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BackgroundNasopharyngeal carcinoma (NPC) is a highly invasive and metastatic cancer, with diverse molecular characteristics and clinical outcomes. This study aims to dissect the molecular heterogeneity of NPC, followed by the construction of a microRNA (miRNA)-based prognostic model for prediction of distant metastasis.MethodsWe retrieved two NPC datasets: GSE32960 and GSE70970 as training and validation cohorts, respectively. Consensus clustering was employed for cluster discovery, and support vector machine was used to build a classifier. Finally, Cox regression analysis was applied to constructing a prognostic model for predicting risk of distant metastasis.ResultsThree NPC subtypes (immunogenic, classical and mesenchymal) were identified that are molecularly distinct and clinically relevant, of which mesenchymal subtype (~ 36%) is associated with poor prognosis, characterized by suppressing tumor suppressor miRNAs and the activation of epithelial-­mesenchymal transition. Out of the 25 most differentially expressed miRNAs in mesenchymal subtype, miR-142, miR-26a, miR-141 and let-7i have significant prognostic power (P < 0.05).ConclusionsWe proposed for the first time that NPC can be stratified into three subtypes. Using a panel of 4 miRNAs, we established a prognostic model that can robustly stratify NPC patients into high- and low- risk groups of distant metastasis.Electronic supplementary materialThe online version of this article (10.1186/s12929-018-0417-5) contains supplementary material, which is available to authorized users.

Introduction: Our previous study found that miR-145 was downregulated in non-small cell lung cancer (NSCLC) tissues and it could inhibit cell proliferation in transfected NSCLC cells. This study aimed to investigate the potential anticancer effect and tumor suppressive function of miR-145, to identify its gene targets, as well as to assess the role of its gene targets as prognostic biomarkers for NSCLC patients. Materials and Methods: Cell proliferation, transwell migration, and invasion assays were performed on three miR-145 mimics transfected NSCLC cells. The effect of miR-145 in the cell cycle in vitro was assessed by flow cytometry analysis. In vivo transfection of miR-145 was performed to examine the cancer growth in eight BALB/c nude mice. Luciferase reporter assay was used to identify the gene targets. To reveal the role of the identified gene targets in patient survival, the clinical data of 500 lung adenocarcinoma patients from The Cancer Genome Atlas (TCGA) database was used for Kaplan-Meier survival analysis. Results: Upregulation of miR-145 significantly inhibited proliferation, migration, and invasion in NSCLC cells in vitro. Flow cytometry analysis revealed that the transfection of miR-145 into NSCLC cells did not show any effect in the cell cycle. After 37 days, transfection of miR-145 showed a reduction in tumor size (49% smaller) and tumor weight (57% lighter) than mimic negative in vivo. MicroRNA target prediction database miRTarBase and luciferase reporter assays identified GOLM1 and RTKN as the direct targets of miR-145. TCGA survival analysis found that the high expressions of GOLM1 (p &lt; 0.0001) and RTKN (p &lt; 0.0045) were significantly correlated with poor overall survival in lung adenocarcinoma patients. Conclusion: Our results revealed that miR-145 inhibited proliferation, migration, and invasion, but did not affect cell cycle regulation in NSCLC. It also caused a reduction in tumor growth and weight. Most importantly, we identified two direct targets (GOLM1 and RTKN) of miR-145, in which RTKN was first identified as a miR-145 target in NSCLC. Moreover, both GOLM1 and RTKN play an imperative role in patients’ overall survival. Taken together, our findings suggest that miR-145 may serve as a molecular therapeutic target for NSCLC, and its gene targets GOLM1 and RTKN may serve as prognostic biomarkers. Their potential clinical applications warrant further investigation. Citation Format: William C. Cho, Chi F. Wong, Leo K. Li, Alvin H. Fong. miR-145 and its gene targets as therapeutic target and prognostic biomarkers for non-small cell lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3756.

Introduction: Currently, the three primary methods of detecting ALK rearrangement are fluorescent in situ hybridization, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR). The former two methods use tissue as the laboratory materials, but some patients do not have sufficient tissue to perform the tests. The RT-PCR method can be conducted by both tissue and blood, but its detection of ALK rearrangement seems not sensitive enough. Digital PCR (dPCR) is a biotechnology refinement of conventional PCR methods that may be a more precise method than RT-PCR. Materials and Methods: An EML4-ALK variant 1 (v1) synthetic sequence, a variant 2 (v2) synthetic sequence, and a variant 3 (v3) non-small cell lung cancer (NSCLC) cell line (NCI-H2228) were used to evaluate the performance of our dPCR assay. To examine the performance PCR on clinical samples, we used both dPCR and RT-PCR assays to quantify the expression levels of EML4-ALK variants (v1, v2, and v3) in the circulating tumor RNAs derived from 414 plasma samples of 30 NSCLC patients during their ALK inhibitor treatment. Results: We have designed a multiplex dPCR assay for the detection of EML4-ALK in a v1 synthetic sequence (with 8,000 copies), a v2 synthetic sequence (with 10,000 copies), and a v3 cell line (with 100 copies). Our results showed high specificities and precisions (8,757 ± 592 copies, 11,070 ± 7,791 copies, and 93 ± 14 copies, respectively) for detecting all three variants simultaneously. On the other hand, our results observed good linearities (R2 = 0.9996, 0.9985, and 0.9991, respectively) in all three variants. To determine our dPCR assay's limit of detection for v1 and v2 synthetic sequences (each with four copies), our assay successfully measured the copy numbers of both v1 and v2 (4.1 ± 1.6 and 3.9 ± 1.8 copies, respectively). Comparing the sensitivities of our dPCR assay with RT-PCR assay, dPCR was able to detect down to 2 copies (for v1 and v2) and 20 pg of cDNA (for v3) while RT-PCR could only detect 200 copies (for v1 and v2) and 200 pg of cDNA (for v3). After confirming the promising performance of our dPCR assay, we tried to detect EML4-ALK variants by both dPCR and RT-PCR assays in the plasma samples of our recruited patients but mostly negative. Only one sample detected 39.2 copies of EML4-ALK v3 using dPCR assay. Conclusion: dPCR assay is a precise method with good performance at detecting EML4-ALK variants in synthetic sequences and cell line, which is more sensitive than RT-PCR. However, EML4-ALK variants could not be detected in the majority of our plasma samples. This may be due to insufficient circulating RNAs for detection. In addition, RNA may be degraded by RNase in plasma and that platelet may be a better alternative than plasma for the detection of RNAs in patient samples. Additional testing is warranted to detect EML4-ALK variants in the platelet samples of another NSCLC patient cohort. Citation Format: William C. Cho, Alvin H. Fong, King Y. Fung, Jeannie Y. Chik, Jacky Y. Li. Feasibility of digital PCR for the detection of ALK rearrangement in the plasma of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 468.

Introduction: Androgen receptor splice variant 7 (AR-V7) is found to be associated with resistance to hormonal therapy in castration-resistant prostate cancer (CRPC). This study aims to develop a minimally-invasive approach for the direct quantification of AR-V7 in blood and evaluates whether AR-V7 can act as a predictor of progression in CRPC patients undergoing hormonal therapy. Materials and Methods: A prostate cancer cell line (VCaP) was used to test the assay’s limit of detection. The detection of AR-V7 was then evaluated in 126 serial plasma samples and 77 serial platelet samples, prospectively collected from 30 patients with CRPC before and during hormonal therapy (abiraterone or enzalutamide) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and droplet digital polymerase chain reaction (ddPCR). Results: RT-qPCR and ddPCR were first performed on VCaP to measure a range of 20 fg to 20 ng VCaP cDNA. ddPCR was able to detect down to 2 pg while RT-qPCR was only able to detect 200 pg for AR-V7 and total AR. RT-qPCR and ddPCR were then performed on the plasma samples of CRPC patients. Our results observed that RT-qPCR was unable to detect AR-V7 from these plasma samples, whereas ddPCR yielded a higher detection rate of 13%. Plasma with 22Rv1 and VCaP RNA spike-in revealed that RNase in plasma degraded spike-in RNA. We further detected AR-V7 in platelet samples, our results found that RT-qPCR was able to detect AR-V7 from 56% of the platelet samples and ddPCR yielded a much higher detection rate of 97%. Finally, we quantified the AR-V7 levels of 22 available platelet samples taken at the point closest to the patients’ progression time to evaluate the correlation of AR-V7 with the patient’s progression records. In receiver operating characteristic curve analysis, the area under the curve was 0.656. Conclusion: Our results showed that platelet samples had higher detection sensitivity than plasma samples, this might be due to the possibility of RNA degradation by RNase during processing in plasma samples. Performing RT-qPCR and ddPCR on platelet samples, a higher detection rate was observed in ddPCR when compared with RT-qPCR. We successfully develop a detection test to assess AR-V7 by highly sensitive ddPCR in the platelet of CRPC patients, with a high detection rate of 97%. We also reveal that there is a certain correlation between the ddPCR detection for AR-V7 in patients’ platelet samples with the patients’ progression status, indicating that AR-V7 may act as a potential predictor of progression in CRPC patients undergoing hormonal therapy. Citation Format: William C. Cho, King Y. Fung, Alvin H. Fong, Timothy T. Chan. Development of AR-V7 blood-based detection test by digital PCR in castration-resistant prostate cancer patients undergoing hormonal therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5139.