lung cancer is one of most common cancers in the world , representing 17.1% of all cancers in men , 6.7% in women , and 12.2% in both sexes . of the histological types , adenocarcinoma ( ac ) has remained the most prevalent among women over the past three decades , with incidence rates increasing slowly over time in many countries . in contrast , squamous cell carcinoma ( sqcc ) has historically been the predominant tumour type in men , but the incidence has declined and converged with the corresponding incidence in women , which has remained fairly stable . traditionally , lung carcinoma was classified into histological types using standard histological techniques . the most critical step in histopathological diagnosis was to distinguish small cell carcinoma ( smcc ) from the other lung carcinomas , which were collectively called the non - small cell lung carcinomas ( nsclcs ) ; patients with the former were referred to chemotherapy , whereas patients with the latter were potentially eligible for surgery or different chemotherapies . over the past few years , the emergence of targeted or combination treatment strategies has created new demands on histopathological diagnostics , as it is now recognised that the efficacy and toxicity of some new drugs are related to the histological type . consequently , the exact determination of histological type by a pathologist has become essential to making clinical decisions . in this context , the integration of conventional histomorphological analysis with an immunohistochemical ( ihc ) panel that includes markers of squamous ( i.e. , p63 , cytokeratin ( ck ) 5/6 ) and glandular ( ttf-1 , ck7 ) cell differentiation allows more accurate identification of the histotype [ 512 ] . moreover , recent studies have demonstrated that the antibody panel approach may help to refine lung cancer classification in fine needle aspiration ( fna ) cell blocks as well as in biopsy material ; remarkably , this approach reduces the proportion of cancers diagnosed as nsclcs and thus improves the quality of treatment decisions [ 11 , 13 ] . the aims of this study were as follows : ( i ) to assess changes in lung cancer incidence in the south of switzerland after the progressive introduction from 2003 to 2009 of ancillary ihc studies for the diagnosis of lung cancers ( panel systematically applied from 2010 ) and ( ii ) to evaluate potential changes in short - term survival trends of the four major lung cancer histotypes . all patients with invasive lung cancer diagnosed by cytology or histology in the south of switzerland between 1996 and 2010 were selected from the files of the population - based ticino cancer registry . the registry is located in the southern , italian - speaking region of switzerland and is closely connected to and part of the regional institute of pathology , which serves the entire region . lung tumour topography and lung tumour histotypes were classified using the international classification of diseases for oncology ( icd - o - iii ) and the who classification of tumours of the lung [ 15 , 16 ] . the tumour site was classified according to the extended icd - o version proposed by wagner . case registration and comparability , validity , and consistency checks were performed according to the international agency for research on cancer ( iarc ) guidelines and the recommendations of the european network of cancer registries ( encrs ) [ 1820 ] . lung cancers were categorised into four histotypes : ac , sqcc , smcc , and large cell carcinoma / non - small - cell lung cancer ( lcc / nsclc ) [ 15 , 16 ] . we included nsclc , not formally classified by who , in the lcc histotype , as considered by default  poorly differentiated carcinomas not otherwise specified ( nos ) , lymphomas , neuroendocrine tumours ( i.e. , carcinoids and large cell neuroendocrine carcinomas ) , and mesenchymal tumours were excluded from the present study . tumours were staged according to the cancer staging manual of the american joint committee on cancer ( ajcc ) , version 5 , until 2002 and according to version 6 from 2003 onwards . the tumours were then categorised as  limited disease  ( any t , any n , and m0 ) or  extensive disease  ( any t , any n , and m1 ) [ 16 , 21 , 22 ] . all histological evaluations were made at the local institute of pathology by the same core group of pathologists . ihc studies for histotyping lung cancers ( i.e. , ttf-1 , p63 , ck 5/6 and 7 ) were progressively introduced from 2003 to 2009 . since 2010 a diagnostic algorithm is applied to all new cases systematically . mean and median values are provided for quantitative variables ; proportions represent qualitative variables . differences among lung cancer histotypes were evaluated using 1-way analysis of variance ( anova ) for patient age ( years ) . a chi - square test was conducted to assess the relationship between the different lung cancer histotypes and main clinicopathological characteristics such as sex , histological grade , and tumour extent at diagnosis . european age - standardised incidence rates were calculated by the direct standardization method to remove the strong correlation between patient age and tumour occurrence . trends in incidence were measured as the estimated annual percentage change ( apc ) and the 95% confidence interval ( ci ) , which were calculated by fitting a linear regression to the natural logarithm of the age - adjusted rates ( r ) , with year of diagnosis as the regressor variable as follows : ln(r ) = m(year ) + b. from m , the slope of the regression line , the apc was calculated as follows : apc = 100  ( e  1 ) [ 2426 ] . two - year overall survival ( os ) ( follow - up on december 31 , 2010 ) was analysed for patients with a 24-month complete follow - up ( incidence period 19962008 ) using the kaplan - meier method . the logrank test was used to detect statistically significant differences in survival . the two - year survival trend analysis considered three incidence periods : 19962001 ( before the introduction of the ihc panel ) , 20022004 ( transitional period ) , and 20052008 ( after the introduction of the complete ihc panel ) . the statistical analysis was conducted in sas version 9.1 ( sas institute inc , cary , nc , usa ) . of 2844 lung cancers that occurred in the south of switzerland between 1996 and 2010 , 2585 ( 90.9% ) were confirmed by cytological or histological assay and were therefore selected for inclusion in the present study . the 118 cases that did not meet the inclusion criteria ( i.e. , carcinomas nos , lymphomas , neuroendocrine , and mesenchymal tumours ) were excluded from the analysis , and the remaining 2467 cases constitute the basis of the present study . the prevalence of each of the four lung cancer histotypes was as follows : 997 ( 40.4% ) acs , 522 ( 21.2% ) lcc / nsclcs , 378 ( 15.3% ) smccs , and 570 ( 23.1% ) sqccs . the four lung cancer histotypes differed significantly by mean age at diagnosis ( anova f - test = 7.49 , p < 0.0001 ) , ac being dominant in the youngest group ( 66.0  11.0 years old ) . when subjects were stratified by age group , this result was confirmed ( p < 0.0001 ) . overall , most lung cancers occurred in men ( 69% ) , with a pick of 80.5% in sqcc ( p < 0.0001 ) . in terms of histological grade at diagnosis , 62.6% of ac tumours and 54.2% of sqcc tumours were poorly differentiated ( p = 0.0048 ) . a significant difference was found when lung cancer histotypes were compared by tumour extent at diagnosis ( p < 0.0001 ) : sqcc showed the highest prevalence of limited disease ( 77.2% ) , whereas > 50% of smccs were classified as  extensive . we also detected an overall significant difference ( p < 0.0001 ) in the distribution of histotypes across the two study periods ( table 1 ) . in particular , we observed a higher proportion of ac tumours after 2003 ( 40.9% in 19962003 versus 59.1% in 20042010 , p < 0.0001 ) , corresponding to a percentage increase of 44.5% of the total cases ; in contrast , there was a significant decrease in lcc / nsclc proportion ( 64.0% in 19962003 versus 36.0% in 20042010 , p < 0.0001 ) , corresponding to a percentage decrease of 43.8% . the above - described trends of proportions were confirmed by the trend analysis of incidence rates , which show a significant increase in ac incidence ( figure 1 , apc : 4.6 ; 95% ci : 3.1 ; 6.0 ) accompanied by a decrease in lcc / nsclc incidence , with a significant joinpoint in 2003 ( apc : 14.7 ; 95% ci : 21.6 ; 7.1 ) . we also detected a slight decrease in the incidence of sqcc and smcc , but it was not statistically significant . the trend seen in ac was also observed when we considered only poorly differentiated lung cancers ( apc : 5.1 ; 95% ci : 3.4 ; 6.8 ) ( figure 2 ) . short - term oss by lung cancer histotype are reported in figure 3 ; smccs and lcc / nsclcs show the worst survival probabilities at two years from diagnosis ( p < 0.0001 ) . we also detected an increase in os for sqcc and a decrease for lcc / nsclc , particularly in the last study period , 20052008 ( figure 4 ) . the observed baseline increase in the incidence of ac and decrease in the incidence of sqcc , which were observed in both genders , are similar to the trends observed worldwide and described in many studies [ 2729 ] . the decreased incidence of smoking in men , the increased incidence of smoking in women , accompanied by changes in the composition of cigarettes , and the implementation of filters are suggested to be responsible for the observed changes [ 2 , 30 ] . it has also been postulated that the use of filtered cigarettes has caused an increase in the incidence of ac , which is usually located in the peripheral lung , because smokers now need to inhale more deeply to achieve a comparable effect . in addition to the increasing incidence of ac , our study highlights a significant decrease in the incidence of the lcc / nsclc histotype , with a joinpoint in 2003 . this observation is difficult to explain by changes in exposure to known lung cancer risk factors . more likely it is associated with the progressive introduction ( beginning in 2003 ) of a panel of ihc markers for squamous ( i.e. , p63 , ck 5/6 ) and glandular differentiation ( ttf-1 , ck7 ) . many studies have already described the benefit to diagnosis of a specific panel of markers that can help pathologists to correctly classify lung cancer histotypes [ 3234 ] . in particular , major benefits are obtained in distinguishing poorly differentiated sqcc from ac , especially in small biopsy tissues or cell specimens obtained by bronchus brossage / lavage or fna [ 69 , 35 ] . in recent years , a precise histopathological classification of lung carcinomas has become increasingly important as a way to identify candidates for targeted therapy and to improve the design of clinical trials [ 3 , 12 ] . thus , the observed decrease in lcc / nsclc diagnosis and increase in ac diagnosis will help to better define the target populations that could benefit from these new drugs . concerning potential confounding factors , it is unlikely that who 's 2003 changes in lung cancer classification ( 19962002 , according to the second edition of the international classification of diseases for oncology ( icd - o - ii ) ; 20032010 , according to the third edition ( icd - o - iii ) ) significantly influenced our results . before the introduction of icd - o - iii in 2003 , lcc and nsclc were routinely considered together as a poorly defined and poorly differentiated entity ( since 2003 the nsclc code was not available in icd - o codes , and as consequence lcc diagnosis was treated as a catch - all  ) . in order to avoid classification bias , we pooled these two groups in the second study period as well . another potential confounding factor ( not observed in our study , data not shown ) would be a shift along the study period in diagnostic procedure ( i.e. , cytology versus histology ) , which could cause an increase or decrease in diagnostic accuracy . the significant changes in incidence by histological subtype appear to be followed by changes in the short - term os . this trend , which is most evident for lcc / nsclc and sqcc , is important , as it could have affected the results of some clinical trials , particularly in cases where the diagnosis of lung cancers was based on histological evaluation without ancillary studies , or where ancillary studies were progressively introduced over the study period . in this contest , the worst short - term os for lcc / nsclc in the last period of the study ( figure 4 ) most likely reflects the selection of poorly differentiated carcinomas . additional studies are warranted to clarify this observation as well as the improved short - term os of patients with sqcc that may be related to a general improvement of cancer care . the most significant aspects of this study are the following : ( i ) it includes an up - to - date case study ( 19962010 ) composed of a large number of lung cancer cases , thus reflecting a representative real - world description of a population with universal and homogeneous access to treatment ; ( ii ) all ages were considered , thereby ensuring the observation of a total population at risk ; ( iii ) the panel analyses of diagnostic markers were all carried out by a single laboratory and evaluated on routinely collected tissue by the same core group of pathologists , thus ensuring the reproducibility of results . in conclusion , in this population - based study we observed a statistically significant decrease of lcc / nsclc histotype , which is most likely attributable to the introduction of ihc diagnostic markers . we underscore the need for cautions interpretation of previous studies and clinical trials in which the diagnosis of lung cancer was only based on conventional histomorphological evaluation . in the era of targeted therapy , we advocate not only quality control programs for molecular testing but also a standardisation of histological diagnoses .
purpose . 
 the aim of this study was to assess the impact of immunohistochemical- ( ihc- ) studies on incidence and survival of lung cancer histotypes . 
 patients and methods . 
 lung cancers occurred in southern switzerland between 1996 and 2010 were selected by the ticino cancer registry and categorised into adenocarcinoma ( ac ) , squamous - cell - carcinoma ( sqcc ) , small - cell - carcinoma ( smcc ) , and large - cell carcinoma / non - small - cell lung cancer ( lcc / nsclc ) . 
 incidence rates , annual - percentage - change ( apc ) , and two - year overall survival ( os ) ( follow - up : 31.12.2010 ) were performed . results . 
 2467 cases were selected : 997 ( 40.4% ) ac ; 522 ( 21.2% ) lcc / nsclc , 378 ( 15.3% ) smcc , and 570 ( 23.1% ) sqcc . 
 trend - analysis showed significant increase in ac ( apc : 4.6 ; 95% ci : 3.1 ; 6.0 ) and decrease of lcc / nsclc , with significant joinpoint in 2003 ( apc : 14.7 ; 95% ci : 21.6 ; 7.1 ) . improved os and decreased os were detected in sqcc and lcc / nsclc , respectively . 
 conclusions . 
 this study highlights that diagnosis with ancillary immunohistochemical studies will change incidence and survival of precisely defined lung cancer subtypes . 
 it calls attention to the need for cautious interpretation of studies and clinical trials , where the diagnosis was based on histology unaccompanied by ihc studies , and to the need of standardised diagnostic procedures .