Biomarkers and prognostic factors of PD-1/PD-L1 inhibitor-based therapy in patients with advanced hepatocellular carcinoma.

Systemic therapies using programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) inhibitors have demonstrated commendable efficacy in some patients with advanced hepatocellular carcinoma (HCC); however, other individuals do not respond favorably. Hence, identifying the biomarkers, the prognostic factors, and their underlying mechanisms is crucial. In this review, we summarized the latest advancements in this field. Within the tumor microenvironment, PD-L1 expression is commonly utilized to predict response. Moreover, the characteristics of tumor-infiltrating lymphocytes are associated with the effectiveness of immunotherapy. Preclinical studies have identified stimulatory dendritic cells, conventional dendritic cells, and macrophages as potential biomarkers. The emergence of single-cell sequencing and spatial transcriptomics has provided invaluable insights into tumor heterogeneity through the lens of single-cell profiling and spatial distribution. With the widespread adoption of next-generation sequencing, certain genomic characteristics, including tumor mutational burden, copy number alterations, specific genes (TP53, CTNNB1, and GZMB), and signaling pathways (WNT/ß-catenin) have been found to correlate with prognosis. Furthermore, clinical features such as tumor size, number, and metastasis status have demonstrated prognostic value. Notably, common indicators such as the Child-Pugh score and Eastern Cooperative Oncology Group score, which are used in patients with liver diseases, have shown potential. Similarly, commonly employed laboratory parameters such as baseline transforming growth factor beta, lactate dehydrogenase, dynamic changes in alpha-fetoprotein (AFP) and abnormal prothrombin, CRAFITY score (composed of C-reactive protein and AFP), and immune adverse events have been identified as predictive biomarkers. Novel imaging techniques such as EOB-MRI and PET/CT employing innovative tracers also have potential. Moreover, liquid biopsy has gained widespread use in biomarker studies owing to its non-invasive, convenient, and highly reproducible nature, as well as its dynamic monitoring capabilities. Research on the gut microbiome, including its composition, dynamic changes, and metabolomic analysis, has gained considerable attention. Efficient biomarker discovery relies on continuous updating of treatment strategies. Next, we summarized recent advancements in clinical research on HCC immunotherapy and provided an overview of ongoing clinical trials for contributing to the understanding and improvement of HCC immunotherapy.

Radiation Therapy With Combination Therapy of Immune Checkpoint Inhibitors and Antiangiogenic Therapy for Hepatocellular Carcinoma.

PURPOSE: Immune checkpoint inhibitors (ICIs) combined with antiangiogenic therapy have limited efficacy in treating advanced hepatocellular carcinoma (HCC). The synergistic effect of systemic therapy and radiation therapy (RT) might resolve this problem. We aimed to investigate the effect of RT on the treatment outcomes of ICIs and antiangiogenic combination therapy in patients with advanced-stage HCC. METHODS AND MATERIALS: This retrospective observational study analyzed the medical records of 194 patients with Barcelona Clinic Liver Cancer stage C HCC who were admitted to our center from August 2018 to June 2022 and received ICIs combined with antiangiogenic therapy as the first-line treatment. Patients who were administered RT for tumor thrombus or symptomatic metastases within 8 weeks of the commencement of combination therapy were allocated to the RT group, whereas those who did not receive RT were assigned to the non-radiation therapy (NRT) group. Propensity score matching was used to mitigate selection bias. The primary endpoints were progression-free survival (PFS) and overall survival (OS). The secondary endpoints included objective response rate, disease control rate (DCR), local PFS, out-of-field PFS, and treatment-related adverse events. RESULTS: A total of 76 patients diagnosed with advanced-stage HCC and treated with ICIs and antiangiogenic therapy were included in the study, with 33 patients in the RT group and 43 patients in the non-RT group. After propensity score matching, 29 matched patient pairs were generated. The median follow-up was 15.5 months, and the RT sites were mainly located on the tumor thrombus (55.2%) and extrahepatic metastatic lesions (48.3%). The median PFS was 8.3 months (95% CI, 5.4-11.3) in the RT group and 4.2 months (95% CI, 3.4-5.0) in the NRT group (P < .001). The median OS was not reached in the RT group and was 9.7 months (95% CI, 4.1-15.3) in the NRT group (P = .002). The objective response rate was 75.9% (95% CI, 56.5-89.7) in the RT group and 24.1% (95% CI, 10.3-43.5) in the NRT group (P < .001). The DCR was 100% in the RT group and 75.9% (95% CI, 56.5-89.7) in the NRT group (P = .005). The median local PFS and out-of-field PFS were 13.2 months (95% CI, 6.3-20.1) and 10.8 months (95% CI, 7.0-14.7), respectively. RT was an independent prognostic factor for PFS (hazard ratio = 0.33; 95% CI, 0.17-0.64; P < .001) and OS (hazard ratio = 0.28; 95% CI, 0.11-0.68; P = .005), respectively. The rates of any grade treatment-related adverse events were similar between the 2 groups. CONCLUSIONS: In comparison to the combination of ICIs and antiangiogenic therapy, the inclusion of RT has been observed to improve the DCR and survival outcomes in patients with advanced-stage HCC. The safety profile of this triple therapy was satisfactory.

The MT1 receptor as the target of ramelteon neuroprotection in ischemic stroke.

Stroke is the leading cause of death and disability worldwide. Novel and effective therapies for ischemic stroke are urgently needed. Here, we report that melatonin receptor 1A (MT1) agonist ramelteon is a neuroprotective drug candidate as demonstrated by comprehensive experimental models of ischemic stroke, including a middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia in vivo, organotypic hippocampal slice cultures ex vivo, and cultured neurons in vitro; the neuroprotective effects of ramelteon are diminished in MT1-knockout (KO) mice and MT1-KO cultured neurons. For the first time, we report that the MT1 receptor is significantly depleted in the brain of MCAO mice, and ramelteon treatment significantly recovers the brain MT1 losses in MCAO mice, which is further explained by the Connectivity Map L1000 bioinformatic analysis that shows gene-expression signatures of MCAO mice are negatively connected to melatonin receptor agonist like Ramelteon. We demonstrate that ramelteon improves the cerebral blood flow signals in ischemic stroke that is potentially mediated, at least, partly by mechanisms of activating endothelial nitric oxide synthase. Our results also show that the neuroprotection of ramelteon counteracts reactive oxygen species-induced oxidative stress and activates the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Ramelteon inhibits the mitochondrial and autophagic death pathways in MCAO mice and cultured neurons, consistent with gene set enrichment analysis from a bioinformatics perspective angle. Our data suggest that Ramelteon is a potential neuroprotective drug candidate, and MT1 is the neuroprotective target for ischemic stroke, which provides new insights into stroke therapy. MT1-KO mice and cultured neurons may provide animal and cellular models of accelerated ischemic damage and neuronal cell death.

Efficacy and safety of subsequent radiotherapy in patients with advanced-stage hepatocellular carcinoma treated with immune checkpoint inhibitors.

Background: The development of immunotherapy resistance is associated with a poor prognosis in patients diagnosed with hepatocellular carcinoma (HCC) who are undergoing treatment with immune checkpoint inhibitors (ICI). This study aimed to evaluate the efficacy and safety of subsequent radiotherapy (RT) for patients with advanced-stage HCC who had lesion enlargement or new lesions (NLs) during ICI therapy. Methods: This retrospective observational study enrolled 36 patients with advanced-stage HCC who underwent subsequent RT for lesion enlargement or NLs during ICI therapy from two centers. The primary endpoints were progression-free survival (PFS) and overall survival (OS). The secondary endpoints included objective response rate (ORR), disease control rate (DCR), 1- and 2-year local control (LC) rates, in-field PFS (IFPFS), out-field PFS (OFPFS), and safety. Results: The median follow-up time was 15.3 months. The median PFS was 7.4 months [95% confidence interval (CI): 3.1-11.7 months], and the median OS was 18.8 months (95% CI: 17.1-20.5 months). ORR and DCR were 38.9% and 72.2%, respectively. In addition, the median IFPFS was 17.8 months (95% CI: 11.5-24.2 months), median OFPFS was 7.9 months (95% CI: 3.4-12.5 months), and estimated 1- and 2-year LC rates were 67.1% and 31.9%, respectively. The most common treatment-related adverse events (all grades) were diarrhea (33.3%), rash (30.6%), and malaise (27.8%); a total of 14 (38.9%) patients developed grade 3-4 AEs. Conclusions: Subsequent RT showed reliable antitumor effects and an acceptable safety profile in patients with advanced-stage HCC who had unsatisfactory response to ICI therapy; therefore, it could serve as an optional salvage strategy.

Real-world cohort study of PD-1 blockade plus lenvatinib for advanced intrahepatic cholangiocarcinoma: effectiveness, safety, and biomarker analysis.

BACKGROUND: In clinical practice, some patients with advanced intrahepatic cholangiocarcinoma (ICC) cannot tolerate or refuse chemotherapy due to the toxicity, necessitating alternative treatments. PD-1 blockade combined with lenvatinib showed promising results in phase II studies with small sample size, but there is a lack of data on the routine use with this regimen. This study aimed to evaluate the effectiveness and safety of the regimen in patients with advanced ICC, and to identify predictors for treatment response and prognosis. METHODS: We conducted a retrospective cohort study of patients treated with PD-1 inhibitors plus lenvatinib for advanced ICC between July 2017 and August 2022. The study endpoints were progression-free survival (PFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR), and safety. Biomarker analysis for CA19-9 and PD-L1 expression was performed. Exploratory analysis for genetic alternation was conducted. RESULTS: The study included 103 patients. It demonstrated a median PFS of 5.9 months and a median OS of 11.4 months. ORR was 18.4% and DCR was 80.6%. The incidence of grade 3 or 4 adverse events was 50.5%. Positive PD-L1 expression (TPS ≥ 1%) was associated with higher ORR (P = 0.013) and prolonged PFS (P = 0.023). Elevated CA19-9 (> 37 U/ml) was associated with decreased ORR (P = 0.019), poorer PFS (P = 0.005) and OS (P = 0.034). Patients with IDH1 mutations exhibited a favorable response to the treatment (P = 0.011), and patients with TP53 mutations tended to have worse OS (P = 0.031). CONCLUSIONS: PD-1 blockade plus lenvatinib is effective and safe in routine practice. PD-L1 expression and CA19-9 level appear to predict the treatment efficacy. IDH1 mutations might indicate a better treatment response. CLINICAL TRIAL REGISTRATION: NCT03892577.

The efficacy and safety of bevacizumab as a salvage therapy for patients with advanced hepatocellular carcinoma targeting immune tolerance.

As is well understood that malignant tumour progression requires additional blood vessels to provide the nutrients necessary for growth. Many patients with advanced hepatocellular carcinoma (aHCC) experience disease progression after treatment with lenvatinib (Lenva) and immune checkpoint inhibitors (ICIs). Therefore, we designed a double-arm retrospective study to evaluate the antitumour activity of additional bevacizumab (Beva, an anti-vascular endothelial growth factor-targeting drug) as a means to reduce the blood vessels needed for tumour growth. Compared with the control group, the group that received Beva had prolonged progression-free survival (PFS) and a trend toward a benefit for overall survival duration. This study aimed to evaluate the anticancer effect of Beva in patients with aHCC who experienced tumour progression after treatment with Lenva+ICIs. From April 2021 to March 2023, we retrospectively included 20 patients as the experimental group and 21 patients as the control group. The patients in the experimental group experienced disease progression after receiving targeted therapy and ICIs, after which we added Beva to the treatment. The patients in the control group only received targeted therapy and ICIs. The efficacy endpoints were overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR), which were evaluated according to RECIST v1.1. Adverse events were assessed using NCI-CTCAE v5.0. Ultimately, 20 patients with aHCC in the experimental group of received Beva after disease progression, compared with 21 patients in the control group. The median OS was 12.6 mo (95% CI: 6.8-18.7) vs. 9.3 mo (95% CI: 4.3-14.4), and the median PFS was 6.9 mo (95% CI: 6.4-7.4) vs. 4.1 mo (95% CI: 2.4-5.8). The ORR for all patients was 5%, and the DCR for all patients was 70.0%. The median follow-up time for all patients was 7.5 mo (95% CI: 5.0-10.0). All patients had adverse events, but no fatal adverse events were observed. In conclusion, Bevacizumab is a drug resistant treatment option for patients with advanced hepatocellular carcinoma after Lenva+PD-1/PD-L1 treatment.

Dysregulation and oncogenic activities of ubiquitin specific peptidase 2a in the pathogenesis of hepatocellular carcinoma.

Ubiquitin specific peptidase 2a (USP2a) plays critical roles in protein degradation and other cellular activities. Currently, our understanding on USP2a dysregulation in subjects with hepatocellular carcinoma (HCC) and its roles in HCC pathogenesis is limited. In this study, we found that USP2a mRNA and protein levels were significantly upregulated in HCC tumors from both human and mice. USP2a overexpression in HepG2 and Huh 7 cells significantly increased cell proliferation while inhibition of USP2a activity by chemical inhibitor or stable knockout of USP2 by CRISPR markedly reduced cell proliferation. In addition, USP2a overexpression significantly augmented the resistance while knockout of USP2a markedly increased the susceptibility of HepG2 cells to bile acid-induced apoptosis and necrosis. Consistent with the oncogenic activities detected in vitro, overexpression of USP2a promoted de novo HCC development in mice with significantly increased tumor occurrence rates, tumor sizes and liver/body ratios. Further investigations with unbiased co-immunoprecipitation (Co-IP)-coupled proteomic analysis and Western blot identified novel USP2a target proteins involved in cell proliferation, apoptosis, and tumorigenesis. Analysis of those USP2a target proteins revealed that USP2a's oncogenic activities are mediated through multiple pathways, including modulating protein folding and assembling through regulating protein chaperones/co-chaperones HSPA1A, DNAJA1 and TCP1, promoting DNA replication and transcription through regulating RUVBL1, PCNA and TARDBP, and altering mitochondrial apoptotic pathway through regulating VDAC2. Indeed, those newly identified USP2a target proteins were markedly dysregulated in HCC tumors. In summary, USP2a was upregulated in HCC subjects and acted as an oncogene in the pathogenesis of HCC through multiple downstream pathways. The findings provided molecular and pathogenesis bases for developing interventions to treat HCC by targeting USP2a or its downstream pathways.

Progress in immunotherapy for neuroendocrine neoplasm of the digestive system.

Neuroendocrine neoplasms (NENs) are rare heterogeneous tumors that can develop in almost any organ, with the digestive organs, including the gastrointestinal tract and pancreas being the most commonly affected sites. Despite the fact that advances in initial therapies have progressed, there is presently no recognized effective treatment for advanced NEN. Immune checkpoint inhibitors (ICIs) have shown superior efficacy in treating several types of solid tumors. Despite their successful role in the treatment of partial NENs, such as small cell lung cancer, and Merkel cell carcinoma, the role of ICIs in most of the NENs remains limited. Nevertheless, due to their specific anti-tumor mechanisms and acceptable safety profile, ICIs are a promising avenue for further study in NENs therapy. Recent clinical trials have illustrated that combination therapy with ICI is more efficient than monotherapy, and multiple clinical trials are constantly ongoing to evaluate the efficacy and safety of these combination therapies. Therefore, the purpose of this review is to provide a comprehensive summary of the clinical progress of immunotherapy in NENs affecting the digestive system, with a specific emphasis on the application of programmed cell death protein 1/programmed death receptor ligand 1 inhibitor. Furthermore, this review has an exploration of the potential beneficiary population and the inherent value of utilizing immunotherapy in the management of NENs.

Golden Buckwheat Extract-Loaded Injectable Hydrogel for Efficient Postsurgical Prevention of Local Tumor Recurrence Caused by Residual Tumor Cells.

To prevent local tumor recurrence caused by possible residual cancer cells after surgery, avoid toxicity of systemic chemotherapy and protect the fragile immune system of postsurgical patients, an increasing amount of attention has been paid to local anti-cancer drug delivery systems. In this paper, golden buckwheat was first applied to prevent post-operative tumor recurrence, which is a Chinese herb and possesses anti-tumor activity. Golden buckwheat extract-loaded gellan gum injectable hydrogels were fabricated via Ca2+ crosslinking for localized chemotherapy. Blank and/or drug-loaded hydrogels were characterized via FT-IR, TG, SEM, density functional theory, drug release and rheology studies to explore the interaction among gellan gum, Ca2+ and golden buckwheat extract (GBE). Blank hydrogels were non-toxic to NIH3T3 cells. Of significance, GBE and GBE-loaded hydrogel inhibited the proliferation of tumor cells (up to 90% inhibition rate in HepG2 cells). In vitro hemolysis assay showed that blank hydrogel and GBE-loaded hydrogel had good blood compatibility. When GBE-loaded hydrogel was applied to the incompletely resected tumor of mice bearing B16 tumor xenografts, it showed inhibition of tumor growth in vivo and induced the apoptosis of tumor cells. Taken together, gellan gum injectable hydrogel containing GBE is a potential local anticancer drug delivery system for the prevention of postsurgical tumor recurrence.

Near-Infrared Light-Driven Shape-Programmable Hydrogel Actuators Loaded with Metal-Organic Frameworks.

Shape-programmable hydrogel-based soft actuators that can adaptively respond to external stimuli are of paramount significance for the development of bioinspired aquatic smart soft robots. Herein, we report the design and synthesis of near-infrared (NIR) light-driven hydrogel actuators through in situ photopolymerization of poly(N-isopropylacrylamide) (PNIPAM) hydrogels loaded with metal-organic frameworks (MOFs) onto the surface of the poly(dimethylsiloxane) (PDMS) thin film. The MOFs can not only function as an excellent photothermal nanotransducer but also accelerate the adsorption/desorption of water due to their porous nanostructure, which speeds up the response rate of the actuators. Shape-programmable hydrogel actuators are fabricated by tailoring the patterning of PDMS thin film, and thus different shape-morphing modes such as directional bending and chiral twisting are observed under the NIR light irradiations. As the proof-of-concept demonstrations, an artificial hand, biomimetic mimosa, and flower are conceptualized with light-driven MOF-containing hydrogel actuators. Interestingly, we are able to achieve an octopus-inspired light-driven soft swimmer upon cyclic NIR illumination due to the fast photoresponsiveness of as-prepared hydrogel actuators. This work can offer insights for fabricating programmable and reconfigurable smart aquatic soft actuators, thus shining a light into their potential applications in emerging fields including soft robots, biomedical devices, and beyond.

Dysregulation and activities of ubiquitin specific peptidase 2b in the pathogenesis of hepatocellular carcinoma.

Ubiquitin specific peptidase-2 (USP2) plays important roles in a myriad of cellular activities through deubiquitinating target proteins and its implications in various diseases, especially cancers, are starting to emerge. Our current understanding on USP2 expression in subjects with hepatocellular carcinoma (HCC) and its roles in the pathogenesis of HCC is limited. In this study, we found that USP2 protein and mRNA levels were significantly dysregulated in HCC tumor (HCC-T) when compared to adjacent non-tumor (HCC-NT) or normal liver tissues from both human and mouse HCC model. Among the USP2 isoforms, USP2b was the predominant isoform in the normal liver and markedly down-regulated in HCC-T tissues in both human and mice. Data from overexpression, chemical inhibition and knockout studies consistently demonstrated that USP2b promoted cell proliferation, colony formation and wound healing in HepG2 and Huh 7 cells. On the other hand, USP2b exhibited proapoptotic and pronecrtotic activities through enhancing bile acid-induced apoptosis and necrosis in both HepG2 and Huh 7 cells. Unbiased proteomic analysis of USP2-knockout (KO) and parental HepG2 cells resulted in identification of USP2-regulated downstream target proteins involved in cell proliferation, apoptosis, and tumorigenesis, including serine/threonine kinase 4 (STK4), epidermal growth factor receptor (EGFR), dipeptidyl peptidase 4 (DPP4) and fatty acid binding protein 1 (FABP1). In conclusion, USP2b expression was dysregulated in subjects with HCC and contributed to the pathogenesis of HCC by promoting cell proliferation and exerting proapoptotic and pronecrotic activities. The findings provide the molecular basis for developing therapies for HCC through modulating USP2b expression or activities.

Reward Design for Customer Referral Programs: Reward-Product Congruence Effect and Gender Difference.

Referral reward design is the core component of customer referral programs, which are often applied to recruit new customers. This research investigates the effectiveness of utilitarian vs. hedonic rewards in terms of referral generation. Through one field study and two laboratory studies, we demonstrate a reward-product congruency effect; that is, utilitarian rewards, compared with hedonic rewards, yield a higher referral likelihood for utilitarian products, while the opposite holds true for hedonic products. However, such a congruency effect would be crippled by gender segmentation. When males make referral decisions toward hedonic products, the effectiveness of utilitarian rewards is at least equal to that of hedonic rewards. When females make referral decisions toward utilitarian products, there is no difference in effectiveness between utilitarian and hedonic rewards. These findings provide novel insights into referral reward design.

Near-Infrared Light-Driven Shape-Morphing of Programmable Anisotropic Hydrogels Enabled by MXene Nanosheets.

Herein, we report near-infrared (NIR) light-driven shape-morphing of programmable MXene-containing anisotropic hydrogel actuators that are fabricated through in situ free-radical copolymerization of a judiciously designed MXene nanomonomer with thermosensitive hydrogel network. A low electric field (few V mm-1 ) was found to enable a spatial distribution of MXene nanosheets and hence introduce anisotropy into the hydrogel network. Programmable anisotropic hydrogel actuators were developed by controlling ITO electrode pattern, direct-current (DC) electric field direction and mask-assisted photopolymerization. As a proof-of-concept, we demonstrate NIR light-driven shape morphing of the MXene-containing anisotropic hydrogel into various shapes and devise a four-arm soft gripper that can perform distinct photomechanical functions such as grasping, lifting/lowering down and releasing an object upon sequential NIR light exposure.

The Significance of 8-oxoGsn in Aging-Related Diseases.

Aging is a common risk factor for the occurrence and development of many diseases, such as Parkinson's disease, Alzheimer's disease, diabetes, hypertension, atherosclerosis and coronary heart disease, and cancer, among others, and is a key problem threatening the health and life expectancy of the elderly. Oxidative damage is an important mechanism involved in aging. The latest discovery pertaining to oxidative damage is that 8-oxoGsn (8-oxo-7,8-dihydroguanosine), an oxidative damage product of RNA, can represent the level of oxidative stress. The significance of RNA oxidative damage to aging has not been fully explained, but the relationship between the accumulation of 8-oxoGsn, a marker of RNA oxidative damage, and the occurrence of diseases has been confirmed in many aging-related diseases. Studying the aging mechanism, monitoring the aging level of the body and exploring the corresponding countermeasures are of great significance for achieving healthy aging and promoting public health and social development. This article reviews the progress of research on 8-oxoGsn in aging-related diseases.

Expression of p42.3 in non-small cell lung cancer.

BACKGROUND: Lung cancer is the most malignant tumor with the highest morbidity and mortality. This study aimed to investigate the role of the expression and the significance of the p42.3 gene in non-small cell lung cancer (NSCLC). METHODS: Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) were analyzed based on the biological information data of The Cancer Genome Atlas (TCGA). Furthermore, 142 postoperative tumor tissue and normal tissue samples (70 cases of LUAD and 72 cases of LUSC) from NSCLC patients admitted to our hospital from 2005 to 2009 were retrospectively collected. Paraffin-embedded tissues were used to make the tissue microarrays (TMA), and the expression of the p42.3 protein was detected by immunohistochemical staining. RESULTS: The expression of p42.3 in both LUAD and LUSC was significantly upregulated (P<0.01) compared with the normal lung tissues. The p42.3 expression was significantly higher than that of LUAD (P<0.01) in the LUSC group. LUSC had a lower level of p42.3 DNA methylation and a higher level of p42.3 DNA amplification than LUAD. The expression rate of p42.3 protein decreased in patients 70 years or older (P=0.029). High expression of the p42.3 protein was an independent factor for worse pathological differentiation (P=0.043). CONCLUSIONS: Both genetic and epigenetic alterations contributed to dysregulated p42.3 in NSCLC. Despite the temporary absence of TCGA-LUSC (TCGA data on LUSC) survival information, we observed that the up-regulated expression of p42.3 in LUSC was significantly higher than that in LUAD by analyzing the public database and reviewing the real-world data. Furthermore, a high expression of p42.3 protein was significantly correlated with poor differentiation of tumor tissues. Therefore, the prognostic value of p42.3 in LUSC deserves further study.