of 2,310 patients , 401 patients underwent a second biopsy or resection for recurrent or metastatic tumors . out of 401 patients , a total of six patients ( 1.5% ) with primary lung adc showed transformed sclc morphology in second biopsy .
all patients were treated in the department of oncology , samsung medical center ( seoul , korea ) .
clinical and follow - up data were obtained through a retrospective analysis of the medical records , including age , sex , smoking history , treatment , clinical course and follow - ups .
all patients were followed until march 2016 with median follow - up period of 39.2 months .
the study was approved by the institutional review board at samsung medical center ( 2014 - 14 - 08610 ) .
dna was extracted from sections of formalin - fixed , paraffin - embedded ( ffpe ) tissue that was also used for histologic diagnosis .
manual microdissection was performed if tumor cell percentages were less than 70% in available samples .
genomic dna was extracted using qiagen dna ffpe tissue kit ( qiagen , hilden , germany ) according to the manufacturer s instructions . in cases of lung adc , routine
testing for the egfr mutation was performed in the pathology laboratory using peptide nucleic acid  mediated clamping polymerase chain reaction ( pcr ) mutation detection kit
as previously described , and results were retrieved from electronic medical records . for one sclc sample , the egfr mutation was detected using targeted sequencing via illumina hiseq 2500 ( illumina inc .
, san diego , ca , usa ) , which was performed for clinical trial enrollment . for the rest of sclc samples
, the egfr mutation was newly evaluated using cobas test , a real - time pcr test as previously described .
egfr mutation results were available for all samples except for one that had no residual tumor . in the current study , we used representative ffpe tissue sections for immunohistochemical staining ( ihc ) .
ihc for cd56 and thyroid transcription factor 1 ( ttf-1 ) was performed for sclc or combined tumors .
staining was performed on 3-m - thick sections from each case using a biotin - avidin - peroxidase method on a bond - max autostainer ( leica , wetzlar , germany ) after retrieval with t / e buffer ( cd56 ) or citrate buffer ( ttf-1 ) .
we used primary antibodies to cd56 ( 1:200 , novocastra , newcastle upon tyne , uk ) and ttf-1 ( 1:100 , dako , glostrup , denmark ) .
during a 6-year period ( 20102015 ) , there were a total of 2,310 diagnoses of pulmonary adc in our institute .
of 2,310 patients , 401 patients underwent a second biopsy or resection for recurrent or metastatic tumors . out of 401 patients , a total of six patients ( 1.5% ) with primary lung adc showed transformed sclc morphology in second biopsy .
all patients were treated in the department of oncology , samsung medical center ( seoul , korea ) .
clinical and follow - up data were obtained through a retrospective analysis of the medical records , including age , sex , smoking history , treatment , clinical course and follow - ups .
all patients were followed until march 2016 with median follow - up period of 39.2 months .
the study was approved by the institutional review board at samsung medical center ( 2014 - 14 - 08610 ) .
dna was extracted from sections of formalin - fixed , paraffin - embedded ( ffpe ) tissue that was also used for histologic diagnosis .
manual microdissection was performed if tumor cell percentages were less than 70% in available samples .
genomic dna was extracted using qiagen dna ffpe tissue kit ( qiagen , hilden , germany ) according to the manufacturer s instructions . in cases of lung adc , routine
testing for the egfr mutation was performed in the pathology laboratory using peptide nucleic acid  mediated clamping polymerase chain reaction ( pcr ) mutation detection kit
as previously described , and results were retrieved from electronic medical records . for one sclc sample , the egfr mutation was detected using targeted sequencing via illumina hiseq 2500 ( illumina inc . , san diego , ca , usa ) , which was performed for clinical trial enrollment . for the rest of sclc samples ,
the egfr mutation was newly evaluated using cobas test , a real - time pcr test as previously described .
egfr mutation results were available for all samples except for one that had no residual tumor .
in the current study , we used representative ffpe tissue sections for immunohistochemical staining ( ihc ) .
ihc for cd56 and thyroid transcription factor 1 ( ttf-1 ) was performed for sclc or combined tumors .
staining was performed on 3-m - thick sections from each case using a biotin - avidin - peroxidase method on a bond - max autostainer ( leica , wetzlar , germany ) after retrieval with t / e buffer ( cd56 ) or citrate buffer ( ttf-1 ) .
we used primary antibodies to cd56 ( 1:200 , novocastra , newcastle upon tyne , uk ) and ttf-1 ( 1:100 , dako , glostrup , denmark ) .
sample information and pathologic features are summarized in table 1 . of the initial samples with diagnosis of adc ,
the histology of the six adcs was acinar ( n=4 ) , mixed acinar and papillary ( n=1 ) , and mixed acinar and solid ( n=1 ) .
of samples that showed transformation to sclc upon second biopsy , four showed pure sclc morphology and two showed combined adc and sclc morphology . in two cases , adc components demonstrated acinar morphology ( fig .
ttf-1 was expressed in four of six cases and cd56 was expressed in all five available cases .
the clinical information and egfr mutation status of the six patients are summarized in table 2 .
initial treatments included complete resection and adjuvant chemotherapy for case 1 ( ct2no ) , palliative chemotherapy for case 2 ( ct3n1m1 ) and case 4 ( ct1n0m1 ) , egfr tki for case 3 ( ct2n3m1 ) , incomplete resection and palliative chemotherapy for case 5 ( ct1n0m1 ) , and complete resection for case 6 ( ct1no ) .
the sites of distant metastasis were as follows ; brain ( cases 2 and 4 ) , pleura ( case 3 , 4 , and 5 ) , bone ( case 4 ) , and liver ( case 4 ) . of the six patients with adc in initial biopsy , four
( cases 14 ) harbored an egfr mutation ( l858r mutation , n=1 ; exon 19 deletion , n=3 ) .
all four , along with case 5 who had wild - type egfr but was enrolled in a clinical trial of gefetinib , were treated with egfr tkis .
cases 1 and 3 were treated with irressa , and afatinib was also added for case 1 .
case 2 was treated with afatinib only , and cases 4 and 5 were treated with gefetinib only .
all five patients who underwent tki treatment were female non - smokers ( table 2 ) . despite tki treatment
, all five patients showed disease progression , upon which a second biopsy was performed .
the interval between initial biopsy and second biopsy ranged from 5 to 50 months with mean of 25.3 months .
after confirmation of transformation of sclc on second biopsy , four patients received further treatment .
the treatment option for two patients ( cases 1 and 3 ) was switched to etoposide and cisplatin , and one of them ( case 3 ) showed partial response .
case 5 died due to disease progression and the other patients were alive in the short - term follow - up period .
egfr mutation status was compared between initial and second samples , and all pairs showed the same egfr status . the original egfr mutation in cases 14
was retained in all transformed sclc samples , while cases 5 and 6 showed no egfr mutation in transformed sclc samples .
sample information and pathologic features are summarized in table 1 . of the initial samples with diagnosis of adc ,
the histology of the six adcs was acinar ( n=4 ) , mixed acinar and papillary ( n=1 ) , and mixed acinar and solid ( n=1 ) .
of samples that showed transformation to sclc upon second biopsy , four showed pure sclc morphology and two showed combined adc and sclc morphology . in two cases , adc components demonstrated acinar morphology ( fig .
ttf-1 was expressed in four of six cases and cd56 was expressed in all five available cases .
the clinical information and egfr mutation status of the six patients are summarized in table 2 .
initial treatments included complete resection and adjuvant chemotherapy for case 1 ( ct2no ) , palliative chemotherapy for case 2 ( ct3n1m1 ) and case 4 ( ct1n0m1 ) , egfr tki for case 3 ( ct2n3m1 ) , incomplete resection and palliative chemotherapy for case 5 ( ct1n0m1 ) , and complete resection for case 6 ( ct1no ) .
the sites of distant metastasis were as follows ; brain ( cases 2 and 4 ) , pleura ( case 3 , 4 , and 5 ) , bone ( case 4 ) , and liver ( case 4 ) . of the six patients with adc in initial biopsy , four
( cases 14 ) harbored an egfr mutation ( l858r mutation , n=1 ; exon 19 deletion , n=3 ) .
all four , along with case 5 who had wild - type egfr but was enrolled in a clinical trial of gefetinib , were treated with egfr tkis .
cases 1 and 3 were treated with irressa , and afatinib was also added for case 1 .
case 2 was treated with afatinib only , and cases 4 and 5 were treated with gefetinib only .
all five patients who underwent tki treatment were female non - smokers ( table 2 ) . despite tki treatment
, all five patients showed disease progression , upon which a second biopsy was performed .
the interval between initial biopsy and second biopsy ranged from 5 to 50 months with mean of 25.3 months .
after confirmation of transformation of sclc on second biopsy , four patients received further treatment .
the treatment option for two patients ( cases 1 and 3 ) was switched to etoposide and cisplatin , and one of them ( case 3 ) showed partial response .
case 5 died due to disease progression and the other patients were alive in the short - term follow - up period .
egfr mutation status was compared between initial and second samples , and all pairs showed the same egfr status .
the original egfr mutation in cases 14 was retained in all transformed sclc samples , while cases 5 and 6 showed no egfr mutation in transformed sclc samples .
transformation of nsclc to sclc was recently proposed as a mechanism of resistance to tki therapy .
identification of histologic transformation may be an important factor in determining a patient s treatment plan due to the differences between nsclc and sclc . while most reports of transformation of adc to sclc were identified in egfr mutant patients related to tki treatment , it is not certain whether transformation is exclusively related to egfr mutation or egfr tki treatment . here
, we report six cases of adc which showed histologic transformation to sclc over a 6-year period at a single institute . similar to previous reports ,
four cases in our series were adc with egfr activating mutations that underwent tki treatment and were subsequently found to have sclc transformation on second biopsy .
however , we also identified two additional cases of sclc transformation that had no egfr mutation , and one of these cases underwent initial tki treatment .
egfr tkis are now being used worldwide for first - line treatment in a subset of lung cancers bearing egfr - activating mutations , and they have demonstrated dramatic therapeutic efficacy .
one of mechanism of resistance to egfr inhibitors is the histological transformation of adc to sclc .
although the presence of combined adc and sclc histology at initial diagnosis is a possibility , genomic sequencing of egfr mutations shows that both the original tumor and transformed sclc at the time of resistance share the original egfr - activating mutation , thus supporting the conclusion that these were not independent tumors [ 6 - 11 ] .
however , the small biopsy size represents only a portion of tumors , and sclc components may become dominant at the time of disease progression .
of the six patients with initially diagnosed with adc in our report , two were diagnosed using surgically resected samples rather than needle biopsies .
for these two cases , the possibility of combined histology at the initial biopsy can be excluded . in case 6 , it was difficult to distinguish between sclc transformation and second primary sclc considering the early stage of initial adc . in our series ,
this suggests that transformation to sclc is not unique to tumors bearing egfr mutations , nor does it exclusively result from tki treatment .
transformation to sclc is also reported as a mechanism of acquired resistance to crizotinib in alk rearranged lung tumors .
in addition , transformation to large cell neuroendocrine carcinoma was identified as an acquired resistance mechanism to egfr tkis and crizotinib .
recent studies suggest that alveolar type ii cells can give rise to both adc and sclc , so egfr - mutant lung cancers derived from alveolar type ii cells may have the potential to transform into sclc during the disease progression . in sum
, it seems that acquisition of neuroendocrine phenotype , which includes sclc transformation , can occur in the progression of disease in both egfr - mutant and egfr  wild - type nsclcs .
second biopsies are not routinely performed for lung cancer patients when patients showed resistance to tki treatment .
therefore , the incidence of transformation to sclc in nsclcs can not be accurately calculated .
an acquired tki resistance arising from the histological transformation to sclc has been reported to be as high as 3% . in our institute ,
the incidence of transformation to sclc identified in the second biopsy of total adcs was 1.5% . recently in our institute , there were two cases of sclc transformation in egfr mutant adc during treatment with azd9291 , an oral irreversible egfr tki with selectivity for activating egfr mutations and the t790 m resistance mutation .
identification of small cell component is important as the treatment option can be switched to etoposide and cisplatin against sclc . in conclusion , we report six cases of lung cancer demonstrating transformation from adc to sclc .
four cases were egfr - mutant tumors from female non - smokers who underwent tki treatment , and the egfr mutation was retained in the transformed sclc tumors .
the other two adcs were egfr  wild - type , and one of these patients received egfr tki treatment .
the neuroendocrine phenotype can thus be acquired during adc disease progression independent of egfr tki treatment .
 background : epidermal growth factor receptor ( egfr ) tyrosine kinase inhibitors ( tkis ) are considered the first line treatment for a subset of egfr - mutated non - small cell lung cancer ( nsclc ) patients . 
 although transformation to small cell lung cancer ( sclc ) is one of the known mechanisms of resistance to egfr tkis , it is not certain whether transformation to sclc is exclusively found as a mechanism of tki resistance in egfr - mutant tumors.methods:we identified six patients with primary lung adenocarcinoma that showed transformation to sclc on second biopsy ( n = 401 ) during a 6-year period . 
 clinicopathologic information was analyzed and egfr mutation results were compared between initial and second biopsy samples.results:six patients showed transformation from adenocarcinoma to sclc , of which four were pure sclcs and two were combined adenocarcinoma and sclcs . clinically , four cases were egfr - mutant tumors from non - smoking females who underwent tki treatment , and the egfr mutation was retained in the transformed sclc tumors . 
 the remaining two adenocarcinomas were egfr wild - type , and one of these patients received egfr tki treatment.conclusions:nsclc can acquire a neuroendocrine phenotype with or without egfr tki treatment . 
MATERIALS AND METHODS
Cases
None
Immunohistochemistry
RESULTS
Sample information and histologic features
Clinical information and EGFR status
DISCUSSION