Performance of DNA-based biomarkers for classification of adrenocortical carcinoma: a prognostic study.

OBJECTIVE: Adrenocortical carcinoma (ACC) is a rare aggressive malignancy with heterogeneous clinical outcomes. Recent studies proposed a combination of clinical/histopathological parameters (S-GRAS score) or molecular biomarkers (BMs) to improve prognostication. We performed a comparative analysis of DNA-based BMs by evaluating their added prognostic value to the S-GRAS score. DESIGN AND METHODS: A total of 194 formalin-fixed, paraffin-embedded (FFPE) ACC samples were analysed, including a retrospective training cohort (n = 107) and a prospective validation cohort (n = 87). Targeted DNA sequencing and pyrosequencing were used to detect somatic single-nucleotide variations in ACC-specific genes and methylation in the promoter region of paired box 5 (PAX5). The European Network for the Study of Adrenocortical Tumors (ENSAT) tumour stage, age, symptoms at presentation, resection status, and Ki-67 were combined to calculate S-GRAS. Endpoints were overall (OS), progression-free (PFS), and disease-free survival (DFS). Prognostic role was evaluated by multivariable survival analysis and their performance compared by Harrell's concordance index (C index). RESULTS: In training cohort, an independent prognostic role was confirmed at multivariate analysis for two DNA-based BMs: alterations in Wnt/ß-catenin and Rb/p53 pathways and hypermethylated PAX5 (both P< .05 for PFS and DFS, hazard ratio [HR] 1.47-2.33). These were combined to S-GRAS to obtain a combined (COMBI) score. At comparative analysis, the best discriminative prognostic model was COMBI score in both cohorts for all endpoints, followed by S-GRAS score (C index for OS 0.724 and 0.765, PFS 0.717 and 0.670, and DFS 0.699 and 0.644, respectively). CONCLUSIONS: Targeted DNA-based BM evaluated on routinely available FFPE samples improves prognostication of ACC beyond routinely available clinical and histopathological parameters. This approach may help to better individualise patient's management.

Coincidence of primary adrenocortical carcinoma and melanoma: three CASE reports.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a heterogeneous prognosis, while adrenal metastasis from other primary cancers, including melanoma, may occur more frequently. ACC may rarely occur as part of familial cancer syndromes, but even in sporadic cases, a significant proportion of patients had other malignancies before or after diagnosis of ACC. Herein we present three cases where sporadic ACC was identified in patients with coexistent or previous history of melanoma. CASE DESCRIPTION: Patient 1 - A 37-yr-old man with a superficial spreading BRAF-positive melanoma was found to harbour a progressively growing left adrenal mass. Initially, he was suspected of having adrenal metastasis, but the histology after adrenalectomy confirmed ACC. Patient 2 - A 68-year-old man with a history of recurrent BRAF-positive melanoma was diagnosed with disseminated metastatic melanoma recurrence, including a rapidly enlarging left adrenal mass. Consequently, he underwent left adrenalectomy, and histology again confirmed ACC. Patient 3 - A 50-yr-old man was referred with histological diagnosis of metastatic ACC. He had a background history of pT1 melanoma. We undertook targeted sequencing of ACC tissue samples in all cases. Somatic variants were observed in the known driver genes CTNNB1 (Patient 1), APC and KMT2D (Patient 2), and APC and TP53 (Patient 3). Germline TP53 variants (Li-Fraumeni syndrome) were excluded in all cases. Retrospective review of our patient cohort in the last 21 years revealed a frequency of 0.5% of histologically diagnosed melanoma metastasis among patients referred for adrenal masses. On the other hand, 1.6% of patients with histologically confirmed ACC had a previous history of melanoma. CONCLUSION: Sporadic ACC can occur in the background of melanoma, even if adrenal metastasis might appear to be the most likely diagnosis. Coexistent primary adrenal malignancy should be considered and investigated for in all patients with a history of melanoma with suspicious adrenal lesions.

Chronic inflammation and other changes are significant components of clinically fibrotic strictures in Crohn's disease: a histological study of resected strictures clinically characterized as noninflamed.

BACKGROUND: Strictures related to Crohn's disease due to fibrosis are a result of an exaggerated tissue remodelling response to inflammation, characterized by accumulation of collagen-rich extracellular matrix produced by mesenchymal cells. OBJECTIVES: The objective of this study was to characterize histological changes seen in resected 'fibrotic' strictures to better understand individual components of intestinal stenosis. METHODS: We identified patients undergoing surgery for ileal Crohn's disease secondary to symptomatic stricturing disease (Montreal B2) using the histopathology database at Queen Elizabeth Hospital in Birmingham, UK, between 2012 and 2017. Phenotypic data were recorded and resection specimens reviewed. Two independent pathologists applied the semiquantitative scoring system previously developed by us to the microscopic images. Data were analyzed using the possible maximum total score (%PMTS). RESULTS: Forty-eight patients (M = 25) were included. with median disease duration of 7 years (range 0.25-39 years); nearly two-thirds had ileocolonic distribution (L3). In this cohort, despite presurgery diagnosis of noninflamed fibrosis, chronic inflammation was noted to be a prominent component of all strictures. The histological scoring showed presence of several other prominent findings such as muscular hyperplasia and volume expansion.There was statistically significant positive correlation between chronic inflammation and fibrosis and muscular hyperplasia. CONCLUSION: The histological features of Crohn's disease-related strictures show multiple changes in multiple layers and not simply fibrosis. In our cohort, despite the observation prior to surgery that strictures were clinically considered fibrotic, the finding of chronic inflammation as a dominant component at a histological level in the resection is important. The findings might suggest that one of the main drivers of progressive fibrosis is the inflammatory component, which probably is never fully resolved.

Defining renal phenotype in Alström syndrome.

BACKGROUND: Alström syndrome (AS) is a rare autosomal recessive ciliopathy with a wide spectrum of clinical features, including cone-rod retinal dystrophy, neuronal deafness, severe insulin resistance and major organ failure. The characteristics of renal disease in the syndrome have not been systematically described. The aim of this study is to define the onset and progression of renal disease in AS. METHOD: Prospective observational cohort study. SETTING AND PARTICIPANTS: Thirty-two adult subjects from a national specialist clinic in UK and 86 subjects from an international AS registry were studied. OUTCOMES: First, an international registry cross-sectional study across all age groups to determine change in kidney function was performed. Secondly, a detailed assessment was carried out of adult AS patients with serial follow-up to determine incidence, aetiology and progression of renal disease. ANALYTICAL APPROACH: Generalized estimating equations were used to evaluate the relationship between age and estimated glomerular filtration rate (eGFR). Associations between patient factors and eGFR levels were then assessed in the adult AS cohort. RESULTS: The international registry study of the renal function of 118 subjects with AS (median age 21 years) showed a rapid decline with age, at an average of -16.7 and -10.9 mL/min/1.73 m2 per decade in males and females, respectively. In a UK national cohort of 32 patients with AS (median age 22 years), 20/32 (63%) had chronic kidney disease (CKD) Stage 3 or above based on eGFR <60 mL/min/1.73 m2 or evidence of albuminuria. Hyperuricaemia was noted in 25/32 (79%). Structural abnormalities such as nephrocalcinosis without hypercalcaemia and cysts were observed in 20/32 (63%) subjects. Lower urinary tract symptoms were frequent in 17/19 (70%) of AS patients. Histological evidence showed mixed tubulo-interstitial and glomerular disease. CONCLUSIONS: This is the first study to demonstrate that renal disease is the hallmark of AS, which starts early and progresses with age, leading to a high prevalence of advanced CKD at young age. AS should be considered in the differential diagnosis of rare genetic renal diseases.

Double trouble: two cases of dual adrenal pathologies in one adrenal mass.

Context Adrenal incidentalomas (AI) represent an increasingly common problem in modern endocrine practice. The diagnostic approach to AIs can be challenging and occasionally reveals surprising features. Here we describe two rare cases of complex adrenal lesions consisting of phaeochromocytomas with synchronous metastases from extra-adrenal primaries. Case descriptions Patient 1 - a 65-year-old gentleman with a newly diagnosed malignant melanoma was found to harbour an adrenal lesion with suspicious radiographic characteristics. Percutaneous adrenal biopsy was consistent with adrenocortical adenoma. After excision of the skin melanoma and regional lymphatic metastases, he was followed up without imaging. Three years later, he presented with abdominal discomfort and enlargement of his adrenal lesion, associated with high plasma metanephrines. Adrenalectomy revealed a mixed tumour consisting of a large phaeochromocytoma with an embedded melanoma metastasis in its core. Patient 2 - a 63-year-old lady with a history of NF-1-related phaeochromocytoma 20 years ago and previous breast cancer presented with a new adrenal lesion on the contralateral side. Plasma normetanephrine was markedly elevated. Elective adrenalectomy revealed an adrenal tumour consisting of chromaffin cells intermixed with breast carcinoma cells. Conclusions Adrenal incidentalomas require careful evaluation to exclude metastatic disease, especially in the context of a history of previous malignancy. Adrenal biopsy provides limited and potentially misleading information. Phaeochromocytomas are highly vascularised tumours that may function as a sieve, extracting and retaining irregularly shaped cancer cells, thereby yielding adrenal masses with intriguing dual pathology. Learning points: Adrenal incidentalomas require careful evaluation focused on exclusion of underlying hormone excess and malignant pathology. Adrenal biopsy can be misleading and should only be considered in select cases. Phaeochromocytomas harbouring intratumoural metastases from other, extra-adrenal primary malignancies represent rare pathological entities that highlight the complexities that can be presented by adrenal tumours.

A patient with a germline SDHB mutation presenting with an isolated pituitary macroprolactinoma.

Symptomatic pituitary adenomas occur with a prevalence of approximately 0.1% in the general population. It is estimated that 5% of pituitary adenomas occur in a familial setting, either in isolated or syndromic form. Recently, loss-of-function mutations in genes encoding succinate dehydrogenase subunits (SDHx) or MYC-associated factor X (MAX) have been found to predispose to pituitary adenomas in co-existence with paragangliomas or phaeochromocytomas. It is rare, however, for a familial SDHx mutation to manifest as an isolated pituitary adenoma. We present the case of a pituitary lactotroph adenoma in a patient with a heterozygous germline SDHB mutation, in the absence of concomitant neoplasms. Initially, the adenoma showed biochemical response but poor tumour shrinkage in response to cabergoline; therefore, transsphenoidal surgery was performed. Following initial clinical improvement, tumour recurrence was identified 15 months later. Interestingly, re-initiation of cabergoline proved successful and the lesion demonstrated both biochemical response and tumour shrinkage. Our patient's SDHB mutation was identified when we realised that her father had a metastatic paraganglioma, prompting genetic testing. Re-inspection of the histopathological report of the prolactinoma confirmed cells with vacuolated cytoplasm. This histological feature is suggestive of an SDHx mutation and should prompt further screening for mutations by immunohistochemistry and/or genetic testing. Surprisingly, immunohistochemistry of this pituitary adenoma demonstrated normal SDHB expression, despite loss of SDHB expression in the patient's father's paraganglioma. LEARNING POINTS: Pituitary adenomas may be the presenting and/or sole feature of SDHB mutation-related disease. SDHx mutated pituitary adenomas may display clinically aggressive behaviour and demonstrate variable response to medical treatment.Histological evidence of intracytoplasmic vacuoles in a pituitary adenoma might suggest an SDH-deficient tumour and should prompt further screening for SDHx mutations.Immunohistochemistry may not always predict the presence of SDHx mutations.

Lesson of the month 2: Blunt abdominal trauma: atypical presentation of phaeochromocytoma.

A 26-year-old man presented following blunt abdominal trauma to a regional major trauma centre for emergency embolisation of a retroperitoneal bleed from a presumed renal laceration. Imaging had also revealed a large right suprarenal mass. Embolisation resulted in a hypertensive crisis raising the suspicion of a metabolically active adrenal tumour. The course was further complicated by the development of ischaemic bowel requiring emergency laparotomy. Intraoperatively he became haemodynamically unstable from an actively haemorrhaging lesion. Emergency laparotomy and adrenalectomy was performed as a life-saving procedure. Histology confirmed a phaeochromocytoma. The patient made a gradual recovery and was discharged home with no sequelae. Definitive management of phaeochromocytoma is surgical resection which requires prolonged preoperative optimisation with alpha receptor blockers to adequately control blood pressure and prevent hypertensive crises. Parenteral alpha receptor blockers, such as phentolamine, are optimal treatment for intraoperative hypertensive emergencies, yet they are currently not available in the UK.

Mitotane treatment in patients with metastatic testicular Leydig cell tumor associated with severe androgen excess.

Mitotane (o,p'DDD) is established in the adjuvant and advanced-stage treatment of adrenocortical carcinoma and counteracts both tumor growth and tumor-related steroid production. Both the adrenal glands and the gonads are steroidogenically active organs and share a common embryogenic origin. Here, we describe the effects of mitotane in two patients with metastatic Leydig cell tumor (LCT) of the testes and associated severe androgen excess (serum testosterone 93 and 88 nmol/L, respectively; male reference range 7-27 nmol/L). Both men suffered from severe restlessness, insomnia and irritability, which they described as intolerable and disrupting normal life activities. Urinary steroid profiling by gas chromatography-mass spectrometry (GC-MS) confirmed excess androgen production and revealed concurrent overproduction of glucocorticoids and glucocorticoid precursors, which under physiological conditions are produced only by the adrenal glands but not by the gonads. In a palliative approach, they were commenced on mitotane, which achieved swift control of the hormone excess and the debilitating clinical symptoms, restoring normal quality of life. GC-MS demonstrated normalization of steroid production and decreased 5α-reductase activity, resulting in decreased androgen activation, and imaging demonstrated disease stabilization for 4-10 months. In conclusion, mitotane can be highly effective in controlling steroid excess in metastatic LCTs, with anti-tumor activity in some cases.

The podoplanin-CLEC-2 axis inhibits inflammation in sepsis.

Platelets play a critical role in vascular inflammation through the podoplanin and collagen/fibrin receptors, C-type-lectin-like-2 (CLEC-2) and glycoprotein VI (GPVI), respectively. Both receptors regulate endothelial permeability and prevent peri-vascular bleeding in inflammation. Here we show that platelet-specific deletion of CLEC-2 but not GPVI leads to enhanced systemic inflammation and accelerated organ injury in two mouse models of sepsis-intra-peritoneal lipopolysaccharide and cecal ligation and puncture. CLEC-2 deficiency is associated with reduced numbers of podoplanin-expressing macrophages despite increased cytokine and chemokine levels in the infected peritoneum. Pharmacological inhibition of the interaction between CLEC-2 and podoplanin regulates immune cell infiltration and the inflammatory reaction during sepsis, suggesting that activation of podoplanin underlies the anti-inflammatory action of platelet CLEC-2. We suggest podoplanin-CLEC-2 as a novel anti-inflammatory axis regulating immune cell recruitment and activation in sepsis.

The Spectrum of Renal Allograft Failure.

BACKGROUND: Causes of "true" late kidney allograft failure remain unclear as study selection bias and limited follow-up risk incomplete representation of the spectrum. METHODS: We evaluated all unselected graft failures from 2008-2014 (n = 171; 0-36 years post-transplantation) by contemporary classification of indication biopsies "proximate" to failure, DSA assessment, clinical and biochemical data. RESULTS: The spectrum of graft failure changed markedly depending on the timing of allograft failure. Failures within the first year were most commonly attributed to technical failure, acute rejection (with T-cell mediated rejection [TCMR] dominating antibody-mediated rejection [ABMR]). Failures beyond a year were increasingly dominated by ABMR and 'interstitial fibrosis with tubular atrophy' without rejection, infection or recurrent disease ("IFTA"). Cases of IFTA associated with inflammation in non-scarred areas (compared with no inflammation or inflammation solely within scarred regions) were more commonly associated with episodes of prior rejection, late rejection and nonadherence, pointing to an alloimmune aetiology. Nonadherence and late rejection were common in ABMR and TCMR, particularly Acute Active ABMR. Acute Active ABMR and nonadherence were associated with younger age, faster functional decline, and less hyalinosis on biopsy. Chronic and Chronic Active ABMR were more commonly associated with Class II DSA. C1q-binding DSA, detected in 33% of ABMR episodes, were associated with shorter time to graft failure. Most non-biopsied patients were DSA-negative (16/21; 76.1%). Finally, twelve losses to recurrent disease were seen (16%). CONCLUSION: This data from an unselected population identifies IFTA alongside ABMR as a very important cause of true late graft failure, with nonadherence-associated TCMR as a phenomenon in some patients. It highlights clinical and immunological characteristics of ABMR subgroups, and should inform clinical practice and individualised patient care.

An International Ki67 Reproducibility Study in Adrenal Cortical Carcinoma.

Despite the established role of Ki67 labeling index in prognostic stratification of adrenocortical carcinomas and its recent integration into treatment flow charts, the reproducibility of the assessment method has not been determined. The aim of this study was to investigate interobserver variability among endocrine pathologists using a web-based virtual microscopy approach. Ki67-stained slides of 76 adrenocortical carcinomas were analyzed independently by 14 observers, each according to their method of preference including eyeballing, formal manual counting, and digital image analysis. The interobserver variation was statistically significant (P<0.001) in the absence of any correlation between the various methods. Subsequently, 61 static images were distributed among 15 observers who were instructed to follow a category-based scoring approach. Low levels of interobserver (F=6.99; Fcrit=1.70; P<0.001) as well as intraobserver concordance (n=11; Cohen κ ranging from -0.057 to 0.361) were detected. To improve harmonization of Ki67 analysis, we tested the utility of an open-source Galaxy virtual machine application, namely Automated Selection of Hotspots, in 61 virtual slides. The software-provided Ki67 values were validated by digital image analysis in identical images, displaying a strong correlation of 0.96 (P<0.0001) and dividing the cases into 3 classes (cutoffs of 0%-15%-30% and/or 0%-10%-20%) with significantly different overall survivals (P<0.05). We conclude that current practices in Ki67 scoring assessment vary greatly, and interobserver variation sets particular limitations to its clinical utility, especially around clinically relevant cutoff values. Novel digital microscopy-enabled methods could provide critical aid in reducing variation, increasing reproducibility, and improving reliability in the clinical setting.

Heterogeneous genetic background of the association of pheochromocytoma/paraganglioma and pituitary adenoma: results from a large patient cohort.

CONTEXT: Pituitary adenomas and pheochromocytomas/paragangliomas (pheo/PGL) can occur in the same patient or in the same family. Coexistence of the two diseases could be due to either a common pathogenic mechanism or a coincidence. OBJECTIVE: The objective of the investigation was to study the possible coexistence of pituitary adenoma and pheo/PGL. DESIGN: Thirty-nine cases of sporadic or familial pheo/PGL and pituitary adenomas were investigated. Known pheo/PGL genes (SDHA-D, SDHAF2, RET, VHL, TMEM127, MAX, FH) and pituitary adenoma genes (MEN1, AIP, CDKN1B) were sequenced using next generation or Sanger sequencing. Loss of heterozygosity study and pathological studies were performed on the available tumor samples. SETTING: The study was conducted at university hospitals. PATIENTS: Thirty-nine patients with sporadic of familial pituitary adenoma and pheo/PGL participated in the study. OUTCOME: Outcomes included genetic screening and clinical characteristics. RESULTS: Eleven germline mutations (five SDHB, one SDHC, one SDHD, two VHL, and two MEN1) and four variants of unknown significance (two SDHA, one SDHB, and one SDHAF2) were identified in the studied genes in our patient cohort. Tumor tissue analysis identified LOH at the SDHB locus in three pituitary adenomas and loss of heterozygosity at the MEN1 locus in two pheochromocytomas. All the pituitary adenomas of patients affected by SDHX alterations have a unique histological feature not previously described in this context. CONCLUSIONS: Mutations in the genes known to cause pheo/PGL can rarely be associated with pituitary adenomas, whereas mutation in a gene predisposing to pituitary adenomas (MEN1) can be associated with pheo/PGL. Our findings suggest that genetic testing should be considered in all patients or families with the constellation of pheo/PGL and a pituitary adenoma.

Duodenal duplication cyst with profound elevation of intracystic carbohydrate antigen (CA 19-9) and carcinoembryonic antigen (CEA): a rare but important differential in the diagnosis of cystic tumours of the pancreas.

CONTEXT: Enteric duplication cysts are rare lesions of uncertain incidence and natural history. Pre-operative confirmation of diagnosis can be difficult. This case reports an adult duodenal duplication cyst presenting with grossly elevated intra-lesional levels of tumour markers. CASE REPORT: A 57-year-old female was found to have a complex cystic lesion of the head of the pancreas. Intra-lesional fluid analysis revealed a grossly elevated CA 19-9 and CEA. Resection was undertaken under the assumption that this was a cystic tumour. Macroscopic examination after opening the duodenum revealed a villous, circumferential tumour in the proximal duodenum measuring 4 cm in length. A cystic lesion was present in the medial wall of the tumour and did not communicate with the duodenal lumen. Microscopically, the tumour comprised Brunner's gland hyperplasia with associated mucosal thickening. The wall of the underlying cystic lesion was comprised of muscularis formed by the outer muscle coat of the duodenal wall. The final diagnosis was of a duodenal duplication cyst. There was no evidence of dysplasia or malignancy. CONCLUSION: This is the first report of a duodenal duplication cyst having elevated intra-cyst fluid levels of amylase, carbohydrate antigen CA 19-9 and carcinoembryonic antigen (CEA). Although rare, this is an important differential diagnosis in the management of cystic tumours of the pancreas.