A 74-year-old, non-smoking woman was hospitalized after presenting with weight loss, progressive dysphagia, odynophagia and globus sensation. The patient had an unremarkable medical past and routine laboratory tests, serum tumor markers, a QuantiFERON^® tuberculosis test and antineutrophil cytoplasmic antibodies test came back negative. A total-body computed tomography scan revealed a 5-cm mass situated at the right posterior wall of the hypopharynx (Fig. 1A). No other lesions were identified.

Radiological examinations demonstrated the impossibility to perform a complete surgical resection since the tumor exhibited infiltrative margins and involved adjacent structures. However, the patient was symptomatic due to the progressive occlusion of the pharynx, which could result in a life-threating condition.

Diagnostic and palliative surgical resection was performed, and the specimen was fixed in 10% neutralized formalin for 24 h at room temperature and embedded in paraffin blocks, which were subsequently cut into 3-µm sections for routine hematoxylin-eosin staining. Examination under a light microscope revealed the proliferation of spindle-to-epithelioid cells intermingled with a mixed inflammatory infiltrate represented by lymphocytes, plasma cells and foamy histiocytes (Fig. 1B-F). Mitotic rate was extremely high (>5 mitoses/high-power field) and focal ulceration of the over lining mucosa was observed. Tumor cells strongly expressed smooth-muscle actin, desmin and ALK (clones ALK1, 5A4 and D5F3), while there was no immunoreactivity for pan-cytokeratins, S100 protein, p63, cluster of differentiation (CD) 21, CD35, CD68 and human herpes virus (HHV)-8. In situ hybridization with an Epstein-Barr virus-encoded small RNA probe came back negative. Subsequently, a diagnosis of IMT with epithelioid features was confirmed. In accordance with the guidelines of the Hospital Institutional Review Board, consent for anonymous research use of the tumor specimen and data collection was obtained. The patient refused enrollment in a clinical trial, which included treatment with ALK inhibitor, and subsequently succumbed to disease progression at 11 months post-diagnosis.

A representative neoplastic formalin-fixed paraffin-embedded (FFPE) tissue sample from the 74-year-old woman was used as biological material. NCI-H2228 and MRC5 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and Sigma-Aldrich (Merck Millipore, Darmstadt, Germany), respectively. The cell lines were cultured under the recommended conditions. Briefly, the NCI-H2228 cell line was cultured in RPMI-1640 medium containing 1% antibiotics (penicillin/streptomycin) and 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. The MRC5 cell line was cultured in Eagle's Minimum Essential Medium supplemented with 2 mM glutamine, 1% non essential amino acids and 10% FBS at 37°C in 5% CO₂. All media were purchased from Euroclone (EuroClone SpA, Milan, Italy). RNA and cell blocks from the H2228 and MRC5 cell lines were utilized as positive and negative controls for fluorescent in situ hybridization (FISH) analysis and variants 3a/b EML4-ALK fusion.

Serial 4-µm-thick sections were obtained from FFPE blocks representative of tumor tissue for immunohistochemical analysis. All reactions were performed using a BenchMark XT fully automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ, USA) and antibody incubation was for 8 h at 37°C. The following antibodies were used: Pan-cytokeratins (prediluted; cat. nos. 760–2521 and 790–4555 for clones AE1 and CAM5.2, respectively; Ventana Medical Systems, Inc.), smooth-muscle actin (prediluted; cat. no. 760–2833; Ventana Medical Systems, Inc.), desmin (prediluted; cat. no. 760–2513; Ventana Medical Systems, Inc.), p63 (prediluted; cat. no. 790–4509; Ventana Medical Systems, Inc.), CD21 (prediluted; cat. no. 760–4245; Ventana Medical Systems, Inc.), CD35 (prediluted; cat. no. 760–4439; Ventana Medical Systems, Inc.), HHV-8 (prediluted; cat. no. 760–4260; Ventana Medical Systems, Inc.), ALK (prediluted; cat. nos. 790–2918 and 790–4796 for clones ALK1 and D5F3, respectively; Ventana Medical Systems, Inc.), ALK (1:50 dilution; cat. no. NCL-L-ALK; Novocastra; Leica Microsystems, Inc., Buffalo Grove, IL, USA), S100 (prediluted; cat. no. 760–2523; Ventana Medical System, Inc.) and CD68 (prediluted; cat. no. 790–2931; Ventana Medical Systems, Inc.).

FISH assay was performed on 4-µm FFPE sections using Vysis ALK Break Apart FISH Probe kit (Abbott Molecular, Abbott Park, IL, USA). The hybridized slides were reviewed on an Olympus IX-50 microscope (Olympus Corporation, Tokyo, Japan) at ×100 magnification.

Total RNA extraction from tumor tissue was performed with the RecoverAll™ Total Nucleic Acid Isolation kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. A total of 500 ng RNA was reverse transcribed using the SuperScript^® III First-Strand Synthesis system (Invitrogen; Thermo Fisher Scientific, Inc.) for RT-PCR reactions. To analyze variants 1 and 2, two specific synthetic DNA double strands of the EML4-ALK fusion region were synthesized. Amplified products were characterized by cloning PCR products into the StrataClone™ PCR Cloning Vector pSC-A (Agilent Technologies, Inc., Santa Clara, CA, USA), straight amplifying from crude lysates of single bacterial colonies was performed using the following primers: EML4_exon6_F, 5′-CTGCAGACAAGCATAAAGATG-3′ and ALK_exon20_R, 5′-GCTTGCTCAGCTTGTACTC-3′. Direct sequencing was performed using the BigDye^® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and an ABI PRISM^® 3130 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.).

Fluorescent PCR amplification was performed using a reverse fluorescein amidite-labeled primer specific to exon 20 of ALK and forward primers specific to exons 13–20 and 6 of EML4 to amplify variants 1, 2 and 3a/b, respectively (8). As a control for RNA quality, primers specific to β-2-microglobulin were employed. RT-PCR products were size-fractionated by capillary electrophoresis in an ABI 3130 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.) and results were analyzed with GeneMapper^® software v.4 (Applied Biosystems; Thermo Fisher Scientific, Inc.).

To confirm the results of fluorescent analysis, DNA fragments were analyzed by microchip electrophoresis using an Agilent 2100 Bioanalyzer with the DNA 1000 assay sizing reagent kit (Agilent Technologies, Inc.).

ALK rearrangement was assessed by FISH analysis. Single and multiple copies of the intact ALK fusion together with the abnormal split pattern were observed in the tumor cells of the tissues specimen with a frequency of 65% ALK rearranged gene (combined EML4-ALK fusion and 5′end-ALK deletion), (Fig. 2A).

Two fragments of the expected length (112 and 142 bp) corresponding to the fusion 3a/b variants were detected during the first round of RT-PCR, which is designed to detect the most frequent 1,2 and 3a/b types of transcript. Using specific primers for the amplification of the EML4-ALK 3a/b variants fusion cDNA, the same two pair of PCR products were detected in the NCI-H2228 cell line and in the IMT patient sample. These molecular findings were confirmed using two different fluorescent fragment analysis methods; the first was previously described by Sanders et al (8), (Fig. 2B) and the second uses novel, fast and efficient microchip electrophoresis to carefully detect ALK variation through the use of capillary microelectrophoresis and detection (Fig. 2C). Molecular cloning and direct sequencing of each PCR product confirmed the presence of the variant 3a fusion (linking exon 6 of EML4 to exon 20 of ALK) and the 3b fusion, containing an insertion of 33 bp mapped to intron 6 of EML4 that was located between the same exons of EML4 and ALK (Fig. 2D).