Molecular cytogenetic analysis of five newly established cervical cancer cell lines using G banding and fluorescence in situ hybridization.

Cervical tumors nearly all have complex karyotypes and more precise cytogenetic information is required to establish whether specific rearrangements occur, and if they are related to the type of HPV infection found. The karyotypes of five recently established cervical cancer cell lines, three from squamous cell carcinomas (two HPV 16 +ve and one HPV 18 +ve), one from an adenocarcinoma (HPV -ve), and one from an adenosquamous carcinoma (HPV 16 +ve), have been analysed using fluorescence in situ hybridization (FISH), with 23 chromosome specific paints, YACs and cosmids as probes, in addition to conventional G banding, in order to identify markers and clarify the breakpoints. Chromosomes 1 and 3 were rearranged in all cell lines. Breakpoints in the squamous lines were all in 3q. but in different regions. Small metacentrics involving chromosome 5 were a del(5q) in one line, and a t(X;5) in another, rather than i(5p). The region 6q21 was involved in three cases and chromosome 9 was rearranged in four. An i(8q) was found in three squamous carcinoma cell lines. Structural changes of 11q were found only in two cases, but a marker 11 representing amplification in the 11q14-22 region was duplicated in the adenosquamous line.

Chromosome analysis of parallel short-term cultures from four testicular germ-cell tumors.

Cytogenetic analysis has been carried out on 17 parallel short-term cultures from four malignant testicular teratomas of intermediate differentiation (MTI), two of them combined with seminomas. Clonal development was seen in three tumors, with most of the variation involving different rearrangements of chromosome 1. Two copies of the i(12p), characteristic of testicular germ-cell tumors, were present in the two tumors with yolk sac elements, a single i(12p) and a 12q- were found in an MTI that had metastasized, and a rearrangement of chromosome 12 containing centromeric chromatin from chromosome 18 was found instead of the i(12p) in a mixed tumor. A 13p+ marker containing Q-negative material was seen in two of the tumors, with a der(7)t(Y;7) in one. Chromosomes 1, 3, 6, 7, 8, 12, 17, and X were invariably overrepresented either as complete or partial trisomies, and chromosomes 4, 5, 13, and 18 were underrepresented in all four tumors, strengthening the idea that tumor suppressor genes on the latter four chromosomes, all of which show some loss of heterozygosity with DNA markers, may be important in tumor progression.

Interleukin-6 (IL-6) functions as an autocrine growth factor in cervical carcinomas in vitro.

Many factors have been implicated in the etiology of cervical neoplasia, with human papilloma virus being the latest in a long line of agents that may on their own or in combination exert various initiating and promoting effects on cervical cells, resulting in their transformation. However, for such altered cells to become invasive, it is clear that they must undergo alterations in their rate of turnover, state of differentiation, and motility. We have investigated the production of the multifunctional cytokine interleukin-6 (IL-6) by five new cervical carcinoma cell lines (XH1, EH2, DE3, JE6, and SM7) and the commercially available CaSki cell line, and have studied the effects of this cytokine on the growth of the cells in vitro. All the cell lines produce biologically active IL-6 in amounts varying between 0.35 to 2.0 ng/ml. In the presence of goat anti-sera to IL-6 all the tumor cell lines showed inhibition of growth. IL-6 acts as an autocrine growth factor for in vitro cervical tumor cell growth.

Cytoblock preparations for examination of cervical and other cells.

There are a number of antibodies which may be of value in the investigation of cervical smears, effusion, and cells grown in monolayer culture. The Shandon Cytoblock method was used to prepare discs of such cells suitable both for diagnosis and for a variety of other techniques.

TDM35--a new monoclonal antibody to the XH1 cervical carcinoma cell line. Characterization and immunoperoxidase localization in benign and malignant tissues.

The murine monoclonal IgG1 kappa antibody TDM35 was raised against the cervical carcinoma cell line XH1. The antibody recognizes 18.5-66 kDa NCA-like glycoproteins and immunostains a variety of formalin-fixed, paraffin-embedded normal, benign, and malignant tissues. It is of value in the diagnosis of carcinoma of the exocrine pancreas and it identifies foci of squamous and glandular differentiation in other tumours. TDM35 should form a useful addition to a panel of antibodies for the evaluation of epithelial lesions.

The immunoperoxidase localization of tumour markers in ovarian cancer: the value of CEA, EMA, cytokeratin and DD9.

Primary tumours from 40 patients with epithelial ovarian cancer, treated at St Thomas's Hospital over a 10-year period, were studied for the immunocytochemical expression of the following tumour markers in formalin-fixed paraffin embedded material: carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), cytokeratin (CAM 5.2), and DD9. An indirect immunoperoxidase staining technique was used. All of the tumours were positive for EMA and CAM 5.2, and 30% of them were positive for both CEA and DD9. The absence of CEA and DD9 may be of value in differentiating between metastatic abdominal adenocarcinomas of ovarian origin and those of gastrointestinal origin, but no indication of prognosis was obtained using these epithelial markers. The strong and widespread staining of all the tumours for EMA suggests that this may be a useful marker for detecting metastatic or recurrent disease by immunoscintigraphy.

Primary sarcoma of the lung: potential dangers of immunocytochemistry.

A case is presented of a successfully resected primary pulmonary sarcoma in a 27-year-old man, followed by an eight-year disease-free follow-up. Details of the histological findings are presented, and the factors associated with good prognosis discussed. Immunocytochemical stains were carried out this year when the case was reviewed, and not at the time of resection. Had the immunostains been available at the time, the results would have suggested that the tumour might be a secondary deposit, and so have caused needless anxiety to the patient and his physicians.

Epithelial markers in pancreatic carcinoma: immunoperoxidase localisation of DD9, CEA, EMA and CAM 5.2.

Paraffin wax embedded, formalin fixed sections of 22 adenocarcinomas of the exocrine pancreas were stained with four mouse monoclonal antibodies: DD9-E7, an antibody raised against a human pancreatic tumour xenograft; carcino-embryonic antigen (CEA); epithelial membrane antigen (EMA); and cytokeratin (CAM 5.2). An indirect immunoperoxidase technique without enzyme pre-digestion and an affinity-purified sheep anti-mouse peroxidase conjugate were used. All of the tumours were positive for DD9-E7, EMA, and CAM 5.2. Twenty out of 22 were focally positive for CEA and the staining was often weak. As all of these adenocarcinomas were DD9-E7 positive, absence of staining for DD9-E7 in a tumour makes the diagnosis of adenocarcinoma of the exocrine pancreas very unlikely, and this is of value in distinction from endocrine carcinomas with a marked acinar pattern. The weak CEA staining distinguished pancreatic carcinomas from colorectal tumours. Because the distribution of staining for EMA and CAM 5.2 was no different from that previously seen in adenocarcinomas from other sites, these markers are likely to be of limited value in the differential diagnosis of abdominal adenocarcinomas of uncertain origin.

A novel hybridoma antibody (PASE/4LJ) to human prostatic acid phosphatase suitable for immunohistochemistry.

A murine monoclonal antibody PASE/4LJ to prostatic acid phosphatase (PAP) was used to immunostain a wide variety of sections of benign and malignant tissues (654 blocks). Non-neoplastic adult and fetal prostatic glands, primary and metastatic prostatic carcinomas, and scattered cells in prostatic and penile urethra were positive. Rat, dog and rabbit prostates were negative. Nine of 400 tumours of non-prostatic origin showed some positivity: 6/36 carcinoids, 1/9 islet cell tumours, 1/55 ovarian adenocarcinomas (serous) and one carcinosarcoma of the lung (epithelial portion). Positive staining was seen in islet cells in 4/5 specimens of normal pancreas, and in 4/9 blocks of normal pancreas surrounding a pancreatic tumour. Loops of Henle, maculae densae, and distal tubules in 10/10 fetal and 2/9 adult kidneys were also positive, with proximal tubules and collecting ducts negative. All other 159 blocks of non-neoplastic adult and fetal tissues were negative. The antibody was also affinity purified from ascitic fluid, and shown not to inhibit the enzyme activity of prostatic acid phosphatase.

Use of immunochemistry in Britain: EQA forum antibody usage questionnaire.

A questionnaire was prepared under the auspices of the Department of Health with the aim of defining the extent and nature of immunocytochemistry use within pathology departments. The questionnaire was circulated to 320 pathology laboratories within the United Kingdom, and a total of 178 replies were received, representing a response rate of 56%. One hundred and thirty eight (78%) of the respondents used immunocytochemical techniques: 64 used immunocytochemical kits, including 35 district general hospital and 29 teaching hospital laboratories. An extensive range of antibodies was being used on a variety of tissues, epithelial and lymphoid markers far exceeding all other antibodies. Several differences in the numbers of cases and the types of tissues studied were identified among laboratories. The techniques used, the problems encountered, and the procedures followed with unsatisfactory reagents were also analysed. Finally, an assessment of the resources allocated to immunocytochemistry, both in terms of staff and reagent costs was made. Taking into account the response rate of 56% and the uncertainty that all pathology departments in the United Kingdom had been circulated, the estimated annual total costs for immunocytochemistry for all pathology laboratories in the United Kingdom was 5.4 million pounds.

Response of breast carcinoma to endocrine therapy predicted using immunostained pelleted fine needle aspirates.

Fine needle aspirates from 82 patients with breast carcinoma were fixed in methacarn, double embedded in agar or gelatin, and then in paraffin wax. Sequential sections were stained with monoclonal antibodies to the oestrogen receptor-related protein P29 (antibody D5), carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and cytokeratin (CAM 5.2). Sixty-one of 82 (74%) aspirates provided sections suitable for immunostaining. Twenty-six (43%) were D5 positive, 23 (38%) CEA positive, 59 (97%) EMA positive, and 54 (89%) CAM 5.2 positive. Twenty-six of these patients were treated with some form of endocrine therapy. Twelve (46%) showed positive staining for D5. Eleven (92%) of the 12 D5-positive patients responded or had static disease, and 8% progressed. Of the 14 D5-negative tumours 43% responded or remained static, and 57% progressed. The difference in response between the D5-positive and the D5-negative tumours was significant (P less than 0.05, Fisher's exact test). There was no correlation between staining for CEA, EMA or cytokeratin and response to endocrine therapy.

Stony testicle: case of calcific granulomatous orchitis.

A thirty-three-year-old man presented with a hard testicular mass considered preoperatively to be a malignant testicular tumor. Frozen section revealed calcific granulomatous orchitis, which does not appear to have been previously described in man. Granulomatous orchitis is an important differential diagnosis of testicular lesions. The hard nature of this particular lesion contributed to the preoperative diagnosis of malignancy.

Optimization and batch production of DTPA-labelled antibody kits for routine use in 111In immunoscintigraphy.

A procedure for conjugating antibodies in batches with DTPA is described which yields pharmaceutical grade preparations that can be kept in frozen storage until required. Routine radiolabelling is then simply performed by adding 111In chloride to the sterile 'kit' vial. After 1h, a DTPA solution is added to make the preparation ready for use in patients. The effects of pH, DTPA to antibody ratio, and time of reaction were investigated to optimize the preparation. By this method the antibody-DTPA conjugates may be prepared and the quality control performed at regional nuclear medicine centres where facilities and expertise already exist. These 'kits' can then be distributed to other departments where the 111In is added as and when required in a similar way to the preparation of other radiopharmaceuticals.

Ectopic prostatic glands in bulbar urethra. Immunoperoxidase study.

Ectopic prostatic glands in the bulbar urethra of a sixty-year-old man were identified by an indirect immunoperoxidase stain for prostatic acid phosphatase. Cystoscopically the appearances were those of "urethritis" without the polypoid appearance previously reported in cases of ectopic prostatic tissue.