INVITED REVIEW

Biomarkers for gastric cancer: prognostic, predictiveor targets of therapy?

Cecília Durães & Gabriela M. Almeida & Raquel Seruca &

Carla Oliveira & Fátima Carneiro

Received: 1 October 2013 /Revised: 12 November 2013 /Accepted: 23 December 2013# Springer-Verlag Berlin Heidelberg 2014

Abstract Gastric cancer is an aggressive disease often diag-nosed at an advanced stage. Despite improvements in surgicaland adjuvant treatment approaches, gastric cancer remains aglobal public health problem with a 5-year overall survival ofless than 25 %. This is a heterogeneous disease, both in termsof biology and genetics, and many prognostic biomarkershave been pointed out in the literature; nevertheless, theirapplication remains debatable. In this review, we opted to giverelevance to those biomarkers that have been the subject ofstudies with significant statistical power, which have beenreplicated and have been/are in targeted therapy clinical trialsand, which as a consequence, have their prognostic and/orpredictive value established. Some gastric cancer biomarkersthat may help in defining the course of treatment are alsodiscussed. Accepted practical guidelines, wet-lab protocolsfor the detection of these biomarkers, as well as ongoing andcompleted clinical trials have been compiled. In summary,clinical approaches based on the combination of correct stag-ing with targeted and conventional systemic therapies mayimprove gastric cancer patients’ outcome, but are only in theirinfancy. Some major challenges in identifying reliableprognostic/predictive biomarkers are individual genetic vari-ation and tumour heterogeneity that often influence responseto therapy and drug resistance. Prognostic and predictivebiomarkers may nevertheless be extremely valuable to cor-rectly stratify gastric cancer patients for treatment and, ulti-mately, improve survival.

Keywords Gastric cancer . Biomarkers . Prognostic .

Predictive . Targeted therapy . Patient stratification

Introduction

Gastric cancer (GC), although declining in incidence in thelast decades, is still the fourth most common malignancy andthe second leading cause of cancer-related death worldwide[1, 2]. This disease affects close to onemillion people per year,particularly in Eastern Asian countries and Western Europe[2]. GC is a heterogeneous disease, both in terms of biologyand genetics. Histologically, the main variants are the intesti-nal type, with clearly defined glandular structures and thediffuse type consisting of individually infiltrating neoplasticcells [3, 4]. About 90 % of the GCs are sporadic, whereas theremaining 10 % show familial clustering. The latter includes1–3 % of hereditary forms of intestinal and diffuse GC(HDGC-hereditary diffuse gastric cancer) [5]. Germline mu-tations and large deletions of CDH1 (E-cadherin gene) are theunderlying genetic defect in 45 % of families with clinicaldiagnosis of HDGC [6].

An endoscopic ultrasound performed concomitantly withesophagoduodenoscopy has high diagnostic accuracy for dif-ferent GC stages. A preoperative laparoscopy can also beperformed to improve staging diagnosis [7]. An accuratepreoperative staging of nodal involvement (N stage) is cur-rently a diagnostic challenge in GC, for which new sentinellymph node mapping technologies are being tested [8, 9].Multi-detector computer tomography is the gold standard forthe detection of distant metastases [7].

Despite improvements in surgical and adjuvant treatmentapproaches, GC remains a global public health problem. Sur-gical resection offers the best chance for curative therapy, with5-year survival of ca. 50–70 %, if an early diagnosis is made.However, most newly diagnosed patients present with

C. Durães :G. M. Almeida : R. Seruca : C. Oliveira : F. CarneiroInstitute of Molecular Pathology and Immunology of the Universityof Porto (IPATIMUP), Porto, Portugal

R. Seruca :C. Oliveira (*) : F. Carneiro (*)Pathology and Oncology Department, Faculty of Medicineof the University of Porto, Porto, Portugale-mail: carlaol@ipatimup.pte-mail: fcarneiro@ipatimup.pt

Virchows ArchDOI 10.1007/s00428-013-1533-y

advanced, and unresectable, disease and 5-year survival dropsto 4–10 %. For these patients, chemotherapy is the maintreatment option; nevertheless, the median overall survival(OS) for advanced disease remains below 1 year, comparedwith best supportive care (3–5 months) [10, 11].

The aim of this review is to provide up-to-date informationabout prognostic and predictive biomarkers for GC patients. Acancer prognostic biomarker provides information on thelikely course of the disease. In contrast, a predictive biomarkeris defined as a marker which can be used to identify subpop-ulations of patients who are most likely to respond (or not) to atargeted therapy [12]. The search for cancer biomarkers iscarried out in order to identify tumour cells at early stagesand predict treatment response, ultimately leading to afavourable therapeutic outcome.

The literature concerning GC prognostic biomarkers israther vast. In this review, we opted to give relevance to thosebiomarkers that have been the subject of studies with signif-icant statistical power, which have been replicated and havebeen/are used in targeted therapy clinical trials and, which as aconsequence, have their prognostic and/or predictive valueestablished. Some GC biomarkers are neither drug targetsnor predictive markers. Nevertheless, they may be useful todefine the course of treatment, such as surgery, administrationof systemic therapy and/or radiotherapy and will, therefore, bediscussed (Table 1).

Prognostic and predictive biomarkers for gastric cancer

The TNM staging (tumour, lymph nodes and metastasis) atdiagnosis has been the most important tool used to assessprognosis and predict the need for systemic treatment inresectable GC [13]. However, as this approach has shownlimited success, molecular biomarkers with either prognosticor predictive value have been comprehensively investigated inthe last decade and are the main focus of this review.

TNM staging

The TNM system describes the size and spread of the tumour,whether cancer cells have spread to lymph nodes and whetherthe cancer has spread to a different part of the body. The mainclassifications currently used are the Western Hemispherestaging system developed by the American Joint Committeeon Cancer (AJCC) and the Union for International CancerControl (UICC) [14], and the Japanese staging system, whichis more elaborated and based on anatomic involvement, par-ticularly the lymph node stations [15].

Despite the widespread use of TNM staging to assess GCpatients’ prognosis, and particularly the number of lymphnodes as a poor prognostic factor, prognosis often variesbetween patients at the same tumour stage. Therefore, in

addition to TNM staging, auxiliary methods have been inves-tigated to refine the accuracy of prognostic stratification. Astudy from 2004 established that performance status ≥2, thepresence of metastases (liver and peritoneal) and alkalinephosphatase ≥100 U/L were independent poor prognosticfactors in GC [16]. A prognostic index was constructed withthese factors by classifying patients as having good (no riskfactor), moderate (one or two factors) or poor prognosis (threeor four risk factors) [16] and was later validated in the REAL-2 trial [17].

Although TNM staging per se is insufficient to predict GCprognosis, accurate staging is mandatory to appropriatelytriage GC into potentially curative (resectable) versusnon-resectable categories and implement multimodalitytherapy [18].

Molecular biomarkers: prognostic, predictive and therapytargets

The main genetic alterations that can drive carcinogenesis aremutations (including small insertions/deletions), amplifica-tions and rearrangements. Gene amplification often leads toprotein overexpression.With the exception of dominant drivermutations in solid tumours such as BRAFV600E in melanoma,activating EGFR mutations in lung cancer and HER2 ampli-fication in breast and GC, other potentially driver geneticalterations are relatively rare [19].

Gene amplification is a frequent mode of genetic alterationin GC. Common methods of detecting gene amplificationinclude fluorescence in situ hybridisation (FISH), chromogen-ic ISH (CISH), silver ISH (SISH), real-time quantitative PCR(RT-qPCR) and single-nucleotide polymorphism (SNP)genotyping assay using next-generation sequencing (NGS).The most commonly accepted definition of amplification isgene/chromosome enumeration probe (CEP) ≥2. Protein over-expression is commonly detected by immunohistochemistry(IHC). Such a variety of available methods presents advan-tages towards a wider comprehension of biomarkers; howev-er, it can also bemisleading due to discrepancies obtainedwithdifferent techniques.

Human epidermal growth factor 2 (HER2)

HER2 (encoded by ERBB2, the v-erb-b2 avian erythroblasticleukaemia viral oncogene homolog 2) is one of the fourmembers of the human epidermal growth factor receptorfamily (EGFR or HER1, HER2, HER3 and HER4) in thereceptor tyrosine kinase (RTK) superfamily. Unlike otherHER family members, HER2 does not contain a ligand bind-ing site and signals through heterodimerisation with otherHER family members, primarily EGFR (Fig. 1) [20].

ERBB2 amplification and HER2 overexpression have beenmost widely studied in breast cancer, constituting established

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Tab

le1

Interpretatio

nof

prognosticandpredictiv

evalueof

biom

arkersforadvanced

GC

Biomarkergroup/pathway

Biomarker

Detectio

nmethod

Biomarkerstatus

Prognostic

value

Clin

icaltrial

status

Targeted

therapya

Predictivevalue

Impact

Ref.

Growth

factor

receptors

EGFR

FISH

,SISH,C

ISH,

RT-qP

CR

EGFRam

plification

Yes

PhaseIII

Cetuxim

abNo

Decreased

PFSandOS

[40]

PhaseIII

Panitumum

abNo

Decreased

OS

[41]

IHC,R

T-qP

CR

Overexpression

PhaseIII

Nim

otuzum

abb

Pending

PhaseII/III

Lapatinibb

Pending

FGFR

2FISH

,RT-qP

CR

FGFR2am

plification

Yes

PhaseII

AZD4547

Pending

VEGFR

2FISH

,CISH

VEGFR

2am

plification

Yes

PhaseIII

Ram

ucirum

abYes

Modestincreased

OS

[51]

IHC

Overexpression

HER2

FISH

,SISH,C

ISH,

RT-qP

CR

ERBB2am

plification

NC

FDA-approved

Trastuzum

abb

Yes

IncreasedOS

[24]

PhaseII/III

T-DM1b

Pending

IHC,R

T-qP

CR

Overexpression

PhaseIIa

Pertuzum

abb

Pending

PhaseIII

Lapatinibb

Pending

MET(H

GFR

)FISH

,SISH,C

ISH,

RT-qP

CR

METam

plification

Yes

PhaseII

Rilo

tumum

abPending

IHC,R

T-qP

CR

Overexpression

PhaseIII

Onartuzum

abPending

Growth

factor

ligands

VEGFA

IHC

Overexpression

Yes

PhaseIII

Bevacizum

abNo

IncreasedOSbutn

otsignificant

[49]

PI3K

/AKT

PI3K

ASequencing

PIK3C

Amutations

Yes

PhaseIb

BYL719

Pending

FISH

,RT-qP

CR

PIK3C

Aam

plification

IHC,R

T-qP

CR

Overexpression

mTOR

FISH

MTO

Ram

plification

Yes

PhaseIII

Everolim

usNo

IncreasedOSbutn

otsignificant

[63]

IHC

Overexpression

MSI

BAT-25,B

AT-26,

NR-21,NR-24,

MONO-27

Sequencing

Nucleotideinsertionor

deletio

nYes

––

–[89]

KRAS/MAPK

KRAS

Sequencing

KRASmutations

Yes

––

–[77]

Celladhesion

E-cadherin

Sequencing

CDH1structuralalteratio

nsYes

––

–[81,82]

CDH1mutations

T-DM1trastuzumab

emtansine,OSoverallsurvival,IH

Cim

munohistochem

istry,RT-qP

CRreal-tim

equantitativePCR,FISHfluorescence

insitu

hybridization,

SISH

silver

insitu

hybridisation,

CISH

chromogenicin

situ

hybridisation,MSI

microsatelliteinstability,N

Cnotconsistentamongseries

aMosto

fthesetargeted

therapieshave

been

givenin

combinatio

nwith

conventio

nalchemotherapy

bGCpatientswereselected

basedon

thetargeted

moleculestatus

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biomarkers of poor prognosis. The currently accepted proto-col to detect HER2 aberrations encloses the use of IHC withspecific antibodies and in situ hybridisation techniques (FISH,CISH and SISH) (Fig. 2). Those cancers scoring 3+ by IHCare considered HER2 positive; those scoring 2+ undergofurther confirmation by FISH, CISH or SISH; and thosescoring <2+ by IHC are considered HER2-negative cases.

Using similar scoring systems, amplification and/or overex-pression of ERBB2/HER2 has also been observed in colorectal,lung, gastric and ovarian cancers [21]. In GC, ERBB2 amplifi-cation or HER2 overexpression has been reported in 7–34 % ofthe tumours [21–23], particularly in the gastroesophageal junc-tion carcinomas (proximal) and in intestinal type GC [22–24].

The prognostic value of HER2 positivity in advanced GCis, however, a controversial issue. There are reports indicatingthat ERBB2 amplification is associated with poor prognosisand aggressive disease [21, 23, 25, 26], whereas other reportsshow no difference in prognosis when compared with HER2-negative tumours [27–29]. A recent study showed that HER2

status is not even an independent prognostic biomarker inearly gastroesophageal adenocarcinoma [30].

Despite the controversy regarding HER2 prognostic value,inhibition of HER2 has been tested as a targeted therapyoption in several cancer types. Trastuzumab, a monoclonalantibody that targets HER2, inhibits HER2-mediated signal-ling and prevents cleavage of its extracellular domain [31]. InHER2-positive metastatic breast cancer, trastuzumab im-proves patient outcome and is now the standard of care [32,33]. The Trastuzumab for Gastric Cancer (ToGA,ClinicalTrials.gov Identifier: NCT01041404) phase III inter-national study assessed the efficacy of a combination oftrastuzumab with conventional chemotherapy (cisplatin plus5-fluorouracil or capecitabine) as a treatment for GC patients.This trial has demonstrated that advanced GC patients, strat-ified by HER2 amplification/overexpression, had longer me-dian OS when treated with trastuzumab/chemotherapy versuschemotherapy alone (13.8 versus 11.1 months) [24]. More-over, there was improved predictive value of trastuzumab

Fig. 1 Frontline therapy trials for advanced GC involve antibodiesagainst overexpressed and/or amplified receptor tyrosine kinases (RTKs)or their ligands, and specific small-molecule inhibitors. The targetedRTKs for advanced GC therapy are HER2, EGFR, MET, FGFR2 andVEGFR2. The activation of these RTKs signals through PI3K (among

other pathways) leading to the activation of mTOR, both novel bio-markers under investigation for targeted GC therapy. All these moleculesregulate signalling cascades important for neoplasia, including differen-tiation, proliferation, angiogenesis and survival

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based on HER2 positivity thresholds. The median OS of the“higher HER2 threshold” categories (Very High: IHC3+/FISH+; High: IHC3+ and IHC2+/FISH+) was better than theremainder (IHC0+/FISH+, any IHC1+ and IHC2+/FISH−).The median OS within the whole trastuzumab arm was13.8 months, whereas in the “Very High HER2” and “HighHER2” groups, it was 17.9 and 16 months, respectively.Trastuzumab in combination with cisplatin and afluoropyrimidine is, therefore, currently approved by the Foodand Drug Administration (FDA, USA) and European Medi-cines Agency as first-line treatment of patients with HER2-overexpressing metastatic GC, who have not received priortreatment for metastatic disease.

Several promising GC clinical trials targeting HER2, usingcombinations of different drugs, are simultaneously undergoing.The LoGIC trial (ClinicalTrials.gov identifier: NCT00680901) isinvestigating the effect of lapatinib, a dual EGFR and HER2tyrosine kinase inhibitor. The effect of pertuzumab, amonoclonalantibody that binds to a different part of HER2 than trastuzumab,is also being studied (ClinicalTrials.gov identifier:NCT01461057). There is also an ongoing randomised trial in-vestigating the predictive value of trastuzumab emtansine (T-DM1), an antibody–drug conjugate incorporating HER2-targeted antitumour properties of trastuzumab with the cytotoxicactivity of the microtubule-inhibitory agent DM1(ClinicalTrials.gov identifier: NCT01641939).

Epidermal growth factor receptor (EGFR, HER1)

EGFR (encoded by EGFR) is another member of the HERfamily which is activated by the binding of specific ligands,including EGF and TGFα (Fig. 1). Kiyose et al. [34],employing FISH assays, reported the incidence of EGFRamplification to be 4.9 % in 365 GC samples. Another study,using SNP assays, reported that 7.7 % of GC harboured EGFRamplification [35]. A larger investigation (511 CG samples)examined EGFR overexpression by IHC and FISH and re-ported that 27.4 % of the samples were positive for EGFRprotein expression by IHC (IHC2+ and 3+) while only 2.3 %

were amplified when detected by FISH [36]. EGFR overex-pression has been shown to be a prognostic indicator of worseoutcome in GC [37, 38]. EGFR IHC or FISH positivity hasbeen associated with the presence of lymph node metastasis,higher stage and poor survival, however, after multivariateanalysis, only EGFR IHC positivity remained a poor prog-nostic factor [36].

EGFR has also been used as a target for therapy in GC.Monoclonal antibodies against EGFR (cetuximab orpanitumumab), combined with conventional chemotherapy,have been explored in two randomised GC clinical trials [39].Contrary to the ToGA study [24], the international phase IIIstudy EXPAND (ClinicalTrials.gov Identifier: NCT01611506),which used a combination of cetuximab and conventionalchemotherapy, did not provide benefit in OS of GC patients[40]. In fact, the cetuximab group had an inferior progression-free survival (PFS) (4.4 months) and inferior OS (9.4 months),compared with cisplatin/capecitabine alone (5.6 and10.7 months, respectively). Similarly, in the REAL3 trial(ClinicalTrials.gov Identifier: NCT00824785), addition ofpanitumumab to epirubicin/oxaliplatin/capecitabine resulted ina significantly lower OS (8.8 months) compared withepirubicin/oxaliplatin/capecitabine alone (11.3 months) [41].

As the results from the EXPAND and REAL3 studiessuggest, the addition of anti-EGFR antibodies to chemother-apy does not deliver additional benefit for patients with ad-vanced GC. In both studies, the prevalence of EGFR amplifi-cation or EGFR IHC positivity was not reported. In thissetting, data from single-group studies suggest that EGFRexpression, EGFR copy number and expression of otherEGFR ligands (epiregulin and amphiregulin) or downstreamcomponents of the EGFR-signalling pathway might be candi-date biomarkers for anti-EGFR-antibody efficacy [42–44]. Aphase III trial (ENRICH, ClinicalTrials.gov identifier:NCT01813253) is investigating the addition of the anti-EGFR antibody nimotuzumab to irinotecan as second-linetreatment for IHC 2+/3+ GC patients. The results from thisstudy might elucidate if selecting EGFR IHC 2+/3+ GCpatients for anti-EGFR therapy will improve survival. Indeed,

Fig. 2 HER2 expression and ERBB2amplification in one GC: a IHC. In most cells, the staining is not limited to basolateral membranous reactivity but tothe entire membrane (original magnification×400). b IHC. Higher magnification highlighting completeness of membrane staining (original magnifi-cation×600). c SISH. Uniform ERBB2 gene amplification

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the prognostic and predictive role of EGFR expression hasalready been demonstrated in the FLEX trial for advancednon-small-cell lung cancer (NSCLC) [45]. This study showedthat high EGFR expression is a tumour biomarker that canpredict survival benefit from the addition of cetuximab tochemotherapy, therefore indicating that assessment of EGFRexpression could offer a personalised treatment approach.

Small-molecule inhibitors of EGFR like erlotinib andlapatinib are also in GC clinical trials; however, the numberof patients included was small and/or the series unselected forEGFR overexpression and/or amplification. A phase II clini-cal trial (ClinicalTrials.gov identifier: NCT01123473) inves-tigating lapatinib in combination with chemotherapy selectedpatients for EGFR or HER2 positivity; however, the resultsare still pending.

Angiogenesis pathway biomarkers

Angiogenesis, the growth of new blood vessels, is an impor-tant aspect of tumourigenesis. It is primarily modulated by thevascular endothelial growth factor A (VEGFA) and its recep-tors VEGFRs, in particular types 1 and 2 (Fig. 1). In GC,expression of VEGFA and VEGFR was reported in 40 and36 % of cases, respectively [38]. VEGFA expression in GCand serum has been associated with poor prognosis, lymphnode involvement and metastasis, both in resectable and ad-vanced GC [46]. Targeted inhibition of VEGFA and VEGFRsis possible and has been a commonly employed strategy inoncology to decrease tumour vascular supply and metastasis,leading to tumour shrinkage. The use of monoclonal antibod-ies against VEGFA, such as bevacizumab, has successfullyshowed to improve OS in patients with advanced colorectalcancer [47] and NSCLC [48]. In the AVAGAST GC trial(ClinicalTrials.gov Identifier: NCT00548548), the additionof bevacizumab to cisplatin and capecitabine resulted inhigher overall response rate (46.0 versus 37.4 %) and higherPFS (6.7 versus 5.3 months) [49]. However, OS was notsignificantly improved (12.1 versus 10.1 months). TheAVAGAST trial did not report the level of VEGFR1 orVEGFR2 expression which is neither prognostic for OS norpredictive to bevacizumab response [50]. A recentrandomised trial (REGARD, ClinicalTrials.gov Identifier:NCT00917384), using ramucirumab, a monoclonal antibodyagainst VEGFR2, instead of the VEGFA, successfullyprolonged OS (5.2 versus 3.8 months on placebo) [51].Ramucirumab is the first biological treatment given as a singledrug that has survival benefits in patients with advanced GCor gastroesophageal cancer progressing after first-line chemo-therapy [51]. Although these findings validate VEGFR2 sig-nalling as an important therapeutic target in advanced GC, theOS improvement with ramucirumab was small, and it will beimportant to investigate if there are any biomarkers underlyingits clinical activity, such as overexpression of VEGFR2.

Emerging biomarkers for target therapy in gastric cancer

The poor long-term outcomes associated with current chemo-therapy and scarce targeted therapy treatment for advancedGC suggest a need for other targeted agents that may confer abetter survival benefit. The most relevant novel biomarkers inGC currently being investigated, some of which are already inclinical trials, include the fibroblast growth factor receptor(FGFR), the hepatocyte growth factor receptor (HGFR,MET) and the mammalian target of rapamycin (mTOR).

Fibroblast growth factor receptor (FGFR)

FGFR family members (FGFR1, FGFR2, FGFR3 andFGFR4) belong to the RTK superfamily (Fig. 1). In a recentgenomic survey of GC using high-resolution SNP arrays,FGFR2 copy number gain was found in 9.3 % of tumoursand was more common than EGFR (7.7 %), HER2 (7.2 %) orMET (4.3 %) copy number gains [35]. Although FGFR2FISHwas also performed in the study, it was not reported if all thesamples with FGFR2 copy number gain were also trulyFGFR2-amplified. Another large screening study, includingCaucasian and Korean GC samples [52], found that 5.9 % ofthe GC presented FGFR2 amplification (cut-off: FGFR2/CEP10 >2). The FGFR2 amplification was more common inCaucasians (7.4 %) than in Koreans (4.2 %) and was associ-ated with lymph node metastasis in both cohorts and thediffuse type of GC in Koreans. Moreover, FGFR2 amplifica-tion was found to be a prognostic biomarker of shorter OS inboth cohorts. Jung et al. also showed the presence of FGFR2amplification in Korean GC samples (4.5 %) and its associa-tion with shorter OS [53].

FGFR2 has therefore attracted significant attention as apotential candidate for targeted therapy in GC. A number ofFGFR2 inhibitors are in clinical development, having demon-strated efficacy in cell lines and in human xenograft modelsin vivo [35, 54]. A randomised phase II trial comparing theselective FGFR inhibitor AZD4547 to paclitaxel as second-line treatment of advanced GC, harbouring FGFR2 polysomyor amplification (SHINE, ClinicalTrials.gov identifier:NCT01457846), is ongoing, and it will inform on the valueof this marker as a therapeutic target.

Hepatocyte growth factor receptor (HGFR, MET)

MET (encoded byMET) belongs to the HGFR family (Fig. 1).MET amplification and/or overexpression of its protein prod-uct has long been implicated in the pathogenesis of GC, withmany reports based on gene copy number, RNA expressionand/or protein expression, supporting its role as a poor prog-nosis factor [37, 55–57]. Nevertheless, the prevalence ofMETamplification in GC varies widely in the literature from 0 %[58] to 21 % [55]. This discrepancy is greatly attributed to the

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methodology employed to detect gene amplification/copynumber gain and/or protein expression. MET copy numbergain detected by RT-qPCR is sometimes interpreted as ampli-fication, similarly to the common definition of gene amplifi-cation by FISH (gene/CEP ≥2), and this may bemisleading. Astudy employing NGS SNP assays reported MET amplifica-tion in 4.1 % of GC cases, while using the FISH criterion, thevalue was lower [35]. Another study did not findMETampli-fication in 38 GC using the FISH criterion [58]. A larger studyincluding 216 GC samples showed by RT-qPCR that 10 % ofGC displayed five or more MET copies. However, a FISHancillary analysis of 32 random samples did not show trueMET amplification [37]. Depending on the method used andthe stringiness of the FISH criteria, different MET amplifica-tion prevalences have been further reported [34, 55–57].Nonetheless, in all these studies, the GC patients withpolysomic and/or amplified MET showed poorer DFS andOS in comparison with the non-polysomic MET.

Recently, a randomised phase II metastatic gastroesopha-geal cancer t r ia l (Cl in ica lTr ia ls .gov Ident i f ie r :NCT00719550) using chemotherapy with and withoutrilotumumab, an antibody against the MET ligand HGF, con-firmed a significantly worse prognosis for MET-positive (IHC2+ in ≥50 % of cells) (median OS of 5.7 months) comparedwith MET-negative tumours within the placebo group (medi-an OS of 8.9 months) [18].

There is also an ongoing randomised phase III trial(MetGastric, ClinicalTrials.gov identifier: NCT01662869) in-vestigating whether the addition of onartuzumab, an antibodyagainst MET, to chemotherapy in MET-positive (IHC 2+/3+)and HER2-negative GC patients will significantly improveOS, when compared with chemotherapy alone.

PI3K/mTOR signalling pathway

The phosphatidylinositol-3-kinase (PI3K)/mTOR pathwayrepresents one common final convergence signalling pathwayoriginated by the activation of several RTKs (Fig. 1), such asthe RTKs herein reviewed. Activation of the PI3K/mTORpathway in GC has been demonstrated in preclinical studies[59, 60], and its deregulation has been associated with in-creased lymph node metastasis and decreased survival of GCpatients [61, 62].

Although alteration of PI3K/mTOR related genes and pro-teins has not been formally and specifically associated withGC prognosis, this pathway has been seen as a target fortherapeutic intervention. In particular, the role of everolimus,an mTOR inhibitor, has been investigated in advanced meta-static GC in two randomised trials. The recently publishedphase III randomised study (GRANITE-1, ClinicalTrials.govIdentifier: NCT00879333) [63] compared the use of everoli-mus or placebo plus best supportive care in second-line treat-ment of advanced GC. This strategy did not significantly

improve the median OS for the everolimus treatment arm(5.4 months on everolimus versus 4.3 months on placebo).The median PFS was only modest with estimates of1.7 months in the everolimus arm and 1.4 months in theplacebo arm. These results were interpreted as possibly indi-cating a subgroup treatment effect and to address that a retro-spective identification of specific biomarkers for patient strat-ification is ongoing in the GRANITE-1 study. Another bio-marker retrospective analysis in a different phase II study(ClinicalTrials.gov Identifier: NCT00729482) of everolimusin GC [64] did not yield significant results; thus, furtherinvestigation is warranted to select GC patients that maybenefit from this particular targeted therapy.

Oncogenic mutations in PIK3CA (gene encoding the alphap110 catalytic subunit of PI3K) have been observed in GC,constitutively activating the PI3KA/mTOR pathway. The firststudies in GC reported a PIK3CAmutation frequency of 25 %[65] and 10 % [66]. Other recent studies report PIK3CAmutation frequencies varying from 5.1 to 16 % [67–69].One of these studies reported PIK3CA amplification in 67 %of GC [68], and association with poor prognosis, indicatingthat this is a major mechanism leading to the activation thePI3K/mTOR pathway in GC.

Small molecules inhibiting PI3KA have still to prove theirefficacy. In view of this, a recently initiated trial(ClinicalTrials.gov identifier: NCT01613950) is intended toinvestigate the safety and preliminary efficacy of the PI3KAinhibitor BYL719, in combination with AUY922 (heat shockprotein HSP90A inhibitor), in patients with advanced GC.Only patients with tumours carrying either a molecular alter-ation of PIK3CA, or amplification of ERBB2, are eligible forthis study, therefore allowing assessment of biomarker sub-groups. Additionally, the dual PI3KA and mTOR inhibitorBEZ235 has been shown to decrease cell viability and induceapoptosis in a GC cell line harbouring PIK3CA and KRASmutations [70]. PI3K/mTOR dual inhibitors have not yetentered early-stage clinical trials for advanced GC.

Non-targetable prognostic biomarkers in gastric cancer

In addition to the most frequently used molecular biomarkersin GC, for which specific targeting in combination with con-ventional chemotherapy has been the subject of several clin-ical trials, there are also a series of alterations that, althoughrare and possibly non-targetable, may potentially be used inpatient stratification and towards improvedGC patient therapyselection/personalised therapy.

Mismatch repair (MMR) deficiency leads to a tumourphenotype known as microsatellite instability (MSI), in whichcells accumulate genetic errors rather than correcting thoseerrors. MSI has been reported in 18 to 28% of GC, mainly dueto hypermethylation of the MLH1 promoter [71]. Patients

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bearing GC tumours with MSI generally develop GC later inlife and have favourable prognosis when compared with tu-mours with MMR proficient tumours [72, 73].

KRAS activating mutations (frequently found in codons 12,13 and 61) are present in ca. 4 % of GC [74, 75]. Although notcurrently used as a prognostic biomarker, KRAS mutationsoccur mainly in GC with MSI [72, 74, 76]. A recent reportclaims that the presence of KRASmutations is associated withworse prognosis in patients with proximal GC [77]. Morestudies are needed to clarify whether the presence of KRASmutations may underlie worse prognosis among GCwithMSIphenotype.

Moreover, KRASmutations may have predictive value forresponsiveness to EGFR-tyrosine kinase inhibitors in GC,similarly to what has been shown in colorectal cancer [78,79]. Therefore, KRAS activating mutations possibly constitutea marker for patient stratification. KRAS is, however, consid-ered to be a non-targetable biomarker since attempts to targetthis protein directly with small-molecule inhibitors have, sofar, proved unsuccessful [80].

E-cadherin (encoded by CDH1) dysfunction is one of themost important factors and frequent aberration (ca. 90%) in GCinitiation and progression, so far described. However, only thepresence of E-cadherin structural alterations (in 10 % of GCcases) represents a poor prognostic factor [81, 82]. Thesealterations are, presently, non-targetable as this would requirerestoring E-cadherin expression by gene therapy. Nevertheless,E-cadherin is a potential predictive marker of response totherapy, since its impairment decreases tumour cell sensitivityto conventional and targeted therapies [83–85]. Retrospectivestudies evaluating E-cadherin status in GC samples integratedin specific clinical trials (and correlating them with patientsurvival) are required in order to fully assess this possibility.

Future perspectives

Two major issues contribute towards the general poor prognosisofGCpatients: diagnosis at late stages and the lack of appropriatetherapies. Early diagnosis may be solved with adequate publichealth screening programs, such as those developed in Japan andKorea [86], and improvement in imaging technologies, such asendoscopy coupled with high-resolution microscopy [87].

The combination of correct GC staging with targeted ther-apy and conventional chemotherapy has made worthy prog-ress in the treatment of patients; nevertheless, the most recenttherapy regimens have had little impact on prognosis im-provement and OS. The development of new therapies willonly be possible if the molecular landscaping of GC is knownfor appropriate patient stratification, as has occurred in coloncancer [88]. The use of targeted therapies in combination withconventional chemotherapy offers limited success in GC pa-tients’ survival, as determined in the ToGA trial, in which

trastuzumab administration only improved OS of HER2-positive GC patients by 3 months [24]. Nevertheless, therapyagainst HER2 is the only targeted therapy that is currentlyaccepted as standard of care in GC. This was possible due tothe demonstration that only GCs with very high expression ofHER2 would benefit from this targeted therapy. In all otherclinical trials herein cited and/or published, with the exceptionof the ongoing ENRICH and lapatinib trials using EGFRantibody/inhibitor, GC patients have not been stratified fortreatment according to a similar premise, and this may be oneof the most obvious reasons for trial failure in GC. It isessential to make progress in GC patient molecular stratifica-tion to improve the clinical trial output, to perform retrospec-tive analyses to understand if particular subsets of GC patientsdid in fact respond to targeted treatment and to identify thelandscape of markers that may constitute targets fortherapy in each GC case.

The major challenges in identifying reliable prognosticbiomarkers are inter-individual variability of response totargeted therapy and drug resistance. At present, althoughthe role of many genetic alterations discovered in GC seemsunclear, they represent a promising tool for stratifying patientsaccording to tumour biological behaviour and likelihood ofresponse to systemic therapy. Additionally, independent vali-dation of the most promising prognostic and predictive bio-markers is required before they can be routinely employed inclinical practice. It is also important to undertake retrospectivestudies in which tumour samples from patients that haveundergone GC therapy are mined for biomarkers, or combi-nation of biomarkers, that can be predictive of favourable/unfavourable response towards a certain chemotherapeuticregimen or define the use of multiple therapy regimens in GC.

Acknowledgments The salary support to CD and GMA from POPH-QREN/Type 4.1, European Social Fund and Portuguese Ministry ofScience and Technology (MCTES), SFRH/BPD/62974/2009 andSFRH/BPD/87257/2012, respectively, is acknowledged. IPATIMUP isan Associate Laboratory of the Portuguese Ministry of Science, Technol-ogy and Higher Education and is partially supported by FCT, the Portu-guese Foundation for Science and Technology.

Conflict of interest The authors declare that they have no conflict ofinterest.

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