Noninvasive Targeting System with Three-Dimensionally Printed Customized Device in Stereotactic Brain Biopsy.

OBJECTIVE: Three-dimensional (3D) printed models are used in the medical field. This study aimed to evaluate the feasibility and safety of a 3D-printed guide plate for use in brain biopsy. METHODS: Twelve patients with intracranial lesions were retrospectively reviewed to determine clinical outcomes and technical procedural operability. These patients underwent brain biopsy assisted with the 3D-printed guide plate. Postoperative computed tomography was performed to assess the accuracy and associated complications of this guide plate. RESULTS: All patients received definite diagnoses assisted by this guide plate. The deviations of the entry and target points were 3.93 ± 0.96 mm and 2.59 ± 0.11 mm, respectively. The angle drift of the puncture path was 5.12° ± 0.14°, and the deviation of the puncture depth was 2.35 ± 1.13 mm. The operation time ranged from 38.5 minutes with local anesthesia to 76.2 minutes with general anesthesia. No patient experienced complications. CONCLUSIONS: The 3D-printed guide plate was noninvasive and had acceptable accuracy and the flexibility of frameless systems. The economic and operative benefits of this device supported its status as a powerful tool for brain biopsy in medical facilities in economically disadvantaged areas or institutions without navigation systems.

Three-Field Versus Two-Field Lymphadenectomy in Minimally Invasive Esophagectomy: 3-Year Survival Outcomes of a Randomized Trial.

BACKGROUND: Minimally invasive esophagectomy (MIE) has been used widely for the treatment of esophageal cancer. However, the optimal extent of lymphadenectomy for esophagectomy in MIE remains unclear. This trial aimed to investigate the 3-year survival and recurrence outcomes in a randomized controlled trial comparing MIE with either three-field lymphadenectomy (3-FL) or two-field lymphadenectomy (2-FL). METHODS: Between June 2016 and May 2019, 76 patients with resectable thoracic esophageal cancer were enrolled in a single-center randomized controlled trial and randomly assigned to MIE that included either 3-FL or 2-FL at a 1:1 ratio (n = 38 patients each). The survival outcomes and recurrence patterns were compared between the two groups. RESULTS: The 3-year cumulative overall survival (OS) probability was 68.2 % (95 % confidence interval [CI], 52.72-83.68 %) for the 3-FL group and 68.6 % (95 % CI, 53.12-84.08 %) for the 2-FL group. The 3-year cumulative probability of disease-free survival (DFS) was 66.3 % (95 % CI, 50.03-82.57 %) for the 3-FL group and 67.1 % (95 % CI, 51.03-83.17 %) for the 2-FL group.. The OS and DFS differences in the two groups were comparable. The overall recurrence rate did not differ significantly between the two groups (P = 0.737). The incidence of cervical lymphatic recurrence in the 2-FL group was higher than in the 3-FL group (P = 0.051). CONCLUSIONS: Compared with 2-FL in MIE, 3-FL tended to prevent cervical lymphatic recurrence. However, it was not found to add survival benefit for the patients with thoracic esophageal cancer.

Combinatorial Application of Papain and CD66B for Isolating Glioma- Associated Neutrophils.

BACKGROUND: Stromal cells in the tumor microenvironment play crucial roles in glioma development. Current methods for isolating tumor-associated stromal cells (such as neutrophils) are inefficient due to the conflict between tissue dissociation and cell surface protein protection, which hampers the research on patient-derived stromal cells. Our study aims to establish a novel method for isolating glioma-associated neutrophils (GANs). METHODS: To observe neutrophil-like polymorphonuclear cells, we performed Hematoxylin-Eosin staining on glioma tissues. For isolating single cells from glioma tissues, we evaluated the efficiency of tissue dissociation with FastPrep Grinder-mediated homogenization or proteases (trypsin or papain) digestion. To definite specific markers of GANs, fluorescence-activated cell sorting (FACS) and immunofluorescence staining were performed. FACS and Ficoll were performed for the separation of neutrophils from glioma tissue-derived single-cell or whole blood pool. To identify the isolated neutrophils, FACS and RT-PCR were carried out. RESULTS: Neutrophil-like cells were abundant in high-grade glioma tissues. Among the three tissue dissociation methods, papain digestion produced a 5.1-fold and 1.7-fold more living cells from glioma mass than physical trituration and trypsin digestion, respectively, and it preserved over 97% of neutrophil surface protein markers. CD66B could be adopted as a unique neutrophil surface protein marker for FACS sorting in glioma. Glioma-derived CD66B+ cells specifically expressed neutrophil marker genes. CONCLUSION: A combination of papain-mediated tissue dissociation and CD66B-mediated FACS sorting is an effective novel method for the isolation of GANs from glioma tissues.

Genomic Instability in Cerebrospinal Fluid Cell-Free DNA Predicts Poor Prognosis in Solid Tumor Patients with Meningeal Metastasis.

Genomic instability (GI), which leads to the accumulation of DNA loss, gain, and rearrangement, is a hallmark of many cancers such as lung cancer, breast cancer, and colon cancer. However, the clinical significance of GI has not been systematically studied in the meningeal metastasis (MM) of solid tumors. Here, we collected both cerebrospinal fluid (CSF) and plasma samples from 56 solid tumor MM patients and isolated cell-free ctDNA to investigate the GI status using a next-generation sequencing-based comprehensive genomic profiling of 543 cancer-related genes. According to the unfiltered heterozygous mutation data-derived GI score, we found that 37 (66.1%) cases of CSF and 3 cases (6%) of plasma had a high GI status, which was further validated by low-depth whole-genome sequencing analysis. It is demonstrated that a high GI status in CSF was associated with poor prognosis, high intracranial pressure, and low Karnofsky performance status scores. More notably, a high GI status was an independent poor prognostic factor of poor MM-free survival and overall survival in lung adenocarcinoma MM patients. Furthermore, high occurrences of the co-mutation of TP53/EGFR, TP53/RB1, TP53/ERBB2, and TP53/KMT2C were found in MM patients with a high GI status. In summary, the GI status in CSF ctDNA might be a valuable prognostic indicator in solid tumor patients with MM.

The Presence of Genomic Instability in Cerebrospinal Fluid in Patients with Meningeal Metastasis.

PURPOSE: This study aimed to explore the genomic instability in cerebrospinal fluid (CSF) in patients with meningeal metastasis (MM). MATERIAL AND METHODS: We collected the blood and CSF samples of 15 MM patients and one brain parenchymal metastasis (BPM) patient. A panel of 543 cancer-related genes was conducted to analyze the status of genomic instability in CSF and plasma cell-free DNA (cfDNA) of all patients. Subsequently, nine patients underwent low-depth whole-genome sequencing (WGS) analysis to verify the existence of genomic instability, followed by genomic scoring by the application of aneuploidy scores. Diagnosis-specific graded prognostic assessment (DS-GPA) score was utilized to assess the clinical status of MM patients. RESULTS: There was significant difference in gene mutation between CSF cfDNA and plasma cfDNA in MM patients. Among them, 12 MM patients developed genomic instability in their CSF cfDNA, while the remaining 3 had stable genetic profile. Besides, BPM patients showed genomic stability in his CSF and paraffin-embedded tissue sections. No genomic instability was noticed in plasma cfDNA of all patients. Sensitive mutations on EGFR, ERBB2, ALK and KRAS genes and increased gene copy numbers of MET and ERBB2 were detected in 10 MM patients with genomic instability, as well as the EGFR gene mutation in one MM case with genomic stability. Additionally, MM patients with genomic instability had lower overall survival and higher aneuploidy scores and tumor mutation burden compared with those with genomic stability. Moreover, MM patients with higher DS-GPA scores benefited from better survival. CONCLUSION: Genomic instability existed in the CSF cfDNA rather than plasma cfDNA of MM patients, which might be the underlying cause of the differences in MM.

Alectinib Together with Intracranial Therapies Improved Survival Outcomes in Untreated ALK-Positive Patients with Non-Small-Cell Lung Cancer and Symptomatic and Synchronic Brain Metastases: A Retrospective Study.

PURPOSE: The performance of alectinib and crizotinib in untreated anaplastic lymphoma kinase (ALK)-positive patients with non-small-cell lung cancer (NSCLC) and symptomatic and synchronic brain metastases is largely unknown. This retrospective study assessed the effectiveness of alectinib and crizotinib, together with intracranial therapies in a cohort of these patients. PATIENTS AND METHODS: This study included 34 previously untreated ALK-positive NSCLC patients with three or fewer intracranial metastases. Of these patients, 13 received oral alectinib 600 mg twice daily, and 21 received oral crizotinib 250 mg twice daily, until progressive disease, unacceptable toxicity, or death. All intracranial metastases were treated with craniotomy, CyberKnife, or both. RESULTS: Median overall progression-free survival (PFS) was 32.8 months (95% CI 24.4-41.2 months) in patients treated with alectinib and 8.0 months (95% CI 7.3-8.7 months) in patients treated with crizotinib. Median PFS of brain lesions was not yet reached with alectinib (95% CI 30.1 months-not estimated) and was 8.5 months (95% CI 7.2-12.3 months) with crizotinib. Median PFS of lung lesions was 38.5 months (95% CI 27.5-49.5 months) with alectinib and 9.2 months (95% CI 7.4-11.0 months) with crizotinib. Median overall survival was not yet reached with alectinib (95% CI 31.0 months-not estimated) and 30.3 months (95% CI 27.3-37.1 months) with crizotinib. CONCLUSION: Compared with crizotinib, alectinib showed superior efficacy and lower toxicity in the treatment of ALK-positive patients with NSCLC and symptomatic and synchronic brain metastases. The inclusion of intracranial therapies such as craniotomy or CyberKnife further improved the brain PFS and overall survival of these patients.

Immune response pathways enriched in breast cancer samples with brain metastasis.

BACKGROUND: Breast cancer (BC) is the most common form of cancer in women. BC brain metastasis (BM) is associated with poor prognosis, especially for Triple negative breast cancer (TNBC). However, the driver genes of this clinical characteristic are poorly understood. METHODS: This study conducted a transcriptome-wide analysis of gene expression levels in BCBM samples from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. Clinical data and gene expression matrix of TNBC samples were collected. Differential analysis and functional enrichment of metastasis vs. non metastasis data samples were conducted. Genes associated with overall survival and BM event was scanned. RESULTS: Up-regulation in 120 genes and down-regulation in 56 genes were found in TNBC metastasis data. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) clustering using expression alternated genes showed unique immune-gene enrichment in BM samples. Immune response category GO:000695 was found as the most significant term associated with metastasis event. KEGG pathways including cytokine pathways and Primary immunodeficiency were significantly changed in metastasis samples. ESR1 and FYB2 genes expression changes were found to be linked to survival or BM events. CONCLUSIONS: Our results suggest that data-mining on the immune microenvironment of BM might be useful in future study.

lnc-REG3G-3-1/miR-215-3p Promotes Brain Metastasis of Lung Adenocarcinoma by Regulating Leptin and SLC2A5.

This study aims to explore the role and mechanism of specific lncRNA in brain metastasis (BM) from lung adenocarcinoma (LADC), providing an effective biomarker for early diagnosis and targeted therapy of BM from LADC. Based on the gene expression profiles of lncRNA and mRNA in LADC and BM tissues detected by Gene Chip, lnc-REG3G-3-1 was selected, and the related genes, including miR-215-3p, leptin, and SLC2A5, were identified by data analysis. Human LADC cell lines A549 and H1299 were cultured. Dual-luciferase and endogenous validation experiments were used to confirm the regulation between these genes. Real-time quantitative reverse transcription-polymerase chain reaction and Western blotting were used to detect gene expression. The tumor metastasis-related gene function of lnc-REG3G-3-1 and miR-215-3p in H1299 cells was verified by Transwell invasion, migration assays, and scratch testing. Nude mice xenograft tumors constructed with decreased lnc-REG3G-3-1 confirmed the influences on gene expression in vivo. lnc-REG3G-3-1 was highly expressed in BM tissues that originated from LADC compared with that in primary cancer tissues. lnc-REG3G-3-1 reduced miR-215-3p expression, thereby regulating the target genes leptin and SLC2A5 and the signaling pathways, taking part in the lnc-REG3G-3-1/miR-215-3p axis in the process of BM from LADC. lnc-REG3G-3-1, leptin, and SLC2A5 through regulating signaling pathways may be jointly involved in the regulation of the biological process of BM in patients with LADC.

TNFα inhibitor C87 sensitizes EGFRvIII transfected glioblastoma cells to gefitinib by a concurrent blockade of TNFα signaling.

OBJECTIVE: More than half of human glioblastomas show EGFR gene amplification and mutation, but EGFR inhibitors have not been effective in treating EGFR-positive glioblastoma patients. The mechanism behind this type of primary resistance is not well understood. The aim of this study was to investigate gefitinib resistance in glioblastoma, and explore ways to circumvent this significant clinical problem. METHODS: MTT method was used to test the cell viability after EGFR-positive glioblastoma cells were treated with indicated drugs; real-time quantitative PCR method was included to detect the TNFα mRNA levels in glioma tissues and cell lines. ELISA was introduced to measure the TNFα protein levels in cell culture supernatant of glioblastoma cells treated with gefitinib. Western blot was used to detect the activity change of intracellular kinases in drug-treated glioblastoma cells. Two mouse xenograft tumor models were carried out to evaluate the in vivo effects of a combination of EGFR and TNFα inhibitors. RESULTS: We found that glioblastoma resistance to gefitinib may be mediated by an adaptive pro-survival TNFα-JNK-Axl signaling axis, and that high TNFα levels in the glioblastoma microenvironment may further intensify primary resistance. A combination of the TNFα-specific small-molecule inhibitor C87 and gefitinib significantly enhanced the sensitivity of glioblastoma cells to gefitinib in vitro and in vivo. CONCLUSIONS: Our findings provide a possible explanation for the primary resistance of glioblastoma to EGFR inhibitors and suggest that dual blockade of TNFα and EGFR may be a viable therapeutic strategy for the treatment of patients with chemotherapy-refractory advanced glioblastoma.

KDELR2 Promotes Glioblastoma Tumorigenesis Targeted by HIF1a via mTOR Signaling Pathway.

The KDEL (Lys-Asp-Glu-Leu) receptors (KDELRs), proteins with seven transmembrane domains, are primarily responsible for endoplasmic reticulum (ER) homeostasis. Recent studies have found additional function of KDELRs in growth, cellular secretory traffic, immune response, and autophagy; however, its role in tumorigenesis is still poorly understood. Here, we showed that KDELR2 is highly expressed in glioblastoma (GBM) tissues. Reviewing the expression of KDELR2 in TCGA and REMBRANDT database, we found that higher expression of KDELR2 is associated with shorter survival of GBM patients. We explored the effect of KDELR2 on tumorigenesis in GBM cells and animal model (nude mice), and identified KDELR2 as oncogene promoting cell proliferation. Additionally, KDELR2 expression in GBM cells correlated positively with HIF1alpha (HIF1α) expression, and we demonstrated by ChIP-qPCR and luciferase reporter assay that the upstream region of the KDELR2 gene is directly targeted by HIF1alpha. Taken together, our data suggest that KDELR2 is a target gene downstream of HIF1-alpha driving the malignancy of GBM and could eventually serve as a therapeutic target for the treatment of GBM patients.

Prognostic factors and outcome of surgically treated patients with brain metastases of non-small cell lung cancer.

BACKGROUND: Brain metastases (BM) are a common consequence of lung cancer and surgery is effective; however, the factors affecting survival after surgery are unclear. The aim of this study was to identify the outcomes and prognoses of post-metastasectomy patients with BM from non-small cell lung cancer (NSCLC) at a single institution over a 15-year period. METHODS: NSCLC patients who had undergone BM surgery were retrospectively identified. Survival was analyzed using the Kaplan-Meier curve, and univariate and multivariate factors associated with survival were identified using the Cox proportional hazards model. RESULTS: The median overall survival was 9.8 months, 18 (14.8%) patients survived > 24 months, and 6 (4.9%) > 36 months. The one and two-year survival rates were 41% and 18.6%, respectively. Univariate analysis revealed that recursive partitioning analysis (RPA) classification, Karnofsky Performance Scale (KPS) scores, BM number, extracranial metastasis status, different lesion locations, resection extent, postoperative treatment, and salvage therapy after recurrence significantly influenced patient survival. The different treatment modalities for primary lesions also affected postoperative survival. KPS ≥ 70, RPA class I/II, and postoperative chemotherapy were independent factors that decreased the risk of death from BM. Interestingly, the initial onset of intracranial lesions could increase the risk of death from BM. CONCLUSION: A KPS score ≥ 70, RPA class I/II, and postoperative chemotherapy could benefit post-metastasectomy patients with BM from NSCLC. Conversely, the initial onset of intracranial lesions is an unfavorable factor that increases the risk of death. These findings support the use of personalized therapy for patients with BM from NSCLC.

[Clinical features and prognostic factors of brain metastasis from colorectal cancer].

OBJECTIVE: The aim of this study was to analyze the clinical features and prognostic factors in patients with brain metastasis from colorectal cancer (CRC). METHODS: Clinical materials of 45 colorectal cancer patients who developed brain metastasis were collected, and the data and follow-up data of those patients were retrospectively analyzed. RESULTS: Most brain metastases were from rectal cancer (64.4%), and 80.0% of the 45 cases had extracranial metastases. The most common extracranial metastatic site was the lung (57.8%), followed by the liver (35.6%). All the brain metastases in patients with liver metastases were supratentorial, while in contrast, 44.8% of the patients without liver metastasis had subtentorial metastasis, showing a significant difference between them (P<0.05). The interval time from diagnosis of CRC to the development of brain metastases in case of Dukes D stage was 12.0 months, significantly shorter than that in the cases of Dukes A stage (24.0 months), B (36.0 months) and C (29.0 months) (P<0.05). The interval time was also shorter in the patients who developed extracranial metastasis within one year than those more than one year (12.0 months vs. 38.0 months)( P<0.05). The median survival time of patients with brain metastasis from colorectal was 6.0 months, with a 1-year survival rate of 21.1% and 2-year survival rate of 3.3% only. Univariate analysis showed that the median survival of patients with a KPS score of ≥70 was 8.0 months, significantly higher than 2.0 months in those with a KPS score of <70 (P<0.05). The median survival of patients with one or two brain metastases was 8.0 months, significantly higher than 4.0 months of those with >2 brain metastases (P<0.05). The median survival time after diagnosis of brain metastasis was 4.0 months for those who received monotherapy (only steroids, only chemotherapy or only radiotherapy), significantly shorter than 10.0 months of patients who received chemoradiotherapy, and 12.0 months of those who underwent surgery (P<0.05). Comparing each two differently treated groups, the survival time of surgery combined with chemotherapy or radiotherapy group was significantly different from that of all of other groups (P<0.05). The median survival time of chemoradiotherapy group was longer than that of monotherapy, but the difference was not significant (P>0.05). Multivariate analysis showed that brain metastases >2 and treatment modality type are independent prognostic factors for survival. CONCLUSIONS: Patients initially diagnosed with a Dukes D stage primary colorectal tumor and occurrence of extracranial metastasis (especially, pulmonary metastasis) within one year are associated to an increased risk of brain metastases and have a shorter survival time. Most brain metastases in patients with liver metastases are supratentorial, while many patients without liver metastasis have subtentorial metastasis. Brain metastases >2 and the type of treatment modality are independent prognostic factors for survival. The prognosis of patients who received chemoradiotherapy is better than those treated only with chemotherapy or radiotherapy. Some subsets of patients may benefit from surgery plus chemotherapy/radiotherapy.

[Robo1 expression in non-small cell lung cancer and its brain metastasis].

OBJECTIVE: To detect the expression of Robo1 in lung cancer tissues, adjacent non-cancerous tissues as well as lung cancer brain metastasis, and explore the correlation of Robo1 expression to lung cancer brain metastasis. METHODS: SP (streptavidin-peroxidase) staining method was used to examine the Robo1 expression in specimens from 80 cases of NSCLC, 52 cases of adjacent non-cancerous tissues and 72 cases of lung cancer with single brain metastasis (without metastasis in other organs). The Robo1 expression was further examined in 17 self control cases with lung cancer tissues and their brain metastasis tissues. The results were assessed by Kaplan-Meier analysis and log-rank test. RESULTS: The positive expression rate of Robo1 among adjacent non-cancerous tissues, lung cancers tissues and the lung cancer brain metastasis tissues were 1.9% (1/52), 13.8% (11/80) and 40.3% (29/72), respectively, and significant differences were detected among them (P < 0.05). During the 17 self control cases, the positive expression rate of Robo1 in lung cancer tissue and their brain metastasis tissues were 17.6% and 64.7%, respectively, with a significant difference between them (P < 0.01). Among the 72 cases of lung cancer brain metastasis, the median survival time of cases with positive Robo1 expression was 10 months, significantly shorter than that of cases with negative expression of Robo1 (17 months, P < 0.05). CONCLUSIONS: The positive expression rate of Robo1 was increased in sequence from the lowest in adjacent non-cancerous tissues, intermediate in the lung cancer tissues to highest in the lung cancer brain metastasis tissues. The expression of Robo1 in lung cancer brain metastasis is negatively correlated with the prognosis of patients with lung cancer brain metastasis. Robo1 may promote the genesis and progression of lung cancer and lung cancer brain metastasis as a cancer-promoting oncogene.

[Expression of Notch1, MMP-2 and MMP-9 and their significance in glioma patients].

OBJECTIVE: To study the expression of Notch1, MMP-2 and MMP-9 in glioma patients and their relationship with progression and prognosis of gliomas. METHODS: Sixty-four cases of glioma were included in this study. There were four cases of grade 1 tumor, twenty-five cases of grade 2, nine cases of grade 3, and twenty-six cases of grade 4. Immunohistochemistry (SP staining method) was used to detect the expression of Notch1, MMP-2 and MMP-9 in glioma tissues and adjacent non-tumor tissues, and the patients were followed up. RESULTS: Notch1, MMP-2 and MMP-9 were detected in glioma tissues but not in adjacent non-tumor tissues. The expression of Notch1 was increased with the pathological grade of the gliomas (r = 0.262, P < 0.05). The survival time of patients with strong expression of Notch1 was 31.0 months, significantly shorter than that of patients with non-strong positive (negative, weak and moderately) Notch1 expression (53.0 months, P < 0.05). Significant difference in survival time was observed between patients with negative and positive expression of MMP-9 (P < 0.05). CONCLUSIONS: Notch1, MMP-2 and MMP-9 are closely correlated with the progression and prognosis of malignant gliomas. Notch1 may participate in the expression regulation of MMP-2 and MMP-9. Compared with MMP-2, MMP-9 may play a more important role in determining the prognosis of malignant glioma. Notch1 and MMP-9 may become new biological markers for prognosis of patients with malignant glioma.