Biomineralization Process of CaCO3 Precipitation Induced by Bacillus mucilaginous and Its Potential Application in Microbial Self-healing Concrete.

Microbial induced calcium carbonate precipitation (MICP) is widely common in nature, which belongs to biomineralization and has been explored carefully in recent decades. The paper studied the effect of temperature, initial pH value and Ca2+ concentration on bacterial growth and carbonic anhydrase activity, and then revealed the biomineralization process through the changes of Ca2+ concentration and calcification rate in alkali environment. Meanwhile, microbial healing agent containing spores and calcium nitrate was prepared and used for the early age concrete cracks repair. The self-healing efficiency was assessed by crack closure rate and water permeability repair rate. The experimental results showed that when the optimal temperature was 30 °C, the pH was 8.0-11.0, and the optimal Ca2+ concentration was 0-90 mM, the bacteria could grow better and the carbonic anhydrase activity was higher. Compared with reference, the crack closure rate with the crack width up to 0.339 mm could reach 95.62% and the water permeability repair rate was 87.54% after 28 d healing time of dry-wet cycles. XRD analysis showed that the precipitates at the crack mouth were calcite CaCO3. Meanwhile, the self-healing mechanism of mortar cracks was discussed in detail. In particular, there is no other pollution in the whole mineralization process, and the self-healing system is environmentally friendly, which provides a novel idea and method for the application of microbial self-healing concrete.

Evolutionary characterization of lung adenocarcinoma morphology in TRACERx.

Lung adenocarcinomas (LUADs) display a broad histological spectrum from low-grade lepidic tumors through to mid-grade acinar and papillary and high-grade solid, cribriform and micropapillary tumors. How morphology reflects tumor evolution and disease progression is poorly understood. Whole-exome sequencing data generated from 805 primary tumor regions and 121 paired metastatic samples across 248 LUADs from the TRACERx 421 cohort, together with RNA-sequencing data from 463 primary tumor regions, were integrated with detailed whole-tumor and regional histopathological analysis. Tumors with predominantly high-grade patterns showed increased chromosomal complexity, with higher burden of loss of heterozygosity and subclonal somatic copy number alterations. Individual regions in predominantly high-grade pattern tumors exhibited higher proliferation and lower clonal diversity, potentially reflecting large recent subclonal expansions. Co-occurrence of truncal loss of chromosomes 3p and 3q was enriched in predominantly low-/mid-grade tumors, while purely undifferentiated solid-pattern tumors had a higher frequency of truncal arm or focal 3q gains and SMARCA4 gene alterations compared with mixed-pattern tumors with a solid component, suggesting distinct evolutionary trajectories. Clonal evolution analysis revealed that tumors tend to evolve toward higher-grade patterns. The presence of micropapillary pattern and 'tumor spread through air spaces' were associated with intrathoracic recurrence, in contrast to the presence of solid/cribriform patterns, necrosis and preoperative circulating tumor DNA detection, which were associated with extra-thoracic recurrence. These data provide insights into the relationship between LUAD morphology, the underlying evolutionary genomic landscape, and clinical and anatomical relapse risk.

Study protocol of an open-label prospective phase II umbrella study of precise neoadjuvant therapy for patients with stage II-IIIB resectable non-small cell lung cancer (PURPOSE).

Background: The outcomes of locally advanced non-small cell lung cancer (LA-NSCLC) are unfavorable mainly due to a high risk of cancer recurrence. Only around 5% of patients can benefit from perioperative chemotherapy which is the current standard treatment. Recently, promising results with neoadjuvant targeted and immune-therapy therapy have been seen. However, most clinical trials are looking for patients eligible for certain drugs, instead of seeking suitable treatments for certain patients. Therefore, it is necessary to look for more efficient perioperative therapies to increase resectability, reduce recurrence and improve prognosis. Methods/Design: The study is an open-label, prospective, phase II, umbrella trial, enrolling patients diagnosed with treatment-naïve potentially resectable Stage II-IIIB NSCLC. Next-generation sequencing (NGS) using a 68-gene panel is performed for biopsies of tumor tissues from eligible patients. Enrolled patients are then stratified into six independent cohorts based on the status of gene mutations and PD-L1 status in tumor tissues, that is, ①EGFR 19del group, ②EGFR 21 L858R group, ③EGFR rare mutation group, ④Other driver mutation group, ⑤Drive mutation-negative group with PD-L1≥1%, ⑥Drive mutation-negative group with PD-L1<1%. A Simon's two-stage design is performed in each cohort independently and patients receive corresponding standard therapies accordingly. We aim to enroll 26 patients in each cohort and totally 156 patients will be enrolled. The primary endpoint is objective response rate (ORR). Secondary endpoints include oncological prognosis and perioperative outcomes. Exploratory endpoint is to investigate patient-specific minimal residual disease (MRD) in predicting treatment efficacy and oncological prognosis. Discussions: This is the first umbrella trial focusing on the safety and efficacy of precise neoadjuvant therapy for patients diagnosed with potentially resectable LA-NSCLC based on NGS results. The results of this trial would help improve overall treatment results in LA-NSCLC patients. Trial registration: Chinese Clinical Trial Registry. Trial registration number: ChiCTR2100053021. Advantages and limitations of this study: There is no neoadjuvant umbrella trial focusing on LA-NSCLCs. This is the first neoadjuvant umbrella trial, using a precise individualized approach and seeking suitable drugs for LA-NSCLC patients, with the aim to improve overall treatment outcomes. Clinical trial registration: https://www.chictr.org.cn/, identifier ChiCTR2100053021.

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355.

Different dissecting orders of the pulmonary bronchus and vessels during right upper lobectomy are associated with surgical feasibility and postoperative recovery for lung cancer patients.

BACKGROUND: Right upper lobectomy (RUL) for lung cancer with different dissecting orders involves the most variable anatomical structures, but no studies have analyzed its effects on postoperative recovery. This study compared the conventional surgical approach, VAB (dissecting pulmonary vessels first, followed by the bronchus), and the alternative surgical approach, aBVA (dissecting the posterior ascending arterial branch first, followed by the bronchus and vessels) on improving surgical feasibility and postoperative recovery for lung cancer patients. METHODS: According to the surgical approach, consecutive lung cancer patients undergoing RUL were grouped into aBVA and VAB cohorts. Their clinical, pathologic, and perioperative characteristics were collected to compare perioperative outcomes. RESULTS: Three hundred one patients were selected (109 in the aBVA cohort and 192 in the VAB cohort). The mean operation time was shorter in the aBVA cohort than in the VAB cohort (164 vs. 221 min, P < 0.001), and less blood loss occurred in the aBVA cohort (92 vs. 141 mL, P < 0.001). The rate of conversion to thoracotomy was lower in the aBVA cohort than in the VAB cohort (0% vs. 11.5%, P < 0.001). The mean duration of postoperative chest drainage was shorter in the aBVA cohort than in the VAB cohort (3.6 vs. 4.5 days, P = 0.001). The rates of postoperative complications were comparable (P = 0.629). The median overall survival was not arrived in both cohorts (P > 0.05). The median disease-free survival was comparable for all patients in the two cohorts (not arrived vs. 41.97 months) and for patients with disease recurrences (13.25 vs. 9.44 months) (both P > 0.05). The recurrence models in two cohorts were also comparable for patients with local recurrences (6.4% vs. 7.8%), distant metastases (10.1% vs. 8.3%), and both (1.8% vs. 1.6%) (all P > 0.05). CONCLUSIONS: Dissecting the right upper bronchus before turning over the lobe repeatedly and dissecting veins via the aBVA approach during RUL would promote surgical feasibility and achieve comparable postoperative recovery for lung cancer patients.

Mixed Responses to Systemic Therapy Revealed Potential Genetic Heterogeneity and Poor Survival in Patients with Non-Small Cell Lung Cancer.

BACKGROUND: A subset of patients with non-small cell lung cancer (NSCLC) fosters mixed responses (MRs) to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) or chemotherapy. However, little is known about the clinical and molecular features or the prognostic significance and potential mechanisms. METHODS: The records of 246 consecutive patients with NSCLC receiving single-line chemotherapy or TKI treatment and who were assessed by baseline and interim positron emission tomography/computed tomography scans were collected retrospectively. The clinicopathological correlations of the MR were analyzed, and a multivariate analysis was performed to explore the prognostic significance of MR. RESULTS: The overall incidence of MR to systemic therapy was 21.5% (53/246) and predominated in patients with stage IIIB-IV, EGFR mutations and those who received TKI therapy (p < .05). Subgroup analyses based on MR classification (efficacious versus inefficacious) showed significant differences in subsequent treatment between the two groups (p < .001) and preferable progression-free survival (PFS) and overall survival (OS) in the efficacious MR group. Multivariate analyses demonstrated that the presence of MR was an independent unfavorable prognostic factor for PFS (hazard ratio [HR], 1.474; 95% confidence interval [CI], 1.018-2.134; p = .040) and OS (HR, 1.849; 95% CI, 1.190-2.871; p = .006) in patients with NSCLC. Induced by former systemic therapy, there were more T790M (18%), concomitant EGFR mutations (15%), and changes to EGFR wild type (19%) in the MR group among patients with EGFR mutations, which indicated higher incidence of genetic heterogeneity. CONCLUSION: MR was not a rare event in patients with NSCLC and tended to occur in those with advanced lung adenocarcinoma treated with a TKI. MR may result from genetic heterogeneity and is an unfavorable prognostic factor for survival. Further studies are imperative to explore subsequent treatment strategies. The Oncologist 2017;22:61-69Implications for Practice: Tumor heterogeneity tends to produce mixed responses (MR) to systemic therapy, including TKI and chemotherapy; however, the clinical significance and potential mechanisms are not fully understood, and the subsequent treatment after MR is also a clinical concern. The present study systemically assessed patients by PET/CT and differentiated MR and therapies. The study identified a relatively high incidence of MR in patients with advanced NSCLC, particularly those treated with targeted therapies. An MR may be an unfavorable prognostic factor and originate from genetic heterogeneity. Further studies are imperative to explore subsequent treatment strategies.

Potential Predictive Value of TP53 and KRAS Mutation Status for Response to PD-1 Blockade Immunotherapy in Lung Adenocarcinoma.

Purpose: Although clinical studies have shown promise for targeting programmed cell death protein-1 (PD-1) and ligand (PD-L1) signaling in non-small cell lung cancer (NSCLC), the factors that predict which subtype patients will be responsive to checkpoint blockade are not fully understood.Experimental Design: We performed an integrated analysis on the multiple-dimensional data types including genomic, transcriptomic, proteomic, and clinical data from cohorts of lung adenocarcinoma public (discovery set) and internal (validation set) database and immunotherapeutic patients. Gene set enrichment analysis (GSEA) was used to determine potentially relevant gene expression signatures between specific subgroups.Results: We observed that TP53 mutation significantly increased expression of immune checkpoints and activated T-effector and interferon-γ signature. More importantly, the TP53/KRAS comutated subgroup manifested exclusive increased expression of PD-L1 and a highest proportion of PD-L1+/CD8A+ Meanwhile, TP53- or KRAS-mutated tumors showed prominently increased mutation burden and specifically enriched in the transversion-high (TH) cohort. Further analysis focused on the potential molecular mechanism revealed that TP53 or KRAS mutation altered a group of genes involved in cell-cycle regulating, DNA replication and damage repair. Finally, immunotherapeutic analysis from public clinical trial and prospective observation in our center were further confirmed that TP53 or KRAS mutation patients, especially those with co-occurring TP53/KRAS mutations, showed remarkable clinical benefit to PD-1 inhibitors.Conclusions: This work provides evidence that TP53 and KRAS mutation in lung adenocarcinoma may be served as a pair of potential predictive factors in guiding anti-PD-1/PD-L1 immunotherapy. Clin Cancer Res; 23(12); 3012-24. ©2016 AACR.

Intraoperative frozen sections of the regional lymph nodes contribute to surgical decision-making in non-small cell lung cancer patients.

BACKGROUND: Individualization of pulmonary parenchymal resection and lymphadenectomy in lung cancer patients will likely become more important as surgical innovation. This study explored the utility of intraoperative pathological frozen sections of regional lymph nodes in non-small cell lung cancer (NSCLC) patients. METHODS: Patients with NSCLC underwent intraoperative sampling of N1 station lymph nodes depending on the location of the tumor, any other suspicious lymph nodes were also biopsied. The contribution of frozen-section analysis to surgical decision-making was evaluated. RESULTS: Of 74 lung cancer patients who underwent intraoperative frozen section analysis of lymph nodes, the positive rate was 18/74 (24.3%). The extents of agreement between preoperative N staging (cN) and intraoperative N staging (sN), cN staging and postoperative N staging (pN), and sN staging and pN staging were 62.2% (46/74), 63.5% (47/74), and 71.6% (53/74), respectively. When frozen section was combined with evaluation of pulmonary function and intrathoracic adhesions, surgical strategies were modified during operations in 18 cases (5 sN-positive, 13 sN-negative). Of these patients, five underwent extensive pulmonary parenchymal resection, and four had conservative lung parenchymal resection. In nine patients, the extent of lymph node dissection (LND) was changed. CONCLUSIONS: Intraoperative frozen section of regional lymph nodes led to 24.3% operative strategies modification in lung cancer. Frozen section analysis may make an important contribution to surgical decision-making in terms of pulmonary parenchymal resection and LND.

Right upper lobectomy performed as dividing posterior ascending artery-bronchus-pulmonary vessels is alternative to primary indolent scar carcinomas.

We describe a case of pulmonary indolent malignancy requiring a strategic surgery and introduce an alternative technique of right upper lobectomy via video-assisted thoracic surgery (VATS) for primary lung cancer patients. A 42-year-old male non-smoker was referred to the hospital following the detection of an opacity with a cystic airspace in the right upper lobe during a routine physical examination. During a regular follow-up over 3.5 years, the solid component enlarged and the cystic wall thickened. Based on a suspicion of indolent scar carcinoma, a right upper lobectomy was performed using VATS. The preoperative diagnosis was clinical T1bN0M0, stage Ia primary lung cancer. Our surgical procedure, posterior single-direction aBVA, consists of dividing the posterior ascending artery branch and then the right upper bronchus, followed by the right upper pulmonary vessels. By efficiently reducing the operation time and blood loss, our method is potentially superior to conventional right upper lobectomy.

Rare discrepancies in a driver gene alteration within histologically heterogeneous primary lung cancers.

OBJECTIVES: Most lung adenocarcinomas consist of mixtures of histological subtypes harboring different frequencies of driver gene mutations. However, little is known about intratumoral heterogeneity(ITH) within histologically heterogeneous primary lung cancers. Investigating key driver genes in respective morphological pattern is crucial to personalized treatment. METHODS: Morphologically different areas within the same surgically resected adenocarcinomas were extracted from tissues to analyze gene status in each growth pattern. Driver genes, epidermal growth factor receptor (EGFR), KRAS and EML4-ALK, were assessed by assays with different sensitivities. RESULTS: Seventy-nine consecutive eligible patients harboring a driver gene (EGFR=65; KRAS=10; EML4-ALK=4) were enrolled. For EGFR mutations, ITH occurred in 13.3% (8/60) by direct sequencing (DS) and 1.7% (1/60) by amplification refractory mutation system (ARMS) (P=0.016) among adenocarcinomas, but consistent within five adeno-squamous cell carcinomas by both methods. ITH among KRAS mutations were detected in 20% (2/10) by DS, whereas consistent (10/10) by high resolution melting. No discrepancies in EML4-ALK rearrangements existed according to fluorescence in situ hybridization. CONCLUSION: Rare ITHs of EGFR/KRAS/EML4-ALK alterations within histologically heterogeneous primary lung adenocarcinomas existed by methods with higher sensitivity. Discrepancies might be due to abundance of mutant tumor cells and detection assays.

Neoadjuvant and adjuvant epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy for lung cancer.

The Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis and the meta-analysis of individual participant data reported by non-small cell lung cancer (NSCLC) Meta-analysis Collaborative Group in neo-adjuvant setting validated respectively that adjuvant and neoadjuvant chemotherapy would significantly improve overall survival (OS) and recurrence-free survival for resectable NSCLC. However, chemotherapy has reached a therapeutic plateau. It has been confirmed that epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeting therapy provides a dramatic response to patients with advanced EGFR-mutation positive NSCLC. Researchers have paid more attention to exploring applications of TKIs to early resectable NSCLCs. Several studies on adjuvant TKI treatment concluded its safety and feasibility. But there existed certain limitations of these studies as inference factors to interpret data accurately: the BR19 study recruited patients among which almost 52% had stage IB and only 15 (3.0%, 15/503) had been confirmed with EGFR-mutant type; retrospective studies performed at Memorial Sloan Kettering Cancer Center (MSKCC) selected EGFR mutant-type NSCLC patients but couldn't avoid inherent defects inside retrospective researches; the RADIANT study revised endpoints from targeting at EGFR immunohistochemistry (IHC)+ and/or fluorescence in situ hybridization (FISH)+ mutation to only EGFR IHC+ mutation, leading to selective bias; despite that the SELECT study validated efficacy of adjuvant TKI and second round of TKI after resistance occurred, a single-arm clinical trial is not that persuasive in the absence of comparison with chemotherapy. Taking all these limitations into account, CTONG1104 in China and IMPACT in Japan have been conducted and recruiting patients to offer higher level of evidences to explore efficacy of preoperative TKI therapy for early resectable EGFR mutation positive NSCLC patients (confirmed by pathological results of tumor tissue or lymph node biopsy). On the other hand, case reports and several phase II clinical trials with small sample size tried to elbow their way on respect of preoperative TKI treatment and advised that TKI tended to improve response rate. However, no data on survival rate was present. The first phase II study of biomarker-guided neoadjuvant therapy for stage IIIA-N2 NSCLC patients stratified by EGFR mutation status, sponsored by CSLC0702, showed erlotinib tended to improve response rate, but failed to show benefits of disease-free survival (DFS) or OS. Subsequently, CTONG1103 was designed to investigate efficacy of erlotinib vs. combination of gemcitabine/cisplatin (GC) as neoadjuvant treatment in stage IIIA-N2 NSCLC with sensitizing EGFR mutation in exon 19 or 21. All these ongoing trials should be worthy of our expect to provide convincing evidences for customized therapy for patients with resectable NSCLC.

Research, evidence, and ethics: new technology or grey medicine.

Major pioneering advances of medicine in history tend to manifest in two directions that seem divergent but actually unified with dialectics: one is the important biological principle revealed by in-depth studies from the clinic to the laboratory based on individual cases; the other is the colonial generality displayed by epidemiologic data from large-scale samples. Although advances predominated, we human beings were paying dearly for it due to serious incidents of endangering ourselves and defects of restrictions of laws and ethics. Subsequently, the Nuremberg Code, Declaration of Helsinki and Belmont Report came into light and constrained human experiments and clinical trials. However, the development of such laws and regulations in China is lagging behind and renders China as a breeding ground for gray medicine. There are three lessons we can learn from painful histories and apply to individualized treatment of lung cancer. Firstly, the abuse of Avastin beyond its indications reflected the similar situation of tyrosine kinase inhibitors in lung cancer due to different molecular types and stages of tumors; secondly, the black market of stem cell therapy in China reminds us how to identify the boundaries of clinical trials and clinical treatment, in similar to the cellular immunotherapy of tumors; thirdly, the theory of Xiao's Reflex Arc emerged us to rethink the level of the validity of clinical evidences, which can provide hints related to video-assisted thoracoscopic surgeries (VATSs). In conclusion, clinical applications of new techniques and treatment regimens should follow three points: identify indications and contraindications clearly, obtain informed consent and permission of patients and supervise effectively according to laws and ethics.

Clinical trials in lung cancer surgery and research cooperation.

BACKGROUND: With China having an increasingly huge burden of lung cancers, the number of Chinese studies was the second largest all over the world. METHODS: We used the advanced search option available on the ClinicalTrials.gov to review clinical trials regarding surgical issues. In the drop down menus, we chose "all studies" for recruitment status and "interventional" for study type and all trials reported until Aug, 2013 on the website were included. RESULTS: Clinical trials on lung cancer surgeries (658 records) mainly aim at surgical procedures and (neo)adjuvant clinical studies, among which phase III trials account for 15.5%. Only 34.9% (230 records) trials were completed, and 43 studies presented their results on ClinicalTrials.gov. The median time to completion (MTC) of phase III surgical procedure trials is 9.4 years. The MTCs of phase III neo-adjuvant and adjuvant trials have not been reached, but definitely are longer than 10 years. In comparison, the MTC of phase III trials in the first-line treatment are only 4.3 years. COMMENTS: We summarized the characteristics of these trials using real-world case examples. Our analyses reveal that it is critically needed for regulatory authorities, clinical trial sponsors, collaborative research groups, and academic institutions to work together to build up the infrastructure and research cooperation for clinical trials with a surgical component. In 2007, a national collaborative clinical research group, Chinese Thoracic Oncology Group (CTONG), was established. CTONG is a network of researchers, physicians and health-care professionals in public institutions throughout China and currently has a growing membership of 25 hospitals. A CTONG-sponsored trial (CTONG1104) is discussed to illustrate our experience in surgical clinical trials. In summary, it is imperative for investigators to collaborate in cooperative clinical trials in order to expedite applications of therapies from clinical researches to cancer treatment. Since cancer treatment is multidisciplinary, current surgical trials should have multiple treatment combinations while retaining a surgical focus, and be carried out systematically with the cooperation of extensive monitoring and coordinating systems.