Identification of metastatic papillary thyroid carcinoma in FNA specimens using thyroid peroxidase immunohistochemistry.

INTRODUCTION: We evaluated immunohistochemical staining for thyroid peroxidase (TPO), a glycoprotein found in the apical plasma membrane of thyroid follicular cells, as a marker for metastatic PTC in FNA samples and compared results with thyroglobulin (Tg) and thyroid transcription factor 1 (TTF1) staining. METHODS: Cell block sections prepared from 100 FNA specimens were stained with a rabbit monoclonal antibody to TPO (EP159). The FNAs included 64 metastatic malignancies from non-thyroid primary sites, including 18 lung, and 36 cases of thyroid tumours (29 PTC, six cases of medullary thyroid carcinoma and one thyroid anaplastic carcinoma). Thyroid tumours were stained with TTF1 and Tg in addition to TPO. All cases of metastatic lung carcinoma also had TTF-1 staining results. RESULTS: TPO staining was negative in all non-thyroid malignancies. Ninety percent (26/29) of PTC were positive. All positive cases showed strong cytoplasmic staining, although 54% (14/26) showed positivity in less than half of the cells. By comparison, Tg staining of TPC cases was present in 62% and TTF-1 in 100%. In addition to showing higher sensitivity, interpretation of staining results with TPO was generally easier with than Tg. All metastatic lung adenocarcinomas were positive for TTF-1 and TPO negative. The six medullary cancers showed positivity in 17%, 0% and 83% with TPO, Tg and TTF-1, respectively. CONCLUSIONS: TPO (mAb EP159) may be a useful addition to immunohistochemical panels for FNA specimens where metastatic PTC is a consideration, particularly in cases where metastatic lung carcinoma features in the differential diagnosis.

The significance of extracellular mucin in breast fine needle aspiration specimens.

OBJECTIVE: To determine if all breast fine needle aspiration (FNA) specimens with abundant extracellular mucin require excisional biopsy. METHODS: Fifty cases of breast FNA containing extracellular mucin, reported with a recommendation for biopsy and with histological follow-up were reviewed. Cellular features were noted, and the cases reclassified ignoring the presence of mucin and then correlated with histological outcome. RESULTS: Subsequent histology showed benign changes in 20% (10/50), two cases (4%) of atypical ductal hyperplasia (ADH) and 76% (38/50) to be malignant lesions, including three cases of ductal carcinoma in situ (DCIS). When the FNA cases were reviewed disregarding the presence of mucin, 11 cases were reclassified as benign and one case that contained mucin-like material but no epithelial cells reviewed as non-diagnostic. All cases of invasive mixed and mucinous carcinoma (MC) would have been reported with a recommendation for histological examination. In addition, the three cases each of fibroadenoma (FA) and a benign mucocoele-like lesion (MLL) were correctly identified as benign. However, two cases each of DCIS and ADH would have been reported benign with no recommendation for further histology. CONCLUSIONS: Breast FNA specimens with mucinous material may arise from sampling of a range of benign and malignant processes; however, a biopsy should be recommended, even in cases of low cellularity, owing to sampling problems and the frequent co-occurrence of significant lesions. FNA is highly accurate at predicting carcinoma with mucinous differentiation although it is not possible to reliably predict if the lesion represents pure MC or a mixed carcinoma.

Rapid on-site evaluation of fine needle aspiration specimens by cytology scientists: a review of 3032 specimens.

OBJECTIVES: To determine: (1) the accuracy of cytology scientists at assessing specimen adequacy by rapid on-site evaluation (ROSE) at fine needle aspiration (FNA) cytology collections; and (2) whether thyroid FNA with ROSE has lower inadequacy rates than non-attended FNAs. METHODS: The ROSE of adequacy for 3032 specimens from 17 anatomical sites collected over a 20-month period was compared with the final report assessment of adequacy. ROSE was performed by 19 cytology scientists. The report profile for 1545 thyroid nodules with ROSE was compared with that for 1536 consecutive non-ROSE thyroid FNAs reported by the same cytopathologists during the study period. RESULTS: ROSE was adequate in 75% (2276/3032), inadequate in 12% (366/3032) and in 13% (390/3032) no opinion was rendered. Of the 2276 cases assessed as adequate by ROSE, 2268 (99.6%) were finally reported as adequate for assessment; eight specimens had adequacy downgraded on the final report. Fifty eight per cent of cases with a ROSE assessment of inadequate were reported as adequate (212/366), whereas 93% (363/390) with no opinion rendered were reported as adequate. The overall final report adequacy rate for the 3032 specimens was 94% (2843/3032). Confirmation of a ROSE of adequacy at reporting was uniformly high amongst the 19 scientists, ranging from 98% to 100%. The inadequacy rate for thyroid FNAs with ROSE (6%) was significantly (P < 0.0001) lower than for non-ROSE thyroid FNAs (17%). A significantly (P = 0.02) higher proportion of adequate ROSE thyroid specimens was reported with abnormalities, compared with non-ROSE thyroid collections. CONCLUSIONS: Cytology scientists are highly accurate at determining specimen adequacy at ROSE for a wide range of body sites. ROSE of thyroid FNAs can significantly reduce inadequate reports.

The varied presentation of metastatic melanoma in fine needle aspiration cytology of the breast.

OBJECTIVE: To identify cytomorphological patterns of metastatic melanoma (MM) in breast fine needle aspiration (FNA) specimens and highlight the differential diagnoses and features most useful in identifying MM. METHODS: The clinical, radiological and FNA findings of 16 cases were reviewed. Cytological features evaluated related to cell arrangement, size and shape of cells, nuclear and cytoplasmic features, and the presence or absence of necrosis. RESULTS: The series consisted of 14 females and two males, ranging in age from 24 to 83 years (mean = 50 years). A previous history of melanoma was available in 12/16 (75%) cases at the time of FNA reporting; however the clinical/radiological impression in 4/16 cases was of a breast cyst. The cases were classified into six morphological variants: classical (8/16), pseudopapillary (3/16), spindle-cell (1/16), melanin-rich (1/16), pleomorphic (2/16) and lymphoma-like (1/16). The varying patterns raised a wide range of differential diagnoses; however, discohesion, binucleation and granular cytoplasm were the major features seen in 94% of all cases. In 14/16 cases (88%), plasmacytoid cells, prominent nucleoli and cytoplasmic vacuolation were identified. Melanin and multinucleation were detected in 44% of cases and intranuclear cytoplasmic invaginations in 63%. Necrosis was present in more than half of the cases (56%). CONCLUSION: MM should be considered in the differential diagnosis of breast FNA specimens when atypical cells are seen that present as plasmacytoid cells in a dispersed or pseudopapillary pattern, or as spindle, pleomorphic or pigmented cells. These features, combined with clinical history and immunocytochemistry, may assist in correctly identifying MM and directing optimal treatment.

The value of calretinin and cytokeratin 5/6 as markers for mesothelioma in cell block preparations of serous effusions.

OBJECTIVE: To determine the value of calretinin and cytokeratin (CK) 5/6 in discriminating mesothelioma from adenocarcinoma in serous effusion specimens. METHODS: A total of 101 recent, histologically or clinically confirmed malignant effusions with immunostained cell block preparations were reviewed. The cases consisted of 34 mesotheliomas and 67 adenocarcinomas. This included 17 ascitic fluid and 84 pleural fluid samples. The adenocarcinomas included metastatic carcinomas from the breast (12), lung (19), stomach (3), colon (1), pancreas (2), ovary (6) endometrium (1) and 23 histologically confirmed metastases from unknown primary sites. The cases were assessed as negative or positive (>5% of cells stained). The staining pattern was recorded as cytoplasmic, cell membrane, nuclear or cytoplasmic and nuclear staining. RESULTS: Calretinin staining was present in 97% (33/34) of the mesothelioma cases with a majority of them showing both cytoplasmic and nuclear staining (29/33). Only 3% (2/67) of adenocarcinomas were positive for calretinin, one being a lung adenocarcinoma and the other an adenocarcinoma of unknown primary site in an ascitic fluid. Cytokeratin 5/6 staining was also present in 33/34 (97%) of mesothelioma cases. Six (9%) adenocarcinomas were positive, including metastases from the lung (1), breast (1), ovary (2) and unknown primary site (2). Four of the six adenocarcinoma cases positive for CK5/6 were in ascitic fluids. No cases of mesothelioma were negative for both calretinin and CK5/6. Only one adenocarcinoma case, (which was from unknown primary site in an ascitic fluid sample), was positive for both markers. CONCLUSIONS: The results confirm that calretinin and CK 5/6 are useful markers for mesothelioma in effusion specimens. CK5/6 staining may be less useful for peritoneal fluid specimens where metastatic adenocarcinomas may be more likely to express the antigen. Further study of ascitic/peritoneal specimens is warranted. However, positive staining, particularly for both antigens, is highly indicative of a mesothelial origin for cells. The two markers make a useful addition to EMA and the panel of adenocarcinoma markers routinely applied to effusion specimens.

Peritoneal washing cytology.

Peritoneal washing cytology (PWC) is a useful indicator of ovarian surface involvement and peritoneal dissemination by ovarian tumours. It may identify subclinical peritoneal spread and thus provide valuable staging and prognostic information, particularly for non-serous ovarian tumours. The role of PWC as a prognostic indicator for endometrial carcinoma is less clear, due in part to the questionable significance of identifying endometrial tumour cells in the peritoneum. Detection of metastatic carcinoma in PWC is based on the recognition of non-mesothelial cell characteristics. However a number of conditions such as reactive mesothelial cells, endometriosis and endosalpingiosis may mimic this appearance. Cells from these conditions may have a similar presentation in PWC to that of serous borderline tumours and low-grade serous carcinoma. The presence of cilia, lack of single atypical cells, prominent cytoplasmic vacuolation, marked nuclear atypia or two distinct cell populations are features favouring a benign process. Attention to these features along with close correlation with clinical history and the results of surgical pathology should help avoid errors. Additional assistance may be provided by the use of cell blocks and special stains.

Improving cervical cytology screening in a remote, high risk population.

OBJECTIVE: To evaluate the use of the ThinPrep method to reduce rates of unsatisfactory Papanicolaou (Pap) smears in women in remote communities. DESIGN: Prospectively collected samples were split and screened conventionally and by ThinPrep at the Queensland Cytology Service. PATIENTS: Three hundred women having cervical smears taken by a Mobile Women's Health Service nurse or at the antenatal and sexual health clinics of a remote north Queensland community. MAIN OUTCOME MEASURE: Number of Pap smears reported as unsatisfactory for evaluation and requiring a repeat smear request. RESULTS: 17.3% of conventionally prepared smears were technically unsatisfactory, compared with 6.3% prepared with ThinPrep. The overall rate of unsatisfactory smears was only 4.3% when both ThinPrep and conventional smears were assessed for a combined report. CONCLUSION: A significant reduction in the proportion of unsatisfactory Pap smears is possible with the ThinPrep method. Targeted use of ThinPrep in communities with high rates of unsatisfactory smears may prove cost-effective.

Monoclonal antibody recognizing a core epitope on mucin.

Monoclonal antibody TH1 (IgM) was prepared by immunizing mice with deglycosylated (TFMSA-treated) cystic fibrosis mucin. TH1 reacted strongly with TFMSA treated cystic fibrosis mucin but not with the fully glycosylated mucin, indicating reactivity with a core mucin epitope. TH1 showed no reactivity with ovine mucin (98% of glycans as sialyl-Tn) but reacted strongly with desialylated ovine mucin, indicating the epitope for this mab was the Tn-antigen (O-linked GalNAc). However, TH1 showed no reactivity with Tn-positive red blood cells, and the binding of TH1 was not inhibited by GalNAc at 2.5 mg/ml, illustrating the importance of the peptide sequence to which the GalNAc is attached. TH1 stained the majority of cancers of the colon, lung, stomach, ovary, breast, and cervix, and the cellular distribution of this antigen in normal tissue suggested reactivity with immature mucin. This antibody appears to be a useful reagent for the detection of immature mucin.

The sensitivity of rapid (partial) review of cervical smears.

Rapid review involves a daily rapid (e.g. 30 s) review of all smears not normally double-screened. It has been suggested that the method may increase the sensitivity of cervical cytology by identifying abnormalities not reported on initial screening, true false negatives (TFN). Rapid screening is reported to have high sensitivity for cervical neoplasia when used as a preview tool. To be effective, however, in a review mode it must be able to detect TFN. Several studies have found that many TFN result from factors such as low numbers of abnormal cells or subtle expression of diagnostic criteria. Studies on the sensitivity of rapid screening for detecting TFN would therefore provide a more reliable estimate of its value as a review tool. The sensitivity of rapid re-screening was evaluated using a test set of 200 cases. Each of 15 screeners rapidly reviewed (30 s partial screen) the set over a 2-week period. The set consisted of 129 normal, 28 low-grade squamous lesions (CIN I), 37 high-grade lesions (CIN II, III and adenocarcinoma in situ (AIS)) and six invasive carcinomas. The abnormals included 20 TFN cases. The median sensitivity for abnormalities was 62%. Rapid review was more sensitive for CIN II and CIN III (67%) and invasive carcinoma (66.7%) than for CIN I (53%). Great variation was apparent in the sensitivity for individual screeners, with a range of 41-86% for all abnormalities. The sensitivity for TFN cases varied even more (10-75%, median 35%) and for most screeners was significantly (P < 0.05) lower than for cases which were detected on initial screen (53-90%, median 70.6%). Following this trial rapid review was used routinely for a period of 3 months. In this time 11,413 cases were rapidly reviewed. This led to the full review of 415 slides (3.5%) and the identification of 16 cases of undetected CIN (12 CIN I, three CIN II, one CIN III). Based on current estimates of our laboratory false-negative rate this represents between a quarter and half of the TFN cases of CIN that probably occurred in this period. In conclusion, rapid screening is likely to be significantly less sensitive when used in a review rather than a preview mode. In routine practice the method requires a daily commitment of screener time, but does provide a higher yield of TFN smears than does random review, and allows amendment of these results prior to reporting.

Immunocytochemical staining of cytologic specimens. How helpful is it?

The contribution of immunocytochemical (ICC) staining to the diagnosis of a range of cytologic specimens reported over a 20-month period was retrospectively assessed. A total of 194 cases (1.6% of total workload) were stained for diagnostic purposes in this period. Immunocytochemical staining was determined to be helpful in 75.8% of cases. In body fluid cases (105), staining was most often performed to assist with the discrimination of mesothelial cells and metastatic malignancy. Immunocytochemical staining was helpful in 82% of fluid specimens, with the preliminary diagnosis confirmed in 64%, refined in 8%, and revised in 10% of cases. In fine-needle aspiration (FNA) specimens, staining was helpful in 69%, resulting in a refinement of diagnosis in 55%, and confirming the preliminary diagnosis in 14%. The preliminary diagnosis was revised in a single FNA case. Immunocytochemical staining was particularly valuable in assisting the subclassification of poorly differentiated malignancies. A more precise diagnosis was possible following consideration of ICC results in 68% of cases with a preliminary diagnosis of poorly differentiated malignancy. Immunocytochemical staining was also helpful in identifying the primary site of metastatic carcinoma in six FNA cases. It is concluded that the selective use of well-chosen panels of antibodies can be very helpful in resolving diagnostic difficulties in cytologic specimens. In particular, ICC may be an invaluable aid to the diagnosis of difficult serous effusion specimens and in the sub-typing of poorly differentiated malignancy.

Chronic radiation effects: a correlative study of smears and biopsies from the cervix and vagina.

Cervicovaginal smears and biopsies from patients treated with radiotherapy for cervical carcinoma were examined morphologically and immunochemically to provide information on the tissue derivation of cells characteristic of chronic radiation effect in postirradiation smears. In biopsies, stromal changes, such as fibrosis, vessel changes, and atypical fibroblasts were most common. Ulceration, leucocytic infiltration, multinucleated giant cells, regenerative epithelium, and atypical glandular epithelial cells were also present in some specimens. These changes were reflected in smears collected from the same patients, where multinucleated giant cells, repair cells, and large atypical cells were often present. Correlation of smears and biopsies suggest that repair cells are collected from areas of epithelial regeneration and glandular radiation atypia. Sampling of ulcerative or eroded tissue may produce smears with multinucleated giant cells, atypical stromal cells, endothelial cells, and numerous macrophages. Correct recognition of these cell types and smear patterns may assist in avoiding false positive diagnoses.

Monoclonal antibodies recognising sialyl-Tn: production and application to immunochemistry.

In order to develop reagents that can detect the exposed sialyl-Tn antigen (NeuAc alpha 2,6GalNAc alpha 1-O-Ser/Thr) on tumour-associated mucins, we have prepared monoclonal antibodies (mabs 3C2 and 3D1, both IgM) against ovine submaxillary mucin (OSM; > 98% of glycans as sialyl-Tn). These mabs showed strong reactivity with OSM and bovine submaxillary mucin (BSM; 50% of glycans as sialyl-Tn) but did not react with desialylated OSM or BSM. Sialic acid at 1 mg/ml did not significantly inhibit mab binding to OSM, suggesting that the linkage to GalNAc may be important for mab binding. 3C2 and 3D1 also showed similar reactivity to sialyl-Tn reactive mab B72.3, and detected B72.3 captured OSM in a sandwich ELISA. In Western blotting of mucus from a patient with a mucinous ovarian tumour, the mabs reacted with high molecular weight (> 200 kDa) species. In immunohistochemistry, these mabs showed strong reactivity with most cancers of the colon, lung, and stomach, and also some tumours of the ovary and breast. There was only limited reactivity in normal tissue from these sites. The antibodies should be useful reagents for the detection of the sialyl-Tn antigen in human cancers.

The value of direct fluorescent antibody (DFA) testing for the detection of Pneumocystis carinii in cytological specimens.

A direct fluorescent antibody (DFA) method was compared with methenamine silver staining (MSS) for the detection of Pneumocystis carinii in 384 cytological specimens. DFA testing was more sensitive than the MSS, with P. carinii detected in 31 specimens with DFA and 24 with the MSS. Results of the two methods disagreed in 17 specimens, all of which were sputa. Twelve sputum specimens were DFA positive/MSS negative and five were MSS positive/DFA negative. It is concluded that the DFA technique, although relatively expensive, is simple to perform and offers superior sensitivity to the MSS. However, in sputum specimens the combined use of DFA and MSS leads to optimal sensitivity for the detection of P. carinii.

Establishment of a cell line from a hepatocellular carcinoma from a patient with hemochromatosis.

We describe the establishment and characterization of a novel hepatoma cell line. This cell line, designated RBHF-1, was established from a hepatocellular carcinoma of a 67-yr-old man with a history of genetic hemochromatosis. At this writing, the cells have been maintained in RPMI-1640 tissue-culture medium and fetal calf serum without any additional supplements for 30 mo. The cells form colonies on soft agar and are not tumorigenic in nude mice. The cell line is polymorphic and displays characteristics of mature hepatocytes by synthesizing albumin, alpha 2-macroglobulin, fibronectin and alpha-fetoprotein. Cytogenetic analysis shows multiple chromosomal aberrations, with a consistent deletion in the long arm and deletions or rearrangements in the short arm of chromosome 1. There is no evidence for hepatitis B or hepatitis C virus infection of the cell line. The cells contain no detectable intracellular iron after staining with Perls' stain. Unlike other hepatoma cell lines, there is no detectable binding of epidermal growth factor to RBHF-1 cells. This is the first cell line to be established from a patient with hemochromatosis, and it provides a potentially important model for the study of hepatocyte transformation in association with iron overload.

Markers for metastatic adenocarcinoma in serous effusion specimens.

A variety of stains have been proposed as useful adjuncts to the morphologic diagnosis of serous effusion specimens. This study evaluates the sensitivity and specificity of nine stains for the detection of metastatic adenocarcinoma in effusions. Mucin cytochemistry with the Periodic acid-Schiff reaction after diastase digestion (PASd) and the mucicarmine method, as well as immunochemical staining for five glycoprotein antigens, was undertaken on 153 effusion specimens with histological and/or clinically confirmed diagnoses. Immunochemical stains included three antibodies to CEA (Dako polyclonal, 4E7 and A5B7) and antibodies to HEA (Ber-EP4), TAG 72 (B72.3), CD15 (LeuM1) and Epithelial membrane antigen (EMA) were evaluated. The sensitivity of the stains for adenocarcinoma (102 cases) was: PASd 37%; mucicarmine 24%; polyclonal anti-CEA 69%; 4E7 52%; A5B7 21%; EMA 91%; B72.3 44%; Ber-EP4 32%; LeuM1 24%. With the exception of EMA, none of the stains reacted with any cases of benign mesothelial cells (11 cases), reactive mesothelial cells (34 cases), or with six cases of mesothelioma. However, EMA staining was present in two cases (6%) of reactive mesothelial cells and all cases of mesothelioma. The optimal combination of stains for use in a panel was polyclonal anti-CEA/B72.3/PASd. Combined results from these three stains yielded a sensitivity of 83% for adenocarcinoma with no false positive results. It is concluded that special staining may provide valuable information to assist in the classification of difficult effusion cases.

Evaluation of a method for cryogenic storage of cytological specimens.

A technique for long-term storage of cytological specimens at -70 degrees C was evaluated with a range of nongynaecological cytology specimens. The effects of frozen storage on the cellularity, morphology, ultrastructure, and reactivity to cytochemical and immunochemical stains were investigated. These parameters were compared in preparations made from specimens processed conventionally and after frozen storage. No significant deterioration in the quality of subsequent preparations was apparent after storage. The method is technically simple and has proven successful for storing a range of specimens, including serous effusions, urine samples, and fine-needle aspirates. This technique allows storage of material until initial microscopic evaluation is complete. If required, the relevant adjuvant procedure, such as cell blocking, may then be carried out using the stored material. Wastage of cytological material on unnecessary preparations is minimised, and material may be stored over years for research or confirmation procedures.