Efficacy of oral metformin in a patient with metastatic adrenocortical carcinoma: Examination of mechanisms and therapeutic implications
Rare Tumors Volume 10: 1-7 C The Author(s) 2018 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/2036361317749645
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RD Peixoto1,2,3, LM Gomes4, TT Sousa1,2, DJ Racy4, M Shigenaga5 and RA Nagourney6
Abstract
Although rare, adrenocortical carcinoma is among the most common tumors found in children with Li-Fraumeni syndrome and Li-Fraumeni-like syndrome, associated with germ-line mutations in the TP53 gene. In southern Brazil, one form of Li-Fraumeni syndrome, associated with childhood adrenocortical carcinoma, is caused by a mutation in the R337H TP53 tetramerisation domain and is attributed to a familial founder effect. Adrenocortical carcinoma is considered an aggressive neoplasm, usually of poor prognosis and is generally unresponsive to systemic chemotherapy. Optimal treatment regimens remain to be established. We report the case of a young woman with metastatic adrenocortical carcinoma, who achieved stable disease with mitotane, cisplatin, doxorubicin, and etoposide as first-line therapy, but then had an objective response to oral metformin that lasted 9 months. The presence of the R337H TP53 mutation suggests a mechanism for the observed response to metformin.
Keywords
Adrenocortical carcinoma, metformin, Li-Fraumeni syndrome
Date received: 2 May 2017; accepted: 6 October 2017
Introduction
Adrenocortical carcinoma (ACC) is a rare endocrine malig- nancy with an estimated annual prevalence of 0.5-2 cases per million population.1 Most cases present as either locally advanced or metastatic disease.1 Despite aggressive multi- modality therapy, the prognosis of metastatic disease remains poor. The incidence of ACC is typically bimodal with most cases presenting in children <5 years of age or in adults in their fourth and fifth decades of life, affecting women slightly more often than men, with a ratio of 1.2- 1.5:1.2,3 Most ACC occurs as sporadic tumors, but a small percentage is associated with the rare hereditary Li-Fraumeni syndrome (LFS).4 Recognizing a 10-fold higher incidence of ACC in southern Brazil, Ribeiro and co-investigators identified a unique germ-line mutation in TP53 that encodes
for an arginine to histidine amino acid substitution at posi- tion 337.5 Unlike the more common TP53 mutations that cluster in the DNA binding and transactivational domains, the R337H mutation occurs in the tetramerisation domain
‘Hospital Alemão Oswaldo Cruz, São Paulo, Brazil 2Oncoclínicas, São Paulo, Brazil
3Universidade Nove de Julho, São Paulo, Brazil
4Hospital São José, São Paulo, Brazil
5Children’s Hospital Oakland Research Institute, Oakland, CA, USA 6Rational Therapeutics, Long Beach, CA, USA
Corresponding author:
RD Peixoto, Hospital Alemão Oswaldo Cruz, São Paulo, Rua Treze de Maio, 1815, São Paulo 01327-001, Brazil. Email: renatadalpino@gmail.com
HE
alpha inibina
Ki-67
resulting in a conditional mutant protein whose function is pH dependent.6,7
The pathogenesis of ACC is not completely understood. Similar to benign adrenal tumors, ACC can be either hor- monally active (functional) or hormonally inactive (non- functional) with approximately 70% of ACCs hormonally active, most often presenting with Cushing’s syndrome.8,9 When hormonally inactive, ACC commonly presents with abdominal pain.10 Complete surgical resection, when feasi- ble, is the optimal therapeutic strategy for improving sur- vival and offers the only possibility for cure.10 Despite treatment, patients with stage-IV ACC have a disease-spe- cific 5-year survival of only 13%.11 Stage-IV ACC is defined as metastatic disease as per the European Network for the Study of Adrenal Tumors (ENSAT) classification.11 The limited efficacy of standard-of-care therapy has led to the search for new treatment options. As metformin has become the focus of a growing number of oncology trials, this class of drugs offered a novel approach for the manage- ment of a patient with advanced ACC.
Case report
A 26-year-old previously healthy female sought medical attention in January 2015 with a 2-month history of weight gain, facial acne, and hirsutism. She was seen by an endocri- nologist who obtained a 24-h urinary cortisol at 483 mcg/mL (normal range: 10-100mcg/mL). Computed tomography (CT) scan of the thorax and abdomen revealed multiple lobu- lated pulmonary nodules of soft tissue density measuring between 5 and 15 mm as well as a solid heterogeneous mass in the left adrenal gland measuring 14×8×7cm, with dys- trophic calcifications, contrast enhancement, central area of necrosis, and extension into the left adrenal vein. The lesion was in contact with the superior kidney pole, displacing it inferiorly, but without signs of invasion. There were three nodular foci in the liver compatible with hemangiomas. Magnetic resonance imaging (MRI) of the abdomen con- firmed a solid lobulated mass in the left adrenal, measuring 14×9×7cm, with high signal on T2 sequences and heteroge- neous contrast enhancement. There was invasion into the left
adrenal vein with associated thrombosis. On 28 January 2015, left adrenalectomy with en bloc radical nephrectomy and adrenal vein tumor thrombus excision was performed. Postoperative recovery was uneventful. Histopathologic exam confirmed a 13× 10×5 cm ACC with solid and diffuse architecture pattern, high nuclear grade, mitotic rate of more than 15 per 10 high power fields with atypical mitosis, exten- sive necrosis, clear cell component of 25%, the presence of capsular invasion, and neoplastic invasion into the left adre- nal vein. Immunohistochemistry showed positivity for alpha-inhibin and synaptophysin, as well as a Ki-67 of 40% (Figure 1). The patient was first seen at our Oncology Center in February 2015. Due to the high mitotic count and Ki-67, she was started on mitotane combined with chemotherapy (doxorubicin, cisplatin, and etoposide). After three cycles, a positron emission tomography-CT (PET-CT) scan demon- strated stable disease. However, due to the side effects (espe- cially nausea, vomiting, malaise, and alopecia), the patient decided to stop chemotherapy.
To explore additional treatment options, the patient trav- elled to California and underwent a surgical biopsy of a peripheral lung nodule that provided viable tissue for the ex vivo analysis of programmed cell death (EVA/PCD). This phenotypic platform provides drug response profiles that have been shown to correlate significantly with response, time to progression, and survival.12 The platform has been the subject of prior review with methods described in previ- ous publications.13-16 In summary, sterile surgical speci- mens are submitted from the operating room in modified RPMI 1640 media. Following mechanical and enzymatic disaggregation, micro-spheroids of desired size (50-70 cells) are isolated by Ficoll-Hypaque density gradient. Cell suspensions are washed twice and adjusted to desired cell density with viability determined by initial Trypan blue. A volume of 90 uL of cell suspensions are then distributed into 96-well plates with cytotoxic agents distributed as 10 uL aliquots with continuous drug exposures for 72h. Cell death events are examined using various endpoints including adenosine triphosphate (ATP) content by lucif- erase, mitochondrial metabolism by 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), caspase
activation, and delayed loss of membrane integrity meas- ured by staining and morphology. As metformin actions are metabolic in nature, we utilize the membrane integrity end- point to measure its activity to prevent possible artifacts being introduced by non-lethal perturbations in mitochon- drial signal.
Phenformin had previously been selected in our plat- form as the index biguanide. Active concentration ranges (500-31 µM) have been determined in training sets and calibrated to provide clinically relevant signals in over 350 prior phenformin human tumor analyses. Five-point dose- response curves, in duplicate, were averaged and interpo- lated to provide the patient LC50 value of 39.7 uM. This fell in the most sensitive range for phenformin as deter- mined by modified Z-score. The results also identified activity for the cyclin-dependent kinase 4/6 inhibitor, pal- bociclib, and the phosphoinositol a-specific kinase inhibi- tor BYL-719 with moderate activity for everolimus and sunitinib. Phenformin was included in this patient’s tumor as a comparator molecule in ongoing research being con- ducted at the time on a structurally unrelated inhibitor of mitochondrial complex I (unreported observation). Based upon this favorable finding with phenformin, the structur- ally similar and clinically available biguanide, metformin, was recommended.
In June 2015, the patient was started on oral metformin at 500mg twice daily and continued on mitotane. After 3 months, a repeat CT scan of the thorax showed marked reduction in the size of the dominant nodules, with com- plete disappearance of several smaller nodules (Figures 2 and 3). The measurable response continued for approxi- mately 9 months.
With her family history positive for a retroperitoneal sarcoma (mother) and a lung cancer (grandmother), the patient was referred for genetic counseling and tested
positive for LFS as a pathogenic mutation was found in TP53: c.1010G>A (p.Arg337His) (het). Exon codons and the exon-intron boundaries of the CHEK2 (22q12.1) and TP53 (17p13.1) genes were amplified and processed for next-generation sequencing (NGS). The sequencing results were analyzed in the Variant Caller software (Ion Torrent; Life Technologies) and compared with the version of the GRCh37/HG19 genome. The nomenclature of the detected mutations was made following the recommendations of HUman Genome Organization (HUGO) and Human Genome Variation Society (HGVS). The reference sequences of the detected genes were NM_000546.5 (TP53) and NM_007194.3 (CHEK2). In this analysis, a pathogenic mutation was detected according to the infor- mation deposited in the consulted databases (Clin Var, dbSNP, and/or IARC LOVD).
Written informed consent for patient information and images was obtained from the patient before publication. Hospital Alemão Oswaldo Cruz does not require ethical approval for reporting individual cases or case series.
Discussion
Surgery is the only curative treatment for ACC. Despite aggressive treatment, 70%-85% of the patients who undergo adrenalectomy develop local recurrence or distant metastases resulting in a 5-year overall survival of 16%- 35% for those with complete resection which falls to less than 1 year for patients with incomplete resection.17 The most common sites of distant metastases are lungs and liver.17
Mitotane is currently a cornerstone in the management of metastatic ACC. Most experts recommend its use either as monotherapy or combined with cytotoxic chemotherapy.18,19 Although the optimal therapeutic strategy is not yet well
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established due to the rarity of this disease, the combination of etoposide, doxorubicin, and cisplatin (EDP) with or with- out mitotane is among the most widely used regimen. In a phase-II trial of 72 ACC patients,20 this combination pro- vided a response rate of 48%, while another small study reported response in 15 of 28 (53%) ACC patients.21 In a subsequent phase-III trial (FIRM-ACT), 304 patients were randomized to receive mitotane either in combination with EDP or with streptozocin. Patients in the EDP-mitotane group achieved a significantly higher response rate than those in the streptozocin-mitotane group (23.2% vs 9.2%, p<0.001) and longer median progression-free survival (5.0 months vs 2.1 months, p<0.001), although there was no significant difference in overall survival between the groups (14.8 months vs 12.0 months, p=0.07).22
The limited efficacy of available systemic chemother- apy has led to a search for new treatment options based on the underlying molecular mechanisms involved in ACC. Several targets and agents have been explored.23 Unfortunately, the epithelial growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib did not show efficacy as a single agent in ACC.24 Similarly, sunitinib exhibited only modest activity as a single agent in mitotane-exposed ACC patients.25 Other pathways such as those involving fibro- blast growth factor receptor (FGFR) and Wnt-B-catenin signaling cascades and loss of p53 function have been implicated in ACC tumorigenesis.26 The development of strategies targeting these pathways should be exploited in the future.
Insulin growth factor 2 (IGF-2) is known to be upregu- lated in ACC. IGF-2 signaling is mediated through its inter- action with the IGF-1 receptor (IGF-1R), which leads to downstream activation of mechanistic target of rapamycin (mTOR).26 Cixutumumab is a new, fully human IgG1 mon- oclonal antibody directed against the IGF-1R. An ongoing phase-I, dose-expansion study with 26 patients with heavily pretreated metastatic ACC is evaluating the combination of
temsirolimus and cixutumumab.27 Preliminary results indi- cate that 11 patients (42%) had durable (>6months) stable disease. Although there were no partial or complete responses, this trial suggests that there might be a role for targeting the IGF-2 pathway in ACC.27 However, an initial single-arm phase study for safety evaluation with cixutu- mumab and mitotane which should be followed by a rand- omized phase was terminated before randomization due to slow accrual and limited efficacy. Although therapeutic effects were observed in 8 of 20 patients, including one par- tial response and seven stable diseases, the relatively low response rate casts doubt on the efficacy of this regimen.28
The biguanides (metformin, phenformin, and buformin) are organic compounds, originally extracted from the French lilac (Galega officinalis), that were introduced into the treatment of diabetes mellitus in the 1950s. Metformin, the only commercially available biguanide, is a well-estab- lished and effective agent for the management of type 2 diabetes mellitus. Its hypoglycemic and insulin-lowering properties may play a role in its anticancer activity since insulin has mitogenic and pro-survival effects. In addition, tumor cells often express high levels of the insulin receptor, indicating a potential sensitivity to the growth-promoting effects of the hormone.29,30 Although the mechanisms of action of metformin are not fully understood, it has been suggested that inhibition of mitochondrial complex I may downregulate mTOR via the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK).31 The observed tumor regression in this case may also reflect inhibition of the IGF-2 pathway, as this is commonly upregulated in ACC.
Metformin has been shown to induce significant growth inhibitory effects in several cancer cell and mouse tumor models.32 In cell culture, metformin inhibits the prolifera- tion of a range of cancer cells including breast, endometrial, ovarian, prostate, colon, pancreatic, gastric, and glioma.32 The effects of metformin on cancer cell proliferation are
associated with AMPK activation, reduced mammalian tar- get of rapamycin (mTOR) signaling, and protein synthesis, as well as a variety of other responses including decreased EGFR, Src, and mitogen-activated protein kinase (MAPK) activation, decreased expression of cyclins, and increased expression of p27.32
P53-null (p53(-/-)) colon cancer cells are presumed to be susceptible to metformin-induced apoptosis owing to their inability to undergo the metabolic alterations imposed by metformin in the absence of p53 which is a crucial con- troller of several aspects of metabolism.33 In a study with HCT116 p53+/+ and p53-/- cell lines, metformin-treated cells compensated for the suppression of oxidative phos- phorylation (OXPHOS) by increasing their rate of glycoly- sis in a p53-dependent manner. It suggests that metformin treatment forces a metabolic conversion that p53(-/-) cells are unable to execute, highlighting the therapeutic potential of metformin in the treatment of p53-deficient tumors.33 In a mouse model of LFS that expresses mutant p53, met- formin inhibited OXPHOS mitochondrial respiration, pro- viding an intriguing evidence of how this drug may operate in inhibiting cancer progression in p53-inactivated tumors related to LFS.34 Mechanistically, inhibition of mitochon- drial function in this mouse model increased autophagy and decreased the aberrant proliferation signaling caused by mutant p53.34
Other investigators have reported that functional P53 is essential for the metformin to induce growth inhibition, senescence, and apoptosis in breast cancer cells.35 They used nutlin-3a and CP/31398 to reactivate p53 and enhance the anti-tumor effect of metformin. Results in prostate can- cer that combined metformin with 2-deoxyglucose also identified the p53 dependence of metformin-induced apop- tosis.36 This raises an alternative mechanism of action for the biguanides that may be operative in the patient reported.
In line with our findings of successful treatment with metformin, Italian investigators have previously reported the activity for metformin in two ACC cell lines, H295R and SW13.37 Using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo- lium (MTS) assay, they identified an LC50 in the H295R cell line between 23.8 and 58.6mM, similar to the LC50 identified in this patient’s primary culture of 39.6mM. In H295R, metformin was shown to induce inhibition of extracellular-signal-regulated kinase (ERK) 1/2 and mTOR, associated with the stimulation of AMPK that led to apop- tosis as measured by Annexin V and caspase-3, a reduction in Ki-67 and growth inhibition in a xenograft mouse model. Of interest, the H295R cell line carries p53 deletions in codons 8 and 9 that determine a frame shift at codon 261 resulting in a stop codon at position 271.38 In the H295R cell model, it appears that metformin exerts its actions inde- pendent of p53 function.
Recently, morphoproteomic analysis with the use of bright-field microscopy and immunohistochemistry directed
against various protein analytes was performed for a case of a young woman with chemoresistant ACC who had meta- static disease to the liver (resected) and left lung.39 Morphoproteomic profiling suggested a combination of metformin, melatonin, and a statin as maintenance therapy following resection, which she used for 2 years, when a new metastatic lesion was found in the right lung out and biop- sied. By comparison with the pretreatment specimen from the liver, the metastatic tumor from the right upper lobe of the lung with the patient on maintenance therapy with met- formin and melatonin showed molecular and morphometric evidence of growth inhibition. Whether this patient had LFS was not reported.39
Our case differs from the aforementioned report in which we have observed apoptotic morphology in our patient’s ACC cells in primary culture following 72h of exposure to phenformin, the closely related biguanide. It was on the basis of observed degree of apoptotic morphol- ogy in the patient’s tumor micro-spheroids that metformin was recommended for therapy. While the exact mecha- nisms by which metformin exerted its anti-tumor effects in this patient remain unclear, the choice of metformin repre- sents an interesting clinical application of a phenotypic platform for the process of drug selection. As this approach is mechanism agnostic, it has the capacity to discern clini- cally relevant drug activities regardless of their putative modes of action. In this instance, the end result, tumor cytolysis, provided the needed insight for treatment selec- tion resulting in the effective application of a novel and well-tolerated therapy for a patient with a rare malignancy for which few good treatment options exist. To our knowl- edge, this is the first report of response to metformin in a patient with metastatic ACC and LFS.
In conclusion, the course of our patient was remarkable for the means by which metformin was identified and for the impressive and durable degree of cytoreduction achieved with this simple, non-toxic intervention. Our results support a role for metformin in the treatment of met- astatic ACC, consistent with the studies reported by Poli et al.37 Clinical trials applying metformin in patients with ACC will enable investigators to better interrogate mecha- nism of action and the p53 dependence of metformin activ- ity in patient samples.
As ACC occurs at demonstrably higher incidence in the southern Brazilian provinces of Parana and Sao Paulo, met- formin may offer a unique and well-tolerated intervention for patients who carry the TP53 R337H mutation, and fur- ther studies in ACC are warranted. Discussions are under- way to investigate the clinical study of metformin in other patients in southern Brazil who are afflicted with this rare form of LFS-associated ACC.
Acknowledgements
Laboratórios Progenética, Hermes Pardini, Av. Presidente Vargas, 962 - 3o andar - Centro Rio de Janeiro - RJ - 20071- 002 - Fone
(21) 2540-5529 progenetica@progenetica.com.br - www.proge- netica.com.br
Conflict of interest
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
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