DELFMUT: duplex sequencing-oriented depth estimation model for stable detection of low-frequency mutations.

Duplex sequencing technology has been widely used in the detection of low-frequency mutations in circulating tumor deoxyribonucleic acid (DNA), but how to determine the sequencing depth and other experimental parameters to ensure the stable detection of low-frequency mutations is still an urgent problem to be solved. The mutation detection rules of duplex sequencing constrain not only the number of mutated templates but also the number of mutation-supportive reads corresponding to each forward and reverse strand of the mutated templates. To tackle this problem, we proposed a Depth Estimation model for stable detection of Low-Frequency MUTations in duplex sequencing (DELFMUT), which models the identity correspondence and quantitative relationships between templates and reads using the zero-truncated negative binomial distribution without considering the sequences composed of bases. The results of DELFMUT were verified by real duplex sequencing data. In the case of known mutation frequency and mutation detection rule, DELFMUT can recommend the combinations of DNA input and sequencing depth to guarantee the stable detection of mutations, and it has a great application value in guiding the experimental parameter setting of duplex sequencing technology.

ARID1A deficiency promotes progression and potentiates therapeutic antitumour immunity in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND: Exploring predictive biomarkers and therapeutic strategies of ICBs has become an urgent need in clinical practice. Increasing evidence has shown that ARID1A deficiency might play a critical role in sculpting tumor environments in various tumors and might be used as pan-cancer biomarkers for immunotherapy outcomes. The current study aims to explored the immune-modulating role of ARID1A deficiency in Hepatitis B virus (HBV) related hepatocellular carcinoma (HBV-HCC) and its potential immunotherapeutic implications. METHODS: In the current study, we performed a comprehensive analysis using bioinformatics approaches and pre-clinical experiments to evaluate the ARID1A regulatory role on the biological behavior, and immune landscape of Hepatitis B virus (HBV) related hepatocellular carcinoma (HBV-HCC). A total of 425 HBV-related hepatocellular carcinoma patients from TCGA-LIHC, AMC and CHCC-HBV cohort were enrolled in bioinformatics analysis. Immunohistochemical staining of HBV-HCC specimens and ARID1A deficiency cellular models were used to validate the results of the analysis. RESULTS: Our results have shown that ARID1A deficiency promoted tumor proliferation and metastasis. More importantly, ARID1A deficiency in HBV-HCC was associated with the higher TMB, elevated immune activity, and up-regulated expression of immune checkpoint proteins, especially TIM-3 in HBV-HCC. Further, the expression of Galectin-9, which is the ligand of TIM-3, was elevated in the ARID1A knockout HBV positive cell line. CONCLUSION: To conclude, we have shown that the ARID1A deficiency was correlated with more active immune signatures and higher expression of immune checkpoints in HBV-HCC. Additionally, the present study provides insights to explore the possibility of the predictive role of ARID1A in HBV-HCC patients responsive to immunotherapy.

Molecular features of gastroenteropancreatic neuroendocrine carcinoma: A comparative analysis with lung neuroendocrine carcinoma and digestive adenocarcinomas.

Objective: There is an ongoing debate about whether the management of gastroenteropancreatic (GEP) neuroendocrine carcinoma (NEC) should follow the guidelines of small-cell lung cancer (SCLC). We aim to identify the genetic differences of GEPNEC and its counterpart. Methods: We recruited GEPNEC patients as the main cohort, with lung NEC and digestive adenocarcinomas as comparative cohorts. All patients undergone next-generation sequencing (NGS). Different gene alterations were compared and analyzed between GEPNEC and lung NEC (LNEC), GEPNEC and adenocarcinoma to yield the remarkable genes. Results: We recruited 257 patients, including 99 GEPNEC, 57 LNEC, and 101 digestive adenocarcinomas. Among the mutations, KRAS, RB1, TERT, IL7R, and CTNNB1 were found to have different gene alterations between GEPNEC and LNEC samples. Specific genes for each site were revealed: gastric NEC ( TERT amplification), colorectal NEC ( KRAS mutation), and bile tract NEC ( ARID1A mutation). The gene disparities between small-cell NEC (SCNEC) and large-cell NEC (LCNEC) were KEAP1 and CDH1. Digestive adenocarcinoma was also compared with GEPNEC and suggested RB1, APC, and KRAS as significant genes. The TP53/ RB1 mutation pattern was associated with first-line effectiveness. Putative targetable genes and biomarkers in GEPNEC were identified in 22.2% of the patients, and they had longer progression-free survival (PFS) upon targetable treatment [12.5 months vs. 3.0 months, HR=0.40 (0.21-0.75), P=0.006]. Conclusions: This work demonstrated striking gene distinctions in GEPNEC compared with LNEC and adenocarcinoma and their clinical utility.

SETD2 variation correlates with tumor mutational burden and MSI along with improved response to immunotherapy.

BACKGROUND: SETD2 protects against genomic instability via maintenance of homologous recombination repair (HRR) and mismatch repair (MMR) in neoplastic cells. However, it remains unclear whether SETD2 dysfunction is a complementary or independent factor to microsatellite instability-high (MSI-H) and tumor mutational burden-high (TMB-H) for immunocheckpoint inhibitor (ICI) treatment, and little is known regarding whether this type of dysfunction acts differently in various types of cancer. METHODS: This cohort study used multidimensional genomic data of 6726 sequencing samples from our cooperative and non-public GenePlus institute from April 1 through April 10, 2020. MSIsensor score, HRD score, RNAseq, mutational data, and corresponding clinical data were obtained from the TCGA and MSKCC cohort for seven solid tumor types. RESULTS: A total of 1021 genes underwent target panel sequencing reveal that SETD2 mutations were associated with a higher TMB. SETD2 deleterious mutation dysfunction affected ICI treatment prognosis independently of TMB-H (p < 0.01) and had a lower death hazard than TMB-H in pancancer patients (0.511 vs 0.757). Significantly higher MSI and lower homologous recombination deficiency were observed in the SETD2 deleterious mutation group. Improved survival rate was found in the MSKCC-IO cohort (P < 0.0001) and was further confirmed in our Chinese cohort. CONCLUSION: We found that SETD2 dysfunction affects ICI treatment prognosis independently of TMB-H and has a lower death hazard than TMB-H in pancancer patients. Therefore, SETD2 has the potential to serve as a candidate biomarker for ICI treatment. Additionally, SETD2 should be considered when dMMR is detected by immunohistochemistry.

Fabrication and Separation of EGaIn Microparticles from Human Blood Based on Dielectrophoresis Force and a W-Type Electrode.

Harmful particles such as heavy metal particles in the human body can cause many problems such as kidney stones, gallstones, and cerebrovascular diseases. Therefore, it is critical to separate them from the blood and perform a systematic analysis as early as possible. Here, we apply eutectic gallium indium (EGaIn) microparticles as a model to study the separation of particles from blood, thanks to their properties of low toxicity, excellent degradability, and negligible vapor pressure. In particular, the dielectrophoresis (DEP) separation method is employed to separate EGaIn of different sizes and characteristics in blood. First, the screen-printing method is used to create EGaIn microparticles with diameters of 15, 23, 18, and 11 µm. According to the lifetime test, these microparticles can last more than 1 month, as evidenced by their surface oxidation characteristics. Moreover, a DEP platform with W-type electrodes is developed to sort EGaIn particles from whole human blood. The results show that a sorting efficiency of 95% can be attained, which is similar to the separation efficiency of 98% achieved by finite element analysis (FEA) using COMSOL software based on the orthogonal array experiment method. The proposed study successfully validates the use of the DEP method to separate particles from human blood, providing insights into heavy metal particle separating, drug screening, and cell sorting and potentially broadening the applications in environmental analysis, food engineering, and bioengineering.

Utility of cell-free DNA from bronchial washing fluid in diagnosis and genomic determination for radiology-suspected pulmonary nodules.

BACKGROUND: Bronchial washing fluid (BWF) is a less-invasive specimen. Due to the limited sensitivity of BWF cellular component diagnosis, the aim of this study was to explore the potential role of BWF supernatant as a source of liquid biopsy of lung cancer. METHODS: This prospective study enrolled 76 suspected and 5 progressed lung cancer patients. Transbronchial biopsy tissues, BWF supernatant (BWF_Sup) and BWF precipitant (BWF_Pre) were tested by a targeted panel of 1021 genes. RESULTS: BWF_Sup cell-free DNA (cfDNA) was superior to tissue biopsy and BWF_Pre in determining mutational allele frequency, tumour mutational burden, and chromosomal instability. Moreover, BWF_Sup and BWF_Pre achieved comparable efficacy to tissue samples in differentiating malignant and benign patients, but only BWF_Sup persisted differentiated performance after excluding 55 malignancies pathologically diagnosed by bronchoscopic biopsy. Among 67 malignant patients, 82.1% and 71.6% of tumour-derived mutations (TDMs) were detected in BWF_Sup and BWF_Pre, respectively, and the detectability of TDMs in BWF_Sup was independent of the cytological examination of BWF. BWF_Sup outperformed BWF_Pre in providing more subclonal information and thus might yield advantage in tracking drug-resistant markers. CONCLUSIONS: BWF_Sup cfDNA is a reliable medium for lung cancer diagnosis and genomic profiles and may provide important information for subsequent therapeutic regimens.

Multiomics Analysis Reveals Distinct Immunogenomic Features of Lung Cancer with Ground-Glass Opacity.

Rationale: Ground-glass opacity (GGO)-associated lung cancers are common and radiologically distinct clinical entities known to have an indolent clinical course and superior survival, implying a unique underlying biology. However, the molecular and immune characteristics of GGO-associated lung nodules have not been systemically studied. Objectives: To provide mechanistic insights for the treatment of these radiologically distinct clinical entities. Methods: We initiated a prospective cohort study to collect and characterize pulmonary nodules with GGO components (nonsolid and part-solid nodules) or without GGO components, as precisely quantified by using three-dimensional image reconstruction to delineate the molecular and immune features associated with GGO. Multiomics assessment conducted by using targeted gene panel sequencing, RNA sequencing, TCR (T-cell receptor) sequencing, and circulating tumor DNA detection was performed. Measurements and Main Results: GGO-associated lung cancers exhibited a lower tumor mutation burden than solid nodules. Transcriptomic analysis revealed a less active immune environment in GGO components and immune pathways, decreased expression of immune activation markers, and less infiltration of most immune-cell subsets, which was confirmed by using multiplex immunofluorescence. Furthermore, T-cell repertoire sequencing revealed lower T-cell expansion in GGO-associated lung cancers. HLA loss of heterozygosity was significantly less common in lung adenocarcinomas with GGO components than in those without. Circulating tumor DNA analysis suggested that the release of tumor DNA to the peripheral blood was correlated with the tumor size of non-GGO components. Conclusions: Compared with lung cancers presenting with solid lung nodules, GGO-associated lung cancers are characterized by a less active metabolism and a less active immune microenvironment, which may be the mechanisms underlying their indolent clinical course. Clinical trial registered with www.clinicaltrials.gov (NCT03320044).

The prevalence of HLA-I LOH in Chinese pan-cancer patients and genomic features of patients harboring HLA-I LOH.

HLA-I LOH may facilitate immune evasion. However, large population studies on the prevalence of HLA-I LOH across different cancer types and in relation to mutational profiles are lacking, in particular, in the Chinese population. In this study, analysis was performed in 1504 advanced pan-cancer patients and 134 early-stage non-small-cell lung cancer patients using a 1021-gene panel. The consistency between the 1021-gene panel and whole-exome sequencing was evaluated in 45 samples, where concordant results were obtained in 95.6% (43/45) of the samples. Analytical results revealed that the prevalence of HLA-I LOH in tumor tissue presents considerable differences across cancer types. HLA-I LOH was relevant to genomic instability, reflected in higher tumor mutation burden level. HLA-I LOH occurs more frequently in MSS samples than in MSI-H samples. The alteration frequencies of p53 pathway, RTK/RAS pathway, Notch pathway, Hippo pathway, and Nrf2 pathway in HLA-I LOH group were significantly higher than that in HLA-I stable group (p < .0001, p < .0001, p = .032, p = .013, p = .003, respectively). In DNA damage response pathways, alterations in the checkpoint factor pathway and Fanconi anemia pathway are enriched in HLA-I LOH group (p < .0001, p = .023, respectively). Besides, HLA-I LOH was accompanied by higher mutation rates of several tumor suppressors, including TP53 and LRP1B. These results may shed light on follow-up tumor immunology research.

Serial circulating tumor DNA identification associated with the efficacy and prognosis of neoadjuvant chemotherapy in breast cancer.

BACKGROUND: Circulating tumor DNA (ctDNA) provides a promising noninvasive alternative to evaluate the efficacy of neoadjuvant chemotherapy (NCT) in breast cancer. METHODS: Herein, we collected 63 tissue (aspiration biopsies and resected tissues) and 206 blood samples (baseline, during chemotherapy (Chemo), after chemotherapy (Post-Chemo), after operation (Post-Op), during follow-up) from 32 patients, and preformed targeted deep sequencing with a customed 1021-gene panel. RESULTS: As the results, TP53 (43.8%) and PIK3CA (40.6%) were the most common mutant genes in the primary tumors. At least one tumor-derived mutation was detected in the following number of blood samples: 21, baseline; 3, Chemo; 9, Post-Chemo; and 5, Post-Op. Four patients with pathologic complete response had no tissue mutation in Chemo and Post-Chemo blood. Compared to patients with mutation-positive Chemo or Post-Chemo blood, the counterparts showed a superior primary tumor decrease (median, 86.5% versus 54.6%) and lymph involvement (median, 1 versus 3.5). All five patients with mutation-positive Post-Op developed distant metastases during follow-up, and the sensitivity of detecting clinically relapsed patients was 71.4% (5/7). The median DFS was 9.8 months for patients with mutation-positive Post-Op but not reached for the others (HR 23.53; 95% CI, 1.904-290.9; p < 0.0001). CONCLUSIONS: Our study shows that sequential monitoring of blood ctDNA was an effective method for evaluating NCT efficacy and patient recurrence. Integrating ctDNA profiling into the management of LABC patients might improve clinical outcome. TRIAL REGISTRATION: This prospective study recruited LABC patients at Peking Union Medical College Hospital (ClinicalTrials.gov Identifier: NCT02797652).

Joule-Heated Layered Double Hydroxide Sponge for Rapid Removal of Silica from Water.

Dissolved silica is a major concern for a variety of industrial processes owing to its tendency to form complex scales that severely deteriorate system performance. In this work, we present a pretreatment technology using a Joule-heated sponge to rapidly remove silica from saline waters through adsorption, thereby effectively mitigating silica scaling in subsequent membrane desalination processes. The adsorbent sponge is fabricated by functionalizing two-dimensional layered double hydroxide (LDH) nanosheets on a porous, conductive stainless-steel sponge. With the application of an external voltage of 4 V, the Joule-heated sponge achieves 85% silica removal and 95% sponge regeneration within 15 min, which is much more efficient than its counterpart without Joule-heating (360 min for silica adsorption and 90 min for sponge regeneration). Material characterization and reaction kinetics analysis reveal that electrostatic interactions and "memory effect"-induced intercalation are the primary mechanisms for silica removal by the LDH nanosheets. Moreover, Joule-heating reduces the boundary layer resistance on nanosheets and facilitates intraparticle diffusion of dissolved silica, thereby increasing silica removal kinetics. Joule-heating also enhances the release of silicate ions during the regeneration stage through exchange with the surrounding anions (OH- or CO32-), resulting in a more efficient sponge regeneration. Pretreatment of silica-rich feedwaters by the Joule-heated sponge effectively reduces reverse osmosis membrane scaling by amorphous silica scale, demonstrating great potential for silica scaling control in a broad range of engineered processes.

Impact of T-cell receptor and B-cell receptor repertoire on the recurrence of early stage lung adenocarcinoma.

Surgical resection is the only curative treatment for patients with early stage non-small cell lung cancer. However, approximately 33% of non-small cell lung cancer patients recur with the stage I disease, which may be attributed to a deficiency in antitumor immunity. In the present study, for early stage lung adenocarcinoma patients with early recurrence and early non-recurrence, we investigated the quantity of tumor-infiltrating T and B cells by immunohistochemistry, as well as the genes in the complementarity determining region 3 of the T-cell receptor ß chain and the B-cell receptor immunoglobulin heavy chain. A decreased number of tumor-infiltrating lymphocytes cells (CD3+, CD4+, CD8+ and CD20+) was present in early recurrence patients. A significant increase in oligoclones and a reduction in T-cell receptor diversity were observed in the early recurrence group. Furthermore, there was a preference for V, J gene, and VJ gene combinations in patients with early recurrence versus non-recurrence, suggesting that this may be a new biomarker for the recurrence of early stage lung adenocarcinoma. These data indicate that T and B cell receptor repertoires influence the depth of human adaptive immune responses, and in addition to the quantity of tumor infiltrating T and B cells, may contribute to the prevention of early stage lung adenocarcinoma recurrence after surgical resection. Our study illustrates the potential value of the immune repertoire for predicting clinical efficacy and patient outcomes.

Accurately estimating the length distributions of genomic micro-satellites by tumor purity deconvolution.

BACKGROUND: Genomic micro-satellites are the genomic regions that consist of short and repetitive DNA motifs. Estimating the length distribution and state of a micro-satellite region is an important computational step in cancer sequencing data pipelines, which is suggested to facilitate the downstream analysis and clinical decision supporting. Although several state-of-the-art approaches have been proposed to identify micro-satellite instability (MSI) events, they are limited in dealing with regions longer than one read length. Moreover, based on our best knowledge, all of these approaches imply a hypothesis that the tumor purity of the sequenced samples is sufficiently high, which is inconsistent with the reality, leading the inferred length distribution to dilute the data signal and introducing the false positive errors. RESULTS: In this article, we proposed a computational approach, named ELMSI, which detected MSI events based on the next generation sequencing technology. ELMSI can estimate the specific length distributions and states of micro-satellite regions from a mixed tumor sample paired with a control one. It first estimated the purity of the tumor sample based on the read counts of the filtered SNVs loci. Then, the algorithm identified the length distributions and the states of short micro-satellites by adding the Maximum Likelihood Estimation (MLE) step to the existing algorithm. After that, ELMSI continued to infer the length distributions of long micro-satellites by incorporating a simplified Expectation Maximization (EM) algorithm with central limit theorem, and then used statistical tests to output the states of these micro-satellites. Based on our experimental results, ELMSI was able to handle micro-satellites with lengths ranging from shorter than one read length to 10kbps. CONCLUSIONS: To verify the reliability of our algorithm, we first compared the ability of classifying the shorter micro-satellites from the mixed samples with the existing algorithm MSIsensor. Meanwhile, we varied the number of micro-satellite regions, the read length and the sequencing coverage to separately test the performance of ELMSI on estimating the longer ones from the mixed samples. ELMSI performed well on mixed samples, and thus ELMSI was of great value for improving the recognition effect of micro-satellite regions and supporting clinical decision supporting. The source codes have been uploaded and maintained at https://github.com/YixuanWang1120/ELMSI for academic use only.

Assessing tumor heterogeneity using ctDNA to predict and monitor therapeutic response in metastatic breast cancer.

Tumor heterogeneity was associated with treatment outcome of metastatic cancers but few studies have examined whether tumor heterogeneity in circulating tumor DNA (ctDNA) can be used to predict treatment outcome. ctDNA analysis was performed in 37 HER2-positive metastatic breast cancer patients treated with pyrotinib. Patients with high tumor heterogeneity had significantly worse PFS outcomes, with a median PFS of 30.0 weeks vs. 60.0 weeks for patients with low tumor heterogeneity (hazard ratio [HR], 2.9; p = 0.02). Patients with trunk resistance mutations receiving pyrotinib monotherapy had worse outcomes (HR, 4.5; p = 0.03), with a median PFS of 7.8 weeks vs. 27.4 weeks for those with branch resistance mutations or without any resistance mutations in baseline ctDNA. Longitudinal monitoring of 21 patients during treatment showed that the molecular tumor burden index ([mTBI] a measure of the percentage of ctDNA in samples) was positively correlated with tumor size as evaluated by computed tomography (p < 0.0001, Pearson r = 0.52) and detected disease progression 8-16 weeks earlier. Our current findings suggested that ctDNA could be used to assess tumor heterogeneity and predict treatment outcomes. Furthermore, the mTBI is better for assessing therapeutic response than single gene mutations and might supplement the current therapeutic response evaluation system.

A hybrid electrically-and-piezoelectrically driven micromixer built on paper for microfluids mixing.

This study aims to explore the channel patterns and the characteristic parameters of the zigzag microchannel based on microfluidic paper-based analytical devices (µPADs), in which the mixing efficiency and speed can be greatly enhanced. Better mixing of the solutions was obtained by adding a simple directing electric field to the optimized structure of the zigzag microchannel on paper-based chips instead of the traditional complex devices. A higher mixing efficiency was reached when the direct-current (DC) power supply reached 20 V. Meanwhile, a piezoelectric transducer (PZT) driver was used in the mixing experiment with the paper-based zigzag microchannel. The results show that the mixing efficiency reached a maximum value when the input voltage and frequency were 30 V and 150 Hz, respectively. These paper-based devices meet the requirements of the biochemical analysis field because they are low cost, easy to operate, and have high efficiencies, giving them good prospects for future applications. Graphical Abstract.

Preoperative evaluation of microvascular invasion with circulating tumour DNA in operable hepatocellular carcinoma.

BACKGROUND & AIMS: Microvascular invasion (MVI) is a critical prognostic factor for operable hepatocellular carcinoma (HCC). This study aimed to explore the performance of circulating tumour DNA (ctDNA) in evaluating MVI status preoperatively. METHODS: Seventy-three HCC patients were enrolled and randomly divided into a training cohort and a validation cohort in a 2:1 ratio, and preoperative blood and surgical tissue samples were obtained. Genomic alterations were analysed using targeted deep sequencing with a 1021-gene panel. RESULTS: In training cohort, 260 somatic mutations were identified in 40 blood samples (81.6%). CtDNA mutation was verified in paired tissue sample in 39 patients (97.5%). In univariate analysis, ctDNA allele frequency (AF) and largest tumour diameter were associated with the presence of MVI, but ctDNA AF was the only independent risk factor in multivariate analysis. With the cut-off value of 0.83%, ctDNA AF determined the presence of MVI with the sensitivity of 89.7% and specificity of 80.0% in the training cohort, and the sensitivity of 78.6% and the specificity of 81.8% in the validation cohort. In preoperative evaluation, ctDNA AF, AFP level and BCLC staging were associated with recurrence-free survival in both univariate and multivariate analysis. CONCLUSIONS: CtDNA can serve as an independent risk factor of MVI for operable HCC and help determining precise treatment strategies. The integration of ctDNA in the management of operable HCC may achieve better clinical outcomes.

Comprehensive genomic variation profiling of cervical intraepithelial neoplasia and cervical cancer identifies potential targets for cervical cancer early warning.

BACKGROUND: To better understand the pathogenesis of cervical cancer (CC), we systematically analysed the genomic variation and human papillomavirus (HPV) integration profiles of cervical intraepithelial neoplasia (CIN) and CC. METHODS: We performed whole-genome sequencing or whole-exome sequencing of 102 tumour-normal pairs and human papillomavirus probe capture sequencing of 45 CCs, 44 CIN samples and 25 normal cervical samples, and constructed strict integrated workflow of genomic analysis. RESULTS: Mutational analysis identified eight significantly mutated genes in CC including four genes (FAT1, MLL3, MLL2 and FADD), which have not previously been reported in CC. Targetable alterations were identified in 55.9% of patients. In addition, HPV integration breakpoints occurred in 97.8% of the CC samples, 70.5% of the CIN samples and 42.8% of the normal cervical samples with HPV infection. Integrations of high-risk HPV strains in CCs, including HPV16, 18, 33 and 58, also occurred in the CIN samples. Moreover, gene mutations were detected in 52% of the CIN specimens, and 54.8% of these mutations occurred in genes that also mutated in CCs. CONCLUSION: Our results lay the foundation for a deep understanding of the molecular mechanisms and finding new diagnostic and therapeutic targets of CC.

Co-mutational assessment of circulating tumour DNA (ctDNA) during osimertinib treatment for T790M mutant lung cancer.

Osimertinib is designed to target the secondary resistant EGFR T790M mutant and has shown outstanding clinical efficacy. However, the prognostic prediction of osimertinib patients is a big problem in clinical practice. The resistance mechanism of osimertinib is also not fully understood. NGS and a 1021 gene capture panel were used to analyse the somatic mutation profile of thirty-six lung adenocarcinoma patients' serial ctDNA samples. Progression-free survival of subgroup patients is analysed. Patients harbour TP53 mutations and patients with higher TMB value in pre-treatment samples showed a shorter PFS. Moreover, compared to CT evaluation, ctDNA changes generally correlated with treatment responses in most patients. Novel resistance mechanisms are discovered including EGFR mutations and alternative activation pathway. Our results implied a broad potential of ctDNA as an adjuvant tool in practical clinical management of NSCLC patients. ctDNA could help with clinical practice during osimertinib treatment, regarding monitoring tumour response, detecting development of heterogeneity, identifying potential resistance mechanisms, predicting treatment efficacy and patient outcome.

Intratumor heterogeneity comparison among different subtypes of non-small-cell lung cancer through multi-region tissue and matched ctDNA sequencing.

Understanding of intratumor heterogeneity (ITH) among different non-small cell lung cancer (NSCLC) subtypes is necessary. Whether circulating tumor DNA (ctDNA) profile could represent these ITH is still an open question. We performed 181 multi-region tumor tissues sequencing and matched ctDNA sequencing from 32 operative NSCLC to compare ITH among different NSCLC subtypes, including EGFR-mutant lung adenocarcinoma (LUAD), KRAS-mutant LUAD, EGFR&KRAS-wild-type LUAD, and lung squamous cell carcinoma (LUSC), and examine potential value of ctDNA for ITH analysis. ITH is evaluated by ITH index (ITHi). If the somatic genetic alteration is shared by all the tissue regions, it is defined as trunk mutation. Otherwise, it is called branch mutation. The ITHi will be higher, if the tumor has less trunk mutations. We found EGFR-mutant LUAD showed significantly higher ITHi than KRAS-mutant LUAD/wild-type LUAD (P = 0.03) and numerically higher ITH than LUSC. For trunk mutations, driver mutations were identified at a higher proportion than passenger mutations (60% vs. 40%, P = 0.0023) in overall, especially in EGFR-mutant LUAD (86% vs. 14%, P = 0.0004), while it was opposite in KRAS-mutant LUAD (40% vs. 60%, P = 0.18). For branch mutations, the proportions of driver mutations and passenger mutations were similar for each NSCLC subtype. ctDNA analysis showed unsatisfactory detections of tumor-derived trunk and branch mutations (43% vs. 23%, P = 4.53e-6) among all NSCLC subtypes. In summary, EGFR-mutant LUAD has the highest ITH than other NSCLC subtypes, offering further understanding of tumorigenesis mechanisms among different NSCLC subtypes. Besides, ctDNA maybe not an appropriate method to reflect ITH.

Prevalence of recurrent oncogenic fusion in mismatch repair-deficient colorectal carcinoma with hypermethylated MLH1 and wild-type BRAF and KRAS.

Oncogenic fusions are rare in colorectal carcinomas, but may be important for prognosis and therapy. An effective strategy for screening targetable oncogenic fusions in colorectal carcinomas is needed. Here, we investigate molecular genetic alterations in colorectal carcinomas based on their DNA mismatch repair status, and to effectively screen for targetable oncogenic fusions in colorectal carcinomas. In this retrospective study, the initial cohort included 125 consecutive mismatch repair-deficient and 238 randomly selected mismatch repair-proficient colorectal carcinomas diagnosed between July 2015 and December 2017 at Peking Union Medical College Hospital. Targeted sequencing was performed. MLH1 promoter hypermethylation analysis was further employed for subgrouping dMMR colorectal carcinomas. Clinicopathological characteristics, molecular features, and survival outcome of colorectal carcinomas harboring oncogenic fusions were assessed. A multicenter cohort comprised of 227 colorectal carcinomas with dual loss of MLH1/PMS2 was used to validate the efficacy of the proposed screening strategy for oncogenic fusions. Of the 363 patients in the initial cohort, 11(3.0%) harbored oncogenic fusions and were all mismatch repair-deficient colorectal carcinomas with hypermethylated MLH1 and wild-type BRAF and KRAS, comprising 55% (11/20) of this subgroup. These patients with oncogenic fusions showed poorer 3-year cancer-specific survival compared with other Stage III/IV mismatch repair-deficient colorectal carcinoma patients (40% vs. 97%), and significantly higher CD274(PD-L1) expression in tumor cells compared with other dMMR colorectal carcinoma patients (46% vs. 6.1%, P < 0.001). An easy-to-perform and cost-efficient strategy for screening targetable fusions was proposed based on the current molecular testing algorithms for colorectal carcinomas, and validated in an independent multicenter cohort. In conclusion, oncogenic fusions were highly enriched and frequently detected in mismatch repair-deficient colorectal carcinomas with MLH1 hypermethylation and wild-type BRAF and KRAS, and were associated with poor prognosis and high tumor CD274(PD-L1) expression.

Silica Removal Using Magnetic Iron-Aluminum Hybrid Nanomaterials: Measurements, Adsorption Mechanisms, and Implications for Silica Scaling in Reverse Osmosis.

Composite magnetic aluminum hydroxide at iron oxide nanomaterials, Al(OH)3@Fe3O4, with a well-defined core-shell structure, were used as pretreatment adsorbents for the removal of silica in brackish water. The Al(OH)3 outer shell confers silica adsorption capacity, and the superparamagnetic Fe3O4 core allows material separation and magnetic recovery. The as-prepared nanomaterials (2 g L-1) remove ∼95 and ∼80% silica from Si-rich solutions with 0.5 and 2 mM initial silica concentrations, respectively. Regeneration under basic conditions was evaluated, and post-adsorption treatment with 0.05 M NaOH yielded optimal material reusability. After four regeneration cycles, the Al(OH)3@Fe3O4 nanomaterials retain their magnetic property while still being able to remove ∼40% silica from solutions at an adsorbent concentration of 2 g L-1. The mechanism of silica adsorption onto the surface of the nanomaterials was probed using several spectroscopic techniques. ATR-FTIR (attenuated total reflection-Fourier transform infrared) integrated with two-dimensional correlation analysis shows that silica species vary from Q2 to Q4 with adsorption time corresponding to silica polymerization. 29Si solid-state NMR spectra show an upfield chemical shift displacement of the Q2 signal, which indicates the formation of Q4 units, suggesting silica polymerization onto the Al(OH)3 shell. In addition, a laboratory-scale reverse osmosis setup was used to evaluate Al(OH)3@Fe3O4 as pretreatment materials for silica removal. Results show that silica scaling was significantly alleviated, and water recovery was enhanced when feed waters were pretreated with the magnetic nanomaterials. Taken together, our study highlights the promise of magnetic Al(OH)3@Fe3O4 nanomaterials in treating brackish water and achieving higher water recovery for inland desalination.