Image cytometric bcl-2:bax and bcl-2:bcl-x ratios in invasive breast carcinoma: correlation with prognosis.

BACKGROUND: The bcl-2 family of proteins are important regulators of apoptosis. Some of the members, such as bcl-2 and bcl-x(L), inhibit cell death, whereas others, such as bax and bcl-x(S), promote cell death. We evaluated the ratios of bcl-2:bax and bcl-2:bcl-x expression by image cytometry in invasive breast carcinoma to determine prognostic significance. DESIGN: Five-micron sections of formalin-fixed, paraffin-embedded tissue from 88 invasive breast carcinomas were immunostained using steam antigen retrieval, an avidin biotin-complex technique with automated stainer and primary antibodies against bcl-2 (1/160; Dako, Carpenteria, CA), bax (1/1,500; PharMingen, San Diego, CA), and bcl-x (1/1,500; PharMingen). Positive controls were tonsil (bcl-2) and normal breast (bax and bcl-x) tissue samples. Immunostain was measured in 15 high power fields as percentage positive area (PPA) in nuclei and cytoplasm using the CAS 200 image analyzer (Becton Dickinson, San Jose, CA). RESULTS: Median follow-up was 105 months (range 11-130). Significantly improved disease-free survival was found in patients with a bcl-2:bcl-x ratio > or = 1 by univariate and multivariate analyses. The bcl-2:bax ratio was not predictive of overall or disease-free survival. A significant difference in overall and disease-free survival was found between carcinomas with positive and negative bcl-2 expression by univariate analysis; by multivariate analysis, bcl-2 expression was an independent prognostic factor for disease-free survival. The 5-year survival rates were 77% and 50% in patients with bcl-2-positive and bcl-2-negative carcinomas, respectively. CONCLUSION: A bcl-2:bcl-x ratio > or = 1, assessed by image cytometry, is significantly associated with improved disease-free survival in patients with invasive breast carcinoma. Significantly increased overall and disease-free survival is associated with positive bcl-2 expression.

Flow and image cytometry in thymic neoplasia: correlation with clinical outcome.

OBJECTIVE: To compare nuclear DNA by flow (FCM) and image cytometry (ICM) in thymic neoplasms and to relate results to clinical outcome. STUDY DESIGN: DNA ploidy of 44 thymomas and 6 thymic carcinomas was studied by FCM and ICM of single nuclear suspensions from paraffin blocks. RESULTS: By FCM, 33 thymomas (75%) and one thymic carcinoma (17%) were diploid; 6 thymomas (14%) and 4 thymic carcinomas (67%) were aneuploid. By ICM, 36 thymomas (82%) were diploid; 7 thymomas (16%) and 6 thymic carcinomas (100%) were aneuploid. Mean follow-up in 44 cases was 46.2 months (range, 1-162). Ten patients with persistent/recurrent disease included four with thymic carcinoma, who died of the disease (two aneuploid by both techniques, two aneuploid by ICM with unsatisfactory/diploid FCM). Four had invasive thymoma and recurrence after 13-150 months (two diploid and two aneuploid by both methods), one had diploidy and noninvasive thymoma that recurred at 92 months, and one had an epithelial thymoma that recurred at 144 months (aneuploid by FCM, diploid by ICM). CONCLUSION: The results obtained in this preliminary, retrospective study show a high concordance between FCM and ICM; aneuploidy correlated with poor outcome by both methodologies. While these findings are encouraging, larger numbers of cases will be needed to define the role of FCM and ICM in predicting outcome in thymic tumors.

Bcl-2: bax and bcl-2: Bcl-x ratios by image cytometric quantitation of immunohistochemical expression in ovarian carcinoma: correlation with prognosis.

Bcl-2 is a proto-oncogene which is involved in prolonging cell survival by inhibiting programmed cell death. Bax and bcl-x are members of the bcl-2 family; when overexpressed, they can counteract the ability of bcl-2 to inhibit apoptosis. This suggests a model in which the ratios of bcl-2 to bax and bcl-x can be used to determine response to therapy and prognosis. The expression of bcl-2, bax and bcl-x was studied in 50 ovarian carcinomas. The percentage of positive area immunostained (PPA) in the nucleus and cytoplasm of each ovarian carcinoma was quantitated in 15 high power fields by image cytometry. The ratios were obtained by dividing the PPA of bcl-2 by the PPA of bax and bcl-x. 17 of 50 ovarian carcinomas (34%) stained positively for bcl-2, 39 for bax (78%) and 47 for bcl-x (94%). Although there is no significant statistical correlation between expression of bcl-2, bax or bcl-x and grade (P = 0.15; P = 0. 47; P = 0.56), stage (P = 0.71; P = 0.6; P = 0.42), and overall or disease-free survival (P = 0.26; P = 0.55; P = 0.16), increased bcl-2 expression was demonstrated in patients with shortened overall and disease-free survival. Also, increased expression of bax and bcl-x was associated with increased overall and disease-free survival. Bcl-2:bax and bcl-2:bcl-x ratios less than 1 are associated with survival advantage, although not statistically significant (P = 0.83; P = 0.93). Image cytometric measurement of bcl-2, bax, and bcl-x expression is feasible. There is a tendency for their expression to correlate with prognosis in ovarian carcinomas.

Sex and androgenic steroid receptor expression in hepatic adenomas.

Sex hormones and anabolic-androgenic steroids are implicated in the development and progression of hepatic adenomas (HA). We studied the expression of their receptors in HA and adjacent liver. Archival tissue sections of 27 HA (16 resections, four needle biopsies, seven aspirations) from 18 patients, and the adjacent liver, were immunostained with monoclonal antibody to estrogen receptor (ER, 1/80) (Dako, Carpinteria, CA), progesterone receptor (PR, 1/50) (BioGenex, San Ramon, CA), and androgen receptor (AR, 1/80) (BioGenex). An avidin-biotin complex technique was used with microwave antigen retrieval. Nuclear expression was assessed as 1+ to 3+ intensity, with semiquantitation of the percentage of nuclei immunopositive. Five percent or more nuclei immunopositive was regarded as positive. The 18 patients included 16 females of 34 years mean age (range, 16 to 49) with an available history of oral contraceptives in five; the two men were 24 and 30 years, with no history of androgenic steroids. ER, PR, and AR were present in seven (26%) (1+/-2+ intensity, 5% to 10% of nuclei) of HA, seven (26%) (1+/-2+ intensity, 5% to 30% of nuclei) and nine (33%) (1+/-3+ intensity, 5% to 80% of nuclei), respectively. In the adjacent liver in 11 cases, there were one (9%) ER, (2+ intensity, 5% of nuclei), four (36%) PR (1+/-2+ intensity, 5% to 20% of nuclei), and two (18%) AR (2+/-3+ intensity, 10% of nuclei). Receptors are present and may mediate the action of sex hormones or androgenic steroids on HA and adjacent liver, but in less than one third of patients. This may have therapeutic implications.

Image cytometric measurement of nuclear proliferation markers (MIB-1, PCNA) in astrocytomas. Prognostic significance.

Proliferative activity in astrocytomas, measured by image cytometry, was related to prognosis. Fifty-eight astrocytic neoplasms (grade 1 or 2, 11; grade 3, 31; glioblastoma multiforme, 16) were immunostained for Ki-67 (MIB-1; 1:50) and proliferating cell nuclear antigen (PCNA; prediluted). Proliferative activity (nuclear immunostain) was measured as the percentage positive nuclear area by image cytometry. With a median of 12.0% and 24.0% for MIB-1 and PCNA, respectively, for all astrocytomas, the mean percentage positive nuclear area for MIB-1 and PCNA was, respectively, 3.06% and 13.11% in low-grade (1 or 2) astrocytomas, 14.34% and 29.68% in high-grade (3) astrocytomas, and 18.77% and 44.11% in glioblastoma multiforme (grade 4). One-way analysis of variance showed a significant correlation between the histologic grade and MIB-1 and between the histologic grade and PCNA. The Cox Proportional Hazards Regression Model showed a statistically significant correlation between survival and MIB-1 and between survival and PCNA. Increasing proliferation correlated with shortened survival. Proliferation in astrocytomas, measured as MIB-1 and PCNA by image cytometry, correlates significantly with histologic grade and patient survival, providing useful additional information for determining the diagnosis and prognosis.

Prognostic indicators in male breast carcinoma.

Twenty-nine male breast cancers (MBC) were studied to determine the relationship between expression of several prognostic factors and clinical outcome. Immunohistochemistry employing a labeled streptavidin-biotin method was used to detect the presence of estrogen (ER) and progesterone receptors (PR), cathepsin D (CD), c-erbB-2 oncoprotein, epidermal growth factor receptor (EGFR), and p53; results were visually semiquantitated. DNA ploidy was evaluated by image analysis (CAS 200) of 5 µm fixed embedded Feulgenstained tissue sections. For proliferating cell nuclear antigen (PCNA), nuclear immunostain was quantitated as percentage positive nuclear area (PPNA) by image cytometry (CAS 200). The frequency of expression was ER, 26/29 (89.7%); PR, 19/29 (65.5%); CD, 25/29 (86.2%); c-erbB-2, 5/29 (17.2%); EGFR, 4/29 (13.8%); and p53, 9/29 (31%). Twenty-one (72.4%) were aneuploid; the mean PPNA for PCNA was 37.87% (control 13%). Of 20 patients, 10 (50%) MBC had lymph node metastases; 6 (21%) had distant metastases to lung (1) and bone (5). Five of the patients died of MBC. Excluding the patients with only ductal carcinoma in situ, the 1-and 5-year survival rates were 90.5% and 56.3%, respectively. In this comprehensive study of a large number of available prognostic markers, their frequency (with the exception of higher ER and CD) and prognostic significance were similar to that in female breast carcinoma. Among clinical and standard pathologic unfavorable prognostic indicators, age ≥ 62 years was significant (p = .004). Trends toward reduced survival were associated with axillary lymph node metastases (p = .145), ER negativity (p = .058), PR negativity (p = .116), and aneuploid DNA content (p = .201).

Tumor angiogenesis as a prognostic factor in laryngeal cancer.

BACKGROUND: Lymph node metastasis is the single greatest predictor of recurrence in laryngeal cancer. Prognostic factors are needed to target patients who may benefit from adjuvant therapy. Tumor angiogenesis correlates with metastasis in breast, bladder, and oral cavity cancer and may have prognostic value in other tumors. METHODS: In order to examine the relationship of tumor angiogenesis to recurrence, 51 patients with squamous cell carcinoma of the larynx were reviewed. In a blinded design, previously sectioned slides were chosen for advanced tumor and highest vessel concentration. Samples were cut and immunocytochemically stained for CD-31 (an endothelial marker). A computer image analyzer quantitated the percent area of staining. Variables were statistically examined against recurrence. RESULTS: Patients were stratified by percent tumor staining. Nodal involvement was seen in 9 (36%) patients with tumor staining < or = 20% and in 20 (77%) with tumor staining > 20% (P = 0.003). Patients with < or = 20% staining and without metastasis had a 13% rate of recurrence whereas patients with > 20% staining and without metastasis had a 67% rate of recurrence (P = 0.025). CONCLUSIONS: Though nodal status was suggestive of predictability, only angiogenesis is a statistically significant predictor of recurrence in node negative patients (P = 0.025). Angiogenesis shows strong correlation with regional recurrence and may be used as an independent prognostic indicator to determine clinically node negative patients who may be at higher risk for metastasis and require adjuvant therapy.

Image cytometric nuclear DNA quantitation of paragangliomas in tissue sections. Prognostic significance.

OBJECTIVE: Image cytometric quantitation of nuclear DNA of paragangliomas may provide prognostic information that cannot be obtained from histopathologic study. Flow cytometry has demonstrated DNA aneuploid tumors to have a higher risk of progression than diploid neoplasms. STUDY DESIGN: DNA ploidy of 56 paragangliomas was assessed by image cytometry of 5-micron, Feulgen-stained, formalin-fixed, paraffin-embedded tissue sections. RESULTS: Thirty-three (59%) paragangliomas were diploid and 23 (41%) aneuploid. Of the 30 adrenal pheochromocytomas, 15 (50%) were diploid. Thirteen (93%) of the 14 carotid body tumors were diploid. Five of seven (71%) glomus jugulare tumors and two of five (40%) extraadrenal paragangliomas were aneuploid. During a mean follow-up of 57 months (range, 1 month to 36 years) of 44 patients with 47 paragangliomas, 33 (75%) were alive and without disease; 7 (16%), including 1 glomus jugulare, 2 carotid body and 4 pheochromocytoma patients, developed recurrences/metastases. By multivariate analysis, image cytometric DNA ploidy was predictive of disease-free survival for adrenal pheochromocytomas. No significant differences in overall survival, disease-free survival or recurrence/metastasis rate were noted between other diploid and aneuploid tumors. CONCLUSION: Aneuploidy suggests a risk of early recurrence for adrenal pheochromocytomas.

Image cytometric comparison of proliferating cell nuclear antigen and MIB-1 staining in hepatocellular carcinoma and adjacent liver tissue.

Studies of cellular proliferation may aid in elucidation of mechanisms of hepatocarcinogenesis and provide potential prognostic information in cases of hepatocellular carcinoma. Increased proliferation has been reported in the liver tissue adjacent to hepatocellular carcinoma and may be a risk factor for recurrence after tumor resection. We quantitated proliferating cell nuclear antigen (PCNA) clone PC10 and MIB-1 nuclear immunostain by image cytometry in fixed, paraffin-embedded tissue sections of normal, cirrhotic, and dysplastic livers with and without co-existent hepatocellular carcinoma and in the hepatocellular carcinoma. Fifteen high power fields were analyzed in each case using the CAS 200 image analyzer and results were expressed as the percent positive nuclear area. Cirrhotic liver showed significantly less proliferation adjacent to hepatocellular carcinoma than without hepatocellular carcinoma. There was a similar, but not significant, tendency for dysplastic liver. Increasing mean proliferation was noted from normal to cirrhotic to dysplastic liver to low grade to high grade hepatocellular carcinoma. These findings were similar using both PC10 and MIB-1. There tended to be slightly more staining with PC10 than with MIB-1, but the mean percent positive nuclear area was significantly greater with PC10 only in high grade hepatocellular carcinoma. Comparison of individual cases, however, yielded significant correlation between the two antibodies in the low grade and high grade hepatocellular carcinomas, but not in the benign livers. These findings demonstrate that image cytometric quantitation of PC10 and MIB-1 immunostain may be comparable measures of cellular proliferation and that there is no increase in proliferation in the hepatic tissue adjacent to hepatocellular carcinoma.

DNA index and ploidy distinguish normal human parathyroids from parathyroid adenomas and primary hyperplastic parathyroids.

BACKGROUND: The goal of this study was to identify factors that might aid in diagnosis and intraoperative management of hyperparathyroidism. METHODS: We analyzed biopsy specimens of 242 parathyroids from 159 patients by use of flow cytometry and image cytometry (ICM) for DNA index (DI), defined as the content of nuclear DNA compared with that expected for a DNA diploid standard, for proliferative index (PI), and for ploidy (diploid versus aneuploid or tetraploid). RESULTS: True normal and normal parathyroids from patients with solitary adenomas were uniformly diploid. Abnormal ploidy (aneuploidy or tetraploidy) was identified frequently in adenomas and occasionally in hyperplasias with the exception that multiple endocrine neoplasia (MEN) biopsy specimens were uniformly diploid. DI for adenomas was similar to that for hyperplasias, and DI of both was higher than for normal glands. ICM-DI correlated positively with flow cytometry-DI and patient age and inversely with serum parathyroid hormone. PI was relatively low in all groups but was higher for hyperplasias versus normal parathyroids from patients with solitary adenomas and MEN versus non-MEN. PI correlated inversely with patient age. CONCLUSIONS: DI by ICM differentiates normal from abnormal parathyroids. DI might influence extent of resection in two- and three-gland hyperplasia and selection of the most appropriate gland for autografting and cryopreservation in patients with four-gland hyperplasia.

Histopathology, morphometry, and nuclear DNA content of iris melanocytic lesions.

PURPOSE: To compare the histologic, morphometric and nuclear DNA content of a group of benign and malignant melanocytic lesions of the iris. METHODS: Forty-four surgically excised melanocytic lesions of the iris were histologically classified as nevus or melanoma. Morphometric analysis using a digital filar micrometer (LaSICO 1602N-10 and 5-4A) measured the mean size of the 10 largest nucleoli, and Feulgen staining and image cytometry (CAS 200 Cell Analysis Systems) analyzed the nuclear DNA ploidy in the lesions. Patient follow-up information was obtained whenever possible. RESULTS: Sixteen cases were histologically classified as nevi and twenty-eight cases as melanoma. The mean of the 10 largest nucleoli of the nevi was smaller than the mean among the melanomas (1.772 microns [SD = 0.366] and 2.773 microns [SD = 0.565], respectively). Feulgen staining revealed that all lesions were diploid, with the exception of two hyperdiploid and two hypodiploid melanomas. Of the patients with follow-up information available, none with nevi developed a metastasis and two with melanoma died of metastatic disease. CONCLUSIONS: The histologic classification of iris melanocytic lesions (i.e., nevus versus melanoma) correlates to nucleolar size (P < 0.001) but not to nuclear DNA ploidy.

DNA image cytometric analysis of paraffin-embedded sections of small renal cortical neoplasms.

DNA ploidy analysis of neoplasms is most frequently performed by flow cytometry of single nuclear suspensions or image analysis of Feulgen-stained cytologic specimens. To assess the prognostic significance of ploidy in renal adenomas (less than 3 cm in diameter), nuclear DNA content of 21 renal cortical neoplasms was analyzed by image cytometry (CAS 200, Cell Analysis Systems, Inc., Elmhurst, IL) on Feulgen-stained 5 microns fixed embedded tissue sections. Five (36%) of 14 small neoplasms (so-called renal adenomas; (mean diameter, 7.2 mm; range, 2-24 mm) were in the aneuploid range. Four (57%) of seven carcinomas (mean diameter, 6.3 cm; range, 3.5-10 cm) were aneuploid, a frequency not significantly different from that in the adenomas (P = 0.4). Mean DNA index (1.38) for aneuploid adenomas and carcinomas was identical. Diploid renal adenomas (mean diameter, 5.9 mm) were smaller, although not significantly so (P > 0.05), than the aneuploid ones (mean, 9.7 mm). Size of diploid (mean, 6.7 cm) and aneuploid (mean, 6.1 cm) renal carcinomas was similar. Clinical follow-up studies to evaluate utility of DNA ploidy and proliferative activity in prognosis and management of small renal cortical neoplasms are indicated.

Tumor angiogenesis as a prognostic factor in oral cavity tumors.

BACKGROUND: Lymph-node metastasis is the single greatest predictor of survival in patients with oral cavity cancers. Tumor angiogenesis has been correlated with metastasis in breast cancer and may have prognostic value in other tumors. PATIENTS AND METHODS: Sixty-six patients with clinically node-negative oral cavity squamous cell cancers were reviewed. Samples were cut and stained for factor VIII. The percentage of area of tissue stained for factor VIII was quantitated by a computerized image analyzer. Tumor depth was measured with an ocular micrometer to the nearest 0.1 mm. Variables were statistically examined against regional recurrence. RESULTS: The probability of metastasis (%) was 2 for tumor staining of < or = 10% and 93 for tumor staining > 10% (P < 0.0001). The tumor depth was < or = 4 mm in 10 and > 4 mm in 83 (P < 0.0001). Patients with < or = 4 mm and < or = 10% staining had a 2% rate of recurrence, and patients with > 4 mm and > 10% staining had a 100% rate of recurrence (P < 0.0001). CONCLUSION: Although tumor thickness was suggestive of predictability, only angiogenesis was a statistically significant predictor of recurrence in a multivariate analysis (P < 0.0001). Angiogenesis showed a strong correlation with regional recurrence and may be used as an independent prognostic indicator.

c-erbB-2 oncoprotein expression and amplification in pleomorphic adenoma and carcinoma ex pleomorphic adenoma: relationship to prognosis.

The c-erbB-2 protooncogene has been shown to be overexpressed and/or amplified in carcinomas of the breast, ovary, pancreas, and other organs. Several studies of human breast carcinoma noted an association of c-erbB-2 overexpression and amplification with poor prognosis. Recent studies have demonstrated c-erbB-2 protein expression in a variety of salivary gland neoplasms, most notably pleomorphic adenoma (PA), carcinoma ex pleomorphic adenoma (CAexPA), and adenocarcinoma. In this study, we analyzed c-erbB-2 expression in 15 PAs and 13 CAexPAs, using immunohistochemistry. In addition, differential polymerase chain reaction was used to evaluate c-erbB-2 gene amplification in these tumors. Low-level c-erbB-2 immunoreactivity was detected in three of 15 PAs. Among the CAexPAs, low-, intermediate-, and high-level immunoreactivity was seen in two, three, and two cases, respectively. The only cases showing c-erbB-2 amplification were the two CAexPA cases with high-level immunoreactivity. Based on statistical analysis of the 10 CAexPA patients with known outcome, no significant association of prognosis with c-erbB-2 expression or amplification was apparent.

Comparative image cytometric DNA ploidy of liver cell dysplasia and hepatocellular carcinoma.

Large-cell liver cell dysplasia (LCD), suggested to be a preneoplastic change that progresses to hepatocellular carcinoma (HCC), has a reported frequency of DNA aneuploidy by flow cytometry intermediate between that of nonneoplastic liver (0%) and HCC (80%). We assessed DNA ploidy by image cytometry of Feulgen-stained 5-microns sections of 30 livers with LCD and of 60 HCCs (29 with LCD in adjacent nonmalignant liver). All 30 LCDs were aneuploid, 27 (90%) of which were multiploid--11 (41%) with hyperdiploid and hypertetraploid peaks. Forty-eight (80%) HCCs were aneuploid; in nine of 20 (42%) with a hyperdiploid peak, a hyperdiploid peak was also present in the LCDs, but in none was there less than 0.24 between DNA indices. Besides the 12 (20%) diploid HCCs, a diploid peak was present in four heterogenous, three multiploid, and six HCCs with two phenotypes and two genotypes, one of which was diploid. One aneuploid/hyperdiploid peak in each of 22 nonneoplastic and 24 cirrhotic livers did not have a corresponding LCD or HCC aneuploid peak. These data do not suggest that dysplastic hepatocytes form a single mutant clone that progresses to HCC.

Immunohistochemical p53 in hepatocellular carcinoma and liver cell dysplasia.

Mutant tumor-suppressor gene p53 is reported in over 50% of hepatocellular carcinomas (HCC). We studied 60 HCC, 30 with large cell liver cell dysplasia (LCD), suggested to be a preneoplastic change progressing to HCC, in the adjacent non-neoplastic liver. Immunohistochemistry was performed for the presence of mutant p53 and hepatitis B surface (HBs) and core (HBc) antigens, using a labeled streptavidin-biotin technique with monoclonal (1/20) and polyclonal (1/40) anti-p53 and with anti-HBs (prediluted) and anti-HBc (1/400). Twenty-nine (48%) HCC were p53 immunopositive, with both antibodies in 9, 17 with monoclonal p53 only, and 3 with polyclonal p53 only. p53 immunoreactivity was present in 3 of 19 (16%) non-neoplastic livers, 4 of 20 (20%) cirrhotic livers, and one of 30 (3%) LCD. HBs and HBc, respectively, were present in 0% and 5% non-neoplastic livers, 20% and 10% cirrhotic livers, 7% and 10% LCD, and 3% and 5% HCC. None of the p53-positive HCC had HBV markers in adjacent liver. This frequency (48%) of p53 in HCC is similar to that in other countries. The data suggest a role for p53 mutations in hepatocarcinogenesis, even in the absence of HBV infection, apparently not progressing through LCD but occurring as a late event.

Evaluation of c-erbB-2 amplification in breast carcinoma by differential polymerase chain reaction.

Previous studies have found that amplification and overexpression of the c-erbB-2 oncogene in mammary ductal adenocarcinomas predicts decreased disease-free or overall survival. Information regarding the prognostic and pathogenetic significance of oncogene amplification has been limited by difficulty obtaining sufficient quantities of high molecular weight DNA for Southern blot analysis. Differential polymerase chain reaction (PCR) has been suggested as an alternative method for evaluating gene amplification and can be performed using formalin-fixed paraffin-embedded specimens. The authors of this study used differential PCR to detect c-erbB-2 gene amplification and immunohistochemistry to evaluate c-erbB-2 expression. A highly significant degree of concordance (P < .002) between c-erbB-2 amplification and expression was observed. Abnormalities of c-erbB-2 copy number or expression were more common in tumors with higher histologic grade, and trends were noted toward association with other prognostically unfavorable biologic markers, such as reduced progesterone receptor content and DNA aneuploidy.

Liver cell dysplasia in alpha-1-antitrypsin deficiency.

Large cell liver cell dysplasia (LCD), a suggested preneoplastic change progressing to hepatocellular carcinoma, has been reported associated with alpha-1-antitrypsin deficiency which in some countries has an increased frequency of hepatocellular carcinoma. We examined the nonneoplastic liver from 13 alpha-1-antitrypsin deficiency patients for LCD and, using a labeled streptavidin-biotin technique, for immunohistochemical markers: AAT (1/200), hepatitis B surface (HBsAg, prediluted) and core (HBcAg, 1/400) antigens, and monoclonal (1/20) and polyclonal (1/40) mutant p53, a tumor suppressor gene. There were eight males and five females ranging from 2 mo to 76 yr (mean 40 yr). Nine livers showed cirrhosis, one chronic persistent hepatitis, one portal fibrosis, and two cholestatic hepatitis (in the two infants). The nine cases with LCD included five males and four females of mean age 46 yr (range, 17-71), eight with cirrhosis and one with portal fibrosis. Only one liver with LCD and cirrhosis had HBcAg in cirrhotic and dysplastic cells. No patient had developed hepatocellular carcinoma. All 13 livers were immunonegative for HBsAg and mutant p53, and immunopositive for AAT present in normal, cirrhotic, and dysplastic liver cells. Thus, LCD was identified in 82% of adult alpha-1-antitrypsin deficiency livers (69% including infantile patients), 89% with cirrhosis, and none with malignancy. HB expression was rarely present; serology for HB and/or hepatitis C was positive in 46% adults. Immunoreactive AAT was present in dysplastic cells. p53 gene mutations do not appear to have a role in the pathogenesis of LCD in alpha-1-antitrypsin deficiency.

Immunohistochemical phenotype of ovarian granulosa cell tumors: absence of epithelial membrane antigen has diagnostic value.

Granulosa cell tumors (GCTs) represent 1.5% to 3% of primary and 6% to 10% of malignant ovarian neoplasms, and present little diagnostic difficulty in the typical case; however, other primary or metastatic tumors may mimic their various histologic patterns. For this reason, immunohistochemistry can be used to supplement routine histology to help determine a final tissue diagnosis. Previous reports on the utility of antibodies to intermediate filaments vary, as some investigators found keratin to be uniformly negative in GCTs while others reported immunoreactivity for keratin in 20% to 68% of cases. To determine the immunophenotype of granulosa cell tumors and to discover which antibodies are useful in differentiating GCTs from histologic look-alikes, we studied 52 GCTs, including 24 typical cases, 23 cases in which the diffuse pattern predominated, and five juvenile cases, with a panel of commercially available antibodies using an automated immunohistochemistry system. Immunoreactivity for granulosa cells in GCTs was as follows: 17 cases (32.7%) reacted with cytokeratin AE1/AE3, six cases (11.5%) reacted with cytokeratin MAK-6, three cases (5.8%) reacted with cytokeratin CAM 5.2, no case (0%) reacted with epithelial membrane antigen, 52 cases (100%) reacted with vimentin, no case (0%) reacted with desmin, 48 cases (92.3%) reacted with smooth muscle actin, and 26 cases (50%) reacted with S-100 protein. No attempt was made to quantify staining of background thecoma-like or fibroma-like elements in GCTs. Immunoreactivity was independent of the histologic subtype of GCT. Cytokeratin immunoreactivity showed a globoid pattern of staining and was consistent with the expression of 52.5-kD and 45-kD cytokeratins (8 and 18 of Moll's classification). For this reason, the presence of cytokeratin immunoreactivity by itself cannot be used to differentiate a primary or metastatic carcinoma from a GCT. The presence of smooth muscle actin and the absence of epithelial membrane antigen immunoreactivity are additional features that are characteristic of a GCT. S-100 protein immunoreactivity is a finding limited exclusively to GCTs among sex cord stromal tumors, and its presence may have some role in differentiating between Sertoli-stromal cell tumors and GCTs. Since epithelial membrane antigen immunoreactivity is present in many of the histologic look-alikes of GCTs, such as metastatic or primary carcinoma, the absence of staining in GCT has diagnostic value.

DNA ploidy of ameloblastoma and ameloblastic carcinoma of the jaws. Analysis by image and flow cytometry.

Ameloblastomas, the second most common odontogenic tumor, behave as benign locally aggressive tumors. Ameloblastic carcinomas, on the other hand, show histologic features of malignancy and may metastasize. By image cytometric analysis of Feulgen-stained sections from decalcified, formalin-fixed, paraffin-embedded tumors, we compared nuclear DNA content of 17 primary and five recurrent ameloblastomas and five ameloblastic carcinomas. Of the primary ameloblastomas, 14 (82%) were diploid; three (60%) of the five recurrent ameloblastomas were diploid. No significant difference in ploidy between primary and recurrent ameloblastomas or among plexiform, follicular, and acanthomatous types of ameloblastoma was demonstrated. Of the five ameloblastic carcinomas, four (80%) were aneuploid; ploidy was not significantly correlated with the incidence of metastasis. Flow cytometry was performed on three carcinomas; 100% concordance between image and flow cytometric data was seen. Aneuploidy is significantly more common in ameloblastic carcinomas than in ameloblastoma and is a strong predictor for malignant potential.