SummaryPeroxisomal division comprises three steps: elongation, constriction, and fission. Translocation of dynamin-like protein 1 (DLP1), a member of the large GTPase family, from the cytosol to peroxisomes is a prerequisite for membrane fission; however, the molecular machinery for peroxisomal targeting of DLP1 remains unclear. This study investigated whether mitochondrial fission factor (Mff), which targets DLP1 to mitochondria, may also recruit DLP1 to peroxisomes. Results show that endogenous Mff is localized to peroxisomes, especially at the membrane-constricted regions of elongated peroxisomes, in addition to mitochondria. Knockdown of MFF abrogates the fission stage of peroxisomal division and is associated with failure to recruit DLP1 to peroxisomes, while ectopic expression of MFF increases the peroxisomal targeting of DLP1. Co-expression of MFF and PEX11β, the latter being a key player in peroxisomal elongation, increases peroxisome abundance. Overexpression of MFF also increases the interaction between DLP1 and Pex11pβ, which knockdown of MFF, but not Fis1, abolishes. Moreover, results show that Pex11pβ interacts with Mff in a DLP1-dependent manner. In conclusion, Mff contributes to the peroxisomal targeting of DLP1 and plays a key role in the fission of the peroxisomal membrane by acting in concert with Pex11pβ and DLP1.
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Background: Epidermal growth factor receptor (EGFR) mutations are important biomarkers in the treatment of patients with advanced or metastatic diseases. The therascreen EGFR Rotor-Gene Q (RGQ) PCR Kit ® (Qiagen, Inc.) is an approved diagnostic test for EGFR mutations in non-small cell lung cancer (NSCLC). This study aims to investigate the diagnostic capability of a loop-mediated isothermal amplification (LAMP) assay as an accurate, efficient, and cost-effective alternative to the therascreen assay.Methods: EGFR mutations were investigated by LAMP and therascreen assays using tissue samples that were surgically resected or biopsied from 117 consecutive patients with NSCLC tumors. The EGFR status from the LAMP assay was compared with that of the therascreen assay. Next-generation sequencing (NGS) was performed to confirm EGFR status of tumors that did not match in both assays. To establish an optimal LAMP AUC value, receiver operating characteristics (ROC) curve analysis was performed within tumors with exon 19 deletion or L858R point mutation.Results: Of the 117 tumors assayed, 45 tumors with EGFR mutations and 68 tumors with EGFR wild type were matched in both assays, four tumors having mismatched EGFR statuses. NGS further confirmed that two of the four discordant tumors had the same EGFR status that was determined by the LAMP assay. The AUC values were 0.973 (95% CI: 0.929-1.00) in exon 19 deletion, and 0.952 (95% CI: 0.885-1.00) in L858R point mutation. In exon 19 deletion, sensitivity, specificity, and accuracy were 89.3%, 98.9%, and 96.6%, respectively, and 94.7%, 95.9%, and 95.7%, respectively, in L858R using AUC value of 0.222. Conclusions:The LAMP assay compared favorably with the therascreen assay and has potential as an effective, simple, rapid, and low-cost diagnostic alternative. Based on these results, a liquid biopsy LAMP system should be developed for point-of-care testing of oncogenes in the near future.
Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n = 36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.
Liquid biopsy has been adapted as a diagnostic test for EGFR mutations in patients with advanced or metastatic non-small cell lung cancer (NSCLC). Loop-mediated isothermal amplification (LAMP) has been widely used for the rapid detection of pathogens through DNA amplification. This study investigated the efficacy of an EGFR-LAMP assay using plasma samples of patients with resected NSCLC tumors. The EGFR status was investigated using both LAMP and next-generation sequencing (NGS) assays in cases that met the following criteria: (1) pulmonary adenocarcinoma with EGFR mutation detected by the Therascreen EGFR PCR Kit and (2) preoperative plasma samples contained enough DNA for the LAMP and NGS experiments. Among 51 specimens from patients with EGFR-mutated tumors or metastatic lymph nodes, the LAMP assay detected 1 EGFR mutation that was also detected in the NGS assay. However, a plasma sample that demonstrated EGFR wild type in the LAMP assay showed an EGFR mutant status in NGS. The detection rates (1.9% in LAMP and 3.9% in NGS) were very low in both assays, demonstrating a similar performance in detecting EGFR mutations in NSCLC tumors; therefore, it could be a more suitable test for the advanced stage, not the early stage. Notably, the LAMP assay was more time-saving, cost-effective, and straightforward. However, further investigation is required to develop a more sensitive assay.
of study drug and samples were obtained 8 hours post dosing. Both dose levels showed robust induction of p21, a p53-regulated mediator of cell cycle arrest, in bone marrow cells on IHC evaluation. In Part 2, 23 subjects allocated to 8 groups received a single 0.3 mg/kg dose of ALRN-6924. Samples were obtained at 4, 8, 12, 16, 20, 24, 36 and 48 post infusion. The 0.3 mg/kg dose showed excellent tolerability: subjects experienced only mild, transient AEs. Robust p21 induction was observed in bone marrow cells, with peak expression between 4 hr and 16 hr following ALRN-6924 administration.Conclusions: ALRN-6924 is safe and effective in inducing cell cycle arrest in bone marrow cells. The results support the evaluation of ALRN-6924 as a chemoprotective drug in cancer patients receiving chemotherapy.
Background The 21-gene Recurrence Score (RS) (Oncotype DX®) is one of the most frequently used multigene assays to predict prognosis and response to treatment for estrogen receptor-positive breast cancer. Although the result of two prospective randomized trials, TAILORx and RxPONDER trial have increasingly established the clinical utility of Oncotype DX®, the cost and testing period are disadvantages especially in Asian countries. To overcome these disadvantages, we developed a new multi-gene assay using Loop-Mediated Isothermal Amplification (LAMP). We have previously selected a twenty-five genes set through comprehensive gene expression analysis of breast cancer samples from more than 500 cases (patent pending). Although these twenty-five genes are different from those used in conventional assays, their physiological functions are similar to those of Oncotype DX®, and it is expected that the results will be equivalent to Oncotype DX® as a predictor of prognosis and chemotherapeutic efficacy. This new assay can be performed in own institution, and the result can be obtained less than one hour. It can lead to significantly reduce costs and testing time compared to conventional assays. Based on these backgrounds, we constructed the prediction algorithm based on the new gene expression profiles using LAMP and evaluated its performance in this study. Methods Total RNA was extracted using Maxwell® RSC RNA FFPE Kit (Promega) from FFPE tumor samples of postoperative breast cancer tissue which Oncotype DX® have already tested in our institution between January 2009 to January 2021and was quantified by Reverse-transcription LAMP enabling one-step reaction from reverse transcription to amplification under isothermal condition (63°C). Samples were divided into three groups, low-RS (RS 0-10), RS-intermediate (RS 11-25) and RS-high (RS ≥26) based on Oncotype DX®-RS. Using the obtained mRNA amplification detection time as an explanatory variable and Oncotype DX®-RS as an objective variable, a prediction algorithm based on expression profiles based on LAMP method was constructed and evaluated its performance with 4-fold cross-validation. The prediction algorithm was trained using extreme gradient boosting (XGBoost) algorithm that parameters used default of R package “xgboost”. We also performed the quality analysis of mRNA excluded from this analysis due to poor mRNA quality. The percentage of mRNA above 200nt (DV200) was calculated by electrophoresis, and the correlation between the degree of mRNA degradation and the number of years of storage was calculated. Results Of the 221 cases which have tested Oncotype DX® during the study period, 90 samples were used in the analysis, and the remaining samples were excluded from this study due to poor mRNA quality. Of the 90 samples, 19 cases were RS-low, 42 cases were RS-intermediate, and 30 cases were RS-high, respectively. The correlation coefficient between Oncotype DX®-RS and our LAMP method-based predicted RS was r=0.911 (95%CI 0.900−0.921, p-value < 0.001). The overall concordance rate with the predicted risk of recurrence (high/intermediated/low-RS) was 0.9343 (95%CI: 0.9174-0.9486, p-value < 0.001). Regarding the quality analysis of mRNA excluded from this analysis, we found the inverse correlation between the degree of mRNA degradation and the storage periods, namely higher DV200 with shorter periods. In particular, there was less mRNA degradation in specimens that had been stored for less than one year. Conclusion Our new gene expression profiling by the LAMP method suggested to have same discriminately ability with Oncotype DX® to predict the risk of recurrence in early breast cancer patients. The duration of mRNA storage and the fixation time in the FFPE preparation process are suggested to be important for maintaining the quality of mRNA. We are planning to further studies with increase sample size and analyze the correlation with prognosis. Citation Format: Yasue Tsuchida, Takaaki Ueda, Yuka Nagatake, Satoru Michiyuki, Miku Hattori, Masaki Sato, Norihiro Tomita, Naoki Kanomata, Hideko Yamauchi. Clinical utility of twenty-five gene-expression profiling using LAMP method in early stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-07-05.
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