A
Histopathology
Histopathology 2018, 72, 82-96. DOI: 10.1111/his.13255
REVIEW
Challenges in surgical pathology of adrenocortical tumours
Lori A. Erickson (D
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA
Erickson L A
(2018) Histopathology 72, 82-96. https://doi.org/10.1111/his.13255
Challenges in surgical pathology of adrenocortical tumours
Adrenocortical carcinomas are rare tumours that can be diagnostically challenging. Numerous multipara- metric scoring systems and diagnostic algorithms have been proposed to differentiate adrenocortical adenoma from adrenocortical carcinoma. Adrenocor- tical neoplasms must also be differentiated from other primary adrenal tumours, such as phaeochromocy- toma and unusual primary adrenal tumours, as well as metastases to the adrenal gland. Myxoid, oncocytic and sarcomatoid variants of adrenocortical tumours must be recognized so that they are not confused with other tumours. The diagnostic criteria for onco- cytic adrenocortical carcinoma are different from
those for conventional adrenocortical carcinomas. Adrenocortical neoplasms in children are particularly challenging to diagnose, as histological features of malignancy in adrenocortical neoplasms in adults may not be associated with aggressive disease in the tumours of children. Recent histological and immunohistochemical studies and more comprehen- sive and integrated genomic characterizations con- tinue to advance our understanding of the tumorigenesis of these aggressive neoplasms, and may provide additional diagnostic and prognostic util- ity and guide the development of therapeutic targets.
Keywords: adrenal, adrenocortical, carcinoma, Ki67, molecular, myxoid, oncocytic, steroidogenic factor, TP53, ß-catenin
Introduction
The diagnosis of adrenocortical neoplasms is often straightforward, but the diagnosis of some adrenocor- tical carcinomas can be very difficult. Unlike adreno- cortical adenomas, which are common and often found incidentally, adrenocortical carcinomas are rare, with an annual incidence of 0.7 to 2 per mil- lion.1,2 Adrenocortical carcinomas have been identi- fied as adrenal incidentalomas.3 However, a large study of 1049 adrenal incidentalomas identified no adrenocortical carcinomas.4 Adrenocortical carcino- mas are more common in females than in males, and hormonally functional tumours are more common in females. Overall, 42-57% of adrenocortical carcino- mas are hormonally functional.5 9 The most common
Address for correspondence: L A Erickson, Department of Labora- tory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. e-mail: erickson.lori@mayo.edu
presentation of patients with hormone excess is hypercortisolism. Adrenocortical carcinomas occur in a bimodal distribution in adults in the fifth decade and in children.10,11 Diagnosing adrenocortical tumours in children is particularly challenging, as the histological features associated with aggressive behaviour in adults may not have the same signifi- cance in children. Diagnosing the oncocytic variant of adrenocortical carcinoma can also be difficult, and the criteria are different from those of conventional adrenocortical carcinoma.12 Other histological vari- ants, such as myxoid or sarcomatoid adrenocortical carcinomas, must also be recognized so that they are not mistaken for other tumours.13-15 Adrenocortical carcinomas can be very aggressive, with the 5-year survival rate being dependent on stage: 66-82% for stage I, 58-63% for stage II, 24-50% for stage III, and 0-17% for stage IV.16-19 The lung and liver (40-90%) are the most common metastatic sites, followed by bone (5-20%).20 Interestingly, 5% of
patients have a contralateral adrenal tumour that can be synchronous or metachronous, and differenti- ating this tumour from a metastasis may not be pos- sible. Adrenocortical carcinomas may occur in syndromes such as Li-Fraumeni syndrome, Beck- with-Wiedemann syndrome, Lynch syndrome, multi- ple endocrine neoplasia type I, familial adenomatous polyposis, and neurofibromatosis type I.21,22 Thus, genetic counselling, germline testing, and even immunohistochemical testing of the tumour, such as for Lynch syndrome, may be helpful in screening for these syndromes. Recent studies, both histological and immunohistochemical, and more comprehensive and integrated genomic characterizations have advanced our understanding of the pathogenesis of these tumours.
Diagnosis of adrenocortical carcinoma
Although the diagnosis of most adrenocortical neo- plasms is straightforward, the diagnosis of adrenocor- tical carcinoma can be difficult. Adrenocortical carcinomas are usually large tumours, often measur- ing 100-140 mm, and may show necrosis.8,23-25 Only 3% of carcinomas are <40 mm.26,27 However, the size can vary greatly, with carcinomas as small as 10 mm having been reported.23 In a study of 376 adrenocortical carcinomas and 44 adenomas with tumour size available, carcinomas (120 ± 56 mm) were larger than adenomas (42 ± 19 mm).27 For adrenocortical carcinomas presenting with local dis- ease, the sensitivities, specificities and likelihood ratios of tumour size to predict malignancy were: 96%, 52% and 2.0, respectively, for tumours ≥40 mm; 90%, 80% and 4.4 for tumours ≥60 mm; 77%, 95% and 16.9 for tumours ≥80 mm; and 55%, 98% and 24.4 for tumours ≥100 mm.27 The probability of malignancy increased from 10% for tumours ≥40 mm to 19% for tumours ≥60 mm to 47% for tumours ≥80 mm.27 The number of genetic aberra- tions detected increases with tumour size and malig- nancy.28 In a study of 107 adrenocortical tumours, the average weight of tumours with benign beha- viour (Weiss score of <3) was significantly lower than that of tumours with malignant behaviour (Weiss score of >3).29 However, a study of 2248 cases found that tumour size at presentation did not relate to the likelihood of non-localized disease at presentation, and, by multivariate analysis, tumour size was not found to be predictive of overall survival in resected localized carcinomas.3º Most adrenocortical adeno- mas weigh <50 g, but weight can vary greatly. A tumour weight of >50 g or >100 g is worrying with
regard to malignancy. Adrenocortical carcinomas often weigh >100 g, but tumours of <50 g have metastasized.31,32 Thus, neither size nor weight alone is diagnostic of malignancy. Most adrenocortical car- cinomas are solid tumours; they may be lobulated and have fibrous bands, can be well circumscribed or infiltrative, and frequently show necrosis. Rarely, adrenocortical carcinomas can be cystic.33 In a study from Mayo Clinic, two of 41 grossly cystic adrenal lesions were cystic adrenocortical carcinomas.33 Interestingly, adrenocortical carcinomas may not show a histological stromal response.
The difficulty in the diagnosis of some adrenocorti- cal neoplasms is reflected in the number of multipara- metric scoring systems and diagnostic algorithms that have been proposed. The most widely used system was introduced by Weiss in 1984.34 Nine histological parameters [nuclear grade (Fuhrman III or IV), mito- tic rate of >5/50 high-power fields (HPFs), abnormal mitoses, ≥25% clear cells, >1/3 diffuse architecture, necrosis, venous invasion, sinusoidal invasion, and capsular invasion] were used to classify 43 adult adrenocortical tumours with a minimum of 5 years of follow-up.34 Each feature was scored 0 if absent and 1 if present. Eighteen of 19 patients with a score of ≥4 developed recurrence or metastases, whereas all 24 patients with a score of ≤2 had a benign clinical outcome.34 No single morphological feature was diag- nostic for malignancy, but the most significant were a mitotic rate of >5/50 HPFs, atypical mitoses, and venous invasion.34
In 1985, Van Slooten35 developed a weighted index of seven differently weighted histological parameters (regressive changes such as necrosis, haemorrhage, fibrosis, or calcification; preservation of normal structure; moderate to marked nuclear atypia; moderate to marked nuclear hyperchromasia; abnor- mal nucleoli; mitotic rate of >2/10 HPFs; capsular and/or vascular invasion) with an overall calculated score from 0 to 28.4, and a threshold for malignancy of >8 in tumours with and without metastases within 10 years of primary surgery.35 As single parameters, tumour weight and mitotic activity had the highest discriminatory value.35
In a subsequent study of 42 adrenocortical carcino- mas by Weiss in 1989, 11 features were evaluated, and the threshold was lowered from a Weiss score of 4 to a Weiss score of 3, as a patient with a Weiss score of 3 had recurrence and died of disease (Table 1).36 In this study, only mitotic rate had a strong statistical association with patient outcome. Carcinomas with >20 mitoses per 50 HPFs were des- ignated as high grade, and patients had a median
| Weiss criterion | Description |
|---|---|
| Nuclear grade (Fuhrman grade III or IV) | High nuclear grade: enlarged, oval to lobated nuclei with coarsely granular to hyperchromatic chromatin and easily discernible, prominent nucleoli. Overall grade is based on the most histologically abnormal area present, even if only focal |
| Mitotic rate of >5/50 high-power fields | Evaluated in 10 high-power fields in the area of greatest mitotic activity in each of five slides. If fewer than five slides were available for a case, a correspondingly greater number of fields per slide were used, to give a total of 50 high-power fields |
| Atypical mitotic figures | Abnormal chromosomal distribution or an excessive number of mitotic spindles with a multipolar morphological appearance |
| Clear cells constitute ≤25% of the tumour | Pale cells with lipid-rich, finely vacuolated cytoplasm resembling cells of the zona fasciculata |
| Diffuse architecture in more than one- third of the tumour | Patternless sheets of cells involving more than one-third of the tumour |
| Necrosis | Necrosis requires a confluent area of cells, as isolated apoptotic cells are not sufficient |
| Venous invasion | The tumour appears as plugs or polypoid luminal projections covered by a layer of endothelial cells-should be distinguished from free-floating tumour cells, which may be artefacts |
| Sinusoidal invasion | Endothelium-lined vascular channel that lacks a supportive smooth muscle wall-must be unequivocal invasion |
| Capsular invasion | Tumour extending into or completely traversing the capsule with an associated stromal reaction |
*The presence of three or more features correlates with malignancy.
survival of 14 months; and carcinomas with ≤20 mitoses per 50 HPFs were designated as low grade, and patients had a median survival of 58 months.36
In 2002, the Weiss system was revisited by Aubert et al., who compared 24 adrenocortical carcinomas with distant metastases, gross local invasion or recur- rence matched by functional status with 25 benign tumours (Table 2).24 The presence of three or more Weiss criteria had a specificity of 96% and a sensitiv- ity 100% for malignancy. A modified system based on the most reliable criteria (2 x mitotic rate + 2 x cytoplasm + abnormal mitoses + necro- sis + capsular invasion) was developed with a possi- ble 7 points, with a similar threshold for malignancy of three or more criteria.24
In 2007, van’t Sant HP et al. evaluated 79 adreno- cortical tumours (17 adenomas, 24 non-metastatic carcinomas, and 19 carcinomas with metastatic dis- ease and/or local recurrence) to compare the prognos- tic value of the Weiss revisited index and the Van Slooten index.37 Both indices were shown to have equal validity, and both showed a correlation with sur- vival for metastasizing adrenocortical carcinomas.37
In 2009, a simplified diagnostic algorithm that included evaluation of the reticular network and prognostic stratification based on 92 adrenocortical carcinomas and 47 adenomas was proposed.38 This
simplified system, utilizing histochemical staining to demonstrate disruption of the reticular network and at least one of three additional parameters (mitotic index of >5/50 HPFs, necrosis, and vascular inva- sion), had a sensitivity of 100% and specificity of 100% for adrenocortical carcinoma (Table 3).38 Mul- tivariate analysis also showed that stage III/IV and a mitotic count of >9/50 HPFs had a strong adverse impact on disease-free and overall survival, which led to the development of three risk groups.38 In a subse- quent multicentre validation study of 184 adrenocor- tical carcinomas and 61 adenomas, the reticulin algorithm classified 178 as carcinomas and 67 as adenomas, and confirmed interobserver reproducibil- ity of the reticulin stain and algorithm.25
The Helsinki score was published in 2015 as a novel model for prediction of metastases in adrenocortical carcinomas.39 The study included 177 consecutive adult primary adrenocortical tumours operated on at Helsinki University Central Hospital from 1990 to 2003, with a minimum of 5 years of follow-up.39 The Weiss score, Weiss score revisited by Aubert and prolif- eration index determined by computer-assisted image analysis were determined. The nine Weiss criteria and proliferation index were evaluated with stepwise regression analysis to develop a calculation: 3 x mi- totic rate (>5/50 HPFs) + 5 x necrosis + proliferation
Table 2. Modified Weiss (‘Weiss revisited’) system24*
Mitotic rate of >5/50 higher-power fields
Atypical mitotic figures
Clear cells constituting ≤25% of the tumour
Necrosis
Capsular invasion
*Five criteria, each 0 or 1 (2 x mitotic rate + 2 x cytoplasm + abnormal mitoses + necrosis + capsular invasion), for a total of 7 possible points, with 3 points being diagnostic of malignancy.
Table 3. Reticulin algorithm25,38
Disruption of reticulin network (highlighted by histochemical staining) and at least one of the following parameters
Mitotic rate of >5/50 high-power fields
Necrosis
Vascular invasion
Table 4. Helsinki score39,40*
3 x mitotic rate (>5/50 HPFs) + 5 x necrosis + proliferation index in the most proliferative area of the tumour
HPF, High-power field.
*With a cutoff of 8.5, this scoring system had 100% sensitivity and 99.4% specificity for diagnosing metastatic adrenocortical carcinoma.39
index in the most proliferative area of the tumour (Table 4).39 With a cutoff of 8.5, this scoring system had 100% sensitivity and 99.4% specificity for diag- nosing metastatic adrenocortical carcinoma.39 A vali- dation study of the prognostic role of the Helsinki score was published in 2016.40 The authors evaluated 225 adrenocortical carcinomas by using the Weiss score and the Helsinki score. By Cox multivariate anal- ysis, the Helsinki score and Weiss score were predictors of poor prognosis. The Helsinki score with a threshold of 28.5 points and the Ki67 index with a threshold of 20.5% were the best predictors of disease-related death. 40
Oncocytic adrenocortical carcinomas
Oncocytic adrenocortical tumours are uncommon and unique tumours composed predominantly or entirely of oncocytic cells (Figure 1). More than 100 cases have now been reported in the literature, but, other than case reports, most studies are of small
series of cases or subsets of oncocytic adrenocortical tumours in larger series of conventional adrenocorti- cal tumours.12,40-44 Oncocytic adrenocortical tumours can be functional, but less often than con- ventional adrenocortical tumours. The distinction of oncocytic adrenocortical adenoma from carcinoma differs from that of conventional adrenocortical neo- plasms. Utilization of the Weiss or modified Weiss sys- tems used for conventional adrenocortical tumours results in overdiagnosis of some tumours as malig- nant. In 1998, Lin reported seven adrenocortical neo- plasms (five oncocytomas and two oncocytic neoplasms of uncertain malignant potential) that ran- ged in size from 50 to 135 mm.42 All of the neo- plasms were composed exclusively of oncocytes, and all showed nuclear atypia, either focal or general- ized.42 The oncocytomas had very low to absent mitotic activity and no necrosis, and the two onco- cytic neoplasms of uncertain malignant potential had increased mitotic activity and necrosis but no inva- sion or metastases.42 The Ki67 index showed increased proliferative activity in the oncocytomas and oncocytic tumours of uncertain malignant poten- tial. Although the follow-up period was fairly short (<2 years), all six patients with clinical follow-up information available were alive without evidence of disease. They concluded that oncocytic adrenocortical neoplasms can become large before they become detected by radiological studies, and that the majority are benign and should not be misdiagnosed as carci- noma.42 Hoang et al. described four oncocytic adrenocortical tumours, ranging in size from 85 to 170 mm, and all showed diffuse proliferation of polygonal cells with large nuclei, prominent nucleoli, and abundant granular eosinophilic cytoplasm. 45 Large tumour size, extracapsular extension necrosis, vascular invasion and metastasis were features of malignant tumours, whereas cytological atypia and mitotic rate could not discriminate benign from malignant tumours. 45
In 2004, Bisceglia et al. reported 10 adrenocortical oncocytic tumours composed entirely or predomi- nantly of oncocytes, and proposed a classification sys- tem for oncocytic adrenocortical tumours.12 Bisceglia et al. compared the nine histological parameters of the Weiss system, and found that two parameters (clear cell composition ≤25% and high nuclear grade) were present in all of the tumours, and that diffuse architecture was present in all but one of the tumours.12 Utilizing the six remaining Weiss parame- ters (mitotic rate of >5/50 HPFs, atypical mitoses, venous invasion, necrosis, capsular invasion, and sinusoidal invasion) and an additional criterion of
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‘large size and huge weight’ (>100 mm and/or >200 g), Bisceglia et al. proposed an algorithm to classify oncocytic adrenocortical tumours.12 Major criteria included high mitotic rate (>5/50 HPFs), atypical mitoses, and venous invasion, and minor cri- teria included large size and huge weight (>100 mm and/or >200 g), necrosis, capsular invasion, and sinusoidal invasion (Table 5).12 One major criterion indicates malignancy, one to four minor criteria indi- cate uncertain malignant potential (borderline), and the absence of all major and minor criteria indicates benign.12 Bisceglia proposed categories for oncocytic neoplasms as a pure oncocytic tumour (>90% onco- cytic cells), a mixed oncocytic tumour (clear cell com- ponent of 10-50%), and an ordinary adrenocortical tumour with focal (<50%) oncocytic change.46 Thus, it is important to extensively sample oncocytic adrenocortical neoplasms so that the appropriate diagnostic criteria are utilized, and the biological behaviour may vary according to the amount of oncocytic component.46
In 2011, 13 additional oncocytic adrenocortical neoplasms were classified (three benign, two border- line, and eight malignant) according to the criteria and algorithm proposed in 2004 by Bisceglia, which
became known as the Lin-Weiss-Bisceglia criteria. 44 In this series, seven of the 13 (54%) tumours were functional.44 The Lin-Weiss-Bisceglia criteria were also retrospectively applied to all cases published with sufficient information in the literature by that time, and this confirmed the validity of the Lin-Weiss-Bis- ceglia criteria and algorithm.44 Overall, 30% of the tumours were hormonally functional. The overall median survival for patients with oncocytic adreno- cortical carcinomas is 58 months, suggesting a more favourable prognosis than that for conventional carci- nomas (14-32 months).44
In 2011, Duregon et al. evaluated 27 oncocytic adrenocortical tumours (15 pure and 12 mixed or focal) with a ‘reticulin algorithm’ that combines retic- ulin staining with three Weiss parameters (high mito- tic index, necrosis, and/or vascular invasion).41 They found that 12 cases were malignant according to the Lin-Weiss-Bisceglia system for pure tumours and the original Weiss system for mixed or focal tumours. Extensive or focal disruption of the reticulin network was identified in 16 of the 27 cases. In 14 of the 16 reticulin-disrupted cases, a high mitotic index, necro- sis, and/or vascular invasion was identified, and these 14 cases were considered to be malignant according
Table 5. Diagnostic algorithm for oncocytic adrenocortical tumours 12,46*
Major criteria
1. High mitotic rate
2. Atypical mitoses
3. Venous invasion
Minor criteria
1. Large size (>100 mm) and/or high weight (>200 g)
2. Necrosis
3. Capsular invasion
4. Sinusoidal invasion
*One major criterion indicates malignancy, one to four minor crite- ria indicate uncertain malignant potential (borderline), and the absence of all major and minor criteria indicates benign.
to the reticulin algorithm.41 They also noted that the pure oncocytic adrenocortical tumours were more indolent than conventional carcinomas.41
In 2016, Duregon et al. validated the prognostic role of the Helsinki score (3 points for a mitotic count of >5/50 HPFs plus 5 points for necrosis plus abso- lute value of the Ki67 proliferation index) in 225 adrenocortical carcinomas.40 Of the 225 adrenocorti- cal tumours, 24 (10%) were purely oncocytic tumours with a Weiss score of ≥3.40 With the Lin- Weiss-Bisceglia classification, the 24 pure oncocytic tumours were reclassified as: one benign, five uncer- tain malignant potential, and 18 malignant.40 The Helsinki score was applied to these cases also, and, when the proposed Helsinki score cut-offs (<13 versus ≥19) were applied, 15 (12 alive with no disease and three alive with disease) were in a group with a bet- ter prognosis; no case had a score between 13 and 19, and nine cases (five alive with no disease, two alive with disease, and two dead from disease) were in the group with a worse prognosis. Cases with a Helsinki score of ≥19 had decreased survival (mean 32 months) as compared with those with a score of <13 (mean 74 months). 40
Myxoid adrenocortical carcinoma
Myxoid adrenocortical carcinomas are uncommon tumours described as case reports and in small series (Figure 1).13,14,47-58 These tumours are often func- tional.13 In a study of 14 myxoid adrenocortical tumours from the Mayo Clinic (six adenomas and eight carcinomas), all patients with adenomas were alive
without disease. Four patients with carcinomas had died of disease, two were alive with metastatic disease and one was alive without disease at 5 years of follow- up.55 The amount of myxoid change ranged from 10% to 95% of the tumours.55 The immunophenotype of these tumours is similar to that of other adrenocortical tumours (positive for vimentin, synaptophysin, a-inhi- bin, and melan A, and can be positive for keratin). 13,55 The myxoid areas are positive for Alican blue, and gen- erally negative for periodic acid Schiff and muci- carmine.55 Neurofilament and CD56 have also been noted in the myxoid areas.13 In a study of 14 myxoid adrenocortical tumours (12 carcinomas and two bor- derline tumours), the amount of myxoid component ranged from 5% to 90%.13 Two distinct growth pat- terns were identified. One showed focal myxoid changes (5-20%) in tumours (four cases) otherwise similar to conventional adrenocortical carcinoma with large atypical cells with eosinophilic cytoplasm and dif- fuse or nodular architecture, which were referred to as conventional adrenocortical carcinomas with focal myxoid degenerative changes.13 One of the four patients with conventional adrenocortical carcinoma with focal myxoid degenerative change died of disease, and the other three were alive with disease at follow- up. Among tumours with the other pattern (10 cases), most had a prominent myxoid stromal component (although it ranged from 5% to 90%) with small cells with mild atypia arranged in cords and microcysts, and these were referred to as myxoid adrenocortical carcinomas.13 Six of the 10 patients with tumours showing these features died of disease, one was alive with disease and three were alive with no evidence of disease at follow-up. Although most of the tumours in this study had malignant features, one of two cases with a Weiss score of 1 was associated with a fatal out- come.13 Thus, the behaviour of these tumours, even with a low Weiss score, is difficult to predict with cer- tainty. A recent study validating the prognostic role of the Helsinki score in 225 adrenocortical tumours included 17 myxoid adrenocortical tumours.40 With proposed Helsinki score cut-offs (<13 versus ≥19), three myxoid cases (two without evidence of disease and one alive with disease had Helsinki scores of <13, and 14 (one without evidence of disease, eight alive with disease, and six who had died of disease) had a Helsinki score of ≥19.40
Sarcomatoid adrenocortical carcinoma
Sarcomatoid adrenocortical carcinoma (adrenocortical carcinoma with sarcomatoid areas) and adrenocorti- cal carcinosarcomas are rare types of adrenocortical
carcinoma, with the literature consisting primarily of case reports.15,59-68 These tumours are considered to be particularly aggressive, even more so than conven- tional adrenocortical carcinoma.59 These tumours may or may not be functional. They are composed of morphologically malignant epithelial and specialized mesenchymal cells (Figure 1). In addition to cases with spindle components, some may have heterolo- gous differentiation with a rhabdomyosarcomatous component or a chondrosarcomatous and osteosarco- matous component. 61,67,69
Recently, a study of six cases (three previously reported) detailed the clinicopathological features of the cases, and showed that Wnt-ß-catenin signalling pathway dysregulation and mutational inactivation of TP53 are common genetic events in sarcomatoid adrenocortical carcinomas, a subset of which are monoclonal in origin. They also found the tumours to be enriched for epithelial-mesenchymal transition- related markers and stem cell factors that may be associated with the poor prognosis of these tumours and may provide possible therapeutic targets.15
Adrenocortical carcinomas in children
Adrenocortical carcinoma is rare in children. A SEER database query for the years 1973-2008 identified 85 patients (57 females and 28 males) aged <20 years with adrenocortical carcinoma.7º An annual incidence of 0.21 per million was calcu- lated.7º These tumours are more common in females than in males.70-73 Adrenocortical carcinomas in children often present with symptoms of endocrine dysfunction, but can present as an asymptomatic mass.10,71-75 Adrenocortical carcinomas occur spo- radically, but can occur in syndromes.
Most benign adrenocortical tumours are readily diagnosed in children, but differentiating some benign adrenocortical tumours from adrenocortical carcino- mas in children can be very challenging. Features of malignancy in adult tumours may not be associated with aggressive behaviour in tumours in children.
Wieneke et al. studied 83 adrenocortical tumours in children aged <20 years (50 females and 33 males).76 The tumours ranged in size from 20 to 200 mm (mean of 88 mm).76 According to the crite- ria for adult adrenocortical tumours, nine tumours would be classified as adenomas and 74 as carcino- mas.76 However, only 23 of these tumours were asso- ciated with clinically malignant behaviour. Nine features were associated with malignant clinical behaviour (tumour weight >400 g, tumour size >105 mm, vena cava invasion, capsular or vascular
invasion, extension into periadrenal soft tissue, con- fluent necrosis, >15 mitoses/20 HPFs, and atypical mitoses).76 However, tumours with metastases had anywhere from one to nine features, whereas those with a good clinical outcome had no features to seven features. Interestingly, vena cava invasion, necrosis and increased mitotic activity were indepen- dently suggestive of malignancy. A three-tier system was proposed, in which tumours with two or fewer criteria were classified as having a benign long-term clinical outcome, those with three criteria as being indeterminate for malignancy, and those with four or more criteria as having a poor clinical outcome (Table 6).76 Subsequent studies confirmed this three- tier scoring system to be reproducible in predicting behaviour in paediatric adrenocortical tumours.77-80
In a multi-institutional study of 13 paediatric adrenocortical tumours evaluated according to Wie- neke’s three-tier system and standard Weiss criteria, the majority (76.9%) presented with endocrine dys- function.78 According to Wieneke’s three-tier system, the tumours were assigned to a benign (n = 6), an intermediate (n = 1) or a malignant (n = 6) group. However, if the standard Weiss criteria had been used, three cases with benign clinical behaviour would be assigned to a malignant group. Their study validates the Wieneke scoring system in predicting malignancy in paediatric adrenocortical tumours.78 They also found that younger age at presentation was associated with a better prognosis.78 In a study of 17 paediatric adrenocortical tumours (nine adeno- mas, seven carcinomas, and one designated as inter- mediate for malignancy), the most significant
Table 6. Diagnostic criteria for paediatric adrenocortical tumours76
Tumour weight >400 g
Tumour size >105 mm
Vena cava invasion
Capsular invasion
Vascular invasion
Extension into periadrenal soft tissue or adjacent organs
Necrosis
>15 mitotic figures/20 high-power fields
Atypical mitotic figures
Up to two features indicates benign, three features indicate indeterminate, and four or more features indicate a poor clinical outcome.
markers in favour of malignancy were capsular and venous invasion, followed by a mitotic rate of >15/20 HPFs. p53 was overexpressed in 86% of carcinomas, and a significant correlation between the Ki67 index and Wieneke scoring system was noted. The authors suggested that immunohistochemical staining for Ki67 and p53 may be helpful ancillary tests.79
A variety of prognostic features have been found to be significant in paediatric adrenocortical carcinomas, such as functional status, patient age, tumour size and/or weight, invasion, mitotic rate, p53, and p57. Adrenocortical carcinomas in children are often func- tional, and children may present with Cushing syn- drome, virilization, precocious puberty, or feminization.10,71-75 A large single-institution study did not find the functional status of adrenocortical tumours to have prognostic significance,75 but others have found a presentation of endocrine dysfunction to be prognostically significant.73 In a study of 85 adrenocortical carcinomas, younger children (≤4 years) were more likely to have more favourable features, localized disease, smaller tumours (<100 mm) and better 5-year survival than older children (5-19 years).70 After accounting for tumour size, only age maintained statistical significance, as the most significant predictors of cancer-specific death were older age (5-19 years) and distant disease.7º In a study of 58 Italian paediatric adrenocortical carci- nomas, 24 cases had material available for histologi- cal review, and were classified according to the Wienecke criteria. The Wienecke scoring system was a strong predictor of prognosis.80 The Wieneke score, tumour volume of <200 cm3 and age <4 years posi- tively correlated with overall and event-free survival. Whereas patient age <4 years was a good prognostic feature, patients aged ≥12 years had a 9.44-fold higher risk of worse event-free survival than those aged <4 years, and a 7.79-fold higher risk of worse overall survival.80 TP53 mutation was also more fre- quent in malignant tumours.8º In a study of 29 pae- diatric adrenocortical carcinomas, larger tumour size and higher mitotic rate were significant predictors of tumour recurrence, and older age, higher mitotic rate and necrosis were independent predictors of tumour- related death.81 In a study of 111 adrenocortical car- cinomas from the National Cancer Data Base in patients aged <18 years, the median age was 4 years, the majority of patients were female (69%), the med- ian tumour size was 95 mm, 19 of 62 patients with available data presented with metastases, and overall 1- and 3-year survival rates were 70% and 64%, respectively.82 Age ≥4 years, tumour size ≥100 mm, extension of tumour into surrounding tissue and
metastatic disease were all significantly associated with decreased survival. Among patients who under- went surgical procedures, margin status was also found to be associated with survival.82 In a retrospec- tive international analysis of 82 paediatric adrenocor- tical carcinomas in Europe from 2000 to 2013, multivariable analysis showed that the main factors associated with progression-free survival were tumour volume >200 cm3 and distant metastases.83
Loss of heterozygosity of 11p15 containing the p57 gene has been suggested to be helpful in differentiat- ing adrenocortical neoplasms.84 YAP1 overexpression has been identified as a marker of poor prognosis in adrenocortical tumours.85 TP53 germline mutation is a cause of Li-Fraumeni syndrome, which is a syn- drome associated with adrenocortical carcinoma.86 In one study, 88 consecutive patients with adrenocorti- cal carcinoma underwent germline TP53 sequencing, and germline mutations were present in 50% of chil- dren.86 The mutations did not correspond to conven- tional hotspot mutations, and there was a wide range of mutant protein function.86 TP53 mutations were prevalent in children with adrenocortical carcinoma, but decreased with age.86 Many studies have found p53 expression to be frequent in malignant adreno- cortical tumours.79,80 Insulin-like growth factor 1 receptor overexpression, decreased expression of HLA- DRA, HLA-DPA1, and HLA-DPB1, and major histo- compatibility complex class II expression have also been suggested as a possible markers of aggressive disease in paediatric adrenocortical tumours.87-89
Proliferative activity
Mitotic rate is an integral component in diagnostic algorithms separating benign from malignant adreno- cortical tumours (Figure 1; Tables 1-6). On compar- ison of the Hough, van Slooten and Weiss systems with a stepwise discriminant diagnostic system, mito- tic figure counting was the most important malig- nancy criterion.90 Mitotic rate is also important in grading adrenocortical tumours. Weiss et al. desig- nated adrenocortical carcinomas with >20 mitoses as high grade and those with ≤20 mitoses as low grade.36 Erickson et al. also used mitotic activity in grading adrenocortical tumours.8 Volante et al. strati- fied adrenocortical tumours into three risk groups that included mitotic activity, and found that a mito- tic rate of >9/50 HPFs and stage III/IV were adverse prognostic factors. 38
Proliferative activity as measured with Ki67 (MIB1) is studied and utilized in evaluating adrenocortical tumours (Figure 1). A study showed the following
Ki67 index values: normal adrenal glands, 1.9; hyperplasias, 3.47; adenomas, 2.11; and carcinomas, 11.94.91 A cut-off of >5% is a sensitive and specific indicator of malignancy in adrenocortical tumours.91 A combination of insulin-like growth factor 2 (IGF2) and Ki67 index has also been found to be sensitive and specific for malignancy in adrenocortical tumours.92 Ki67 indices have prognostic significance in adrenocortical carcinomas. In a study of 319 adrenocortical carcinomas and an independent vali- dation cohort of 250 cases from three European cen- tres, age, tumour size, venous tumour thrombus and Ki67 index were significant prognostic markers.93 Of these, the Ki67 index was the single most important factor predicting recurrence in patients following RO resection.93 Another study of 17 adrenocortical carci- nomas found that a Ki67 index ≥7% was associated with shortened disease-free survival.94 In a retrospec- tive study of 86 adrenocortical carcinomas, multivari- able analysis showed that tumour size, Ki67 index, stage and resection status were independently associ- ated with overall survival.95 A study of 52 adreno- cortical carcinomas evaluated phospho-histone H3, and compared manual and computerized counting with standard manual haematoxylin and eosin-based counting and the Ki67 index.96 They found excellent interobserver agreement. They confirmed traditional mitotic count to be a strong predictor of overall sur- vival and better than phospho-histone H3-based eval- uation, but not as strong as the Ki67 index.96 They divided the adrenocortical carcinomas into three prognostic groups based on the Ki67 index (low, <20%; intermediate, 20-50%; and high, >50%). Overall, the Ki67 index was superior to mitotic count (irrespective of method of estimation) in the prognos- tic stratification of adrenocortical carcinomas for overall survival.96 A study comparing methods for measuring Ki67 proliferation indices in 18 adrenocor- tical carcinomas by seven pathologists using micro- scopes (manual analysis) and digital image analysis and by selecting ‘hotspots’ and ‘average’ indices found that digital image analysis significantly corre- lated with manual analysis in hotspots but not in the average fields.97 The hotspots had significantly and consistently higher Ki67 indices than the average areas by both methods.97 Patients with carcinomas with Ki67 indices <10% had significantly better out- comes than those with Ki67 indices >10% by manual analysis in hotspots. Overall, the Ki67 index in hot- spots measured by manual analysis best reflected the clinical and pathological features of the tumours.97 The authors suggested that digital image analysis needs improvement, including correction of its
overestimation of the value by counting of non- tumour cells and nuclear segmentation in areas of high cell density.97 In an international Ki67 repro- ducibility study of 76 adrenocortical carcinomas anal- ysed by 14 observers, each using their method of preference (eyeballing, formal manual counting, and digital image analysis), the interobserver variation was significant in the absence of any correlation between the various methods.98 When 61 static images were distributed to the observers with instruc- tions to follow a category-based scoring approach, low levels of interobserver and intraobserver concor- dance were detected. An open source virtual applica- tion was utilized in 61 virtual slides, and the software Ki67 values were validated by digital image analysis. Dividing the cases into three classes with cut-offs of 0%, 15%, and 30% or 1%, 10%, and 20% showed significantly different overall survival rates.98 They concluded that, as the current methods for Ki67 scoring assessment vary greatly, and interobserver variation limits its clinical utility, digital microscopy- enabled methods may be helpful in reducing variation, increasing reproducibility and improving reliability in the clinical setting.98
Immunophenotype
Immunophenotypic studies are important in differen- tiating adrenocortical tumours from phaeochromocy- tomas and from metastases to the adrenal gland. In a review of 300 consultation cases, the most common pitfall (10 cases) was distinguishing adrenocortical carcinoma from phaeochromocytoma and vice versa.99 Both adrenocortical tumours and phaeochro- mocytomas are positive for synaptophysin, but adrenocortical tumours, unlike phaeochromocytomas, are negative for chromogranin. S100 usually high- lights sustentacular cells in phaeochromocytomas and is negative in adrenocortical tumours. Unlike phaeochromocytomas, adrenocortical tumours are usually positive for Cam 5.2, although other keratins may be weakly positive or negative. Adrenocortical tumours are often positive for a-inhibin, particularly functional tumours, and calretinin.100,101 Steroido- genic factor 1 is also a marker of adrenocortical derivation, and the antibody N1665 may have prog- nostic significance and be a possible therapeutic target.102,103 Adrenocortical carcinomas must be dif- ferentiated from metastases to the adrenal glands, as metastases to the adrenal gland are more common than adrenocortical carcinoma. Both adrenocortical tumours and metastatic melanoma are positive for Melan A (Mart1), but adrenocortical tumours are
negative for S100 and other melanocytic markers.104 Melan A (Mart1) and a-inhibin are helpful in differ- entiating adrenocortical from renal and hepatic tumours.104 In a study of Melan A (Mart1) in adrenocortical tumours, phaeochromocytomas, and metastatic and extra-adrenal carcinomas, all adreno- cortical tumours were positive for Melan A (Mart1), and no phaeochromocytoma or metastastatic carci- noma was positive for Melan A (Mart1). Only one of 269 extra-adrenal carcinomas was positive for Melan A (Mart1). Metastatic carcinomas, such as those from the breast and lung, often metastasize to the adrenal gland. Breast cancers are usually positive for keratin 7 and negative for Melan A (Mart1), a-inhibin, and thyroid transcription factor 1 (TTF1). Lung adenocar- cinomas are usually positive for keratin 7 and TTF1. Immunohistochemical studies are also useful in dis- tinguishing mesenchymal and lymphoproliferative neoplasms from adrenocortical carcinomas.
In addition to Ki67, as discussed previously, other immunohistochemical markers, such as topoiso- merase IIa, p53, E-cadherin, retinoblastoma, CDK4, p27, and HER-2/neu, among many others, have been evaluated for differentiating benign from malignant or as prognostic markers in adrenocortical tumours, but overlap limits their practical utility.105-108 In addition to Ki67, ß-catenin is also used in the evalua- tion of adrenocortical tumours. As the prevalence of Lynch syndrome in patients with adrenocortical car- cinoma is reported to be 3.2%, immunohistochemical screening of all adrenocortical carcinomas has been suggested for Lynch syndrome patients.109
Genetic abnormalities
Adrenocortical tumours, both benign and malignant, show genetic chromosomal aberrations. Carcinomas have more aberrations (gains involving 4, 5, 7, 12, 16, 19 and 20, and losses involving 1p, 2q, 11q, 13, 17p and 22) than adenomas, which often show gains in the 9q34 region, which includes the stromal cell- derived factor-1 (SDF-1) locus, that may be associated with SDF-1 overexpression.28,110-112 Copy-number estimates at 5q, 7p, 11p, 13q, 16q and 22q and gene expression studies have showed differences between adrenocortical carcinomas and adenomas.112,113 IGF2 and steroidogenesis clusters of genes may also be predictive of malignancy.114 In a study of 153 adrenocortical tumours, clustering analysis of gene expression profiling discriminated adenomas from car- cinomas, and identified carcinoma groups with sur- vival differences.115 In a study of 33 adrenocortical carcinomas, 22 adenomas, and 10 non-tumor tissues,
2875 differentially expressed genes were identified, and carcinoma groups were identified with survival differences.116
Li-Fraumeni syndrome is caused by inactivating mutations of TP53 (17q13). Adrenocortical carci- noma is a significant component of Li-Fraumeni syn- drome. The prevalence of TP53 mutations in children with adrenocortical carcinoma is reported to range from 50% to 97%.117 In a study from the University of Michigan Endocrine Oncology Clinic, four of 53 (7.5%) unselected patients with adrenocortical carci- noma were found to have TP53 mutations.117 Three of the four were aged >18 years (3/53, 5.8%); thus, genetic counselling and germline TP53 testing may be helpful for all patients with adrenocortical carci- noma, as basing a decision on age restriction or strength of family history would not identify some mutation carriers.117 Approximately 20-30% of spo- radic adult adrenocortical carcinomas have somatic TP53 mutations, and allelic loss of 17p13 can occur in sporadic adult adrenocortical carcinomas.118,119 Although TP53 mutations are associated with larger, more aggressive tumours, more advanced stage and poor outcome in adults with adrenocortical carcino- mas, they are common in paediatric adrenocortical carcinomas, and are not predictive of outcome in children.118,120,121 TP53 and CTNNB1 mutations are associated with poor-outcome and have been found to be mutually exclusive in these tumours.121
Two of the most common alterations in adrenocor- tical carcinoma are IGF2 overexpression and constitu- tive activation of the Wnt-B-catenin pathway. Beckwith-Wiedemann syndrome is also associated with adrenocortical carcinoma and with 11p15 alter- ations. IGF2, CDKN1C/p57 and H19 are located on 11p15.21 IGF2 overexpression and down-regulation of CDKN1C and H19 can also occur in sporadic adrenocortical tumours.122 Carcinomas have been found to show higher expression of IGF2, MALD2L1 and CCNB1 and lower expression of ABLIM1, NAV3, SEPT4 and RPRM than adenomas.123 The Wnt-ß- catenin pathway is activated in both adrenocortical adenomas and carcinomas, and ß-catenin activation is associated with decreased survival in adrenocortical carcinomas.124 Activation of the Wnt-ß-catenin path- way is usually due to mutations in the ß-catenin gene (CTNNB1). CTNNB1 mutations and abnormal ß-catenin occur in benign and malignant adrenocorti- cal tumours.125 Activation of the Wnt signalling pathway is common in adrenocortical adenomas and carcinomas.126 In a study of 100 adrenocortical car- cinomas, abnormal cytoplasmic and/or nuclear ß-catenin immunohistochemical staining was
identified in 51 adrenocortical carcinomas, suggesting Wnt-B-catenin pathway alteration.124 CTNNB1 mutations were identified in 36 cases, all of which showed abnormal ß-catenin immunohistochemical staining, suggesting that activation of the Wnt-ß- catenin pathway may be due to activating mutations of CTNNB1 in 70% of cases. They also found that CTNNB1 mutations were most common in large and non-secreting adrenocortical carcinomas, and sug- gested that Wnt-ß-catenin pathway activation may be associated with less differentiated carcinomas.124
Recently, comprehensive genomic characterization of 91 adrenocortical carcinomas (84 usual type, four oncocytic, two sarcomatoid, and one myxoid) was completed as part of the Cancer Genome Atlas.127 Known adrenocortical cancer driver genes were expanded to include PRKAR1A, RPL22, TERF2, CCNE1, and NF1.127 The genes altered most fre- quently by somatic mutations, copy-number alter- ations and epigenetic silencing were TP53 (21%), ZNRF3 (19%), CDKN2A (15%), CTNNB1 (16%), TERT (14%), and PRKAR1A (11%).127 Most of the alter- ations were mutations or copy-number changes, but deletion and epigenetic silencing by promoter DNA methylation was frequent in CDKN2A.127 Massive DNA loss followed by whole-genome doubling was fre- quent and associated with an aggressive clinical course.127 Three adrenocortical carcinoma subtypes with distinct clinical outcomes and molecular alter- ations were identified.127 Implementation of the three- class grading system using DNA-methylation profiles may eventually be utilized in the clinical setting.127
Conclusion
Diagnosing adrenocortical neoplasms can be chal- lenging, as reflected in the number of classification schemes. The Weiss system or modified Weiss system is most often used in the diagnostic setting, but new systems, such as the Helsinki system, continue to evolve. Although adrenocortical carcinomas are rare, they are in the differential diagnosis of every adreno- cortical tumour and metastasis to the adrenal gland that a surgical pathologist encounters. Diagnosing adrenocortical tumours with unusual features, such as oncocytic, myxoid, and sarcomatoid, can be partic- ularly challenging. Oncocytic adrenocortical tumours are classified according to what has become known as the Lin-Weiss-Bisceglia system.12 Myxoid or sarco- matoid adrenocortical carcinomas must also be recog- nized so that they are not mistaken for other tumours.13-15 Ancillary studies, such as those
involving Ki67, TP53, and ß-catenin, continue to grow in importance. Because adrenocortical carcinomas can be associated with syndromes such as Li-Fraumeni syndrome, Beckwith-Wiedemann syndrome, multiple endocrine neoplasia type I, Lynch syndrome, familial adenomatous polyposis, and neu- rofibromatosis I, genetic counselling, germline testing and even immunohistochemical testing of the tumour, such as in screening for Lynch syndrome, may be helpful. As more comprehensive and inte- grated genomic characterizations continue to advance our understanding of the pathogenesis of these tumours, molecular screening for personalized treat- ment will become more common, and additional therapeutic targets will hopefully emerge.
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