SUMMARY:Superficial transitional cell carcinomas (TCC) of the urinary bladder have been shown to be monoclonal. However, no combined study of clonality and tumor suppressor genes (TSG) is available to date for muscle-invasive TCC. Forty-four muscle-invasive TCC of the urinary bladder selected from women were included in this study. Tumor cells located above and below the muscularis mucosa zone were systematically microdissected and used for DNA extraction. Hha-I digested and undigested samples were used to study the methylation pattern of androgen receptor alleles and undigested samples were used for microsatellite analysis of TSG (TP53, RB1, WT1, and NF1). Both loss of heterozygosity (LOH) and single nucleotide polymorphism (SNP) analyses were performed using optimized denaturing gradient gel electrophoresis. The expression of p53, pRB, and p21 WAF1 was assessed by immunohistochemistry. Appropriate controls were run in every case. All except two TCC showed a monoclonal pattern with the same allele inactivated in both compartments. Microsatellite analysis of TSG revealed the same LOH/SNP pattern in both tumor compartments in 30 cases (involving more than 1 TSG locus in 8) and genetic heterogeneity in 14 cases. From the latter group, 9 cases expressed more genetic changes in the deep compartment (involving TP53 gene in all cases, WT1 gene in 2, and NF1 in 1), whereas in 4 cases the superficial compartment showed more genetic changes (three involving NF1 and one involving both RB and TP53). No statistical difference in the immunoexpression was detected, although it tended to be higher in the superficial compartment than in the deep compartment. These concordant data in polymorphic DNA regions indicate that bladder-muscle-invasive TCC are monoclonal proliferations with homogeneous tumor cell selection. Heterogeneous tumor cell selection by topography defined two different genetic compartments: superficial, NF1-defective, and deep, TP53-defective. No differences in the immunohistochemical expression were observed, precluding a more extensive clinical application. (Lab Invest 2000, 80:279-289).

Clonal overgrowths represent the hallmark of neoplastic proliferations, and their demonstration has been proved useful clinically for the diagnosis of malignant lymphomas based on the detection of specific and dominant immunoglobulin and/or T-cell receptor gene rearrangements. Nonrandom genetic alterations can also be used to test clonal expansions and the clonal evolution of neoplasms, especially analyzing hypervariable deoxyribonucleic acid (DNA) regions from patients heterozygous for a given marker. These tests rely basically on the demonstration of loss of heterozygosity (LOH) resulting from either hemizygosity (nonrandom interstitial DNA deletions) or homozygosity of mutant alleles observed in neoplasms. LOH analyses identify clonal expansions of a tumor cell population, and point to monoclonal proliferation when multiple and consistent LOH are demonstrated. Based on the methylation-related inactivation of one X chromosome in female subjects, X-linked markers (e.g., androgen receptor gene) will provide clonality information using LOH analyses after DNA digestion with methylation-sensitive restriction endonucleases. Therefore, both non-X-linked and X-linked analyses give complementary information, related and not related to the malignant transformation pathway respectively. Applied appropriately, these tools can establish the clonal evolution of tumor cell populations (tumor heterogeneity), identify early relapses, distinguish recurrent tumors from other metachronic neoplasms, and differentiate field transformation from metastatic tumor growths in synchronic and histologically identical neoplasms.

Neoplasms result from the progressive and convergent selection of cell populations, but several factors should be considered. On one hand, selection will determine tumor progression and cellular heterogeneity. On the other hand, cellular selection must be related to cell kinetics process.

Neoplastic transformation is a multistep process that results in a continuous spectrum from the normal (physiological) state to a fully established neoplasm. The gold standard for diagnosis of papillary thyroid carcinoma is conventional histology, the essential element being the characteristic nuclear features, regardless of whether papillary structures are present or not. However, other criteria are being used increasingly in the diagnosis of neoplasms, including immunohistochemical staining and molecular profile. The RET/PTC gene rearrangement is highly specific for papillary thyroid carcinoma and is associated with the characteristic nuclear features seen in papillary thyroid carcinoma. There is an overlap in the morphological features, immunohistochemical staining pattern, and most importantly, molecular profile between papillary thyroid carcinoma and Hashimoto's thyroiditis. Although considered a 'benign' condition, Hashimoto's thyroiditis almost always harbours a genetic rearrangement that is strongly associated with and is highly specific for papillary thyroid carcinoma. Submicroscopic foci of papillary thyroid carcinoma must be present in Hashimoto's thyroiditis, although the clinical behaviour is still benign. Further studies are required to predict which foci will progress to papillary thyroid carcinoma.

We evaluated 71 muscle-invasive transitional cell carcinomas (TCCs) of the bladder by tumor compartments. Kinetic parameters included mitotic figure counting, Ki-67 index, proliferation rate (DNA slide cytometry), and apoptotic index (in situ end labeling [ISEL] of fragmented DNA using digoxigenin-labeled deoxyuridine triphosphate and Escherichia coli DNA polymerase [Klenow fragment]). At least 50 high-power fields per compartment were screened from the same tumor areas; results are expressed as percentage of positive neoplastic cells. Mean and SD were compared by tumor compartment. DNA was extracted from microdissected samples (superficial and deep) and used for microsatellite analysis of TP53 and NF1 by polymerase chain reaction-denaturing gradient gel electrophoresis. Significantly higher marker scores were revealed in the superficial compartment than in the deep compartment. An ISEL index of less than 1% was revealed in 63% (45/71) of superficial compartments and 86% (61/71) of deep compartments. Isolated NF1 alterations were observed mainly in superficial compartments, whereas isolated TP53 abnormalities were present in deep compartments. Lower proliferation and down-regulation of apoptosis define kinetically the deep compartment of muscle-invasive TCC of the bladder and correlate with the topographic heterogeneity, NF1-defective in superficial compartments and TP53-defective in deep compartments.

Multiple microsatellite alterations and topographic intratumor heterogeneity characterize malignant pheochromocytomas, suggesting a multistep tumorigenesis through somatic topographic down-regulation of MMR proteins. Locally invasive pheochromocytomas reveal topographic heterogeneity and single-locus microsatellite alterations, especially involving NF1.

The relationship among histological features, cell kinetics, and clonality has not been studied in adrenal medullary hyperplasias (AMHs) and phaeochromocytomas (PCCs). Thirty-four PCCs (23 sporadic and 11 MEN-2A (multiple endocrine neoplasia type 2A)-related tumours, the latter associated with AMH) from females were included in this study. Representative samples were histologically evaluated and microdissected to extract DNA and evaluate the methylation pattern of the androgen receptor alleles. At least two tissue samples (from the peripheral and internal zones in each tumour) were analysed with appropriate tissue controls run in every case. The same areas were selected for MIB-1 staining and in situ end labelling (ISEL). Malignant PCCs were defined by histologically confirmed distant metastases. All monoclonal AMH nodules from the same patient showed the same X-chromosome inactivated. Six sporadic PCCs revealed liver metastases (malignant PCC) and eight additional sporadic PCCs showed periadrenal infiltration (locally invasive PCC). All informative PCCs were monoclonal, except for five locally invasive PCCs and one benign PCC that revealed polyclonal patterns. Those cases also showed a fibroblastic stromal reaction with prominent blood vessels, focal smooth muscle differentiation, and significantly higher MIB-1 (126.8+/-29.9) and ISEL (50.9+/-12.8) indices. Concordant X-chromosome inactivation in nodules from a given patient suggests that MEN-2A AMH is a multifocal monoclonal condition. A subgroup of PCCs characterized by balanced methylation of androgen receptor alleles, high cellular turnover, and stromal proliferation also shows locally invasive features.

Low concordance in grading atypical (dysplastic) melanocytic nevi (AMN) has been reported, and no systematic evaluation is available. We studied 123 AMN with architectural and cytologic atypia (40 associated with atypical-mole syndrome), classified according to standard criteria by 3 independent observers. Histologic variables included junctional and dermal symmetry, lateral extension, cohesion and migration of epidermal melanocytes, maturation, regression, nuclear features, nuclear grade, melanin, inflammatory infiltrate location, and fibroplasia. AMN (43 junctional and 80 compound) were graded mild (31), moderate (61), and severe (31). AMN-severe correlated with 3 or more nuclear abnormalities (especially pleomorphism, heterogeneous chromatin, and prominent nucleolus) and absence of regression, mixed junctional pattern, and suprabasilar melanocytes on top of lentiginous hyperplasia. AMN-severe diagnostic accuracy was 99.5% using these criteria, but only the absence of nuclear pleomorphism differentiated AMN-mild from AMN-moderate. No architectural features distinguishing AMN-mild from AMN-moderate were selected as significant by the discriminant analysis. AMN from atypical-mole syndrome revealed subtle architectural differences, but none were statistically significant in the discriminant analysis. Histologic criteria can reliably distinguish AMN-severe but fail to differentiate AMN-mild from AMN-moderate. AMN from atypical-mole syndrome cannot be diagnosed using pathologic criteria alone.