(1) Background: Early predictive markers to track treatment responses are needed for advanced esophageal squamous cell carcinoma (ESCC) patients. We examined the prognostication and risk stratification role of liquid biopsy serial monitoring for this deadly cancer. (2) Methods: Circulating tumor cells (CTCs) and plasma cell-free DNA (cfDNA) were isolated from 60 ESCC patients treated by chemotherapy (CT) at five serial timepoints: baseline (CTC1/cfDNA1), CT pre-cycle III (CTC2/cfDNA2), CT post-cycle IV, end of CT and relapse. (3) Results: In 45/57 ESCC patients with evaluable CTC counts at CT pre-cycle III, positive CTC2 (≥3 CTCs) is independently associated with response at interim reassessment and progression-free survival (PFS) in multivariate analysis. In 42/57 ESCC patients with changes of CTC1/CTC2 and cfDNA1/cfDNA2, patients categorized into four risk groups based on the number of favorable and unfavorable changes of CTC1/CTC2 and cfDNA1/cfDNA2, were independently associated with overall survival (OS) by multivariate analysis. (4) Conclusions: CTC counts at pre-cycle III are independently associated with response at interim reassessment and PFS. Combined changes of CTC counts and cfDNA levels from baseline to pre-cycle III are independently associated with OS. Longitudinal liquid biopsy serial monitoring provides complementary information for prediction and prognosis for CT responses in advanced ESCC.
// Arthur Kwok Leung Cheung 1,2 , Joseph Chok Yan Ip 1 , Adrian Chi Hang Chu 1 , Yue Cheng 1,2 , Merrin Man Long Leong 1 , Josephine Mun Yee Ko 1 , Wai Ho Shuen 1,4 , Hong Lok Lung 1,2 and Maria Li Lung 1,2,3 1 Department of Clinical Oncology, University of Hong Kong, Hong Kong (SAR), People’s Republic of China 2 Centre for Cancer Research, University of Hong Kong, Hong Kong (SAR), People’s Republic of China 3 Centre for Nasopharyngeal Carcinoma Research, University of Hong Kong, Hong Kong (SAR), People’s Republic of China 4 Division of Medical Oncology, National Cancer Centre, Singapore Correspondence to: Maria Li Lung, email: // Keywords : PTPRG, Akt, EGFR, nasopharyngeal carcinoma, tumor suppressor Received : January 13, 2015 Accepted : April 03, 2015 Published : April 19, 2015 Abstract Protein Tyrosine Phosphatase, Receptor Type G (PTPRG) was identified as a candidate tumor suppressor gene in nasopharyngeal carcinoma (NPC). PTPRG induces significant in vivo tumor suppression in NPC. We identified EGFR as a PTPRG potential interacting partner and examined this interaction. Dephosphorylation of EGFR at EGFR-Y1068 and -Y1086 sites inactivated the PI3K/Akt signaling cascade and subsequent down-regulation of downstream pro-angiogenic and -invasive proteins (VEGF, IL6, and IL8) and suppressed tumor cell proliferation, angiogenesis, and invasion. The effect of Akt inhibition in NPC cells was further validated by Akt knockdown experiments in the PTPRG-down-regulated NPC cell lines. Our results suggested that inhibition of Akt in NPC cells induces tumor suppression at both the in vitro and in vivo levels, and also importantly, in vivo metastasis. In conclusion, we confirmed the vital role of PTPRG in inhibiting Akt signaling with the resultant suppression of in vivo tumorigenesis and metastasis.
Abstract Background and Aims The genetic etiology of NPC and mechanisms for inherited susceptibility remain unclear. Only modest low-penetrance effects of cancer-predisposing common variant SNPs were previously identified in the few large-scale NPC association studies reported. Most NPC association studies focused on single or limited candidate genes with modest sample sizes. Systematic and comprehensive study designs for evaluation of higher order gene-gene interactions are scanty. A large-scale NPC case-control SNP association study was performed to examine the genetic risk factors for NPC development. In order to elucidate the genetic susceptibility for NPC, we hypothesized that heritable risk is attributable to cumulative effects of common low-risk variants, especially for genes in DNA repair pathways. Material and Methods Genotype screening of 377 SNPs and validation in an 81-SNP panel of DNA repair and other genes in 2349 individuals from Hong Kong (1177 cases and 1172 controls) using iPLEX Gold assays with the MassARRAY platform were performed for association analyses for risk allele identification and pathway-based studies for NPC. All statistical tests were two-sided. Results Three SNPs (rs401681, rs6774494, rs3757318) corresponding to TERT/CLPTM1L (OR 0.77, 95% CI 0.68-0.88), MDS1-EVI1 (OR 0.79, 95% CI 0.69-0.89), and CCDC170 (OR 0.76, 95% CI 0.66-0.86) conferred modest protective effects individually for NPC risk by the logistic regression (pBonferroni <0.05) (Table II). Stratification of NPC according to familial status identified rs2380165 in BLM (OR 1.49, 95% CI 1.20-1.86) association with family history-positive NPC (FH+NPC) patients (pBonferroni <0.05) (Table III). Higher order multiple SNPs pathway-based analysis revealed that the combined adverse effects of TERT-CLPTM1L and double-strand break repair (DSBR) (HR, NHEJ, FA-BS, and FA-HR pathways) conferred significant elevated risk up to OR 5.6 (95% CI 2.67-11.78) in FH+NPC and 2.4 (95% CI 1.28-4.48) in sporadic NPC patients carrying five adverse alleles in TERT/NHEJ and TERT/FA/BS pathways, respectively. (Fig. 2,3) Conclusions Three SNPs corresponding to MDS1-EVI1, TERT/CLPTM1L, and CCDC170 conferred modest protective effects individually for NPC risk in Hong Kong patients. Importance of pathway-based analysis for the cumulative increased risk associations with TERT locus and DNA repair pathways for DBS repair play a critical role in determining genetic susceptibility to NPC. A SNP in BLM is associated with FH+NPC patients. Pathway-based analysis also showed that the cumulative effect is more pronounced in FH+NPC patients. Aknowledgements Hong Kong Research Grants Council Area of Excellence grant (AoE/M-06/08) Citation Format: Maria L. Lung, Josephine Ko, Wei Dai, Elibe Wong, Dora Kwong, Wai Tong Ng, Anne Lee, Roger Ngan, Chun Chung Yau, Stewart Tung. Adverse effects of TERT-CLPTM1L and double-strand breaks repair contribute to risk for nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4148. doi:10.1158/1538-7445.AM2014-4148
ADAMTS metalloprotease family member ADAMTS9 maps to 3p14.2 and shows significant associations with the aerodigestive tract cancers esophageal squamous cell carcinoma (ESCC) and nasopharyngeal carcinoma (NPC). However, the functional impact of ADAMTS9 on cancer development has not been explored. In this study, we evaluated the hypothesized antiangiogenic and tumor-suppressive functions of ADAMTS9 in ESCC and NPC, in stringent tumorigenicity and Matrigel plug angiogenesis assays. ADAMTS9 activation suppressed tumor formation in nude mice. Conversely, knockdown of ADAMTS9 resulted in clones reverting to the tumorigenic phenotype of parental cells. In vivo angiogenesis assays revealed a reduction in microvessel numbers in gel plugs injected with tumor-suppressive cell transfectants. Similarly, conditioned medium from cell transfectants dramatically reduced the tube-forming capacity of human umbilical vein endothelial cells. These activities were associated with a reduction in expression levels of the proangiogenic factors MMP9 and VEGFA, which were consistently reduced in ADAMTS9 transfectants derived from both cancers. Taken together, our results indicate that ADAMTS9 contributes an important function in the tumor microenvironment that acts to inhibit angiogenesis and tumor growth in both ESCC and NPC.
: Circulating tumor cells (CTCs) and disseminated tumor cells (DTCs), traveling in our blood circulation as single cells or rarely as circulating tumor microemboli (CTM), are considered as the metastatic seeds for spreading of solid tumors. CTCs are highly heterogenous. Only the aggressive CTC subpopulations with cancer stem cell (CSC)-like and partial epithelial-mesenchymal transition (EMT) properties survive and undergo clonal evolution during disease progression. The real-time non-invasive monitoring of CTCs alone or combined with cell-free DNA (cfDNA) hold great potential as predictive or prognostic biomarkers of treatment response. This review summarizes the CTC enrichment strategies and findings suggestive for its potential clinical utility in tracking of minimal disease burden and prognostication of nasopharyngeal carcinoma (NPC). Although the NPC CTC field studies are still in their infancy, with technological advancement in CTC isolation platforms and sensitive multi-omics profiling, the potential clinical application of CTC analysis on its own or in combination with other sources of liquid biopsy holds great promise in future translation into the clinic, as non-invasive real-time serial monitoring of treatment of advanced NPC patients. CTC studies, especially at the single cell level, are anticipated to provide knowledge for identification of new therapeutic targets and drug resistance mechanisms, novel biomarkers for prognostication and prediction of treatment responses for promising translation into precision personalized medicine.
Abstract Introduction: Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in Northern China, where there is evidence for a significant familial association. We performed targeted next-generation sequencing (NGS) analysis on familial ESCC germline samples compared to non-cancer controls from the same high-risk region and compiled a list of candidate cancer predisposition genes. Interestingly, genes related to the Fanconi Anemia (FA) - BRCA pathway are enriched in the list. Among these FA-BRCA genes, Fanconi anemia complementation group D2 (FANCD2) was one of the top candidates, as it also had a high frequency of somatic mutations in ESCC tumor specimens. Therefore, we aim to characterize the role of FANCD2 in tumor development and explore its translational value. Methods: We knocked out the FANCD2 gene in ESCC cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique to evaluate its potential oncogenic function in ESCC. Cell proliferation was measured by a MTT 2D clonogenic assay in vitro. Subcutaneous injection of the FANCD2 knockout ESCC cells into BALB/c-nude mice in vivo was performed to assess its functional impact on tumorigenesis. The single cell gel electrophoresis/comet assay was used to investigate the genome stability. Results: The FANCD2 knockout efficiency was confirmed by western blotting. Surprisingly, in vitro functional analyses showed that ESCC cells with FANCD2 knockout survive, with a greatly reduced growth rate and colony-forming ability. Consistent with the in vitro data, ESCC cells with FANCD2 knockout form significantly smaller subcutaneous tumors in nude mice. By applying the comet assay to examine the genome integrity, ESCC cells with FANCD2 knockout show significantly greater damage to the genome. Conclusion: These results suggest that FANCD2 plays an important role in supporting ESCC tumor growth. We attribute this to its core function in DNA repair ability and genome integrity maintenance. Acknowledgement: We acknowledge the grant support from the Hong Kong Research Grants Council Collaborative Research Fund (C7031.15G to M.L.L.). Citation Format: Lisa Chan Lei, Valen Zhuoyou Yu, Lvwen Ning, Josephine Mun-Yee Ko, Li Dong Wang, Maria Li Lung. Functional characterization of FANCD2 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1357.
Background: Circulating tumor cells (CTCs) are released from primary or metastatic tumors into the cancer patient's bloodstream and may establish secondary tumors in distant body sites. Their frequencies are thought to have great prognostic impact and their mutation signatures may yield pointers in treatment decisions. However, CTCs are extremely rare in the blood and are challenging to sequence. Current next-generation-sequencing (NGS) technologies and analysis procedures must be carefully strategized to maximize sensitivity, while maintaining specificity and controlling false discovery rates.Aim of the study: This study aims to establish a robust and sensitive NGS bench work and analysis pipeline for genetic analysis of CTCs with good positive predictive value (PPV) and low false discovery rate (FDR), which can ultimately be translated into clinical application. We will compare and evaluate the performance of two NGS platforms for CTC analysis in the Hong Kong cancer patients.Methods: This study will streamline the sequencing and analyses of two NGS approaches, i.e. amplicon-based or hybridization capture-based, for their application for CTC analyses. Firstly, a cancer cell line with known mutations will be spiked into normal blood cells at different percentages and sequenced with both approaches independently. The amplicon-based platform covers 60 cancer relevant and actionable genes, while the customized capture kit for the hybridization-based platform includes 245 cancer relevant genes curated in our group. Both platform's sensitivity, specificity, PPV and FDR will be evaluated. Secondly, CTCs from several cancer types with high incidence in Asia will be enriched by size, enumerated for comparative analysis, and sequenced. Sequencing results will be used to refine the analysis pipelines for both platforms. Results of sequencing analyses, including potential druggable targets and prognostic markers identified, will be determined.Results: We successfully detected over 80% of known mutations at as low as 1.25% CTC purity, and with FDRs below 10% at 2.5% CTC purity by the hybridization-based method. We detected damaging missense, stopgain, or splicing mutations with the amplification-based platform in all 19 samples sequenced so far. More than 80% of blood samples from cancer patients had at least 5 CTCs detected. Preliminary comparison of the sequencing results of one high CTC purity sample showed that more than 50% of variants detected by this platform was also identified by hybridization-based sequencing.Conclusion: Our study sheds light on studying the genomic profiles of the rare CTCs for clinical application and identifying the potential actionable targets in Asian cancer patients.Acknowledgements: This study is supported by the HKU Platform Technology Fund and the Hong Kong Research Grants Council Area of Excellence grant AoE/M-06/08 to MLL.Citation Format: Chi Shan Candy Lam, Wei Dai, Josephine Mun Yee Ko, Maria Lung. Evaluation of two next-generation sequencing platforms for genomic analysis in circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5099.
The patients with dual oesophageal squamous cell carcinoma (ESCC) and hypopharyngeal cancer (HPC) have poor prognosis; their underlying genetic pathogenesis is unclear. We hypothesise that development of synchronous ESCC/HPC depends on multicentricity or independent origin, rather than multifocality due to local or lateral spreading.Multiple region whole-exome sequencing (M-WES) and clonality analysis were used to assess clonal relationship and spatial inter- or intra-tumour heterogeneity (ITH) in 62 tumour regions from eight dual ESCC/HPC and ten ESCC patients.All synchronous ESCC/HPC patients had COSMIC 16 mutation signatures, compared to only 40% ESCC in the current study (p = 0.013) and public data set (n = 165, p = 0.003). This alcohol consumption-related mutation signature 16, commonly involved in multiple alcohol-related cancers, was significantly associated with drinking and alcohol metabolism-related ADH1B rs1229984. The mutational landscape and copy number profiles were completely distinct between the two primary tumours; clonality analysis further suggested the two primary tumours shared no or only one clone accompanying independent subclone evolution. M-WES strategy demonstrated higher sensitivity and accuracy for detection of mutational prevalence and the late branch mutations among different regions in the ESCC tumours, compared to traditional sequencing analysis based on single biopsy strategy. Patients with high ITH assessed by cancer cell fraction analysis after M-WES were significantly associated with both relapse and survival.Our hypothesis-generating M-WES ITH assessment data have implications for prognostication. Collectively, our findings support multicentric independent clonal evolution, the field cancerisation theory, and suggest novel insights implicating an aetiologic role of alcohol metabolism in dual ESCC/HPC carcinogenesis.
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