Molecular and phenotypic analysis of poorly differentiated sinonasal neoplasms: an integrated approach for early diagnosis and classification

Abstract

Primary poorly differentiated (small round and non-small) sinonasal neoplasms comprise histogenetically and biologically diverse entities with overlapping morphologic features. Because of the limited initial biopsy tissue materials, differential diagnostic difficulties may arise and complicate timely management of some cases. We used immunohistochemical and molecular marker analyses in a large cohort of these tumors to optimize their early diagnosis and classification. Fifty-two tumors of the skull base and sinonasal regions and, for comparison, 19 poorly differentiated neoplasms of other head and neck sites were analyzed by a panel of immunohistochemical markers including those of epithelial, mesenchymal, melanocytic, and neuroectodermal origin using tissue microarray. Reverse transcriptase-polymerase chain reaction analysis of messenger RNA for EWS-FLI1 and PAX-FKHR fusion transcripts and the human achaete-scute homolog-1 gene was performed on 24 of the 52 sinonasal tumors and the 19 tumors of other sites for comparison. The immunohistochemical results substantiated the phenotypic assessment and the initial diagnosis in 49 of the 52 tumors. In 4 instances the integrated markers and phenotypic analyses led to reclassification of 3 tumors and confirmed the histogenesis of a mesenchymal tumor with aberrant cytokeratin expression. Molecular analysis of the EWS-FLI1 fusion gene transcript revealed 4 (9.3%) of the 43 tumors to be positive; all were Ewing sarcomas. The human achaete-scute homolog-1 gene transcript was identified in 10 (23.8%) of 42 tumors: 3 of 6 neuroblastomas, all 4 neuroendocrine carcinomas, and 1 each in sinonasal undifferentiated carcinoma, rhabdomyosarcoma, and melanoma. The PAX-FKHR fusion transcript was not detected in any tumors. We conclude that (1) an integrated morphologic and biomarker algorithm may better optimize the early diagnosis of poorly differentiated sinonasal and skull-base tumors; (2) molecular analysis may assist in future biological stratification of certain classes of these tumors; and (3) the human achaete-scute homolog-1 gene transcript is a nonspecific marker for the diagnosis of neuroblastoma.

Fig. 1

Histologic and immunohistochemical comparison of primitive sinonasal and skull base tumors (cytokeratin negative tumors): olfactory neuroblastoma (ONB), rhabdomyosarcoma (Rhabdo), Ewing’s sarcoma (Ewings), and melanoma. A1–D1: Hematoxylin and eosin-stained sections are shown for each tumor along with markers expressed in each tumor type. A1–A3: Olfactory neuroblastoma showing diffuse expression of synaptophysin (Synp) (A2) and scattered chromogranin (Chromo) (A3). B1–B3: Rhabdomyosarcoma expressing desmin (B2) and myogenin (nuclear expression) (B3). C1–C3: Ewing’s sarcoma expressing CD99 (C2) and negative for cyto-keratin (C3). D1–D3: Melanoma expressing scattered S100 (D2) and pan-melanoma (Pan-Mel) markers (Mart1, HMB45, and tyrosinase) (D3).

Fig. 2

Discrepant immunohistochemical expression Rhabdomyosarcoma A–D: (A) Hematoxylin and eosin-stained section (H&E); (B) aberrant cytokeratin expression; (C) desmin positivity; and (D) myogenin nuclear positivity confirming diagnosis. An initial Ewing’s sarcoma reclassified as SNUC E–G: (E) H&E staining, (F) keratin expression, and (G) CD99 expressing weak reactivity.

Fig. 3

Molecular analysis for the EWS-FLI-1 fusion transcript in Ewing’s sarcomas. EWS-FLI-1 transcript is present in samples 2, 3, 8, and 10. The amplified transcript of the corresponding house-keeping gene, phosphoglycerate kinase (PGK), is also shown. P= Positive control, RB-SJH30 cell line and N= Negative control, without RNA.

Fig. 4

Molecular analysis for the hASH1 gene transcript. N = negative control without RNA; numbers 1, 6, 9, and 10 are neuroendocrine carcinomas; 2–5, 7, and 8 are olfactory neuroblastomas; P= positive control (H720 small cell carcinoma cell line); numbers 11–21 are non-neuroendocrine tumors. Tumors positive for hASH1 transcript: numbers 1, 4–10; 14 (basal cell carcinoma); 16 (rhabdomyosarcoma); 20 (melanoma); and 21 (SNUC).

Fig. 5

Diagnostic flow chart for immunohistochemical evaluation of poorly differentiated skull-base tumors. An initial panel (A) of keratin, synaptophysin (Synap), desmin, and melanin markers allows for the classification of most neoplasms. Panel B includes confirmatory/ancillary markers and molecular studies for rhabdomyosarcoma (Rhabdo), MyoD or Myogenin and PAX-FKHR for the alveolar type, and Ewing’s Sarcoma/Peripheral Neuroectodermal tumor (EWS/PNET), CD99 and EWS-FLI1. Olfactory neuroblastoma (ONB); neuroendocrine carcinoma (NEC); squamous carcinoma (SCC); sinonasal undifferentiated carcinoma (SNUC).