Development and validation of a liquid chromatography coupled to a diode array detector (LC-DAD) method for measuring mitotane (DDD) in plasma samples.

INTRODUCTION: Mitotane (o,p'-DDD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration. OBJECTIVE: To develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples. METHOD: Drug-free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of mitotane (DDD). The p,p'-DDD was used as an Internal Standard (IS) and was added at 25.0 µg/mL concentration to all samples, standards and controls. Samples were submitted to protein precipitation with acetonitrile and then centrifuged. 50 uL of the supernatant was injected into an HPLC system coupled to a Diode Array Detector (DAD). DDD and IS were detected at 230 nm in a 12 min isocratic mode with a solvent mixture of 60 % acetonitrile and 40 % formic acid in water with 0.1 % pump mixed, at 0.6 mL/min flow rate, in a reversed-phase (C18) chromatographic column kept at 28°C. The sensitivity, selectivity, precision, presence of carry-over, recovery and matrix-effect, linearity, and method accuracy were evaluated. RESULTS: The present study's method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4'-DDD (internal standard) with retention times of 6.0 min, 6.4 mim, respectively, with resolutions higher than 1.0. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.00-50.00 µg/mL for mitotane (R2 > 0.9987 and a 97.80 %‒105.50 % of extraction efficiency). Analytical sensitivity was 0.98 µg/mL. Functional sensitivity (LOQ) was 1.00 µg/L, intra-assay and inter-assay coefficient of variations were less than 9.98 %, and carry-over was not observed for this method. Recovery ranged from 98.00 % to 117.00 %, linearity ranged from 95.00 % to 119.00 %, and high accuracy of 89.40 % to 105.90 % with no matrix effects or interference was observed for mitotane measurements. Patients' sample results were compared with previous measurements by the GC-MS method with a high correlation (r = 0.88 and bias = -10.20 %). CONCLUSION: DDD determination in plasma samples by the developed and validated method is simple, robust, efficient, and sensitive for therapeutic drug monitoring and dose management to achieve a therapeutic index of mitotane in patients with adrenocortical cancer.

Two-dimensional chromatography for enantiomeric analysis of mitotane and its metabolite o,p'-DDA in patients with adrenocortical carcinoma indicates enantioselective metabolism.

Mitotane is a chiral drug used to treat adrenocortical carcinoma, being metabolized to the o,p'-dichlorodiphenyl acetic acid (o,p'-DDA), also a chiral compound. Despite of its therapeutic significance, the overall ratios and enantiomers have not been known. In this study, we analyzed the enantiomers of mitotane and o,p'-DDA in the plasma of patients by a newly developed chiral-phase method employed in two-dimensional chromatography. Important differences were observed in the ratio of (S)/(R)-mitotane, which varied substantially from 1:1.2 to 1:10 whereas the (S)/(R)-o,p'-DDA ratio was relatively conserved, at approximately 2:1. These findings provide evidence for the enantioselective metabolism and provide a method for further analyses of mitotane and metabolites, which can explain the variation in the therapeutic response.

SEOM clinical guideline on heritable TP53-related cancer syndrome (2022).

Li-Fraumeni syndrome is caused by heterozygous germline pathogenic variants in the TP53 gene. It involves a high risk of a variety of malignant tumors in childhood and adulthood, the main ones being premenopausal breast cancer, soft tissue sarcomas and osteosarcomas, central nervous system tumors, and adrenocortical carcinomas. The variability of the associated clinical manifestations, which do not always fit the classic criteria of Li-Fraumeni syndrome, has led the concept of SLF to extend to a more overarching cancer predisposition syndrome, termed hereditable TP53-related cancer syndrome (hTP53rc). However, prospective studies are needed to assess genotype-phenotype characteristics, as well as to evaluate and validate risk-adjusted recommendations. This guideline aims to establish the basis for interpreting pathogenic variants in the TP53 gene and provide recommendations for effective screening and prevention of associated cancers in carrier individuals.

[Adrenocortical carcinoma in women of reproductive age]. / Carcinoma adrenocortical en mujeres en edad fértil.

Adrenocortical carcinomas are rare tumors, usually hyperfunctioning, with poor overall survival. Frequent age of presentation is described in adults between 40 and 60 years of age, predominantly female. Two unusual cases of adrenal carcinoma diagnosed in young women are presented. The first one was discovered in the second trimester of gestation, with signs and symptoms of hypercortisolism and localized adrenal lesion, which was resolved with complete resection by week 20 of pregnancy. In the second case, the patient begined with clinical manifestations of rapidly progressive virilization, the biochemical pattern being pure hyperandrogenism. In both cases, despite complete resection, the high Ki67 as the main prognostic factor leaded to categorization as "high risk of recurrence". In addition, pregnancy and glucocorticoid secretory pattern have been associated as additional risk factors of recurrence. This is particularly high within the first two years after diagnosis. There is controversy about the use of adjuvant mitotane in these patients, and the general recommendation is to be started no longer than 3 months after surgery. However, the available evidence does not suggest that its use is ineffective beyond that period. Limitations, such as the course of pregnancy and the immediate puerperium, as well as the difficulty of accessing this medication in our environment, prevented the early initiation of adjuvant treatment with mitotane in both cases, although there is still concern whether its administration would still be appropriate.

Los carcinomas adrenocorticales son tumores infrecuentes, habitualmente hiperfuncionantes y con una supervivencia global pobre. La edad frecuente de presentación se describe en adultos entre 40 a 60 años, con predominio en sexo femenino. Se presentan dos casos inusuales de carcinoma adrenal diagnosticados en mujeres en edad fértil. El primero de ellos se descubrió en el segundo trimestre de gestación, con un cuadro de hipercortisolismo y lesión adrenal localizada, que resolvió con resección completa hacia la semana 20. En el segundo, la paciente debutó con manifestaciones clínicas de virilización rápidamente progresiva, siendo el hiperandrogenismo puro el patrón bioquímico hallado. En ambos casos, a pesar de haberse realizado la resección completa, el Ki67 elevado como principal factor pronóstico condujo a categorizarlas como de "alto riesgo de recurrencia". Asimismo, se ha asociado a la gestación y al patrón secretor de glucocorticoides como factores adicionales de mayor riesgo de recurrencia. Este es particularmente elevado dentro de los dos primeros años posteriores al diagnóstico. Existe aún controversia sobre el uso de mitotane adyuvante en estos pacientes, y su inicio está recomendado hasta los tres meses del postquirúrgico. Sin embargo, la evidencia disponible no permite suponer la falta de eficacia si se utiliza fuera de ese período. Los limitantes, como fueron el curso de la gestación y el puerperio inmediato, así como la dificultad para el acceso a la medicación en nuestro medio, impidieron el inicio precoz del tratamiento adyuvante en ambos casos, aunque surge la inquietud de si aún sería oportuna su instauración.

Rectal leiomyosarcoma as the initial phenotypic manifestation of Li-Fraumeni-like syndrome: a case report and review of the literature.

BACKGROUND: Leiomyosarcoma is a rare malignant tumor of smooth muscle origin and represents 10-20% of all soft tissue sarcomas. Primary colon and rectal sarcomas constitute < 0.1% of all large bowel malignancies. In Li-Fraumeni syndrome, sarcomas are the second most frequent cancer (25%). Li-Fraumeni syndrome is a genetic disease with a familial predisposition to multiple malignant neoplasms. This syndrome has an autosomal dominant pattern of inheritance and high penetrance characterized by germline TP53 mutations. Patients with a history of cancer who do not meet all the "classic" criteria for Li-Fraumeni syndrome are considered to have Li-Fraumeni-like syndrome. To the best of our knowledge, this article is the first report of a patient with rectal leiomyosarcoma as the initial phenotypic manifestation of Li-Fraumeni-like syndrome. The authors also present a literature review. CASE PRESENTATION: A 67-year-old Brazilian woman underwent anterior rectosigmoidectomy and panhysterectomy secondary to rectal leiomyosarcoma. She subsequently developed carcinomatosis and died 2 years after the operation. Her family medical history consisted of a daughter who died at 32 years of age from breast cancer, a granddaughter diagnosed with adrenocortical carcinoma at 6 years of age and two siblings who died from prostate cancer. A genetic study was carried out to identify a pathogenic variant of Li-Fraumeni syndrome. In the DNA extracted from the peripheral blood leukocyte, restriction fragment length polymorphism was analyzed to search for mutations in the TP53 gene. The DNA sequencing identified the germline pathogenic variant p. R337H heterozygous in exon 10 of TP53. The patient was classified as having Li-Fraumeni-like syndrome. CONCLUSION: In patients with rectal leiomyosarcoma, it is advisable to investigate the family history of cancer and perform genetic studies to screen for Li-Fraumeni syndrome.

Vitamin D receptor activation is a feasible therapeutic target to impair adrenocortical tumorigenesis.

OBJECTIVE: To evaluate the therapeutic potential of vitamin D receptor (VDR) signaling in adrenocortical carcinoma (ACC) cells. METHODS: We evaluated VDR's methylation pattern in H295R ACC cells, and investigated the effects of calcitriol and seocalcitol treatments on adrenocortical tumorigenesis. RESULTS: VDR was hypermethylated and underexpressed in basal H295R cells. Treatments with calcitriol and seocalcitol restored VDR signaling, resulted in antiproliferative effects, and impaired Wnt/B-catenin signaling. RNAseq of treated cells demonstrated VDR activation on steroid hormones biosynthesis and Rap1 signaling, among others. In vivo, seocalcitol constrained the growth of H295R xenografts and reduced autonomous tumor steroid secretion without hypercalcemia-associated side effects. CONCLUSIONS: H295R cells present VDR hypermethylation, which can be responsible for its underexpression and signaling inactivation under basal conditions. VDR signaling promoted antiproliferative effects in vitro and in vivo, suggesting that it may be a potential therapeutic target for ACC and a valuable tool for patient's clinical management.

N-type calcium channel v2.2 is a target of TCF21 in adrenocortical carcinomas.

Transcription factor 21 (TCF21) directly binds and regulates SF1 mRNA expression in tumor and normal adrenocortical cells, and both are involved in the development and steroidogenesis of the adrenal cortex. TCF21 is a tumor suppressor gene and its expression is reduced in malignant tumors. In adrenocortical tumors, it is less expressed in adrenocortical carcinomas (ACC) than in adrenocortical adenomas (ACA) and normal tissues. However, a comprehensive analysis to identify TCF21 targets has not yet been conducted in any type of cancer. In this study, we performed Chromatin Immunoprecipitation and Sequencing (ChIP-Seq) in an adrenocortical carcinoma cell line (NCI-H295R) overexpressing TCF21, with the aim of identifying TCF21 new targets. The five most frequently identified sequences corresponded to the PRDM7, CNTNAP2, CACNA1B, PTPRN2, and KCNE1B genes. Validation experiments showed that, in NCI-H295R cells, TCF21 negatively regulates the expression of the CACNA1B gene. Recently, it was observed that the N-type calcium channel v2.2 (Cav2.2) encoded by the CACNA1B gene is important in Angiotensin II signal transduction for corticosteroid biosynthesis in NCI-H295R adrenocortical carcinoma cells. Indeed, TCF21 inhibits CACNA1B and Cav2.2 expression in NCI-H295R. In addition, in a cohort of 55 adult patients with adrenocortical tumors, CACNA1B expression was higher in ACC than ACA and was related to poor disease-free survival in ACC patients. These results suggest a mechanism of steroidogenesis control by TCF21 in adrenocortical tumor cells, in addition to the control observed through SF1 inhibition. Importantly, steroid production could impair tumor immunogenicity, contributing to the immune resistance described in adrenal cancer.

Histopathological Criteria for Paediatric Adrenocortical Carcinoma.

INTRODUCTION: Adrenocortical carcinoma (ACC) is diagnosed in paediatric patients at 5 months after symptom onset on average, and 38% die during the first 2.5 years of follow-up. This study aimed to compare the accuracy of Weiss, Van Slooten, and Wieneke histopathological ACC classifications for predicting follow-up prognosis in a paediatric population. METHODS: Data were retrieved from medical records of 57 patients aged <18 years who underwent surgical treatment for ACC with surgical follow-up over 6 months or death due to ACC. They were classified into either good (without recurrence/death due to ACC) or poor (with recurrence/death due to ACC) prognosis group. Two expert pathologists classified the ACC surgical specimens according to the Weiss, Van Slooten, and Wieneke criteria. RESULTS: The median follow-up duration was 126 (18-225) months in 38 males (66.7%) and 19 females (33.3%) (median age: 3 [1-6.5] years). The good prognosis group was younger than the poor prognosis group (median age: 3 [1.5-6.2] years vs. 5 [2-10] years). Seventeen (29.8%) patients in the poor prognosis group died due to ACC within the first 50 months of surgical follow-up; the earliest death occurred in the fourth follow-up month, and the majority of deaths occurred within 24 months of follow-up. The accuracies of Weiss, Van Slooten, and Wieneke classification systems were 40%, 47%, and 77%, respectively. DISCUSSION/CONCLUSION: The Wieneke classification showed the best accuracy but was not sufficiently precise to establish reliable prognosis for ACC in the paediatric population. The Wieneke classification had approximately 95% sensitivity and negative predictive value.

Carcinoma corticosuprarrenal virilizante en un niño / Virilizing adrenocortical carcinoma in a child

Introducción: Los tumores suprarrenales en niños son poco frecuentes y el carcinoma suprarrenal representa menos de un 10 %. En el prepúber, la manifestación más típica es el desarrollo de pubertad precoz. Objetivo: Describir las características clínicas, los procederes diagnósticos y terapéuticos de un paciente con carcinoma adrenal en edad pediátrica. Presentación de caso: Paciente de 8 años, masculino y de piel blanca con antecedentes de salud. Acude a la consulta por crecimiento de vello pubiano y aumento del pene en longitud y grosor de aproximadamente 2 años de evolución. En el examen físico se constatan aumento de la velocidad de crecimiento y signos sugestivos de virilización (voz gruesa, vello axilar, vello sexual púbico y genitales externos estadio III de Tanner). Se realizaron estudios hormonales que corroboraron el hiperandrogenismo por secreción endógena autónoma, con niveles de gonadotropinas suprimidas, niveles de testosterona y dehidroepiandrosterona elevados. También se realizaron estudios imagenológicos que evidenciaron edad ósea acelerada y la existencia de un tumor. Se realizó una adrenalectomía izquierda y se confirmó por anatomía patológica el carcinoma corticosuprarrenal virilizante izquierdo en estadío 2. Inició un tratamiento con quimioterapia por dicho diagnóstico y actualmente se mantiene en seguimiento. Conclusiones: Los carcinomas corticosuprarrenales en niños son mayoritariamente funcionantes y constituyen una de las causas de pubertad precoz periférica. Estos son infrecuentes y agresivos, por lo que la realización de estudios genéticos en familias con síndromes hereditarios contribuiría a su diagnóstico precoz para un adecuado tratamiento y mejor pronóstico(AU)

Introduction: Adrenal tumors in children are rare and adrenal carcinoma represents less than the 10 percent. In the prepubescent, the most typical manifestation is the development of early puberty. Objective: Describe the clinical characteristics and diagnostic and therapeutic procedures of a patient with adrenal carcinoma in a pediatric age. Case presentation: 8-year-old male, white-skinned patient with a history of health conditions. He attentds to the consultation due to pubic hair growth and penis enlargement in length and thickness of approximately 2 years of evolution. Physical examination shows increased growth rate and signs suggestive to virilization (deep voice, axillary hair, pubic sexual hair and external genitalia in Tanner's stage III). Hormonal studies were carried out that corroborated hyperandrogenism by autonomic endogenous secretion, with suppressed gonadotropin levels, elevated testosterone and dehydroepiandrosterone levels. Imaging studies were also performed that showed accelerated bone age and the existence of a tumor. A left adrenalectomy was performed and stage 2 left virilizing adrenocrotical carcinoma was confirmed by pathological anatomy studies. He began chemotherapy treatment for this diagnosis and is currently being followed up. Conclusions: Adrenocortical carcinomas in children are mostly functioning and are one of the causes of peripheral early puberty. These are uncommon and aggressive, so genetic studies in families with hereditary syndromes would contribute to their early diagnosis for adequate treatment and better prognosis(AU)

Vitamin D receptor hypermethylation as a biomarker for pediatric adrenocortical tumors.

Objective: Pediatric adrenocortical tumors (pACT) display complex genomic backgrounds, lacking robust prognostic markers and targeted therapeutic options. Vitamin D3 receptor (VDR) promoter hypermethylation and underexpression were reported in adrenocortical carcinomas from adult patients. In this study, we aimed to investigate VDR expression levels and methylation status in pACT and their clinical and prognostic significance. Design: Retrospective cross-sectional study enrolling pediatric patients with ACT from two tertiary referral institutions. Methods: We evaluated clinicopathological features, VDR mRNA (qPCR) and protein (immunohistochemistry) expression, and VDR-wide methylation of ACT samples from 108 pediatric patients. Fourteen pediatric and 32 fetal and postnatal normal adrenals were used as controls. Results: Unlike in pre- and post-natal normal adrenals, most pACT lacked nuclear VDR expression and had reduced mRNA levels, especially the carcinomas. Unsupervised analysis of VDR methylation data revealed two groups of pACT with distinct disease features and outcomes. Tumors with high VDR methylation presented lower mRNA levels, and the respective patients presented advanced disease and reduced disease-free and overall survival. Conclusions: VDR has a role in normal adrenocortical development and homeostasis, which is impaired during tumorigenesis. VDR hypermethylation and underexpression may be both predictive and prognostic biomarkers for pACT.

Cytoreductive Surgery of the Primary Tumor in Metastatic Adrenocortical Carcinoma: Impact on Patients' Survival.

CONTEXT: The role of cytoreduction of adrenocortical carcinoma (ACC) remains poorly understood. OBJECTIVE: To analyze the impact of cytoreductive surgery of the primary tumor in patients with metastatic ACC. DESIGN AND SETTING: We performed a multicentric, retrospective paired cohort study comparing the overall survival (OS) in patients with metastatic ACC who were treated either with cytoreductive surgery (CR group) or without cytoreductive surgery (no-CR group) of the primary tumor. Data were retrieved from 9 referral centers in the American-Australian-Asian Adrenal Alliance collaborative research group. PATIENTS: Patients aged ≥18 years with metastatic ACC at initial presentation who were treated between January 1, 1995, and May 31, 2019. INTERVENTION: Performance (or not) of cytoreductive surgery of the primary tumor. MAIN OUTCOME AND MEASURES: A propensity score match was done using age and the number of organs with metastasis (≤2 or >2). The main outcome was OS, determined from the date of diagnosis until death or until last follow-up for living patients. RESULTS: Of 339 patients pooled, 239 were paired and included: 128 in the CR group and 111 in the no-CR group. The mean follow-up was 67 months. Patients in the no-CR group had greater risk of death than did patients in the CR group (hazard ratio [HR] = 3.18; 95% CI, 2.34-4.32). Independent predictors of survival included age (HR = 1.02; 95% CI, 1.00-1.03), hormone excess (HR = 2.56; 95% CI, 1.66-3.92), and local metastasis therapy (HR = 0.41; 95% CI, 0.47-0.65). CONCLUSION: Cytoreductive surgery of the primary tumor in patients with metastatic ACC is associated with prolonged survival.

Adrenocortical carcinoma, case report.

The adrenocortical carcinoma is rare and aggressive. It has a bimodal presentation, predominantly female, > 20% of cases will be diagnosed incidentally. 43-year-old male, with colic pain in the left flank, weight loss and intermittent fever. Computed tomography with a tumor on the left adrenal with liver metastases, block resection surgery was performed, pathological report of adrenocortical carcinoma with a 7 points of Weiss score and Ki67 40%. Adrenocortical carcinoma is a rare and aggressive neoplasm; the clinical presentation is variable. Systemic therapy is important even in patients with localized disease and independent of surgical approach.

El carcinoma corticoadrenal es una neoplasia rara, altamente agresiva, de distribución bimodal, con predominio en el sexo femenino, de la cual el 20% de los casos se diagnostican de manera incidental. Se presenta el caso de un varón de 43 años con dolor de tipo cólico en el flanco izquierdo, pérdida de peso y fiebre intermitente. La tomografía computarizada mostró un tumor en la glándula suprarrenal izquierda y metástasis hepática. Se realizó adrenalectomía radical en bloque, con reporte anatomopatológico de carcinoma corticoadrenal, Weiss de 7 puntos y Ki67 40%. El carcinoma corticoadrenal es una neoplasia agresiva y de presentación clínica variable. La terapia sistémica es importante incluso en pacientes con enfermedad localizada e independientemente de la cirugía.

MSI2 expression in adrenocortical carcinoma: Association with unfavorable prognosis and correlation with steroid and immune-related pathways.

Adrenocortical carcinoma (ACC) is a rare, but highly aggressive cancer of the adrenal cortex with a generally poor prognosis. Despite being rare, completely resected ACCs present a high risk of recurrence. Musashi-2 (MSI2) has recently been recognized as a potential prognostic biomarker and therapeutic target in many cancers. However, no studies have evaluated the clinical significance of MSI2 expression in ACC. Here, we addressed MSI2 expression and its association with ACC prognosis and clinicopathological parameters. MSI2 expression was analyzed in TCGA, GSE12368, GSE33371, and GSE49278 ACC datasets; and its correlation with other genes and immune cell infiltration were investigated by using the R2: Genomics Analysis and Visualization Platform and TIMER databases, respectively. Enrichment analysis was performed with the DAVID Functional Annotation Tool. Kaplan-Meier curves, log-rank tests, and Cox regression analyses were used to explore the prognostic role of MSI2 in ACC. Our findings demonstrated the potential value of MSI2 overexpression as an independent predictor of poor prognosis in patients with completely resected ACC (hazard ratio 6.715, 95% confidence interval 1.266 - 35.620, p =.025). In addition, MSI2 overexpression was associated with characteristics of unfavorable prognosis, such as cortisol excess (p = .002), recurrence (p =.003), and death (p =.015); positively correlated with genes related to steroid biosynthesis (p < .05); and negatively correlated with immune-related pathways (p < .05). Our findings demonstrate that MSI2 has value as a prognostic marker for completely resected ACC and reinforce the investigation of its role as a possible therapeutic target for patients with ACC.

Carcinoma adrenocortical: Presentación y desenlaces en una institución oncológica / Adrenocortical carcinoma: Presentation and outcomes at an oncological institute

Resumen Introducción: El carcinoma adrenocortical es una neoplasia endocrina infrecuente pero con un comportamiento altamente agresivo y pobre pronóstico. Dado su baja prevalencia, la experiencia de los centros de referencia es fundamental para aumentar el conocimiento de esta entidad. Métodos: Se elaboró una serie de casos de pacientes con carcinoma adrenocortical, tratados en una institución oncológica de referencia entre enero de 2007 y diciembre de 2017. Se describieron las características clínicas e histopatológicas de los pacientes. Se estimó el tiempo de supervivencia libre de progresión y el tiempo de supervivencia global (SG) de forma gráfica y con funciones de tiempo al evento mediante la función de Kaplan-Meier. Resultados: Se identificaron 19 pacientes, 14 de los cuales fueron mujeres con edad media al diagnóstico de 43.4 años (rango 20 - 65). El 58% de los pacientes tuvo secreción hormonal, siendo el síndrome de Cushing el predominante. 7 pacientes tuvieron compromiso metastásico al momento del diagnóstico. Todos los pacientes fueron llevados a adrenalectomía y el estado postquirúrgico en 10 pacientes fue R0. Al final del periodo de estudio, 11 pacientes estaban vivos. La mediana de supervivencia libre de progresión fue de 18 meses +/- 7.86 y la mediana de supervivencia global fue de 30 meses +/-19.80. Conclusión: En la población de pacientes analizada, se encontraron desenlaces de supervivencia libre de progresión y supervivencia global similares a lo reportado en centros de alta experiencia en patología adrenal.

Abstract Introduction: Adrenocortical carcinoma is a rare endocrine neoplasm, but with highly aggressive behavior and a poor prognosis. Given its low prevalence, the experience of reference centers is essential to characterize the factors associated with this disease. Methods: It is a case series of patients with adrenocortical carcinoma, treated at a reference oncology institute between January 2007 and December 2017. The clinical and histopathological characteristics of patients are described. Progression-free survival and overall survival (OS) were estimated graphically and with time-to-event data using the Kaplan-Meier function. Results: 19 patients were identified; 14 of them were women with a mean age at diagnosis of 43.4 years (range 20-65). 58% of the patients had hormone secretion, with Cushing's syndrome being the predominant one. 7 patients had metastatic compromise at the time of diagnosis. All patients underwent adrenalectomy, and R0 was the post-surgical status in 10 of them. At the end of the study period, 11 patients were alive. The median progression-free survival was 18 months +/- 7.86, and the median overall survival was 30 months +/- 19.8. Conclusion: In the analyzed patient population, outcomes of progression-free survival and overall survival were similar to that reported at centers with extensive experience in adrenal disease.

Adrenocortical Carcinoma Steroid Profiles: In Silico Pan-Cancer Analysis of TCGA Data Uncovers Immunotherapy Targets for Potential Improved Outcomes.

Despite progress in understanding the biology of adrenocortical carcinoma (ACC), treatment options have not dramatically changed in the last three decades, nor have we learned how to avoid some of its long-term side effects. Our goal was to improve the understanding of immune pathways that may include druggable targets to enhance immune responses of patients with ACC, focusing on immune evasion and the activation of immune cells against ACC. Our strategy was aimed at improving insight regarding gene expression without steroid interference. Using approaches based on high and low steroid phenotypes (HSP and LSP, respectively), we characterized immune pathways using The Cancer Genome Atlas (TCGA) ACC cohort data. Although previous studies have suggested that patients with ACC receive minimal benefit from immunotherapy, high expression of immune modulators was noted in patients with LSP, suggesting the activation of these biomarkers may be an important adjuvant therapy target after clearance of excess glucocorticoids. In addition, patients with LSP ACC had higher immune cell infiltration than patients with HSP ACC and other cancer subtypes. Our findings can be summarized as follows (1): we confirmed and improved the definition of two immune response pathways to ACC (HSP and LSP) based on in silico transcriptome analysis (2), we demonstrated the steroid profile should be considered, otherwise analyses of ACC immune characteristics can generate confounding results (3), among the overexpressed immunotherapy targets, we demonstrated that LSP was rich in PDCD1LG2 (PD-L2) and both HSP and LSP overexpressed CD276 (B7-H3), which was associated with resistance to anti-PD1 therapy and may have accounted for the modest results of previous clinical trials, and (4) identification of patients with LSP or HSP ACC can be used to help determine whether immunotherapy should be used. In conclusion, we highlighted the differences between LSP and HSP, drawing attention to potential therapeutic targets (CD276, PDCD1, and PDCD1LG2). Treatments to reduce immune evasion, as well as the use of other natural and pharmacological immune activators, should include prior pharmacological inhibition of steroidogenesis. Attempts to combine these with tumor cell proliferation inhibitors, if they do not affect cells of the immune system, may produce interesting results.

In vitro cytotoxic effect of a chitin-like polysaccharide produced by Mortierella alpina on adrenocortical carcinoma cells H295R, and its use as mitotane adjuvant.

This study presents an in vitro evaluation of the antitumor potential of a chitin-like exopolysaccharide (EPS, produced by Mortierella alpina) on Adrenocortical carcinoma cells (ACC) compared to mitotane, a commercial drug commonly used in ACC treatment, and known for its side effects. Techniques of cellular viability determination such as MTT and fluorescence were used to measure the cytotoxic effects of the EPS and mitotane in tumoral cells (H295R) and non-tumoral cells (VERO), observing high cytotoxicity of mitotane and a 10% superior pro-apoptotic effect of the EPS compared to mitotane (p < 0.05). The cytotoxic effect of the EPS was similar to the effect of 50 µM mitotane on tumoral cells (p < 0.05). A decrement of the lysosomal volume was also noted in tumoral cells treated with the EPS. To enhance the antitumor effect, a combination of mitotane at a lower dosage and the EPS (as adjuvant) was also tested, showing a slight improvement of the cytotoxicity effect on tumoral cells. Therefore, the results indicate a cytotoxic effect of the EPS produced by Mortierella alpina on adrenocortical carcinoma, and a possible application in biomedical formulations or additional treatments.

Clinical Impact of Pathological Features Including the Ki-67 Labeling Index on Diagnosis and Prognosis of Adult and Pediatric Adrenocortical Tumors.

Adrenocortical tumors (ACT) in the adult and pediatric population are generally considered distinct entities due to differences in molecular events related to tumorigenesis, clinical presentation, and outcome. Furthermore, pathological criteria used for diagnosis and prognostication of ACT in adults are usually inadequate for predicting the biological behavior of ACT in children. Here, we analyzed 146 adult and 44 pediatric (< 15y/o) ACT with long-term clinical follow-up and furthered current evidence on the clinical and pathological differences between pediatric and adult tumors. Predilection for female over male gender was observed in both cohorts, but more so in adults (84% vs. 61%, p = 0.003). Cushing syndrome was more frequent in adults (p < 0.001), whereas virilization, either isolated (p < 0.001) or combined to Cushing (p = 0.047), was more common in children. The Ki67 labelling index (LI) of pediatric adenomas and carcinomas was much higher than their corresponding tumors in adults (p < 0.001). Despite these differences, pathological analyses including the evaluation of Ki67 greatly improved patient prognostication in both age cohorts. Indeed, increased Weiss scores and Ki67 indexes correlated with poor overall- and disease-free survival in adult patients with carcinoma. Among the proliferative indexes tested, Ki67 LI ≥ 10% showed the highest hazard ratio (HR) for recurrence and the Ki67 LI ≥ 3% showed the highest HR for survival. In pediatric tumors, the Wieneke score (p < 0.001) and the Ki67 LI (p < 0.001) showed high accuracy for predicting biological behavior, and increased scores/indexes correlated with worse overall and disease-free survival. In this age cohort, Ki67 LI < 10% was able to rule out malignant behavior, whereas Ki67 LI ≥ 15% may be used to predict the patients with higher risks of recurrence and/or poor outcome.

Low Protein Expression of both ATRX and ZNRF3 as Novel Negative Prognostic Markers of Adult Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignancy that is associated with a dismal prognosis. Pan-genomic studies have demonstrated the involvement of ATRX and ZNRF3 genes in adrenocortical tumorigenesis. Our aims were to evaluate the protein expression of ATRX and ZNRF3 in a cohort of 82 adults with ACC and to establish their prognostic value. Two pathologists analyzed immuno-stained slides of a tissue microarray. The low protein expression of ATRX and ZNRF3 was associated with a decrease in overall survival (OS) (p = 0.045, p = 0.012, respectively). The Cox regression for ATRX protein expression of >1.5 showed a hazard ratio (HR) for OS of 0.521 (95% CI 0.273-0.997; p = 0.049) when compared with ≤1.5; for ZNRF3 expression >2, the HR for OS was 0.441 (95% CI, 0.229-0.852; p = 0.015) when compared with ≤2. High ATRX and ZNRF3 protein expressions were associated with optimistic recurrence-free survival (RFS) (p = 0.027 and p = 0.005, respectively). The Cox regression of RFS showed an HR of 0.332 (95%CI, 0.111-0.932) for ATRX expression >2.7 (p = 0.037), and an HR of 0.333 (95%CI, 0.140-0.790) for ZNRF3 expression >2 (p = 0.013). In conclusion, low protein expression of ATRX and ZNRF3 are negative prognostic markers of ACC; however, different cohorts should be evaluated to validate these findings.