Patent Application: US-29017305-A

Abstract:
the invention relates to a set of isolated marker genes comprising at least one gene identified as having differential expression as between patients who are responders and non responders to an erbb receptor tyrosine kinase inhibitor ; said gene set comprising one or more genes selected from at least the group consisting of the 51 genes listed herein including gene - specific oligonucleotides derived from said genes ; and uses of such sets in diagnostic applications .

Description:
the invention will be described in more detail and illustrated by the following examples which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention . certain elements of the invention are also described in more detail below . these are , according to the context of the embodiments described herein , a group of genes which can be used in classification or categorisation of patent response according to the invention . genes that are either expressed at a higher or lower level as between groups of responders or nonresponders . objective tumour responses according to union international contre le cancer / world health organization ( u icc / who ) criteria are categorised as follows : complete response ( cr ): no residual tumour in all evaluable lesions ; partial response ( pr ): residual tumour with evidence of chemotherapy - induced 50 % or greater decrease under baseline in the sum of all measurable lesions and no new lesions ; stable disease ( sd ) residual tumour not qualified for cr ; and progressive disease ( pd ): residual tumour with evidence of 25 % or greater increase under baseline in the sum of all measurable lesions or appearance of new lesions . as defined herein , non responders are pd . the present invention is particularly effective for determining those patients which are cr or pr this family includes egf , erbb2 ( her ), erbb3 ( note that erbb3 does not have a functional kinase domain ) and erbb4 as described in the background to the invention above . these are intended to be unique to the respective genes so that , for example , fragments of the gene that uniquely identify the gene . advantageously , a gene - specific oligonucleotide is between 5 and 50 nucleotides in length , preferably about 15 to 30 nucleotides , and most preferably about 23 nucleotides . array technology and the various techniques and applications associated with it are described generally in numerous textbooks and documents . gene array technology is particularly suited to the practice of the present invention . methods for preparing microarrays are well known in the art . these include lemieux et al ., ( 1998 ), molecular breeding 4 , 277 - 289 , schena and davis . parallel analysis with biological chips . in pcr methods manual ( eds . m . innis , d . gelfand , j . sninsky ), schena and davis , ( 1999 ), genes , genomes and chips . in dna microarrays : a practical approach ( ed . m . schena ), oxford university press , oxford , uk , 1999 ), the chipping forecast ( nature genetics special issue ; january 1999 supplement ), mark schena ( ed . ), microarray biochip technology , ( eaton publishing company ), cortes , 2000 , the scientist 14 [ 17 ]: 25 , gwynne and page , microarray analysis : the next revolution in molecular biology , science , aug . 6 , 1999 ; and eakins and chu , 1999 , trends in biotechnology , 17 , 217 - 218 . the technology is described in pct / us01 / 10063 and us 2002 090979 and references therein . major applications for array technology include the identification of sequence ( nucleotide sequence / nucleotide sequence mutation ) and the determination of expression level ( abundance ) of nucleotide sequences . gene expression profiling may make use of array technology , optionally in combination with proteomics techniques ( celis et al , 2000 , febs lett , 480 ( 1 ): 2 - 16 ; lockhart and winzeler , 2000 , nature 405 ( 6788 ): 827 - 836 ; khan et al ., 1999 , 20 ( 2 ): 223 - 9 ). other applications of array technology are also known in the art ; for example , nucleotide sequence discovery , cancer research ( marx , 2000 , science 289 : 1670 - 1672 ; scherf , et al , 2000 , nat genet ; 24 ( 3 ): 236 - 44 ; ross et al , 2000 , nat genet . march 2000 ; 24 ( 3 ): 227 - 35 ), snp analysis ( wang et al , 1998 , science , 280 ( 5366 ): 1077 - 82 ), drug discovery , pharmacogenomics , disease diagnosis ( for example , utilising microfluidics devices : chemical & amp ; engineering news , feb . 22 , 1999 , 77 ( 8 ): 27 - 36 ), toxicology ( rockett and dix ( 2000 ), xenobiotica , 30 ( 2 ): 155 - 77 ; afshari et al ., 1999 , cancer res1 ; 59 ( 19 ): 4759 - 60 ) and toxicogenomics ( a hybrid of functional genomics and molecular toxicology ). the goal of toxicogenomics is to find correlations between toxic responses to toxicants and changes in the nucleotide sequencetic profiles of the objects exposed to such toxicants ( nuwaysir , et al ( 1999 ), molecular carcinonucleotide sequencesis , 24 : 153 - 159 ). in general , any library may be arranged in an orderly manner into an array , by spatially separating the members of the library . examples of suitable libraries for arraying include nucleic acid libraries ( including dna , nucleotide sequence , oligonucleotide , etc libraries ), peptide , polypeptide and protein libraries , as well as libraries comprising any molecules , such as ligand libraries , among others . accordingly , where reference is made to a “ library ” such reference includes reference to a library in the form of an array . the members of a library are generally fixed or immobilised onto a solid phase , preferably a solid substrate , to limit diffusion and admixing of the samples . in particular , the libraries may be immobilised to a substantially planar solid phase , including membranes and non - porous substrates such as plastic and glass . furthermore , the samples are preferably arranged in such a way that indexing ( i . e . reference or access to a particular sample ) is facilitated . typically the samples are applied as spots in a grid formation . common assay systems may be adapted for this purpose . for example , an array may be immobilised on the surface of a microplate , either with multiple samples in a well , or with a single sample in each well . furthermore , the solid substrate may be a membrane , such as a nitrocellulose or nylon membrane ( for example , membranes used in blotting experiments ). alternative substrates include glass , or silica based substrates . thus , the samples are immobilised by any suitable method known in the art , for example , by charge interactions , or by chemical coupling to the walls or bottom of the wells , or the surface of the membrane . other means of arranging and fixing may be used , for example , pipetting , drop - touch , piezoelectric means , ink - jet and bubblejet technology , electrostatic application , etc . in the case of silicon - based chips , photolithography may be utilised to arrange and fix the samples on the chip . the samples may be arranged by being “ spotted ” onto the solid substrate ; this may be done by hand or by making use of robotics to deposit the sample . in general , arrays may be described as macroarrays or microarrays , the difference being the size of the sample spots . macroarrays typically contain sample spot sizes of about 300 microns or larger and may be easily imaged by existing gel and blot scanners . the sample spot sizes in microarrays are typically less than 200 microns in diameter and these arrays usually contain thousands of spots . thus , microarrays may require specialised robotics and imaging equipment , which may need to be custom made . instrumentation is described generally in a review by cortese , 2000 , the scientist 14 [ 11 ]: 26 . techniques for producing immobilised libraries of dna molecules have been described in the art . generally , most prior art methods describe how to prepare single - stranded nucleic acid molecule libraries , using for example masking techniques to build up various permutations of sequences at the various discrete positions on the solid substrate . u . s . pat . no . 5 , 837 , 832 describes an improved method for producing dna arrays immobilised to silicon substrates based on very large scale integration technology . in particular , u . s . pat . no . 5 , 837 , 832 describes a strategy called “ tiling ” to prepare specific sets of probes at spatially - defined locations on a substrate which may be used to produced the immobilised dna libraries of the present invention . u . s . pat . no . 5 , 837 , 832 also provides references for earlier techniques that may also be used . to aid detection , targets and probes may be labelled with any readily detectable reporter such as a fluorescent , bioluminescent , phosphorescent , radioactive reporter . labelling of probes and targets is disclosed in shalon et al ., 1996 , genome res 6 ( 7 ): 639 - 45 . the materials for use in the methods of the present invention are ideally suited for preparation of kits . a set of instructions will typically be included . the present invention employs , unless otherwise indicated , conventional techniques of chemistry , molecular biology , microbiology , recombinant dna and immunology , which are within the capabilities of a person of ordinary skill in the art . such techniques are explained in the literature . see , for example , j . sambrook , e . f . fritsch , and t . maniatis , 1989 , molecular cloning : a laboratory manual , second edition , books 1 - 3 , cold spring harbor laboratory press ; ausubel , f . m . et al . ( 1995 and periodic supplements ; current protocols in molecular biology , ch . 9 , 13 , and 16 , john wiley & amp ; sons , new york , n . y . ); b . roe , j . crabtree , and a . kahn , 1996 , dna isolation and sequencing : essential techniques , john wiley & amp ; sons ; m . j . gait ( editor ), 1984 , oligonucleotide synthesis : a practical approach , irl press ; and , d . m . j . lilley and j . e . dahlberg , 1992 , methods of enzymology : dna structure part a : synthesis and physical analysis of dna methods in enzymology , academic press . each of these general texts is herein incorporated by reference . in a specific embodiment of the invention , a cdna microarray system representing 27 , 648 genes was used to select a set of genes predicating the responsiveness to gefitinib for advanced nsclc . statistical analysis of the expression profiles identified dozens of genes differentially expressed between responders and non - responders to gefitinib . a drug response scoring ( drs ) system based on the expression of these genes successfully predicted the response to gefitinib therapy . a phase ii clinical study was carried out comprising a multi - center trial to explore the dominant biological factors responsible for clinical anti - tumor effect , adverse drug reactions ( adr ) and pharmacokinetics of zd1839 dosed 250 mg daily in patients with advanced non - small - cell lung cancer who have failed previous chemotherapy . the primary endpoint was to clarify a gene - expression profile that could determine in advance a potential anti - tumor effect of gefitinib . at the start of the study , the sample size was estimated using studies conducted thus far as a rationale . 12 , 13 since the response rate for gefitinib has been less than 20 % in patients with lung cancer , 8 - 10 about 50 patients were estimated to be required to obtain learning cases estimated above . patients whose locally advanced ( stage iiib ) or metastasized ( stage iv ) nsclcs were resistant to one or more regimens of conventional chemotherapy were enrolled in this trial . inclusion criteria were ( 1 ) age greater than 20 years , ( 2 ) performance status ( ps ) 0 - 2 , ( 3 ) adequate liver and kidney function tests . all patients were treated with 250 mg of gefitinib orally once a day at the tokushima university or kinki university hospitals in japan . the treatment was continued until the patient was dropped from the study due to ( 1 ) progression of disease , ( 2 ) intolerable toxicity , or ( 3 ) withdrawal of consent . objective tumor responses were assessed every 4 weeks after the beginning of treatment , according to criteria outlined by the union international contre le cancer / world health organization ( uicc / who ). response categories were as follows : complete response ( cr ), no residual tumor in any evaluable lesion ; partial response ( pr ), residual tumor with evidence of 50 % or greater decrease under baseline in the sum of all measurable lesions , and no new lesions ; progressive disease ( pd ), residual tumor with evidence of 25 % or greater increase under baseline in the sum of all measurable lesions , or appearance of new lesions ; and stable disease ( sd ), residual tumor not qualified for cr , pr , or pd . all evaluable lesions were measured bi - dimensionally ( sum of products of longest diameter and its longest perpendicular of measurable lesions ) using the same techniques as baseline , e . g . plain x - ray , ct , or mri . at the end of 4 - month treatment ( or withdrawal ), the best overall response was evaluated for each patient based on definitions as follows : cr , patients who qualified for cr at two sequential examination points with an interval of at least 28 days between them ; pr , patients judged as pr or better at two sequential examination points with an interval of at least 28 days between them ; sd , patients who were sd or better at two sequential examination points at least 28 days apart but who did not qualify as cr or pr . the first judgment of an sd case must be done at or after the first tumor assessment point ( 28 days after randomization ); pd , the patients determined as pd at or before the first tumor assessment point ( 28 days after randomization ); unknown , the patient does not qualify for a best response of increased disease , and all objective statuses after baseline ( before randomization ) and before progression are unknown . prior to the gefitinib treatment , tumor specimens were taken by trans - bronchial ( tbb ), skin , or lymph - node biopsy with written informed consent from each patient . ethics approval was obtained from the ethics committee of the individual institutes . biopsy samples were frozen immediately , embedded in tissuetek oct medium ( sakura , tokyo , japan ), and stored at − 80 ° c . all samples were examined microscopically , and samples from 28 patients ( 17 learning and 11 test cases ) that contained enough cancer cells for analysis of expression profiles were initially selected for further analysis . for validation of the prediction system , a blinded set of samples from 5 newly enrolled cases ( 4 pd and 1 sd ) were also added to the 11 test cases . clinical and histological information about these patients is summarized in table 1 - 3 . in view of significant differences in the proportions of cancer cells and various types of parenchymal cells that are present from one tumor to another , microdissection is a necessary means of obtaining precise gene - expression profiles on cdna microarrays . therefore we stained 8 μm - thick frozen sections with hematoxylin and eosin and collected cancer cells selectively , using the μcut laser - microbeam microdissection system ( molecular machines & amp ; industries ag , glattbrugg , switzerland ). 14 in this system tissue sections are mounted on a thin supporting polyethylene membrane that will be cut together with the target tissue ; a pulsed - ultraviolet ( uv ) narrow - beam - focus laser cuts out cancer cells along a pre - selected track that can be observed on a video screen . the material to be extracted is never directly exposed to the laser but only circumscribed by it ; unlike other lmm systems , this one allows recovery of dissected cells to proceed without radiation . moreover , the membrane protects the tissue on the slide against cross - contamination . using this system we were able to isolate small areas of tissue rapidly , and to isolate single cells from histological sections ( fig1 ). total rna was extracted from individual microdissected populations of cancer cells using rneasy mini kits and rnase - free dnase kits ( qiagen , hilden , germany ) according to the manufacturer &# 39 ; s protocols . total rnas were subjected to t7 - based rna amplification , as described previously . 15 two rounds of amplification yielded 40 - 200 μg of arna ( amplified rna ) (& gt ; 100 , 000 - fold ) from each sample . as a control probe , normal human lung poly ( a ) + rna ( bd biosciences clontech , palo alto , calif . and biochain , hayward , calif ., usa ) was amplified in the same way . aliquots ( 2 · 5 μg ) of mrna from individual samples and from the control were reversely transcribed in the presence of cy5 - dctp and cy3 - dctp respectively . our “ genome - wide ” cdna microarray system contains 27 , 648 cdnas selected from the unigene database of the national center for biotechnology information . 15 fabrication of the microarray , hybridization , washing , and detection of signal intensities were described previously . 15 to normalize the amount of mrna between tumors and controls , the cy5 / cy3 ratio for each gene &# 39 ; s expression was adjusted so that the averaged cy5 / cy3 ratio of 52 housekeeping genes was equal to one . we assigned a cutoff value to each microarray slide using analysis of variance , and the cy5 / cy3 ratio of the gene was calculated as follows : ( 1 ) if cy5 ( cancer sample ) was lower than the cut off level , then the cy5 / cy3 ratio of the gene was substituted by 2 - 5 percentile among the cy5 / cy3 ratios of other genes whose cy5 and cy3 were higher than the cut off level ; ( 2 ) if cy3 ( control sample ) was lower than the cut off level , then the cy5 / cy3 ratio of the gene was substituted by 97 · 5 percentile among the cy5 / cy3 ratios of other genes whose cy5 and cy3 were higher than the cut off level ; ( 3 ) if both cy5 and cy3 were lower than the cut off level , then the cy5 / cy3 ratio of the gene was left blank . to discover genes that might be associated with sensitivity to gefitinib , individual measurements of about 27 , 648 genes were compared between the two groups of patients , one classified as responders to gefitinib ( pr ) and the other as non - responders ( pd ). to reduce the dimensionality of the number of potent genes that could discriminate between the two classes , we extracted only genes that fulfilled two criteria : 1 ) signal intensities were higher than the cut - off level in at least 60 % of either group , and 2 ) 1 med pr − med pd |≦ 1 , where med indicates the median calculated from log - transformed relative expression ratios in each group . then random - permutation tests were applied to estimate the ability of individual genes to distinguish between the two classes ( pr and pd ); mean ( μ ) and standard deviations ( σ ) were calculated from the log - transformed relative expression ratios of each gene in both groups . a discrimination score ( ds ) for each gene was defined as follows : the samples were randomly permutated 10 , 000 times for each pair of groups . since the ds dataset of each gene showed a normal distribution , we calculated a p - value for the user - defined grouping . we calculated the drug response scores for gefitinib ( gefitinib response scores , or grs ) reflecting the expression levels of candidate prediction - genes according to procedures described previously . 16 - 18 each gene ( gi ) votes for either responder ( pr ) or non - responder ( pd ) depending on whether the expression level ( xi ) in the sample is closer to the mean expression level of one group or the other in reference samples . the magnitude of the vote ( vi ) reflects the deviation of the expression level in the sample from the average of the two classes : we summed the votes to obtain total votes for responders ( v pr ) and non - responders ( v pd ), and calculated grs values as follows : grs =(( v pr − v pd )/( v pr + v pd ))× 100 , where the grs value reflects the margin of victory in the direction of either responder or non - responder . grs values range from − 100 to 100 ; the higher an absolute value of grs , the stronger the prediction . the prediction scores of all samples were obtained by a leave - one - out approach , in which one sample at a time was removed from the sample set ; permutational p - values and mean values of the two classes were calculated for each gene using the remaining samples . the drug - response of the withheld sample was predicted by calculating the prediction score . to evaluate the reliability of the prediction system , we calculated a “ classification score ” ( cs ) using the grs values of responders and non - responders in each gene set , as follows : a larger value of cs indicates better separation of the two groups by the prediction system . we used web - available software (“ cluster ” and “ treeview ”) written by m . eisen ( http :// genome - www5 . stanford . edu / microarray / smd / restech . html ) to create a graphic representation of the microarray data and to create a dendrogram of hierarchical clustering . before the clustering algorithm was applied , the fluorescence ratio for each spot was first log - transformed and then the data for each sample were median - centered to remove experimental biases . aliquots ( 5 · 0 μg ) of the same arna hybridized to the microarray slides from individual samples and from the normal control lung were reversely transcribed using oligo ( dt ) 12 - 18 primer and superscript ii reverse transcriptase ( invitrogen , carlsbad , calif ., usa ). semi - quantitative rt - pcr experiments were carried out with the following sets of synthesized primers specific to the 12 top - ranked genes used for establishing a grs or with beta - actin ( actb )- specific primers as an internal control : flj22662 , 5 ′- gccataagtggtcccacagt - 3 ′ and 5 ′- gtcttctagtccgtcatctccct - 3 ′; amphiregulin ( areg ), 5 ′- ccatagctgcctttatgtctgc - 3 ′ and 5 ′- ctttttaccttcgtgcaccttt - 3 ′, coronin , actin binding protein , ic ( coroic ), 5 ′- taatctgctgaggaccttttgtc - 3 ′ and 5 ′- taattcactgtcctcttctggga - 3 ′; apoptosis , caspase activation inhibitor ( aven ), 5 ′- gctcacagcagtaaatgccta - 3 ′ and 5 ′- tgctatgctgtaaacactggcta - 3 ′; dual specificity phosphatase 3 ( dusp3 ), 5 ′- ggatcctttattggtggtagagc - 3 ′ and 5 ′- ccagagtgaccctgaagataaat - 3 ′; dj473b4 , 5 ′- acctgattctctaggtgcagttt - 3 ′ and 5 ′- gtcgtttcaaccaggtagttttg - 3 ′; pleckstrin homology - like domain , family a , member 2 ( phlda2 ), 5 ′- gggcgccttaagttattgga - 3 ′ and 5 ′- ggatggtagaaaagcaaactgg - 3 ′; rna binding motifprotein 7 ( rbm7 ), 5 ′- tgtaatggagattgtacaggttg - 3 ′ and 5 ′- aggaacagtacaaatgctgtggt - 3 ′; bx092512 ( est ), 5 ′- gcactccttgaaggtacactaac - 3 ′ and 5 ′- atttgtattcactcagccatgc - 3 ′; oncostatin m receptor ( osmr ), 5 ′- acccaacttcaaaactaggactc - 3 ′ and 5 ′- acagcttgatgtcctttctatgc - 3 ′, glutamate - cysteine ligase , catalytic subunit ( gclc ), 5 ′- tcatgaaaggcactgagttttg - 3 ′ and 5 ′- gttagctgaagcagctttattgc - 3 ′; collagen , type iv , alpha 3 binding protein ( col4a3bp ), 5 ′- atatgcacaatcctggaagtga - 3 ′ and 5 ′- tgccttactagcattaccaccat - 3 ′; actb , 5 ′- gaggtgatagcattgctttcg - 3 ′ and 5 ′- caagtcagtgtacaggtaagc - 3 ′. pcr reactions were optimized for the number of cycles to ensure product intensity within the logarithmic phase of amplification . we did phosphor imager quantification analysis ( molecular imager fx : bio - rad laboratories , hercules , calif ., usa ), and rt - pcr band intensities were quantitatively compared with normalized cy5 / cy3 ratio of gene expression from the microarray data . rt - pcr was performed to screen the mutation at entire region of codon 709 - 870 ( from p - loop to activation loop ) of egfr which was recently reported as a hot spot of mutation , 18 using three primer sets : fragment - 1 , 5 ′- tcttacacccagtggagaagc - 3 ′ and 5 ′- gtctttgtgttcccggacat - 3 ′; fragment - 2 , 5 ′- actatgtccgggaacacaaa - 3 ′ and 5 ′- ttccgtcatatggcttgg - 3 ′; fragment - 3 , 5 ′- cgtcgctatcaaggaattaagag - 3 ′ and 5 ′- gtagctccagacatcactctggt - 3 ′. rt - pcr products from 19 nsclc patients treated with gefitinib were analyzed by direct sequencing . to confirm the differential expression of areg and transforming growth factor - alpha ( tgfa ) proteins , both of which encode the ligand for egfr and other erbb members , and other 3 candidate markers ( a disintegrin and metalloproteinase domain 9 ( adam9 ), d9 antigen ( p24 ), and osmr ), which are also known to relate to the egfr signalling , for predicting responders vs non - responders to gefitinib , we stained clinical tissue sections obtained by fiberscopic transbronchial biopsy ( tbb ) and lymph - node biopsy using envision + kit / hrp ( dakocytomation , glostrup denmark ). briefly , after endogenous peroxidase and protein blocking reactions , anti - human areg polyclonal antibody ( neo markers , fremont , calif ., usa ), anti - human tgfa monoclonal antibody ( calbiochem , darmstadt , germany ), anti - human adam9 monoclonal antibody ( r & amp ; d systems inc . minneapolis , minn ., usa ), anti - human cd9 monoclonal antibody ( novocastra laboratories ltd , newcastle upon tyne , uk ), or anti - human osmr monoclonal antibody ( santa cruz biotechnology , inc ., santa cruz , calif ., usa ), was added , and then hrp - labeled anti - rabbit or anti - mouse igg as the secondary antibody . substrate - chromogen was then added and the specimens were counterstained with hematoxylin . frozen tissue samples from 11 patients were selected for analysis of immunohistochemistry . positivity of immunostaining was assessed semi - quantitatively by scoring intensity as absent or positive by three independent investigators without prior knowledge of the clinical follow - up data . cases were accepted only as positive if reviewers independently defined them thus . serum was obtained from an independent set of 35 lung - adc patients who were treated with gefitinib based on the same protocol as this clinical study at hiroshima university hospital in japan ( 5 for pr , 10 for sd , and 20 for pd ). the sera of all the patients were obtained with informed consent at the time of diagnosis and every 4 weeks after the beginning of treatment , and stored at − 80 ° c . the serum tgfa levels were measured by an elisa using a commercially available enzyme test kits ( tgf - alpha elisa kit : oncogene rsearch products , san diego , calif ., usa ). human nsclc ( adenocarcinoma ) cell lines pc - 9 , nci - h358 , and nci - h522 were purchased from the american type culture collection ( atcc ; rockville , md ., usa ). to detect expression of areg in these nsclc cells , total rna from each line was reverse - transcribed for single - stranded cdnas using oligo ( dt ) 12 - 18 primer and superscript ii ( invitrogen ). semi - quantitative reverse transcriptase - pcr ( rt - pcr ) was carried out as described previously . 14 gefitinib ( 4 -( 3 - chloro - 4 - fluoroanilino )- 7 - methoxy - 6 -( 3 - morpholinopropoxy ) quinazoline : zd 1839 , iressa ), an inhibitor of epidermal growth factor receptor tyrosine kinase , was provided by astrazeneca pharmaceuticals ( macclesfield , uk ). the drug was dissolved in dmso at a concentration of 10 mm and kept at − 20 ° c . we performed flow - cytometry to determine the sensitivity of lung adenocarcinoma cell lines to gefitinib treatment . cells were plated at densities of 5 × 10 cells / 100 - mm dish and treated with 1 · 0 μm of gefitinib in appropriate serum - free medium . the cells were trypsinized 72 hours after the treatment , collected in pbs , and fixed in 70 % cold ethanol for 30 min . after treatment with 100 μg / ml rnase ( sigma - aldrich co ., st . louis , mo ., usa ), the cells were stained with 50 μg / ml propidium iodide ( sigma - aldrich co .) in pbs . flow cytometry was performed on a becton dickinson facscan and analyzed by modfit software ( verity software house , inc ., topsham , me ., usa ). the percentages of nuclei in g0 / g1 , s , and g2 / m phases of the cell cycle and sub - g1 population were determined from at least 20 , 000 ungated cells . to investigate whether areg functions as an autocrine anti - apoptotic factor in lung adenocarcinoma cells treated with gefitinib , we carried out the following assay . first , gefitinib - sensitive pc - 9 cells , which do not express areg , were cultured in serum - free medium for at least 8 hours prior to gefitinib treatment . these cells were then incubated with 0 · 5 or 1 · 0 μm of gefitinib for 72 hours in media that were either serum - free or supplemented with 10 % fcs , or in serum - free conditioned medium collected from 72 - hour cultures of areg - expressing cells ( nci - h358 or nci - h522 ). each medium was replaced once with the same medium containing gefitinib at the 48 - hour time point . to detect the response of each cell line to gefitinib , viability was evaluated by mtt assays using cell counting kits ( wako , osaka , japan ). to confirm the autocrine effect of areg on the gefitinib - resistance of nsclc cells , we cultured pc - 9 cells for 72 hours in serum - free medium containing 1 · 0 μm of gefitinib and recombinant areg protein ( genzyme - techne , minneapolis , minn ., usa ) in final concentrations of 1 - 100 ng / ml . cell viability was evaluated by mtt assays . a possible effect of areg itself on the viability of nsclc cells was evaluated also , by culturing the pc - 9 cells in serum - and gefitinib - free medium containing only recombinant areg protein . mtt assays were performed as above . of the 53 patients enrolled in this trial , 46 had tumors diagnosed as adenocarcinomas ( 86 · 8 %); five were squamous - cell carcinomas ( 9 · 4 %); two were large cell carcinomas ( 3 · 8 %). fifteen patients achieved a pr and nobody revealed a cr ; 17 patients were classified as sd , and 19 as pd . no clinical - response data were available for two of the patients . the tumor - response rate ( cr + pr / cr + pr + sd + pd ) for this treatment was 29 · 4 %, and the disease control rate ( cr + pr + sd / cr + pr + sd + pd ) was 62 · 8 % ( table 1 ). tumor samples were collected from 43 patients . samples from 32 of those 43 contained sufficient numbers of cancer cells for analysis of expression profiles on our cdna microarray . the numbers of samples that were judged to be suitable for further microarray analysis , were 8 for pr , 7 for sd , and 13 for pd ( table 2 ). 17 of the 28 samples were analyzed as learning cases ( 7 for pr and 10 for pd ), and 11 were as test cases ( 1 for pr , 3 for pd , and 7 for sd ) for establishing a predictive scoring system for the efficacy of gefitinib treatment . for further validation of the prediction system , another blinded set of samples from 5 newly enrolled test - cases ( 4 for pd and 1 for sd ) were obtained and added finally to the initial 11 test cases above . we attempted to extract genes that were differentially expressed between tumors from seven patients in the pr group ( defined as responders ) and those from 10 patients in the pd group ( defined as non - responders ) by comparing expression levels of 27 , 648 genes . ( tables 2 , 3 ). we carried out a random - permutation test to distinguish between the two subclasses defined by tumor response , and identified 51 genes whose permutational p - values were less than 0 · 001 ( table 4 ). expression levels of 40 genes were higher , and those of the other 11 were lower , in the non - responders . establishment of a predictive scoring system for the efficacy of gefitinib treatment based on the expression profiles of the 51 genes selected above , we tried to establish a predictive scoring system for the efficacy of gefitinib treatment . prediction scores , termed gefitinib response score ( grs ), were calculated according to procedures described previously ( see methods ). to determine the number of candidates that provided the best separation of the two groups , we ranked the 51 genes on the basis of the significance of their permutational p - values and calculated prediction scores by the leave - one - out test , in decrements of 1 starting from the bottom of the rank - ordered list ( 51 , 50 , 49 , 48 etc .). we calculated a classification score ( cs ), a standard we had previously defined for evaluation of the ability to discriminate two classes , for each set of genes . 17 as shown in fig2 a , we obtained different prediction scores when the number of discriminating genes was changed . we obtained the best cs , meaning the best separation of responders from non - responders , when we calculated the scores using only the 12 top - ranked genes in our candidate list . hierarchical clustering analyses using all 51 genes , or only the top 12 , classified all 17 cases into one of two groups according to the response to gefitinib ( fig2 , b ). the two groups were most clearly separated when we used the top 12 genes for cluster analysis . finally , we established a numerical drug - response - scoring algorithm that might be clinically applicable for predicting sensitivity of an individual nsclc to gefitinib , on the basis of expression levels of the 12 selected genes . to validate this prediction system we investigated 8 additional (“ test ”) nsclc cases ( 1 for pr and 7 for pd ) that were completely independent of the 17 “ learning ” cases used for establishing the system . we examined gene - expression profiles in each of those samples and then calculated grs on the basis of the expression levels of the 12 discriminating genes . as shown in fig2 c , scores obtained by the grs system were concordant with the clinical responses to gefitinib in all eight “ test ” cases . grs values for the eight test - sd patients were calculated according to the predictive scoring system established above . although the values were widely distributed from − 83 · 0 ( predicted as non - responder ) to 61 · 6 ( responder ), the scores of patients who retained sd status throughout the observation period were likely to be higher than those of patients who had been judged as sd at a certain time - point of the study but showed progression of the disease within three or four months after the start of treatment ( fig2 , c ). although the grs system was established on the basis of gene - expression profiles that distinguished between patients with pr and patients with pd ( without sd ) in tumor response , these results suggest that the grs serves in classifying sd patients into groups according to their response to gefitinib . to confirm differential expression of the top 12 predictive genes between pr and pd cases , expression values derived from microarray data were correlated with values from semi - quantitative rt - pcr of rnas from the same patients ( 5 pr and 7 pd ) ( fig3 , a , table 5 , a ). spearman rank correlations were positive for all of the 12 genes and significantly positive for seven of 12 genes . to validate differential expression of the predictive protein markers between pr and pd cases , we carried out immunohistochemical staining with five different antibodies for areg , tgfa , adam9 , cd9 , and osmr , all of which were known to be involved in the ligand - egfrs signalling and whose permutational p - values were less than 0 · 01 . we first stained paired tumor tissue sections obtained by tbb and lymph - node biopsy from the same patients using these 5 antibodies . no intra - patient differences on protein expression of these five markers were observed in three different patients ( fig3 , b ). we also validated the microarray data with the five markers in 11 nsclc samples ( 5 for pr and 6 for pd ). the results were consistent with the microarray data ( fig3 , c , table 5 , b ). to further evaluate the availability of the prediction system in routine clinical situations , we detected tgfa protein using elisa in serum samples from 5 pr , 10 sd , and 20 pd patients that were independently collected for serological test and were not enrolled in microarray analysis . the serum levels of tgfa were 19 · 0 ± 2 · 8 pg / ml ( mean ± se ) in pd patients , 13 · 9 ± 1 · 9 pg / ml in sd patients , and 12 · 8 ± 1 · 4 pg / ml in pr patients ( fig4 ). twelve of 20 serum samples from pd patients were positive for tgfa and all samples from pr patients were negative , when 16 · 0 pg / ml was used as a cutoff . areg , a ligand for egfr and other erbb members was significantly over - expressed in non - responders but not ( or hardly ) detectable in responders . to investigate whether areg protein leads to resistance of nsclcs to gefitinib therapy when it is secreted in an autocrine manner , we performed the following biological analyses . we initially identified expression of areg mrna in lung - adenocarcinoma cell lines nci - h358 and - h522 , but not in pc - 9 , by means of rt - pcr experiments ( fig5 , a ). next , we performed flow - cytometric analysis 72 hours after treatment of pc - 9 cells with 1 · 0 μm of gefitinib , and found that gefitinib increased the percentages of nuclei in sub - g1 ( 24 %) compared with cells with no treatment ( 6 %) ( data not shown ). this result suggested that gefitinib might induce apoptosis in pc - 9 cells . we then analyzed the viability of pc - 9 cells , which are gefitinib - sensitive and do not express areg , after culture in serum - free medium or in serum - free , conditioned medium obtained from nci - h358 or - h522 cells grown in the presence or absence of 0 · 5 or 1 · 0 μm of gefitinib . as shown in fig5 b , the viability of pc - 9 cells incubated in the serum - free , conditioned medium containing gefitinib was greater than that of pc - 9 cells grown in serum - free medium with the same concentrations of gefitinib . as the supplier of gefinitib has reported previously , the anti - tumor effect of gefitinib decreases in the presence of 10 % fcs , suggesting that this assay should be suitable for quantitative measurement of gefitinib dosage and activity . to investigate whether areg , secreted in an autocrine manner , inhibits apoptosis of nsclc cells treated with gefitinib , we cultured pc - 9 cells in serum - free medium containing recombinant areg protein at final concentrations of 1 - 100 ng / ml , in the presence or absence of 1 · 0 μm gefitinib . the viability of pc - 9 cells incubated with both areg and 1 · 0 μm gefitinib was increased in comparison to cells incubated with 1 · 0 μm gefitinib only , in an areg - dose - dependent manner ( fig5 , c ). on the other hand , recombinant areg alone had no effect on the viability of pc - 9 cells ( fig5 , c ). this observation appeared to indicate that areg inhibits the apoptosis induced by gefitinib , but does not in itself affect cell viability . immunostaining for areg is shown in fig6 . a large body of evidence supports the view that molecules in the egfr autocrine pathway are involved in a number of processes important to cancer formation and progression , including cell proliferation , angiogenesis , and metastatic spread . 5 therapeutic blockade of specific signalling , therefore , could be a promising strategy for cancer treatment . gefitinib , a synthetic anilinoquinazoline , inhibits the tyrosine kinase activity of egfr by competing with adenosine triphosphate for a binding site on the intracellular domain of the receptor . 7 in phase ii trials ( ideal 1 and ideal 2 ), use of gefitinib as a 2nd -, 3rd -, or 4th - line monotherapy for advanced nsclc achieved tumor - response rates of nearly 20 %, 8 - 10 which were superior to those achieved with conventional cytotoxic agents . multivariate analysis of patients in the ideal 1 study suggested that the response rate in females might be higher than in males , and higher in patients with adenocarcinomas than in patients with squamous - cell carcinomas ( odds ratios 2 · 7 and 3 · 5 respectively ). 9 recent study suggested that individuals in whom gefitinib is efficacious are more likely to have adenocarcinomas of the bronchioloalveolar subtype and to be never smokers ( odds ratios 13 · 5 and 4 · 2 respectively ). 19 the higher tumor - response rate ( 29 · 4 %) documented in the clinical trial reported here might reflect a higher proportion of patients with adenocarcinoma ( 46 adenocarcinomas , five squamous - cell carcinomas and two large - cell carcinomas ) than has been the case in other studies . the clinicopathological determinants of gefitinib sensitivity including bronchioloalveolar carcinoma ( bac ) features are predictve to a certan extent , 9 , 10 , 19 , 20 however , previous reports and our observations obviously suggest that no factors can perfectly predict the response of nsclc to gefitinib treatment . therefore novel methods to discriminate responders from non - responders in advance could allow a more focused use of gefitinib in clinical settings . by statistical analysis of gene - expression profiles of advanced nsclcs obtained on cdna microarrays , we identified dozens of genes associated with sensitivity to gefitinib . we introduced a prediction - scoring system based on expression of the 12 genes that had shown the most significant differences in expression levels between responder ( pr ) and non - responder ( pd ) groups . this set of genes was selected from expression profiles of lung adenocarcinomas ; however , the grs system successfully classified all eight of our “ test ” pr and pd cases in accord with their clinical responses to gefitinib , and one of them was a squamous - cell carcinoma . moreover , this system was likely to separate intermediate tumor responses ( sd ) into two groups , one representing patients who succeeded in maintaining the tumor - static effect for a long period and the other representing patients who failed to do so . in practical terms , we need to predict the chemosensitivity of individual tumors using the minimally invasive techniques available at every hospital , because patients with advanced nsclcs are rarely candidates for surgical resection of their tumors . therefore we have tried to establish a prediction system that requires only the amount of cancerous tissue that can be obtained by , for example , flexible bronchofiberscopy . by verifying individual steps of the method , we were able to precisely profile gene expression in biopsy specimens as small as 1 mm . relevant microarray results were confirmed by semi - quantitative rt - pcr for 12 genes that showed the most significant differences to establish a grs system . furthermore , we validated the effectiveness of antibodies for 5 different biomarkers ( areg , tgfa , adam9 , cd9 , and osmr ), all of which were reported to be involved in the ligand - egfr signalling , for discriminating potential responders from non - responders , in both tbb and lymph - node biopsy samples . moreover , we were able to detect serum tgfa proteins in lung - adc patients by elisa . further evaluation of these markers for clinical use are necessary , however , the limited number of genes required for prediction should eventually enable laboratories to diagnose in advance the efficacy of gefitinib treatment for an nsclc patient , using routine procedures such as serological examinations of blood , pcr experiments , or immunohistochemical analysis of biopsy specimens . to our knowledge , this is the first report about gene - expression profiles of unresectable “ advanced ” lung cancers , although profiles of surgically resected specimens of “ early ” lung cancers have been reported . 21 , 22 however , about 70 % of tumors in patients diagnosed with ncslc are already locally advanced or metastatic , which generally renders them resistant to conventional therapeutic modalities . therefore the genes listed here should be useful for disclosing molecular mechanisms of lung - cancer progression and may be potential targets for drug development . gefitinib was developed as a “ selective ” inhibitor of egfr - tk ; however , no clear association between the level of egfr activation and response to gefitinib has been found in vitro or in vivo . 7 , 23 in clinical trials , gefitinib has been more effective against adenocarcinomas than against squamous - cell carcinomas , 9 , 10 although over - expression of egfr is less frequent in adenocarcinomas . 24 therefore , it is important to identify which individual tumors are good targets for this treatment . in our analysis using clinical samples , the difference in egfr protein expression between responders and non - responders were not statistically significant . on the other hand , amphiregulin ( areg ) and transforming growth factor alpha ( tgfa ), both of which encode the ligand for egfr and other erbb members , were significantly over - expressed in non - responders but not ( or hardly ) detectable in responders p = 0 · 0000000000093 and 0 · 0095 respectively ; table 4 ). the significance of the ligands and the egfr autocrine loop in growth and survival of lung - cancer cells is indisputable , 24 - 26 but the role of areg in formation and progression of cancers is poorly understood . however , several lines of evidence suggest that over - expression of areg is associated with shortened survival of patients with nsclc . 24 moreover , anti - apoptotic activity of areg in human lung - adenocarcinoma cells was reported recently . 25 to investigate whether the anti - apoptotic activity of areg leads to resistance of nsclc cells to gefitinib therapy , we performed a biological assay using a gefitinib - sensitive but areg - non - expressing nsclc cell line , pc - 9 . we found that the anti - tumor activity of gefitinib on pc - 9 cells was dramatically decreased by autocrine secretion of areg . this evidence strongly suggests that although growth - factor signalling by the egfr is markedly complicated at every step because of the multiplicity of ligands , dimerization partners , effectors , and downstream pathways , 26 areg might be a principal activator of the ligands - receptor autocrine growth pathway that leads to cancer progression and resistance to gefitinib . several elements associated with the egfr - tk pathway are present on our list of differentially - expressed genes . for example , genes encoding dual specificity phosphatase 3 ( dusp3 ), adam9 , cd9 , and osmr were expressed predominantly in non - responders ( p = 0 · 00000000094 , 0 · 01 , 0 · 000022 , and 0 . 0000011 , respectively ). dusp3 gene modulates egfr signalling by dephosphorylating mitogen activated protein kinase ( mapk ), a key mediator of signal transduction , 27 and adam9 is involved in activation of egfr signalling by shedding the ectodomain of prohb - egf ( pro heparin - binding epidermal growth factor - like growth factor ). 28 cd9 physically interacts with transmembrane tgfa . cd9 expression strongly decreases the growth factor - and pma - induced proteolytic conversions of transmembrane to soluble tgfa and strongly enhances the tgfa - induced egfr activation . 29 osmr is reported to be constitutively associated with erbb2 in breast cancer cells . 30 although other target molecules for gefitinib have been suggested , our results suggest that egfr signalling is at least one of the important processes involved in response to this drug . since gefitinib can induce apoptosis of some cancer cells in vivo , other molecules with anti - apoptotic activity , as well as areg , may contribute to a tumor &# 39 ; s resistance to the drug . aven ( apoptosis , caspase - activation inhibitor ), which was specifically expressed in our non - responders ( p = 0 · 00000000042 ), is known to enhance the anti - apoptotic activity of bcl - xl and to suppress apaf - 1 - mediated caspase activation . 31 on the other hand , mechanisms regulating drug transport should also affect drug resistance . gclc ( glutamate - cysteine ligase , catalytic subunit ), which plays an important role in cellular detoxification of anticancer drugs such as cisplatin , etoposide and doxorubicin , 32 was over - expressed in our group of non - responders ( p = 0 · 00000012 ). as these genes correlated negatively with responses to chemotherapy in our panel of tumors ( i . e . the higher the expression of these genes , the greater the resistance to gefitinib ), they might be involved in the mechanism ( s ) leading to that resistance . it should be noted also that the functions of nearly half of our candidate prediction - 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(*) for further validation of the grs , another blinded set of samples from 5 newly enrolled cases ( 4 pd and 1 sd ) were also added to these 28 cases later .