Case report: Rechallenge with EGFR-TKIs after immunotherapy in EGFR-mutated non-small cell lung cancer with leptomeningeal metastasis.

Rechallenge of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) after PD-1 blockade failure was an effective therapy for non-small cell lung cancer (NSCLC) patients with resistance to EGFR-TKIs. The third-generation TKIs, like osimertinib and furmonertinib, can reach higher concentration in the cerebrospinal fluid (CSF) than other TKIs, and exhibit a beneficial effect in NSCLC patients with leptomeningeal metastases (LM) harboring sensitive EGFR mutation. Here, we report that two-stage IV pulmonary adenocarcinoma patients with LM harboring an EGFR L858R mutation benefit from the third-generation EGFR-TKIs rechallenge after immune checkpoint inhibitor (ICI) and anti-angiogenic agent combination therapy. Complete response (CR) to partial response (PR) of central nervous system (CNS) response was achieved immediately after the administration of furmonertinib and osimertinib. We conducted next-generation sequencing (NGS) and IHC to elucidate the evolution of driver mutations and the immune microenvironment. In conclusion, these two cases might provide a therapeutic strategy for further clinical practice. More research was needed to elucidate the resistance mechanisms and improve current treatment strategies in EGFR-mutated patients with LM.

Genomic profiling of non-small cell lung cancer with the rare pulmonary lymphangitic carcinomatosis and clinical outcome of the exploratory anlotinib treatment.

Background: To evaluate the potential treatment for patients with non-small cell lung cancer (NSCLC) and rare malignant pulmonary lymphangitis carcinomatosis (PLC), our study provided a genomic profile and clinical outcome of this group of patients. Methods: We retrospectively reviewed patients with NSCLC who developed PLC. The genomic alterations, tumor mutation burden (TMB), and microsatellite instability (MSI) based on DNA-based next-generation sequencing were reviewed and compared in a Chinese population with lung adenocarcinomas (Chinese-LUAD cohort). Clinical outcomes after exploratory anlotinib treatment and factors influencing survival are summarized. Results: A total of 564 patients with stage IV NSCLC were reviewed, and 39 patients with PLC were included. Genomic profiling of 17 adenocarcinoma patients with PLC (PLC-LUAD cohort) revealed TP53, EGFR, and LRP1B as the three most frequently altered genes. EGFR was less mutated in PLC-LUAD than Chinese-LUAD cohort of 778 patients (35.3% vs. 60.9%, P = 0.043). BRIP1 was mutated more often in the PLC-LUAD cohort (11.8% vs. 1.8%, P= 0.043). Two patients presented with high tumor mutational burden (TMB-H, 10 mutations/MB). Combing alterations in the patient with squamous cell carcinoma, the most altered pathways of PLC included cell cycle/DNA damage, chromatin modification, the RTK/Ras/MAPK pathway and VEGF signaling changes. Fourteen of the participants received anlotinib treatment. The ORR and DCR were 57.1% and 92.9%, respectively. Patients achieved a median progression-free survival of 4.9 months and a median overall survival of 7 months. The adverse effects were manageable. In patients with adenocarcinoma, the mPFS (5.3 months vs. 2.6 months) and mOS (9.9 months vs. 4.5 months) were prolonged in patients receiving anlotinib treatment compared to those receiving other treatment strategies (P < 0.05). Conclusion: Patients with PLC in NSCLC demonstrated distinct genetic alterations. The results improve our understanding of the plausible genetic underpinnings of tumorigenesis in PLC and potential treatment strategies. Exploratory anlotinib treatment achieved considerable benefits and demonstrated manageable safety.

Case report: Almonertinib in combination with bevacizumab for leptomeningeal metastases from epidermal growth factor receptor-mutation non-small cell lung cancer: Case series.

Leptomeningeal metastasis (LM) is a lethal complication of advanced non-small cell lung cancer (NSCLC) with rapid deterioration and poor prognosis. It has no standard treatment for epidermal growth factor receptor mutation (EGFRm) NSCLC, and improving the clinical outcomes for patients with LM has become an urgent problem in clinical treatment. Both almonertinib and bevacizumab are capable of crossing the blood-brain barrier with comparable central nervous system effectiveness. To date, the almonertinib treatment in combination with bevacizumab in EGFRm NSCLC with LM has not been studied. We herein present five cases to further evaluate the effectiveness and tolerability of almonertinib in combination with bevacizumab for patients with EGFRm NSCLC and LM. For the first time, we report that almonertinib plus bevacizumab can not only effectively improve the neurological symptoms caused by LM but also prolong the survival time of patients with limited and controllable side effects, which provided a novel therapeutic approach for LM from EGFRm NSCLC.

A novel intergenic region ALK fusion is targetable by alectinib in a non-small cell lung cancer patient with brain metastasis.

Anaplastic lymphoma kinase ( ALK ) rearrangement defines a unique nonsmall cell lung cancer (NSCLC) molecular subtype, of which the patients could potentially benefit from anti- ALK therapies. So far, the outcomes of the canonical echinoderm microtubule-associated protein-like ( EML-ALK ) patients subjected to ALK inhibitors are well established. However, given the increasing complexity of ALK fusion partners, as detected by high-throughput sequencing, the responses of those with rare ALK fusion events remain to be explored. Here, we report a lung adenocarcinoma patient with brain metastasis harboring an ARHGAP5 downstream intergenic region ALK fusion, as detected by using DNA-based next-generation sequencing, who experienced a partial response to alectinib treatment. While whole- transcriptome RNA sequencing (RNA-seq) failed to identify potential ALK fusion transcripts, subsequent targeted deep RNA-seq revealed the expression of EML4-ALK transcripts in the tumor tissue. Given the increasing application of the ALK-tyrosine kinase inhibitors (TKIs), it is extremely crucial to define the patients who could be suitable for this treatment in clinic. The present case has provided supporting evidence that noncanonical ALK rearrangements on the genomic level are often functionally relevant and targetable by ALK-TKI, particularly in cases with sub-optimal quantity and quality for RNA validation.

Lung adenocarcinoma patients with KEAP1 mutation harboring low immune cell infiltration and low activity of immune environment.

BACKGROUND: Kelch-like ECH-associated protein 1 (KEAP1) has been identified as a cancer driver gene in lung adenocarcinoma (LUAD), and increased evidence has given us clues about the association of KEAP1 mutation and immune characteristics. We assessed the association between KEAP1 mutation and tumor microenvironment in LUAD systematically. METHODS: With the data collected from The Cancer Genome Atlas (TCGA), we evaluated the association of KEAP1 mutation with tumor infiltrating leukocytes (TILs), including dendritic cell, CD8 T cell, CD4 T cell, neutrophil, B cells, and macrophage. Expression differences of the markers of those immune cells were also measured. We further compared the expression of antigen presentation genes and chemokines and the enrichment score of immune-related pathways. RESULTS: KEAP1 mutation had significant association with lower TILs and cytotoxic T lymphocyte. Strikingly, almost all of antigen presentation genes and chemokine showed lower expression in KEAP1-mutated tumors. Moreover, most of immune-related pathways were less active in KEAP1-mutated tumors. As expected, KEAP1-wild type LUADs favored better overall survival after immunotherapy. Finally, one patient harboring KEAP1 mutation along with a lack of immune cells infiltration in tumor microenvironment failed to respond to checkpoint inhibitor despite high tumor mutational burden (TMB). CONCLUSIONS: KEAP1 mutation has a significant effect on the tumor immune milieu of LUAD and may play as a predictive biomarker of immunotherapy for LUAD patients.

Association of TP53 Mutations with Response to Anlotinib Treatment in Advanced Non-Small Cell Lung Cancer.

Multitargeted antiangiogenic drugs have demonstrated significant antitumor activity against a variety of solid tumors. Anlotinib, a novel oral multitargeted antiangiogenic tyrosine kinase inhibitor, was approved as a third-line treatment for advanced NSCLC in China. However, predictive biomarkers are currently insufficient and are urgently required. Herein, we report three pre-treated cases of advanced NSCLC with TP53 mutations, wherein these patients showed partial response to anlotinib. Moreover, the three patients have achieved a progression-free survival of 8, 6.5, and 5 months, respectively. The main toxicities were hypertension, hand-foot syndrome and fatigue. In conclusion, TP53 mutations may represent a biomarker for predicting salutary effects of anlotinib.

Genome-wide characterization and evolutionary analysis of heat shock transcription factors (HSFs) to reveal their potential role under abiotic stresses in radish (Raphanus sativus L.).

BACKGROUND: Abiotic stresses due to climate change pose a great threat to crop production. Heat shock transcription factors (HSFs) are vital regulators that play key roles in protecting plants against various abiotic stresses. Therefore, the identification and characterization of HSFs is imperative to dissect the mechanism responsible for plant stress responses. Although the HSF gene family has been extensively studied in several plant species, its characterization, evolutionary history and expression patterns in the radish (Raphanus sativus L.) remain limited. RESULTS: In this study, 33 RsHSF genes were obtained from the radish genome, which were classified into three main groups based on HSF protein domain structure. Chromosomal localization analysis revealed that 28 of 33 RsHSF genes were located on nine chromosomes, and 10 duplicated RsHSF genes were grouped into eight gene pairs by whole genome duplication (WGD). Moreover, there were 23 or 9 pairs of orthologous HSFs were identified between radish and Arabidopsis or rice, respectively. Comparative analysis revealed a close relationship among radish, Chinese cabbage and Arabidopsis. RNA-seq data showed that eight RsHSF genes including RsHSF-03, were highly expressed in the leaf, root, cortex, cambium and xylem, indicating that these genes might be involved in plant growth and development. Further, quantitative real-time polymerase chain reaction (RT-qPCR) indicated that the expression patterns of 12 RsHSF genes varied upon exposure to different abiotic stresses including heat, salt, and heavy metals. These results indicated that the RsHSFs may be involved in abiotic stress response. CONCLUSIONS: These results could provide fundamental insights into the characteristics and evolution of the HSF family and facilitate further dissection of the molecular mechanism responsible for radish abiotic stress responses.

Transcriptome-based gene expression profiling of diploid radish (Raphanus sativus L.) and the corresponding autotetraploid.

Polyploidy is an important evolutionary factor in most land plant lineages which possess more than two complete sets of chromosomes. Radish (Raphanus sativus L.) is an economically annual/biennial root vegetable crop worldwide. However, the expression patterns of duplicated homologs involved in the autopolyploidization remains unclear. In present study, the autotetraploid radish plants (2n = 4x = 36) were produced with colchicine and exhibited an increase in the size of flowers, leaves, stomata and pollen grains. The differential gene expression (DGE) profiling was performed to investigate the differences in gene expression patterns between diploid and its corresponding autotetraploid by RNA-Sequencing (RNA-Seq). Totally, 483 up-regulated differentially expressed genes (DEGs) and 408 down-regulated DEGs were detected in diploid and autotetraploid radishes, which majorly involved in the pathways of hormones, photosynthesis and stress response. Moreover, the xyloglucan endotransglucosylase/hydrolase (XTH) and pectin methylesterases (PME) family members related to cell enlargement and cell wall construction were found to be enriched in GO enrichment analysis, of which XTH family members enriched in "apoplast" and "cell wall" terms, while PME family members enriched in "cell wall" term. Reverse-transcription quantitative PCR (RT-qPCR) analysis indicated that the expression profile of DEGs were consistent with results from the RNA-Seq analysis. The DEGs involved in cell wall construction and auxin metabolism were predicted to be associated with organs size increase of autotetraploid radishes in the present study. These results could provide valuable information for elucidating the molecular mechanism underlying polyploidization and facilitating further genetic improvements of important traits in radish breeding programs.

Genome-wide characterization and expression profiling of NAC transcription factor genes under abiotic stresses in radish (Raphanus sativus L.).

NAC (NAM, no apical meristem; ATAF, Arabidopsis transcription activation factor and CUC, cup-shaped cotyledon) proteins are among the largest transcription factor (TF) families playing fundamental biological processes, including cell expansion and differentiation, and hormone signaling in response to biotic and abiotic stresses. In this study, 172 RsNACs comprising 17 membrane-bound members were identified from the whole radish genome. In total, 98 RsNAC genes were non-uniformly distributed across the nine radish chromosomes. In silico analysis revealed that expression patterns of several NAC genes were tissue-specific such as a preferential expression in roots and leaves. In addition, 21 representative NAC genes were selected to investigate their responses to heavy metals (HMs), salt, heat, drought and abscisic acid (ABA) stresses using real-time polymerase chain reaction (RT-qPCR). As a result, differential expressions among these genes were identified where RsNAC023 and RsNAC080 genes responded positively to all stresses except ABA, while RsNAC145 responded more actively to salt, heat and drought stresses compared with other genes. The results provides more valuable information and robust candidate genes for future functional analysis for improving abiotic stress tolerances in radish.

PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli.

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli.