Skip to main content
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 May 12.
Published in final edited form as: Hum Pathol. 2013 Nov 12;45(4):701–708. doi: 10.1016/j.humpath.2013.11.001

Molecular Characterization of Gallbladder Cancer using Somatic Mutation Profiling

Milind Javle *, Asif Rashid *, Chaitanya Churi *, Siddhartha Kar *, Mingxin Zuo *, Agda Karina Eterovic *, Graciela M Nogueras-Gonzalez *, Siraj Ali **, Filip Janku *, Rachna Shroff *, Thomas A Aloia *, Jean-Nicholas Vauthey *, Steven Curley *, Gordon Mills *, Ivan Roa ***
PMCID: PMC4428571  NIHMSID: NIHMS660933  PMID: 24508317

Abstract

Gallbladder cancer is relatively uncommon with high incidence in certain geographic locations, including Latin America, East and South Asia and Eastern Europe. Molecular characterization of this disease has been limited and targeted therapy options for advanced disease remain an open area of investigation. In the present study, surgical pathology obtained from resected gallbladder cancer cases (n=72) was examined for the presence of targetable, somatic mutations. All cases were formalin-fixed and paraffin-embedded (FFPE). Two approaches were used: a) mass spectroscopy-based profiling for 159 point (‘hot-spot’) mutations in 33 genes commonly involved in solid tumors and b) next-generation sequencing (NGS) platform that examined the complete coding sequence of in 182 cancer-related genes. Fifty-seven cases were analyzed for hotspot mutations and 15 for NGS. Fourteen hotspot mutations were identified in nine cases. Of these, KRAS mutation was significantly associated with poor survival on multivariate analysis. Other targetable mutations included PIK3CA (N=2) and ALK (N=1). On NGS, 26 mutations were noted in 15 cases. P53 and PI3 kinase pathway (STK11, RICTOR,TSC2) mutations were common. One case had FGF10 amplification while another had FGF3-TACC gene fusion, not previously described in gallbladder cancer. In conclusion, somatic mutation profiling using archival FFPE samples from gallbladder cancer is feasible. NGS, in particular may be a useful platform for identifying novel mutations for targeted therapy.

Keywords: gallbladder neoplasms, mutational analysis, DNA Sequencing

INTRODUCTION

Gallbladder cancer affects over 140,000 patients annually worldwide and over 100,000 will die each year from this disease.(1) Women are affected more than men and in the U.S.; Hispanic population and Alaskan natives have a disproportionately high incidence of gallbladder cancer.(2) There is a remarkable geographic variation with the highest incidence rates reported in India, Korea, Japan, Czech Republic, Slovakia, Spain, Columbia, Chile, Peru, Bolivia, and Ecuador. Etiologies include chronic cholelithiasias, Salmonella infections, toxin exposure, obesity and rarely due to genetic diseases like Hereditary Non-Polyposis Cancer Coli (HNPCC) and type 1 neurofibromatosis. Gallbladder cancer is thought to be at least partly the consequence of chronic inflammation-induced genetic changes.

The current molecular profiling data of gallbladder cancer are limited to small case series or case reports that include one or more oncogenes. High-throughput screening for targetable mutations in this disease is lacking. An understanding of the molecular characteristics and heterogeneity of gallbladder cancer is critical towards improving the treatment paradigm for this disease. An impetus for such characterization is the potential of targeted therapies directed against the products of these molecular aberrations including the tumor proteomic profile. Once the underlying molecular abnormalities of a cancer are identified, targeted inhibitors can be discovered and result in incremental benefit even in genetically heterogeneous malignancies. For instance, in lung cancer the identification of echinoderm microtubule associated protein like 4 - anaplastic lymphoma kinase (EML4-ALK) mutation has led to a targeted approach with crizotinib and tumors with epidermal growth factor receptor (EGFR) mutations to the development of erlotinib or gefitinib.(3) High-throughput technologies that can rapidly screen for somatic mutations in archival formalin-fixed, paraffin-embedded specimens are critical for this effort. The Sequenom Massarray™ system is ideally suited for the detection of low abundance mutations and can be customized towards targeted therapeutics.(4, 5) In the present study, we used the high-throughput Sequenom MassArray™ approach to investigate mutations in 33 genes in a cohort of gallbladder cancer cases to determine the frequency of genetic mutations in this population. We also explored next generation sequencing (NGS) to examine a wider panel of genetic aberrations in a limited number of gallbladder cancer cases.

MATERIAL AND METHODS

Tumor samples

Surgically resected, formalin fixed paraffin embedded (FFPE) specimens were obtained for 72 patients with gallbladder cancer. The paraffin embedded blocks were sectioned, and hematoxylin & eosin (H&E) stained slides were reviewed by surgical pathology to confirm the tumor content in each section. Ten serial sections (4µm) were cut from selected tissue blocks and areas with tumor tissue were micro dissected from those slides using the H&E slides as templates. Approval for the study was obtained from the institutional review board at MD Anderson Cancer Center.

DNA Extraction

The samples were deparaffinized using xylene washes followed by ethanol (100%) washes. DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer protocol. DNA was quantitated using the NanoQuant system (Tecan Group, Männedorf, Switzerland).

Sequenom MassArray

Hotspot mutational analysis was performed using the Sequenom MassARRAY™ using the iPLEX™ technology (Sequenom, Inc, San Diego, CA). This technology allows for parallel high-throughput screening while using minimal DNA obtained from FFPE specimens (6). Mutations were screened by using amplification through polymerase chain reaction (PCR) and single-base primer extension where the wild type or mutated base was identified by mass spectrometry. Briefly, for each mutation site, PCR and extension primers were designed using Sequenom, Inc. Assay Design. PCR reactions were run following manufacture’s protocol. After PCR, amplicons were cleaned using EXO-SAP® kit (Sequenom) in a GeneAmp 9700 thermocycler (Applied Biosystems). Then the primer was then extended by IPLEX™ chemistry, desalted using Clean Resin (Sequenom), and spotted onto SpectroChip matrix chips (Sequenom) using a nanodispenser (Samsung). Chips were run in duplicate on a Sequenom MassArray Matrix-assisted laser desorption/ionization-Time of Flight (MALDI-TOF) MassArray system. We used Sequenom Typer Software for visual inspection and interpretation of mass spectra. Reactions where the mutant peak represented more than 10% of the wild type peak were scored as positive. The data analysis was performed using MassArray TYPER 4.0 genotyping software (Sequenom) where the SNP calls were divided in 3 groups: conservative, moderate and aggressive calls, depending on the level of confidence.

The Sequenom panel used here was previously designed by the Characterized Cell Line Core (Core Shared Resources – CCSG) at MD Anderson Cancer Center with the aim of detecting somatic DNA alterations in cancer samples. The Sequenom panel was designed based on data form the Catalogue of Somatic Mutations in Cancer (COSMIC) and the Cancer Genome Atlas (TCGA) that reported those alterations (and others in the panel) as somatic mutations previously. A total of 159 point mutations in 33 genes frequently mutated in solid tumors including were analyzed. The analytical sensitivity of the assay [limit of detection (LOD) 5%–10% of mutant DNA in total DNA] is higher than conventional Sanger sequencing (LOD: 10%–20%) and similar to pyrosequencing (LOD: 5%–10%). The advantages offered by the MassARRAY system include high-throughput screening for many hot-spot mutations in parallel, use of minimal DNA isolated from formalin-fixed paraffin-embedded tissues, ability to detect coexisting multiple mutations, and cost and time effectiveness. Appendix 1 lists the genes and mutations investigated in this study.

Next Generation Sequencing

The pathologic diagnosis of each case of gallbladder cancer was confirmed on routine hematoxylin- and eosin-stained slides. All samples sent for DNA extraction contained a minimum of 20% DNA derived from tumor cells. DNA was extracted from 40 mm of FFPE tissue using the Maxwell 16 FFPE Plus LEV DNA Purification kit (Promega™) and quantified using a standardized PicoGreen fluorescence assay (Invitrogen™). Library construction was performed as described previously, using 50–200 ng of DNA sheared by sonication to B100–400 bp before end-repair, dA addition and ligation of indexed, Illumina™ sequencing adaptors (7, 8). Enrichment of target sequences (3320 exons of 182 cancer-related genes and 37 introns from 14 genes recurrently rearranged in cancer representing approximately 1.1Mb of the human genome) was achieved by solution-based hybrid capture with a custom Agilent SureSelect™ biotinylated RNA baitset (8). The selected libraries were sequenced on an Illumina HiSeq 2000 platform using 49149 paired-end reads. Sequence data from genomic DNA was mapped to the reference human genome (hg19) using the Burrows-Wheeler Aligner™ and were processed using the publicly available Sequence Alignment/Map (SAMtools), Picard and Genome Analysis Toolkit (9, 10). Point mutations were identified by a Bayesian algorithm; short insertions and deletions determined by local assembly; gene copy number alterations (amplifications) by comparison to process matched normal controls; and gene fusions/rearrangements were detected by clustering chimeric reads mapped to targeted introns as described previously (11).

Statistical Analysis

Given the limited number of cases analyzed for NGS, only the cases analyzed for hotspot mutations (n=57) were analyzed for their association with survival. Overall survival (OS) was calculated as the number of months from surgery (or core biopsy) to death or last follow-up date. Patients who were alive at their last follow-up were censored on that date. Time to Progression (TTP) was calculated as the number of months from surgery (or core biopsy) to progression. Patients without tumor progression at their last follow-up were censored on that date. The Kaplan-Meier product limit method was used to estimate the median OS for each clinical/demographic factor.(12) Univariate Cox proportional hazards regression was used to model the association between potential predictors and OS. Multivariate Cox proportional hazards regression was used to model all the statistically significant variables in the univariate setting. Backwards selection method was used to remove variables that did not remain significant in the multivariate model.(13) For each factor, medians, hazard ratios (HR), their 95% confidence intervals (CI), and proportional hazards regression p-values are presented in tables. Similar analyses were performed for time to progression. Statistical significance was considered at P-values of <0.05.

Statistical analysis was performed using STATA/SE version 12.1 statistical software (Stata Corp. LP, College Station, TX).

RESULTS

Fifty-seven cases of gallbladder cancer were analyzed for hotspot mutations and 15 for NGS. Patient demographics are described in Table 1. Fourteen hotspot mutations (Table 2) were identified from eleven different tumors within this sample set, with three cases demonstrating more than 1 mutation. IDH1 mutations were the most frequent (n=4). The others identified included mutations of KRAS (n=3), NRAS (n=3), PIK3CA (n=2) and MET (n=1). Of these, IDH1 and MET may represent germline polymorphisms rather than somatic mutations as discussed below. Figure 1 demonstrates the PIK3CA, IDH1 and KRAS mutations. Figures 2A–2D depict the histologies (H&E) of four gallbladder cancer cases along with their corresponding mutations. A total of 36/57 (63.2%) patients enrolled in the study have expired to date. A univariate survival analysis on these data demonstrated a significant relationship of overall survival with six factors. The overall risk of mortality was associated with treatment with chemotherapy (HR: 2.84; 95%CI: 1.23–6.53; p=0.014), lymphatic infiltration (HR: 2.72; 95%CI: 1.22–6.04; p=0.014), venous infiltration (HR: 2.27; 95%CI: 1.08–4.79; p=0.031), perineural infiltration (HR: 2.14; 95%CI: 1.06–4.33; p=0.033), positive KRAS mutation (HR: 3.56; 95%CI: 1.06–11.92; p=0.040), and with a positive IDH1 mutation (HR: 4.04; 95%CI: 1.35–12.13; p=0.013) (Fig.3a). In addition, patients who had chemotherapy were at greater risk of progressing than non-treated patients (HR: 13.82; 95%CI: 1.84–103.84; p=0.011).

Table 1.

Summary Statistics of Patient Demographics and Tumor Characteristics

CHARACTERISTICS Type of Analysis

Hotspot Mutation Analysis
(N = 57)
Next Generation Sequencing
(N=15)

Age (years) MEDIAN (RANGE)
62 (30–84)
MEDIAN (RANGE)
62 (48–78)

N (%) N (%)

Sex
  Male 25 (44%) 5 (33%)
  Female 32 (56%) 10 (67%)

Ethnicity
  Asian 1 (2%) 1 (7%)
  Hispanic 8 (14%) 1 (7%)
  Black 5 (9%) 0 (0%)
  White 43 (75%) 13 (86%)

Type of Surgery
  None 3 (5%) 4 (27%)
  Simple (Laparoscopic) 31 (54%) 6 (40%)
  Radical 23 (41%) 5 (33%)

Adjuvant Therapy
  Chemotherapy 25 (44%) 13 (87%)
  Chemotherapy & Radiation 11 (19%) 2 (13%)
  None 21 (37%) 0 (0%)

Histological Type
  Adenocarcinoma 51 (90%) 15 (100%)
  Adenosquamous 4 (7%) 0 (0%)
  Carcinosarcoma 2(3%) 0 (0%)

Degree of Differentiation
  Poor 16 (28%) 7 (47%)
  Moderate 38 (67%) 6 (40%)
  Well 2 (4%) 2 (13%)
  N/A 1 (2%) 0 (0%)

Lymphatic Infiltration*
  No 21 (39%) 1 (10%)
  Yes 33 (61%) 9 (90%)

Venous Infiltration*
  No 22 (41%) 1 (10%)
  Yes 32 (59%) 9 (90%)

Perineural Infiltration*
  No 29 (54%) 3 (30%)
  Yes 25 (46%) 7 (70%)

N=Patient numbers;

*

Surgical samples only (Hotspot N=54, NGS N=10)

Table 2.

Genetic Mutations identified through Hotspot Analysis

Sample ID Histology Mutations
13 Adenocarcinoma IDH1_V178I_G532A*
PIK3CA_H1047RL_A3140GT
26 Adenosquamous KRAS_G12DAV_G35ACT
NRAS_Q61RPL_A182GCT
32 Adenocarcinoma IDH1_V178I_G532A*
34 Adenocarcinoma NRAS_G12DAV_G35ACT
42 Adenocarcinoma IDH1_V178I_G532A*
44 Adenocarcinoma KRAS_G12DAV_G35ACT
MET_N375S_A1124G*
46 Adenocarcinoma KRAS_G13DAV_G38ACT
47 Adenocarcinoma IDH1_V178I_G532A*
49 Adenocarcinoma PIK3CA_M1043I_G3129ATC
56 Adenocarcinoma ALK_F1174L_C3522AG
57 Adenocarcinoma NRAS_G12DAV_G35ACT
*

Most likely to represent genomic variation (SNP)

Figure 1.

Figure 1

Peaks for PIK3CA, IDH1 and KRAS mutations (Sequenom Massarray)

Figure 2.

Figure 2

2a) IDH1 mutation and association with overall survival.

2b) KRAS mutation and association with overall survival

Figure 3.

Figure 3

Schematic of FGFR3-TACC3 Fusion Gene in Gallbladder Cancer

A multivariate analysis of overall survival was also performed using backward elimination methods. Overall survival was seen to be associated with patients age 62–79 (HR: 5.93; 95%CI: 1.76 – 20.00; p=0.004), and age ≥ 70 (HR: 3.84; 95%CI: 1.19 – 12.39; p=0.024), clinical stages 3a, 3b, 4a & 4b (HR: 2.60; 95%CI: 1.03–6.59; p=0.044), venous infiltration (HR: 3.42; 95%CI: 1.46–8.03; p=0.005) and KRAS (HR: 8.91; 95%CI: 1.99–39.94; p=0.004-Fig.3b).

On NGS, 26 mutations were noted in 15 cases (Tables 3). P53 was most common and there was relative preponderance of mutations involving the PI3 kinase pathway: STK11, RICTOR, TSC2. Two cases had FGF pathway aberrations: FGF10 amplification and one case of FGF3-TACC fusion gene (Fig 4). Two cases are illustrated wherein the mutational data were utilized for targeted therapeutics with success (Fig 5a; Fig 5b).

Table 3.

Genetic Alterations Identified Through NGS (N=15)

GENE Alterations (With allele frequency or copy number)
TP53 V274F (10%)
R282G (50%)
R213* (29%)
Y220C (2%)
R342* (24%)
C141* (21%)
Splice site 559+1G>T (21%)
F109V (46%)
V272L
STK11 R86* (11%)
E120* (15%)
K62fs*98 (6%)
CCNE1 Amplification (copy no 11×)
Amplification (copy no 13×)
MDM2 Amplification (copy no 6×)
Amplification (copy no 16×)
MYC Amplification (copy no 12×)
Amplification (copy no 7×)
RICTOR Amplification (copy no 12×)
Amplification (copy no 7×)
APC S2113fs*25 (21%)
ARID1A G284fs*78 (18%)
AURKA S398L (48%)
CDKN2A Truncation - exon 1
CDKN2A/B Loss
Loss
CRKL Amplification (copy no 12×)
FGF10 Amplification (copy no 7×)
FGFR3-TACC FGFR3-TACC3 fusion, Amplification (copy no 8×)
KRAS G12C, 3%
MCL1 Amplification (copy no 8×)
Amplification (copy no 8×)
PRKAR1A R97* (33%)
SMAD4 Truncation
SMARCA4 D558fs*6 (26%)
TSC2 Loss
BAP1 splice site 438-1delGTTTTTCCCC AG, 10%
1delGTTTTTCCCC AG, 10%
ERBB2 Amplification (copy no 20×)
Amplification (copy no 9×)
PIK3CA Amplification (copy no 7×)
ZNF703 Amplification (copy no 7×)

Figure 4.

Figure 4

Illustrations of mutational data successfully utilized for targeted therapeutics.

4a) Erlotinib in combination with gemcitabine and oxaliplatin before therapy and 4 months post therapy.

4b) Trastuzumab in combination with 5-fluorouracil, leucovorin and oxaliplatin as second-line therapy before therapy and 3 months post-therapy.

Figure 5.

Figure 5

Representative histopathology of samples with corresponding mutations used for Sequenom analysis and NGS.

5A) KRAS

5B) TP53, ERBB2

5C) FGFR3-TACC3, CCNE1, MCL1, MYC, TP53

5D)ARID1A

DISCUSSION

Gallbladder cancer has been referred to as an ‘orphan’ cancer, given its relative infrequency in the Western population. Molecular research in this disease has lagged behind the commoner gastrointestinal cancers, such as colorectal and gastric cancer. The known genetic alterations include mutations of K-RAS (in 3–40%, more likely in East Asia), PI3KCA (12%), p53 (40%) and BRAF (33%) oncogenes, and amplification of Her-2/ Neu (15%).(14, 15) Other genetic alterations described include loss of expression fragile histidine triad (FHIT) gene, microsatellite instability, overexpression of P13-K/Akt, VEGF and p21.(16, 17) Key limitations of the above data include the small number of cases tested, geographic and ethnic variation.

The present study, to our knowledge represents the largest number of surgically resected gallbladder cancer cases that had somatic mutation profiling. All of our specimens were FFPE and therefore we chose a platform that had non-fastidious DNA requirements and could detect low-abundance mutations. Sequenom Massarray technique is ideal in this situation for profiling single nucleotide mutations and polymorphisms. A limitation of this retrospective study is that we did not have parallel blood or normal tissue to assess if the mutations we noted were germline or somatic. We have used preselected panels, which included targetable oncogenes from the COSMIC and TCGA database. While the plan was to include somatic mutations only, in these panels, subsequent studies have reported that at least two of the genetic alterations (IDH1 and met) were germline.

Sanger sequencing has been effectively used for somatic mutation discovery. However, when there is a heterogeneous mixture of cancerous and normal tissue, Sanger sequencing may be unable to detect low frequency mutations. In one published study, sequencing failed to detect EGFR (Epidermal Growth Factor Receptor) mutations in tumors with roughly 10% allele frequencies.(18) Clinical somatic mutation detection will require high degree of sensitivity than standard sequencing. The Massarray™ system combines PCR with matrix-assisted laser desorption/ ionization time of flight mass spectrometry for rapidly multiplexed nucleic acid analysis. Furthermore, this system can rapidly profile hundreds of mutations in FFPE samples with as little as 5% mutation abundance with a short turn-around time. However, the podisadvantages of this approach is that these multiplex genomic tests only detect the expression of pre-selected hotspot mutations and do not lead to the discovery of novel targets. This limitation is particularly relevant to the less common tumors, such as gallbladder cancer.

Our findings indicated that IDH1_V178I was the commonest DNA variation on Sequenom Massarray. It is estimated that another mutation on IDH1_R132 occurs in upto 20% of high grade glioma and this mutation is associated with a better prognosis and response to therapy.(19) On the other hand, the same somatic mutation in acute myeloid leukemia is associated with a poor prognosis and lack of complete response, particularly in otherwise cytogenetically normal cases.(20) A poor prognosis was noted in our study with IDH1_V178I mutation. In a prior study, IDH1 mutations (IDH1_R132) were noted in cholangiocarcinoma, but none were noted in the 25 cases of gallbladder cancer studied. (21) Isocitrate dehydrogenase (IDH) catalyzes the conversion of isocitrate to α-ketoglutarate and mutations in this pathway is a relevant target for therapy given the development of IDH inhibitors.(22) These mutations also conferred an enzymatic gain-of-function: the novel NADPH-dependent reduction of α-ketoglutarate to the normally trace metabolite R(−)-2-hydroxyglutarate (2-HG), which is oncogenic.(23) Measurement of intracellular 2-HG can therefore be used to assess the functional impact of the mutation. In case of IDH1_V178I, no elevation of 2-HG was noted, which raises the question of whether this mutation represents a non-functional polymorphism or if the functional oncogenic effect includes a non 2-HG metabolic pathway. Several SNPs related to the lipid metabolism, estrogen receptor and DNA repair have been associated with survival in gallbladder cancer (2426). One case had ALK mutation (ALK_F1174L_C3522AG), which has not yet been described in this disease and offers effective targeted therapy options.

The next generation sequencing approach offers several advantages over the traditional methods, including the ability to simultaneously sequence hundreds of genes in a single test, have a higher depth of coverage and thereby heightened sensitivity for mutation detection, ideal for ‘precision medicine’.(27) In addition, these technologies can detect deletions, amplifications, translocations and base substitutions at a relatively rapid rate. The disadvantage includes cost, high computational requirements and high tissue requirement that make the technology unsuitable for smaller biopsies, circulating tumor cells and circulating plasma DNA. A notable finding in our study was the relatively common occurrence PI3-kinase pathway mutations (TCS2, STK11, RICTOR), which opens potential options for targeted therapies directed against these proteins. Deshpande et al, had noted the relative frequency of PI3KCA mutations in this population.(21) Other targetable mutations included AURKA and BAP, which may potentially be treated with aurora kinase inhibitors and DNA damaging agents [such as cisplatin and poly ADP ribose polymerase (PARP) inhibitors], respectively.

A novel finding in our study was the detection of fusion between Fibroblast Growth Factor Receptor (FGFR3) and Transforming Acidic Coiled-Coil (TACC) [in-frame fusion between exons 1–17 of FGFR3 (containing the kinase domain) and exons 11 to the C-terminus of TACC3 (containing the coiled coil TACC domain)]. The FGFR family plays an important role in cellular proliferation and angiogenesis and gain of function mutations in FGFRs have been reported in several malignancies.(28) FGFR3 mutation or amplification has not been reported in gallbladder cancer to our knowledge. Similar fusions between FGFR3 and TACC3 have recently been reported in a small percentage of glioblastomas.(29) These fusions have also recently been described in cholangiocarcinoma, are proven to be oncogenic and the resulting tumors may be susceptible to FGFR inhibitors.(30)

In conclusion, gallbladder cancer is amenable to precise interventions with targeted therapies and novel sequencing techniques may provide prognostic and therapeutic opportunities.

Acknowledgments

Funding Disclosures: Supported by a grant from Global Academic Programs, MD Anderson Cancer Center and Project Fondecyt 1120208.

Appendix 1

Appendix 1.

GENES AND MUTATIONS INVESTIGATED

AKT1_E17K_G49A FGFR1_S125L_C374T MET_Y1248C_A3743G
AKT2_E17K_G49A FGFR2_N549KK_T1647GA MET_Y1248HD_T3742CG
AKT3_E17K_G49A FGFR2_S252W_C755G MET_Y1253D_T3757G
ALK_F1174CS_T3521GC FGFR3_G370C_G1108T MGA_T1747N_C5421A
ALK_F1174L_C3522AG FGFR3_G380R_G1138A NRAS_A146T_G436A
ALK_F1174LIV_T3520CAG FGFR3_G697C_G2089T NRAS_G12DAV_G35ACT
ALK_F1245C_T3734G FGFR3_K650MT_A1949TC NRAS_G12SRC_G34ACT
ALK_F1245L_C3735AG FGFR3_R248C_C742T NRAS_G13DAV_G38ACT
ALK_F1245VI_T3733GA FGFR3_S249C_C746G NRAS_G13SRC_G37ACT
ALK_I1171N_T3512A FGFR3_Y373C_A1118G NRAS_Q61EKX_C181GAT
ALK_R1275QL_G3824AT FOXL2_C134W_C402G NRAS_Q61HHQ_A183TCG
BCOR_N1407STI_A4220GCT GNA11_Q209LP_A626TC NRAS_Q61RPL_A182GCT
BRAF_D594GV_A1781GT GNA11_R183C_C547T PDGFRA_D842V_A2525T
BRAF_E586K_G1756A GNAQ_Q209H_A627T PDGFRA_D842YN_G2524TA
BRAF_G464EVA_G1391ATC GNAQ_Q209LPR_A626TCG PDGFRA_N659K_C1977A
BRAF_G466EVA_G1397ATC GNAS_R201H_G602A PDGFRA_N659Y_A1975T
BRAF_G466R_G1396CA GNAS_R201SC_C601AT PDGFRA_V561D_T1682A
BRAF_G469EVA_G1406ATC GRM3_E870K_G2608A PIK3CA_A1046V_C3137T
BRAF_G469R_G1405CA IDH1_G70D_G209A PIK3CA_C420R_T1258C
BRAF_K601E_A1801G IDH1_R132CGS_C394TGA PIK3CA_E110K_G328A
BRAF_L597RQ_T1790GA IDH1_R132HL_G395AT PIK3CA_E418K_G1252A
BRAF_V600_G1800 IDH1_V178I_G532A PIK3CA_E453K_G1357A
BRAF_V600EAG_T1799ACG_F IDH2_R140LQ_G419TA PIK3CA_E542KQ_G1624AC
BRAF_V600EAG_T1799ACG_R IDH2_R140W_C418T PIK3CA_E542VG_A1625TG
BRAF_V600LM_G1798TA IDH2_R172GW_A514GT PIK3CA_E545AGV_A1634CGT
CC2D1A_L913V_C3036G IDH2_R172MK_G515TA PIK3CA_E545D_G1635CT
CDK4_R24C_C70T IDH2_R172S_G516T PIK3CA_E545KQ_G1633AC
CDK4_R24H_G71A JAK2_V617F_G1849T PIK3CA_F909L_C2727G
CSMD1_A409S_G1225T KIT_D816GVA_A2447GTC PIK3CA_G118D_G353A
CSMD1_Q3005X_C9013T KIT_D816HNY_G2446CAT PIK3CA_H1047RL_A3140GT_F
CTNNB1_D32AGV_A95CGT KIT_K642E_A1924G PIK3CA_H1047RL_A3140GT_R
CTNNB1_D32HNY_G94CAT KIT_L576P_T1727C PIK3CA_H1047Y_C3139T
CTNNB1_G34EVA_G101ATC KIT_N566D_A1696G PIK3CA_H701P_A2102C
CTNNB1_H36PRY_A107CGT KIT_N822KNK_T2466GCA PIK3CA_K111N_G333C
CTNNB1_I35NST_T104AGC KIT_N822YHD_A2464TCG PIK3CA_M1043I_G3129ATC
CTNNB1_S33APT_T97GCA KIT_R634W_C1900T PIK3CA_M1043V_A3127G
CTNNB1_S37CFY_C110GTA KIT_V559ADG_T1676CAG PIK3CA_N345K_T1035A
CTNNB1_S45APT_T133GCA KIT_V560DGA_T1679AGC PIK3CA_P539R_C1616G
CTNNB1_S45CFY_C134GTA KIT_V825A_T2474C PIK3CA_Q060K_C178A
CTNNB1_T41APS_A121GCT KIT_Y553N_T1657A PIK3CA_Q546EK_C1636GA
CTNNB1_T41INS_C122TAG KRAS_A146PT_G436CA PIK3CA_Q546LPR_A1637TCG
EGFR_G719CS_G2155TA KRAS_G10R_G28A PIK3CA_R088Q_G263A
EGFR_K860I_A2579T KRAS_G12DAV_G35ACT PIK3CA_S405F_C1214T
EGFR_L858R_T2573G KRAS_G12SRC_G34ACT PIK3CA_T1025SA_A3073TG
EGFR_L861QR_T2582AG KRAS_G13DAV_G38ACT PIK3CA_Y1021C_A3062G
EGFR_S720P_T2158C KRAS_G13SRC_G37ACT PIK3CA_Y1021HN_T3061CA
EGFR_T790M_C2369T KRAS_Q61EKX_C181GAT PPP2R1A_W257G_T769G
EGFR_T854I_C2561T KRAS_Q61HHQ_A183CTG RAF1_A319S_G955T
EGFR_Y813C_A2438G KRAS_Q61LPR_A182TCG RAF1_L613V_C1837G
EPHA3_K761NN_G2283TC MAP2K2_E207KQ_G619AC RAF1_N115S_A344G
FBXO4_L23Q_T68A MAP2K7_D290D_C870T RAF1_Q335H_G1005C
FBXO4_P76T_C226A MAP2K7_R162H_G485A RAF1_S259A_T775G
FBXO4_S8R_C24AG MAP2K7_S271T_T811A RAF1_Y340D_T1018G
FBXW7_R465C_C1393T MAP2K7_S311L_C932T RET_M918T_T2753C
FBXW7_R465HL_G1394AT MET_H1112RL_A3335GT RPL22_K15TRM_A44CGT
FBXW7_R479QL_G1436AT MET_H1112Y_C3334T SFRS9_Y192X_C722A
FBXW7_R505CS_C1513TA MET_M1268T_T3803C SMO_A324T_G970A
FBXW7_R505HLP_G1514ATC MET_N375S_A1124G SRC_Q531X_C1591T
FBXW7_S582L_C1745T MET_R988C_C2962T TGM2_S212P_T734C
FGFR1_P252T_C754A MET_T1010I_C3029T

Appendix 2

APPENDIX 2.

GENETIC MUTATIONS SEQUENCED USING NGS

182 genes sequenced across entire coding sequence
Gene Gene Gene Gene Gene
ABL1 CDK6 FLT4 MEN1 PTPN11
ABL2 CDK8 FOXP4 MET PTPRD
AKT1 CDKN2A GATA1 MITF RAF1
AKT2 CDKN2B GNA11 MLH1 RARA
AKT3 CDKN2C GNAQ MLL RB1
ALK CEBPA GNAS MPL RET
APC CHEK1 GPR124 MRE11A RICTOR
AR CHEK2 GUCY1A2 MSH2 RPTOR
ARAF CRKL HQXA3 MSH6 RUNX1
ARFRP1 CRLF2 HRAS MTOR SMAD2
ARID1A CTNNB1 HSP9OAA1 MUTYI-1 SMAD3
ATM DDR2 IDH1 MYC SMAD4
ATR DNMT3A IDH2 MYCL1 SMARCA4
AURKA DOT1L IGF1R MYCN SMARCB1
AURKS EGFR IGF2R NFl SMO
BAP1 EPI-1A3 IKBKE NF2 SOX1O
BCL2 EPF-1A5 IKZF1 NKX2-1 SOX2
BCL2A1 EPHA6 INHBA NOTCH1 SRC
BCL2L1 EPHA7 INSR NPM1 STAT3
BCL2L2 EPHB1 IRS2 NRAS STK11
BCL6 EPHB4 JAK1 NTRK1 SUFU
BRAF EPHB6 JAK2 NTRK2 T5X22
BRCA1 ERBB2 JAK3 NTRK3 TET2
BRCA2 ERBB3 JUN PAK3 TGFBR2
CARD11 ERBB4 KDM6A PAX5 TNFAIP3
CBL ERCC2 KDR PDGFRA TNKS
CCND1 ERG KIT PDGFRB TNKS2
CCND2 ESR1 KRAS PHLPP2 TOP1
CCND3 EZH2 LRP1B PIK3CA TP53
CCNE1 FANCA LRP6 PIK3CG TSC1
CD79A FBXW7 LTK PIK3R1 TSC2
CD79B FGFR1 MAP2K1 PKHD1 USP9X
CDH1 FGFR2 MAP2K2 PLCG1 VHL
CDH2 FGFR3 MAP2K4 PRKDC WT1
CDH2O FGFR4 MCL1 PTCH1
CDH5 FLT1 MDM2 PTCH2
CDK4 FLT3 MDM4 PTEN
14 genes sequenced across selected iritrons
Gene
ALK
BCR
BRAF
EGFR
ETV1
ETV4
ETV5
ETV6
EWSR1
MLL
RAF1
RARA
RET
TMPRSS2

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of Interest: None

References

  • 1.Wistuba II, Gazdar AF. Gallbladder cancer: lessons from a rare tumour. Nature reviews Cancer. 2004;4:695–706. doi: 10.1038/nrc1429. [DOI] [PubMed] [Google Scholar]
  • 2.Zatonski WA, Lowenfels AB, Boyle P, Maisonneuve P, Bueno de Mesquita HB, Ghadirian P, Jain M, Przewozniak K, Baghurst P, Moerman CJ, Simard A, Howe GR, McMichael AJ, Hsieh CC, Walker AM. Epidemiologic aspects of gallbladder cancer: a case-control study of the SEARCH Program of the International Agency for Research on Cancer. J Natl Cancer Inst. 1997;89:1132–1138. doi: 10.1093/jnci/89.15.1132. [DOI] [PubMed] [Google Scholar]
  • 3.Trial watch: success for crizotinib in ALK-driven cancer. Nat Rev Drug Discov. 2010;9:908. doi: 10.1038/nrd3328. [DOI] [PubMed] [Google Scholar]
  • 4.Stemke-Hale K, Shipman K, Kitsou-Mylona I, de Castro DG, Hird V, Brown R, Flanagan J, Gabra H, Mills GB, Agarwal R, El-Bahrawy M. Frequency of mutations and polymorphisms in borderline ovarian tumors of known cancer genes. Mod Pathol. 2013;26:544–552. doi: 10.1038/modpathol.2012.194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Wang LE, Ma H, Hale KS, Yin M, Meyer LA, Liu H, Li J, Lu KH, Hennessy BT, Li X, Spitz MR, Wei Q, Mills GB. Roles of genetic variants in the PI3K and RAS/RAF pathways in susceptibility to endometrial cancer and clinical outcomes. Journal of cancer research and clinical oncology. 2012;138:377–385. doi: 10.1007/s00432-011-1103-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Andreou A, Kopetz S, Maru DM, Chen SS, Zimmitti G, Brouquet A, Shindoh J, Curley SA, Garrett C, Overman MJ, Aloia TA, Vauthey J-N. Adjuvant chemotherapy with FOLFOX for primary colorectal cancer is associated with increased somatic gene mutations and inferior survival in patients undergoing hepatectomy for metachronous liver metastases. Annals of Surgery. 2012;256:642–650. doi: 10.1097/SLA.0b013e31826b4dcc. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ross JS, Cronin M. Whole cancer genome sequencing by next-generation methods. Am J Clin Pathol. 2011;136:527–539. doi: 10.1309/AJCPR1SVT1VHUGXW. [DOI] [PubMed] [Google Scholar]
  • 8.Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe DB, Lander ES, Nusbaum C. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol. 2009;27:182–189. doi: 10.1038/nbt.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–2079. doi: 10.1093/bioinformatics/btp352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–1303. doi: 10.1101/gr.107524.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Lipson D, Capelletti M, Yelensky R, Otto G, Parker A, Jarosz M, Curran JA, Balasubramanian S, Bloom T, Brennan KW, Donahue A, Downing SR, Frampton GM, Garcia L, Juhn F, Mitchell KC, White E, White J, Zwirko Z, Peretz T, Nechushtan H, Soussan-Gutman L, Kim J, Sasaki H, Kim HR, Park SI, Ercan D, Sheehan CE, Ross JS, Cronin MT, Janne PA, Stephens PJ. Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nat Med. 2012;18:382–384. doi: 10.1038/nm.2673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Kaplan EL, Meier P. Nonparametric estimation from incomplete observations. Journal of the American Statistical Association. 1958;53:457–481. [Google Scholar]
  • 13.Cox DR. Regression models and life tables (with discussion) Journal of the Royal Statistical Society B. 1972;34:187–220. [Google Scholar]
  • 14.Zhu AX, Hezel AF. Development of molecularly targeted therapies in biliary tract cancers: reassessing the challenges and opportunities. Hepatology. 2011;53:695–704. doi: 10.1002/hep.24145. [DOI] [PubMed] [Google Scholar]
  • 15.Rashid A. Cellular and molecular biology of biliary tract cancers. Surg Oncol Clin N Am. 2002;11:995–1009. doi: 10.1016/s1055-3207(02)00042-x. [DOI] [PubMed] [Google Scholar]
  • 16.Kawamoto T, Krishnamurthy S, Tarco E, Trivedi S, Wistuba II, Li D, Roa I, Roa JC, Thomas MB. HER Receptor Family: Novel Candidate for Targeted Therapy for Gallbladder and Extrahepatic Bile Duct Cancer. Gastrointest Cancer Res. 2007;1:221–227. [PMC free article] [PubMed] [Google Scholar]
  • 17.Roa JC, Anabalon L, Roa I, Melo A, Araya JC, Tapia O, de Aretxabala X, Munoz S, Schneider B. Promoter methylation profile in gallbladder cancer. J Gastroenterol. 2006;41:269–275. doi: 10.1007/s00535-005-1752-3. [DOI] [PubMed] [Google Scholar]
  • 18.Zhang HP, Ruan L, Zheng LM, Bai DY, Zhang HF, Liao YQ, Ding Y. Screening for EGFR mutations in lung cancer by a novel real-time PCR with double-loop probe and Sanger DNA sequencing. Zhonghua zhong liu za zhi [Chinese journal of oncology] 2013;35:28–32. doi: 10.3760/cma.j.issn.0253-3766.2013.01.006. [DOI] [PubMed] [Google Scholar]
  • 19.Labussiere M, Sanson M, Idbaih A, Delattre JY. IDH1 gene mutations: a new paradigm in glioma prognosis and therapy? Oncologist. 2010;15:196–199. doi: 10.1634/theoncologist.2009-0218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Marcucci G, Maharry K, Wu YZ, Radmacher MD, Mrozek K, Margeson D, Holland KB, Whitman SP, Becker H, Schwind S, Metzeler KH, Powell BL, Carter TH, Kolitz JE, Wetzler M, Carroll AJ, Baer MR, Caligiuri MA, Larson RA, Bloomfield CD. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2010;28:2348–2355. doi: 10.1200/JCO.2009.27.3730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Borger DR, Tanabe KK, Fan KC, Lopez HU, Fantin VR, Straley KS, Schenkein DP, Hezel AF, Ancukiewicz M, Liebman HM, Kwak EL, Clark JW, Ryan DP, Deshpande V, Dias-Santagata D, Ellisen LW, Zhu AX, Iafrate AJ. Frequent mutation of isocitrate dehydrogenase (IDH)1 and IDH2 in cholangiocarcinoma identified through broad-based tumor genotyping. Oncologist. 2012;17:72–79. doi: 10.1634/theoncologist.2011-0386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yen KE, Bittinger MA, Su SM, Fantin VR. Cancer-associated IDH mutations: biomarker and therapeutic opportunities. Oncogene. 2010;29:6409–6417. doi: 10.1038/onc.2010.444. [DOI] [PubMed] [Google Scholar]
  • 23.Ward PS, Lu C, Cross JR, Abdel-Wahab O, Levine RL, Schwartz GK, Thompson CB. The potential for isocitrate dehydrogenase mutations to produce 2-hydroxyglutarate depends on allele specificity and subcellular compartmentalization. The Journal of biological chemistry. 2013;288:3804–3815. doi: 10.1074/jbc.M112.435495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Andreotti G, Chen J, Gao YT, Rashid A, Chen BE, Rosenberg P, Sakoda LC, Deng J, Shen MC, Wang BS, Han TQ, Zhang BH, Yeager M, Welch R, Chanock S, Fraumeni JF, Jr, Hsing AW. Polymorphisms of genes in the lipid metabolism pathway and risk of biliary tract cancers and stones: a population-based case-control study in Shanghai, China. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2008;17:525–534. doi: 10.1158/1055-9965.EPI-07-2704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Park SK, Andreotti G, Sakoda LC, Gao YT, Rashid A, Chen J, Chen BE, Rosenberg PS, Shen MC, Wang BS, Han TQ, Zhang BH, Yeager M, Chanock S, Hsing AW. Variants in hormone-related genes and the risk of biliary tract cancers and stones: a population-based study in China. Carcinogenesis. 2009;30:606–614. doi: 10.1093/carcin/bgp024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Srivastava K, Srivastava A, Mittal B. Polymorphisms in ERCC2, MSH2, and OGG1 DNA repair genes and gallbladder cancer risk in a population of Northern India. Cancer. 2010;116:3160–3169. doi: 10.1002/cncr.25063. [DOI] [PubMed] [Google Scholar]
  • 27.Garraway LA, Verweij J, Ballman KV. Precision oncology: an overview. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2013;31:1803–1805. doi: 10.1200/JCO.2013.49.4799. [DOI] [PubMed] [Google Scholar]
  • 28.Eswarakumar VP, Lax I, Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 2005;16:139–149. doi: 10.1016/j.cytogfr.2005.01.001. [DOI] [PubMed] [Google Scholar]
  • 29.Singh D, Chan JM, Zoppoli P, Niola F, Sullivan R, Castano A, Liu EM, Reichel J, Porrati P, Pellegatta S, Qiu K, Gao Z, Ceccarelli M, Riccardi R, Brat DJ, Guha A, Aldape K, Golfinos JG, Zagzag D, Mikkelsen T, Finocchiaro G, Lasorella A, Rabadan R, Iavarone A. Transforming fusions of FGFR and TACC genes in human glioblastoma. Science. 2012;337:1231–1235. doi: 10.1126/science.1220834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Wu YM, Su F, Kalyana-Sundaram S, Khazanov N, Ateeq B, Cao X, Lonigro RJ, Vats P, Wang R, Lin SF, Cheng AJ, Kunju LP, Siddiqui J, Tomlins SA, Wyngaard P, Sadis S, Roychowdhury S, Hussain MH, Feng FY, Zalupski MM, Talpaz M, Pienta KJ, Rhodes DR, Robinson DR, Chinnaiyan AM. Identification of Targetable FGFR Gene Fusions in Diverse Cancers. Cancer Discov. 2013;3:636–647. doi: 10.1158/2159-8290.CD-13-0050. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES