BACKGROUND
despite progress in the treatment of several cancers over recent decades, the lack of clinically relevant tumour models for individual subtypes of human cancer has proven to be a major impediment in the development of effective anti-cancer therapies  <cit> .
approaches that facilitate development of novel rational therapies targeting specific tumours  remain an urgent priority.
traditional models of human cancer involving the analysis of immortalised cell lines have given way in recent years to more clinically relevant studies in models that mirror the features of primary tumours  <cit> .
the two main approaches have been the generation of primary tumour-derived cell lines, and the generation of mouse models, either via transgenic approaches or through the engraftment of primary human tumour into immune-compromised mouse models  <cit> .
mouse models have been used extensively in this regard, for preclinical testing of drug efficacy and toxicity prior to establishing clinical trials.
a broad panel of xenografts with known treatment responsiveness, and well-defined molecular profiles, would provide an excellent adjunct to these models  <cit> .
mouse xenograft models of haematological malignancies, established by the transplantation of donor cells into non-obese diabetic/severe combined immunodeficient  or nod/scid/il- <dig> receptor gamma chain−/−  mice, are recognised as one of the most clinically relevant systems for investigating leukaemia biology and testing new treatments .
this is due to the faithful recapitulation of many aspects of the human disease, including kinetics of engraftment in the bone marrow , with subsequent infiltration of the spleen, peripheral blood and other organs  <cit> .
for these reasons, patient-derived xenografts  are considered superior to in vitro immortalised cancer cell lines that show many differences to primary tumours, including gene expression, drug responsiveness and epigenetic profiles  <cit> , which is most likely due to the selective processes associated with long term culturing.
pdxs have become increasingly popular as evidence mounts that they accurately recapitulate many of the features of patient tumours, such as tumour microenvironment, differentiation state and morphology, architecture and in some instances molecular signatures of the original patient tumour .
to establish the relevance of pdx models to primary tumours, high density molecular profiling of gene expression and epigenetic markers should be performed.
this was recently demonstrated for gene expression both between two tissue types, bone marrow and spleen and between independently engrafted mice for t-all  <cit> .
as a first step towards examining the equivalence of epigenetic profiles between primary tumour and xenograft, we carried out parallel dna methylation and gene expression profiling on a panel of childhood b-cell precursor acute lymphoblastic leukaemia  selected by their clinical responses to prednisolone.
this panel consisted of five individuals who had a good response to prednisolone  and five who had a poor response .
by comparing dna methylation and gene expression profiles between primary and derived, single-passaged xenograft lines, we report the stability of both gene expression and dna methylation in the xenograft, further highlighting their potential for exploring gene expression and epigenetic changes associated with responses to established and novel drugs.
methods
patient samples, characteristics and xenograft model generation
all experimental studies were approved by the human research ethics committee and the animal care and ethics committee of the university of new south wales.
written informed consent was obtained from the parents or guardians of paediatric all patients for use of biopsy samples in research, with the exception of samples obtained prior to may  <dig> , for which a waiver had been issued by the human research ethics committee.
a total of  <dig> xenograft lines were generated from children diagnosed with bcp-all.
individuals were selected based on their response to prednisolone.
we classified prednisolone poor responders  as patients with a peripheral blast count of ≥  <dig> × 109/l on day  <dig> following induction treatment with prednisolone and a single intrathecal dose of methotrexate, while a prednisolone good responder  demonstrated a day  <dig> peripheral blast count of <  <dig> × 109/l .
xenografts were established in nod/scid or nsg mice using direct explants of patient bm biopsies, exactly as described previously  <cit> .
when mice were highly engrafted with leukaemia human cd45+, mononuclear cells were isolated from spleens by facs at >90% purity and cryopreserved for subsequent experiments.table  <dig>
patient demographics of xenografts used in this study
ppr
<dig>
ppr
<dig>
ppr
<dig>
ppr
<dig>
ppr
<dig>
pgr
<dig>
pgr
<dig>
pgr
<dig>
pgr
<dig>
pgr
<dig>
genomic dna and total rna extraction
genomic dna was extracted from the primary bone marrow biopsies used for xenografting and from cells harvested from the spleens of engrafted animals for each xenograft using standard phenol/chloroform extraction and isopropanol precipitation.
total rna was extracted using trizol reagent  according to manufacturer’s instructions.
quality and yield were measured using a nanodrop spectrophotometer.
sodium bisulphite conversion of genomic dna
genomic dna was converted for dna methylation analysis using the methyleasy xceed kit  according to manufacturer’s instructions.
converted dna was used for downstream illumina infinium dna methylation beadarray analysis and sequenom epittyper validation.
genome-scale dna methylation analysis
converted genomic dna was processed and analysed for illumina infinium humanmethylation <dig> beadarray  according to manufacturer’s instructions .
this beadarray platform interrogates  <dig>  cpg sites across the human genome.
the arrays were scanned using an illumina beadarray reader and subsequently processed using the illumina genomestudio v. <dig> software package.
the bioconductor lumi package was used for downstream data processing and normalisation  <cit> .
briefly, dna probe methylation data were quality checked and then colour balance adjusted, background corrected and scaled based on the mean of all probes, using the methylation simple scaling normalization  implemented within the lumi package.
cpg sites with at least one sample having a detection p-value >  <dig>  were excluded from subsequent analyses, leaving  <dig>  cpg sites.
differential methylation analysis was performed using the limma package from bioconductor  <cit> .
significantly differentially methylated probes were selected based on a benjamini-hochberg adjusted p-value <  <dig> .
the methylation microarray data have been deposited into gene expression ominibus  with the identifier gse <dig>
gene expression illumina array analysis
total rna was extracted from the primary and xenograft tumours and amplified using the illumina totalprep rna amplification kit .
the amplified total rna was analysed using illumina wg-6_v <dig> chips  according to manufacturer’s instructions.
the sample probe profiles with no normalisation or background correction were exported from beadstudio , and the data were pre-processed using quantile normalisation.
probes with detection p-value greater than  <dig>  on all arrays were deemed as non-expressed probes and filtered out.
differential gene expression was determined using limma with the positive false discovery rate  correction for multiple testing .
the gene expression microarray data have been deposited into gene expression ominibus  with the identifier gse <dig>
sequenom massarray epityper analysis
primers  were designed to generate pcr amplicons from bisulphite converted genomic dna suitable for sequenom epityper chemistry as per the manufacturer’s protocol.
samples were analysed using maldi-tof mass spectrometry, dna methylation information was collected using epityper viewer software .
non-analysable and poor quality cpg sites were removed from downstream analysis as previously described  <cit> .
RESULTS
xenograft models of bcp-all are an accurate reflection of dna methylation and gene expression status of the corresponding primary tumour
one sample in our analysis, all28p, failed to meet array quality metrics .
therefore, the matching xenograft pair, all28x along with all28p gene expression data was removed from subsequent analysis.
all <dig> was also removed from the dna methylation and gene expression correlation analysis herein.
plotting the beta values of the entire data set revealed similar dna methylation profiles between primary tumour tissue and the matching xenograft from each of the  <dig> patients in our study.
similarly, gene expression levels between primary tumour tissue and xenograft were also comparable .
for genome-scale dna methylation, pearson’s correlation coefficients between matching primary and xenograft samples ranged between  <dig> - <dig>  while correlation coefficients between individuals ranged between  <dig> - <dig> .
for genome-wide gene expression, pearson’s correlation coefficients between primary and xenograft samples ranged between  <dig> - <dig>  and between individuals was greater than  <dig> - <dig>  .
gene expression profiles between individuals were more correlated than their dna methylation profiles.
consistent with this observation, unsupervised hierarchical clustering of the most variable dna methylation and gene expression across all samples revealed clustering of matching primary and xenograft samples.
this implies that the profiles from the xenografts recapitulate the profile of the primary tumour .figure  <dig>
comparisons of dna methylation and gene expression profiles between primary tumour tissue and xenografts.
scatterplots of dna methylation and gene expression array results from all <dig> showing high correlation between primary and xenograft tumours.
heatmap plot of pearson’s correlation coefficients of all primary and xenograft samples analysed for dna methylation and gene expression.
coefficients greater than  <dig>  and  <dig>  between matching primary and xenograft tumours were observed for dna methylation and gene expression respectively.
heatmap plot of the most variable dna methylation and gene expression probes.
a high level of similarity between matching primary and xenograft tumours resulted in all pairs clustering together.
green sample bar depicts ppr, blue sample bar depicts pgr.
to identify differential dna methylation between primary tumour and matching xenograft samples we applied a linear model with empirical bayes estimation and found  <dig> probes to be differentially methylated between matching primary tumour and xenograft sample after correction for multiple testing .
the majority of these probes demonstrated a small change in dna methylation with the average difference across individuals ranging from  <dig>  to  <dig> % .
we also looked for differential gene expression between matching primary and xenograft cell lines again applying a linear model with empirical bayes estimation on the genome-scale gene expression microarray results.
we found  <dig> probes from  <dig> genes to be differentially expressed between primary and xenograft lines .
however, as we observed with dna methylation, the differences in expression of these probes between primary and xenograft were minimal with an average fold difference in expression between primary and xenograft tumours of  <dig>  .
using david , the differentially methylated and differentially expressed genes between primary and matching xenograft lines were found to be mainly involved in haematological and cell signalling processes that could be accounted for given the cellular origins of the primary  and xenograft  samples.
given the relatively small number of differentially methylated probes  and differentially expressed probes , and the minimal absolute differences in dna methylation and expression , our results indicate that xenograft models largely recapitulate the dna methylation and gene expression profile of the corresponding primary tumour.
this highlights the potential utility of xenograft cell lines for modelling primary disease.
molecular biomarkers associated with prednisolone response
we then sought to identify differential dna methylation and gene expression associated with prednisolone poor  and prednisolone good  responders and included primary and xenograft samples in our analysis.
after correction for multiple testing,  <dig> dna methylation probes were differentially methylated between ppr and pgr .
gene expression analysis revealed  <dig> genes differentially expressed between ppr and pgr .
from these lists, we did not find any commonly annotated genes associated with prednisolone response between the top differentially methylated and top differentially expressed probes.
differential dna methylation segregated ppr from pgr by supervised hierarchical clustering and may serve as potential biomarkers for prednisolone response .
however, interrogating gene expression alone did not accurately segregate ppr from pgr .
functional annotation of differentially methylated genes annotated to these probes identified a number of apoptotic, cell signalling/structure pathways that did not reach statistical significance .table  <dig>
differential probes associated with prednisolone response
heatmap plot of the most significant dna methylation and gene expression probes distinguishing prednisolone good responders  from poor responders  after limma analysis .
dna methylation probes distinguished pgr from ppr while gene expression probes did not.
we then determined the relationship between dna methylation and gene expression in association with prednisolone response.
plotting the average dna methylation and gene expression differences between ppr and pgr revealed  <dig> probes annotated to  <dig> genes that were more highly expressed and less methylated in ppr samples compared to pgr samples .
conversely,  <dig> probes annotated to  <dig> genes were less highly expressed and more methylated in ppr samples compared to ppr .
with the exception of expression probes annotated to pawr, mtx <dig> and myo3a no other gene expression and dna methylation probes reached statistical significance .
dna methylation probes associated with pawr, mtx <dig> and myo3a demonstrated an average difference of > <dig>  between groups but did not reach significance with limma analysis between ppr and pgr.figure  <dig>
scatterplot of the average dna methylation and gene expression difference between ppr and pgr samples reveals  <dig> genes with a negative association between gene expression and dna methylation.
probes both differentially methylated and expressed in association with prednisolone response
validation of dna methylation biomarkers associated with prednisolone response
from our array analysis, the dna methylation changes segregated samples by prednisolone response.
we validated  <dig> of these probes using sequenom epityper chemistry on both primary and xenograft samples by selecting from our limma analysis, those also associated with changes in gene expression .
of the assays containing the  <dig> probes of interest,  <dig> regions continued to discriminate samples according to prednisolone response .
these were associated with the genes caps <dig> and arhgap <dig> , arx and hoxb <dig> .
primary and matching xenograft samples showed similar dna methylation levels in all cases.figure  <dig>
validation of dna methylation across four probes,
caps <dig>  arhgap <dig>  arx
and
hoxb6
using sequenom epityper chemistry.
discussion
it is becoming clear that the complexity of genetic, epigenetic, and subsequent gene expression disruption associated with human cancer is immense.
as such, many mouse models of tumourigenesis are limited in their capacity to faithfully mimic human disease.
in light of this, patient derived tumour tissue xenograft models are increasingly recognised as offering the most robust approach for testing tumour responses to various chemotherapeutic regimens, evaluating the efficacy of novel therapeutic agents, analysing the process of tumour progression at the cellular and molecular level and the identification of new therapeutic targets  <cit> .
however, as with most mouse xenograft models, the stability of molecular profiles  that regulate all aspects of tumour function remains to be determined.
confirmation of this stability is crucial in order identify molecular responses to treatment within the xenograft that could be extrapolated back to patients.
here, we have determined the stability of genome wide dna methylation and gene expression profiles between primary tumour cells and matching xenograft tumour cells from a small number of paediatric all cases with differential response to prednisolone.
a high correlation in both dna methylation and gene expression profiles was observed in all cases, confirming the stability of these molecular features of primary tumours in the mouse system.
differences in dna methylation and gene expression between primary and xenograft samples were negligible in magnitude  and comprised of a small fraction of probes for each array platform.
the differentially methylated genes include myod <dig>  gpr <dig> and slc27a <dig> .
many genes associated with minor expression differences were part of the globin gene family and genes involved in oxygen transport and include hbb, ahsp, hbd, hba <dig> .
this is likely to have arisen by the differences in cellular composition as the primary tumour samples contained a milieu of haematopoietic cells, including human erythrocytes that were absent in the xenograft samples that comprised of mononuclear cells derived from the murine spleen.
given the high degree of correlation and clustering of matching primary and xenograft samples after unsupervised hierarchical clustering of the most varied probes for dna methylation and gene expression, the xenografts described in this study are an accurate reflection of their corresponding primary tumours.
while a number of candidate genes whose dna methylation and/or gene expression status were associated with prednisolone response, given the small sample numbers and inherent genetic heterogeneity of the tumours, the significance of these genes remains unclear.
using hierarchical clustering, the most significant probes for dna methylation discriminated prednisolone response while the gene expression probes did not , reflecting the more variable nature of gene expression compared to dna methylation  <cit> .
using sequenom, we were able to replicate dna methylation changes at four genes associated with prednisolone response indicative of a potential dna methylation biomarker.
taking methylation and expression status together,  <dig> genes were differentially regulated between good and poor responders to prednisolone .
while the genes were found to be part of apoptotic and cell signalling pathways, their significance remains unclear given the small numbers in each group.
pawr demonstrated significant overexpression and hypomethylation across pprs compared to pgrs.
this is a wt <dig> interacting protein that also functions as a transcriptional repressor with pro-apoptotic functions and tumour resistance  <cit> .
while the down regulation of pawr confers poor prognosis in a range of solid tumours  <cit> , its role in haematological malignancy is less clear, with expression detectable in a range of leukaemias  <cit> .
our results warrant further investigation of pawr to determine a potential role in prednisolone response and responses to other novel drug regimens in an expanded xenograft cohort.
another gene with potential interest is pou4f <dig>  which appears to be differentially regulated according to prednisolone response .
however in our analysis, statistical significance was not achieved with the modest sample size of our panel.
pou4f <dig> has been shown to have a role in regulating the expression of b-cell markers in t positive acute myeloid leukaemia .
its role in b-cell all response to prednisolone remains unclear and could be a potential gene target for further characterisation in an expanded b-cell all xenograft panel.
while our study did not identify statistically significant genes associated with prednisolone response, we present here a first pass analysis using low-resolution microarray platforms to interrogate dna methylation and gene expression across our model system.
we demonstrate that our b-cell all xenograft panel recapitulates the dna methylation and gene expression profiles of the primary tumour and will facilitate future genome-wide interrogation of gene expression and dna methylation using next generation sequencing methodology.
CONCLUSIONS
patient-derived tumour xenograft models offer superior utility as preclinical models over cell line systems with their ability to recapitulate the milieu and microenvironment of the primary tumour.
however, the extent of gene expression and epigenetic stability within the xenograft has remained unclear at least in the haematological setting.
we have demonstrated that the gene expression and dna methylation profiles of cells taken from the spleens of engrafted mice are highly correlated to the original primary tumour.
given the similarity to the primary tumour, our study confirms the opportunity to investigate gene expression and dna methylation biomarkers in response to novel treatment strategies.
availability of supporting data
the data sets supporting the results of this article are included within the article and its additional files.
all microarray data presented in this paper have been deposited into gene expression omnibus  with the identifiers gse <dig> and gse <dig>
electronic supplementary material
additional file 1: table s1: sequenom epityper primers used in this study.
additional file 2: table s2: differentially methylated probes between primary and xenograft tumours.
additional file 3: figure s1: heatmap plot of the most significant dna methylation  and gene expression  probes differentiating primary to xenograft tumours.
while the samples clustered accordingly, the magnitude of dna methylation and gene expression differences across these probes were minimal.
additional file 4: table s3: differentially expressed probes between primary and xenograft tumours.
additional file 5: table s4: david ontology list of functional pathways of genes found to be associated with prednisolone response.
additional file 6: figure s2: sequenom validation of  <dig> probes identified as significantly differentially methylated between primary and xenograft tumours.
the green side column depicts pgr samples, while red depicts ppr samples.
dna methylation of these probes were able to separate tumours on prednisolone response, with  <dig>  giving the most discriminatory power.
competing interests
the authors declare no actual or perceived competing interests.
authors’ contributions
ncw, vab, jmc, rs and rbl designed the study; ncw, vab and jm performed the experiments; ncw, vab, jm, mp-b and jn analysed data; ncw, vab, rs and rbl interpreted the data and wrote the manuscript.
all authors read and approved the final manuscript.

 despite progress in the treatment of several cancers over recent decades, the lack of clinically relevant tumour models for individual subtypes of human cancer has proven to be a major impediment in the development of effective anti-cancer therapies. approaches that facilitate development of novel rational therapies targeting specific tumours remain an urgent priority. traditional models of human cancer involving the analysis of immortalised cell lines have given way in recent years to more clinically relevant studies in models that mirror the features of primary tumours. the two main approaches have been the generation of primary tumour-derived cell lines, and the generation of mouse models, either via transgenic approaches or through the engraftment of primary human tumour into immune-compromised mouse models. mouse models have been used extensively in this regard, for preclinical testing of drug efficacy and toxicity prior to establishing clinical trials. a broad panel of xenografts with known treatment responsiveness, and well-defined molecular profiles, would provide an excellent adjunct to these models. mouse xenograft models of haematological malignancies, established by the transplantation of donor cells into non-obese diabetic/severe combined immunodeficient or nod/scid/il- receptor gamma chain−/− mice, are recognised as one of the most clinically relevant systems for investigating leukaemia biology and testing new treatments. this is due to the faithful recapitulation of many aspects of the human disease, including kinetics of engraftment in the bone marrow, with subsequent infiltration of the spleen, peripheral blood and other organs. for these reasons, patient-derived xenografts are considered superior to in vitro immortalised cancer cell lines that show many differences to primary tumours, including gene expression, drug responsiveness and epigenetic profiles, which is most likely due to the selective processes associated with long term culturing. pdxs have become increasingly popular as evidence mounts that they accurately recapitulate many of the features of patient tumours, such as tumour microenvironment, differentiation state and morphology, architecture and in some instances molecular signatures of the original patient tumour. to establish the relevance of pdx models to primary tumours, high density molecular profiling of gene expression and epigenetic markers should be performed. this was recently demonstrated for gene expression both between two tissue types, bone marrow and spleen and between independently engrafted mice for t-all. as a first step towards examining the equivalence of epigenetic profiles between primary tumour and xenograft, we carried out parallel dna methylation and gene expression profiling on a panel of childhood b-cell precursor acute lymphoblastic leukaemia selected by their clinical responses to prednisolone. this panel consisted of five individuals who had a good response to prednisolone and five who had a poor response. by comparing dna methylation and gene expression profiles between primary and derived, single-passaged xenograft lines, we report the stability of both gene expression and dna methylation in the xenograft, further highlighting their potential for exploring gene expression and epigenetic changes associated with responses to established and novel drugs. methods patient samples, characteristics and xenograft model generation all experimental studies were approved by the human research ethics committee and the animal care and ethics committee of the university of new south wales. written informed consent was obtained from the parents or guardians of paediatric all patients for use of biopsy samples in research, with the exception of samples obtained prior to may, for which a waiver had been issued by the human research ethics committee. a total of xenograft lines were generated from children diagnosed with bcp-all. individuals were selected based on their response to prednisolone. we classified prednisolone poor responders as patients with a peripheral blast count of ≥  × 109/l on day following induction treatment with prednisolone and a single intrathecal dose of methotrexate, while a prednisolone good responder demonstrated a day peripheral blast count of <  × 109/l. xenografts were established in nod/scid or nsg mice using direct explants of patient bm biopsies, exactly as described previously. when mice were highly engrafted with leukaemia human cd45+, mononuclear cells were isolated from spleens by facs at >90% purity and cryopreserved for subsequent experiments.table patient demographics of xenografts used in this study ppr ppr ppr ppr ppr pgr pgr pgr pgr pgr genomic dna and total rna extraction genomic dna was extracted from the primary bone marrow biopsies used for xenografting and from cells harvested from the spleens of engrafted animals for each xenograft using standard phenol/chloroform extraction and isopropanol precipitation. total rna was extracted using trizol reagent according to manufacturer’s instructions. quality and yield were measured using a nanodrop spectrophotometer. sodium bisulphite conversion of genomic dna genomic dna was converted for dna methylation analysis using the methyleasy xceed kit according to manufacturer’s instructions. converted dna was used for downstream illumina infinium dna methylation beadarray analysis and sequenom epittyper validation. genome-scale dna methylation analysis converted genomic dna was processed and analysed for illumina infinium humanmethylation beadarray according to manufacturer’s instructions. this beadarray platform interrogates cpg sites across the human genome. the arrays were scanned using an illumina beadarray reader and subsequently processed using the illumina genomestudio v. software package. the bioconductor lumi package was used for downstream data processing and normalisation. briefly, dna probe methylation data were quality checked and then colour balance adjusted, background corrected and scaled based on the mean of all probes, using the methylation simple scaling normalization implemented within the lumi package. cpg sites with at least one sample having a detection p-value >  were excluded from subsequent analyses, leaving cpg sites. differential methylation analysis was performed using the limma package from bioconductor. significantly differentially methylated probes were selected based on a benjamini-hochberg adjusted p-value < . the methylation microarray data have been deposited into gene expression ominibus with the identifier gse gene expression illumina array analysis total rna was extracted from the primary and xenograft tumours and amplified using the illumina totalprep rna amplification kit. the amplified total rna was analysed using illumina wg-6_v chips according to manufacturer’s instructions. the sample probe profiles with no normalisation or background correction were exported from beadstudio, and the data were pre-processed using quantile normalisation. probes with detection p-value greater than on all arrays were deemed as non-expressed probes and filtered out. differential gene expression was determined using limma with the positive false discovery rate correction for multiple testing. the gene expression microarray data have been deposited into gene expression ominibus with the identifier gse sequenom massarray epityper analysis primers were designed to generate pcr amplicons from bisulphite converted genomic dna suitable for sequenom epityper chemistry as per the manufacturer’s protocol. samples were analysed using maldi-tof mass spectrometry, dna methylation information was collected using epityper viewer software. non-analysable and poor quality cpg sites were removed from downstream analysis as previously described.  xenograft models of bcp-all are an accurate reflection of dna methylation and gene expression status of the corresponding primary tumour one sample in our analysis, all28p, failed to meet array quality metrics. therefore, the matching xenograft pair, all28x along with all28p gene expression data was removed from subsequent analysis. all was also removed from the dna methylation and gene expression correlation analysis herein. plotting the beta values of the entire data set revealed similar dna methylation profiles between primary tumour tissue and the matching xenograft from each of the patients in our study. similarly, gene expression levels between primary tumour tissue and xenograft were also comparable. for genome-scale dna methylation, pearson’s correlation coefficients between matching primary and xenograft samples ranged between - while correlation coefficients between individuals ranged between -. for genome-wide gene expression, pearson’s correlation coefficients between primary and xenograft samples ranged between - and between individuals was greater than -. gene expression profiles between individuals were more correlated than their dna methylation profiles. consistent with this observation, unsupervised hierarchical clustering of the most variable dna methylation and gene expression across all samples revealed clustering of matching primary and xenograft samples. this implies that the profiles from the xenografts recapitulate the profile of the primary tumour.figure comparisons of dna methylation and gene expression profiles between primary tumour tissue and xenografts. scatterplots of dna methylation and gene expression array results from all showing high correlation between primary and xenograft tumours. heatmap plot of pearson’s correlation coefficients of all primary and xenograft samples analysed for dna methylation and gene expression. coefficients greater than and between matching primary and xenograft tumours were observed for dna methylation and gene expression respectively. heatmap plot of the most variable dna methylation and gene expression probes. a high level of similarity between matching primary and xenograft tumours resulted in all pairs clustering together. green sample bar depicts ppr, blue sample bar depicts pgr. to identify differential dna methylation between primary tumour and matching xenograft samples we applied a linear model with empirical bayes estimation and found probes to be differentially methylated between matching primary tumour and xenograft sample after correction for multiple testing. the majority of these probes demonstrated a small change in dna methylation with the average difference across individuals ranging from to %. we also looked for differential gene expression between matching primary and xenograft cell lines again applying a linear model with empirical bayes estimation on the genome-scale gene expression microarray results. we found probes from genes to be differentially expressed between primary and xenograft lines. however, as we observed with dna methylation, the differences in expression of these probes between primary and xenograft were minimal with an average fold difference in expression between primary and xenograft tumours of. using david, the differentially methylated and differentially expressed genes between primary and matching xenograft lines were found to be mainly involved in haematological and cell signalling processes that could be accounted for given the cellular origins of the primary and xenograft samples. given the relatively small number of differentially methylated probes and differentially expressed probes, and the minimal absolute differences in dna methylation and expression, our results indicate that xenograft models largely recapitulate the dna methylation and gene expression profile of the corresponding primary tumour. this highlights the potential utility of xenograft cell lines for modelling primary disease. molecular biomarkers associated with prednisolone response we then sought to identify differential dna methylation and gene expression associated with prednisolone poor and prednisolone good responders and included primary and xenograft samples in our analysis. after correction for multiple testing, dna methylation probes were differentially methylated between ppr and pgr. gene expression analysis revealed genes differentially expressed between ppr and pgr. from these lists, we did not find any commonly annotated genes associated with prednisolone response between the top differentially methylated and top differentially expressed probes. differential dna methylation segregated ppr from pgr by supervised hierarchical clustering and may serve as potential biomarkers for prednisolone response. however, interrogating gene expression alone did not accurately segregate ppr from pgr. functional annotation of differentially methylated genes annotated to these probes identified a number of apoptotic, cell signalling/structure pathways that did not reach statistical significance.table differential probes associated with prednisolone response heatmap plot of the most significant dna methylation and gene expression probes distinguishing prednisolone good responders from poor responders after limma analysis. dna methylation probes distinguished pgr from ppr while gene expression probes did not. we then determined the relationship between dna methylation and gene expression in association with prednisolone response. plotting the average dna methylation and gene expression differences between ppr and pgr revealed probes annotated to genes that were more highly expressed and less methylated in ppr samples compared to pgr samples. conversely, probes annotated to genes were less highly expressed and more methylated in ppr samples compared to ppr. with the exception of expression probes annotated to pawr, mtx and myo3a no other gene expression and dna methylation probes reached statistical significance. dna methylation probes associated with pawr, mtx and myo3a demonstrated an average difference of > between groups but did not reach significance with limma analysis between ppr and pgr.figure scatterplot of the average dna methylation and gene expression difference between ppr and pgr samples reveals genes with a negative association between gene expression and dna methylation. probes both differentially methylated and expressed in association with prednisolone response validation of dna methylation biomarkers associated with prednisolone response from our array analysis, the dna methylation changes segregated samples by prednisolone response. we validated of these probes using sequenom epityper chemistry on both primary and xenograft samples by selecting from our limma analysis, those also associated with changes in gene expression. of the assays containing the probes of interest, regions continued to discriminate samples according to prednisolone response. these were associated with the genes caps and arhgap, arx and hoxb. primary and matching xenograft samples showed similar dna methylation levels in all cases.figure validation of dna methylation across four probes, caps arhgap arx and hoxb6 using sequenom epityper chemistry. discussion it is becoming clear that the complexity of genetic, epigenetic, and subsequent gene expression disruption associated with human cancer is immense. as such, many mouse models of tumourigenesis are limited in their capacity to faithfully mimic human disease. in light of this, patient derived tumour tissue xenograft models are increasingly recognised as offering the most robust approach for testing tumour responses to various chemotherapeutic regimens, evaluating the efficacy of novel therapeutic agents, analysing the process of tumour progression at the cellular and molecular level and the identification of new therapeutic targets. however, as with most mouse xenograft models, the stability of molecular profiles that regulate all aspects of tumour function remains to be determined. confirmation of this stability is crucial in order identify molecular responses to treatment within the xenograft that could be extrapolated back to patients. here, we have determined the stability of genome wide dna methylation and gene expression profiles between primary tumour cells and matching xenograft tumour cells from a small number of paediatric all cases with differential response to prednisolone. a high correlation in both dna methylation and gene expression profiles was observed in all cases, confirming the stability of these molecular features of primary tumours in the mouse system. differences in dna methylation and gene expression between primary and xenograft samples were negligible in magnitude and comprised of a small fraction of probes for each array platform. the differentially methylated genes include myod gpr and slc27a. many genes associated with minor expression differences were part of the globin gene family and genes involved in oxygen transport and include hbb, ahsp, hbd, hba. this is likely to have arisen by the differences in cellular composition as the primary tumour samples contained a milieu of haematopoietic cells, including human erythrocytes that were absent in the xenograft samples that comprised of mononuclear cells derived from the murine spleen. given the high degree of correlation and clustering of matching primary and xenograft samples after unsupervised hierarchical clustering of the most varied probes for dna methylation and gene expression, the xenografts described in this study are an accurate reflection of their corresponding primary tumours. while a number of candidate genes whose dna methylation and/or gene expression status were associated with prednisolone response, given the small sample numbers and inherent genetic heterogeneity of the tumours, the significance of these genes remains unclear. using hierarchical clustering, the most significant probes for dna methylation discriminated prednisolone response while the gene expression probes did not, reflecting the more variable nature of gene expression compared to dna methylation. using sequenom, we were able to replicate dna methylation changes at four genes associated with prednisolone response indicative of a potential dna methylation biomarker. taking methylation and expression status together, genes were differentially regulated between good and poor responders to prednisolone. while the genes were found to be part of apoptotic and cell signalling pathways, their significance remains unclear given the small numbers in each group. pawr demonstrated significant overexpression and hypomethylation across pprs compared to pgrs. this is a wt interacting protein that also functions as a transcriptional repressor with pro-apoptotic functions and tumour resistance. while the down regulation of pawr confers poor prognosis in a range of solid tumours, its role in haematological malignancy is less clear, with expression detectable in a range of leukaemias. our results warrant further investigation of pawr to determine a potential role in prednisolone response and responses to other novel drug regimens in an expanded xenograft cohort. another gene with potential interest is pou4f which appears to be differentially regulated according to prednisolone response. however in our analysis, statistical significance was not achieved with the modest sample size of our panel. pou4f has been shown to have a role in regulating the expression of b-cell markers in t positive acute myeloid leukaemia. its role in b-cell all response to prednisolone remains unclear and could be a potential gene target for further characterisation in an expanded b-cell all xenograft panel. while our study did not identify statistically significant genes associated with prednisolone response, we present here a first pass analysis using low-resolution microarray platforms to interrogate dna methylation and gene expression across our model system. we demonstrate that our b-cell all xenograft panel recapitulates the dna methylation and gene expression profiles of the primary tumour and will facilitate future genome-wide interrogation of gene expression and dna methylation using next generation sequencing methodology.  patient-derived tumour xenograft models offer superior utility as preclinical models over cell line systems with their ability to recapitulate the milieu and microenvironment of the primary tumour. however, the extent of gene expression and epigenetic stability within the xenograft has remained unclear at least in the haematological setting. we have demonstrated that the gene expression and dna methylation profiles of cells taken from the spleens of engrafted mice are highly correlated to the original primary tumour. given the similarity to the primary tumour, our study confirms the opportunity to investigate gene expression and dna methylation biomarkers in response to novel treatment strategies. availability of supporting data the data sets supporting the results of this article are included within the article and its additional files. all microarray data presented in this paper have been deposited into gene expression omnibus with the identifiers gse and gse electronic supplementary material additional file 1: table s1: sequenom epityper primers used in this study. additional file 2: table s2: differentially methylated probes between primary and xenograft tumours. additional file 3: figure s1: heatmap plot of the most significant dna methylation and gene expression probes differentiating primary to xenograft tumours. while the samples clustered accordingly, the magnitude of dna methylation and gene expression differences across these probes were minimal. additional file 4: table s3: differentially expressed probes between primary and xenograft tumours. additional file 5: table s4: david ontology list of functional pathways of genes found to be associated with prednisolone response. additional file 6: figure s2: sequenom validation of probes identified as significantly differentially methylated between primary and xenograft tumours. the green side column depicts pgr samples, while red depicts ppr samples. dna methylation of these probes were able to separate tumours on prednisolone response, with giving the most discriminatory power. competing interests the authors declare no actual or perceived competing interests. authors’ contributions ncw, vab, jmc, rs and rbl designed the study; ncw, vab and jm performed the experiments; ncw, vab, jm, mp-b and jn analysed data; ncw, vab, rs and rbl interpreted the data and wrote the manuscript. all authors read and approved the final manuscript. 
as a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale dna methylation and gene expression profiling on a panel of paediatric b-cell precursor acute lymphoblastic leukaemia tumours that were stratified by prednisolone response. in order to demonstrate the potential utility of epigenetic analyses in bcp-all xenografts, we identified dna methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. we found high correlations in dna methylation and gene expression profiles between matching primary and xenograft tumour samples with pearson’s correlation coefficients ranging between and. we identified genes showing an inverse relationship between dna methylation and gene expression in association with prednisolone response. acute lymphoblastic leukaemiaxenograftsgenome-wide dna methylationmicroarray analysis of gene expressionglucocorticoid resistanceissue-copyright-statement© the author 2014 insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. the findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric bcp-all propagated in mouse xenograft models. further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses.
 patient-derived tumour xenografts are an attractive model for preclinical testing of anti-cancer drugs. insights into tumour biology and biomarkers predictive of responses to chemotherapeutic drugs can also be gained from investigating xenograft models. as a first step towards examining the equivalence of epigenetic profiles between xenografts and primary tumours in paediatric leukaemia, we performed genome-scale dna methylation and gene expression profiling on a panel of paediatric b-cell precursor acute lymphoblastic leukaemia tumours that were stratified by prednisolone response.  we found high correlations in dna methylation and gene expression profiles between matching primary and xenograft tumour samples with pearson’s correlation coefficients ranging between and. in order to demonstrate the potential utility of epigenetic analyses in bcp-all xenografts, we identified dna methylation biomarkers that correlated with prednisolone responsiveness of the original tumour samples. differential methylation of caps arhgap arx and hoxb were confirmed by locus specific analysis. we identified genes showing an inverse relationship between dna methylation and gene expression in association with prednisolone response. pathway analysis of these genes implicated apoptosis, cell signalling and cell structure networks in prednisolone responsiveness.  the findings of this study confirm the stability of epigenetic and gene expression profiles of paediatric bcp-all propagated in mouse xenograft models. further, our preliminary investigation of prednisolone sensitivity highlights the utility of mouse xenograft models for preclinical development of novel drug regimens with parallel investigation of underlying gene expression and epigenetic responses associated with novel drug responses. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords acute lymphoblastic leukaemiaxenograftsgenome-wide dna methylationmicroarray analysis of gene expressionglucocorticoid resistanceissue-copyright-statement© the author 2014 