcinomas arising in the thymus, in contrast to 6 (25%) of 24 benign and invasive thymomas arising in the thymus. Similarly, all of the five cases of ITET/CASTLE, both cervical thymic carcinomas, but not the cervical thymoma, were immunoreactive for mcl-1, as were 9 (90%) of 10 thymic carcinomas, in contrast to 6 (33%) of 18 benign and invasive thymomas. We conclude that dual immunoreactivity for bcl-2 and mcl-1 is a feature of malignant neoplasms with thymic differentiation in general, both within the thymus and at ectopic sites.     

Exhaustive scanning approach to screen all the mitochondrial tRNA genes for mutations and its application to the investigation of 35 independent patients with mitochondrial disorders

There have been several reports that thymoma in human is a progressive disease, and that thymoma and thymic carcinoma form a continuum. We established a stable line of SV40T transgenic mice, which consistently produced thymic epithelial tumours progressing to thymic carcinoma within a predictable time span. Using this animal model and a morphological approach, thymic epithelial tumour progression was studied with reference to sequential changes at different time points in animals aged from 3 to 32 weeks. At all ages, SV40T was expressed in the nuclei of thymic epithelial cells; in these transgenic mice we observed the entire spectrum from cortical type thymoma to thymic carcinoma. Thymic size tended to increase with ageing in SV40T TG mice. While younger mice had predominantly cortical (organoid) or cortical thymoma, older mice had well-differentiated thymic carcinoma (WDTC) or poorly differentiated thymic carcinoma. When SV40T TG mice (248 line) reached a certain age, carcinoma of the thymus was present in all of them. Cortical-type thymoma became malignant within a predictable time span, suggesting a cortical thymoma-carcinoma sequence. When the mice were 9 weeks of age, the thymuses formed gross masses compatible with cortical thymoma. At 14 weeks of age, WDTC appeared against the background of cortical thymoma. Poorly differentiated thymic carcinoma was found after 15 weeks and affected all animals over 23 weeks of age. Most thymic carcinomas coexisted in varying proportions with cortical-type thymoma. Medullary thymomas did not develop in the mice, and no transition from medullary-type thymomas to thymic carcinomas was observed. In this SV40T transgenic mouse model, thymic carcinoma is clearly preceded by cortical-type thymoma. These transgenic mice may provide an interesting model for the progression from cortical thymoma to WDTC and/or high-grade carcinoma.     

Molecular biology and immunology of acetylcholine receptor in relation to myasthenia gravis

In search for the myasthenogenic sites in the molecular structure of acetylcholine receptor (AChR) alpha-subunit, the conformation-dependent B-cell epitopes, and the MHC class II-restricted, immune cofactors-modified T-cell epitopes were studied. Using the peptides synthesized corresponding to AChR amino acid sequence, the alpha 183-200 (as an antigen to raise "blocking antibody") and the alpha 70-90, alpha 125-147 and alpha 67-76 with alpha 107-116 (as antigens to raise "binding antibody") were found immunogenic in the induction of the disease in animals. Phenotypic changes in the T-cell lineages in the thymus were discussed. An impairment of excitation-contraction coupling in some of myasthenic muscles was attributed to a defect caused by antibodies raised against ryanodine receptor protein. Myasthenia gravis patients' sera containing anti-ryanodine receptor antibodies inhibited the calcium-induced release of calcium in response to caffeine in human rhabdomyosarcoma cell line. Buffalo/Mna rats with spontaneous benign thymoma showed (1) ryanodine receptor expressed in the thymic epithelial cells, (2) anti-ryanodine receptor antibodies in serum, and (3) reduced twitch and tetanic force without abnormality in synaptic transmission and muscle membrane properties. It is suggested that thymic epithelial cell and skeletal muscle share common ryanodine receptor antigen. The finding seen in this rat strain can be a counterpart of the feature reflecting an immune attack directed against a compartment of skeletal muscle in myasthenia gravis.     

Multilocular thymic cysts associated with thymoma. A case report

The association of multilocular thymic cysts (MTC) with thymoma is exceedingly rare, and the pathogenesis of this combination is controversial. We describe the case of a 42-year-old man with an anterior mediastinal mass found to contain MTC and thymoma. A multilocular cystic mass, measuring 13 x 6.5 x 2 cm, was found in the right lobe of the thymus, and contained a 4.7 x 2 cm thymoma in its center. Microscopic thymomas, lipomatously involuted remaining thymic tissue, and lymphoid follicles with germinal centers were found in the walls of MTC as well as in the left thymic lobe. Non-specific chronic inflammation was also present in the walls. In addition, microcysts, which were only found at the periphery of the thymoma and covered with epithelium, might have been formed secondarily by dilatation of the perivascular spaces and of Hassall's corpuscles. These findings suggest that a chronic inflammatory process was responsible for the early formation and enlargement of this patient's MTC, and that while the cavities of the MTC expanded to various degrees, the thymoma, which originated from one of the microscopic thymomas in the walls of MTC, increased in size, and grew to involve the remaining thymic tissue.     

HIDA kinetics in children

We have investigated the immunohistochemical expression of p53, mdm2, p21/waf1 and bcl-2 proteins in 31 thymic epithelial tumours comprising five medullary thymomas (MDT), four mixed thymomas (MT), 12 cortical thymomas (CT), eight predominately cortical thymomas (PCT) and two well-differentiated thymic carcinomas (WDTC). We have found p53, mdm2, p21 and bcl-2 protein expression in 25/31, 8/31, 5/31 and 10/31 thymic epithelial tumours, respectively. Coexpression of p53 and mdm2 proteins was found in eight cases (three CT, four PCT and one WDTC). Five of them were also p21 positive and three p21 negative. Discordant p53+/mdm2-/p21- protein expression was found in 19 cases (three MDT, three MT, nine CT, three PCT and one WDTC). Mdm2 and p21 proteins were not expressed in the absence of p53 protein. Coexpression of bcl-2 and p53 proteins was found in seven cases (three MDT, three MT and one WDTC). Eighteen cases were p53+/bcl-2- (10 CT, seven PCT and one WDTC) and three cases (two MDT and one MT) were bcl-2+/p53-. Our findings indicate that in thymomas, p53 expression is more frequently associated with cortical histotypes while bcl-2 expression is strongly associated with medullary and mixed histotypes. In addition, there is an inverse correlation between p53 and bcl-2 protein expression in thymomas. Coexpression of p53/mdm2/p21 proteins may reflect thymomas with wild-type (wt), p53 gene since mdm2 and p21 proteins are inducible by wt p53 gene. However, in view of previous findings that p53 mutation is an early event in thymomas, the possibility of p53 gene mutation with p53-independent mdm2 and p21 expression should be considered in these cases. Discordant p53+/mdm2-/p21- protein expression may represent thymomas with p53 gene mutations unable to activate expression of mdm2 and p21 proteins.     

Thymoma-associated myasthenia gravis. Transplantation of thymoma and extrathymomal thymic tissue into SCID mice

To study the possible role of thymomas and of extrathymomal thymic tissue in the development and maintenance of myasthenia gravis, we transplanted fragments of either tissue into SCID mice and monitored the production of anti-acetylcholine receptor antibodies in the recipients. Furthermore, the transplants were characterized by immunohistochemistry. Unlike after transplantation of thymus with lymphofollicular hyperplasia that induced high titers of anti-acetylcholine receptor antibodies, thymoma transplants never produced autoantibodies. Mice transplanted with extrathymomal thymic tissue also failed to produce anti-acetylcholine receptor antibodies except one group that received transplants containing hyperplastic extrathymomal tissue. These findings may explain the refractoriness of thymomatous myasthenia to thymectomy.     

p53 alteration, proliferating cell nuclear antigen, and nucleolar organizer regions in thymic epithelial tumors

We examined p53 protein expression, p53 gene mutation, proliferating cell nuclear antigen (PCNA), and argyrophilic nuclear organizer regions (AgNOR), in 30 patients with surgically-treated thymic tumors (26 thymoma and 4 thymic carcinoma cases). p53 expression ratio with DO-1 was divided as p53 negative (0% positivity), low expressor (<10% positivity), high expressor (>10% positivity). The incidence of p53 low and high expressor in thymoma were 19% (5/26) and 8% (2/26), respectively. p53 immunopositivity in thymoma was significantly correlated with PCNA labeling index (LI). p53 expression ratio in invasive thymoma (33%) tended to be higher than that in non-invasive thymoma (18%). p53 expression was detected in one of the thymic carcinoma. There were no p53 gene mutations in 15 invasive thymoma, although one of four (25%) thymic carcinomas showed two point mutations. p53 gene alterations seem to be associated with malignant activity of tumor cells, and therefore detection of p53 gene mutations is useful as a diagnostic factor.     

Thymic carcinoma: current staging does not predict prognosis

BACKGROUND: Thymic carcinomas are currently staged by Masaoka classification, a staging system for thymomas. We retrospectively evaluated surgical patients with thymic carcinoma to determine prognostic factors and to evaluate the usefulness of Masaoka staging in this disease. METHODS: Our computerized tumor registry yielded 118 patients with thymoma. Review of pathologic material revealed 43 cases of thymic carcinoma. Collection of data was by review of hospital and physician charts and telephone contact with patients. Analysis of prognostic factors was performed in patients undergoing complete resection by the method of Kaplan-Meier and Cox proportional hazards regression. RESULTS: Between 1949 and 1993, 43 patients underwent surgery for thymic carcinoma. Overall survival was 65% at 5 years and 35% at 10 years. Overall recurrence was 65% at 5 years and 75% at 10 years. On univariate analysis, survival was not dependent on age, sex, tumor size, or Masaoka stage but was dependent on innominate vessel invasion. By multivariate analysis, survival was dependent only on innominate vessel invasion. CONCLUSIONS: Patients with thymic carcinoma have a high rate of recurrence. Tumor invasion of the innominate vessels is associated with a particularly poor prognosis. Although Masaoka staging is useful in staging patients with thymoma, it does not appear to predict outcome for patients with thymic carcinoma.     

A 37-amino acid sequence in the skeletal muscle ryanodine receptor interacts with the cytoplasmic loop between domains II and III in the skeletal muscle dihydropyridine receptor

Interactions between the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor or RyR1) and the loop linking domains II and III (II-III loop) of the skeletal muscle L-type Ca2+ channel (dihydropyridine receptor or DHPR) are critical for excitation-contraction coupling in skeletal muscle. The DHPR II-III loop was fused to glutathione S-transferase- or His-peptide and used as a protein affinity column for 35S-labeled in vitro translated fragments from the N-terminal three-fourths of RyR1. RyR1 residues Leu922-Asp1112 bound specifically to the DHPR II-III loop column, but the corresponding fragment from the cardiac ryanodine receptor (RyR2) did not. The use of chimeras between RyR1 and RyR2 localized the interaction to 37 amino acids, Arg1076-Asp1112, in RyR1. The RyR1 922-1112 fragment did not bind to the cardiac DHPR II-III loop but did bind to the skeletal muscle Na+ channel II-III loop. The skeletal DHPR II-III loop double mutant K677E/K682E lost most of its capacity to interact with RyR1, suggesting that two positively charged residues are important in the interaction between RyR and DHPR.     

Immunoreactivity of a new CD5 antibody with normal epithelium and malignant tumors including thymic carcinoma

CD5, first recognized on subsets of lymphocytes, also is detected in thymic carcinoma but not in thymoma or other malignant tumors. We studied CD5 expression in 73 cases of malignant tumors of various organs, 22 cases of thymoma, and 7 cases of thymic carcinoma by immunohistochemistry using the new monoclonal anti-CD5 antibody, NCL-CD5-4C7, with a pressure cooker antigen retrieval method. All cases of thymic carcinoma showed positive staining for CD5, predominantly on the cell membrane. Two of 4 cases of atypical thymoma also showed focal positivity, whereas the other types of thymoma were negative. CD5 was detected in cases of malignant tumors other than squamous cell carcinoma and in the normal epithelium of their counterparts. Squamous cell carcinomas of various organs were negative for CD5. Malignant mesothelioma showed peculiar intracytoplasmic staining in contrast to the other tumors. The NCL-CD5-4C7 positivity in thymic epithelial tumors may support the hypothesis suggesting progression of atypical thymoma to thymic carcinoma. NCL-CD5-4C7 may be useful in the differential diagnosis of mediastinal tumors, especially between thymic carcinoma and metastatic squamous cell carcinoma of various primary sites, and for distinguishing malignant mesothelioma from adenocarcinoma of the lung by the different staining pattern.     

FK-binding protein is associated with the ryanodine receptor of skeletal muscle in vertebrate animals

The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.     

DNA sequence recognition by bis-linked netropsin and distamycin derivatives

The dihydropyridine receptor (DHPR), a voltage-gated L-type Ca2+ channel, and the Ca2+ release channel/ryanodine receptor isoform-1 (RyR1) are key molecules involved in skeletal muscle excitation-contraction coupling. We have reported age-related decreases in the level of DHPR expression in fast- and slow-twitch muscles from Fisher 344 cross Brown Norway (F344BNX) rats (Renganathan, Messi and Delbono, J. Membr. Biol. 157 (1997) 247-253). Based on these studies we postulate that excitation-contraction uncoupling is a basic mechanism for the decline in muscle force with aging (Delbono, Renganathan and Messi, Muscle Nerve Suppl. 5 (1997) S88-92). In the present study, we extended our studies to older ages and we intended to prevent or retard excitation-contraction uncoupling by restricting the caloric intake of the F344BNX rats from 16 weeks of age. Three age groups, 8-, 18-, and 33-month old caloric restricted rats, were compared with ad libitum fed animals. The number of DHPR and RyR1 and DHPR/RyR1 ratio (an index of the level of receptors uncoupling) in skeletal muscles of 8-month and 18-month rats was not significantly different in either ad libitum fed or caloric restricted rats. However, the age-related decrease in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in 33-month old ad libitum fed rats was absent in 33-month old caloric restricted rats. These results suggest that caloric restriction prevents age-related decreases in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in fast- and slow-twitch rat skeletal muscles.