Molecular profiles in foregut oncology

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Cancer Genetics ■■ (2016) ■■–■■

Molecular profiles in foregut oncology Prashant Sukharamwala, Daniel Hennessey, Thomas Wood, Shelly Singh, Carrie Ryan, Alexander Rosemurgy * Florida Hospital Tampa, 3000 Medical Park Drive Suite 310, Tampa, FL 33613, USA Oncology is and will continue to evolve resulting from a better understanding of the biology and intrinsic genetic profile of each cancer. Tumor biomarkers and targeted therapies are the new face of precision medicine, so it is essential for all physicians caring for cancer patients to understand and assist patients in understanding the role and importance of such markers and strategies to target them. This review was initiated in an attempt to identify, characterize, and discuss literature supporting clinically relevant molecular markers and interventions. The efficacy of targeting specific markers will be examined with data from clinical trials focusing on treatments for esophageal, gastric, liver, gallbladder, biliary tract, and pancreatic cancers. Keywords Cancer, neoplasm, liver, digestive system, tumor marker © 2016 Elsevier Inc. All rights reserved.

Introduction Cancer, characterized by an aberrant overgrowth of cells, is a substantial disease making it the second leading cause of death behind heart disease (1). Before the term cancer was established, it was referenced during the pyramid age in Egypt as bulging breast tumors. These records were documented in The Edwin Smith Surgical Papyrus dating back to 3000 BC (2). Hippocrates described non-ulcer and ulcer forming tumors with the terms carcinos and carcinoma, which is a Greek description for being crab-like. The Roman physician Celsus later translated the Greek term into cancer which is Latin for crab. Another Greek physician Galen used the word oncos, Greek for swelling, to describe tumors. A new era of treating diseases with chemicals began in the early 1900s when German chemist Paul Ehrlich coined the term chemotherapy (3). The first four decades of the 20th century were devoted to developing cancer models and thereafter research was dedicated to finding effective chemotherapies. In 1943 nitrogen mustard rolled out as the first cancer treatment, antifolates came in 1948, thiopurines in 1951, 5-Fluorouracil in 1957, methotrexate in 1958, adjuvant chemotherapy in 1968–1975, imatinib or Gleevac in 1996, the first monoclonal antibody in 1997,

Received September 30, 2015; received in revised form September 30, 2015; accepted September 19, 2016. * Corresponding author. E-mail address: [email protected] (A. Rosemurgy). 2210-7762/$ - see front matter © 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cancergen.2016.09.008

tyrosine kinase inhibitors in 2005, and ultimately target specific screening began in 2007 (3). The 1940s also included the advent of tumor markers, which are cancer cell specific. Advances in cancer genetics allowed tumor markers to be used as diagnostic and monitoring tools, and more recently predictors of treatment efficacy. Oncology continues to evolve as a result of ongoing scientific inquiry, technical innovation, and evidence based combination treatments which led to precision healthcare. Personalized chemotherapeutic regimens are based on individual variations in genes, presence or absence of tumor markers, tumor immunostains, and more accurate diagnosis by modern imaging modalities. For example, monoclonal antibodies directed against a wide range of oncoprotein biomarkers allow specific and targeted therapy based on tumor type and molecular characteristics. It is essential for all physicians caring for cancer patients to understand the role and importance of molecular tumor markers as the biology of each cancer is becoming well known. These physicians should also assist patients in understanding the critical role of the specific markers. The goals of this review are to identify, characterize, and discuss literature supporting clinically relevant molecular markers and interventions as a means to assist physicians in forming a treatment plan targeting specific aberrations. This review presents the relevant biological tumor markers, pathogenesis, immunohistochemical and histological definition, and current targeted therapies for esophageal, gastric, liver, gallbladder, biliary tract, and pancreatic neoplasms.

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Biological tumor markers ADAM-17 ADAM enzymes are zinc dependent cell surface proteins which cleave various other proteins (4). The cleaved proteins can then have autocrine, juxtacrine, paracrine, or endocrine downstream effects. ADAM-17 is a tumor necrosis factor (TNF)-α converting enzyme (TACE), which cleaves and converts TNF-α into a soluble form.

P. Sukharamwala et al. ulation of the tyrosine kinase and subsequent intracellular signal transduction. Alterations in these signaling pathways can ultimately lead to carcinogenesis.

ERCC1 Excision repair cross complementary 1 (ERCC1) is a DNA repair enzyme which has ATP-dependent DNA helicase activity. ERCC1 is involved in transcription coupled nucleotide excision repair and defects in this gene are associated with multiple syndromes (10).

ABCG8 hENT1 Adenosine triphosphate-binding cassette transporter (ABCG8) is among a class of ATP binding cassette transporters which regulate molecular movement across the cell membrane. The mechanism of ABCG8 is to pump plant sterols and cholesterol out of the intestinal epithelium and back into the lumen of the gastrointestinal tract. ABCG8 is indicated in gallstone formation because gallstones are primarily composed of cholesterol (5).

AXL AXL is a receptor tyrosine kinase (RTK) with oncogenic potential and transforming activity. The AXL extracellular domain consists of two immunoglobulin-like domains. Upregulation of AXL and binding to its ligand, growth arrest-specific 6 (GAS6), has been associated with cell survival, proliferation, and migration (6).

BCL-2 BCL-2 is an anti-apoptotic protein found on the outer membrane of mitochondria. It prevents activation of pro-apoptotic proteins BAX and BAK, also found on the mitochondria membrane. Apoptosis is induced by BAX and BAK aggregating to form a pore that releases intermembrane proteins and triggers the caspase cascade (7).

COX

Human equilibrative nucleoside transporter 1 (hENT1) gene encodes a transmembrane glycoprotein located in the cellular and mitochondrial membranes. hENT1 functions by mediating the cellular uptake of nucleosides required for cells lacking de novo nucleoside synthesis pathways also mediating the uptake of cytotoxic nucleosides used in cancer treatment (11–13).

HER2 Human epidermal growth factor receptor 2 (HER2) is involved in cell growth and differentiation. HER2 belongs to the epidermal growth factor receptor family because it has intrinsic tyrosine kinase activity, but does not bind directly to ligands (14,15). It forms a heterodimer by tightly binding other ligand bound EGFR family members.

IGF-1 Insulin-like growth factor-1 (IGF-1) and its tyrosine kinase receptor transduce cellular signals for mobility, adhesiveness, vascularization, proliferation, apoptosis, migration and differentiation (16). IGF-1 is known to have paracrine and autocrine activity dependent upon nutritional status and insulin plays a role in its synthesis, secretion and activity.

KRAS

Cyclooxygenases (COX-1 and COX-2) are involved in the formation of prostaglandins from arachidonic acid. COX-1 is constitutively expressed while COX-2 is induced, but both can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs). COX-2 is strongly associated with inflammation, while the constitutive aspect of COX-1 provides prostaglandins to the stomach and intestine for the maintenance of mucosal epithelium integrity (8).

KRAS is a member of the family of RAS proto-oncogenes which encodes a GTPase. Mutant KRAS is known to activate downstream signal transduction pathways that stimulate tumorigenesis and resistance to therapies. Such pathways and intermediates include the phosphatidylinositol 3-kinase pathway, mitogen-activated protein kinase (MAPK) MEK, and mammalian target of rapamycin (mTOR) (17).

EGFR

Mesothelin

The gene expressing epidermal growth factor receptor (EGFR) is one of the most common to be affected in human cancers. EGFR binds to ligands such as EGF and transforming growth factor (TGF)-α (9). EGFR has an extracellular ligand-binding region, a single membrane-spanning region, and a cytoplasmic region containing a tyrosine kinase domain. Ligand binding causes receptor homo- and hetero-dimerization leading to stim-

Mesothelin is found on human mesothelial cell membranes, and plays a role in cell adhesion, recognition, cell survival, migration, invasion, and tumor progression. It is the C-terminal cleavage product of a precursor protein initially identified in a human pancreatic cancer cell line (18). The N-terminal cleavage product, megakaryocyte proliferation factor, is not currently a reliable serum tumor marker for pancreatic adenocarcinoma.

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NF-κB Nuclear Factor-kappa B (NF-κB) is a transcription factor with involvement in multiple signal transduction pathways such as the MAPK and AKT/mTOR pathways. It controls over 150 genes associated with stress, cell survival, proliferation, and the immune system. NF-κB is activated by reactive oxygen species, hypoxia, hyperoxia, anoxia, cytokines, UV radiation, dsRNA, protein kinase C activators, MAPK activators and bacterial or viral products (19).

p15 and p16 p15 stimulation activates Gap 1 (G1) phase arrest within the cell cycle as a regulator before DNA synthesis (20). p16 is also involved in G1 phase arrest through inhibition of D type cyclin dependent kinases (CDK) (21). Cyclins bind to specific CDKs in each phase and activate them to allow cell cycle progression. Aberrations of p16 no longer inhibit CDKs allowing for unregulated DNA synthesis. Methylation of p16 occurs in the early stages of cancer transformation, while homozygous deletion is a late event.

p21 P21 or cyclin dependent kinase inhibitor 1A (CDKNA1) is a gene induced by wild-type p53 and mediates arrest of the G1 phase. The inhibitory effect of p21 allows for DNA repair during G1 arrest and before the synthesis phase (22).

p53 p53 is a tumor suppressor that regulates the cell cycle with roles in cycle progression, apoptosis, and DNA repair. Specifically, its two main functions include initiation of cell-cycle arrest and suppression of BCL-2 expression. TP53 is the most frequently mutated gene in human cancers with nearly 80% being point mutations and are prognostic factors in multiple cancers (23). Alterations of TP53 occur in the early stages of cancer transformation and are associated with tumor progression.

PML Promyelotic leukemia (PML) is a tumor suppressor gene known to have reduced expression in cancers. In addition to tumor suppression, PML also plays a role in apoptotic regulation, cell growth, and DNA repair (24).

RASSF1A Ras association domain family 1A (RASSF1A) is a tumor suppressor that prevents cell proliferation, arrests the cell cycle and mediates apoptosis (25). Hypermethylation of the promoter sequence is what determines inactivation of RASSF1A.

3 junction with cyclin dependent kinases and inhibitors of cyclin dependent kinases, RB regulation of the cell cycle is bypassed in cancers (26).

RRM1 The ribonucleotide reductase subunit M1 (RRM1) gene expresses one of two subunits that compose ribonucleosidediphosphate reductase. The reductase enzyme is necessary for the production of deoxyribonucleotides prior to DNA synthesis in the S phase of the cell cycle. The subunit is a major factor in gemcitabine efficacy, and defects of the gene are attributed to many cancers and syndromes (27).

SMAD4 SMAD4, also called deleted in pancreatic carcinoma locus 4 (DPC4), plays a critical role in the tumor-suppressive functions of the transforming growth factor (TGF)-β pathway. This signaling pathway contains both proliferative and antiproliferative signals (28). Pancreatic intraepithelial neoplasia (PanIN) precursor lesion, PanIN-3, specimens lose SMAD4 expression in comparison to PanIN-1 and PanIN-2 neoplastic lesions (29).

SPARC Secreted Protein Acidic Rich in Cysteine (SPARC) is a calcium binding protein involved in extracellular matrix interaction with the microenvironment. It is associated with accumulation of albumin and albumin-bound proteins in cells. The mechanisms of action for SPARC include angiogenesis during wound healing, cell adhesion, tissue remodeling, cell migration and proliferation (30). SPARC may also be involved in metastasis of cancers and impact cell permeability.

TGF-β TGF-β regulates multiple cell processes such as cell proliferation, apoptosis, differentiation, migration, tumorigenesis and metastasis (31). TGF-β is an anti-proliferating molecule and an abnormal TGF-β signal transduction leads to stimulation of cancer cell replication and evasion of apoptosis.

TNF-α Tumor necrosis factor (TNF)-α has multiple roles with one being a pro-inflammatory cytokine. Apart from its involvement in the innate immune response, apoptosis is induced by TNF-α binding to its receptor (32).

TOPO1 Retinoblastoma The retinoblastoma protein (RB) regulates the G1-S check point in the cell cycle and activity of transcription factors. In con-

Topoisomerase 1 (TOPO1) separates and ligates DNA during the replication process. This allows the strands to pass through one another by altering the topological state of DNA (33).

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TYMS Thymidylate Synthase (TS or TYMS) catalyzes the methylation of deoxyuridylate to deoxythymidylate using the cofactor methylenetetrahydrofolate, which regulates the concentration of dTMP used for DNA replication and repair (34). The enzyme is a specific target for cancer therapies such as fluorouracil and folate analogs.

VEGF Vascular endothelial growth factor (VEGF) is one of the most important factors in regulating vasculogenesis and angiogenesis. Transcriptional induction of VEGF is regulated by tumor hypoxia factors that bind the VEGF promoter. The normal function of VEGF is to stimulate the formation of new blood vessels and maintain immature vessels. Tumor blood vessels stimulated by VEGF result in prolific and unorganized growth with blind ends, increased permeability, poorly perfused tumors, and a positive feedback of VEGF production (35). Overexpression is correlated with the extent of microvessel density, distant metastasis, poor survival rate and advanced stage of disease. Abnormal vasculature of tumors result in increased interstitial fluid pressure, and for these reasons VEGF is a potential independent prognostic factor that may be a helpful prognostic biomarker (35).

Esophageal cancer Pathogenesis Adenocarcinoma Esophageal cancer is diagnosed yearly in approximately half a million people and is the sixth leading cause of cancer death worldwide (36). Esophageal carcinoma has varying pathogenesis based on its anatomic location and is commonly categorized into squamous cell carcinoma and adenocarcinoma, with both accounting for 98% of esophageal cancers (37). Adenocarcinoma tends to affect the lower esophagus or gastroesophageal junction and is associated with gastroesophageal reflux disease and obesity (38). Gastroesophageal reflux disease chronically leads to irritation of the esophagus, which can result in premalignant precursors, termed Barrett’s esophagus. In Barrett’s esophagus, an alteration occurs in which the normal squamous epithelium of the esophagus is replaced by columnar epithelium. This alteration increases the risk for further dysplastic changes or carcinoma, once the muscularis propria is crossed (39). In recent years, there has been a trend toward esophageal adenocarcinoma becoming the predominant esophageal cancer, although worldwide this is not the case (38). Despite numerous modalities of current treatments, there has only been a minimal increase in the 5-year survival over the past decade. Esophageal cancer still portends significant mortality, and for this reason, new treatments are continuously being studied. COX-2, HER2 and VEGF are current targets of esophageal therapeutics and overexpressed in dysplasia and adenocarcinoma. IGF-1 and its tyrosine kinase receptor, IGF-1R, have been reported to be associated with esophageal cancer and Barrett’s esophagus. Related malignancies of esophageal adenocarcinoma are tumor growth from overexpression of IGF-

P. Sukharamwala et al. 1R, prevention of apoptosis via the IGF-1 autocrine loop, and mitogenic effects of IGF-1 and IGF-2.

Squamous cell carcinoma Squamous cell carcinoma typically affects the upper esophagus with the pathology being linked to tobacco and alcohol abuse. The overall risk of developing squamous cell carcinoma substantially increases with the use of such substances. IGF binding protein-3 (IGFBP3) is a regulator of IGF-1 and 2 and commonly noted to be overexpressed in squamous cell carcinoma of the esophagus in conjunction with EGFR overexpression. The level of serum IGF-1 and IGFBP3 appears to be correlated with tumor invasion, poor prognosis, and low patient survival rate (36). p53 mutations are noted in approximately 50–80% of esophageal cancers. Studies have shown that p53 antibody in the serum of patients with squamous cell carcinoma is a helpful prognostic marker and detector of p53 mutations. p16 mutations are found in 10% of normal esophageal tissue, with increased expression noted in 88% of the squamous cell carcinoma cases. p15 mutations are noted in 92% of squamous cell carcinoma cases with inactivation occurring simultaneously with p16 via promoter methylation and homozygous deletion (20,21). The retinoblastoma protein (RB) is implicated in the development and progression of esophageal cancer. RB mutations are rare in squamous cell carcinoma, but several studies indicated that alterations in p16 and p53 lead to the blockage of RB function. Loss of retinoblastoma heterozygosity plays an essential role in the inactivation of the RB gene and is associated with p53 alterations. It is suggested that involvement of RB with p53 inactivation may be the major event in the development and progression of esophageal squamous cell carcinoma (36).

Immunohistochemistry and histological definition Adenocarcinoma Cell proliferation markers of Barrett’s mucosa and esophageal adenocarcinoma detected by immunohistochemical stainings are cyclin D1, p53, and Ki67. p53 was present in 87% of adenocarcinomas compared to 9–55% of dysplastic and 0% of metaplastic mucosa (40). Cyclin D1, involved in cell cycle regulation, has been associated with an increased risk of progression from Barrett’s esophagus to adenocarcinoma. Cell cycle deregulation and increased cell surface expression of Ki67 has been recognized in high grade dysplasia. Along with these stained markers, the most recent and accurate modality of esophageal adenocarcinoma detection along with N and T staging is endoscopic ultrasound (41).

Squamous cell carcinoma Keratins are intermediate filaments that comprise cytoskeletal proteins with 30 subtypes identified (CK1–30). CK10, 18, and 19 were associated with esophageal squamous cell carcinoma. Receptors in the EGFR family are HER1 and HER2, with HER1 overexpressed in 29–92% of well differentiated squamous cell carcinoma tumor samples. A study (n = 14) showed HER1 overexpression to be associated with minimal response to chemoradiotherapy and a useful marker of poor prognosis (42).

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Therapy targets Adenocarcinoma EGFR EGFR expression is present in 30–70% of esophageal carcinomas, and EGF overexpression has been detected in squamous cell carcinomas and adenocarcinomas derived from Barrett’s esophagus (39,43). EGFR expression portends worse patient survival, poor prognosis and minimal response to esophageal carcinoma related chemotherapy. Most common agents targeting EGFR include tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs). mAbs bind to the extracellular domain of the receptor and compete with other ligands. On the intracellular region of the receptor, TKIs compete with ATP by binding to the tyrosine kinase portion and blocking downstream pathways (36). There have been several phase II trials testing various mAb based drugs, such as cetuximab, panitumumab, nimotuzumab. Cetuximab was the first EGFR inhibitor (EGFRI) and is one such therapy being studied as a second line agent both alone and in combination with irinotecan. In esophageal adenocarcinoma, the therapy has shown a decrease in response rate and overall survival time (44). However, additional studies have shown a trend toward greater time to progression when cetuximab was used for maintenance therapy with chemotherapy versus chemotherapy alone without maintenance treatment. Overall, response rate results are contraindicatory, and when used as a monotherapy appear to have minimal activity in gastroesophageal junction cancers. Panitumumab is a second-generation EGFR-I and a fully human immunoglobulin G2 mAb. A phase III randomized trial added panitumumab to epirubicin, ox, and cape (EOX) to treat an unselected population with gastroesophageal junction cancer. Results showed an increased risk of death, worse overall survival and a trend toward shorter overall progression free survival when compared to EOX alone (45). In 2012, a randomized phase II trial of panitumumab in combination with chemotherapy showed better median time to progression, but no recommendations on its use have been made (45). Preliminary results on studies related to nimotuzumab were presented in the ASCO annual meeting in 2011 and did not show clear benefit. Additional studies suggest trends toward a potential benefit in treating patients with EGFR targets. Gefitinib has been involved in several phase II studies of esophageal adenocarcinomas, with varying results. Reports of combinatorial gefitinib and radiotherapy showed a synergistic effect and an additive effect in human gastroesophageal carcinoma cell lines (46). In patients with locally advanced esophageal and gastroesophageal junction cancer, neoadjuvant gefitinib combined with cisplatin, 5-Fluorouracil (5-FU) and radiotherapy increased overall survival but did not increase complete response rates. Lapatinib is a dual EGFR and HER2 inhibitor and has not shown significantly positive results with gastroesophageal junction cancer (39). Phase I trials are now ongoing for esophageal cancer. AXL receptor tyrosine kinase is determined to be an adverse prognostic factor in esophageal adenocarcinoma (6). Small molecule inhibitors of AXL were analyzed for which blockade of AXL function abrogated phosphorylation of HER2 at the lapatinib residue and indicative of receptor crosstalk.

5 HER2 HER2 is associated with 30–80% of esophageal adenocarcinoma and squamous cell carcinoma and presence of HER2 expression is associated with lower tumor grade malignancy, less invasiveness, fewer malignant nodes, and the presence of adjacent Barrett’s esophagus. HER2-positivity confers improved survival in esophageal adenocarcinomas associated with Barrett’s esophagus along with a greater level of amplification compared to no presence of Barrett’s esophagus (14). Based on research and regarding prognostic impact, HER2 amplification was found to have a Barrett’s esophagus status dependence (14). Trastuzumab, or herceptin, is a monoclonal antibody that targets HER2 and is effective only in cancers with HER2 overexpression. ToGA, a phase III openlabel, randomized-controlled trial, tested capecitabine plus cisplatin or FU plus cisplatin compared to chemotherapy plus intravenous trastuzumab. The study resulted in a median overall survival of 13.8 months in those assigned to trastuzumab plus chemotherapy compared to 11.1 months in those assigned chemotherapy alone. The authors concluded that Trastuzumab in combination with chemotherapy can be considered a new standard option for patients with HER2-positive gastroesophageal junction cancer (15,44). Lapatinib is recognized in research as a dual TKI of EGFR and HER2. However, lapatinib has shown a lack of response in gastroesophageal junction cancer (37). VEGF VEGF is overexpressed in 30%–60% of esophageal cancer tumors, with upregulation observed in Barrett’s esophagus, esophageal adenocarcinoma, and gastroesophageal junction tumors. VEGF inhibitors consist of VEGF TKIs such as sorafenib, sunitinib, cediranib, apatinib, and telatinib and VEGF mAbs such as bevacizumab and ramucirumab (47). Bevacizumab is currently under review for treatment of esophageal cancer and is a direct inhibitor of VEGF. 774 patients in a phase III study with no prior therapy and having inoperable, locally advanced, or metastatic stomach or gastroesophageal junction adenocarcinoma were randomized to treatment with capecitabine and cisplatin plus or minus bevacizumab. The study resulted in no survival benefit with addition of bevacizumab (48). A randomized phase III study showed that ramucirumab yields overall survival improvement in patients with gastric and gastroesophageal junction adenocarcinoma (49). Agents including sunitinib and sorafenib are currently being studied in esophageal cancer (50). COX-2 Upregulation of COX-2 appears to play an important role in the transition of Barrett’s metaplasia to esophageal carcinoma. Meta-analyses have shown that patients who take NSAIDS can have up to a 40% lower rate of esophageal cancer (51). High COX-2 is noted to be expressed in 25% of esophageal adenocarcinoma patients (52). COX-2 overexpression also correlates with invasion, metastatic potential and resistance to radiotherapy of esophageal cancer as well as cancer proliferation through EGFR stimulation (51). A phase II study by the Hoosier Oncology Group reported a series of 31 patients who received combination cisplatin, 5-FU and celecoxib prior to surgery. Of the 22 patients who underwent surgery, five had a pathologic complete response. An Australian group reported a series of 13 patients who received standard cisplatin, 5-FU, celecoxib and radiation. The clinical complete response rate was 54%, all of which were complete responses, compared to six patients who underwent surgery having one

ARTICLE IN PRESS 6 pathologic complete response. The trial was closed early due to safety concerns with celecoxib. In another trial, celecoxib was given with cisplatin, irinotecan and radiation as preoperative treatment for locally advanced esophageal cancer. Of 36 patients, 33 patients had grade 3–4 toxicity and 25 completed treatment and underwent surgery. The overall pathologic complete response rate was 35%. A Dutch group recently reported a study in which neo-adjuvant COX-2 inhibition selectively down-regulates hepatocyte growth factor receptor (MET) expression as well as COX-2 expression. MET has been shown to be independently associated with a poor prognosis in esophageal cancer (49,53). Studies have also noted COX-2 to be an independent prognostic factor in both esophageal adenocarcinoma and squamous cell carcinoma (54). Trials using COX-2 inhibitors have generally come to a standstill because of increased cardiovascular risk (55). Squamous cell carcinoma EGFR Cetuximab is FDA approved for the treatment of locally advanced head and neck squamous cell carcinoma and has shown good results. A study including patients with squamous cell carcinoma used a combination of cetuximab, cisplatin and 5-FU compared to cisplatin and 5-FU alone. The results showed that cetuximab could be safely combined with and possibly increase the efficacy of a cisplatin and 5-FU combination. Another study showed that in vitro cell lines from squamous cell carcinoma of the esophagus induced EGFR activation when treated with cisplatin, but cisplatin induction of EGFR activation was inhibited by combination treatment with cetuximab (56). The standard treatment of concurrent chemoradiotherapy for patients with locally advanced esophageal squamous cell carcinoma is sometimes intolerable in patients with malnutrition or advanced age. A study consisting of 18 patients intolerant to chemoradiotherapy received concurrent erlotinib and radiotherapy and resulted in a median overall survival of 21.1 months and progression free survival of 12 months (57). Another study observing 30 patients with metastatic cancer of the esophageal and gastroesophageal junction who received erlotinib daily showed erlotinib to have limited activity (46). However, time to progression was noted to be longer in patients with squamous cell carcinoma, but studies suggest that erlotinib does not predict outcome. Fatty acid synthase Because of the hypoxic conditions in the tumor, anaerobic metabolism predominates, which causes lipid combustion and beta oxidation. Fatty acid synthase (FAS) makes lipids from fatty acids and is expressed in many cancers. Very high levels of expression exist in both esophageal adenocarcinoma and squamous cell carcinoma (58). FAS levels can be detected in the circulation of cancer patients and can act as a potential tumor marker. Anti-FAS therapy, acting to inhibit FAS activity, has been shown to suppress cancer cell proliferation with agents such as the antibiotic Cerulenin, small interfering RNA specific for the FAS gene, a pancreatic lipase inhibitor Orlistat, and C75 which is a synthetic small-molecule inhibitor of type 1 mammalian FAS mimicking the mechanism of action of cerulenin (36). HIF-1α HIF-1α is implicated in the progression of esophageal squamous cell carcinoma and is the most critical factor

P. Sukharamwala et al. in hypoxia tolerance of tumors. In vivo inhibition of HIF-1α via YC-1 or knockdown with short hairpin RNA plasmids suppressed tumorigenicity, increased apoptosis, and downregulated expression of VEGF, matrix metalloproteinase 2 (MMP2) and BCL2 (59). VEGF, MMP2, and BCL2 play a major role in angiogenesis, invasion and metastasis, and apoptosis suppression, respectively. This study provides future target possibilities for esophageal squamous cell carcinoma therapies.

Gastric cancer Pathogenesis Adenocarcinoma Gastric or stomach cancer tends to imply adenocarcinoma because 90–95% of gastric cancers are adenocarcinoma and located in the inner lining mucosa. Gastric cancer incidence has declined over the decades, but remains a leading cause of mortality worldwide, especially in developing nations. The common tumor markers and targets of gastric cancer treatments include HER2, VEGF, and EGFR. KRAS mutations, although rare and not used as a prognostic biomarker in gastric cancer, can serve to establish responsiveness to anti-EGFR treatment. 90% of gastric cancers contain dysfunctional E-cadherin, but only structural modifications, present in 10% of cases, proved to be a poor prognostic marker. Gastrointestinal stromal tumors Gastrointestinal stromal tumors (GISTs) are a rare type of stomach cancer, and at a molecular level are characterized by the presence of the KIT protein and mutations in the KIT gene. Thus, the KIT protein is found in greater than 95% of GISTs, while mutations in the KIT gene are present in 80– 85% of cases (60). 5–10% of GISTs have mutations in the homologous gene platelet derived growth factor receptor alpha (PDGFRA) (60). Overall, 80–95% of GISTs have mutations in either the KIT or PDGFRA gene. For the 5% of GISTs that fail to express KIT protein, mutational analysis of both the KIT and PDGFRA genes may confirm the diagnosis. Another study by the European Group on Tumor Markers recommended that staining for KIT protein should be used as a diagnostic aid for GIST. However, its absence does not exclude GIST. Mutational analysis of KIT or PDGFR genes may be considered, if the sample is KIT protein-negative.

Immunohistochemistry and histological definition Adenocarcinoma The International Agency for Research on Cancer stated Helicobacter pylori as the primary cause of gastric cancers, which induces an inflammatory response to gastric tissue. Histological analysis of gastric metaplasia shows absence of a brush border along with cytoplasmic mucin droplets. Normal gastric mucins have a neutral pH and are stained magenta by acid-Schiff staining. Metaplasia analysis shows mucins to be more acidic with a pH of 2.5 and stain blue with Alcian blue (61). Using the two staining methods can differentiate between normal and metaplastic tissue. Other associated gastric cancer mutations include E-cadherin, or CDH1, and DNA mismatch repair mechanisms.

ARTICLE IN PRESS Molecular profiles in foregut oncology Gastrointestinal stromal tumor Other markers that may aid the diagnosis of GISTs include DOG1, CD34, S100, desmin, PS100 and smooth muscle actin (62). Measurement of markers, however, complements but does not replace histopathology in the diagnosis of GIST. Not all GISTs harbor mutations of the KIT and PDGFRA genes, and approximately 10–15% of GISTs lack such mutations. A panel of immunohistochemical stains has been traditionally used in GIST diagnosis. The results of studies have suggested that in most cases of suspected GIST a combination of CD117 and DOG1 immunostaining should be sufficient to confirm the diagnosis. In cases that fail to stain for either CD117 or DOG1 further immunostaining with a broader panel of antibodies, including muscle (SMA and desmin) and neural markers (S100), together with molecular analysis, should be considered (62).

Therapy targets Adenocarcinoma Gastric cancer tends to also be diagnosed in a later stage because of a later symptom onset. Increased staging accuracy of gastric cancer has been shown by using endoscopic ultrasound with esophagogastroduodenoscopy. Early detection offers a 5 year survival of 50–70%, with surgical resection offering the best therapy. However, the 5 year survival drops to 4–10% in advanced unresectable patients, with chemotherapy being the best option (63). A study of 556 patients with gastric or gastroesophageal junction tumors were randomized to surgery plus adjuvant therapy versus surgery alone. There was a significant benefit in overall survival and disease free survival in the adjuvant therapy arm, which consisted of 5-FU, leucovorin and radiotherapy (64). A MAGIC trial randomized 503 patients to perioperative chemotherapy or surgery alone and resulted in increased progressive free survival and overall survival in the arm receiving perioperative chemotherapy. Chemotherapy consisted of epirubicin, cisplatin and infused 5-FU (65). HER2 Gastric cancer prognosis based on HER2 has yet to be determined, but many studies have shown HER2 to be overexpressed. A phase III ToGA study demonstrated the first survival benefits in gastric cancer with combinatorial trastuzumab. The study compared trastuzumab with chemotherapy, consisting of cisplatin with either capecitabine or 5-FU, versus chemotherapy alone. Favoring the combination therapy arm, the median overall survival was 13.8 months versus 11.1 months, and the median progressive free survival was 6.7 months versus 5.5 months. A phase II trial studied patients with HER2 amplifications treated with lapatinib, but was discontinued due to a low response rate and slow enrollment. The complete response of gastro-esophageal cancer was 6.25% (66). VEGF VEGF expression has been associated with a poor prognosis, and it is expressed in 40% of gastric cancer cases and its receptor expressed in 36% of cases (67). AVAGAST is a trial that studied the addition of bevacizumab to cisplatin and capecitabine. The results showed a higher overall response rate of 46% versus 37.4% and a higher progressive free sur-

7 vival at 6.7 months versus 5.3 months, but the overall survival was not significantly improved at 12.1 months versus 10.1 months (68). The receptor of VEGF has also been targeted by a recent randomized trial, which increased overall survival to 5.2 months versus 3.8 months using ramucirumab (n = 238) and a placebo (n = 117), respectively (69). EGFR EXPAND, an international phase III study, showed that addition of cetuximab to cisplatin and capecitabine had no benefit in overall survival. Versus chemotherapy alone the combination decreased progression free survival and decreased overall survival at 5.6 months versus 10.7 months. The REAL3 trial compared the addition of panitumumab to epirubicin, oxaliplatin, and capecitabine, which had significantly lower overall survival at 8.8 months compared to chemotherapy alone at 11.3 months. An ongoing phase II study is treating patients with gastric and gastroesophageal junction cancer with or without lapatinib added to epirubicin hydrochloride, cisplatin, and fluorouracil or capecitabine (63). Gastrointestinal stromal tumor Complete R0 resection of primary localized GISTs remains the principle treatment, but patients often have recurrence and a 5-year survival of 54%. Unresectable or metastatic GISTs have a poor prognosis with a median survival of 1.5 years (70). Imatinib, or Gleevac, inhibits the tyrosine kinase of KIT and PDGFRA and was first used to treat GISTs in 2000. 80% of patients treated with imatinib resulted in a partial response or stable disease, and the 2-year survival in advanced GIST is 75–80%. Sunitinib inhibits the tyrosine kinase of VEGFR, KIT and PDGFRA and is used in patients who gained resistance or intolerance to imatinib. Receptor tyrosine kinase inhibitor, or tyrosine kinase inhibitor (TKI), therapy revolutionized the treatment of GISTs but the tumors of patients eventually develop resistance requiring a need for additional therapeutic targets in advanced GIST. PI3K/AKT/mTOR This pathway allows for cell survival and proliferation with hyperactivation caused by constitutive receptor tyrosine kinase signaling (71). mTOR inhibition using rapamycin analogs in combination with imatinib have produced modest results. A phase I/II study using everolimus and imatinib resulted in a median progression free survival of 1.9 months. This was after progression on imatinib and 3.5 months after therapy with imatinib and another TKI. In vitro studies using imatinib resistant GIST cells showed PI3K inhibition with LY294002 resulted in 40–74% reduction in cellular division and 3–4 fold increase in apoptosis (72). Conversely, mTOR inhibition only showed 5–20% cell division reduction without any apoptosis activity. PI3K/AKT pathway inhibitors are an active area of investigation and may be a more effective treatment than mTOR inhibition. Heat shock protein 90 Heat shock protein 90 (HSP90) is a conserved chaperone that stabilizes protein folding and assembly in times of physiologic stress, such as hypoxia, temperature changes, and acidosis. Early HSP90 inhibitors showed little benefit in that they prevented proliferation in KIT-dependent cell lines but not

ARTICLE IN PRESS 8 KIT-independent cell lines. Newer HSP90 inhibitors, such as IPI-493 and -504, STA-9090 and others, are actively being studied and were well-tolerated in the phase I trials. However, a phase III study treating advanced and unresectable GIST, in patients whose disease progressed on imatinib and sunitinib, with IPI-504 was discontinued early due to deaths on the IPI504 arm. The deaths were considered drug related including renal failure, liver failure, metabolic acidosis, and other complications. The phase I trial resulted in 38% or 11 patients (n = 29) with a partial metabolic response on PET. As of 2013, a patient initially responding to IPI-504 has been continuing on treatment for 5 years.

Liver cancer Pathogenesis Hepatocellular carcinoma Estimated by the American Cancer Society, over 35,600 new cases of primary liver and intrahepatic bile duct cancer will be diagnosed in the United States throughout 2015. The most common type of liver cancer is hepatocellular carcinoma. It comprises 80% of primary liver cancers and one of the most frequent cancers worldwide. Prognosis remains poor for advanced stages of hepatocellular carcinoma, with a 5 year survival rate of 7%. Interruption of many signaling pathways, such as the JAK/STAT, MAPK, and WNT/β-catenin, has been a target strategy of hepatocellular carcinoma treatment (73). Alpha-fetoprotein (AFP) has been widely used as a surveillance test for hepatocellular carcinoma, with studies including patients with cirrhosis. A multi-institutional case control study compared the two biomarkers des-gamma carboxyprothrombin (DCP) and lectin-bound AFP (AFP-L3) as potential surveillance markers. A total of 836 patients were enrolled with 417 (50%) being cirrhosis controls and 419 (50%) hepatocellular carcinoma cases. Of the hepatocellular carcinoma cases 208 (49.6%) had early stage hepatocellular carcinoma (n = 77 very early, n = 131 early). AFP had the best area under the receiver operating characteristic curve (0.80, 95% CI: 0.77–0.84) followed by DCP (0.72, 95% CI: 0.68–0.77) and AFP-L3% (0.66, 95% CI: 0.62–0.70) for early stage hepatocellular carcinoma (74,75). The optimal AFP cutoff value was 10.9 ng/mL, leading to a sensitivity of 66%. When only those with very early hepatocellular carcinoma were evaluated, the area under the curve for AFP was 0.78 (95% CI: 0.72–0.85) leading to a sensitivity of 65% at the same cutoff. The authors concluded that AFP was more sensitive than DCP and AFPL3 for the diagnosis of early and very early stage HCC at a new cutoff of 10.9 ng/mL (75).

Therapy targets VEGF Multiple studies have shown VEGF to be one of the important factors contributing to the angiogenesis of hepatocellular carcinoma. Also, VEGF promotes hepatocellular carcinoma development and metastasis, and the serum VEGF level has been reported as a significant independent prognostic factor in hepatocellular carcinoma patients. A high serum level of VEGF is a predictor of poor outcome after resection of he-

P. Sukharamwala et al. patocellular carcinoma. Serum VEGF, but not endostatin, may be a useful prognostic marker in patients with hepatocellular carcinoma (76–78). Sorafenib, an oral multikinase inhibitor of VEGF, PDGFR, and RAF, may be effective in hepatocellular carcinoma. In patients with advanced hepatocellular carcinoma, median survival and the time to radiologic progression was nearly 3 months longer for patients treated with sorafenib than for those given placebo. Median overall survival and time to progression was statistically significant in patients with advanced hepatocellular carcinoma who received sorafenib compared to placebo (79,80). EGFR In addition to angiogenesis, EGF and its receptor, EGFR, also play a crucial role in the proliferation of hepatocellular carcinoma (81). A phase II study reported single-agent erlotinib was well tolerated and had modest disease control benefit in hepatocellular carcinoma. A combination of bevacizumab and erlotinib failed to show clinical activity in sorafenib refractory hepatocellular carcinoma population. A phase III randomized, controlled, double blind trial, SEARCH, compared sorafenib plus erlotinib to sorafenib plus placebo. The results did not prolong time to progression at 3.2 months versus 4 months or overall survival at 9.5 months versus 8.5 months. AKT pathway Erlotinib and lapatinib are known to prevent downstream signaling by the receptor of EGF. Everolimus, temsirolimus, and sirolimus inhibit mTOR, which regulates protein synthesis, increases cell proliferation, and increases cell survival by inactivating the apoptotic factor BAD. A phase I and phase II study of everolimus for hepatocellular carcinoma resulted in a median progression free survival of 3.8 months and overall survival of 8.4 months (82). Ongoing trials include a phase III study comparing everolimus and placebo, and a phase II study of sorafenib and everolimus versus sorafenib alone to determine efficacy.

Gallbladder cancer Pathogenesis The American Cancer Society estimates nearly 11,000 new cases of gallbladder cancer in 2015, with the disease being rare and usually found in advanced stages. Prognosis is dependent upon the stage of detection with only 20% found early because symptom onset occurs in later stages. Gallbladder cancer is, however, associated with chronic cholecystitis and cholelithiasis and tends to be a more rare disease (5). Because of the rarity, limited knowledge exists on effective therapies. Mutated or deleted tumor suppressor genes are sometimes associated with poor prognosis, such as decreased survival, but these genes have no targeted therapies currently available. Presence of COX2 at the invasive front of gallbladder cancer specimens was associated with decreased overall survival. It has been shown that upregulation of TNF can be used as a marker for the stage of gallbladder carcinoma progression. The presence of TNF mRNA expression in cells of gallbladder mucosa ranged from 0% in hyperplasia to 20% in dysplasia and 90% in carcinoma (32). Increased expression of ADAM-17 is associated with a higher grade and stage

ARTICLE IN PRESS Molecular profiles in foregut oncology of tumors along with lower survival. 31% (n = 61) of gallbladder cancer cases contain a mutation in TP53, and this subset had a shorter survival after complete resection when treated with chemotherapy (6.5 vs 17.3 months, p < 0.01). However, there was still positive immunohistochemistry (IHC) staining for p53 independent of gene mutation, demonstrating that IHC is not reliable to determine integrity of p53 in gallbladder cancer (83). Hypermethylation of promoter region 1 in RASSF1A was observed in 23% (n = 22) of gallbladder carcinomas, 12.5% (n = 8) of gallbladder adenomas, and 0% (n = 26) of nonneoplastic tissues in a Korean study. Hypermethylation of promoter region 2 was seen in 36% of gallbladder carcinoma specimens, 25% of gallbladder adenomas, and 8% of nonneoplastic tissues. KRAS mutations were rare occurring in 4.5% of carcinomas, but 25% of adenomas demonstrated KRAS abnormalities. Normal expression of PML resulted in favorable 5 year survival rates, and ABCG8 is responsible for increased gallstone formation leading to increased inflammation. Using a Kaplan–Meier estimate, 42% (n = 101) of cases had high PML expression versus 13% (n = 28) having low PML expression. The low expression cases had significantly lower median survival at 9 months compared to 24 months (24).

Immunohistochemistry/histological definition In a molecular profile case study of the gallbladder, gallbladder cancer was positive for P-cadherin in 64% (n = 39) of the cases and positive for CD24 in 46% (84). Another study found that 78% (n = 207) of patients who underwent surgery for gallbladder cancer stained positive for CD24, which is associated with poor prognosis (85). The CD24 antigen could represent a potential mAb target in patients with gallbladder cancer overexpressing CD24. A study observing cytokeratin (CK) 7 and 20 found gallbladder cancer was positive for CK7 in 69% and positive for CK20 in 28%. Gallstone-associated chronic cholecystitis was positive for CK7 in 100% and positive for CK20 in 52% (86). Gallbladder cancers, when compared to normal gallbladder mucosa, express MUC1 and downregulate MUC6 (87). CDX2 is associated with multiple GI cancers, more commonly seen in colorectal and intestinal-type gastric cancers than pancreatic. Expression in gallbladder epithelium is seen in gallbladder intestinal metaplasia. One report stated that CDX2 was upregulated in metaplastic and dysplastic gallbladder specimens but not in normal gallbladder epithelium (88). Gallbladder adenocarcinoma demonstrates 29–36% CDX2 expression, but reports vary on the clinical significance of this expression with only one of two studies demonstrating prognostic differences (89,90).

9 allowing for crosslinks between bases, usually at locations where adenine and guanine are located next to each other on the same strand (92). Gemcitabine is a cytidine analog which is transported across the cell membrane by nucleoside transporters. The active drug metabolites are formed by intracellular phosphorylation to form gemcitabine diphosphate and triphosphate. The triphosphate metabolite inhibits DNA synthesis by incorporating into DNA, which terminates chain elongation and induces a programmed cell death via apoptosis. ABC-01 was a phase II trial which proved beneficial ramifications with cisplatin and gemcitabine combinations. ABC02 (n = 410) was a phase III randomized controlled multicenter trial enrolling patients with locally advanced or metastatic cholangiocarcinoma, gallbladder, or ampullary cancer. Treatment lasted 24 weeks and consisted of either gemcitabine or a combination of gemcitabine and cisplatin. There were 327 deaths and 362 patients with disease progression for which 278 died. In the drug combination group, the median survival was 11.7 months vs 8.1 months in the single agent gemcitabine group (p < 0.0001) (91). EGFR 38% of gallbladder cancers express EGFR, and erlotinib and gefitinib are drugs targeting EGFR (9). These agents received FDA approval for non-small cell lung cancer which progresses on first line chemotherapy, and erlotinib is approved in combination with gemcitabine for advanced pancreatic cancer (9). Erlotinib is an inhibitor of the tyrosine kinase domain of the EGFR in ERBB family members. Erlotinib monotherapy was used in a phase II study as neoadjuvant and adjuvant treatment. A low response rate of 8% was shown but had a high percentage, 17%, of patients free of symptom progression for 24 weeks or longer (50). The intensity and presence or absence of EGFR expression seemed to not affect the prognosis after this therapy. VEGF Vessel normalization is a process of pruning the tumor vasculature, reducing vessel tortuosity, and decreasing interstitial fluid pressure all resulting from anti-VEGF treatment. Advanced disease stage and poor prognosis has been associated with expression levels of VEGF. Sorafenib is a multikinase inhibitor preventing cellular proliferation by targeting the MAPK pathway. A phase II study, Bay 43-9006, using sorafenib to treat patients with unresectable or metastatic gallbladder cancer or cholangiocarcinoma had a 6% (n = 36) response rate and 6 month median overall survival (93).

Therapy targets

Bile duct cancer

The standard of care for gallbladder cancer tends to be combination therapy with gemcitabine plus cisplatin or oxaliplatin (91). Cisplatin is a molecule consisting of a platinum ion surrounded by two amines and two chlorides. Oxaliplatin and carboplatin have carboxylates substituting the chlorides, which are also the leaving group because the amine ligands form stronger interactions with the platinum ion. The two chloride ions are intracellularly displaced from the cisplatin molecule and cisplatin therefore becomes a reactive species. The reactive species form bonds with DNA, in the cis geometry,

Pathogenesis Cancer of the biliary tract is another rarity and known to have a poor prognosis because of its late stage diagnosis. Cholangiocarcinomas are associated with chronic inflammation, reflux of pancreatic secretions, and the presence of an abnormal pancreaticobiliary junction. Recent advancements in imaging technologies have improved algorithms for diagnosing cholangiocarcinoma. Based on their anatomical location, cholangiocarcinomas represent a heterogeneous group of

ARTICLE IN PRESS 10 malignancies often classified as intrahepatic or extrahepatic. However, a need exists for therapies that improve patient survival, especially advanced-stage patients not amenable to surgical intervention. Many patients are not suitable candidates for hepatic resection because symptom onset does not usually occur until advanced stages. To date, hepatic resection is the only therapy associated with prolonged survival, due to a lack of efficacious chemotherapy regimens. Somatic mutation analysis has become a useful tool in selecting personalized therapy for many solid tumors. There is a potential for new broad-based molecular techniques to profile tumors and identify potential therapeutic targets. Some biological markers of biliary tract carcinogenesis and potential therapeutic targets are iNOS, COX-2, MCL-1, HER2, β-catenin, MUC1, VEGF, TGF-β and EGFR (94). These molecules play a role in apoptosis, tumor vascularization, proliferation, and infiltration. Inflammatory cytokines induce expression of iNOS leading to NO formation and oxidative stress of the bile duct epithelium. Increased DNA damage and suppression of DNA repair can then stimulate mutations. Cancer cell proliferation and prolonged survival is caused by arachidonic acid metabolism. The arachidonic acid pathway is induced by iNOS stimulating COX-2, which forms prostaglandin E2 (PGE2). MCL-1 is a tumor marker promoting cell survival with its degradation inhibited by EGFR transactivation. HER2, known to be constitutively expressed in biliary tract cancer, is an oncogene activated by interacting with β-catenin and MUC1. HER2 is overexpressed by genomic amplification in 10% of gallbladder cancers (9). HER2 activation regulates production of MUC1, VEGF, and telomerase, which control vascularization and apoptosis. Vascularization can also be promoted by TGF-β induction of VEGF. The common mutations in samples of cholangiocarcinoma are KRAS, EGFR, and BRAF. KRAS mutation is one of the most frequently (10%–45%) altered genes in cholangiocarcinoma (95). As reported in many series, the incidence of KRAS mutation in intrahepatic cholangiocarcinoma ranges from 0% to 100%. The mutation is reported to be 100% in English patients and 4%–60% among Japanese and Thai patients (96–98). The variable incidence of KRAS mutations in intrahepatic cholangiocarcinoma may reflect the anatomical location of the disease. This is because KRAS mutation is crucial for carcinogenesis originating downstream of the biliary tree. In fact, KRAS point mutation is frequent in the periductal infiltrating type but not in the mass forming type. This suggests that KRAS mutation is important in intrahepatic cholangiocarcinoma arising near the hepatic hilus. About 31% of patients with intrahepatic cholangiocarcinoma associated with primary sclerosing cholangitis have KRAS mutation (97). A multi-institutional phase II trial tested a combination of bevacizumab and erlotinib, which showed clinical activity with infrequent grade 3 and 4 adverse effects in patients with advanced biliary cancers. On the basis of preliminary molecular analysis, presence of a KRAS mutation may alter erlotinib efficacy (99).

P. Sukharamwala et al. hepatic cholangiocarcinoma. In the nuclei of carcinoma cells, p53 protein was accumulated and immunohistochemically detectable in 25%–75% of intrahepatic cholangiocarcinoma cases (98,100). The positivity for p53 in nonpapillary intrahepatic cholangiocarcinoma was significantly higher than in intraductal papillary intrahepatic cholangiocarcinoma (65% vs 30%, p = 0.05) (98). Furthermore, p53 protein expression was significantly correlated with the progression of intraductal papillary biliary neoplasms. Dysplastic extrahepatic biliary tract epithelium was positive for P-cadherin in 91% of the cases and positive for CD24 in 71%. Normal and inflamed biliary tract epithelium were negative for P-cadherin and CD24 (84).

Therapy targets EGFR There are not many chemotherapy agents known to treat cholangiocarcinoma, but the predominant targets used for intervention of biliary tract cancer are EGFR, HER2, and VEGF/ R. Erlotinib is an orally active tyrosine-kinase inhibitor of EGFR. A phase II study using erlotinib for unresectable biliary tract cancer reported a 17% (n = 17) response rate with a median overall survival of 7.5 months (101). However, a randomized multicenter phase III study looked at progression free survival in 84 patients who received gemcitabine and oxaliplatin versus 96 patients who received gemcitabine and oxaliplatin with erlotinib. The median progression free survival was longer in the chemotherapy plus erlotinib group compared with chemotherapy alone, 5.8 months versus 4.2 months. However, this difference was not statistically significant (HR 0.80, 95% CI 0.61–1.03; p = 0.087). Additionally, there was no difference in overall survival between treatment groups, but erlotinib caused more diarrhea and toxic effects of the skin than did chemotherapy alone (102). Lapatinib is a dual TKI of the ERBB family members EGFR and HER2. A phase II study of lapatinib for hepatocellular carcinoma and biliary tract cancer resulted in no response in patients with biliary tract cancer and overall median survival of 5.2 months (103). Cetuximab is an anti-EGFR antibody, and a phase II study of gemcitabine and oxaliplatin (GEMOX) and cetuximab resulted in a response rate of 63% (n = 30) and a median overall survival of 15.2 months (104). The response rate for the combination therapy was 10% for complete and 53% for partial response. Cetuximab is a chimerized IgG1 monoclonal antibody which blocks binding of EGF and TGF-α to the EGF receptor. A phase II trial investigating cetuximab in combination with gemcitabine and oxaliplatin (GEMOX) for treating biliary tract cancer showed an objective response rate of 63% (105). Recently, a randomized phase II trial BINGO failed to show a significant improvement by adding cetuximab to the GEMOX regimen (106). A phase III randomized control trial of GEMOX with and without erlotinib in adenocarcinoma of the biliary tract, gallbladder, or ampulla of Vater was conducted. Progressive free survival was not prolonged, but there was significant benefit in progressive free survival of the GEMOX plus erlotinib arm for adenocarcinoma of the biliary tract.

Immunohistochemistry and histological definition Inactivation of TP53 is frequently involved in the carcinogenesis and progression of neoplasms arising in various organs, and it is also an important step in the development of intra-

VEGF Bevacizumab is an anti-VEGF recombinant humanized mAb. A phase II study using gemcitabine, oxaliplatin, and bevacizumab showed great results with a response rate of

ARTICLE IN PRESS Molecular profiles in foregut oncology 40% (n = 35) and median overall survival of 12.7 months (107). Another phase II multicenter study tested inhibition of both VEGF and EGFR with a combination of bevacizumab and erlotinib. The trial consisted of patients with unresectable biliary cancer receiving biweekly dosing of bevacizumab and daily erlotinib. The outcome was a response rate of 12% (n = 53) and a median overall survival of 9.9 months (108). In another phase II study, a combination of gemcitabine and oxaliplatin plus bevacizumab (GEMOX-B) showed significant antitumor activity and a tolerable safety profile in patients with advanced biliary tract cancer. Phase III studies of bevacizumab in addition to chemotherapy have shown a survival benefit in patients with several types of malignancies, including colorectal and lung cancers. VEGF is also related to cancer progression in biliary-tract cancer, and anti-angiogenic treatment using bevacizumab is expected to enhance the efficacy of chemotherapy. Other targeted therapies have been explored in phase II trials: the VEGF-targeting antibody bevacizumab combined with GEMOX led to a 7.0 months median progression free survival (108). A phase II study of sorafenib in patients with advanced biliary tract carcinomas resulted in a 2% (n = 46) response rate and 4.4 month median overall survival (109).

Pancreatic cancer

11 agnosis confirmation by immunohistochemistry. 89.29% of the tumors stained positive for CK19, or 25 of the 28 cases. 23 cases or 82.14% were positive for CD10. Therapy targets Receptor tyrosine kinase Many phase II and III studies are ongoing with agents that target multiple receptors. Sunitinib is one such drug targeting PDGFR, KIT, RET, CSF-1R, FLT3 and VEGFR 1–3, which has direct antitumor and antiangiogenic effects. A study of 66 patients with pancreatic NET had a median time of tumor progression of 7.7 months and a 1-year survival of 81.1%. A randomized phase III double blind study of sunitinib and placebo was closed early due to greater risk of progression and death in patients on the placebo arm. However, median progression free survival was 11.4 months with sunitinib versus 5.5 months on placebo and the European Commission approved sunitinib for use in 2010 on patients with unresectable or metastatic well differentiated pancreatic NET with disease progression (113). Additional drugs have been tested but lack the efficacy of sunitinib. Sorafenib selectively inhibits VEGFR, PDGFR, FLT3, KIT, and RET plus the RAF kinases BRAF and RAF-1 that activate the RTKs. 61% or 14 of 23 patients were progression free at 6 months (114). Gefitinib and bevacizumab achieve partial responses but convincing data and trials are lacking.

Endocrine tumors Pathogenesis According to the American Cancer Society, the rate of pancreatic cancer has been slowly increasing since 2000 and projects around 48,900 new cases and 40,500 deaths in 2015. Endocrine tumors tend to be the least common pancreatic cancer, with Chromogranin A (CgA) thought to be the optimal marker for most neuroendocrine tumors (NETs), also called islet cell tumors. CgA was confirmed to be the best tumor marker currently available for identifying patients suffering from NETs of the gastroenteropancreatic system, lung carcinoids and neuroblastomas, as it is independent of the biological characteristics of the tumor. CgA evaluation is recommended in the follow-up of patients with such tumors. Bernini et al. showed CgA was abnormal in 23 of 28 neuroendocrine cases (82% sensitivity), and in 6 cases it was the only circulating marker of disease. In gastroenteropancreatic tumors, CgA measurement identified all cases (100% sensitivity) (110). Specificity of CgA in patients with essential hypertension was 98%. CgA determination showed high sensitivity in identifying gastroenteropancreatic tumors, and, in association with catecholamines, detecting patients with pheochromocytoma. CgA sometimes appeared to be the only circulating marker of disease. Since the specificity of CgA proved to be excellent, this assay may be useful for diagnosis both of functioning and non-functioning NETs (110). Immunohistochemistry and histological definition Many NETs stain positive for neuron specific enolase, synaptophysin, and CD56. However, markers specifically for pancreatic NETs are CK19, CD10, CD99, COX2 and p27 (111). CK19 and CD99 have prognostic significance in predicting survival and biologic behavior (112). A study consisting of 28 patients that underwent endoscopic ultrasound guided fine needle aspiration cytology for diagnosis of NETs also had di-

sVEGFR2 Soluble receptor for vascular endothelial growth factor 2 (sVEGFR2) was evaluated in predicting clinical outcome in patients with metastatic NETs treated with pazopanib, a multi-tyrosine kinase and VEGF inhibitor (115). sVEGFR2 levels were found to be decreased at 12 weeks (median decrease: 20%, p < 0.0001), as well as with a longer duration of treatment (p = 0.0046). Patients with a greater decrease in sVEGFR2 (more than 20% decrease vs less than 20%) trended toward an improved mean progression free survival (12.6 vs 9.1 months, p = 0.067). mTOR Everolimus is a rapamycin derivative that inhibits the proliferative activity of mTOR, attenuates phosphorylation of downstream AKT targets, and induces cell death via apoptosis in vitro and in vivo (116). The everolimus activity was confirmed in a randomized double blind phase III trial (n = 410) evaluating everolimus plus best supportive care (n = 207) versus placebo plus best supportive care (n = 203) in patients with progressive well differentiate malignant pancreatic NET (117). The median progression free survival was 11 months versus 4.6 months with placebo, and the regulatory submission for everolimus to treat patients with pancreatic NET is in the process.

Exocrine tumors Pathogenesis Adenocarcinoma After diagnosis, pancreatic cancer has a short five year survival rate inversely proportional to stage. According to a study relating diabetes to pancreatic cancer, approximately 40% of pancreatic cancer patients had been diagnosed with diabetes one year prior and 16% within two years (118,119). Many biomarkers are known for pancreatic cancer, but KRAS is a predominant mutation found in 75– 90% of pancreatic carcinoma specimens. KRAS mutation

ARTICLE IN PRESS 12 causes constitutive activation and subsequent downstream signaling for cell proliferation. Early PanIN-1 specimens demonstrate this mutation before any architectural atypia, suggesting that it occurs early in the development of carcinoma. In metastatic colorectal cancer and non-small cell lung cancer, KRAS mutations are associated with resistance to epidermal growth factor receptor based monoclonal antibodies (e.g. panitumumab and cetuximab). However, there is no clear role for the testing of KRAS in pancreatic cancer due to a lack of available monoclonal antibodies. EGFR, or HER1, is overexpressed in 49% of pancreatic adenocarcinoma samples by IHC and 46% had an EGFR gene amplification by fluorescence in-situ hybridization (120). In one study, HER2 was overexpressed in up to 80% of pancreatic ductal adenocarcinomas (121). Conversely, a study using gene amplification showed HER2 overexpression in 16% of pancreatic ductal adenocarcinomas and IHC techniques resulted in 2+ or 3+ staining in 20% (122). Study results with HER2 overexpression are conflicting, but a review of HER2 overexpression in solid tumors states that pancreatic adenocarcinomas have generally low expression (123). A study at Johns Hopkins Hospital utilized 299 pancreatic cancer specimens and by IHC tested for SPARC. Both tumor cells and peritumoral fibroblasts were examined. SPARC expression in peritumoral fibroblasts was associated with shorter median survival. The median survival for patients expressing SPARC was 15 months versus 30 months for non expression, which was statistically significant. Tumor cell expression was not associated with outcome due to a myriad of contributing factors, such as tumor size and grade, positive lymph nodes, margin status, and age (30). Conversely, a phase I/II trial of nab-paclitaxel and gemcitabine observed SPARC expression in stromal cells and found it associated with longer overall survival (124). Deactivation of gemcitabine is a result of the enzyme cytidine deaminase, which is what nab-paclitaxel inhibits (125). The largest randomized phase III clinical trial, n = 861, treats previously untreated advanced pancreatic cancer patients with gemcitabine and nabpaclitaxel versus single agent gemcitabine (126). In favor of the combinatorial therapy, the median overall survival was 8.5 versus 6.7 months (hazard ratio [HR]: 0.72, 95% CI: 0.62– 0.84, p = 0.001), the median progression free survival was 5.5 versus 3.7 months (HR: 0.69, 95% CI: 0.58–0.82, p = 0.001), and 1 year survival was 35% versus 22%. The role of SPARC expression was not analyzed in this study and should be clarified. Mesothelin is abnormally expressed in squamous cell carcinomas of the esophagus, lung, and cervix. It has been identified as a target for investigational immunotherapies because of monoclonal antibody affinity (18). A study of pancreatic ductal adenocarcinoma specimens associated overexpression of mesothelin with lower disease-specific survival based on immunohistochemistry analysis (OR = 12.47, p = 0.01) (127). The odds ratio attributed to mesothelin expression was significant in this investigation, while traditional pathologic features such as size, lymph node metastases, resection margin status, and grade were not associated with long or short disease-specific survival. In a separate investigation, it was found that both mesothelin and MUC16 (also known as CA125) were overexpressed in invasive pancreatic adenocarcinoma but not overexpressed in normal pancreatic tissue or PanIN-3 (128). It has been proposed that pancre-

P. Sukharamwala et al. atic cancer invasion is enhanced by the interaction between mesothelin, MUC16, and matrix metalloproteinase-7 (MMP7). Depletion of MMP-7, however, may eliminate mesothelinmediated pancreatic cancer invasion (129). Deleted or functionally inactivated tumor suppressor genes have been shown to be more prevalent in advanced precursor lesions than KRAS (PanIN-2 and PanIN-3). Specific examples include TP53, TP16, and SMAD4. TP53 is one of the most commonly inactivated genes in oncology. Cells without this protein have decreased response to a number of cell stressors that would typically cause apoptosis, and more than 50% of pancreatic cancer specimens demonstrate inactivated p53. PanIN-2 and PanIN-3 lesions have demonstrated TP53 genetic mutations, pointing to a role later in the development of invasive cancer. p16 is a part of a tumor suppressor pathway involving the RB protein. The cascade involved in RB activation is a normal part of cellular surveillance and apoptosis but includes CDK4 and cyclin D. TP16 functional deletion is the most common genetic abnormality involved in this pathway seen in 80–95% of sporadic pancreatic cancers. PanIN-2 and PanIN-3 lesions have demonstrated TP16 functional deletions emphasizing the role in later stages of the neoplasia-carcinoma sequence. SMAD4 mediates the TGF-β signaling pathway and was first identified in pancreatic cancer in 1996. While inactivation of the gene is strongly correlated to the loss of protein expression, a study found that SMAD4 is associated with histopathological grades of pancreatic cancer (130). 188 intraepithelial neoplasms were examined by IHC for which 31% (9/29) of PanIN-3 lesions did not express the protein and the remaining 159 PanIN-1 and -2 expressed SMAD4. Ampullary Loss of the E-cadherin/β-catenin complex is related to poor prognosis in ampullary cancer. Loss of β-catenin is a predictor of recurrence in multivariate analysis. p53 in endoscopic biopsy specimens was of preoperative diagnostic value for carcinoma of the ampulla of Vater. The p53 positive tumors had a relatively higher malignant potential compared to p53 negative samples. The MIB-1/Ki-67 LI was useful in differentiating non-tumorous lesions from adenomas with low- or highgrade atypia. The MIB-1/Ki-67 LI had a prognostic value because clinicopathological factors of ampullary carcinoma correlated with MIB-1/Ki-67 LI. The molecular events leading to p53 accumulation in tumors of the ampulla of Vater occur early in the neoplastic process (131). Tumors of the ampulla of Vater with biopsies negative for malignancy but positive for p53 are very likely to be carcinomas. Immunohistochemistry and histological definition Adenocarcinoma Histologically, pancreatic ductal adenocarcinoma is typically a moderately well differentiated adenocarcinoma with tubular and ductal structures formed by mucus-secreting columnar cells. Invasive carcinoma is often associated with adjacent PanIN precursor lesions. IHC stains most often demonstrate CK7+, CK8+, MUC1+, CEA+. Pertinent negative stains are synaptophysin along with other neuroendocrine markers, AFP, and MUC2 (132). Ampullary It was found that positive CK17 staining, in particular the diffuse staining pattern, may aid in differential diagnosis of pancreaticobiliary adenocarcinomas from

ARTICLE IN PRESS Molecular profiles in foregut oncology extra-pancreaticobiliary nonmucinous adenocarcinomas. Coexpression of MUC1, MUC5AC, and CK17 is a unique feature of pancreaticobiliary adenocarcinomas and has not been reported in other types of tumors. MUC1+/CK17+ can be used as positive markers and MUC2+/CDX2+ can be used as negative markers for pancreaticobiliary adenocarcinomas in distinguishing pancreaticobiliary adenocarcinoma from the intestinal-type adenocarcinoma of duodenal papillary origin (133). CDX2 is a sensitive and specific marker for colorectal adenocarcinoma, but expression is decreased among higher grade and stage tumors and not invariably present in metastases from positive primaries. CDX2 may also be helpful in distinguishing adenocarcinomas of the ampulla from those arising in the pancreas and biliary tree. Two studies discriminated intestinal type carcinomas and pancreaticobiliary type carcinomas by the expression of apomucin MUC2 (134,135). The prognostic significance of some immunohistochemical markers such as Ki67 and p53 was evaluated in ampullary carcinoma (136–138). A separate study found a positive correlation between proliferating cell nuclear antigen and patient survival, and established that with a greater Ki67 index there came a worse 5-year survival (137). Therapy targets Proteins RRM1 overexpression is associated with gemcitabine resistance of pancreatic cancer (27). Molecular sequencing of human pancreatic cancer cell lines, which displayed gemcitabine resistance through in vitro analysis, showed overexpression of RRM1 by 4.5 times. 18 patients with recurrent pancreatic cancer consented to analysis of tumor specimens classified by RRM1 RNA overexpression. Patients with tumors overexpressing RRM1 were more likely to have disease progression despite gemcitabine therapy, and had shorter overall survival after gemcitabine treatment. ERCC1 overexpression is associated with resistance to platinum-based chemotherapy drugs in non-small cell lung cancer and metastatic breast cancer due to its role in DNA cross linking repair (10). Literature is lacking on the availability to determine the role of ERCC1 testing for platinumbased chemotherapy regimens in exocrine tumors. TS or TYMS, the target enzyme for 5-FU, generates dTMP for use in de novo DNA synthesis. High TS expression, measured by IHC, was associated with poor prognosis in pancreatic cancer. However, low TS expression was associated with a lack of survival advantage of 5-FU adjuvant chemotherapy (34). hENT1 is responsible for transferring gemcitabine into cells. Studies are conflicting on the significance of the expression of this protein, with some studies demonstrating improved response to gemcitabine with overexpression of hENT1 (11,13). However, other studies demonstrated no association between high hENT1 expression and improved survival with gemcitabine (12). TOPO1 is a prime contributor to DNA transcription. Irinotecan is an inhibitor of TOPO1 and functionally creates double stranded DNA breaks in affected cells. In patients with metastatic colorectal cancer, expression of TOPO1 has been associated with improved survival when treated with irinotecan (33). EGFR Cetuximab showed initial promise as a targeted therapy that enhanced the effectiveness of standard chemotherapeu-

13 tic agents in advanced exocrine pancreatic cancer. However, multiple randomized trials in advanced stages failed to show effectiveness. A phase III study showed no benefit of cetuximab when added to gemcitabine alone. Two phase II studies demonstrated no benefit when combined with gemcitabine and oxaliplatin, and a separate phase II study showed no benefit when combined with gemcitabine and cisplatin. Clinical trials of panitumumab in combination chemotherapy or chemoradiation regimens are ongoing for pancreatic cancer, but no published studies exist at this time. Erlotinib demonstrated increased overall survival when used in combination with gemcitabine for locally advanced or metastatic pancreatic adenocarcinoma (139). Lapatinib, the dual tyrosine kinase inhibitor for HER2 and EGFR, used in a cell line study demonstrated dose-dependent response of pancreatic cancer cell lines, including cell lines with erlotinib resistant KRAS mutations (140). Enhanced activity of lapatinib is demonstrated when cell lines are concomitantly treated with trametinib, an agent that blocks the downstream MAPK enzyme, MEK (141). HER2 In pancreatic cancer samples, 40% contained HER2 expression with 16% overamplification. Pancreatic cancer cell lines responded to trastuzumab by showing cell growth inhibition and decreased signal transduction. However, this has not translated into significant benefit.

Conclusion Chemotherapy strategies first began as an all out attack on newly forming or rapidly dividing cells, but much more collateral damage occurs with this approach. The more resources and research put into understanding the aberrant cancer cell mechanisms has allowed for a more targeted approach resulting in less severe side effects. This targeted approach after tissue analysis is now recognized as personalized or precision medicine and has many benefits. Tumor markers add confirmation along with imaging modalities for more accurate diagnosis of cancer type and precise staging. Efficacy of chemotherapeutics can be determined by proteins that are over or underexpressed. For example, the RAS proteins are effectors commonly mutated downstream of the EGF receptor. If a drug targets EGFR and the RAS protein is mutated to become constitutively active, that drug efficacy would be low compared to a drug targeting the overactive RAS protein and turning it off. Target specific treatments have been shown to be safe and efficacious. For example, the mAb cetuximab targets EGFR and is one such drug better used for maintenance therapy with chemotherapy in esophageal adenocarcinoma. Reports of combinatorial gefitinib and radiotherapy showed a synergistic effect and an additive effect in human esophageal carcinoma cell lines (46). However, significantly positive results are lacking and recommendations are against gefitinib-based gene target therapy for carcinomas of the gastroesophageal junction (142). Erlotinib was found to have limited activity in esophageal cancer and limited to patients with squamous cell (46). In addition, time to progression was noted to be longer in patients with squamous cell carcinoma, and studies suggest that erlotinib does not predict outcome. Trastuzumab in combination with chemotherapy could be considered a new standard of care for patients with HER2 positive advanced gastric or

ARTICLE IN PRESS 14 gastroesophageal junction cancer. Ramucirumab targets VEGF and yields overall survival improvement in patients with gastric and gastroesophageal junction adenocarcinoma. Trastuzumab and cisplatin with either capecitabine or 5-FU demonstrated by a ToGA trial the first survival benefits in gastric cancer diagnosed in later stages. KRAS tends to be an indicator of antiEGFR therapy, and anti-angiogenic therapy tends to allow better delivery of the drug to the tumor site. In hepatocellular carcinoma, signaling pathways are becoming the target strategy for therapy. The pathways under review are JAK/STAT, MAPK, and WNT/β-catenin. VEGF is a known prognostic factor, with both VEGF and EGFR being therapeutic targets. Dominant gallbladder therapeutic targets are EGFR, HER2, and VEGF/ R, with the standard of care being combinatorial gemcitabine and cisplatin or oxaliplatin. Some biological markers of biliary tract carcinogenesis and future potential therapeutic targets are iNOS, COX-2, MCL-1, HER2, β-catenin, MUC1, VEGF, TGF-β and EGFR (94). A high median overall survival was achieved with the use of GEMOX and cetuximab in biliary cancers. Gemcitabine, oxaliplatin, and bevacizumab also produced a high overall survival rate. In pancreatic cancer, future drugs may inhibit production of RRM1 allowing gemcitabine to properly target the cancer cells. Recently, annexin A8 was found to be an adverse prognostic factor and potential therapeutic target for pancreatic cancer (143). Erlotinib demonstrated increased overall survival when used in combination with gemcitabine for locally advanced or metastatic pancreatic adenocarcinoma. Many studies found that because SPARC is a means for nab-paclitaxel delivery, SPARC positive pancreatic cancer patients showed improved survival with a nab-paclitaxel and gemcitabine combination. 5-FU along with oxaliplatin and irinotecan (FOLFIRINOX) also had increased overall survival, because higher toxicity was restricted to younger and fit patients. Nab-paclitaxel and gemcitabine are used more exclusively on the elderly patients. With many possibilities and combinations of interventions available, continued studies will allow for generalizable knowledge to progress our current understanding of target therapy efficacies and help find a more efficient amalgamation causing less collateral damage.

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