Feasibility of immunocytochemical detection of tumor markers (XIAP, phosphohistone H1 and p63) in FNA cellblock samples from head and neck squamous cell carcinoma.

One of the major handicaps in contemporary clinical oncology is the inability to predict the responsiveness of any individual's malignancy to specific therapies. The purpose of this study was to test the feasibility of immunocytochemically detecting markers that may be affected by therapy or are predictive of therapeutic responsiveness, including phosphohistone H1 (anti-p-H1 MoAb 12D11) and X-linked inhibitor of apoptosis (XIAP) in small samples obtained via fine-needle aspiration (FNA) biopsy procedure, thus improving therapeutic monitoring. p63, a squamous stem cell regulatory protein, was also examined. These three markers were studied in FNA cell block samples of head and neck squamous cell carcinoma (HNSCC). Twenty-eight alcohol-fixed formalin-postfixed paraffin-embedded cell-block samples from FNAs of patients with HNSCC were subjected to antigen retrieval and then incubated with anti-XIAP, anti-p-H1, and anti-p63, and developed using EnVision-Plus reagents and diaminobenzidine as chromagen; Granular or heterogeneous cytoplasmic staining for XIAP and nuclear staining for p63 and p-H1 were considered positive. Among the 28 cases studied, the overall positive rates for XIAP, p-H1, and p63 were 60.7%, 96.4%, and 92.8%, respectively. The staining intensity for XIAP: + 70.6%, ++ 23.5%, +++ 0%, and ++++ 5.9%; for p-H1: + 48.1%, ++ 11.1%, +++37.0%, and ++++ 3.7%; and for p63: + 11.5%, ++ 23.1%, +++ 53.9%, and ++++ 11.5%. The expression of p-H1 and p63 appeared to be higher and stronger than that of XIAP in HNSCC. This study demonstrated the feasibility of monitoring expression of three tumor markers using FNA samples. p-H1 and XIAP may be useful for monitoring actions of cyclin-dependent kinase inhibitors, XIAP-lowering, and/or apoptosis-inducing drugs, respectively. Future studies will focus on the impact of therapies upon these staining profiles.

GLUT1 and p63 expression in endometrial intraepithelial and uterine serous papillary carcinoma.

AIMS: To analyse the expression patterns of GLUT1, p63 and p53 and the possible correlation between these markers in uterine serous papillary carcinoma (USPC) and endometrial intraepithelial carcinoma (EIC). METHODS AND RESULTS: Fourteen cases of USPC, 12 of which also showed EIC, were examined for GLUT1, p63 and p53 immunoreactivity. Four-micrometre sections from formalin-fixed paraffin-embedded tissue were immunostained using commercially available primary antibodies and Dako Envision Plus reagents for visualization. Membranous GLUT1 immunoreactivity was observed in all cases, including all 14 invasive tumours (100%) and 11 of 12 associated EICs (92%), and was confined to neoplastic cells. In USPC, staining tended to be strongest in superficial antistromal regions. p63 positivity was found in 8/14 (57%) USPCs and 9/12 (75%) associated EICs. In 11 cases p53 was overexpressed in both invasive USPC (11/14; 79%) and EIC (11/12; 92%). p53+ USPCs tended to be positive for p63, whereas p53- USPCs were also negative for p63. CONCLUSIONS: GLUT1 is expressed in the vast majority of USPC and EIC, suggesting a biological role during the early steps of carcinogenesis in endometrial serous neoplasms. GLUT1 expression may be induced by hypoxia-related as well as other mechanisms.

Immunohistochemical detection of GLUT1, p63 and phosphorylated histone H1 in head and neck squamous intraepithelial neoplasia: evidence for aberrations in hypoxia-related, cell cycle- and stem-cell-regulatory pathways.

AIM: Most epithelial malignancies are characterized by multistep progression from preinvasive/intraepithelial neoplasia to invasive malignancy. Detection and grading of early squamous intraepithelial neoplasia may at times be problematic. The aim of this study was to examine the ability of immunomarkers GLUT1, phospho-histone H1 and p63 to detect such early lesions. METHODS: Sections of formalin-fixed paraffin-embedded tissue from 27 cases of squamous intraepithelial neoplasia, 26 associated with invasive carcinoma, were immunostained with anti-p63 (4A4; Santa Cruz), anti-GLUT1 (Chemicon) and anti-phospho-histone H1 (monoclonal 12D11) and compared with normal, hyperplastic and immature squamous metaplastic epithelium. RESULTS: Normal epithelium displayed phospho-histone H1 in scattered parabasal cells; p63 in the basal one-quarter to one-half of epithelium; and GLUT1 negativity or weak/equivocal mid-epithelial GLUT1+ foci. In hyperplasia phospho-histone H1+ cells were also limited to the parabasal layer; p63 positivity was essentially identical to that in normal epithelium; GLUT1 characteristically stained basal cells in rete-like areas. p63 staining in squamous intraepithelial neoplasia (grade 1) was indistinguishable from normal epithelial staining; in contrast, squamous intraepithelial neoplasia (grade 3) was readily apparent, with up to full-thickness p63 positivity. Squamous intraepithelial neoplasia (grade 1) was readily distinguishable from normal epithelium with both phospho-histone H1 and GLUT1 immunostaining; both markers were detected in cell layers above the parabasal layer. With both, progressively higher cell layers stained in proportion to the severity of the intraepithelial neoplasia, up to full thickness positivity in grade 3 squamous intraepithelial neoplasia. Squamous metaplasia and grade 3 squamous intraepithelial neoplasia were not distinguishable with p63 (both showed full-thickness staining) but were readily distinguishable with GLUT1 and phospho-histone H1 stains. GLUT1 appeared to be the most sensitive marker for all grades of intraepithelial neoplasia. CONCLUSION: Altered expression of all three markers was a common finding in squamous intraepithelial neoplasia, hence, dysregulation of cell cycle-promoting cyclin-dependent kinases (phospho-histone H1), altered stem cell regulatory pathways (p63) and enhancement of hypoxia-sensing pathways (GLUT1) are all early alterations in the progression of squamous malignancy of head and neck origin. A panel of all three may be a useful means of detecting squamous intraepithelial neoplasia.

Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinization.

Immunodetection of GLUT1, p63 and phospho-histone H1 in invasive head and neck squamous carcinoma: correlation of immunohistochemical staining patterns with keratinizationAims : To examine invasive head and neck squamous carcinomas for expression of GLUT1, a glucose transporter and marker of increased glucose uptake, glycolytic metabolism and response to tissue hypoxia; p63, a p53 homologue that is a marker of the undifferentiated proliferative basaloid phenotype; and phospho-histone H1, a marker of activation of the cell cycle-promoting cyclin-dependent kinases 1 and 2. Methods : Routinely processed slides from 34 invasive squamous carcinomas, including 25 with intraepithelial components, were immunostained with anti-GLUT1 (Chemicon), anti-p63 (4A4, Santa Cruz), and antiphospho-histone H1 (monoclonal 12D11). Results : In keratinizing carcinomas, all three markers were most commonly immunodetected peripherally, with loss of expression in central keratinized zones. In contrast, in non-keratinizing carcinomas, p63 and phospho-histone H1 expression was most commonly observed throughout tumour nests and anti-GLUT1 stained in a pattern suggestive of hypoxia-induced expression ('antistromal' staining), in which cells at the tumour-stromal interface were GLUT1- and cells in central, perinecrotic zones showed progressive induction of GLUT1. Intraepithelial components also displayed basal and 'antibasal' GLUT1 staining patterns, homologous to the pro- and antistromal patterns in invasive carcinoma; basal patterns in intraepithelial lesions appeared to be more predictive of keratinizing invasive carcinoma and antibasal intraepithelial staining more predictive of non-keratinizing poorly differentiated carcinomas. Conclusions : Keratinizing and non-keratinizing squamous carcinomas differ in expression patterns of GLUT1, p63 and phospho-histone H1. In the former, all three markers were typically suppressed in conjunction with keratinization; in the latter, GLUT1 expression was more likely to occur in a hypoxia-inducible pattern and expression of p63 and phospho-histone H1 was unsuppressed. GLUT1 expression patterns in intraepithelial lesions may be predictive of the differentiation status of the associated invasive carcinoma.

Thyroid cytology and the risk of malignancy in thyroid nodules: importance of nuclear atypia in indeterminate specimens.

Fine needle aspiration (FNA) cytology is the best test for malignancy in thyroid nodules. However, cytologic interpretation of FNA specimens is often difficult, especially in the presence of indeterminate microfollicular cytologic patterns, which are thought to suggest follicular neoplasm (adenoma or carcinoma). To assess the risk of malignancy associated with specific cytologic patterns, we correlated preoperative FNA cytologic patterns (n = 484 reports including repeat aspirations) with final histological diagnoses for 368 surgical thyroid specimens obtained during the period 1994-1998. The overall prevalence of malignancy in the surgical specimens was 31% (113 cancers, including 96 papillary and 9 follicular carcinomas). For nodules with benign FNA cytologic diagnoses of nodular goiter and chronic thyroiditis there was a low risk of malignancy (6/99, or 6.1%). Nodules with indeterminate cytologic patterns in the absence of nuclear atypia (i.e., microfollicles without nuclear atypia) had a similarly low malignancy risk (3/46, or 6.5%). In contrast, 31/52 nodules with cytologic nuclear atypia consistent with follicular neoplasm were malignant (60%), including specimens with or without microfollicular cytology. Nodules with frankly malignant cytologic patterns were almost invariably cancer (54/55), and cytologic diagnoses of papillary carcinoma were confirmed at surgery in all 49 cases. These results indicate that indeterminate microfollicular cytologic patterns in the absence of nuclear atypia are associated with a low risk of malignancy, at least in this series. This finding suggests that many nodules with such microfollicular cytology might be managed conservatively with observation. In contrast, cytologic nuclear atypia consistent with a follicular neoplasm confers a high risk of cancer. In addition, frankly malignant cytologic diagnoses, especially papillary carcinoma, are highly reliable, and thus may be used as a guide for planning surgery appropriate for thyroid cancer.

Immunohistochemical staining of GLUT1 in benign, hyperplastic, and malignant endometrial epithelia.

BACKGROUND: Aberrant expression of the facilitative glucose transporter, GLUT1, is found in a wide spectrum of epithelial malignancies. The current study describes an immunohistochemical study of GLUT1 expression in benign, hyperplastic, and malignant endometrial epithelia. METHODS: One hundred sixteen formalin fixed, paraffin embedded sections of benign, hyperplastic, and malignant endometrial epithelium were immunostained with rabbit anti-GLUT1 antibody using the streptavidin-biotin method. RESULTS: Proliferative, secretory, and atrophic endometrium were not stained with GLUT1 antiserum. Rare, minute foci of tubal metaplasia stained positively. Simple and complex endometrial hyperplasias were consistently GLUT1 negative. All specimens of atypical hyperplasia had foci that were positive for GLUT1. All endometrial adenocarcinomas were GLUT1 positive. CONCLUSIONS: The results of the current study appear to indicate that 1) aberrant overexpression of GLUT1 is a consistent feature of endometrioid adenocarcinoma; 2) GLUT1 immunostaining may be useful in distinguishing benign hyperplasias from hyperplasias that are associated strongly with malignancy; and 3) some or all cases of atypical hyperplasia may be neoplastic rather than hyperplastic, given the existence of a molecular defect common to this hyperplastic subtype and endometrioid adenocarcinoma.

Fine-needle aspiration diagnosis of synovial chondromatosis of the tibiofibular joint.

We report a case of synovial chondromatosis of the tibiofibular joint in a 25-year-old woman that was diagnosed by fine-needle aspiration (FNA). The patient presented with pain in the left knee and a mass in the popliteal fossa. Synovial chondromatosis usually presents with joint symptoms and is often associated with intra-articular loose bodies, whereas presentation as a soft tissue mass is unusual and may raise the clinical suspicion of malignant neoplasm. The diagnosis is commonly confirmed by histopathologic examination of biopsy or excision of the specimen. To the best of our knowledge, this is the first case of synovial chondromatosis of a large joint successfully diagnosed by FNA. Two cases of synovial chondromatosis of the temporomandibular joint have been reported in which the diagnosis was suspected on the basis of FNA. In both these cases, the final diagnosis was established by histopathology of the excised specimens.

Cytologic findings in the differential diagnosis of C-cell hyperplasia and medullary carcinoma by fine needle aspiration. A case report.

BACKGROUND: The cytologic features of C-cell hyperplasia of the thyroid have not been previously addressed in the literature. We describe the first case, to our knowledge, of C-cell hyperplasia that was suggested by fine needle aspiration. CASE: Cellular material was obtained from a nonnodular region of the thyroid gland in a 67-year-old male with chronic diarrhea, unexplained elevated serum calcitonin, no clinically detectable thyroid mass and no known medical or family history of an endocrine disorder. Aspiration yielded a scant bimodal cell population composed of benign follicular cells and a second population of larger cells, later confirmed as C-cells via immunohistochemistry. Although the diagnosis of medullary carcinoma was entertained, the absence of a discrete mass clinically and the presence of two interspersed, distinct cell populations suggested the alternate diagnosis, C-cell hyperplasia, which was confirmed by subsequent thyroidectomy. CONCLUSION: C-cell hyperplasia can mimic medullary carcinoma biochemically, and this case suggests the possible role of fine needle aspiration of the thyroid to distinguish between the two. In patients with elevated serum calcitonin and absence of a discrete thyroid nodule, the finding of clusters of calcitonin-positive cells intermixed with normal follicular cells by fine needle aspiration may provide a means of making a presurgical diagnosis of C-cell hyperplasia.

GLUT1 glucose transporter expression in colorectal carcinoma: a marker for poor prognosis.

BACKGROUND: Malignant cells exhibit increased glycolytic metabolism, and in many cases increased glucose transporter gene expression. The authors hypothesized that GLUT1 glucose transporter expression is increased in colorectal carcinoma, and that the degree of expression might have prognostic significance. METHODS: GLUT1 glucose transporter immunostaining was studied in normal colon and benign colon adenomas and in 112 colorectal carcinomas from patients for whom long term clinical outcome was known. RESULTS: GLUT1 immunostaining was absent in normal colorectal epithelium and tubular adenomas, and absent or only weakly apparent in tubulovillous adenomas. The majority of carcinomas (101 of 112; 90%) had GLUT1 immunostaining. Tumors from 92 patients had low GLUT1 expression (< 50% of cells were GLUT1 positive) and 19 of these patients (21%) died of disease during follow-up. In contrast, tumors from 20 patients had high GLUT1 expression (> 50% of cells were GLUT1 positive) and 9 of these patients (45%) died of disease during follow-up. Disease specific mortality was greater in patients with high GLUT1 tumors (relative risk of 2.4; P=0.02). In a multivariate analysis to assess whether high GLUT1 staining correlated with increased mortality independently of Dukes stage, the risk of death from colon carcinoma in the group with high GLUT1 staining was 2.3 times that in the group with low GLUT1 staining, a difference that approached statistical significance (P=0.07). CONCLUSIONS: GLUT1 glucose transporter expression is associated strongly with neoplastic progression in the colon, and assessment of the extent of GLUT1 immunostaining in colorectal carcinoma identifies patients with a poorer prognosis.

GLUT1 glucose transporter: a highly sensitive marker of malignancy in body cavity effusions.

Malignant cells exhibit increased rates of glycolysis and glucose uptake, and several types of cancer have been reported to overexpress the GLUT1 glucose transporter. The diagnosis of malignancy in body cavity effusions remains a dilemma in certain cases, despite recent progress in diagnostic immunocytochemistry. We used immunostaining to detect the facilitative glucose transporter, GLUT1, in cytologic preparations of body cavity effusions and washes. With the use of standard avidin-biotin immunostaining for GLUT1, we examined cell blocks of body cavity effusions or washings from 31 carcinomas, 1 lymphoma, and 25 benign effusions or washes. GLUT1 staining occurred in the malignant cell population in 29 (93.5%) of 31 carcinomatous effusions or washes. The characteristic staining pattern consisted of dense, linear staining of the plasma membrane, with accentuation at cell-cell borders, with or without cytoplasmic staining. Erythrocytes showed positive GLUT1 membrane staining, consistent with previous reports. Of 25 benign effusions, 20 were nonstaining (excepting erythrocytes), and 5 contained rare single mesothelial cells, with equivocal to very weak membrane staining. Staining of these cells was readily distinguishable from the characteristic strong staining of malignant cells, and these cells were easily distinguished from tumor cells by their benign morphologic characteristics. At least three of these latter five specimens were from patients with cirrhosis. In all of the other cases, mesothelial cells, histiocytes, and other inflammatory cells did not stain. These findings suggest that GLUT1 immunostaining could be useful in diagnostic cytopathology. The findings also suggest that enhanced glycolysis, which requires increased glucose transport, might be a survival adaptation for tumor cells in effusions, a significant number of which are hypoxic.

GLUT1 glucose transporter expression in benign and malignant thyroid nodules.

Malignant cells exhibit increased rates of glycolysis and glucose uptake, the latter of which is mediated by glucose transport proteins. Because several types of cancer have been shown to express high levels of the GLUT1 glucose transporter isoform, we hypothesized that expression of GLUT1 might distinguish malignant from benign thyroid tissue. Archival thyroid tissue obtained at surgery was immunostained for GLUT1 protein. There were 38 benign cases (24 follicular adenoma, 1 Hürthle cell adenoma, 8 nodular goiter, 3 Hashimoto's thyroiditis, 2 Graves' disease) and 28 cases of thyroid cancer (17 papillary and its follicular variant, 6 follicular, 1 Hurthle cell, 2 anaplastic, 2 medullary). Normal thyroid tissue adjacent to nodules showed no thyrocyte staining in any case. No GLUT1 staining was seen in thyrocytes in benign nodular tissue, except for a single case of Hashimoto's thyroiditis in which a few Hurthle cells showed weak staining. Among the thyroid cancers, 13 of 28 (46%) showed tumor cell GLUT1 staining in at least some areas. This included 9 of 17 cases of papillary carcinoma and its follicular variant, 2 of 6 cases of follicular carcinoma and 2 of 2 cases of anaplastic carcinoma. Tumor cell GLUT1 staining was seen in two patterns: circumferential plasma membrane staining focally within the tumor, or asymmetric staining of the basilar aspect of tumor cells adjacent to stroma in some cases of papillary carcinoma. We conclude that GLUT1 expression is frequently detectable by immunostaining in thyroid cancer, but not in benign nodules or normal thyroid. GLUT1 expression may be a clinically useful molecular marker for thyroid cancer.

Reproducibility and accuracy of interactive segmentation procedures for image analysis in cytology.

The segmentation of nuclear images is a crucial step in the development of procedures using image analysis for the cytological diagnosis of cancer. The purpose of this study is to evaluate the reproducibility and accuracy of several interactive segmentation methods which can be used in this context. Four methods were studied: a thresholding-based method enabling selection of intensity histogram contrast and brightness, manual tracing with a stylus, and arc- and ellipse-fitting routines. Features of nuclear size and shape were derived from nuclei segmented on repeated occasions by several individuals. Variance component models provided a statistical framework for evaluating the intraobserver and interobserver variability of these measurements in terms of their intraclass correlation coefficients. Of the methods tested, the arc-fitting segmentation method gave the most reproducible results, and thresholding the least. Reproducibility was generally very high both between individuals and for repeated segmentations by a single individual. Accuracies of area measurements for the various methods, as determined with respect to point counting, paralleled the reproducibilities of the methods. Sample size requirements were observed to be more dependent on the biological variability of the tissue sampled than on the particular segmentation method or on the number of individuals performing segmentation.

Value of GCDFP-15 (BRST-2) as a specific immunocytochemical marker for breast carcinoma in cytologic specimens.

OBJECTIVE: The diagnosis of metastatic mammary carcinoma by morphologic criteria alone can be difficult, depending on the site of metastasis and state of cell differentiation. Numerous histopathologic studies have shown GCDFP-15 (BRST-2) to be a specific marker for breast cancer in surgical specimens. To date, no studies have been done to evaluate its utility in cytologic preparations. STUDY DESIGN: To evaluate the usefulness of GCDFP-15 as a marker in the cytologic diagnosis of breast carcinoma, we studied 23 cases of mammary carcinoma and compared them with 20 cases of tumors of nonmammary origin (lung, ovary, liver, colon, stomach and bladder). "Bench top" fine needle aspirates from unfixed surgical specimens of breast carcinoma, cytocentrifuge samples from body cavity fluids and cerebrospinal fluids with morphologically proven metastatic carcinoma were studied. RESULTS: Expression of BRST-2 was found in 56.5% of primary and recurrent or metastatic breast carcinomas. All the nonmammary carcinomas studied were negative. Staining was found to be strongly dependent on the means of cell fixation. Slides fixed in 10% formalin and Bouin's solution gave optimal results. Except in two cases, which showed focal immunostaining, all specimens fixed in alcohol were negative. CONCLUSION: Our results support the diagnostic value of GCDFP-15 in recognizing tumors of breast origin and suggest that in clinical situations in which metastatic breast carcinoma is suspected, a portion of the cytologic specimen should be fixed with an optimal fixative for BRST-2 detection.

Cytology of extrapulmonary Pneumocystis carinii infection in the acquired immunodeficiency syndrome.

Rare cases of extrapulmonary Pneumocystis carinii (EPPC) have been seen in patients with acquired immunodeficiency syndrome (AIDS). We report seven such diagnoses of nonpulmonary P carinii (PC) from four AIDS patients between 1986 and 1989. The specimens included fine needle aspirate of liver, spleen, periarticular tissue and pleura as well as ankle fluid, pleural fluid and ascites. In some, but not all, cases the patients had concurrent or previous episodes of PC pneumonia. In all cases the typical granular, eosinophilic aggregates of PC cysts were noted on routine Papanicolaou staining, leading to the definitive detection of PC cysts with Grocott silver stain. In most cases, evidence for granulomalike and neovascularized tissue reaction was present in cytologic material. One specimen demonstrated concurrent acid fast bacilli. In the setting of AIDS, cytology of effusions and masses should include an evaluation for EPPC.

Immunocytochemical detection of cyclin, a proliferation-associated protein, in cytologic preparations.

Cyclin is a nuclear protein associated with DNA-polymerase delta, whose expression correlates with cell proliferation in vitro. To assess the value of cyclin staining in diagnostic cytology, an anticyclin monoclonal antibody was used to survey cyclin expression in cytologic preparations obtained as "bench top" aspirates from surgically resected specimens. The tissues assayed included carcinomas and normal and benign proliferative tissues of renal, mammary, prostatic and colonic origin. Staining was performed via the avidin-biotin-complex immunoperoxidase method. The staining of tumor cells was nuclear, with sparing of the nucleoli; the results were variable in different areas of a given tumor and varied significantly between tumors of the same histopathologic type. Benign proliferative tissues also showed staining. Nonproliferative tissues, such as renal tubules adjacent to a renal cell adenocarcinoma, were largely, but not entirely, nonstaining. The percentage of cyclin-positive nuclei was sometimes much higher than the typical percentages of tumor cells found in S phase. This observation was confirmed in two cases in which cyclin staining was much greater than the percentage of S-phase cells detected by flow cytometry. This suggests either stabilization of the protein beyond S phase in cells and/or dysregulation of cyclin expression in malignant cells. The viability of unfixed surgically resected tissue may also have affected the detection of cyclin, a problem that should not exist with clinically aspirated tissue fixed immediately after aspiration. These preliminary observations suggest that the selective use of cyclin staining may facilitate cytologic diagnoses. Furthermore, the wide range of cyclin expression within tumors of one histologic type suggests that cyclin expression may serve as a new parameter for investigating tumor behavior and prognosis.

Fine needle aspiration biopsy diagnosis of tuberculous lymphadenitis in patients with and without the acquired immune deficiency syndrome.

All Bellevue Hospital cases from a recent 27-month period whose fine needle aspiration (FNA) samples of cervical or supraclavicular masses showed acute and/or granulomatous inflammation were reviewed. The 30 patients included 8 with the acquired immune deficiency syndrome (AIDS), 3 with the AIDS-related complex (ARC), 2 with AIDS risk factors and 17 without known risk factors for AIDS. Of these, mycobacterial infections had been diagnosed in 22 patients: 18 by cultures positive for Mycobacterium tuberculosis and 4 by positive staining for acid-fast bacilli. In addition to the presence of neutrophils, two criteria for the diagnosis of mycobacterial infection were identified on the routinely stained FNA smears: caseous material and granulomas. Caseous material was the most sensitive and specific criterion. Granulomas were often present in patients with mycobacterial infection, but were also occasionally present in patients with other processes. The differences in cytologic specimens between AIDS and non-AIDS patients are discussed. The findings suggest that FNA is a safe and sensitive technique for the diagnosis of mycobacterial lymphadenitis in AIDS patients and that purulent aspirates from appropriate patient populations should prompt the use of special stains and cultures to rule out mycobacterial infection.

Fine needle aspiration biopsy of cystic benign lymphoepithelial lesion of the parotid gland in patients at risk for the acquired immune deficiency syndrome.

Cystic benign lymphoepithelial lesion (BLL), a previously rare lesion of the parotid gland consisting of marked lymphoid hyperplasia with accompanying squamous-lined cysts, has recently been described in patients with the acquired immune deficiency syndrome (AIDS) or AIDS risk factors. Thirteen fine needle aspiration (FNA) samples of parotid gland masses from patients with AIDS (one case), AIDS risk factors (five cases) or denial of AIDS risk factors (two cases) and a histopathologic diagnosis of BLL were examined. The FNA features that correlated best with the histopathologic findings were (1) a heterogeneous lymphoid population, (2) scattered single and/or clustered foamy macrophages and (3) superficial and/or anucleated squamous cells. Most aspirates showed some combination of these three components. The differential diagnostic considerations, the clinical and radiologic correlations and the relationship of this lesion to HIV infection are discussed. Patients with parotid masses whose aspirates consist of some combination of squamous cells, lymphocytes and foamy macrophages should be questioned for possible AIDS risk factors.

Immunohistochemical localization of basic fibroblast growth factor in astrocytomas.

Because of the prominent neovascularization observed in the growth of brain tumors, we studied the occurrence of basic fibroblast growth factor (bFGF), a potent angiogenic factor in astrocytomas, the most aggressive of which often have marked vascular hyperplasia. Using immunohistochemical methods, we examined 21 examples of such tumors, 7 glioblastomas multiforme, 7 anaplastic astrocytomas, and 7 low grade astrocytomas. Using polyclonal and affinity-purified rabbit antisera to human bFGF, we detected immunoreactive bFGF in all cases of glioblastoma multiforme. bFGF was present in both endothelial cells and neoplastic astrocytes. In 4 of 7 anaplastic astrocytomas, the tumor astrocytes had bFGF immunoreactivity and, in 5 of 7 cases, endothelial cells were also immunopositive. In glioblastomas multiforme and anaplastic astrocytomas, capillaries adjacent to tumor showed bFGF immunoreactivity, whereas capillaries distant from the tumors were not immunostained. In low grade astrocytomas, astrocytic cells were weakly immunoreactive in 2 of 7 cases, and in only 1 of the 7 cases capillaries were immunostained. In each grade, reactive astroglial cells showed variable bFGF immunoreactivity. The immunostaining was not seen with the flow-through fraction obtained after affinity purification of the bFGF antiserum with pure recombinant bFGF. These results suggest a possible role for bFGF in tumor growth and in angiogenesis in astrocytomas.

Regulation of protein kinase C activity in neuronal differentiation induced by the N-ras oncogene in PC-12 cells.

Expression of the N-ras oncogene under the control of the glucocorticoid-responsive promoter in the pheochromocytoma cell line UR61, a subline of PC-12 cells, has been used to investigate the differentiation process to neuronal cells triggered by ras oncogenes (I. Guerrero, A. Pellicer, and D. E. Burstein, Biochem. Biophys. Res. Commun. 150:1185-1192, 1988). Using ras-inducible cell lines, we observed that expression of the oncogenic N-ras p21 protein interferes with the ability of phorbol esters to induce downregulation of protein kinase C. This effect was associated with the appearance of immunologically detectable protein kinase C as well as the activity of the enzyme as analyzed either by binding of [3H]phorbol-12,13-dibutyrate in intact cells or by in vitro kinase activity. These results indicate a relationship between ras p21 and protein kinase C in neuronal differentiation in this model system. Comparison to the murine fibroblast system suggests that this relationship may be functional.

Potentiation of oncogenic N-ras-induced neurite outgrowth and ornithine decarboxylase activity by phorbol dibutyrate and protein kinase inhibitor H-8.

A recombinant N-ras oncogene, under the transcriptional control of a corticosteroid-inducible mouse mammary tumor virus (MMTV) promoter, has been stably transfected into a PC12 rat pheochromocytoma subline. This cell line, designated UR61, undergoes N-ras-induced neurite outgrowth and cessation of division when treated with dexamethasone (Guerrero et al.: Biochemical and Biophysical Research Communications 150:1185-1192, 1988). We have employed the UR61 cell line as a model for ras oncogene-induced neuronal differentiation. In UR61 cells, dexamethasone-induced expression of the recombinant N-ras gene resulted in time-dependent expression of ornithine decarboxylase enzyme (ODC) activity. Prompted by recent reports of possible functional (Lacal et al.: Molecular and Cellular Biology 7:4146-4149, 1987; Wolfman and Macara: Nature 325: 359-361, 1987) and direct (Jeng et al.: Biochemical and Biophysical Research Communications 145:782-788, 1987) interactions between oncogene ras-coded p21 and protein kinase C (PK-C; Ca++/phospholipid-dependent protein kinase), we employed the protein kinase inhibitor H-8 (N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride) and phorbol 12,13-dibutyrate (PDBu) to investigate this putative interaction in the UR61 cells, where ODC activity and neurite outgrowth were used as indicators of oncogenic N-ras action. Treatment of UR61 cells with PDBu depleted cells of PK-C and failed to promote neurite outgrowth but enhanced N-ras-induced neurite outgrowth and ODC activity. H-8, which suppressed ODC induction by forskolin and phorbol myristate acetate, enhanced both N-ras-induced ODC activity and neurite outgrowth. Inhibition of ODC activity by difluoromethylornithine (DFMO) did not suppress oncogenic ras-induced neurite outgrowth, suggesting that these two ras-triggered events are mechanistically independent. These findings suggest that certain actions of N-ras can occur in cells depleted of PK-C, and thus, the role of PK-C in ras-induced differentiation differs from its role in ras-induced mitogenesis and transformation.