Molecular prognostic factors in patients with pancreatic cancer

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10.1517/14728222.11.12.1553 © 2007 Informa UK Ltd ISSN 1472-8222 1553

1. Introduction

2. Materials and methods

3. Expert opinion

Oncologic, Endocrine & Metabolic

Molecular prognostic factors in patients with pancreatic cancer Giuseppe Tonini † , Francesco Pantano , Bruno Vincenzi , Armando Gabbrielli , Roberto Coppola & Daniele Santini † University Campus Bio-Medico of Rome , Medical Oncology , Via Emilio Longoni, 83, 00155, Rome , Italy

Pancreatic cancer is the fourth most common cause of cancer death in Western society and is a leading cause of cancer death worldwide. Its incidence and mortality rates are almost identical. Surgery is the only treatment theorically curative, but < 20% of all patients admitted with ductal adenocarcinoma of the pancreas undergo resection and at best, 25% of those survive for 5 years. The identification of prognostic factors that are able to stratify patient populations recognizing those that could able to benefit by a radical surgical treatment and/or a chemotherapeutic treatment. This paper is a not only a detailed review of existing studies evaluating pancreatic cancer biomarkers, but also a critical evaluation of the real clinical use of these kinds of prognostic factors with the purpose to help discriminate between a variety of factors which, so far, can be con-sidered really useful in everyday clinical practice. Although no single marker has been shown to be perfect in predicting patient outcome, a profile based on the best of these markers may prove useful in directing patient therapy. The markers with the strongest evidence as independent predictors of patient outcome include, p16, MMP7 and vascular endothelial growth factor.

Keywords: genes , outcome , pancreatic cancer , prognosis , protein , survival

Expert Opin. Ther. Targets (2007) 11(12):1553-1569

1. Introduction

Pancreatic cancer is the fourth most common cause of cancer death in Western society and is a leading cause of cancer death worldwide. Its incidence and mortality rates are almost identical. The 5-year survival rate is ∼ 5%, and the median survival time is 3 – 6 months. Surgery is the only treatment theorically curative but < 20% of all patients admitted with ductal adenocarcinoma of the pancreas undergo resection, and at best 25% of those survive for 5 years [1-3] . Furthermore, the majority of patients who undergo resection die because of early liver metastasis. Thus, pancreatic ductal adenocarcinoma is one of the most malignant and aggressive cancers and predicting prognosis for patients with pan-creatic cancers may identify those who could benefit from aggressive intervention including surgery and/or chemotherapy. Beside the classical clinical–pathologic factors it was necessary to look for new type of biologic prognostic factors. The molecular biology of pancreatic cancer and its progression is characterized by aberrant activity of several regulatory pathways, both within the pancreas cells and in the surrounding tissue. Variations at the DNA, RNA and/or protein levels of molecules involved in these pathways are all potential candidate markers of prognosis. Finally, the study of molecular markers in pancreatic cancer can be important not only because of potential relationships with outcome but also because the association of a molecule with adverse outcome might suggest a key

Molecular prognostic factors in patients with pancreatic cancer

1554 Expert Opin. Ther. Targets (2007) 11(12)

role for a given molecule in the disease, providing putative targets for new molecular-based therapies.

2. Materials and methods

In order to review the literature for prognostic biomarkers in pancreatic cancer, the authors did a search on PubMed, a service of the National Library of Medicine, which includes over 14 million citations for biomedical articles dating back to the 1950s [201] . The authors used the key phrases ‘pan creatic cancer prognosis protein expression’ and ‘pancreatic cancer prognosis gene expression’. In the process of extracting these papers and reviewing them, the authors screened the references to select other papers that appeared to be appropriate for this review. The authors used the following criteria to select papers for more detailed reviews: (i) the marker was grouped in oncogenes and tumor suppressor genes, apoptosis genes, growth factors and angiogenesis factors; (ii) the study included stage I and II patients; (iii) an attempt was made to quantify the biomarker; and (iv) an attempt was made to control for known clinical factors that are associated with outcome. The main method used to evaluate the effect of a marker on clinical outcome is the analysis of survival curves using Kaplan–Meier analyses. Only studies that used survival curve methods to evaluate the impact of the marker on patient outcome were selected.

2.1 Oncogenes and tumor suppressor genes The cell cycle is normally regulated by proto-oncogenes and onco-suppressor genes, with opposed functions on cellular growth: stimulation and inhibition, respectively. Oncogenes are defined as dominant genes that can induce or maintain cellular transformation [4] .

They are often related to normal cellular genes, called proto-oncogenes, which have critical functions. Proto-oncogenes are usually strictly regulated by other genes that either promote or inhibit their transcription. Alterations that strengthen proto-oncogene activities promote unrestrained tumoral proliferation. Proto-oncogenes can activate cancer transformation through two main ways: genetic mutation, inducing an altered protein with a constantly activated function, or overexpression of a structurally normal gene after genic amplification, rearrange-ment or loss of transcriptional control [5] . Tumor suppressor genes, on the othe hand, encode for proteins whose normal function is to inhibit cell transformation and whose inacti-vation is advantageous for tumor cell growth and survival [6] . A variety of mechanisms result in the inactivation of tumor suppressor genes, including intragenic mutations, chromosomal deletions and loss of expression by methylation-mediated transcriptional silencing or increased proteolysis, genetic mutations inactivating onco-suppressor genes free the cells from proliferation restrictions and so induce an uncontrolled cellular growth, typical of the neoplasms [7] . Table 1 summarizes the prognostic role of several genes involved in cell cycle regulation.

2.1.1 p53 The p53 gene encodes a 53 kDa nuclear phosphoprotein. p53 inhibits cell growth through activation of cell cycle arrest and apoptosis [8,9] . The p53 gene is mutated in > 50% of human cancers and pancreatic cell lines showed this mutations at frequencies of 95%. The transcription factor p53 regulates an essential growth checkpoint that both protects against genomic rearrangement or the accumulation of mutations, and suppresses cellular transformation caused by oncogene activation or the loss of tumor suppressor pathways. p53 is stabilized and activated by extracellular stress including γ -irradiation and intracellular stress, such as deregulation of cellular oncogenes. Once activated, p53 can induce cell cycle arrest or apoptosis. Loss of p53 is associated with aneuploidy, an outstanding feature of pancreatic cancer, indicating that p53 function maintains genomic stability [10] .

Several studies analyzed p53 in pancreatic cancer as prognostic markers. The available data, so far, show that the evidences relating p53 expression to survival are conflicting. For the most part infact, studies have failed to demonstrate a convincing correlation of p53 mutation with decreased survival [11-28] .

2.1.2 p16 and Smad4 INK4a/ARF is an onco-suppressor gene that plays a key role in pancreatic cancer carcinogenesis. INK4a/ARF is frequently inactivated in many neoplasia and is also inactivated in ∼ 90% of pancreatic adenocarcinoma. Nearly half of these inactivations were by intragenic mutation of p16 and the remainder were by homozygous deletion of the gene [29] . Homozygous deletion of p16 INK4A /p14 ARF locus is a characteristic genetic alteration observed in 80 – 95% of human pancreatic cancer and usually occurs in later stages of the tumor progression model. This locus on chromosome 9q21 encodes the two related tumor suppressor genes INK4a and ARF , whos coding sequence partially overlap. INK4a and ARF are generated by the use of a different first exon and an alternative reading frame in exon 2. Whereas INK4a regulates cell cycle progression as an inhibitor of the cyclin D/CDK 4/6 kinase complex, ARF directly interacts with murine double minute (Mdm) 2/HDM 2 to block the interaction with p53 by localizing Mdm 2/HDM 2 to the nucleolus and by directly inhibiting E3 ubiquitin ligase activity of the Mdm 2/HDM 2, thereby, contributing to p53 activation [30] . Several studies confirmed the importance of p16 expression as prognostic factor in pancreas cancer [31-35] .

Signaling by transforming growth factor (TGF)- β ligands requires two transmembrane serine-threonine kinase receptors (type II and type I). The ligand brings the two receptors together in a complex and the constitutively active type II receptor kinase phosphorylates and activates the type I receptor kinase, which in turn activates downstream signaling pathways. Several signaling pathways originate

Tonini, Pantano, Vincenzi, Gabbrielli, Coppola & Santini

Expert Opin. Ther. Targets (2007) 11(12) 1555

Table 1 . Oncogenes and tumor suppressor genes.

Molecular prognostic factor

n Method Survival p Value Ref.

p53 protein 133 IHC High p53 expression correlated with decreased survival

p = 0.05 Campani et al. [19]

p53 gene 37 Direct sequencing High p53 expression correlated with decreased survival

p = 0.02 Nakamori et al. [27]

p53 protein and gene 69 IHC and western blotting

High p53 expression correlated with decreased survival

p < 0.05 Yokoyama et al. [91]

p16 protein and gene 60 IHC and PCR Lower survival in patients with p16 mutation or hypermethylation

p < 0.05 Ohtsubo et al. [21]

p16 protein and gene 62 IHC and PCR p16 protein mutation associated with decreased survival

p < 0.05 Gerdes et al. [31]

p16 protein 20 IHC Lower survival in cases with no p16 expression

p < 0.05 Hu et al. [21]

DPC4 protein 129 IHC Loss of DPC4 expression associated with longer survival

p = 0.0257 Biankin et al. [32]

SMAD4 protein and gene 249 IHC and gene sequencing

SMAD4 preservation resulted in longer survival times

p = 0.03 Tascilar et al. [40]

p27 protein 143 IHC p27 expression resulted in longer survival times

p = 0.03 Juuti et al. [52]

p27 protein 46 IHC Loss of p27 expression associated with poorer survival

p = 0.024 Lu et al. [53]

K-Ras gene mutation 101 Dot blot hybridisation K-ras mutation associated with lower survival

p < 0.05 Finkelstein et al. [50]

Cyclin D1 82 IHC Lower survival in cyclin D1 positive patients

p < 0.05 Gansauge et al. [57]

Cyclin D1 mRNA 32 Reverse PCR Lower survival in cyclin D1 positive patients

p < 0.007 Kornmann et al. [59]

Cyclin D1 protein and gene 67 IHC and PCR Lower survival in cyclin D1 positive patients

p < 0.01 Gansauge et al. [56]

IHC: Immunohistochemical method; PCR: Polymerase chain reaction; SSCP: Single-strand conformation polymorphism.

from the type I receptor, the most prominent one being the Smad pathway. Characterization of Smad functions has segregated them into three groups: receptor-regulated Smads (Smads 1,2,3,5 and 8), common partner Smad (Smad4) and inhibitory Smads (Smads 6 and 7). Normally, ligand-induced TGF- β receptor activation results in the formation of heterodimeric complexes of Smad3 with Smad4, leading to the translocation of these complexes into the nucleus, where Smad4 activates transcription of cell cycle inhibitory factors p21 WAF1/CIP1 [25] and p15 INK4B [36] . Homozygous deletion or mutation of deleted in pancreatic cancer, locus 4 ( DPC4 ), the gene encoding Smad4 located on chromosome 18q, has been reported in 55% of pancreatic ductal adenocarcinomas. Loss of Smad4 may lead to upregulation of the Rb pathway, consequent progression from the G1 to the S phase of the cell cycle, and hence increased cellular proliferation.

Smad4 , in particular, (also termed DPC4 ) has the charac-teristics of a classic tumor suppressor gene, being mutated or deleted in ∼ 50% of pancreatic carcinomas and 15% of colorectal cancers. Since the discovery as a tumor suppressor, much interest has focused on the role of Smad4/DPC4 as a mediator of TGF- β anti-proliferative signals, which were originally thought to account for most, if not all, of its antioncogenic activity. However, several recent studies have confirmed that suppression of tumor formation and metastasis through this protein is more complex, involving inhibition of tumor angiogenesis and changes in the expression and activity of genes implicated in the control of cell adhesion and invasion [36-38] .

The three studies that analyzed DPC4/Smad4 in pancreas cancer as prognostic markers showed conflicting results. Infact, Biankin et al. its suggested his clinical usefulness in predicting poorer prognosis, whereas Tascilar et al. obtained



opposing results correlating tumor DPC4/Smad4 expression with a better outcome [32,39,40] . The possible explanation is that immunohistochemical analysis may not differentiate between wild type and mutated SMAD4 proteins. So, further studies of the influence of DPC4 /Smad4 expression in pancreatic adenocarcinoma are essential to its further evaluation as a prognostic and therapeutic marker.

2.1.3 K-ras The K-ras gene is the locus for the c-K-ras proto-oncogene, lying on chromosome 12p12, and is ∼ 45,000 base pairs in lenth. It encodes for a 2.0 kb transcript, which is highly conserved across species, and is translated into the p21-ras protein [41] . These proteins are located in the plasma membrane and could transduce growth and differentiation signals from activated receptors to protein kinases within the cell. p21-ras proteins are in a weak GTP-bound (active state), thereby, altering transduction into the cell [42] . K - ras mutations are found in 80 – 90% of pancreatic cancer patients. The majority of mutations have been found at K-ras codons 12 and 13. These mutations consist of single base pair substitutions that led to the change of one amino acid in the protein, resulting in the K-ras protein product (p21-ras) permanent activation, which may promote cell proliferation [43-45] . So far, despite the several studies [46-52] that investigated its role as prognostic marker in pancreatic cancer, there is no evidence of its clinical use in offering prognostic information.

2.1.4 p27Kip1 and cyclin D1 p27Kip1 is a cyclin-dependent kinase (CDK) inhibitor that regulates cell cycle progression from the G1 phase to the S phase by binding to the cyclin E/cdk2 complex, thereby inhibiting this kinase. In pancreatic cancer few studies demonstrated a correlation between its expression and a better prognosis [53,54] .

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through the G 1 -S phase of the cell cycle.

Overexpression of cyclin D1 is known to correlate with early onset of cancer and risk of tumor progression and metastasis [55] . So far, data on the impact of cyclin D1 on prognosis for pancreatic cancer are limited, but there is some evidence that cyclin D1 expression may influence survival [56-61] . In one study in particular, Gansauge et al. demonstrated the value of cyclin D2 in detecting patients with poorer prognosis.

2.1.5 Apoptosis Apoptosis was originally defined as a physiologic or programmed form of cell death that affects scattered cells in a tissue and has a characteristic and stereotypical morphology, including cell shrinkage, retention of organelles and nuclear chromatin condensation, which occur in response to a

variety of stress-related stimuli [62] . Although apoptosis can be triggered by several different stimuli, apoptotic signaling within the cell is transduced mainly via two defined molecular pathways: the death receptor pathway (also called the extrinsic pathway) and the mitochondrial pathway (also called the intrinsic pathway). The end point of both the intrinsic and extrinsic pathways is activation of a wide variety of intracellular proteases (especially a group of proteolytic enzymes called caspases) and endonucleases that ultimately degrade the cellular constituents [63,64] . Table 2 shows the relationship between genes and protein involved in apoptosis and pancreatic cancer prognosis.

2.1.6 B-cell lymphoma-2 gene family and BNIP3 The B-cell lymphoma-2 (Bcl-2) gene family consists of several members, including Bcl-2, Bcl-xL, mcl-1 and bax. They function as blockers or promoters of apoptosis. Bcl-2, which functions as an antiapoptotic factor, is located on chromosome 18q21, which consists of three exons separated by large introns. Its antiapoptotic functions extend cell survival in both normal and tumor cells by inhibiting different cell death mechanisms, such as those induced by ionising radiation and anticancer drugs. In contrast with the apoptosis inhibitor Bcl-2, the bax gene is a promoter of apoptosis. The protein encoded by the bax gene shares 21% amino acid sequence homology with Bcl-2, mainly within two highly conserved regions called the Bcl-2 homology 1 and 2. Bcl-2 and Bax form homodimers and/or heterodimers. The ratio between Bcl-2 and Bax determines cell survival or death following an apoptotic signal. An excess of Bcl-2 homodimers inhibits apoptosis, whereas an excess of Bax homodimers promotes cell death [65] . Several studies [66-77] investigated the role of Bcl-2 as prognostic factors in pancreatic cancer finding, par-adoxically, that Bcl-2 expression is in most cases correlated with a better survival. These results underline how poor our comprehension, so far, of the role of members of the Bcl-2 family. Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a mitochondrial pro-apoptotic protein that has a single Bcl-2 homology 3 (BH3) domain and a C-terminal transmembrane (TM) domain. Although it belongs to the Bcl-2 family and can heterodimerize with Bcl-2, its pro-apoptotic activity is distinct from those of other members of the Bcl-2 family. For example, cell death mediated by BNIP3 is independent of caspases and shows several characteristics of necrosis. BNIP3 plays an important role in hypoxia-induced death of normal and malignant cells. Its expression is markedly increased in the hypoxic regions of some solid tumors and appears to be regulated by hypoxia-inducible factor (HIF) [78] . Erkan et al. demonstrated the use of BNIP3 as a molecular prognostic factor correlating with a worsened prognosis in pancreatic cancer [79] .

2.1.7 Survivin Survivin has recently been identified as a novel inhibitor of apoptosis (IAP). It blocks common downstream elements of



both the mitochondrial and the death receptor pathway, by directly inhibiting terminal effector caspase-3, -7 and -9 activity [80,81] . Thus, it inhibits apoptosis pathway differently from Bcl-2, which blocks mitochondrial cytochrome c release into the cytosol, resulting in the inhibition of mitochondrial apoptotic pathway. The prognostic role in pancreas cancer is been evaluated by Tonini et al. , finding survivin an independent prognostic factor for survival [82] .

2.1.8 Cell–cell and cell–matrix interactions Multiple and diverse cell adhesion molecules take part in intercellular and cell–extracellular matrix (ECM) interactions of cancer. A growing body of evidence indicates that alter-ations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of cancer. Loss of intercellular adhesion and the desquamation of cells from the underlying lamina propria allows malignant cells to escape from their site of origin, degrade the ECM, acquire a more motile and invasion phenotype, and finally, invade and metastasize. A crucial role in this process is attributed to interaction with the ECM. Requisite for neoplastic cell and capillary or inflammatory cell invasion during carcinogenesis processes is the remodeling events that occur within the stroma or ECM [83] . The ECM is a complex meshwork of collagens, fibrillar glycoproteins and proteoglycans that determines tissue architecture and conditions many biologic activities. The ECM is involved in both normal and pathologic processes. Components of the ECM provide a large variety of specific signals that directly influence cell proliferation, migration, morphology, differentiation and biosynthetic activities [84] . In addition, the ECM plays an essential role in cell survival, as loss of adhesive contact results in apoptosis termed anoikis [85] . Therefore, alterations of the ECM in tumors might lead to abnormal host and cancer cell functions and even to cancer progression. Quantitative changes in matrix components may be related to an imbalance between their synthesis and

degradation. Tumor cells may directly alter the adjacent matrix by changing the production of matrix proteins or proteolytic enzymes. Alternatively, the desmoplastic response may depend on specific interactions between tumor cells and host fibroblastic cells. Table 3 shows a summary about the prognostic role of several molecular factors involved in cell–cell and cell–matrix interaction

2.1.9 Matrix metalloproteases, ADAM9 and APN/CD13 The matrix metalloproteases (MMPs) are a family of zinc-containing proteolytic enzymes that break down proteins in the ECM in both physiologic and pathologic conditions. The four main subgroups, organized in part according to substrate specificity in vitro and in part by association with the cell membrane, are the collagenases (MMPs 1,8 and 13), the gelatinases (MMPs 2 and 9), stromelysins (MMPs 3,10,11 and 18) and the membrane-bound MMPs (MMPs 14 – 17) [86] . Loss of the tight control of MMP activity in cancer is thought to contribute to excessive destruction of the ECM, neo-vascularization, tumor spread and metastases.

Most studies have found that increased MMP expression in the pancreas correlates with poorer prognosis. In parti-cular, MMP-7, or matrilysin, has consistently been reported to have a negative impact on survival [87-94] .

The a disintegrin and metalloprotease (ADAM) family of proteins is a group of metalloproteases that belong to the zinc protease superfamily. ADAMs are transmembrane proteins that contain both disintegrin and metalloprotease domains and, therefore, have both cell adhesive and protease activities. Thus, ADAMs have the potential to be key modulators of cell–matrix interactions through the activities of their constituent domains. Grutzmann et al. found cytoplasmic ADAM9 overexpression is associated with poor differentiation and shortened survival [95] .

APN/CD13 is a surface glycoprotein encoded by a gene located on the long arm of chromosome 15 at bands

Table 2 . Apoptosis.



Bcl-2 protein 97 IHC Lower survival in Bcl-2 positive patients p = 0.05 Sun et al. [65]

Bcl-2 protein 67 IHC Higher survival in Bcl-2 positive patients p = 0.0379 Magistrelli et al. [76]

Bax 67 IHC Higher survival in Bax positive patients p = 0.0311 Magistrelli et al. [76]

Bcl-2 protein 59 IHC Higher survival in Bcl-2 positive patients p = 0.002 Dong et al. [75]

Bax 59 IHC Higher survival in Baxpositive patients p < 0.05 Dong et al. [75]

Bax protein and mRNA 60 IHC and northern blot

Strong predictor of survival p < 0.039 Friess et al. [70]

Survivin 47 IHC Lower survival in survivin positive patients p = 0.02 Kami et al. [80]

Survivin 67 IHC Lower survival in survivin positive patients p = 0.0009 Tonini et al. [81]

IHC: Immunohistochemical method.



q25-26. This 967 amino acid integral membrane protein has a large extracellular C-terminal domain containing a pentapeptide motif, which is characteristic of members of the zinc-binding metalloprotease superfamily APN/CD13 has been implicated in the tumor invasion and can be considered as an independent prognostic factors in pancreatic cancer [96] .

2.1.10 Dysadherin, syndecan-1 and thrombospondin-1 Dysadherin, an antiadhesion molecule (a novel cancer-associated cell membrane glycoprotein) was originally characterized and denominated by Hirohashi et al. It encodes 178 amino acids, including a putative signal sequence, an O -glycosylated extracellular domain, a single transmembrane domain and a short cytoplasmic tail. Overexpression of dysadherin downregulated the protein expression and function of E-cadherin in a post trans-criptional manner in cells and promoted metastasis to the liver in an animal model.

Dysadherin is found to be a positive marker of poor prognosis in pancreatic cancer [97] .

The syndecans are a four member family of trans-membrane cell surface proteoglycans (PGs) that bear heparan sulfate glycosaminoglycan (GAG) chains. The syndecans are expressed on virtually all cell types throughout development and adulthood, and their expression can be altered under certain pathophysiologic conditions, including the processes of tumor onset, progression and metastasis.

Syndecan-1, in particular, is highly expressed at the basolateral surface of epithelial cells where it is thought to act as a transmembrane receptor that participates in cell–cell and cell–matrix interactions, cell proliferation and cell migration with the actin cytoskeleton, and to modulate growth factor signalling [98] .

Juuti et al. suggested that stromal syndecan-1 expression is an independent prognostic marker in pancreatic cancer, whereas epithelial syndecan-1 expression predicts better prognosis in resectable disease [99] .

Thrombospondins (TSPs) are a family of extracellular proteins recognized as an endogenous constituent of the ECM in many human tissues. They are adhesive proteins that promote cell–cell and cell–matrix interactions. TSP-1, the first family member to be identified, is a 450 kDa adhesive glycoprotein that was initially discovered in platelets and, subsequently, in a variety of cell types, including endothelial cells, fibroblasts, smooth muscle cells, keratinocytes, macrophages and neutrophils. TSP has been implicated in the regulation of cell growth and proliferation, cell motility cytoskeletal organization, inflammation and wound healing, and the development and differentiation of cell types. This could increase the likelihood of the tumor gaining and the capacity to induce blood vessel formation, thereby making the ‘angiogenic switch’ and progressing towards greater malignancy. TSP was found to be a positive marker of poorer outcome in one study conducted by Tobita et al. [100] .

Table 3 . Extracellular matrix and tumor–stroma interaction.

Molecular prognostic factor n Method Survival p Value Ref.

MMP-7 39 IHC Lower survival time in MMP-7 positive patients

– Nakamura et al. [87]


p = 0.022 Yammamoto et al. [88]


Ito et al. [91]

ADAM9 59 IHC Lower survival time in ADAM9 positive patients

p < 0.05 Grutzmann et al. [95]

TSP1 77 IHC Higher survival time in TSP1 positive patients

– Tobita K et al. [101]

APN/CD13 50 IHC Lower survival time in positive APN/CD13 patients

p = 0.009 Ikeda et al. [95]

Dysadherin 125 IHC Lower survival time in positive Dysadherin patients

– Shimamura et al. [96]

Syndecan-1 144 IHC Higher survival time in stromal Syndecan-1 negative patients

p = 0.002 Juuti et al. [99]

RECK 50 IHC Higher survival time in RECK positive patients

p = 0.0463 Masui et al. [102]

ADAM: A disintegrin and metalloprotease; IHC: Immunohistochemical method; MMP: Matrix metalloprotease; RECK: Reversion-inducing-cysteine-rich protein with kazal motifs; TSP1: Thrombospondin 1.



2.1.11 Mucin 4, reversion-inducing-cysteine-rich protein with kazal motifs, pigment epithelium-derived factor and tissue factor Mucin 4 (MUC 4) belongs to MUC family, a heterogeneous group of glycoproteins with various roles both in homeostasis and carcinogenesis, is a membrane mucin that was proven to be an intramembrane ligand for receptor tyrosine kinase ErbB2 and related to regulation of p27, which is a CDK inhibitor involved in the control of G1 and S phases of the cell cycle. MUC 4 was evaluated as a prognostic factor and its expression was correlated to a poorer prognosis [101] .

The reversion-inducing-cysteine-rich protein with kazal motifs ( RECK ) gene encodes a membrane-anchored glycoprotein with multiple epidermal growth factor-like repeats and serine protease inhibitor-like domains and is associated with the cell membrane through a C-terminal glycosylphosphatidylinositol modification. The RECK gene is widely expressed in various human tissues and non-neoplastic cell lines. Restored expression of RECK in malignant cells results in the suppression of their invasive and metastatic activities with concomitant suppression of proteolytic activity of MMP-9, MMP-2 and MT1-MMP, suggesting a role for RECK in the regulation of MMPs and tumor invasiveness. In pancreatic cancer, RECK expression is correlated with better prognosis [102] .

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein expressed in fetal and adult liver, adult testis, ovaries, placenta, brain and pancreas. PEDF resembles, in sequence and structure, members of the serine protease inhibitor (serpin) family, but lacks protease inhibitor activity itself. PEDF also inhibits endothelial cell migration and proliferation, and has also been shown to inhibit choroidal and retinal neovascularization. In an endothelial cell migration assay, PEDF was more potent than any of the other known inhibitors of angiogenesis, being more than twice as potent as angiostatin and more than seven times as potent as endostatin. In pancreatic cancer PEDF expression correlates with better survival [103] .

Tissue factor (TF) is a transmembrane glycoprotein that functions as a cellular receptor for coagulation factor VII (FVII) and modulates it to produce the activated form, FVIIa. The TF/FVIIa complex is regarded as the initiator of the extrinsic blood coagulation cascade, which ultimately leads to the generation of thrombin. In addition to its role as a hemostatic initiator, the binding of FVIIa with TF has been suggested to be involved in intracellular signaling mechanisms, such as the MAPK pathway and the Src family member/PI3K/Rac-dependent signaling pathway, at least in some cell types. Transfection of TF promoted the metastasis of melanoma in a mouse model and enhanced primary tumor growth in a pancreatic adenocarcinoma cell line. Nitori et al. demonstrated its clinical usefulness as prognostic factor in pancreas cancer patients [104] .

2.1.12 Galectin-3 Galectin-3 is a member of a family of β -galactoside-binding animal lectins. Although the precise function of galectin-3 remains unknown, there is some evidence that galectin-3 plays a role in cell–cell adhesion, cell–ECM interactions, cellular proliferation, cellular differentiation and apoptosis. High galectin-3 expression in pancreatic cancer is associated with a better survival [105] .

2.2 Growth factors It is well known that cell proliferation is the result of an interplay between cell signaling and transcriptional regulation [106,107] . According to this model, mitogenic growth factors and antiproliferative peptides traduce signals emanating from cell surface transmembrane receptors that recruit a distinct set of intracellular ‘signaling proteins’. These proteins then act on nuclear transcription factors to ultimately modify the expression of genes responsible for cell proliferation. Therefore, it appears that molecular alterations play a key role in the pathogenesis of cancer [108] . Growth factors and their receptors have important functions in the process of tumor progression [109] . These changes stimulate tumor growth and enhance the metastatic behavior of pancreatic cancer cells and, thereby, may contribute to shorter postoperative survival, following tumor resection.

2.2.1 Transforming growth factor- β TGF- β belongs to a family of homologous polypeptides that includes three major isoforms (TGF- β 1, TGF- β 2, TGF- β 3) [110] . It has been reported that TGF- β influence different cell functions, including growth, proliferation and differentiation. It can influence cancer growth in various ways, for example, by stimulating angiogenesis, suppressing cancer-directed immune functions, increasing the expression of adhesion molecules and ECM components. Human pancreatic cancer cells may exhibit loss of responsiveness to TGF- β -mediated growth inhibition as a consequence of altered TGF- β expression, as well as a result of postreceptor alterations. It has also been demonstrated that TGF- β induced cell cycle arrest can be partially attributed to the regulatory effects of TGF- β on both the expression and activity of CDK inhibitors, such as p21 and p27 [111] . There is conflicting data in the literature about the correlation of TGF- β 1 expression with survival in pancreatic cancer [112] . In most series, the presence of TGF- β 1 is associated with shorter postoperative survival, although in some other studies TGF- β 1 expression showed a significant correlation with longer survival time. On the other hand, there are reports indicating that TGF- β expression has no association with survival [113,114] .

2.2.2 Epidermal growth factor receptor The epidermal growth factor (EGF) receptor is a 170 kDa glycoprotein on the cell surface of a variety of cell types and is characterized by its ligand-dependent tyrosin kinase



activity. The EGF receptor, also known as human EGF receptor 1 (HER 1), is closely related to several other receptors, including HER 2 (c-ErbB2), HER 3 (c-ErbB3) and HER 4 (c-ErbB4) [115,116] . These four growth factor receptors are composed of an extracellular ligand-binding domain with cysteine-rich regions, a transmembrane domain and an intracellular domain with tyrosine kinase activity. After ligand binding to the extracellular domain, the EGF receptors are activated by homo- and/or heterodimerization with auto- and transphosphorylation on tyrosine residues at the intracellular domain. The phosphorylated receptors then transmit signals through a variety of intracellular substrates, depending partly on the cell type, the ligand and the participating EGF receptors [117] .

Prognostic significance of EGF receptors expression has been widely studied but most of the evidence, suggests that overexpression EGF receptor is not significantly associated with survival in pancreatic cancer patients [118-132] .

2.2.3 Vascular endothelial growth factor The main functions of vascular endothelial growth factor (VEGF) are to promote survival, induce proliferation and enhance migration and invasion of endothelial cells, which contribute to angiogenesis. It regulates these functions

by interacting with its tyrosine kinase receptors and trans-mitting signals to various downstream proteins. Elevated VEGF production by tumors is associated with increased tumor vascularity, metastasis, chemoresistance and poorer prognosis compared with VEGF-negative tumors. Circulating VEGF is elevated in breast, lung and gastrointestinal cancers [133] . VEGF expression can act as a prognostic factor where high levels of VEGF in the circulation or tumor tissue is negatively correlated to survival [134-137] .

2.3 Other molecular prognostic factors 2.3.1 CDX2 Caudal type homeobox 2 (CDX2) belongs to the group of homeobox genes and is characterized by structural and functional similarities to the homeobox gene caudal expressed in Drosophila melanogaster . All homeobox genes code for a so-called homeodomain, a typical amino acid sequence ( ∼ 60 amino acids), which binds DNA and controls the transcription of several genes. CDX2 represents a transcription factor for various genes and is an important regulator of cell differentiation [138-140] .

Matsumoto et al. evaluated the role of expression of Cdx2 in pancreatic cancer finding that Cdx2-positive patients have a significantly better outcome than their counterparts [141] .

Table 4 . Growth factors.



TGF- β 1 62 IHC TGF- β positive tumors found to have better prognosis

p < 0.05 Hashimoto et al. [111]

TGF- β R-2 receptor 42 IHC Higher levels of TGF- β R-2 resulted in lower survival times

p < 0.05 Wagner et al. [112]

TGF- β 1 42 IHC TGF- β 1 expression associated with better survival

p < 0.05 Coppola et al. [113]

TGF- β 1 60 IHC and northern blot

Expression of TGF- β isoforms associated with decreased post-operative survival

p < 0.05 Friess et al. [114]

EGFR 76 IHC Cytoplasmic EGFR associated with lower survival

p < 0.001 Ueda et al. [120]

Serum and tissue c-ErbB2 protein

100 IHC Serum c-ErbB2 levels correlated with lower survival

p < 0.01 Okada et al. [130]

HER-2 21 IHC Ovexpression linked to lower survival p = 0.02 Lei et al. [131]

VEGFR-II 24 IHC and northern blot

Poorer survival in VEGFR-II positive patients p < 0.05 Buchler et al. [134]

VEGF 142 IHC Lower survival in High or moderate VEGF expression patients

p < 0.05 Seo et al. [136]

VEGF 40 IHC and PCR for gene expression

Positive VEGF expression associated with lower survival

p = 0.0443 Ikeda et al. [137]

VEGF-C and -D 58 IHC Lower survival in high expression VEGF c-d patients

p = 0.017 Kurahara et al. [133]

EGFR: Epidermal growth factor receptor; FISH: Fluorescence in-situ hybridisation; IHC: Immunohistochemical method; PCR: Polymerase chain reaction; RT PCR: Real time polymerase chain reaction; TGF- β : Transforming growth factor- β ; VEGF: Vascular endothelial growth factor.



2.3.2 MAGEA3 The MAGE gene family is a kind of tumor-specific antigen gene that is expressed in malignant tumors of various origins, but not in normal tissues, except in the testis and placenta. MAGE genes code for peptide segments on HLA recognized by cytotoxic T lymphocytes. The MAGEA subfamily, which is comprised of 12 genes, has had one or more of its antigens detected in various gastrointestinal malignancies, including pancreatic cancer. MAGEA3 in particular was identified by Kim et al. as a significant prognostic factor for poor survival in pancreatic cancer [142] .

2.3.3 Receptor binding cancer antigen expressed on SiSo cells 1 Receptor binding cancer antigen expressed on SiSo cells (RCAS1) is a novel tumor-associated antigen, highly expressed in many kinds of tumors, such as those of the lung, esophagus, stomach and liver and pancreas. RCAS1 is present as a type II transmembrane protein on the cell surface and also as a secreted soluble protein. The antigen acts as a ligand for a putative receptor present on the cells of the immune system, such as natural killer cells and activated T cells. Binding of the RCAS1 ligand induced apoptosis of these receptor expressing cells, so that RCAS1 probably plays an important role in the evasion of host immune sur-veillance by tumour [143] . Hiraoka studied the prognostic value of RCAS1 expression in pancreatic cancer finding that RCAS1 might be a significant tumor marker for pancreatic adenocarcinoma and an unfavorable predictor for prognosis of patients who have undergone surgical resection [144] .

2.3.4 Skp2 Skp2 is one of the founding members of the F-box protein family that serves as the ubiquitylation substrate recruiting subunit of the SCF-Skp2-Roc1 complex. The best-established substrate of Skp2 is the CKI p27, although Skp2 can also promote ubiquitylation and

degradation of other proteins including the p27 family members p21 and p57. Since p27 is a negative regulator of cell proliferation, the p27 ubiquitylation activity of Skp2 suggested that it is a proliferation-stimulating protein. Indeed, Skp2 exhibits proliferation-stimulating activity in various experimental assays and is found overexpressed in various human cancers [145] .

Einama et al. found that high Skp2 expression might be clinically useful for prognostication of poorer outcome in patient with pancreatic carcinoma [146] .

2.3.5 Hypoxia-inducible factor-1 HIF-1, composed of HIF- α and HIF- β subunits, is a heterodimeric transcriptional activator. In response to hypoxia, stimulation of growth factors, and activation of oncogenes as well as carcinogens, HIF-1 α is overexpressed and/or activated and targets those genes which are required for angiogenesis, metabolic adaptation to low oxygen and promotes survival. HIF-1 is overexpressed in many different types of tumors and has been correlated with unfavorable prognosis in pancreas cancer [147] .

2.3.6 PGP9.5 PGP9.5 is a ubiquitin hydrolase and widely expressed in neuronal tissues at all stages of neuronal differentiation. Ubiquitin carboxyl-terminal hydrolases are enzymes involved in the translational processing of pro-ubiquitin gene products, as well as in the release of ubiquitin from tagged proteins. These enzymes play an important role in the cellular proteolytic pathway that regulates many cellular processes, including the cell cycle and cell death. In fact, ubiquitination of cellular proteins, and targeting them for subsequent degradation via ubiquitin-mediated proteolysis, is potentially an important mechanism that regulates cell cycle genes. In tumors, increased de-ubiquitination of cyclins by PGP9.5 could contribute to the uncontrolled growth of somatic cells.

Table 5 . Other molecular prognostic factors.



Cdx2 41 IHC Higher survival in Cdx2 positive patients p = 0.015 Matsumoto et al. [124]

MAGEA3 57 Quantitative RT-PCR

Lower survival in MAGE3A positive patients p = 0.032 Kim et al. [142]

RCAS1 80 IHC Lower survival in RCAS1 positive patients p = 0.0012 Hiraoka et al. [144]

Skp2 46 IHC Lower survival in Skp2 positive patients p = 0.0140 Einama et al. [146]

PGP9.5 68 IHC Higher survival in PGP9.5 negative patients p = 0.0006 Tezel et al. [148]

S100A6 60 IHC Lower survival in positive S100A6 patients p = 0.003 Vimalachandran et al. [149]

Akt/PKB 65 IHC Lower survival in Skp2 positive patients p < 0.05 Yamamoto et al. [151]

Cav-1 79 IHC Lower survival in Cav-1 positive patients p = 0.0008 Suzuoki et al. [152]

Cav-1: Caveolin-1; IHC: Immunohistochemical method; RCAS: Receptor binding cancer antigen expressed on SiSo cells; RT-PCR: Real time polymerase chain reaction.



In pancreatic cancer, patients with PGP9.5 expression had significantly shorter survival times than those without PGP9.5 [148] .

2.3.7 S100A6 S100A6 is a low molecular weight calcium binding protein, acting as a calcium sensor. It belongs to a family of proteins of the EF-hand type known as the S100 protein family. Members of this family can interact with effector proteins, thereby regulating enzyme activities, dynamics of cytoskeleton constituents, cell growth and differentiation, and calcium homeostasis. So, S100A6 has been implicated in several cellular processes, even if its role is not yet completely clear. S100A6 expression is associated with poor survival in pancreatic cancer patients [149] .

2.3.8 Akt/PKB Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members, known as PKB α /Akt1, PKB β /Akt2 and PKB γ /Akt3 in mammalian cells. Similar to many other protein kinases, Akt/PKB contains a conserved domain structure, including a specific PH domain, a central kinase domain and a carboxyl-terminal regulatory domain that mediates the interaction between signaling molecules. Akt/PKB plays important roles in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions, including nutrient metabolism, cell growth, apoptosis and survival [150] . One study conducted by Yamamoto et al. showed that activated Akt expression is an independent prognostic factor for pancreatic cancer [151] .

2.3.9 Cav-1 Caveolae are specialized lipid raft microdomains that are functionally implicated in pinocytosis, transcytosis and endocytosis, cholesterol homeostasis, cell transformation, and signal transduction modulating cell growth, survival, adhesion and migration. Typically, caveolae require the presence of functionally highly conserved, hairpin loop-shaped, 22 – 24 kDa, oligomeric proteins, termed caveolins, in parti-cular caveolin-1 (Cav-1). Dysregulation of Cav-1 is associated with several human malignancies. In pancreatic cancer, Cav-1 expression is a marker of a poorer outcome [152] .

2.3.10 P-glycoprotein The MDR phenotype is most often due to overexpression of drug efflux pumps in the plasma membrane of cancer cells. P-glycoprotein, a 170 kDa transmembrane glycoprotein, encoded by the mdr1 gene, is the best characterized drug efflux pump so far and is a member of the ATP-binding cassette transporter family. A wide range of anticancer drugs have been described to be substrates for P-glycoprotein. Overexpression of P-glycoprotein has been shown to

confer MDR in cultured cells and has also been implicated in the clinical MDR. In addition, P-glycoprotein overexpression correlates with poor prognosis for a number of human cancers [153] .

Unfortunately, this parameter in pancreatic cancer seems not to be useful in detecting prognosis. In fact, Lu et al. demonstrated that survival of patients with high expression of MDR1/P-glycoprotein was not significantly different from that of patients without detectable MDR1/P-glycoprotein expression [154] .

Paradoxically another study showed even that MDR1/P-glycoprotein expression in pancreatic cancer without chemotherapy, inversely correlates with biologic aggressiveness and is an independent indicator of favorable prognosis [155] .

3. Expert opinion

The identification of prognostic factors, that are able to stratify patients that potentially can benefit by an aggressive treatment, is all along an aim of anticancer research both in clinical and, in recent years, in the biologic field. The advance of molecular biology techniques led to the study of the genetic alterations at the base of carcinogenesis and, consequently, to the research of new class of molecular prognostic factors. In other cancers, breast cancer for example, the study of molecular prognostic factor is already routine, leading to considerable improvement of therapeutic management. Establishing a prognostic score could ‘tailor’ the therapeutic management of the different sub-population recognizing those that really could able to benefit by a radical surgical treatment and/or chemotherapeutic treatment. A critical evaluation of the real clinical use of this kind of prognostic factors could help to discriminate among a variety of factors those that, so far, can be considered really useful in every-day clinical practice. Despite this, p53 and K-Ras are two of the most studied prognostic markers in pancreatic cancer, the data available suggest their poor clinical usefulness, as well as Smad4, which showed conflicting results in the three studies that evaluated its effectiveness. p27Kip1 and cyclin D1 seem to correlate with prognosis, but the data are still too limited, whereas p16 could be considered among oncogenes and oncosuppressor gene a good candidate as prognostic marker in clinical practice.

As for apoptosis genes, Bcl-2 has failed to demonstrate a sure correlation with survival, but there are some very early but promising data about the role of survivin in offering prognostic informations. The poor prognosis of pancreatic cancer is also dependent on its invasive and metastatic capabilities; so many gene products involved in cell–cell and cell–matrix interaction has been widely studied. So far, only MMP-7 demonstrated in convincing way, the faculty to predict prognosis. Moreover, as regards growth factors, TGF- β showed an ambiguous value in predicting outcome,



but consequently cannot be considered a fully reliable marker and EGF, despite of the large number of studies carried out, failed to demonstrate a clinical use in predicting prognosis. Instead, VEGF expression proved to be a relevant predictor of a poorer outcome and this has justified the recent intensification of the studies involving anti-angiogenetic agents. Finally, further studies are necessary to confirm the real use of the other molecular prognostic factors reviewed in this article that have demonstrated their capacity to predict prognosis only in single studies.

The outcome of pancreatic cancer therapy continues to be disappointing, with poor long-term survival, even in patients with respectable disease at presentation. The high relapse rate, despite aggressive standard-of-care surgery, means that the majority of patients will require some form of systemic therapy over the course of their illness. The standard for the past decade has been single-agent gemcitabine, which was approved primarily on the basis of quality-of-life benefits. Numerous other targeted agents have been evaluated in combination with gemcitabine, Preclinical studies have used several strategies for inhibiting activated Ras. To become activated, Ras must be farnesylated, which allows prenylation and attachment to the plasma membrane. The farnesyl transferase inhibitors (FTIs) (e.g., tipifarnib) were designed to abrogate Ras signalling [156] . The drug was further investigated in a randomized, double-blinded, placebo-controlled study in which gemcitabine monotherapy was compared with gemcitabine plus tipifarnib in 688 patients with newly diagnosed disease. This study found no statistically significant overall survival difference between arms or in the secondary end points of objective disease response and progression-free survival. It was hypothesized that tipifarnib was not effective because Ras proteins can also be activated by geranylgeranylation, a process very similar to farnesylation that is not inhibited by tipifarnib. Alternative strategies to inhibiting Ras signaling include targeting down-stream signaling mediators. Activated K-ras mediates signal-ing through MAPK (Ras-MAPK), phosphoinositide 3-kinase (PI3K), and Ral-GDS. Ongoing studies are inves tigating the possibility of targeting these signaling molecules [157,158] . Also, the way to target MMP results in no clinical benefit.

A Phase III study was performed in which 414 patients with advanced pancreatic cancer were randomized to marimastat, an oral MMP inhibitor or standard-dose

gemcitabine (1000 mg/m 2 ). No improvement in median survival was reported with marimastat versus gemcitabine (p = 0.19) and a trend toward inferior outcomes with marimastat was noted in comparison with gemcitabine [159] . Marimastat was also evaluated in combination with gemcitabine in a study of 239 patients with unresectable pancreatic cancer, but investigators reported no improvement in overall survival or response rate over gemcitabine alone [160] . On the contrary, a small but statistically signi-ficant increase in overall survival, was observed in a Phase III international, multicenter, double-blinded, controlled study, which randomized 569 chemotherapy-naive patients with metastatic pancreatic cancer to standard-dose gemcitabine plus erlotinib, a small-molecule EGFR tyrosine kinase inhibitor, or placebo. This was the first study to demonstrate a survival benefit when a second drug was added to gemcitabine in this setting [161] . Moreover, a positive correlation between rash and clinical outcomes with EGFR-targeted therapy (erlotinib) has been recently demonstrated. Therefore, the potential of using rash as a surrogate marker for efficacy of EGFR inhibitors in pancreatic cancer therapy exists and needs to be further explored [162] . Combination therapy also led to improve-ments in the secondary end points of progression-free survival and rate of disease control. In preclinical studies, downregulation of MUC4 was associated with reduced tumor growth and metastases, as well as decreased expression of HER2/neu [163] . Targeting MUC4 as a therapeutic strategy is evolving in pancreatic cancer as well as other solid malignancies [164] . Finally, despite the prognostic value of VEGF, targeting angiogenesis through VEGF inhibition with bevacizumab, so far, showed no clinical benefit in a randomized, Phase III study that enrolled 602 patients [165] . Given the extensive genetic abnormalities present in pancreatic and other cancers, it is reasonable to assume that targeting a single pathway will be of limited use because of signaling redundancy. Use of drug combinations or multitargeted molecular agents should result in much greater clinical efficacy by simultaneously inhibiting several of the multiple pathways driving oncogenesis. This approach represents an appropriate way to move forward in developing therapeutic strategies against this disease. Agents targeting many of the pathways critical to pancreatic cancer tumorigenesis are presently being investigated and offer hope for a better outcome in the future.



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Website 201. http://www.ncbi.nlm.nih.gov/

entrez/query.fcgi

Affi liation Giuseppe Tonini†1 MD PhD, Francesco Pantano1, Bruno Vincenzi1, Armando Gabbrielli2, Roberto Coppola3 & Daniele Santini1 †Author for correspondence 1 Campus Bio-Medico University, Medical Oncology, Via Emilio Longoni, 83, 00155, Rome, Italy Tel: +39 06 22541853 ; Fax: +39 06 22541520 ; E-mail: [email protected] 2 University Campus Bio-Medico of Rome, Digestive Endoscopy, Via Emilio Longoni, 83, 00155, Rome, Italy 3 University Campus Bio-Medico of Rome, Surgery, Via Emilio Longoni, 83,

00155, Rome, Italy

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Molecular prognostic factors in patients with pancreatic cancer