Inhibition of phorbol ester-receptor binding by a factor from human serum.

The inhibition of receptor binding of [3H]phorbol-12,13-dibutyrate (PDBu) by a factor from human serum was characterized. The serum factor inhibited [3H]PDBu binding in intact monolayer cultures of the rat embryo cell line CREF N and in a subcellular system containing membranes from these cells. Inhibition occurred at both 37 and 4 degrees C and was rapid and reversible. An analysis of [3H]PDBu binding in the presence of the serum factor indicated that inhibition of [3H]PDBu binding by the serum factor was noncompetitive. Using gel filtration to separate the serum factor from free [3H]PDBu, we obtained evidence that the serum factor does not act by binding or trapping the [3H]PDBu. Unlike the phorbol ester tumor promoters, the serum factor alone did not stimulate the release of choline or arachidonic acid from cellular phospholipids, nor did it inhibit the binding of 125I-labeled epidermal growth factor to cellular receptors. The factor did, however, antagonize the inhibition of epidermal growth factor binding induced by PDBu. Sera from pregnant women were, in general, more inhibitory of [3H]PDBu binding than were those from nonpregnant women, which were more inhibitory than those from men. During these studies we found that CREF N cells responded to being grown in the presence of PDBu by partial down regulation of the phorboid receptor. The 50% effective dose for down regulation was 8 nM PDBu, and the maximum effect occurred after 6 h. Taken together, our results indicate that the serum factor inhibits [3H]PDBu binding by a direct physical effect at the level of the phorboid receptors or their associated membranes. It would appear that if this factor acts in vivo, then it might antagonize certain effects of this class of tumor promoters.

Binding of phorbol dibutyrate and epidermal growth factor to cultured human epidermal cells.

Primary cell cultures of normal human epidermal keratinocytes and melanocytes and human cell lines established from a primary melanoma (SK-PM-4) and metastatic melanomas (HO#1, SK-MEL21, and SK-MEL37) contain specific and saturable receptors for the tumor promoter phorbol dibutyrate (PDBu). Scatchard analyses of the keratinocytes revealed two classes of binding sites: 1) a high-affinity class (affinity constant = 37 nM; 1.3 X 10(6) sites/cell) and a low-affinity class (affinity constant = 4,880 nM; 7 X 10(7) sites/cell). The melanoma cultures, likewise, showed high- and low-affinity classes of PDBu binding sites. However, the affinity constant values and total numbers of sites in the melanoma cells were lower than the corresponding values in the keratinocytes. The binding of [3H]PDBu to human keratinocytes was inhibited by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate and teleocidin but not by phorbol, which lacks tumor-promoting activity. Human serum also inhibited binding. Specific receptors for epidermal growth factor (EGF) were demonstrated in the keratinocytes and primary melanoma cultures. In contrast, three metastatic melanoma cultures gave negligible levels of EGF binding. Among the various cell types, the extent of [3H]PDBu binding did not correlate with the extent of EGF binding, indicating that these two substances occupy distinctly separate types of receptors.

Treatment for breast cancer and blood levels of chlorinated hydrocarbons.

Small studies have examined, with conflicting results, whether breast cancer risk is increased among women exposed to high levels of chlorinated hydrocarbons, as measured in breast fat tissue or peripheral blood collected prior to treatment (pretreatment blood). For a population-based, case-control study, collection of pretreatment blood is a labor-intensive effort. An alternative is to collect blood from cases at interview, as is done for controls, after breast cancer treatment has commenced (posttreatment blood). It is unknown whether treatment affects blood levels of the organochlorines 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) or polychlorinated biphenyls (PCBs). The purpose of this study was to determine whether pretreatment versus posttreatment blood samples yielded significantly different estimates of cumulative exposure to DDE and PCBs among newly diagnosed breast cancer patients. Two-ml blood samples were collected prior to and after treatment for breast cancer from 22 nonfasting women, ages 45-87 years, newly diagnosed with invasive disease. Treatment was defined as major surgery (mastectomy or node removal), radiation, hormones (tamoxifen), or chemotherapy. Pretreatment and posttreatment blood samples were assayed for DDE and PCBs in blinded, matched pairs. The reported concentrations (volume basis) were adjusted for estimated total plasma lipids. For DDE, mean differences in unadjusted [0.99 ng/ml; 95% confidence interval (CI), -0.36 to 2.34 ng/ml] and lipid-adjusted (0.05 microgram/g lipid; 95% CI, -0.04 to 0.13 microgram/g lipid) levels were small. For PCBs, the unadjusted (0.68 ng/ml; 95% CI, 0.05 to 1.30 ng/ml) and adjusted (0.070 microgram/g lipid; 95% CI, -0.009 to 0.149 microgram/g lipid) mean differences were of borderline statistical significance. The mean percent change in lipid-adjusted organochlorine levels did not vary substantially between treatment groups, except for those patients receiving chemotherapy [n = 5; 15.8% (DDE), 29.4% (PCBs)]. Adjusted mean differences also increased with increasing time between the pretreatment and posttreatment blood draws. In multiple regression models that included treatment, age, race, stage, and time between blood draws, only chemotherapy appeared to predict the percent change in adjusted pretreatment and posttreatment levels of DDE or PCBs (P = 0.10 and 0.06, respectively). Posttreatment blood samples drawn within 3 months of pretreatment samples, with the exception of those drawn after the commencement of chemotherapy, provide similar measures of DDE body burden levels among breast cancer cases. The use of blood samples collected after treatment, rather than before treatment, for characterizing PCB levels may lead to misclassification of exposure.

Late fatal adenovirus pneumonitis in a lung transplant recipient.

Adenovirus (ADV) is increasingly recognized as a cause of morbidity and mortality in transplant recipients, but ADV pneumonitis has rarely been reported after lung transplantation. The few reported instances of ADV pneumonitis occurred mostly in children immediately after lung transplantation suggesting "primary" infection. We report a fatal case of ADV pneumonitis occurring in an adult, 4 years after unilateral lung transplantation, in whom the premortem diagnosis was not determined. Autopsy revealed severe necrotizing bronchitis, bronchiolitis, and interstitial pneumonitis. Characteristic smudgy intranuclear inclusions, immunohistochemistry for viral protein, in situ hybridization for viral genome, and postmortem lung cultures established ADV as the etiologic agent. ADV can cause fatal, occult respiratory infection in adult lung transplant recipients, remote from transplant surgery.

Metastatic calcification in skin: exclusive involvement of eccrine sweat ducts. A case report.

A remarkable distribution of metastatic calcium in skin involving exclusively the excretory duct of eccrine sweat glands was observed in an adult male with hypercalcemia. An explanation is offered for the unusual distribution of calcium in the skin.

Malignant granular cell tumor: a case report and review of the recent literature.

We report a case of an extremely rare neoplasm, malignant granular cell tumor (MGCT). The tumor occurred in the infratemporal fossa of a 30-year-old man, extended to the left orbital base, into the foramen ovale, and invaded the mandible. A granular cell tumor (GCT) was diagnosed by fine-needle aspiration and core needle biopsy of the mass. The patient underwent a radical subtotal debulking procedure followed by radiotherapy. He is alive with recurrent disease 12 months after presentation. Cytologically, the aspirated material was abundantly cellular showing large polygonal cells with ample granular eosinophilic cytoplasm, eccentric nuclei, and often prominent nucleoli. Histologically, the tumor consisted of solid sheets of similar cells that stained strongly with S-100 protein, neuron-specific enolase (NSE), and vimentin. There was moderate nuclear pleomorphism and broad zones of necrosis. Four mitotic figures per 100 high-power field (HPF) were counted. By electron microscopy, the cytoplasm of the tumor cells was filled with lysosomes. Although, some observers advocate that the diagnosis of a MGCT should be reserved for cases in which lymph node and/or distant organ metastasis is evident, we believe malignancy ought to be considered in any GCT with aggressive clinical course defined by persistent local recurrence and destruction of neighboring structures. Nuclear pleomorphism, necrosis, and presence of any mitotic activity should indicate malignancy.

Blackened bronchoalveolar lavage fluid in crack smokers. A preliminary study.

A retrospective study was performed on heavily pigmented pulmonary cytologic specimens from 14 hospital patients to determine the clinical features distinguishing these cases. The lavage fluid or sputum in each case was turbid and gray or black, exceeding the blackness usually seen in heavy tobacco smokers dwelling in the same urban environment. Excessive carbonaceous material was observed in the cytoplasm of pulmonary alveolar macrophages or the extracellular compartment of the smears. The latter feature is not seen in cigarette smokers. Many other pigmentary sources were ruled out, including melanin, hemosiderin, medicinal charcoal, India ink, and hematoxylin crystals. The common feature of the patients was that they recently or currently smoked the crack form of cocaine heavily; five patients also had positive toxicologic results for cocaine at admission. The authors suggest that blackened bronchoalveolar lavage fluid indicates the possibility of crack cocaine smoking and the associated sequelae, particularly when the carbonaceous material is present in the extracellular compartment.

Additional mimics of mucinous mammary carcinoma: fibroepithelial lesions.

To determine the origin and nature of mucinlike material in fine-needle aspiration (FNA) smears of the breast in noncancerous breast lesions, we studied breast FNA smears from four patients. All smears contained epithelial cells floating in a mucinlike background, which raised suspicion for mucinous (colloid) carcinoma. Mucicarmine stain was performed on one smear from each case. Subsequent tissue biopsy specimens were studied using mucicarmine, periodic acid-Schiff with and without diastase, and alcian blue stains at pH 2.7 and 0.9 on selected tissue sections. Correlation of the cytologic and histologic findings of each lesion was performed. The mucinlike background in all four FNA smears stained strongly with mucicarmine. Corresponding biopsy specimens revealed pseudoangiomatous hyperplasia in the first case, fibroadenoma and atypical ductal hyperplasia in the second, benign phyllodes tumor in the third, and fibroadenoma in the fourth. Each lesion in cases 1 to 3 was associated closely with fibrocystic changes. In case 4, cystic changes were located within the fibroadenoma. On tissue sections of all four cases, the cyst contents and 10% to 50% of normal lobule and duct contents stained with mucicarmine, indicating that the cyst contents were the most probable source of mucin in the FNA smears. The presence of pools of mucicarmine-positive material in FNA smears of the breast is not an exclusive feature of mucinous carcinoma; mucicarmine-positive mucin can arise from benign cystic changes as well as from normal lobules and ducts.

Comparison of needle aspiration and solid biopsy technics in the flow cytometric study of DNA distributions of surgically resected tumors.

Flow cytometric analysis (FCM) of DNA content of nuclei was performed on simultaneously obtained tissue samples and needle aspirates from 37 primary colorectal cancers and from 21 other tumors. There was a marked increase in the proportion of the nondiploid cell population in 18 of 58 aspirates when compared with the corresponding tissue samples, presumably because of selective aspiration of tumor cells. The difference was significant at level alpha much less than 0.01 by a paired t-test and was most pronounced in tumors wherein a nondiploid population constituted more than 20% of the sample. The difference did not correlate with the grade or stage of the tumor. These observations suggest that the sampling of surgically resected tissue specimens for DNA analysis by FCM is performed best by needle aspiration, which may increase the yield of nondiploid cells, does not interfere with histologic diagnosis, and may prove especially useful in the analysis of small surgical specimens.

Evaluation of esophageal cytology using a neural net-based interactive scanning system (the PAPNET system): its possible role in screening for esophageal and gastric carcinoma.

A neural net-based, semiautomated, interactive computerized cell analysis system (The PAPNET system, Neuromedical Systems, Suffern, NY) was used to examine cells from 138 esophageal smears obtained by lavage, brushings, or balloon from as many patients. From each smear, trained human observers examined 128 cell images selected by the machine. Abnormal cells were identified in all 35 patients with cancer, whether esophageal, gastric, oral, or metastatic. Further, in 11 smears, the displayed images allowed the recognition of effects of radiotherapy and, in 14 smears, the diagnosis of a specific tumor type, such as squamous cell carcinoma (8 patients) or adenocarcinoma (6 patients). In 3 additional cases, the diagnosis of "carcinoma, not further specified," was established. One case of esophageal carcinoma in situ, not previously recognized on a smear or in the biopsy specimen, and one case of gastric adenocarcinoma, not recognized in the smear, were identified in PAPNET-generated images. The possible application of the apparatus to the triage of smears and population screening for esophageal and gastric carcinoma precursors is discussed.

DNA ploidy of spindle cell soft-tissue tumors and its relationship to histology and clinical outcome.

With the use of fresh cell suspensions, the DNA ploidy of 11 benign and 27 malignant spindle cell soft-tissue lesions was determined by flow cytometry (37 cases) and/or image cytometry (35 cases). Of the 27 malignant lesions, 10 were low- or intermediate-grade sarcomas, and 17 were high-grade sarcomas. In the malignant lesions, the DNA ploidy was correlated with the histologic grade and the clinical outcome. Of the 11 benign lesions, six had a diploid DNA ploidy pattern, and five were nondiploid by either flow cytometry or image cytometry. All benign cases had a favorable outocome regardless of ploidy. Eight of the 10 low- or intermediate-grade malignant lesions were diploid, whereas two were nondiploid. Of the 17 high-grade sarcomas, 15 were nondiploid by either method of measurement and nine by both. Of the 10 diploid malignant tumors, only one had an unfavorable outcome (malignant mesothelioma with biopsy only), whereas of the 17 malignant lesions that were nondiploid, five had no evidence of recurrent disease, three cases recurred, eight patients died of disease, and one patient died of an unrelated cause. We concluded that (1) ploidy does not differentiate benign from malignant spindle cell soft-tissue tumors, (2) the nondiploid DNA pattern is more common in high-grade than in low- or intermediate-grade sarcomas, and (3) there is a statistically significant relationship between nondiploidy and a worse clinical outcome in sarcomas.

Carbon-laden macrophages in pleural fluid of crack smokers.

Carbon-laden macrophages in bronchoalveolar lavage have been noted to be associated with a history of crack smoking. We report herein the finding of carbon-laden macrophages in four cytological preparations of pleural fluid from two crack smokers. The etiology of the two patients' pleural effusions differed; neither had a bronchopleural fistula. Patient 1 had AIDS, Pneumocystis carinii pneumonia, and Kaposi's sarcoma of the right lung with an associated bilateral pleural effusion. Patient 2 was HIV seropositive, had pulmonary tuberculosis, hepatitis A, B, and C, cardiomyopathy, pulmonary embolism, and bilateral pleural effusions, the latter of which were probably due to cardio-pulmonary dysfunction. An additional two crack smokers with pleural effusions due to malignancy, one primary pulmonary adenocarcinoma and the other metastatic melanoma, did not have carbon-like material in their pleural fluid cytology. We hypothesize that intracellular accumulation of carbonaceous material in the lung parenchyma and pleural space occurs when normal clearance mechanisms are overwhelmed.

Fine-needle aspiration diagnosis of enterogenous cyst of the orbit: a case report.

Enterogenous cyst is a benign lesion derived from misplaced endodermal epithelium. We report the aspiration cytology findings of an orbital cyst from a 32-year-old woman. The smears contain benign-appearing cuboidal glandular cells with focal mucinous features. Carcinoembryonic antigen level in the cyst fluid was markedly elevated. These findings are consistent with recurrence of the enterogenous cyst initially diagnosed 7 years earlier.

Open neural tube defects: immunocytochemical demonstration of neuroepithelial cells in amniotic fluid.

Cytologic evaluation of second trimester amniotic fluid (AF) is a rapid, inexpensive adjunct to prenatal diagnosis of open neural tube defects (ONTDs). Our goal was to determine whether the neural-appearing cells and/or large foamy macrophages in the AF of anencephalics are indeed of neural and/or glial origin. In two second trimester patients with elevated serum alpha-fetoprotein (AFP) and polyhydramnios, fetal sonogram studies showed anencephaly; amniocentesis was performed for AF-AFP, cytogenetic, and cytologic studies. AF sediment smears were initially Papanicolaou-stained; next, the same smears were immunoperoxidase (IP)-stained for glial fibrillary acidic protein (GFAP). If GFAP negative, slides were restained for synaptophysin (SYN) and neuron-specific enolase (NSE). Both AFs contained small neural-appearing cells (5-10 microns) singly and in clusters, with dense, round, homogeneous nuclei, an occasional nucleolus, and scant cytoplasmic rim. These were GFAP negative and SYN and NSE positive; the large vacuolated, lipid-laden macrophages (20-40 microns) were negative for all three IP stains. In conclusion, positive IP staining for SYN and NSE supports the morphologic impression that small dark cells in AF are of neural origin, while negative IP staining of large foamy macrophages suggests nonneural, nonglial origin.

Biliary stent replacement cytology.

Using fluoroscopic guidance, polyethylene biliary stents are replaced endoscopically or percutaneously when bile duct stenosis recurs. To improve the sensitivity of conventional biliary cytology, we examined cells recovered from removed stents. Biliary stents removed endoscopically from each of 11 patients were rinsed with saline; next, the rinse was centrifuged and the sediment smeared and Papanicolaou stained. Three patients with choledocholithiasis had biliary stent replacement cytology (BSRC) to exclude a neoplastic etiology. Eight patients with clinicoradiologic evidence of hepatobiliary or pancreatic carcinoma had BSRCs performed for pathologic documentation of carcinoma. BSRC from six of eight patients with clinicoradiologically malignant biliary strictures contained malignant cells, predominantly in loose clusters, but also singly (sensitivity 75%, specificity 100%; positive predictive value 75%, negative predicative value 60%). Reparative epithelial atypia was also present in all cases. BSRC from two patients with clinicoradiological evidence of carcinoma of the biliary region and from three with choledocholithiasis contained only bile pigment, leukocytes, and benign epithelial cells. The sampling of cells which have accumulated on, or in biliary stents, improves the sensitivity of biliary cytology. This is most applicable when 1) a patient is inoperable, 2) tissue biopsy is neither feasible nor diagnostic, 3) prior brush, suction, percutaneous, or endoscopic needle aspiration cytology is inconclusive, and 4) permanent metal stent is needed.

Transvaginal sonographic characterization combined with cytologic evaluation in the diagnosis of ovarian and adnexal cysts.

A transvaginal sonographic (TVS) scoring system using morphologic features has been developed at our institution to maximize discrimination between benign and malignant ovarian and adnexal cysts. Low (4-7) or intermediate (8-9) scores have been found to correlate with benignity, hence TVS-guided or laparoscopically directed needle aspiration of low-scoring lesions may safely be performed. High-scoring lesions (10-14) are often malignant, therefore in situ needle aspiration of such lesions is not recommended. The aim of our study was to correlate the results of TVS characterization of ovarian and adnexal cysts with the aspiration cytologic evaluation. Twenty-three of the 43 cysts studied were aspirated in situ from the patient; 20 were aspirated from resected surgical specimens. Thirty-six benign cysts had TVS scores ranging from 4 to 12, with a median score of 7. All 25 cysts that were benign by TVS and/or histology were also cytologically benign as well as an additional 11 cysts that were not resected (TVS scores: 4 to 9). Seven cytologically and histologically malignant cysts had high TVS scores (TVS scores 10-14; median = 12). The combination of TVS and needle aspiration cytology is valuable, particularly in the diagnosis of cysts having low or intermediate TVS scores and benign cytology. Aspiration of cysts or masses with high TVS scores is not recommended. This combined evaluation may allow a more limited surgical approach, such as operative laparoscopy, or, in some cases, obviate the need for operative treatment altogether.

Megakaryocytes and ganglion cells mimicking cancer in fine needle aspiration of the prostate.

In six cases, fine needle aspiration (FNA) directed at prostatic lesions produced specimens containing megakaryocytes or ganglion cells, which are sources of potential diagnostic error in the interpretation of aspirates of the prostate gland. In three cases, apparent accidental penetration of the tip of the transrectal FNA biopsy needle into the ischium instead of the prostate resulted in the aspiration of megakaryocytes and other bone marrow elements; the megakaryocytes mimicked anaplastic cancer. Three additional FNAs of the prostate produced specimens containing a small number of ganglion cells mimicking well-differentiated adenocarcinoma; the accompanying strands of wavy neural tissue served as a clue to the ganglionic origin of these potentially misleading cells. The paucity of "atypical" cells and the sparsity or absence of prostatic epithelium in the aspirates were additional clues to the cytologist that the findings represented an anatomic misplacement of the FNA biopsy needle tip, rather than a prostatic cancer.

Cytologic manifestation of an unusual bacterial form, Simonsiella species.

Puzzling rodlike structures overlying benign squamous cells in exfoliative cytologic specimens from the upper gastrointestinal and respiratory tracts were initially considered to be fungi, protozoa, bronchodilator crystals, hemoglobin tactoids or plastic fragments. Their morphologic similarity to Simonsiella, a gram-negative bacteria frequently found in the oral cavity, was ultimately recognized. Further studies of smears and cultures obtained from the oral cavities of the authors and from the American Type Culture Collection confirmed the nature of the original findings. These giant bacterial forms were usually found in caterpillarlike side-by-side arrangements of 10 to 12 organisms. Cytologists should be aware of their appearance to avoid possible confusion with pathogenic organisms.

DNA ploidy of ovarian and adnexal cyst fluid. A useful adjunct to cytology.

It is increasingly common for cytology laboratories to receive ovarian, adnexal and pelvic cyst fluids obtained via sonographically directed aspiration and laparoscopic techniques, especially from women who are desirous of preserving fertility or who are undergoing in vitro fertilization (IVF). Accurate characterization of such cysts is a worthwhile goal, given the superior prognosis for ovarian carcinomas that are diagnosed at an early stage. In an effort to improve upon the false-negative diagnosis rate associated with cytology, we evaluated DNA ploidy as a possible adjunctive criterion. We examined 55 benign, 3 borderline and 6 malignant aspirates received by our cytopathology laboratory; 35 were aspirated directly from the patient from clinically and ultrasonographically benign cysts, and 29 were aspirated from surgically removed benign (20) and malignant (9) cysts. Adjunctive DNA ploidy and cell cycle analysis was performed using the Cell Analysis Systems CAS-200 on Feulgen-stained cytologic smears of the 64 cyst fluids. Adequate material for DNA analysis was obtained from 33/35 in situ aspirated cysts and from 19/29 surgical specimen cysts. Forty-seven of 52 cytologically benign cysts were diploid. Of the 5 nondiploid benign cysts, 3 were follicle cysts (2 from hormonally stimulated IVF patients and 1 from a postpartum patient), and 1 was a benign cystic teratoma. Their nondiploid DNA pattern or tetraploidy may be due to a high proliferative index. The fifth nondiploid benign aspirate was from a resected benign epithelium-lined cyst; its DNA histogram contained a conspicuous tetraploid population. All 9 malignant cysts were cytologically malignant. Of the 3 borderline cysts, 1 was nondiploid, and 2 were diploid. All 6 fully malignant cysts were nondiploid; 2 of them were tetraploid. Based on our results, we conclude that DNA ploidy analysis of cells derived from ovarian and adnexal cyst aspirates is feasible (in 95% of cases) and relatively specific (90%) and has a relatively high negative predictive value (92%). The results are not sufficiently predictive of the histology of the lesion to warrant therapeutic intervention based on ploidy alone (sensitivity of nondiploid results, 78%; positive predictive value of nondiploid results, 58%). Nondiploidy should suggest consideration, but is not conclusive, of a malignancy diagnosis. There may even be prognostic implications to the ploidy pattern, particularly in borderline tumors, in which nondiploidy portends a poor prognosis.

Aspiration cytology of ovarian cysts in in vitro fertilization patients.

Transvaginal ultrasonography is routinely performed in the course of egg retrieval in patients presenting for in vitro fertilization (IVF) and results in the discovery of occult, subclinical ovarian cysts that would otherwise have gone undetected. This study (1) evaluated the cellular composition of the cyst fluids based on a comparison with cells obtained from cysts of known and documented histologic type, and (2) attempted to determine if the cytologic evaluation of cyst fluid was necessary to exclude occult ovarian cancer. During the 1.8 years of our study, 931 patients underwent 1,544 ultrasound-guided ovum retrievals; during this same period, 90 specimens of ovarian cyst fluid were examined. Of them, none contained cancer cells. A single case of cystic ovarian cancer was detected by ultrasound but was not aspirated. The cytologic diagnosis of endometriosis was established in 12 specimens from 10 patients, 5 of whom did not have a previously documented clinical diagnosis of endometriosis. The role of routine ovarian cyst fluid cytology as part of an IVF protocol may be of limited value in cancer diagnosis. However, until the incidence of ovarian cancer in the subset of women with both infertility and ovarian cysts is known, it would seem prudent to continue to examine any voluminous or discolored ovarian cyst fluid obtained from IVF patients. The presence of ovarian cysts did not affect the clinical pregnancy rate per retrieval.