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Abstract
The most recent WHO classification of endocrine and neuroendocrine tumours has brought about significant changes in the diagnosis and grading of these lesions. For instance, pathologists now have the ability to stratify subsets of thyroid and adrenal neoplasms using various histological features and composite risk assessment models. Moreover, novel recommendations on how to approach endocrine neoplasia involve additional immunohistochemical analyses, and the recognition and implementation of these key markers is essential for modernising diagnostic capabilities. Additionally, an improved understanding of tumour origin has led to the renaming of several entities, resulting in the emergence of terminology not yet universally recognised. The adjustments in nomenclature and prognostication may pose a challenge for the clinical team, and care providers might be eager to engage in a dialogue with the diagnosing pathologist, as treatment guidelines have not fully caught up with these recent changes. Therefore, it is crucial for a surgical pathologist to be aware of the knowledge behind the implementation of changes in the WHO classification scheme. This review article will delve into the most significant diagnostic and prognostic changes related to lesions in the parathyroid, thyroid, adrenal glands and the gastroenteropancreatic neuroendocrine system. Additionally, the author will briefly share his personal reflections on the clinical implementation, drawing from a couple of years of experience with these new algorithms.
- WHO
- update
- thyroid
- parathyroid
- adrenal
- neuroendocrine
- review
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Introduction
Endocrine pathology is an evolving field, in which morphological assessment integrated with immunohistochemical analysis has become vital to safely render diagnoses and prognosticate lesions. Unlike many other pathology subspecialties, distinguishing malignant potential of a given tumour without phenotypic evidence of invasive behaviour is challenging in endocrine pathology, as cytology alone may not suffice to render a diagnosis. Hence, the endocrine pathologist must integrate findings from gross handling, microscopic assessment, immunohistochemical investigations and sometimes even molecular genetics to accurately pinpoint the correct diagnosis.
With the introduction of the fifth edition of the WHO classification of endocrine and neuroendocrine tumours in 2022, pathologists face an expanded spectrum of recognised endocrine lesions, placing even greater emphasis on our ability to accurately rule out morphological mimics.1 Additionally, heightened research efforts have driven the development of multiparameter grading schemes and novel immunohistochemical markers to assess the prognosis of various endocrine tumours. Another distinctive aspect of endocrine pathology is the broad distribution of patient ages, with substantial subsets of younger subjects exhibiting hereditary diseases. This characteristic is reflected in modern endocrine pathology through the association of syndromic cases with specific morphological, expressional and genetic findings. Therefore, endocrine pathology has undergone a well-deserved modernisation, and practising colleagues around the world are expected to stay informed and update their knowledge to enhance the services provided to our patients. In this brief review, the author will detail some of the most important changes and additions to the framework of diagnostic and prognostic workup of endocrine and neuroendocrine tumours.
Unveiling parathyroid puzzles: tackling atypical features
The majority of parathyroid tumours are benign adenomas, posing no significant diagnostic challenges for experienced pathologists. In most instances, immunohistochemistry is not necessary for a correct diagnosis, although there are exceptions. The adenomas are well-defined, rarely exhibit cytological abnormalities, and lack invasive features.2 3 This is in stark contrast to malignant parathyroid carcinomas, typically large tumours associated with elevated PTH and calcium levels in the blood prior to surgery.4 Microscopically, these tumours often display unusual cytological abnormalities, occasional mitoses, necrosis and always present invasive features.2 3 A significant subset of these tumours exhibits mutations in CDC73, resulting in the immunohistochemical loss of the corresponding protein parafibromin.5–9 As such mutations are rare in sporadic adenomas, parafibromin has become a marker for identifying parathyroid tumours with malignant potential.7 10 11
The most notable change in the new classification is the clarification of the atypical parathyroid tumour (APT) concept.2 Various criteria are now provided for this diagnosis, with a fundamental requirement being the absence of invasive features, accompanied by several atypical histological findings such as fibrous bands, unclear relationship between tumour and capsule, atypical mitoses, necrosis, etc.2 Moreover, the previously accepted terminology ‘atypical adenoma’ is no longer recommended, as subsets of these tumours in fact harbour malignant potential, which therefore contradicts the adenoma nomenclature.2 APTs can be assessed for parafibromin expression; the lack of this marker indicates an underlying mutation and an increased risk of future recurrences (figures 1 and 2).12 13 The new classification emphasises several markers, including galectin-3, PGP9.5 and Ki-67, which can aid in assessing APTs.2 14–18 While most lesions within this category are clinically benign, those lacking parafibromin show a risk of recurrence and should be monitored.13 Additionally, patients with an absence of parafibromin should undergo clinical genetics investigation (figure 1).2
Schematic overview of parathyroid neoplasms and recommendations for triaging cases to parafibromin immunohistochemistry. Flow chart illustrating the current diagnostic workflow for parathyroid neoplasia. Parathyroid carcinomas necessitate evidence of one or more of the following criteria: angioinvasion, as indicated by tumour infiltration within a vessel wall accompanied by thrombus formation; lymphatic invasion; perineural invasion; infiltration into adjacent anatomical structures or histologically/cytologically documented metastatic disease. All patients with parathyroid carcinoma should undergo CDC73 gene testing of germline DNA. Atypical parathyroid tumours with demonstrable parafibromin (PFIB) expression have an exceedingly low risk of recurrence, while cases with aberrant staining have an unneglectable risk of relapse, as well as a possibility of harbouring an underlying CDC73 gene mutation. Parathyroid adenomas are usually not screened for PFIB immunoreactivity, however rare cases with distinct morphology may require investigation. Created using BioRender.com.
Microscopic features of parafibromin-deficient parathyroid carcinoma. (A) H&E stain depicting main morphological attributes of a parafibromin-deficient parathyroid tumour growing in solid formations, exhibiting an eosinophilic cytoplasm with an evident perinuclear halo. Nuclear pleomorphism is also evident. (B) The same tumour demonstrating negative immunoreactivity for parafibromin. Note the positive stromal cells acting as internal positive controls. (C) This tumour exhibited venous invasion (black arrows), thus fulfilling criteria for parathyroid carcinoma. Not the associated tumour thrombus. (D) Double-stain immunohistochemistry with pan-cytokeratin in red and CD31 (endothelial cell marker) in brown highlighting the intravascular tumour deposits.
Recent advancements in our understanding of parathyroid tumours have led to the identification of specific morphological attributes associated with parafibromin deficiency and CDC73 gene mutations, and phenotypic clues include sheet-like growth of eosinophilic cells with nuclei exhibiting a coarse chromatin, perinuclear clearing, microcystic change and an arborising vasculature (figure 2).19 20 These histological clues thus serve as a valuable tool for pathologists to triage cases for parafibromin immunohistochemistry, as an aberrant staining in this context may indicate an underlying CDC73 gene mutation regardless of the final diagnosis.
It is important to emphasise that there is no defined set of aberrant clinical, histological and immunohistochemical factors that unequivocally determine whether a parathyroid tumour should be diagnosed as APT or not. Instead, a comprehensive evaluation involves weighing various factors collectively before reaching a diagnosis.2 In my personal opinion, pathologists frequently overcall atypical features, potentially interpreting focal fibrosis and the identification of a single mitosis as sufficient criteria. In such cases, an immunohistochemical workup may be deemed necessary to conclusively rule out malignant potential, as the recommended markers almost always indicate a benign course for these types of cases (unpublished observations). As of today, numerous laboratories do not include parafibromin in their routine clinical repertoire, although the number of institutions offering it is steadily increasing each year. Nevertheless, it remains a frequent subject of consultation from other hospitals, and the analyses can be crucial when evaluating parathyroid tumours with atypical features. Even so, it is important to remember that aberrant parafibromin immunoreactivity by itself is not a definite criterion for malignancy, as CDC73 gene mutations may occur also in parathyroid tumours without recurrences even after decades of clinical follow-up.10 Therefore, parafibromin expression must be related to clinical and histological factors before a final diagnosis is put forward. The proliferation marker Ki-67 also holds additional value in this context, with very high indices serving as an indicator for pathologists to investigate further.2 3 However, the foundation of all work on these tumours lies in morphological assessment, where multilevel sectioning proves to be a powerful tool for identifying vascular invasion. In this regard, endothelial cell markers and CD61 can also be valuable in confirming intravascular tumour burden and in vivo phenomena, respectively (figure 2). This is particularly helpful in cases when tumour cells are intermingled with excessive peliosis, which may mimic vascular thrombi.2
Triaging thyroid tumours: navigating nomenclature shifts and grading updates
The chapters on thyroid tumours have undergone extensive changes in the new 2022 WHO classification.21 Recent research in histology, molecular analyses and associations with patient outcomes has resulted in nomenclature changes and grading models that better reflect the biological behaviour of certain entities. Similarly, increased knowledge of other rare thyroid tumours has prompted nomenclature changes to emphasise that these entities have an unclear histogenesis. These changes place greater demands on the pathologist to carefully evaluate these lesions using routine morphology and, at times, additional analyses. While some parts of thyroid pathology have become more complicated and laborious with the advent of novel nomenclature, other areas have been simplified. Consequently, the umbrella term thyroid follicular nodular disease has been introduced to consolidate several different terms previously used to describe benign thyroid conditions, including monoclonal, benign tumours that are not easily separated from the ambiguous terms ‘hyperplastic nodule’ and ‘adenomatoid nodule’.21 22
The 2022 WHO classification underscores the heterogeneous nature of papillary thyroid carcinoma (PTC) manifested through various subtypes with variations in cytological, structural, genetic and clinical characteristics.21 PTC subtypes display varying degrees of PTC-related nuclear atypia, with BRAF-driven PTCs generally exhibiting more pronounced nuclear features compared with RAS-driven PTCs.21 23 24 Recognising PTC subtypes is crucial as they are associated with diverse prognoses. For instance, tall cell PTCs and columnar cell PTCs are often linked to poorer outcomes, while the minimally invasive encapsulated follicular variant PTC (IEFVPTC) and Warthin-like PTC subtypes are associated with indolent behaviour.21 Hence, morphology alone can provide valuable prognostic information for the treating physician. Given this, histological subtyping should be regarded as a cornerstone in the diagnostic workup of PTCs, even for subcentimetre lesions if feasible. As thyroid cancer guidelines increasingly emphasise high-risk histology, endocrinologists and oncologists will need this information to plan treatment and follow-up effectively.
In addition to recommend PTC subtyping, the 2022 WHO classification also endorses triaging of follicular variant PTC into infiltrative follicular variant PTC (IFVPTC) and invasive encapsulated follicular variant PTC (IEFVPTC), in which the latter should be subdivided into minimally invasive, encapsulated angioinvasive and widely invasive, similar to the subdivision of FTCs proposed in the former WHO classification (figure 3).21 22 While IFVPTC represents an invasive malignancy exhibiting all the classical features of PTC except papillae (such as florid nuclear atypia, psammoma bodies, a fibrous stroma, lymphatic invasion, often driven by BRAF mutations, RET translocations, NTRK or ALK fusions), IEFVPTCs are RAS-driven and resemble FTC, with a well-defined border and local invasion (through capsule and/or into veins) (figures 3 and 4).21 Simply put, while IFVPTC most likely represents a variant of PTC, IEFVPTCs could in theory, be considered as FTCs with nuclear atypia. It will be intriguing to observe whether the latter diagnosis persists in future WHO classifications or if it transitions to the FTC category.
Novel aspects of thyroid tumour classification. The subclassification of papillary thyroid carcinoma (PTC) into invasive encapsulated follicular variant PTC and infiltrative follicular variant PTC, based on overall architecture, nuclear atypia and genetics, is clinically significant. The former entity behaves akin to follicular thyroid carcinoma in terms of prognosis and metastatic routes, while the latter entity behaves more similarly to PTC in general, with local metastases to regional lymph nodes. Follicular thyroid carcinoma and other subtypes of PTC are introduced in this illustration for comparison. RAS-like: RAS gene mutations, PAX8::PPARG fusions, rare mutations in PTEN, DICER1 and BRAF K601E. BRAF-like: BRAF V600E mutations, RET translocations, NTRK fusions, ALK fusions. Created using BioRender.com. N.A, not available.
Microscopic features of infiltrative follicular variant papillary thyroid carcinoma (IFVPTC) and minimally invasive encapsulated follicular variant papillary thyroid carcinoma (miEVPTC). (A–C) The IFVPTC exhibits irregular contours with diffuse infiltration of the surrounding parenchyma (A), displays florid nuclear atypia (B) and usually carries a BRAF V600E mutation, as illustrated by the BRAF VE1 immunohistochemistry (C). (D–F) The miEVPTC is demarcated and encapsulated, with focal capsular invasion (D). Nuclear atypia is more subtle, but still present (E) and the entity is RAS driven and therefore lacks BRAF V600E mutations (F).
In terms of grading, a novel nomenclature has been introduced to precisely identify differentiated thyroid carcinoma (PTC, follicular thyroid carcinoma (FTC) and oncocytic thyroid carcinoma (OTC)) exhibiting a particularly aggressive clinical course. The 2022 WHO classification introduces the term ‘high-grade thyroid carcinoma’, encompassing two distinct tumorous classifications with similar prognoses: differentiated high-grade thyroid carcinoma (DHGTC) and poorly differentiated thyroid carcinoma (PDTC).21 22 Under the DHGTC umbrella, we encounter PTCs, FTCs and OTCs showcasing an elevated mitotic index (≥5 mitoses per 2 mm2) and/or tumour necrosis (figure 5). These variables intricately intertwine, resulting in worse outcomes compared with differentiated thyroid carcinoma lacking these features. On the other front, PDTCs constitute a malignant, dedifferentiated entity, displaying a dominant solid, trabecular or insular growth pattern, accompanied by an increased mitotic index (≥3 mitoses per 2 mm2) and/or tumour necrosis (the so-called ‘Turin criteria’).25 The absence of PTC-related changes and the notable lack of the classic micro-acinar growth pattern of FTCs are essential to the diagnosis. DHGTCs and PDTCs exhibit some differences on the genetic level, potentially linked to the type of differentiated thyroid carcinoma from which they derive. While DHGTC appears to be preferentially associated with BRAF mutations and a PTC origin, PDTCs often exhibit RAS mutations, suggesting an ancestry related to FTCs or IEFVPTCs.21 Unveiling additional aberrations signalling a bleak prognosis, such as mutations of the TERT promoter and the TP53 gene, further contributes to the intricate tapestry of high-grade thyroid carcinoma.21
Differentiated high-grade thyroid carcinoma. (A) Solid papillary thyroid carcinoma (PTC) with focal necrosis (asterisk), thus the tumour was classified as a differentiated high-grade thyroid carcinoma (DHGTC). Note the retained nuclear atypia, which is an exclusion criterion for poorly differentiated thyroid carcinoma. (B) Tall cell PTC with nine mitotic figures per 2 mm2, thus classified as DHGTC. Note the mitotic figure (circle) and apparent nuclear features of PTC (arrows). Well-differentiated thyroid carcinoma with increased mitotic counts and/or tumour necrosis exhibits a poor prognosis, thus there is a clinical need to identify these lesions at the histopathological level.
Anaplastic thyroid carcinoma (ATC) represents the undifferentiated end point of thyroid carcinoma, indicating a uniformly dismal prognosis for nearly all patients. While curative surgery has been reported for small tumours, disease dissemination to distant sites renders ATC challenging to cure or control, with only a few case reports highlighting patients achieving complete remission.26–28 The 2022 WHO classification now recognises squamous cell carcinoma of the thyroid gland as a histological variant of ATC rather than a distinct entity. This decision is based on the observation that both tumour types coexist with a differentiated thyroid carcinoma component, exhibit BRAF mutations, may express thyroid-related lineage markers (particularly PAX8, but also TTF1) and share similar patient outcomes.21 29 30 Moreover, pathologists can provide substantial value to patients by promptly testing all anaplastic carcinomas for the presence of BRAF V600E mutations. A positive finding may serve as motivation for combinatory treatments using BRAF and MEK inhibitors.31 This testing can be conducted through standard sequencing or via immunostaining against the mutated protein (BRAF VE1).21
Medullary thyroid carcinoma is the most common neuroendocrine tumour (NET) of the thyroid gland and intensified research during the latest years has propelled the recognition of various histological and immunohistochemical features associated with worse outcomes. In recent international studies, a two-tiered grading system for MTC has been proposed, grounded in a robust correlation between unfavourable patient outcomes and certain histological features, including tumour necrosis, elevated mitotic count or an elevated Ki-67 proliferation index.32–36 According to this methodology, MTCs are categorised as high-grade tumours if they exhibit at least one of the following parameters: tumour necrosis, mitotic count ≥5 per 2 mm2 and/or a Ki-67 proliferation index ≥5%. The grading system has been suggested as an independent predictor of poorer patient outcomes regardless of TNM stage and genetic status. Thus, the 2022 WHO classification recommends this algorithm to better assess the prognosis for MTC patients.21
Finally, certain entities have been categorised under the term ‘thyroid tumours of uncertain histogenesis’. Although rare, these lesions serve as important differential diagnoses to bear in mind when evaluating thyroid tumours with atypical histology. In the fifth edition of the WHO classification of thyroid tumours, the previously designated entity ‘cribriform-morular variant of PTC’ has been rebranded as ‘cribriform-morular thyroid carcinoma’.21 This change in nomenclature reflects the general absence of thyroid lineage marker expression, aberrant expression of hormone receptors and distinct genetics characterised by APC mutations and aberrant Wnt signalling, prompting its removal from the PTC umbrella.37 This tumour type is particularly important for practising pathologists to recognise due to its association with familial adenomatous polyposis, as identifying it could potentially lead to the detection of a syndromic condition.21 On a similar note, the precise origin of the rare entity ‘sclerosing mucoepidermoid carcinoma with eosinophilia’ remains unknown. These tumours typically lack follicular cell markers such as PAX8 and thyroglobulin expression. Genetic analyses have failed to identify mutations or fusions associated with thyroid-type or salivary gland-type carcinomas, commonly observed in the salivary gland counterpart. As of this, sclerosing mucoepidermoid carcinoma with eosinophilia is also considered a tumour of uncertain histogenesis.38 39
In all, the contemporary histological examination of thyroid tumours necessitates a collaborative approach involving morphological, immunohistochemical and occasionally molecular analyses to enhance diagnostic and prognostic capabilities. Although the alterations introduced by the 2022 WHO classification will undoubtedly amplify the workload for pathologists, it must be juxtaposed with the augmented benefits for patients. This also underscores the growing imperative for updated and specialised endocrine pathologists to deliver high-resolution diagnostics, aligning with the global trend towards increased subspecialisation in our field.
Delineating the adrenal cortex: revamped approaches with functional immunohistochemistry and enhanced malignancy assessment
The chapters on adrenocortical lesions have undergone several important and clinically motivated changes in nomenclature.40 Monoclonality studies have led to the discouragement of the term ‘nodular adrenocortical hyperplasia’ for sporadic, non-producing lesions, as they are thought to represent neoplastic events rather than hyperplastic lesions, the latter being a term which would suggest a response to a physiological event. Instead, the term ‘sporadic nodular adrenocortical disease’ is recommended for these cases.40 Overall, there have been various nomenclature changes concerning cortisol-producing lesions that better reflect the clinical differences of the diagnostic groups. The umbrella term ‘adrenal cortical nodular disease’ now includes sporadic nodular adrenal cortical disease and a pragmatic subdivision into bilateral micronodular adrenocortical disease and bilateral macronodular adrenocortical disease, two entities with a high proportion of patients exhibiting germline mutations in different risk genes.40 41
The new WHO classification has also adopted the so-called HISTALDO classification for the evaluation of primary hyperaldosteronism, where immunohistochemistry against CYP11B2 (aldosterone synthase) is essential to effectively identify nodules with aldosterone production, which is not easily done by routine morphology.42 The basic idea is that multiple aldosterone-producing nodules are associated with the risk of bilateral disease and, thus, biochemical relapse, while single focal manifestations with CYP11B2 positivity indicate a cured patient. Several studies have highlighted the value of implementing this marker in clinical practice, in which cases previously classified as adenomas were discovered to be multifocal CYP11B2-positive lesions not identified by routine histology alone (figure 6).43–45 Similarly, cases with clearly identifiable multiple nodules on H&E were found to be solitary CYP11B2-expressing lesions with non-producing ‘background’ nodules of lesser importance. Therefore, the introduction of CYP11B2 clearly improves the identification of patients requiring biochemical surveillance for future recurrences on the contralateral adrenal, as well as aiding in the identification of patients with bona fide adenomas cured by surgery and in little need of clinical follow-up. The remaining issue lies in delineating what constitutes normal activity of the zona glomerulosa (ZG). It is not uncommon to identify submillimetre CYP11B2-expressing areas within the ZG in a case otherwise showing a clear-cut adenoma. Consequently, diagnostic confusion between normal ‘background’ activity and multiple aldosterone-producing micronodules (MAPMNs) is expected, and well-designed studies targeting this dilemma are highly warranted. Some authors advocate for the use of a size-based ‘B2 ratio’ (the ratio of sizes of the largest and second-largest CYP11B2-expressing nodule) to detect adenoma from MAPMNs, but this observation has not yet been assessed in other studies.45
CYP11B2 as a facilitating marker for aldosterone-producing adrenal cortical lesions. (A) 11 mm solitary and encapsulated adrenal cortical nodule in a patient with primary hyperaldosteronism (PA). There are no additional nodules. (B) CYP11B2 immunostaining identifies this lesion as aldosterone-producing. The final diagnosis was aldosterone-producing adrenal cortical adenoma. This patient is most likely cured by adrenalectomy. (C) Multiple cortical expansions were noted in this adrenal specimen from a patient with PA. (D) The nodules were CYP11B2 positive, and a diagnosis of multiple aldosterone-producing nodules was put forward. This is entitled ‘non-classic histology’ as per the HISTALDO algorithm, and approximately 50% of patients with PA with this histological appearance will recur biochemically.
The fifth edition of WHO also includes a description of different adrenal cysts, divided into pseudocyst, endothelial cyst, epithelial cyst and parasitic cyst.40 The distinction may require immunohistochemical or histochemical analysis, where CD31 visualises endothelial cysts, calretinin identifies epithelial cysts and PAS can confirm parasitic cysts. As adrenal cysts may mimic tumour conditions from a radiological perspective, it is important to be able to safely assess these lesions in order to rule out clinically important differential diagnoses. Importantly, rare subsets of adrenal tumours may present with a cystic appearance, it is valuable for the endocrine pathologist to be able to readily discern cystic neoplasms from true adrenal cysts.46
Another clinically important distinction is between adrenal cortical adenoma (ACA) and adrenal cortical carcinoma (ACC). The classic pathological criteria for diagnosing ACCs have not changed, but the 2022 WHO classification emphasises the use of multiparameter testing and also lists a number of biomarkers that can be used diagnostically and predictively.40 In terms of algorithms, adult, non-oncocytic ACCs may be assessed using the Weiss and modified Weiss criteria, while oncocytic ACCs (defined by >90% of tumour cells being oncocytic) should be assessed using the Lin-Weiss-Bisceglia criteria.40 Importantly, the novel WHO classification also endorses the use of the reticulin and Helsinki algorithms, which are models that may be used for conventional, oncocytic or myxoid ACCs.47 48 The main features of these multiparameter algorithms are highlighted in figure 7. In short, the reticulin algorithm is based on the disturbed meshwork of reticular fibres in ACCs compared with ACAs and requires a Gordon-Sweet histochemical stain. The outcome of this staining is then incorporated into a model that also takes into account mitoses, necrosis and vascular invasion.48 The Helsinki algorithm makes use of the Ki-67 proliferation index as a model to score tumours, with points also being given for mitoses and necrosis; thus, this is the first algorithm incorporating immunohistochemistry into the model.47 In terms of biomarkers, the novel WHO classification emphasises the value of IGF2 and Ki-67 immunohistochemistry in supporting a diagnosis of ACC.40 49 Additionally, it highlights the use of P53 and beta-catenin to identify ACCs with a higher risk of tumour progression. Moreover, the importance of identifying the adrenocortical origin of an adrenal non-functioning neoplasm is also acknowledged.
Schematic overview of risk assessment models for adrenal cortical carcinoma. UMP, uncertain malignant potential. Created using BioRender.com.
Pheochromocytoma and paraganglioma: mastering terminology and prognostication
Pheochromocytoma and abdominal paraganglioma (PPGL) represent keratin-negative neuroendocrine tumours originating from the adrenal medulla and parasympathetic ganglia, respectively. While they display some malignant potential, the overall risk of distant metastasis is low. These lesions exhibit intricate genetics, and a considerable number of patients manifest syndromic disease. Thanks to intensified research in modern molecular genetics, it has become possible to identify specific gene patterns strongly associated with the risk of disease progression and worse outcomes. Consequently, certain surrogate markers can now be analysed to predict genetic background and the risk of future relapse.
The fifth edition of the WHO classification reinforces the need for genetic screening of all patients with PPGL, given the massive hereditary involvement.50 The new classification also includes recommendations for significant biomarkers associated with the genetic profile of PPGLs. These markers may guide genetic testing, confirm the pathogenicity of molecular abnormalities and offer predictive and prognostic information. For example, immunohistochemistry for SDHA, SDHB, CAIX, FH and alpha-inhibin may all suggest an underlying mutation in a gene regulating the pseudo-hypoxic pathway.51–55 These genetic changes are associated with an increased risk of metastases, and the analyses are therefore clinically motivated (figure 8). Additionally, the staining outcomes may provide assistance to clinical geneticists when a variant of uncertain significance is detected in patient germline tissues, as absent expression may support a damaging mechanism.
Worrisome features of pheochromocytoma and paraganglioma. (A) Profound nuclear pleomorphism and increased mitotic figures (circles). (B) Loss of SDHB immunoreactivity indicates an underlying SDHB, SDHC or SDHD gene mutation, which are genetic events coupled to an aggressive clinical course. Note the positive stromal tissue. (C) Geographic tumour necrosis (marked by asterisk) is a histological feature over-represented in metastatic pheochromocytoma and paraganglioma. (D) Although these tumours are not graded as neuroendocrine tumours of the gastroenteropancreatic system, Ki-67 could be useful, as an elevated proliferation index could indicate worse outcomes. (E) Venous invasion (arrow) is an adverse histological feature.
Although the value of histological risk classification systems like ‘Pheochromocytoma of the Adrenal gland Scaled Score’ (PASS) and ‘the Grading of Adrenal Pheochromocytoma and Paraganglioma’ (GAPP) in predicting metastatic potential is debated, a combined assessment of various clinical, histological and genetic factors is likely necessary for optimal prognostication.56 57 In fact, the fifth WHO classification does not endorse the use of PASS or GAPP due to limited reproducibility and a lack of large series highlighting clinical benefits.50 While the PASS score may be useful as a rule-out algorithm based on its high negative predictive value for the absence of metastatic behaviour, its positive predictive value to identify metastatic cases is limited and the model also suffers from a general lack of definitions of its histological parameters.58 The GAPP score, a younger model with promising results in terms of predictive power, may be challenging to implement and compare between institutions due to the inclusion of biochemistry into the algorithm, and certain subgroups of pheochromocytoma with specific molecular backgrounds may yield false high-risk predictions.59 Even so, this algorithm has been validated as a reproducible model in independent cohort, thereby acknowledging the value of this model with its standardised histological parameters and inclusion of immunohistochemistry.60 Therefore, the 2022 WHO classification does not endorse current grading systems but does not discourage their use in individual practices either. Future research, combining histology, immunohistochemistry and molecular analyses, may potentially lead to even better risk stratification of PPGLs (figure 8). For instance, SDHB immunohistochemistry is a potent tool to screen for underlying SDHB, SDHC or SDHD gene mutations, linked to an increased risk of metastases. Additionally, Ki-67 may serve as an indicator of cases at risk of dissemination, although clinically meaningful grading cutoffs are yet to be established (figure 8).61 62 The proliferation index could therefore be considered as a continuous variable associated with risk of poorer patient outcomes. Potential molecular markers of value may include TERT promoter mutations and telomerase activation, ATRX mutations, high mutational burden and MAML3 gene fusions, as these events have been associated with a worse outcome.50 51 63 64
Among the infra-diaphragmatic paragangliomas, there have also been some nomenclature changes. Lesions in the duodenum and cauda equina are now referred to as separate entities rather than variants of paragangliomas, based on their distinct morphology, protein expression and genetics. The terminology ‘paraganglioma-like neuroendocrine neoplasia’ (NEN) encompasses cauda equina NET, previously called ‘cauda equina paraganglioma’, and composite gangliocytoma/neuroma and neuroendocrine tumour, previously called ‘gangliocytic paraganglioma’.50 The change in nomenclature is supported by positive keratin expression in these two entities, thereby distinguishing them from conventional paraganglioma. Moreover, these lesions do not exhibit the same morphology or genetic aberrancies as PPGLs in general.
Epithelial neuroendocrine neoplasia: a mixed bag of goodies
For the first time, the WHO classification of endocrine tumours now includes descriptions of NEN across all organs, not limited solely to endocrine glands.65 This encompasses NENs arising in various sites such as the gastrointestinal tract, lung, upper airways, urogenital system, breast and skin. The functional aspect of NENs is reinforced, and endocrine pathologists are recommended to visualise hormone production via immunohistochemistry, given the fact that not all patients with NEN have undergone proper preoperative hormone screening—which could therefore help in identifying disease that was clinically unknown. In terms of biomarkers, the second-generation neuroendocrine marker INSM1 is acknowledged for its excellent sensitivity towards NENs alongside synaptophysin; however, the reduced specificity is also mentioned.65–68
Moreover, the concept of stratifying highly proliferative (Ki-67 index >20%) NENs into grade 3 NET or neuroendocrine carcinoma (NEC), introduced for pancreatic NENs in the former WHO classification, is now solidified and recommended for other anatomical sites/primaries. A joint evaluation to distinguish between these entities is necessary, incorporating clinical presentation (hormone hypersecretion more common in NET G3 than NEC), imaging (NET G3 usually detected by 68Ga-DOTATATE PET/CT, while NECs require PET), morphology (well-differentiated histology more common in NET G3 than NEC) and immunohistochemistry (NET G3 often express SSRT2, SSRT5 which NECs lack, while NECs usually exhibit aberrant P53 or Rb staining).65
Finally, the diagnostic algorithm for mixed endocrine non-neuroendocrine neoplasms (MINENs) is simplified, where true MINENs must consist of two morphologically distinct tumour entities, one of which is neuroendocrine and stains positive for several neuroendocrine markers. Epithelial neoplasms with a single morphological appearance should be classified based on the neuroendocrine marker staining outcome categorised as either ‘carcinoma with interspersed neuroendocrine cells’, ‘carcinoma with isolated synaptophysin expression’ or ‘amphicrine carcinoma’ (an exceedingly rare entity exhibiting positivity for both neuroendocrine and non-neuroendocrine markers in the same cell).65 69
Concluding remarks and reflections
The latest WHO classification of endocrine and neuroendocrine tumours signifies a significant advancement in the field of endocrine pathology, providing a much-needed rejuvenation. This updated classification places considerable emphasis on the seamless integration of histopathological findings with clinical data, along with the utilisation of biomarkers to enhance diagnostic accuracy and prognostic assessment. Although the molecular era looms on the horizon, the latest edition of the classification merely hints at the potential of molecular biomarkers, rather than fully embracing them. However, ongoing research endeavours aimed at refining our understanding of molecular mechanisms underlying these tumours hold promise for their integration into future classifications. The evolving landscape of endocrine pathology places heightened demands on pathologists to precisely stratify tumours both histologically and according to grading criteria. This refinement is long-awaited and contributes significantly to the identification of high-risk patients. Therefore, it is imperative that the novel aspects of this classification are thoroughly disseminated among our colleagues and effectively communicated to our peers in surgery, endocrinology and oncology. By fostering interdisciplinary collaboration and knowledge exchange, we can optimise patient care and further advance the field of endocrine pathology.
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References
Footnotes
Handling editor Munita Bal.
Contributors CCJ wrote the manuscript and created all figures.
Funding This study was funded by the Swedish Cancer Society (Senior Clinical Investigator Award).
Competing interests None declared.
Provenance and peer review Commissioned; internally peer reviewed.