primary malignant tumors of the hypopharynx are predominantly squamous cell carcinomas ( sqccs ) , as primary small - cell carcinoma ( smcc ) of the hypopharynx is rare .
combined primary smcc and sqcc of the hypopharynx , referred to as composite tumor of the hypopharynx , is even more rare , with only 3 cases previously reported ( 13 ) .
we herein report a the case of a patient with primary combined smcc with an sqcc element and investigate the expression of specific proteins for molecular - targeted therapy .
a 74-year - old man , with a ~50-year history of excessive alcohol consumption and smoking , presented with a 3-month history of throat pain and hoarseness .
on hypopharyngoscopy , a tumor was identified in the right anterior wall of the piriform sinus ( fig .
a pathologist analyzed the biopsy sample and diagnosed the lesion as sqcc , as no smcc component was identified in the biopsy specimen .
the right vocal cord was not fixed . a contrast computed tomography ( ct ) scan of the neck revealed a heterogeneously enhanced tumor sized 251738 mm extending through the right piriform sinus ( fig .
moreover , fluorodeoxyglucose positron emission tomography ( fdg - pet ) revealed high - level accumulation in the primary tumor , with a maximum standardized uptake value of 14.6 .
there was no evidence of cervical lymph node metastasis , primary lung tumor , or distant metastasis .
the patient was diagnosed with hypopharyngeal cancer classified as t2n0m0 , according to the 2009 union for international cancer control staging system ( 4 ) .
we performed a tracheostomy , total laryngectomy , right hemithyroidectomy and bilateral lateral neck dissection ( level ii 
microscopically , two components were identified in the lesion , namely a poorly differentiated sqcc and an smcc containing chromatin - rich nuclei with scanty cytoplasm ( fig .
pathologically , multiple metastatic cervical lymph nodes were identifed bilaterally ( right side , levels ii and iii ; and left side , level iii ) .
however , after the first course , adjuvant chemotherapy was discontinued due to prolonged bone marrow suppression , although the therapy was effective .
however , despite treatment , distant metastases in the thoracic vertebrae ( t5 , 9 and 10 ) were confirmed by ct 6 months later . the patient 's condition started to deteriorate and he succumbed to the disease 7 months after the initial treatment . on gross examination of the laryngectomy specimen ,
the tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology , ranging from ulcerated , to nodular , to plaque - like growth .
the tumor infiltrated 1.3 cm in depth , but did not involve the thyroid cartilage , vocal cord , or trachea .
microscopic sections from the larynx revealed a combined tumor , composed predominantly of smcc localized in the upper side and sqcc localized in the lower side ( figs . 1 and 2a ) .
we observed 2 components in the lesion : a poorly differentiated sqcc and a small - sized smcc with finely granular , hyperchromatic nuclei , inconspicuous nucleoli and scanty cytoplasm .
the metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels ( fig .
diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis ( fig .
the sqcc component was negative for neuroendocrine markers , including cd56 , synaptophysin and chromogranin a , although it was positive for ck 34e12 ( fig .
immunoreactivity to ki-67 was seen in the nuclei of tumor cells . in the smcc component ,
the ki-67 labeling index was 50.2% , while that in the sqcc component was 47.5% ( fig .
to predict the prognosis of this patient , we evaluated the expression of epidermal growth factor receptor ( egfr ) .
furthermore , to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc , the expression of proteins such as platelet - derived growth factor receptor  ( pdgfr ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) and kit was examined .
on immunohistochemical analysis , the smcc element was strongly positive for kit and pdgfr ; however , egfr and vegfr2 were not expressed ( fig .
the sqcc element was mildly positive for pdgfr and egfr ; however , kit and vegfr2 were not expressed ( fig .
genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit ( qiagen , hilden , germany ) , according to the manufacturer 's protocol . a molecular genetic analysis of kit ( exons 9 , 11 , 13 and 17 ) and pdgfr ( exons 12 , 14 and 18 ) was performed using the polymerase chain reaction ( pcr ) direct sequencing method , as previously reported ( 58 ) .
pcr products were extracted and subjected to a computed automatic dna sequencing ( abi prism 3100 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) .
analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position .
comparison of this sequence variation with the single - nucleotide polymorphism ( snp ) database revealed the presence of a known snp at codon 567 of pdgfr ( 9 ) .
the hpv status was determined using the hpv typing set ( takara bio . , tokyo , japan ) , a pcr primer set specifically designed to identify hpv genotypes 16 , 18 , 31 , 33 , 35 , 52 and 58 in genomic dna .
the pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide .
our case was negative and not considered as a high - risk hpv status ( fig .
on gross examination of the laryngectomy specimen , a mass involving the right piriform sinus was palpable .
the tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology , ranging from ulcerated , to nodular , to plaque - like growth .
the tumor infiltrated 1.3 cm in depth , but did not involve the thyroid cartilage , vocal cord , or trachea .
microscopic sections from the larynx revealed a combined tumor , composed predominantly of smcc localized in the upper side and sqcc localized in the lower side ( figs . 1 and 2a ) .
we observed 2 components in the lesion : a poorly differentiated sqcc and a small - sized smcc with finely granular , hyperchromatic nuclei , inconspicuous nucleoli and scanty cytoplasm .
the metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels ( fig .
diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis ( fig .
the sqcc component was negative for neuroendocrine markers , including cd56 , synaptophysin and chromogranin a , although it was positive for ck 34e12 ( fig .
immunoreactivity to ki-67 was seen in the nuclei of tumor cells . in the smcc component ,
the ki-67 labeling index was 50.2% , while that in the sqcc component was 47.5% ( fig .
furthermore , to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc , the expression of proteins such as platelet - derived growth factor receptor  ( pdgfr ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) and kit was examined .
on immunohistochemical analysis , the smcc element was strongly positive for kit and pdgfr ; however , egfr and vegfr2 were not expressed ( fig .
the sqcc element was mildly positive for pdgfr and egfr ; however , kit and vegfr2 were not expressed ( fig .
genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit ( qiagen , hilden , germany ) , according to the manufacturer 's protocol . a molecular genetic analysis of kit ( exons 9 , 11 , 13 and 17 ) and pdgfr ( exons 12
, 14 and 18 ) was performed using the polymerase chain reaction ( pcr ) direct sequencing method , as previously reported ( 58 ) .
pcr products were extracted and subjected to a computed automatic dna sequencing ( abi prism 3100 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) .
analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position .
comparison of this sequence variation with the single - nucleotide polymorphism ( snp ) database revealed the presence of a known snp at codon 567 of pdgfr ( 9 ) .
tokyo , japan ) , a pcr primer set specifically designed to identify hpv genotypes 16 , 18 , 31 , 33 , 35 , 52 and 58 in genomic dna .
the pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide .
our case was negative and not considered as a high - risk hpv status ( fig .
on gross examination of the laryngectomy specimen , a mass involving the right piriform sinus was palpable .
the tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology , ranging from ulcerated , to nodular , to plaque - like growth .
the tumor infiltrated 1.3 cm in depth , but did not involve the thyroid cartilage , vocal cord , or trachea .
microscopic sections from the larynx revealed a combined tumor , composed predominantly of smcc localized in the upper side and sqcc localized in the lower side ( figs . 1 and 2a ) .
we observed 2 components in the lesion : a poorly differentiated sqcc and a small - sized smcc with finely granular , hyperchromatic nuclei , inconspicuous nucleoli and scanty cytoplasm .
the metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels ( fig .
diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis ( fig .
the sqcc component was negative for neuroendocrine markers , including cd56 , synaptophysin and chromogranin a , although it was positive for ck 34e12 ( fig .
immunoreactivity to ki-67 was seen in the nuclei of tumor cells . in the smcc component ,
the ki-67 labeling index was 50.2% , while that in the sqcc component was 47.5% ( fig .
furthermore , to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc , the expression of proteins such as platelet - derived growth factor receptor  ( pdgfr ) , vascular endothelial growth factor receptor 2 ( vegfr2 ) and kit was examined .
on immunohistochemical analysis , the smcc element was strongly positive for kit and pdgfr ; however , egfr and vegfr2 were not expressed ( fig .
the sqcc element was mildly positive for pdgfr and egfr ; however , kit and vegfr2 were not expressed ( fig .
genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit ( qiagen , hilden , germany ) , according to the manufacturer 's protocol . a molecular genetic analysis of kit ( exons 9 , 11 ,
13 and 17 ) and pdgfr ( exons 12 , 14 and 18 ) was performed using the polymerase chain reaction ( pcr ) direct sequencing method , as previously reported ( 58 ) .
pcr products were extracted and subjected to a computed automatic dna sequencing ( abi prism 3100 genetic analyzer ; applied biosystems , carlsbad , ca , usa ) .
analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position .
comparison of this sequence variation with the single - nucleotide polymorphism ( snp ) database revealed the presence of a known snp at codon 567 of pdgfr ( 9 ) .
tokyo , japan ) , a pcr primer set specifically designed to identify hpv genotypes 16 , 18 , 31 , 33 , 35 , 52 and 58 in genomic dna .
the pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide .
our case was negative and not considered as a high - risk hpv status ( fig .
in the head and neck , smcc most commonly arises in the larynx , but has also been reported in the sinonasal tract and salivary glands ( 10,11 ) .
combined smcc of the larynx has only been reported in 17 cases in the literature to date .
moreover , the majority of the patients succumbed to the disease within 2 years of diagnosis , even with adjuvant radiation and chemotherapy ( 1214 ) . despite the rarity of these tumors , the clinical behavior of combined smcc of the larynx appears to be similar to that of the hypopharynx .
metastasis from a primary lung smcc must be carefully distinguished from a primary hypopharyngeal or laryngeal smcc by imaging studies of the lung ( 15 ) . in this case ,
fdg - pet revealed high - level accumulation in the hypopharynx , without evidence of a lung tumor or distant metastasis . a chest contrast ct scan confirmed the absence of a primary lung tumor . according to the world health organization classification of head and neck tumors in 2005 , neuroendocrine carcinomas may be classified as typical carcinoid , atypical carcinoid , and smcc ( 16 ) .
neuroendocrine types of smcc associated with an sqcc component are referred to as combined carcinomas .
neuroendocrine neoplasms of the hypopharynx and larynx constitute a morphologicaly heterogeneous group of tumors , with considerable differences in clinical behavior and very different treatment strategies ( 12 ) .
combined smcc of the hypopharynx is extremely rare and is often difficult to pathologically diagnose prior to surgical treatment .
moreover , the prognosis is very poor due to early metastasis , without established treatment regimen ( 3 ) .
of the 4 reported cases of combined smcc of the hypopharynx , including our case , 3 succumbed to the disease within 1 year ( table i ) .
multi - modality therapy should be performed for combined smcc of the head and neck region .
the present case reports new findings : positive expression of kit and pdgfr in the smcc element and positive expression of egfr and pdgfr in the sqcc element .
furthermore , to the best of our knowledge , the present study is the first to report a kit and pdgfr mutation analysis in combined hypopharyngeal smcc .
kit and pdgfr , both mapped to chromosome 4q12 , have structural similarities with other pdgfr family members .
a majority of gastrointestinal stromal tumors ( gists ) display a gain - of - function mutation in the kit proto - oncogene that encodes the kit protein .
the majority of mutations occur in kit exon 11 , but mutations may also be found in exon 9 and rarely in exons 13 and 17 .
the pdgfr gene is also very similar to the kit gene , and pdgfr mutations have been found in exons 12 , 14 and 18 .
terada et al ( 18 ) reported kit protein expression in 100% and pdgfr expression in 65% of small - cell lung carcinoma cases ; however , there were no genetic mutations of kit and pdgfr in small - cell lung carcinoma ( 18 ) . in a previously reported case of esophageal combined smcc , kit and pdgfr
were expressed in the smcc component , but not in the sqcc component ( 5 ) .
it was found that smcc of the lung and extrapulmonary organs expressed kit and pdgfr , but there were no mutations in these genes within the hot spots of gists .
no biomarker has been yet proven to predict response to targeted therapy in either smcc or sqcc . moreover
, a predictive biomarker in one cancer type may not be helpful in another cancer type , suggesting that different mechanisms may be involved in combined smcc .
to the best of our knowledge , a molecular - targeted therapy for treating combined smcc of the hypopharynx and larynx has not yet been reported .
our case is the first to report kit and pdgfr expression in combined smcc in the hypopharynx and larynx ; if similar results are obtained from larger series , these data may have management and prognostic implications through the possible use of targeted biological therapy in these tumors . at present ,
smcc treatment remains a significant challenge for the oncologists , with several targeted agents under evaluation .
however , there is no effective molecular - targeted therapy for smcc . evaluating the expression of the kit and pdgfr genes within hypopharyngeal smcc
may facilitate the development of novel agents for the molecular - targeted therapy of this tumor
. however , further investigations are necessary regarding these proteins expression and clinical application of molecular targeted therapy for hypophageal combined smcc .
 we herein report a very rare case of combined small - cell carcinoma ( smcc ) of the hypopharynx , with a squamous cell carcinoma ( sqcc ) element . a 74-year - old man presented with a 3-month history of throat pain and hoarseness . 
 on hypopharyngoscopy , a tumor was identified in the right anterior wall of the piriform sinus and , following examination of a biopsy sample , the lesion was diagnosed as sqcc . 
 total laryngectomy with bilateral neck dissection was performed and the malignancy was diagnosed as combined smcc . 
 one month after surgery , concomitant chemoradiotherapy with cisplatin and etoposide was administered . 
 immunohistochemically , the smcc element was positive for cd56 and ki-67 ( 50.2% ) , whereas the sqcc element was positive for cytokeratin 34e12 and ki-67 ( 47.5% ) . 
 furthermore , the smcc element was positive for kit and platelet - derived growth factor  ( pdgfr ) , while the sqcc element was positive for epidermal growth factor receptor ( egfr ) and pdgfr. by genetic analysis , a silent mutation in the pdgfr gene was recognized . 
 the expression of kit , pdgfr and egfr in this case provided evidence that combined smcc may be a candidate for molecular - targeted therapy , although further investigations are required . 
Introduction
Case report
None
Pathology
DNA extraction, genetic analysis and human papillomavirus (HPV) polymerase chain reaction
Discussion