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Cancer Biomarkers 00 (2012/2013) 1–12 1DOI 10.3233/CBM-130327IOS Press

Predictive factors to targeted treatment ingastrointestinal carcinomas

Nicola Silvestrisa,!, Ilaria Marechb, Anna Elisabetta Brunettic, Amalia Azzaritid, Gianmauro Numicoe,Giuseppe Cicerof , Sabina Delcuratoloc, Raffaele De Lucag, Claudia Burzh and Vito LorussoaaMedical Oncology Unit, National Cancer Research Centre “Giovanni Paolo II”, Bari, ItalybInterventional Radiology Unit with Integrated Section of Translational Medical Oncology, National CancerResearch Centre “Giovanni Paolo II”, Bari, ItalycScientific Direction, National Cancer Research Centre “Giovanni Paolo II”, Bari, ItalydClinical and Preclinical Pharmacology Laboratory, National Cancer Research Centre “Giovanni Paolo II”, Bari,ItalyeMedical Oncology Department, Ospedale U. Parini, Aosta, ItalyfClinical Experimental Oncology Laboratory, National Cancer Research Centre “Giovanni Paolo II”, Bari, ItalygDepartment of Surgical Oncology, National Cancer Research Centre “Giovanni Paolo II”, Bari, ItalyhThe Oncology Institute “Prof. Dr. Ion Chiricuta”, Cluj-Napoca, Romania

Abstract. Most cancers are traditionally treated with either chemotherapeutic agents, radiotherapy, or both. Identification ofspecific molecular characteristics of tumors and the advent of molecular-targeted drugs not only enhance the efficacy but alsodecrease the toxicity of treatment. These new therapies may target pathways critical to tumor development or specific drivermutations in cancer cells. This understanding of the molecular pathways of cancer cells has led to the ability to predict cancerdevelopment, behaviour and prognosis, as well as response or resistance to current therapeutic agents. As a result, pathologicanalyses play a vital role in the detection of cancer biomarkers, which are important not only in the diagnosis of cancers but alsoin the selection of appropriate therapeutic agents and in the development of new targeted therapies.

Keywords: Gastrointestinal cancer, targeted therapy, EGFR, HER2, VEGF

1. Introduction1

In recent years, there has been considerable interest2

in understanding the mechanisms underlying the de-3

velopment and progression of tumors. The identifica-4

tion of specific pathways has led to the development of5

drugs that are able to block specific molecular targets.6

Among these, inhibitors of the epidermal growth fac-7

tor receptor and HER2/neu pathways now play a ma-8

jor role in the management of gastrointestinal cancers,9

in addition to other solid malignancies. In this review,10

!Corresponding author: Nicola Silvestris, Medical and Experi-mental Oncology Unit, National Cancer Research Centre “GiovanniPaolo II”, Viale Orazio Flacco, 65, 70124 Bari, Italy. Tel.: +39 0805555442; Fax: +39 080 5555416; Mobile: +39 3332228492; E-mail:n.silvestris@oncologico.bari.it.

recent advances in the field of targeted therapy of gas- 11

trointestinal cancers are discussed. We also focus on 12

the mechanisms of primary and secondary resistance, 13

as well as on the emergence of new predictive mark- 14

ers of response to treatment. The development of bio- 15

logic agents for use in highly selected patient subsets 16

should represent a new paradigm for the treatment of 17

gastrointestinal cancers. 18

2. Colorectal cancer 19

In spite of the progress in gastrointestinal can- 20

cer research, the translation of genetic discoveries 21

into diagnostic tests that predict response to therapy 22

for metastatic colorectal cancer (mCRC) patients has 23

been disappointing. Among several cancer gene muta- 24

ISSN 1574-0153/12/13/$27.50 c" 2012/2013 – IOS Press and the authors. All rights reserved

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2 N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas

tions involved in CRC development, only KRAS sta-25

tus currently is validated as a predictive biomarker26

of response to anti-epidermal growth factor receptor27

(EGFR) therapy in this subset of patients [60]. In fact,28

tumor KRAS mutations, which are found in approxi-29

mately 40% of cases, have been widely demonstrated30

to be major predictive markers of resistance to cetux-31

imab or panitumumab, thereby opening the way to in-32

dividualized treatment for patients with mCRC [66,33

83]. Other oncogenic mutations such as BRAF or34

PIK3CA mutations or loss of PTEN expression also35

may be interesting additional predictive markers of re-36

sponse to anti-EGFR monoclonal antibodies (mAbs)37

but require further evaluation before being incorpo-38

rated into clinical practice [82].39

2.1. EGFR40

EGFR is a member of the ErbB family of trans-41

membrane receptor tyrosine kinases. Its activation oc-42

curs by several ligands (such as EGF, TGF-alfa cy-43

tokines, hormones, drugs and others growth factors)44

binding to the extracellular domain through the au-45

tophosphorylation of key tyrosine residues in the in-46

tracellular domain. EGFR activation results in sig-47

nal transduction through the pathways of MAP kinase48

and PI3K/AKT, leading to proliferation, angiogene-49

sis and migration [82]. EGFR, evaluated by immuno-50

histochemistry (IHC), is over-expressed in 60–80% of51

CRC [95]. The malignancy signal mediated by EGFR52

is abrogated by two monoclonal antibodies: cetuximab53

a chimeric IgG1, and the fully humanized IgG2 panitu-54

mumab selectively binding to the extracellular domain55

of the EGFR [82]. This leads to inactivation of KRAS,56

preventing the phosphorylation cascade of RAF, MEK,57

ERK and of multiple nuclear transcription factors that58

inhibit DNA synthesis and cell proliferation (Fig. 1).59

Moroni et al. assumed a correlation between EGFR60

gene copy number (GCN) and clinical response to61

anti-EGFR therapy in CRC [58]. Some patients with62

mCRC, who achieved a good clinical response to anti-63

EGFR treatment (cetuximab or panitumumab), had a64

significantly increased EGFR copy number on assess-65

ment of individual tumor samples by FISH. The re-66

sults of the study were not strong enough to produce a67

firm conclusion due to the very low number of patients68

(n = 31). Conversely, Italiano et al. demonstrated that69

neither EGFR expression assessed by IHC nor EGFR70

gene copy gain assessed by FISH were significantly71

correlated with response rate, progression-free sur-72

vival, or overall survival of patients previously treated73

with cetuximab because EGFR FISH analysis did not 74

appear to be a sufficiently robust test for selecting can- 75

didate CRC patients for cetuximab therapy [40]. Re- 76

cently, Yang et al., assuming EGFR gene copy num- 77

ber (GCN) as a potential predictive biomarker for the 78

treatment of (mCRC) with anti-EGFR MAbs, con- 79

cluded that although increased EGFR GCN is a posi- 80

tive predictive factor to anti-EGFR MAb therapy (par- 81

ticularly in patients with wild type KRAS), the util- 82

ity of this biomarker would be limited by laboratory 83

techniques such as the heterogeneous scoring system 84

and the poor reproducibility of EGFR GCN enumer- 85

ation [95]. Chung et al. showed that patients who do 86

not express EGFR by IHC had a good clinical outcome 87

to anti-EGFR mAb treatments [12]. Furthermore, in 88

patients with KRAS wild type, neither EGFR protein 89

expression levels nor a specific EGFR CA polymor- 90

phism in intron 1 predicted clinical outcomes to cetux- 91

imab treatment [65]. Further studies will be necessary 92

to confirm whether the EGFR mutation status could 93

be an important determinant in the resistance to anti- 94

EGFR mAb treatment [62]. In conclusion, the level of 95

protein expression of EGFR or EGFR mutation sta- 96

tus currently does not predict response to anti-EGFR 97

mAbs. 98

2.2. KRAS 99

The RAS protein, encoded by KRAS and belong- 100

ing to a GTPase protein family, is key in the activation 101

of epidermal growth factor receptor signaling (EGFR). 102

KRAS is a proto-oncogene encoded on the short arm of 103

chromosome 12. KRAS-mutations, occurring in about 104

35–42% of CRC, constitute more than 97% of the ob- 105

served genetic events (predominately within codons 12 106

and 13 of exon 2), leading to constitutive activation 107

of the RAS/RAF pathway and neoplastic transforma- 108

tion [57]. Cetuximab and panitumumab are approved 109

for use in combination with chemotherapy (5-FU, ox- 110

aliplatin or irinotecan based) for stage IV mCRC [60] 111

or as a single agent for the treatment of chemotherapy- 112

resistant mCRC [95]. Early studies suggested that pa- 113

tients harboring a KRAS mutation had a worse prog- 114

nosis with increased risk of recurrence (p = 0.007) and 115

death (p = 0.004) compared with patients with wild 116

type KRAS [3]. The KRAS gene is mutated, mainly in 117

the codons 12 and 13, and to a lesser degree in 61 and 118

146 [27]. It has been shown that mutations in codons 119

12, 13, and 61 all predict a lack of response to cetux- 120

imab. Conversely, it has been demonstrated that muta- 121

tions in both codon 13 and 146 do not affect cetuximab 122

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N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas 3

EGF and other ligands

EGFR

Fig. 1. MAP kinase and PI3K/AKT pathways. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/CBM-130327)

efficacy [18]. In addition, it has been suggested by De123

Roock et al. [18] and recently confirmed by Tejpar et124

al. [87] that patients carrying the KRAS G13D muta-125

tion benefitted significantly from first line chemother-126

apy plus cetuximab. However, only when the results127

of the ongoing randomized studies in this patient sub-128

set are available will the use of anti-EGFR mAbs be129

allowed in patients carrying the G13D mutation. To130

date, KRAS remains the only molecular marker used131

to select patients who are eligible for mAb treatment,132

and only mutations in codons 12 and 13 are investi-133

gated in routine clinical practice. It is noteworthy that134

fewer than one-half of the KRAS wild type patients re-135

spond to anti-EGFR therapy [16] and that a significant136

proportion of patients with wild type KRAS remain137

unresponsive to the combination of anti-EGFR MAbs138

plus chemotherapy, which increased response rate by139

only 10–20% as compared to chemotherapy alone [66].140

Thus, it is important to identify other biomarkers that141

can be used jointly with KRAS in predicting treatment142

response, as anti-EGFR therapy is associated with a143

significant increase in toxicity and cost.144

A focus on accordance between the genotype in the145

primary tumor and metastases from the same patient146

has gained attention recently, as mutation status in the147

primary tumor might not reflect the mutational situa-148

tion in metastases of the patient [10,15]. The concor-149

dance of KRAS status in primary tumor and metasta-150

sis seems to be 93% with a range from 70% to 100%.151

Moreover, hepatic metastases more frequently demon-152

strate concordance to the primary tumor than lymph 153

node metastases: on average 95% vs. 84%, respec- 154

tively. 155

2.3. BRAF 156

The BRAF gene encodes a serine/threonine protein 157

kinase that also belongs to the RAS/RAF kinase path- 158

way. Mutations in BRAF, leading to constitutive acti- 159

vation of the BRAF kinase activity, are less prevalent in 160

CRC tumors than KRAS mutations with a frequency of 161

10% vs. 30%, respectively. Mutations in KRAS and the 162

downstream effector BRAF, which are higher in lymph 163

node metastases, are mutually exclusive [30], although 164

some overlap has been shown in about 3 to 9% of 165

cases [78]. As for KRAS, mutations in BRAF also pre- 166

dict a negative response to anti-EGFR therapy, as well 167

as reduced survival [17,69]. In this regard, an Italian 168

group retrospectively analyzed tumor responses, time 169

to progression, overall survival, and the mutational sta- 170

tus of KRAS and BRAF in 113 tumors treated with 171

anti-EGFR mAbs. Activating mutations in BRAF are 172

observed in about 10% of CRC in the kinase domain 173

of the gene, and the most frequent is the substitu- 174

tion of valine by glutamate in position 600 [21]. The 175

BRAF V600E mutation was detected in 11 of 79 pa- 176

tients who had wild type KRAS. None of the BRAF- 177

mutated patients responded to the anti-EGFR treat- 178

ment, and none of the responders carried BRAF mu- 179

tations (p = 0.029). Moreover, patients carrying the 180

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4 N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas

BRAF mutation had significantly shorter progression-181

free survival (p = 0.011) and overall survival (p <182

0.0001) than patients with wild type BRAF [21]. A183

recent Slovenian study showed BRAF V600E muta-184

tion in 5.1% of patients [24]. In a retrospective analy-185

sis by Fariña-Sarasqueta et al. [25] it was demonstrated186

that the BRAF V600E mutation was an independent187

prognostic factor for the survival of patients with colon188

cancer in stages II and III, while the KRAS muta-189

tions did not have any effect on the overall survival190

of these patients [25]. In the Crystal study, the BRAF191

V600E mutation was correlated to a poor prognosis192

regardless of treatment. In the presence of this muta-193

tion, chemotherapy with cetuximab conferred a clini-194

cal benefit only in patients not previously treated [89].195

The overall concordance in primary tumor and metas-196

tasis of the hotspot mutation V600E in BRAF inves-197

tigated across seven studies was reported to be 98%198

(91–100%). When evaluating BRAF mutated primary199

tumors only, concordance was 70% (50–100%) [72].200

2.4. PIK3CA and AKT pathways201

In addition to signaling downstream through BRAF,202

KRAS also activates the AKT pathway. PTEN and203

PIK3CA have opposing roles in this pathway, convert-204

ing PIP3 (phosphatidylinositol 3-phosphate) to PIP2205

(phosphatidylinositol 2-phosphate). Elevated levels of206

PIP3 result in hyperphosphorylation of AKT, either207

due to loss of PTEN activity or to activation of muta-208

tions in PIK3CA. Hyperphosphorylated AKT inhibits209

the cell from undergoing apoptosis. In patients with210

metastatic CRC KRAS wild type, the presence of211

PTEN loss or activating mutations in PIK3CA pre-212

dicts a reduced overall survival and no response to anti-213

EGFR treatment [74]. Several compounds in clinical214

development selectively inhibit mutated BRAF [13].215

However, these agents should be avoided in cancers216

with wild type BRAF or those that are KRAS mu-217

tated, as these will have increased activity through218

BRAF [37]. Further downstream of the EGFR signal-219

ing cascade from BRAF is the mitogen-activated pro-220

tein kinase kinase (MEK). MEK inhibitors have been221

suggested for patients who are mutant for either KRAS222

or BRAF [22]. In vitro BRAF mutant cell lines were223

sensitive to MEK inhibitors. However, in vivo low re-224

sponse rate and ocular toxicity have blocked further225

clinical development of these agents [51]. BRAF mu-226

tated cell lines were found to be more sensitive to MEK227

inhibitors than KRAS mutated cell lines, which might228

be due to the fact that KRAS has several downstream229

effectors [11]. However, BRAF gene amplification has 230

been demonstrated to result in resistance to both MEK 231

and BRAF inhibitors. In KRAS wild type patients the 232

presence and concordance of mutations in BRAF and 233

PIK3CA from primary tumor and metastases has been 234

investigated [10]. Cejas et al. showed that one out of 235

70 patients presented a mutation of BRAF (1.4%) in 236

the primary tumor, and the mutational status was the 237

same in the corresponding liver metastasis. A muta- 238

tion of PIK3CA was observed in 7% (five patients) of 239

the primary tumors, whereas 10% of the metastases 240

were mutated. Discordance of PIK3CA mutations was 241

detected in four primary-metastasis pairs (94%) [10]. 242

Retrospective analyses reported that PIK3CA muta- 243

tions conferred poor prognosis in KRAS wild type pa- 244

tients [74]. Investigation on PTEN loss in primary tu- 245

mors and metastases from the same patient reported 246

PTEN loss to have low concordance (range 47–89%) 247

across seven studies [65]. This large range might be 248

caused by the fact that no standard method for de- 249

tecting PTEN expression was used. Di Nicolantonio 250

et al. [21] found that the probability of response to 251

anti-EGFR treatment (panitumumab or cetuximab) in 252

absence of genetic alterations in KRAS, BRAF, and 253

PIK3CA and PTEN genes was 51%. This probability 254

of response dropped to 4% among patients with one of 255

these alterations and to 0% for patients with more than 256

two alterations (p < 0.0001). Similarly, progression- 257

free survival and overall survival were increasingly 258

worse for patients with tumors harboring 0, 1, or 2 or 259

more alterations (p < 0.001) [47]. The development of 260

therapy-related resistance during anti-EGFR antibody 261

treatment in metastatic CRC has been investigated in 262

the light of the treatment-induced shift in mutation sta- 263

tus for KRAS and BRAF. KRAS and BRAF mutation 264

status was shown to be highly concordant in primary 265

tumors before and after anti-EGFR therapy, indicating 266

that therapy-induced resistance is not likely induced by 267

mutations in the hotspot regions of these genes [31]. 268

However, more recently, a study found that 35% of 269

24 patients receiving monotherapy with panitumumab, 270

whose tumors were initially KRAS wild type, devel- 271

oped detectable mutations in KRAS in their sera. Three 272

of these patients developed multiple different KRAS 273

mutations [19]. This supports the theory that the emer- 274

gence of KRAS mutations is a mediator of acquired 275

resistance to the EGFR blockade and that these muta- 276

tions can be detected by a blood test. 277

2.5. Chromosomal aberration 278

In literature it is reported that allelic deletions in- 279

volving chromosomes 18q and 17p occur in more than 280

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N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas 5

70% of CRC [44]. In patients with stage II and III281

CRC, some retrospective studies showed strong cor-282

relations between genomic deletion events on chro-283

mosome arm 18q and increased risk of developing284

metastases, reduced overall survival and progression285

free survival [91]. A number of the deleterious ef-286

fects for such a large chromosomal deletion are eas-287

ily understandable, given that the cancer genes DCC288

(Deleted Colorectal Cancer encoding for cell adhesion289

molecule), SMAD2 and SMAD4 (transcription fac-290

tors) are located in the region of deletion [26]. Iso-291

lated genomic deletions of SMAD2 or SMAD4 are292

not sufficient to account for the prognostic significance293

of 18q deletion [47]. This suggests that other candi-294

date colorectal cancer genes may exist in the 18q re-295

gion [47]. A number of other studies have failed to cor-296

relate the 18q deletion with poor prognosis, complicat-297

ing the possible clinical utility of 18q [14]. The Eastern298

Cooperative Oncology Group (ECOG) study 5202 is299

an ongoing prospective clinical trial that is randomiz-300

ing to observation vs. chemotherapy patients with stage301

II disease based on 18q status and microsatellite in-302

stability (MSI), with the aim of prospectively evalu-303

ating the prognostic value of molecular markers [64].304

The tumor suppressor gene p53 (called the “guardian305

of the genome”) is located on 17p and is mutated (so-306

matic point mutations) in 40% to 60% of CRC [64].307

P53 status has been rigorously analyzed as both a prog-308

nostic and predictive marker of prognosis and therapy309

outcome of CRC, with conflicting results. Therefore,310

the disparities in reporting results make it difficult to311

validate its prognostic significance [59]. This could be312

the direct result of the assorted methodologies lacking313

adequate sensitivity to assess p53 mutations, lack of314

concordance among studies, and the limited examina-315

tion of both alleles of the gene. Danner et al. compared316

chromosomal aberrations in primary tumors to cor-317

responding pulmonary metastases [15]. In this study,318

chromosomal instability was similar in tumors from319

the same patient, suggesting that chromosomal abnor-320

malities occur prior to the metastatic process. Danner321

et al. reported that the most frequent alteration was the322

loss of 5q, involving the APC gene, whose abnormality323

so far has not been correlated with survival [15]. More-324

over, Kim SH et al. recently demonstrated that mCRC325

patients, previously treated with FOLFIRI plus cetux-326

imab and with promoter CpG island hypermethylation327

of p16, had reduced time to progression and overall328

survival, irrespective of KRAS mutation [46].329

2.6. Microsatellite Instability (MSI) 330

MSI is characterized by a change in length of DNA 331

microsatellites due to the insertion or deletion of re- 332

peating units and caused by defects in mismatch re- 333

pair (MMR) genes such as MLH1, MSH2, or MSH6, 334

or methylation of the MLH1 promoter. 335

Studying colon cancer in hereditary nonpolyposis 336

colorectal cancer (HNPCC) families first identified 337

germline mutations in the MMR genes. Subsequently, 338

scientists found that sporadic colon cancers also dis- 339

play somatic mutations in the MMR genes. Accord- 340

ing to the Bethesda Criteria, a diagnosis of MSI in- 341

volves the examination of a reference panel of five mi- 342

crosatellite loci – D5S346, D2S123, D17S250, BAT25, 343

and BAT26. The detection of MSI in two or more of 344

the above loci is considered high instability (MSI-H). 345

Microsatellite-stable tumors show no evidence of in- 346

stability in any of the five loci. Low-MSI (MSI-L) tu- 347

mors exhibit changes in only one of the loci [8]. In 348

current clinical practice, PCR amplification with com- 349

mercially available kits followed by fluorescent cap- 350

illary electrophoresis is used to assess the lengths of 351

the mononucleotide or dinucleotide repeat elements in 352

these five loci from tumor and normal tissue. About 353

15% of colorectal cancers present MSI. Tumors with 354

MSI-H are more proximal, poorly differentiated and 355

mucinous, and show lymphocytic infiltration. Clinical 356

studies have suggested that MSI is associated with im- 357

proved prognosis [45] but decreased response to 5- 358

FU-based chemotherapy [6,68,73]. Ribic et al. evalu- 359

ated increased survival in 570 patients with low-MSI 360

or microsatellite-stable tumors receiving 5-FU-based 361

adjuvant chemotherapy compared with surgery alone 362

(hazard ratio = 0.72; p = 0.04). Conversely, 5-FU ther- 363

apy on patients with MSI-H tumors showed no bene- 364

fit compared with surgery alone, and the patients had 365

worse survival rates after chemotherapy [68]. Gryfe 366

and Gallinger showed that patients in stage II and III 367

CRC with MSI-H had improved survival and that MSI 368

phenotypes correlated to better recurrence-free sur- 369

vival than MSS phenotypes [12]. Several studies con- 370

cluded that CRC patients exhibiting MSI had a signif- 371

icantly better prognosis compared with those with in- 372

tact MMR but did not benefit from the administration 373

of 5-FU therapy in the adjuvant setting [91]. The pres- 374

ence of a mutation in transforming growth factor-beta- 375

RII (TGF-!-RII) was shown to improve survival in pa- 376

tients who also possess MSI-H. The 5-year survival 377

rate for patients with MSI-H tumors and the TGF-!- 378

RII mutation was 74% following adjuvant 5-FU–based 379

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6 N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas

therapy compared to 46% in patients with MSI-H tu-380

mors lacking the mutation in TGF-!-RII. Interestingly,381

61% of stage III colon cancers in this study exhibited382

the TGF-!-RII mutation, indicating that this high fre-383

quency mutation may be useful in combination with384

MSI status as a prognostic marker for adjuvant ther-385

apy [91].386

2.7. Thymidylate synthase and ethylene387

tetrahydrofolate reductase388

Theoretically, responsiveness to chemotherapies389

may be impacted by a patient’s germline variations390

that affect drug metabolism. For example, almost all391

chemotherapy regimens for colorectal cancer use 5-392

FU. One mechanism by which 5-FU exerts its anti-393

cancer effect is through the inhibition of thymidylate394

synthase (TS), which is encoded by the TYMS gene.395

TS catalyzes the reductive methylation of deoxyuridine396

monophosphate to deoxythymidine monophosphate397

using 5,10-methylenetetrahydrofolate as the methyl398

donor. Methylenetetrahydrofolate reductase (MTHFR;399

MTHFR gene) regulates the amount of 5,10-methylen-400

etetrahydrofolate by irreversibly converting it to 5-401

methylhydrofolate. The above process provides the402

sole de novo source of thymidylate, which is necessary403

for DNA replication and repair [33]. Thus, one would404

expect that mutations that alter TS or MTHFR activ-405

ity would affect chemotherapeutic outcomes. Unfor-406

tunately, despite being grounded in theoretical logic,407

the effects of TYMS [7] and MTHFR [7,24] polymor-408

phisms have yielded conflicting results in clinical stud-409

ies. As in the case of TP53, these studies have suffered410

from a lack of consistency in experimental design. Het-411

erogeneity of study populations, methodologies in de-412

tecting the polymorphisms and measurements of out-413

comes has made a direct comparison across studies dif-414

ficult.415

2.8. Predictive factors of antiangiogenic drugs416

A recent promising approach in the treatment of pa-417

tients with colorectal cancer is anti-angiogenic therapy.418

This approach is based on a) targeting one or more sol-419

uble ligands of the VEGF family, b) inhibiting specif-420

ically one or more receptors of the VEGF receptor421

family, and 3) inhibiting indirectly VEGF receptor(s).422

To date, bevacizumab and aflibercept have been uti-423

lized for the treatment of colorectal cancer patients,424

with only the former registered for use in clinical prac-425

tice [4,84].426

Bevacizumab is a humanized monoclonal antibody 427

inhibiting vascular endothelial growth factor A (VEGF 428

-A), while aflibercept is a dual VEGF-A and placen- 429

tal growth factor (PIGF) inhibitor. As known, VEGF- 430

A and PIGF promote angiogenesis, and consequently 431

their inhibition halts the formation of new blood ves- 432

sels that supply blood to the tumors. Other antian- 433

giogenic therapies, including both monoclonal anti- 434

body and tyrosine kinase inhibitors (TKI) such as ra- 435

mucirumab, regorafenib, brivanib alaninate, cediranib, 436

sunitinib, and vatalanib, are currently in late-stage clin- 437

ical trials for metastatic colorectal cancer (mCRC). 438

Some of them showed negative results in phase III tri- 439

als [20]. Biological markers to identify subsets of pa- 440

tients more likely to respond to a given anti-angiogenic 441

therapy, detect early clinical benefit or emerging re- 442

sistances, and decide whether to change therapy in 443

second-line treatments are an urgent need in clinical 444

practice. Unfortunately, despite the considerable effort 445

made by clinicians and scientists in this field through 446

preclinical and clinical studies, no validated predic- 447

tive biomarkers are available [67]. A revision of the 448

literature data on promising predictive factors for an- 449

tiangiogenic drug response showed that there could be 450

several: a physiological response to these drugs (hy- 451

pertension); circulating biomarker, including both pro- 452

teins and cells; tumoral biomarkers meaning tumor tis- 453

sues and/or genetic characteristics; genetic polymor- 454

phisms; and imaging parameters (radiographic mark- 455

ers). Among the proposed biological markers related to 456

antiangiogenic drug response, the circulating ones are 457

the most numerous with pharmacological studies sug- 458

gesting that their baseline levels are less predictive that 459

their variation occurring during therapy [41]. 460

Focusing attention on colorectal cancer patients, evi- 461

dence has stressed the role of the circulating tumor pro- 462

teins VEGF and PIGF as predictive factors. The mea- 463

surement of serum VEGF as a surrogate biomarker for 464

bevacizumab is controversial [34,92,94], and the feasi- 465

bility of the immunodepletion of plasma samples that 466

measure only changes of free VEGF levels in cancer 467

patients after treatment with bevacizumab has been in- 468

vestigated with promising results [53,54,81]. The ex- 469

tent of increase in PIGF levels in plasma was associ- 470

ated with a better outcome in patients with rectal can- 471

cer treated with bevacizumab and chemo-radiotherapy; 472

however, only further investigation will enable clini- 473

cians to distinguish between a predictive and/or prog- 474

nostic value for this factor [92]. Recently, Abajo et 475

al. reported that other circulating biomarkers should 476

be considered for antiangiogenic drug(s); high serum 477

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N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas 7

levels of EGF and macrophage-derived chemokine to-478

gether with low levels of interleukins (IL) 10, 6 and 8479

were associated with bevacizumab response in mCRC480

patients [1]. Clinicians’ attention for predicting the re-481

sponse to bevacizumab has focused on the evaluation482

of soluble VEGFRs. However, Willett et al. did not483

completely validate sVEGFR1 together with plasma484

VEGF, PlGF, and IL-6 as biomarkers of response for485

bevacizumab when given in association with 5FU and486

radiotherapy in locally advanced rectal cancer patients,487

and further evaluations are needed [93]. Recently, the488

investigation of genetic variants in the VEGF path-489

way related to antiangiogenic drug response has been490

carried out by several research teams who have re-491

ported the association of VEGF genotype with be-492

vacizumab efficacy in irinotecan-based chemotherapy493

combined with this antibody in metastatic colorec-494

tal cancer patients; results showed VEGF-2578AA495

and VEGF-1154AA as predictive of the overall sur-496

vival [48]. Lambrechts et al. reported that the presence497

of the single nucleotide polymorphism rs9582036 in498

VEGFR1 correlates with the increased expression of499

the receptor and poor outcome of bevacizumab treat-500

ment, suggesting further evaluation to confirm the pre-501

dictive value of this novel biomarker [50].502

Finally, the search for potential noninvasive and503

quantitative biomarkers of antiangiogenic drug re-504

sponse also has focused on radiological methods, even505

if the validation of these measurements as biomark-506

ers with predictive value needs further exploration.507

Among them, changes in blood flow, blood volume,508

or permeability by dynamic magnetic resonance imag-509

ing and computed tomography have been demonstrated510

to occur after treatment with bevacizumab or anti-511

VEGFR TKIs in clinical studies, while magnetic reso-512

nance spectroscopy is still in evaluation for its ability513

to predict response to antiangiogenic agents [42].514

3. Gastric cancer515

The prognosis of patients with advanced gastric can-516

cer (AGC) is poor, with a 5-year survival rate of 10–517

15% and an overall survival of 11.9 months for patients518

who undergo palliative chemotherapy. This compares519

to four to five months for those receiving the best sup-520

portive care [32]. In the last decade, the development521

of targeted therapies has represented a new hope in the522

therapeutic management of AGC.523

3.1. HER2 pathway 524

Preclinical trials demonstrated the antitumor activ- 525

ity of trastuzumab in cell lines expressing the HER2 526

receptor [55,86]. The HER2 protein is a transmem- 527

brane receptor tyrosine kinase belonging to the fam- 528

ily of receptors for epidermal growth factor, which in- 529

cludes four receptor subtypes (HER1 or EGFR, HER2, 530

HER3, and HER4) [2]. All of these receptors share the 531

same functional structure; they are formed by an ex- 532

tracellular ligand-binding domain, a helical transmem- 533

brane segment, and an intracellular protein tyrosine ki- 534

nase domain. Phosphorylation of the receptor stimu- 535

lates an intracellular signalling cascade of events that 536

ultimately modulates the expression of key genes in- 537

volved in multiple cellular processes. PI3K and MAP 538

kinase (MAPK) are the two most important down- 539

stream pathways activated by HER members [70,80]. 540

HER2 does not have a specific ligand, even if its het- 541

erodimerization with other receptors of the HER fam- 542

ily is responsible for the activation of downstream 543

pathways involved in the growth and proliferation of 544

the tumor cell [38]. Many studies have examined the 545

rate of EGFR expression in gastric cancer, with overex- 546

pression being almost exclusively determined by gene 547

amplification [35]. At present, evidence regarding the 548

potential prognostic role of EGFR is limited and con- 549

troversial [29]. More is known about the biological role 550

and pattern of expression of HER2. To date, there are 551

conflicting opinions about the prognostic significance 552

of the receptor [28]. Its predictive role has been found 553

however, as the addition of trastuzumab to chemother- 554

apy with platinum and fluoropyrimidine has signifi- 555

cantly increased the overall survival of patients with 556

AGC. In the Trastuzumab for Gastric Cancer (ToGA) 557

trial, the addition of trastuzumab to chemotherapy sig- 558

nificantly improved overall survival compared with 559

chemotherapy alone in patients with HER2+ AGC, 560

achieving a median overall survival of 13.8 months in 561

the trastuzumab plus chemotherapy group [5]. 562

The simultaneous inhibition of multiple receptors 563

could be an attractive strategy because interactions be- 564

tween HER2 and EGFR provide a mechanism for sig- 565

nal augmentation. Lapatinib is a small molecule TKI 566

with dual activity able to block the intracellular do- 567

main of both EGFR and HER2 [71]. The phase II 568

trial of lapatinib monotherapy as first-line therapy in 569

47 patients with AGC demonstrated excellent toler- 570

ability and moderate activity (median time to treat- 571

ment failure was 1.9 months and overall survival was 572

4.8 months) [39]. TYTAN (Lapatinib [Tykerb] with 573

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8 N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas

paclitaxel [Taxol] in Asian ErbB2+ [HER2+] Gas-574

tric Cancer Study), is an ongoing, open-label, ran-575

domized phase III study of second-line therapy of576

paclitaxel with or without lapatinib in patients with577

HER2+ GC [75]. The ongoing LOGIC Trial, a phase578

III study in 410 patients, aims to evaluate if lapa-579

tinib, in combination with oxaliplatin and capecitabine,580

is able to improve the progression free survival of581

HER2+ AGC or esophagogastric junction tumors com-582

pared to chemotherapy alone.583

HER2 overexpression can be determined by IHC584

using a mAb or by the detection of HER2 gene am-585

plification through fluorescent in situ hybridization586

(FISH) [96]. Increased expression of HER2 has been587

detected in 13–23% of patients with GC [9]. However,588

no definitive biomarkers allow for the selection of pa-589

tients who will respond to trastuzumab. It is known that590

the levels of HER2 expression might predict the re-591

sponse to trastuzumab, as reported in the post hoc anal-592

ysis of overall survival in the “strongly HER2-positive”593

group in the ToGA trial [5].594

Despite the initial efficacy demonstrated by the im-595

proved response rate and progression-free and overall596

survival in the ToGA trial, some patients with HER2+597

disease demonstrated primary resistance, and the re-598

maining developed secondary resistance [5]. In cancer599

cells treated with kinase inhibitors such as gefitinib and600

erlotinib, the diminished activity of HER2 is compen-601

sated by HER3 expression, which results in tumor cell602

survival [77]. Recent studies have shown that membra-603

nous HER3 and HER4 proteins can move to the nu-604

cleus and that this event may play an important role605

in cancer progression [61,90]. Therefore, the develop-606

ment of new predictive markers that analyze dimers607

rather than isolated markers could introduce a new ap-608

proach to understanding these interactions and aid in609

understanding the importance of the HER family with610

regard to the biology and behavior of GC.611

3.2. VEGF pathway612

The role of VEGF has been studied in several can-613

cers, including GC. Some studies have confirmed the614

negative prognostic significance of the growth factor,615

whose presence correlates with a lack of response to616

chemotherapy, as well as with a more aggressive be-617

havior of the tumor. However, other authors do not con-618

firm the data [43,88]. Lieto et al. examined the expres-619

sion of both VEGF and EGFR by IHC and found a cor-620

relation with tumor progression, with the ability to in-621

filtrate the network vascular blood and lymphatic cir-622

culation, and especially with a reduced long-term sur- 623

vival [52]. These data suggest that it is necessary to 624

clarify both the molecular mechanisms and the behav- 625

ior of the individual tumor with the aim of improving 626

therapeutic opportunities. 627

Bevacizumab combined with cisplatin and irinote- 628

can reported response rates of 67%, a time to pro- 629

gression of 8.3 months, and a median overall sur- 630

vival of 12.3 months in 47 patients [79]. Bevacizumab- 631

related toxicities were gastric perforation (6%), my- 632

ocardial infarction (2%), and thromboembolic events 633

(25%). Enzinger et al. reported similar outcomes with 634

bevacizumab combined with docetaxel, cisplatin, and 635

irinotecan, with partial response and stable disease 636

rates of 63% and 30%, respectively; thromboembolic 637

events were seen in 9% of patients [23]. On the basis of 638

results from these phase II studies, a phase III random- 639

ized, double-blind, contrast study (AVAGAST) was 640

conducted internationally [63]. This study included 641

774 patients with previously untreated and locally 642

advanced or metastatic GC or esophagogastric junc- 643

tion cancer. Patients were treated with capecitabine 644

and cisplatin in combination with either bevacizumab 645

or placebo. The median rate of overall survival was 646

10.1 months for the placebo group and 12.1 months 647

for the bevacizumab group (HR = 0.87; P = 0.1002), 648

failing to meet the primary endpoint. Nevertheless, sig- 649

nificant improvement in progression free survival and 650

overall response rate was noted in the bevacizumab 651

group. 652

New therapeutic approaches have seen the use of 653

drugs capable of blocking the receptor for VEGF 654

or the pathway downstream of the receptor. Ramu- 655

cirumab [49], a humanized mAb directed against 656

VEGFR2, currently is being tested in several ongo- 657

ing clinical trials. These include a randomized phase 658

II trial in which the Ramucirumab is associated with 659

mFOLFOX6 chemotherapy regimen (oxaliplatin, 5- 660

FU, folic acid) compared to placebo in patients with 661

AGC; a randomized phase III trial of combination 662

of paclitaxel with or without Ramucirumab after fail- 663

ure of first-line therapy with platinum and fluoropy- 664

rimidine; and another phase III study comparing Ra- 665

mucirumab with best supportive care. Early studies 666

with TKIs such as sorafenib, sunitinib, and cediranib 667

have not seen encouraging results [56,76,85]. Addi- 668

tional studies with these TKI are needed in AGC. Other 669

VEGFR TKIs such as axitinib, vatalinib, semaxinib, 670

vandetanib, and pazopanib, as well as aflibercept (anti 671

VGFR-A and placenta growth factor), are under in- 672

vestigation in phase I and II trials in patients with ad- 673

vanced solid tumors, but none of these agents has yet 674

been evaluated in GC cancer. 675

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N. Silvestris et al. / Predictive factors to targeted treatment in gastrointestinal carcinomas 9

4. Conclusions676

This review describes the complete scenario of new677

molecular therapies in mCRC and GC, as well as pre-678

dictive markers that guide the choice of the most suit-679

able therapy for individual patients. In particular, the680

treatment of mCRC has undergone major develop-681

ments in recent years due to the use of target drugs682

that have minimized systemic toxicity. These new tar-683

geted therapies bring promise for improving our abil-684

ity to prevent, diagnose, and treat cancer. With the ad-685

dition of these new targeted drugs, the median survival686

for patients with metastatic CRC has now increased to687

almost two years [36]. In patients with GC, the success688

of the ToGA trial and, more recently, the failure of the689

antiangiogenic agent bevacizumab in a large phase III690

study in unselected patients [63] clearly show that the691

only hope of achieving better treatment modalities for692

this disease will come from the use of targeted agents693

in selected patient populations. Therefore, it is time694

to move from the identification of predictive mark-695

ers in small subgroups of responsive patients in phase696

III trials to molecularly driven phase I and II studies.697

In these small trials, understanding the key signaling698

pathways that are relevant in preclinical models could699

provide the rationale for the focused development of700

new agents.701

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