To investigate the activity of etoposide, doxorubicin, and cisplatin plus mitotane in the management of advanced adrenocortical carcinoma (ACC) patients, 72 patients with measurable disease not amenable to radical surgery were enrolled in a prospective, multicenter phase II trial. EDP schedule (etoposide 100 mg/m2 on days 5–7, doxorubicin 20 mg/m2 on days 1 and 8, and cisplatin 40 mg/m2 on days 1 and 9) was administered intravenously every 4 weeks. Concomitantly, patients were given up to 4 g/day of oral mitotane. Five patients achieved a complete response and 30 a partial response, for an overall response rate of 48.6% (95% CI: 37.1–60.3). Median time to progression in responding patients was 18 months. The EDP regimen was well tolerated, leukopenia being the dose limiting toxicity. One toxic related death due to septic shock, however, was registered. Radical surgical resection of residual disease after chemotherapy was performed in 10 patients. The overall survival of patients attaining a disease free status (clinical complete responders+radically resected) was significantly higher than that of patients with partial response or no response (P<0.002). Androgen secretion was associated with long survival, while glucocorticoid secretion was associated with poor prognosis both in univariate and multivariate analysis. In conclusion, EDP plus mitotane is an active and manageable combination scheme for ACC patients. Surgical resection of residual disease subsequent to chemotherapy leads to a more favourable outcome. The natural history of the disease is significantly influenced by the secretory status of the tumor.
Alfredo Berruti, Massimo Terzolo, Paola Sperone, Anna Pia, Silvia Della Casa, David J Gross, Carlo Carnaghi, Paolo Casali, Francesco Porpiglia, Franco Mantero, Giuseppe Reimondo, Alberto Angeli, and Luigi Dogliotti
Michael Solarski, Fabio Rotondo, William D Foulkes, John R Priest, Luis V Syro, Henriett Butz, Michael D Cusimano, and Kalman Kovacs
In this review, the importance of the DICER1 gene in the function of endocrine cells is discussed. There is conclusive evidence that DICER1 mutations play a crucial role in the development, progression, cell proliferation, therapeutic responsiveness and behavior of several endocrine tumors. We review the literature of DICER1 gene mutations in thyroid, parathyroid, pituitary, pineal gland, endocrine pancreas, paragangliomas, medullary, adrenocortical, ovarian and testicular tumors. Although significant progress has been made during the last few years, much more work is needed to fully understand the significance of DICER1 mutations.
Adwitiya Kar, Yu Zhang, Betelehem W Yacob, Jordan Saeed, Kenneth D Tompkins, Stacey M Bagby, Todd M Pitts, Hilary Somerset, Stephen Leong, Margaret E Wierman, and Katja Kiseljak-Vassiliades
Adrenocortical carcinoma (ACC) is an aggressive orphan malignancy with less than 35% 5-year survival and 75% recurrence. Surgery remains the primary therapy and mitotane, an adrenolytic, is the only FDA-approved drug with wide-range toxicities and poor tolerability. There are no targeted agents available to date. For the last three decades, H295R cell line and its xenograft were the only available preclinical models. We recently developed two new ACC patient-derived xenograft mouse models and corresponding cell lines (CU-ACC1 and CU-ACC2) to advance research in the field. Here, we have utilized these novel models along with H295R cells to establish the mitotic PDZ-binding kinase (PBK) as a promising therapeutic target. PBK is overexpressed in ACC samples and correlates with poor survival. We show that PBK is regulated by FOXM1 and targeting PBK via shRNA decreased cell proliferation, clonogenicity and anchorage-independent growth in ACC cell lines. PBK silencing inhibited pAkt, pp38MAPK and pHistone H3 altering the cell cycle. Therapeutically, targeting PBK with the small-molecule inhibitor HITOPK032 phenocopied PBK-specific modulation of pAkt and pHistone H3, but also induced apoptosis via activation of JNK. Consistent with in vitro findings, treatment of CU-ACC1 PDXs with HITOPK032 significantly reduced tumor growth by 5-fold (P < 0.01). Treated tumor tissues demonstrated increased rates of apoptosis and JNK activation, with decreased pAkt and Histone H3 phosphorylation, consistent with effects observed in ACC cell lines. Together these studies elucidate the mechanism of PBK in ACC tumorigenesis and establish the potential therapeutic potential of HITOPK032 in ACC patients.
Antonio M Lerario, Kazutaka Nanba, Amy R Blinder, Sachiko Suematsu, Masao Omura, Tetsuo Nishikawa, Thomas J Giordano, William E Rainey, and Tobias Else
Somatic variants in genes that regulate intracellular ion homeostasis have been identified in aldosterone-producing adenomas (APAs). Although the mechanisms leading to increased aldosterone production in APA cells have been well studied, the molecular events that cause cell proliferation and tumor formation are poorly understood. In the present study, we have performed whole-exome sequencing (WES) to characterize the landscape of somatic alterations in a homogeneous series of APA with pathogenic KCNJ5 variants. In the WES analysis on 11 APAs, 84 exonic somatic events were called by 3 different somatic callers. Besides the KCNJ5 gene, only two genes (MED13 and ZNF669) harbored somatic variants in more than one APA. Unlike adrenocortical carcinomas, no chromosomal instability was observed by the somatic copy-number alteration and loss of heterozygosity analyses. The estimated tumor purity ranged from 0.35 to 0.67, suggesting a significant proportion of normal cell infiltration. Based on the results of PureCN analysis, the KCNJ5 variants appear to be clonal. In conclusion, in addition to KCNJ5 somatic pathogenic variants, no significant somatic event that would obviously explain proliferation or tumor growth was observed in our homogeneous cohort of KCNJ5-mutated APA. The molecular mechanisms causing APA growth and tumorigenesis remain to be elucidated.
Teresa M Seccia, Ambrogio Fassina, Gastone G Nussdorfer, Achille C Pessina, and Gian Paolo Rossi
Aldosterone-producing adrenocortical carcinoma (APAC) is a rare cause of hypertension often diagnosed late because of paucity of information. Thus, we delineated its clinical course and survival rates based on two cases referred to us that featured diverging clinical courses, and on a scrutiny of the literature since 1955 when the first case of APAC was identified. Data on demography, imaging results, hormonal assessment, histology, and clinical course were extracted independently by the investigators. We included in our database 58 cases, most presenting with Conn’s syndrome. Plasma aldosterone levels were on average increased 14-fold; plasma renin activity was suppressed in 55% of cases. The tumor showed extremely variable size and weight, and no gender or side preference. Metastases were present in 10% of all cases at initial diagnosis and in an additional 48% of cases at follow-up. Median survival was 546 days (95% confidence interval (CI): 240–851); median time to either recurrence or death was 212 days (95% CI: 29–395). No clinical or histological signs predicted survival with Cox regression analysis. We concluded that, although an ominous course with a poor survival rate is common, no sign accurately predicts the course of APAC. Thus, molecular studies to identify diagnostic markers of survival are mandatory.
Vassiliki Kotoula, Elias Sozopoulos, Helen Litsiou, Galinos Fanourakis, Triantafyllia Koletsa, Gerassimos Voutsinas, Sophia Tseleni-Balafouta, Constantine S Mitsiades, Axel Wellmann, and Nicholas Mitsiades
The serine/threonine kinase B-Raf plays a key role in the Ras/Raf/MEK/ERK pathway that relays extracellular signals for cell proliferation and survival. Several types of human malignancies harbor activating BRAF mutations, most frequently a V600E substitution. The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small cell lung carcinomas harboring activating EGFR TK domain somatic mutations. We evaluated the presence of mutations in BRAF (exons 11 and 15), KRAS (exons 1 and 2), NRAS (exons 1 and 2), and EGFR (exons 18–21) in adrenal carcinomas (35 tumor specimens and two cell lines) by DNA sequencing. BRAF mutations were found in two carcinomas (5.7%). Four carcinomas (11.4%) carried EGFR TK domain mutations. One specimen carried a KRAS mutation, and another carried two NRAS mutations. No mutations were found in the two adrenocortical cell lines. BRAF- and EGFR-mutant tumor specimens exhibited stronger immunostaining for the phosphorylated forms of the MEK and ERK kinases than their wild-type counterparts. EGFR-mutant carcinomas exhibited increased phosphorylation of EGFR (Tyr 992) compared with wild-type carcinomas. We conclude that BRAF, RAS, and EGFR mutations occur in a subset of human adrenocortical carcinomas. Inhibitors of the Ras/Raf/MEK/ERK and EGFR pathways represent candidate targeted therapies for future clinical trials in carefully selected patients with adrenocortical carcinomas harboring respective activating mutations.
T M A Kerkhofs, M H T Ettaieb, I G C Hermsen, and H R Haak
Cancer of the adrenal cortex (ACC) is a rare endocrine malignancy with limited treatment options. Patients typically present with autonomous hormonal overproduction and/or a large abdominal mass. Hormonal assays and medical imaging can be diagnostic, but urinary steroid profiling might be a more sensitive technique to assess malignancy in adrenal tumours. The stage of the disease at diagnosis is the most important prognostic factor. The current staging system needs refinement, especially to separate aggressive from indolent disease in stage IV patients and to select patients who need adjuvant treatment after complete surgical resection. Regarding the latter, assessing the proliferation index Ki-67 seems the best tool currently available. Genomic profiling is expected to become of clinical relevance in the future. Medical therapy is centred on the adrenolytic drug mitotane, which carries considerable toxicity and is not easy to manage. Its tolerability and long plasma level build-up phase may be improved by therapeutic drug monitoring based on pharmacokinetic modelling and intensive counselling of patients. Current chemotherapy regimens can offer disease stabilization in about 50% of patients, but an objective response should be expected in <25%. Research on targeted therapy and immunotherapy is difficult in this rare disease with often heavily pre-treated patients and has not yet been successful. Quality of care should be ensured by treating patients in centres with established experience in multidisciplinary oncologic care, who adhere to prevailing guidelines and state-of-the-art in diagnostic and treatment concepts. International collaboration in fundamental research and clinical trials is the key to further elucidate the pathogenesis and to improve patient care.
K E Lines, P Filippakopoulos, M Stevenson, S Müller, H E Lockstone, B Wright, S Knapp, D Buck, C Bountra, and R V Thakker
Medical treatments for corticotrophinomas are limited, and we therefore investigated the effects of epigenetic modulators, a new class of anti-tumour drugs, on the murine adrenocorticotropic hormone (ACTH)-secreting corticotrophinoma cell line AtT20. We found that AtT20 cells express members of the bromo and extra-terminal (BET) protein family, which bind acetylated histones, and therefore, studied the anti-proliferative and pro-apoptotic effects of two BET inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, using CellTiter Blue and Caspase Glo assays, respectively. JQ1 and PFI-1 significantly decreased proliferation by 95% (P < 0.0005) and 43% (P < 0.0005), respectively, but only JQ1 significantly increased apoptosis by >50-fold (P < 0.0005), when compared to untreated control cells. The anti-proliferative effects of JQ1 and PFI-1 remained for 96 h after removal of the respective compound. JQ1, but not PFI-1, affected the cell cycle, as assessed by propidium iodide staining and flow cytometry, and resulted in a higher number of AtT20 cells in the sub G1 phase. RNA-sequence analysis, which was confirmed by qRT-PCR and Western blot analyses, revealed that JQ1 treatment significantly altered expression of genes involved in apoptosis, such as NFκB, and the somatostatin receptor 2 (SSTR2) anti-proliferative signalling pathway, including SSTR2. JQ1 treatment also significantly reduced transcription and protein expression of the ACTH precursor pro-opiomelanocortin (POMC) and ACTH secretion by AtT20 cells. Thus, JQ1 treatment has anti-proliferative and pro-apoptotic effects on AtT20 cells and reduces ACTH secretion, thereby indicating that BET inhibition may provide a novel approach for treatment of corticotrophinomas.
Giada Poli, Daniele Guasti, Elena Rapizzi, Rossella Fucci, Letizia Canu, Alessandra Bandinelli, Nicoletta Cini, Daniele Bani, Massimo Mannelli, and Michaela Luconi
At present, mitotane (MTT) represents the first-line pharmacological approach for the treatment of advanced adrenocortical carcinoma (ACC). Despite clear evidence that the drug can reduce the clinical signs of steroid excess in secreting ACC, the mechanism mediating the possible toxic effect of MTT on tumor cells still remains obscure. This study investigated the intracellular events underlying the toxic effect of MTT by studying qualitative and quantitative alterations in mitochondrial morphology and functions in human adrenocortical cancer cell lines, H295R and SW13. Increasing concentrations of MTT resulted in rapid intracellular accumulation and conversion of the drug. Cytostatic and cytotoxic effects were evident at doses corresponding to the therapeutic window (30–50 μM) through an apoptotic mechanism involving caspase 3/7. Electron microscopic analysis of cell mitochondria displayed MTT-induced dose- and time-dependent alterations in the morphology of the organelle. These alterations were characterized by a marked swelling and a decrease in the number of respiratory cristae, accompanied by a significant depolarization of the mitochondrial membrane potential, finally leading to the disruption of the organelle. A drastic reduction of oxygen consumption was observed due to mitochondrial membrane damage, which was accompanied by a decrease in the levels of VDAC1 integral membrane channel. These findings contribute to better understand the intracellular mechanism of action of MTT in ACC cells, showing that its cytotoxic effect seems to be mainly mediated by an apoptotic process activated by the disruption of mitochondria.
Paola Sperone, Anna Ferrero, Fulvia Daffara, Adriano Priola, Barbara Zaggia, Marco Volante, Daniele Santini, Bruno Vincenzi, Giuseppe Badalamenti, Chiara Intrivici, Sabrina Del Buono, Silvia De Francia, Emmanouil Kalomirakis, Riccardo Ratti, Alberto Angeli, Luigi Dogliotti, Mauro Papotti, Massimo Terzolo, and Alfredo Berruti
Adrenocortical carcinoma (ACC) is a rare neoplasm characterized by poor prognosis. First-line systemic treatments in advanced disease include mitotane, either alone or in combination with chemotherapy. Studies evaluating second-line therapy options have obtained disappointing results. This trial assessed the activity and toxicity of gemcitabine plus metronomic fluoropyrimidines in heavily pretreated advanced ACC patients. From 1998 to 2008, 28 patients with advanced ACC progressing after mitotane plus one or two systemic chemotherapy lines were enrolled. They received a combination of i.v. gemcitabine (800 mg/m2, on days 1 and 8, every 21 days) and i.v. 5-fluorouracil protracted infusion (200 mg/m2/daily without interruption until progression) in the first six patients, or oral capecitabine (1500 mg/daily) in the subsequent patients. Mitotane administration was maintained in all cases. The rate of non-progressing patients after 4 months of treatment was 46.3%. A complete response was observed in 1 patient (3.5%); 1 patient (3.5%) obtained a partial regression, 11 patients (39.3%) obtained a disease stabilization and 15 patients (53.7%) progressed. Treatment was well tolerated, with grade III and IV toxicities consisting of leukopenia in six patients (21.4%), thrombocytopenia in one patient (3.5%), and mucositis in one patient (3.5%). Median time to progression and overall survival in the patient population were 5.3 (range: 1–43) and 9.8 months (range: 3–73) respectively. Gemcitabine plus metronomic fluoropyrimidines is a well-tolerated and moderately active regimen in heavily pretreated ACC patients.