DNA and kinetic heterogeneity during the clonal evolution of adrenocortical proliferative lesions

Abstract

Monoclonal adrenocortical lesions show inverse correlation between proliferation and apoptosis, with proliferation being the single most important criterion of malignancy in adrenal lesions. No study yet has evaluated the variability of proliferation regarding the clonal pattern and diagnosis in adrenocortical nodular hyperplasias (ACNHs), adrenocortical adenomas (ACAs), and adrenocortical carcinomas (ACCs). We studied 69 ACNHs, 64 ACAs, and 23 ACCs (World Health Organization criteria) from 156 females. Clonality HUMARA test (from microdissected DNA samples), DNA content and proliferation analysis (slide and flow cytometry), and mitotic figure (MF) counting/50 high-power fields (HPFs) were performed in the same areas. Heterogeneity was assessed by 5cER (percentage of nonoctaploid cells with DNA content exceeding 5c) and standard deviation of MF/HPF. Statistics included analysis of variance/Student t tests regarding the clonal patterns and diagnosis. Polyclonal patterns were observed in 48 of 62 informative ACNHs and 7 of 56 informative ACAs, and monoclonal in 14 of 62 ACNHs, 49 of 56 ACAs, and 21 of 21 ACCs, with all hyperdiploid lesions (14 ACCs and 13 ACAs) being monoclonal. The standard deviation of MF/HPF progressively increased in ACNH-ACA-ACC (0.048 +/- 0.076, 0.110 +/- 0.097, 0.506 +/- 0.291, respectively; P = .0023), but did not differentiate ACNH/ACA. Only tetraploid percentage (P = .0496) and 5cER (P = .0352) distinguished polyclonal (3.64 +/- 2.20 and 0.14 +/- 0.15) from monoclonal (7.25 +/- 7.52 and 1.00 +/- 1.74) benign lesions. Malignancy significantly correlated with a low diploid percentage and high tetraploid percentage. Cell kinetic heterogeneity is the hallmark of adrenocortical neoplasms: tetraploid/hypertetraploid cell accumulation characterizes monoclonal lesions (suggesting nondisjunctional mitoses), whereas heterogeneously distributed mitotic figures and decreased diploid percentage define ACCs.

Adrenocortical Carcinoma

“…Over 30 years ago, cytogenetic and flow cytometry techniques began to be applied to study ACC (179). One of the first genetic assessments with flow cytometry revealed aneuploidy (a genomic aberration consistently observed in most cancers) in 4 of 4 ACCs, yet only diploidy or tetraploidy in normal adrenal cortices and benign adrenal tumors (180).…”

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

“…7,19,20,29 Immunohistochemical Detection of Ki-67, Cell Cycle Regulators, and TERT Sections were mounted on positively charged slides (Superfrost Plus, Fisher Scientific, Fair Lawn, NJ, USA), baked at 601C for 2 h, and processed as described. 22,24,30 After routine dewaxing and rehydration, endogenous peroxidase quenching, and antigen heat retrieval, the slides were transferred to a moist chamber. Nonspecific binding was blocked with polyclonal horse serum and sections incubated with monoclonal primary antibodies ( Table 3).…”

“…19,20 However, the association of multiple genetic alterations would become statistically less probable as the number of molecular markers increases and is useful to test clonal expansions in tumors. 7,18 Although genetic abnormalities are probably asymmetrically acquired, there is a correlation with tumor cell topography in both luminal and solid organs, 1,[19][20][21][22][23] which has not been assessed in skin melanocytic lesions. Any potential topographic segregation of tumor cells will influence interpretations of the results, supporting a linear model if progressive grading is parallel to intraepithelial-to-invasive topography of tumor cells.…”

Insights into the role of genetic alterations in adrenocortical tumorigenesis

“…In particular, clonal composition of ACTs tumors has been determined by the patterns of X-chromosome inactivation in females heterozygous for X-linked polymorphisms. From the three studies carried out thus far, one may conclude that ACCs are more often monoclonal whereas ACAs may be either polyclonal or monoclonal (Beuschlein et al, 1994;Gicquel et al, 1994b;Blanes and Diaz-Cano, 2006). The genetic heterogeneity evidenced in ACAs may be explained either by different pathological mechanisms or, by different stages of a common multistep process.…”