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Background: In the large amount of hematuria patients, a considerable part of them originate in upper tract urinary carcinoma(UTUC). The most important in the evaluation of hematuria is to find accurate and economical method to discriminate between non-malignant and malignant causes. Liquid biopsy for urine samples can be helpful for UTUC diagnosis in patients with hematuria and rule out individuals with low-risk. Methods: In this study, we combined high-throughput sequencing of 17 genes and methylation analysis for ONECUT2 CpG sites as a liquid biopsy panel. We developed a prediction model which contained several significant features to evaluate the risk of UTUC in hematuria patients. Findings: 94 healthy, 70 UTUC- and 70 UTUC+ cases’ samples are qualified for this study, our liquid biopsy panel resulting in a sensitivity of 94% (66/70), and a specificity of 96% (158/164), the positive predication value of 92% and a negative predication value of 98% in this study. With age as additional factor, our model was able to stratify patients into a low or high-risk group with an accuracy score at 94%. Further validation in a large prospective cohort will answer whether this early screening tool can also be used in monitoring postoperative patients who need serials of follow-ups. Interpretation: Urine based liquid biopsy can assist evaluating upper tract urinary carcinoma risk of patients with hematuria and outperformed FISH for accurately ruling out patients who do not have UTUC. Funding Statement: In this research, no funding or other financial supports was received. Declaration of Interests: The authors declared that they have no conflicts of interest to this study. Ethics Approval Statement: Informed written consent was obtained from patients at PLA General Hospital and the study was approved by the Committees on Clinical Research Ethics of the Chinese PLA General Hospital.

Background: Upper tract urinary carcinoma (UTUC) includes renal pelvic cancer and ureteral cancer. For its diagnosis, ureteroscopy is the only method to obtain pathological result before surgery. However, there are also some problems with ureteroscopy. In addition to the trauma and infection risks, the possibility of exudation and adhesion of surrounding tissues after ureteroscopy is significantly increased, hypertension in the cavity may cause tumor spread, and the use of ureteroscopy and biopsy may cause recurrence of bladder tumors after surgery. These unfavorable factors make clinically urgent need for a sensitive and non-invasive method for the diagnosis of UTUC. Materials and Methods: In this study, 150 hematuria patients with upper tract urinary disease and 100 healthy people were enrolled. Among the patients, 75 were pathologically confirmed with upper tract urinary carcinoma while 75 with benign urinary diseases. A Genetron Urinary Assay was designed to maximize the number of unique driver gene variants of UTUC by a limited number of amplicons. Next-generation sequencing libraries were constructed using multiplex PCR methods. We also detected CpG-sites on ONECUT2 gene by performing bisulfite-specific real-time PCR for bisulfite converted DNA. Furthermore, we developed a prediction model based on several significant features to evaluate the risk of UTUC. Results: 234 samples were available for analysis. Our liquid biopsy assay demonstrated a sensitivity of 94%, a specificity of 96%, a positive predication value (PPV) of 92% and a negative predication value(NPV) of 98% in this study. By using a decision tree model, we found age, gene mutations of HRAS, TERT and TP53 and methylation of ONECUT2 did place at some key nodes to classify the risk of patients of UTUC, and age around 48.5, the frequencies of mutated genes above certain value and ΔCt value of ONECUT2 about 7.6 would be good cutoffs. This model was able to stratify patients into a low or high-risk group with an accuracy score at 94%. After further analysis, we found that ONECUT2 methylation and TP53 mutations were more common in high grade UTUC (p Conclusion: In this study, Genetron Urinary Assay has a good performance in the diagnostic of UTUC. Its high sensitivity, high specificity, and non-invasiveness make it a potential clinical alternative to ureteroscopy. Further validation in a large prospective cohort of wide population is necessary to prove the true clinical value of this newly developed method. Citation Format: Xin Ma, Yansheng Xu, Hongzhao Li, Xing Ai, Jiangping Gao, Yiming Liang, Qiaosong Zheng, Kaisheng Mao, Tonghui Ma, Xu Zhang. A urine-based liquid biopsy method for detection of upper tract urinary carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2293.

Objective We previously postulated that 2-deoxyglucose (2-DG) activates multiple pro-survival pathways through IGF1R to negate its inhibitory effect on glycolysis. Here, we evaluated whether IGF1R inhibitor synergizes with 2-DG to impede the growth of non-small cell lung cancer (NSCLC). Materials and methods The activation of IGF1R signaling was assessed by the phosphorylation of IGF1R and its downstream target AKT using immunoblot. Drug dose response and combination index analyses were carried out according to the method of Chou and Talalay. Flow cytometry was used to evaluate cell cycle progression. Apoptosis was monitored by caspase-3/PARP cleavages or Annexin V staining. A subcutaneous xenograft model was used to assess this combination in vivo. Results 2-DG induces the phosphorylation of IGF1R in its kinase domain, which can be abolished by the IGF1R inhibitor BMS-754807. Furthermore, the combination of 2-DG and BMS-754807 synergistically inhibited the survival of several non-small cell lung cancer (NSCLC) cell lines both in vitro and in vivo. The mechanistic basis of this synergy was cell line-dependent, and LKB1-inactivated EKVX cells underwent apoptosis following treatment with a subtoxic dose of 2-DG and BMS-754807. For these cells, the restoration of LKB1 kinase activity suppressed apoptosis induced by this combination but enhanced G1 arrest. In H460 cells, the addition of 2-DG did not enhance the low level of apoptosis induced by BMS-754807. However, treatment with 0.75 μM of BMS-754807 resulted in the accumulation of H460 cells with 8n-DNA content without affecting cell density increases. Hence, H460 cells may escape BMS-754807-induced G2/M cell cycle arrest through polyploidy. The inclusion of 2-DG blocked formation of the 8n-DNA cell population and restored G2/M phase cell cycle arrest. Conclusion The combination of 2-DG and IGF1R inhibitor BMS-754807 may be used to suppress the proliferation of NSCLC tumors through different mechanisms.

The tumor suppressor LKB1 has recently been shown to be involved in the regulation of microtubule dynamics, thus cancer cells with inactivated LKB1 may have developed a means to overcome dysregulated microtubule functions, making them intrinsically resistant to microtubule targeting agents. Here, we generated isogenic LKB1-wild type and mutant non-small cell lung cancer (NSCLC) cell lines to evaluate the role of LKB1 in paclitaxel resistance.SRB, flow cytometry and immunoblotting were used to assess cell proliferation and apoptosis in NSCLC cell lines after paclitaxel treatment. Expression of LKB1 was restored in LKB1-null cells by retrovirus infection and was reduced in LKB1-wild type cells by shRNA knock down.The restoration of LKB1 in LKB1-null cells failed to promote paclitaxel-induced apoptosis in both p53-wild type and p53-mutant backgrounds, indicating that LKB1 was not required for paclitaxel-induced apoptosis. Interestingly, the re-establishment of LKB1 expression led to the up-regulation of class III beta-tubulin and MDR1 in EKVX cells. The up-regulation of MDR1 protein and transcripts in EKVX cells was specifically associated with the expression of wild-type LKB1 and mainly responsible for the increased cellular resistance to paclitaxel. However, the presence of LKB1 protein was not required to maintain this increased MDR1 expression even though there was no genetic amplification or promoter de-methylation of the ABCB1 locus in EKVX-LKB1-WT cells. These data suggest that LKB1 does not promote paclitaxel-induced apoptosis in most NSCLC cell lines. In contrast, in some NSCLC, the presence of LKB1 may facilitate increases in either MDR1 or class III beta-tubulin expression which can lead to paclitaxel resistance.

LKB1 is frequently inactivated in non-small cell lung cancers, probably because of its role in suppressing tumor metastasis. Here, we identified a novel LKB1 mutation in the EKVX lung adenocarcinoma cell line, where serine216 in the kinase domain was mutated to phenylalanine. Genomic sequencing revealed this change to be homozygous, suggesting that the other allele was probably eliminated through a loss of heterozygosity mechanism. We also demonstrated that EKVX cells are defective in energetic stress-induced AMPK phosphorylation, which can be restored by the re-establishment of wild-type but not mutant LKB1 expression. This is consistent with the role of LKB1 as an upstream kinase for AMPK in energetic stress conditions. Interestingly, expression of wild-type LKB1 in EKVX cells was found to be resistant to microtubule targeting agents, such as paclitaxel or vinblastine by SRB analysis. Cell cycle analysis indicated that while the parental or LKB1-mutant expressing cells enter mitotic arrest after 25nM paclitaxel treatment, EKVX-LKB1 cells failed to enter G2/M arrest. Immunofluorescence analysis also revealed a decrease in microtubule bundling after 50nM paclitaxel treatment in EKVX cells with wild-type LKB1 expression. This phenomenon, however, was not observed in other natural LKB1-null A549 or H460 cells. Therefore, our data suggest that the re-establishment of LKB1 expression might lead to resistance to microtubule targeting agents in a subset of non-small cell lung cancer cells with LKB1 mutation. Citation Format: Kaisheng Mao, Fakeng Liu, Xiuju Liu, Yulong He, Fadlo Khuri, Adam Marcus, Mingsong Li, Wei Zhou. Restoration of LKB1 defect in EKVX cells leads to resistance to microtubule targeting agents. [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 773. doi:10.1158/1538-7445.AM2014-773