Guanshan Zhu

EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples

Aims Activating mutations in the gene encoding epidermal growth factor receptor (EGFR) can confer sensitivity to EGFR tyrosine kinase inhibitors such as gefitinib in patients with advanced non-small-cell lung cancer. Testing for mutations in EGFR is therefore an important step in the treatment-decision pathway. We reviewed reported methods for EGFR mutation testing in patients with lung cancer, initially focusing on studies involving standard tumour tissue samples. We also evaluated data on the use of cytology samples in order to determine their suitability for EGFR mutation analysis. Methods We searched the MEDLINE database for studies reporting on EGFR mutation testing methods in patients with lung cancer. Results Various methods have been investigated as potential alternatives to the historical standard for EGFR mutation testing, direct DNA sequencing. Many of these are targeted methods that specifically detect the most common EGFR mutations. The development of targeted mutation testing methods and commercially available test kits has enabled sensitive, rapid and robust analysis of clinical samples. The use of screening methods, subsequent to sample micro dissection, has also ensured that identification of more rare, uncommon mutations is now feasible. Cytology samples including fine needle aspirate and pleural effusion can be used successfully to determine EGFR mutation status provided that sensitive testing methods are employed. Conclusions Several different testing methods offer a more sensitive alternative to direct sequencing for the detection of common EGFR mutations. Evidence published to date suggests cytology samples are viable alternatives for mutation testing when tumour tissue samples are not available.

Translating the Therapeutic Potential of AZD4547 in FGFR1-Amplified Non–Small Cell Lung Cancer through the Use of Patient-Derived Tumor Xenograft Models

Purpose: To investigate the incidence of FGFR1 amplification in Chinese non–small cell lung cancer (NSCLC) and to preclinically test the hypothesis that the novel, potent, and selective fibroblast growth factor receptor (FGFR) small-molecule inhibitor AZD4547 will deliver potent antitumor activity in NSCLC FGFR1–amplified patient-derived tumor xenograft (PDTX) models. Experimental Design: A range of assays was used to assess the translational relevance of FGFR1 amplification and AZD4547 treatment including in vitro lung cell line panel screening and pharmacodynamic (PD) analysis, FGFR1 FISH tissue microarray (TMA) analysis of Chinese NSCLC (n = 127), and, importantly, antitumor efficacy testing and PD analysis of lung PDTX models using AZD4547. Results: The incidence of FGFR1 amplification within Chinese patient NSCLC tumors was 12.5% of squamous origin (6 of 48) and 7% of adenocarcinoma (5 of 76). AZD4547 displayed a highly selective profile across a lung cell line panel, potently inhibiting cell growth only in those lines harboring amplified FGFR1 (GI50 = 0.003–0.111 μmol/L). AZD4547 induced potent tumor stasis or regressive effects in four of five FGFR1-amplified squamous NSCLC PDTX models. Pharmacodynamic modulation was observed in vivo, and antitumor efficacy correlated well with FGFR1 FISH score and protein expression level. Conclusions: This study provides novel epidemiologic data through identification of FGFR1 gene amplification in Chinese NSCLC specimens (particularly squamous) and, importantly, extends the clinical significance of this finding by using multiple FGFR1-amplified squamous lung cancer PDTX models to show tumor stasis or regression effects using a specific FGFR inhibitor (AZD4547). Thus, the translational science presented here provides a strong rationale for investigation of AZD4547 as a therapeutic option for patients with squamous NSCLC tumors harboring amplification of FGFR1. Clin Cancer Res; 18(24); 6658–67. ©2012 AACR.

The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.

Aims To evaluate the suitability of malignant pleural effusion (MPE) and plasma as surrogate samples for epidermal growth factor receptor (EGFR) mutation detection, and compare three different detection methods. Methods Matched tissue and plasma samples were collected from patients with advanced non-small cell lung cancer (NSCLC) (stage IIIB/IV adenocarcinoma/adenosquamous carcinoma), with matched MPE samples collected from a subgroup. DNA was extracted from tissue, MPE cell block, MPE supernatant and plasma before mutation detection by amplification refractory mutation system (ARMS) (all samples), Sanger sequencing and mutant-specific immunohistochemistry (IHC) (tissue and MPE cell blocks only). Results Sensitivity of MPE cell block, MPE supernatant and plasma versus tissue: 81.8% (9/11), 63.6% (7/11) and 67.5% (27/40); specificity was 80.0% (8/10), 100% (10/10) and 100% (46/46), respectively. Sensitivity of Sanger sequencing versus ARMS: 81.8% (27/33) for tissue, 40% (4/10) for MPE cell blocks; specificity was 100% (36/36 and 12/12) for both. Sensitivity of mutant-specific IHC versus ARMS: 54.8% (17/31) for tissue, 50.0% (6/12) for MPE cell blocks; specificity was 97.1% (34/35) and 100% (14/14), respectively. Conclusions MPE and plasma are valid surrogates for NSCLC tumour EGFR mutation detection when tissue is not available. ARMS is most suitable for mutation detection in tissue and MPE cell blocks; however, mutant-specific IHC could be a complementary method when DNA-based molecular testing is unavailable.

Plasma EGFR T790M ctDNA status is associated with clinical outcome in advanced NSCLC patients with acquired EGFR-TKI resistance

EGFR T790M mutation occurs in half of non-small cell lung cancer (NSCLC) patients with acquired EGFR-TKI (TKI) resistance, based on tumor re-biopsies using an invasive clinical procedure. Here, we dynamically monitored T790M mutation in circulating tumor DNA (ctDNA) using serial plasma samples from NSCLC patients receiving TKI through Droplet Digital PCR (ddPCR) method and the associations between overall survival (OS) starting from initial TKI treatment and the T790M ctDNA status detected in plasma were analyzed. Among 318 patients, 117 who acquired TKI resistance were eligible for the analysis. T790M ctDNA was detected in the plasma of 55/117 (47%) patients. Almost half of the T790M ctDNA positive patients were identified at a median time of 2.2 months prior to clinically progressive disease (PD). Furthermore, within the patients receiving TKI treatment at 2nd line or later, the T790M ctDNA positive group had significantly shorter OS than the negative group (median OS: 26.9 months versus NA, P = 0.0489). Our study demonstrates the feasibility of monitoring EGFR mutation dynamics in serial plasma samples from NSCLC patients receiving TKI therapy. T790M ctDNA can be detected in plasma before and after PD as a poor prognostic factor.

Establishment of patient-derived non-small cell lung cancer xenograft models with genetic aberrations within EGFR, KRAS and FGFR1: useful tools for preclinical studies of targeted therapies

BackgroundPatient-derived tumor xenograft models have been established and increasingly used for preclinical studies of targeted therapies in recent years. However, patient-derived non-small cell lung cancer (NSCLC) xenograft mouse models are relatively few in number and are limited in their degree of genetic characterization and validation. In this study, we aimed to establish a variety of patient-derived NSCLC models and characterize these for common genetic aberrations to provide more informative models for preclinical drug efficacy testing.MethodsNSCLC tissues from thirty-one patients were collected and implanted into immunodeficient mice. Established xenograft models were characterized for common genetic aberrations, including detection of gene mutations within EGFR and KRAS, and genetic amplification of FGFR1 and cMET. Finally, gefitinib anti-tumor efficacy was tested in these patient-derived NSCLC xenograft models.ResultsTen passable patient-derived NSCLC xenograft models were established by implantation of NSCLC specimens of thirty-one patients into immunodeficient mice. Genetic aberrations were detected in six of the models, including one model with an EGFR activating mutation (Exon19 Del), one model with KRAS mutation, one model with both KRAS mutation and cMET gene amplification, and three models with FGFR1 amplification. Anti-tumor efficacy studies using gefitinib demonstrated that the EGFR activating mutation model had superior sensitivity and that the KRAS mutation models were resistant to gefitinib. The range of gefitinib responses in the patient-derived NSCLC xenograft models were consistent with the results reported from clinical trials. Furthermore, we observed that patient-derived NSCLC models with FGFR1 gene amplification were insensitive to gefitinib treatment.ConclusionsTen patient-derived NSCLC xenograft models were established containing a variety of genetic aberrations including EGFR activating mutation, KRAS mutation, and FGFR 1 and cMET amplification. Gefitinib anti-tumor efficacy in these patient-derived NSCLC xenografts containing EGFR and KRAS mutation was consistent with the reported results from previous clinical trials. Thus, data from our panel of patient-derived NSCLC xenograft models confirms the utility of these models in furthering our understanding of this disease and aiding the development of personalized therapies for NSCLC patients.

Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models.

BackgroundTrastuzumab is currently approved for the clinical treatment of breast and gastric cancer patients with HER-2 positive tumors, but not yet for the treatment of esophageal carcinoma patients, whose tumors typically show 5 ~ 35% HER-2 gene amplification and 0 ~ 56% HER-2 protein expression. This study aimed to investigate the therapeutic efficacy of Trastuzumab in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models.MethodsPDECX models were established by implanting patient esophageal squamous cell carcinoma (ESCC) tissues into immunodeficient (SCID/nude) mice. HER-2 gene copy number (GCN) and protein expression were determined in xenograft tissues and corresponding patient EC samples by FISH and IHC analysis. Trastuzumab anti-tumor efficacy was evaluated within these PDECX models (n = 8 animals/group). Furthermore, hotspot mutations of EGFR, K-ras, B-raf and PIK3CA genes were screened for in the PDECX models and their corresponding patient’s ESCC tissues. Similarity between the PDECX models and their corresponding patient’s ESCC tissue was confirmed by histology, morphology, HER-2 GCN and mutation.ResultsNone of the PDECX models (or their corresponding patient’s ESCC tissues) harbored HER-2 gene amplification. IHC staining showed HER-2 positivity (IHC 2+) in 2 PDECX models and negativity in 3 PDECX models. Significant tumor regression was observed in the Trastuzumab-treated EC044 HER-2 positive model (IHC 2+). A second HER-2 positive (IHC 2+) model, EC039, harbored a known PIK3CA mutation and showed strong activation of the AKT signaling pathway and was insensitive to Trastuzumab treatment, but could be resensitised using a combination of Trastuzumab and AKT inhibitor AZD5363. In summary, we established 5 PDECX mouse models and demonstrated tumor regression in response to Trastuzumab treatment in a HER-2 IHC 2+ model, but resistance in a HER-2 IHC 2+/PIK3CA mutated model.ConclusionsThis study demonstrates Trastuzumab-induced tumor regressions in HER-2 positive tumors, and highlights PIK3CA mutation as a potential resistance mechanism to Trastuzumab treatment in pre-clinical patient-derived EC xenograft models.

Whole genome gene copy number profiling of gastric cancer identifies PAK1 and KRAS gene amplification as therapy targets.

Gastric cancer is the second leading cause of death from cancer worldwide, with an approximately 20% 5‐year survival rate. To identify molecular subtypes associated with the clinical prognosis, in addition to genetic aberrations for potential targeted therapeutics, we conducted a comprehensive whole‐genome analysis of 131 Chinese gastric cancer tissue specimens using whole‐genome array comparative genomic hybridization. The analyses revealed gene focal amplifications, including CTSB, PRKCI, PAK1, STARD13, KRAS, and ABCC4, in addition to ERBB2, FGFR2, and MET. The growth of PAK1‐amplified gastric cancer cells in vitro and in vivo was inhibited when the corresponding mRNA was knocked down. Furthermore, both KRAS amplification and KRAS mutation were identified in the gastric cancer specimens. KRAS amplification was associated with worse clinical outcomes, and the KRAS gene mutation predicted sensitivity to the MEK1/2 inhibitor AZD6244 in gastric cancer cell lines. In summary, amplified PAK1, as well as KRAS amplification/mutation, may represent unique opportunities for developing targeted therapeutics for the treatment of gastric cancer.

The essential role of TNIK gene amplification in gastric cancer growth.

Traf2- and Nck-interacting kinase (TNIK) is one of the germinal center kinase family members involved in cytoskeleton organization and neuronal dendrite extension. Emerging evidence supports that TNIK is essential for activation of WNT signaling pathway in colon cancer growth. To search for novel genetic aberrations that drive carcinogenesis, we performed microarray-based comparative hybridization assay for gene copy number variations in primary tumor samples. Our data showed that TNIK gene was amplified in 7% (8/106) of Chinese gastric cancer patients. Theses amplifications were confirmed by fluorescence in situ hybridization analysis. PAMC82 human gastric cancer and T47D human breast cancer cell lines with TNIK amplification were identified to further understand the function of TNIK gene amplification. RNA-interference-mediated silencing of TNIK resulted in significant inhibition of cell growth and induction of cell death in TNIK-amplified, but not in TNIK-non-amplified, cell lines tested. This selective sensitivity to the TNIK inhibition was also observed under the effect of a small-molecule TNIK inhibitor. Furthermore, our data indicated that TNIK’s role in gastric cancer growth was not dependent on Wnt signaling but rather was involved in AKT activation and cell autophagy. Together, our results suggest that TNIK is a novel therapeutic target in gastric cancer and TNIK amplification can be potentially used for patient selection.

Identification of HBV-MLL4 Integration and Its Molecular Basis in Chinese Hepatocellular Carcinoma

To gain molecular insights of HBV integration that may contribute to HCC tumorigenesis, we performed whole transcriptome sequencing and whole genome copy number profiling of hepatocellular carcinoma (HCC) samples from 50 Chinese patients. We identified a total of 33 HBV-human integration sites in 16 of 44 HBV-positive HCC tissues, which were enriched in HBV genotype C-infected patients. In addition, significantly recurrent HBV-MLL4 integration (18%; 8/44) was found in this cohort of patients. Using long-range PCR and Sanger sequencing, we comprehensively characterized gDNA and cDNA sequences that encode for the HBV-MLL4 transcripts, and we revealed that HBV integration into MLL4 exons led to much higher mRNA expression of MLL4 than the integration into MLL4 introns due to an alternative splicing mechanism. Moreover, the HBV-MLL4 integration occurred almost exclusively in CTNNB1 and TP53 wild-type patients. The integration was also associated with a distinct gene expression profile. In conclusion, this is the first report on the molecular basis of the MLL4 integration driving MLL4 over-expression. HBV-MLL4 integration occurred frequently in Chinese HCC patients, representing a unique molecular segment for HCC with HBV infection.

An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma

To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break‐apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break‐apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break‐apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs.