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.

[Reevaluation of cytological smears in patients with cervical cancer. Regional quality assurance program with the cooperation of the Austrian Society for Cytology, the Carinthian Medical Association and the Carinthian Ministry of Health]. / Reevaluation von Zervixabstrichen bei Zervixkarzinompatientinnen. Regionales Qualitätssicherungsprogramm unter Mitwirkung der Osterreichischen Gesellschaft für Zytologie, der Kärntner Arztekammer und der Landessanitätsdirektion Kärntens.

BACKGROUND AND METHODS: To evaluate the reasons for the occurrence of invasive cervical cancer in Carinthia despite cytological screening, all 132 patients diagnosed with cervical cancer in the years 2000-2002 were recorded and all gynecological cytological smears made within the 5 years prior to the diagnosis of cancer were reevaluated. RESULTS: Within the 5 years prior to diagnosis, no gynaecological cytological smear was found for 50% of the patients diagnosed with cervical cancer in the years 2000-2002. In the year 2002, a total of 53 patients were reported to have cervical cancer and 78 smears were reevaluated. Of all the smears primarily diagnosed as negative, 49% were found to be positive (> or =Pap III) after reevaluation and 92% of all smears "correctly" diagnosed as negative showed quality deficiencies. The interobserver variability (kappa-statistics) showed a moderate value when the primary screening results were compared with the reevaluation. The interobserver variability within the group of reevaluators was also moderate.

Cytopathology in Austria.

Cervical cancer screening developed rapidly during the 1970s. Today, approximately 1.5 million smears are taken annually, so 50% of the target population are screened every year, 30% are cytologically underserved (24% never had a smears, 6% only once). This figure correlates with the fact that there are still 30% deaths from cervical cancer compared with 1960. Since 1998 a voluntary quality assurance programme was introduced by the Austrian Society of Cytology, based on comparison of results reported from participating laboratories.

Results of preoperative ultrasound guided fine needle aspiration biopsy of solitary thyroid nodules as compared with the histology. A retrospective analysis of 538 patients.

AIM: The goal of this study was to assess the accuracy and limitations of ultrasound guided fine-needle aspiration biopsy (ug-FNAB) of solitary thyroid nodules. METHODS: The ug-FNAB results of 538 patients with solitary thyroid nodules, who afterwards underwent thyroid surgery, were compared retrospectively with the histology. Patients with multinodular goiter were excluded from the study. Ug-FNAB was performed on growing and/or hypoechoic and/or hypofunctional nodules. The ug-FNAB results were grouped as follows: group 1: malignant (n = 44); group 2: malignancy cannot be ruled out (n = 173); group 3: non-malignant (n = 296), group 4: inadequate (n = 25). RESULTS: When the cytological results of group 1 and group 2 were interpreted as being malignant and those of group 3 as being benign, sensitivity, specificity and accuracy of ug-FNAB were 96.7%, 65.8% and 69.5% respectively. The 62 thyroid carcinomas (TC) biopsied presented in 59 cases a suspicious or malignant cytology (95.2%). The smallest TC diagnosed by ug-FNAB had a diameter of 0.5 cm and 36.4% of all papillary TC < or = 1 cm displayed stage pT4. The histology verified a TC in 18 cases out of the 173 ug-FNABs in group 2. Non-malignant ug-FNABs were confirmed by histology in 294 patients (99.3%) in group 3. In 4.65% of the ug-FNABs inadequate material was aspirated. CONCLUSION: Nodules with non-suspicious ug-FNAB results can be safely followed-up by sonography, as the cytological diagnoses were verified in more than 99% by histology. Papillary TC can be diagnosed with ug-FNAB very accurately. As stage pT4 was present in more than one third of patients with papillary TC < or = 1 cm, ug-FNAB is also recommended for thyroid nodules 0.5-1 cm in diameter located adjacent to the thyroid capsule. However, microfollicular proliferations remain the limitation of ug-FNAB, as the cytology cannot distinguish between benign adenoma and follicular TC.

Value of ultrasound-guided fine-needle aspiration biopsy of thyroid nodules in an endemic goitre area.

The aim of this study was to determine the value, advantages and limitations of ultrasound-guided fine-needle aspiration biopsy (US-FNAB) in an endemic goitre area. US-FNAB was performed on all outpatients who presented with hypoechoic and/or hypofunctional and/or growing nodules. A total of 4518 US-FNABs were performed and 718 patients from this series underwent surgery. Cytological results of the primarily performed US-FNAB of these patients were compared retrospectively with the histological results. US-FNAB results were grouped as (1) non-malignant (n = 303), (2) non-malignant follicular proliferation (n = 177), (3) malignancy cannot be ruled out (n = 133), (4) malignant (n = 61), (5) inadequate (n = 34), and (6) sampling error; biopsy of a non-malignant nodule (n = 10). Nodules as small as 5 mm in diameter could be biopsied, gaining representative material. US-FNAB found a malignant or suspicious cytology in 65 out of 87 cases with malignant histology (74.71%). Diagnosis of early tumour stages was often possible: 12 of 18 thyroid carcinomas biopsied and smaller than 10 mm in diameter had malignant or suspicious cytology (groups 3 and 4). US-FNAB was performed incorrectly within non-malignant nodules in ten patients (1.39%) with multinodular goitre (ten papillary carcinomas, nine smaller than 10 mm). Regarding the cytology of groups 1 and 2 as benign and those of groups 3 and 4 as malignant, US-FNAB performance was as follows: sensitivity 87.84%, specificity 78.50%, negative predictive values 98.13%, positive predictive values 33.51% and accuracy 79.53%. Biopsies with inadequate material were obtained in 4.73% of all biopsies. No major adverse effects occurred. Re-biopsies in 61 cases did not alter the cytological outcome in those cases where adequate material was obtained. US-FNAB is a valuable method in the pre-operative assessment of thyroid nodules in order to select patients for surgery, as malignancy can often be detected even in early tumour stages. However, even with ultrasonographic guidance, the minimal tumour size detectable by US-FNAB is around 5 mm. The cytological interpretation in cases with regression and microfollicular proliferation also sets limits on the method. However, patients with non-malignant cytologies can be followed up safely by sonography due to the high NPV of US-FNAB as long as thyroid nodules do not become larger. Re-biopsies seem to be of limited value as long as adequate material was obtained by US-FNAB.

Comparison of different tracers in the follow up of differentiated thyroid carcinoma.

In the follow up of differentiated thyroid carcinoma (DTC) several scintigraphic methods are used in addition to the serum thyroglobulin and ultrasonography of the neck. Iodine-131 whole body scintigraphy (WBS), which is performed since many years, is able to detect iodine positive recurrence, lymph node metastases and distant metastases in a very specific way. However, the problem of I-131 WBS is the fact that only 67% of metastases from DTC accumulate iodine. Therefore non specific radionuclides like TI-201 or tracers such as Tc-99m Sestamibi or Tc-99m Tetrofosmin and new metabolic tracers like F-18 FDG were introduced in the diagnostic work up to detect iodine negative metastases as well. This study describes the comparison of different tracers in 35 patients with elevated thyroglobulin and suspicion of metastatic disease or already known metastases from DTC.

Thallium-201 uptake with negative iodine-131 scintigraphy and serum thyroglobulin in metastatic oxyphilic papillary thyroid carcinoma.

We report a case of a 48-yr-old woman who underwent surgery because of papillary oxyphilic thyroid carcinoma pT3. After total thyroidectomy, we administered 2960 MBq (131)I for ablation of the residual tissue. initial follow-up visits showed no clinical, radiological or scintigraphic evidence of residual or metastatic thyroid tissue. Serum thyroglobulin levels (Tg) and (131)I whole-body scintigraphy were negative. Three years after thyroidectomy, the patient experienced seizures, and as a consequence a brain tumor was removed. It was an undetected metastasis of the primary thyroid carcinoma. Histological examinations showed that neither the primary tumor nor the metastasis produced any Tg. With this fact in mind and the knowledge of negative (131)I whole-body scans we had to concentrate on radiological (CT and MRI scans) and nonspecific scintigraphic methods such as 201TI whole-body scintigraphy in our management of the patient. Further follow-up demonstrated multiple metastasis by 201TI whole-body scan (mediastinum, bones and soft tissue), and most of them have been removed by surgery. This case report demonstrates that, in addition to (131)I whole-body scans and measurement of serum Tg, the use of nonspecific tracers like 201TI is important to detect (131)I and/or Tg negative metastases.

Technetium-99m-tetrofosmin uptake in sarcoidosis stage I.

The uptake of 99mTc-tetrofosmin in enlarged lymph nodes, of the lung hilus, in the case of sarcoidosis Stage I (histopathologically confirmed by mediastinoscopic biopsy) is demonstrated. On a routine chest radiograph of a 78-yr-old woman, hilar lymphadenopathy was first detected. In the following mammography, disseminated micro calcifications were found in the left breast and a 99mTc-tetrofosmin study was performed for detection of breast cancer. Scintigraphy using 99mTc-tetrofosmin showed clear uptake in the hilar lymph nodes, but not in the left breast. The 99mTc-tetrofosmin uptake in the hilar lymph nodes was due to sarcoidosis confirmed by histology. Therefore, 99mTc-tetrofosmin scintigraphy may be useful in patients with suspected sarcoidosis, especially in Stage I.

Radioimmunoscintigraphy with Tc-99m labeled monoclonal antibody 170H.82 in suspected primary, recurrent, or metastatic breast cancer.

Radioimmunoscintigraphy (RIS) with Tc-99m labeled 170H.82, an intact murine monoclonal antibody (MAb) was performed in 27 patients with suspected primary, recurrent, or metastatic breast cancer. For radioimmunoimaging the authors used a double-headed, high-resolution gamma camera. The lesions detected by RIS were evaluated by histopathology, and for metastatic disease by CT, MRI, and bone scan. If the evaluation was made lesion by lesion, RIS with Tc-99m MAb 170H.82 showed 23 true-positive results, 3 false-negative results, 9 true-negative results, 3 false-positive results for primary and recurrent disease; sensitivity was 88% and specificity was 75%. In patients with distant metastases, detection rate was 67% (12 true-positive results and 6 false-negative results). All patients had normal baseline values for human anti-mouse antibodies (HAMA) and 6 weeks after RIS with 2 mg MAb, only 2 of 23 patients had developed a HAMA response (9%). No adverse effects were observed. The authors' clinical experience shows that RIS with MAb 170H.82 is a valuable additional method for detecting breast cancer, especially in patients with uncertain primaries or scar tissue after breast conserving treatment in which differential diagnosis is very difficult.

Cytomorphology of anaplastic ependymoma in a pleural effusion. A case report.

In a 42-year-old women a recurrent anaplastic ependymoma of the frontal lobe developed intracerebral metastases and, after implantation of an atrioventricular shunt, also metastasized to the lung, pleural cavity and mediastinal lymph nodes. In routinely stained smear preparations of the pleural effusion sediment, the typical cytologic features of an anaplastic ependymoma were found--papilliform cell clusters with acellular axes ("pseudorosettes"), incomplete true ependymal rosettes, and epithelioid and tanycytoid tumor cells with signs of anaplasia. A peculiar matrix material was a characteristic feature of this case and, to our knowledge, was not reported previously. It consisted of refringent, fibrillar strands composed of tightly packed, roundish to rodshaped granules. This matrix seemed to be associated with the cytoplasmic processes of the tumor cells.

[Squamous cell carcinoma of the thyroid originating from a thyroglossal duct cyst]. / Plattenepithelkarzinom der Schilddrüse ausgehend von einer Ductus thyreoglossus Zyste.

A 73 y old female was found to suffer from a squamous cell carcinoma with a focal thymus-like differentiation in a thyroglossal duct cyst. A neoplasma of this type in a goitre with Graves' disease is extremely rare. Sonographic, radiological, scintigraphic and microscopic findings are discussed.

[Abdominal wall implantation metastasis 2 years after apparently uneventful laparoscopic cholecystectomy]. / Bauchdeckenimplantationsmetastase zwei Jahre nach scheinbar unauffälliger laparoskopischer Cholecystektomie.

An extraordinary rare case of implant metastasis at the laparoscopic working port, two years after an apparent uncomplicated laparoscopic cholecystectomy is presented. The histological reexamination of the gallbladder with the detection of a minimal carcinomatous lymphangiosis in newly prepared sections, the exclusion of other primary tumors and also the tumor-free interval of six months until now, after radical excision of the abdominal wall, are strongly suggestive for an intraoperative tumor cell dissemination into the abdominal wall by a histologically unrecognized carcinoma of the gallbladder.

Immunostaining of human autopsy aortas with antibodies to modified apolipoprotein B and apoprotein(a).

A systematic immunohistochemical study of different stages of atherosclerosis in human aortas was performed using several antibodies. Because oxidation of lipoproteins could be a key event in atherogenesis, an antibody against apolipoprotein B (apoB) from low-density lipoprotein (LDL) modified with the lipid peroxidation-specific aldehyde, 4-hydroxynonenal (4-HNE) (anti-4-HNE-apoB), was raised in rabbits. This antibody recognizing 4-HNE protein adducts was used in concert with an antibody to apo(a) from lipoprotein(a), considered also potentially atherogenic, as well as with an antibody and a monoclonal antibody (mAb) to apoB. Autopsy material from 12 corpses was investigated. The immunohistochemical investigation by the alkaline-phosphatase technique included control specimens regarding postmortem artifacts by autolysis and oxidation. The results from six specimens from five corpses are presented. A positive staining with the antibody to apoB but not with anti-4-HNE-apoB was seen in the normal intima. The thickened intima of early, transitional, and advanced atherosclerotic lesions and atheromata showed a predominantly extracellular staining with all antibodies and the applied mAb. To test the specificity of the staining, antibodies preadsorbed by the appropriate antigens and nonimmune sera were used, giving negative results. These findings indicated a colocalization of epitopes derived from lipid peroxidation of polyunsaturated fatty acids and epitopes specific for apoB and apo(a) during atherogenesis in humans.

A combined biopsy-plugging device based on the Menghini- or Trucut needle for percutaneous liver biopsy: clinical experience.

Impaired blood clotting precludes percutaneous liver biopsy for histologic examination of liver tissue. The transjugular or laparoscopic approach are ways to reduce the risk of bleeding. These techniques, however, are laborious and confined to specialized centers. Methods of plugging the needle track with sealant presented so far are hampered by the need to leave either the cannula in situ or the need for a second percutaneous approach for the application of the sealant. We have tested a combined plugging-biopsy device allowing to perform the biopsy as a one-step procedure in patients with impaired clotting under laparoscopic vision control. In 37 patients either a modified Trucut or Menghini needle was tested. Handling of the Trucut needle proved easier. Tissue yield was satisfactory with both needles. Only one episode of bleeding was observed with a prototype Menghini needle. We conclude, that the combined plugging-biopsy device is a safe and reliable tool for obtaining liver tissue in patients with impaired blood coagulation.

Early osteoinduction in rats is not altered by fibrin sealant.

The angiogenic and fibrogenic activities of fibrin sealant (Tisseel) suggest that it could enhance osteoinductivity during the first days after implantation. To test this hypothesis, the osteoinductivity of sealed and unsealed bone matrix gelatin was tested by implantation into rat abdominal wall muscle pouches for two to ten days. A sponge of collagen with or without fibrin sealant was used for control. Alkaline phosphatase (ALPase) was employed to quantify osteoinduction, and myeloperoxidase (MPO) was used to determine the inflammatory reaction. Plain histologic analysis of bone matrix gelatin implants at Days 2, 4, and 10 did not demonstrate any significant differences between sealed and nonsealed implants. Histologic analysis at Day 21 confirmed the osteoinductivity of the implant. The combination of bone matrix gelatin and fibrin sealant induced an early significant increase in ALPase activity, which could be interpreted as early induction of histologically undetectable osteogenesis. A simultaneous increase in the level of MPO suggested that the bone matrix gelatin/fibrin sealant-induced ALPase reaction might be caused by an inflammatory process, but neutrophil counting did not correlate to the MPO data. Morphometry, however, seems to be insufficient to assay a heterogenous histologic distribution of singular cells. It is proposed that the ALP/MPO ratio may be used as an inflammation-independent marker of osteogenesis. This study does not support the notion of an osteoinductive potency of fibrin sealant per se; however, it is clear that fibrin sealant does not impair bone matrix gelatin-dependent osteoinduction.

Immunohistochemical detection of tumor necrosis factor-alpha, other cytokines and adhesion molecules in human livers with alcoholic hepatitis.

This immunohistochemical study was designed to investigate the possible contribution to and topographical distribution of some important cytokines, such as tumour necrosis factor alpha (TNF alpha) and interleukins, in acute alcoholic hepatitis. The well-known inductive capacity of these cytokines with respect to the expression and/or up-regulation of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1) and endothelial leukocyte adhesion molecule-1 (ELAM-1), was a further point to be studied. Moreover, the proposed induction of adhesion molecules might also be associated with the activation and attraction of a special population of inflammatory cells characteristic for alcoholic hepatitis. Frozen liver samples from patients who died with signs of acute alcoholic hepatitis were evaluated using the alkaline phosphatase anti-alkaline phosphatase immunostaining technique and also single and double indirect immunofluorescence. In acute alcoholic hepatitis TNF alpha could be detected predominantly in ballooned hepatocytes, which often contained alcoholic hyalin (Mallory bodies). Moreover, TNF alpha showed a co-distribution with ICAM-1 expressed in the membranes of hepatocytes and with the occurrence of CD11b positive polymorphonuclear leukocytes (neutrophils) suggesting a possible major role of the beta 2-integrin Mac-1 as a ligand for ICAM-1. No induction of ELAM-1 could be found. In alcoholic hepatitis cytokines may be responsible for the induction of the adhesion molecule ICAM-1 on hepatocytic membranes and activate a defined population of inflammatory cells, thus contributing to the characteristic histological picture of acute alcoholic hepatitis with its concentration of neutrophils especially in areas with ballooned Mallory body-containing hepatocytes. Our results are in line with clinical findings showing high levels of TNF alpha and interleukin-1 in sera of patients with alcoholic hepatitis and with the already reported expression of ICAM-1 on hepatocytes.

Cryopreservation of cytological specimens for immunocytochemistry.

A method has been established for storage and preservation of cytological specimens in liquid nitrogen and further processing for immunocytochemistry as smears prepared from thawed cells or cryo-sections of frozen cell pellets. For the experiments cultured cells of a T-lymphoblastic leukemia cell line (ATCC CCL 119) and blood cells of the buffy coat of healthy humans were treated with a cryo-solution (fetal calf serum +5% dimethylsulfoxid) and after freezing stored in liquid nitrogen. Alternatively, cells preincubated with cryo-solution followed by suspension in fetal calf serum without cryo-additive were frozen and stored in liquid nitrogen for the production of cryo-sections. Indirect immunofluorescence and alkaline phosphatase--antialkaline phosphatase based immunoreactions were performed for the decoration of various surface antigens with a panel of monoclonal antibodies. All immunoreactions were repeated at least three times and the stored cell preparations were investigated after different periods of storage (up to four months). The immunoreactions of fresh cells in suspension (which were used as controls) were comparable with those of cryopreserved cells, e.g. cells on smears after thawing and on cryo-sections of cell pellets. The strongest immunoreactions were achieved on fixed cryo-sections. The maintenance of cell morphology of smears from cryopreserved cells was slightly better than of cells from cryo-sections. In our hands the preparation of cell pellets, which are suitable for the storage in liquid nitrogen and the production of cryosections, is a very useful method for immunocytochemical investigations of cytological specimens especially in situations where immunoreactions cannot be performed on fresh material.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcoholic liver disease. Parenchyma to stroma relationship in fibrosis and cirrhosis as revealed by three-dimensional reconstruction and immunohistochemistry.

Severe ethanol-induced liver damage is characterized by fibrous dissociation of liver cell plates leading to many apparently isolated hepatocytes. Three-dimensional reconstruction, however, revealed hepatocytes that were surrounded by connective tissue as endpoints of "parenchymal pillars" or in association with liver cell plates and bile ductules. Double immunofluorescence studies displayed the expression of cytokeratin (CK) 7 in bile ducts, including bile ductules, but also in some hepatocytes still organized in liver cell plates. The other bile duct, typical CK, namely CK 19, was only detectable in few hepatocytes. However, the expression of CK 7 and/or CK 19 was less frequent in hepatocytes that were closely associated with bile ductules. CK 7 and CK 19 were also found in some, but not all, Mallory bodies. These observations indicate that the expression of these two CKs is neither related to a transformation of hepatocytes to bile duct-like structures ("ductal metaplasia") nor to the formation of Mallory bodies. Furthermore, double immunofluorescence studies revealed small groups of hepatocytes and bile ductules that were encircled by basement membrane material, thus suggesting the formation of "secretory units."