General morphological and biological features of neoplasms: integration of molecular findings.

This review highlights the importance of morphology-molecular correlations for a proper implementation of new markers. It covers both general aspects of tumorigenesis (which are normally omitted in papers analysing molecular pathways) and the general mechanisms for the acquired capabilities of neoplasms. The mechanisms are also supported by appropriate diagrams for each acquired capability that include overlooked features such as mobilization of cellular resources and changes in chromatin, transcription and epigenetics; fully accepted oncogenes and tumour suppressor genes are highlighted, while the pathways are also presented as activating or inactivating with appropriate colour coding. Finally, the concepts and mechanisms presented enable us to understand the basic requirements for the appropriate implementation of molecular tests in clinical practice. In summary, the basic findings are presented to serve as a bridge to clinical applications. The current definition of neoplasm is descriptive and difficult to apply routinely. Biologically, neoplasms develop through acquisition of capabilities that involve tumour cell aspects and modified microenvironment interactions, resulting in unrestricted growth due to a stepwise accumulation of cooperative genetic alterations that affect key molecular pathways. The correlation of these molecular aspects with morphological changes is essential for better understanding of essential concepts as early neoplasms/precancerous lesions, progression/dedifferentiation, and intratumour heterogeneity. The acquired capabilities include self-maintained replication (cell cycle dysregulation), extended cell survival (cell cycle arrest, apoptosis dysregulation, and replicative lifespan), genetic instability (chromosomal and microsatellite), changes of chromatin, transcription and epigenetics, mobilization of cellular resources, and modified microenvironment interactions (tumour cells, stromal cells, extracellular, endothelium). The acquired capabilities defining neoplasms are the hallmarks of cancer, but they also comprise useful tools to improve diagnosis and prognosis, as well as potential therapeutic targets. The application of these concepts in oncological pathology leads to consideration of the molecular test requirements (Molecular Test Score System) for reliable implementation; these requirements should cover biological effects, molecular pathway, biological validation, and technical validation. Sensible application of molecular markers in tumour pathology always needs solid morphological support.

Trichilemmal carcinoma with neuroendocrine differentiation.

We report a 12-mm nodular, cream-coloured skin lesion that appeared on the left nasal ala in an 81-year-old man. This trabecular infiltrative tumour showed keratin microcysts, stromal hyalization, cytoarchitectural malignancy features, colonizing melanocytes, and immunoexpression of epithelial membrane antigen, cytokeratin 15/20, chromogranin, synaptophysin and CD56. To our knowledge, this is the first documented case of a trichilemmal carcinoma with neuroendocrine differentiation and melanocyte colonization, which is suggested by the trabecular growth pattern and requires immunohistochemical confirmation. The colonization of the epithelial nests by nonatypical dendritic or spindle melanocytes is a clue to morphological recognition of pilar neoplasms, along with the presence of stromal induction (CD34-positive peritumoral spindle cells), catagen-like apoptotic bodies, calcifications, keratin microcysts and cell balls.

Microsatellite abnormalities and somatic down-regulation of mismatch repair characterize nodular-trabecular muscle-invasive urothelial carcinoma of the bladder.

AIMS: To correlate histological infiltration patterns with genetic and mismatch repair (MMR) profiles in muscle-invasive bladder urothelial carcinomas (UroC). METHODS AND RESULTS: Infiltration patterns were assessed in the deep compartment of muscle-invasive UroC (nodular-trabecular, 45 cases; infiltrative, 27 cases). Tumour compartment (superficial and deep to muscularis mucosa) analysis included: microsatellite pattern of TP53, RB1, WT1 and NF1 by polymerase chain reaction/denaturing gradient gel electrophoresis; mitotic, Ki67, in situ end labelling (ISEL) indices and DNA ploidy. MMR was assessed by MLH1 and MSH2 sequencing and immunohistochemistry in UroC with two or more abnormal microsatellite loci. Statistical differences were tested using anova and Fisher's exact tests. Infiltrative UroC showed lower Ki67 index 14.94 +/- 4.28, ISEL index 14.1 +/- 10.0 and shorter median survival (20 months) than nodular-trabecular UroC (Ki67 index 20.65 +/- 4.94, ISEL 20.2 +/- 22.7, 37-month survival, respectively). The genetic profile was significantly different for RB1 (P = 0.0003) and NF1 (P = 0.0023) only, being more frequently abnormal in nodular-trabecular UroC. A significant decrease in MLH1 or MSH2 protein expression with no gene mutations was identified in UroC with microsatellite abnormalities and a nodular-trabecular growth pattern. CONCLUSIONS: Somatic down-regulation of MMR proteins in nodular-trabecular muscle-invasive UroC results in RB1/NF1 microsatellite abnormalities, correlating with higher cellular turnover and longer survival.

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. METHODS: We selected 27 small-cell MCCs (expressing one epithelial and two neural markers, with consistent ultrastructural findings) to evaluate mitotic figure counting, Ki-67 index, apoptosis index based on the in situ end labelling of fragmented DNA (using Escherichia coli DNA polymerase I, Klenow fragment), DNA ploidy, and BCL2 and TP53 immuno-expression. At least 50 high-power fields were screened per topographic compartment (superficial or papillary dermis, and deep or reticular dermis), recording average and standard deviation for each variable. Variables were statistically compared in each tumour compartment using analysis of variance and Student's t-test (significant if P < 0.05). RESULTS: MCCs revealed superficial aneuploid DNA content, and no topographic differences for proliferation markers. Apoptosis showed significantly lower values in the deep compartment (average, P = 0.0050, and standard deviation, P = 0.0074), correlating with increased BCL2 and TP53 immuno-expressions. CONCLUSIONS: High homogeneously distributed proliferation and superficial aneuploid DNA content defines MCCs. Apoptosis follows proliferation in superficial compartments, being less variable and proliferation independent in deep compartments, where it is inversely correlated with BCL2/TP53 expression.

Cytoarchitectural and kinetic features in the histological evaluation of follicular thyroid neoplasms.

AIMS: The diagnosis of follicular thyroid carcinomas is mainly based on capsular and vascular invasion. The aim of this study was to determine the diagnostic relevance of nuclear features, inflammation and stromal changes. METHODS AND RESULTS: Anisokaryosis, chromatin pattern, nucleolus, nuclear pleomorphism, nuclear/cytoplasmic ratio, necrosis, stromal changes and tumour interstitial lymphocytes (TIL) were analysed in adenomatous hyperplastic nodules (39), adenomas (43) and carcinomas (28 minimally invasive, 48 widely invasive and 27 anaplastic). Ki67 immunostaining, in situ end labelling (ISEL) for apoptosis and the Ki67/ISEL index were analysed by topographical compartments. Variables were compared by histological diagnosis using Fisher's exact test, analysis of variance and Student's t-tests and considered significant if P < 0.05. TIL were absent in 96% of neoplasms and 54% of adenomatous hyperplastic nodules. Conspicuous nucleoli, increased nuclear-cytoplasmic ratio and coexistent apoptosis-myxoid changes distinguished minimally invasive carcinomas from adenomas. The most specific variables of high-grade carcinoma were vasculonecrotic patterns, nuclear hyperchromatism and pleomorphism. A kinetic advantage predominated in the internal compartments of benign lesions and in the peripheral compartments of malignant lesions. CONCLUSIONS: Follicular carcinomas show up-regulation of proliferation markers and the distinctive topographical kinetic profiles provide a basis for the distinction between benign and malignant and an explanation for the circumscription and encapsulation of benign lesions.

Malignant melanoma in an 8-year-old Caribbean girl: diagnostic criteria and utility of sentinel lymph node biopsy.

The incidence of malignant melanoma (MM) is continuing to rise, although childhood MM remains rare. We describe an 8-year-old Afro-Caribbean girl who developed a non pigmented lesion on the tip of her left thumb, which persisted despite treatment in primary care with cryotherapy. At biopsy she was found to have an acquired acral MM. She underwent amputation of the distal phalanx of her thumb, together with positive sentinel lymph node (SLN) biopsy and subsequent axillary lymph node clearance and adjuvant chemotherapy. MMs are very rare in this age and skin-type group, therefore requiring strict diagnostic criteria. These criteria include the distinction from MM mimics, especially Spitz tumours, and an appropriate use of staging techniques such as SLN biopsy to influence management.

Hashimoto's thyroiditis shares features with early papillary thyroid carcinoma.

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.

Contribution of the microvessel network to the clonal and kinetic profiles of adrenal cortical proliferative lesions.

Monoclonal adrenocortical lesions have been characterized by an inverse correlation between proliferation and apoptosis, and polyclonal lesions show a direct correlation. Their relationship with the vascular pattern remains unknown in adrenocortical nodular hyperplasias (ACNHs), adenomas (ACAs), and carcinomas (ACCs). We studied 20 ACNHs, 25 ACAs, and 10 ACCs (World Health Organization classification criteria) from 55 women. The analysis included X-chromosome inactivation assay (on microdissected samples), slide and flow cytometry, and in situ end labeling. Endothelial cells were stained with anti-CD31, and the blood vessel area and density were quantified by image analysis in the same areas. Appropriate tissue controls were run in every case. Regression analyses between kinetic and vascular features were performed in both polyclonal and monoclonal lesions. Polyclonal patterns were observed in 14 of 18 informative ACNHs and 3 of 22 informative ACAs, and monoclonal patterns were seen in 4 of 18 ACNHs, 19 of 22 ACAs, and 9 of 9 ACCs. A progressive increase in microvessel area was observed in the ACNH-ACA-ACC transition but was statistically significant between benign and malignant lesions only (191.36 +/- 168.32 v 958.07 +/- 1279.86 microm(2); P < .0001). In addition, case stratification by clonal pattern showed significant differences between polyclonal and monoclonal benign lesions; 6% of polyclonal and 57% of monoclonal lesions had microvessel area >186 microm(2) (P = .0000008). Monoclonal lesions showed parallel trends (but with opposite signs) for microvessel area and density in comparison with proliferation and apoptosis, whereas polyclonal lesions showed inverse trends. In conclusion, the kinetic advantage of monoclonal adrenal cortical lesions (increased proliferation, decreased apoptosis) is maintained by parallel increases in microvessel area and density.

Germline RET 634 mutation positive MEN 2A-related C-cell hyperplasias have genetic features consistent with intraepithelial neoplasia.

C-cell hyperplasias are normally multifocal in multiple endocrine neoplasia type 2A. We compared clonality, microsatellite pattern of tumor suppressor genes, and cellular kinetics of C-cell hyperplasia foci in each thyroid lobe. We selected 11 females from multiple endocrine neoplasia type 2A kindred treated with thyroidectomy due to hypercalcitoninemia. C-cell hyperplasia foci were microdissected for DNA extraction to analyze the methylation pattern of androgen receptor alleles and microsatellite regions (TP53, RB1, WT1, and NF1). Consecutive sections were selected for MIB-1, pRB1, p53, Mdm-2, and p21WAF1 immunostaining, DNA content analysis, and in situ end labeling. Appropriate tissue controls were run. Only two patients had medullary thyroid carcinoma foci. Nine informative C-cell hyperplasia patients showed germline point mutation in RET, eight of them with the same androgen receptor allele preferentially methylated in both lobes. C-cell hyperplasia foci showed heterogeneous DNA deletions revealed by loss of heterozygosity of TP53 (12 of 20), RB1 (6 of 14), and WT1 (4 of 20) and hypodiploid G0/G1 cells (14 of 20), low cellular turnover (MIB-1 index 4.5%, in situ end labeling index 0.03%), and significantly high nuclear area to DNA index ratio. MEN 2A (germline point mutation in RET codon 634) C-cell hyperplasias are monoclonal and genetically heterogeneous and show down-regulated apoptosis, findings consistent with an intraepithelial neoplasia. Concordant X-chromosome inactivation and interstitial gene deletions suggest clone expansions of precursors occurring at a point in embryonic development before divergence of each thyroid lobe and may represent a paradigm for other germline mutations.

Molecular and kinetic features of transitional cell carcinomas of the bladder: biological and clinical implications.

Molecular and kinetic analyses have contributed to our understanding of the biology of transitional cell carcinomas (TCC) of the bladder. The concordant pattern of X-chromosome inactivation of multiple TCCs appearing at different times and at different sites and concordant genetic abnormalities in a subset of muscle-invasive TCC strongly support a monoclonal origin and a homogeneous tumor cell selection throughout the neoplasm. However, topographic intratumor heterogeneity results from the accumulation of genetic lesions in tumor suppressor genes, predominantly neurofibromatosis (NF)-1-defective in the superficial compartment and tumor protein p53 (TP53)-defective in the deep one, with lower proliferation and down-regulation of apoptosis in the latter. TCCs follow the general concept of multistep carcinogenesis and proceed through two distinct genetic pathways responsible for generating different TCC morphologies. These are the inactivation of cyclin-dependent kinase inhibitors (p15, p16, and p21WAF/CIP1) in low-grade TCC and early TP53-mediated abnormalities in high-grade TCC. TCC progression correlates with genetic instability and accumulation of collaborative genetic lesions mainly involving TP53, retinoblastoma (RB)-1, and growth factors. Distinctive genetic (low incidence of RB-1 and NF-1 abnormalities) and kinetic (slower cell turnover) profiles also correlate with a "single-file" infiltration pattern and poor survival in muscle-invasive TCCs. The underlying molecular changes of carcinoma in situ involve multiple and more extensive deletions (normally TP53-defective) than coexistent invasive TCC, suggesting an independent genetic evolution, while low-grade dysplasia is mainly polyclonal and shows a low rate of gene deletions.

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.

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.