gall bladder cancer: a cancer awaiting targeted therapy

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Cite this article: Roa I, Game A, de Toro G, Ibacache G, de Aretxabala X, et al. (2016) Gallbladder Cancer: A Cancer Awaiting Targeted Therapy. JSM Surg Oncol Res 1(2): 1007.

*Corresponding authorIván Roa E, Senador Estebanez 645, Temuco, Chile, Tel: 99203107, Email:

Submitted: 19 October 2016

Accepted: 18 November 2016

Published: 18 November 2016

ISSN: 2578-3688

Copyright© 2016 Roa et al.

OPEN ACCESS

Keywords•Gallbladder cancer•HER2/NEU•PTEN•PI3k•KRAS•IDH1•Targeted therapy

Short Communication

Gallbladder Cancer: A Cancer Awaiting Targeted TherapyIván Roa1,2*, Anakaren Game3, Gonzalo de Toro4, Gilda Ibacache5, Xabier de Aretxabala6, and Milind Javle7

1Creative Bioscience Santiago, Chile2Centro de Diagnostico Histopatologia-Citopatologia, Chile 3Interna de Medicina. Universidad del Desarrollo. Santiago, Chile4Pathology Unit Hospital de Puerto Montt, Puerto Montt, Chile5Centro de Diagnostico Histopatologia-Citopatologi, Chile6Departamento de Cirugía, Clínica Alemana de Santiago, Chile7UT-MD Anderson Cancer Center, USA

Abstract

Introduction: Gallbladder cancer (GBC) is an orphan disease with few studies aimed at incorporating these patients into targeted therapy.

Aims: To study the molecular alterations in advanced GBC that would justify the use of targeted therapy.

Materials and Methods: Overexpression of HER2/NEU, PTEN inactivation and point mutations of PI3K, KRAS and IDH1-2 were determined in patients with GBC using immunohistochemistry, direct sequencing and mass spectrometry.

Results: Overexpression of HER2/NEU was observed in 13.8% and PTEN inactivation in 3.9% of advanced GBC. Mutations were demonstrated in the PI3K (E542K, E545G, E545K, H1047L, H1047R), KRAS (G12D, G13D) and IDH1 (V178I) genes in 16.9%, 5.2% and 7%, respectively. PTEN inactivation and KRAS and IDH1 mutations were associated with a worse prognosis (p= 0.03; p=0.02 and p=0.009), respectively.

Conclusion: At least 4 genes represent metabolic pathways treatable with targeted therapy and that together represent around 45% of patients with advanced GBC who could benefit from targeted therapy.

INTRODUCTIONChile has the highest incidence of and mortality from

gallbladder cancer (GBC) in the world, in both genders, and it represents the second cause of death by malignant tumors in women in Chile [1-3]. GBC is in 20th place among all malignant tumors with an incidence of 2.2 × 106 inhabitants and a mortality in 22nd place (0.7 × 106) (http://globocan.iarc.fr last access December 2015).

Of the multiple molecular alterations observed in GBC, it has not yet been possible to pinpoint which ones are the “driver” genes or controllers of the neoplastic process and to differentiate them from the “passenger” genes, those observed mainly in sporadic malignant tumors like GBC in which epigenetic alterations predominate [4-6]. The most frequently mutated genes in GBC are: TP53 (41%), CDKN2A (28%) KRAS (19%), TERT (8%), CTNNB1 (8%) and PI3K (7%) (http://cancer.sanger.ac.uk/cosmic last access February 2016). The signaling pathway of the ERBB family [7,8] is one of the most frequently mutated in GBC [9,10]. These receptors participate in regulating cell proliferation,

differentiation and survival [11,12]. Their amplification mainly translates into protein over expression [13]. The receptor HER2/NEU, after dimerization activates a large variety of downstream pathways such as RAS-RAF-MEK-ERK1/2 or PI3k-AKT-MTOR with great influence on cell proliferation [14,15]. The overexpression and amplification of HER2/NEU has been demonstrated in gastric, esophageal and endometrial cancer, among others associated with a poor prognosis [16-19]. On the other hand, PTEN (phosphatase and tensin homolog) is a tumor suppressor gene that encodes a protein with phosphatase function that inactivates substrates like PI3K [20,21]. In many malignant tumors such as thyroid, endometrium, brain, prostate and melanomas [22], there can be PTEN functional or monoallelic loss, more than a biallelic mutation [20,21,23]. The absence of the PTEN functional protein permits the activation of PI3k with an even greater intensity than the activating mutation of PI3K itself [24,25]. The uncontrolled production of PIP3 is one of the most important effectors of the PI3K/AKT pathway with mTOR stimulating protein synthesis that regulate apoptosis [26]. The immunohistochemical expression of the PTEN protein is considered a good way to evaluate the functional state of the gene [27-29].

http://globocan.iarc.fr

http://cancer.sanger.ac.uk/cosmic



Roa et al. (2016)Email: [email protected]

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JSM Surg Oncol Res 1(2): 1007 (2016) 2/9

The PI3K pathway regulates numerous cell processes [30], including metabolism, survival, proliferation, apoptosis, growth and migration [31-33]. Most PI3K mutations occur in exons 9 and 20, which encode the kinase domains and in the p110 α subunit, respectively. The PI3K pathway is activated through various mechanisms, including PTEN loss, EGFR mutations, HER2/NEU amplifications and mutation of PI3K [34,35]. Between 2% and 30% of malignant tumors present mutations of the PI3K pathway[36-41]. By contrast, KRAS is a potent oncogene that actively participates in multiple tumors in the human being [42]. Its mutations can activate and interact with multiple metabolic pathways, among which the main ones are PI3K/PDK1/ AKT and BRAF/MEK1/MEK2/MAP [43]. The close relation existing between KRAS and HER2/NEU, cyclin-dependent kinases, BRAF and MEK1/MEK2, among others [43] has enabled the use of inhibitors of those pathways where KRAS participates in their activation [44-46]. One of the main utilities to determine the presence of KRAS-activating mutations is to predict the resistance to EGFR inhibiters [47-49]. Finally, the isocitrate dehydrogenase (IDH) genes encode for three enzymes whose activities are dependent on nicotinamide adenine dinucleotide (NADP) [50,51]. IDH1 and IDH2 are mutated in a wide variety of solid and hematological tumors [52], such as low-grade gliomas, secondary glioblastomas, acute myeloid leukemia, oral squamous cell carcinoma, cholangiocarcinomas, etc. [51,53-57]. The mutations of IDH1 and 2 rarely occur in the same tumor and are mutually exclusive of the EGFR and PTEN mutations [58,59]. It has been demonstrated that they have a role as prognostic biomarkers in several types of cancers, such as intrahepatic cholangiocarcinoma [60]. In GBC the information is still limited and it has been suggested that some of these molecular determinations could be used for the selection of patient groups in advanced stages that could receive targeted therapy [10,61-66].

The aim of this work is to summarize the molecular alterations that we have demonstrated in advanced GBC and which include the overexpression of HER2/NEU, PTEN inactivation and

mutations of the PI3K, KRAS and IDH1 genes and thus determine the existence of patient groups who could benefit from targeted therapy.

MATERIALS AND METHODSRepresentative samples of patients with GBC at different

stages with at least five years of clinical follow-up were selected. For each molecular test, the number of patients varies according to the type of technique, tissue availability, quantity and quality of extracted genomic DNA. Overexpression of HER2/NEU and PTEN inactivation were identified by immunohistochemistry, PI3K mutations gene by direct capillary sequencing and KRAS, IDH1 and IDH2 point mutations by mass spectrometry. All the studies were conducted with anonymized samples of patients with GBC of tissues fixed in formalin and embedded in paraffin. This work was approved by the Ethics Committee of the National Fund for Scientific and Technological Development (FONDECYT), Faculty of Medicine of the Clínica Alemana-Universidad del Desarrollo and MD Anderson Cancer Center, respectively.

Immunohistochemical expression of HER2/NEU and PTEN

187 and 108 cases of GBC respectively were included. A representative inclusion of the tumor was selected from each one. As a control for HER2/NEU, 75 non-tumor gallbladder samples were used. The immunostaining of PTEN was done only in the cases where there was tumor and non-tumor mucosa in the same sample. 4µ-thick histological sections were used and antigens were recovered using a microwave oven and in buffer citrate pH 6.0 and washed in PBS at pH 7.4. The monoclonal antibody anti-ErbB2 (NCL-CB11) NCL-CB11 (Novocastra, Newcastle, United Kingdom) was used in a dilution of 1/40 and the monoclonal antibody PTEN (D4.3) XP® (Rabbit mAb Cell Signaling, Boston, MA, USA) in a dilution of 1:125, incubated at room temperature for 60 minutes and developed with the Super Picture Polymer Detection Kit™ (Zymed SanFrancisco, CA, USA)

Figure 1 Immunohistochemical expression of HER2/NEU y PTEN.A: HER2/NEU negative control in chronic cholecystitis. B: strongly positive (3+) in all tumor cells of GBC. C: PTEN intense positivity in adenocarcinoma and stromal cells. D: malignant glandular groups PTEN negative with strong positivity in stromal cell (internal control)



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in a Dakoautostainer™. Positivity for HER2/NEU was measured according to the recommendation suggested for breast cancer by the CAP/ASCO guidelines (67), and for PTEN it was estimated according to the scale used by other authors [28,68]: intensity 0 = (negative), 1 + (weak), 2+ (moderate or intense) based on the comparison of non-tumor and tumor mucosa. Tests were performed with homologous scales that included factors like staining intensity and an estimation of the percentage of positive cells. The resulting score made it possible to conduct an ROC analysis to determine the greatest degree of sensitivity and specificity of the immunohistochemical staining [27,69]. There were no significant differences in terms of the semi-quantitative estimation of positivity initially described.

Study of Mutations of PI3K KRAS, NRAS, IDH1 and IDH2

DNA extraction: Representative areas of the tumor were selected and 10 sections were created of 10µ, in addition to a final section that was stained with hematoxylin eosin. The selection and demarcation of the tumor area and dissection were done under a microscope (Figure 2A). All samples included at least 30% tumor cells. The DNA extraction for direct sequencing was done using the AxyPrep Multisource Genomic DNA Miniprep kit Axygen® (Axygen Biosciences, Union City, CA, USA) as per the manufacturer’s instructions. For the study of point mutations by mass spectrometry (Sequenom MassArray®) of KRAS, NRAS, IDH1 and IDH2, the QIAamp DNA Mini® kit (Qiagen, Valencia, CA, USA) was used according to the manufacturer’s instructions.

PI3K Mutations: Direct capillary sequencing was performed on 130 GBC in search of point mutations in the 9 exons (E542K, E545K and E545G) and exon 20 (H1047R and H1057L) of the PI3K

gene. Previously reported primer sequences were used [70]. The program consisted of: initial denaturation at 95ºC for 5 minutes, followed by 35 cycles consisting of 95ºC for 45 seconds, 55ºC for 45 seconds and 72ºC for 45 seconds. Final elongation was at 72ºC for 10 minutes. PCR products were run in 2% agarose gel and stained with ethidium bromide. DNA was purified using the MinElute PCR Purification Kit (Qiagen Chatsworth, CA, USA) following the manufacturer’s instructions. The sequencing took place in an ABI PRISM BigDyeTM Terminator Cycle Sequencing Kit with AmpliTaq DNA polymerase (FS enzyme) (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. As a positive control, cell lines with constituent mutations in exon 9 (bladder cancer TCCSUP HTB-5 pE545K c1633G>A) and in exon 20 (breast cancer HCC1954 CRL-2338 c3140A>G H1047R) (ATCC, Manassas, VA) were used. Electropherograms were aligned and read on the ClustalW (EMBL-EBI) Blast platform (EMBL-EBI COSMIC version 75, http://www.ebi.ac.uk/Tools/msa/clustalw2, last access December 2, 2015; program currently out of service)

KRAS, NRAS, IDH1 and IDH2 Mutations: The somatic point mutations previously reported in 57 cases of GBC were examined by mass spectrometry Sequenom MassArray® (Sequenom, Inc, San Diego, CA, USA) (MD Anderson Cancer Center). The panel mutations of the study genes was designed on the database of the somatic mutations catalog of Atlas Genome Cancer (https://cgwb.nci.nih.gov/ last access July 2013). In the study of these three genes a total of 17 of the frequently observed point mutations was included in KRAS (A146PT, G10R, G12DAV, G12SRC, G13DAV, G13SRC, Q61EKX, Q61HHQ, Q61LPR), IDH1 (G70D, R132CGS, R132HL, V178-I) and IDH2 (G70D, R132CGS,

Figure 2 Selection of tumor areas and PI3K y KRAS Mutation Analysis.GBC paraffin block and their corresponding histological section. The tumor location has been demarcated and easily identifiable dissectible area (arrow). B: Electropherogram showing a point mutation (arrow) of PI3K (E542K G> A). C: KRAS mutation (G12D), recognizable due mass difference and intensity (mass spectrometry) between tumor (arrow) and non-tumor cell population.

http://www.ebi.ac.uk/Tools/msa/clustalw2

http://www.ebi.ac.uk/Tools/msa/clustalw2

https://cgwb.nci.nih.gov/

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R132HL, V178I). The detection limit was between 5% and 10% of mutated DNA. The data were analyzed on the Mass Array TYPER 4.0 genotyping software program (Sequenom).

Statistical Analysis: This was done by means of a chi-squared test and Fisher’s exact test for the contingency tables (p < 0.05) as well as an analysis of variance for the averages and Kaplan-Meier actuarial survival curves with a log rank test of significance, using the statistical programs WinSTAT® version 9.1 (R. Fitch Software, Bad Krozingen, Germany) and STATA/SE® version 12.1 (Stata Corp. LP, College Station, TX).

RESULTS

Over expression of HER2/NEU

The group comprised 187 patients with GBC, the general characteristics of whom are summarized in (Table 1). 88% of the cases were women with an average age of 61.6 years (SD ± 13.5 years) and 22 cases were men with an average age of 69.0 years (SD ± 14.3 years). The immunohistochemical staining in the 75 controls (gallbladders without tumor) was negative in 68 cases (91%) and seven cases (9%) were 2+ or ambiguous. No positive case (3+) was observed in the control group. In the GBC cases, 66.8% were negative (125 cases); 38 cases (20.3%) ambiguous and 24 cases (12.8%) were positive (3+) (Table 2) (Figure 1A,1B). HER2/NEU over expression was observed in 7.1% of the early carcinomas (mucosal and muscular) and in 13.8% of the advanced (subserosal or beyond) (p = 0.2). HER2/NEU overexpression was more frequent in the well differentiated tumors (17.4%) than the less differentiated ones (10.3%) (p = 0.3).

PTEN Inactivation

This group included 108 GBC in which there was tumor and non-tumor mucosa in the same sample. Immuno histochemical staining in the 108 tumors and their respective internal controls is summarized in (Table 3).

In 96.3% (104 cases) the non-tumor mucosa showed moderate or intense positivity (2+) and only 3.7% (4 cases) showed weak positivity (1+). No negative cases were observed in this group. However, in three cases (2.8%) of GBC there was an absence of PTEN expression or inactivation (Figure 1C-1D). The three cases were advanced GBC, so the frequency of PTEN inactivation in this group reached 4.1% (one subserosal case and two serosal cases). In GBC a positive (1+) or weak stain was observed in 54.6% of the cases and moderate or intense positivity (2+) in 42.6%, which might suggest a degree of partial or incomplete PTEN activity.

PI3K Mutation

130 GBC were studied. 22 cases (16.9%) with point mutations of PI3K were found (Figure 2B). All the mutations were missense substitutions. In 63.6% (14 cases) the mutations were located in exon 9 with the following distribution: E542K (64%), E545K (29%) and E545G (7%). The eight remaining cases were located in exon 20 (37.4%): H1047L (50%) and H1047R (50%). (Table 4) is a summary according to the type of mutation, change of amino acid and position. In 22% of the early GBC and in 14.6% of the advanced GBC PI3K Mutation were observed, which could suggest its appearance in the early stages of the development

of this neoplasia. No differences were found in the frequency and distribution of the mutations in terms of gender, age, tumor location or degree of histological differentiation.

KRAS and IHD1 Mutation

57 GBC were studied, of which all were advanced carcinomas. 3.5% of the tumors were well differentiated, with 66.7% being moderately or 28.1% being poorly differentiated. Point mutations in KRAS and IHD1 Mutation were detected by mass spectrometry (Figure 2C). Point mutations were detected in KRAS in 3 cases (5.2%) (G12DAV, G12DAV and G13DAV) and in IHD1 in 4 cases (7%), all V178I. In the case of IDH1 these mutations could represent a polymorphism from germinal series more than somatic mutations.

Molecular Markers and Survival

Actuarial survival of the total patient group with GBC at five years was 100% in the patients with mucosal carcinomas; 93% in the muscular carcinomas; and 32% and 5% in the subserosal and serosal carcinomas, respectively. At 70 months of follow-up all the patients with serosal carcinomas had died. The patients with overexpression of HER2/NEU showed an actuarial survival of 34% at five years compared to 41% of the patients who were negative. This difference was not significant (p = 0.2). Figure 3 illustrates the actuarial survival and its relation to PTEN inactivation and the presence of point mutations in PI3K, KRAS and IHD1. No significant differences were found in the survival and presence of point mutations in PI3K (p = 0.3). However, in the patients with PTEN inactivation, they had all died before

Table 1: General features of the GBC patients.Age n years SDFemale 165 61.6 ± 13.5Male 22 69.1 ± 14.3Total 187 62.5 ±14.4

Minimum 16 years

Maximum 90 years

LOCALIZATION n %Fundus 44 23.5Body 36 19.2Neck 11 6.2Diffuse 23 12.3Unaparent 73 38.8

187 100HISTOLOGICAL TYPE n %Adenocarcinoma 187 100HISTOLOGICAL DIFFERENTIATION n %

Well 40 21.4Moderate 104 55.4Poorly 43 23.2 187 100INFILTRATION LEVEL n %Mucosal 14 7.5Muscular 14 7.5Subserosal 125 66.8Serosal 34 18.2Total 187 100



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10 months (p = 0.03). The three patients who presented KRAS mutations and the four with IDH1 mutations had also died before 12 months of follow-up (p = 0.03 and p = 0.009, respectively).

DISCUSSIONOur results show a series of molecular alterations present in

GBC that could contribute to a better understanding of the de-velopment of this malignant tumor. These findings would also

Table 2: Immunohistochemical expression of HER2/NEU and PTEN. 2A Staining Intensity HER2 Type of lesion 0-1+ 2+ 3+ Negative Equivocal Positive n n n Total % 3+Non-Tumoral 68 7 0 75 0Gallbladder cancer 125 38 24 187 12.8 2B Staining Intensity PTENType of lesion 0 1+ or 2+ Negative Positive n % n %Non-tumoral 0 0 108 100Gallbladder cancer 3 3.7 105 96.3

Table 3: Summary of PI3K mutations.

Exon Mutation Position n Percentage Case number Mutation Cosmid ID Aachange Base change Type

9 E542K 9 40.9 1,2,5,7,8,15,18,21,22 GAA>AAA 760 Glutamic acid/ Lysine 1624 G>A Substitution -

Missense

9 E545G 1 4.6 11 GAG>GGG 764 Glutamic acid/ Lysine 1634 A>G Substitution -

Missense

9 E545K 4 18.2 12,17,19,20 GAG>AAG 763 Glutamic acid/ Lysine 1633 G>A Substitution -

Missense

20 H1047L 4 18.2 3,9,10,16 CAT>CTT 776 Histidine/Leucine 3140 A>T Substitution -

Missense

20 H1047R 4 18.2 4,6,13,14 CAT>CGT 775 Histidine/Arginine 3140 A>G Substitution -

Missense

Figure 3 Actuarial Survival.

justify their use as potential therapeutic targets for targeted therapy as with other malignant tumors [71,72]. We observed the overexpression of HER2/NEU in 13.8% of advanced GBC. Other authors have reported an overexpression of HER2/NEU in GBC between 2% and 25% [73-78]. However, the results are not comparable given the absence of standardized criteria in the evaluation of the positivity of HER2/NEU for GBC [73-77,78,79]. Therefore, we used the currently most widely accepted criterion



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as it is breast cancer [67]. As in patients with breast and stomach cancer, a group of patients with advanced GBC presenting over-expression of HER2/NEU would be eligible to receive treatment with monoclonal antibodies such as trastuzumab, pertuzumab, ramucirumab, or tyrosine kinase inhibitors like lapatinib [80,81]. The relationship between the overexpression of the protein and the degree of amplification of HER2/NEU with techniques like FISH or CISH, a relationship observed in over 85% of breast and stomach cancers [82-84], is yet to be established. It must also be pointed out that in 20% of GBC, staining considered ambiguous or inconclusive was observed, cases that would require confir-mation through the amplification of the HER2/NEU gene [85]. On the other hand, the absence of PTEN expression or inactiva-tion in 4.1% of advanced GBC would suggest it is a rare and de-layed phenomenon (loss of its expression was not shown in any of the 35 early carcinomas studied) [56,75,78]. The variability in the intensity of the positive PTEN staining in GBC (54.5% light staining 42.6% moderate or intense staining) could translate dif-ferent degrees of the gene’s activity or inactivation, which could be only monoallelic [20,22,23]. Other authors have reported PTEN inactivation between 0% and 5% in GBC [56,75,86], and thus its inactivation would not be of great importance in the deregulation of the PI3K/AKT/MTOR pathway in GBC. Other mechanisms of PTEN inactivation [21,68] cannot be ruled out. For those tumors with loss of PTEN function, experimental tar-geted therapies have been developed, such as the incorporation of lethal synthetic aberrations like NLK, PLK4 and MLK, the use of demethylating agents or agents able to derepress PTEN phos-phatase activity like P-REX2a and SIPL1; however, direct activa-tors have not had the expected results [87,88]. Thus, molecules that interfere with or inactivate the PI3K/AKT/MTOR pathway downstream have been used with greater success in tumors with loss of PTEN activity [21-23]. On the other hand, the PI3K gene has been found to be mutated in around 16.9% of GBC (14.6% of advanced GBC); therefore, it is a frequently mutated gene in GBC after TP53 (40%), CDKN2A (29%), KRAS (18%) and SMAD4 (18 %) (http://cancer.sanger.ac.uk/cosmic last access December 2015). Thus, GBC would be one of the malignant tumors with a high frequency of PI3K mutation, after prostate cancer (29%), breast cancer (27%), endometrial cancer (23%), and malignant tumors of the urinary tract (17%) (http://cancer.sanger.ac.uk/ cosmic last access December 2015). The frequency of somatic mutations in our report is slightly higher than other published works: Riener 1/23 (4%) [89], Pignochino en 1/13 (7.6%)(75), Javle (2/57 3,5%) [64], Despande (4/32, 12.5%) [90], Kumari (2/49 4.1%) [91], and the explanation for this is yet to be proven without being able to rule out population variations that could explain these differences [92,93]. The presence of mutations in early carcinomas (22%) could indicate that they are an early event in gallbladder carcinogenesis. For this pathway, a variety of inhibiters and blockers have been developed, many of them cur-rently in successful clinical use, such as everolimus, tesirolimus, idelalisib to name a few (94-96). With respect to KRAS, we found it mutated in 5.2% of advanced GBC. Despite it being one of the most extensively studied oncogenes and frequently mutated in a wide range of human neoplasias, specific inhibiters have failed to produce the expected result. Nevertheless, the interaction and redundancy of KRAS with the RAS/BRAF/MEK/ERK pathways, or RAF, MTOR, PI3K, PI3K/MTOR, AKT and MEK have enabled a con-

siderable number of molecules to be used to block this metabolic pathways, such as KRAS (sorafenib), MEK (selumetinib, trametin-ib), BRAF (dabrafenib, vemurafenib), MET (onartuzumab, tivan-tinib, of MTOR (temsirolimus), PIK3 (idelalisib, buparlisib), and AKT1 (TAS-117, AZD536) inhibitors, among others (http://www.cancer.gov/about-cancer/treatment/types/targeted-therapies/targeted-therapies-fact-sheet last access April 2016). Finally, the mutations observed in the IDH1 gene (IDH1_V178I) in 7% of ad-vanced GBC leave open the suggestion of their role in the devel-opment of this neoplasia. Similar findings have been reported in other biliary tract cancers [56]. IDH1 mutations have been dem-onstrated in up to 20% of high-grade gliomas, associated with a better response to treatment and better prognosis [57,97,98]. The mutation observed in GBC (IDH1_V178I) is not associated with an elevation of the 2-hydroxyglutarate (2-HG), which raises the question of whether this mutation represents a non-func-tional polymorphism, or whether the functional oncogenic effect could include a non-2-HG metabolic pathway [50]. The meaning and importance of the IDH1 mutations in GBC have not yet been determined; nevertheless, the four patients who presented this alteration had a worse prognosis and very low survival. The de-velopment of specific inhibiters like dasatinib, or IDH1 (AG-120) or IDH2 (AG-221) inhibiters are already in the clinical trial phase [60]. In summary, we can report that in advanced GBC we ob-served the overexpression of HER2/NEU in 13.8%; PTEN inacti-vation in 3.9%, and mutation of PI3K, KRAS and IDH1 in 14.6%, 7% and 5.2%, respectively. These alterations together represent 44.5% of advanced GBC. This group of patients could be poten-tially treatable with targeted therapy. Our preliminary results justify the initiation of clinical tests on patients with advanced GBC in high-risk countries like Chile with the aim of offering new life expectancies for this mortal disease.

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gall bladder cancer: a cancer awaiting targeted therapy

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