Neoadjuvant Chemoimmunotherapy Increases Tumor Immune Lymphocytes Infiltration in Resectable Non-small Cell Lung Cancer.

BACKGROUND: Neoadjuvant chemoimmunotherapy treatment (NCIT) has achieved great success for non-small cell lung cancer (NSCLC); however, the intrinsic mechanism underlying this treatment remains unclear. METHODS: Thirty-two patients with stage IIA-IIIC NSCLC who underwent surgery after NCIT were included in this retrospective study. Multiplex immunofluorescence (mIF) staining and image analysis assays were performed on the samples collected before and after NCIT for each patient. RNA analyses was applied to confirm the mIF results. RESULTS: Among the enrolled patients, 14 achieved major pathological response or pathological complete response (pCR) and were defined as the 'response' group, whereas 18 patients did not respond well to NCIT and were defined as the 'nonresponse' group. The results of the mIF assays revealed an overall increase in tumor immune lymphocytes (TILs) after NCIT in the stroma area (p = 0.03) rather than the tumor area (p = 0.86). The percentage of CD8+ T cells and tertiary lymphoid structure counts in both the response and nonresponse groups increased significantly after NCIT compared with before NCIT. CD3+ T cells and FOXP3+ cells decreased significantly in the response group but remained unchanged or increased in the nonresponse group. A comparison of the response and nonresponse groups showed that CD3, FOXP3+ and CD8+/PD-1+ cells before NCIT may serve as predictors of the response to neoadjuvant immunotherapy. The RNA analyses confirmed the mIF results that TILs were elevated after NCIT. CONCLUSIONS: The infiltration of immune cells before NCIT was correlated with pathologic complete response, which enhanced the TILs as a promising predictor for selecting patients who were more likely to benefit from NCIT.

A durable response to savolitinib in a patient with lung adenocarcinoma harboring two novel MET exon 14 skipping sites.

MET exon 14 ( METex14 ) skipping variants are oncogenic drivers in non-small-cell lung cancer. Several METex14 skipping alterations have been identified, but different mesenchymal-epithelial transition (MET) exon splicing variants tend to present different clinical outcomes. Here, we reported that a patient with lung adenocarcinoma harbored two novel METex14 skipping mutations (c.2888-35_2888-16del and c.2888-4T>G) identified by the tissue-based next-generation sequencing (NGS) and received savolitinib treatment after chemotherapy failed with brain metastasis. The patient responded well to savolitinib until disease progression in brain lesions and achieved a progress-free survival (PFS) of over 19.7 months. Considering the durable response for extracranial lesions and the same METex14 skipping sites identified by circulating tumor DNA-based NGS, the patient was still given savolitinib combined with stereotactic body radiation therapy for brain lesions. An extracranial PFS of 28 months was achieved. This is the first report of a patient with lung adenocarcinoma harboring two novel METex14 skipping mutations that responded to the MET inhibitor savolitinib. Our case may provide evidence for the treatment of patients with two novel METex14 skipping variants and offer a therapy regimen for those with intracranial progression.

ctDNA-adjusted bTMB as a predictive biomarker for patients with NSCLC treated with PD-(L)1 inhibitors.

BACKGROUND: In non-small cell lung cancer (NSCLC) patients receiving immune checkpoint inhibitors (ICIs), higher blood tumor mutational burden (bTMB) was usually associated with better progression-free survival (PFS) and objective response rate (ORR). However, the association between bTMB and overall survival (OS) benefit remains undefined. It has been reported that patients harboring a high level of circulating tumor DNA (ctDNA) had poor survival. We hypothesized that ctDNA-adjusted bTMB might predict OS benefit in NSCLC patients receiving ICIs. METHODS: Our study was retrospectively performed in three cohorts, including OAK and POPLAR cohort (n = 853), Shanghai and Wuhan (SH&WH) cohort (n = 44), and National Cancer Center (NCC) cohort (n = 47). Durable clinical benefit (DCB) was defined as PFS lasting ≥ 6 months. The cutoff value of ctDNA-adjusted bTMB for DCB prediction was calculated based on a receiver operating characteristic curve. Interaction between treatments and ctDNA-adjusted bTMB was assessed. RESULTS: The bTMB score was significantly associated with tumor burden, while no association was observed between ctDNA-adjusted bTMB with tumor burden. In the OAK and POPLAR cohort, significantly higher ORR (P = 0.020) and DCB (P < 0.001) were observed in patients with high ctDNA-adjusted bTMB than those with low ctDNA-adjusted bTMB. Importantly, the interactions between ctDNA-adjusted bTMB and treatments were significant for OS (interaction P = 0.019) and PFS (interaction P = 0.002). In the SH&WH cohort, the interactions between ctDNA-adjusted bTMB and treatment were marginally significant for OS (interaction P = 0.081) and PFS (interaction P = 0.062). Similar result was demonstrated in the NCC cohort. CONCLUSIONS: Our study indicated that ctDNA-adjusted bTMB might predict OS benefit in NSCLC patients receiving ICIs. The potential of ctDNA-adjusted bTMB as a noninvasive predictor for immunotherapy should be confirmed in future studies.

Quick remedy commits and their impact on mining software repositories.

Most changes during software maintenance and evolution are not atomic changes, but rather the result of several related changes affecting different parts of the code. It may happen that developers omit needed changes, thus leaving a task partially unfinished, introducing technical debt or injecting bugs. We present a study investigating "quick remedy commits" performed by developers to implement changes omitted in previous commits. With quick remedy commits we refer to commits that (i) quickly follow a commit performed by the same developer, and (ii) aim at remedying issues introduced as the result of code changes omitted in the previous commit (e.g., fix references to code components that have been broken as a consequence of a rename refactoring) or simply improve the previously committed change (e.g., improve the name of a newly introduced variable). Through a manual analysis of 500 quick remedy commits, we define a taxonomy categorizing the types of changes that developers tend to omit. The taxonomy can (i) guide the development of tools aimed at detecting omitted changes and (ii) help researchers in identifying corner cases that must be properly handled. For example, one of the categories in our taxonomy groups the reverted commits, meaning changes that are undone in a subsequent commit. We show that not accounting for such commits when mining software repositories can undermine one's findings. In particular, our results show that considering completely reverted commits when mining software repositories accounts, on average, for 0.07 and 0.27 noisy data points when dealing with two typical MSR data collection tasks (i.e., bug-fixing commits identification and refactoring operations mining, respectively).

Efficient DNA interrogation of SpCas9 governed by its electrostatic interaction with DNA beyond the PAM and protospacer.

Streptococcus pyogenes Cas9 (SpCas9), a programmable RNA-guided DNA endonuclease, has been widely repurposed for biological and medical applications. Critical interactions between SpCas9 and DNA confer the high specificity of the enzyme in genome engineering. Here, we unveil that an essential SpCas9-DNA interaction located beyond the protospacer adjacent motif (PAM) is realized through electrostatic forces between four positively charged lysines among SpCas9 residues 1151-1156 and the negatively charged DNA backbone. Modulating this interaction by substituting lysines with amino acids that have distinct charges revealed a strong dependence of DNA target binding and cleavage activities of SpCas9 on the charge. Moreover, the SpCas9 mutants show markedly distinguishable DNA interaction sites beyond the PAM compared with wild-type SpCas9. Functionally, this interaction governs DNA sampling and participates in protospacer DNA unwinding during DNA interrogation. Overall, a mechanistic and functional understanding of this vital interaction explains how SpCas9 carries out efficient DNA interrogation.

Comutations in DDR Pathways Predict Atezolizumab Response in Non-Small Cell Lung Cancer Patients.

The presence of comutations (co-mut+) in DNA damage response and repair (DDR) pathways was associated with improved survival for immune checkpoint inhibitor (ICI) therapy in non-small cell lung cancer (NSCLC). However, it remains unknown whether co-mut+ status could be a predictive biomarker for immunotherapy. We aimed to explore the predictive role of co-mut+ status in the efficacy of ICIs. A total of 853 NSCLC patients from OAK and POPLAR trials were included in the analyses for the relationship between co-mut status and clinical outcomes with atezolizumab treatment. In co-mut+ NSCLC patients, significantly prolonged progression-free survival (PFS) (p = 0.004) and overall survival (OS) (p < 0.001) were observed in atezolizumab over docetaxel. The interaction between co-mut status and treatment was significant for PFS (p for interaction = 0.010) and OS (p for interaction = 0.017). In patients with negative or low programmed death receptor-ligand 1 expression, co-mut+ status still predicted improved clinical outcomes from atezolizumab therapy. These findings suggested that co-mut status may be a promising predictor of ICI therapy in NSCLC.

A novel partially open state of SHP2 points to a "multiple gear" regulation mechanism.

The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.

Conformational Dynamics of Nonenveloped Circovirus Capsid to the Host Cell Receptor.

Circovirus, comprising one capsid protein, is the smallest nonenveloped virus and induces lymphopenia. Circovirus can be used to explore the cell adhesion mechanism of nonenveloped viruses. We developed a single-molecule fluorescence resonance energy transfer (smFRET) assay to directly visualize the capsid's conformational feature. The capsid underwent reversible dynamic transformation between three conformations. The cell surface receptor heparan sulfate (HS) altered the dynamic equilibrium of the capsid to the high-FRET state, revealing the HS-binding region. Neutralizing antibodies restricted capsid transition to a low-FRET state, masking the HS-binding domain. The lack of positively charged amino acids in the HS-binding site reduced cell surface affinity and attenuated virus infectivity via conformational changes. These intrinsic characteristics of the capsid suggested that conformational dynamics is critical for the structural changes occurring upon cell surface receptor binding, supporting a dynamics-based mechanism of receptor binding.

Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation.

Staphylococcus aureus Cas9 (SaCas9) is an RNA-guided endonuclease that targets complementary DNA adjacent to a protospacer adjacent motif (PAM) for cleavage. Its small size facilitates in vivo delivery for genome editing in various organisms. Herein, using single-molecule and ensemble approaches, we systemically study the mechanism of SaCas9 underlying its interplay with DNA. We find that the DNA binding and cleavage of SaCas9 require complementarities of 6- and 18-bp of PAM-proximal DNA with guide RNA, respectively. These activities are mediated by two steady interactions among the ternary complex, one of which is located approximately 6 bp from the PAM and beyond the apparent footprint of SaCas9 on DNA. Notably, the other interaction within the protospacer is significantly strong and thus poses DNA-bound SaCas9 a persistent block to DNA-tracking motors. Intriguingly, after cleavage, SaCas9 autonomously releases the PAM-distal DNA while retaining binding to the PAM. This partial DNA release immediately abolishes its strong interaction with the protospacer DNA and consequently promotes its subsequent dissociation from the PAM. Overall, these data provide a dynamic understanding of SaCas9 and instruct its effective applications.

The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation.

Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.