Bellizzi, Andrew M. MD*,†
The neuroendocrine epithelial neoplasms (NENs), including well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs), are characterized by the expression of keratin intermediate filaments and the production of peptide hormones and/or biogenic amines. Whereas PDNECs are biologically aggressive and highly lethal, with localized Merkel cell carcinoma (MCC) somewhat of an exception, WDNETs exhibit a range of “indolent” biologic potentials—from benign to widely metastatic and eventually fatal. Within each of these 2 groups of tumors, there is substantial morphologic overlap. The PDNECs are composed of “small,” intermediate, or large cells, often exhibit diffuse growth patterns, and are highly proliferative. The WDNETs, regardless of site of origin, are characterized by a series of growth patterns collectively referred to as “organoid” and frequently demonstrate finely granular chromatin. The range of biologies referenced above for the WDNETs can to a large extent be predicted based on anatomic site. In addition, the therapeutic armamentarium for metastatic WDNETs is rapidly expanding, and the efficacy of individual agents is related to site of origin.
When faced with a metastatic WDNET of unknown origin (generally to the liver), an all-too-familiar diagnostic scenario, pathologists have long been content to levy a diagnosis to the effect of “metastatic neuroendocrine tumor” and to move on with their day. This practice is no longer sufficient. Patients and their treating surgical oncologists, endocrinologists, and medical oncologists deserve some attempt at assigning site of origin, as well as comment on the degree of proliferation. These parameters are of significant prognostic and therapeutic importance. In the skin, MCC must be distinguished from spread of a visceral PDNEC. Most pathologists are familiar with the application of cytokeratin (CK) 20 and thyroid transcription factor-1 (TTF-1) immunohistochemistry (IHC) in this setting. Fewer are familiar with the frequency of TTF-1 expression in PDNECs, regardless of site of origin. At least 2 other markers are useful in this setting, including one that directly reflects the etiopathogenesis of MCC.
Regarding the WDNETs, a number of markers, including several transcription factors, have emerged that are very useful in assigning site of origin. Most of these antibodies are “workhorses” in diagnostic IHC (eg, TTF-1, CDX2), others are emerging “powerhouses” (eg, polyclonal PAX8), and a few others are “classics” [eg, progesterone receptor (PR), S-100], perhaps with a “new trick up their sleeves.” Pathologists are, in general, unfamiliar with this application of IHC or will make assumptions about the expression pattern of a marker based on data from adenocarcinoma (eg, they assume that CDX2 will be expressed by tumors throughout the intestine).
This review intends to prove the thesis that determining the site of origin of a NEN is clinically vital and that diagnostic IHC is well suited to the task. It will begin by reviewing current World Health Organization (WHO) terminology as well as an embryology-based and a histologic pattern–based classification that I find useful in organizing my thoughts about these lesions. It will present (1) population-based data on the relative frequency and biology of WDNETs arising at various anatomic sites, as a framework for, in the metastatic setting, conceptualizing the pretest probabilities of spread from various sites; (2) the frequency of metastases of unknown primary in several recent series; and (3) the limitations of current imaging techniques, as a means of defining the scope of the problem. It will go on to discuss the contemporary therapeutic significance of site of origin. The heart of this review is a synthesis of data compiled from >100 manuscripts on the expression of individual markers in WDNETs and PDNECs, as regards site of origin. These will include proteins I consider “key markers” and others that are either useful “secondary markers,” potentially very useful markers that need to be further vetted, or ones that are widely applied despite a lack of efficacy in this setting. It will conclude with my approach to the metastatic NEN of unknown origin.
World Health Organization Classification
The WHO Classification of Tumours of the Digestive System presents a nomenclature for gastroenteropancreatic (GEP) NENs that supplants the WHO 2000 Classification.1 Morphologically well-differentiated NENs are referred to simply as “neuroendocrine tumors” (NETs), whereas poorly differentiated examples are classified as “neuroendocrine carcinomas” (NECs). The “well-differentiated endocrine carcinoma” of WHO 2000 no longer exists, with the current classification of a NEN based on its appearance, rather than whether or not it has metastasized. In this classification, the term “NET” implies a well-differentiated tumor (although WDNET will be used often throughout this review for clarity).
As discussed above, NETs are biologically heterogenous, and it is well recognized that a group of tumors characterized by tumor necrosis, increased mitotic activity, and increased tumor fraction participating in the cell cycle [ie, increased proliferation index (PI); determined by the percentage of tumor cells expressing Ki-67 by IHC] carve out a “middle path” between less-proliferative NETs and PDNECs. In WHO 2010, this is captured in the grade of a NET. By definition, grade (G)1 tumors demonstrate <2 mitotic figures per 10 high-power fields (HPF) and/or a Ki-67 index of ≤2%, whereas G2 is assigned on the basis of a mitotic count of 2 to 20/10 HPF and/or a Ki-67 index of 3% to 20%. The NECs can be described as being of “large cell” or “small cell type” (the WHO 2010 term “NEC” implies a poorly differentiated tumor, although PDNEC will be used for clarity). NECs are G3, with a mitotic count >20/10 HPF and/or a Ki-67 index of >20%.
The most recent WHO Classification of pulmonary NENs is contained in the 2004 WHO Classification of Tumours: Pathology and Genetics of Tumours of the Lung, Pleura, Thymus and Heart.2 In this system, the term “carcinoid tumor” persists. WDNETs of the lung are classified as “typical carcinoid” (TC) or “atypical carcinoid” (AC) on the basis of mitotic rate and the presence or absence of tumor necrosis. TCs possess well-differentiated histology, demonstrate <2 mitotic figures per 2 mm2 (generally related as 10 HPF but actually closer to 8 HPF on many modern microscopes), lack necrosis, and measure ≥0.5 cm. A diagnosis of AC is rendered when a morphologically similar tumor contains 2 to 10 mitotic figures per 2 mm2 and/or necrosis.
It is noteworthy that Ki-67 PI is not presently a component of the classification of pulmonary NENs (although it may be part of the next one). Despite this fact, Ki-67 IHC has been shown to highlight PIs for TC and AC analogous to those seen with G1 and G2 GEP NETs, respectively (reviewed in Rekhtman3). As has been pointed out by several authors, Ki-67 IHC is often a useful diagnostic adjunct in these tumors, particularly in crushed small biopsies in which mistaking a carcinoid tumor for small cell lung carcinoma (SCLC) is a genuine pitfall of serious clinical significance.3–5
The PDNECs include SCLC and large cell neuroendocrine carcinoma (LCNEC). To qualify as poorly differentiated, a pulmonary NEN must demonstrate at least 11 mitotic figures per 2 mm2, although examples usually exhibit much higher mitotic rates. [It is noteworthy that the current mitotic “floor” for a pulmonary PDNEC (≥11/2 mm2) is lower than that for a GEP NEC (>20/10 HPF).] Compared with SCLC, LCNEC is more apt to demonstrate an organoid (rather than diffuse) architecture, is composed of larger cells with more abundant cytoplasm, typically contains prominent nucleoli, and may or may not exhibit “neuroendocrine chromatin.” Because of substantial morphologic overlap with large cell (undifferentiated) carcinoma, a diagnosis of LCNEC formally requires the immunohistochemical demonstration of expression of at least 1 general neuroendocrine marker.2
Although WDNETs and PDNECs predominate in the GEP system and lung, respectively, similar-appearing tumors occur in every organ system. These have generally been classified by appropriating the terminology used for GEP or pulmonary tumors. As most of the relevant WHO Blue Books are still in their third editions, the descriptor “carcinoid tumor” (a term emphatically stricken from the record for GEP NETs) still abounds. At some sites WDNETs are all classified as well-differentiated NECs, based on the premise that all WDNETs are potentially malignant.6 These semantics should not interfere with the simple facts that the NENs, regardless of anatomic site, can be separated into well-differentiated, generally indolent tumors and poorly differentiated, aggressive ones, and that, for the well-differentiated tumors, evidence of increased cell turnover suggests the possibility of more aggressive behavior. A couple tumors deserve special mention here. MCC is a PDNEC of the skin, and medullary thyroid carcinoma is defined as a “malignant tumor of the thyroid gland showing C-cell differentiation.”7
Williams and Sandler Classification
Based on observations of the histology, histochemical features, biochemical content, and biology of WDNETs, Williams and Sandler,8 in an article published in The Lancet in 1963, proposed that tumors be grouped into those that are foregut, midgut, and hindgut derived. The familiar “midgut carcinoid” is characterized by a nested architecture, often contains brightly eosinophilic cytoplasmic granules that are argentaffin positive (ie, they reduce silver salts to metallic silver), has a high 5-hydroxytrypamine (serotonin) content, and is frequently associated with carcinoid syndrome. Foregut-derived and hindgut-derived tumors share several characteristics that distinguish them from midgut ones. They have a tendency toward trabecular architecture, are nonargentaffin, and contain little, if any, serotonin. Compared to hindgut tumors, foregut ones are more apt to be associated with carcinoid syndrome and often secrete 5-hydroxytryptophan.
Transcription factor expression is tightly regulated in time and space and drives embryonic patterning. Thus, the Williams and Sandler classification provides an intuitively appealing framework upon which to hang the observation that jejunal, ileal, and appendiceal WDNETs, all midgut derived, cluster together in terms of transcription factor expression and that pancreaticoduodenal tumors, foregut derived and arising from intimately developmentally related organs, would, by transcription factor expression patterns, be essentially indistinguishable. Similarly, the classification may be useful to predict the expression profiles of cecal and other colonic tumors, for which little data exist. However, it is insufficient to explain all observations—for example, why the transcription factor signature of pancreaticoduodenal tumors seems to have more in common with hindgut-derived rectal tumors than with fellow foregut-derived gastric ones.
Soga and Tazawa Classification
Soga and Tazawa,9 in a manuscript published in Cancer in 1971 (based on their observations in 62 tumors), described 5 architectural patterns in “carcinoid tumors” (types A to D and mixed) and correlated them with site of origin (Figs. 1A–D). Type A tumors are composed of solid nests (ie, insular). Type B tumors are trabecular or “ribbon-like,” with the cords of cells having a tendency to anastomose. Type C tumors contain tubular, acinar, or rosette-like structures (ie, pseudoglandular). Type D tumors exhibit “lower or atypical differentiation” (ie, “diffuse” or “undifferentiated”). Mixed tumors exhibit any combination of patterns A to D. In their series, type A histology was typical of “midgut carcinoids,” foregut-derived tumors tended to be type B, and hindgut tumors were apt to be mixed.
In the Armed Forces Institute of Pathology (AFIP) series of 167 jejunoileal WDNETs, 93% were type A.10 The association with type A histology is less strong in the appendix, where Burke et al11 described, among 86 pure WDNETs, 50% type A and 24% mixed tumors, as well as 20% “tubular” carcinoids and 6% “clear cell” carcinoids. While the type A, mixed, and clear cell tumors were argentaffin positive and produced serotonin [characteristics of enterochromaffin (EC)-cell WDNETs], the tubular carcinoids reacted with antibodies to glucagon. The latter tumor type may be related to rare appendiceal WDNETs composed of L cells (enteroglucagon cells), which express glucagon-like peptides and peptide YY; these minute, indolent tumors are distinctly trabecular (type B).12 Small, incidentally discovered rectal WDNETs are also characteristically composed of L cells and demonstrate a type B growth pattern.13 Type C histology is typical of somatostatin-producing D-cell tumors of the duodenum.14 Garbrecht et al15 found that, among 82 non-multiple endocrine neoplasia type 1 (MEN1)-related duodenal NENs, 26% predominantly or exclusively expressed somatostatin; among these, 60% demonstrated at least a component of type C growth. Also of note, 71% of these D-cell tumors occurred in the ampulla and 58% contained psammoma bodies. A type C component was noted in only 22% of pancreatic D-cell tumors, which were much less common than in the duodenum (4%; 21/541). Outside of the jejunoileum (type A) and rectum (type B), WDNETs are often mixed.9,16
FREQUENCY, BIOLOGY, AND IMAGING OF WELL-DIFFERENTIATED NEUROENDOCRINE TUMORS
Population-based Data Regarding Relative Frequency and Biology
In 2008, Yao et al17 published an analysis of the epidemiology of WDNETs based on 35,825 tumors reported to the Surveillance, Epidemiology, and End Results (SEER) Program from 1973 to 2004. This study expanded on previous reports of US population–based cohorts published in 1975 and 2003.18,19 Data from this study regarding relative tumor frequency, proportion presenting with distant metastasis, and median overall survival—segregated by anatomic site—are summarized in Table 1. The 5 most frequent primary sites included lung (27%), rectum (15%), jejunoileum (13%), pancreas (6.4%), and stomach (6%), accounting for two thirds of all tumors. Among these, pancreatic tumors most often presented with distant metastasis (64%) and were associated with the shortest median survival (42 mo). By comparison, jejunoileal tumors were metastatic half as often (30%) and associated with twice as long overall survival (88 mo). Lung tumors were metastatic at a rate of 28% but were associated with long survival (193 mo), only exceeded by rectal (240 mo) and appendiceal (median survival not reached) tumors. A significant weakness of the SEER data set is that it relies on the reporting of tumors to the registry, with the authors acknowledging that “many small, benign-appearing tumors likely are excluded.” This may explain why appendiceal tumors, for example, perceived as relatively common among WDNETs, represented only 3%.
Frequency and Biology of Metastatic Tumors of Unknown Origin
Data from recent studies reporting the proportion of metastatic tumors of unknown origin among all WDNETs are summarized in Table 2. The frequency has ranged from 9% (population-based cohort)23 to 19% (single-center, referral-based cohort).20 The median of these studies is 11%. In the SEER series, the frequency of tumors of “other/unknown” origin was exceeded only by that of bronchopulmonary and rectal primaries.17
Notable among these studies is one from Kirshbom et al20 that reported on the clinical and biochemical characteristics of 143 metastatic WDNETs of unknown primary (among >750 total WDNETs) seen by J.M. Feldman at Duke University Medical Center/Durham Veterans Affairs Hospital over a nearly 30-year period. Patients with unknown primary tumors had high levels of urine 5-hydroxyindoleacetic acid (main serotonin metabolite) and urine, platelet, and serum serotonin levels on par with those seen in patients with metastatic midgut WDNETs (and greater than those seen in patients with locoregional midgut tumors and much greater than patients with foregut or hindgut tumors). The survival curves for unknown primary and metastatic midgut tumors were also quite similar, with 10-year survivals of 22% and 28%, respectively, and convergence of survival around 15 to 20 years. These findings led the authors to suggest that most WDNETs of unknown origin may represent metastases from midgut primaries.
A retrospective analysis of the University of California, San Francisco (UCSF) experience with liver metastases from WDNETs lends additional support to Kirshbom and colleagues’ hypothesis.25 Wang and colleagues identified 123 patients diagnosed between 1993 and 2008, in whom hepatic metastasis was the initial disease presentation in 64% (n=79). Among 71 patients presenting with metastatic disease, adequate medical records, and in whom a workup was undertaken, the primary tumor was successfully localized [making use of various combinations of computed tomography (CT), somatostatin receptor imaging (SRI), positron emission tomography (PET), magnetic resonance imaging, and upper and lower endoscopy] in 56 (79%): pancreas 27, small intestine 17, colon 7, lung 2, stomach 1, kidney 1, presacral 1. The remaining 15 patients underwent surgical exploration to detect an occult primary, which successfully identified 12 ileal and 1 jejunal tumor (13/15; 87%).
Imaging of Well-Differentiated Neuroendocrine Tumors
In addition to conventional imaging modalities, WDNETs can be visualized using nuclear medicine techniques that take advantage of distinctive biochemical properties of this class of tumors, including increased glucose metabolism, uptake of biogenic amines, and frequent expression of somatostatin receptors (SSTRs). The first of these is the basis of PET imaging with the radiotracer 18F-fluoro-2-deoxy-d-glucose, which is more pertinent to the highly metabolically active PDNECs. The uptake of radioiodine-labeled metaiodobenzylguanidine (MIBG) by the majority of WDNETs is the basis of the MIBG scan, as well as MIBG-radionuclide therapy. Although this modality has been overtaken by SRI in NENs, MIBG-radionuclide therapy remains a mainstay of the treatment of advanced pheochromocytoma/paraganglioma.26
Somatostatin is an inhibitory peptide hormone produced in regions of the brain and by D cells in the stomach, intestine, and pancreas. In the gastrointestinal (GI) tract, somatostatin suppresses the release of several hormones including gastrin, cholecystokinin, vasoactive intestinal peptide, insulin, and glucagon. It mediates its effects through SSTRs, which exist as 6 subtypes (SSTR1, 2A, 2B, 3 to 5). Frequent SSTR expression by NENs is the basis of the OctreoScan, in which the somatostatin analogue octreotide is coupled to 111In through the chelator diethylene triamine pentaacetic acid. Of the SSTR subtypes, a positive scan has been shown to most closely relate to SSTR2A expression,27,28 which can be detected immunohistochemically in 60% to 90% of NENs (see below).29–34 Newer radiotracers couple octreotide or octreotate to 68Ga via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) (eg, DOTATOC, DOTANOC, DOTATATE). These are used with PET and hybrid PET/CT imaging (as opposed to planar imaging or single photon–emission CT with the 111In compound) and hold promise for superior spatial resolution, decreased patient exposure to radiation, and shorter scanning times.35,36
A common response to the suggestion that pathologists should participate in assigning the site of origin of a metastatic WDNET of unknown primary is that we should “let radiology do it.” In the UCSF experience discussed above, although CT successfully detected pancreatic WDNETs (pancNETs) in every case, the sensitivities of CT and SRI (presumably OctreoScan) for detecting tubal gut primaries were only 34.6% and 26.2%, respectively.25 Savelli et al37 found that among 36 patients presenting with a metastatic NET of unknown primary (among 428 GEP NETs; 8.4%), OctreoScan successfully localized the primary in 39% (9 midgut, 3 pancreas, 1 cecum, 1 colon). More recently, Prasad et al35 reported that DOTANOC PET/CT identified a primary site in 59% of 59 patients (14 jejunoileum, 16 pancreas, 2 colorectum, 2 lung, 1 paraganglioma). Thus, traditional SRI may be useful in assigning the primary site in less than half of cases. Newer PET tracers appear more sensitive, but they are not widely available. An additional consideration is cost. OctreoScan is available at most US hospitals with a Nuclear Medicine section, is charged at about $8000, and demonstrates a sensitivity of 20 mm (the mean size of the occult small intestinal primaries detected by surgical exploration in Wang and colleagues’ series was 13.8 mm). In contrast, DOTA scans, with a sensitivity of 4 mm, are, at present, available at only 4 US centers, through the Food and Drug Administration’s Investigational New Drug program (T.M. O’Dorisio, written personal communication). Diagnostic IHC is nearly universally available and is much less expensive than either of these imaging techniques (or a high-resolution CT for that matter); the Medicare Fee Schedule for Current Procedural Terminology code 88342 is around $100.38
THERAPEUTIC SIGNIFICANCE OF DETERMINING THE SITE OF ORIGIN IN WELL-DIFFERENTIATED NEUROENDOCRINE TUMORS
Key therapeutic determinants in WDNETs include tumor site and size, anatomic extent of disease (local, regional, metastatic), presence of a functional syndrome, and, in metastatic disease not amenable to surgery with curative intent, site of origin of the primary tumor. Therapeutic options include endoscopic removal (for select gastroduodenal and rectal tumors), surgery, ablative/locoregional techniques (for liver metastases), antisecretory treatment (for functioning tumors), and antiproliferative treatment. In patients with locoregional disease, the tumor is generally excised. Metastatic disease to the liver, depending on the pattern and extent of spread, is also potentially resectable.39–42 Adjuvant therapy following an R0 or R1 resection is not standard of care.43 Patients with diffuse hepatic involvement or extrahepatic disease are candidates for antiproliferative therapy. Even patients with advanced metastatic disease may have their primary tumor resected; this is particularly the case for jejunoileal tumors, which have a tendency to cause obstruction, bleeding, and ischemia (due to mesenteric vascular elastosis).
Antiproliferative treatment options are very different in metastatic midgut and pancNETs (Table 3). Classes of agents include those also used in antisecretory therapy, systemic chemotherapeutics, small molecular inhibitors, and peptide receptor–targeted radiotherapeutics. The somatostatin analogue octreotide is the mainstay of antisecretory therapy in functioning tumors (including patients with carcinoid syndrome); interferon-α is a second-line agent due to its unfavorable side-effect profile. These agents also have weak antiproliferative activity and are utilized in advanced metastatic disease of midgut origin.44,45 Metastatic pancNETs often respond to streptozocin-based systemic chemotherapy.46 Alternatively, temozolomide, another alkylating agent, may be used.47 Intriguingly, in 1 retrospective study, immunohistochemically detectable deficiency of the DNA-repair enzyme O(6)-methylguanine DNA methyltransferase, observed in half of pancreatic and in no jejunoileal tumors, predicted treatment response.48 The response rate of midgut tumors to both of these agents has been shown to be poor.48,49
There is surging clinical interest in directed biological therapy. Small-molecule inhibitors have been increasingly evaluated in clinical trials. The mTOR inhibitor everolimus was recently shown in a phase 3 randomized control trial to prolong progression-free survival in patients with advanced pancNETs by 6.4 months (vs. best supportive care).50 A similar trial in advanced midgut tumors fell just short of reaching statistical significance, but given the paucity of therapeutic alternatives, everolimus may be considered.51 A phase 3 trial of the multiple receptor tyrosine kinase inhibitor sunitinib (inhibits platelet-derived growth factor receptors, vascular endothelial growth factor receptors, and KIT, among others) in advanced pancNETs was similarly positive,52 while efficacy was not demonstrated in midgut tumors.53 Finally, although peptide receptor–targeted therapy (eg, 90Y-DOTATOC) holds promise in both advanced midgut and pancreatic tumors, it is not widely available in the United States.54,55
THERAPEUTIC SIGNIFICANCE OF DETERMINING THE SITE OF ORIGIN IN POORLY DIFFERENTIATED NEUROENDOCRINE CARCINOMAS
In the skin, the critical distinction is between MCC and metastatic visceral PDNEC. Clinically localized MCC is treated with excision and sentinel lymph node biopsy.56 A positive sentinel lymph node biopsy prompts completion lymphadenectomy and/or radiation therapy. The primary tumor bed may also be treated with radiation therapy, especially in patients considered at high risk for local recurrence (eg, positive margins, large tumor size, lymphovascular space invasion). At other potentially metastatic sites (eg, liver), determination of the site of origin of a PDNEC is mainly of academic interest, as all metastatic tumors, regardless of origin, are treated with platinum-based “SCLC” chemotherapy (ie, cisplatin or carboplatin and etoposide).57–59
KEY MARKERS IN DETERMINING THE SITE OF ORIGIN OF A WELL-DIFFERENTIATED NEUROENDOCRINE TUMOR
Caudal type Homeobox 2 (CDX2)
CDX2, one of 3 human homologues of the Drosophila gene Caudal, is a homeodomain-containing transcription factor critical in gut development and patterning and in the maintenance of its phenotype.60,61 It is normally expressed by epithelial cells of the tubal gut distal to the stomach.62 CDX2 expression can also be found in pancreatic centroacinar and intercalated and intralobular duct cells and in scattered cells in the larger ducts.63 Downstream targets include MUC2, sucrose-isomaltase, and lactase. Induction of CDX2 expression is characteristic of gastric intestinal metaplasia64 and Barrett's esophagus.65Cdx2 homozygous–null mouse embryos fail to implant, as Cdx2 expression is required for the development of trophoectoderm. In a conditional knockout (that overcomes the implantation block) the tubal gut, which is lined by squamous instead of columnar epithelium, ends in a blind pouch at the cecum.66
Diagnostic pathologists routinely use CDX2 as a marker of intestinal-type adenocarcinomas. Aside from colorectal and appendiceal tumors, in which expression is detected in most cases (>90%), it is often, although variably, reported in tumors arising elsewhere in the gut and in other tumors with intestinal-type differentiation (eg, mucinous ovarian tumors).63,67–69 Two early IHC surveys of CDX2 expression reported positivity in a significant fraction of intestinal WDNETs.63,68 Subsequently, CDX2 expression has been shown to be highly sensitive and fairly specific for a midgut origin (ie, jejunoileum or appendix). Nearly all of the data regarding CDX2 expression in WDNETs are based on staining with the mouse monoclonal antibody (MoAb) CDX2-88. A recent study demonstrated high sensitivity and superior specificity for a jejunoileal origin (appendiceal tumors were not studied) with the rabbit MoAb EPR2764Y.70
Data from 14 studies assessing CDX2 expression in primary and metastatic WDNETs are summarized in Table 4. Expression has been detected in 90% (177/197) of primary and 91% of metastatic (129/142) jejunoileal tumors (Figs. 2A, B). Similarly, CDX2 positivity has been noted in 93% (67/72) of appendiceal primaries; only a handful of metastases have been tested (83%; 5/6). CDX2 expression has been detected in 31% (14/45) of duodenal, 14% (10/74) of gastric, and 29% (32/110) of rectal primaries. A large number of pancreatic tumors have been assessed, with CDX2 expression found in 16% (66/415) of primary and 18% (15/84) of metastatic tumors. Only 3% (8/233) of pulmonary carcinoids have been positive. The specificity of CDX2 in this diagnostic context is probably increased by taking quantity and intensity of staining into account. Whereas midgut tumors generally demonstrate diffuse, strong staining, expression in the foregut and hindgut GEP tumors is often weak and patchy.
One additional anatomic site deserves special mention here. WDNETs of the ovary often arise in the background of a teratoma and are divided into insular, trabecular, strumal, and mucinous types. Rabban et al83 found that 5 of 7 (71%) ovarian WDNETs with an insular growth pattern expressed CDX2, while none of 8 (0%) tumors with a trabecular growth pattern did. Thus, in the ovary, CDX2 is not helpful in distinguishing a primary insular WDNET from a midgut metastasis; interestingly, these 2 tumor types are essentially histologically identical. Also of note, tumors in this study did not express TTF-1.
Thyroid Transcription Factor-1
Initially identified based on its ability to bind to the thyroglobulin (TG) gene promoter,84 TTF-1 (also known as NK2 homeobox 1) is the best known of several human NKX homeodomain–containing transcription factors.85,86 It is critical to lung, thyroid, and brain (diencephalon) development, and it is expressed by type II pneumocytes, a subpopulation of nonciliated bronchiolar epithelial cells, follicular and parafollicular (C) cells, and neuroblasts in areas of the developing diencephalon including the hypothalamus and neurohypophysis.87–89 In addition to TG, TTF-1 promotes the expression of thyroperoxidase, thyrotropin receptor, surfactant proteins, and Clara cell secretory protein. A mouse knockout is athyroid, lung development distal to the lobar bronchi fails to take place, the ventral forebrain is abnormally formed, and the pituitary is missing.90
TTF-1 is most commonly used in diagnostic pathology as a marker of lung adenocarcinoma and thyroid tumors.91–95 In the setting of a WDNET, TTF-1 has been found to be a variably sensitive, although incredibly specific, marker of lung origin. Although unlikely to present as a metastasis from an occult primary, expression is also detected in nearly all medullary thyroid carcinomas, which may additionally mark for calcitonin and carcinoembryonic antigen.89,96–99 Nearly all studies of TTF-1 immunoexpression in NENs have used the same mouse MoAb 8G7G3/1 initially described by Holzinger et al100 in 1996, which was produced against full-length recombinant rat protein. A few more recent studies have also used the mouse MoAb SPT24, raised against a 123 amino acid sequence from the N-terminal region of human TTF-1. The distinction is not inconsequential, with SPT24 generally demonstrating increased sensitivity in tumor types in which TTF-1 expression is typical, while some studies have also demonstrated SPT24 positivity in occasional tumors in which TTF-1 expression is considered unusual.101,102 For example, Compérat et al101 found SPT24 and 8G7G3/1 positivity in 84% versus 65% of 86 lung adenocarcinomas and 10% versus 0% of 41 lung metastases of colorectal origin, respectively.
In 21 studies using the 8G7G3/1 clone, TTF-1 expression has been detected in 32% of 557 lung carcinoid tumors (Table 5 and Figs. 3A–C). Including 1 additional study in which a small number of TCs were examined with a rabbit polyclonal antibody, rates of positivity in individual studies have ranged from 0% to 95%, with a median of 32%. ACs (mean 32%; median 40%) are possibly somewhat more likely to be positive than TCs (mean 28%; median 21%). Spindle cell carcinoids frequently express TTF-1 (mean and median 69%). Thirteen of 23 (57%) metastases have been positive. In contrast, TTF-1 expression by a GEP WDNET is exceptional, reported to date in 0.6% of 708 primary tumors and 0% of 220 metastases. In 3 studies comparing SPT24 with 8G7G3/1, SPT24 was more frequently expressed in lung carcinoids than was 8G7G3/1. For example, Matoso et al102 found 61% (SPT24) versus 17% (8G7G3/1) of 23 TCs to be positive. In 1 study also evaluating GEP tumors, this increased sensitivity was not at the expense of specificity, with La Rosa et al112 not detecting SPT24 positivity in any (0%) of 103 tumors. The huge range of positivity (0% to 95%) cannot be explained by differing antibody clones (it preceded the first reports of SPT24 expression in this class of tumors), use or nonuse of heat-induced epitope retrieval (use of which is described in most of these studies), or differing retrieval solutions (although a variety are described, citrate has been most frequently used and has been associated with both extremes of this range).
Paired Box Gene 8 (PAX8)
The transcription factor PAX8, 1 of 9 paired box genes, is essential for thyroid development,115,116 participates with PAX2 in kidney development,117,118 and was found, through microarray-based gene expression profiling, to be highly expressed in surface ovarian carcinomas.119 As such, detection of PAX8 expression has emerged as an important diagnostic tool to identify carcinomas of the thyroid, Müllerian system, and kidney.120–122 As application of PAX8 IHC became more prevalent, a few groups recognized strong expression in islets of Langerhans and pancNETs and reported on the usefulness of PAX8 expression to suggest the pancreatic origin of a WDNET (Table 6 and Figs. 4A, B).
In 7 studies using a commercially available polyclonal PAX8 antibody, expression has been detected in 145 of 209 (69%) primary and 46 of 84 (55%) metastatic pancNETs. Although only a few duodenal (14 primaries) and relatively few rectal (48 primaries) tumors have been studied, PAX8 expression (detected by the same antibody) appears typical of these sites as well (79% and 58%, respectively). PAX8 positivity has not been noted in 85 primary and 67 metastatic ileal tumors, and it has ranged from 8% to 17% in tumors from the lung, stomach, and appendix. Also of note, neither PAX8, PAX2, CDX2, nor TTF-1 were found to be expressed in a series of 9 renal WDNETs.135
Interestingly, up to 2010, the pancreatic developmental biology literature was quiet on PAX8. Instead, PAX4 and PAX6 had been shown critical to islet development, with the PAX4 knockout mouse lacking mature β and δ cells,136 the PAX6 knockout lacking α-cells,137,138 and the combined PAX4/PAX6 knockout lacking any pancreatic neuroendocrine cells.137 The most frequently used PAX8 antibody is polyclonal, raised against an N-terminal peptide that is highly conserved across the PAXs. It has been demonstrated that PAX8 mRNA is expressed at very low levels in human islets (relative to PAX6 and PAX4), that a MoAb directed against the divergent C-terminal region does not react with non-neoplastic islets or pancNETs, and that the polyclonal PAX8 antibody cross-reacts with PAX6.129
The authors of the above-referenced study suggest that their results “cast doubts on the value of Pax8 as a pancreatic neuroendocrine tumor marker.”129 In a letter published in the American Journal of Surgical Pathology, the same group warns of the potential for “flawed diagnostics.”139 Another group reports on the development of a PAX8 MoAb, which they suggest “offers a significant advantage by simplifying interpretation and enabling a more confident and accurate diagnosis.”130 In my opinion, abandoning the polyclonal PAX8 antibody in this context is akin to throwing out the proverbial baby with the bathwater. As a GI pathologist, I embrace polyclonal carcinoembryonic antigen IHC’s cross-reactivity with biliary glycoprotein to highlight the bile canaliculi diagnostic of hepatocellular differentiation. Am I to divert my eyes from granular cytoplasmic staining for TTF-1, suggesting the possibility, again, of hepatocellular differentiation (another cross-reactivity, this time with carbamoyl phosphate synthetase I)? Even if polyclonal PAX8 does cross-react with PAX6, the antibody remains an excellent marker of pancreatic (and probably duodenal and rectal) WDNETs, better, in fact, than PAX6 itself [in Lorenzo et al’s129 study, while polyclonal PAX8 highlighted 7 of 9 (78%) pancNETs, a PAX6 MoAb was positive in only 3 (33%)]. It can be argued that this cross-reactivity adds value above and beyond that seen with the recently developed PAX8 monoclonal.
Insulin Gene Enhancer Binding Protein Isl-1 (Islet 1)
The homeodomain-containing transcription factor Islet 1 was initially isolated based on its ability to bind the insulin gene enhancer.140 In the adult rat, Islet 1 expression was detected in α, β, and δ cells of the islets of Langerhans, cells in the anterior and intermediate lobes of the pituitary, parafollicular (C) cells, chromaffin cells and some neurons in the adrenal medulla, some sensory neurons in the dorsal root ganglia, and subsets of motor neurons and central nervous system nuclei involved in autonomic and endocrine control.141 In the developing embryo, Islet 1 is expressed in mesenchymal cells of the dorsal pancreatic bud, as well as islet cells. An Islet 1 knockout mouse is characterized by failed exocrine development from the dorsal but not the ventral pancreatic bud and a complete absence of differentiated islet cells.142 Given its importance in pancreatic, and in particular islet, development, Islet 1 expression has been explored as a marker of pancNETs. As with polyclonal PAX8, in addition to expression in this group of tumors, Islet 1 positivity is also typical of duodenal and rectal WDNETs (Table 6).
In a total of 5 studies investigating nearly 700 WDNETs, Islet 1 expression has been detected in 209 of 255 (82%) primary and 47 of 67 (70%) metastatic pancNETs. Similarly, 92% of 25 duodenal and 86% of 22 rectal primaries have been positive. Although only a handful of metastases from the duodenum and rectum have been analyzed, the results are similar. Islet 1 expression is uncommon to rare at other sites. Twelve percent (12%) of 94 pulmonary and 17% of 42 appendiceal primaries have been positive, whereas the few metastases studied have not. Islet 1 expression has not been described in a gastric tumor, and although 3% of 69 primary ileal tumors have been positive, all 68 metastases studied to date have been negative. A recent study has highlighted frequent expression in medullary thyroid carcinoma (9/9; 100%).131 In the same study, Islet 1 expression was also seen in most PDNECs including MCCs and SCLCs, as well as neuroblastomas and paragangliomas/pheochromocytomas. (Of note, this lack of “anatomic site specificity” for a transcription factor in PDNECs has a precedent; see directly below.) Compared with PAX8, Islet 1 boasts superior sensitivity for a pancreaticoduodenal or rectal primary. Its principal disadvantage is that it is a “unitasker” (ie, it has no other defined diagnostic application at this time).
KEY MARKERS IN DETERMINING THE SITE OF ORIGIN OF A POORLY DIFFERENTIATED NEUROENDOCRINE CARCINOMA
Thyroid Transcription Factor-1
Although most familiar as a marker of lung adenocarcinoma, the first reports of TTF-1 expression in human tumors actually found more frequent expression in pulmonary small cell carcinomas than in adenocarcinomas. In their initial description of the 8G7G3/1 MoAb, Holzinger et al100 found 5 of 6 (83%) SCLCs and 7 of 11 (64%) adenocarcinomas to be positive, whereas Fabbro et al,103 using a rabbit polyclonal antibody, reported TTF-1 expression in 10 of 10 (100%) SCLCs and only 3 of 11 (27%) adenocarcinomas. TTF-1 is also expressed, although much less frequently, by pulmonary LCNECs. Although the reported rates have been highly variable, TTF-1 is also expressed by at least a significant minority of extrapulmonary PDNECs. However, expression by a cutaneous PDNEC (ie, MCC) is exceptional. As such, TTF-1 IHC is an essential component of the small panel of stains useful in distinguishing visceral from cutaneous tumors, with expression effectively ruling out the diagnosis of MCC.
Data from 57 studies examining TTF-1 expression in PDNECs are summarized in Table 7. Expression has been detected in 698 of 846 (83%) SCLCs and 101 of 282 (36%) pulmonary LCNECs; the median reported rates for these 2 tumor types are 86% and 41%, respectively (Figs. 5A, B). Two recent studies have compared the performance of the 8G7G3/1 and SPT24 clones, and, as with lung carcinoids, positivity is more frequent with SPT24. For example, Masai et al114 detected TTF-1 expression in 86% versus 68% of SCLCs and 47% versus 23% of pulmonary LCNECs. TTF-1 positivity has been reported in 200 of 550 (36%) extrapulmonary PDNECs; rates in individual studies have ranged from 0% to 83%, with a median of 33%. TTF-1 expression has been reported in only 2 of 260 (0.8%) MCCs, each as a single occurrence in 2 of 15 studies.
Using a combination of 2-dimensional gel electrophoresis, immunoblotting, and tryptic peptide mapping, in 1985 Moll and Franke203 identified a distinct cytoskeletal protein of molecular weight 46,000 in 9 of 9 (100%) MCCs and in none of 8 (0%) SCLCs. The protein colocalized with one they had previously described in human intestinal cells, GI tract adenocarcinomas, and a human colon cancer cell line, which they had designated protein IT.204 Several years later, protein IT was proven to be a keratin and designated CK20.205 CK20 is normally expressed by gastric foveolar epithelium, intestinal epithelium, urothelial umbrella cells, and by Merkel cells in the skin. Diagnostically, it is generally used as a marker of intestinal adenocarcinomas, and it is expressed by a smaller number of gastroesophageal; pancreatobiliary; mucinous ovarian and lung; and urothelial carcinomas.186,194 Germane to this discussion, CK20 expression is seen in the vast majority of MCCs, while expression is unusual in SCLC. CK20 is similarly uncommon in extrapulmonary PDNECs, with the exception of those arising in the major salivary glands, which have been described as “Merkel cell–like.”
CK20 expression has been noted in 416 of 472 (88%) MCCs, with rates ranging from 67% to 100% in 24 studies (median 91%) (Table 7). Expression is typically perinuclear/dot-like but may also be diffuse (Fig. 6). CK20 positivity has been reported in 18 of 373 (5%) SCLCs and 1 of 10 (10%) pulmonary LCNECs. Eleven of these 18 were seen in 1 study (11/33; 33%).145 Excluding this outlier study, the rate is 2% (7/340); the median frequency in 19 studies is 0%. CK20 expression has been found in 19 of 30 (63%) major salivary gland tumors and 20 of 331 (6%) nonmajor salivary gland ones. In the largest series of major salivary gland small cell carcinomas (SCNECs), CK20 expression was noted in 11 of 15 (73%) tumors; TTF-1 expression was restricted to 3 of the 4 CK20-negative cases.157
OTHER MARKERS OF INTEREST IN WELL-DIFFERENTIATED NEUROENDOCRINE TUMORS
Cytokeratins 7 and 20
A good deal of effort has been put forth to define the expression of various keratin types in carcinomas from diverse anatomic sites, with the goal of applying these data to questions of tumor type (eg, adenocarcinoma vs. squamous cell carcinoma) and, especially, site of origin. I have seen pathologists attempt to extrapolate the findings from what principally has been the study of non-neuroendocrine carcinomas to WDNETs. In fact, there is actually a relative paucity of data on the expression of individual keratins in these tumors, with CK7 and CK20, given their wide clinical application, having received the most attention (there is a separate literature on the role of CK19 as a prognostic marker in pancNETs). Although only up to a few hundred tumors have been studied, the results have been fairly consistent, such that conclusions can be drawn (Table 8).
CK7 is frequently, although not invariably, expressed by carcinoid tumors of the lung. In 5 studies including a total of 87 tumors, frequency of expression in individual studies has ranged from 4% to 66% (median 30%); overall, 40% of pulmonary WDNETs have expressed CK7. Expression is less frequent in both tubal gut (11/131; 8%) and pancNETs (8/37; 22%), with the caveat that relatively few pancreatic tumors have been examined. Although CK7 expression is more common in lung tumors, given more frequent metastasis from ileal and pancreatic primaries, CK7 positivity at a metastatic site probably would be most often seen with spread from the pancreas. CK7 IHC is neither sensitive nor specific enough to be useful in this diagnostic application.
CK20 is expressed by around a quarter of tubal gut WDNETs, perhaps slightly less often in pancNETs, and almost never in carcinoid tumors of the lung. In 7 studies including a total of 114 tubal gut tumors, frequency of expression in individual studies has ranged from 15% to 100% (median 24%); overall, expression has been noted in 24%. Of note, the 2 earliest studies describing CK20 positivity in 100% and 86% of 2 and 7 tumors, respectively, reported expression in rare cells only.184,187 For pancNETs, frequency of expression in 4 studies has ranged from 12% to 33% (median 17%); in total, 9 of 49 (18%) tumors have been CK20 positive. Chan et al80 recently reported CK20 expression in 1 of 29 (3%) bronchopulmonary carcinoids; 56 tumors in 4 previous studies had all been negative. Although relatively insensitive as a marker of GEP WDNETs, CK20 expression argues against a pulmonary origin.
Neuroendocrine Secretory Protein 55
Neuroendocrine secretory protein 55 (NESP55) is 1 of 5 main transcripts of the GNAS locus. It is a member of the granin family (like the more familiar chromogranin) and is found in dense-core secretory granules in the adrenal medulla, pituitary, and several parts of the brain.209 A few studies have suggested that NESP55 expression in a WDNET is in keeping with a pancreatic origin. Jakobsen et al210 found NESP55 expression in 14 of 25 (56%) pancNETs and 0 of 15 (0%) ileal WDNETs. NESP55 expression was also detected in non-neoplastic islets, most frequently in β cells. Also of note, 19 of 19 (100%) pheochromocytomas were positive. Similarly, Srivastava et al211 described NESP55 expression in 14 of 19 (74%) pancNETs, 10 of 10 (100%) pheochromocytomas, and 0 of 11 (0%) ileal WDNETs. In addition, there was no staining in 4 gastric tumors and focal staining (<5% of cells) in 1 of 4 rectal (25%) and 1 of 15 (7%) lung tumors. Srivastava and Hornick subsequently extended this finding in a larger group of tumors, reporting expression in 16 of 39 (41%) pancNETs and no expression in gastric (0/5), duodenal (0/5), ileal (0/31), or appendiceal tumors (0/11). As in the prior study, expression was noted in a rare rectal (1 of 12; 8%) and lung (1 of 20; 5%) tumor.78 Recently, Denby et al81 reported NESP55 expression in 5 of 16 (31%) and 0 of 16 (0%) metastatic pancreatic and tubal gut WDNETs, respectively.
Of note, all 4 of these studies made use of a polyclonal rabbit antibody gifted by the same Austrian investigator. An internet search identified several commercial sources of anti-NESP55, including at least 1 claiming to have been tested in IHC of formalin-fixed, paraffin-embedded tissue. Although the specificity of these commercial antibodies for pancNETs would have to be verified, NESP55 represents an attractive option in this setting. A drawback of this marker is its relatively limited clinical utility beyond this specific application.
Pancreatic and Duodenal Homeobox 1
Pancreatic and duodenal homeobox 1 (PDX1) is another homeodomain-containing transcription factor, initially described based on its ability to bind to and transactivate the insulin and somatostatin gene promoters.212,213 In the developing embryo, PDX1 is expressed in the dorsal and ventral pancreatic buds and the intervening duodenum; in the pancreas, expression is gradually extinguished in acinar and ductal epithelium, such that, by around birth, PDX1 is relatively islet-restricted.214 PDX1 knockout mice demonstrate severe pancreatic hypoplasia, duodenal malformation, absence of Brunner glands, and decreased numbers of duodenal enteroendocrine cells.214,215 In the adult, PDX1 expression has been noted in islets of Langerhans, centroacinar cells, rare intralobular and interlobular ductal cells, and duodenal epithelium, and not in pancreatic acinar parenchyma.134 Given its importance in pancreaticoduodenal development and its continued expression in islets and the duodenum, PDX1 has been explored as a marker of WDNETs of these organs (Table 6).
There are very little data regarding PDX1 expression outside of the pancreas—where it has been demonstrated in 114 of 220 (54%) primary tumors and 10 of 10 (100%) metastases (data from 1 study are excluded from these totals because primaries and metastases were not reported separately). Although in 3 studies PDX1 was expressed by most duodenal tumors [16/19 (84%) primaries and 2/2 (100%) metastasis],78–80 in a single study that only looked at functioning gastrinomas, PDX1 was not expressed at all (15 primaries and 3 metastases).132 Hermann et al79 achieved a very different result, with PDX1 expression noted in 10 of 10 (100%) gastrin-expressing duodenal tumors [5 of these patients had Zollinger-Ellison syndrome (ZES)]. PDX1 expression appears to be relatively uncommon in lung primaries (3/49; 6%) and, importantly, has not been observed in 37 jejunoileal primaries and 12 metastases (0%). There are almost no data from the stomach (3/5 primaries; 60%) and little data from the appendix (7/17 primaries; 41%) or rectum (2/14 primaries; 14%). PDX1 may have some role as a marker of pancreaticoduodenal WDNETs. It may be less frequently positive in rectal tumors than the other similar markers PAX8 and Islet 1, although additional data would be welcomed.
Commercial antibodies to a wide variety of peptide hormones are available. A partial list of those normally expressed in the GEP system is provided in Table 9. Aside from gastrin, which is often used in gastric mucosal biopsies as part of the workup of a suspected case of autoimmune atrophic gastritis, these are generally considered of limited clinical utility. As such, most laboratories will only carry a few of these. Of note, functional syndromes are defined based on the presence of characteristic symptoms, rather than predominant or even exclusive immunohistochemical expression of a peptide hormone. Numerous studies have demonstrated that WDNETs, regardless of site of origin, tend to express >1 hormone.14,216–222 Furthermore, metastases may produce fewer or even additional peptides than matched primary tumors.219 I have used peptide hormone IHC in the workup of a WDNET in 3 contexts: (1) to distinguish pancreatic neuroendocrine (pseudo)hyperplasia, which should express some combination of insulin, glucagon, somatostatin, and pancreatic polypeptide, from a microadenoma (defined as a nonfunctioning pancNET <0.5 cm), which tends to express a single hormone; (2) in operated patients with multiple simultaneous tumors and a functional syndrome (MEN1-ZES), to help determine, for example, which of the resected tumors (if any) is the source of the syndrome; (3) in patients with metachronous tumors, in which the first was known to express a dominant hormone (Figs. 7A–D).
Progesterone receptor IHC is a standard-of-care predictive marker in breast cancer and is widely used as part of a panel to help determine the site of origin of a carcinoma of unknown primary. In the setting of a WDNET, PR expression has been proposed as indicative of pancreatic origin. Doglioni et al223 reported PR expression in 40% to 75% of non-neoplastic islet cells, especially prevalent in α cells, and 7 of 18 (39%) pancNETs. Estrogen receptor (ER) expression was not detected. In a follow-up study from the same group, PR expression was detected in 56 of 96 (58%) pancNETs, 0 of 29 (0%) tubal gut WDNETs, and 1 of 15 (7%) lung TCs.224 Tempering enthusiasm for the marker, expression was much more frequent in benign tumors (72%) than in malignant ones (31%; defined in this study by the presence of local invasion or metastasis), and expression was detected in only 1 of 7 (14%) liver metastases.
This finding has apparently received relatively little attention, as only 2 other relevant published studies were identified. Zhao et al225 found (focal) PR and ER expression in 1 of 42 (2%) ovarian carcinoid tumors. Sica et al226 studied PR and ER expression in pulmonary NENs and found more frequent PR expression than did the Milanese group: 11 of 42 (26%) TCs and 2 of 7 (29%) ACs. In addition, ER expression was seen in over half the carcinoids. Given the wide availability of PR IHC and its potential application as a secondary marker in assigning the site of origin in a WDNET, PR expression in these tumors warrants additional investigation.
Prostatic Acid Phosphatase
Prostatic acid phosphatase (PrAP) is often used along with prostate-specific antigen to support a diagnosis of prostate cancer, either in the workup of a carcinoma of unknown primary or in the setting of a high-grade tumor in the prostate or bladder for which urothelial carcinoma also enters the differential diagnosis. The frequent expression of PrAP by rectal WDNETs (leading to an incorrect diagnosis of prostate cancer in a small, crushed rectal biopsy) represents a “classic pitfall” in diagnostic IHC. In the AFIP series of 81 rectal (and 3 sigmoid) WDNETs, PrAP expression was detected in 82%.222
PrAP expression is not restricted to rectal tumors, although at most other sites it appears more infrequent. In the AFIP series of 51 jejunoileal WDNETs, PrAP was expressed in 10 (20%).10 In an earlier study, the same group reported expression in 15% of nonrectal WDNETs (including gastric, small intestinal, and appendiceal tumors).227 Azumi et al228 found even less frequent expression in 28 foregut-derived and midgut-derived tumors (focal, weak expression in 1 lung tumor). In contrast, frequent expression has been reported in insulinomas229,230 and strumal carcinoids,231–233 although this finding has not been well vetted in these tumor types. Again, given wide availability and apparent differential expression across sites of origin, PrAP may have some use as a secondary marker in this context.
The detection of S-100 protein expression, including as a marker of melanocytic and Schwannian differentiation, is a pillar of diagnostic IHC. S-100 is also expressed by sustentacular cells (eg, in pheochromocytoma/paraganglioma), relevant to this discussion. The presence of S-100-positive sustentacular cells has been touted as characteristic of an appendiceal WDNET, especially in the differential diagnosis of an ileal tumor, which otherwise would stain similarly for the markers already discussed. For example, Lundqvist and Wilander234 described this pattern of S-100 expression of 11 of 12 (92%) appendiceal tumors and not in small intestinal (0/11) or cecal (0/10) tumors. Similarly, Moyana and Satkunam235 found sustentacular cells in 8 of 8 (100%) appendiceal WDNETs and 0 of 8 (0%) jejunoileal ones. Sustentacular cells are also characteristic, although less frequently seen, in bronchopulmonary carcinoids. Barbareschi et al236 found them in 18 of 46 (39%) tumors; recently, using the more specific marker SOX10, Tsuta et al237 detected sustentacular cells in 51 of 78 (65%) pulmonary carcinoids (more frequently in spindle cell/peripheral tumors) and 0 of 35 (0%) pulmonary PDNECs. One important study casts doubt on the appendiceal specificity of sustentacular cells in midgut WDNETs, with Al-Khafaji et al238 finding S-100-positive sustentacular cells in 3 of the 11 (27%) jejunoileal WDNETs they studied.
I recently encountered an appendiceal WDNET metastatic to the uterus, in which sustentacular cells were demonstrable in both the primary and metastatic tumor (Figs. 8A, B). That sustentacular cells were present in the metastasis is intriguing, and the possibility that S-100 IHC might have some usefulness in assigning the site of origin of a WDNET is considered, although, obviously an appendiceal tumor presenting as a metastatic WDNET of unknown primary would be very unusual. S-100 could also be potentially useful in distinguishing a tumor arising at the base of the appendix from an ileocecal one.
Somatostatin Receptor 2A
As discussed above, most NENs, even poorly differentiated examples, express SSTRs. Of these, the 2A subtype is the most relevant, as the somatostatin analogues used clinically (SRI, octreotide/lanreotide therapy, and peptide receptor radionuclide therapy) have the greatest affinity for this receptor. SSTR2A IHC may have a role in selecting patients for somatostatin analogue therapy in instances in which SRI is unavailable or is negative (particularly in small tumors).45 Until recently, application was limited by the fact that the best characterized antibody, which is polyclonal, was not commercially available. A recently developed, commercially available MoAb (clone UMB-1) compares favorably to this antibody and gold standard receptor autoradiography. Körner et al239 recently reported optimization of a staining protocol with this antibody. Heat-induced epitope retrieval in a pressure cooker with citrate buffer was shown to perform best. Quality and intensity of staining are evaluated, with only membranous staining considered truly positive. Staining is either faint (1+), strong but with incomplete membrane staining (2+), or strong with complete membrane staining (3+). The group recommended that staining of >10% of tumor cells (of any intensity) be considered positive; staining of 1% to 10% of tumors cells be considered inconclusive, with the caveat that 1+ staining in this setting is likely negative; and that no tumor staining be considered negative. The >10% cutoff was 86% sensitive, 95% specific, and had a 95% positive predictive value for high receptor levels by autoradiography. Of 27 tumors with no staining, 96% had no or low receptor levels by autoradiography. In the future, alternative somatostatin analogue–based therapies may be developed that preferentially target other or a wider range of SSTRs.
Vesicular Monoamine Transporter 2
Vesicular monoamine transporters (VMATs) are integral membrane proteins involved in loading biogenic amines into synaptic vesicles. Two human isoforms exist, with VMAT2 expression in the tubal gut relatively restricted to the histamine-producing, EC-like (ECL) cells of gastric corpus mucosa.240 In contrast, only rare VMAT1-expressing neuroendocrine cells are found in the stomach, while the majority of neuroendocrine cells in the small and large intestine are VMAT1 positive. As such, VMAT2 has been advocated as a marker of ECL-cell gastric WDNETs. VMAT2 IHC is considered a considerable improvement over histamine IHC, which is practically limited by the low histamine content of ECL cells and its rapid dissolution in liquid fixatives.
Rindi et al241 reported diffuse, strong VMAT2 expression in 16 of 16 (100%) ECL-cell tumors, the presence of only rare VMAT2-positive cells in 12 of 21 (57%) EC-cell (ie, serotonergic) tumors and 9 of 11 (82%) pancNETs, and absent expression (0%) in 5 gastrin, 4 somatostatin, and 3 enteroglucagon-expressing tumors. Like NESP55, VMAT2 expression is also characteristic of pheochromocytoma [6 of 6 (100%) in this series; of note, both VMAT 1 and 2 are highly expressed in the adrenal medulla]. Similarly, Jakobsen and colleagues found VMAT2 expression in 11 of 12 (92%) gastric WDNETs and less frequent and generally focal expression in appendiceal (4/21; 19%), rectal (2/22; 9%), and pancreatic (8/24; 33%) tumors. VMAT2 expression was also detected in 34 of 52 (65%) ileal tumors, and in 7 (13%) it was diffuse. Anlauf and colleagues performed a focused investigation of non-neoplastic pancreas and pancNETs. In the non-neoplastic pancreas, VMAT2 expression was restricted to β cells. It was also seen in 15 of 44 (34%) insulinomas and 12 of 48 (25%) other functioning and nonfunctioning tumors.242 These studies serve to highlight high sensitivity but lack of specificity for an ECL-cell primary (although diffuse, strong expression may be moderately specific). In fact, a recent study has suggested VMAT2 be regarded as a “general neuroendocrine marker.”243 And unfortunately, although high-level expression is typical of the ECL-cell tumors driven by hypergastrinemic states (ie, those arising in autoimmune atrophic gastritis and combined MEN1-ZES), it is also usually encountered in sporadic gastric WDNETs, as well.241,244,245 Given the evidence, VMAT2 expression appears to be more scientifically interesting than diagnostically useful.
OTHER MARKERS OF INTEREST IN POORLY DIFFERENTIATED NEUROENDOCRINE CARCINOMAS
Merkel Cell Polyomavirus Large T Antigen
Drs Yuan Chang and Patrick Moore, having previously discovered the human herpesvirus that causes Kaposi sarcoma, hypothesized that, given its epidemiology (ie, associations with older age and immune suppression), MCC might also be attributable to an infectious agent. Utilizing a technique they dubbed “digital transcriptome subtraction,” which entailed (1) sequencing tens of millions of bases from cDNA libraries of 4 MCCs, (2) aligning those sequences with National Center for Biotechnology Information reference sequences, and (3) looking for homology with infectious agents in the rare nonaligned sequences, they discovered a single sequence with high homology to known polyomaviruses.246 They subsequently identified the virus, which they termed Merkel cell polyomavirus (MCPyV) in 8 of 10 (80%) tumor samples. MCPyV is clonally integrated into tumor DNA and is associated with characteristic mutations in the large T antigen (LT Ag), which abolish the virus’s ability to effectively replicate while maintaining LT Ag’s oncogenic properties.247 To date, MCPyV has been detected by polymerase chain reaction by ∼20 groups analyzing ∼600 MCCs, generally in the 80% range initially reported by Chang and Moore. A notable outlier was a cohort of Australian tumors, in which virus was detected in only 5 of 21 (24%).248
Chang and Moore’s group developed a MoAb to residues 116 to 129 of the LT Ag (clone CM2B4), which has emerged as a fairly sensitive and highly specific marker of MCC. In 8 studies including 313 pure MCCs, LT Ag expression has been detected in 60% (Table 10). As with the polymerase chain reaction data, there is a single outlier, again an Australian study in which only 19 of 89 (21%) tumors were positive.253 Excluding this study, 168 of 224 (75%) tumors have been CM2B4 positive. About 5% of MCCs are associated with actinic keratosis, squamous cell carcinoma in situ, a component of squamous cell carcinoma or basal cell carcinoma, or have foci of intratumoral squamous differentiation (so-called “combined MCC”). Interestingly, these tumors have been uniformly negative for LT Ag (0/43; 0%). These findings suggest that, although most MCCs are MCPyV related, a subset including the combined tumors and some pure tumors are not, and are instead more likely ultraviolet radiation–associated (explaining enrichment in the Australian cohorts). Nearly 400 non-MCCs, including a large number of PDNECs (predominantly pulmonary but also CK20-positive tumors from the salivary gland and uterine cervix) and hematolymphoid tumors have been studied. Overall, 7 of 387 tumors (2%) have been positive, including 6 of 23 (26%) SCLCs from 1 study.254 Outside of this 1 study, CM2B4 IHC has been extremely specific, with only 1 other group reporting a single positive case (of 74 SCNECs; 1.3%).253
The CM2B4 clone is now commercially available from Santa Cruz Biotechnology (Santa Cruz, CA). It would appear useful to support a diagnosis of MCC in the up to 10% of CK20-negative tumors and to argue against a diagnosis of MCC in other CK20-positive SCNECs (especially primary parotid tumors). CM2B4 negativity appears too frequent for it to exclude a diagnosis of MCC (especially in heavily sun-exposed populations). Rodig et al256 recently described a new MoAb (Ab3) raised against the N-terminal 260 residues of the LT Ag with improved sensitivity. Ab3 IHC was positive in 56 of 58 (97%) MCCs and 0 of 18 (0%) GI and lung PDNECs, while CM2B4 positivity was noted in only 81% of the same MCCs. Ab3’s performance in an Australian group of tumors would be of interest. The antibody is not yet commercially available.
Neurofilaments (NFs) are 1 of 6 types of intermediate filaments. NF IHC has applications in neuropathology and soft tissue pathology, in which it is used to highlight axons. NF IHC has been suggested as a marker of MCC, especially in its differential diagnosis with SCLC. In the 5 largest series directly comparing NF expression in MCC and SCLC, expression has been noted in 67 of 93 (72%) and 0 of 143 (0%) tumors, respectively.166,189,190,257,258 Expression is typically perinuclear/dot-like.
As a note of caution, NF expression has been detected by some investigators in SCLC, and it can be seen in a subset of extrapulmonary SCNECs. In 1983, Lehto et al259 first described detection of NF expression in SCLCs (6/6, 100%; using a rabbit polyclonal antibody) and actually suggested its utility in distinguishing this tumor type from other lung tumors (all 22 of which were negative). In a follow-up study, van Muijen et al260 failed to detect NF expression with 2 different MoAbs in a series of 9 SCLCs. The largest study to detect NF expression in SCLC was reported by Shy et al,261 who found 69% of 67 tumors to be positive (utilizing an MoAb from Immunotec). These discrepant results may, at least in part, be attributable to differences in the antibodies used to study NF expression.
Nagao et al157 detected NF in 6 of 15 (40%) SCNECs of the major salivary glands, and McCluggage et al177 found NF in 7 of 21 (33%) PDNECs of the uterine cervix. Interestingly, at both of these sites primary PDNECs with a MCC-like phenotype (ie, CK20 positive) are not uncommon, and while Nagao and colleagues’ NF-positive cases coexpressed CK20, McCluggage and colleagues’ did not. Otherwise, there are little data on NF expression in extrapulmonary PDNECs. Gaffey et al262 failed to detect NF in any (0%) of 19 colorectal tumors, as did Shin et al193 and Zamboni et al263 in 9 and 3 breast and ampullary tumors, respectively; most recently, Lewis et al179 described expression in 1 of 14 (7%) head and neck mucosal-based tumors.
Like MCPyV IHC, detection of NF may be useful to support a diagnosis of MCC. Individual laboratories would need to verify a lack of significant expression in SCLC. Additional data on NF expression in extrapulmonary PDNECs, some of which are clearly positive, are of interest.
APPROACH TO A SUSPECTED METASTATIC NEN
An algorithmic approach to a suspected metastatic WDNET, with an emphasis on assigning site of origin, is presented in Figure 9. The first considerations are to distinguish WDNET from other non-neuroendocrine tumor types (eg, adenocarcinoma, squamous cell carcinoma) and from PDNEC. As illustrated in Figure 1C, WDNETs, especially those with type C histology, are occasionally mistaken for adenocarcinomas. I have a very low threshold for performing IHC for broad-spectrum keratins (eg, AE1/AE3) and general neuroendocrine markers (eg, synaptophysin and chromogranin). As discussed above and illustrated in Figure 3, WDNETs are sometimes mistaken for PDNECs, especially in crushed small biopsies. Ki-67 IHC guards against this pitfall and allows for assignment of WHO G1 or G2 (with the caveats that this grading scheme only formally applies to GEP tumors and that I have sometimes seen grade discordance between metastases and matched primaries, with the metastases generally of higher grade). For tumors with a PI>20% an alternative diagnosis (eg, PDNEC) should be considered, although tumors with well-differentiated histology but a PI in the G3 range have been described.131,264 There are very limited data on transcription factor IHC in these rare tumors, although in 1 recent report Islet 1 was expressed in 5 of 7 (71%) well-differentiated/G3 pancreatic NENs versus only 1 of 13 (8%) pancreatic PDNECs.131
Once the diagnosis of metastatic WDNET is secured, attention can turn to assigning site of origin. Any available clinical information, especially the results of imaging studies, is welcome. If the site of origin is “occult” at presentation, given multiple lines of evidence, including the Duke biochemical and survival data and the UCSF surgical exploration experience, I believe a jejunoileal primary to be most likely. I examine the tumor with this thought in mind, seeking out characteristic histologic features including the presence of a monomorphous population of round (rather than spindled) cells, perhaps punctuated by an occasional larger nucleus; moderate (rather than voluminous) amounts of cytoplasm, ideally with scattered eosinophilic cytoplasmic granules; and disposition in a type A pattern (although I have certainly seen types B, C, D, and mixed patterns in metastases from midgut primaries).
For a metastatic WDNET of unknown origin, I perform CDX2, TTF-1, and polyclonal PAX8 IHC. For tumors with a known or suspected origin, I may use 1 or more markers to confirm the clinical impression (eg, only CDX2 in a suspected midgut metastasis). I do not have Islet 1 IHC in my laboratory at this time, although in the algorithm a positive reaction with the polyclonal PAX8 antibody or for Islet 1 are considered equivalent. I have divided the results of this core panel into 4 patterns. The presence of diffuse, strong CDX2 expression, with negative results for TTF-1 and polyclonal PAX8 (or Islet 1) is strong support for a midgut origin, while TTF-1 positivity, with negative results for the other markers, strongly supports a lung origin. Polyclonal PAX8 (or Islet 1) positivity is less specific, as it may be seen with pancreaticoduodenal or rectal tumors; it also may be accompanied by CDX2 expression, which is typically weak and patchy. Given WDNET biology, though, I interpret polyclonal PAX8 (or Islet 1)-positive tumors to be likely pancreatic in origin. Upper and lower endoscopy can be pursued to exclude duodenal and rectal primaries, respectively. The final, “triple-negative” pattern (although CDX2 may actually be “weak, patchy positive”) is noninformative. I report it as “unusual for a midgut tumor (although it may be seen in up to 10%) and in keeping with, although not ‘diagnostic of,’ a pancreatic origin” (as up to half of the metastases from the pancreas are polyclonal PAX8 negative—an argument for bringing Islet 1 IHC online). For the final 2 patterns, additional IHC is of interest, and I have occasionally attempted PR or PrAP IHC in the setting of a nondiagnostic immunophenotype. Finally, it is not unlikely that additional markers will emerge in the near future that will further inform the assignment of site of origin.
As with the well-differentiated tumors, the first step in working up a PDNEC is to distinguish it from other tumor types. This is especially important in the liver, where metastatic adenocarcinomas are so frequent. Adenocarcinomas often contain minor populations of neuroendocrine cells (on the order of 1% to 5% of total cells), and the results of synaptophysin and chromogranin IHC are sometimes “overinterpreted” in this setting. Occasional poorly differentiated tumors will have extensive neuroendocrine marker expression (usually synaptophysin rather than chromogranin), while lacking the histologic features of a PDNEC. These have been referred to as “non–small cell carcinomas with (occult) neuroendocrine differentiation.” It is important to distinguish these from true PDNECs, as they are unlikely to respond to “small cell” chemotherapy. Although the histomorphology of SCNEC and LCNEC differs, a common feature, and one that generally distinguishes them from other high-grade carcinomas, is monomorphism.
In the skin, once the diagnosis of a PDNEC is made, the key distinction is between MCC and metastatic visceral PDNEC. This is usually easily accomplished with the mini-IHC panel of CK20 and TTF-1. CK20 positivity essentially confirms a diagnosis of MCC, while TTF-1 positivity refutes it. Given negativity for both markers, correlation with imaging studies is recommended, since, if a PDNEC is metastatic to the skin, it is apt to be detectable elsewhere. NF and MCPyV (CM2B4) IHC may also be helpful in this setting. The latter of these may also be useful in the head and neck, as parotid gland tumors are often CK20 positive but MCPyV negative.
IHC is not useful in assigning the site of origin of a visceral PDNEC. Pathologists often infer a lung origin based on TTF-1 expression, but expression is also seen in around 35% of extrapulmonary tumors. In fact, given a morphologic impression of SCNEC and negative chromogranin and synaptophysin immunostains, I have occasionally used TTF-1 expression to support the tumor diagnosis. Regardless of the site of origin, metastatic PDNECs are generally treated with “small cell” chemotherapy.
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