Assessing Biological Aggression in Adrenocortical Neoplasia
CrossMark
Isobel C. Mouata, Thomas J. Giordano, MD, PHDa,b,c,*
KEYWORDS
· Adrenocortical neoplasia · Adrenocortical adenomas · Adrenocortical carcinomas
· Histologic grading · Molecular grading
ABSTRACT
P athologists are highly skilled at the evalua- tion of adrenal neoplasms. Occasional adre- nocortical tumors can be diagnostically challenging and supplementary tools can assist in these cases. Histologic and molecular studies support a model that includes 2 broad classes of adrenocortical carcinoma with distinct somatic genetic alterations and clinical outcomes. Patholo- gists should endeavor to grade adrenocortical car- cinomas to assign each case into one of these 2 classes. Mitotic grading by mitotic counting and Ki-67 immunohistochemistry represent the most practicable and informative methods currently available.
OVERVIEW
The incidence of tumors of the adrenal cortex has steadily increased over the past few decades, largely due to increased use of clinical imaging studies. Fortunately, a vast majority of these inci- dentally discovered masses (so-called adrenal in- cidentalomas) are benign. Of these benign lesions, adrenocortical adenomas represent the most common pathologic entity. Conversely, adrenocortical carcinomas are rare tumors with malignant potential and an incidence of 1 to 2 cases per million.
When faced with the diagnostic challenge of an adrenal specimen with a mass, surgical patholo- gists must address several fundamental questions.
First, broadly speaking, Is it a neoplasm? And, if so, What is the origin/differentiation of the neoplasm (ie, Is it a cortical or medullary tumor?)? Perhaps it is neither, such as a mesenchymal neoplasm or a metastasis. Second, once determining that a mass is of cortical origin, Does it possess malig- nant potential (ie, the ability to invade and meta- stasize)? If so, a diagnosis of carcinoma is appropriate. Alternatively, if the mass is cortical and lacks malignant potential, a diagnosis of adre- nocortical adenoma should be rendered. In those cases of adrenocortical carcinoma, the remaining questions are related to stage and grade. Is the tu- mor confined to the adrenal gland (stages I and II) or is there extra-adrenal extension (stage III)? Finally, what is the biological aggression of the mass (ie, What is the grade of the neoplasm?)? Sur- gical pathologists have historically focused most of their attention on the first questions, What is the origin of the mass? Is it carcinoma? and What is the stage of disease? Increasing attention is being paid, however, to the final question of tumor grade and, by extension, determining what is the most informative grading method. This article briefly re- views the literature on these issues, with emphasis on histologic and molecular grading of adrenocor- tical carcinoma.
DIFFERENTIAL DIAGNOSIS OF ADRENAL MASSES
Compared with other organs, the differential diag- nosis of the solitary adrenal mass is relatively
a Department of Pathology, University of Michigan Health System, Ann Arbor, MI, USA; b Department of In- ternal Medicine, University of Michigan Health System, Ann Arbor, MI, USA; ” Comprehensive Cancer Center, University of Michigan Health System, Ann Arbor, MI, USA
* Corresponding author. Department of Pathology, University of Michigan Health System, 1150 West Medical Center Drive, MSRB1, 4520D, Ann Arbor, MI 48109-5602.
E-mail address: giordano@umich.edu
http://dx.doi.org/10.1016/j.path.2014.08.003
Key Features OF ADRENOCORTICAL NEOPLASIA
Clinical, genetic, and radiologic
· Adrenocortical neoplasia can be hormonally functional or nonfunctional.
· Adrenocortical neoplasia is sporadic in most cases; syndromic forms are recognized (eg, Li-Fraumeni).
· Adrenocortical neoplasia is often incidentally discovered by imaging.
· Adenomas are often homogenous on imaging.
· Carcinomas are often heterogeneous with necrosis on imaging.
Gross pathology
· Adenomas tend to be small (<5 cm) and homogenous.
· Carcinomas tend to be large (>5 cm) with clonal nodularity and necrosis.
Histopathology for diagnosis
· Adenomas can be lipid rich, have a nested growth pattern, be low nuclear grade, have low mitotic counts, and lack invasion, necro- sis, and atypical mitotic figures.
· Carcinoma are usually lipid poor, have a diffuse growth pattern, have high nuclear grade, have abundant mitoses, and display vascular invasion, necrosis, and atypical mitotic figures.
· There is immunoreactivity for a-inhibin, ste- roidogenesis factor (SF)-1, melan-A, and synaptophysin.
· Tumors are nonimmunoreactive for chro- mogranin and most keratins.
· Ki-67 index can be useful for diagnosis. Histopathology for prognosis
· Mitotic grade defines 2 classes of carcinoma (low grade and high grade).
. Immunoreactivity for p53 and nuclear ß-catenin supports a diagnosis of high-grade carcinoma.
· Ki-67 index can define prognostic subgroups of carcinoma.
restricted. Adrenal masses are broadly divided into 4 categories: cortical, medullary, miscella- neous primary tumors, and metastases. Patholo- gists are highly skilled at differentiating between
!
Pitfalls IN ASSESSMENT OF ADRENOCORTICAL NEOPLASIA
! Confusing cortical and medullary tumors
! Undersampling the capsule of a large cortical tumor, leading to an underappreciation of invasion
! Misinterpreting hemorrhage and organiza- tion in an adenoma as evidence of carcinoma
! Overdiagnosis of carcinoma based on large tumor size in the absence of supporting fea- tures of carcinoma
! Not grading carcinomas by mitotic counts and/or Ki-67 index
! Undergrading carcinoma diagnosed by needle biopsy
these diagnostic possibilities purely on histologic grounds. Occasional cases are challenging and for those cases a variety of diagnostic immunohis- tochemical tools are available.1-3 In general,
| Differential Diagnosis OF ADRENOCORTICAL NEOPLASIA | |
| Adrenocortical adenoma | Absence of Weiss parameters (invasion, high nuclear grade, mitoses, necrosis, and so forth); immunoprofile of a cortical tumor |
| Adrenocortical carcinoma | Presence of Weiss parameters (invasion, high nuclear grade, mitoses, necrosis, and so forth); immunoprofile of a cortical tumor |
| Medullary tumors | Nested growth pattern Amphophilic cytoplasm Chromogranin immunoreactivity |
| Miscellaneous primary tumors | Histopathology and immunprofile reflect the type of tumor |
| Adrenal metastases | Histopathology and immunprofile reflect the origin of the tumor |
cortical tumors selectively express the following proteins, for which commercially established anti- bodies that robustly work with formalin-fixed paraffin-embedded tissues are available: a-inhibin,4-7 SF-1,1 melan-A,8,9 and calretinin.6,10 Expression of intermediate filaments is largely restricted to vimentin,11 because cortical tumors poorly express most keratins despite being carci- nomas. 12 Medullary tumors express S100 in a sus- tenacular pattern and diffusely express markers of neuroendocrine differentiation, such as chromog- ranin A.13,14 Synaptophysin, a marker of neuroen- docrine differentiation, is coexpressed in cortical and medullary neoplasms,13 despite cortical tu- mors not being part of the neuroendocrine family of tumors.
Miscellaneous primary tumors and metastases have immunohistochemical profiles that reflect the specific type of tumor that is present. For example, leiomyomas of the adrenal gland ex- press smooth muscle markers (eg, actins), angio- sarcomas express vascular markers (eg, CD31), metastatic carcinomas express epithelial markers (keratins), and nonadrenal tissue-specific markers that reflect the organ of origin (eg, TTF1 expres- sion in metastatic lung adenocarcinoma) and lym- phomas express a variety of lymphoid markers depending on the lymphoma phenotype (eg, CD20). The combination of routine histologic assessment and immunohistochemistry, as needed for select cases, is highly effective in determining the type or origin of the vast majority of adrenal tumors.
ASSESSMENT OF MALIGNANT POTENTIAL IN ADRENOCORTICAL TUMORS BY ROUTINE HISTOLOGY AND IMMUNOHISTOCHEMISTRY
Once a pathologist has determined that a given adrenal tumor is of adrenocortical origin, the ass- essment of its malignant potential becomes para- mount; this decision fundamentally determines the treatment plan. There was a time when patholo- gists found this differential diagnosis especially challenging and a certain level of mysticism sur- rounded the diagnosis of adrenocortical tumors. This situation was greatly impacted by 3 landmark studies that systematically examined multiple clinical and histologic features to derive character- istics associated specifically with malignant behavior.15-17 Of these systematic studies, the Weiss system has obtained the widest accep- tance by practicing pathologists, 18 largely a reflec- tion of its ease of use. This system recently marked its 25th anniversary since its inception and has been incorporated into many molecular
studies. 19-22 Modifications designed to streamline the scoring parameters have been introduced23,24 as well as similar systems for pediatric cases25 and oncocytic tumors.26,27 The main morphologic features of the Weiss system are illustrated in Fig. 1.
In addition, recent work has demonstrated that carcinomas have selective loss of their reticulin network compared with adenomas.28,29 This knowledge provides another tool for the workup of adrenocortical neoplasms, allowing for addi- tional corroboration of diagnosis.
Beyond these histologic assessments, a diag- nostic role for the evaluation of the degree of tu- mor cell proliferation in adrenocortical tumors has emerged. Most such studies have focused on Ki-67 expression as assessed by MIB1 immuno- histochemistry.20,23,30-33 High expression of Ki-67 demonstrates high proliferative activity, thus supporting a diagnosis of adrenocortical carcinoma. Although no single immunohisto- chemical marker in isolation can distinguish ade- noma and carcinoma in all cases, the Ki67 index adds value in a large majority of cases and has become a useful tool in the multifactorial evalua- tion of the separating adenomas and carcinomas (Fig. 2).
ASSESSMENT OF MALIGNANT POTENTIAL IN ADRENOCORTICAL TUMORS BY MOLECULAR METHODS
Adrenocortical neoplasms, despite their rare na- ture, have been extensively studied by a variety of molecular methods. Numerous individual molecular features have been investigated for their diagnostic potential as well as more recent comprehensive and genome-wide molecular characterizations. For example, individual markers shown to have utility in the diagnostic separation of adenoma and carcinoma include alteration of the of IGF2 locus, resulting in sub- stantially increased expression in approximately 90% of adrenocortical carcinomas.34 Looking back, the degree of progress has been remark- able. A complete review is beyond the scope of this article, but several recent reviews are available.35-37
One of the first genome-wide gene expression profiling studies of adrenocortical neoplasms came from the authors’ laboratory and clearly showed that adenoma and carcinomas displayed profoundly distinct gene expression.38 This study, although limited by a small number of tumors, clearly demonstrated the diagnostic potential of gene expression in adrenocortical tumors.
A
B
C
D
E
Numerous similar studies confirmed and expanded these initial findings (reviewed by Assis and col- leagues39,40). It is now accepted that adenomas and carcinomas have distinct gene expression profiles. The authors’ group has attempted to
distill these results into a molecular reverse tran- scription-polymerase chain reaction assay that can be used to confirm a diagnosis of carcinoma when needed and evaluate diagnostically difficult cases with equivocal features. 41
A
B
C
D
ASSESSMENT OF BIOLOGICAL AGGRESSION IN ADRENOCORTICAL CARCINOMAS BY ROUTINE HISTOLOGY AND IMMUNOHISTOCHEMISTRY
Once a diagnosis of adrenocortical carcinoma has been rendered, it is also the role of the pathologist to grade the carcinoma in an effort to predict its clinical course. This has been attempted using a variety of methods:
· Mitotic grade
· Stereoidogenic factor-1 (SF-1)
. Other proliferation-based scoring methods
MITOTIC GRADE
First, the Weiss score, as discussed previously, originally designed used to assess the malignant potential of a given tumor, also possesses prog- nostic significance42; high Weiss score tumors generally display aggressive clinical behaviors. In
the course of developing the Weiss system, Dr Weiss and colleagues noted that, among the indi- vidual Weiss parameters, mitotic count possessed the highest prognostic significance. This observa- tion led them to propose a 2-tier grading system based solely on mitotic rate. Low-grade carci- nomas were designated as having fewer than 20 mitoses per 50 high-power fields, whereas high- grade carcinomas had greater than 20 mitoses per 50 high-power fields. At the authors’ center, this mitotic grading has become fully entrenched and essential to individualizing the treatment plan.43,44 In the authors’ opinion, mitotic grading of adrenocortical carcinoma is the gold standard against which other molecular grading methods must be judged, especially given the low cost of routine mitotic grading.
STEREOIDOGENIC FACTOR-1
Given its essential role in adrenal development, 45 there has been much interest in the expression of stereoidogenic factor-1 (SF-1) as a diagnostic
and prognostic marker.46,47 Validation studies confirm the expression of stereoidogenic factor 1 and reinforce its potential role as a prognostic marker, even in models that include Ki-67 index and mitotic count data.48
RELATED PROLIFERATION-BASED SCORING METHODS
Similar to its diagnostic role in separating adenoma and carcinoma, assessment of proliferation by alternative methods has been well studied. A recent article by Duregon and colleagues49 compared 3 related methods for assessing proliferative activity in adrenocortical carcinoma: mitotic counts, phos- phorhistone H3-specific immunohistochemistry, and Ki-67 expression by immunohistochemistry. The antibody against phosphohistone H3 targets the phosphorylated form of the H3 histone protein, which is preferentially detected during mitotic chro- mosome condensation.5º Accordingly, immuno- histochemical detection of phosphohistone H3 represents an alternative method for assessing the mitotic rate and has been deployed in studies of a wide range of tumor types, including other endocrine tumors.51 In directly comparing these approaches in a cohort of 52 adrenocortical carci- nomas using both manual and automated methods, they demonstrated that the Ki-67 index was the strongest predictor of overall survival. Moreover, they showed the Ki-67 index could divide the cases in 2 or 3 classes with significantly different survivals.
ASSESSMENT OF BIOLOGICAL AGGRESSION IN ADRENOCORTICAL CARCINOMAS BY MOLECULAR METHODS
Many studies have attempted to derive clinically meaningful classification schemes of adrenocor- tical carcinoma using molecular data. Among these, most have focused on genome-wide mea- surement of gene expression (ie, the transcrip- tome). Two such studies from independent groups (the European Network for the Study of Ad- renal Tumors and the University of Michigan) divided adrenocortical carcinomas into 2 primary groups with distinctly different outcomes. 19,52
In the case of the University of Michigan cohort, the subgroups reflected the mitotic grade. A gene expression derived measure, however, did provide incremental prognostic information in an outcome model that included tumor stage and mitotic grade, suggesting that gene expression data might yield a more refined grading.
Integration of information regarding the common somatic mutations observed in adrenocortical
carcinoma provides a genetic framework for the 2-class model. Low- and high-grade tumors have approximately equal numbers of cases with altered IGF2 expression, 19,52 suggesting that alter- ation of the IGF2 locus with subsequent increase of IGF2 expression is an early event in adreno- cortical carcinoma development. Conversely, somatic alterations of TP53 and CTNNB1 are en- riched in high-grade adrenocortical carcinomas,53 suggesting these mutations are late events in adrenocortical carcinoma development and may be defining alterations of high-grade tumors.54 Beyond TP53 and CTNNB1, alterations of RB1 have been found associated with adrenocortical carcinomas with aggressive clinical behavior55 and may represent an alternative path for the development of high-grade disease.
Other genomic technologies have been used to derive classification schemes of adrenocortical carcinoma, the results of which generally support a low- and high-grade model. Genome-wide assessment of copy number changes did reveal diagnostic significance and divided the carci- nomas into 2 groups with distinct survival.56 Simi- larly, DNA methylation studies have also divided adrenocortical carcinoma into 2 groups with distinct survival.57 A recent integrated multiplat- form genomic analysis confirmed 2 classes of adrenocortical carcinoma with distinct mutational profiles and clinical outcomes.58
ROUTINE GRADING OF ADRENOCORTICAL CARCINOMA
Given the consistent results from many studies that indicate adrenocortical carcinomas segregate into 2 groups with different clinical outcomes, it should become incumbent on pathologists to assign each case to an indolent (low-grade) or aggressive (high-grade) category. It remains to be determined exactly what is the most informa- tive grading method that also can be easily de- ployed in all pathology laboratories, although recent studies suggest the Ki-67 index may be the most informative grading method. 49
Once a diagnosis of adrenocortical has been rendered, mitotic grading by mitotic counting is required whenever sufficient tumor is available.43 In cases of core biopsies where sampling errors are common, a tentative mitotic count and grade can be rendered along with a cautionary comment that the tumor grade might be higher than what is represented by the core biopsy.
Beyond mitotic counting, immunohistoche- mistry for Ki-67, p53, and ß-catenin can be infor- mative and these markers can be performed
whenever tissue blocks are available. In particular, assessment of proliferation by Ki-67 can be useful when reviewing previously diagnosed cases in which there is a discrepancy between pathologists in mitotic counts. Suboptimal histologic prepara- tions can make identification of mitotic figures difficult, resulting in discordant mitotic counts. In such cases, demonstration of a low or high Ki-67 labeling index can help resolve the discrepancy and thereby assist in accurate mitotic grading. Beyond diagnostically challenging cases, if the Ki-67 index is further shown the most informative prognostic biomarker, then this will quickly become the standard of care in addition to mitotic grade by mitotic counting.
Routine immunohistochemical assessment of p53 and ß-catenin are a bit more controversial. On one hand, having strong nuclear staining for either of these markers suggests an underlying ge- netic mutation in TP53 or in one of the genes of the wnt pathway, usually CTNNB1. These findings are strongly associated with aggressive behavior39 and support a diagnosis of high-grade adrenocor- tical carcinoma. On the other hand, knowledge of these results does not have a direct impact on the treatment plan; therefore, it has been argued that these stains do not provide independent data and lack medical value.
SUMMARY
Pathologists are highly skilled at the evaluation of adrenal neoplasms. Occasional adrenocortical tu- mors can be diagnostically challenging, however, and supplementary tools can assist in these cases. Histologic and molecular studies support a model that includes 2 broad classes of adreno- cortical carcinoma with distinct somatic genetic alterations and clinical outcomes. Pathologists should endeavor to grade adrenocortical carci- nomas to assign each case into one of these 2 classes. Mitotic grading by mitotic counting and Ki-67 immunohistochemistry represent the most practicable and informative methods currently available.
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