Aberrant epigenetics is a hallmark of cancer, and endocrine-related tumors are no exception. Recent research has been identifying an ever-growing number of epigenetic alterations in both genomic DNA methylation and histone post-translational modification in tumors of the endocrine system. Novel microarray and ultra-deep sequencing technologies have allowed the identification of genome-wide epigenetic patterns in some tumor types such as adrenocortical, parathyroid, and breast carcinomas. However, in other cancer types, such as the multiple endocrine neoplasia syndromes and thyroid cancer, tumor information is limited to candidate genes alone. Future research should fill this gap and deepen our understanding of the functional role of these alterations in cancer, as well as defining their possible clinical uses.
Sandra Rodríguez-Rodero, Elías Delgado-Álvarez, Agustín F Fernández, Juan L Fernández-Morera, Edelmiro Menéndez-Torre, and Mario F Fraga
Sara Jung, Zoltan Nagy, Martin Fassnacht, Gerard Zambetti, Max Weiss, Martin Reincke, Peter Igaz, Felix Beuschlein, and Constanze Hantel
Systemic therapy of adrenocortical carcinoma (ACC) is limited by heterogeneous tumor response and adverse effects. Recently, we demonstrated anti-tumor activity of LEDP-M (etoposide, liposomal doxorubicin, liposomal cisplatin, mitotane), a liposomal variant of EDP-M (etoposide, doxorubicin, cisplatin, mitotane). To improve the therapeutic efficacy and off-target profiles of the clinical gold standard EDP-M, we investigated liposomal EDP-M regimens in different preclinical settings and in a small number of ACC patients with very advanced disease. Short- and long-term experiments were performed on two ACC models (SW-13 and SJ-ACC3) in vivo. We evaluated the anti-tumoral effects and off-target profiles of EDP-M, LEDP-M and a novel regimen L(l)EDP-M including liposomal etoposide. Furthermore, the role of plasma microRNA-210 as a therapeutic biomarker and first clinical data were assessed. Classical and liposomal protocols revealed anti-proliferative efficacy against SW-13 (EDP-M P < 0.01; LEDP-M: P < 0.001; L(l)EDP-M: P < 0.001 vs controls), whereas in SJ-ACC3, only EDP-M (P < 0.05 vs controls) was slightly effective. Long-term experiments in SW-13 demonstrated anti-tumor efficacy for all treatment schemes (EDP-M: P < 0.01, LEDP-M: P < 0.05, L(l)EDP-M P < 0.001 vs controls). The analysis of pre-defined criteria leading to study termination revealed significant differences for control (P < 0.0001) and EDP-M (P = 0.003) compared to L(l)EDP-M treatment. Raising its potential for therapy monitoring, we detected elevated levels of circulating microRNA-210 in SW-13 after LEDP-M treatment (P < 0.05). In contrast, no comparable effects were detectable for SJ-ACC3. However, overall histological evaluation demonstrated improved off-target profiles following liposomal regimens. The first clinical data indicate improved tolerability of liposomal EDP-M, thus confirming our results. In summary, liposomal EDP-M regimens represent promising treatment options to improve clinical treatment of ACC.
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.
Régia Caroline Peixoto Lira, Paola Fernanda Fedatto, David Santos Marco Antonio, Letícia Ferro Leal, Carlos Eduardo Martinelli, Margaret de Castro, Silvio Tucci, Luciano Neder, Leandra Ramalho, Ana Luiza Seidinger, Izilda Cardinalli, Maria José Mastellaro, José Andres Yunes, Silvia Regina Brandalise, Luiz Gonzaga Tone, Sonir Roberto Rauber Antonini, and Carlos Alberto Scrideli
Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patient’s clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1μM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.
Meenu Jain, Lisa Zhang, Mei He, Ya-Qin Zhang, Min Shen, and Electron Kebebew
Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with no effective therapy for patients with unresectable disease. The aim of the current study was i) to evaluate TOP2A expression and function in human adrenocortical neoplasm and ACC cells and ii) to determine the anticancer activity of agents that target TOP2A. TOP2A mRNA and protein expression levels were evaluated in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 ACCs). In vitro siRNA knockdown of TOP2A in ACC cell lines (NCI-H295R and SW13) was used to determine its effect on cellular proliferation, cell cycle, anchorage-independent growth, and cellular invasion. We screened 14 TOP2A inhibitors for their anticancer activity in ACC cells. TOP2A mRNA and protein expression was significantly higher in ACC than in benign and normal adrenocortical tissue samples (P<0.05). Knockdown of TOP2A gene expression in ACC cell lines significantly decreased cell proliferation, anchorage-independent growth, and invasion (P<0.05). A screening assay in NCI-H295R cells showed that 11 of 14 TOP2A inhibitors had antiproliferative activity, 5 of the 14 TOP2A inhibitors had a higher antiproliferative activity than mitotane, and aclarubicin was the agent with the highest activity. Aclarubicin was validated to significantly decrease proliferation and tumor spheroid size in both NCI-H295R and SW13 ACC cell lines (P<0.05). Our results suggest that TOP2A is overexpressed in ACC, regulates cellular proliferation and invasion in ACC cells, and is an attractive target for ACC therapy. Of the TOP2A inhibitors screened, aclarubicin is a good candidate agent to test in future clinical trials for patients with locally advanced and metastatic ACC.
D E Schteingart, G M Doherty, P G Gauger, T J Giordano, G D Hammer, M Korobkin, and F P Worden
Adrenocortical carcinomas are rare, highly malignant tumors that account for only 0.2% of deaths due to cancer. Given the limited number of patients seen in most medical centers with this diagnosis, series usually reported are small and clinical trials not randomized or blinded. In an attempt to answer important questions concerning the management of patients with adrenal cancer, a consensus conference was organized and held at the University of Michigan in Ann Arbor, MI, 11–13 September 2003, with the participation of an international group of physicians who had reported on the largest series of patients with this disease and who had recognized basic and clinical research expertise in adrenal cortical cancer. Totally 43 questions were addressed by the presenters and recommendations discussed in plenary and breakout sessions. Evidence for the recommendations of this conference was at the 2–4+ level and based on available literature and participants’ experience.
In addition to setting up guidelines in specific areas of the diagnosis and treatment of adrenal cancer, the conference recommended and initiated the planning of an international prospective trial for treatment of patients with adrenal cancer in stages III and IV. In terms of new therapies, first trials of dendritic cell therapy in human subjects with adrenal cancer have been started, but it is too early to comment on efficacy. Different strategies of immunotherapy, including DNA vaccination are currently being tried in animal models. There are no clinical gene therapy trials for human adrenal cortical cancer. The adrenals are a preferred target for adenovirus and the results of gene therapy in preclinical studies are promising. In addition, there is evidence that histone deacetylase inhibitors can further enhance the rate of adenoviral infectivity in human adrenal cancer cells. Testing of retroviral vectors, non-viral vectors, small interfering RNA technology, and combined approaches could be performed in various laboratories. Anti-angiogenic substances have only been applied in preclinical studies. The use of these and other agents in the treatment of adrenal cancer should be hypothesis-driven and based on a thorough analysis of tumor biology.
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.
Debbie L Hay, Christopher S Walker, and David R Poyner
Adrenomedullin (AM), adrenomedullin 2 (AM2/intermedin) and calcitonin gene-related peptide (CGRP) are members of the calcitonin family of peptides. They can act as growth or survival factors for a number of tumours, including those that are endocrine-related. One mechanism through which this occurs is stimulating angiogenesis and lymphangiogenesis. AM is expressed by numerous tumour types and for some cancers, plasma AM levels can be correlated with the severity of the disease. In cancer models, lowering AM content or blocking AM receptors can reduce tumour mass. AM receptors are complexes formed between a seven transmembrane protein, calcitonin receptor-like receptor and one of the two accessory proteins, receptor activity-modifying proteins (RAMPs) 2 or 3 to give the AM1 and AM2 receptors respectively. AM also has affinity at the CGRP receptor, which uses RAMP1. Unfortunately, due to a lack of selective pharmacological tools or antibodies to distinguish AM and CGRP receptors, the precise receptors and signal transduction pathways used by the peptides are often uncertain. Two other membrane proteins, RDC1 and L1/G10D (the ‘ADMR’), are not currently considered to be genuine CGRP or AM receptors. In order to properly evaluate whether AM or CGRP receptor inhibition has a role in cancer therapy, it is important to identify which receptors mediate the effects of these peptides. To effectively distinguish AM1 and AM2 receptors, selective receptor antagonists need to be developed. The development of specific CGRP receptor antagonists suggests that this is now feasible.
Helene Myrtue Nielsen, Alexandre How-Kit, Carole Guerin, Frederic Castinetti, Hans Kristian Moen Vollan, Catherine De Micco, Antoine Daunay, David Taieb, Peter Van Loo, Celine Besse, Vessela N Kristensen, Lise Lotte Hansen, Anne Barlier, Frederic Sebag, and Jörg Tost
Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.
S G Creemers, L J Hofland, E Korpershoek, G J H Franssen, F J van Kemenade, W W de Herder, and R A Feelders
Adrenocortical carcinoma (ACC) is a rare disease with a poor prognosis. Discrimination between ACCs and adrenocortical adenomas (ACAs) remains challenging, with the current gold standard being the Weiss score, consisting of several histopathological characteristics. However, new markers like Ki67, a marker for proliferation, and the staining of reticulins are promising not only as it comes to identifying malignancy but also as prognostic markers in patients with ACC. Currently, surgery is still the only curative treatment for ACC. Mitotane, an adrenolytic drug, is used in the adjuvant setting and in case of metastatic or advanced disease. Patients with progressive disease are frequently treated with mitotane, alone or in combination with etoposide, doxorubicine and cisplatin. Radiotherapy is indicated in selected cases. The low response rates and high toxicity of the systemic therapies emphasize the need for markers that enable the identification of responders and non-responders. Consequently, research is focusing on predictive factors varying from the expression of DNA repair genes to clinical patient characteristics. Subgroups of ACC with different prognosis have been identified based on transcriptome characteristics. As a conclusion from large molecular studies, ACCs appear to harbor many abnormalities compared to ACAs. Altered pathways driving ACC pathogenesis include the IGF, TP53 and the Wnt signaling pathway, allowing these as new potential targets for medical therapy. However, despite efforts in preclinical and clinical studies investigating efficacy of targeting these pathways, most novel therapies appear to be effective in only a subset of patients with ACC. New treatment concepts are therefore urgently needed.