Spatial and temporal heterogeneity of tumor immune microenvironment between primary tumor and brain metastases in NSCLC.

BACKGROUND: Brain metastasis is a common outcome in non-small cell lung cancer, and despite aggressive treatment, its clinical outcome is still frustrating. In recent years, immunotherapy has been developing rapidly, however, its therapeutic outcomes for primary lung cancer and brain metastases are not the same, suggesting that there may be differences in the immune microenvironment of primary lung cancer and brain metastases, however, we currently know little about these differences. METHODS: Seventeen paired samples of NSCLC and their brain metastases and 45 other unpaired brain metastases samples were collected for the current study. Immunohistochemical staining was performed on all samples for the following markers: immune checkpoints CTLA-4, PD-1, PD-L1, B7-H3, B7-H4, IDO1, and EphA2; tumor-infiltrating lymphocytes (TILs) CD3, CD4, CD8, and CD20; tumor-associated microglia/macrophages (TAMs) CD68 and CD163; and tumor proliferation index Ki-67. The differences in expression of these markers were compared in 17 paired samples, and the effect of the expression level of these markers on the prognosis of patients was analyzed in lung adenocarcinoma brain metastases samples. Subsequently, multiplex immunofluorescence staining was performed in a typical lung-brain paired sample based on the aforementioned results. The multiplex immunofluorescence staining results revealed the difference in tumor immune microenvironment between primary NSCLC and brain metastases. RESULTS: In 17 paired lesions, the infiltration of CTLA-4+ (P = 0.461), PD-1+ (P = 0.106), CD3+ (P = 0.045), CD4+ (P = 0.037), CD8+ (P = 0.008), and CD20+ (P = 0.029) TILs in brain metastases were significantly decreased compared with primary tumors. No statistically significant difference was observed in the CD68 (P = 0.954) and CD163 (P = 0.654) TAM infiltration between primary NSCLC and paired brain metastases. In all the brain metastases lesions, the expression of PD-L1 is related to the time interval of brain metastases in NSCLC. In addition, the Cox proportional hazards regression models showed high expression of B7-H4 (hazard ratio [HR] = 3.276, 95% confidence interval [CI] 1.335-8.041, P = 0.010) and CD68 TAM infiltration (HR = 3.775, 95% CI 1.419-10.044, P = 0.008) were independent prognosis factors for lung adenocarcinoma brain metastases patients. CONCLUSIONS: Both temporal and spatial heterogeneity is present between the primary tumor and brain metastases of NCSLC. Brain metastases lesions exhibit a more immunosuppressive tumor immune microenvironment. B7-H4 and CD68+ TAMs may have potential therapeutic value for lung adenocarcinoma brain metastases patients.

Primary intracranial sarcomas: a clinicopathological investigation.

Background: The purpose of this study is to present a series of primary intracranial sarcomas (PIS), a rare type of tumor of the central nervous system, in order to improve our understanding of the disease. These tumors are heterogeneous and prone to recurrence after resection, exhibiting a high mortality rate. As PIS has yet to be understood and studied on a large scale, it is vital for further evaluation and research. Methods: Our study included 14 cases of PIS. The patients' clinical, pathological, and imaging features were retrospectively analyzed. Additionally, targeted DNA next-generation sequencing (NGS) was applied for the 481-gene panel to detect gene mutations. Results: The average age for PIS patients was 31.4 years. Headache (7, 50.0%) was the most common symptom leading to the hospital visit. Twelve cases had PIS located in the supratentorial area and two in the cerebellopontine angle region. The maximum tumor diameter ranged from 19.0 mm to 130.0 mm, with an average diameter of 50.3 mm. Pathological types of tumors were heterogeneous, with chondrosarcoma being the most common, followed by fibrosarcoma. Eight of the 10 PIS cases that underwent MRI scanning showed gadolinium enhancement; 7 of these cases were heterogeneous, and 1 of them was garland-like. Targeted sequencing was performed in two cases and identified mutations in genes such as NRAS, PIK3CA, BAP1, KDR, BLM, PBRM1, TOP2A, DUSP2, and CNV deletions of SMARCB1. Additionally, the SH3BP5::RAF1 fusion gene was also detected. Of the 14 patients, 9 underwent a gross total resection (GTR), and 5 chose subtotal resection. Patients who underwent GTR displayed a trend toward superior survival. Among the 11 patients with available follow-up information, one had developed lung metastases, three had died, and eight were alive. Conclusion: PIS is extremely rare compared to extracranial soft sarcomas. The most common histological type of intracranial sarcoma (IS) is chondrosarcoma. Patients who underwent GTR of these lesions showed improved survival rates. Recent advancements in NGS aided in the identification of diagnostic and therapeutic PIS-relevant targets.

ZDHHC15 promotes glioma malignancy and acts as a novel prognostic biomarker for patients with glioma.

BACKGROUND: Glioma is the most common and aggressive tumor in the adult brain. Recent studies have indicated that Zinc finger DHHC-type palmitoyltransferases (ZDHHCs) play vital roles in regulating the progression of glioma. ZDHHC15, a member of the ZDHHCs family, participates in various physiological activities in the brain. However, the biological functions and related mechanisms of ZDHHC15 in glioma remain poorly understood. METHODS: Data from multiple glioma-associated datasets were used to investigate the expression profiles and potential biological functions of ZDHHC15 in glioma. Expression of ZDHHC15 and its association with clinicopathological characteristics in glioma were validated by quantitative reverse transcription PCR (RT-qPCR) and immunohistochemical experiments. GO enrichment analysis, KEGG analysis, GSEA analysis, CCK-8, EdU, transwell, and western blotting assays were performed to confirm the functions and mechanism of ZDHHC15 in glioma. Moreover, we performed Kaplan-Meier analysis and Cox progression analysis to explore the prognostic significance of ZDHHC15 in glioma patients. RESULTS: ZDHHC15 expression was significantly up-regulated in glioma and positively associated with malignant phenotypes. Results from the GO and KEGG enrichment analysis revealed that ZDHHC15 was involved in regulating cell cycle and migration. Knockdown of ZDHHC15 inhibited glioma cell proliferation and migration, while overexpression of ZDHHC15 presented opposite effects on glioma cells. Besides, results from GSEA analysis suggested that ZDHHC15 was enriched in STAT3 signaling pathway. Knockdown or overexpression of ZDHHC15 indeed affected the activation of STAT3 signaling pathway. Additionally, we identified ZDHHC15 as an independent prognostic biomarker in glioma, and higher expression of ZDHHC15 predicted a poorer prognosis in glioma patients. CONCLUSION: Our findings suggest that ZDHHC15 promotes glioma malignancy and can serve as a novel prognostic biomarker for glioma patients. Targeting ZDHHC15 may be a promising therapeutic strategy for glioma.

First report of Fusarium oxysporum Causing Fusarium Dieback on Archidendron clypearia in China.

Archidendron clypearia (Jack) Benth. (common name: Monkey-pod) is a perennial arbor tree belonging to the family Fabaceae. It is used to produce a traditional medicine to treat a variety of heat toxicity symptoms in China. The distribution of A. clypearia mainly include Guangdong, Fujian, and Zhejiang provinces in China as well as other countries in Southeast Asia. In 2018, about 30 Monkey-pod trees were observed showing typical dieback symptoms in a commercial plantation (114°36'09.401″E, 22°58'38.553″N) of Huizhou, Guangdong Province, China. In addition, white cylindrical sawdust exudates from exit holes of ambrosia beetles were observed on the trunks and main branches of the diseased trees. Discoloration was seen around the exit holes of the beetles by splitting the wood. Samples of symptomatic and asymptomatic wood tissues of branches, trunks, and roots were collected from 11 trees. To identify the pathogen, the samples were disinfected with a sodium hypochlorite solution containing 50 g/l chlorine for 60 s, rinsed three times with sterile distilled water, and placed on potato dextrose agar (PDA) for culture at 25℃ for 14 days. Beetles from exit holes of the pathogen-infected wood tissues were also collected and identified as Xyleborus affinis by their cytochrome c oxidase I (COI) sequences (GenBank accession numbers: MT921005 and MT921006) as well as morphological features. After two weeks incubation, 37 single-spore isolates were obtained (31 from galleries of branches and trunks, and 6 from mycangia and the body surface of beetles). Colonies on PDA were fusarium-like, pale orange, floccose with abundant aerial mycelia, and growing at the rate of 3.0 to 4.5 mm/d at 25℃. Conidiophores from the aerial mycelia often had single phialides. Microconidia were hyaline, 0-1 septate, (4-)6-12(-16) × 2-4 µm (n=100), Macroconidia were hyaline, ellipsoidal to falcate, 2-4 septate, (10-)25-45(-50) × 3-6 µm (n=100), with basal foot cells shaped to pointed and apical cells tapered and curved. Two representative isolates (CYML 367, 368) were used for molecular identification and pathogenicity tests. The internal transcriptional spacer region (ITS), ß-tubulin (TUB), and translation elongation factor-1α (TEF 1-α) regions were amplified with primers ITS1F/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1-728F (Carbone and Kohn 1999) /EF-2 (O'Donnell et al.1998), respectively. DNA extraction and PCR conditions were followed the methods described by Yin et al. (2019). The amplified fragments were sequenced. The results of BLAST demonstrated that the fragment sequences of ITS (468 bp), TEF 1-α (616 bp), and TUB (295 bp) of isolates CYML367 and CYML368 had 100% similarity to correspondence sequences from the Fusarium oxysporum Schlecht. emend. Snyder & Hansen species complex (GenBank accession numbers MT032694, JF740777, and MN451171, respectively). Sequences generated from this study were deposited into GenBank under accession numbers MN826824 and MN826825 (ITS), MN839680 and MN839681 (TUB), and MN839682 and MN839683 (TEF 1-α). Pathogenicity tests were conducted on 2-year-old seedlings (60-70 cm height, 1.0-1.2 cm in diameter) of A. clypearia with the representative isolates. Ten seedlings were inoculated with each isolate. The stem surface was disinfected with 75% ethanol for 30 s, and rinsed with sterilized water, wounded by removing part of the phloem and xylem, and placed with a mycelial plug (5 mm diameter) from the margin of a 7-day-old PDA plate with mycelia facing the cambium. The inoculated wounds were wrapped with a medical sterile gauze to prevent desiccation and contamination. Control plants were inoculated with non-colonized PDA plugs. These inoculated and control plants were incubated in a chamber at 25℃, 50% humidity, 12 h light/12 h dark cycle. After 3 weeks, all inoculated plants displayed Fusarium dieback symptoms similar to those observed on the original diseased trees in the plantation. The average lesion lengths (1.2/1.7 cm) caused by two isolates were all significantly longer than the wounds in the negative controls (P<0.05). The same fungus was re-isolated from the symptomatic plants, thus fulfilling the Koch's postulates. Further studies should be conducted on the potential vector association, transmission route, and management strategies of this pathogen on A. clypearia in growing regions of China. To our best knowledge, this is the first report of F. oxysporum causing dieback on A. clypearia in China.