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Chronic hepatitis B is a widespread viral illness with the serious sequelae of cirrhosis and hepatocellular carcinoma. Current therapy with interferon is not universally efficacious, and this has led to the evaluation of other antiviral agents. A recent Phase II trial of the nucleoside analogue, fluoroiodoarabinofuranosyluracil (fialuridine, FIAU) was halted because of the sudden development of severe multisystem toxicity characterized by hepatic failure, lactic acidosis, and pancreatitis, which resulted in the deaths of five patients. We systematically evaluated pre- and post-therapy biopsy, explant, and autopsy specimens from the 15 patients involved in this trial to define the hepatic changes of fialuridine toxicity and to determine whether the degree of pre-existing hepatitis contributed to the severity of toxicity. Severe hepatotoxicity from fialuridine was characterized by hepatomegaly with diffuse, predominantly microvesicular steatosis, hepatocellular glycogen depletion, marked bile ductular proliferation, and cholestasis. Ultrastructural examination revealed intracytoplasmic lipid droplets and marked mitochondrial injury. Patients in whom severe toxicity did not develop mainly showed changes caused by the underlying chronic hepatitis B alone. There was a subtle increase in the amount of microvesicular steatosis in two of six patients with mild or no symptoms of toxicity. The microscopic and ultrastructural pattern of injury and systemic symptoms in patients with fialuridine toxicity are consistent with severe mitochondrial and metabolic derangements. Similar hepatic pathologic findings have been reported rarely for other antiviral nucleoside analogues, which suggests that the mechanisms of toxicity might be related.

The deregulation of cyclin D1 (BCL-1, PRAD1, CCND1) protein, normally synthesized in the G1 phase of the cell cycle, has been implicated in the pathogenesis of some malignant neoplasms, including invasive mammary carcinomas. We used rabbit polyclonal antibody 19 to detect cyclin D1 in 55 infiltrating ductal carcinomas and compared the findings to six important clinicopathologic parameters and cyclin D1 gene amplification. Nuclear immunoreactivity of variable intensity for cyclin D1 was present in 35% of the neoplasms, whereas immunoreactivity of normal mammary epithelial nuclei was absent. No significant correlations were observed between immunoreactivity and patient age, axillary lymph node status, estrogen receptors, progesterone receptors, histologic grade, or any of its three components. There was a correlation between cyclin D1 immunostaining and tumor size (P = 0.013). Fourteen of 15 tumors 2 cm or less were negative, whereas 7 of 12 neoplasms larger than 4 cm were immunopositive. Fifteen percent of the invasive carcinomas had cyclin D1 gene amplification. Of these eight tumors, six showed cyclin D1 immunoreactivity (P = 0.017). In this study, cyclin D1 was detected immunohistochemically in approximately one-third of infiltrating ductal carcinomas; approximately one-third of these had detectable cyclin D1 gene amplification. These results further implicate cyclin D1 in breast tumorigenesis and are additional evidence for the role of cell cycle regulatory proteins in invasive mammary carcinoma.

Amplification of chromosome 11q13 leads to overexpression of a G1 cyclin gene, cyclin D1 (PRAD-1, CCND-1), in many non-cervical human carcinomas. Homology between cyclin D1 and human papillomavirus oncoprotein E7 binding sites for the retinoblastoma tumor-suppressor protein suggests that human papillomavirus oncoproteins cyclin D1, and other cell cycle regulatory proteins may act through a common mechanism in the pathogenesis of human cervical squamous cell carcinoma. We have examined 48 cases of cervical neoplasia by RNA-mRNA in situ hybridization for human papillomavirus mRNA and cyclin D1 mRNA and by immunohistochemistry for cyclin D1 protein expression. Hybridization demonstrated human papillomavirus RNA in all 48 cases with types 6, 16, or 18 in 2, 26, and 20 cases, respectively. Immunohistochemical detection using anti-cyclin D1 rabbit polyclonal antibody 19 demonstrated appropriate cyclin D1 expression at constitutive low levels in normal squamous epithelium and low-grade intraepithelial lesions. Immunohistochemical staining failed to demonstrate significant protein expression in any of the high-grade or invasive lesions. In contrast to the immunohistochemical results, in situ hybridization demonstrated cyclin D1 mRNA overexpression in three of five cases of low-grade squamous intraepithelial lesion, one of eight cases of high-grade squamous intraepithelial lesion, 14 of 18 cases of invasive squamous cell carcinoma, two of five cases of adenocarcinoma in situ, one of seven cases of invasive adenocarcinoma, and two of five cases of small cell undifferentiated carcinoma. Transcriptional activation of cyclin D1 can occur in vivo in human papillomavirus-associated invasive cervical carcinoma, but it does not seem to result in a steady state, increased level of cyclin D1 protein expression. These data indicate a limited role for cyclin D1 protein in the pathogenesis of human papillomavirus-associated invasive cervical squamous carcinoma. They support a model in which human papillomavirus proteins can circumvent cellular requirements for cyclin D1 in human cervical neoplasia.

Granulosa cell tumors with bizarre nuclei (GCT-BN) are rare lesions with a prognosis apparently similar to that of conventional granulosa cell tumors (GCT-NOS). The immunohistochemical features of GCT-BN have not been described, and the exact nature of the bizarre nuclei (BN) is unclear. Thirteen GCT-BN were studied with antibodies to cytokeratin, vimentin, epithelial membrane antigen, muscle-specific actin, alpha smooth muscle actin, desmin, and S-100 protein. Six cases were also examined by fluorescence in situ hybridization for trisomy 12, a nonrandom chromosomal aberration found in a proportion of ovarian sex-cord stromal tumors. Histologically, 12 tumors (86%) contained BN areas interspersed with large areas of GCT-NOS. The remaining tumor contained only microscopic foci of GCT-NOS. Immunohistochemically, the tumors stained for vimentin (13 tumors), S-100 protein (11 tumors), muscle-specific actin (10 tumors), cytokeratin (eight tumors), alpha smooth muscle actin (eight tumors), and desmin (one tumor), but none stained for epithelial membrane antigen. Immunostaining results for the BN and GCT-NOS areas were concordant in eight (73%) of the 11 tumors in which both areas could be independently assessed. The remaining three tumors (27%) showed discordant results for only one of the eight markers used. In five patients, trisomy 12 was detected by fluorescence in situ hybridization in areas of BN but not in areas of GCT-NOS present in the same tumor. Trisomy 12 was also present in another BN tumor in which the foci of GCT-NOS were too small to be evaluated. We conclude that within GCT-BN, areas with BN are immunohistochemically similar to areas of GCT-NOS present in the same tumor. The finding of trisomy 12 in areas with BN but not GCT-NOS in the same tumor, however, suggests that cells with BN represent a genetically distinct clone of tumor cells arising within GCT-NOS.

Glutathione S-transferase pi, a drug-detoxifying isoenzyme of potential prognostic value for subsets of patients with mammary cancer, was studied by immunohistochemistry and Southern blot analysis in 58 infiltrating ductal carcinomas. The results were compared with the findings of six important clinicopathologic parameters. Cytoplasmic immunoreactivity for glutathione S-transferase pi was absent in 40%, 1+ in 26%, 2+ in 15%, and 3+ in 19% of the cases. There were no significant correlations between the level of immunostaining and patient age, tumor size, axillary lymph node status, nuclear pleomorphism, or tubule formation, but there was a trend with mitotic count (P = 0.06). Immunoreactivity was associated with histologic grade (P = 0.02) and inversely correlated with estrogen (P = 0.006) and progesterone (P = 0.02) receptor content. Ten percent of the cases showed modest levels of glutathione S-transferase pi gene amplification, but no significant correlations were observed between glutathione S-transferase pi amplification and any of the clinicopathologic parameters or level of immunostaining. The results indicate that increased immunohistochemical staining for glutathione S-transferase pi occurs in high-grade, estrogen and progesterone receptor-negative neoplasms. As glutathione S-transferase pi gene amplification appears unassociated with immunopositivity, other mechanisms are responsible for the production of this isoenzyme in infiltrating ductal carcinomas.

To assess the interobserver reproducibility for the diagnosis of medullary carcinoma of the breast (MC), 53 previously diagnosed MCs were independently assessed by six observers for growth pattern, nuclear grade (NG), inflammation, tumor margin, intraductal component, and glandular features. Tumors were reclassified as MC, atypical MC, or infiltrating ductal carcinoma according to the histopathologic criteria of Ridolfi et al. (Cancer 40:1365, 1977), Wargotz and Silverberg (Hum Pathol 19:1340, 1988), and Pedersen et al. (Br J Cancer 63:591, 1991). NG was the most reproducible parameter, and tumor margin was the least, with consensus agreement by four of six observers for 49 (92%) and 26 (49%) of cases, respectively. Utilizing the histopathologic criteria proposed by Ridolfi et al., Wargotz and Silverberg, and Pedersen et al., consensus diagnoses were achieved in 37 cases (70%), 46 cases (87%), and 51 cases (96%), respectively. A consensus diagnosis of MC in all three systems was unassociated with tumor size, axillary lymph node status or overall survival (median follow-up: 89 mo). The consensus (or better) reclassification of 44/53 (83%), 35/53 (66%), and 27/53 (51%) previously diagnosed MC as atypical MC or infiltrating ductal carcinoma by the criteria of Ridolfi et al., Wargotz and Silverberg, and Pedersen et al., respectively, suggests that MC was previously over-diagnosed. While the scheme of Pedersen et al. is the most reproducible, additional follow-up information is necessary to determine the biological significance of this classification system. To minimize these difficulties in practice, pathologists should carefully adhere to published criteria and indicate the classification system utilized.

A B-cell anaplastic large cell lymphoma, confirmed by immunohistochemistry and Southern blot immunoglobulin gene rearrangement analysis, contained neoplastic cells that were immunoreactive for cytokeratin using antibodies CAM 5.2, M20, MAK 6, and KS-B17.2. Bands corresponding to cytokeratin 18 and cytokeratins 18 and 8 were seen on Western blot immunoanalysis using antibodies KS-B17.2 and CAM 5.2. The lymph node also contained cytokeratin-positive extrafollicular fibroblastic reticulum cells. Although it is possible that the presence of cytokeratin in the cells of anaplastic large cell lymphoma represented phagocytosed filaments from the reticulum cells, it is more likely that the cytokeratins were synthesized by the malignant cells. The finding of cytokeratin in anaplastic large cell lymphoma, although infrequent, adds to the confusion in the diagnosis of this pleomorphic neoplasm.

To determine the frequency and clinicopathologic correlates of chromosome 11q13 amplification in breast carcinoma, DNA from 50 invasive tumors was analyzed by Southern blot hybridization using probes for the 11q13 loci bcl-1, PRAD1, hst1, EMS1, and GST-pi, as well as oncogenes c-erbB-2 and c-myc. Sixteen tumors (32%) were amplified for one or more loci. Seven tumors (14%) showed amplification of 11q13 loci; six of these were coamplified with either c-erbB-2 (three), c-myc (two), or both (one). Nine additional tumors (18%) were amplified for c-erbB-2, and three of these were coamplified with c-myc. None showed c-myc amplification alone. Tumors with 11q13 amplification showed equivalent degrees of bcl-1, PRAD1, hst1, and EMS1 amplification, delineating an approximately 800-kb amplicon. GST-pi was inconsistently amplified, evidence that it lies outside the amplicon defined by bcl-1 and EMS1. Amplification of 11q13 was unassociated with patient age, tumor size, axillary lymph node status, hormone receptors, DNA content, histologic grade, mitotic rate, nuclear pleomorphism, or tubule formation. c-myc amplification correlated with the lack of tubule formation (p = 0.04), whereas c-erbB-2 amplification correlated with axillary lymph node positivity (p = 0.04), high histologic grade (p = 0.003), increased nuclear pleomorphism (p = 0.008), and a high mitotic rate (p = 0.02). The frequency of coamplification of 11q13 loci, c-myc, and c-erbB-2 contradicts previous proposals that amplification of these genes occurs in independent subsets of breast carcinoma. Extended clinical followup will be necessary to determine whether 11q13 amplification has prognostic utility in invasive breast cancer.

Fifty-two infiltrating breast carcinomas with medullary features (BCMF) were studied immunohistochemically to determine the immunophenotype of the mononuclear tumor inflammatory cells (MTIC) in formalin-fixed, paraffin-embedded material. The neoplasms were also examined for Epstein-Barr virus (EBV) DNA by the polymerase chain reaction (PCR). BCMF were independently classified as medullary carcinoma (MC) or infiltrating ductal carcinoma (IDC) by six observers according to the criteria of Pedersen et al. DNA from 35 BCMF was successfully amplified using PCR, but all were negative for EBV DNA. These included, by 4/6 consensus diagnosis, 16 MC, 18 IDC, and one BCMF which failed to achieve consensus diagnosis. MTIC were present to a mild degree in 19 BCMF (37%) and to moderate to severe degrees in 33 (63%). MTIC were predominantly (> or = 75%) lymphocytic in 31 BCMF (13 MC, 16 IDC, two without consensus diagnostic agreement), and plasmacytic in 10 (six MC, four IDC); equal proportions of lymphocytes and plasma cells occurred in 11 (six MC, five IDC). Lymphocytic MTIC were mostly CD45RO+/CD3+ T-cells in nearly all cases, and showed a predominant CD3+/CD4+ and CD3+/CD4- immunophenotype in 36% and 64% of cases, respectively. Natural killer cells (CD57+) and histiocytes (MAC 387+) were virtually absent. The number, cell type, and T-cell subsets of the MTIC were unrelated to consensus diagnosis, axillary lymph node status, or overall survival. EBV is unassociated with MC, despite the histologic similarities of MC to EBV-associated lymphoepithelial lesions of other organs.(ABSTRACT TRUNCATED AT 250 WORDS)

The monoclonal antibody mES 13 was previously produced against bacterially expressed BALB ras p21 and was reported to have both membrane and cytoplasmic reactivity in formalin-fixed, paraffin-embedded tissue sections. In the current study, the cytoplasmic reactivity of mES 13 is investigated and demonstrated to be mitochondrial. Immunoelectron microscopic studies showed specific labeling of mitochondria without labeling of other organelles. In normal tissues, the antibody strongly labeled tissues known to have large amounts of mitochondria such as renal tubules, hepatocytes, and myocardium. The pattern of reactivity of tumors generally mimicked that of normal tissues, with carcinomas and melanomas usually showing stronger staining than sarcomas and lymphomas. Two granular cell tumors were negative. Among renal neoplasms, mES 13 strongly labeled renal oncocytomas and granular cell renal cell carcinomas and showed weaker staining of clear cell and chromophobe cell tumors. The mES 13 antibody should be useful in the characterization and diagnosis of tumors in which oncocytoma is in the differential diagnosis, especially when only paraffin-embedded tissue is available for study.