Open Access

A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4‑ALK fusion gene

  • Authors:
    • Lucia Anna Muscarella
    • Giulio Rossi
    • Domenico Trombetta
    • Annamaria La Torre
    • Leonarda Di Candia
    • Maria Cecilia Mengoli
    • Angelo Sparaneo
    • Vito Michele Fazio
    • Paolo Graziano
  • View Affiliations

  • Published online on: December 14, 2016     https://doi.org/10.3892/ol.2016.5504
  • Pages: 593-598
  • Copyright: © Muscarella et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Inflammatory myofibroblastic tumors (IMT) in the head and neck region are rare neoplasms that generally mimic benign/low‑grade neoplasms. Overexpression of anaplastic lymphoma kinase (ALK) has been reported in 50% of IMT cases, secondary to ALK activation by structural rearrangements in the ALK gene, which results in a fusion protein with echinoderm microtubule associated protein like 4 (EML4) in ~20% of cases. The present study describes a case of a 74‑year‑old woman with a malignant IMT in the right posterior hypopharynx harboring a previously unreported chromosomal rearrangement resulting in EML4 and ALK gene fusion. Strong ALK immunoreactivity was observed in neoplastic cells, while fluorescent in situ hybridization combined with fluorescent fragment analysis and direct sequencing identified the first case of the 3a/b variants of the EML4‑ALK fusion gene in IMT. The results of the current study highlight the uncommon occurrence of ALK‑positive IMT in the head/neck region and demonstrate the importance of integrating different molecular methodologies to identify unequivocal gene fusion characterization.

Introduction

Inflammatory myofibroblastic tumors (IMT) are distinctive tumor entities described in almost all anatomical sites under several definitions, including inflammatory pseudotumor and plasma cell granuloma. IMT generally shows a benign clinical outcome and is common in children and young adults, although the age of occurrence is extremely broad (1). Presenting symptoms are related to the site of the primary tumor origin, although fever, weight loss, malaise and night sweats are also reported. Surgical resection is the mainstay of treatment, while chemoradiotherapy is generally ineffective (1).

Approximately half of IMT patients carry rearrangements of the anaplastic lymphoma kinase (ALK) gene (25). ALK is a tyrosine kinase receptor, which is typically expressed in the central nervous system (3,4). Fusion of the ALK gene with different partners may result in the overexpression of ALK and activation of its kinase domain (68). Identification of ALK fusion genes may support a diagnosis of IMT, and additionally allow initiation of an effective treatment regimen with ALK inhibitors (6,7). IMTs occurring in the head and neck region are extremely rare and are more commonly located in the larynx, orbit, paranasal sinus, trachea and parotid gland. The present study describes the characterization of the 3a/b variants of the echinoderm microtubule associated protein like 4 (EML4)-ALK gene fusion, which has not been reported previously in IMT of the hypopharynx.

Materials and methods

Case description

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.

Tissue specimen and cell lines

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.

Immunohistochemistry

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.).

Cytogenetics and ALK break apart FISH assay

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.

RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)

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 fragment analysis

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.).

Results

Detection of ALK involvement in IMT

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).

Identification of the EML4-ALK 3a/b fusion variants

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).

Discussion

The World health organization (WHO) classification of soft tissue tumors defines IMT as the neoplastic counterpart among the wastebasket group of lesions previously known as inflammatory pseudotumors (9). IMT represents a proliferation of myofibroblastic cells, often intermingled with lymphocytes, plasma cells and histiocytes, which eventually forms a highly aggressive lesion (9). IMT is ubiquitous and margin-free surgical resection is considered the only curative treatment. By contrast, aggressive or metastatic lesions have no standard therapy and chemotherapy is usually ineffective.

In the head and neck region, the larynx is the most common location of IMT, while a few cases in the hypopharynx have been reported (10,11). Clinical and radiological differential diagnosis is broad and includes infections (tuberculosis and mycoses), Wegener's granulomatosis, amyloidosis and other malignancies, such as carcinoma, melanoma and salivary gland tumors (10,11).

Up to 70% of IMTs harbour ALK gene rearrangements resulting in the formation of a chimeric fusion protein, which is detectable by FISH or immunohistochemistry (25). ALK is a receptor-type protein tyrosine kinase that is currently being analysed by oncologists as an essential growth driver, which defines its potential susceptibility to ALK inhibitors (7,1215). The oncogenic function of ALK occurs as a result of it forming a fusion protein with various gene partners through chromosomal translocations, most commonly TPM3 or TPM4 in IMT, resulting in oncogenic activation of ALK (3). Alternatively, a small inversion [inv2(2)(p21p23)] on the short arm of chromosome 2 leads to a functional EML4-ALK fusion-type tyrosine kinase (322) (Table I). Initially described in non-small cell lung carcinoma (15), this abnormality was also identified in ~20% of ALK-rearranged pulmonary IMT cases (5). To date, a total of 10 cases of EML4-ALK fusions in IMT have been reported (3,5,9). The present study described a EML4-ALK rearranged IMT located in the hypopharynx. By using two different fluorescent fragmentation analysis methods, the specific 3a/b variants were identified within the RNA of the tumor specimen and confirmed by standard Sanger sequencing. This description of the ALK fusion 3a/b variants in an IMT case, together with the few cases of ALK-EML4 fusions reported in literature, corroborates the existing hypothesis that identical ALK fusions detected in different tumor types may drive an inappropriate activation of the same kinase signalling pathway, which could be oncogenic in disparate cellular lineages. In addition, the present case also noted the epithelioid appearance of the tumor cells, which could potentially lead to a dismal clinical course. Notably, Mariño-Enríquez et al (23) described ALK-positive intra-abdominal IMT composed predominantly of sheets of round-to-epithelioid cells significantly associated with aggressive course with rapid local recurrences.

Table I.

ALK fusion partners in IMT.

Table I.

ALK fusion partners in IMT.

Authors, yearIMT locationPatient age/genderALK fusionRefs.
Lawrence et al, 2000Abdomen23 years/FTMP3-ALK(3)
Lung30 years/FTMP3_ALK(3)
Abdomen1 year/MTMP4-ALK(3)
Bridge et al, 2001Neck3 years/FCLTC-ALK(16)
Pelvis37 years/MCLTC-ALK(16)
Cools et al, 2002Abdomen4 months/MCARS-ALK(17)
Ma et al, 2003Abdomen1 year/M RANBP2-ALK(18)
Abdomen7 months/M RANBP2-ALK(18)
Debiec-Rychter et al, 2003Bladder46 years/M ATIC-ALK(19)
Panagopoulos et al, 2006Abdomen23 years/M SEC31L1-ALK(20)
Takeuchi et al, 2011Lung45 years/M PPFIBP1-ALK(21)
Lung34 years/F PPFIBP1-ALK(21)
Wang et al, 2012Neck7 years/F DCTN1-ALK(22)
Lovly et al, 2014Lung38 years/F EML4-ALK(12)
Mesentery11 years/F LMNA-ALK(12)
Shoulder1 year/F PRAKAR1A-ALK(12)
Bladder26 years/FFN1-ALK(12)
Pelvis14 years/MTFG-ALK(12)

[i] ALK, anaplastic lymphoma kinase; IMT, inflammatory myofibroblastic tumors; F, female; M, male.

In conclusion, to the best of our knowledge, the present study describes for the first time the presence of the EML4-ALK 3a/b variant in a case of malignant IMT of the hypopharynx. This was achieved by integrating different methodologies, which is considered the most suitable molecular approach for gene fusion characterization. Furthermore, the identification of ALK rearrangement in cases of malignant IMTs may offer a rationale to adopt selective targeted therapies in this ‘orphan’ tumor.

Acknowledgements

The present study was supported by the Italian Ministry of Health (grant nos. RC1503LO51, 2010-2316264 and RC1502AP18), the ‘5×1000′ voluntary contributions and Associazione Italiana per la Ricerca sul Cancro (grant no. 12983).

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Spandidos Publications style
Muscarella LA, Rossi G, Trombetta D, La Torre A, Di Candia L, Mengoli MC, Sparaneo A, Fazio VM and Graziano P: A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4‑ALK fusion gene. Oncol Lett 13: 593-598, 2017
APA
Muscarella, L.A., Rossi, G., Trombetta, D., La Torre, A., Di Candia, L., Mengoli, M.C. ... Graziano, P. (2017). A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4‑ALK fusion gene. Oncology Letters, 13, 593-598. https://doi.org/10.3892/ol.2016.5504
MLA
Muscarella, L. A., Rossi, G., Trombetta, D., La Torre, A., Di Candia, L., Mengoli, M. C., Sparaneo, A., Fazio, V. M., Graziano, P."A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4‑ALK fusion gene". Oncology Letters 13.2 (2017): 593-598.
Chicago
Muscarella, L. A., Rossi, G., Trombetta, D., La Torre, A., Di Candia, L., Mengoli, M. C., Sparaneo, A., Fazio, V. M., Graziano, P."A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4‑ALK fusion gene". Oncology Letters 13, no. 2 (2017): 593-598. https://doi.org/10.3892/ol.2016.5504