Xiaoming Song

A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy

A preclinical trial identified 4 of 20 (20%) gastric cancer (GC) patient-derived xenografts responded to cetuximab. Genome-wide profiling and additional investigations revealed that high EGFR mRNA expression and immunohistochemistry score (3+) are associated with tumor growth inhibition. Furthermore, EGFR amplification were observed in 2/4 (50%) responders with average copy number 5.8 and >15 respectively. Our data suggest that a GC subtype with EGFR amplification and overexpression benefit from cetuximab treatment.

Overcoming erlotinib resistance with tailored treatment regimen in patient‐derived xenografts from naïve Asian NSCLC patients

Overall benefits of EGFR‐TKIs are limited because these treatments are largely only for adenocarcinoma (ADC) with EGFR activating mutation. The treatments also usually lead to development of resistances. We have established a panel of patient‐derived xenografts (PDXs) from treatment naïve Asian NSCLC patients, including those containing “classic” EGFR activating mutations. Some of these EGFR‐mutated PDXs do not respond to erlotinib: LU1868 containing L858R/T790M mutations, and LU0858 having L858R mutation as well as c‐MET gene amplification, both squamous cell carcinoma (SCC). Treatment of LU0858 with crizotinib, a small molecule inhibitor for ALK and c‐MET, inhibited tumor growth and c‐MET activity. Combination of erlotinib and crizotinib caused complete response, indicating the activation of both EGFR and c‐MET promote its growth/survival. LU2503 and LU1901, both with wild‐type EGFR and c‐MET gene amplification, showed complete response to crizotinib alone, suggesting that c‐MET gene amplification, not EGFR signaling, is the main oncogenic driver. Interestingly, LU1868 with the EGFR L858R/T790M, but without c‐met amplification, had a complete response to cetuximab. Our data offer novel practical approaches to overcome the two most common resistances to EGFR‐TKIs seen in the clinic using marketed target therapies.

Abstract 1611: Hepatocellular carcinoma xenograft models and biomarkers are powerful tools for the evaluation of the new drugs targeting HCC

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Hepatocellular Carcinoma (HCC) is a major health problem, as the fifth most common cancer and the third most common cause of cancer death worldwide. Many pharmaceutical companies lead the effort to design new medicines on this cancer type. The overwhelming need to improve preclinical models of HCC requires robust models that closely mimic the disease population and therefore have the potential to better predict clinical outcome with novel therapies. Here we successfully validated subcutaneous and orthotopic xenograft models derived from HepG2, SK-HEP-1, Hep3B, PLC/PRF/5, HUH-1, HUH-7 and MHCC97H cell lines and also directly from HCC patient tumors (HuPrime®). Four HCC serum markers (AFP, α-fetoprotein; AFP-L3; DCP, Des-gamma-Carboxyprothrombin; GP73, Golgi Protein-73) have been used or will be potentially used clinically in detection of this disease. AFP is the most widely used biomarker in HCC, somehow it is not always correlated well with the liver cancer progress, even it gives false result, and some liver lesion or liver cirrhosis can also produce high level of AFP; while GP73 is reported a promising alternative as HCC serum marker. AFP and GP73 were tested at both mRNA level and protein level. They were also compared in the sensitivity and correlation with the tumor burden using the blood samples from the orthotopic xenograft models. Our results demonstrated that biomarkers released by the tumor models could well monitor the tumor take rate, growth and response to treatments. Furthermore, Sorafenib, one popular anti- HCC medicine, was tested, and the serum biomarkers could be utilized for confidence in evaluating the efficacy of Sorafenib. In conclusion, the use of HCC xenograft models combined with the biomarker strategies is helpful in maximizing the value of in vivo research and designing clinical trials Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2011-1611

Abstract A91: Activating EGFR mutation, G719A, was selected in the c-MET amplified NSCLC-PDX-LU1901 during the treatments by c-MET inhibitors and the resistance development.

We previously described the establishment of an Asian NSCLC patient derived xenograft (PDX), LU1901 (large cell carcinoma, or LCC), from a surgically removed patient tumor tissue, along with large panel of other NSCLC-PDX (1). LU1901 was found to have amplified c-MET gene and responded to multiple c-MET inhibitors, including crizotinib (near-complete regression) (1). Interestingly, we recently also observed that the TKI treatments, including crizotinib, induced rapid development of resistance against the TKIs (LU1902, LU1903) (2). However, efforts so far have yet to demonstrate mechanism causing the induced resistance (2). We are currently investigating many aspects of the resistant tumors, including histopathology and molecular features, e.g., further c-MET mutation, c-MET activation status, and inhibition by c-MET-TKI, etc. By hotspot sequencing of EGFR gene of LU1901 and LU1903 recently (Sanger Method), we found a known activating mutation, G719A in exon 18, was selected during the treatment that was also accompanied by the c-MET TKI resistance development. G719A has been found in small subset of patients (∼3%) (3), and has been associated with increased sensitivity to EGFR-TKIs (4), suggesting it could be a new driver mutation gained in resistant LU1903. At present, our working hypothesis is that the selection of G719A mutation of EGFR rendered LU1903 resistant to c-MET-TKI. We have planned further experiments to verify this hypothesis. As a first step, we performed RNAseq of the TKI-sensitive parental LU1901, and found that G719A seemed to already exist, although at much lower frequency as compared to wild type allele at this location. This preliminary results suggests that G719A is a pre-exist mutation in a small subset of LU1901 tumor cells which was rapidly selected out during c-MET TKI treatment. Our current working hypothesis is that this selection could be responsible for the resistance development. If our working hypothesis is eventually proven, it will provide a new important mechanism for the development of c-MET TKI resistancy that can occur in the clinic during MET based therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A91. Citation Format: Mengmeng Yang, Jie Cai, Sheng Guo, Shijun Yu, Wubin Qian, Xiaoming Song, Youzhu Wang, Jean-Pierre Wery, Henry Li. Activating EGFR mutation, G719A, was selected in the c-MET amplified NSCLC-PDX-LU1901 during the treatments by c-MET inhibitors and the resistance development. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A91.

Abstract B43: A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy.

Gastric carcinoma (GC) is a common cancer of high mortality and particularly prevalent in East Asia. There are only a few treatment options: surgery, chemotherapy, as well as Herceptin® for a small subset of patients with erbb2 amplification. Cetuximab has been approved for CRC (colorectal) and SCCHN, but not yet for gastric carcinoma. Cetuximab, combined with capecitabine/cisplatin, has recently been tested in Phase III clinical trial (EXPAND) in GC patients (1), but failed to demonstrate benefit over the existing chemotherapy (capecitabine/cisplatin). One of the reasons for the failure is only small subset of patients responding to cetuximab, which constitutes little effects on large trial populations, while the identity of the responders yet to be revealed. One of the keys to a successful search for effective treatment is the development of experimental model that truly mimics patient conditions. Patient derived xenograft (PDX or HuPrime®) is believed to best mimic human cancers (2). Recently, we have established a cohort of ∼70 GC xenograft (PDX) models from Asian and Caucasian patients. In the present study, we conducted a mouse clinical trial (MCT, or PDX trial) to test cetuximab in a cohort of GC-PDX, aiming at identifying those responsive to cetuximab. Interestingly, our trial result identified 4 of 20 (20%) GC-PDX responded to cetuximab. Genome-wide profiling and additional investigations revealed that high EGFR mRNA expression and immunohistochemistry score (3+) are associated with tumor growth inhibition. Furthermore, EGFR amplification were observed in 4/4 (50%) responders by DNAchip analysis or 2/4 confirmed with FISH analysis, with average copy number 5.8 and >15 respectively. Our data suggest that a GC subtype with EGFR amplification and overexpression benefit from cetuximab treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B43. Citation Format: Lianhai Zhang, Jie Yang, Yiyou Chen, Jie Cai, Xiaoming Song, Xuesong Huang, Jianyun Deng, Dawei Chen, Mengmeng Yang, Shuangxi Li, Aiwen Wu, Ziyu Li, Zhongwu Li, Yiqian Liu, Jean-Pierre Wery, Henry Li, Jiafu Ji. A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B43.

Abstract C14: Squamous non-small cell lung cancer (NSCLC-SCC) patient-derived xenografts (PDX) from Asian patients have high response rate (RR) to cetuximab than those of adenocarcinoma.

Cetuximab was approved for advanced colorectal carcinoma (CRC), but yet for NSCLC, although some trials [e.g. FLEX (1) and BMS099 (2)] demonstrated benefit in subsets of patients. Recent retrospective study on Caucasian patients among the FLEX study cohort revealed that high EGFR expressors, uniquely defined by high-IHC score, are likely to benefit from the treatment (3). In particular, this was confirmed in a 50-patient derived xenografts model trial, mouse clinical trial (MCT or “Patient Avatar Trial”), in a similar format of FLEX, first time demonstrating relevance of human trial and MCT. The goal of the present study is identify the subset of Asian NSCLC patients that respond to cetuximab in a MCT. We have established a large panel of PDXs from NSCLC Asian patients of different histology (4): squamous cell carcinoma (SCC), adenocarcinoma (ADC), pleiomorphic carcinoma (PLC), and large cell carcinoma (LCC). We tested cetuximab activity in a randomly selected 32 PDX in a MCT. While NSCLC had overall engraftment rate of 25%, SCC interestingly, had higher rate of ∼31%, near-double ∼17% of ADC, which may have metastasis and prognosis implications. A subset of PDXs were found to respond to cetuximab (10/32) (31%, defined by %ΔT/ΔC 25%) include ADC (7/22), LCC (2/22) and SCC (12/22). Among the 12 non-/poor-responsive SCC, one has KRAS-G12D mutation, one has ALK-EML4 fusion and one has c-MET gene amplification. Among the SCC responders are ones with activating EGFR mutations. SCC has strikingly higher cetuximab response rate (RR) than ADC, with possible positive factors of EGFR mutation and negative factors of KRAS mutation, ALK fusion and c-met amplification. If proven true clinically, cetuximab could become a new therapy for NSCLC-SCC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C14. Citation Format: Mengmeng Yang, Jie Cai, Sheng Guo, Xuesong Huang, Jie Yang, Xiaoming Song, Dawei Chen, Likun Zhang, Jiahua Jiang, Jean-Pierre Wery, Henry Li. Squamous non-small cell lung cancer (NSCLC-SCC) patient-derived xenografts (PDX) from Asian patients have high response rate (RR) to cetuximab than those of adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C14.

Abstract 1614: Murine matrigel plug assay for quick evaluation of anti-angiogenesis drugs.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Angiogenesis plays important roles in disease processes such as tumor development and progression, as well as many physiological conditions. A few cancer drugs were successfully approved based on anti-angiogenesis mechanisms. More candidates are in different evaluation stages, including clinical phase II stages. Among many assays used for angiogenesis evaluation, Murine Matrigel Plug Assay (MMPA) is a relatively simple, highly effective in vivo assay to evaluate anti-angiogenesis responses of the test articles. Because of its simplicity and short assay duration, the assay is scalable for efficient screening of large number of test articles. However, the analysis of the plugs, usually with H&E staining and microvessel counting, is time consuming and error prone, hampers the usage of this method as a general screening assay for test articles. Here, we set out to test a number of combinations of assay conditions, including mouse strains, angiogenesis inducers, varying days of treatment, and different routes of SOC drug treatments. More importantly, we tested plug analysis methods including H&E staining, CD31 IHC staining, as well as hemoglobin concentration analysis. We demonstrated that hemoglobin concentration analysis results are corroborated by IHC staining as well as H&E staining, in most of the conditions tested. Hemoglobin assay is less time consuming and easier to scale up. Thus MMPA followed by hemoglobin analysis may be suitable for large scale screening studies for angiogenesis modulators. Interestingly, CTX, one of the popular anti-cancer therapeutics, was shown in MMPA as a good anti-angiogenesis agent. In conclusion, the MMPA described may greatly facilitate the preclinical development of cancer therapeutics that target the angiogenesis. Citation Format: Yanmei Sun, Xiaoming Song, Nan Li, Jie Cai, Qian Shi, Taiping Chen. Murine matrigel plug assay for quick evaluation of anti-angiogenesis drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1614. doi:10.1158/1538-7445.AM2013-1614

Abstract 337: A NSCLC PDX with EML4-ALK fusion mutation responds to crizotinib.

Non-small cell lung cancer (NSCLC) has the highest mortality among all malignances while with limited treatment options. Among the most significant treatment advancement in recent years are the introduction of target therapies of EGFR-TKIs (Erlotinib and Gefitinib) for adenocarcinoma with EGFR activating mutations, and Crizotinib for NSCLC with ALK activation (EML4-ALK fusion)1. ALK activation are associated with a subset of NSCLC patients (3∼5%).Crizotinib is considered not a particularly potent ALK inhibitor, with IC50 at range of tens of nanomoles. It may not be an ideal agent for treating NSCLC with activating ALK. Therefore, a new ALK inhibitor with better potency and specificity could be of particularly medical importance. Currently, the industry is actively searching for such new agents. A relevant experimental model could also greatly facilitate evaluating and validating these new drug candidates. Patient derived xenografts (PDX) (including NSCLC PDX) mirror patients’ histopathology and genetic profiles2, thus known as “patient avatar”. It has improved predictive power as preclinical cancer models, and enables discovery of predictive biomarkers for targeted therapeutics. We have established a large collection of NSCLC PDX models (NSCLC-HuPrime®) by engrafting naive patient tumor tissues into immune-compromised mice2. Among these models, we have identified one model, LU1656, of NSCL carcinosarcoma histology and with EML4-ALK fusion mutation. This mutation apparently causes over-expression of ALK as measured at mRNA level. This model has wild-type KRAS, c-MET and EGFR genes. Treatment of LU1656 with Crizotinib caused significant but not complete tumor response, suggesting ALK activation is one of the oncogenic drivers of this model. In addition, LU1656 also responded to Cetuximab, but respond poorly to Erlotinib. It would be interesting to examine the combination of these treatments. This model is, to our knowledge, the first reported PDX model with EML4-ALK fusion mutation. We believe that LU1656 would be a useful model to evaluate investigational ALK inhibitors, and new combination treatment regimens of Crizotinib and standard of care chemotherapies. Citation Format: Jie Cai, Mengmeng Yang, Jianyun Deng, Xiaoming Song, Xuesong Huang, Taiping Chen, Jean Pierre Wery, Yiyou Chen, Henry QX Li. A NSCLC PDX with EML4-ALK fusion mutation responds to crizotinib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 337. doi:10.1158/1538-7445.AM2013-337

Abstract 2731: Classification of HCC patient derived xenograft (PDX) models by transcriptome and microRNome profiling, and their resemblance to patients.

Hepatocellular carcinoma (HCC) is a disease of diverse types. Transcriptome analysis recently classified HCC into three major categories with distinct clinical parameters as well as cellular differentiation1. They are S1 (stem-like group with activation of the WNT pathway and TGF-β), S2 (activation of MYC and AKT), and S3 (hepatocyte differentiation). Patient derived xenografts (PDXs) have been suggested to be predictive experimental models (“Patient avatars”) because they have not been in vitro manipulated, and have the original patients’ histopathological and genetic profiles2,3. In order to assess the resemblance of these “avatar” to patients, we performed transcriptome profiling on a collection of HCC-PDXs using the same algorithm1. Twenty-two HCC-PDX models were classified into 3 groups, among which S1 and S2 are more closely related. Using the 572 genes generated from the algorithm, the same classifications were obtained by hierarchical clustering and principal component analysis (PCA), except for two outliers. The results demonstrate that our cohort of HCC-PDXs can be divided into the same three subclasses as those from patient samples1. In addition, Luk et al., recently classified HCC using expression levels of microRNA cluster at 14q32.2 locus, in which a stem-like HCC phenotype was associated with high expression4. We thus also used this criterion to classify our collection of HCC PDXs. The two resulting categories, 14q32.2-hi and 14q32.2-lo, can be compared to the S1, S2, S3 subclasses determined by mRNA profiling described above, in which S1 belongs to 14q32.2-lo and S2/3 belong to 14q32.2-hi (except LI1025, LI1078). The serum α-fetoprotein (AFP) and tumor AFP-mRNA levels were found to be strongly and positively correlated, and also associated with S21 and 14q32.2-hi4, consistent with previous reports1. Six stem-like markers were found not to be associated with S11, nor with 14q32.2-hi4, thus different from the report by Luk et al4. Activation of c-MET was found exclusively in S1 models, consistent with one of previous observations5, but not the other4. When all these models were treated with Sorafenib, a multi-kinase inhibitor, there seemed to be no correlation in the tumor responses between the subclasses. In summary, the data provided insights into the classifications of HCC by mRNA expression1 and 14q32.2 microRNA expression4, partially consistent with existing reports1,2. The results also show distinctive characteristics of our HCC-PDXs that merit further studies. Citation Format: Sheng Guo, Wubin Qian, Jie Cai, Dawei Chen, Jie Yang, Zhun Wang, Xiaoming Song, Taiping Chen, Jean Pierre Wery, Yiyou Chen, Henry QX Li. Classification of HCC patient derived xenograft (PDX) models by transcriptome and microRNome profiling, and their resemblance to patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2013-2731

Abstract 5630: Treatments of NSCLC-PDX with c-MET gene amplification by c-MET inhibitors lead to rapid development of drug resistance.

Drug resistance is one of important factors limiting effect of most cancer therapies. Understanding mechanisms causing resistance can facilitate overcoming resistance. c-MET gene activation, via either activating mutations or gene amplification, is an oncogenic driver for some non-small cell lung cancers (NSCLC) in patients as well as in patient derived xenograft (PDX) (1). c-MET gene activation is also one of the two most dominant TKI-resistance seen among the patients treated with EGFR-TKI (tyrosine kinase inhibitors) (1, 2). Therefore, c-MET inhibitor could become an important treatment option for NSCLC patients whose c-MET is either de nova activated or activated during TKI treatment (1). We recently demonstrated that Crizotinib (PF2341066), an approved drug against NSCLC with ALK-fusion and a known c-MET inhibitor, can effectively treat NSCLC with c-MET gene amplification, including those resistant to EGFR-TKIs (1), in PDXs. Specifically, we described two models LU1901 and LU2503 – both with amplified gene, accompanied with over-expression, of c-MET, and both sensitive to Crizotinib (complete regression in LU2503, while near-complete regression in LU1901) (1), confirming the c-MET activation as dominant oncogenic driver for these two tumors. In the present study, interestingly, the treated LU1901 tumors subsequently re-emerge and re-grow even in the presence of continuous treatment. In particular, the re-growed LU1901 become completely resistant to Crizotinib (we call LU1901R), even after it was re-engrafted. Pharmacodynamic analysis revealed that the LU1901R tumor has similar level of phosphorylation (or activation) of c-MET as the parental LU1901. Crizotinib treatment caused nearly-similar inhibition of c-MET in both LU1901 and LU1901R, as well as the downstream signaling, including pMEK and pAKT by Crizotinib in both LU1901 and LU1901R tumors along with c-MET inhibition. All these suggest that the threshold of c-MET activation for prompting tumor growth/survival is significantly lower in LU1901R than LU1901. We are currently characterizing the LU1901R in order to identify the mechanisms of resistance, and attempt to find approach to overcome them. In summary, we have successfully developed c-MET inhibitor resistant models that may reflect resistance emergence in the clinics. These models could be particularly useful in investigating clinical resistant mechanisms and evaluating new therapy that can overcome resistance. Citation Format: Mengmeng Yang, Xiaoming Song, JIe Cai, Youzhu Wang, Taiping Chen, Jean-Pierre Wery, Yiyou Chen, Henry Li. Treatments of NSCLC-PDX with c-MET gene amplification by c-MET inhibitors lead to rapid development of drug resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5630. doi:10.1158/1538-7445.AM2013-5630