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.

Clinical significance and molecular annotation of cellular morphometric subtypes in lower-grade gliomas discovered by machine learning.

BACKGROUND: Lower-grade gliomas (LGG) are heterogeneous diseases by clinical, histological, and molecular criteria. We aimed to personalize the diagnosis and therapy of LGG patients by developing and validating robust cellular morphometric subtypes (CMS) and to uncover the molecular signatures underlying these subtypes. METHODS: Cellular morphometric biomarkers (CMBs) were identified with artificial intelligence technique from TCGA-LGG cohort. Consensus clustering was used to define CMS. Survival analysis was performed to assess the clinical impact of CMBs and CMS. A nomogram was constructed to predict 3- and 5-year overall survival (OS) of LGG patients. Tumor mutational burden (TMB) and immune cell infiltration between subtypes were analyzed using the Mann-Whitney U test. The double-blinded validation for important immunotherapy-related biomarkers was executed using immunohistochemistry (IHC). RESULTS: We developed a machine learning (ML) pipeline to extract CMBs from whole-slide images of tissue histology; identifying and externally validating robust CMS of LGGs in multicenter cohorts. The subtypes had independent predicted OS across all three independent cohorts. In the TCGA-LGG cohort, patients within the poor-prognosis subtype responded poorly to primary and follow-up therapies. LGGs within the poor-prognosis subtype were characterized by high mutational burden, high frequencies of copy number alterations, and high levels of tumor-infiltrating lymphocytes and immune checkpoint genes. Higher levels of PD-1/PD-L1/CTLA-4 were confirmed by IHC staining. In addition, the subtypes learned from LGG demonstrate translational impact on glioblastoma (GBM). CONCLUSIONS: We developed and validated a framework (CMS-ML) for CMS discovery in LGG associated with specific molecular alterations, immune microenvironment, prognosis, and treatment response.

Genetic alteration and clonal evolution of primary glioblastoma into secondary gliosarcoma.

AIMS: Secondary gliosarcoma (SGS) rarely arises post treatment of primary glioblastoma multiforme (GBM), and contains gliomatous and sarcomatous components. The origin and clonal evolution of SGS sarcomatous components remain uncharacterized. Therapeutic radiation is mutagenic and can induce sarcomas in patients with other tumor phenotypes, but possible causal relationships between radiotherapy and induction of SGS sarcomatous components remain unexplored. Herein, we investigated the clonal origin of SGS in a patient with primary GBM progressing into SGS post-radiochemotherapy. METHODS: Somatic mutation profile in GBM and SGS was examined using whole-genome sequencing and deep-whole-exome sequencing. Mutation signatures were characterized to investigate relationships between radiochemotherapy and SGS pathogenesis. RESULTS: A mutation cluster containing two founding mutations in tumor-suppressor genes NF1 (variant allele frequency [VAF]: 50.0% in GBM and 51.1% in SGS) and TP53 (VAF: 26.7% in GBM and 50.8% in SGS) was shared in GBM and SGS. SGS exhibited an overpresented C>A (G>T) transversion (oxidative DNA damage signature) but no signature 11 mutations (alkylating-agents - exposure signature). Since radiation induces DNA lesions by generating reactive oxygen species, the mutations observed in this case of SGS were likely the result of radiotherapy rather than chemotherapy. CONCLUSIONS: Secondary gliosarcoma components likely have a monoclonal origin, and the clone possessing mutations in NF1 and TP53 was likely the founding clone in this case of SGS.

Impact of beta-2 microglobulin expression on the survival of glioma patients via modulating the tumor immune microenvironment.

AIMS: High immune cell infiltration in gliomas establishes an immunosuppressive tumor microenvironment, which in turn promotes resistance to immunotherapy. Hence, it is important to identify novel targets associated with high immune cell infiltration in gliomas. Our previous study showed that serum levels of beta-2 microglobulin (B2M) in lower-grade glioma patients were lower than those in glioblastoma patients. In the present study, we focused on exploring the roles of B2M in glioma immune infiltration. METHODS: A large cohort of patients with gliomas from the TCGA, CGGA, and Gravendeel databases was included to explore differential expression patterns and potential roles of B2M in gliomas. A total of 103 glioma tissue samples were collected to determine the distributions of B2M protein levels by immunofluorescent assays. Kaplan-Meier survival analysis and meta-analysis were used for survival analysis. GO(Gene-ontology) enrichment analysis, co-expression analysis, KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis, and immune infiltration analysis were performed to explore roles and related mechanisms of B2M in glioma. RESULTS: We found that both B2M mRNA and protein levels were abnormally upregulated in glioma samples compared with those from normal brain tissue. B2M expression was correlated with tumor grade and was downregulated in IDH1 mutant samples. Furthermore, B2M was a moderately sensitive indicator for predicting the mesenchymal molecular subtype of gliomas. Interestingly, glioma patients with lower B2M expression had remarkably longer survival times than those with higher B2M expression. Moreover, meta-analysis showed that B2M was an independent predictive marker in glioma patients. The results of GO enrichment analysis revealed that B2M contributed to immune cell infiltration in glioma patients. In addition, results of KEGG pathway analysis and co-expression analysis suggested that B2M may mediate glioma immune infiltration via chemokines. CONCLUSIONS: We conclude that B2M levels are critical for the survival times of glioma patients, at least in part due to mediating high immune infiltration.

Dominant expression of 85-kDa form of cortactin in colorectal cancer.

PURPOSE: Cortactin is commonly expressed in several human cancers, which may alter their invasive or metastatic properties. Eighty five kilodalton form (p85) and 80-kDa form (p80) of cortactin are two separate bands in SDS-PAGE representing different conformational states. The objective of this study was to investigate cortactin expression in colorectal cancer (CRC). EXPERIMENTAL DESIGN: Cortactin expression was studied in an eight paired laser capture microdissection (LCM) CRC tissues and matched non-cancerous epithelia by immunoblotting. The expression in 58 CRC and two cell lines, HCT8 and HCT116, was studied respectively by immunohistochemistry and confocal laser scanning immunofluorescence. RESULTS: Dominant expression of p85 was identified in LCM-procured CRC tissues compared with equal intensity of p85 and p80 forms in non-cancerous tissues, while the amount of total cortactin was approximate. Immunohistochemistry analysis demonstrated that cortactin located in the cytoplasm of tumor cells and adjacent non-cancerous cells, and its expression was negatively correlated with TNM staging and lymphatic invasion status. However, the invasion fronts in 3 of 58 primary tumors and 28 of 39 available lymph node metastases were intensively stained. Further, immunofluorescence analysis showed that cortactin was distributed in cytoplasm and enriched in the front of the extending lamellipodia at adhering side of cultured cancer cells. CONCLUSIONS: Our results demonstrated the dominant expression of p85 form of cortactin in CRC for the first time. The enrichment of cortactin in the invasion front of some tumor cells and in the extending lamellipodia of cultured cancer cells suggests that cortactin may help cancer cell movement.

[Expression of NFkappaB p65 and its target genes in gastric cancer and precancerous lesions].

OBJECTIVE: To study the expression of NFkappaB p65 and its target genes in intestinal metaplasia (IM), dysplasia (Dys), gastric cancer (GC) infected with Helicobacter pylori (Hp) and explore the mechanism of infection by cytotoxin-associated antigen A expressing Hp (CagA(+)Hp) in the development of gastric cancer. METHODS: CagA antibody in blood sample of 289 patients was determined by ELISA. Hp was detected by rapid urease test and Warthin starry staining. Expression of NFkappaB p65 and its target genes in IM, Dys and GC was examined by immunohistochemistry. RESULTS: In IMI approximately II, IMIII, DysI, DysII approximately III and GC, the expression of NFkappaB p65 was significantly higher in patients with CagA(+)Hp infection than those without CagA Hp infection. In IMIII and DysII approximately III, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in patients with CagA Hp infection than those without CagA Hp infection. In gastric cancer infected with CagA(+)Hp, the expression of NFkappaB p65, c-myc, CyclinD(1) and bcl-xl was significantly higher in intestinal type than in diffuse type. CONCLUSION: There are different mechanisms in intestinal type and diffuse type in the development of gastric cancer. The occurrence of intestinal type gastric cancer is associated with CagA(+)Hp infection which by NFkappaB p65 upregulating the expression of c-myc, CyclinD(1),bcl-xl in patients with IMIII, DysII approximately III. It may be an effective method to prevent gastric cancer by inhibiting NFkappaB p65.

[Relationship between Helicobacter pylori infection and expression of c-myc, Bcl-2, and Bax protein in different gastric mucosa lesions].

BACKGROUND & OBJECTIVE: Helicobacter pylori (HP) has been believed to be a carcinogen of gastric carcinoma. However, its mechanism was yet not clearly understood. This study was designed to investigate the relationship between HP infection and gastric epithelial cell proliferation as well as apoptosis in different gastric mucosa lesions and elucidate the probable mechanism of gastric carcinogenesis relating with HP infection. METHODS: A total of 272 cases were available for the study including 42 cases of chronic gastritis (CG), 46 cases of intestinal metaplasia I or II (IM I- II), 25 cases of intestinal metaplasia III (IM III), 21 cases of mild dysplasia (Dys I), 54 cases of modest or severe dysplasia (Dys II- III), and 84 cases of gastric cancer (GC). HP infection was detected by Warthin-Starry bacterium staining method and streptavidin-peroxidase (SP) immunohistochemical method. HID-AB(pH2.5)- PAS method was used to define the quality of mucus. The expression of c-myc, Bcl-2, and Bax were detected using SP immunohistochemical method. The chi-square test and the Fisher's exact probability test were used to compare the frequencies. RESULTS: (1)The expression of c-myc and Bcl-2 increased as gastric mucosa lesions developed from CG,IM,Dys to GC,but the expression of Bax decreased. The expression of c-myc was significantly higher in GC than that in Dys II- III and IM III(all P< 0.01), but the expression of Bax was significantly lower in GC than that in Dys II- III and IM III(P< 0.05 or P< 0.01). (2)The expression of c-myc in IM III and Dys II- III with HP infection was 62.50% and 66.67%,respectively, significantly higher than that without infection(11.11%,27.78%,all P< 0.05). The expression of Bax in CG, IM I- II and IM III with HP infection were 87.10%, 81.25%, and 62.50%, respectively, significantly higher than those without infection (54.55%, 42.86%, 11.11%, all P< 0.05). Furthermore HP infection was associated with the expression of Bcl-2 in IM III, Dys II- III and GC (P< 0.05 or P< 0.01). CONCLUSION: HP infection can cause serious imbalance between cell proliferation and apoptosis in the precancerous lesions (IM III and Dys II- III), giving chances for gastric carcinogenesis.

[Multidrug resistance and its relationship with neuroendocrine differentiation in non-small cell lung carcinoma].

OBJECTIVE: To study the multidrug resistance (MDR) of non-small lung cancer (NSCLC) and its relationship with neuroendocrine (NE) differentiation. METHODS: NSCLC samples from 113 untreated patients were analyzed immunohistochemically with antibodies to glutathion-s-transferase-pi (GST-pi), multidrug resistance associated protein (MRP), lung resistance associated protein (LRP), neuro-specific enolase (NSE), synaptophysin (SYN) and chromogranin (CgA). RESULTS: (1) The expression of the three proteins was significantly associated with the type of lung carcinoma (P < 0.05), but not with the differentiation and lymph node metastasis. The expression of GST-pi was significantly related with MRP, MRP and LRP (P < 0.05). (2) The positive rates of the NE markers were: NSE, 53.1%; SYN, 26.6%; CgA, 6.2%; and 21.2% for at least two markers. The expression of at least 2 markers was associated with the degree of differentiation (P < 0.05), but not with the type of lung cancer and lymph node metastasis. (3) The expression of the three multidrug resistance related proteins in the positive group for at least 2 markers was significantly lower than that in the negative group (P < 0.05). CONCLUSIONS: The over-expressions of GST-pi, MRP and LRP are important causes of primary multidrug resistance in NSCLC. The differentiation of NE may be one of the factors involved in multidrug resistance.

[Relationship between Helicobacter pylori infection and expressions of tumor suppressor genes in gastric carcinoma and related lesions].

BACKGROUND & OBJECTIVE: Helicobacter pylori (Hp) are believed to be a carcinogen of gastric carcinoma. However, its mechanism was yet not clearly understood. p53, p21WAF1, and p16 are main negative regulator genes of cell cycle. This study was designed to investigate the relationship between these 3 genes and Hp infection. METHODS: The authors examined the expression of these 3 tumor suppressor genes and Hp infection in 65 cases with chronic atrophic gastritis (CAG), 93 cases with intestinal metaplasia(IM), 94 cases with gastric epithelial dysplasia (GED) and 60 cases with gastric carcinoma (GC) using HID-AB (pH 2.5)-PAS, SP immunohistochemistry staining, and Warthin-Starry staining. RESULTS: For CAC, IM stage I-II, IM stage III, GED stage I, GED stage II-III, and GC, the positive expression rates of p53 were 0, 1.64%, 6.25%, 5.45%, 23.08%, and 70.00%, respectively (increased with pathological process); the positive expression rates of p21WAF1 were 100%, 95.08%, 100%, 100%, 71.79%, and 45.00%, respectively; the positive expression rates of p16 were 83.08%, 81.97%, 78.13%, 89.09%, 69.23%, and 40.00%, respectively. All the expression of these 3 genes showed significant difference between GED stage II-III and GED stage I, GC and GED stage II-III. In the same pathological changes, the positive expression rate of these 3 genes was higher in Hp infection group than in no Hp infection group, while there was no significant difference (P > 0.05). CONCLUSION: The mutation of p53 and inactivation of p21WAF1 and p16 play an important role in carcinogenesis of stomach. However, Hp infection was not associated with the abnormal expression of these 3 genes.