Gastric cancer—molecular and clinical dimensions

Department of Gastrointestinal Medical Oncology (R. Wadhwa, S. Song, Y. Yao, J. A. Ajani ),

Department of Systems Biology (J.?S. Lee ), Department of

Epidemiology (Q. Wei ), University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard (FC10.3022), Houston, TX 77030, USA.

Correspondence to: J. A. Ajani

jajani@https://www.doczj.com/doc/8d14967670.html,

Gastric cancer—molecular and clinical dimensions

Roopma Wadhwa, Shumei Song, Ju-Seog Lee, Yixin Yao, Qingyi Wei and Jaffer A. Ajani

Abstract | Gastric cancer imposes a considerable health burden around the globe despite its declining incidence. The disease is often diagnosed in advanced stages and is associated with a poor prognosis for patients. An in-depth understanding of the molecular underpinnings of gastric cancer has lagged behind many other cancers of similar incidence and morbidity, owing to our limited knowledge of germline susceptibility traits for risk and somatic drivers of progression (to identify novel therapeutic targets). A few germline (PLCE1) and somatic (ERBB2, ERBB3, PTEN, PI3K/AKT/mTOR, FGF , TP53, CDH1 and MET ) alterations are emerging and some are being pursued clinically. Novel somatic gene targets (ARID1A, FAT4, MLL and KMT2C ) have also been identified and are of interest. Variations in the therapeutic approaches dependent on geographical region are evident for localized gastric cancer—differences that are driven by preferences for the adjuvant strategies and the extent of surgery coupled with philosophical divides. However, greater uniformity in approach has been noted in the metastatic cancer setting, an incurable condition. Having realized only modest successes, momentum is building for carrying out more phase III comparative trials, with some using biomarker-based patient selection strategies. Overall, rapid progress in biotechnology is improving our molecular understanding and can help with new drug discovery. The future prospects are excellent for defining biomarker-based subsets of patients and application of specific therapeutics. However, many challenges remain to be tackled. Here, we review representative molecular and clinical dimensions of gastric cancer.

Wadhwa, R. et al. Nat. Rev. Clin. Oncol. advance online publication 24 September 2013; doi:10.1038/nrclinonc.2013.170

Introduction

Globally, the incidence of gastric cancer ranks as the fourth most frequent cancer in men and fifth in women, but its death rate is comparable to lung cancer—the most common cancer worldwide.1 Histologically, gastric cancer can be mostly diffuse or intestinal type. Diffuse gastric cancer (DGC) represents poorly differentiated cancer cells interspersed with stromal cells. By contrast, intestinal gastric cancer (IGC) comprises cancer cells that form gland-like tubular structures with limited stromal components.2

In 2008, approximately 989,600 (8% of all cancers) new cases of gastric cancer worldwide and 738,000 (10% of all cancer deaths) gastric-cancer-specific deaths were reported. Of these, 70% of deaths occurred in develop-ing nations—with China reporting approximately 40%—owing to the high incidence of gastric cancer in these regions,1 with Asia, Eastern Europe and South America being most affected. Despite these seemingly worrying numbers, the incidence of gastric cancer globally has been declining since World War II;3 several factors that have contributed to the decline include improved living standards 3–6 and early detection strategies, which have reduced the death rate in Japan.7 Helicobacter pylori infection as a risk factor is of importance for preven-tive strat e gies by developing strategic elimination of

the bacteria in high-risk areas.8 Furthermore, major molecular biology advances have identified small subsets of gastric cancers defined by biomarkers. Of these, the over e xpression of HER2 protein and amplification of its gene ERBB2 have led to new treatment approaches in gastric cancer, such as trastuzumab. At present, addi-tional biomarkers (for example, c-MET, PI3K/Akt/mTOR and HER3) are currently being explored. Greater understanding of the molecular biology and immune biology of gastric cancer is still lacking, but likely to be highly rewarding.

This Review focuses on the aspects of the emerging molecular biology of gastric cancer that might lead to improved therapeutics. We discuss the complexity of genomic alterations that drive the disease and show how these present potential opportunities for exploit-ation. The details of molecular signalling are largely not covered if their translational value is unclear. We discuss the role of tumour-initiating cells and the role of many oncogenes in conferring secondary resistance to therapy. In addition, we emphasize the progress—or lack thereof—made in the clinical arena by outlining recent phase III trial results. We aim to highlight advances in molecular and clinical research that reflect the current understanding of gastric cancer, rather than provide an encyclopaedic reference. Details of preventive strategies, impact of new classifications and nuances of surgery and radiotherapy are beyond the scope of this Review.

Competing interests

The authors declare no competing interests.

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Helicobacter pylori as a risk factor

The risk factors for gastric cancer include old age, smoking, alcohol consumption, above normal body weight, high salt and fat consumption, low vegetable and fruit consumption, low economic status, perni-cious anaemia, other chronic gastric diseases and H. pylori infection.6,9 Of these risk factors, H. pylori is the most fascinating (Figure 1) and best studied. The bacterium is associated with a range of effects, from chronic inflamma t ion to epigenetic modulations that can promote t r ansformation, invasion and metastasis of host cells.

H. pylori infection increases the risk of gastric cancer threefold to sixfold,10 and is associated with distal gastric cancer and IGC.11 Chronic active gastritis is an integral part of bacteria-related gastric cancer:11,12 the organism attaches to gastric epithelial cells, leading to inflammation and an increase in the reactive oxygen and nitrogen species, which causes tissue damage.13,14 Furthermore, H. pylori also induces the expression of Toll-like receptors (TLRs), which leads to proliferation of the gastric epithelium.15 The oncogene CagA (now known as S100A8) is produced by carcinogenic H. pylori strains;12,16,17 the gene is included in the cag patho g enicity island and is translocated by the bacterium into the host epithelial cytosol.16,18 Once in the host cell, CagA is phosphory l ated by Src and c-Abl kinases19,20 to its active form to exert a range of effects,such as forming a complex with the SRC homology 2-domain (SH2)-containing tyrosine phosphatase SHP-2 (encoded by PTPN11), resulting in cytoskeletal reorganization that can induce malignant transformation.21 CagA activates the ERK/MAP kinase cascade, resulting in ETS domain-containing protein Elk-1 (Elk-1) phosphorylation and increased proto-oncogene c-Fos (c-Fos) transcription.22 In addition, CagA promotes invasion through activation of the hepatocyte growth factor receptor c-MET, which leads to an oncogenic response.23 CagA also induces E-cadherin-mediated impairment of cell adhesion junctions leading to cytoplasmic and nuclear accumu-lation of β-catenin.24 The protein CagA can bind Crk adaptor (p38) proteins (Crk-II, Crk-I and Crk-L)25 and kinase PAR1,26 eliciting loss of cell polarity, which is an oncogenic event. Furthermore, CagA stimulates the inflammatory pathway by inducing the secretion of cytokines IL-8, IL-1 and TNF-α27–29 through NF-κB in the epi t helium.30 Proinflammatory IL-1 gene cluster polymorphisms (in IL1B, encoding IL-1β, and in the loci for IL1RN and its receptor antagonist) increase the risk of noncardia gastric cancer (that is, cancer that affects the lower stomach).31,32 CagA also upregulates cyclo-oxygenase 2 (COX-2),27,33 which in turn induces the expression of oncogenic prostaglandins.34,35

Aside from its CagA-mediated effects, H. pylori reduces the expression of the proapoptotic FAS-associated factor 1 (encoded by FAF1).36 The bacteri u m also mediates increases in another oncoprotein, a q uaporin-3 (encoded by AQP3),37alters DNA methyl-ation of E-cadherin (encoded by CDH1), which is an oncogenic event,38,39 and promotes methylation of the tumour suppressor TFF240 and RUNX3,41 as well as likely promoting the methylation of six other tumour suppres-sors (FLNC, HAND1, THBD, ARPC1B, HRASLS and LOX).42 Finally, H. pylori can lead to aberrant expres-sion of the nucleic acid editing enzyme activation-induced cytidine deaminase (AID), which can promote carcino g enesis through gene editing.43 These elucidated pathways and mechanisms show that H. pylori is carcino-genic, albeit in susceptible individuals. These improved and detailed understandings of H.pylori-induced host events will lead to plausible targets for drug development to reduce the risk of gastric cancer.

and its several virulence factors, such as CagA, interact with gastric epithelial cells to induce chronic inflammation, mucosal damage and multiple alterations in gene expression and genetic and epigenetic changes, eventually leading to gastric carcinogenesis. Abbreviations: COX-2, cyclooxygenase-2; CpG island, areas of cytosine and guanine repeats; LPS, lipopolysaccharide; RNS, reactive nitrogen species; ROS, reactive oxygen species; VacA, vacuolating cytoxin A.

The genomic profile of gastric cancer

The regions that have a high prevalence of H. pylori—for example, South America, Asia, Africa and Eastern Europe—have few cases of gastric cancer, which implies that genetic susceptibility might be a critical factor to consider.44 Indeed, several studies of single nucleotide polymorphisms (SNPs) and genome-wide association studies (GWAS) have revealed some plausible genes implicated in gastric cancer.

Rare germline mutations (<0.001% of the general popu lation)in CDH1 have been implicated in familial cases of gastric cancer.45,46 Intuitively, SNPs can facilitate gastric cancer such that one adverse allele contributes weakly, but multiple adverse alleles can considerably increase the risk.47 Prior investigations of SNPs have focused on genes involved in mucosal protection

against H. pylori infection (for example, IL1B, IL1RN and TNFA), carcino g en metabolism (for example, CYP2E1 and GSTM1), deoxynucleotide synthesis (for example, MTHFR and TYMS), DNA repair (for example, MTHFR and XRCC1) and tumour suppressors (for example, TP53 and CDH1). However, these studies have had limited yield and none can be used clinically because SNP studies require customized approaches (with a priori assumptions that alterations in certain functional SNPs would increase susceptibility to gastric cancer). In spite of the correlations between certain SNPs and gastric cancer, prospective validation requiring large, population-based studies have been lacking as they are labour-intensive and resource-intensive.

GWAS have been used to scan the whole genome to identify SNPs that are implicated in the disease (Table 1). Such studies have identified genes not previously known to be involved in gastric cancer—for example, PLCE1 (encoding pancreas-enriched phospholipase C) has an oncogenic role in skin and intestinal cancers,48 but is now thought to have a role in gastric cancer. A Japanese research group documented that SNPs in PSCA (encod-ing prostate stem-cell antigen) was associated with diffuse gastric cancer.49,50 The researchers genotyped 188 cases and 752 controls for 85,576 SNPs and then valid data were obtained from 749 cases and 750 controls for 2,753 SNPs. Although the intronic rs2976392 SNP in PSCA was identi f ied as the risk allele and the SNP was in disequilibrium with rs2294008 (located in exon 1), the function of PSCA in gastric tissue is unclear, although its possible tumour-suppressing function in the gastric epi-thelium has been suggested.51 A second study included 1,077 oesophageal cancer (which bears many similari-ties to gastric cardia cancer in terms of environmental risk factors) cases and 1,733 controls and identified 18 ‘hits’ that were validated in 2,766 cases of gastric cardia cancer; PLCE1 rs2274223 and C20orf54 rs1304295 SNPs were shown to be associated with gastric cancer risk.48 Another study of 2,240 gastric cancer cases and 3,302 controls identified the PLCE1 rs2274223 SNP as a risk allele for gastric cardia cancer.52 Indeed, PLCE1 SNPs have been shown to be associated with risk of gastric cancer53,54 and poor prognosis in Chinese patients,55 but not white patients.56 The fourth GWAS included 1,006 cases and 2,273 controls in China and validated

the genomic data in 3,288 cases and 3,069 controls; SNP

rs13361707 (located between PTGER4 and PRKAA1)

and the ZBTB20 rs9841504 SNP were associated with

increased risk of gastric cancer.57

Cancer stem cells and aberrant pathways

Gastric carcinogenesis is complex and not fully character-

ized.58 Although IGC develops after systematic progres-

sion from the preneoplastic stages, DGC is thought to

arise de novo as the result of downregulation (mutation or

promoter methylation) of CDH1,59,60 thereby permitting tumorigenesis and progression. Nevertheless, accumu-

lated genetic alterations—mutations, amplifications,

insertions, deletions and recombinations—contribute to

gastric cancer,61,62 with more alterations accumulating as

the cancer progresses (Table 2). Furthermore, increasing

evidence points to the existence of gastric cancer stem

cells (CSCs) that initiate malignancy by self-renewal and differentiation. Although the origin of human gastric

CSCs is still unclear, the mesenchymal stem cells in the

bone marrow have been suggested to have a role.63,64

Despite the efforts to genotype IGC and DGC (and iden-

tify novel subtypes),65–76 clinically relevant and robust

molecular subtypes have yet to emerge; in one study, IGC

and DGC were found not to be genotypically distinct, but

did respond differently on the basis of their histopatho-

logical characteristics.73 However, these are preliminary

results. Further detailed analyses of the implicated cells

and pathways might point to viable targets that can

be exploited in gastric cancer; indeed, some of these

s t rategies have been studied in other cancer types.

CSCs are known to undergo epithelial– m esenchymal

transition (EMT), activate oncogenic pathways77 and

activate embryogenic signalling pathways,78 which

are essential for the self-renewal and maintenance of

tumours. EMT leads to the CSC-like phenotypes.79 CSCs

are generally resistant to stress caused by reactive oxygen

species, which in turn makes them relatively resistant to

radiotherapy and chemotherapy. Additionally, CSCs have

self-renewal capacities. These cells depend on the Wnt,

Notch and Hedgehog pathways for maintenance.80 Four

pleiotropic transcriptional factors (Snail, Slug, Twist and

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Zeb1/2) orchestrate the EMT and related processes,81 whereas c-MET and TGF-β signalling can be critical for transformation to EMT.82 c-MET activation can induce the reprogramming transcription factors known to support embryonic stem cells and induce differentiated cells to form the pluripotent stem (iPS) cells.83

TGF-β can be pro-oncogenic by inducing matrix deposition, immunosuppression and EMT.84–86 TGF-β signalling drives EMT and CSC self-renewal by target-ing microRNAs (miRNAs),81 upregulating Snail family members and repressing E-cadherin.87 Downstream of c-MET and the TGF-β receptor, PI3K/Akt/mTOR signal l ing conveys prosurvival messages that lead to CSC expansion and maintenance.88 Consequently, there has been interest in targeting mTOR with metformin to inhibit CSCs in breast cancer (and possibly other cancers).89 Ras, along with Hedgehog, helps to maintain CSCs:90,91 Ras alters cellular metabolism to provide an advantage to CSCs to gather biomass to support cellular division. Ras also facilitates tolerance to DNA damage during cell cycling by inhibiting the activity of tumour suppressors. Hedgehog components facilitate rapid cell division in CSCs, as is their function in develop i ng embryos. Furthermore, TGF-β is overexpressed in DGC92 and stimulates collagen synthesis and subsequent fibro-sis. TGF-β can be antiapoptotic through trans a ctivation of EGFR.93 The bone morphogenetic proteins (BMPs), which are members of the TGF-β superfamily, activate PI3K/Akt.94 Given the considerable interest in the inhibi-tion of angiogenesis, VEGF and VEGFR overexpression, which is common in IGC through activation of NF-κB by H. pylori, is a potential target.95,96

G astric CSCs express CD133, CD44, aldehyde dehydro g enase 1 (ALDH1) and ATP-binding cassette sub-family G member 2 (ABCG2); CD44 and ALDH1 are associated with therapy resistance and might be valid therapeutic targets.97 CD44 imparts stem-cell-like proper-ties by promoting synthesis of intracellular reduced gluta-thione.98 Among the other plausible targets in gastric cancer, HER family members have been of interest.99 The oncogenic properties of HER are conferred through the RAS/MEK/MAPK and PI3K/Akt/mTOR pathways.100,101 Overexpression of HER2 caused by ERBB2 amplifica-tion is more prevalent in IGC than DGC.102,103 Thus, confirm i ng the HER2 status in patients with IGC is more important than in patients with DGC; in DGC HER over-expression is rare and might not be driving the cancer. HER2 interacts with EGFR (also known as HER1), HER3104 and insulin-like growth factor 1 (IGF-1R),105 which are amplified, overexpressed106,107 or acquire acti-vating mutations in cancer.108–110ERBB3 mutations have been reported in 10% of gastric cancers.111 Constitutive activation of c-MET can trigger prolifer-ation and antiapoptotic signals.112 Amplification or over-expression of c-MET, rather than mutation of its gene, can activate receptor tyrosine kinase function.113,114 c-MET overexpression or amplification is common in DGC as well as IGC.115–117 Amplified c-MET crosstalk can acti-vate EGFR, HER2 and HER3 to establish a s i gnalling network leading to constitutive PI3K/Akt signal-ling.118–120 This information enables the understanding of the mechanisms of primary and second a ry resistance when the c-MET pathway is inhibited and can lead to ration a l therapeutic strategies in the future, including dual pathway inhibition. Gastric cancers with c-MET overexpression coexpress EGFR, HER3 or both,121 which is clinically relevant for strategies looking to inhibit both targets concurrently.117,120,121 Indeed, the PI3K/Akt/mTOR pathway is frequently altered as a result of amplification or overexpression (PIK3CA, AKT1), activating muta-tions (PIK3CA)59,122 or loss of PTEN.123 Overexpression of phosphory l ated mTOR can occur in DGC;59 HER3 and FGFR amplification in DGC is another mechanism for PI3K/Akt activation.124,125

Although HER, c-MET, PI3K/Akt/mTOR, VEGFR and VEGF have been targeted in gastric and other cancer types, TGF-β and E-cadherin are not targetable because loss of function of a gene is difficult to restore in patients (although it can be achieved in cell lines). Several novel targets worth mentioning include chromatin modifiers, such as ARID1A, MLL3, MLL and FAT4 (cell adhesion), which are of increasing interest and importance.75,126 Frequent mutations in the genes mentioned above have been observed in 47% of gastric cancers and can poten-tially be therapeutic targets. As we uncover the complex-ity of RNA regulation of intracellular processes, more targets are likely to emerge, for example, targeting the mutations in the spliceosome machinery.

MicroRNAs

miRNAs have a role in tumorigenesis, tumour pro-gression and metastasis, and will—in all likelihood—c o ntinue to gain importance in diagnostics, monitoring during therapy and as therapeutic targets (Figure 2).127 Oncogenic microRNAs (oncomiRs) are overexpressed and inhibit tumour suppressors, leading to cell prolifer-ation, invasion and reduced apoptosis. For example, over-expression of miR-296-5p in gastric cancer cells increased cell prolifer a tion and inhibition of apoptosis via the repres-sion of tumour suppressor CDX1.128 Furthermore, over-expression of miR-301a directly targets tumour suppressor RUNX3,129 whereas miR-17-5p/20a targets tumour protein p53-inducible nuclear protein 1 (TP53INP1).130 miR-18a levels were correlated with those of survivin (also known as baculoviral inhibitor of apoptosis repeat-containing 5; BIRC5), Bcl-xL (B-cell lymphoma-extra large) and c-Myc, all three being downstream transcriptional targets of STAT3. STAT3-induced transcription can be negatively regulated by PIAS3. Thus, miR-18a acts as an oncomiR by negatively regulating PIAS3.131 miRNA-372 is onco-genic as it targets TNFAIP1 and modulates NF-κB signal-ling in gastric cancer cells.132IRX1, a newly identified tumour suppressor gene, is inacti v ated by miR-544.133 miR-10b is highly expressed in IGC and is associated with the depth of invasion, lymph-node involvement and metastatic progression.134

Tumour suppressor microRNAs (tsmiRs), by contrast,

are downregulated miRNAs that facilitate the activity of target oncogenes. miR-195 and miR-378 have been shown to be downregulated in gastric cancer (in vivo and in vitro); their target oncogenes are CDK6 and VEGF.135 The oncogene target of miR-133b is FGFR1, which is often amplified in DGC.136 The target of miR-29c is Mcl?1; acti-vation of this miRNA by celecoxib represses Mcl?1 and promotes apoptosis of gastric cancer cells.137 miR-34a is down r egulated in gastric cancer cells; its oncogene target is BIRC5.138 miR-145 suppresses protein C-ets-1 (ETS1), also known as p54, by binding to its three-prime untranslated region (3'-UTR) and reducing the onco-genic processes.139 Let-7i is frequently down r egulated in most tumours and is prognostic of lymphatic invasion, nodal metastasis and poor pathological tumour response in patients with gastric cancer.140 Furthermore, ZFX has a role the maintenance of CSCs and is the target of miR-144 in gastric cancer.141 miR-101 targets the 3'-UTR of PTGS2 (also known as COX2) mRNA and its down-regulation in gastric cancer correlates with over e xpression of COX-2 and cell prolifer a tion.142 Finally, miRNA-146a inhibits NF-κB by targeting CARD10 and COPS8 in gastric cancer.143

All miRNAs are stable in serum, plasma, gastric juice and other bodily fluids, which makes them ideal bio-markers for the disease.144 Indeed, miR-21 and miR-106a were shown to be overexpressed in the gastric juice of patients compared with normal controls.145 Additionally, levels of miR-421 in the gastric juice of patients was higher than in controls (P <0.001) and it resulted in the earlier diagnosis of gastric cancer than by serum carcino-embryonic antigen.146 Similarly, plasma miR-106b, miR-20a and miR-221 levels were elevated in patients

compared with healthy controls (P <0.05).147 In another

study, plasma levels of miRNA-199a-3p were associ-

ated with tumour invasion, lymph-node involvement

and

metasta ses.148 Circulating miR-17-5p and miR-20a

(miR-17-5p/20a) have also been detected in patients

with gastric cancer and both of these miRNAs corre-

lated with overall survival and relapse-free survival.149

Finally, the miR-200c expression level in the blood in

patients with gastric cancer were significantly higher

than in the blood of normal controls (P =0.018).150 Clearly,

research on several miRNAs holds promise and needs to

be fully developed.

Mechanisms of resistance

Treatment resistance is inevitable; the major reasons

for treatment failure in patients are the occurrence of

primary and secondary resistance. For example, HER2

is the only validated biomarker in gastric cancer, and

HER2-overexpressing tumours regularly become resist-

ant to targeted agents, such as trastuzumab. Accordingly, understanding the underlying mechanisms of resistance

is important. In HER2-overexpressing tumours, the

predominant mechanism of resistance is compensa-

tory signalling by other cell surface receptors.151 HER2-overexpressing cells—when inhibited—reprogramme

other oncogenes, including IGF1R, MET, GDF15 (encod-

ing growth differentiation factor 15) and other members

of the ERBB family.152 The IGF1R-mediated resistance

to HER2 targeting involves the PI3K pathway, leading to

enhanced degradation of p27Kip1,105,153,154 whereas activated

Figure 2 | microRNA targets and functions in gastric cancer. Some oncomiRs are overexpressed in tumours and inhibit tumour suppressors, leading to cell proliferation, invasion and reduced apoptosis. By contrast, tsmiRs normally target oncogenes that are downregulated in tumours and facilitate the activity of their target oncogenes. Abbreviations: EMT, epithelial–mesenchymal transition; miR, microRNA; oncomiRs, oncogenic microRNAs; tsmiRs, tumour-suppressor microRNAs.

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c-MET mediates resistance in gastric cancer cells155,156 by restoring shared downstream signalling in the MAPK and Akt pathways.155 Increased levels of EGFR and HER3 ligands can overcome HER2 inhibition.88,124,152 Constitutively activated p95HER2, a truncated HER2 receptor, is the most intriguing mechanism of resistance in response to the blockade of the extracellular domain of HER2.157 This method of conferring resistance to trastu-zumab is mediated by the regulatory DNA that engages a specific protease gene to truncate the receptor. Thus, resist-ance is not caused by acquiring a mutation in the ERBB2 gene itself, rather another gene modifies the receptor and renders the truncated receptor constitutively active. Membrane mucins, such as mucin-4, interact with HER2 in HER2-overexpressing breast cancer cells, resulting in epitope masking that blocks trastuzumab binding.158 Other factors that confer resistance to trastuzumab include focal adhesion kinase (FAK), Src and altera-tions in cell-cycle regulators.159 Constitutive activation of PI3K caused by activating PIK3CA mutations,61 reduced PTEN expression160 or deregulated signalling can induce resistance to HER2 inhibition.61,161,162 Finally, STAT3 activation can mediate resistance as a result of produc-tion of IL-6.163 These data suggest that cancer cells have many redundant mechanisms to overcome resistance to therapy. Accordingly, we have considerable work ahead of us, including exploring antibody–drug c o njugates and immune modulation therapies.

Clinical considerations

Much clinical development is underway in the setting of gastric cancer, which includes many of the targets dis-cussed herein. Many of the recently completed phase III trials should have an impact on patient care and future research directions (Figure 3). Notably, any discussion regarding clinical developments and the future of gastric cancer care must be made with consideration of the regional differ e nces, both in terms of epidemiology and standard practice. The epidemiology and predominant location of primary gastric cancer varies geographically164 because of variations in genetic susceptibility, predomi-nance of certain histological phenotypes (for example,

FGFR2 amplification (9%), VEGF/VEGFR overexpression (36–40%), EGFR amplification and overexpression (27–44%), HER-2 amplification and overexpression (7–34%), c-MET amplification (10–15%), kRAS mutation (2–20%), Raf mutation (0–3%),

PI3K mutation (4–36%), phospho-Akt expression (29–86%), phospho-mTOR expression (60–88%), PTCH1 overexpression (16%), SMO overexpression (12%) and HER3 mutations (10%, not shown). *No clinical trials of these agents have yet been reported in gastric cancer. ?No known numbers or percentages for these genes and pathways. Abbreviations: EGFR, epidermal growth factor receptor; FGFR, fibroblast growth factor receptor; GLI, glioma-associated oncogene family zinc finger 1; HDAC, histone deacetylase; HER, human epidermal growth factor receptor; HGF, hepatocyte growth factor;

Hh, Hedgehog; IGFR, insulin-like growth factor receptor; MMP, matrix metalloproteinase; mTOR, mammalian target

of rapamycin; PDGFR, platelet-derived growth factor receptor; Ptch-1, protein patched homolog 1; Smo, smoothened; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

IGC is frequent in the endemic areas) and carcinogenic determinants, including H. pylori.165 Gastric cardia cancer is more common in the West, whereas noncardia gastric cancer is more common in the endemic regions. Besides these differ e nces, the surgical approach is more comprehensive in Asia—where a D2 dissection (lymph node dissection that includes regional nodes outside the perigastric area) is routine166—than in the West, where D0 (without formal node dissection) or D1 (dissection limited to perigastric nodes) approaches are preferred. Other regional differences are reflected in the adjuvant strategy for localized gastric cancer (LGC). In the West, postoperative chemoradiotherapy or preoperative chemo-therapy has been embraced as the adjunctive strategy for patients with localized gastric cancer.167 By contrast, in Southeast Asia, two prospective trials (ACTS-GC168 and CLASSIC)169 established the benefit of post o perative adju-vant chemo t herapy. Many of these factors might account for d i fferences in survival of patients in different regions.166

Localized gastric cancer

LGC can be staged as clinical T1 (cT1) or higher with or without regional nodes. Once a patient is diagnosed with LGC, a multidisciplinary evaluation (by medical oncolo-gists, surgical oncologists, surgeons, gastroenterologists, pathologists, radiation oncologists, geneticists [if appro-priate] and nutritionists) is highly recommended prior to initiating any therapy.170 Endoscopic resection for a T1 lesion, when feasible, is recommended. For tumours not amenable to effective endoscopic therapy, surgery should be considered. However, adjunctive therapies (preoperative and postoperative) have contributed to the higher cure rates than those obtained by surgery.167,171–173 Indeed, in North America and Europe, results from the INT-0116167 and Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC)174 trials have established specific adjunctive strategies. In Asia, post-operative adjuvant chemotherapy has been established as the standard of care.168,169

Postoperative adjuvant chemoradiotherapy

Despite safety concerns, such as haematological effects (leukopenia) and treatment-related deaths (cardiac events, pulmonary fibrosis and sepsis as a complication of myelosuppression) during the INT-0116 phase III trial (conducted in the USA) of observation after surgery versus adjuvant chemoradiotherapy (5- f luorouracil, leuco v orin and radiotherapy) after surgery, chemoradio-therapy improved the 5-year cure rate by ~10%.167 This advantage prevailed with longer follow-up durations.175 However, a subsequent US-based trial coordinated by Cancer and Leukemia Group B produced negative results when investigating the adjuvant chemotherapy strategy (epirubicin, cisplatin and 5- f luorouracil).176 Similarly, the ARTIST trial used the INT-0116 strategy, but differed in two aspects: the control group was treated with chemo-therapy and all patients had undergone a D2 gastrec-tomy.177 The trial failed to demonstrate a benefit for those receiving c hemoradiotherapy,178 raising more questions than it answered.Postoperative chemotherapy

The benefits of adjuvant chemotherapy after D2 gastrec-

tomy were first established in Japan using S-1 (an oral fluoropyrimidine) as the adjuvant.168 The Adjuvant Chemotherapy Trial of TS-1 for Gastric Cancer (ACTS-

GC)168 randomly assigned 1,059 patients to 1 year of S-1

or observation. The primary analysis demonstrated a 33%

improvement in overall survival for the S-1 group, results

that prevailed after a longer follow-up duration (5 years).178

A second Asian study, the Capecitabine and Oxaliplatin

Adjuvant Study in Stomach Cancer (CLASSIC trial) ran-

domly assigned 1,035 patients who had undergone D2

gastrectomy to capecitabine plus oxaliplatin for 6 months

or observation,169 documenting benefit for chemotherapy

for the end point of disease- f ree survival (at 3 years; hazard

ratio [HR]=0.56, 95% CI, 0.44–0.72; P <0.0001). A meta-

analysis based on data from 3,710 patients showed a 7%

improvement in overall survival for fluorouracil-based

postoperative chemotherapy when compared with surgery

alone,173 but this evidence is not robust; the analysis com-

bined many studies from different time periods with

differing eligibility criteria and therapeutic approaches,

making it difficult to make a firm conclusion.

Perioperative or preoperative chemotherapy

The MAGIC trial, which randomly assigned 504 patients

(74% of whom had gastric cancer), established the evi-

dence for perioperative chemotherapy (epirubicin, cispla-

tin and 5-fluorouracil) for gastroesophageal cancer in

the West.174 A second, French trial (in which 25% of the

patients had gastric cancer) terminated early because of

the lack of interest, but demonstrated benefit for preopera-

tive chemotherapy (5-fluorouracil and cisplatin).172Several

trials using a variety of adjuvant strategies are currently

ongoing (Table 3).179

Advanced gastric cancer

Most targeted therapies investigated in unenriched (that

is, patients are not selected based on a biomarker) patient

populations have produced disappointing results (Table 3).

Despite these results, the infrastructure for conduct i ng

regional or global phase III trials has ripened and timely

patient accrual is less of a problem. However, many ques-

tions regarding the lack of progress for patients with

advanced gastric cancer remain. Glaring unanswered ques-

tions include: would further understanding of molecular

biology of gastric cancer lead to selection of appropriate

patient subsets and, therefore, more effective therapy? Are

regional differ e nces in gastric cancers (and patient genet-

ics) so substantial that global trials in unenriched popula-

tions will not be fruitful? Do second-line and third-line

therapies produce substantial survival advantages as

to jeopardize the primary end point of overall survival

required by most regulatory agencies for first-line phase III

trials? Certainly, many other unknown factors are likely to

contribute to the disappointing results observed thus far in

advanced gastric cancer. We stress that use of a single bio-

logical agent (such as trastuzumab) in enriched populations

(HER2-overexpressing tumours) provides only marginal

advantage because of the presence of primary resistance

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or development of secondary resistance. In today’s era of advanced bio t echnology, we are perhaps witnessing the end of empirical investigative strat e gies whereby the new agent is investigated in patients with similar stages with the assumption that the genomic makeup of all tumours and genetics of all patients is similar, which will certainly impact future trials in this cancer type.

First-line therapy

Level 1 evidence for an advantage in overall survival is available for only a few therapeutic agents (docetaxel,180 cisplatin181 and trastuzumab).182 Most trials have been disappointing with the exception of the trastuzumab trial that investigated a HER2-enriched population.182 However, the longer follow-up duration has reduced the hazard ratio for overall survival.183 In the primary analysis,182 the hazard ratio for overall survival was 0.74, but reduced to 0.80. The difference in median overall survival was reduced from 2.7 months to merely 1.4 months, representing an approxi-mate 50% decrease in the effect of trastuzumab, which suggests that only a few patients benefit. Notable among trials with disappointing results are two randomized studies investi g ating the value of EGFR inhibition (EXPAND and REAL-3).184,185 In the EXPAND trial,184 patients were

administered capecitabine and cisplatin, with (experimen-tal) or without (control) cetuximab. Median progression-free survival was 4.4 months in the experimental group compared with 5.6 months in the control group. In the REAL-3 trial,185 panitumumab was added to epicrubicin, oxaliplatin and capecitabine. The experimental (four-agent) group had shorter overall survival (8.8 months) com-pared with the control (three-agent) group (11.3 months; HR 1.37). In both trials, the results demonstrated that the addition of anti-EGFR antibodies (cetuximab or panitu-mumab) to chemo t herapy did not confer a benefit. Except for the ToGA trial,182 all studies investigating a biological agent were c o nducted in an unenriched population. Amongst the failed trials in advanced gastric cancer, was the AV AGAST trial, which was conducted in 774 patients who were randomly assigned to chemotherapy with or without bevacizumab (which targets VEGF-A). This trial did not meet its primary end point of overall survival.186 Retrospective biomarker evaluation of plasma VEGF-A and neuropilin-1 levels seemed to be associated with beva-cizumab efficacy; high plasma VEGF-A and low tumour n e uropilin- 1 levels seemed to correlate with improved overall survival, but neither of these biomarkers is vali-dated.187 Another noteworthy trial is the First line Advanced Gastric Cancer Study (FLAGS), conducted in >1,000 patients who were randomly assigned to receive S-1 plus cisplatin versus 5-fluorouracil plus cisplatin, which also did not meet its primary end point of an overall survival advantage.188

Second-line therapy

A small phase III Arbeitsgemeinschaft Internistische Onkologie (AIO) trial of 40 patients randomly assigned to irinotecan with best supportive care (BSC) or BSC alone showed that overall survival could be extended using irino-tecan plus BSC.189 The randomized GRANITE-1 study (NCT00879333)190 included >600 patients and compared everolimus with placebo in the second-line or third-line settings, but did not achieve its primary end point of improved overall survival.191 However, the REGARD trial (NCT00917384)192 that compared BSC with or without ramucirumab (which targets VEGFR2) in 345 patients demonstrated a marginal improvement in overall survival for ramucirumab (P =0.047),193 suggesting that ramuci-rumab has only a borderline effect. More impressively, the randomized Cougar-02 trial of approximately 186 patients assigned to BSC or docetaxel plus BSC demon s trated a signifi c ant prolongation of overall survival in the combi-nation arm. The patients who received docetaxel survived 44% longer than those who did not receive docetaxel; overall survival was 5.2 months versus 3.6 months, respec-tively (P =0.01, HR 0.67).194 Lapatinib—a dual inhibitor of HER2 and EGFR—was investigated in a phase III study (TYTAN) in >300 patients assigned to either lapatinib or placebo, but the primary end point of prolonged overall survival was not achieved.195

Targeting the c-MET pathway in these patients is also of interest. In a small study with crizotinib, two out of four patients with five or more MET copy number gains had a longer response duration than those with fewer than five copies of the gene.196 Furthermore, rilotumumab (AMG 102), a fully human monoclonal antibody, demon-

strated longer overall survival for patients whose tumours

had high total c-MET expression.197

Conclusions

Despite considerable advances in biotechnology that have

improved our understanding of cancer, immense com-

plexities confront us. Although gastric cancer lags behind

many other tumour types in terms of genetic sequenc-

ing and specially designed therapies, more progress is

anticipated. Greater understanding of the pathogenesis

of IGC by H. pylori is poised to help with increasingly

sophisticated preventive strategies. Germline susceptibil-

ity investigations have uncovered novel genes, but clinical implementation has proven problematic and more work

is needed. Clinically, pro g ress has been slow, but adjuvant

strategies are now frequently employed in patients with

LGC around the world, which should be considered an

important advancement. Future progress will be propelled

by further improvements in biotechnologies to produce

better b i omarkers and drugs.

Given our current knowledge, patients who are ≤45 years

old should be recommended genetic counselling followed

by genetic testing, if needed. Patients with advanced gastric

cancer could benefit from HER2/neu testing.151 The value

of assessing other somatic alterations in gastric cancer is

currently ongoing, but results from ongoing large-scale

genomic sequencing projects will yield insights into the

landscape of genomic alterations. Banking of research blood

(and other bodily fluids) as well as tumour tissue from all

patients for studies should enable detailed examination of

this heterogeneous cancer, and is highly desirable but chal-

lenging. Interrogation of blood for specific alterations in

circulating DNA, RNA, peptides, proteins and tumour cells

holds particular promise for the early detection of gastric

cancer and its m o nitoring during therapy and surveillance.

All patients with LGC must undergo multidisciplinary

evaluation and discussion by experts representing each

major discipline. LGC is a complex disease and patients

should be treated only by experienced high-volume physi-

cians and at centres with excellent infrastructure. Patients

with clinical T1aN0 gastric cancer should be evaluated for

endoscopic therapy. Patients with clinical >T1bN0 gastric

cancer should be offered adjunctive therapy (preoperative,

postoperative or both). Suitable patients with advanced

disease should be offered second- l ine systemic therapy. Investigations of therapeutic agents that take advantage

of the host immune system and mechanisms (including

a n

tibody–drug conjugates) hold immense promise.

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Acknowledgements

The authors are supported by grants from the

Caporella, Cantu, Smith, Park, Fairman, Frazier, Sultan,

Oaks, Vansteklenberg and Milrod families, by the

Kevin Fund, Schecter Private Fund and the Rivercreek

Foundation and a Multidisciplinary Research Grant from

University of Texas MD Anderson Cancer Center.

J. A. Ajani is also supported by grants RO1CA138671

and ROaCA172741 awarded by the National Cancer

Institute, Bethesda, MD, USA. The authors also thank

the editorial staff of Nature Reviews Clinical Oncology for

their editorial assistance in considerably improving the

clarity and organization of the manuscript.

Author contributions

All authors researched the data, discussed the

article’s content, wrote the manuscript and edited

it before submission.

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