Alfredo Blanes

PCR Techniques for Clonality Assays

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.

Kinetic Profiles by Topographic Compartments in Muscle- Invasive Transitional Cell Carcinomas of the Bladder Role of TP53 and NF1 Genes

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.

Critical Analysis of Histologic Criteria for Grading Atypical (Dysplastic) Melanocytic Nevi

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.

Clonality as Expression of Distinctive Cell Kinetics Patterns in Nodular Hyperplasias and Adenomas of the Adrenal Cortex

Although histopathologic criteria for adrenal cortical nodular hyperplasias (ACNHs) and adenomas (ACAs) have been developed, their kinetics and clonality are virtually unknown. We studied 20 ACNHs and 25 ACAs (based on World Health Organization criteria) from 45 females. Representative samples were histologically evaluated, and the methylation pattern of the androgen receptor alleles was analyzed on microdissected samples. Consecutive sections were selected for slide cytometry, flow cytometry, and in situ end labeling (ISEL). Apoptosis was studied by flow cytometry (nuclear area/DNA content plotter analysis) and by ISEL. Appropriate tissue controls were run in every case. Polyclonal gel patterns were revealed in 14/18 informative ACNHs and in 3/22 informative ACAs, whereas monoclonal gel patterns were observed in 4/18 ACNHs and 19/22 ACAs. Overlapping proliferation rates (PRs) were observed in both clonal groups, and apoptosis was detected only in G(0)/G(1) cells, especially in monoclonal ACNHs (3/4; 75%) and in polyclonal ACAs (2/3; 67%). Significantly higher PRs were observed in ACNHs with polyclonal patterns and G(0)/G(1) apoptosis and in ACAs regardless of clonality pattern and presence of G(0)/G(1) apoptosis. All except one ACNH (19/20; 95%) and 15/25 ACAs (60%) showed diploid DNA content, whereas the remaining cases were hyperdiploid. A direct correlation between PR and ISEL was observed in polyclonal lesions (PR = 29.32 ISEL - 1.93), whereas the correlation was inverse for monoclonal lesions (PR = -9.13 ISEL + 21.57). We concluded that only simultaneous down-regulated apoptosis and high proliferation result in selective kinetic advantage, dominant clone expansion, and unbalanced methylation patterns of androgen receptor alleles in ACNHs and ACAs.

Molecular evolution and intratumor heterogeneity by topographic compartments in muscle-invasive transitional cell carcinoma of the urinary bladder

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 p21WAF1 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.

Histologic Criteria for Adrenocortical Proliferative Lesions Value of Mitotic Figure Variability

This study compared 3 systems and a newly designed stepwise discriminant diagnostic system (SDDS) to assess accuracy, reproducibility, and reliability in adrenocortical nodular hyperplasia (ACNH; n = 82), adenoma (ACA; n = 78), and carcinoma (ACC; n = 32) (diagnoses according to World Health Organization criteria; median follow-up, 135 months). In cross-validations, we studied cortex appearance, growth pattern, cytoplasmic features, nuclear parameters, mitotic figure counting (MFC), necrosis, invasion, and stromal-inflammatory reactions. The SDDS independent predictors were MFC/high-power field SD, anisokaryosis, cortex appearance, and stromal reaction, correctly classifying 100% of ACNH, 91% of ACA, and 88% of ACC cases. The Hough system correctly classified 78% of ACNH, 81% of ACA, and 84% of ACC cases; the Weiss and van Slooten systems correctly classified 100% of ACNH, 0% of ACA, and 92% of ACC cases. MFC variability is the most important adrenocortical malignancy criterion. Accurate malignancy diagnosis requires multiple variable evaluations, provided by SDDS (the most specific system) and the Weiss or van Slooten system (the most sensitive). SDDS is the most useful system for distinguishing tumors from ACNH (myxoid stroma and anisokaryosis).

Merkel Cell (Neuroendocrine) Carcinoma of the Vulva: A Case Report with Immunohistochemical and Ultrastructural Findings and Review of the Literature

A new case of primary Merkel cell carcinoma (MCC) of the vulva is reported and the literature reviewed for noting its clinical presentation, microscopic, immunohistochemical and ultrastructural features, as well as for establishing the role of immunohistochemistry in the ultimate diagnosis of this uncommon and aggressive tumor. The lesion occurred in a 79 year old patient. Histologically, the tumor was composed of intradermal small cells with high mitotic index and frequent apoptosis. The immunohistochemical study showed positivity for wide spectrum and low molecular weight cytokeratins, epithelial membrane antigen, neurofilaments, neuron specific enolase and chromogranin A. Electron microscopy revealed intermediate filaments in a typical globular paranuclear arrangement. The coexpression of cytokeratins (including cytokeratin 20) and neurofilaments, both in typical globular paranuclear arrangement, made possible the diagnosis of MCC, differentiating it from other malignant small cell tumors such as neuroendocrine metastatic carcinoma.

Clonal patterns in phaeochromocytomas and MEN-2A adrenal medullary hyperplasias: histological and kinetic correlates {

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. Copyright

Differential kinetic features by tumour topography in cutaneous small-cell neuroendocrine (Merkel cell) carcinomas

Background/Objectives  Merkel cell carcinomas (MCC) reveal epithelial and neuroendocrine differentiation, but its topographic cell kinetics remains unknown. This study analyses proliferation, apoptosis, and DNA ploidy by topography, features that can help planning therapeutic protocols. This study topographically analyses proliferation, apoptosis, and DNA ploidy.

Heterogeneous topographic profiles of kinetic and cell cycle regulator microsatellites in atypical (dysplastic) melanocytic nevi.

Atypical (dysplastic) melanocytic nevi are clinically heterogeneous malignant melanoma precursors, for which no topographic analysis of cell kinetic, cell cycle regulators and microsatellite profile is available. We selected low-grade atypical melanocytic nevi (92), high-grade atypical melanocytic nevi (41), melanocytic nevi (18 junctional, 25 compound) and malignant melanomas (16 radial growth phase and 27 vertical growth phase). TP53, CDKN2A, CDKN1A, and CDKN1B microsatellite patterns were topographically studied after microdissection; Ki-67, TP53, CDKN2A, CDKN1A, and CDKN1B expressions and DNA fragmentation by in situ end labeling for apoptosis were topographically scored. Results were statistically analyzed. A decreasing junctional–dermal marker expression gradient was observed, directly correlating with atypical melanocytic nevus grading. High-grade atypical melanocytic nevi revealed coexistent TP53–CDKN2A–CDKN1B microsatellite abnormalities, and significantly higher junctional Ki67–TP53 expression (inversely correlated with CDKN1A–CDKN1B expression and in situ end labeling). Malignant melanomas showed coexistent microsatellite abnormalities (CDKN2A–CDKN1B), no topographic gradient, and significantly decreased expression. Melanocytic nevi and low-grade atypical melanocytic nevi revealed sporadic junctional CDKN2A microsatellite abnormalities and no significant topographic kinetic differences. High-grade atypical melanocytic nevi accumulate junctional TP53–CDKN1A–CDKN1B microsatellite abnormalities, being progression TP53-independent and better assessed in the dermis. Melanocytic nevi and low-grade atypical melanocytic nevi show low incidence of microsatellite abnormalities, and kinetic features that make progression unlikely.