Immunohistochemistry in the diagnosis and classification of neuroendocrine neoplasms: what can brown do for you?

Abstract

This review is based on a presentation given at the Hans Popper Hepatopathology Society companion meeting at the 2019 United States and Canadian Academy of Pathology Annual Meeting. It presents updates on the diagnosis and classification of neuroendocrine neoplasms, with an emphasis on the role of immunohistochemistry. Neuroendocrine neoplasms often present in liver biopsies as metastases of occult origin. Specific topics covered include 1. general features of neuroendocrine neoplasms, 2. general neuroendocrine marker immunohistochemistry, with discussion of the emerging marker INSM1, 3. non-small cell carcinoma with (occult) neuroendocrine differentiation, 4. the WHO Classification of neuroendocrine neoplasms, with discussion of the 2019 classification of gastroenteropancreatic neoplasms, 5. use of Ki-67 immunohistochemistry, 6. immunohistochemistry to assign site of origin in neuroendocrine metastasis of occult origin, 7. immunohistochemistry to distinguish well-differentiated neuroendocrine tumor G3 from poorly differentiated neuroendocrine carcinoma, 8. lesions frequently misdiagnosed as well-differentiated neuroendocrine tumor, and 9. required and recommended data elements for biopsies and resections with associated immunohistochemical stains. Next-generation immunohistochemistry, including lineage-restricted transcription factors (e.g., CDX2, islet 1, OTP, SATB2) and protein correlates of molecular genetic events (e.g., p53, Rb), is indispensable for the accurate diagnosis and classification of these neoplasms.

Keywords: Carcinoma of Unknown Primary; Differential Diagnosis; INSM1; Immunohistochemistry; Ki-67; Neuroendocrine; World Health Organization Classification.

Figure 1. Somatostatin Receptor (SSTR) Expression by Neuroendocrine Neoplasms:

Along with general neuroendocrine marker expression and the production of peptide hormones and/or biogenic amines, somatostatin receptor expression is a characteristic feature of neuroendocrine neoplasms (NEN). (A) At Iowa, we have been routinely performing SSTR2A immunohistochemistry on NENs since the Fall of 2014. Diffuse, strong membrane expression is seen in nearly all enterochromaffin-cell tumors (depicted here), 80–90% of pancreatic tumors, and fewer bronchopulmonary tumors and poorly differentiated neuroendocrine carcinomas. (B) Somatostatin receptor expression is the basis of NEN functional imaging and octreotide and peptide receptor radionuclide therapy. This DOTATATE scan demonstrates several liver metastases.

Figure 2. Chromogranin A (CgA) Expression by Small Cell Neuroendocrine Carcinoma:

(A) This crushed small cell neuroendocrine carcinoma demonstrates (B) rather extensive, though punctate CgA expression. CgA is a component of neuroendocrine dense core granules, which are often few in number in this tumor type. When assessing CgA-positivity, I pay more attention to the proportion of cells with signal than the total cross-sectional area occupied by signal.

Figure 3. Extended Immunohistochemistry to Support a Diagnosis of Small Cell Neuroendocrine Carcinoma (NEC):

(A) The morphologic impression in this metastasis from a patient with a lung mass was small cell NEC. (B) Keratin AE1/AE3 is positive, while both (C) chromogranin A (depicted) and synaptophysin were entirely negative. (D) INSM1 demonstrates multifocal positivity, (E) Rb expression is lost (with intact staining in endothelial cell nuclei), and (F) CXCR4 shows diffuse, strong membrane staining. INSM1 is more sensitive than traditional general neuroendocrine markers in NEC; Rb inactivation, a molecular genetic hallmark of small cell lung cancer, is a sensitive NEC marker; CXCR4 overexpression is seen in ≥80% of NECs; in my anecdotal experience these latter two markers are reasonably specific for NEC.

Figure 4. Non-Small Cell Carcinoma with (Occult) Neuroendocrine Differentiation:

(A) This metastasis from a woman with a history of breast cancer simultaneously expresses (B) synaptophysin, (C) GATA-3, and (D) ER. Ten to twenty percent of non-neuroendocrine carcinomas from diverse anatomic sites demonstrate some degree of general neuroendocrine marker expression, ranging from rare cells to diffuse, strong staining (as in this example). Occult neuroendocrine differentiation has no clear prognostic or therapeutic implication. The diagnosis of “poorly differentiated carcinoma with neuroendocrine differentiation” is strongly discouraged, as it engenders confusion in the treating clinician. Breast cancers with occult neuroendocrine differentiation are typically of luminal A type and are treated as such.

Figure 5. Well-Differentiated Neuroendocrine Tumor (NET) G3:

(A) Metastatic tumor demonstrating juxtaposition of well- (left) and “less well”-differentiated (right) components with (B) corresponding Ki-67. (C) At higher power the well-differentiated component demonstrates extensive polarized eosinophilic cytoplasmic granularity and prominent pseudoglands (i.e., a readily recognizable midgut NET) with a (D) corresponding Ki-67 proliferation index of 0.75%. (E) The “less well”-differentiated component demonstrates a (F) Ki-67 proliferation index of 35%. Five percent of NETs are morphologically well-differentiated but with “G3 range”-proliferation indices and/or mitotic counts. Some examples demonstrate substantial morphologic overlap with large cell neuroendocrine carcinoma. Presence of an adjacent G1/G2 NET supports the diagnosis of NET G3. In this case, I had also performed p53 (wild-type), Rb (intact), and CDX2 (expressed throughout, though with diminished intensity in the G3 component), which further support the diagnosis of NET G3 (not depicted).

Figure 6. Mixed Neuroendocrine-Non-Neuroendocrine Neoplasm (MiNEN):

(A) This colon cancer arising in association with an adenoma is composed of (B) conventional adenocarcinoma and (C) poorly differentiated neuroendocrine carcinoma (NEC) components. (D) CDX2 is expressed by the background colonic mucosa, adenoma, and adenocarcinoma but is nearly absent in the NEC, while (E) synaptophysin shows the opposite pattern. (F) Rb loss is confined to the NEC component. The WHO 2019 term “MiNEN” replaces the term “mixed adenoneuroendocrine carcinoma (MANEC),” as the non-neuroendocrine component of mixed tumors may be squamous and the neuroendocrine component may be NET, though the combination depicted in this example is the most common one in the West.

Figure 7. Use of Ki-67 Immunohistochemistry Beyond Gastroenteropancreatic Well-Differentiated Neuroendocrine Tumor:

(A) Fine-needle aspiration of a lung mass reveals carcinoid tumor but there are only a couple HPF for mitotic counting. The medical oncologist asked us to perform a Ki-67 to get a better sense of whether this was typical carcinoid or atypical carcinoid tumor, which would impact treatment planning. (B) The Ki-67 proliferation index is 15%; although Ki-67 is not part of the WHO Classification of lung neuroendocrine neoplasms, this result is essentially diagnostic of atypical carcinoid tumor. Subsequent DOTATATE scan revealed extensive hilar and mediastinal adenopathy. This Ki-67-immunostained slide reveals weak and strong, nucleolar and diffuse-pattern staining, all of which are considered positive.

Figure 8. Morphologic Clues to Well-Differentiated Neuroendocrine Tumor Site of Origin:

(A) Tumor with predominantly nested architecture, minor pseudogland component, and heavy eosinophilic cytoplasmic granularity diagnostic of an enterochromaffin-cell tumor; tumors with this morphology typically arise in the ileum, though this is actually a testicular primary. (B) Trabecular architecture is most commonly seen in tumors of rectal origin. (C) Pseudoglandular architecture is typical of ampullary tumors, which are apt to be mistaken for adenocarcinoma in forceps biopsy. (D) Spindle cell morphology is often seen in lung tumors that metastasize; small foci of necrosis like this are more common in lung (in this case diagnostic of atypical carcinoid tumor) than gastroenteropancreatic tumors.

Figure 9. University of Iowa Immunohistochemical Algorithm for Well-Differentiated Neuroendocrine Tumor Site of Origin.

Figure 10. CDX2-Negativity in Enterochromaffin (EC)-Cell Well-Differentiated Neuroendocrine Tumors (NET):

CDX2 is the best widely available EC-cell NET marker, expressed by 90% of tumors of midgut origin. (A) Clonal loss of CDX2 in a primary tumor, possibly due to promoter methylation. (B) Weak, patchy CDX2-positivity in a whole section from a liver resection; this tumor appeared to be CDX2-negative in tissue microarray.

Figure 11. Simplified Immunohistochemical Algorithm for Well-Differentiated Neuroendocrine Tumor Site of Origin.

Figure 12. Immunohistochemistry for Merkel Cell Carcinoma:

(A) Monomorphous high-grade tumor with powdery chromatin demonstrates (B) dot-like CK20, (C) dot-like and diffuse neurofilament, and (D) nuclear Merkel cell polyomavirus large T antigen (clone CM2B4) expression. CK20 is expressed by 90% of Merkel cell carcinomas and is otherwise uncommonly expressed by neuroendocrine carcinomas (NEC) except for those arising in the parotid; neurofilament is expressed by 75% of tumors and appears reasonably specific. Up to 80% of Merkel cell carcinomas are polyomavirus-positive; the remainder are UV-light associated. Most CM2B4-positive Merkel cell carcinomas are CK20-positive, somewhat limiting this marker's diagnostic utility. Polyomavirus-driven Merkel cell carcinomas are remarkably monomorphous compared to all other NECs (i.e., you should be able to predict the result of the CM2B4 based on morphology!).

Figure 13. Poorly Differentiated Neuroendocrine Carcinomas (NEC) Demonstrate Marked Transcription Factor Lineage Infidelity:

(A) This endometrial NEC co-expresses (B) GATA-3, (C) SATB2, (D) p40, (E)MYC, and, naturally, does not express (F) PAX8. NECs tend to express multiple transcription factors independent of site of origin. Expression is typically patchy but can be rather extensive. The only transcription factors with a role in NEC site of origin assignment are TTF-1 and ASCL1 (for visceral origin) and strong SATB2 (for cutaneous origin).

Figure 14. Immunohistochemical Algorithm for Morphologically Ambiguous G3 Neuroendocrine Epithelial Neoplasms.

Figure 15. Immunohistochemistry to Distinguish Well-Differentiated Neuroendocrine Tumor (NET) G3 from Poorly Differentiated Neuroendocrine Carcinoma (NEC):

(A) This patient had a lung mass, and I favored a diagnosis of large cell NEC in this liver metastasis. (B) p53 demonstrates wild-type pattern staining and (C) Rb expression is intact, favoring NET. The tumor was found to express OTP (not depicted), further supporting the diagnosis. (D) This large cell NEC demonstrates (E) missense-mutation pattern p53 and (F) Rb loss, supporting the morphologic impression. The NET G3 versus NEC immunohistochemical classifier is predicated on NEC genetics.

Figure 16. Rb Immunohistochemistry and the Relationship Between Non-Neuroendocrine and Neuroendocrine Carcinomas:

(A) This gastric large cell neuroendocrine carcinoma (NEC) arises in association with columnar dysplasia; (B) both components demonstrate Rb inactivation. In the GI tract, NECs arise from non-neuroendocrine precursors; many castration-resistant prostate cancers have a NEC phenotype; small cell lung cancer may arise from EGFR-mutant lung adenocarcinoma in the setting of anti-EGFR therapy. In these contexts, Rb inactivation, though insufficient on its own, appears to be permissive to the development of the NEC phenotype.

Figure 17. Lesions Apt to be Mistaken for Well-Differentiated Neuroendocrine Tumor (NET):

Each of these tumors demonstrates relatively monomorphous cytomorphology and general neuroendocrine marker-positivity (in many instances quite strong). (A) Gastric glomus tumor with (B) variable but overall rather extensive synaptophysin-positivity; (C) smooth muscle actin-positivity supports the diagnosis. (D) Solid pseudopapillary tumor with (E) modest synaptophysin-positivity; (F) nuclear beta-catenin-positivity supports the diagnosis in the appropriate morphologic context. (G) This mixed acinar-neuroendocrine carcinoma has the H&E-appearance of acinar cell carcinoma but (H) demonstrates diffuse, strong synaptophysin-positivity; (I) trypsin-positivity supports the morphologic impression. (J) This pancreatoblastoma, with morules evident at the right center and upper left, was misdiagnosed as a NET based on (K) this strongly positive chromogranin A; (L) heterogeneous nuclear beta-catenin staining, which has a predilection for the morules, is typical; the tumor also demonstrated multifocal trypsin-positivity (not depicted). (M) Adrenal cortical carcinoma demonstrates (N) diffuse, strong synaptophysin-positivity; (O) melan A-positivity supports the diagnosis. I always consider adrenal cortical carcinoma in presumed NETs in which immunohistochemistry-based site of origin assignment is uncertain and in tumors described as “retroperitoneal” in location.

Figure 18. Immunohistochemistry for Paraganglioma/Pheochromocytoma:

(A) This paraganglioma demonstrates characteristic zellballen architecture; (B) strong GATA-3-positivity supports the diagnosis in this well-fixed biopsy of a metastatic tumor; (C) loss of SDHB expression, with intact staining in endothelium and sustentacular cells, also supports a diagnosis of paraganglioma or pheochromocytoma and suggests the possibility of hereditary paraganglioma-pheochromocytoma syndrome. (D) This pheochromocytoma is the “essence of amphophilic;” (E) GATA-3-positivity is barely discernable in this resected tumor; (F) diffuse, strong tyrosine hydroxylase expression is seen in 100% of pheochromocytomas and 40% of paragangliomas and staining is very robust.