HER2/neu-directed therapy for biliary tract cancer

Background Biliary cancers are highly aggressive tumors that are often diagnosed an advanced disease stage and have a poor outcome with systemic therapy. Recent efforts towards molecular characterization have identified a subset of biliary patients that have HER2/neu amplification or mutation. HER2/neu amplification is associated with response to HER2/neu-directed therapy in breast and gastric cancers. However, the efficacy of HER2/neu-targeted therapy in biliary cancers is unknown. Patients and methods We retrospectively reviewed cases of advanced gallbladder cancer and cholangiocarcinoma with HER2/neu genetic aberrations or protein overexpression who received HER2/neu-directed therapy between 2007 and 2014. Clinical data were retrieved from medical records, and imaging studies were independently reviewed. Results Nine patients with gallbladder cancer and five patients with cholangiocarcinoma had received HER2/neu-directed therapy (trastuzumab, lapatinib, or pertuzumab) during the study period. In the gallbladder cancer group, HER2/neu gene amplification or overexpression was detected in eight cases. These patients experienced disease stability (n = 3), partial response (n = 4), or complete response (n = 1) with HER2/neu-directed therapy. One patient had HER2/neu mutation and experienced a mixed response after lapatinib therapy. The duration of response varied from 8+ to 168 weeks (median 40 weeks), and three patients are still on therapy. One patient developed HER2/neu amplification as a secondary event after FGFR-directed therapy for FGF3-TACC3 gene fusion. The cholangiocarcinoma cases treated in this series had a higher proportion of HER2/neu mutations, and no radiological responses were seen in these patients despite HER2/neu-directed therapy. Conclusions HER2/neu blockade is a promising treatment strategy for gallbladder cancer patients with gene amplification and deserves further exploration in a multi-center study.


Introduction
Gallbladder cancer is one of the most aggressive solid tumors and represents an important cause of cancerrelated mortality in South America and South Asia, while occurring less frequently in the United States and Western Europe. Late stage at presentation, early occurrence of liver and peritoneal metastases, and poor outcome with standard chemotherapy contribute to the poor prognosis of gallbladder cancer. Understanding the molecular characterization of this disease may help alter the dismal outcome with targeted therapeutics [1,2]. An estimated 12-15 % of gallbladder cancers have HER2/neu amplification or protein overexpression using commonly accepted criteria for positivity [2][3][4]. Cholangiocarcinoma is increasing in incidence in the Western world. Recent efforts at molecular characterization have identified distinct subsets with prognostic and therapeutic implications [5]. HER2/neu mutation or amplification has also been reported in cholangiocarcinoma [6][7][8]. These patients may be candidates for HER2/neu-directed therapy.
The HER2/neu gene is located on the 17q12-q21 chromosomal region and acts as an oncogene in several human cancers [9]. HER2/neu protein overexpression, either as the product of gene amplification or transcriptional deregulation, is observed in approximately 20 % of breast and ovarian cancers and 12 % of gastric cancers [10]. The overexpression and amplification of HER2/neu has also been demonstrated in gastricesophageal and endometrial cancer, and in these tumors, it is also usually associated with a worse prognosis [11][12][13][14]. The HER2/ neu receptors after dimerization can transactivate a number of downstream pathways like RAS-RAF-MEK-ERK1/ 2 or PI3k-AKT-mTOR resulting in cancer cell proliferation and cell survival [15,16].
Several therapeutic agents have been developed for HER2/neu positive breast cancer: these include monoclonal antibodies such as trastuzumab and pertuzumab, small molecule tyrosine kinase inhibitors like lapatinib, and chemo-immunotherapy conjugates like ado-trastuzumab emtansine (T-DM1). These have substantially changed the management of breast and gastric cancers and have already been incorporated into standardized treatment algorithms [11,17,18]. However, data in regards to the efficacy of HER2/neu targeted therapy in biliary cancers are scarce.

Case selection
All patients selected had pathologically confirmed gallbladder or biliary adenocarcinoma, had received HER2/ neu-directed therapy, and had a minimum of 3 months of follow-up. Clinical, pharmacy, and pathology records were reviewed to identify cases that were HER2/neu positive and had received HER2-directed therapy. The study was approved by the Institutional Review Board of MD Anderson Cancer Center. Patient demographics, clinical data, survival data, and treatment history were retrieved from medical records.

Tumor samples
Paraffin embedded blocks were sectioned, and hematoxylin and eosin (H&E) stained slides were reviewed by surgical pathology to confirm the tumor content in each section. Ten serial sections (4 μm) were cut from selected tissue blocks and areas with tumor tissue were macrodissected from those slides using the H&E slides as templates.

DNA extraction
The pathologic diagnosis of each case was confirmed on routine H&E slides. All samples sent for DNA extraction contained a minimum of 20 % DNA derived from tumor cells. DNA was extracted from 40 mm of fresh frozen, paraffin-embedded (FFPE) tissue using the Maxwell 16 FFPE Plus LEV DNA Purification kit (Promega Corporation, Madison, WI, USA) and quantified using a standardized PicoGreen fluorescence assay (Invitrogen, Carlsbad, CA, USA).

Next-generation sequencing (NGS)
For subjects with adequate biopsy tissue, an amplicon library was generated from 10 ng of DNA from each sample using the Ion Ampliseq Cancer Panel ( . Primers for PCR amplification included the 190-primer pair pool provided by the vendor with an additional primer pair that was custom added to cover the "hotspot" location on codon 17 of AKT1. Following PCR amplification of target sequences, barcodes were ligated to the amplicons using the Ion Xpress Barcode Adaptors Kit (Life Technologies, Carlsbad, CA, USA). Library quantification was then performed using the Bioanalyzer High Sensitivity DNA Chip (Agilent Technologies, Santa Clara, CA, USA). The library was diluted in nuclease-free water to obtain a final concentration of 16 pM. Emulsion PCR was performed manually using the Ion Xpress Template Kit (Life Technologies, Carlsbad, CA, USA) followed by manual breaking of the emulsion to isolate the ion spheres (ISPs). The quality of the DNA following PCR was measured using the Qubit IonSphere Quality control kit (Life Technologies, Carlsbad, CA, USA).
Selective ISPs with DNA were isolated and sequenced on an Ion 316 Chip (four samples/chip) or a Ion 318 Chip (eight samples/chip) using the vendor-provided sequencing kit (Life Technologies, Carlsbad, CA, USA). Successful sequencing of a sample required at least 300,000 reads with a quality score of AQ20 (1 misaligned base per 100 bases). For a wild-type call, a minimum coverage of 250× was required. As tumor specimens were admixed with normal tissue, a minimum coverage of 500× with at least 10 % frequency was used as cutoff for a variant to be considered true. All variants detected by Ion PGM with at least 10 % frequency were selected for confirmation by alternate platforms. Further details regarding the methodology and analysis have been described previously [19]. For four samples, NGS was performed by Foundation Medicine using the Illumina Hiseq 2000 Platform (Illumina, San Diego, CA, USA) for 236 targetable GAs; the methods have been described previously [20,21].

Immunohistochemistry for HER2/Neu
Tissues were fixed in formalin and embedded in paraffin using standard techniques. The 4-μm-thick histologic sections obtained from the TMAs were deparaffinized and hydrated in decreasing alcohol concentrations. Antigens were recovered by exposure to microwaves in citrate buffer (pH 6.0) and washed in PBS (pH 7.4). The monoclonal antibody anti-ErbB2 (NCL-CB11; Novocastra, Leica Biosystems, Wetzlar, Germany) was used at a dilution of 1:40. The primary antibody was incubated at room temperature for 60 min and then incubated with the complex Super Picture Polymer Detection Kit (Zymed Laboratories Inc., San Francisco, CA, USA) in a Dako autostainer (Dako, Glostrup, Denmark). Standard criteria for HER2/Neu scoring were used [3]. Fluorescent in situ hybridization (FISH) was performed using methods. Scoring was conducted according to the College of American Pathologists/American Society of Clinical Oncology (CAP/ ASCO) criteria for breast cancer [22].

Results
In the gallbladder cancer group, there were seven females and two males, with a median age of 63 years. One patient was Asian while others were Caucasian (Table 1). All had metastatic disease; seven received trastuzumab either alone (n = 2) or in combination with chemotherapy (n = 5). Other HER2-directed therapies were lapatinib and pertuzumab. One patient who received lapatinib had a mixed response; this patient had a HER2/neu mutation V777L. All other cases had HER2/neu amplification or overexpression. In all these cases, trastuzumab was associated with partial response (n = 4), stable disease (n = 3), or complete response (n = 1). (Figures 1,  2, 3, and 4 illustrate the responses seen in this group). The duration of response varied from 8+ to 168 weeks (median 40 weeks), and three patients are still on therapy. In two cases, therapy was administered neoadjuvantly for T4 disease and was followed by surgical resection. In one case, disease recurrence occurred 14 months after surgical resection, which was again successfully salvaged with trastuzumab ( Fig. 1). Details regarding prior therapy administered, concurrent chemotherapy, and duration of treatment are described in Table 2.
One patient had FGF3-TACC3 fusion gene and had received prior therapy with the FGFR-directed agents pazopanib and dovitinib. Repeat biopsy after these therapies indicated that the tumor had developed HER2/neu amplification as a secondary event, while this was absent at initial diagnosis. This finding was confirmed by FISH analysis of the tumor biopsy before and after the FGFRdirected therapy. In this case, disease stability or response was noted after FGFR-directed therapy as well as from trastuzumab. This was also accompanied by a CA19-9 response (Fig. 5a-c).
In the cholangiocarcinoma group, the median age was 59 years; all patients were Caucasian with stage IV disease. Two patients had HER2/neu mutations (V777L and S310F) while the others had amplification using FISH or NGS (Table 3). Associated mutations noted on NGS along with response data are depicted in Table 4. All but one had received prior therapy, and two patients received no concurrent chemotherapy. In case of cholangiocarcinoma, no responses occurred from trastuzumab therapy, unlike the case with gallbladder cancer. In all cases, HER2/neu-directed therapies were well-tolerated without any notable cardiac events despite the length of therapy.

Discussion
HER2/neu gene is a key driver of tumorigenesis and its overexpression as a result of gene amplification is a critical target for therapy in breast cancer. Other solid tumors with reported overexpression of this gene include gastric adenocarcinoma (11 %), pulmonary adenocarcinoma (28 %), colorectal adenocarcinomas (17 %), pulmonary squamous (11 %), and pancreatic adenocarcinoma (7 %). There are several existing reports of HER2/neu overexpression in gallbladder cancer and the incidence has varied widely, depending upon the method used for assessment and scoring technique. Our group recently studied HER2/neu expression in 187 cases of gallbladder cancer; this is the largest reported series to date using the commonly accepted CAP/ASCO criteria. We noted than 13 % of patients have HER2/neu overexpression (3+ by immunohistochemistry). Yoshikawa and colleagues reviewed 236 cases of surgically resected cholangiocarcinoma and reported a 0.9 and 8.5 % incidence of HER2/ neu expression in intra-and extrahepatic cholangiocarcinoma, respectively. In their series, early stage disease The mass directly invades the liver, with a 2.9 × 3.9 cm liver mass (arrow). b After 3 months of trastuzumab and FOLFOX, the polypoid gallbladder mass is no longer visualized, and the liver mass decreased to 1.2 × 1.1 cm (arrow). The patient was then treated with en bloc cholecystectomy and extended right hepatectomy, followed by capecitabine and trastuzumab for 7 months. c After 6 months of observation (14 months after surgery), the patient had small volume recurrence to retroperitoneal lymph nodes (large arrowheads). After treatment with FOXFOX and trastuzumab for 3 months, d a previously seen 1-cm retroperitoneal lymph node is nearly imperceptible and well-differentiated tumors had a higher incidence of HER2/neu positivity. Data with regard to the prognostic value of HER2/neu overexpression in biliary cancers is mixed, with some studies suggesting a worse prognosis [23,24], while others suggest the contrary [25,26]. NGS for HER2/neu gene amplification may be regarded as the gold standard. However, this technology is dependent upon the quality of the pathological sample and DNA degradation may result from formalin fixation. In our retrospective series, the criteria for HER2/neu positivity were stringent and included 3+ protein expression on immunohistochemistry, FISH ratio higher than 2.2 or HER2/neu genetic aberration in a Clinical Laboratory Improvement Amendment (CLIA) compliant laboratory. Slamon and others have described the remarkable success of HER2/neu-targeted therapy for breast cancer [27][28][29]. In gastric cancer, the trastuzumab for gastric adenocarcinoma (ToGA) trial established the benefit of trastuzumab in combination with a fluoropyrimidine plus cisplatin in a randomized phase 3 trial of gastric cancer patients with overexpression or gene amplification of HER2/neu [30]. It is logical therefore to investigate HER2/ neu targeting in other cancers that have gene amplification or overexpression.
Although the number of cases in this retrospective review is limited, to our knowledge, this is the largest reported case series of gallbladder cancer with HER2/ neu amplification or overexpression treated with targeted therapy. Our experience demonstrates that HER2/ neu-directed therapy appears to be beneficial for gallbladder cancer cases with HER2/neu amplification. Only one case in our series failed to respond, this tumor had HER2/neu mutation (V777L in the kinase domain). Two prior case reports have described responses with trastuzumab in subjects with gallbladder cancer. In the first, a 45-year-old woman received paclitaxel and trastuzumab and a dramatic regression of her lung metastases occurred [31]. In the second, a 61-year-old woman also with gallbladder cancer had a remarkable response to weekly paclitaxel and trastuzumab, which persisted when trastuzumab was continued as a single agent [32]. An estimated 80-100,000 cases of gallbladder cancer are  diagnosed worldwide annually, and the benefit of targeted therapy for those with HER2/neu amplification would be substantial.
In this review, we also report a patient with gallbladder cancer with FGF3-TACC3 fusion gene with negative HER2/neu gene amplification on NGS (confirmed by IHC and FISH, both being negative) who was treated with targeted FGFR inhibitors (dovitinib followed by pazopanib) resulting in initial response. She underwent repeat tumor biopsy on disease progression that then showed HER2/neu amplification on NGS (confirmed by IHC and FISH). In this case, we hypothesize that the HER2/neu amplification was a secondary event leading to resistance to FGFR inhibitors. Interestingly, FGFR2 addiction has been reported to be a mechanism leading to lapatinib resistance in breast cancer [33]. However, to our knowledge this is the first report of HER2/neu amplification as a mechanism of resistance to FGFR inhibitors. This finding Fig. 5 A 73-year-old female with metastatic retroperitoneal lymphadenopathy from gallbladder carcinoma. Axial contrast-enhanced CT images demonstrate a a 1.9-cm lymph node (arrowheads) posterior to the left renal vein. After 2 months of trastuzumab and pertuzumab, b the lymph node decreased to 1.2 cm. c Her CA 19-9 response to HER2/neu inhibition after prior FGFR inhibitor therapy In contrast, trastuzumab was less effective in the case of cholangiocarcinoma and there were no responses noted in our series. Two of these cases with cholangiocarcinoma had HER2/neu mutations, which are not known to be responsive to trastuzumab, but may potentially respond to tyrosine kinase inhibitors like lapatinib, neratinib, and canertinib [34]. HER2/neu-targeted therapy has also not proven to be effective despite the presence of gene amplification in diseases like colon and pancreas cancers. The reason for this lack of efficacy is not known. Junttila et al. demonstrated that trastuzumab disrupts ligand-independent ErbB2/ErbB3/PI3K complexes blocking AKT signaling; when PI3K is mutated or dysfunctional, complex disruption does not inhibit AKT [35]. Another possible explanation could involve the higher rate of KRAS mutations seen in intrahepatic cholangiocarcinomas as compared with gallbladder cancers, possibly mediating resistance to upstream HER2 blockade [2,5,36].
In summary, HER2/neu-directed therapy is a promising avenue for patients with gallbladder cancer with gene amplification and should be further explored in an international clinical trial.