REVIEW URRENT C OPINION
Gallbladder cancer in Chile: what have we learned? Iva´n Roa a and Xabier de Aretxabala b
Purpose of review Gallbladder cancer (GBC) should be considered an orphan disease in oncology and represent a unique carcinogenetic model. This review will analyse some of the current aspects of GBC. Recent findings Chile has the highest incidence and mortality of GBC in the world. Most patients are diagnosed in advanced stages with few treatment options. During the last two decades, little progress has been made in early diagnosis and treatment. At the molecular level, recent access to next-generation sequencing and other techniques for detecting the mutations of multiple genes have made advances in this area. Summary The use of therapies targeted according to the detection of specific molecular alterations is in the early stages of evaluation and could represent a significant advance in the treatment of a large number of patients from developing countries. Keywords carcinogenesis, gallbladder neoplasm, molecular pathology
INTRODUCTION Gallbladder cancer (GBC) is an infrequent malignant tumour; however, Chile has the highest incidence and mortality of GBC in the world. Surgical curative treatment is only possible in early cases, those frequently found during cholecystectomies for symptomatic lithiasis; yet, most patients are diagnosed in advanced stages with few treatment options. There has been no significant progress in early diagnosis of this neoplasm, or in understanding its pathogenesis. At molecular level, the exome or whole genome sequencing for detecting the mutations of multiple genes have made advances in this area. HER2/neu gene amplification and mutations in the EGFR, KRAS, PI3K, IDH1 genes are the most frequent molecular abnormalities. In GBC, evidence indicates the participation of metabolic pathways that may be susceptible to targeted therapy, among which we can mention: RAS/RAF/ MAPkinase, EGFR/HER2, PI3k/AKT/MTOR, ERK/ MAP, JAK/STAT1, Receptor/NF-KappaB, TGF-Beta/ BMP, WNT and DNA repair mechanisms, apoptosis and inflammation regulation (XRCC1, OGG1, ERCC2, MSH2, CASP8, TLR2, TLR4 and PTGS2). Chronic inflammation and gallstones seem to be the most determinant factors in the pathogenesis of GBC and require further study to attain primary prevention of this disease. The cholecystectomy programme for symptomatic patients started in
Chile focusing on specific age groups, which is a reasonable alternative in terms of the epidemiological findings.
GENERAL AND EPIDEMIOLOGICAL ASPECTS For many years, Chile has had the highest incidence and mortality of GBC in the world, in both sexes, women in particular, and it represents the second cause of death by malignant tumours in women 4–5 : 1 compared with men [1,2] (http://globoc an.iarc.fr/Default.aspx. 2012). Other Indo-American countries such as Bolivia, Mexico, Ecuador follow in frequency, and elsewhere India and Pakistan [3] (http://globocan.iarc.fr/Default.aspx. 2012). It ranks 20th among all malignant tumours, with an incidence of 2.2 106 inhabitants and 22nd for mortality (0.7 106) and prevalence at 5 years of 16.8 106 inhabitants (http://globocan.iarc.fr/Defa ult.aspx. 2012).
a
Creative Bioscience and bDepartamento de Cirugı´a, Clı´nica Alemana, Santiago, Chile Correspondence to Dr Iva´n Roa, Bustos 2569, Providencia, 7511032 Santiago, Chile. Tel: +56 022761328; e-mail: [email protected] Curr Opin Gastroenterol 2015, 31:269–275 DOI:10.1097/MOG.0000000000000164
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Biliary tract
KEY POINTS GBC is an unusual tumour in developed countries. The highest incidence and mortality are observed in IndoAmerican countries such as Chile, Bolivia, Mexico, Ecuador, and India and Pakistan. Chronic inflammation and gallstones seem to be the most determinant factors in the pathogenesis of GBC, and dysplasia-carcinoma sequence is the most biologically plausible carcinogenic pathway. Exome or whole genome sequencing has demonstrated that HER2/neu gene amplification and EGFR, KRAS, PI3K, IDH1 gene mutations are the most frequent molecular abnormalities. The molecular profile and clinical trials are required to validate target therapy in advanced cases. Prophylactic cholecystectomy in patients with symptomatic stones in high-risk populations could contribute cost-effectiveness to reduce its incidence and high mortality.
manifestations of the disease. Both conditions determine that in countries such as Chile, thousands of patients undergo surgery to solve the problem of lithiasis [2]. The preoperative diagnosis of GBC is rare and does not exceed 15%. This means that most of the cancers are discovered during surgery or in the subsequent anatomopathological study of the surgical specimen [12,13]. In 2006, Chile instituted a GBC secondary prevention programme in the public healthcare system, promoting preventive cholecystectomies for patients with symptomatic lithiasis between 35 and 49 years of age [14]. In this group, in addition to solving the problem of symptomatic lithiasis and its complications, it is expected to have an effect on the age group with the frequency of GBC in the early stages (mucosal and intramuscular carcinomas). It is hoped that an impact on mortality will be seen starting in 2015. We must point out that in our series of 1366 GBCs studied over 20 years, 25.3% of the patients were detected in initial stages, wherein the simple cholecystectomy was curative in around 90% of the cases [15 ]. Gallstones are considered the most important risk factor for GBC [4,16]. The highest incidence rates of GBC and frequency of lithiasis are observed in the indigenous populations of America and India (Mapuche women of Chile, Pima Indians in the United States and eastern India) with a prevalence of 49.4, 75.8 and 21.6% of gallstones, respectively [4,16]. In our studies, over 90% of GBCs have been linked to the presence of gallstones. A relation has been shown between the size and volume of the gallstones and the frequency of cancer [17]. Thus, larger stones with a greater volume may reflect a longer period present in the gallbladder lumen, thereby prolonging its irritative–inflammatory–carcinogenic effect on the gallbladder mucosa [18]. Almost all GBCs are associated with chronic inflammation of the gallbladder wall; yet, the inflammation–cancer connection has not been proven unequivocally [19]. Activation of inflammatory pathways such as COX-2, mediators such as nuclear factor kappa B, formation of reactive oxygen species and nitrogen, inflammatory cytokines (interleukin6, IL6), prostaglandins and microRNAs may occur through changes in the proliferation and apoptosis processes [20]. This may lead to an increase in the basal rate of somatic mutations, DNA methylation, as well as angiogenesis processes with potential neoplastic transformation [20,21]. The anomalous pancreaticobiliary junction has been reported particularly in Far Eastern countries [22], but in the West, this does not seem to be significant in gallbladder carcinogenesis [23]. This malformation may be more associated with carcinomas of the bile ducts [24,25] with frequent &
GBC has a markedly irregular distribution and incidence on the different continents, being high in Latin America and Asia, moderate in Central and Eastern Europe (Hungary, Germany and Poland), and low in the Mediterranean countries of Europe and the United States [4]. Among the different races, the Mapuche Indians of Chile show the highest incidence and mortality of GBC (12.3 106 in men and 27.3 106 in women), followed by the Indians of New Mexico with an annual average rate of 8.9 106. In Asia, there has also been an increase in the frequency of GBC in women in northern India and Pakistan, as well as in men in Korea (8.1 106 in men and 5.6 106 in women) [1,3,5]. In general, GBC should be considered an orphan disease in oncology. There is significantly less scientific information about GBC than other malignant tumours (around 0.3% of indexed publications). If molecular aspects of GBC are included, this corresponds to approximately 0.1% of available scientific information. The most important risk factors for GBC include gallstones, being female, age, nutritional conditions such as obesity and parity [4–7,8 ]. Other factors such as chronic inflammation of the gallbladder wall, adenomas and an anomalous pancreaticobiliary junction have also proven to be high-risk factors for developing a GBC [1,3–5,8 ,9,10], as well as the cholecystectomy rate [5,9,11]. GBC is expressed clinically in advanced stages; the presence of gallstones and chronic inflammation are the most frequently associated &&
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Gallbladder cancer in Chile Roa and de Aretxabala
mutations in the KRAS and p53 genes [26]. Nor has a clear connection been established between environmental factors such as heavy metals (chromium, lead, arsenic, zinc) [27] and occupational factors (miners exposed to radon) with GBC [28,29]. Excessive consumption of hot red pepper has been reported with a greater relative risk of cancer (2.9 times) in the population of southern Santiago; however, this association has not been shown in other studies [30,31]. Obesity with BMI more than 30 kg/m2 increases the risk of developing different types of cancers, including GBC [4,32]. It has been reported that for every five-point increase in BMI, the relative risk of developing GBC increases 1.59 times for women and 1.09 for men [33,34]. Other elements associated with obesity such as metabolic syndrome and diabetes mellitus may be a risk factor for developing gallstones. There may be a higher risk in diabetic patients, even in the absence of gallstones [34,35].
CARCINOGENIC PATHWAYS: DYSPLASIACARCINOMA VS. ADENOMA-CARCINOMA SEQUENCE In glandular epithelial tumours or adenocarcinomas of the digestive tract, two carcinogenic models are recognized: dysplasia-carcinoma and adenoma-carcinoma sequencing [36]. The former is based on alterations of the mucosal epithelium that undergoes a metaplasia secondary to chronic irritation and inflammation, transforms to dysplasia and then a carcinoma in situ (CIS). The adenoma-carcinoma model proposes the malignant transformation of a benign tumour or adenoma. In the gallbladder, there is morphological and molecular evidence to support the existence of both biological models (Fig. 1) [37 ,38]. Atypical intraepithelial lesions or dysplasias are considered the preneoplastic lesions of this organ [39]. CIS may evolve into an intramucosal carcinoma and then an invasive carcinoma [3,37 ,40]. Chronic inflammation may cause damage from epithelial regeneration with adaptive changes such as metaplasias. Most early carcinomas show metaplastic foci in adjacent mucosa [41]. The importance of the metaplasia and its connection to dysplasia has not been clarified completely in the gallbladder. In our series of 210 early carcinomas, which were completely mapped, in more than 80% of the cases, there were intraepithelial lesions (CIS and dysplasias) adjacent to the tumour [42,43]. These findings strongly suggest that the malignant transformation in the gallbladder starts from the epithelium and not from an adenoma [37 ,43]. Adenomas are rare in the gallbladder (0.001% of the cholecystectomies for &&
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symptomatic lithiasis). Some isolated cases have revealed the presence of adenomatous foci in gallbladder adenocarcinomas or malignant foci in adenomas [36,44]. Of the 210 early carcinomas mentioned, the remnant foci of an adenoma were found in only six cases (2.8%). Also, in our series, in 25% of the adenomas (eight out of 32 cases), there were adenocarcinoma foci [43,45]. The ages of patients with adenomas, adenomas with malignant changes and adenocarcinomas (50, 58 and 64 years, respectively) also suggest their progression over time [46].
MORPHOLOGICAL ALTERATIONS OF GALLBLADDER CANCER In a recent study, the authors summarized the morphological characteristics observed in 1366 GBCs studied in the same institution for 20 years with a standardized study protocol [15 ]. Worthy of note among the conclusions: the gallbladder length in the cancers was greater than the gallbladder without tumour (10.8 vs. 9.01 cm P ¼ 0.00001). The relative risk of GBC in women older than 55 years with lengths over 9.5 cm was more than five times that of the controls. The thickness of the gallbladder wall was seen to be increased in 97% of the GBCs, there being a relation between wall thickness and survival (P ¼ 0.0001). GBCs with a wall of more than 10 mm thick had a survival of 14% at 24 months follow up. Cholesterolosis had a negative association with GBC; the patients with cholesterolosis were 11.3 times less likely to have a cancer than patients who did not. In 38.8% (368 cases), it was not possible to establish the presence of a tumour on the mucosa surface during the macroscopic examination to any degree of certainty, as the alterations observed in chronic cholecystitis were indistinguishable in these cases. On the contrary, the location of the tumour was also shown to be an independent prognostic factor, with the prognosis being worse in those located on the serosa side than those on the hepatic side (P ¼ 0.0006) in the pT2 stages [47]. Tumour involvement of the Rokitansky– Aschoff sinuses is a factor to consider, as a CIS may transform into an advanced carcinoma (pt2) [48,49]. Among the histological elements, the most important independent prognostic factors in GBC are the level of tumour infiltration in the wall, blood vessel and lymphatic involvement (P < 0.001). Blood vessel involvement had 100% mortality [15 ]. &
&
MOLECULAR ALTERATIONS IN GALLBLADDER CANCER Just as in other epithelial malignant tumours of the digestive tract, the GBC is the result of multiple
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(a)
(b)
(c)
(d)
FIGURE 1. Histological sequence from dysplasia to adenocarcinoma of the gallbladder. Histologic features of normal gallbladder epithelium (a). Dysplasia with pseudo stratification, nuclear enlargement, hyperchromasia and loss of polarity (b). Carcinoma in situ with larger nuclei, irregular chromatin distribution and presence of nucleoli (c). Mucosal carcinoma with micro papillary projections and frequent atypical mitosis in upper portions of the epithelium (d).
genetic alterations involving multiple genes from multiple metabolic pathways, the importance of which has not yet been clearly established (Fig. 2) [5,50–53]. There is a consensus that the most frequently altered processes are those that regulate growth signals, the evasion of apoptosis, the potential for limitless replication, angiogenesis, tissue invasion and the generation of metastasis [54,55]. Other alterations that can produce functional changes in genes are altering their activity at the level of transcription and regulating posttranscriptional processes [56]. DNA hypermethylation of the gene promoter areas, loss of heterozygosity (LOH) and histone acetylation and so on are also frequent gene inactivation mechanisms [57–61]. Most of the molecular information available on GBC corresponds to studies of individual genes or segments of metabolic pathways. Frequent mutations have been found in the oncogenes KRAS, BRAF, EGFR and PIK3CA [62–64], as well as tumour suppressor genes such as CDKN2A, TP53 and SMAD4 [59,65,66]. As with other cancers, GBC presents numerous somatic mutations, of which only a small subgroup contributes to tumour progression. The distinction between mutations in the ‘driver’ genes 272
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(controllers) of the multiple mutations in neutral or ‘passenger’ genes, which participate in only a small way in cancer development, has been made only recently [67,68]. The few studies conducted by means of exome sequencing, ultrasequencing or mass spectrometry on GBC [69–73] show mutation patterns defined by the overwhelming prevalence of C > T mutations at TCN sites. Frequently mutated genes in GBC include TP53 (47.1%), KRAS (7.8%) and ERBB3 (11.8%). Nevertheless, the ErbB signalling that includes EGFR, ERBB2, ERBB3, ERBB4 is one of the most frequently altered pathways in 21 of 57 cases (36.8%) [69,71]. Patients with a mutation of the ErbB pathways had a worse prognosis [69]. Our work on 187 cases of GBC revealed ERBB2 overexpression in 13.8% of the advanced GBCs, suggesting the usefulness of blockers for this pathway in these patients [74]. The large number of pathways and altered genes involved as well as the complexity of the interrelations between genes that cooperate or are antagonized is exemplified in the report of a patient with three metachronic epithelial cancers (breast, gallbladder and lung), whose GBC was studied via Volume 31 Number 3 May 2015
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Histopathological and molecular events in dysplasia–carcinoma sequence of GBC
P53 COX-2 TSG methylation LOH
HER2/neu EGFr PI3k p16ink4
KRAS IDH1 CD44 Cancer
Normal mucosa
Age
Chronic inflammation metaplasia
Dysplasia-CIS
Early carcinoma
(46.5)
(51.9–56.8)
(61.5)
Advanced carcinoma
(62.9)
FIGURE 2. Dysplasia-carcinoma sequence: scheme of molecular alterations frequently observed at different histological stages in the development and progression of cancer of the gallbladder.
next-generation sequencing and in which 24 545 single nucleotide polymorphisms (SNPs) were detected, of which 10 868 (44.27%) were SNPs in coding regions and 1077 (4.38%) were in untranslated regions (UTRs). Four thousand four hundred and eighty mutations were observed in 3367 genes with at least 30 proteins truncated and 10 mutations that generated different protein isoforms. Substitutions or premature terminations were found in 132 proteins coded by cancer-related genes. In the Ras-MAPK pathway, 18 genes were homozygotically mutated, 15 growth factors/cytokines and their receptors, nine transcription factors, six proteins in the Wnt signalling pathway [70], which gives an account of the complexity of the problem and what remains to be ascertained. Recently, therapeutic use has been suggested for some molecular determinations, such as amplification of the HER2/neu gene and mutations in the EGFR, KRAS, PI3K, IDH1 genes, the determination of which could be used routinely in targeted therapy for these patients [69,72,74]. In GBC, evidence indicates the participation of metabolic pathways that may be susceptible to targeted therapy, among which we can indicate the following: RAS/RAF/MAP kinase [75,76], EGFR/HER2 [77], Pi3k/AKT/MTor [76,78,79], ERK/MAPK [80,81], JAK/STAT1 [82], receptor/NF-KappaB [83], TGF-Beta/BMP [79,84], WNT, DNA repair mechanisms and apoptosis and inflammation regulation (XRCC1, OGG1, ERCC2, MSH2, CASP8, TLR2, TLR4 and PTGS2) [56,85,86].
CONCLUSION GBC continues to be a public health issue in some countries such as Chile. The advances achieved in the understanding of the carcinogenesis have been insufficient. The curative nature of treatment is only possible in early cases, those frequently found during cholecystectomies for symptomatic lithiasis. Surgical treatment has proven to be of more use in the intermediate stages of the disease. Access to new molecular biology techniques in developing countries will make it possible to determine the molecular profile of high-risk populations and to initiate valid clinical trials for these populations. The cholecystectomy programme for symptomatic patients started in Chile focusing on specific age groups is a reasonable alternative in terms of the epidemiological findings and these results are still being assessed and may be exportable to other high-risk populations. Chronic inflammation and gallstones seem to be the most determinant factors in the pathogenesis of GBC and require further study to attain primary prevention of this disease. GBCs reported in the absence of gallstones for now continue to be a mystery. Acknowledgements We would like to thank Dr Helen Lowry for her assistance in the manuscript translation. Financial support and sponsorship This manuscript was financed by FONDECYT project 1120208.
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Conflicts of interest There are no conflicts of interest.
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Gallbladder cancer in Chile Roa and de Aretxabala 59. Kagohara LT, Schussel JL, Subbannayya T, et al. Global and gene-specific DNA methylation pattern discriminates cholecystitis from gallbladder cancer patients in Chile 10.2217/fon 14. 165. Future Oncol 2015; 11:233–249. 60. Black JC, Whetstine JR. Chromatin landscape: methylation beyond transcription. Epigenetics 2011; 6:9–15. 61. Letelier P, Brebi P, Tapia O, Roa JC. DNA promoter methylation as a diagnostic and therapeutic biomarker in gallbladder cancer. Clin Epigenetics 2012; 4:11. 62. Deshpande V, Nduaguba A, Zimmerman SM, et al. Mutational profiling reveals PIK3CA mutations in gallbladder carcinoma. BMC Cancer 2011; 11:60. 63. Pignochino Y, Sarotto I, Peraldo-Neia C, et al. Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas. BMC Cancer 2010; 10:631. 64. Riener MO, Bawohl M, Clavien PA, Jochum W. Rare PIK3CA hotspot mutations in carcinomas of the biliary tract. Genes Chromosomes Cancer 2008; 47:363–367. 65. Rai R, Tewari M, Kumar M, et al. p53: its alteration and gallbladder cancer. Eur J Cancer Prev 2011; 20:77–85. 66. Roa JC, Anabalon L, Roa I, et al. Promoter methylation profile in gallbladder cancer. J Gastroenterol 2006; 41:269–275. 67. Ji X, Tang J, Halberg R, et al. Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study. BMC Cancer 2010; 10:426. 68. Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science 2013; 339:1546–1558. 69. Li M, Zhang Z, Li X, et al. Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway [10. 1038/ng. 3030]. Nat Genet 2014. 70. He ML, Chen Y, Chen Q, et al. Multiple gene dysfunctions lead to high cancersusceptibility: evidences from a whole-exome sequencing study. Am J Cancer Res 2011; 1:562–573. 71. Jiao Y, Pawlik TM, Anders RA, et al. Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet 2013; 45:1470–1473. 72. Javle M, Rashid A, Churi C, et al. Molecular characterization of gallbladder cancer using somatic mutation profiling. Hum Pathol 2014; 45:701–708. 73. Kumari N, Corless CL, Warrick A, et al. Mutation profiling in gallbladder cancer in Indian population. Indian J Pathol Microbiol 2014; 57:9–12.
74. Roa I, de Toro G, Schalper K, et al. Overexpression of the HER2/neu gene: a new therapeutic possibility for patients with advanced gallbladder cancer. Gastrointest Cancer Res 2014; 7:42–48. 75. Pai RK, Mojtahed K. Mutations in the RAS/RAF/MAP kinase pathway commonly occur in gallbladder adenomas but are uncommon in gallbladder adenocarcinomas. Appl Immunohistochem Mol Morphol 2011; 19:133–140. 76. Faris JE, Zhu AX. Targeted therapy for biliary tract cancers. J Hepatobiliary Pancreat Sci 2012; 19:326–336. 77. Subbiah IM, Subbiah V, Tsimberidou AM, et al. Targeted therapy of advanced gallbladder cancer and cholangiocarcinoma with aggressive biology: eliciting early response signals from phase 1 trials. Oncotarget 2013; 4:153–162. 78. Leal P, Garcia P, Sandoval A, et al. AKT/mTOR substrate P70S6K is frequently phosphorylated in gallbladder cancer tissue and cell lines. Onco Targets Ther 2013; 6:1373–1384. 79. Zong H, Yin B, Zhou H, et al. Inhibition of mTOR pathway attenuates migration and invasion of gallbladder cancer via EMT inhibition. Mol Biol Rep 2014; 41:4507–4512. 80. Li Q, Yang Z. Expression of phospho-ERK1/2 and PI3-K in benign and malignant gallbladder lesions and its clinical and pathological correlations. J Exp Clin Cancer Res 2009; 28:65. 81. Wu XS, Wang XA, Wu WG, et al. MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/MAPK pathway. Cancer Biol Ther 2014; 15:806–814. 82. Zhang P, Jiang G, Gao J, et al. SAHA down-regulates the expression of indoleamine 2,3-dioxygenase via inhibition of the JAK/STAT1 signaling pathway in gallbladder carcinoma cells. Oncol Rep 2013; 29:269–275. 83. Iwase R, Haruki K, Fujiwara Y, et al. Combination chemotherapy of nafamostat mesylate with gemcitabine for gallbladder cancer targeting nuclear factor-kB activation. J Surg Res 2013; 184:605–612. 84. Koninger J, di Mola FF, Di Sebastiano P, et al. Transforming growth factor-beta pathway is activated in cholecystolithiasis. Langenbecks Arch Surg 2005; 390:21–28. 85. Huang M, Shen A, Ding J, Geng M. Molecularly targeted cancer therapy: some lessons from the past decade. Trends Pharmacol Sci 2014; 35:41–50. 86. Roa I, de Aretxabala X, Lantadilla S, Munoz S. ERCC1 (excision repair crosscomplementing 1) expression in pT2 gallbladder cancer is a prognostic factor. Histol Histopathol 2011; 26:37–43.
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Gallbladder cancer in Chile: what have we learned? Iva´n Roa a and Xabier de Aretxabala b
Purpose of review Gallbladder cancer (GBC) should be considered an orphan disease in oncology and represent a unique carcinogenetic model. This review will analyse some of the current aspects of GBC. Recent findings Chile has the highest incidence and mortality of GBC in the world. Most patients are diagnosed in advanced stages with few treatment options. During the last two decades, little progress has been made in early diagnosis and treatment. At the molecular level, recent access to next-generation sequencing and other techniques for detecting the mutations of multiple genes have made advances in this area. Summary The use of therapies targeted according to the detection of specific molecular alterations is in the early stages of evaluation and could represent a significant advance in the treatment of a large number of patients from developing countries. Keywords carcinogenesis, gallbladder neoplasm, molecular pathology
INTRODUCTION Gallbladder cancer (GBC) is an infrequent malignant tumour; however, Chile has the highest incidence and mortality of GBC in the world. Surgical curative treatment is only possible in early cases, those frequently found during cholecystectomies for symptomatic lithiasis; yet, most patients are diagnosed in advanced stages with few treatment options. There has been no significant progress in early diagnosis of this neoplasm, or in understanding its pathogenesis. At molecular level, the exome or whole genome sequencing for detecting the mutations of multiple genes have made advances in this area. HER2/neu gene amplification and mutations in the EGFR, KRAS, PI3K, IDH1 genes are the most frequent molecular abnormalities. In GBC, evidence indicates the participation of metabolic pathways that may be susceptible to targeted therapy, among which we can mention: RAS/RAF/ MAPkinase, EGFR/HER2, PI3k/AKT/MTOR, ERK/ MAP, JAK/STAT1, Receptor/NF-KappaB, TGF-Beta/ BMP, WNT and DNA repair mechanisms, apoptosis and inflammation regulation (XRCC1, OGG1, ERCC2, MSH2, CASP8, TLR2, TLR4 and PTGS2). Chronic inflammation and gallstones seem to be the most determinant factors in the pathogenesis of GBC and require further study to attain primary prevention of this disease. The cholecystectomy programme for symptomatic patients started in
Chile focusing on specific age groups, which is a reasonable alternative in terms of the epidemiological findings.
GENERAL AND EPIDEMIOLOGICAL ASPECTS For many years, Chile has had the highest incidence and mortality of GBC in the world, in both sexes, women in particular, and it represents the second cause of death by malignant tumours in women 4–5 : 1 compared with men [1,2] (http://globoc an.iarc.fr/Default.aspx. 2012). Other Indo-American countries such as Bolivia, Mexico, Ecuador follow in frequency, and elsewhere India and Pakistan [3] (http://globocan.iarc.fr/Default.aspx. 2012). It ranks 20th among all malignant tumours, with an incidence of 2.2 106 inhabitants and 22nd for mortality (0.7 106) and prevalence at 5 years of 16.8 106 inhabitants (http://globocan.iarc.fr/Defa ult.aspx. 2012).
a
Creative Bioscience and bDepartamento de Cirugı´a, Clı´nica Alemana, Santiago, Chile Correspondence to Dr Iva´n Roa, Bustos 2569, Providencia, 7511032 Santiago, Chile. Tel: +56 022761328; e-mail: [email protected] Curr Opin Gastroenterol 2015, 31:269–275 DOI:10.1097/MOG.0000000000000164
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Biliary tract
KEY POINTS GBC is an unusual tumour in developed countries. The highest incidence and mortality are observed in IndoAmerican countries such as Chile, Bolivia, Mexico, Ecuador, and India and Pakistan. Chronic inflammation and gallstones seem to be the most determinant factors in the pathogenesis of GBC, and dysplasia-carcinoma sequence is the most biologically plausible carcinogenic pathway. Exome or whole genome sequencing has demonstrated that HER2/neu gene amplification and EGFR, KRAS, PI3K, IDH1 gene mutations are the most frequent molecular abnormalities. The molecular profile and clinical trials are required to validate target therapy in advanced cases. Prophylactic cholecystectomy in patients with symptomatic stones in high-risk populations could contribute cost-effectiveness to reduce its incidence and high mortality.
manifestations of the disease. Both conditions determine that in countries such as Chile, thousands of patients undergo surgery to solve the problem of lithiasis [2]. The preoperative diagnosis of GBC is rare and does not exceed 15%. This means that most of the cancers are discovered during surgery or in the subsequent anatomopathological study of the surgical specimen [12,13]. In 2006, Chile instituted a GBC secondary prevention programme in the public healthcare system, promoting preventive cholecystectomies for patients with symptomatic lithiasis between 35 and 49 years of age [14]. In this group, in addition to solving the problem of symptomatic lithiasis and its complications, it is expected to have an effect on the age group with the frequency of GBC in the early stages (mucosal and intramuscular carcinomas). It is hoped that an impact on mortality will be seen starting in 2015. We must point out that in our series of 1366 GBCs studied over 20 years, 25.3% of the patients were detected in initial stages, wherein the simple cholecystectomy was curative in around 90% of the cases [15 ]. Gallstones are considered the most important risk factor for GBC [4,16]. The highest incidence rates of GBC and frequency of lithiasis are observed in the indigenous populations of America and India (Mapuche women of Chile, Pima Indians in the United States and eastern India) with a prevalence of 49.4, 75.8 and 21.6% of gallstones, respectively [4,16]. In our studies, over 90% of GBCs have been linked to the presence of gallstones. A relation has been shown between the size and volume of the gallstones and the frequency of cancer [17]. Thus, larger stones with a greater volume may reflect a longer period present in the gallbladder lumen, thereby prolonging its irritative–inflammatory–carcinogenic effect on the gallbladder mucosa [18]. Almost all GBCs are associated with chronic inflammation of the gallbladder wall; yet, the inflammation–cancer connection has not been proven unequivocally [19]. Activation of inflammatory pathways such as COX-2, mediators such as nuclear factor kappa B, formation of reactive oxygen species and nitrogen, inflammatory cytokines (interleukin6, IL6), prostaglandins and microRNAs may occur through changes in the proliferation and apoptosis processes [20]. This may lead to an increase in the basal rate of somatic mutations, DNA methylation, as well as angiogenesis processes with potential neoplastic transformation [20,21]. The anomalous pancreaticobiliary junction has been reported particularly in Far Eastern countries [22], but in the West, this does not seem to be significant in gallbladder carcinogenesis [23]. This malformation may be more associated with carcinomas of the bile ducts [24,25] with frequent &
GBC has a markedly irregular distribution and incidence on the different continents, being high in Latin America and Asia, moderate in Central and Eastern Europe (Hungary, Germany and Poland), and low in the Mediterranean countries of Europe and the United States [4]. Among the different races, the Mapuche Indians of Chile show the highest incidence and mortality of GBC (12.3 106 in men and 27.3 106 in women), followed by the Indians of New Mexico with an annual average rate of 8.9 106. In Asia, there has also been an increase in the frequency of GBC in women in northern India and Pakistan, as well as in men in Korea (8.1 106 in men and 5.6 106 in women) [1,3,5]. In general, GBC should be considered an orphan disease in oncology. There is significantly less scientific information about GBC than other malignant tumours (around 0.3% of indexed publications). If molecular aspects of GBC are included, this corresponds to approximately 0.1% of available scientific information. The most important risk factors for GBC include gallstones, being female, age, nutritional conditions such as obesity and parity [4–7,8 ]. Other factors such as chronic inflammation of the gallbladder wall, adenomas and an anomalous pancreaticobiliary junction have also proven to be high-risk factors for developing a GBC [1,3–5,8 ,9,10], as well as the cholecystectomy rate [5,9,11]. GBC is expressed clinically in advanced stages; the presence of gallstones and chronic inflammation are the most frequently associated &&
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Gallbladder cancer in Chile Roa and de Aretxabala
mutations in the KRAS and p53 genes [26]. Nor has a clear connection been established between environmental factors such as heavy metals (chromium, lead, arsenic, zinc) [27] and occupational factors (miners exposed to radon) with GBC [28,29]. Excessive consumption of hot red pepper has been reported with a greater relative risk of cancer (2.9 times) in the population of southern Santiago; however, this association has not been shown in other studies [30,31]. Obesity with BMI more than 30 kg/m2 increases the risk of developing different types of cancers, including GBC [4,32]. It has been reported that for every five-point increase in BMI, the relative risk of developing GBC increases 1.59 times for women and 1.09 for men [33,34]. Other elements associated with obesity such as metabolic syndrome and diabetes mellitus may be a risk factor for developing gallstones. There may be a higher risk in diabetic patients, even in the absence of gallstones [34,35].
CARCINOGENIC PATHWAYS: DYSPLASIACARCINOMA VS. ADENOMA-CARCINOMA SEQUENCE In glandular epithelial tumours or adenocarcinomas of the digestive tract, two carcinogenic models are recognized: dysplasia-carcinoma and adenoma-carcinoma sequencing [36]. The former is based on alterations of the mucosal epithelium that undergoes a metaplasia secondary to chronic irritation and inflammation, transforms to dysplasia and then a carcinoma in situ (CIS). The adenoma-carcinoma model proposes the malignant transformation of a benign tumour or adenoma. In the gallbladder, there is morphological and molecular evidence to support the existence of both biological models (Fig. 1) [37 ,38]. Atypical intraepithelial lesions or dysplasias are considered the preneoplastic lesions of this organ [39]. CIS may evolve into an intramucosal carcinoma and then an invasive carcinoma [3,37 ,40]. Chronic inflammation may cause damage from epithelial regeneration with adaptive changes such as metaplasias. Most early carcinomas show metaplastic foci in adjacent mucosa [41]. The importance of the metaplasia and its connection to dysplasia has not been clarified completely in the gallbladder. In our series of 210 early carcinomas, which were completely mapped, in more than 80% of the cases, there were intraepithelial lesions (CIS and dysplasias) adjacent to the tumour [42,43]. These findings strongly suggest that the malignant transformation in the gallbladder starts from the epithelium and not from an adenoma [37 ,43]. Adenomas are rare in the gallbladder (0.001% of the cholecystectomies for &&
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symptomatic lithiasis). Some isolated cases have revealed the presence of adenomatous foci in gallbladder adenocarcinomas or malignant foci in adenomas [36,44]. Of the 210 early carcinomas mentioned, the remnant foci of an adenoma were found in only six cases (2.8%). Also, in our series, in 25% of the adenomas (eight out of 32 cases), there were adenocarcinoma foci [43,45]. The ages of patients with adenomas, adenomas with malignant changes and adenocarcinomas (50, 58 and 64 years, respectively) also suggest their progression over time [46].
MORPHOLOGICAL ALTERATIONS OF GALLBLADDER CANCER In a recent study, the authors summarized the morphological characteristics observed in 1366 GBCs studied in the same institution for 20 years with a standardized study protocol [15 ]. Worthy of note among the conclusions: the gallbladder length in the cancers was greater than the gallbladder without tumour (10.8 vs. 9.01 cm P ¼ 0.00001). The relative risk of GBC in women older than 55 years with lengths over 9.5 cm was more than five times that of the controls. The thickness of the gallbladder wall was seen to be increased in 97% of the GBCs, there being a relation between wall thickness and survival (P ¼ 0.0001). GBCs with a wall of more than 10 mm thick had a survival of 14% at 24 months follow up. Cholesterolosis had a negative association with GBC; the patients with cholesterolosis were 11.3 times less likely to have a cancer than patients who did not. In 38.8% (368 cases), it was not possible to establish the presence of a tumour on the mucosa surface during the macroscopic examination to any degree of certainty, as the alterations observed in chronic cholecystitis were indistinguishable in these cases. On the contrary, the location of the tumour was also shown to be an independent prognostic factor, with the prognosis being worse in those located on the serosa side than those on the hepatic side (P ¼ 0.0006) in the pT2 stages [47]. Tumour involvement of the Rokitansky– Aschoff sinuses is a factor to consider, as a CIS may transform into an advanced carcinoma (pt2) [48,49]. Among the histological elements, the most important independent prognostic factors in GBC are the level of tumour infiltration in the wall, blood vessel and lymphatic involvement (P < 0.001). Blood vessel involvement had 100% mortality [15 ]. &
&
MOLECULAR ALTERATIONS IN GALLBLADDER CANCER Just as in other epithelial malignant tumours of the digestive tract, the GBC is the result of multiple
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(a)
(b)
(c)
(d)
FIGURE 1. Histological sequence from dysplasia to adenocarcinoma of the gallbladder. Histologic features of normal gallbladder epithelium (a). Dysplasia with pseudo stratification, nuclear enlargement, hyperchromasia and loss of polarity (b). Carcinoma in situ with larger nuclei, irregular chromatin distribution and presence of nucleoli (c). Mucosal carcinoma with micro papillary projections and frequent atypical mitosis in upper portions of the epithelium (d).
genetic alterations involving multiple genes from multiple metabolic pathways, the importance of which has not yet been clearly established (Fig. 2) [5,50–53]. There is a consensus that the most frequently altered processes are those that regulate growth signals, the evasion of apoptosis, the potential for limitless replication, angiogenesis, tissue invasion and the generation of metastasis [54,55]. Other alterations that can produce functional changes in genes are altering their activity at the level of transcription and regulating posttranscriptional processes [56]. DNA hypermethylation of the gene promoter areas, loss of heterozygosity (LOH) and histone acetylation and so on are also frequent gene inactivation mechanisms [57–61]. Most of the molecular information available on GBC corresponds to studies of individual genes or segments of metabolic pathways. Frequent mutations have been found in the oncogenes KRAS, BRAF, EGFR and PIK3CA [62–64], as well as tumour suppressor genes such as CDKN2A, TP53 and SMAD4 [59,65,66]. As with other cancers, GBC presents numerous somatic mutations, of which only a small subgroup contributes to tumour progression. The distinction between mutations in the ‘driver’ genes 272
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(controllers) of the multiple mutations in neutral or ‘passenger’ genes, which participate in only a small way in cancer development, has been made only recently [67,68]. The few studies conducted by means of exome sequencing, ultrasequencing or mass spectrometry on GBC [69–73] show mutation patterns defined by the overwhelming prevalence of C > T mutations at TCN sites. Frequently mutated genes in GBC include TP53 (47.1%), KRAS (7.8%) and ERBB3 (11.8%). Nevertheless, the ErbB signalling that includes EGFR, ERBB2, ERBB3, ERBB4 is one of the most frequently altered pathways in 21 of 57 cases (36.8%) [69,71]. Patients with a mutation of the ErbB pathways had a worse prognosis [69]. Our work on 187 cases of GBC revealed ERBB2 overexpression in 13.8% of the advanced GBCs, suggesting the usefulness of blockers for this pathway in these patients [74]. The large number of pathways and altered genes involved as well as the complexity of the interrelations between genes that cooperate or are antagonized is exemplified in the report of a patient with three metachronic epithelial cancers (breast, gallbladder and lung), whose GBC was studied via Volume 31 Number 3 May 2015
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Gallbladder cancer in Chile Roa and de Aretxabala
Histopathological and molecular events in dysplasia–carcinoma sequence of GBC
P53 COX-2 TSG methylation LOH
HER2/neu EGFr PI3k p16ink4
KRAS IDH1 CD44 Cancer
Normal mucosa
Age
Chronic inflammation metaplasia
Dysplasia-CIS
Early carcinoma
(46.5)
(51.9–56.8)
(61.5)
Advanced carcinoma
(62.9)
FIGURE 2. Dysplasia-carcinoma sequence: scheme of molecular alterations frequently observed at different histological stages in the development and progression of cancer of the gallbladder.
next-generation sequencing and in which 24 545 single nucleotide polymorphisms (SNPs) were detected, of which 10 868 (44.27%) were SNPs in coding regions and 1077 (4.38%) were in untranslated regions (UTRs). Four thousand four hundred and eighty mutations were observed in 3367 genes with at least 30 proteins truncated and 10 mutations that generated different protein isoforms. Substitutions or premature terminations were found in 132 proteins coded by cancer-related genes. In the Ras-MAPK pathway, 18 genes were homozygotically mutated, 15 growth factors/cytokines and their receptors, nine transcription factors, six proteins in the Wnt signalling pathway [70], which gives an account of the complexity of the problem and what remains to be ascertained. Recently, therapeutic use has been suggested for some molecular determinations, such as amplification of the HER2/neu gene and mutations in the EGFR, KRAS, PI3K, IDH1 genes, the determination of which could be used routinely in targeted therapy for these patients [69,72,74]. In GBC, evidence indicates the participation of metabolic pathways that may be susceptible to targeted therapy, among which we can indicate the following: RAS/RAF/MAP kinase [75,76], EGFR/HER2 [77], Pi3k/AKT/MTor [76,78,79], ERK/MAPK [80,81], JAK/STAT1 [82], receptor/NF-KappaB [83], TGF-Beta/BMP [79,84], WNT, DNA repair mechanisms and apoptosis and inflammation regulation (XRCC1, OGG1, ERCC2, MSH2, CASP8, TLR2, TLR4 and PTGS2) [56,85,86].
CONCLUSION GBC continues to be a public health issue in some countries such as Chile. The advances achieved in the understanding of the carcinogenesis have been insufficient. The curative nature of treatment is only possible in early cases, those frequently found during cholecystectomies for symptomatic lithiasis. Surgical treatment has proven to be of more use in the intermediate stages of the disease. Access to new molecular biology techniques in developing countries will make it possible to determine the molecular profile of high-risk populations and to initiate valid clinical trials for these populations. The cholecystectomy programme for symptomatic patients started in Chile focusing on specific age groups is a reasonable alternative in terms of the epidemiological findings and these results are still being assessed and may be exportable to other high-risk populations. Chronic inflammation and gallstones seem to be the most determinant factors in the pathogenesis of GBC and require further study to attain primary prevention of this disease. GBCs reported in the absence of gallstones for now continue to be a mystery. Acknowledgements We would like to thank Dr Helen Lowry for her assistance in the manuscript translation. Financial support and sponsorship This manuscript was financed by FONDECYT project 1120208.
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Conflicts of interest There are no conflicts of interest.
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Gallbladder cancer in Chile Roa and de Aretxabala 59. Kagohara LT, Schussel JL, Subbannayya T, et al. Global and gene-specific DNA methylation pattern discriminates cholecystitis from gallbladder cancer patients in Chile 10.2217/fon 14. 165. Future Oncol 2015; 11:233–249. 60. Black JC, Whetstine JR. Chromatin landscape: methylation beyond transcription. Epigenetics 2011; 6:9–15. 61. Letelier P, Brebi P, Tapia O, Roa JC. DNA promoter methylation as a diagnostic and therapeutic biomarker in gallbladder cancer. Clin Epigenetics 2012; 4:11. 62. Deshpande V, Nduaguba A, Zimmerman SM, et al. Mutational profiling reveals PIK3CA mutations in gallbladder carcinoma. BMC Cancer 2011; 11:60. 63. Pignochino Y, Sarotto I, Peraldo-Neia C, et al. Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas. BMC Cancer 2010; 10:631. 64. Riener MO, Bawohl M, Clavien PA, Jochum W. Rare PIK3CA hotspot mutations in carcinomas of the biliary tract. Genes Chromosomes Cancer 2008; 47:363–367. 65. Rai R, Tewari M, Kumar M, et al. p53: its alteration and gallbladder cancer. Eur J Cancer Prev 2011; 20:77–85. 66. Roa JC, Anabalon L, Roa I, et al. Promoter methylation profile in gallbladder cancer. J Gastroenterol 2006; 41:269–275. 67. Ji X, Tang J, Halberg R, et al. Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study. BMC Cancer 2010; 10:426. 68. Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science 2013; 339:1546–1558. 69. Li M, Zhang Z, Li X, et al. Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway [10. 1038/ng. 3030]. Nat Genet 2014. 70. He ML, Chen Y, Chen Q, et al. Multiple gene dysfunctions lead to high cancersusceptibility: evidences from a whole-exome sequencing study. Am J Cancer Res 2011; 1:562–573. 71. Jiao Y, Pawlik TM, Anders RA, et al. Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas. Nat Genet 2013; 45:1470–1473. 72. Javle M, Rashid A, Churi C, et al. Molecular characterization of gallbladder cancer using somatic mutation profiling. Hum Pathol 2014; 45:701–708. 73. Kumari N, Corless CL, Warrick A, et al. Mutation profiling in gallbladder cancer in Indian population. Indian J Pathol Microbiol 2014; 57:9–12.
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