all the samples were collected from patients undergoing surgical ablation of larynx squamous cell carcinoma at the head and neck surgery division of the department of ophthalmology , otorhinolaryngology and head & neck of ribeirao preto medical school , usp ( brazil ) .
inclusion criteria were : histopathological diagnosis of lscc , elective surgeries for lscc in patients without previous treatments and patients ' allowance to donate part of their tumor for genetic studies .
exclusion criteria were : doubtful diagnosis of lscc , unavailable post - surgical follow - up , patients without complete clinical data or signed agreement for collection of samples .
a total of twenty nine lscc tumor samples and thirteen adjacent non - neoplastic tissues proceed for the transcriptome analysis . clinical information and tnm staging classification of the lscc patients
most of the patients were male ( 96.8% ) , all of them were smokers and alcoholic , including both current and former .
thirty five percent of the tumors were originated from glottis , followed by larynx ( 29.0% ) and supraglottis ( 22.6% ) .
patients were further classified according to the tnm system : 48.4% of the patients were in early staging and 51.6% in late staging .
this study was approved by the ethics committee of ribeirao preto medical school , university of sao paulo ( usp ) ( proc .
after lscc histopathological confirmation and microdissection of the tumors from their non - neoplastic adjacent tissue , the samples were store in liquid nitrogen .
after extraction , the rna was purified with rneasy kit ( qiagen , usa ) and quantified with nanodrop spectrophotometer ( thermo scientific , usa ) .
its quality was evaluated by 1.5% agarose gel electrophoresis ( 28s and 18s ribosomal rna detection ) . for the microarray analysis
it was used the whole human genome oligo microarray kit 4    44k ( agilent , g4112f , usa ) .
cdna was hybridized to the microarray chip in the fluidics station 450 system ( affymetrix , usa ) , using the quick amp labeling one - color kit ( agilent , usa ) . as an internal mrna control ,
the one color rna spike - in kit was used ( agilent , usa ) .
agilent feature extraction software ( version 9.5.3.1 ) was used to analyze acquired array images .
plain text files were loaded and processed into r with the bioconductor package agi4    44pre - process .
the cv.rep.probes function was used to estimate the percent of coefficient of variation ( % cv ) for replicated non - control probes , inferring the reproducibility of the arrays ( fig .
the gprocessedsignal and the gbgused values of each array were loaded as foreground and background signals , respectively into an rglist object .
the following flags were used to filter probes : control , well above bg , is found , well above neg ctrls , is saturated , population outlier , and non uniform outlier . additionally probe signals with at least 90% of good features in an experimental condition were selected .
it was performed by looking for arrays ( a and b ) , which the sum ( s ) of the distances ( d ) to all other arrays , sa  =  b 
 dab was exceptionally large ; calculating the kolmogorov 
smirnov statistic ( ka ) between each array and the pooled data distribution ; and computing hoeffding 's statistic ( da ) on the joint distribution of a and m for each array ( m  =  log2(i1 )    log2(i2 ) ; a  =  1/2 ( log2(i1 )  +  log2(i2 ) ) , where i1 is the intensity of the array studied , and i2 is the intensity of a  pseudo-array that consists of the median across arrays ) .
stagewise normalization is recommended when data include technical and biological replicates producing a better median chip .
then , quantile normalization methods were applied , within groups and subsequently between all arrays .
each set of replicated non - control probes has been collapsed into a single value computed as the median of the probes intensities belonging to the same set . principal component analysis ( pca )
was applied to classify samples by tumor and adjacent non - neoplastic tissue as demonstrated on fig .
we compared the gene expression between tumor and adjacent non - neoplastic tissue using a non - paired - t test analysis . false discovery rate ( fdr ) adjusted p - value  <  1e    07 and fold change ( fc )  >  10 were accepted to consider genes to be differentially expressed , identifying a total of 81 probes above this cut - off ( fig .
3 ) . then we investigated the biological process of upregulated and downregulated genes in tumor compared to normal samples with web - based gene set analysis toolkit ( webgestalt ) .
p - value was calculated using hypergeometric distribution and fdr as multiple hypothesis test correction method .
tissue morphogenesis ( go : 0009888 ) and differentiation ( go : 0032332 ) are the main biological process in upregulated genes while cytokines and growth factor genes are highly expressed in adjacent non - neoplastic patients , suggesting a role on lscc tumorigenesis .
all the samples were collected from patients undergoing surgical ablation of larynx squamous cell carcinoma at the head and neck surgery division of the department of ophthalmology , otorhinolaryngology and head & neck of ribeirao preto medical school , usp ( brazil ) .
inclusion criteria were : histopathological diagnosis of lscc , elective surgeries for lscc in patients without previous treatments and patients ' allowance to donate part of their tumor for genetic studies .
exclusion criteria were : doubtful diagnosis of lscc , unavailable post - surgical follow - up , patients without complete clinical data or signed agreement for collection of samples .
a total of twenty nine lscc tumor samples and thirteen adjacent non - neoplastic tissues proceed for the transcriptome analysis . clinical information and tnm staging classification of the lscc patients
most of the patients were male ( 96.8% ) , all of them were smokers and alcoholic , including both current and former .
thirty five percent of the tumors were originated from glottis , followed by larynx ( 29.0% ) and supraglottis ( 22.6% ) .
patients were further classified according to the tnm system : 48.4% of the patients were in early staging and 51.6% in late staging .
this study was approved by the ethics committee of ribeirao preto medical school , university of sao paulo ( usp ) ( proc .
after lscc histopathological confirmation and microdissection of the tumors from their non - neoplastic adjacent tissue , the samples were store in liquid nitrogen .
after extraction , the rna was purified with rneasy kit ( qiagen , usa ) and quantified with nanodrop spectrophotometer ( thermo scientific , usa ) .
its quality was evaluated by 1.5% agarose gel electrophoresis ( 28s and 18s ribosomal rna detection ) . for the microarray analysis
it was used the whole human genome oligo microarray kit 4    44k ( agilent , g4112f , usa ) .
cdna was hybridized to the microarray chip in the fluidics station 450 system ( affymetrix , usa ) , using the quick amp labeling one - color kit ( agilent , usa ) . as an internal mrna control ,
the one color rna spike - in kit was used ( agilent , usa ) .
agilent feature extraction software ( version 9.5.3.1 ) was used to analyze acquired array images .
plain text files were loaded and processed into r with the bioconductor package agi4    44pre - process .
the cv.rep.probes function was used to estimate the percent of coefficient of variation ( % cv ) for replicated non - control probes , inferring the reproducibility of the arrays ( fig .
the gprocessedsignal and the gbgused values of each array were loaded as foreground and background signals , respectively into an rglist object .
the following flags were used to filter probes : control , well above bg , is found , well above neg ctrls , is saturated , population outlier , and non uniform outlier .
additionally probe signals with at least 90% of good features in an experimental condition were selected .
it was performed by looking for arrays ( a and b ) , which the sum ( s ) of the distances ( d ) to all other arrays , sa  =  b 
 dab was exceptionally large ; calculating the kolmogorov 
smirnov statistic ( ka ) between each array and the pooled data distribution ; and computing hoeffding 's statistic ( da ) on the joint distribution of a and m for each array ( m  =  log2(i1 )    log2(i2 ) ; a  =  1/2 ( log2(i1 )  +  log2(i2 ) ) , where i1 is the intensity of the array studied , and i2 is the intensity of a
stagewise normalization is recommended when data include technical and biological replicates producing a better median chip .
then , quantile normalization methods were applied , within groups and subsequently between all arrays .
each set of replicated non - control probes has been collapsed into a single value computed as the median of the probes intensities belonging to the same set .
principal component analysis ( pca ) was applied to classify samples by tumor and adjacent non - neoplastic tissue as demonstrated on fig .
2 . we compared the gene expression between tumor and adjacent non - neoplastic tissue using a non - paired - t test analysis . false discovery rate ( fdr ) adjusted p - value  <  1e    07 and fold change ( fc )  >  10 were accepted to consider genes to be differentially expressed , identifying a total of 81 probes above this cut - off ( fig .
then we investigated the biological process of upregulated and downregulated genes in tumor compared to normal samples with web - based gene set analysis toolkit ( webgestalt ) .
p - value was calculated using hypergeometric distribution and fdr as multiple hypothesis test correction method .
tissue morphogenesis ( go : 0009888 ) and differentiation ( go : 0032332 ) are the main biological process in upregulated genes while cytokines and growth factor genes are highly expressed in adjacent non - neoplastic patients , suggesting a role on lscc tumorigenesis .
we describe detailed technical methods to reproduce the analysis of whole human genome oligo microarray kit ( agilent , g4112f , usa ) from twenty - nine lscc tumor samples and thirteen adjacent non - neoplastic tissues .
lscc is the second most common malignancies of the head and neck tumors , and this high frequency may be associated with the lifestyle of patients , , .
the high quality gene expression data are concordant with important clinical parameters , demonstrating the relevant expression profile signature of differentially expressed genes .
the following is the supplementary data related to this article.supplemental table 1tumor sample description . tumor sample description
 laryngeal squamous cell carcinoma ( lscc ) is one of the most common malignancies of the head and neck tumors zhang et al . , 2013 [ 1 ] ) . 
 previous studies have associated its occurrence with social activities , such as tobacco and alcohol consumption ( hashibe et al . , 2007a [ 2 ] ; hashibe et al . , 2007b [ 3 ] ; shangina et al . , 2006 
 [ 4 ] ) . here 
 , we performed a genome - wide gene expression profiling in thirty - one patients positively diagnosed for lscc , in order to investigate new targets involved in tumorigenesis . 
Direct link to deposited data
Experimental design, materials and methods
Study population and clinical data
Microarray experiments
Quality control and signal pre-processing
Normalization
Differentially expressed genes and pathways analysis
Discussion