Cristina L. Ronchi

Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome

BACKGROUNDnCorticotropin-independent Cushings syndrome is caused by tumors or hyperplasia of the adrenal cortex. The molecular pathogenesis of cortisol-producing adrenal adenomas is not well understood.nnnMETHODSnWe performed exome sequencing of tumor-tissue specimens from 10 patients with cortisol-producing adrenal adenomas and evaluated recurrent mutations in candidate genes in an additional 171 patients with adrenocortical tumors. We also performed genomewide copy-number analysis in 35 patients with cortisol-secreting bilateral adrenal hyperplasias. We studied the effects of these genetic defects both clinically and in vitro.nnnRESULTSnExome sequencing revealed somatic mutations in PRKACA, which encodes the catalytic subunit of cyclic AMP-dependent protein kinase (protein kinase A [PKA]), in 8 of 10 adenomas (c.617A→C in 7 and c.595_596insCAC in 1). Overall, PRKACA somatic mutations were identified in 22 of 59 unilateral adenomas (37%) from patients with overt Cushings syndrome; these mutations were not detectable in 40 patients with subclinical hypercortisolism or in 82 patients with other adrenal tumors. Among 35 patients with cortisol-producing hyperplasias, 5 (including 2 first-degree relatives) carried a germline copy-number gain (duplication) of the genomic region on chromosome 19 that includes PRKACA. In vitro studies showed impaired inhibition of both PKA catalytic subunit mutants by the PKA regulatory subunit, whereas cells from patients with germline chromosomal gains showed increased protein levels of the PKA catalytic subunit; in both instances, basal PKA activity was increased.nnnCONCLUSIONSnGenetic alterations of the catalytic subunit of PKA were found to be associated with human disease. Germline duplications of this gene resulted in bilateral adrenal hyperplasias, whereas somatic PRKACA mutations resulted in unilateral cortisol-producing adrenal adenomas. (Funded by the European Commission Seventh Framework Program and others.).

Mutations in the deubiquitinase gene USP8 cause Cushing's disease

Cushings disease is caused by corticotroph adenomas of the pituitary. To explore the molecular mechanisms of endocrine autonomy in these tumors, we performed exome sequencing of 10 corticotroph adenomas. We found somatic mutations in the USP8 deubiquitinase gene in 4 of 10 adenomas. The mutations clustered in the 14-3-3 protein binding motif and enhanced the proteolytic cleavage and catalytic activity of USP8. Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling. USP8 mutants enhanced promoter activity of the gene encoding proopiomelanocortin. In summary, our data show that dominant mutations in USP8 cause Cushings disease via activation of EGF receptor signaling.

Linsitinib (OSI-906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomised, phase 3 study

BACKGROUNDnAdrenocortical carcinoma is a rare, aggressive cancer for which few treatment options are available. Linsitinib (OSI-906) is a potent, oral small molecule inhibitor of both IGF-1R and the insulin receptor, which has shown acceptable tolerability and preliminary evidence of anti-tumour activity. We assessed linsitinib against placebo to investigate efficacy in patients with advanced adrenocortical carcinoma.nnnMETHODSnIn this international, double-blind, placebo-controlled phase 3 study, adult patients with histologically confirmed locally advanced or metastatic adrenocortical carcinoma were recruited at clinical sites in nine countries. Patients were randomly assigned (2:1) twice-daily 150 mg oral linsitinib or placebo via a web-based, centralised randomisation system and stratified according to previous systemic cytotoxic chemotherapy for adrenocortical carcinoma, Eastern Cooperative Oncology Group performance status, and use of one or more oral antihyperglycaemic therapy at randomisation. Allocation was concealed by blinded block size and permuted block randomisation. The primary endpoint was overall survival, calculated from date of randomisation until death from any cause. The primary analysis was done in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00924989.nnnFINDINGSnBetween Dec 2, 2009, and July 11, 2011, 139 patients were enrolled, of whom 90 were assigned to linsitinib and 49 to placebo. The trial was unblinded on March 19, 2012, based on data monitoring committee recommendation due to the failure of linsitinib to increase either progression-free survival or overall survival. At database lock and based on 92 deaths, no difference in overall survival was noted between linsitinib and placebo (median 323 days [95% CI 256-507] vs 356 days [249-556]; hazard ratio 0·94 [95% CI 0·61-1·44]; p=0·77). The most common treatment-related adverse events of grade 3 or worse in the linsitinib group were fatigue (three [3%] patients vs no patients in the placebo group), nausea (two [2%] vs none), and hyperglycaemia (two [2%] vs none). No adverse events in the linsitinib group were deemed to be treatment related; one death (due to sepsis and megacolon) in the placebo group was deemed to be treatment related.nnnINTERPRETATIONnLinsitinib did not increase overall survival and so cannot be recommended as treatment for this general patient population. Further studies of IGF-1R and insulin receptor inhibitors, together with genetic profiling of responders, might pave the way toward individualised and improved therapeutic options in adrenocortical carcinoma.nnnFUNDINGnAstellas.

Major Prognostic Role of Ki67 in Localized Adrenocortical Carcinoma After Complete Resection

BACKGROUNDnRecurrence of adrenocortical carcinoma (ACC) even after complete (R0) resection occurs frequently.nnnOBJECTIVEnThe aim of this study was to identify markers with prognostic value for patients in this clinical setting.nnnDESIGN, SETTING, AND PARTICIPANTSnFrom the German ACC registry, 319 patients with the European Network for the Study of Adrenal Tumors stage I-III were identified. As an independent validation cohort, 250 patients from three European countries were included.nnnOUTCOME MEASUREMENTS AND STATISTICAL ANALYSISnClinical, histological, and immunohistochemical markers were correlated with recurrence-free (RFS) and overall survival (OS).nnnRESULTSnAlthough univariable analysis within the German cohort suggested several factors with potential prognostic power, upon multivariable adjustment only a few including age, tumor size, venous tumor thrombus (VTT), and the proliferation marker Ki67 retained significance. Among these, Ki67 provided the single best prognostic value for RFS (hazard ratio [HR] for recurrence, 1.042 per 1% increase; P < .0001) and OS (HR for death, 1.051; P < .0001) which was confirmed in the validation cohort. Accordingly, clinical outcome differed significantly between patients with Ki67 <10%, 10-19%, and ≥20% (for the German cohort: median RFS, 53.2 vs 31.6 vs 9.4 mo; median OS, 180.5 vs 113.5 vs 42.0 mo). Using the combined cohort prognostic scores including tumor size, VTT, and Ki67 were established. Although these scores discriminated slightly better between subgroups, there was no clinically meaningful advantage in comparison with Ki67 alone.nnnCONCLUSIONnThis largest study on prognostic markers in localized ACC identified Ki67 as the single most important factor predicting recurrence in patients following R0 resection. Thus, evaluation of Ki67 indices should be introduced as standard grading in all pathology reports of patients with ACC.

Mitotane Inhibits Sterol-O-Acyl Transferase 1 Triggering Lipid-Mediated Endoplasmic Reticulum Stress and Apoptosis in Adrenocortical Carcinoma Cells

Adrenocortical carcinoma (ACC) is a rare malignancy that harbors a dismal prognosis in advanced stages. Mitotane is approved as an orphan drug for treatment of ACC and counteracts tumor growth and steroid hormone production. Despite serious adverse effects, mitotane has been clinically used for decades. Elucidation of its unknown molecular mechanism of action seems essential to develop better ACC therapies. Here, we set out to identify the molecular target of mitotane and altered downstream mechanisms by combining expression genomics and mass spectrometry technology in the NCI-H295 ACC model cell line. Pathway analyses of expression genomics data demonstrated activation of endoplasmic reticulum (ER) stress and profound alteration of lipid-related genes caused by mitotane treatment. ER stress marker CHOP was strongly induced and the two upstream ER stress signalling events XBP1-mRNA splicing and eukaryotic initiation factor 2 A (eIF2α) phosphorylation were activated by mitotane in NCI-H295 cells but to a much lesser extent in four nonsteroidogenic cell lines. Lipid mass spectrometry revealed mitotane-induced increase of free cholesterol, oxysterols, and fatty acids specifically in NCI-H295 cells as cause of ER stress. We demonstrate that mitotane is an inhibitor of sterol-O-acyl-transferase 1 (SOAT1) leading to accumulation of these toxic lipids. In ACC tissue samples we show variable SOAT1 expression correlating with the response to mitotane treatment. In conclusion, mitotane confers adrenal-specific cytotoxicity and down-regulates steroidogenesis by inhibition of SOAT1 leading to lipid-induced ER stress. Targeting of cancer-specific lipid metabolism opens new avenues for treatment of ACC and potentially other types of cancer.

Expression of excision repair cross complementing group 1 and prognosis in adrenocortical carcinoma patients treated with platinum-based chemotherapy

Therapeutic progress in adrenocortical carcinoma (ACC) is severely hampered by its low incidence. Platinum-based chemotherapies are the most effective cytotoxic treatment regimens in ACC but response rates remain <50%. In other tumor entities, expression of excision repair cross complementing group 1 (ERCC1) predicts resistance to platinum compounds. Therefore, we correlated ERCC1 protein expression and clinical outcome. We have retrolectively established adrenal tissue microarrays and analyzed prospectively samples from 163 ACCs, 15 benign adrenal adenomas, and 8 normal adrenal glands by immunohistochemistry for ERCC1 protein expression. Detailed clinical data were available by the German ACC Registry. ERCC1 protein was highly expressed in all normal adrenal glands, 14 benign tumors (93%) and in 75 ACCs (47%). In ACC, no differences in baseline parameters were found between patients with and without ERCC1 expression. Detection of ERCC1 was not correlated with survival in patients who never received platinum-based chemotherapy. In platinum-treated patients (n=45), objective response to platinum compounds was observed in 3/21 patients (14.3%) with high ERCC1 expression and in 7/24 patients (29.2%) with low ERCC1 expression (P=0.23). ERCC1 expression was strongly correlated with overall survival after platinum treatment (median: eight months in patients with high ERCC1 versus 24 months in low ERCC1 expression, hazard ratio (HR) 2.95 (95% confidence interval (CI) 1.4-6.2), P=0.004). Multivariate analysis confirmed that high ERCC1 expression was a predictive factor for poor prognosis in platinum treated patients (HR 2.2, 95% CI 1.0-4.5, P=0.038). Our findings suggest that ERCC1 expression is the first factor for predicting survival in ACC patients treated with platinum-based chemotherapy.

Prognostic Role of Overt Hypercortisolism in Completely Operated Patients with Adrenocortical Cancer

BACKGROUNDnAlthough prognostic parameters are important to guide adjuvant treatment, very few have been identified in patients with completely resected adrenocortical carcinoma (ACC).nnnOBJECTIVEnTo assess the prognostic role of clinical symptoms of hypercortisolism in a large series of patients with completely resected ACC.nnnDESIGN, SETTING, AND PARTICIPANTSnA total of 524 patients followed at referral centers for ACC in Europe and the United States entered the study. Inclusion criteria were ≥18 yr of age, a histologic diagnosis of ACC, and complete surgery (R0). Exclusion criteria were a history of other malignancies and adjuvant systemic therapies other than mitotane.nnnINTERVENTIONnAll ACC patients were completely resected, and adjuvant mitotane therapy was prescribed at the discretion of the investigators.nnnOUTCOME MEASUREMENTS AND STATISTICAL ANALYSISnThe primary end point was overall survival (OS). The secondary end points were recurrence-free survival (RFS) and the efficacy of adjuvant mitotane therapy according to cortisol secretion.nnnRESULTS AND LIMITATIONSnOvert hypercortisolism was observed in 197 patients (37.6%). Patients with cortisol excess were younger (p=0.002); no difference according to sex and tumor stage was observed. The median follow-up of the series was 50 mo. After adjustment for sex, age, tumor stage, and mitotane treatment, the prognostic significance of cortisol excess was highly significant for both RFS (hazard ratio [HR]: 1.30; 95% confidence interval [CI], 1.04-2.62; p=0.02) and OS (HR: 1.55; 95% CI, 1.15-2.09; p=0.004). Mitotane administration was associated with a reduction of disease progression (adjusted HR: 0.65; 95% CI, 0.49-0.86; p=0.003) that did not differ according to the patients secretory status. A major limitation is that only symptomatic patients were considered as having hypercortisolism, thus excluding information on the prognostic role of elevated cortisol levels in the absence of a clinical syndrome.nnnCONCLUSIONSnClinically relevant hypercortisolism is a new prognostic factor in patients with completely resected ACC. The efficacy of adjuvant mitotane does not seem to be influenced by overt hypercortisolism.

Prognostic factors in stage III–IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study

BACKGROUNDnThe clinical course of advanced adrenocortical carcinoma (ACC) is heterogeneous. Our study aimed primarily to refine and make headway in the prognostic stratification of advanced ACC.nnnPATIENTS AND METHODSnPatients with advanced ENSAT ACC (stage III or stage IV) at diagnosis registered between 2000 and 2009 in the ENSAT database were enrolled. The primary end point was overall survival (OS). Parameters of potential prognostic relevance were selected. Univariate and multivariate analyses were carried out: model 1 before surgery; model 2 post-surgery.nnnRESULTSnFour hundred and forty-four patients with advanced ENSAT ACC (stage III: 210; stage IV: 234) were analyzed. After a median follow-up of 55.2 months, the median OS was 24 months. A modified ENSAT (mENSAT) classification was validated: stage III (invasion of surrounding tissues/organs or the vena renalis/cava) and stage IVa, IVb, IVc (2, 3 or >3 metastatic organs, including N, respectively). Two- or 5-year OS was 73%, 46%, 26% and 15% or 50%, 15%, 14% and 2% for stages III, IVa, IVb and IVc, respectively. In the multivariate analysis, mENSAT stages (stages IVa, IVb, or IVc, respectively) were significantly correlated with OS (P < 0.0001), as well as additional parameters: age ≥ 50 years (P < 0.0001), tumor- or hormone-related symptoms (P = 0.01 and 0.03, respectively) in model 1 but also the R status (P = 0.001) and Grade (Weiss >6 and/or Ki67 ≥ 20%, P = 0.06) in model 2.nnnCONCLUSIONnThe mENSAT classification and GRAS parameters (Grade, R status, Age and Symptoms) were found to best stratify the prognosis of patients with advanced ACC.

Single Nucleotide Polymorphism Array Profiling of Adrenocortical Tumors - Evidence for an Adenoma Carcinoma Sequence?

Adrenocortical tumors consist of benign adenomas and highly malignant carcinomas with a still incompletely understood pathogenesis. A total of 46 adrenocortical tumors (24 adenomas and 22 carcinomas) were investigated aiming to identify novel genes involved in adrenocortical tumorigenesis. High-resolution single nucleotide polymorphism arrays (Affymetrix) were used to detect copy number alterations (CNAs) and copy neutral losses of heterozygosity (cnLOH). Genomic clustering showed good separation between adenomas and carcinomas, with best partition including only chromosome 5, which was highly amplified in 17/22 malignant tumors. The malignant tumors had more relevant genomic aberrations than benign tumors, such as a higher median number of recurrent CNA (2631 vs 94), CNAs >100 Kb (62.5 vs 7) and CN losses (72.5 vs 5.5), and a higher percentage of samples with cnLOH (91% vs 29%). Within the carcinoma cohort, a precise genetic pattern (i.e. large gains at chr 5, 7, 12, and 19, and losses at chr 1, 2, 13, 17, and 22) was associated with a better prognosis (overall survival: 72.2 vs 35.4 months, P=0.063). Interestingly, >70% of gains frequent in beningn were also present in malignant tumors. Notch signaling was the most frequently involved pathway in both tumor entities. Finally, a CN gain at imprinted “IGF2” locus chr 11p15.5 appeared to be an early alteration in a multi-step tumor progression, followed by the loss of one or two alleles, associated with increased IGF2 expression, only in carcinomas. Our study serves as database for the identification of genes and pathways, such as Notch signaling, which could be involved in the pathogenesis of adrenocortical tumors. Using these data, we postulate an adenoma-carcinoma sequence for these tumors.

PKA catalytic subunit mutations in adrenocortical Cushing’s adenoma impair association with the regulatory subunit

We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.