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Review
Neuroendocrine tumours—challenges in the diagnosis and classification of pulmonary neuroendocrine tumours
  1. M A den Bakker1,2,
  2. F B J M Thunnissen3
  1. 1Department of Pathology, Maasstad Hospital, Rotterdam, The Netherlands
  2. 2Department of Pathology Erasmus MC, Rotterdam, The Netherlands
  3. 3Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
  1. Correspondence to Dr M A den Bakker, Department of Pathology, Maasstad Ziekenhuis, PO Box 3100, Rotterdam 3007 AC, The Netherlands; bakkerma{at}maasstadziekenhuis.nl, m.denbakker{at}erasmusmc.nl

Abstract

Pulmonary neuroendocrine (NE) proliferations are a diverse group of disorders which share distinct cytological, architectural and biosynthetic features. Tumours composed of NE cells are dispersed among different tumour categories in the WHO classification of tumours and as such do not conform to a singular group with regards to treatment and prognosis. This is reflected by the highly variable behaviour of NE proliferations, ranging from asymptomatic, for instance in diffuse idiopathic pulmonary NE cell hyperplasia and tumourlets, to highly malignant cancers such as small cell lung cancer and large cell NE carcinoma. In this review NE proliferations are described as distinct entities ranging from low grade lesions to high grade cancers. The differential diagnoses are considered with each of the entries. Finally, mention is made of tumours which may show some NE features.

  • Neuroendocrine
  • LUNG
  • HISTOPATHOLOGY

https://doi.org/10.1136/jclinpath-2012-201310

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    Cytological features, shared by cells in all neuroendocrine (NE) proliferations, include a dispersed, slightly coarse, ‘stippled’ chromatin pattern and, with the exception of large cell neuroendocrine carcinoma (LCNEC), absence of, or only indistinct nucleoli (figure 1A,B). Architectural growth patterns are a characteristic feature and include distinct growth patterns such as nesting, ribbons, trabeculae, palisading of cells at the periphery of nests and the presence of rosettes. NE tumours are commonly cellular neoplasms with a highly vascular stroma. In lower grade NE tumours such as typical carcinoids (Tca) and atypical carcinoids (Aca) stromal calcification and ossification are not uncommon (figure 2).

    Figure 1
    Figure 1

    Typical cytology of low grade NE tumour. (A) Monomorphic round nuclei with slightly coarse chromatin, often referred to as ‘pepper and salt’, indistinct nucleoli, sharp nuclear borders; bland eosinophilic cytoplasm (resin embedded section, ultra-thin hematoxylin eosin (HE) section). A hint of nesting and palisading trabecular growth is seen. (B) Spindle cell morphology in low grade NE proliferation (tumourlet in diffuse idiopathic pulmonary neuroendocrine cell hyperplasia ). Similarly there is little variation in nuclear morphology (HE stain of paraffin-embedded section).

    Figure 2
    Figure 2

    Medium power view of a typical carcinoid tumour with stromal ossification. A nested growth pattern is seen with ramifying delicate fibrovascular stromal septa and centrally lamellar bone (HE stain).

    Further confirmation of NE differentiation is accomplished by demonstration of the production of specific proteins which are identified by immunohistochemistry. Although electron microscope demonstration of dense core granules was considered a suitable method to support NE differentiation, this modality has been superseded by immunohistochemistry. The use of immunohistochemistry is now a routine procedure in pathology laboratories although the choice of antibodies to demonstrate NE differentiation does vary. As in all biomarker applications sensitivity and specificity of markers often require a trade-off which in part may be circumvented by the use of multiple markers. Markers commonly used to demonstrate NE differentiation listed by increasing sensitivity and decreasing specificity include chromogranin-A, synaptophysin and CD56 (neural cell adhesion molecule).1 ,2 Other NE markers with considerably lower specificity and associated limited diagnostic use are neurone-specific enolase, protein gene product 9.5 and CD57. The staining pattern of non-NE markers may at times indicate NE differentiation, such as punctuate cytoplasmic staining seen with cytokeratin antibodies. Parenthetically, immunohistochemistry should ideally be used to confirm morphological pointers to NE differentiation. The routine use of NE markers in tumours lacking morphological evidence of NE differentiation is discouraged, as the designation of intrinsically non-NE tumours as showing NE differentiation bears no relation to tumour behaviour or prognosis.1 ,3 Thus, with the sole exception of LCNEC, demonstration of NE differentiation is not required to establish a diagnosis of a NE tumour.

    NE proliferations may present with signs and symptoms common to other pulmonary neoplasms but may also manifest by the production of bioactive substances which may cause paraneoplastic syndromes or the carcinoid syndrome. With the exception of diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH), which is associated with the development of carcinoids, there is no known precursor lesion for NE tumours. Moreover, the cell of origin has not been conclusively identified although it would seem that the bronchial basal cell is the most likely candidate.4 NE cells are an intrinsic component of the pulmonary epithelium, forming a dispersed population comprising approximately1% of all epithelial cells in the adult lung.5 NE cell hyperplasia, defined as more than 3% NE cells or clusters of three or more NE cells, may be seen in pulmonary conditions characterised by hypoxia such as chronic obstructive lung disease (COPD) or prolonged stay at high altitude. NE hyperplasia does not constitute a risk factor for the development of NE neoplasms.

    Tumourlets

    Tumourlets are small collections of NE cells usually seen in a background of NE cell hyperplasia and which are encountered in lung tissue removed from patients with some form of chronic lung disease. By definition these small foci of NE cells extend through the basement membrane but by definition do not exceed 5mm in size, if larger than 5 mm these are diagnosed as carcinoid tumours. The arbitrary size cut-off point implies that there is a grey zone where uncertainty exists over tumourlets and neoplasia, which is exemplified by the fact that metastasis has been reported in patients with tumourlets.6 Tumourlets may be seen in lung tissue with carcinoids and tumourlets combined with NE hyperplasia are a typical component of DIPNECH (see below) (figure 3). Cytologically the cells of NE hyperplasia and tumourlets are indistinguishable from those in carcinoids and are monomorphic often slightly spindled cells with regular oval nuclei with typical ‘NE type chromatin’; mitotic activity and necrosis are absent.

    Figure 3
    Figure 3

    Low power view of a synaptophysin stain of a carcinoid tumourlet in diffuse idiopathic pulmonary neuroendocrine cell hyperplasia.

    Small collections of NE cells may be confused with minute meningothelial nodules (MMNs), which similarly to low grade NE proliferations are composed of bland, slightly spindled cells (figure 4). The cells of MMNs show evidence of true meningothelial differentiation on the immunohistochemical and ultrastructural level. Cytologically the cells of MMNs resemble NE cells but do not stain for NE markers, with the exception of CD56.7 ,8 MMNs are incidental findings with no clinical significance, although a rare disseminated form (pulmonary meningotheliomatosis) has been described.9

    Figure 4
    Figure 4

    Medium power view of an incidentally discovered ‘minute meningothelial nodule’ (MMN). These small proliferations closely approximate the appearance of low grade NE proliferations with regular cytomorphology and a nested growth pattern. However, MMNs do not stain for NE markers. (HE stain).

    Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia

    In contrast to tumourlets and NE hyperplasia, DIPNECH is a primary disorder that is not related to pre-existing lung disease. The entity was fully described in 1992, although earlier reports describe an identical process.10 ,11 DIPNECH may be discovered incidentally in patients under investigation for another form of pulmonary disease (for instance shortness of breath or metastatic disease). Symptomatic patients with DIPNECH present with cough and dyspnoea and show evidence of airway restriction and obstruction, in part owing to obliterative bronchiolitis which has been suggested to be caused by fibrogenic substances released from NE cells, resulting in fibrosis in the subepithelial stroma underlying the NE hyperplasia. DIPNECH usually runs an indolent or stable course but progressive disease with obliterative bronchiolitis requiring lung transplantation has been reported.12 The NE proliferations encountered in DIPNECH are low-grade lesions with a continuum from NE cell hyperplasia, tumourlets and carcinoid tumours (typical and atypical forms, see below)12–14 (figure 5). As stated above, the diagnosis of DIPNECH is often established while investigations for other pulmonary disease are performed or when resection of a carcinoid tumour reveals background NE cell hyperplasia in otherwise normal lung.

    Figure 5
    Figure 5

    Medium power view of NE cell hyperplasia in diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. The complete spectrum for low grade NE proliferations, from hyperplasia to atypical carcinoids may be seen in this disorder. In this view the basement membrane has not been breached and the lesion falls well under the size limit for a tumourlet. (HE stain).

    Carcinoid tumours

    Carcinoid tumours are neoplastic proliferations of NE cells with typical NE cytology and growth patterns. The size of the tumours by definition exceeds 5 mm and mitotic activity does not exceed 10 mitoses in 2 mm2. Pulmonary carcinoids are further subdivided into Tca and Aca, a subdivision which is based on a review series taking prognosis into account. Tca are defined as having less than 2 mitoses per 2 mm2 and no necrosis, while Aca have between 2–10 mitoses and/or punctuate necrosis. Although the 5-year survival rates for Tca and Aca are similar, the 10-year survival rates are distinctly different (87% vs 35%).15 ,16 However, reported survival rates vary considerably in series with some series even reporting similar survival rates for Tca and Aca.16 It has been suggested that the variability in survival rates may be caused by interobserver variability in distinguishing Aca and Tca.17 Recent work by Swarts et al, (manuscript submitted) indicated that the interobserver agreement for the morphological separation of Aca and Tca was only fair (κ=0.32). The difficulty in the separation Aca and Tca most likely stems from difficulty in counting mitoses, in which apoptotic cells and degenerated cells may be counted as mitoses or conversely mitosis may be missed in poorly preserved or poorly fixed tumour samples. Furthermore, in the work by Swarts et al, it appeared that identification of necrosis varied between observers. However, application of ki-67 as a surrogate marker for proliferation, similar to gastrointestinal NE tumours, significantly correlated with prognosis. Emerging new data shows promising results for nuclear expression of orthopedia homeobox as marker segregating Tca and Aca.18 The strict cut-off points for Tca and Aca introduces a further complication when mitotically active (>10 mitoses per 2 mm2) well differentiated tumours, that is, showing carcinoid morphology, are encountered. According to current definitions these should be diagnosed as LCNEC (described below), but are most likely behave in a fashion in keeping with Aca. Although different molecular aberrations have been identified, these have so far not been able to confidently segregate Tca and Aca.

    Carcinoids are not caused by smoking but generally do present with typical pulmonary ‘cancer’ symptoms including haemoptysis, shortness of breath or pneumonia. In addition, symptoms caused by bioactive substances elaborated by the tumours may be present, such as adrenocorticotropic hormone (ACTH), calcitonin, growth-hormone-releasing hormone (GHRH) and 5-hydroxytryptamine (HT) which may cause the carcinoid syndrome.19 Pulmonary carcinoid tumours may occur as part of type 1 multiple endocrine neoplasia, while sporadic pulmonary carcinoids frequently show inactivation of the type 1 multiple endocrine neoplasia gene.20 ,21

    The morphology of Tca and Aca is similar with typical NE architecture and cytology, although peripherally located carcinoids are more frequently composed of spindled NE cells in comparison to centrally located bronchial carcinoids. Carcinoid tumours are cellular tumours which show a variety of growth patterns. Nested, trabecular, pseudoglandular and solid patterns may form and pseudorosettes are a common finding. The stromal component varies but is commonly highly vascular. Stromal hyalinisation, calcification and ossification may be observed and amyloid deposition is not uncommon (figure 6). Carcinoids stain strongly with NE markers with minor non-significant differences between Aca and Tca.

    Figure 6
    Figure 6

    Macroscopy of a centrally located carcinoid tumour. This tumour was biologically active, producing GHRH. Macroscopically carcinoids are generally well demarcated, soft beige-pink tumours, commonly with a bronchial component extending in the pulmonary parenchyma.

    The differential diagnosis primarily includes metastases from NE tumours elsewhere, in particular from those originating in the gastrointestinal tract. Staining for TTF-1 and CDX-2 solves this possibility as non-pulmonary carcinoids are TTF-1 negative and pulmonary carcinoids are CDX-2 negative.22 ,23 TTF-1 positivity has been reported in up to 95% of pulmonary carcinoids, with stronger staining in spindle cell carcinoids.22–24 However, choice of TTF-1 antibody has been shown to influence staining percentages.25 A further problem may arise in small crushed biopsies where the cytological details and mitotic figures of a possible carcinoid tumour cannot be adequately assessed. Smearing of nuclei may then lead to an erroneous diagnosis of small cell lung cancer (SCLC). In this setting ki-67 (MIB-1) staining is valuable as SCLC will show a proliferation index >50% while carcinoids have a proliferation index <20%.26

    Although rare, pulmonary paraganglioma, either primary or metastatic, share many similarities with carcinoid tumours. Although many reported cases of pulmonary paraganglioma probably represent NE tumours, bona fide cases do exist and may be functional. Carcinoid tumours and pulmonary paraganglioma share similar architectural and cytological features with a nested growth pattern and an overlapping immunohistochemical profile with chromogranin, synaptophysin, CD56 staining.27–29 However, TTF-1 and, in most cases, cytokeratin staining is negative. The presence of S-100 positive sustentacular cells has been described in pulmonary carcinoids and cannot be used to differentiate from paraganglioma.30

    Intrapulmonary glomangioma is a rare tumour in the lung, but may (especially on frozen section) be considered in the differential diagnosis of carcinoid. The positive staining of vimentin and smooth muscle differentiation and lack of NE, TTF-1 and cytokeratin staining leads to correct diagnosis.31

    Small cell lung carcinoma

    SCLC is a highly malignant NE carcinoma which is usually disseminated at the time of diagnosis. A morphological identical tumour may arise in other organs which commonly have an identical immunohistochemical profile, including TTF-1 positivity.32–34 The classical cytomorphological features of SCLC include a high nuclear-cytoplasmic ratio, absent or indistinct nucleoli and moulding of nuclei, which show little variability in size (figure 7A). The cells are arranged in sheets and may form (pseudo)rosettes. The cells are fragile and easily damaged resulting in encrustation of nuclear material on blood vessel walls, referred to as the Azzopardi phenomenon (figure 8). Necrosis is common and the mitotic rate is typically very high (>70/10HPF). Compared with biopsy specimens, the cytological features in resection specimens reveals slightly larger tumour cells with often more vesicular appearing nuclei and occasional nucleoli.35 The fragility of SCLC cells results in artefactual changes when these tumours are biopsied. Cell nuclei are often disrupted with smearing of nuclear material, hampering histological and cytological assessment. Distinguishing SCLC from lymphoid infiltrates is facilitated by including lymphoid markers in an immunohistochemical panel. Low-grade NE tumours may be segregated by Ki-67 (Mib-1) staining.26 Other tumours composed of small cells may on occasion enter the differential diagnosis, in particular metastases from so-called ‘small blue round cell tumours’. However, these tumours generally arise in the paediatric age group and are identified by appropriate immunohistochemical stains. A small cell variant of squamous cell carcinoma may mimic SCLC, particularly in biopsies where tell-tale signs of squamous differentiation are lacking. Staining for p63, combined with negativity for NE markers, is useful to establish the diagnosis36 (figure 9A,B).

    Figure 7
    Figure 7

    High grade neuroendocrine carcinomas. (a) Small cell lung cancer (SCLC), composed of closely packed fusiform cells with barely discernible cytoplasm. The nuclei are hyperchromatic and devoid of nucleoli. Centrally a rosette is seen. Resection specimen, HE stain. (b) High grade neuroendocrine carcinoma which will create discussion as to its classification as SCLC or large cell neuroendocrine carcinoma (LCNEC). Although the nuclei are monomorphic, the cells have more cytoplasm resulting in a lower nuclear-cytoplasmic ratio. Resection specimen, HE stain. (c) LCNEC, with larger pleomorphic nuclei which vary in size and shape. The cells have easily identifiable eosinophilic cytoplasm. However, nuclei are indistinct. LCNEC requires immunohistochemical confirmation as a neuroendocrine tumour. Resection specimen, HE stain.

    Figure 8
    Figure 8

    The so-called Azzopardi phenomenon, named for Dr Azzorpardi who described this feature in 1959, results from precipitation of DNA from tumour cells on the connective tissue in vessel walls, although it is not specific for small cell lung cancer (SCLC) and may be seen in other cellular tumours with easily damaged cells. However, in the setting of a primary pulmonary neoplasm it is typical for SCLC.

    Figure 9
    Figure 9

    Small cell variant of squamous cell carcinoma, a mimic of small cell lung cancer. Obviously this tumour will not stain for NE markers but is correctly identified by p63 positivity. (a) HE stain, (b) p63 immunoperoxidase stain.

    The distinction between SCLC and LCNEC (see below) can be difficult as the distinguishing features overlap and are prone to interobserver variability (figure 7A−C). This is particularly relevant in cytological specimens.37

    SCLC may occur combined with other lung cancer subtypes, including LCNEC (figure 10). This is considered divergent differentiation rather than collision, which finds support in the identification of similar genetic aberrations in discrete tumour components.38 The presence of any amount of a differentiated carcinoma (adenocarcinoma, squamous carcinoma or other type of carcinoma) is acceptable to diagnose combined SCLC, except for the presence of an undifferentiated large cell component which must comprise at least 10% of the tumour.35 ,39

    Figure 10
    Figure 10

    Combined small cell lung cancer (SCLC)-adenocarcinoma case. Note that there are subtle changes in the nuclear morphology as the SCLC component merges with the glandular component.

    Large cell neuroendocrine carcinoma

    LCNEC is a relatively recently described form of high grade lung cancer with morphological and cytological NE features which approximate and even overlap with those of SCLC but which are qualitatively different. It is the only entity within the WHO lung cancer classification which requires immunohistochemical confirmation. These tumours were set aside from Aca and SCLC on the other hand, because of morphological and prognostic differences.40 Paradoxically, despite the apparent difference implied by their names, cell size is not a reliable feature to segregate SCLC and LCNEC.41–43

    LCNECs show NE growth patterns and NE cytology although less well developed than in lower grade NE tumours and SCLCs (figure 7C). Cytological features typical for LCNEC include increased cell size, polygonal cell shape, presence of nucleoli (often) and discernible cytoplasm resulting in a higher nucleocytoplasmic ratio. In contrast to SCLC, moulding is uncommon. Despite these differences some cases of LCNEC remain difficult to classify, owing to the nature of morphological diagnostic features which overlap with those of SCLC.17 ,44 ,45 It has been suggested to group SCLC and LCNEC in a single group of high grade NE carcinomas as these tumours share many morphological, cytological, prognostic, predictive and molecular features.16 ,35 ,46–51 This is further extended by the suggestion, which is not generally accepted, to categorise NE tumours into hyperplasias (tumourlets), low grade (grade I) NE carcinomas (Tca), intermediate grade (grade II) NE carcinomas (Aca) and high grade (grade III) NE carcinomas (including SCLC, LCNEC and combined cases). A recent retrospective analysis concluded that LCNEC clinically and biologically compared better to large cell carcinoma (LCC) than to SCLC.52 However, clinical (survival) rather than biological aspects were addressed in this study and the study did not take the considerable impact of imprecise categorisation as a result of interobserver variability into account, rendering the conclusions tenuous. Despite the morphological overlap between SCLC and LCNEC, gene expression studies, partly supported by RT-qPCR and immunohistochemical studies provide some support for the segregation of LCNEC and SCLC as distinct entities.53–56 Nevertheless, the data on molecular segregation of SCLC and LCNEC is conflicting. A very recent large study in which somatic copy number alterations, analysed by single nucleotide polymorphism arrays combined with analysis of frequently mutated genes did not show significant differences between SCLC and LCNEC (Seidel et al, personal communication, manuscript submitted). Conversely, another recent study using gene expression arrays on well characterised SCLC and LCNEC cases identified CDX-2 as a marker for LCNEC and brain angiogenesis inhibitor 3 as a specific marker for SCLC. The findings were corroborated with RT-qPCR and immunohistochemistry.53

    As may be inferred from the discussion above, the differential diagnosis of LCNEC will often include SCLC. In addition other forms of non-small carcinomas need to be distinguished from LCNEC. Basaloid carcinoma and basaloid squamous carcinoma in particular show some morphological overlap with LCNEC on account of the nested growth pattern, high grade nuclear features and necrosis. However, NE markers, which are required for a diagnosis of LCNEC, are not expressed in these entities.

    In addition to the primary lung tumours discussed above other tumours in the lung have been described which may to varying extent show morphological and immunohistochemical features, in particular peripheral neuroectodermal tumour (PNET), pulmonary blastoma, desmoplastic round cell tumour, and large cell carcinoma with rhabdoid phenotype.

    Although large cell carcinoma with rhabdoid phenotype is included in the list of tumours showing NE differentiation, the evidence to support this is meagre.57–59 Similar to conventional non-small cell carcinoma NE expression is often sought for by immunohistochemistry while the true meaning of this finding is uncertain.

    PNET may occur in adults and several examples of primary pulmonary PNET have been reported.60 However, the age of presentation in a recent series, including the referenced cases herein, was considerably lower than for pulmonary carcinoma. Histologically SCLC may be confused with PNET. NE markers may be positive and cytokeratin staining is well known in PNET.61 However, CD99 does not stain SCLC and conversely TTF-1 has not been reported in PNET. Judicious use of immunohistochemistry and attention to clinical facts (age, smoking history) will avoid this potential pitfall.

    Pulmonary blastoma is a rare form of sarcomatoid carcinoma occurring in adults, which has been divided into a monophasic (epithelial only) form, composed of fetal-like glandular proliferation (fetal-type adenocarcinoma) and a biphasic tumour with a glandular component and a malignant stromal component which may show divergent mesenchymal differentiation (figure 11). Both forms may harbour solid epithelial nodules (morules) which mark with synaptophysin and/or chromogranin, in addition to CD10.62 ,63 As discussed above, these tumours are not to be regarded as true NE tumours.

    Figure 11
    Figure 11

    Pulmonary blastoma, which may show some evidence of NE differentiation but are not considered NE tumours. In this example an ‘immature’ appearing glandular component is combined with a malignant stroma component. HE stain.

    Desmoplastic small round cell tumour is a very rare highly malignant tumour typically arising in the abdominal or pelvic soft tissues of young adults predominantly male individuals. Like PNET (and SCLC) it forms part of the spectrum of small blue round cell tumours and typically is composed of cellular solid nests of monomorphic small cells set in sclerotic collagenous stroma. Primary pulmonary desmoplastic small round cell tumor (DSCRT) is very rare with only single case reports published, while pleural examples have been more frequently reported.64 ,65 The tumour shows a mixed epithelial/mesenchymal immunoprofile, staining for cytokeratin, epithelial membrane antigen (EMA), desmin and vimentin. In addition synaptophysin and chromogranin may be positive in 29% and 10%, respectively.66 ,67 Although the morphology and immunohistochemistry is typical, the diagnosis may be confirmed by demonstrating an EWS-WT1 gene fusion which typifies this tumour.

    Key message

    • Tumours with neuroendocrine differentiation are primarily recognized by distinct cytomorphology and architectural growth patterns.

    • Occasionally SCLC and LCNEC may show overlapping morphological features, to date there are no established supplemental techniques which improve on standard histological diagnosis.

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    Footnotes

    • Contributors MAdB wrote the draft of this manuscript and provided the illustrations. FBJMT corrected the draft versions and supplied background information.

    • Competing interests None.

    • Provenance and peer review Not commissioned; externally peer reviewed.