Diagnosis of papillary thyroid carcinoma is facilitated by using an RT-PCR approach on laser-microdissected archival material to detect RET oncogene activation.

OBJECTIVE: The purpose of this study was to investigate the value of the expression of the RET oncogene (rearranged during transfection) in papillary thyroid carcinomas (PTC) and its variants in the differential diagnosis of thyroid neoplasias. According to the literature RET oncogene activation by chromosomal rearrangements has been exclusively implicated in PTCs. METHODS: To establish the incidence of RET activation in PTCs we used 5- to 10-microm sections from archival paraffin blocks. Either parts of the tissue slices were manually dissected or a few distinct cells were microdissected by laser-mediated manipulation with the Robot-MicroBeam system. RNA was extracted from paraffin-embedded thyroid tumors and the corresponding normal tissue. RT and nested PCR were performed using primers for RET/PTC1, PTC2 and PTC3, or for RET exons 12 and 13. PCR products were resolved by gel electrophoresis. RESULTS: We detected RET transcription in approximately 85% of the PTCs including follicular variants and in isolated cells of the same tissues, but not in nonmalignant thyroid tissue. CONCLUSIONS: Our method may serve as an additional diagnostic tool to characterize ambiguous neoplasias and to identify especially nonpapillary, i.e. follicular tumors, as papillary carcinomas. Additionally, this study has demonstrated that expressed genes can be analyzed from routine histopathological tissue slides or pooled single cells. Large retrospective studies can also be performed with this method.

FDG PET evaluation of granular cell tumor of the breast.

Granular cell tumor is a rare, usually benign neoplasm of neural origin that may arise in virtually any site and, when situated in the breast, can mimic breast carcinoma. We describe a case of granular cell tumor of the breast in a 57-yr-old woman. Clinical evaluation, mammography, sonography and MRI suggested a carcinoma with infiltration of skin and muscle. However, the tumor did not display increased glucose metabolism on PET. Clinical findings, imaging results, histological characteristics and surgical management are discussed.

Combined amyloidosis of the upper and lower respiratory tract.

Pulmonary and laryngeal manifestations of localized and organ-limited amyloidosis are sometimes seen, although pulmonary and laryngeotracheal amyloidosis are not always associated. Diagnosis can only be established histologically by the characteristic green birefringence in polarized light after Congo red staining and by immunohistochemical techniques. We describe the case of a 77-year-old woman who presented with hoarseness and an unproductive cough due to extensive amyloid deposits in both the upper and lower respiratory tract, immunohistochemically proven as the A lambda-type.

Contrast-enhanced MR imaging of the breast: comparison of two different doses of gadopentetate dimeglumine.

PURPOSE: To optimize detection and diagnosis of breast lesions with contrast-enhanced magnetic resonance (MR) imaging. MATERIALS AND METHODS: A three-dimensional fast low-angle shot pulse sequence was used for a group comparison consisting of 76 high-dose (0.16 mmol gadopentetate dimeglumine per kilogram of body weight) and 76 low-dose (0.1 mmol/kg) examinations. Intraindividual comparisons were possible in a subgroup of 20 patients. RESULTS: Enhancement with the high dose was about 1.5 times higher for benign and malignant tissues. With the lower dose, no false-positive findings could be avoided and definition of a threshold that excluded false-negative findings was problematic. Conspicuity of malignant lesions was much improved with the higher dose (a good to excellent rating in 81% vs 26% with the lower dose). Three small foci were visible only with the higher dose. CONCLUSION: The higher dose of contrast material allowed much better results. Dose comparison studies are also recommended for other techniques.

Contrast-enhanced MRI of the breast after limited surgery and radiation therapy.

OBJECTIVE: Posttherapeutic changes in the breast after tumorectomy (TE) and radiation therapy (RT) may mimic or obscure recurrent or new malignancies and thus interfere with conventional diagnostic studies. We investigated the enhancement of tissue during variable time intervals after therapy with contrast-enhanced MRI in 62 patients. MATERIALS AND METHODS: We report the results of 77 studies in 62 patients undergoing TE and RT. We include only those studies with at least 24 months of clinical and mammographic follow-up (n = 60) or histopathologic results (n = 17). RESULTS: Up to 9 months after therapy, differentiation between posttherapeutic changes and recurrence was frequently impossible because of the strong enhancement. Ten to 18 months after therapy, this posttherapeutic enhancement subsided slowly with some interindividual variations. After 18 months posttherapy, no significant enhancement was encountered in 30 of 32 cases. Diffuse or focal enhancement was present in all recurrent tumors and all recurrences were correctly diagnosed. Furthermore, 4 of 11 recurrences and 10 of 18 single recurrent foci were detected by MR alone, based on focal enhancement. CONCLUSION: Accordingly, contrast-enhanced MR is not recommended during the first 9 months after therapy. Nine to 18 months after therapy, it may be helpful in those two-thirds of cases where the scar does not enhance. If enhancement takes place (one-third of cases), it may represent either scar or tumor, and in such circumstances, enhanced MR is of no value. After 18 months, enhanced MRI has proven a valuable additional tool. By correctly detecting or excluding recurrent tumor, it can significantly improve diagnostic accuracy.

Cytokeratins, smooth muscle actin and vimentin in human normal salivary gland and pleomorphic adenomas. Immunohistochemical studies with particular reference to myoepithelial and basal cells.

The distribution of immunostaining in normal major salivary gland and in 12 pleomorphic adenomas was studied using monospecific monoclonal antibodies to a number of cytokeratins, including cytokeratin 14, to smooth muscle actin and vimentin. A number of these antibodies enabled a distinction to be made between structural components of the normal gland, and to relate this to the different structures of pleomorphic adenomas. In the normal gland, the luminal duct cells expressed cytokeratins 7, 8, 18 and 19. Three antibodies were of particular value for the characterization of normal myoepithelial and basal cells; while the antibody to smooth muscle actin and the cytokeratin antibody Ks8.12 mutually exclusively stained the myoepithelial (basket) cells and the basal duct (light) cells, respectively, the recently established monospecific antibodies to cytokeratin 14 showed specific immunostaining with both cell types. These three antibodies left luminal cells virtually unstained. Ck 13 was found occasionally in single luminal excretory duct cells. Antibodies to cytokeratins 1/2, 10 and 10/11 did not show any staining in the normal gland. In the pleomorphic adenomas, the staining pattern of the two-layered tubular formation resembled that of the normal gland ducts: tumour luminal cells showed the characteristic, although more irregular, expression of cytokeratins 7, 8, 18 and 19; the outer cells resembled normal ductal basal cells with their anti-cytokeratin 14/Ks8.12-epitope staining and in that they virtually lacked staining for smooth muscle actin. Trabecular formations and cells in myxoid areas were reactive with Ks8.12 and for cytokeratin 14, occasionally also for cytokeratins 7, 18 and 19. Epidermoid cell islets expressed mainly cytokeratin 14 and inconsistently the squamous epithelial cytokeratin 13 and the epidermal cytokeratin 10/11. Vimentin was found in cells of myxoid areas. The results support the postulate that some of the normal duct basal cells act as reserve cells and can give rise to tumour formation with a primitive myxoid or trabecular pattern and a more differentiated tubular or epidermoid configuration.

Antibody markers of basal cells in complex epithelia.

In the course of immunohistochemical studies it has become apparent that there is a distinct phenotype of keratin expression that is shared by basal epithelial cells in a variety of different tissues. A basal cell can be defined as a cell in contact with a basal lamina but with no free luminal surface; this distinguishes it from a simple epithelial cell, which has a free luminal surface as well as basal lamina contact, and from stratifying suprabasal keratinocytes, which have neither basal lamina contact nor free luminal surface. All basal cells, whether they are in glandular ductal or secretory epithelia, or in stratified squamous epithelia, express the keratin pair K5 and K14. In this paper we describe monoclonal and polyclonal antibodies that are monospecific for both keratins 14 and 5 or are specific for denaturation-sensitive epitopes unique to basal cells, including five new monoclonal antibodies: LL001 and LL002 (to keratin 14), 2.1.D7 (to keratins 5, 6 and 8), and LH6 and LH8 (conformation-specific basal cell markers). These antibodies have been used to monitor the distribution of the basal cell phenotype and to demonstrate the expression of keratins 5 and 14 in this cell type, in both stratified epithelia and mixed epithelial glands. The consistent association of this keratin pair with basal cells suggests a possible specific function for these keratin in reinforcing epithelia under physical stress, whilst expression of these keratins may conflict with the differentiated functions of most simple epithelial cells.

Amyloid goiter and arthritides after kidney transplantation in a patient with systemic amyloidosis and Muckle-Wells syndrome.

A case of hereditary AA amyloidosis with Muckle-Wells syndrome is described. After a successful kidney transplantation for chronic renal failure due to renal amyloid deposits at age 21, the patient, a white female now 26 years of age, developed a large amyloid goiter as a manifestation of the systemic amyloidosis and recurrent monarthritides. Both observations are novel for this disease. Subtotal thyroidectomy and oral colchicine administration, known to be effective in preventing complications of familial Mediterranean fever, another hereditary type of AA amyloidosis, proved highly effective in the management of this unusual case.

Diversity in immunoreactivity of tumor-derived cytokeratin monoclonal antibodies.

The cytokeratins 8, 18, and 19, expressed in many normal and malignant epithelial cells, were purified from human gastrointestinal tumors and used as immunogen for hybridoma generation. The reactivity pattern of five of the generated ten monoclonal antibodies (MAb) was characterized biochemically and immunohistochemically. All of the generated MAb were reactive with the central rod portion of the cytokeratins, as determined after partial enzymatic degradation, and displayed characteristic reactivity patterns. MAb TS 4 exhibits pan-epithelial immunohistochemical reactivity staining of all epithelial structures, including all layers of epidermis and non-keratinizing squamous epithelium and myoepithelial cells. The determinant involved is present on several different cytokeratins, i.e., nos. 1, 5, 7, 8, and 15, as determined by immunoblotting experiments from different tissues and cell lines. MAb TS 1, TS 3, and TS 7 reveal pluri-epithelial reactivity pattern immunohistochemically, similar to TS 4, but they are unreactive with whole epidermis and with superficial cell layers of non-keratinizing squamous cells. MAb TS 1 was found to be highly specific and reactive only with cytokeratin 8. Furthermore, the TS 1 MAb alone can precipitate the antigen, indicating reactivity with repetitive epitopes on cytokeratin 8. MAb TS 3 and 7 bind to cytokeratins 7 and 8. Finally, MAb TS 8 was found to be immunohistologically the most restricted, in general lacking reactivity to hepatocytes, pancreatic and salivary gland acinar cells, proximal renal tubules, and luminal cells of the epididymis. TS 8 was mainly reactive with cytokeratin 19 and showed weak binding to cytokeratin 8 and 14.

Distribution of tissue polypeptide antigen (TPA) in normal mucosa, in colitis ulcerosa, in adenomas and in carcinomas of the human colorectum. An immunohistochemical study.

65 carcinomas with their normal resection margins, 30 adenomas of the colorectum, and also ulcerative colitis biopsies from 10 cases were analysed immunohistochemically for pattern and intensity of expression of Tissue Polypeptide Antigen (TPA). In normal colon, and in well- and moderately-differentiated carcinomas, a cell membrane type staining pattern was predominant. In ulcerative colitis, in carcinoma cell groups within mucus of mucinous carcinomas or in single cells at the invasion front of all grades of carcinomas, a strong cytoplasmic type staining pattern was found. The cytoplasmic pattern was also found in poorly differentiated carcinomas, but with weaker staining intensity. The relationship between staining intensity and pattern and carcinoma grade was significant, whereas a similar relationship with the Dukes stages was not significant.

Immunohistochemical study of granular cell tumours. Demonstration of neurone specific enolase, S 100 protein, laminin and alpha-1-antichymotrypsin.

Nine granular cell tumours were investigated with poly- or monoclonal antisera to neurone specific enolase (NSE), glial enolase (GE), S 100 protein, alpha-1-antichymotrypsin, lysozyme, laminin, neurofilament (NF), glial fibrillary acidic protein (GFAP), brain creatine kinase (CK), different cytokeratins (Keratin Dako, PKK1), tissue polypeptide antigen (TPA), carcinoembryonic antigen (CEA), desmin, myoglobin and leukocyte common antigen (LCA), using immunoperoxidase-methods on formalin fixed paraffin embedded sections. While five tumours from adults show specific cytoplasmic staining for NSE and S 100, three congenital tumours, two from the gingiva and one from palatine, show only a weak reaction for NSE, reflecting a possible origin from mature and immature Schwann cells, respectively. However, one subcutaneous tumour from near the clavicule of a ten year old girl differs from the other eight tumours by its specific cytoplasmic staining for alpha-1-antichymotrypsin only, supporting the view that there are granular cell tumours of histiocytic origin. In addition, the five adult NSE-S100 tumours show strong laminin-immunostaining around the single small or syncytial granular cells, whereas pericellular laminin is not detectable in the histiocytic nor in the three congenital tumours. None of the tumours shows any staining for lysozyme, epithelial, muscular, leukocyte, neurofilament or glial antigens.

Distribution of tissue polypeptide antigen (TPA) in normal human tissues: Immunohistochemical study on unfixed, methanol-, ethanol-, and formalin-fixed tissues.

The distribution of tissue polypeptide antigen (TPA) was studied in unfixed, methanol-, 95% ethanol-1% acetic acid (EA)-, and formalin-fixed paraffin-embedded sections of all adult human tissues using an indirect immunoperoxidase method. The specific staining patterns were virtually identical in unfixed and alcohol-fixed tissues, but in formalin-fixed tissues this similarity was found only after fixation for up to 24 hr and pretreatment with protease for 15 min. Although prolongation of formalin fixation beyond 48 hr increasingly diminished the TPA reactivity, TPA could still be demonstrated in tissues fixed in formalin for up to 6 months. TPA was found to be a cytoplasmic constituent of almost all adult human duct and cavity lining, simple, and stratified epithelia. TPA was not demonstrated in epidermis, renal proximal convoluted and testicular tubules, basket-like myoepithelial cells, nor in most glandular acini, including hepatocytes and pancreatic acinar cells. The TPA staining was also negative in all non-epithelial tissues, including lymph nodes and bone marrow. The well-defined epithelial distribution and the comparable demonstrability in differently preserved tissues make TPA a useful tool for the identification of cells of epithelial character.

Immunohistochemical identification and crossreactions of amyloid-A fibril protein in man and eleven other species.

Antisera were prepared in rabbits, sheep or chicken against purified amyloid fibril protein AA from man, mouse, stone marten, dog, cow and hamster. These antisera were tested by immunodiffusion against all purified antigens and applied to tissue sections containing amyloid from man, mouse, hamster, guinea pig, rabbit, cat, dog, mink, stone marten, pine marten, cow and horse. The binding of the antibodies to amyloid in tissue sections was assessed by the indirect immunoperoxidase method. The strongest reactions in the immunodiffusion and immunohistochemical methods were found between amyloid deposits of members of a given species and an antibody raised against protein AA from the same species. In contrast to the lack of cross-reactivity in immunodiffusion (except in the mouse-man relationship), extensive cross-reactions were observed immunohistochemically in phylogenetically related species, e.g. between stone marten, pine marten and mink, or between hamster and mouse. However, cross-reactions were also observed in combinations such as man-mouse, man-dog, man-cat, mouse-horse, and dog-cow. In addition, individual antisera showed variations in immunohistochemical reactivity with amyloid deposits of different members of one given species. Moreover, antisera prepared in rabbits reacted more restrictedly than those prepared in sheep, while rabbit antisera against any AA-protein did not react with rabbit amyloid. Finally, the widest degree of cross-reactivity including almost all mammalian species investigated was observed with a chicken antiserum to human amyloid AA protein.

Identification of amyloid A protein in a sporadic Muckle-Wells syndrome. N-terminal amino acid sequence after isolation from formalin-fixed tissue.

The amyloid of a patient (WAL) with a sporadic Muckle-Wells syndrome was analyzed in tissue sections and after it had been isolated from formalin-fixed tissue. The predominant amyloid fibril protein was the amyloid A (AA) type determined by the following criteria. (a) Only antiserum against protein AA gave a strong specific reaction, whereas the other antisera with specificity against amyloid of immunoglobulin origin, i.e., A-lambda and A-kappa, did not stain using the indirect immunoperoxidase technique on formalin-fixed, paraffin-embedded tissue sections. (b) In immunodiffusion, the predominant amyloid fibril protein from WAL isolated in pure form from formalin-fixed tissues precipitated in a line of identity with anti-AA and a purified and chemically identified protein AA from another patient. (c) Amyloid fibril protein from WAL had a molecular weight of 8000 to 9000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and, thus, is in the same range as the commonly found protein AA. (d) The N-terminal amino acid sequence analysis was that of protein AA. In addition to the pure amyloid fibril protein AA (WAL), proteins of higher molecular weights with AA-antigenic determinants were also isolated. These proteins may represent protein AA in a complex form or protein AA linked to other proteins. Since an inflammation is the most likely cause of amyloidosis complicating the Muckle-Wells syndrome an antiinflammatory therapy (as in other AA-type amyloidoses) is recommended.

Immuno-electron microscopic identification and classification of amyloid in tissue sections by the postembedding protein-A gold method.

Amyloid deposits from 3 patients with amyloid of the AA, A lambda, or A kappa type were investigated on ultrathin sections using the protein-A gold method and affinity purified amyloid-type-specific immunoglobulins. Anti-AA, -A lambda, and -A kappa reacted specifically with the corresponding amyloid fibril visualized by the agglomeration of the gold colloid particles. Therefore, this method can identify and classify amyloid fibrils and can distinguish them from other fibrillar proteins at the electron microscopic level.

Immunohistochemical analysis of thyroglobulin and keratin in benign and malignant thyroid tumours.

56 thyroid gland tumours and non neoplastic alterations were studied for keratin and thyroglobulin staining, using the indirect immunoperoxidase method on serial formalin fixed paraffin embedded sections. Papillary carcinomas showed a strong reaction with anti-keratin serum but a weak reaction with anti-thyroglobulin serum. Follicular adenomas and carcinomas showed virtually no reaction for keratin but a strong reaction for thyroglobulin. Undifferentiated and medullary carcinomas did not react with either antiserum, except for single cells in two undifferentiated carcinomas which reacted with anti-keratin serum. In nodular goiters, hyperplastic follicles showed little or no reaction with anti-keratin serum and strong reaction with anti-thyroglobulin serum. It is suggested that this virtually type-specific staining for keratin or thyroglobulin may be related to different degrees of cellular differentiation and organelle content in the tumour cells.

Immunohistochemical demonstration of epithelial and urothelial antigens at the light- and electron microscope levels.

Antiserum raised in rabbits against the cytosol fraction from calf bladder epithelium was absorbed with sera, human erythrocytes and non-epithelial tissues until it became specific for a number of epithelia including urothelium, as demonstrated by immunofluorescence and immunoperoxidase techniques. Electron microscope immunocytochemistry showed that, in the urothelium, the majority of reaction product was present in the superficial cells in association with the plasma membrane, cytoplasmic membrane vesicles and cytoskeleton. After a second series of absorptions with epithelial organs other than bladder, the antiserum was rendered urothelium-specific and the strongest reaction was seen in superficial layer cells. Epithelium and urothelium specific antigens were species cross-reactive and could also be demonstrated in foetal and malignant urothelium. In malignantly transformed mouse bladder epithelium cell lines, the epithelial antigens were associated with the inner surface of the plasma membrane and underlying cytoskeletal elements.

Lysozyme (muramidase) and alpha 1-anti-chymotrypsin as immunohistochemical tumour markers.

Since lysozyme and alpha 1-anti-chymotrypsin are constituents of normal histiocytes, their value as tumor cell markers in histiocytes neoplasias has been investigated using the indirect immunoperoxidase method and commercially available specific antisera on formaldehyde-fixed, paraffin-embedded 5 micrometers sections after pretreatment with pronase. The distribution of both markers was determined in 35 cases of malignant fibrous histiocytoma (MFH) and in 13 cases of malignant histiocytosis (MH). In 12 cases of MH both markers were found whereas in MFH alpha 1-antichymotrypsin was demonstrated in 26 and lysozyme in 16 cases only. In general, the staining for alpha 1-anti-chymotrypsin was more intense than the staining for lysozyme. A negative reaction does not exclude the possibility of MH or MFH. The presence of both constituents in tumours, however, can be considered as indicative of histiocytogenic origin and both can be useful markers for distinguishing histiocytic neoplasias from other tumours.