1 Japanese Journal of Clinical Oncology, 2015, 45(4) doi: /jjco/hyv008 Advance Access Publication Date: 10 February 2015 Original Article Original Article Nine cases of carcinoma with neuroendocrine features in the head and neck: clinicopathological characteristics and clinical outcomes Takao Hamamoto 1, *, Satoshi Fujii 2, *, Masakazu Miyazaki 1, Takeshi Shinozaki 1, Toshifumi Tomioka 1, and Ryuichi Hayashi 1 1 Division of Head and Neck Surgery, National Cancer Center Hospital East, Chiba, and 2 Pathology Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, Chiba, Japan *For reprints and all correspondence: Takao Hamamoto, Department of Otorhinolaryngology, Head and Neck Surgery, Hiroshima University Hospital, 1-2-3, Kasumi, Hiroshima , Japan; Division of Head and Neck Surgery, National Cancer Center Hospital East, 6-5-1, Kashiwanoha, Kashiwa, Chiba , Japan. Satoshi Fujii, Pathology Division, Research Center for Innovative Oncology, National Cancer Center Hospital East, 6-5-1, Kashiwanoha, Kashiwa, Chiba , Japan. Received 12 March 2014; Accepted 9 January 2015 Abstract Objective: As neuroendocrine carcinomas in the head and neck region are extremely rare, their clinicopathological characteristics remain largely unknown. Moreover, the 2005 World Health Organization classification criteria for head and neck carcinomas with neuroendocrine features have numerous limitations. Therefore, the clinicopathological features and patient outcomes of these tumors must be clarified. Methods: Between 2007 and 2012, we encountered nine cases of head and neck cancer involving a neuroendocrine carcinoma component. We investigated these tumors according to the 2010 World Health Organization classification criteria for neuroendocrine tumors, and their clinicopathological characteristics and clinical outcomes were examined. Results: Carcinomas with neuroendocrine features were found to have an aggressive clinical course, which corresponded with the Ki-67 index and mitotic count. Conclusions: Owing to the difficulty in appropriately diagnosing head and neck carcinomas with neuroendocrine features using the current classification system, a new classification system should be developed for use in these cases. Key words: neuroendocrine carcinoma, head and neck, small-cell carcinoma, immunohistochemistry Introduction Neuroendocrine carcinomas (NECs) may, on rare occasions, arise in the head and neck region, including in the larynx, nasal cavity or paranasal cavities (1 3). The 2005 World Health Organization (WHO) classification criteria outlined a classification system for NECs in the larynx, in which these tumors were classified into four types: (i) typical carcinoid; (ii) atypical carcinoid; (iii) small-cell carcinoma, neuroendocrine type and (iv) combined small-cell carcinoma, neuroendocrine type, with non-small-cell carcinoma (4). However, the 2005 WHO classification criteria for NECs in the head and neck region have some limitations. First, large cell NEC is wrongly classified as atypical carcinoid, a malignancy commonly confused with carcinoma and which cannot be clearly distinguished from NEC. Additionally, this classification lacks specific definitions on how to diagnose carcinomas with neuroendocrine features and regarding the quantity of neuroendocrine component needed for the tumor to be classified as combined NEC upon immunohistochemical analysis. The Author Published by Oxford University Press. All rights reserved. For Permissions, please 328
2 Jpn J Clin Oncol, 2015, Vol. 45, No In 2010, the WHO released their latest updated classification criteria for NECs in the gastrointestinal tract and pancreas (5). According to these criteria, all neuroendocrine neoplasms are referred to as being potentially malignant. This classification system divides neuroendocrine neoplasms into neuroendocrine tumor (NET) Grade 1, NET Grade 2 and NEC, based on morphological criteria and assessment of the proliferative rate using the Ki-67 index and mitotic counts, as summarized in Table 1. As previous studies have reported that evaluation of the tumor proliferative rate may also be of prognostic significance for tumors in the head and neck region (3,6,7), we recognized that the suitability of the 2010 WHO classification for this region should be investigated. Between 2007 and 2012, we encountered nine cases of carcinoma with neuroendocrine features in our hospital. We investigated the clinical outcomes and clinicopathological characteristics of these tumors. The purpose of this study was to investigate these tumors in view of the 2010 WHO classification, which is based on analyses of the proliferative rate using the Ki-67 index, mitotic counts and the expression of the tumor suppressor p53. Patients and methods Between January 2007 and December 2012, we encountered nine cases of carcinoma with neuroendocrine features at the National Cancer Center Hospital East, Chiba, Japan, which were all diagnosed in accordance with the 2005 WHO classification criteria using primary surgically resected specimens. The clinicopathological records were reviewed and evaluated retrospectively after the institutional review board of the National Cancer Center approved all protocols. Information regarding the patients sex, age, primary tumor site, clinical history, staging evaluation according to the American Joint Committee on Cancer TNM staging manual, treatment history and outcome was collected from documented clinical reports and imaging findings. Histopathological and immunohistochemical analyses were routinely performed in our institution using formalin-fixed and paraffinembedded tissue blocks with surgically resected specimens. Neuroendocrine features can be detected by immunohistochemical staining using neuroendocrine cell differentiation markers such as chromogranin A, synaptophysin, cluster of differentiation (CD)56, ASH1L [(absent, small or homeotic)-like (Drosophila)], and β-1,3-glucuronyltransferase 1, among others (4). However, as these markers are not sufficiently sensitive or specific individually, a combination of markers should be examined to demonstrate the existence of histopathological neuroendocrine features. In our hospital, routine immunohistochemical staining of tumors using anti-chromogranin A (LK2H10, Roche Diagnostics Corporation), synaptophysin (MRQ-40, Roche Diagnostics Corporation), CD56 (MRQ-42, Roche Diagnostics Corporation) and Ki-67 (N1633, DAKO, Glostrup, Denmark) antibodies was performed using the Table 1. World Health Organization classification of neuroendocrine tumors in the gastrointestinal tract and pancreas (2010) Tumor type, grade Mitoses (/10 HPFs) Ki-67 index (%) NET G1 <2 2% G % NEC G3 >20 >20% NET, neuroendocrine tumor; NEC, neuroendocrine carcinoma; HPF, high-power field. automated Benchmark XT platform (Ventana Medical Systems, Tucson, AZ, USA) to make a pathological diagnosis. Moreover, immunohistochemical staining using an anti-p53 polyclonal antibody (RSP53, NICHIREI BIOSCIENCES INC., Tokyo, Japan, Code No ) was performed. All tumor samples and specimens were re-evaluated by experienced pathologists in our institution in accordance with the 2010 WHO classification criteria, as summarized in Table 1. The mitotic count was determined by evaluating hematoxylin and eosin (H&E)-stained sections using a light microscope ( 400 magnification). Mitotic figures were counted at the base of the tumors in the most active area ( hot spots ) in at least 50 high-power fields (HPFs). The number of mitotic figures per 10 HPFs was calculated and presented as number per 10 HPFs. Moreover, Ki-67 expression was quantified using a visual grading system. Ki-67-positive cells were counted, and a percentage was calculated. If the staining was homogenous, the percentage of Ki-67 positive cells among the total number of carcinoma cells counted in the section was determined by counting in 10 randomly selected fields. When hot spots, defined as areas in which Ki-67 positivity was particularly prevalent, were present, the positive rate in the hot spot was recorded as the Ki-67 labeling index. All immunohistochemical staining procedures included an external control (using the same tissue section) to validate the status of Ki-67 protein expression for each case. Regarding the immunohistochemical staining of p53, in cases where >10% of the total tumor cells showed positive p53 expression, the tumor was considered p53 positive. Results All nine patients were Japanese (age range, years); had a suspected malignant tumor, as determined by analysis of the biopsy tissue; and underwent surgery as the primary treatment. The patient and clinical characteristics are summarized in Table 2. The results of the immunohistochemical staining for CD56, synaptophysin, chromogranin A, Ki-67 and p53 and the mitotic count are summarized in Table 3. Case reports Case 1: NEC of the maxillary sinus A 68-year-old man was diagnosed with a maxillary tumor (ct3n2am0) and underwent surgery. He experienced local recurrence and neck lymph node metastasis 5 months and 1 year after the operation, respectively, and hence underwent additional surgeries. Two years later, clinical investigation revealed multiple distant (mediastinum region, lung and bone) metastases. Although he underwent palliative chemotherapy, he died of the disease 6 years after the initial diagnosis. Pathological findings. H&E staining showed a poorly differentiated tumor with large alveolar structure and invasive growth to the subcutaneous tissue. The tumor cells had large, irregular-shaped nuclei, with enlarged nucleoli and eosinophilic granular cytoplasm observed, and showed partial necrosis. The tumor cells consisted of polygonal shaped cells and spindle shaped cells (Fig. 1A). The mitotic rate of the tumor was relatively low (5/10 HPFs). Immunohistochemical analyses showed positive staining for CD56, synaptophysin (Fig. 1B) and chromogranin A. The Ki-67 labeling index was high (40%), and the p53 expression was negative. Based on the above-mentioned findings, NEC was diagnosed. Case 2: NEC of the larynx A 78-year-old man was diagnosed with a larynx tumor (ct2n0m0) and underwent surgery. Two years after the first operation, he
3 330 Neuroendocrine carcinoma in the head and neck Table 2. Patients clinicopathological characteristics Case Age (year) Sex Primary site TNM stage Diagnosis Outcome Follow-up period 1 68 Male Maxilla T3N2aM0 NEC Dead 6 years 2 78 Male Larynx T2N0M0 NEC Alive 6 years 5 months 3 75 Male Hypopharynx T1N0M0 Small-cell carcinoma + SCC Alive 10 months 4 59 Male Oropharynx T2N2bM0 Small-cell carcinoma + SCC Dead 5 months 5 79 Male Larynx T3N2bM0 SCC + NEC Alive with DM 4 years 6 41 Male Unknown T0N2bM0 Spindle cell carcinoma with NEF Dead 11 months 7 66 Male Maxilla T3N0M0 SCC with NEF Dead 1 year 3 months 8 35 Male Maxilla T2N0M0 Adenocarcinoma with NEF Dead 2 years 3 months 9 64 Female Oral cavity T2N2bM0 Undifferentiated carcinoma with NEF Alive 5 years 7 months SCC, squamous cell carcinoma; NEF, neuroendocrine features; DM, distant metastasis. Table 3. Immunohistochemical findings Case CD56 Synaptophysin Chromogranin A Mitotic count (number/10 HPFs) Ki-67 index (%) p53-percentage of positive cells (%) CD56, cluster of differentiation 56. experienced recurrence in the neck lymph nodes and underwent neck dissection. At the latest follow-up, 6 years and 5 months after the initial diagnosis, he was alive with no relapse or evidence of disease. Pathological findings. Oval or polygonal tumor cells with solid growth and invasion into the subcutaneous tissue around the larynx were observed. The tumor cells had granular and eosinophilic cytoplasm and darkly stained nuclei. The mitotic rate was 7/10 HPFs (Fig. 2A). Immunohistochemical analyses showed positive staining with antibodies against synaptophysin and chromogranin A (Fig. 2B). The Ki-67 labeling index was slightly elevated (25%), and p53 expression was negative. Based on the above findings, the tumor was diagnosed as a NEC. Case 3: combined small-cell carcinoma and squamous cell carcinoma of the hypopharynx A 75-year-old man was diagnosed with a hypopharynx tumor (ct1n0m0) and underwent partial hypopharyngectomy and neck lymph node dissection. He received no adjuvant treatment after the operation, and was alive with no evidence of recurrence at the latest follow-up, 10 months after the initial diagnosis. Pathological findings. Macroscopic findings of the tumor showed two kinds of surfaces: an ash-grayish, irregular surface and a yellow flat and smooth surface. The former component consisted of polygonal tumor cells showing a solid growth pattern with keratinizing materials accompanied with intraepithelial spread. Immunohistochemistry of these tumor cells showed no evidence of neuroendocrine features, and this tumor component was accordingly diagnosed as squamous cell carcinoma (Fig. 3A). The latter tumor component was a solid alveolar component composed of closely packed cells with inconspicuous cytoplasm and round, oval-to-spindle shaped nuclei, showing a high nuclear/cytoplasmic (N/C) ratio (Fig. 3B). Immunohistochemical analyses revealed that these tumor cells showed neuroendocrine features; positive CD56, chromogranin A and synaptophysin expression. The Ki-67 index was 70%, and the p53 expression was negative. Based on the above-mentioned findings, the pathological diagnosis of this tumor was combined small-cell carcinoma and squamous cell carcinoma. Case 4: combined small-cell carcinoma and squamous cell carcinoma A 59-year-old man was diagnosed with an oropharyngeal tumor (ct2n2bm0) and underwent surgery and adjuvant radiotherapy. One month post-surgery, clinical investigation revealed multiple liver metastases, and the patient died of the disease 3 months after the initial diagnosis. Pathological findings. That tumor consisted of two co-existing components. The major component consisted of tumor cells with inconspicuous cytoplasm, showing a high N/C ratio (Fig. 4A, right). The tumor cells proliferated with solid growth, and the mitotic rate was very high (94/10 HPFs). Immunohistochemistry of this lesion showed CD56 positivity (Fig. 4B, right), but synaptophysin and chromogranin A negativity. The Ki-67 index was 70%, and the p53 expression was positive. The major tumor component was diagnosed as small-cell carcinoma. The second, minor component consisted of polygonal cells showing keratinizing or stratified differentiation (Fig. 4A, left). Immunohistochemistry of this lesion showed no evidence of neuroendocrine features, and the component was diagnosed as squamous cell carcinoma (Fig. 4B, left). Based on the findings described above, the tumor was diagnosed as combined small-cell carcinoma and squamous cell carcinoma.
4 Jpn J Clin Oncol, 2015, Vol. 45, No Figure 1. Case 1: neuroendocrine carcinoma (NEC) of the maxillary sinus. (A) Hematoxylin and eosin (H&E) staining. (B) Synaptophysin. Figure 2. Case 2: NEC of the larynx. (A) H&E staining. (B) Synaptophysin. Figure 3. Case 3: combined small-cell carcinoma and squamous cell carcinoma of the hypopharynx. (A) H&E staining. (B) H&E staining. Case 5: squamous cell carcinoma with neuroendocrine features A 79-year-old man was diagnosed with a laryngeal tumor (ct3n2bm0) and underwent surgery. Eighteen months post-surgery, he experienced local recurrence and underwent radiotherapy. He was alive with mediastinum lymph node metastasis at the latest follow-up, four years after the initial diagnosis. Pathological findings. The tumor consisted of a solid carcinoma component covered by squamous cell carcinoma showing intraepithelial spread. In the subepithelial part, the tumor cells formed solid growth, and multifocal necrosis was observed. The tumor cells showed moderate sized cytoplasm and irregularly shaped nuclei with increased chromatin (Fig. 5A). The mitotic rate of the tumor cells was high (36/ 10 HPFs). Immunohistochemical staining showed strong reactivity for CD56 (Fig. 5B) and positivity for p53 (90%) in the latter tumor component. High positivity of p53 was also observed in the former squamous cell carcinoma component. The Ki-67 index was 50%. Based on the above-mentioned findings, the pathological diagnosis of this tumor was squamous cell carcinoma with neuroendocrine features.
5 332 Neuroendocrine carcinoma in the head and neck Figure 4. Case 4: combined small-cell carcinoma and squamous cell carcinoma. (A) H&E staining. (B) CD56. Figure 5. Case 5: squamous cell carcinoma with neuroendocrine features. (A) H&E staining. (B) CD56. Figure 6. Case 6: metastatic spindle cell carcinoma with neuroendocrine features. (A) H&E staining. (B) CD56. Case 6: metastatic spindle cell carcinoma with neuroendocrine features A 41-year-old man was diagnosed with an unknown primary tumor (ct0n2bm0) and underwent neck dissection. Six months after the initial operation, follow-up computed tomography (CT) imaging revealed an adrenal gland tumor, multiple liver metastases and peritoneal dissemination. CT-guided fine needle aspiration of the adrenal tumor revealed identical pathological features as the original neck lymph node metastasis. The patient underwent palliative chemotherapy but died of the disease 11 months after the initial diagnosis. Pathological findings. The histological assessment showed spindle cell proliferation with bundle formation and multifocal necrosis (Fig. 6A). The tumor cells showed apoptosis and frequent mitoses (mitotic rate, 11/10 HPFs). Immunohistochemistry showed multifocal positivity for CD56 in the whole tumor (Fig. 6B). The Ki-67 index was 50%, and the
6 Jpn J Clin Oncol, 2015, Vol. 45, No Figure 7. Case 7: squamous cell carcinoma with neuroendocrine features. (A) H&E staining. (B) Synaptophysin. Figure 8. Case 8: adenocarcinoma with neuroendocrine features. (A) H&E staining. (B) CD56. p53 expression was positive. The findings described above were consistent with metastatic spindle cell carcinoma with neuroendocrine features. Case 7: squamous cell carcinoma with neuroendocrine features A 66-year-old man was diagnosed with maxillary carcinoma (ct3n0m0) and underwent partial maxillectomy along with adjuvant chemo-radiotherapy. One year after his first treatment, clinical investigation revealed neck lymph node metastasis and multiple distant (mediastinum, lung and bone) metastases. Although he underwent palliative chemotherapy, he died of the disease 2 years and 3 months after the initial diagnosis. Pathological findings. The tumor consisted of polygonal cells showing solid growth. However, undifferentiated cells with alveolar configuration were also observed (Fig. 7A). The mitotic rate was very high (95/ 10 HPFs). Immunohistochemistry of these undifferentiated cells showed neuroendocrine features; positive expression of CD56 and synaptophysin (Fig. 7B) with a high Ki-67 index (70%). The p53 expression was positive. Accordingly, the tumor was diagnosed as squamous cell carcinoma with neuroendocrine features. Case 8: adenocarcinoma with neuroendocrine features A 66-year-old man was diagnosed with a maxillary tumor (ct2n0m0) and underwent partial maxillectomy. Six months post-surgery, he experienced local recurrence and underwent chemo-radiotherapy. In spite of the chemo-radiotherapy, the tumor enlarged and the patient died of the disease 2 years and 3 months after the initial diagnosis. Pathological findings. The tumor consisted of polygonal tumor cells, which showed a high N/C ratio and a high mitotic rate (88/10 HPFs), with solid alveolar growth and myxomatous stroma. The tumor cells showed a partial duct-like configuration (Fig. 8A) filled with Alcian Blue-periodic acid-schiff-positive mucus materials, with neutrophils infiltrate observed inside of, and around, the tumor alveolar structure. Immunohistochemical analysis revealed that the tumor cells showed neuroendocrine features; positive expression of CD56 (Fig. 8B) and chromogranin A. The Ki-67 index was 60%, and p53 expression was negative. Based on these findings, the tumor was diagnosed as adenocarcinoma with neuroendocrine features. Case 9: undifferentiated carcinoma with neuroendocrine features A 64-year-old woman was diagnosed with oral carcinoma (ct2n2bm0) and underwent surgery. She received no adjuvant treatment and was alive with no evidence of recurrence at the latest follow-up, 5 years and 7 months after the initial diagnosis. Pathological findings. The tumor consisted of polygonal shaped and undifferentiated tumor cells showing solid growth with cobblestone appearance, and ulcer formation was observed. Most of the tumor cells
7 334 Neuroendocrine carcinoma in the head and neck Figure 9. Case 9: undifferentiated carcinoma with neuroendocrine features. (A) H&E staining. (B) CD56. were medium sized, and had oval-round nuclei with increased chromatin and a high N/C ratio (Fig. 9A). Some scattered large tumor cells with abnormal mitosis were observed, and the mitotic rate was very high (69/10 HPFs). No evidence of obvious keratinization or glandular differentiation was found, and the tumor was hence diagnosed as undifferentiated carcinoma. Immunohistochemistry showed neuroendocrine features ( positive CD56 expression) (Fig. 9B), the Ki-67 index was 80%, and p53 expression was negative, suggesting a diagnosis of undifferentiated carcinoma with neuroendocrine features. Discussion We here reported on our experiences with nine cases of carcinoma with neuroendocrine features. The pathological findings of these carcinomas showed various characteristics and could be broadly categorized into three groups: Group 1: tumors composed of pure NEC cells without any other carcinoma component (Cases 1 and 2), Group 2: NEC component and other carcinoma component existing in the same tumor, designated as combined tumor with a demarcated border (Case 3, 4 and 5) and Group 3: tumors diagnosed as ordinary carcinoma accompanied with neuroendocrine features observed upon immunohistochemical analysis (Case 6, 7, 8 and 9). Almost all of these tumors showed aggressive clinical courses with early recurrence and distant metastasis, which had been detected clinically even in the early stages. In the 2005 WHO classification, no standard definition specifically describes the quantity of neuroendocrine components required for classification as NECs. In other words, there is no definition to distinguish between combined carcinoma, in which NEC and another carcinoma coexist (Group 2), and mixed carcinoma, in which the ordinary carcinoma such as squamous cell carcinoma and adenocarcinoma shows neuroendocrine features (Group 3). In recent years, the head and neck pathology community has reached the consensus that the current WHO classification of head and neck carcinomas does not adequately describe the clinicopathological spectrum of NECs in the head and neck (3,8,9), and Xu et al. (10) recently proposed a new classification of NECs based on some elements of the 2010 WHO classification. Although current data are not sufficient to support incorporation of the Ki-67 index as a grading system of NECs in the head and neck region, several recent studies have nonetheless reported high MIB-1 labeling indices of NECs in this region (8,9,11). With regard to the diagnosis of NEC, the 2010 WHO classification appears to be useful for the evaluation of malignant potential based on proliferation kinetics and may also be of prognostic significance. Based on our results herein, we believe that, although head and neck carcinomas with neuroendocrine features are currently not diagnosed fully in accordance with the conventional WHO classification criteria, they could potentially be classified as NECs, based on both the Ki-67 index and mitotic count, which correspond with the 2010 WHO criteria for NECs. In conclusion, based on our experiences in this study, we believe that carcinomas with neuroendocrine features may be diagnosed as NECs. As these carcinomas have an aggressive behavior and poor prognosis, correct classification is crucial for proper disease management. Because there is some difficulty in appropriately diagnosing NEC using the 2005 WHO classification system, we recommend that the new (2010) classification system should instead be adapted for use in the head and neck region. However, further studies are needed to improve our understanding of the clinicopathological features of NEC and to establish the appropriate treatment strategies. Acknowledgements The authors thank Dr T. Mukaigawa (National Cancer Center Hospital East, Chiba, Japan) for his helpful suggestions and S. Yanagi (National Cancer Center Hospital East, Chiba, Japan) for technical support. Conflict of interest statement None declared. References 1. Ferlito A, Devaney KO, Rinaldo A. Neuroendocrine neoplasms of the larynx: advances in identification, understanding, and management. Oral Oncol 2006;42: Ferlito A, Silver CE, Bradford CR, Rinaldo A. Neuroendocrine neoplasms of the larynx: an overview. Head Neck 2009;31: Lewis JS Jr, Ferlito A, Gnepp DR, et al. Terminology and classification of neuroendocrine neoplasms of the larynx. Laryngoscope 2011;121:
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