Phenylethanolamine N-methyltransferase downregulation is associated with malignant pheochromocytoma/paraganglioma

Seung Eun Lee, Ensel Oh, Boram Lee, Yu Jin Kim, Doo-Yi Oh, Kyungsoo Jung, Jong-Sun Choi, Junghan Kim, Sung Joo Kim, Jung Wook Yang, Jungsuk An, Young Lyun Oh, Yoon La Choi, Seung Eun Lee, Ensel Oh, Boram Lee, Yu Jin Kim, Doo-Yi Oh, Kyungsoo Jung, Jong-Sun Choi, Junghan Kim, Sung Joo Kim, Jung Wook Yang, Jungsuk An, Young Lyun Oh, Yoon La Choi

Abstract

Malignant pheochromocytoma/paraganglioma (PCC/PGL) is defined by the presence of metastases at non-chromaffin sites, which makes it difficult to prospectively diagnose malignancy. Here, we performed array CGH (aCGH) and paired gene expression profiling of fresh, frozen PCC/PGL samples (n = 12), including three malignant tumors, to identify genes that distinguish benign from malignant tumors. Most PCC/PGL cases showed few copy number aberrations, regardless of malignancy status, but mRNA analysis revealed that 390 genes were differentially expressed in benign and malignant tumors. Expression of the enzyme, phenylethanolamine N-methyltransferase (PNMT), which catalyzes the methylation of norepinephrine to epinephrine, was significantly lower in malignant PCC/PGL as compared to benign samples. In 62 additional samples, we confirmed that PNMT mRNA and protein levels were decreased in malignant PCC/PGL using quantitative real-time polymerase chain reaction and immunohistochemistry. The present study demonstrates that PNMT downregulation correlates with malignancy in PCC/PGL and identifies PNMT as one of the most differentially expressed genes between malignant and benign tumors.

Keywords: biomarker; endocrine tumors; metastasis; phenylethanolamine N-methyltransferase; pheochromocytoma/paraganglioma.

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1. Heatmap of genomic profiles of…
Figure 1. Heatmap of genomic profiles of the segmented copy number data
Figure 2. Histogram of PNMT expression in…
Figure 2. Histogram of PNMT expression in GSE19987
Two distinct distributions are shown.
Figure 3. Biological functions of genes overexpressed…
Figure 3. Biological functions of genes overexpressed in the PNMT-low group
Overrepresentation of blood vessel development-related functions was observed upon analysis of the 200 highly upregulated genes in the PNMT-low group. Each circle represents a selected enriched gene ontology (GO) term, and darker color represents increased statistical significance.
Figure 4. Correlation between PNMT and RET
Figure 4. Correlation between PNMT and RET
RET showed the highest correlation (r = 0.91) with PNMT in our data (A) Malignant tumors showed low expression of both PNMT and RET. RET showed the highest correlation (r = 0.77) with PNMT in the GSE19987 dataset, which includes hereditary PCC/PGL (B) Tumors harboring RET germline mutations showed high PNMT and RET expression, while SDHx/VHL germline-mutated tumors showed low expression.
Figure 5. Comparison of benign and malignant…
Figure 5. Comparison of benign and malignant PCC/PGL
Comparative quantification of PNMT protein levels by immunohistochemistry (IHC) (A) Correlation between PNMT mRNA and protein levels (B).
Figure 6. Representative PNMT protein levels in…
Figure 6. Representative PNMT protein levels in benign and malignant PCC and PGL
Benign PCC (arrowhead) showed the same immunostaining intensity as the adjacent normal adrenal medulla (arrow) (A) Strong cytoplasmic staining was observed (left upper inset). Malignant PCC (arrowhead) exhibited complete absence of PNMT compared with the adjacent normal adrenal medulla (arrow) (B) Left upper inset represents higher magnification. Benign PGL showed diffuse and strong PNMT staining (C) Malignant PGL showed complete absence of PNMT positivity (D).
Figure 7. Correlation between PASS and PNMT…
Figure 7. Correlation between PASS and PNMT protein levels
Figure 8. PNMT immunostaining in specimens from…
Figure 8. PNMT immunostaining in specimens from patient 7
Primary extra-adrenal pheochromocytoma in the right para-aortic area with complete absence of PNMT positivity (A) Recurrent extra-adrenal pheochromocytoma in the retrocaval space with complete absence of PNMT positivity (B).

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