New Prognostic Indicators in Pediatric Adrenal Tumors: Neuroblastoma and Adrenal Cortical Tumors, Can We Predict When These Will Behave Badly?

Pediatric adrenal tumors are unique entities with specific diagnostic, prognostic, and therapeutic challenges. The adrenal medulla gives rise to peripheral neuroblastic tumors (pNTs), pathologically defined by their architecture, stromal content, degree of differentiation, and mitotic-karyorrhectic index. Successful risk stratification of pNTs uses patient age, stage, tumor histology, and molecular/genetic aberrations. The adrenal cortex gives rise to adrenocortical tumors (ACTs), which present diagnostic and prognostic challenges. Histologic features that signify poor prognosis in adults can be meaningless in children, who have superior outcomes. The key clinical, pathologic, and molecular findings of pediatric ACTs have yet to be completely identified.

Intratumoral heterogeneity of the tumor cells based on in situ cortisol excess in cortisol-producing adenomas; ∼An association among morphometry, genotype and cellular senescence∼.

Cortisol-producing adrenocortical adenomas (CPAs) are associated with ACTH-independent Cushing's syndrome and histologically composed of two cellular subtypes: compact (lipid-poor) and clear (lipid-rich) tumor cells. However, the details of hormonal and biological activities of these tumor cells have remained unknown, especially in CPAs. CPAs frequently harbored unique histological features different from those of aldosterone-producing adenomas (APAs) including a senescent phenotype. Therefore, we explored the association between morphological features and the immunoreactivity of steroidogenic enzymes in CPAs with different genotypes and compared them with cellular senescence markers as well as clinicopathological factors of the cases. Hormonal activities (3ßHSD, CYP21A, CYP17A1, CYP11B1 and DHEA-ST) and cellular senescence markers (p16, p21 and Ki-67) within different morphological features (clear and compact) were evaluated in 40 CPAs. CPA genotypes (PRKACA, GNAS and CTNNB1) were examined by Sanger sequencing and then compared them with the factors above. p21 immunoreactivity was significantly positively correlated with that of CYP21A (p = 0.0110), CYP17A1 (p = 0.0356) and DHEA-ST (p = 0.0420) but inversely with tumor size (p = 0.0015). CYP21A (p = 0.0016), CYP11B1 (p = 0.0001), CYP17A1 (p < 0.0001) and p16 (p = 0.0137) immunoreactivity were all significantly higher in compact cells than those in clear cells. CYP17A1 (p = 0.0056) and 3ßHSD (p = 0.0437) immunoreactivity was significantly higher in PRKACA-mutated than wild type CPAs. p16 immunoreactivity and serum DHEA-S level were both significantly higher in GNAS-mutated than PRKACA-mutated (p = 0.0250) and wild type (p = 0.0180) CPAs. Results of our present study did demonstrate that compact tumor cells were hormonally active and more senescent than clear tumor cells in CPAs. PRKACA- and GNAS-mutated tumor cells were more hormonally active and senescent than those without mutations despite the similar morphological features. We herein proposed a novel histological classification of the tumor cell subtypes based on in situ cortisol excess, genotypes and the status of cell senescence in CPAs.

Targeted RNAseq of Formalin-Fixed Paraffin-Embedded Tissue to Differentiate Among Benign and Malignant Adrenal Cortical Tumors.

Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2, IGF2, etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.

Exploring heterogeneity of adrenal cortical tumors in children: The French pediatric rare tumor group (Fracture) experience.

INTRODUCTION: Pediatric adrenal cortical tumors are characterized by a wide spectrum of behavior. Questions remain regarding intermediate disease stages with isolated tumor rupture or relapse. OBJECTIVES: To describe clinical characteristics, treatment strategy, and outcome of patients depending on disease stage, tumor rupture, or in case of a refractory tumor, to discuss optimal management. MATERIAL AND METHODS: Pediatric patients with histological material reviewed and treated between 2000 and 2018 in 23 French oncology centers were included. RESULTS: Among 95 cases, 59% of patients had stage I tumors (n = 55), 16% had stage II tumors (n = 16), 19% had stage III tumors (n = 17), and 5% had stage IV tumors (n = 5) (missing data: 2). Overall, 27% of patients (n = 25) had an unfavorable histology. Initial tumor resection was performed for 90% of patients (n = 86). Systemic therapies included mitotane in 20 cases and chemotherapy in 13 cases. Among 17 stage III patients, 12 had microscopic residual tumor due to an initial biopsy (n = 5), intraoperative rupture (n = 8), or surgical resection with microscopic residue or tumor spillage surgery (n = 1) (two patients with two modalities). After a median follow-up of 96 months (25-119), four early progressions and two relapses occurred. A total of seven patients died, including five of disease. Stage III diseases due to microscopic residual disease correlated with a worse prognosis: 5-year progression-free survival 44% (95% CI, 22-87%) versus 82% (95% CI, 73-91%) for the whole cohort (P < .0001). Among the 14 patients with refractory disease, only 3 were alive and free of disease after multimodal second-line therapy. CONCLUSIONS: Stage III diseases due to a microscopic residual tumor have a dismal prognosis, arguing for the systematic use of adjuvant therapy. Patients with a relapsed disease should be included in experimental studies.

[Current criteria for the diagnosis of adrenocortical carcinoma]. / Sovremennye kriterii diagnostiki adrenokortikal'nogo raka.

Adrenocortical carcinoma is a rare malignant tumor of the adrenal cortex with an unfavorable prognosis. In 2017, the International Agency for Research on Cancer (IARC) and the World Health Organization (WHO) published the 4th edition of the WHO Classification of Tumors of Endocrine Organs. The updated classification reflects a multidisciplinary experience in diagnosing and predicting the course of adrenal cortex tumors, obtained on the basis of current studies. This paper highlights the key provisions of the updated WHO classification for adrenocortical carcinoma.

A Novel T-Stage Classification System for Adrenocortical Carcinoma: Proposal from the US Adrenocortical Carcinoma Study Group.

BACKGROUND: The 7th AJCC T-stage system for adrenocortical carcinoma (ACC), based on size and extra-adrenal invasion, does not adequately stratify patients by survival. Lymphovascular invasion (LVI) is a known poor prognostic factor. We propose a novel T-stage system that incorporates LVI to better risk-stratify patients undergoing resection for ACC. METHOD: Patients undergoing curative-intent resections for ACC from 1993 to 2014 at 13 institutions comprising the US ACC Group were included. Primary outcome was disease-specific survival (DSS). RESULTS: Of the 265 patients with ACC, 149 were included for analysis. The current T-stage system failed to differentiate patients with T2 versus T3 disease (p = 0.10). Presence of LVI was associated with worse DSS versus no LVI (36 mo vs. 168 mo; p = 0.001). After accounting for the individual components of the current T-stage system (size, extra-adrenal invasion), LVI remained a poor prognostic factor on multivariable analysis (hazard ratio 2.14, 95% confidence interval 1.05-4.38, p = 0.04). LVI positivity further stratified patients with T2 and T3 disease (T2: 37 mo vs. median not reached; T3: 36 mo vs. 96 mo; p = 0.03) but did not influence survival in patients with T1 or T4 disease. By incorporating LVI, a new T-stage classification system was created: [T1: ≤ 5 cm, (-)local invasion, (+/-)LVI; T2: > 5 cm, (-)local invasion, (-)LVI OR any size, (+)local invasion, (-)LVI; T3: > 5 cm, (-)local invasion, (+)LVI OR any size, (+)local invasion, (+)LVI; T4: any size, (+)adjacent organ invasion, (+/-)LVI]. Each progressive new T-stage group was associated with worse median DSS (T1: 167 mo; T2: 96 mo; T3: 37 mo; T4: 15 mo; p < 0.001). CONCLUSIONS: Compared with the current T-stage system, the proposed T-stage system, which incorporates LVI, better differentiates T2 and T3 disease and accurately stratifies patients by disease-specific survival. If externally validated, this T-stage classification should be considered for future AJCC staging systems.

Validation of the prognostic role of the "Helsinki Score" in 225 cases of adrenocortical carcinoma.

Adrenocortical carcinoma patient prognosis is extremely variable and poorly predictable. The newly introduced Helsinki Score is the first so far proposed diagnostic and prognostic system based on the combined evaluation of morphological (mitoses and necrosis) and immunohistochemical (Ki-67) parameters. The aim of the study was to validate the prognostic role of the Helsinki Score for adrenocortical carcinoma characterization. Thus, 225 adrenocortical carcinomas were reclassified using the Weiss Score and the Helsinki Score (3× mitotic count + 5 × necrosis + Ki-67 index). At univariate analysis, statistically significant prognostic values were observed at the log-rank test for mitotic count (cutoff values: <6 and ≥55; P<.0001), Ki-67 (cutoff values: <20 and ≥50; P<.0001), Weiss Score (cutoff values: <5 and ≥8; P<.0001), Helsinki Score (cutoff values: <13 and ≥19; P<.0001), histological variant (conventional versus oncocytic; P=.009), necrosis (P=.001), and stage (P=.005). Cox multivariate analysis using a backward stepwise selection method retained only Helsinki Score and Weiss Score as predictors of poor prognosis (P<.0001 and P=.0005, respectively). Helsinki Score (with a threshold of 28.5 points; area under the curve [AUC]=0.729, 95% confidence interval=0.66-0.79) and Ki-67 (with a threshold of 20.5%; AUC=0.727, 95% confidence interval=0.66-0.79) showed the best and equivalent AUCs predicting disease-related deaths determined using receiver operating characteristic statistics. In conclusion, the Helsinki Score is a valuable system to predict prognosis in adrenocortical carcinoma, outperforming the currently established prognostic parameters.

Dissecting Morphological and Molecular Heterogeneity in Adrenocortical Carcinoma.

Adrenocortical carcinoma is generally considered a single entity by pathologists and clinicians. Nevertheless, the knowledge cumulated along the last decade on the pathological characterization, the clinical outcome and the molecular pathogenesis of adrenocortical carcinoma demonstrate that one of the most relevant emerging issues is related to its heterogeneity. Three major morphological variants have been described (oncocytic, myxoid and sarcomatoid) but are not included in the current WHO classification, yet. Moreover, even "conventional" adrenocortical carcinomas have a high degree of morphological heterogeneity as well as different mitotic/proliferative capacity, either among different cases or within individual lesions. Furthermore, immunohistochemical and molecular studies, based on a wide set of different methodologies, identified novel biomarkers in adrenocortical carcinoma of diagnostic and prognostic relevance, which claimed again the concept that this tumor type represents an heterogeneous group of neoplasms which cannot be considered a unique entity. The integration between morphology, immunophenotype and molecular data is expected in the next years to build a novel concept of adrenocortical carcinoma classification into specific subgroups, as it is currently approached for other types of neoplasms such as breast or lung cancer, which are not merely descriptive, but also characterized by a specific biological and clinical behavior.

Application of Molecular Pathology in Endocrine Pathology.

Rapid growth in knowledge of cell and molecular biology led to the increased usage of molecular techniques in anatomical pathology. This is also due to the advances achieved in the techniques introduced in the last few years which are less laborious as compared to the techniques used at the beginning of the "molecular era". The initial assays were also very expensive and were not performed except for selected centers. Moreover, the clinicians were not sure how to make use of the accumulating molecular information. That situation has also changed and molecular techniques are being performed in a wide variety of medical settings which also has a reflection on the endocrine system pathology among other organ systems. This review will provide an update of genetic changes observed in different endocrine system pathologies and their diagnostic, therapeutic and prognostic values.

[Comparative clinical analysis of histological systems of adrenocortical tumors diagnosis].

Differential diagnosis of adrenocortical cancer (ACC) and cortical adenoma presents certain difficulties since there is no specific histological criterion allowing to distinguish tumors of the adrenal cortex with malignant clinical course. Currently there are offered several systems, and the most widely spread have the index Weiss (IW) and the modified index Weiss (MIW). The accuracy of one or another of the proposed systems remains a matter of debate. There was analyzed own experience on the use of IW and MIW in the diagnosis of 91 cases of the ACC and 13 cases of cortex adenomas of the size at least 5 cm. For the diagnosis of large adenomas sensitivity IW was 77%, MIW--100%. For the diagnosis of metastatic and non-metastatic ACC--100% and 97%, 100% and 86%, respectively (p > 0.05). In multivariate analysis of life expectancy of patients the definition of IW and MIW had a prognostic significance. MIW was less subjective, more simple and convenient to be used and it showed a great informative value at the reclassification of certain "adenomas" into ACC. However to use it on their own, without IW, was impractical as MIW had wider gray area and did not reach the threshold value in some cases of ACC. For the diagnosis of tumors of the adrenal cortex IW remains a standard; when a value was equal of 2 or in cases of doubt it was necessary to calculate MIW as well.

Accuracy of cytology in distinguishing adrenocortical tumors from pheochromocytoma in companion animals.

BACKGROUND: The distinction between adrenocortical tumors and pheochromocytoma can be challenging using clinical findings, diagnostic imaging and laboratory tests. Cytology might be a simple, minimally invasive method to reach a correct diagnosis. OBJECTIVES: The purpose of this study was to assess the accuracy of cytology in differentiating cortical from medullary tumors of the adrenal glands in dogs and cats. METHODS: Cytologic key features of adrenocortical tumors and pheochromocytoma were defined by one reference author. Cytologic specimens from primary adrenal tumors were submitted to 4 cytopathologists who were asked to classify the tumors based on the previously defined key features without knowledge of previous classification. RESULTS: Twenty specimens from histologically confirmed adrenal tumors (Group 1) and 4 specimens from adrenal tumors causing adrenal-dependent Cushing's syndrome (Group 2) were evaluated by the 4 cytopathologists. Accuracy in differentiating cortical from medullary origin ranged from 90% to 100%, with a Kappa coefficient of agreement between cytopathologists of 0.95. CONCLUSIONS: The origin of an adrenal tumor can be easily determined by cytology alone in many cases. However, cytology was not reliable in distinguishing benign from malignant neoplasia. Additional studies are needed to assess possible risks and complications associated with fine-needle biopsy of adrenal tumors in dogs and cats.

[Treatment of adrenocortical carcinomas is challenging]. / Diagnose og behandling af binyrebarkkarcinom.

Adrenocortical carcinomas (ACC) are rare tumours responsible for only 0.02% of the total number of malignant diseases. However the ACC are aggressive with a mean fiveyears survival of 20-50% and are often associated with increased production of adrenocortical hormones. The effect of the treatment is controversial and often based on small retrospective series or expert opinions. Centralization, international attention and collaboration in the treatment of ACC are mandatory. Randomized clinical trials are needed to determine the best treatment strategy in order to increase survival in patients with ACC.

Histologic and immunohistochemical classification of 41 bovine adrenal gland neoplasms.

Tumors of the adrenal glands are among the most frequent tumors in cattle; however, few studies have been conducted to describe their characteristics. The aim of this study was to classify 41 bovine adrenal neoplasms from 40 animals based on macroscopic and histologic examination, including electron microscopy and immunohistochemistry for melan A, synaptophysin, chromogranin A, vimentin, pan-cytokeratin, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase), and Ki-67. The tumors were classified as 23 adrenocortical adenomas, 12 adrenocortical carcinomas, 2 schwannomas, 2 pheochromocytomas (1 malignant), and 1 ganglioneuroma. Five histologic features were characteristic of metastasizing adrenocortical tumors: invasion of the capsule, vascular invasion, diffuse growth pattern, spindle-cell morphology, and nuclear pleomorphism. Adrenocortical tumors with at least 3 of these features were classified as malignant. Immunohistochemically, adrenocortical tumors expressed melan A (16/19), vimentin (14/26), cytokeratin (11/26), and chromogranin A (9/27), whereas pheochromocytomas expressed chromogranin A (2/2), synaptophysin (2/2), and vimentin (1/2). Both schwannomas expressed CNPase. An immunohistochemistry panel consisting of antibodies against melan A, synaptophysin, and CNPase was considered most useful to classify bovine adrenal tumors. However, the distinction between benign and malignant adrenocortical tumors was based on histologic features as in human medicine.

Current update on cytogenetics, taxonomy, diagnosis, and management of adrenocortical carcinoma: what radiologists should know.

OBJECTIVE: A multimodality imaging spectrum of adrenocortical carcinoma, with an emphasis on both anatomic and functional imaging, will be reviewed. Recent advances in the molecular cytogenetics of this tumor and its impact on diagnosis, prognosis, and development of newer targeted therapy will be discussed in this article. CONCLUSION: Adrenocortical carcinomas are rare aggressive tumors associated with a poor prognosis. Awareness of the molecular behavior and spectrum of imaging features of this tumor can help in appropriate patient treatment.

Adrenocortical neoplasia: evolving concepts in tumorigenesis with an emphasis on adrenal cortical carcinoma variants.

Adrenocortical carcinoma (ACC) is a rare, heterogeneous malignancy with a poor prognosis. According to WHO classification 2004, ACC variants include oncocytic ACCs, myxoid ACCs and ACCs with sarcomatous areas. Herein, we provide a comprehensive review of these rare subtypes of adrenocortical malignancy and emphasize their clinicopathological features with the aim of elucidating aspects of diagnostic categorization, differential diagnostics and biological behavior. The issue of current terminology, applied to biphasic tumors with pleomorphic, sarcomatous or sarcomatoid elements arising in adrenal cortex, is also discussed. We additionally present emerging evidence concerning the adrenal cortical tumorigenesis and the putative adenoma-carcinoma sequence as well.

Classification of adrenal cortical tumors: what limits for the pathological approach?

Most adrenocortical tumors are benign; adrenocortical carcinomas are rare but their prognosis is poor and their therapeutics are sparse. In most adrenocortical tumors, the morphological approach in particular by Weiss system, brings sufficient elements to establish the differential diagnosis between a benign and a malignant tumor. But some tumors of Weiss score of 2 or 3 can raise problems: are they benign, malignant or are they of uncertain malignant potential? On the other hand, some Weiss criteria are difficult to evaluate as, for example, sinusoidal invasion. These observations led to the development of other approaches, in particular genetic approaches. These genetics findings already have repercussions for the patients in the development of molecular markers for diagnosis and prognosis and in the future they could help in the development of new morphological approaches, in particular immunohistochemical approaches.

Classification of adrenal cortical tumors: promise of the 'molecular' approach.

Surgical pathologists, using routine histological techniques and tumor morphology, have established the overall taxonomy of adrenal cortical tumors that dictates care of patients with these tumors. However, a novel molecular classification of these tumors would provide numerous benefits to clinicians and patients, including a more accurate classification of diagnostically intermediate tumors, separation of carcinomas into prognostically-relevant subgroups, and optimal selection of therapy. Recent advances towards a molecular classification of adrenal cortical tumors are presented and discussed.

[Current TNM classification systems for adrenocortical carcinoma]. / Aktuelle TNM-Klassifikationssysteme für das Nebennierenkarzinom.

Adrenocortical carcinoma (ACC) is a rare malignancy and often difficult to diagnose. It was only in 2003 that the UICC proposed the first TNM classification for ACC. However, an analysis based on data from the German ACC Registry revealed several shortcomings of this classification; in particular, the outcome of patients with UICC stage II and III was not significantly different. Therefore, the European Network for the Study of Adrenal Tumours (ENSAT) developed a revised staging system, the superiority of which was recently confirmed in an independent American cohort. In the ENSAT classification, stage I (tumors ≤ 5 cm) and II (tumors < 5 cm) are non-infiltrating tumors without positive lymph nodes and distant metastases. Stage III is defined by the presence of positive lymph nodes, infiltration of surrounding tissue, or venous tumor thrombus. Stage IV is restricted to patients with distant metastasis. Since the ENSAT classification better reflects patient prognosis than the UICC classification, its use for future clinical and research purposes is recommended. Furthermore, exact documentation of the resection status is essential for optimal decisions on treatment.

[TNM 2010. What's new?]. / TNM 2010. Was ist neu?

In the seventh edition of the TNM Classification of Malignant Tumours there are several entirely new classifications: upper aerodigestive mucosal melanoma, gastrointestinal stromal tumour, gastrointestinal carcinoid (neuroendocrine tumour), intrahepatic cholangiocarcinoma, Merkel cell carcinoma, uterine sarcomas, and adrenal cortical carcinoma. Significant modifications concern carcinomas of the oesophagus, oesophagogastric junction, stomach, appendix, biliary tract, lung, skin, prostate and ophthalmic tumours, which will be not addressed in this article. For several tumour entities only minor changes were introduced which might be of importance in daily practice. The new classifications and changes will be commented on without going into details.

Improvement of histopathological classification of adrenal gland tumors by genetic differentiation.

PURPOSE: There are often problems in differentiating between benign and malignant adrenal gland tumors by imaging and histopathology. Fine-needle biopsy is possible but not used owing to problems in histopathological differentiation. On account of considerable differences in the therapy and aftercare of benign and malignant adrenal tumors, correct classification of tumor type is of greatest importance. The purpose of this study was to define specific genetic alterations differentiating between adenomas and carcinomas. METHODS: DNA was isolated from tumor areas in paraffin sections and amplified by a modified protocol for DOP-PCR. After labeling of tumor-DNA and normal DNA with biotin-dUTP and digoxigenin-dUTP, respectively, comparative genomic hybridization (CGH) was carried out according to standard protocols. Retrospectively, 26 (16 adenomas and 10 carcinomas) tumors of the adrenal cortex were analyzed. RESULTS: Genetic alterations were found in 5/16 adenomas (31.25%) and in all adrenocortical carcinomas. The mean number of genetic changes per tumor was 8.7 (range 6-12) in carcinomas. The benign cortical tumors present 1.6 changes (range 0-3) per tumor. Only a moderate correlation between number of alterations and size of tumor was seen. Furthermore, specific chromosomal alterations of carcinomas were identified. CONCLUSIONS: Genetic evaluation facilitates differentiation between adrenal gland tumors. Genetic tests should be used in routine diagnostics of adrenal specimens. Potentially, fine-needle biopsy can be established as standard diagnostics of adrenal tumors with unknown genesis.