Rom J Morphol Embryol 2013, 54(3 Suppl):839–843

ISSN (print) 1220–0522 ISSN (on-line) 2066–8279

CCAASSEE RREEPPOORRTT

Non-keratinizing undifferentiated carcinoma of the nasopharynx

E. R. BOIA1,2), MARIOARA BOIA3,4), N. C. BALICA1,2), LAURA-CRISTINA RUSU5), O. MAZILU6), C. SOLOVAN7,8), FLAVIA BADERCA9,10)

1)ENT Department, “Victor Babeş” University of Medicine and Pharmacy, Timisoara

2)ENT Department, Emergency City Hospital, Timisoara

3)Pediatrics Department, “Victor Babeş” University of Medicine and Pharmacy, Timisoara

4)Pediatrics Department, “Louis Ţurcanu” Children Emergency Hospital, Timisoara

5)Department of Technology of Materials and Devices in Dental Medicine 6)Department of Surgical Semiology

7)Department of Dermatology “Victor Babeş” University of Medicine and Pharmacy, Timisoara

8)Department of Dermatology, Emergency City Hospital, Timisoara

9)Department of Microscopic Morphology, “Victor Babeş” University of Medicine and Pharmacy, Timisoara

10)Service of Pathology, Emergency City Hospital, Timisoara

Abstract Nasopharyngeal carcinoma is the predominant tumor type arising in the nasopharynx with cervical lymph nodes present in 60–90% of all cases at the time of presentation. The most frequent pathological varieties include squamous cell carcinoma well-differentiated keratinizing, moderately differentiated non-keratinizing and an undifferentiated type. We present a case of non-keratinizing undifferentiated carcinoma of the nasopharynx with parapharyngeal and middle cranial fossa space involvement in an 18-year-old male who has been admitted in our hospital for recurrent right ear otitis media. Symptoms consisted in mild conductive hearing loss, trigeminal V2 nerve anesthesia, right ear tinnitus, mild dysphagia, mild dysphonia, right hypoglossal nerve paralysis and right Claude Bernard–Horner’s syndrome. Clinical examination revealed no lymph node masses, chest X-ray corresponding to a normal thoracic image. Cranial contrast enhanced CT scan showed a non-homogenous mass of 5.4/4.5/5.5 cm from the level of the right rhinopharyngeal wall, extending in the right parapharyngeal space, invading the right middle cranial fossa. Cranial MRI with contrast enhancement revealed a rhino- and parapharyngeal mass of 5.5/4.6/5.3 cm with intracerebral extension in the right cavernous sinus, right internal carotid artery being engulfed by the tumor mass with partial compression. Several lymph node masses of 1.7/1.2 cm were also revealed. We performed rhinopharyngeal biopsy, right tympanotomy and grommet tube insertion. The tissue specimens were processed with routine histological technique. Subsequent immunohistochemical reactions for pan-cytokeratin AE1/AE3 and leukocytes common antigen were performed. The histological examination of routine stained slides showed that malignant tumor cells had a syncytial pattern of growth in a background of small lymphocytes. The positivity of tumor cells for pan-cytokeratin established the final diagnosis of non-keratinizing undifferentiated carcinoma. The age of onset, the clinical signs and symptoms and minimum involvement of lymph nodes represents the particular aspects of the case.

Keywords: nasopharyngeal carcinoma, pan-cytokeratin, AE1/AE3, CD45, LCA.

Introduction

Nasopharyngeal carcinoma (NPC) is a malignant tumor of nasopharyngeal epithelium. It is the main type in nasopharyngeal malignant tumors in both endemic areas and regions with low incidence. NPC epidemiological studies focused on etiology and biological behavior of the disease were strongly encouraged because of the International Union against Cancer (UICC) Symposium on Cancer of Nasopharynx held in Singapore in 1964. Investigations in the past four decades have produced many important findings in those aspects [1].

NPC has unique epidemiological features, including obvious regional, racial, and familial aggregation.

NPC is a type of tumor with extremely unbalanced endemic distribution. NPC appears in many countries and areas of the five continents. However, the incidence of NPC is lower than 1/105 in most areas. High-incidence areas are centralized in the southern part of China (including Hong Kong). The highest incidence was found in Guangdong Province, and the incidence in male can reach 20–50/100 000 [1].

Etiological factors are dietary environmental factors,

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such as salted fish, a popular dish in southern China, when consumed from a young age, due to its volatile nitrosamines (N-nitroso-dimethylamine and N-nitroso-diethylamine) [2].

Patient, Methods and Results

An 18-year-old male patient was admitted in ENT Department of Emergency City Hospital, Timişoara, Romania, in April 2013. Initial diagnosis was right recurrent otitis media.

Hospital admission symptoms and signs consisted in right mild conductive hearing loss, right trigeminal V2 nerve anesthesia, right ear tinnitus, mild dysphagia, mild dysphonia, right hypoglossal nerve paralysis and right Claude Bernard–Horner’s syndrome. Clinical examination revealed no lymph node masses and chest X-ray – normal thoracic image.

Cranial contrast enhanced CT scan showed a non-homogenous mass of 5.4/4.5/5.5 cm from the level of the right rhinopharyngeal wall, extending in the right para-pharyngeal space, invading the right middle cranial fossa.

Cranial MRI with contrast enhancement revealed a rhino and parapharyngeal mass of 5.5/4.6/5.3 cm with intracranial extension in the right cavernous sinus, right internal carotid artery being engulfed by the tumor mass with partial compression. Several lymph node masses of 1.7/1.2 cm were identified (Figures 1 and 2).

We performed a rhinopharyngeal biopsy, right tympanotomy and grommet tube insertion.

The tissue specimens were processed according with the routine histological technique. The specimens were fixed in 4% (v/v) buffered formalin and paraffin embedded. Three micrometers thick serial sections were stained with Hematoxylin–Eosin.

Additional immunohistochemical reactions for multi-cytokeratin AE1/AE3 (CK AE1/AE3) and CD45 (leukocyte common antigen, LCA) were made in order to establish the correct diagnosis. Sections of 3-µm thick were made and mounted onto Superfrost slides. The deparaffining was followed by rehydration using decreasing concen-trations of alcohol.

For both reactions, we used heat-induced antigen retrieval. Antigen retrieval was done in Epitope Retrieval Solution pH 6 (10×) for CK AE1/AE3 (Novocastra, code RE7113-CE) and in Epitope Retrieval Solution pH 9 (10×) for CD45 (Novocastra, code RE7119-CE), for 30 minutes using microwaves.

The inhibition of the endogenous peroxidase and of other tissue protein was done using the solutions contained in Novolink Max Polymer Detection System (Novocastra) that was used also as detection system. The washing solution was represented by Bond Wash Solution, 10× (Leica).

The antibodies used were multi-CK AE1/AE3 (Novocastra, NCL-L-AE1/AE3, Liquid Monoclonals, clone AE1/AE3) and CD45 (Novocastra, NCL-L-LCA, Liquid Concentrated Monoclonal Antibody, clone X16/99).

For dilution of the antibodies, we used NovocastraTM IHC Diluent, in a ratio of 1:150. The incubation time with primary antibody was 30 minutes for both antibodies.

The antigen–antibody complex was visualized with 3,3’-diaminobenzidine (from Novolink Max Polymer Detection System, Novocastra). The slides were washed in tap water and counterstained with Mayer’s Hematoxylin and then dehydrated, cleared, and mounted. The signal was brown with cytoplasmic distribution for AE1/AE3 and membranar for CD45.

In each determination, external control slides were included.

Histopathological evaluation was performed with Nikon Eclipse E600 microscope and images were acquired using Lucia G system.

The histological examination at scan magnification of routine stained slides showed that malignant tumor cells had a syncytial pattern of growth in a background of small lymphocytes and plasma cells. The tumor was composed of irregular islands or solid sheets intermingled with non-tumoral lymphoid tissue (Figure 3).

At higher magnification, the tumor cells were small or medium in size, round or oval and had a crowded appearance. In some areas of the tumor, the cells were overlapping or had spindled features (Figure 4).

The scant cytoplasm was either eosinophilic or amphophilic. The nuclei were big, vesicular with prominent nucleoli, situated centrally in the nucleolus. A small number of nuclei were better chromatin-rich rather than vesicular (Figures 5 and 6).

The positivity of tumor cells for pan-cytokeratin and the absence of immune reaction for lymphoid marker established the final diagnosis of non-keratinizing undifferentiated carcinoma (Figures 7 and 8).

Radiochemotherapy represented the therapeutic option for this case.

Figure 1 – Cranial MRI with contrast enhancement – right rhino and parapharyngeal mass of 5.5/4.6/5.3 cm.

Figure 2 – Cranial MRI with contrast enhancement – right internal carotid artery being engulfed by the tumor mass with partial compression.

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Figure 3 – Syncytial tumor cells in numerous non-tumor small lymphocytes area (HE stain, ob. 10×).

Figure 4 – Detail: Syncytial tumor cells in numerous non-tumor small lymphocytes area (HE stain, ob. 20×).

Figure 5 – Tumor cells with vesicular nuclei and prominent nucleoli (HE stain, ob. 40×).

Figure 6 – Spindled tumor cells (HE stain, ob. 40×).

Figure 7 – Expression of CK AE1/AE3 in tumor cells (ob. 20×).

Figure 8 – Negative reaction in tumor cells, positive reaction in small lymphocytes (LCA immunostaining, ob. 40×).

Discussion

Nasopharyngeal cancer in the western world is relatively rare and typically presents as advanced stage disease.

In the United States, only 3% of NPC occurs in patients younger than 19 years of age according to Surveillance, Epidemiology and End Results (SEER) data [3], and pediatric NPC is more prevalent among African-Americans

and its geographic distribution favors southern states [4]. Furthermore, approximately 38% of the cases in NPC patients younger than 20 years or between 20 and 40 years were undifferentiated subtype, respectively, whereas patients older than 40 years were often diagnosed with keratinizing squamous cell carcinoma. The randomized study conducted by the Pediatric Oncology Group (POG) enrolled 18 patients from 59 institutions to the POG

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9486 study, and majority of patients from the United States were from Southeastern or Southern-Midwestern institutions. Among all the accrued patients, 65% were reportedly of African-American origin [5].

Ning JP et al. (1990) in a non-Cantonese rural population of southern China showed that approximately 50% of NPC cases had exposure to salted fish. The risk of NPC was found to be increased with earlier age at first exposure, frequency and duration of consumption and the method of cooking the salted fish. These findings further strengthen the hypothesis that the risk of developing NPC is diet related and that Chinese salted fish and other traditional Chinese foods are important potential human nasopharyngeal carcinogens [2].

Yu MC et al. (1990) reported a significant three-fold risk in smokers of 30 and more cigarettes a day, while living with a smoker through childhood constitutes a significant and independent risk factor for NPC. Studies of the possible role of previous ear or nose disease suggested by a history of a chronic ear or nose condition (rhinitis, sinusitis, nasal polyp or otitis media) all seem to indicate a positive and significant association with NPC development [6].

NPC was first described as a separate entity by Regaud C and Schmincke A in 1921 [7, 8].

The World Health Organization (WHO) classifies NPC into two histopathological types based on the degree of differentiation [9].

Type 1: keratinizing squamous cell carcinoma can be histopathologically subdivided in undifferentiated and differentiated. This distinction has no clinical or prognostic significance. The tumor is comprised of small irregular islands, solid or dyscohesive sheets of cells or has trabecular pattern of growth in a stroma with numerous small lymphocytes and plasma cells. The both subtypes can be seen in the same patient. In that case, the tumor will be classified according to the major component. The differentiated subtype and its variants (verrucous carcinoma, papillary squamous cell carcinoma, basaloid squamous cell carcinoma, spindle cell carcinoma, adenosquamous carcinoma, acantholytic squamous cell carcinoma), are seen in 5–10% of cases of NPC and is characterized by well-differentiated cells that produce keratin and demonstrated the presence of intracellular bridges when observed under the electron microscope. The undifferentiated subtype is more common is less differentiated, with highly variable cell types (clear cell, spindle cell, anaplastic) [10, 11]. The tumor has crowded syncytial-appearing being composed of large cells with indistinct cell borders. The nuclei are vesicular, with big nucleoli. Sometimes small areas of squamous differentiation can be observed but this feature was not noted in our case, probably because of the small size of the biopsies.

Type 2: non-keratinizing (cylindrical cell, transitional) carcinoma, varies in cell differentiation (from mature to anaplastic cells), but typically there is a lack of cell maturation, the cells, like in urothelial carcinoma, which is a subtype of this tumor, do not produce keratin. It has a ribbon-like pattern of growth, with small islands or isolated cells invading the stroma. Cytologic atypia can be prominent. Sometimes, mucus-producing cells can be observed. If the tumor is poorly differentiated, the

differential diagnosis with olfactory neuroblastoma and neuroendocrine carcinoma must be made.

Types 2 and undifferentiated form of Type 1 are Epstein–Barr virus (EBV) associated and have better prognoses than type 1 [3–4, 12–14].

NPC is one of the few malignancies in childhood that emerge from the epithelium, and constitutes 1–5% of all pediatric cancers and 20–50% of all primary nasopharyngeal malignant tumors in children [12]. Several studies indicated the distinct clinical features of pediatric NPC when compared with its adult counterpart. Young patients have a higher rate of lymph node metastasis, fewer WHO Type 1 (keratinizing) tumors, and a better prognosis when compared with adults [3].

Khabir A et al. (2000) previously demonstrated that p53 accumulation is much less frequent in younger patients. The same group of researchers investigated Bcl-2 and Bcl-X expression by immunohistochemistry in patients below 30-year-old or those aged over 30 years. The average Bcl-2 score was found to be much lower for patients below 30-year-old, and concluded that this finding strengthened their hypothesis that oncologic mechanisms may be different for pediatric and adult patients. Their comparison of clinical data revealed a major difference between patients below 30-year-old or older in terms of frequency of lymph node involvement also. While all patients below 30-year-old had clinical lymph node invasion, the figure was 66% for patients older than 30-year-old [13]. The same observation was made previously by Maalej M et al. (1995) and they found that almost all the patients in their series with T4N0 disease were over 30-year-old [16].

Historically, radiotherapy alone was used for both early stage and advanced nasopharyngeal carcinoma. Interpreting the results of studies from various parts of the world is difficult, as the prognostic roles of EBV and histological classifications are incompletely understood.

The patient was addressed for radiochemotherapy. The University of Michigan and Washington University have reported reductions in late xerostomia with Intensity-Modulated Radiation Therapy (IMRT) [17]. IMRT has essentially become standard of care for the majority of head and neck cancer sites. IMRT is a useful technique for the nasopharynx, due to the proximity of critical structures to the target. With conventional therapy, there is considerable risk of late morbidity.

While the concomitant chemotherapy and radiation, with or without adjuvant chemotherapy, is the current standard for adult patients with NPC, neoadjuvant chemo-therapy with radiotherapy has gained popularity parallel to other pediatric treatment protocols in various solid tumors.

The ideal radiotherapy in the nasopharyngeal carcinoma implicates a 40 Gy (2 Gy/fraction) from 70 Gy total dose prescribed [9].

Although multimodality treatment has increased the five-year overall survival to 70–90%, late morbidity is a major concern. Any potential reduction in radiation field and doses is desirable due to the significant chronic morbidity among the long-term survivors.

Conclusions

The age of onset, the clinical signs and symptoms and minimum involvement of lymph nodes represent the

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particular aspects of the case. The patient was addressed for radiochemotherapy. NPC is a rare disease in children with distinct epidemiological and etiological features, histopathological characteristics, and clinical presentation. Children with NPC usually have the undifferentiated variant of the disease, which is associated with advanced regional spread and distant metastases.

Acknowledgments This article was supported by Doctoral Grant No.

970/31.01.2013 of “Victor Babeş” University of Medicine and Pharmacy, Timişoara, Romania.

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Corresponding author Flavia Baderca, Lecturer, MD, PhD, Department of Microscopic Morphology, “Victor Babeş” University of Medicine and Pharmacy, 2 Eftimie Murgu Square, 300077 Timişoara, Romania; Phone +40256–202 619, e-mail: flaviabaderca@ yahoo.com Received: July 23, 2013

Accepted: September 20, 2013