Flow cytometric DNA analysis was performed on 19 adrenal pheochromocytomas and 6 extra-adrenal paragangliomas in parallel with clinical and histopathological review to determine the usefulness of this technique to predict biologic behavior of these tumors. In pheochromocytomas and paragangliomas, tetraploidy or near-tetraploidy occurred in 32% and 33% and aneuploidy in 10% and none respectively. A case of malignant pheochromocytoma had diploid DNA content. Occurrence of aneuploidy or tetraploidy is frequent in clinically benign tumors in conjunction with a marked degree of nuclear atypia and cannot be a predictor of malignancy.
Adolescent ; Adrenal Gland Neoplasms/*genetics/pathology ; Adult ; Age Factors ; Child ; DNA, Neoplasm/*analysis ; Female ; *Flow Cytometry ; Human ; Male ; Middle Age ; Mitosis ; Paraganglioma/*genetics/pathology ; Pheochromocytoma/*genetics/pathology ; Ploidies

OBJECTIVESTo investigate the differences in morphology, immunohistochemistry, DNA ploidy status, LOH and MSI of 11q13 and 1p between benign and malignant pheochromocytomas, and to find the marker or markers useful in distinction between benign and malignant pheochromocytoma or for predicting the malignant potential of this tumor.

METHODSTwenty-two cases of clinically documented benign and malignant pheochromocytomas from the files of Peking Union Medical College Hospital were analyzed. Aside from histological study, Ki-67, p53, CgA, S-100, PCNA and survivin immunohistochemistry studies were performed. DNA ploidy status was assessed by flow cytometry on cell suspensions prepared from formalin-fixed, paraffin-embedded sections. Twelve tumors (7 benign and 5 malignant) with paired normal tissues were microdissected. Tumor and normal tissue DNA were extracted. The obtained DNAs and 8 microsatellite markers related to 11q13 and 1q were subjected to PCR amplification for analysis of LOH and MSI.

RESULTSNone of the tumors showed atypical mitosis, only 1 malignant tumor had a mitotic count > 1/10 HPF (2.3/10 HPF). Two malignant tumors exhibited confluent necrosis. Ki-67 index was low in benign tumors (average 0.73%), and high in malignant tumors (average 2.4%). The difference of Ki-67 index between benign and malignant tumors was statistically significant. DNA ploidy status did not correlate with malignancy. Although LOH and/or MSI of 11q13 and 1p were observed in several tumors, a statistically significant difference could not be reached due to the small number of tumors analyzed.

CONCLUSIONOnly Ki-67 index (> 3%) is an useful marker for distinguishing benign from malignant or for predicting the malignant potential of pheochromocytoma.

Adrenal Gland Neoplasms ; genetics ; metabolism ; pathology ; Adult ; Aged ; Biomarkers, Tumor ; metabolism ; DNA, Neoplasm ; genetics ; metabolism ; Female ; Humans ; Ki-67 Antigen ; genetics ; metabolism ; Loss of Heterozygosity ; Male ; Middle Aged ; Neoplasm Metastasis ; Pheochromocytoma ; genetics ; metabolism ; pathology ; Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; Urinary Bladder Neoplasms ; genetics ; metabolism ; pathology

Epigenomic and genomic changes affect gene expression and contribute to tumor development. The histone modifications trimethylated histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) are epigenetic regulators associated to active and silenced genes, respectively and alterations of these modifications have been observed in cancer. Furthermore, genomic aberrations such as DNA copy number changes are common events in tumors. Pheochromocytoma is a rare endocrine tumor of the adrenal gland that mostly occurs sporadic with unknown epigenetic/genetic cause. The majority of cases are benign. Here we aimed to combine the genome-wide profiling of H3K4me3 and H3K27me3, obtained by the ChIP-chip methodology, and DNA copy number data with global gene expression examination in a malignant pheochromocytoma sample. The integrated analysis of the tumor expression levels, in relation to normal adrenal medulla, indicated that either histone modifications or chromosomal alterations, or both, have great impact on the expression of a substantial fraction of the genes in the investigated sample. Candidate tumor suppressor genes identified with decreased expression, a H3K27me3 mark and/or in regions of deletion were for instance TGIF1, DSC3, TNFRSF10B, RASSF2, HOXA9, PTPRE and CDH11. More genes were found with increased expression, a H3K4me3 mark, and/or in regions of gain. Potential oncogenes detected among those were GNAS, INSM1, DOK5, ETV1, RET, NTRK1, IGF2, and the H3K27 trimethylase gene EZH2. Our approach to associate histone methylations and DNA copy number changes to gene expression revealed apparent impact on global gene transcription, and enabled the identification of candidate tumor genes for further exploration.
Adrenal Gland Neoplasms/*genetics/*pathology ; *Epigenesis, Genetic ; Female ; Gene Dosage/genetics ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks/genetics ; Genome, Human/*genetics ; *Genomics ; Histones/metabolism ; Humans ; Lysine/metabolism ; Methylation ; Pheochromocytoma/*genetics/*pathology ; Protein Processing, Post-Translational ; Tumor Suppressor Proteins/genetics/metabolism