all patients were part of the lung cancer group cologne ( http://www.cio-koeln-bonn.de ) cohort and analyzed as part of our routine molecular diagnostics program according to the local ethical guidelines and reviewed by the institutional ethics committee . for squamous cell carcinomas , all consecutive patients with sufficient tumor material from january 2010 until june 2011 were analyzed , and cases with other histological subtypes were selected at random from the same period in time . three - hundred sixty - seven primary pulmonary carcinomas were included : 307 squamous cell carcinomas , 47 adenocarcinomas , and 13 carcinomas of other differentiation types ( i.e. , 6 undifferentiated large cell carcinomas , 4 adenosquamous carcinomas , 2 large cell neuroendocrine carcinomas , 1 combined small cell and squamous carcinoma ) . adenosquamous carcinomas and the combined small cell and squamous carcinoma were included to answer the question whether tumors with only partial squamous differentiation may also be amplified for fgfr1 . since emerging data from expression profiles provide evidence that some pulmonary large cell carcinomas might represent a dedifferentiation end point of squamous carcinomas , we further investigated a small number of these tumors . an additional cohort of 53 primary adenocarcinomas of the lung , which were sent to the lung cancer group cologne for molecular testing from october 2011 until february 2012 , was examined in a confirmatory study . thus , a total of 420 cases were included ( 307 squamous cell carcinomas , 100 adenocarcinomas , and 13 carcinomas of other differentiation types ) . all diagnoses were reviewed by two experienced pathologists and confirmed by immunostainings , if appropriate . for squamous cell carcinomas , these include positive cytokeratin 5/6 ( clone d5&16b4 , cellmarque ; dilution 1:50 ) and p63 ( a4a , zeta corp . ; positive stainings for cytokeratin 7 ( ov - tl 12/30 , dako ; 1:800 ) , ttf1 ( 8g7g3/1 , cellmarque ; 1:200 ) , and napsin a ( rabbit polyclonal antibody , cellmarque ; 1:200 ) were performed . stainings for cytokeratin 20 ( ks20.8 , cellmarque ; 1:400 ) and cdx2 ( cdx2 - 88 , biogenex ; 1:200 ) were used to exclude pulmonary metastases of intestinal adenocarcinomas . diagnoses and grading of tumors were made in accordance with the current who classification system . for fish , tumor tissue from biopsies or surgical resection specimens three to four m tissue sections were mounted on sialinized slides and hybridized overnight with the zytolight spec fgfr1/cen 8 dual color probe ( zytovision , bremerhaven , germany ) . briefly , deparaffinization , protease treatment , and washes were performed on the half - automated vp2000 processor system ( abbott molecular , wiesbaden , germany ) . after pretreatment , the slides were denatured in the presence of 10  l probe for 5  min at 75c and hybridized at 37c overnight . post - hybridization ssc washes were performed at 72c and the slides stained with dapi before analysis . normal tissue including vessels , fibroblasts , or non - tumor lung tissue served as internal positive control . cases were only further evaluated if control tissue nuclei displayed one or two clearly distinct signals of each color . tumor tissue was scanned for amplification hot spots by using  40 or  63 objectives ( dm5500 fluorescent microscope ; leica ) . if the fgfr1 signals were homogeneously distributed , then random areas were used for counting the signals . twenty contiguous tumor cell nuclei from three hot spots or random areas , resulting in a total of 60 nuclei , were individually evaluated with the  100 or  63 objectives by counting green fgfr1 and orange centromer 8 ( cen8 ) signals . the fgfr1/cen8 ratio , the number of cells with 5 and 15 fgfr1 signals and the average fgfr1 copy number per cell were calculated . for statistical analysis having evaluated the first 50 tumors of our series , we became aware of different signal distribution patterns ( figure 1 ) . fgfr1 signals were heterogeneously distributed in most tumors , and small clusters of amplified cells often occurred . we observed high - level cluster amplifications with 15 fgfr1 signals in a small subset of tumors whereas small clusters (  microclusters ' ) with 5 fgfr1 signals on average were found more frequently . the majority of tumors displayed polysomy with > 2 cen8 signals , sometimes leading to an fgfr1/cen8 ratio below 1.0 despite an increase in absolute numbers of fgfr1 signals compared with normal tissue . fgfr1 signal doublets and triplets were counted as one signal , but closely spaced groupings of signals were considered as small clusters of fgfr1 signals according to the reading criteria for most other fish probes ( e.g. , her2 and egfr ) . therefore , we developed the following reading and evaluation strategy that was derived from our observations in lung cancer and fgfr1 data published previously from studies with other tumor entities : 
 assessment of hybridization quality : evaluate only samples and areas with sharp borders of nuclei , no signs of overdigestion , non - overlapping nuclei , bright and specific green and orange signals in internal control tissue and in the tumor area.scanning the slide : scan the entire tumor area for hot spots of increased fgfr1 copy numbers.reading the slide : count 20 tumor cell nuclei in three areas , either in three hot spots or in three random areas in case of homogeneous signal distribution . count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas.count only clearly distinct signals as two separate signals . count fgfr1 signal doublets and triplets as one signal . in cases of signal clusters give cluster estimation in steps of five signals , for example , 15 , 20 , or 25 fgfr1 signals . assessment of hybridization quality : evaluate only samples and areas with sharp borders of nuclei , no signs of overdigestion , non - overlapping nuclei , bright and specific green and orange signals in internal control tissue and in the tumor area . scanning the slide : reading the slide : count 20 tumor cell nuclei in three areas , either in three hot spots or in three random areas in case of homogeneous signal distribution . count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas . count fgfr1 signal doublets and triplets as one signal . in cases of signal clusters give cluster estimation in steps of five signals , for example , 15 , 20 , or 25 fgfr1 signals . cases were considered as fgfr1 positive (  amplified ' ) under one of the following conditions : 
 the fgfr1/cen8 ratio is 2.0;the average number of fgfr1 signals per tumor cell nucleus is 6;the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10%the percentage of tumor cells containing 5 fgfr1 signals is 50%,with ( 13 ) representing a high - level and ( 4 ) a low - level amplification . the fgfr1/cen8 ratio is 2.0 ; the average number of fgfr1 signals per tumor cell nucleus is 6 ; the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10% the percentage of tumor cells containing 5 fgfr1 signals is 50% , in all , 347 out of 367 tumors ( 95% ) were evaluable , that is , 290 squamous cell carcinomas , 44 adenocarcinomas , and 13 carcinomas of other differentiation types . in all , 58 of 290 squamous cell carcinomas ( 20% ) were fgfr1 amplified according to our fish scoring definition , whereas 0/44 adenocarcinomas and 2/13 tumors of other differentiation were amplified . the latter two positive cases were one tumor with adenosquamous differentiation and one undifferentiated large cell carcinoma , in which a high - level amplification was found . in a confirmatory study , 53 adenocarcinomas were additionally examined ; all of them were fish negative . thus , a total of 97 pulmonary adenocarcinomas did not show an amplification of fgfr1 . among the 60 positive tumors , 13 cases revealed low - level amplification as defined by category ( 4 ) of our scoring system ( table 1 ) . thus , low - level amplifications represent 22% of all fish - positive cases . among the squamous cell carcinomas , the average fgfr1 copy number per nucleus ( figure 2 ) ranged from 1.1 to 16.5 ( mean : 3.6 ) . the mean fgfr1/cen8 ratio was 1.4 ( range : 0.48.8 ) , the mean percentages of tumor cells with 5 and 15 fgfr1 copies were 22 and 3% , respectively ( figure 2 ) . fgfr1/cen8 ratio ( t - test , p=0.01 ) , average fgfr1 copy number per nucleus ( t - test , p<0.001 ) , and percentage of tumor cells with 5 fgfr1 copies ( t - test , p<0.001 ) were significantly higher in squamous cell carcinomas compared with all other tumor types . the percentage of tumor cells with 15 fgfr1 copies was significantly higher in squamous cell carcinomas compared with adenocarcinomas ( p<0.001 ) . among evaluable squamous cell carcinomas , a high - level amplification was detected at a frequency of 16% ( n=45 ) and low - level amplification at a frequency of 4% ( n=13 ) . importantly , the fgfr1 amplification status is not exclusively based on an fgfr1/cen8 ratio of 2 , as some tumors fulfill only criteria ( 2 ) and/or ( 3 ) for high - level amplification despite an overall ratio below 2.0 ( table 1 ) . however , in the majority of cases in which a high - level amplification was determined at least two criteria were fulfilled . ratio 2.0 and average number of fgfr1 signals per tumor cell nucleus 6 turned out to be the most consistent criteria for fgfr1 amplification . all fish - positive cases , low- and high - grade amplifications , had at least 50% of tumor cell nuclei containing five or more fgfr1 signals , thus , fulfilling criterion ( 4 ) . preliminary survival data indicate that there is no significant difference between outcome of fgfr1 amplified and non - amplified lung cancer patients . having evaluated the first 50 tumors of our series , we became aware of different signal distribution patterns ( figure 1 ) . fgfr1 signals were heterogeneously distributed in most tumors , and small clusters of amplified cells often occurred . we observed high - level cluster amplifications with 15 fgfr1 signals in a small subset of tumors whereas small clusters (  microclusters ' ) with 5 fgfr1 signals on average were found more frequently . the majority of tumors displayed polysomy with > 2 cen8 signals , sometimes leading to an fgfr1/cen8 ratio below 1.0 despite an increase in absolute numbers of fgfr1 signals compared with normal tissue . fgfr1 signal doublets and triplets were counted as one signal , but closely spaced groupings of signals were considered as small clusters of fgfr1 signals according to the reading criteria for most other fish probes ( e.g. , her2 and egfr ) . therefore , we developed the following reading and evaluation strategy that was derived from our observations in lung cancer and fgfr1 data published previously from studies with other tumor entities : 
 assessment of hybridization quality : evaluate only samples and areas with sharp borders of nuclei , no signs of overdigestion , non - overlapping nuclei , bright and specific green and orange signals in internal control tissue and in the tumor area.scanning the slide : scan the entire tumor area for hot spots of increased fgfr1 copy numbers.reading the slide : count 20 tumor cell nuclei in three areas , either in three hot spots or in three random areas in case of homogeneous signal distribution . count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas.count only clearly distinct signals as two separate signals . count fgfr1 signal doublets and triplets as one signal . in cases of signal clusters give cluster estimation in steps of five signals , for example , 15 , 20 , or 25 fgfr1 signals . assessment of hybridization quality : evaluate only samples and areas with sharp borders of nuclei , no signs of overdigestion , non - overlapping nuclei , bright and specific green and orange signals in internal control tissue and in the tumor area . scanning the slide : reading the slide : count 20 tumor cell nuclei in three areas , either in three hot spots or in three random areas in case of homogeneous signal distribution . count cohesive tumor cells do not selectively consider isolated amplified tumor cells from different areas . count fgfr1 signal doublets and triplets as one signal . in cases of signal clusters give cluster estimation in steps of five signals , for example , 15 , 20 , or 25 fgfr1 signals . cases were considered as fgfr1 positive (  amplified ' ) under one of the following conditions : 
 the fgfr1/cen8 ratio is 2.0;the average number of fgfr1 signals per tumor cell nucleus is 6;the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10%the percentage of tumor cells containing 5 fgfr1 signals is 50%,with ( 13 ) representing a high - level and ( 4 ) a low - level amplification . the fgfr1/cen8 ratio is 2.0 ; the average number of fgfr1 signals per tumor cell nucleus is 6 ; the percentage of tumor cells containing 15 fgfr1 signals or large clusters is 10% the percentage of tumor cells containing 5 fgfr1 signals is 50% , in all , 347 out of 367 tumors ( 95% ) were evaluable , that is , 290 squamous cell carcinomas , 44 adenocarcinomas , and 13 carcinomas of other differentiation types . in all , 58 of 290 squamous cell carcinomas ( 20% ) were fgfr1 amplified according to our fish scoring definition , whereas 0/44 adenocarcinomas and 2/13 tumors of other differentiation were amplified . the latter two positive cases were one tumor with adenosquamous differentiation and one undifferentiated large cell carcinoma , in which a high - level amplification was found . in a confirmatory study thus , a total of 97 pulmonary adenocarcinomas did not show an amplification of fgfr1 . among the 60 positive tumors , 13 cases revealed low - level amplification as defined by category ( 4 ) of our scoring system ( table 1 ) . thus , low - level amplifications represent 22% of all fish - positive cases . among the squamous cell carcinomas , the average fgfr1 copy number per nucleus ( figure 2 ) ranged from 1.1 to 16.5 ( mean : 3.6 ) . the mean fgfr1/cen8 ratio was 1.4 ( range : 0.48.8 ) , the mean percentages of tumor cells with 5 and 15 fgfr1 copies were 22 and 3% , respectively ( figure 2 ) . fgfr1/cen8 ratio ( t - test , p=0.01 ) , average fgfr1 copy number per nucleus ( t - test , p<0.001 ) , and percentage of tumor cells with 5 fgfr1 copies ( t - test , p<0.001 ) were significantly higher in squamous cell carcinomas compared with all other tumor types . the percentage of tumor cells with 15 fgfr1 copies was significantly higher in squamous cell carcinomas compared with adenocarcinomas ( p<0.001 ) . among evaluable squamous cell carcinomas , a high - level amplification was detected at a frequency of 16% ( n=45 ) and low - level amplification at a frequency of 4% ( n=13 ) . importantly , the fgfr1 amplification status is not exclusively based on an fgfr1/cen8 ratio of 2 , as some tumors fulfill only criteria ( 2 ) and/or ( 3 ) for high - level amplification despite an overall ratio below 2.0 ( table 1 ) . however , in the majority of cases in which a high - level amplification was determined at least two criteria were fulfilled . ratio 2.0 and average number of fgfr1 signals per tumor cell nucleus 6 turned out to be the most consistent criteria for fgfr1 amplification . all fish - positive cases , low- and high - grade amplifications , had at least 50% of tumor cell nuclei containing five or more fgfr1 signals , thus , fulfilling criterion ( 4 ) . preliminary survival data indicate that there is no significant difference between outcome of fgfr1 amplified and non - amplified lung cancer patients . we and others recently described therapeutically targetable fgfr1 amplifications in smoking - associated human squamous lung cancers . this is a clinically important finding since worldwide this cohort of patients is steadily increasing in size and current therapies are very limited squamous cell lung carcinomas account for 25% of new lung carcinoma cases and 40  000 deaths per year in the united states . to translate this finding into the clinic patients eligible for treatment with fgfr inhibitors in clinical trials furthermore , their putative response data need to be carefully related to the specific pattern of fgfr1 amplification . in addition , molecular testing needs to be fast and reliable on frequently very small biopsies . therefore , we evaluated 290 squamous cell carcinomas by a dual color fish assay and found that 20% of cases showed high- or low - level amplifications . the percentage is very close to data observed in two cohorts analyzed by snp 6.0 by us ( n=155 , frequency 10% ) and fish ( n=153 , frequency 22% ) and others ( n=57 , frequency 21% ) . importantly , the distribution of fgfr1 copies is heterogeneous in most tumors , and small clusters (  microclusters ' ) as well as high - level amplification in only few isolated tumor cells and co - amplifications of fgfr1 and cen8 signals are common features . our reading and evaluation strategy is able to address the issue of intratumoral heterogeneity and other special features of fgfr1 in pulmonary carcinomas . thus , our scoring system might serve as a standardized screening tool to identify patients for ongoing and upcoming clinical trials with fgfr1 inhibitors . several compounds have already entered early clinical trials giving precise fgfr1 diagnostics high clinical impact . in the setting of these clinical trials , we believe that our fish score might be helpful for identifying patients with significant increase in fgfr1 gene copy numbers . however , it still needs to be clarified by future response data derived from these studies whether our criteria are equally good or even better predictor of response than traditional amplification criteria . thus , our work represents a proposal for standardized screening for clinical trials , which potentially needs to be modified against the background of future clinical response data . however , it appears likely that fgfr1 fish assay is going to represent a companion diagnostic and there is a need for definition of interpretation criteria that could be implemented in the clinical practice . having carefully assessed a large cohort of pulmonary carcinoma cases by applying our reading and evaluation strategy , we were able to divide positive cases into two types of amplification patterns . low amplification levels ( as defined by 5 fgfr1 signals in 50% of tumor cells ) occurred at a frequency of 4% in squamous cell carcinomas and account for 22% of all fish - positive cases . in all , 16% of squamous cell carcinomas ( 78% of fish - positive tumors ) show high - level amplification ( as defined by an fgfr1/cen8 2.0 , or average number of fgfr1 signals per tumor cell nucleus 6 , or the percentage of tumor cells containing 15 fgfr1 signals or large clusters 10% ) . our previous data suggest that cancer cell lines with high focal amplifications respond better to treatment with fgfr inhibitors , which was also confirmed in an independent study . therefore , it may be expected that tumors with high - level fgfr1 amplification will respond better to fgfr inhibitors . very early and preliminary data from a phase i study with a selective pan fgfr inhibitor indicate a partial response in one patient whose pulmonary squamous cell carcinoma has been tested positive by applying the fgfr1 fish score . however , further results from ongoing early clinical trials need to be awaited . in any case , it will be important to record amplification levels obtained by fish in a reproducible manner as proposed by our score to correlate these clinical response data with amplification patterns . we found very rare fgfr1 amplifications in non - squamous lung cancer : only one single case each of undifferentiated large cell carcinoma and adenosquamous carcinoma , but not in adenocarcinomas of the lung . these data indicate that squamous cell carcinomas , adenosquamous , and undifferentiated large cell carcinomas should be included in fgfr1 amplification screening of lung cancer , but cases of pure adenocarcinoma may be omitted .
we recently reported fibroblast growth factor receptor - type 1 ( fgfr1 ) amplification to be associated with therapeutically tractable fgfr1 dependency in squamous cell lung cancer . 
 this makes fgfr1 a novel target for directed therapy in these tumors . to reproducibly identify patients for clinical studies 
 , we developed a standardized reading and evaluation strategy for fgfr1 fluorescence in - situ hybridization ( fish ) and propose evaluation criteria , describe different patterns of low- and high - level amplifications and report on the prevalence of fgfr1 amplifications in pulmonary carcinomas . 
 a total of 420 lung cancer patients including 307 squamous carcinomas , 100 adenocarcinomas of the lung and 13 carcinomas of other types were analyzed for fgfr1 amplification using a dual color fish . 
 we found heterogeneous and different patterns of gene copy numbers . 
 fgfr1 amplifications were observed in 20% of pulmonary squamous carcinomas but not in adenocarcinomas . high - level amplification ( as defined by an fgfr1/centromer 8 ( cen8 ) ratio 2.0 , or average number of fgfr1 signals per tumor cell nucleus 6 , or the percentage of tumor cells containing 15 fgfr1 signals or large clusters 10% ) was detected at a frequency of 16% and low - level amplification ( as defined by 5 fgfr1 signals in 50% of tumor cells ) at a frequency of 4% . 
 we conclude that fgfr1 amplification is one of the most frequent therapeutically tractable genetic lesions in pulmonary carcinomas . 
 standardized reporting of fgfr1 amplification in squamous carcinomas of the lung will become increasingly important to correlate therapeutic responses with fgfr1 inhibitors in clinical studies . 
 thus , our reading and evaluation strategy might serve as a basis for identifying patients for ongoing and upcoming clinical trials .