Practice of HER-2 immunohistochemistry in breast carcinoma in Austria.

Practice and accuracy of immunohistochemistry is known to vary highly. Reliability of HER-2 immunohistochemistry is critical because of its role in patient selection for therapeutical options in breast cancer. Therefore reliability of HER-2 immunohistochemistry in pathology laboratories in Austria was assessed. Ten tissue specimens of invasive ductal breast carcinomas and three cell line samples were tested. Presence/absence of gene amplification was determined by FISH to be used as a gold standard. Laboratories were asked to stain and assess slides using their routine immunohistochemical staining protocol. Overall the study consisted of 311 tests on tissue specimens and 142 on cell lines. In all cases manual scoring was performed. Participation was voluntary and was 94%. Overall sensitivity was 90.5% and specificity 99.2%. Overscoring including true false positive results were found in 6.7% and 6.3% in tissue specimens and cell lines, respectively. False negative determinations were obtained in 1.9% and 2.8% of tissue specimens and cell lines, respectively. HercepTest showed slightly higher reliability in comparison with individualized staining methods. By manual scoring inaccurate scoring affected 12.3% of test results and 62% of the laboratories. In conclusion participation rate and accuracy of HER-immunohistochemistry was high all over the country. Manually performed scoring demonstrated some limitations.

Seminoma metastases mimicking primary pancreatic cancer.

BACKGROUND: A case of seminoma clinical stage III, arising from the right testis and mimicking a primary pancreatic malignancy is reported. CASE REPORT: A 57-year-old male patient presented with obstructive jaundice. He suffered from recurrent abdominal pain and significant weight loss over the past 4 months. Abdominal CT scan showed a tumor in the head of the pancreas and multiple pathologically enlarged peripancreatic lymph nodes. In the laboratory findings there were signs of cholestasis and infection. A laparoscopic biopsy out of a suspicious lesion of the head of the pancreas and a surrounding lymph node was done. Histopathological examination reported metastasis of seminoma in a lymph node. Further laboratory findings showed an elevation of the human placental alkaline phosphatase (HPLAP) and urological examination revealed a suspect right testis. The patient underwent castration of the right testis and histopathological examination confirmed a seminoma. 4 cycles of chemotherapy including cisplatinum, etoposide and bleomycin led into complete response that is still ongoing. CONCLUSION: This case shows a seminoma with metastases at retroperitoneal site, mimicking a primary pancreatic neoplasm. It provides an example of the possibility of an uncommon clinical appearance of seminoma metastases and again underlines the importance of exact radiological and histopathological examination to distinguish between curable and incurable tumor.

Ultraviolet exposure as the main initiator of p53 mutations in basal cell carcinomas from psoralen and ultraviolet A-treated patients with psoriasis.

Basal cell carcinoma, the most frequent skin cancer in humans, is often linked to chronic sun exposure. In psoralen and ultraviolet A-treated psoriatic patients, basal cell carcinomas may occur even more frequently; however, the exact etiology and mechanisms of tumorigenesis in psoriatic patients are unclear because psoralen and ultraviolet A is not only a carcinogen but also an immunosuppressor and because psoralen and ultraviolet A-treated psoriatic patients often have other (co)carcinogenic risk factors (e.g, therapeutic exposure to ultraviolet B, X-ray radiation, arsenic, tar, and/or chemotherapeutic agents such as methotrexate). In this study, we analyzed the DNA of 13 basal cell carcinomas from five psoralen and ultraviolet A-treated psoriatic patients for mutations of the p53 tumor suppressor gene. DNA sequencing revealed a total of 11 mis-sense, two non-sense, and four silent mutations in seven of the 13 basal cell carcinomas (54%). Of the 13 total mis-sense or non-sense mutations, 12 (92%) occurred at dipyrimidine sites and nine (69%) were of the ultraviolet fingerprint type (eight C-->T transitions and one CC-->TT transition). Three of the C-->T transitions occurred at dipyrimidine sites opposite a 5'-TpG sequence (a potential psoralen-binding site and target for psoralen and ultraviolet A mutagenesis). Thus, whether these mutations were induced by ultraviolet or psoralen and ultraviolet A was not clear. In addition, two other mutations (15%) occurred at 5'-TpG sites, one (8%) occurred at a 5'-TpA site (the most frequent site of psoralen binding and mutagenesis in cell and murine studies), and one (8%) involved a G-->T transversion. These results suggest that (i) the major initiator of p53 mutations in basal cell carcinoma in psoralen and ultraviolet A-treated psoriasis patients is environmental and/or therapeutic ultraviolet(B) exposure, and that (ii) psoralen and ultraviolet A itself causes only a smaller portion of p53 mutations in psoralen and ultraviolet A-associated basal cell carcinomas.

Quality assessment in diagnostic molecular pathology: experience from a German-Austrian-Swiss multicenter trial.

In order to assess the current technical standard of diagnostic molecular pathology, we have conducted a multicenter trial with 34 participating pathology laboratories in Germany, Austria and Switzerland. Formalin-fixed, paraffin-embedded tissue blocks were selected from 15 cases, comprising 4 B-cell non-Hodgkin's lymphomas, 4 T-cell non-Hodgkin lymphomas, 4 cases with lymphadenitis, 2 cases with confirmed tuberculosis and 1 case of sarcoidosis. All participating laboratories received one 10-microm section from each of the 15 cases to detect clonality using immunoglobulin heavy chain (IgH) gene or T-cell receptor (TCR)-gamma gene rearrangement analysis in 12 and mycobacterial DNA in 3 cases. In addition, participants had to answer technical questions about the application of internal quality controls and performance of fragment length or sequence analysis. Correct results were reported in 80% and 90% for IgH and TCR-gammagene rearrangement analysis, respectively, and in 83% for mycobacterial DNA analysis. No significant differences in the quality of results were obvious when the individual techniques used for molecular analysis were compared. However, when two independent techniques were used by the same laboratory, a higher rate of correct results was obtained for IgH and TCR rearrangement analysis. In conclusion, this study demonstrates a high technical standard of molecular diagnostic adjuncts among the participating laboratories. Regular multicenter trials with a greater number of participating laboratories working in this field will be indispensable to ensure a continuing or increasing standard in diagnostic molecular pathology.

Primary cutaneous follicle center cell lymphoma with follicular growth pattern.

Cutaneous B-cell infiltrates showing a prominent follicular growth pattern with germinal centers are thought by some authors to represent either marginal zone lymphomas with reactive germinal centers or pseudolymphomas. To establish whether a true primary cutaneous follicular lymphoma exists, we studied biopsies from 15 patients with skin lesions characterized histopathologically by the presence of B-cell infiltrates with follicular pattern. Staging investigations, including bone marrow biopsy, were negative in all patients. All were negative for bcl-2 protein expression and did not present the t(14;18). In all biopsy specimens neoplastic follicles showed 1 or more morphologic or immunophenotypic criteria of malignancy (presence of a reduced mantle zone, absence of tingible body macrophages, reduced proliferation rate). In 9 specimens a monoclonal rearrangement of J(H) genes could be detected by polymerase chain reaction analysis. After laser beam microdissection, a band of the same length could be observed in 6 probes from different follicles from the same specimen, indicating the presence of the same monoclonal population of follicle center cells. Follow-up examinations in all patients revealed no evidence of extracutaneous spread (mean follow-up, 48.7 months). Our study demonstrates that primary cutaneous follicular lymphoma represents a distinct entity of the cutaneous B-cell lymphomas. (Blood. 2000;95:3922-3928)

[Point mutation of thyrotropin receptors as the main cause of autonomously functioning thyroid nodules: experiences in the framework of our Styrian patients]. / Punktmutationen des Thyreotropin-Rezeptors als wesentliche Ursache für die Schilddrüsenautonomie: Erfahrungen im Rahmen unserer steirischen Patienten.

Autonomously functioning thyroid nodules are frequently associated with mutations of the thyrotropin receptor. We analyzed a part of exon 10 of the thyrotropin receptor gene (base pairs 1762-1976) by direct sequencing and found missense mutations in 5 of 14 cases (codons 629, 631, 632, 633). Histologically, 3 of the 14 nodules were adenomas whereas 11 were hyperplasias. Nodules with mutations did not show significant differences from nodules without mutations with respect to age, histology, size, additional (non-functional) nodules and clinical symptomatology. Our results confirm that thyrotropin receptor mutations are involved in the development of autonomously functioning thyroid nodules. In this context, the terms hyperplasia and neoplasia should be reevaluated.

[Lattice corneal dystrophy. Detection of a point mutation in the kerato-epithelin gene]. / Gittrige Hornhautdystrophie. Nachweis einer Punktmutation im Keratoepithelin-Gen.

BACKGROUND: Lattice dystrophy is an autosomal-dominantly inherited disease. A mutation of the gene coding for kerato-epithelin has been found in patients with this stromal dystrophy. In codon 124 a Guanine to Adenine mutation of the nucleotide 417 has been described. We looked for this mutation in a family with lattice dystrophy treated in our clinic. PATIENTS AND METHODS: Using primers specific for kerato-epithelin gene, we amplified the cDNA extracted from lymphocytes of two patients suffering from lattice dystrophy. The polymerase chain reaction (PCR) products were subcloned and sequenced. RESULTS: Guanine to Adenine mutations, as published were detected in both of our patients at codon 124. CONCLUSION: We found the published mutation in both of our patients, indicating that this Guanine to Adenine exchange is pathognomonic for lattice dystrophy.

Factor V Leiden and prothrombin gene G 20210 A variant in children with ischemic stroke.

OBJECTIVE: To investigate if the factor V Leiden mutation (F-V-LM) and/or the prothrombin gene G 20210 A variant (P-G20210A-V) are risk factors for acute stroke in Austrian children. PATIENTS: 33 children with acute ischemic stroke documented by computer tomography and/or magnetic resonance imaging of the brain were enrolled in an open multicenter survey. RESULTS: 6/33 children had F-V-LM (5 heterozygous, 1 homozygous). This represents 18% (95% CI: 6.7-39.9%) of our pediatric stroke population and thus exceeds the expected prevalence in the Austrian population of 4,6% (Fischer's exact test, p = 0.01). F-V-LM was not found in 11 children with neonatal stroke but in 6/22 children with stroke after the neonatal period. 5/6 children with F-V-LM had an underlying disorder that is a risk factor for stroke in children. The P-G20210A-V was detected in 1/26 (3.85%; 95% CI: 0.1-21.4%) patients. Comparison of the prevalence of P-G20210A-V in our study with that in the general population of Austria of 1% revealed no statistical significance (Fischer's exact test, p = 0.38). CONCLUSION: Our data suggest that the F-V-LM is a risk factor for acute stroke in Austrian children beyond the neonatal period. The P-G20210A-V apparently does not represent a risk factor for stroke in Austrian children.

Extranodal follicular dendritic cell tumour of the nasopharynx.

We report the first case of an extranodal follicular dendritic cell (FDC) tumour localized in the nasopharynx of a 44-year-old male patient. The tumour cells were characterized immunohistochemically by strong expression of CD21, HLA-DR and vimentin and focal expression of CD68 and cytokeratin. Electron microscopic examination revealed desmosomal cell junctions between adjacent cell processes. Molecular genetic analysis using polymerase chain reaction (PCR) showed germline configuration of immunoglobulin and T-cell receptor genes. Epstein-Barr virus (EBV) genomes were detectable by PCR. After complete surgical tumour removal and radiotherapy the patient is disease-free 20 months after the initial diagnosis.

Primary cutaneous marginal zone B-cell lymphoma: a recently described entity of low-grade malignant cutaneous B-cell lymphoma.

Recently a new classification of primary cutaneous B-cell lymphomas (PCBCLs) has been proposed by the European Organization for Research and Treatment of Cancer (EORTC)--Cutaneous Lymphoma Project Group. The marginal zone B-cell lymphomas (MZLs) were not included as a distinct entity because of insufficient experience and controversial opinions. We have studied 32 patients (M:F ratio 1.5:1; age range 25-93 years; mean age 49.6 years; median age 50 years) to determine the diagnostic criteria of primary cutaneous MZL and the relationship with other low-grade malignant PCBCLs. For comparison, three patients with immunocytoma were included in the study. Clinically, patients presented with solitary or clustered reddish or red-brown papules, nodules, and plaques, sometimes surrounded by an erythematous halo. Histopathologic sections showed nodular or diffuse infiltrates involving the dermis and subcutaneous fat. Cytomorphologically small to medium-sized cells with indented nuclei and abundant pale cytoplasm (marginal zone cells, centrocyte-like cells) predominated. In addition, scattered blasts, lymphoplasmacytoid cells, and plasma cells were observed below the epidermis and at the periphery of the infiltrates. Reactive germinal centers were present in 75% of the cases. The three cases of immunocytoma showed a more monomorphous pattern with predominance of lymphoplasmacytoid cells. The marginal zone cells showed a CD20+, CD79a+, CD5- and Bcl-2+ immunophenotype. They expressed immunoglobulin G in the majority of the cases. Staining with the monocytoid B cell-related antibody KiM1p gave positive results in all specimens with a typical intracytoplasmic granular pattern. A monoclonal distribution of immunoglobulin light chains was observed in marginal zone cells in 75% of the cases. Germinal centers, when present, were either polyclonal or negative for both kappa and lambda light chains. Monoclonal rearrangement of the JH gene was detected via polymerase chain reaction (PCR) in 18 of 26 investigated specimens. Analysis in 12 patients of the bcl-2/immunoglobulin heavy chain gene rearrangement using PCR yielded negative results. Lesions were treated by surgical excision followed in some patients by local radiotherapy. Systemic antibiotic therapy was administered to three patients, with good response in two. The prognosis is excellent. After a mean follow-up of 47.9 months (range 6-252; median 24) all patients are alive without signs of systemic lymphoma. Primary cutaneous MZL represents a distinct clinicopathologic subtype of low-grade malignant PCBCL.

Genomic organization and molecular characterization of a gene encoding HsPXF, a human peroxisomal farnesylated protein.

A protein modification essential for the cellular sorting of many biologically relevant proteins is the covalent attachment of prenyl lipids by specific transferases. Isoprenylation is known to render protein domains hydrophobic, thereby facilitating the interaction with lipid bilayers and/or membrane proteins. The target for the modification with farnesyl groups is the COOH-terminal sequence CaaX. Among the variety of farnesylated proteins the only one reported so far to be located to peroxisomes is the 37-kDa peroxisomal farnesylated hamster protein PxF. Recently we published data on the cDNA of the human gene HK33 (A. Braun et al., 1994, Gene 146: 291-295), which was revealed to be the human ortholog of PxF and was consequently renamed HsPXF. The genomic structure, molecular characterization, and evolutionary conservation of HsPXF are described herein. The exact location of the gene was defined as chromosome 1q22. The gene spans a region of approximately 9 kb, containing eight exons and seven introns. The 5' upstream region showed two potential Sp1-binding sites and an Alu repetitive sequence. Luciferase reporter activating capacity confirmed the presumed promoter activity of this region. On the transcriptional level, we detected four splice variants originating either from exon skipping or from alternative splicing events. For the HsPXF protein, a carboxyterminal farnesylation at cysteine residues was demonstrated. Through the use of HsPXF-specific antibodies, the protein was shown to be attached to the outer surface of peroxisomes. This localization together with the similarity to a peroxisomal assembly protein from Saccharomyces cerevisiae suggests HsPXF is involved in the process of peroxisomal biogenesis or assembly.

[Lymphoepithelioma-like lung carcinomas]. / Lymphoepitheliomartige Lungenkarzinome.

Having observed 2 cases of lymphoepithelioma-like carcinoma of the lung in a 49-year-old female and in a 66-year-old male patient, we present a review on this entity, which was described for the first time in 1987. Essentially this neoplasm has the same histological appearance as a Schmincke-Regaud tumor, but it is possible that a certain morphological variety exists. In the differential diagnosis, a metastasis of a Schmincke-Regaud tumor and a malignant lymphoma should be considered. Including our 2 cases, a total of 30 cases have been reported: 14 male patients aged between 33 and 73 years and 12 female patients between 38 and 70 years; in 4 cases there was no reference to sex or age. Most of the patients were Asians, mainly Chinese. These tumors presented with nearly the same frequency in both lungs. They mostly appeared as peripheral coin lesions in the chest X-ray study. Lymph node metastases were found in approximately 25% of the cases. Hematogenous metastases seldom occurred and were observed almost only in the skeletal system. In most cases a lobectomy was performed. At present, no exact assertion is possible regarding the prognosis. An association with an Epstein-Barr virus infection was observed in the Asian patients, but not in the Caucasian patients.

[Point mutations of the thyrotropin receptor gene in autonomously functioning thyroid gland nodules: correlation with clinical findings and morphology]. / Punktmutationen des Thyrotropin-Rezeptor-Gens in funktionell-autonomen Knoten der Schilddrüse: Korrelation mit Klinik und Morphologie.

Seventeen autonomously functioning thyroid nodules (AFTN) were analyzed for the most frequent mutations of the thyrotropin (TSH) receptor gene at codons 619, 631, 632 and 633. DNA was extracted from formalin-fixed, paraffin-embedded samples from both nodules and surrounding tissue after micro- or macrodissection. A fragment of the TSH receptor gene (bp 1762-1976) encoding the third cytoplasmic loop and the sixth transmembranous domain was amplified by PCR. Screening for mutations at codons 619, 631, 632 and 633 was performed by restriction enzyme analysis using Asp 718, Hph I, Taq I and EcoR I, respectively. For verification, cases with a mutated restriction site were cloned and sequenced. Mutations were found in 3 AFTNs (18%): in 2 cases at codon 632 and in 1 case at codon 631. The 2 cases with a mutation at codon 632 harbored additional mutations at codon 599 and 640, respectively, detected by sequencing. Normal thyroid tissue outside the AFTNs did not harbor mutant TSH-receptor. Mutations occurred in patients with clinical and subclinical hyperthyroidism. Mutations were associated both with total (2 cases) and partial (1 case) suppression of the surrounding thyroid tissue as noted on the scinti scan. AFTNs with TSH-receptor mutation occurred as single nodules. All 3 nodules with mutations were classified histologically as nodular hyperplasia according to the criteria of the WHO. Although the number of cases is small, our data suggest that AFTNs are clinically and morphologically heterogenous. Since the most common TSH-receptor mutations occur only in a small subset of AFTNs other molecular genetic alterations may be involved in the development of AFTNs.

[Primary malignant non-Hodgkin's lymphoma of the breast: breast preserving therapy in 3 patients]. / Primäres malignes Non-Hodgkin-Lymphom der Mamma: Brusterhaltende Therapie bei 3 Patientinnen.

Primary malignant non-Hodgkin lymphoma is rare. The mammographic appearance is unspecific. The final diagnosis can usually be made after examination of paraffin-embedded tissue only. There exists no therapeutic standard for this disease. In the case of 3 patients treated at our institution, tumorectomy only and adjuvant therapy have been performed because there is no survival advantage for patients who underwent radical surgery.

Specific cutaneous infiltrates of B-cell chronic lymphocytic leukemia: a clinicopathologic and prognostic study of 42 patients.

The clinical and histopathologic features of specific skin infiltrates in patients with B-cell chronic lymphocytic leukemia (B-CLL) have rarely been reported in detail. In this study we analyzed the clinical, histopathologic, immunophenotypic, and molecular features of 84 skin lesions from 42 patients (M:F = 1.3:1; mean age, 66.0 years; range, 42-83 years) with specific cutaneous manifestations of B-CLL. The duration of B-CLL before skin manifestations varied from 0 to 142 months (mean, 39 months). In seven patients (16.7%), skin lesions represented the first sign of disease. Clinical presentations included localized or generalized erythematous papules, plaques, nodules, and large tumors. Ulceration was uncommon. In six patients lesions were confined at the sites of scars from previous herpes zoster (four patients) or herpes simplex (two patients) eruptions. Histologically, three main patterns were recognized: (a) patchy perivascular and periadnexal, (b) nodular-diffuse, and (c) band-like. Cytomorphologically, small monomorphous lymphocytes predominated. Proliferation centers were observed in only four specimens. In two patients presenting with tumors, a high content of large cells with feature of centroblasts and immunoblasts was found (Richter's syndrome). Immunohistologic analyses were performed on paraffin-embedded specimens in 40 biopsies from 20 patients and on cryostat sections in 17 biopsies from 11 patients. Neoplastic B lymphocytes in all cases showed an aberrant phenotype (paraffin sections: CD20+/CD5+/CD43+; cryostat sections: CD19+/CD5+; immunoglobulin light-chain restriction). Proliferation markers (Ki67, PCNA, MIB1) stained 5 to 80% of cells (mean, 25%; median, 20%). Polymerase chain reaction performed in nine cases on paraffin-embedded tissues using consensus primers for immunoglobulin heavy-chain genes showed a monoclonal population of B lymphocytes in all cases. Several discrete bands in addition to the prominent ones were noted in five cases, indicating the additional presence of B lymphocytes whose immunoglobulin genes were not monoclonally but oligoclonally rearranged. Follow-up data could be obtained from 31 patients. The two patients with Richter's syndrome died after 5 and 8 months, respectively. The 5-year survival of patients with small-cell cutaneous B-CLL was 66.6%. Our study indicates that cutaneous specific manifestations of B-CLL present with characteristic histologic, immunophenotypic, and molecular patterns. Prognosis in these patients is probably not affected by skin involvement.

Photoaccentuated erythroderma associated with CD4+ T lymphocytopenia: successful treatment with 5-methoxypsoralen and UVA, interferon alfa-2b, and extracorporeal photopheresis.

We describe a 53-year-old HIV-negative white man who had chronic CD4+ T lymphocytopenia and photoaccentuated erythroderma with lymphoma-like histologic changes. The erythroderma completely responded to 5-methoxypsoralen and UVA (PUVA), interferon alfa-2b, and extracorporeal photopheresis. During therapy opportunistic skin infections, including tinea corporis, warts, and disseminated molluscum contagiosum, developed. Although the patient met the current definition of idiopathic CD4+ T lymphocytopenia (ICTL), we cannot rule out the possibility that this peripheral CD4+ T lymphocytopenia resulted from sequestration of CD4+ T lymphocytes in erythrodermic skin.