pancreatic ductal adenocarcinoma ( pdac ) represents the fourth leading cause of cancer mortality worldwide , with an incidence of approximately 217,000 new cases each year nearly matched by 213,000 deaths ( parkin et  al . , 2001 ) .
several of the most frequent genetic events underlying the initiation and progression of human pancreatic cancer have been identified ( hezel et  al . , 2006 ; maitra and hruban , 2008 ) .
these include activating mutations in the kras proto - oncogene , which occur in > 90% of pdac ( caldas and kern , 1995 ) and are considered as a key driver for pancreatic carcinogenesis , and mutations inactivating the tp53 gene , which occur in 5075% of patients ( redston et  al . , 1994 ) .
moreover , several lines of evidence implicate mutations inactivating the brca2 tumour suppressor in an estimated 520% of familial pdac ( hahn et  al . , 2003 ; couch et  al . , 2007 ) .
germline carriers of deleterious brca2 mutations that commonly truncate the encoded protein exhibit an increased lifetime risk of developing pdac , in addition to their well - known predisposition to cancers of the breast and ovary ( breast cancer linkage consortium , 1999 ) . within high - risk pancreatic cancer kindreds , inherited mutations in brca2 represent the most frequently encountered germline genetic alteration ( hahn et  al . , 2003 ) .
the incidence of germline brca2 mutations in apparently sporadic pancreatic cancers may be as high as in breast or ovarian cancer ( goggins et  al . , 1996 ) .
more recently , palb2 , which encodes a brca2-interacting protein also essential for homology - directed dna repair , has emerged as a pancreatic cancer susceptibility allele ( jones et  al . , 2009 ) .
three new transgenic models for pancreatic adenocarcinoma associated with brca2 inactivation have recently been described ( skoulidis et  al .
one of these models does not incorporate activation of the kras oncogene ( feldmann et  al . , 2011 ) .
in contrast , the other two models ( skoulidis et  al . , 2010 ; rowley et  al . , 2011 ) use a conditional gene - targeted allele developed by tuveson , jacks and colleagues ( jackson et  al . , 2001 ;
johnson et  al . , 2001 ) , in which tissue - specific activation of oncogenic krasg12d is driven on a single allele by loxp - cre mediated recombination , mimicking a genetic event that frequently triggers kras activation in human cancers .
cre recombinase expression is controlled by the pdx1 promoter , which is expressed at e8.5 and required for organogenesis of the pancreas , whereby loss of the gene is associated with an absence of pancreatic formation ( jonsson et  al . , 1994 ; offield et  al . ,
the expression of the pdx1-cre transgene therefore occurs throughout the pancreatic cellular compartment , albeit in a stochastic manner , to trigger kras activation ( hingorani et  al . ,
patients who carry germline mutations affecting brca2 harbour the germline mutant allele in all somatic tissues , whereas the second brca2 allele is wildtype ( wooster et  al . , 1994 ) .
it has been widely believed that loss of the second , wild - type brca2 allele in nascent cancer cells ( termed  loss of heterozygosity or loh ) is necessary for the emergence of tumours in germline mutation carriers . the pancreatic cancer model developed in our laboratory ( skoulidis et  al . , 2010 )
it carries in all somatic tissues a truncated allele of murine brca2 ( brca2 ) , which truncates the gene in an evolutionarily conserved and functionally critical region encoded by exon 11 , resembling deleterious germline mutations found in human carriers ( friedman et  al . , 1998 ) .
the second brca2 allele , brca2 ( jonkers et  al . , 2001 ) can be conditionally disrupted to remove exon 11 by loxp - cre recombination in the pancreas .
this event is driven by pdx1-cre , and therefore occurs in the same tissues which undergo kras activation .
there is evidence that both the brca2 and brca2 alleles can express a truncated protein product ( patel et  al . , 1998 ;
notably , the models developed by rowley et  al . and feldmann et  al .
both exclusively use a strain homozygous for the conditional brca2 allele ; thus , germline heterozygosity for brca2 is not modelled in their experiments .
all three of the new pancreatic cancer models incorporate conditional alleles that inactivate tp53 in the pancreas , to mimic the frequent loss of this tumour suppressor in human pancreatic cancers .
somewhat different tp53 alleles are used by each group , an important distinction given that the nature of tp53 mutations is thought to affect pancreatic cancer development ( olive et  al . , 2004 ) .
two of the studies employ gain - of - function mutants affecting a single allele .
these are either the structural mutant tp53 ( feldmann et  al . , 2011 ) or the contact mutant tp53 ( skoulidis et  al . , 2010 ) .
both these tp53 mutants are associated with the development of carcinomas ( olive et  al . , 2004 ) .
in contrast , the third study uses a null allele of tp53 , wherein exons 210 are deleted by pdx1-cre activation ( rowley et  al . , 2011 ) , in a manner that less faithfully represents human cancer - associated mutations .
thus , it should be clear from the foregoing that these three recently - published models for pancreatic carcinogenesis associated with brca2 inactivation harbour important differences not only in the tissue - specificity , nature and timing of mutant tp53 and brca2 alleles , but also in the presence of mutant kras .
we believe that these distinctions are vital to understanding the marked differences in pancreatic carcinogenesis observed in the studies , the key findings from which are highlighted in table 1 .
brca2 has been believed to follow the classical  two - hit paradigm for tumour suppression ( smith et  al . , 1992 ; collins et  al .
initial studies soon after the discovery of brca2 reported consistent inactivation of the wild - type brca2 allele through loss - of - heterozygosity ( loh ) in breast or ovarian cancer cells from mutation carriers ( collins et  al . , 1995 ; gudmundsson et  al . ,
1995 ) , engendering the widely accepted view that brca2 loh is an essential event in carcinogenesis .
a few notes of dissent have emerged in later studies ( king et  al .
, 2007 ; willems et  al . , 2008 ) , but they have not gained widespread attention . in this context
unexpectedly reveal that brca2 heterozygosity promotes pancreatic cancer development in mice and men . in both the tp53 wildtype and tp53 cohorts from the murine model ,
heterozygosity for brca2 ( through the brca2 genotype ) acts with kras to accelerate the progression and development of pdac .
a similar conclusion is reached from studies on a small number of human pancreatic cancer samples from carriers of the icelandic founder mutation in brca2 , the allele brca2 , which is 5 bp deletion in exon 9 that causes a frame - shift leading to the expression of a very short and unstable protein product ( mikaelsdottir et  al . , 2004 ) .
one possibility is that this genotype causes a mutator phenotype , owing to defects in dna repair arising from a known role of brca2 in homologous dna recombination ( patel et  al . , 1998 ;
however , previous studies on murine embryo fibroblasts ( mefs ) heterozygous for brca2 reveal no statistically significant effects on sensitivity to genotoxic agents ( patel et  al .
neither brca2 mice ( friedman et  al . , 1998 ) , nor strains heterozygous for other brca2 truncation mutants ( connor et  al .
, 2001 ; yan et  al . , 2004 ) , exhibit cancer predisposition .
notably , a lacz mutation reporter gene ( boerrigter et  al . , 1995 ) incorporated into the germline of mice heterozygous for a brca2 truncation similar but not identical to brca2 ( tutt et  al . ,
2002 ) reveals no evident mutator phenotype . on the other hand , mefs from this strain showed a mild alteration in dna repair kinetics during recovery from 4gy of ionizing radiation .
thus , there is little convincing evidence that heterozygosity for these brca2 mutant alleles creates a dna repair defect that could explain heightened cancer predisposition , although the possibility has not yet been conclusively excluded . in this connection , it is important to note that these cellular approaches do not yet account for the cooperative effect of mutant kras on pancreatic carcinogenesis associated with brca2 heterozygosity , as suggested by the murine model developed by skoulidis et  al .
even a subtle increase in mutational load induced by brca2 heterozygosity in mutant kras expressing cells  which might be undetectable in cellular experiments , but significant in  vivo  could plausibly accelerate the progression of pre - malignant pancreatic intra - epithelial ( panin ) lesions ( which occur frequently even in apparently normal pancreatic parenchyma ( hruban et  al . , 2008 ) ) to overt malignancy .
whether different brca2 alleles behave in a manner similar to brca2 is not clear . like brca2 ,
however , the instability of the truncated protein encoded by brca2 ( mikaelsdottir et  al . , 2004 ) suggests that haploinsufficiency for brca2 ( as opposed to any trans - dominant effect of a mutant brca2 protein ) accounts for the phenotypic effects of heterozygosity in patients who carry this icelandic founder mutation .
describe no heterozygous effect in any of their brca2 cohorts despite the presence of mutant kras .
interpretation of this difference is not straightforward , since the brca2 allele engenders brca2 loss only in the cells expressing pdx1-cre , unlike brca2 , which is expressed in all somatic cells .
this raises the possibility that non - cell autonomous effects of brca2 heterozygosity  for example on stromal cells rather than the nascent cancer cells  may account for the cancer - predisposing effect of the brca2 allele .
mitotic functions have also been ascribed to brca2 , and interestingly , defects in g2 checkpoint function ( menzel et  al . , 2011 ) , mitotic checkpoint enforcement ( choi et  al . , 2012 ) and the completion of cell division by cytokinesis ( daniels et  al . , 2004 ;
whether or not these roles for brca2 may explain the effect of heterozygosity in tumour development is yet to be explored .
heterozygosity for the brca2 allele is enough to trigger cytokinetic defects in mefs ( daniels et  al . , 2004 ) , but it is unclear whether the other mitotic functions are perturbed by brca2 heterozygosity .
importantly , recent data from human studies further support that brca2 heterozygosity is enough to promote carcinogenesis . in breast cancers ,
incomplete loss of the remaining wild - type allele has been observed using techniques more sensitive than those applied in the original studies ( king et  al . , 2007 ) .
importantly large - scale , unbiased genomic sequencing of high - grade serous ovarian carcinomas highlighted the retention of the wild - type allele in end stage disease from 25% of germline brca2 carriers ( atlas , 2011 ) .
furthermore , a detailed study of prostate tumour progression in brca2 germline mutation carriers uncovered no loh in high - grade prostatic intraepithelial neoplasias , considered precursor lesions to the development of prostate adenocarcinoma , and up to 55% of the malignant tumours analysed ( willems - jones et  al . , 2012 ) .
collectively , these data suggest that cancers arising in germline brca2 mutation carriers frequently fail to exhibit loss of the wildtype allele , and that failure to exhibit loh occurs in brca2-mutant cancers from several different tissues .
interestingly , these conclusions can be set against the emerging backdrop of ongoing studies on tissue samples from patients with familial forms of pancreatic cancer
. a study of 58 pancreatic intra - epithelial neoplasms and intraductal papillary mucinous neoplasms reveals that somatic losses in brca2 copy number are infrequent ( hong et  al . , 2012 ) .
however , definitive evidence addressing the extent to which the lessons from gemms of brca2-deficient pancreatic cancers can be applied to human neoplasia awaits the results of more extensive genome sequencing studies on pancreatic cancer samples from patients harbouring germline brca2 mutations .
murine pancreatic cancers emerging in brca2 strains in which both brca2 alleles are inactivated in pdx1-cre expressing cells exhibit a preponderance of acinar cell carcinoma histology . correspondingly , 3 of the four human pancreatic cancers from brca2 mutation carriers that exhibited loh were also of the acinar type ( skoulidis et  al . , 2010 ) , which normally accounts for only 12%% of human pancreatic cancers ( hruban , 2007 ) .
this raises the possibility that these genotypes promote the evolution of acinar cell carcinomas rather than pdac .
( 2011 ) also observe differences in the histopathological spectrum of pancreatic malignancies from mice in which brca2 as well as tp53 had been inactivated , when compared to tp53 deficiency alone .
these observations raise the possibility that the nature of brca2 mutations , their timing , or their coincidence with alterations with tp53 may alter the histopathological evolution of pancreatic cancers in mice . however , these observations remain too limited to allow firm conclusions to be drawn , and we draw attention to them here simply to emphasize the need for further studies .
we and others have shown ( patel et  al . , 1998 ; lee et  al . , 1999 ; tutt et  al . , 1999 ) that the genome - wide dna damage that follows homozygous inactivation of brca2 leads to checkpoint activation and cell cycle arrest , rather than the unrestrained cellular proliferation typical of cancer .
we have previously proposed ( venkitaraman , 2009 ) that checkpoint inactivation may therefore be an essential pre - requisite for homozygous brca2 inactivation through loh during carcinogenesis .
the work of skoulidis et  al . provides strong in  vivo evidence for this hypothesis , supported by the observations of rowley et  al . in both murine models ,
bi - allelic brca2 inactivation by itself leads to a loss of exocrine pancreatic parenchyma , a concomitant increase in adipose tissue , and progressive loss of organ functionality .
further demonstrate that pancreatic insufficiency is preceded by the widespread occurrence of dna double - strand breakage marked by h2ax staining in cells lacking both copies of brca2 .
moreover , both skoulidis et  al . and rowley et  al . find that the concomitant inactivation of tp53 function prevents pancreatic insufficiency , and allows rapid pdac development , in the pancreas of mice carrying bi - allelic mutations inactivating brca2 .
when the observations from these studies are synthesized , a picture emerges wherein brca2 heterozygosity in germline mutation carriers may suffice to allow the development of kras - driven pdac .
later inactivation of tp53 or other checkpoint genes may then allow eventual loss of the second brca2 allele : although loh is not an obligate step , it may promote the emergence of advanced cancers .
indeed , inferences from a very small study of just 5 samples from human pancreatic cancer patients support such a scenario , although it remains to be firmly established .
, our results suggest that brca2 heterozygosity suffices for pdac formation driven by mutant kras in mice and men .
however , the rationale for the use of targeted agents such as parp1 inhibitors ( parp1i ) in brca2-deficient cancers is contingent upon bi - allelic brca2 inactivation in the tumour cells ( bryant et  al . ,
therefore , as confirmed in our work ( skoulidis et  al . , 2010 ) , pdac cells that retain a functional brca2 allele are resistant to parpi such as the astrazeneca compound olaparib .
thus , parp1 inhibitors should be reserved for clinical use when brca2 loh can be verified in the tumour , assessment of which emerges as a critical requirement in the design of human clinical trials for the treatment of brca2-deficient cancers .
these findings exemplify how the new generation of gemms for pdac may represent valuable surrogate models for preclinical tests of therapeutic efficacy in patients .
importantly , such models not only allow in  vivo proof of new therapeutic concepts , but may also provide a platform to assess the pharmacodynamic and pharmacokinetic properties of new agents , although species - specific differences may limit such interpretations .
the models also provide a flexible method to assess the impact of therapy on tumour progression using adapted multimodal imaging and drug bioavailability ( including tissue drug penetrance ) analyses .
an important feature that determines if a particular gemm is useful as a preclinical platform is if the model recapitulates a similar clinical response to standard therapy agents in clinical use in human .
for instance , the kpc mouse model is relatively unaffected by gemcitabine similar to the small clinical benefit from this agent in the advanced pancreatic cancer setting in humans ( olive et  al . , 2009 ) .
each gemm can be likened to a patient with a particular tumour type , and hence , can be enrolled into preclinical trial of novel agents ( eklund et  al . , 2013 ; guerra and barbacid , 2013 ) .
such trials are facilitated by the use of adapted imaging techniques to monitor for tumour development and progression .
such utility is beginning to have an impact in the clinical setting . in humans ,
early phase clinical trials have shown promise for the combination of nanoparticle albumin - linked paclitaxel ( nab - paclitaxel ) and gemcitabine in advanced pdac .
frese and colleagues have used the kpc mouse model of pancreatic cancer to provide a mechanistic understanding of the synergistic effect of this combination .
paclitaxel appears to inhibit the breakdown of gemcitabine through modulation of a degradative enzyme , cytidine deaminase through a reactive oxygen species - dependent pathway ( frese et  al . , 2012 ) .
such mechanistic analyses may help to rationalize our clinical strategy of using such drug combination .
for instance , the nab - paclitaxel can be used as an inducting agent followed by gemcitabine to enhance the tumouricidal effect of the latter .
rational clinical trials in man are likely to benefit from the incorporation of an in  vivo component that provides relatively rapid feedback of the predicted response to new agents . the preclinical assessment using gemms can either be used to screen potentially useful therapeutic agents before progressing to clinical trials , or alternatively , to critically assess the mechanisms of action in  vivo once an agent has been found to be effective in a small - scale trial , before progressing to larger phase iii clinical trial .
one potential advantage of such gemms is that unlike human trials , they will allow sequential sampling of appropriate tumour tissues to assess the pharmacodynamic impact of a particular agent . in pancreatic cancer
, several novel agents targeting a diverse range of molecular pathways have been tested in gemms to complement early phase clinical trials ( olive et  al .
, 2009 ; plentz et  al . , 2009 ; cook et  al .
the results of these trials will in due course affirm or refute the value of pdac gemms as a predictive tool for clinical efficacy in human cancers .
the potential value of pdac gemms as surrogates for the preclinical testing of new therapies is critically dependent on how closely these models mimic human pdac .
several studies ( hingorani et  al . , 2003 ; tuveson and hingorani , 2005 ; olive et  al . , 2009 ; plentz et  al . , 2009 ; cook et  al .
, 2012 ) have emphasized the similarities in histopathology , cancer progression , clinical behaviour and even drug pharmacodynamics between pdac gemms and human pdacs .
initial observations suggest that murine kpc pdacs bear resemblance to the human disease insofar as they exhibit a high degree of genomic instability , evident from multiple non - reciprocal chromosomal translocations ( hingorani et  al . , 2005 ) . however , with emerging data from large - scale sequencing of human pdac tumours ( biankin et  al . , 2012 ) , it is now imperative that we further validate the gemms at the genomic level to compare the genomic landscapes of murine and human tumours . because pdac gemms incorporate high - penetrance genetic events such as initiating oncogenes or inactivated tumour suppressor genes from an early stage in a large number of susceptible cells , the resulting stereotypy of the malignancies arising therein may not reflect the heterogeneity likely to be present in human cancers .
importantly , the genetic heterogeneity of human cancers may give rise to differing therapeutic responses to any particular agent due to the differing genetic and epigenetic signatures of the constituent cells .
it is conceivable that individual tumours can take differing genetic  routes to achieve tumoural progression , depending on the type of initiating genetic lesions and secondary genetic hits that occur stochastically .
it is presumed that through inactivation of genes involved in maintaining genomic stability ( e.g. brca2 in models of pancreatic cancer ) may promote the stochastic acquisition of genetic and consequent morphologic heterogeneity due to the expected increase in mutation rate .
however , this point remains to be established in future studies , and also has important implications for the potential value of gemm models in testing new therapeutic approaches against pdac .
 chromosomal instability is a hallmark of human cancer cells , but its role in carcinogenesis remains poorly resolved . 
 insights into this role have emerged from studies on the tumour suppressor brca2 , whose inactivation in human cancers causes chromosomal instability through the loss of essential functions of the brca2 protein in the normal mechanisms responsible for the replication , repair and segregation of dna during cell division . 
 humans who carry heterozygous germline mutations in the brca2 gene are highly predisposed to cancers of the breast , ovary , pancreas , prostate and other tissues . here , we review recent studies that describe genetically engineered mouse models ( gemms ) for pancreatic cancer associated with brca2 mutations . 
 these studies not only surprisingly show that brca2 does not follow the classical knudson  two hit  
 paradigm for tumour suppression , but also highlight features of the interplay between tp53 inactivation and carcinogenesis in the context of brca2 deficiency . 
 thus , the models reveal novel aspects of cancer evolution in carriers of germline brca2 mutations , provide new insights into the tumour suppressive role of brca2 , and establish valuable new preclinical settings for testing approaches to pancreatic cancer therapy ; together , these features emphasize the value of gemms in cancer research . 
Modelling human pancreatic cancer associated with 
None
Pancreatic cancer histopathology and 
Checkpoint inactivation, 
Mouse models for PDAC associated with 