Activation of the mammalian target of rapamycin in malignant pheochromocytoma: a potential therapeutic target
Creators: name: Brimo, Fadi
name: Chaux, Alcides
name: Schultz, Luciana
name: Epstein, Jonathan I.
name: Marzo, Angelo M. De
name: Pedram Argani
name: Netto, George J.
Publisher: name: F1000Research
Year: 2016

description: INTRODUCTION: Activation of the phosphatidylinositol 3’-kinase (PI3K)-protein kinase B (AKT) pathway leads to AKT phosphorylation and activation (phos-AKT). phos-AKT promotes cell cycle progression through p27 depletion, cell proliferation through c-myc up-regulation, and protein translation through the mammalian target of rapamycin (mTOR) activation via its two downstream molecules, phosphorylated S6 protein (phos-S6) and eukaryotic translation initiation factor 4E-binding protein-1 (4E-BP1). Dysregulation of the mTOR pathway is present in several types of malignancies such as small cell carcinoma of the lung, clear cell renal cell carcinoma and urothelial carcinoma in which agents targeting mTOR have been or are currently being evaluated. Although the majority of pheochromocytomas have a benign clinical course, about 10% of cases are malignant. Other than the presence of metastatic disease which indicates malignancy, differentiating benign from potentially malignant tumors in the primary setting is difficult on histological grounds. The most widely used system is the PASS score (Pheochromocytoma of the Adrenal gland Scaled Score) which includes pathological parameters such as necrosis, mitotic count, capsular and vascular invasion. However, the fact remains that close clinical and biochemical follow-up is recommended in all cases due to the known unpredictable behavior of pheochromocytomas. In the metastatic setting, available treatments in the form of surgery, chemotherapy, and radionuclide therapy may improve symptoms and biochemical markers, but the results for the control of tumor bulk are less favorable. Furthermore, responses to treatment are frequently short-lived. We herein assess the status of mTOR pathway components and their prognostic role in a series of benign and malignant pheochromocytomas.<br><br>MATERIALS AND METHODS: Included were 47 in-house pheochromocytoma cases in which surgery was performed at the Johns Hopkins Hospital between 1996 and 2009 and in which paraffin blocks were available. Cases included adrenal primary and metastatic cases. Immunohistochemical staining was performed using a tissue microarray (TMA) in which duplicate tumor samples was used. The expression of the following markers was evaluated: PTEN (nuclear and cytoplasmic), phos-AKT (nuclear), phos-S6 (cytoplasmic), p27 (nuclear), and c-myc (nuclear). For each marker, the percentage of positive cells (extent) and the intensity of staining (0, 1, 2, 3) were recorded and an H-score (intensity by extent) was calculated. For each case, the average H-score of the two TMA spots was used for analysis. The raw H-score of all biomarkers was correlated with the presence of metastases using the Pearson correlation coefficient; mean H-scores were compared using the unpaired Student’s t-test. PTEN was considered to be lost when less than 10% of the cells expressed the marker. Since no immunohistochemical expression was observed with c-myc this marker was excluded from comparison analyses. In the remainder cases (for AKT, p27, and pS6 expression) the cutoff point was established post-analytically, as follows: the H-score distribution (range 0–300) was divided into quartiles. Groups were created by collapsing quartiles in two sets of contiguous categories (positive vs. negative) and each group was evaluated in their ability to predict malignancy (metastatic pheochromocytomas). Finally, the group with the lowest P-value was selected, establishing the highest H-score of the “negative” category as the cutoff point. In all cases, as well as in the evaluation of PTEN expression, the Fisher’s exact test was used.<br><br>CLINICAL RESULTS: The mean age was 45.9 years (range 10-71) with 28 of the patients being males and 19 females (male to female ratio 1.47 : 1). Of the 47 cases, 39 were adrenal primary and eight were metastatic pheochromocytomas.<br><br>PRIMARY PHEOCHROMOCYTOMAS: Post-surgical clinical follow-up was available for 20 of the 39 primary tumors and of all of them had a benign clinical course with a mean follow-up period of 4.4 years (median=4.2 years, range 0.3–10 years). Nineteen patients did not have follow-up; those cases were considered to have an indeterminate clinical course.<br><br>METASTATIC PHEOCHROMOCYTOMAS: The site of the metastatic disease was retroperitoneal lymph nodes in six patients and bone in two patients. None of those cases had a matched primary tumor specimen available for evaluation. Four patients were alive with disease at a mean post-surgical period of 3.4 years (range 0.5-10), two died of disease at 1.5 years and 2 years post-surgery, and in two cases no clinical follow-up was available.<br><br>IMMUNOHISTOCHEMICAL RESULTS: c-myc was negative in all cases. In comparison to benign cases, malignant (metastatic) pheochromocytomas showed lower p27 and higher phos-S6 expression, but this difference was not significant. PTEN expression was similar in the two groups. On the other hand, phos-AKT was significantly overexpressed in malignant pheochromocytomas (P=0.012). Similar findings were obtained when comparing primary (N=39) and metastatic (N=8) tumors. H-score correlation analyses between each biomarker and the others disclosed no significant association.<br><br>CONCLUSION: The significantly higher expression levels of phos-AKT in malignant pheochromocytomas in comparison to benign tumors indicates activation of the mTOR pathway in this subset of pheochromocytomas. mTOR pathway-targeted therapy in malignant pheochromocytomas should be further investigated and pursued as the currently available treatment modalities do not achieve optimal disease control.