Primary malignant epithelial tumors of the liver in children: a study of DNA content and oncogene expression.

Primary malignant epithelial tumors of the liver (PMETL) are rare in the pediatric age group, and very little is known about their biology as compared with adult tumors. The prognostic value of the DNA contents measured by image analysis and expression of oncogene c-erb2 and tumor suppressor gene p53 were studied in 30 cases of PMETL in children, including 24 with hepatoblastomas (HB) and 6 with hepatocellular carcinomas (HCC). p53 overexpression was detected in 12 out of 26 cases (46.0%), or in 3 of 5 HCC and 9 of 21 HB cases. A relatively high concordance of staining was observed with the two antibodies used (clone DO7, Dako and clone DO1, Santa Cruz Biotechnology). c-erb-B2 did not yield the characteristic membrane staining in any of the 27 cases in which reliable staining was obtained. However, 1 out of 4 patients with HCC and 1 of 23 with HB revealed strong granular cytoplasmic staining in several neoplastic cells. Interestingly, these were two of the three aneuploid multiploid cases. DNA histograms of 13 out of 29 cases (54.8%) were classified as DNA aneuploid (5/6 HCC and 8/23 HB): nine were hyperdiploid, one was hypodiploid (1HB), and three were multiploid (2HB and 1HCC). In the HB group, DNA aneuploidy was strongly associated with embryonal histological areas, suggesting that a disturbance in the process of cell differentiation is associated with marked genetic aberrations. Only the group of HB was submitted to univariate analysis of survival by the Kaplan-Meier method for age (< 24 months vs. > or = 24 months), sex, preoperative chemotherapy (yes vs. no), residual disease (metastasis, and/or unresectable tumor), p53 expression by immunohistochemistry (positive vs. negative), and DNA ploidy (diploid vs. aneuploid). Only residual disease at the time of diagnosis (P < 0.017) and preoperative chemotherapy (0.030) were found to be negatively correlated with biological behavior, estimated as overall survival. DNA aneuploidy tumors (P < 0.125) and male patients (P = 0.123) showed a trend toward a more aggressive clinical behavior, although the difference was not statistically significant. Combining DNA ploidy and residual disease, patients were categorized into three groups: group I, patients with no adverse prognostic factors, i.e., diploid tumors without residual disease; group II, patients with only one adverse prognostic factor, i.e., aneuploid tumor or residual disease; and group III, patients with both adverse factors, aneuploid tumors and residual disease at time of diagnosis. A log-rank test comparing the three survival curves showed a statistically significant difference between them (P < 0.003). Although the series of cases is small, the results of this study highlight the importance of including DNA ploidy in the protocols designed for HB in children by international cooperative groups.

Evaluation of a new slide-based laser scanning cytometer for DNA analysis of tumors. Comparison with flow cytometry and image analysis.

DNA measurements generated by a new automated slide-based cytometer, the laser scanning cytometer (LSC), were compared with those produced by commercial flow cytometry (FCM) and image analysis (IA) devices. Laser scanning-cytometric analysis was performed by scanning alcohol-fixed, propidium iodide-stained tumor imprints with a 5-microns spot laser beam. Fifty-three malignant tumors (51 breast carcinomas and 2 lung carcinomas) were studied. Ploidy concordance rates for FCM versus LSC, IA versus LSC, and FCM versus IA were 96%, 91%, and 91%, respectively. Statistically significant agreement between methods was determined by linear regression analysis of DNA indices. Synthesis-phase fractions generated by FCM and LSC also were comparable, as demonstrated by linear regression (r = .83). Mean coefficients of variation for the LSC compared favorably with those for FCM and IA. The few discrepancies in ploidy status between methods could be explained by sampling error, the presence of possible near-diploid aneuploid populations that could not be effectively resolved by one or another modality, and the visual selection bias with IA when small aneuploid cell populations were present. The LSC shares many useful features with FCM, including automation, accuracy of quantitation, rapidity, and generation of reliable information regarding cell proliferation (synthesis-phase fraction). In addition, it has some of the advantages of IA, such as minimal tissue requirement, no need for special preparation, and the potential for visual selection of the cells measured. The LSC holds great promise for use in the clinical laboratory because of these combined characteristics.

DNA ploidy abnormalities in the liver of children with hereditary tyrosinemia type I. Correlation with histopathologic features.

Hereditary tyrosinemia (HT) is an autosomal recessive disorder of tyrosine metabolism that results in cirrhosis and hepatocellular carcinoma early in life, and that may be a useful model of early malignant transformation. This is the first report of DNA ploidy in this disease. The authors studied formalin-fixed liver tissue in three cases (two chronic and one acute) of HT for the presence of DNA aneuploidy by flow cytometric (FCM) analysis and image analysis (IA). In the chronic cases, the authors found DNA aneuploidy by FCM in 2/20 cirrhotic nodules in one case and 1/21 tissue sections in the other. Questionable minor aneuploid peaks were detected by FCM in 2/20 and 2/21 sections in these two cases, respectively. Static DNA measurements by IA were performed on those sections having abnormal histograms as well as in some sections having diploid DNA distribution. By this method, the authors confirmed the results of FCM studies and found additional small aneuploid nodules not detected by FCM, frequently associated with various forms of hepatocellular dysplasia as well as steatosis. In one case of acute HT (autopsy), no definite aneuploid peaks were present in five blocks. By immunohistochemical analysis, the authors found frequent positive staining for alpha-fetoprotein (AFP) in the cirrhotic nodules of the two chronic cases as well as in the steatotic regenerative nodules of the acute case. The expression of AFP may represent disturbed regeneration and maturation of liver cells in this disease. This study shows that DNA ploidy may be a useful marker for early malignant transformation in HT and supports the preneoplastic nature of the hepatocellular dysplasia in this disease.

Localization of nuclear matrix proteins (NMPs) in multiple tissue types with NM-200.4 (an antibody strongly reactive with NMPs found in breast carcinoma).

Nuclear matrix proteins (NMP) are nonhistone proteins found in the nucleus of many eukaryotic cells. Furthermore certain NMPs are reported to be cell-type specific and expressed differentially by malignant cells. To study the specificity of NM-200.4 (an antibody reactive to NMPs extracted from cultured breast carcinoma cells of the T-47D line), cancers and benign tissues from multiple body sites were surveyed. All 17 breast carcinomas showed strong reactivity to tumor cell nuclei. Also nuclei from one of two lung carcinomas, a papillary thyroid carcinoma, an ovarian fibroma, and a lymphoma were strongly reactive. One leiomyosarcoma and a dermoid cyst were negative. Although 1 benign breast with duct hyperplasia showed moderate reactivity, only 1 of 10 benign breast biopsies without hyperplasia showed reactivity. Three of 4 skin biopsies, 2 liver biopsies, 6 of 9 kidney biopsies, and 5 of 10 gastrointestinal mucosal biopsies showed reactivity in benign nuclei. It is concluded that, although breast carcinoma nuclei showed the most consistent reactivity for NM-200.4, both benign and malignant nuclei from other body sites also show reactivity.