ELSEVIER
Perspectives in pathology
The Weiss system for evaluating adrenocortical neoplasms: 25 years later
Sean K. Lau MD*, Lawrence M. Weiss MD
Department of Pathology, City of Hope National Medical Center, Duarte, CA 91010, USA
Received 9 February 2009; revised 4 March 2009; accepted 6 March 2009
Keywords:
Weiss system; Adrenal cortex; Adrenocortical adenoma; Adrenocortical carcinoma
Summary The evaluation and categorization of adrenocortical neoplasms remain among the most challenging areas in adrenal pathology. The Weiss system, first introduced 25 years ago, provides specific guidelines for differentiating adrenocortical adenoma from adrenocortical carcinoma and is considered the standard for determining malignancy in tumors of the adrenal cortex. Considerable advances in the understanding of the pathology of adrenocortical neoplasia have occurred since delineation of the Weiss system, offering alternative approaches to the contemporary assessment of adrenocortical tumors.
@ 2009 Elsevier Inc. All rights reserved.
1. Introduction
Nodules involving the adrenal cortex are considered a relatively common occurrence [1]. These lesions are often detected incidentally during radiologic studies performed for investigation of unrelated clinical conditions. Based on clinical and imaging considerations, a number of these adrenocortical nodules will require surgical resection [1,2]. Consequently, adrenal glands are encountered with increas- ing frequency in many histopathology laboratories.
The pathologic assessment of an adrenocortical tumor often raises diagnostically challenging issues. Proper cate- gorization of the lesion as benign or malignant is often problematic, even for experienced pathologists. This distinc- tion has important clinical implications because a diagnosis of adrenocortical carcinoma carries an extremely poor prognosis [3]. Many adrenocortical carcinomas will show evidence of locoregional invasion or distant metastasis at presentation and are easily diagnosed as such [4]. However, identifying the
malignant potential of an adrenocortical neoplasm localized to the adrenal gland remains difficult.
In 1984, the senior author of this report (L. M. W.) proposed a set of histologic guidelines for evaluating adrenocortical neoplasms [5]. Since that time, the Weiss system has been adopted by pathologists worldwide and has arguably become the standard for the assessment and categorization of neoplasms of the adrenal cortex.
In the ensuing 25 years since the Weiss system was introduced, there has been a tremendous growth in the understanding of the biology and pathology of adrenocortical tumors. This review focuses on some of these advances and their impact on the evaluation of adrenocortical neoplasms and provides an overview and discussion of the Weiss system in the context of these contemporary developments.
2. The Weiss system
The Weiss system was developed following detailed histologic analysis of a series of 43 adrenocortical tumors [5]. All cases occurred in adult patients with a minimum of 5 years clinical follow-up. Nine histologic features were
* Corresponding author.
E-mail address: slau@coh.org (S. K. Lau).
Table 1 Weiss system for separating benign from malignant adrenocortical neoplasms [5,7]
High nuclear grade (grade 3 or 4 according to criteria of Fuhrman et al [6])
Mitotic rate greater than 5 per 50 high-power fields Atypical mitoses Clear cells comprising 25% or less of the tumor Diffuse architecture (greater than one third of the tumor) Necrosis Invasion of venous structures Invasion of sinusoidal structures Invasion of capsule of tumor
NOTE. The presence of 3 or more criteria correlates with subsequent malignant behavior.
determined to be useful in separating clinically benign from malignant neoplasms of the adrenal cortex (Table 1). Definitions of the 9 histopathologic criteria comprising the Weiss system are provided below.
2.1. High nuclear grade
Grading of adrenocortical neoplasms is based on the Fuhrman nuclear grading system that is commonly used to evaluate renal cell carcinomas [6]. Adrenocortical tumors exhibiting high-grade nuclei show features that would meet the definition of grade 3 or grade 4 in the Fuhrman system. As such, to meet the criterion of high nuclear grade, a given adrenocortical tumor should demonstrate enlarged, oval to lobated nuclei with coarsely granular to hyperchro- matic chromatin and easily discernible, prominent nucleoli (Fig. 1A). Because individual tumor cell nuclei in adrenocortical neoplasms are often heterogeneous in appearance, it is important to remember that the overall grade assigned to a given tumor is based on the most histologically abnormal area present, even if only focal.
2.2. Mitotic rate greater than 5 per 50 high-power fields
In the Weiss system, mitotic rate is assessed in a systematic fashion by evaluating 10 high-power fields in the area of the greatest mitotic activity in each of 5 slides (Fig. 1B). If less than 5 slides are available for a given case, evaluating a greater number of fields per slide to satisfy a total of 50 high-power fields is acceptable. Mitotic figures should be clearly recognizable as such and should not be confused with degenerating or karyorrhectic nuclei, which are sometimes seen in adrenocortical tumors.
2.3. Atypical mitoses
Mitotic figures are defined as atypical if they demonstrate an abnormal chromosomal distribution or an excessive
number of mitotic spindles with a multipolar morphologic appearance (Fig. 1C).
2.4. Clear cells comprising less than 25% of the tumor
Adrenocortical tumors can show a variable proportion of clear cells with pale staining, lipid-rich, finely vacuolated
A
B
C
cytoplasm resembling cells of the zone fasciculata of the normal adrenal gland. A predominant clear cell component was a finding more frequently observed in adrenocortical adenomas, whereas adrenocortical carcinomas typically contained few (<25%) clear cells and were composed primarily of cells with nonclear, eosinophilic cytoplasm (Fig. 2A) [5].
A
B
C
2.5. Diffuse architecture
The architecture of an adrenocortical neoplasm is classified as diffuse if greater than one third of the tumor demonstrates a growth pattern characterized by patternless sheets of cells (Fig. 2B). Other organized arrangements of tumor cells, such as nests, cords, or trabeculae, would be considered nondiffuse.
2.6. Necrosis
Necrosis is regarded as present if involving a confluent area of cells (Fig. 2C). The presence of isolated apoptotic cells alone does not satisfy this criterion.
2.7. Invasion of venous structures
In the Weiss system, a vein is defined as an endothelial- lined structure with a wall containing smooth muscle. Tumor invading venous structures often appears as plugs or form polypoid luminal projections covered by a layer of endothelial cells (Fig. 3A). Venous invasion should be distinguished from free-floating tumor cells, which may reflect artifactual implantation of tumor into vascular spaces or retraction artifacts induced by fixation simulating vascular involvement.
2.8. Invasion of sinusoidal structures
A sinusoid is considered an endothelial-lined vascular channel that lacks a supportive smooth muscle wall. As is the case with venous structures, the presence of sinusoidal invasion by tumor should be accepted only when unequivocal, and artifactual vascular involvement excluded (Fig. 3B).
2.9. Invasion of capsule
Capsule invasion is characterized by tumor extending into or completely traversing through the capsule of the adrenal gland, with an associated stromal reaction (Fig. 3C).
A given adrenocortical tumor is assigned a numeric score according to the number of the aforementioned histologic features present. The individual histologic parameters in the Weiss system are not weighted and carry equal value; individual criterion are simply given a score of 1 if present and 0 if absent, yielding an overall score which ranges from 0 to 9. Based on application of this system, 18 of 19 patients with adrenocortical tumors exhibiting 4 or more of the Weiss criteria developed documented recurrence or metastatic disease, whereas all 24 patients with tumors showing 2 or fewer of the Weiss criteria had a benign clinical outcome [5]. In a subsequent study, the threshold for classifying an adrenocortical neoplasm as malignant was lowered from 4 to 3 criteria
A
B
C
based on experience with a patient with an adrenal tumor exhibiting only 3 adverse histologic features who devel- oped local recurrence and ultimately death from disease [7]. Using the presence of 3 rather than 4 criteria as a breakpoint showed similar efficacy in identifying adreno- cortical tumors with a propensity for aggressive biologic behavior; all tumors with less than 3 criteria were observed to behave in a benign fashion, whereas in contrast,
recurrence or metastasis developed in 91% of patients whose tumors demonstrated 3 or more unfavorable morphologic features [7].
Among the histologic parameters comprising the Weiss system, those observed most frequently in adrenocortical carcinomas include a diffuse pattern of growth, necrosis, high-grade nuclear atypia, and a predominance of acidophilic cells [5,7-11], although none of these particular features are considered exclusive to carcinomas, having been well documented in adrenocortical tumors that clinically did not recur or develop metastatic disease [7,9,10,12]. Although no single morphologic feature is diagnostic for malignancy, those criteria considered the most specific for a diagnosis of adrenocortical carcinoma include a mitotic rate of greater than 5 per 50 high-power fields, atypical mitoses, and invasion of venous structures [5,9].
Diagnostic criteria for adrenocortical carcinoma have also been proposed by other investigators based on observed differences between adrenocortical tumors with documented benign or malignant clinical outcome (Tables 2-4) [10,13,14]. Similar to the Weiss system, evaluation of a combination of microscopic features forms the basis for establishing a diagnosis of malignancy. Although these various approaches have excellent predictive value in assessing malignancy in adrenocortical tumors, the advan- tage of the Weiss system is that it is perhaps the simplest to use in daily practice [12,15]. In contrast with other systems for differentiating adrenocortical carcinoma from adrenocor-
| Criteria | Numeric value |
|---|---|
| Histologic criteria | |
| Diffuse growth pattern | 0.92 |
| Vascular invasion | 0.92 |
| Tumor cell necrosis | 0.69 |
| Broad fibrous bands | 1.00 |
| Capsular invasion | 0.37 |
| Mitotic index >1 (no. of mitoses in 100 | 0.60 |
| high-power fields divided by 10) | |
| Nuclear pleomorphism (moderate/marked) | 0.39 |
| Nonhistologic criteria | |
| Tumor mass (>100 g) | 0.60 |
| Urinary 17-ketosteroids | 0.30 |
| (>10 mg/g creatinine per 24 h) | |
| Response to ACTH (17-hydroxysteroids increased | 0.42 |
| 2-fold after 50 µg ACTH, intravenous) | |
| Cushing syndrome with virilism, | 0.42 |
| virilism alone, or no clinical syndrome | |
| Weight loss (>10 lb in last 3 mo) | 2.0 |
NOTE. The sums of the numeric values for criteria present are used to derive nonhistologic and histologic indices of malignancy. The mean histologic index of malignancy associated with benign tumors was 0.17; of indeterminate tumors, 1.00; and of malignant tumors, 2.91.
| Criteria | Discriminating value |
|---|---|
| Regressive changes (necrosis, hemorrhage, fibrosis, or calcification) | 5.7 |
| Loss of normal structure | 1.6 |
| Nuclear atypia (moderate/marked) | 2.1 |
| Nuclear hyperchromasia | 2.6 |
| (moderate/marked) | |
| Abnormal nucleoli | 4.1 |
| Mitotic activity (>2 mitotic | 9.0 |
| figures per 10 high-power fields) | |
| Capsular and/or vascular invasion | 3.3 |
NOTE. Summating the discriminating values of each parameter present generates a histologic index. A histologic index greater than 8 is associated with malignancy.
tical adenoma, the Weiss system does not require assigning weighted values to histologic criteria and does not rely on correlation with clinical data, which is sometimes incomplete or unavailable to the pathologist. Application of the Weiss system has also been shown to be highly reproducible with excellent interobserver agreement [16].
The effectiveness of the Weiss system in separating benign from malignant adrenocortical tumors has been validated by other investigators [10,16,17]. Among 49 adrenocortical tumors, Aubert et al [16] observed that the presence of 3 or more of the Weiss criteria correctly correlated with clinical outcome in 98% of the cases and was significant for malignancy with 100% sensitivity and 96% specificity. Similarly, a recent study demonstrated use of the Weiss system to have a sensitivity of 94% and a specificity of 97% in
Table 4 Stepwise discriminant diagnostic system for adrenocortical proliferative lesions [10]
Adrenocortical carcinoma
>5 MF per 50 HPF or SDMF/HPF>0.3
2-5 MF per 50 HPF or SDMF/HPF 0.2-0.3, high nuclear grade, and diffuse growth pattern
2-5 MF per 50 HPF or SDMF/HPF 0.2-0.3, high nuclear grade, nondiffuse growth pattern, and desmoplastic tissue reaction Adrenocortical adenoma
2-5 MF per 50 HPF or SDMF/HPF 0.2-0.3, high nuclear grade, nondiffuse growth pattern, and myxoid tissue reaction
2-5 MF per 50 HPF or SDMF/HPF 0.2-0.3, low nuclear grade, and anisokaryosis
<2 MF per 50 HPF or SDMF/HPF <0.2, anisokaryosis, and atrophic adrenal cortex adjacent to lesion
Adrenocortical nodular hyperplasia
<2 MF per 50 HPF or SDMF/HPF <0.2, anisokaryosis, and hyperplastic adrenal cortex adjacent to lesion
<2 MF per 50 HPF or SDMF/HPF <0.2, and no anisokaryosis
2-5 MF per 50 HPF or SDMF/HPF 0.2-0.3, low nuclear grade, and no anisokaryosis
Abbreviations: MF, mitotic figures; HPF, high-power fields.
distinguishing malignant from benign adrenocortical neo- plasms [10].
3. Prognostic value of the Weiss system
In addition to effectively distinguishing adrenocortical adenomas from adrenocortical carcinomas, application of the Weiss system may also provide useful information pertaining to the biologic behavior of adrenocortical tumors. In a study of 87 adrenocortical neoplasms, distant metastasis or local recurrence was observed in 91% of tumors exhibiting 3 or more histologic criteria of the Weiss system, whereas all patients with tumors showing less than 3 criteria remained free of disease [7]. A correlation between the Weiss system and clinical outcome was also shown by Lucon et al [17]. In their study, the 5-year survival of patients with tumors characterized by 3 or fewer parameters of the Weiss system was 100% as compared to a survival rate of 62% in patients with tumors displaying greater than 3 criteria. A statistically significant difference in disease-free survival between benign and malignant adrenocortical tumors classified according to the Weiss system has also been demonstrated, with the presence of more than 3 pathologic criteria observed to be strongly associated with shorter disease-free survival and increased risk of relapse [18,19]. Among the individual histologic para- meters of the Weiss system, mitotic rate and atypical mitotic figures appear to be the most important predictors of survival in both patients with and without metastatic disease [7-9,20].
4. The modified Weiss system
Although the popularity of the Weiss system in assessing malignancy in adrenocortical tumors can largely be attributed to its reliability and relative simplicity compared with other proposed systems, several authors have felt recognition and interpretation of some of the histologic criteria of the Weiss system to be difficult and subject to interobserver variability [15,21,22]. In 2002, Aubert et al [16] proposed simplifying the Weiss system by eliminating criteria that were considered to be more subjective or difficult to interpret (Table 5). Using a stepwise regression analysis, a modified scoring system was generated using 5 criteria: 2 mitotic rate + 2 cytoplasm +
Table 5 Modified Weiss system for separating benign from malignant adrenocortical neoplasms [16]
Mitotic rate (>5 per 50 high-power fields)
Cytoplasm (clear cells comprising 25% or less of the tumor) Abnormal mitoses Necrosis
Capsular invasion
NOTE. Modified Weiss scoring system: 2 × mitotic rate +2 × cytoplasm + abnormal mitoses + necrosis + capsular invasion.
A score of 3 or greater correlates with subsequent malignant behavior.
abnormal mitoses + necrosis + capsular invasion. These 5 histologic criteria were included in this system based on high interobserver agreement as measured by k values. For an individual tumor, each criterion is given a score of zero when absent and one when present, yielding an overall score that can range from 0 to 7. Similar to the original Weiss system, a score of 3 or greater is considered indicative of malignancy. This modified system correlated well with the original Weiss system and may be easier to use in practice because only 5 histologic features are assessed rather than 9. A recent study has confirmed the diagnostic value of the modified Weiss system for evaluating malignancy in adrenocortical neo- plasms and has also shown this particular scoring system to be significantly correlated with time of survival in patients with adrenocortical carcinoma [23].
5. Pediatric adrenocortical neoplasms
Adrenocortical tumors are infrequently encountered in the pediatric age group. Because of their rarity, identifying morphologic features of prognostic significance and defining criteria for malignancy for adrenocortical neoplasms occur- ring in this patient population have been difficult. Evidence indicates that classification systems developed for distin- guishing benign from malignant tumors of the adrenal cortex in adults, including the Weiss system, are not entirely applicable to pediatric tumors and do not accurately predict clinical outcome in this particular context. Studies have demonstrated that many pediatric adrenocortical neoplasms that exhibit worrisome histologic findings associated with malignancy in adults, such as necrosis, nuclear pleomorph- ism, capsular invasion, vascular invasion, increased mitotic rate, and even atypical mitotic figures, ultimately prove to behave in a clinically benign manner [24-29]. Thus, histologic features important for establishing a diagnosis of malignancy in adult adrenocortical neoplasms may not carry the same significance in such tumors arising in the pediatric population.
Various pathologic parameters predictive of biologic behavior of adrenocortical tumors in the pediatric age group have been identified by different investigators. Bugg et al [30] proposed categorizing pediatric adrenocortical neoplasms based on evaluation of 4 histologic features of the Weiss system: high nuclear grade, necrosis, mitotic rate greater than 5 per 50 high-power fields, and atypical mitoses. Tumors lacking the aforementioned morphologic findings were classified as adrenocortical adenomas, with the remainder considered to be adrenocortical carcinomas. Carcinomas were further designated as low grade if the tumor displayed between 6 and 20 mitotic figures per 50 high-power fields or high grade if exhibiting greater than 20 mitotic figures per 50 high-power fields. Histologic classification using this modification of the Weiss system was found to be a statistically significant predictor of event- free survival: local recurrence or metastasis was observed in 9 of 16 high-grade adrenocortical carcinomas, 5 of 27 low-
Table 6 Armed Forces Institute of Pathology criteria for separating benign from malignant adrenocortical neoplasms in pediatric patients [27]
Tumor weight >400 g
Tumor size >10.5 cm
Extension into periadrenal soft tissues and/or adjacent organs Invasion into vena cava
Venous invasion
Capsular invasion
Presence of tumor necrosis
>15 mitoses per 20 high-power fields
Presence of atypical mitotic figures
NOTE. The presence of up to 2 criteria is associated with benign outcome; 3 criteria are considered indeterminate for malignancy; and 4 or more criteria are associated with malignancy.
grade adrenocortical carcinomas, and in none of the 11 tumors diagnosed as adrenocortical adenoma [30].
Tumor size has also been emphasized as a useful criterion for discriminating between benign and malignant adreno- cortical tumors in children. Cagle et al [24] found tumor weight to be the only reliable predictor of clinical outcome in pediatric adrenocortical neoplasms, with all tumors in their study exceeding 500 g exhibiting malignant behavior. Similarly, Ribeiro et al [25] reported tumor volume greater than 200 cm3 and tumor weight greater than 80 g to be factors associated with an unfavorable clinical outcome.
Investigators from the Armed Forces Institute of Pathol- ogy have recently assessed the prognostic value of a number of morphologic features in a large series of pediatric adrenocortical neoplasms with extensive clinical follow-up [27]. Several parameters were determined to be significantly associated with a poor clinical outcome, which are listed in Table 6. Among these, vena caval invasion, necrosis, and increased mitotic activity (>15 mitoses per 20 high-power fields) were the only variables independently predictive of malignant clinical behavior based on multivariate analysis. Based on these findings, the authors proposed separating adrenocortical neoplasms occurring in the pediatric age group according to the number of unfavorable pathologic features present in a given tumor into benign (≤2 criteria), indeterminate for malignancy (3 criteria), and malignant (≥4 criteria) categories. Using this particular approach, poor clinical outcome, defined as recurrence, metastasis, and/or death from disease, was observed in 18 of 28 patients with tumors classified as malignant, 3 of 18 patients with tumors classified as indeterminate for malignancy, and in 2 of 37 patients with pathologically benign tumors in their study [27].
6. Oncocytic adrenocortical neoplasms
Oncocytic tumors arising from the adrenal cortex are rare. Similar to oncocytic neoplasms involving other organs, those occurring in the adrenal gland are composed exclusively of cells with copious, densely granular, eosi-
nophilic cytoplasm resulting from the accumulation of mitochondria (Fig. 4) [31-35].
As with nononcocytic adrenocortical neoplasms, the distinction between benign and malignant oncocytic tumors of the adrenal gland can be difficult. Based on our experience with these particular tumors, it became clear that not all criteria of the Weiss system useful for predicting biologic behavior in nononcocytic neoplasms of the adrenal cortex are applicable in the context of oncocytic lesions [32]. In particular, essentially every adrenal oncocytic tumor is composed of cells with eosinophilic cytoplasm (ie, clear cells comprising ≤25% of the tumor), and most will exhibit a diffuse architecture as well as high-grade nuclear atypia in at least a subset of the neoplastic cells [32,34,35]. These parameters appear to be inherent features of adrenocortical oncocytic tumors, and the presence of these histologic findings alone has not been associated with neoplasms that have recurred or metastasized. Thus, in contrast with nononcocytic tumors of the adrenal, these 3 histologic features are not considered useful for predicting prognosis in oncocytic adrenocortical neoplasms.
In collaboration with Bisceglia et al [35], we have recently proposed guidelines for the evaluation of adrenocortical oncocytic tumors based on elements of the Weiss system applied to nononcocytic adrenal neoplasms (Table 7). Criteria for malignancy in oncocytic adrenocortical neoplasms include the presence of any one of the following microscopic features: mitotic rate of greater than 5 mitotic figures per 50 high-power fields, atypical mitoses, or invasion of venous structures. In the absence of the preceding criteria, tumors exhibiting large size (>10 cm and/or >200 g), necrosis, capsular invasion, or sinusoidal invasion are categorized as borderline oncocytic neoplasms of uncertain malignant potential. Adrenal oncocytic tumors lacking any of the aforementioned histologic features are considered benign.
The proposed system for evaluating oncocytic adreno- cortical tumors, unlike the original Weiss system, contains an intermediate category in which tumors can be classified as borderline or of uncertain malignant potential. The presence
Table 7 Criteria for separating benign from malignant oncocytic adrenocortical neoplasms [35]
Major criteria
Mitotic rate >5 per 50 high-power fields Atypical mitoses Venous invasion
Minor criteria
Size >10 cm and/or >200 g
Necrosis Capsular invasion
Sinusoidal invasion
NOTE. Oncocytic adrenocortical carcinoma = presence of any major criterion.
Borderline oncocytic adrenocortical neoplasm of uncertain malignant potential = presence of any minor criterion.
Adrenocortical oncocytoma = absence of all major and minor criteria.
of this diagnostic category is largely a reflection of the fact that there is rather little information available concerning the clinical behavior of oncocytic neoplasms of the adrenal gland. This is mostly attributable to the limited number of reported cases of these particular tumors and absence of extended clinical follow-up data. Allowing for classification of a given tumor as indeterminate for malignancy thus prevents underdiagnosis of a potentially malignant tumor and ensures proper close clinical follow-up. To date, most reported cases that would have been categorized as border- line oncocytic neoplasms using our proposed guidelines have had benign clinical outcomes [36]; however, 2 tumors, one exhibiting large size and the other showing only necrosis, have behaved in a biologically aggressive fashion [37,38].
7. Fine needle aspiration and core needle biopsy
At present, the use of fine needle aspiration (FNA) in the assessment of lesions involving the adrenal gland is limited to specific clinical settings. The primary indication for adrenal FNA is for diagnostic evaluation of suspected metastatic disease in patients with a history of extra adrenal malignancy presenting with a nonfunctional adrenal mass that exhibits equivocal imaging characteristics [1,39,40]. In this context, aspiration cytology is quite effective, with reported sensitivity rates generally exceeding 80% and specificity rates approaching 100% for the detection of malignancy [41-48]. Other accepted indications for FNA of the adrenal gland include evaluation of suspected infectious processes or lymphoma involving the adrenal because a positive diagnosis in these settings would have important therapeutic implications [39].
FNA is not a technique that is widely used for purposes of diagnosis of primary neoplasms of the adrenal cortex. Although adrenocortical adenoma and adrenocortical carci- noma generally differ in their cytomorphology, the 2 lesions not infrequently show overlapping cytologic features, such
that it is felt benign and malignant adrenocortical tumors cannot be reliably differentiated based on cytologic findings alone [40,49].
The cytologic appearance of benign and malignant lesions of the adrenal cortex on aspirate material has been well described. Adrenocortical adenomas are characterized by uniform-appearing cells with round nuclei, fine evenly distributed chromatin, and small to inconspicuous nucleoli arranged in small clusters [43,46,49-52]. The cytoplasm ranges from vacuolated to finely granular and may have a frayed appearance. Naked or stripped nuclei are also usually present and may be numerous [46,48,50-52]. Smears in most cases have a bubbly or foamy background due to the presence of lipid material [46,48,50-52]. Rare cells exhibiting mild anisonucleosis and moderate atypia with nuclear enlarge- ment, hyperchromasia, and prominent nucleoli may be observed [43,49]. Aspirates typically lack necrosis and mitotic figures [49,50].
The cytomorphologic features of adrenocortical carci- noma can vary, depending on the extent of differentiation of the tumor. In general, smears are highly cellular and composed of dyscohesive cells arranged individually and in aggregates. Poorly differentiated tumors show consider- able pleomorphism and are characterized by cells with increased nuclear to cytoplasmic ratios, irregularly thickened nuclear membranes, coarse chromatin, prominent nucleoli, and eosinophilic cytoplasm [43,46,48,51,53,54]. Bizarre- appearing giant cells, multinucleated cells, and spindle cells may be seen [43,46,50,53,56]. Necrosis and mitotic figures are also common findings [43,46,48,49,53,54]. Well-differ- entiated adrenocortical carcinomas can exhibit rather bland cytologic features typified by uniform-appearing cells with vacuolated cytoplasm, simulating the appearance of an adrenocortical adenoma [43,49,54].
Diagnosis of adrenocortical neoplasms by aspirate cytol- ogy is problematic because there is a lack of well-established and clearly defined cytomorphologic criteria to distinguish benign and malignant tumors in this particular setting. Proposed systems for making this distinction have been formulated based on examination of resected adrenal glands [5,10,13,14,16]. As such, many of the histologic criteria that are components of these systems, including capsular and vascular invasion, tumor architecture, and mitotic rate, cannot be readily evaluated in FNA material.
In general, the presence of abundant stripped nuclei and a bubbly appearing lipid-rich background favors a benign diag- nosis, whereas hypercellularity, necrosis, and mitotic figures are cytologic features more commonly present in malignant adrenocortical tumors [43,48-52]. Cellular atypia alone is not considered a reliable indicator of malignancy because adrenocortical adenomas may not infrequently show occa- sional cells with atypical cytomorphology, whereas low-grade or well-differentiated adrenocortical carcinomas often lack appreciable cytologic atypia [43,49,54].
Katz et al [43] have suggested criteria for the diagnosis of adrenocortical lesions based on cytologic features and size
considerations. Adrenocortical nodules less than 3.5 cm with minimal or focal nuclear atypia in the absence of necrosis and mitotic figures are considered benign, whereas nodules exceeding 3.5 cm exhibiting necrosis and mitoses with any degree of nuclear atypia are diagnosed as malignant. Adrenocortical tumors less than 3.5 cm without necrosis and mitotic figures and showing diffuse, severe nuclear atypia are classified as inconclusive for malignancy. This designation is also used for tumors greater than 3.5 cm lacking necrosis and mitoses, regardless of the degree of nuclear atypia present.
More recently, Ren et al [54] retrospectively evaluated aspirate material from 9 primary and 11 metastatic adrenocortical carcinomas. The cytologic features that were most frequently observed among these cases included high cellularity, background necrotic debris, moderate to marked nuclear pleomorphism, mitotic figures, and prominent nucleoli. All cases of adrenocortical carcinoma in their series displayed at least 3 of the 5 aforementioned cytologic features, with necrosis and/or mitotic figures notably present in all samples. Based on these findings, the authors proposed that the identification of at least 3 of these specific cytologic features in an aspirate from an adrenocortical nodule would favor a diagnosis of adrenocortical carcinoma.
The role of core needle biopsy in the diagnostic evaluation of neoplasms of the adrenal cortex has not been well studied. In the context of separating adrenocortical adenoma from adrenocortical carcinoma, this technique would appear to suffer from many of the inherent short- comings associated with FNA. In particular, limited sampling does not permit adequate assessment of many of the histologic features essential for establishing a diagnosis of a malignant adrenocortical tumor [5,10,13,14,16].
The diagnostic value of core needle biopsy of the adrenal gland has been recently evaluated in a large series of adrenal lesions procured as part of the German and Austrian Adrenal Network Multicenter Trial [55]. In a novel approach, tissue was obtained by core needle biopsy ex vivo from surgically resected adrenal specimens and assessed by a single reference pathologist blinded with respect to patient clinical data. Diagnoses rendered based on evaluation of core needle biopsy material were subsequently compared with the final definitive diagnoses made on the resected adrenal specimens in each of the participating study centers. The authors of this study indicated that adrenocortical tumors were classified as benign or malignant using systems proposed by Weiss et al [5,7], Hough et al [13], and van Slooten et al [14], although it is not clear how certain histologic parameters such as vascular invasion, capsular invasion, and extent of mitotic activity were evaluated on core biopsy specimens. None- theless, in their experience, histopathologic analysis of biopsy material yielded a correct diagnosis in 75.6% of cases of adrenocortical adenoma and 76.2% of cases adrenocortical carcinoma [55]. Among the cases of adrenocortical adenoma, 9.3% were misdiagnosed as
malignant based on the microscopic finding of necrosis in the biopsy samples. All adrenocortical carcinomas were correctly recognized as malignant on biopsy, although 23.8% of the cases were misclassified as metastases rather than primary adrenal neoplasms. Although data from this study suggest a potential role for core needle biopsy in classifying adrenocortical tumors, these preliminary obser- vations require validation in an in vivo clinical setting to fully determine the diagnostic sensitivity and specificity of this technique.
8. Immunohistochemistry
A number of studies have evaluated the potential role of immunohistochemistry as an ancillary technique for the assessment of malignancy in adrenocortical neoplasms. These have focused largely on the differential expression of various proteins associated with cell proliferation and tumor suppression as a means of distinguishing adrenocor- tical adenoma from adrenocortical carcinoma [9,56,57]. Among the different proteins evaluated, immunohistochem- ical expression of Ki-67 has been the most extensively investigated in this context [9,16,56-64]. Levels of Ki-67 expression have been demonstrated to be consistently higher in adrenocortical carcinomas compared with adrenocortical adenomas. Increased Ki-67 immunoreactivity has also been significantly associated with shortened disease-free survival and death from disease [58,60,62,64]. However, although it is true that most adrenocortical carcinomas will exhibit a high Ki-67 labeling index, the extent of Ki-67 immunor- eactivity in these tumors can range widely, with some
exhibiting rather low labeling indices that overlap with those typically observed in adrenocortical adenomas [59,63]. As such, there is no standard accepted threshold value of Ki-67 expression for establishing malignancy in adrenocortical tumors, with suggested cutoff points ranging from 2.5% to 10% [9,16,56,57,59-62,64]. For this reason, we do not routinely employ Ki-67 immunohistochemistry in clinical practice to help differentiate benign from malignant adrenocortical tumors, and rely primarily on microscopic features to make this distinction.
Other proteins detectable by immunohistochemical meth- ods with reported potential in separating benign from malignant adrenocortical neoplasms have included topoi- somerase II a, insulin-like growth factor 2 (IGF2), and matrix metalloproteinase type 2 [57,59,63,65,66]. These particular proteins appear to be preferentially expressed by adrenocortical carcinomas compared with adrenocortical adenomas, although the diagnostic use of these biomarkers awaits further validation.
9. Molecular genetics
The past several years has seen remarkable progress in the understanding of the molecular mechanisms implicated in the pathogenesis of tumors of the adrenal cortex [67-69]. Studies addressing the tumorigenesis of adrenocortical neoplasms have identified a variety of molecular genetic alterations, many with the potential to discriminate between adrenocortical adenomas and adrenocortical carcinomas.
Comparative genomic hybridization analysis has demon- strated clear differences in genomic aberrations between
| Table 8 Genes significantly differentially expressed between benign and malignant adrenocortical neoplasms by microarray analysis | ||||||||
|---|---|---|---|---|---|---|---|---|
| References | Up-regulated genes | Down-regulated genes | ||||||
| Giordano et al [74] | IGF2 | UBCH10 | KIAA0101 | SPP1 | ADH1 | ADH2 | TMOD | SDF1 |
| C20ORF1 | KIAA1024 | |||||||
| de Fraipont et al [75] | IGF2 | FGFR1 | FGF4 | TGFß2 | CYP11A | CYP11B1 | CYP17 | CYP21A2 |
| TGFØR1 | MST1R | KCNQ1OT1 | GAPD | HSD3B1 | STAR | INHA | CREM | |
| RB1 | PPM1A | NME1 | S100B | |||||
| TGFØR3 | GPC3 | |||||||
| Velázquez-Fernández | USP4 | UFD1L | INPPL1 | AQP3 | CXCL10 | RARRES2 | ALDH1A1 | CYBRD1 |
| et al [76] | HEFEB | MLLT10 | FFPIA1 | ZNF32 | GSTA4 | CDH2 | ABCG1 | SPTBN1 |
| CDH13 | MCLC | IGF2R | IGF2 | LRRN3 | AEBP1 | KCNJ8 | MTA3 | |
| BIRC3 | NOL1 | CAMK1 | ||||||
| Slater et al [77] | MCOLN3 | FEFR1 | IGF2 | CYFIP2 | ABCB1 | SCARB1 | CCL15 | |
| FOSB | GPI | MGMST1 | ABLIM1 | |||||
| ALDH1A1 | ABCC3 | SLC26A2 | RGN | |||||
| West et al [78] | TRIP | DLL3 | FLJ22814 | DUOX2 | PAH | HLA-DRA | PLAGL1 | CYP11B1 |
| FLJ10458 | TCEB2 | WIT-1 | HLA-DPA1 | HRC | HLA-DRA | EMCN | ||
| CD7 | HLA-DRB1 | HLA-DPB1 | APOE | |||||
| Fernandez-Ranvier et al [79] | SERPING1 | MRPL48 | TM7SF2 | DDB1 | ||||
| NDUSF8 | PRDX5 | |||||||
| Fernandez-Ranvier et al [80] | IL13RA2 | CCNB2 | HTR2B | RARRES2 | SLC16A9 | |||
| de Reyniès et al [81] | DLG7 | PINK1 | ||||||
benign and malignant adrenocortical tumors. Although DNA copy number changes are detectable in both adrenocortical adenomas and adrenocortical carcinomas, the number of such chromosomal changes tends to be higher in malignant tumors and to occur with increasing frequency with greater tumor size [70-72]. Among adrenocortical carcinomas, gains have been reported to affect chromosomes 4, 5, 7, 12, 14, and 19 most commonly, whereas losses are most often seen on chromosomes 1, 2, 11, 17, and 22 [70-72].
Allelic losses at 11q13, 17p13, and 11p15 have been shown consistently in most sporadic adrenocortical carci- nomas but are detected infrequently in benign adrenocor- tical tumors [18,19,61,73]. Loss of heterozygosity at the latter 2 loci has also been reported to be associated with a higher risk of tumor recurrence as well as shorter disease- free survival [18,19]. The IGF2 gene, which maps to the 11p15 locus, has similarly been implicated in the pathogen- esis of adrenocortical neoplasia [18,19,73,74]. IGF2 is frequently overexpressed in adrenocortical carcinomas compared with adrenocortical adenomas, which is thought to be due to paternal isodisomy or loss of imprinting of the 11p15 locus [73].
With the advent of gene microarray technology, several investigators have been able to generate transcriptional signatures of benign and malignant adrenocortical tumors [74-81]. Through utilization of this methodology, it has become clear that adrenocortical adenomas and adrenocor- tical carcinomas are characterized by different gene expres- sion profiles. Although a detailed discussion of the molecular and biologic functions of the candidate genes identified in these various studies is beyond the scope of this review, the genes reported to be significantly differentially expressed in malignant compared with benign adrenocortical tumors are summarized in Table 8. Dysregulation of IGF2 appears to be the most consistent finding among the various microarray studies, which otherwise show significant differences in the specific genes identified as differentially expressed in adrenocortical adenomas and carcinomas. These disparities may be attributed to differences in sample size, microarray platforms, the number of genes analyzed, and criteria used to define differential gene expression. Nonetheless, detection of differential up-regulation or down-regulation of specific genes by microarray gene expression analysis offers a promising alternative means of segregating adrenocortical carcinomas from adrenocortical adenomas, although the diagnostic use of this approach in clinical practice remains to be determined.
10. Conclusions
Twenty-five years have elapsed since the Weiss system was introduced. Since that time, there have been a number of significant developments in the field of adrenocortical neoplasia. There is now evidence to suggest that neoplasms of the adrenal cortex occurring in the pediatric population
and adrenocortical tumors with oncocytic histology are clinicopathologic entities distinct from conventional adult adrenocortical neoplasms. As such, the histologic parameters of the Weiss system for differentiating adenoma from carcinoma are not applicable in these particular settings. Although the criteria used for making this important distinction in pediatric and oncocytic adrenocortical tumors are somewhat similar to those used in the Weiss system, there are significant differences, and in the end, each of these lesions requires a different diagnostic approach.
Although data generated by molecular genetic techniques has undoubtably made important contributions to the pathologic evaluation of adrenocortical neoplasms, at present, histologic assessment as defined by the Weiss system still remains the mainstay for the diagnosis of adrenocortical carcinoma in the adult population. Issues of reproducibility of the Weiss histologic criteria have been largely resolved by introduction of a modified Weiss system, which has been shown to be similarly effective in separating benign and malignant adrenocortical tumors.
The molecular classification of adrenocortical neoplasms based on gene expression analysis has yielded promising results, although further standardization and validation of techniques are required before use in clinical practice.
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