novel biological agents , including anti - epidermal growth factor receptor ( egfr ) , anti - vegf targeting therapies , and small - molecule multikinase inhibitors , have recently changed the standard of care of metastatic colorectal cancer ( mcrc ) patients .
while the use of positive predictive biomarkers for biological therapy would be desirable in order to identify specific subsets of responsive patients , to date the only validated negative predictive biomarker for mcrc is the mutational status of all members of the ras gene family , which identifies mcrc patients not eligible to monoclonal antibody ( moab ) anti - egfr therapies . emphasizing the limitations of negative predictive biomarkers , unfortunately only a subgroup of wt ras mcrc patients respond to anti - egfr drugs ,
being the molecular mechanism / s underlying resistance to anti - egfr treatment not fully understood . activating mutations in other members of the ras - braf - mek and pi3k - akt pathways ,
both acting downstream of the egfr signaling cascade , are being investigated as further potential predictive biomarkers .
apparently , no specific target treatment seems to be available for wt ras and anti - egfr resistant mcrc patients .
indeed , the inhibition of the braf oncoprotein by the small - molecule drug vemurafenib , which is highly effective in melanoma , showed a very limited response in the mcrc setting .
coherently , only a prognostic significance has been attributed to braf mutations in crc , so far .
interestingly however , preclinical studies have indicated that egfr reactivation contributes to insensitivity of braf - mutant crc to vemurafenib .
thus , the association of braf and egfr inhibitors might effectively target braf mutant colon cancers .
we report here the first case of a patient with braf - mutant , amplified egfr ( double positive ) and kras / pik3ca wt , not - amplified her2 ( triple negative ) mcrc whose disease had progressed on standard lines of treatment , but successfully responded to a new combination therapy consisting of vemurafenib ( zelboraf ) and panitumumab ( vectibix ) .
a 55-y - old man was admitted to our oncology department in july 2007 for a poorly - differentiated adenocarcinoma of the transverse colon .
preoperative carcinoembryonic antigen ( cea ) and ca19.9 serum levels were 1.2 ng / ml and 63 u / ml , respectively .
the patient was staged as iiib and adjuvant standard treatment with folfox4 ( 6 mo ) was performed .
eleven months later , the patient developed peritoneal carcinomatosis and was treated with folfiri - bevacizumab ( 9 cycles ) , discontinued for pulmonary embolism , followed by cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy . after a 12 mo disease - free interval , an increment of ca19.9 and a ct scan revealed a peritoneal progression . at this time
the patient was characterized for wild - type kras mutational status and high egfr expression by immunohistochemistry and underwent several lines of treatment , such as irinotecan  cetuximab , a second peritoneal cytoreductive surgery , capecitabine 
every disease progression was exclusively peritoneal and marked by a significant increase in ca19.9 and cea .
an additional line of treatment with regorafenib demonstrated a good control of the disease for 9 mo in an expanded access program .
subsequently , the patient showed a significant rise in serum markers ( ca19.9 and cea ) and a multivisceral disease progression ( peritoneum , liver , and lung ) accompanied by important clinical troubles including diffuse abdominal pain , weight loss , and episodes of sub - ileus . in order to find additional treatment opportunities dictated by tumor biology ,
the molecular profile of the tumor was evaluated on a liver metastasis biopsy performed at the time of the latest progression and on previously collected tumor material ( primary lesion and peritoneal implants ) .
all samples concordantly revealed the following status : non - amplified her2 , kras wt , pik3ca wt , amplified egfr , and mutant braf .
the lack of other established therapeutic alternatives together with the above mentioned molecular characterization and preclinical data , met the ethical constraints of a tailored therapy , and prompted us to propose a vemurafenib - panitumumab combined treatment .
the protocol was approved by the institutional review board ( policlinico umberto i , sapienza university of rome ) on june 2013 , and the patient provided written informed consent .  
patient restaging before vemurafenib  panitumumab treatment showed hepatic , pulmonary and peritoneal disease measurable at spiral ct scan ( fig . 
cea and ca19.9 serum levels were 558 ng / ml and 4800 u / ml , respectively ( fig .  2 ) .
q - pcr analysis of plasma dna indicated the presence of circulating tumor dna ( ctdna ) via the identification of the braf mutation ( fig .  3 ) .
ct scans of the patient before and after panitumumab - vemurafenib treatment for metastatic crc .
tumor masses ( arrow ) can be seen in the liver of the patient before initiation of panitumumab - vemurafenib treatment ( a ) .
the masses ( arrow ) became hypodense , homogenous and significantly reduced in size on ct obtained 3 and 6 mo after treatment ( b and c ) , indicating good response to combination treatment .
( a ) electropherogram showing the heterozygous braf mutation in dna isolated from patient 's crc tissue .
( b ) allele - specific q - pcr detection of the braf mutation in plasma free dna reveals the presence of circulating tumor dna before treatment ( t0 ) but not 12 wk after treatment initiation ( t1 ) .
data are reported as averages of the threshold cycles ( ct ) obtained in two different q - pcr for the braf amplicon and the reference gene amplicon .
the patient was treated with panitumumab 6 mg / kg iv every 14 d and vemurafenib 960 mg orally twice daily . soon after 4 wk , a significant clinical benefit with complete regression of the clinical symptoms occurred .
twelve weeks later , the programmed disease restaging with ct scan showed a strong reduction of all metastatic lesions ( pr according to recist1.1 criteria ) ( fig . 
consistent with the ct scan , tumor markers also showed a significant decrease : cea , 51 ng / ml , ca19.9 , 221 u / ml ( fig .  2 ) .
, the patient is still asymptomatic 28 wk after starting the treatment when the ct scan showed a further reduction of all metastatic lesions ( pr according to recist1.1 criteria ) ( fig . 
tumor markers are stably low ( fig .  2 ) and circulating braf dna absent .
treatment regimen is being well tolerated and the patient only presents minor skin - related toxicity ( maximum grade 2 ) . in particular no cutaneous toxicity
( such as squamous cell carcinomas and keratoacanthomas ) typically associated with vemurafenib are observed .
our work represents the first clinical evidence that the combination of an inhibitor of braf ( vemurafenib ) and an egfr antagonist ( panitumumab ) is well tolerated and results in a highly effective control of the disease , in a mcrc patient extensively pretreated with several standard and off - label lines of treatment .  
braf belongs to a gene family also including craf ( or raf1 ) and araf , encoding serine 
threonine kinases that heterodimerize each other and constitute , together with downstream mek and erk proteins , a powerful mitogen - activated protein kinase ( mapk ) pathway .
raf kinases are activated by several receptor tyrosine kinases ( rtks ) such as egfr .
notably , while mutations of araf and craf are rare in human cancer , mutated braf frequently occurs in papillary thyroid carcinoma , melanoma , hairy cell leukemia , hepatocellular carcinoma , and 11% of crc . to this
regard , braf is an ideal druggable target in braf mutated cancers ( i.e. , crc ) since it is active as a monomer in an upstream egfr / ras - independent way , thus providing an explanation to the observed resistance to inhibitors of egfr / ras pathway ( i.e. , anti - egfr antibodies ) ( fig .  4 , left panel ) .
the resistance to egfr - targeted therapies eventually developing along progression of lung , colorectal , pancreatic , or head and neck cancers , is challenging because of the various mechanisms underlying anti - egfr insensitivity ( summarized in fig .  4 , right panel ) .
understanding these various resistance pathways would provide an opportunity to develop new inhibitors to prevent or overcome therapeutic resistance . in this context , it can be predicted that braf - containing crcs would be inhibitable by braf antagonists ( fig .  4 , right panel ) .
likewise a short - term sensitivity to anti - braf monotherapy has been reported in melanoma .
model of braf - dependent cell growth and mechanisms of resistance to braf inhibition and restoring of drug sensitivity through combination therapies .
braf mutation confers constitutive pathway activation ( red arrows ) independent of upstream rtks ( egfr , igf-1r)/ras signaling ( left panel ) , providing an explanation to the observed insensitivity to anti - egfr antibody treatment ( panitumumab , cetuximab ) in braf mutant crc .
such a process is blunted by braf ( braf ) inhibitors ( blue symbols and drugs ) ( right panel ) .
additional mechanisms of egfr primary or secondary resistance described in lung , head - neck cancer and crc ( i.e. , egfr mutation ; oncogenic shift or activation of a bypass pathway such as kras , braf , pik3ca , secondary egfr mutation or a parallel / alternative pathway [ pten , igf1 ] is also indicated ) .
right panel also illustrates how bypass and resistance to braf - inhibitors may occur through the rescue of the braf - mediated attenuation of erk negative feedback induced by braf inhibitors and subsequent reactivation of ligand - dependent signaling from egfr or igf-1r or via wild type ras / raf or pik3ca / akt or pik3ca or egfr or mek1 or further braf gain - of - function mutations ( pik3ca or asterisks ) in several tumor types .
drugs under development in preclinical models or clinical trials to overcome braf inhibitor resistance are labeled in blue .
several mechanisms have been reported to underlie the resistance to braf inhibitors , as summarized in figure  4 . while inhibitors of mutant braf monomers suppress the erk signaling , they relieve erk - dependent negative feedback and reactivate ligand - dependent egfr / ras signaling upon wild - type raf dimers in melanoma cells .
likewise , recent preclinical evidence of the feedback reactivation of egfr in crc by vemurafenib - mediated blockade of braf has been reported ( fig . 
in turn , this triggers sustained ras- and craf - mediated mapk signaling and cell proliferation . blocking egfr activity with a monoclonal antibody
to egfr has been shown to restore the sensitivity to vemurafenib in preclinical models , providing a strong rationale for the combination of vemurafenib and anti - egfr antibodies in braf - mutated crc .
resistance to braf inhibitors has also been reported through further activation of ras / mapk pathway ( e.g. , mutations of nras or mek1 , loss of nf1 , alternatively spliced variants of braf , amplification of craf or braf ) as well as activation of additional parallel signaling pathways such as the igf1r / pi3k / akt ( e.g. , resulting from pten loss ) or pdgfr pathways ( fig .  4 ) .
although this drug resistance mechanism and its overcoming by combined treatments is potentially operating in several other tumors harboring braf mutations , no clinical efficacy has been reported either .
in addition to the clinical efficacy of the combined therapy with vemurafenib and panitumumab described here , al - marrawi et al . recently reported that treatment of a single patient with a combination of sorafenib and cetuximab led to a mixed radiographic response with some areas showing dramatic improvement and other areas showing stable disease over a 7-mo period .
these findings are consistent with the modest disease control we also observed in our case treated with panitunumab and sorafenib .
however , sorafenib , a multi - kinase inhibitor with limited activity on braf , can not be considered an optimal drug for the specific purpose . indeed , weak inhibition of braf by sorafenib would transactivate raf dimers maintaining a still consistent downstream mek activity .
our case report raises several considerations that need to be exploited in subsequent studies performed on large series . the first one is related to the observation of braf mutation together with egfr amplification , suggesting that vemurafenib might be an effective drug in braf mutant mcrc patients if used in association with egfr inhibitors
this would blunt escaping signaling pathways ( i.e. , pi3k - akt ) downstream of the amplified egfr responsible for the maintenance of the disease progression , suggesting that the dramatic response observed in our case might have been further influenced by egfr amplification .
thus , a careful selection of the patients eligible to this treatment might be performed on the basis of two distinct positive predictive markers .
however , vemurafenib toxicity spectrum , ( e.g. , squamous cell carcinomas / keratoacanthomas , maculopapular rashes , hyperkeratosis ) is significantly distinct from that of panitumumab ( acneiform rash , paronychia , xerosis ) . in the case
described , no cutaneous toxic effects typically associated with vemurafenib were observed . in particular , squamoproliferative lesions appearing at early times ( median time 8 wk ) during vemurafenib treatment
therefore the vemurafenib - panitumumab combination appears to limit the typical toxicity of single agents . besides overcoming braf inhibitor resistance through co - targeting activated parallel or upstream rtk mitogenic or survival pathways ( i.e. , egfr , met , igf1r )
, additional therapeutic strategies include the combined use of mek or erk or akt inhibitors as well as the intermittent administration of braf inhibitors described in preclinical models . however , to date no clinical evidence substantiates this hypothesis .
here we report the first clinical evidence that the combination of an anti - egfr ( panitumumab ) and an inhibitor of braf ( vemurafenib ) is well tolerated and results in a highly effective disease control in an extensively pretreated mcrc patient .
the possibility to select patients eligible to this therapeutic approach on the basis of two distinct positive predictive markers ( egfr gene amplification / overexpression and mutant braf ) and the apparent reciprocal limitation of the side effects of the two drugs are interesting hypotheses to be tested in prospective studies .
furthermore , unraveling the various mechanisms of resistance to braf inhibition is expected to pinpoint novel combination therapies . to this
regard , recent advances in crc patients classification based on gene expression profiles may help identifying new strategies for personalized treatment .
 as the knowledge on cancer genetic alterations progresses , it fosters the need for more personalized therapeutic intervention in modern cancer management . 
 recently , mutations in kras , braf , and pik3ca genes have emerged as important mechanisms of resistance to egfr - targeted therapy in metastatic colorectal cancer ( mcrc ) . 

 here 
 we report the first case of a mcrc patient whose disease had progressed on standard lines of treatment and for which we devised a personalized therapeutic approach consisting of vemurafenib ( zelboraftm ) and panitumumab ( vectibixtm ) , based on the following molecular profile : brafv600e - mutant , amplified egfr ( double positive ) and wt kras , wt pik3ca , not - amplified her2 ( triple negative ) . 
 
 this new combination therapy was well tolerated and resulted in a strong control of the disease . in particular , the vemurafenib - panitumumab combination appears to limit the typical toxicity of single agents , since no cutaneous toxic effects typically associated with vemurafenib were observed . 
 
 here we report the first clinical evidence that the combination of an anti - egfr ( panitumumab ) and an inhibitor of brafv600e ( vemurafenib ) is well tolerated and results in a strong disease control in an extensively pretreated mcrc patient . 
Introduction
Case Report
Discussion
Conclusions and Future Directions