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The Journal of International Medical Research logoLink to The Journal of International Medical Research
. 2019 May 20;47(6):2768–2777. doi: 10.1177/0300060519847796

Dual-targeting strategy using trastuzumab and lapatinib in a patient with HER2 gene amplification in recurrent metachronous metastatic gallbladder carcinoma

Minfeng Ye 1,*, Jieqing Lv 1,*, Guangen Xu 1, Wei Wang 1, Yuanming Jing 1, Aijing Sun 2, Zengxin Lu 3, Xue Wu 4, Yichuan Liu 4, Yang W Shao 4, Fang Liu 2, Feng Tao 1,
PMCID: PMC6567708  PMID: 31106632

Short abstract

Gallbladder carcinoma (GBC) is a rare and highly aggressive tumor. Early diagnosis is challenging, which results in a poor prognosis using systemic therapy. Recent studies have identified a subset of GBC patients with HER2 gene (ERBB2) amplification that could benefit from HER2-targeted therapy. Here, we report one patient with recurrent metachronous GBC with metastasis, who received the combination of trastuzumab and lapatinib. This approach achieved a partial response for both the brain and the lung metastases. This study demonstrated that HER2 inhibition is a promising therapeutic strategy for GBC with HER2 amplification and, combined with lapatinib, it can effectively target brain metastasis.

Keywords: Gallbladder carcinoma, HER2 amplification, dual-targeted therapy, trastuzumab, lapatinib, tumor

Introduction

Anti-HER2 molecular targeted agents are established for treating breast cancer,13 gastric cancer,4 and metastatic colorectal cancer5 in patients with HER2 gene amplification (an increase in the copy number). A combination of two HER2-targeted therapies, such as trastuzumab and lapatinib, has also been demonstrated to have clinical efficacy in advanced HER2-positive metastatic breast cancers2,3 and colorectal cancers.5

Lapatinib (GW572016) is a selective inhibitor of epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinase,6 and trastuzumab is a humanized monoclonal antibody that targets the HER-2 extracellular domain.7 The dual targeting of HER2-positive tumors using trastuzumab and lapatinib results from the primary and acquired resistance of these two drugs, their partly non-overlapping mechanisms of action, and the well characterized synergistic interaction between them in HER2 breast-cancer models. Clinically, trastuzumab primarily induces pro-apoptotic effects, while lapatinib inhibits proliferation.8,9 Clinical evidence shows indirect support for dual HER2 blockade. In patients with refractory breast cancer who are treated with trastuzumab, lapatinib plus trastuzumab prolonged the progression-free survival compared with lapatinib alone. Konecny et al.10 analyzed the combination of trastuzumab plus lapatinib, and they observed synergistic drug interactions in four HER-2-overexpressing breast cancer cell lines. Additionally, similar results were obtained in the study reported by Baselga et al.,11 which suggested complementary mechanisms of action and synergistic anti-tumor activity for the monoclonal antibody in a model of breast cancer that overexpressed HER2. Xu et al.3 conducted a systematic review and meta-analysis of randomized controlled trials (RCTs), and the results showed that lapatinib plus trastuzumab significantly prolonged the pathological complete response, overall survival, and event-free survival with tolerance in HER2-positive breast cancer patients. Moreover, the combination of trastuzumab and lapatinib was also effective and well tolerated in patients with HER2-positive metastatic colorectal cancer refractory disease.5

Gallbladder carcinoma (GBC) is a rare and highly aggressive tumor. Early diagnosis is challenging, which results in a poor prognosis in patients who receive systemic therapy. However, combination therapy with trastuzumab and lapatinib has not been reported in patients with GBC. Here, we present the case of a patient with dual-targeted HER2 therapy including lapatinib that was used to treat advanced GBC with brain metastases.

Case presentation

On 4 January 2016, a 48-year-old Chinese female patient was admitted to the Shaoxing People’s Hospital because of nausea, vomiting, dizziness, headache, crooked mouth, and slurred speech. Magnetic resonance imaging (MRI) showed multi-site metastatic brain tumors (Figure 1a) and bilateral adrenal gland metastasis (data not shown). Chest computed tomography (CT) scans revealed multi-site metastatic lung tumors (Figure 1b). This patient was diagnosed with GBC via cholecystectomy that was performed because of cholecystitis and gallstones in 2010 (pT3N0M0; TNM staging: IIB; Nevin staging: III) (Figure 1e). Lung metastasis was found in 2014, and brain metastasis was detected in 2015. The patient’s medical history is shown in Figure 2.

Figure 1.

Figure 1.

Dual-targeted therapy with trastuzumab and lapatinib were effective in advanced gallbladder carcinoma patients with multi-site brain and lung metastases.

Magnetic resonance imaging showed the status of brain metastases before (a) and after (c) three cycles of dual-HER2 targeted therapy with trastuzumab and lapatinib. Chest computed tomography scans showed the status of lung metastasis before (b) and after (d) three cycles of dual HER2-targeted therapy with trastuzumab and lapatinib. (e) A biopsy identified that the gallbladder tumor was moderate-differentiated adenocarcinoma (hematoxylin and eosin staining; original magnification ×100). (f) A biopsy showed that the metastatic brain tumors were adenocarcinoma (hematoxylin and eosin staining; original magnification ×100).

Figure 2.

Figure 2.

Medical history, clinical interventions, and metastasis development.

One month after admission, the patient underwent surgery to remove one of the largest metastatic tumors (longest diameter in the transverse plane, 38.96 mm) in the left frontal lobe. The biopsy showed that the metastatic tumor was adenocarcinoma (Figure 1f). The patient was diagnosed with the GBC brain metastasis after a comprehensive review. Genetic profiling of 416 cancer-relevant genes was performed using targeted next generation sequencing (NGS; see Materials and Methods) to develop potential molecular therapeutic approaches. GBC primary tumor and brain metastatic tissues, as well as the circulating tumor DNA (ctDNA) in the blood were collected from the patient for genetic profiling. The sequencing results showed a significant increase in the HER2 copy number in the GBC primary tumor (8.6×), brain metastasis (14.6×), and ctDNA (2.5×) (Table 1). Thus, dual-targeted therapy was initiated on 6 March, 2016 using trastuzumab (administered intravenously at an initial dose of 8 mg/kg, followed by subsequent doses of 6 mg/kg once per week for 3 weeks) and lapatinib (administered orally each day at a dose of 1000 mg per day in 21-day treatment cycles), based on previously published work in breast cancer2,3 (i.e., one dose of trastuzumab each week and one dose of oral lapatinib each day). No concurrent chemotherapy was administrated.

Table 1.

Genetic alterations identified in samples before/after HER2-targeted therapy.

Sample type Sample ID Genetic alterations identified MAF (%)/Fold change (×)
Before HER2-targeted therapy
 ctDNA P16020611144 TP53: p.K291X (c.A871T) 4%
CDK12: amplification 2.87×
ERBB2: amplification 2.48×
 FFPE sample of F16020611145 TP53: p.K291X (c.A871T) 31%
  gallbladder tumor CDK12: amplification 9.03×
ERBB2: amplification 8.40×
 FFPE sample of F16020611146 TP53: p.K291X (c.A871T) 72%
  brain tumor CDK12: amplification 11.28×
ERBB2: amplification 14.60×
RB1: Single-copy loss 0.53×
After three cycles of anti-HER2 targeted therapy
 ctDNA P16051918361 TP53: p.K291X (c.A871T) 0.5%
CDK12: amplification Not detected
ERBB2: amplification Not detected
RB1: Single-copy loss Not detected

MAF, mutant allele frequency; ctDNA, circulating tumor DNA; FFPE, formalin-fixed paraffin-embedded.

Three weeks after dual HER2 targeted therapy with trastuzumab and lapatinib, the follow-up chest and brain MRI revealed that the patient achieved a partial response in brain metastatic tumors (Figure 1c) and lung metastatic tumors (Figure 1d), after three cycles of treatment based on the Response Criteria Evaluation in Solid Tumors (RECIST) version 1.1. The MRI results showed that the metastatic tumors had shrunk. A concurrent blood sample was collected for genetic testing of ctDNA using targeted NGS. The HER2 copy numbers did not increase at this time (Table 1), suggesting that the tumors were cleared after trastuzumab and lapatinib dual therapy. It was also accompanied by a carcinoembryonic antigen (CEA) response (CEA levels decreased from 4030 ng/mL to 660 ng/mL). The patient developed grade 3 adverse reactions including fatigue, skin rash, and increased bilirubin concentration, but no grade 4 or 5 adverse reactions were observed. In summary, dual-targeted therapy with trastuzumab and lapatinib offered a chemotherapy-free option and achieved tumor suppression with an acceptable safety profile for this patient with HER2 amplification in GBC.

Materials and methods

Patient information

The patient provided written informed consent. The study was approved by the ethics committee at the institute and was performed in strict accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization and Good Clinical Practice guidelines.

Tumor assessment

Tumor measurements were performed by a radiologist (ZXL) before treatment and were assessed every 2 months in accordance with RECIST (version 1.1). The patient was examined using a CT of the chest, abdomen, or pelvis, and MRI brain scans when clinically indicated in accordance with the protocol. All tumor measurements were reviewed by a radiologist (ZXL) who read the MRI or CT scans, and collected, stored, and interpreted the imaging results.

Adverse reaction assessment

The patient’s health was continuously monitored and graded in accordance with the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Laboratory test assessments (hematology, serum chemistry, urine analysis, and tumor markers) were performed at baseline (day 1 of every cycle), and at the end of treatment. The left ventricular ejection fraction was examined at baseline (day 1 of every cycle) and at the end of treatment.

DNA extraction

Formalin-fixed paraffin-embedded (FFPE) tissues were sectioned, and hematoxylin and eosin (H&E)-stained slides were reviewed by surgical pathologists (FL and AJS). Tumor tissue areas were dissected from ten serial sections (4 μm) guided by H&E slides as templates. The FFPE tissues were deparaffinized with xylene and genomic DNA was extracted using a QIAamp DNA FFPE tissue kit (Qiagen, Valencia, CA, USA). The concentration of DNA was determined using a Qubit dsDNA HS assay kit on the Qubit 3.0 (Life Technologies, Carlsbad, CA, USA), in strict accordance with the manufacturer’s protocol. DNA (A260/280 and A260/230) was measured using the Nanodrop-2000 (Thermo Fisher Scientific, Waltham, MA, USA).

Whole blood (8 mL) was collected using EDTA blood collection tubes (BD Biosciences, San Jose, CA, USA) that were centrifuged within 2 hours at 1800 ×g for 10 minutes at ≥4°C to collect the plasma. ctDNA was extracted from plasma using a QIAamp circulating nucleic acid kit (Qiagen). The purified ctDNA was quantified using a Qubit dsDNA HS assay kit on the Qubit 3.0 Fluorometer (Life Technologies).

Library construction

The sequencing library was prepared using a KAPA Hyper Prep Kit (Kapa Biosystems, Boston, MA, USA), in accordance with the manufacturing protocol. Briefly, 1 μg genomic DNA sample was fragmented into 350 bp in an Adaptive Focused Acoustics (AFA) fiber snap-cap microTUBE using Covaris M220 (Covaris, Woburn, MA, USA) or 10–50 ng ctDNA underwent end repairing, A-tailing, adapter ligation, size selection, and finally PCR amplification. Library concentration was determined using a Qubit dsDNA HS assay kit on the Qubit 3.0 Fluorometer (Life Technologies).

Hybrid capture and ultra-deep next generation sequencing

The 5ʹ-biotinylated probe solution was used as capture probes, which targeted 416 cancer-related genes (Table 2). The capture reaction was performed using the NimbleGen SeqCap EZ hybridization and Wash Kit (Roche) with 1 μg of pooled libraries, 5 μg of human Cot-1 DNA, 1 unit of adapter-specific blocker DNA, and the capture probes. The solution hybridization was performed for 16–18 hours at 65°C. The captured targets were selected by pulling down the biotinylated probe/target hybrids using streptavidin-coated magnetic beads, and the off-target library was removed by washing with wash buffer, followed by PCR amplification of the target-enriched libraries. Sequencing libraries were quantified by qPCR using the KAPA Library Quantification kit (KAPA Biosystems, Boston, MA, USA), sized on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), and then deep-sequenced on an Illumina HiSeq 4000 using the PE150 kit (Illumina Inc., San Diego, CA, USA).

Table 2.

Gene targeted in hybridization capture.

ABCB1 (MDR1) CDK4 ERBB4 HGF MSH2 PTEN STK11
ABCC2 (MRP2) CDK6 ERCC1 HNF1A MSH3 PTPN11 STMN1
ACTB CDK8 ERCC2 HNF1B MSH6 PTPN2 STX11
ADH1B CDKN1B ERCC3 HRAS MTHFR PTPN6 STXBP2
AIP CDKN1C ERCC4 ID3 MTOR PTPRO SUFU
AKT1 CDKN2A ERCC5 IDH1 MUTYH QKI SUZ12
AKT2 CDKN2B ESR1 IDH2 MYC RAC1 SYN3
AKT3 CDKN2C ETV1 IGF1R MYCL1 RAD21 TCN2
ALDH2 CEBPA ETV4 IGF2 MYCN RAD50 TEK
ALK CEP57 EWSR1 IKBKE MYD88 RAD51 TEKT4
AMER1 CHD4 EXT1 IKZF1 MYNN RAD51C TERC
AP3B1 CHEK1 EXT2 IKZF2 NBN RAD51D TERT
APC CHEK2 EZH2 IKZF3 NCSTN RAF1 TET2
AR CKS1B FANCA IL13 NF1 RARA TGFBR2
ARAF CREBBP FANCB IL7R NF2 RASGEF1A TLE1
ARID1A CRKL FANCC INPP4B NFKBIA RB1 TLE4
ARID2 CROT FANCD2 INPP5D NKX2-1 RECQL4 TMEM127
ARID5B CSF1R FANCE IRF1 NOTCH1 RELN TMPRSS2
ASXL1 CSF3R FANCF IRF2 NOTCH2 RET TNFAIP3
ATM CTCF FANCG IRF4 NPM1 RHBDF2 TNFRSF14
ATR CTLA4 FANCI IRF8 NQO1 RHOA TNFRSF17
ATRX CTNNB1 FANCL JAK1 NRAS RICTOR TNFRSF19
AURKA CUX1 FANCM JAK2 NRG1 RNF146 TOP1
AURKB CXCR4 FAT1 JAK3 NSD1 RNF43 TOP2A
AXIN1 CYLD FBXO11 JARID2 NT5C2 ROS1 TP53
AXL CYP19A1 FCGR2B JUN NTRK1 RPTOR TP63
B2M CYP2B6*6 FGF19 KDM2B PAG1 RRM1 TPMT
BAP1 CYP2C19*2 FGFR1 KDM5A PAK3 RTEL1 TRAF2
BARD1 CYP2C9*3 FGFR2 KDR (VEGFR2) PALB2 RUNX1 TRAF3
BCL2 CYP2D6*3 FGFR3 KIF1B PARK2 SBDS TRAF5
BCL2L1 CYP2D6*4 FGFR4 KIT PAX5 SDHA TSC1
BCL2L2 CYP2D6*5 FH KMT2B PBRM1 SDHAF2 TSC2
BCORL1 CYP2D6*6 FIP1L1 KMT2C PC SDHB TSHR
BCL2L11 (BIM) CYP3A4*4 FLCN KRAS PDCD1 SDHC TTF1
BLM CYP3A5*1 FLT1 (VEGFR1) LEF1 PDCD1LG2 (PD-L2) SDHD TUBB3
BMPR1A CYP3A5*3 FLT3 LMO1 PDGFRA SERP2 TYMS
BRAF DAB2 FLT4 (VEGFR4) LSP1 PDGFRB SETBP1 TYR
BRCA1 DAXX GADD45B LYN PDK1 SETD2 U2AF1
BRCA2 DDR2 GATA1 LYST PHF6 SF3B1 UGT1A1
BRD4 DDX1 GATA2 LZTR1 PHOX2B SGK1 UNC13D
BRIP1 DHFR GATA3 MAP2K1 (MEK1) PICK3R1 SH2D1A VEGFA
BTG2 DICER1 GATA4 MAP2K2 (MEK2) PIK3C3 SLX4 VHL
BTK DIS3L2 GATA6 MAP2K4 PIK3CA SMAD2 WISP3
BTLA DLG2 GNA11 MAP3K1 PIK3CD SMAD3 WRN
BUB1B DMNT3A GNA13 MCL1 PIK3R1 SMAD4 WT1
c11orf30 DNM2 GNAQ MDM2 PIK3R2 SMAD7 XIAP
CALR DOCK1 GNAS MDM4 PLCE1 SMARCA4 XPA
CBL DOT1L GPC3 MECOM PLK1 SMARCB1 XPC
CCND1 DPYD GRIN2A MED12 PMS1 SMC1A XPO1
CCNE1 DUSP2 GRM3 MEF2B PMS2 SMC3 XRCC1
CCT6B EBF1 GSTM1 MEN1 POLD1 SMO YAP1
CD22 ECT2L GSTP1 MET POLD3 SOX2 ZAP70
CD274 (PD-L1) EED GSTT1 MGMT POLE SPOP ZBTB20
CD58 EGFR HBA1 MITF POT1 SRC ZNF217
CD70 EGR1 HBA2 MLH1 PPP2R1A SRSF2 ZNF703
CDA EP300 HBB MLL PRDM1 STAG2 ZRSR2
CDC73 EPCAM HDAC1 MLLT10 PRF1 STAT3
CDH1 EPHA3 HDAC2 MLPH PRKAR1A STAT5A
CDK10 ERBB2 (HER2) HDAC4 MPL PRKCI STAT5B
CDK12 ERBB3 HDAC7 MRE11A PTCH1 STIL

Sequence alignment and data processing

Quality control was applied using Trimmomatic.12 High quality reads were mapped to the human genome (hg19, GRCh37 Genome Reference Consortium Human Reference 37) using a modified Burrows-Wheeler Aligner (BWA) version 0.7.1213 with BWA-MEM algorithm and default parameters. The Genome Analysis Toolkit14 (GATK, version 3.4-0) was modified and used to locally realign the BAM files at intervals with insertion/deletion (indel) mismatches and to recalibrate the BAM file read base quality scores. Single nucleotide variants (SNVs) and short indels were identified using VarScan2 2.3.915 with a minimum variant allele frequency threshold set at 0.01 and a p-value threshold for calling variants set at 0.05 to generate variant call format (VCF) files. All SNVs/indels were annotated using ANNOVAR, and each SNV/indel was manually checked using the integrative genomics viewer (IGV).16 Copy number variations (CNVs) were identified using ADTEx 1.0.4.17 The analysis and data are presented as copy number changes in all Tables.

Discussion

Recent studies have identified a subset of patients who are undergoing treatment for advanced GBC with HER2 overexpression or amplification that could potentially benefit from HER2-targeted therapy.6,7 Some studies previously reported the use of HER2-targeted treatment to treat HER2-positive gallbladder cancers. One case included a significant reduction in lung metastasis in patients with HER2-amplified metastatic cholangiocarcinoma that was treated with trastuzumab and paclitaxel.8 Another study retrospectively reviewed GBC patients who received HER2-directed therapy with trastuzumab, lapatinib, or pertuzumab, and showed that HER2 blockage was a promising treatment strategy in HER2-positive GBC patients.7 Most of those patients received a single HER2-targeted reagent combined with chemotherapy.

Clinical trials have demonstrated that lapatinib can be used as a therapeutic option for advanced HER2 alterations in cancers with brain metastasis.9 Considering the multi-site brain and lung metastases of the GBC patients, we administrated dual-targeted HER2 therapy with trastuzumab and lapatinib and achieved PR after 4 months of treatment without concurrent chemotherapy. In summary, this is the first study to administer dual-targeted HER2 therapy including lapatinib to treat patients with advanced GBC with brain metastases. Patient follow-up and further multi-center clinical trials are necessary to investigate the long-term efficacy.

Conclusion

HER2 inhibition is a promising therapeutic strategy for GBC with HER2 amplification and used in combination with lapatinib, it can effectively target brain metastasis.

Declaration of conflicting interest

The authors declare that there is no conflict of interest.

Disclosure

Xue Wu, Yichuan Liu, and Yang W. Shao are the shareholders or employees of Geneseeq Technology Inc.

Funding

This work was supported by National Natural Science Foundation of China (Grants No. 81374014 and 81472210) and Zhejiang Provincial Medical and Healthy Science and Technology Projects (Grant No. 2013KYA228).

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