Genetic Epidemiology of Glioblastoma Multiforme: Confirmatory and New Findings from Analyses of Human Leukocyte Antigen Alleles and Motifs

Human leukocyte antigen (HLA) class I genes mediate cytotoxic T-lymphocyte responses and natural killer cell function. In a previous study, several HLA-B and HLA-C alleles and haplotypes were positively or negatively associated with the occurrence and prognosis of glioblastoma multiforme (GBM).As an extension of the Upper Midwest Health Study, we have performed HLA genotyping for 149 GBM patients and 149 healthy control subjects from a non-metropolitan population consisting almost exclusively of European Americans. Conditional logistic regression models did not reproduce the association of HLA-B*07 or the B*07-Cw*07 haplotype with GBM. Nonetheless, HLA-A*32, which has previously been shown to predispose GBM patients to a favorable prognosis, was negatively associated with occurrence of GBM (odds ratio=0.41, p=0.04 by univariate analysis). Other alleles (A*29, A*30, A*31 and A*33) within the A19 serology group to which A*32 belongs showed inconsistent trends. Sequencing-based HLA-A genotyping established that A*3201 was the single A*32 allele underlying the observed association. Additional evaluation of HLA-A promoter and exon 1 sequences did not detect any unexpected single nucleotide polymorphisms that could suggest differential allelic expression. Further analyses restricted to female GBM cases and controls revealed a second association with a specific HLA-B sequence motif corresponding to Bw4-80Ile (odds ratio=2.71, p=0.02).HLA-A allelic product encoded by A*3201 is likely to be functionally important to GBM. The novel, sex-specific association will require further confirmation in other representative study populations

Intraperitoneal drain placement and outcomes after elective colorectal surgery: international matched, prospective, cohort study

Despite current guidelines, intraperitoneal drain placement after elective colorectal surgery remains widespread. Drains were not associated with earlier detection of intraperitoneal collections, but were associated with prolonged hospital stay and increased risk of surgical-site infections.Background Many surgeons routinely place intraperitoneal drains after elective colorectal surgery. However, enhanced recovery after surgery guidelines recommend against their routine use owing to a lack of clear clinical benefit. This study aimed to describe international variation in intraperitoneal drain placement and the safety of this practice. Methods COMPASS (COMPlicAted intra-abdominal collectionS after colorectal Surgery) was a prospective, international, cohort study which enrolled consecutive adults undergoing elective colorectal surgery (February to March 2020). The primary outcome was the rate of intraperitoneal drain placement. Secondary outcomes included: rate and time to diagnosis of postoperative intraperitoneal collections; rate of surgical site infections (SSIs); time to discharge; and 30-day major postoperative complications (Clavien-Dindo grade at least III). After propensity score matching, multivariable logistic regression and Cox proportional hazards regression were used to estimate the independent association of the secondary outcomes with drain placement. Results Overall, 1805 patients from 22 countries were included (798 women, 44.2 per cent; median age 67.0 years). The drain insertion rate was 51.9 per cent (937 patients). After matching, drains were not associated with reduced rates (odds ratio (OR) 1.33, 95 per cent c.i. 0.79 to 2.23; P = 0.287) or earlier detection (hazard ratio (HR) 0.87, 0.33 to 2.31; P = 0.780) of collections. Although not associated with worse major postoperative complications (OR 1.09, 0.68 to 1.75; P = 0.709), drains were associated with delayed hospital discharge (HR 0.58, 0.52 to 0.66; P < 0.001) and an increased risk of SSIs (OR 2.47, 1.50 to 4.05; P < 0.001). Conclusion Intraperitoneal drain placement after elective colorectal surgery is not associated with earlier detection of postoperative collections, but prolongs hospital stay and increases SSI risk

Profound tumor-specific Th2 bias in patients with malignant glioma

<p>Abstract</p> <p>Background</p> <p>Vaccination against tumor-associated antigens is one promising approach to immunotherapy against malignant gliomas. While previous vaccine efforts have focused exclusively on HLA class I-restricted peptides, class II-restricted peptides are necessary to induce CD4⁺ helper T cells and sustain effective anti-tumor immunity. In this report we investigated the ability of five candidate peptide epitopes derived from glioma-associated antigens MAGE and IL-13 receptor α2 to detect and characterize CD4⁺ helper T cell responses in the peripheral blood of patients with malignant gliomas.</p> <p>Methods</p> <p>Primary T cell responses were determined by stimulating freshly isolated PBMCs from patients with primary glioblastoma (GBM) (n = 8), recurrent GBM (n = 5), meningioma (n = 7), and healthy controls (n = 6) with each candidate peptide, as well as anti-CD3 monoclonal antibody (mAb) and an immunodominant peptide epitope derived from myelin basic protein (MBP) serving as positive and negative controls, respectively. ELISA was used to measure IFN-γ and IL-5 levels, and the ratio of IFN-γ/IL-5 was used to determine whether the response had a predominant Th1 or Th2 bias.</p> <p>Results</p> <p>We demonstrate that novel HLA Class-II restricted MAGE-A3 and IL-13Rα2 peptides can detect T cell responses in patients with GBMs as well as in healthy subjects. Stimulation with a variety of peptide antigens over-expressed by gliomas is associated with a profound reduction in the IFN-γ/IL-5 ratio in GBM patients relative to healthy subjects. This bias is more pronounced in patients with recurrent GBMs.</p> <p>Conclusions</p> <p>Therapeutic vaccine strategies to shift tumor antigen-specific T cell response to a more immunostimulatory Th1 bias may be needed for immunotherapeutic trials to be more successful clinically.</p

Inhibition of caveolin-1 restores myeloid cell function in human glioblastoma.

BACKGROUND: Gliomas are the most common primary brain tumor in both children and adults. The prognosis for glioblastoma (GBM), the most common type of malignant glioma, has remained dismal, with median survival a little over one year despite maximal therapy with surgery, chemotherapy, and radiation. Although immunotherapy has become increasingly successful against many systemic tumors, clinical efficacy against brain tumors has been limited. One reason for this is an incomplete understanding of the local immunologic tumor microenvironment, particularly the function of large numbers of infiltrating myeloid derived cells. Monocytes/microglia are myeloid derived immunomodulatory cells, and they represent the predominant infiltrating immune cell population in gliomas. Our group has previously demonstrated using complementary in vitro and in vivo approaches that GBM tumor cells polarize tumor-associated myeloid cells (TAMs) and suppress their immunostimulatory function. METHODS AND RESULTS: To better understand the mechanisms responsible for this immunosuppression, we used gene expression profiling of stimulated monocytes in the presence or absence of GBM tumor cells. Our analysis identified caveolin-1 (CAV1), a plasma membrane molecule with pleiotropic functions, as significantly up-regulated in monocytes in the presence of GBMs. We validated these findings ex vivo by confirming up-regulation of CAV1 in TAMs isolated from GBMs immediately after surgical resection. Finally, we demonstrate that siRNA inhibition of CAV1 restores myeloid cell function, as measured by TNF-alpha secretion, in the presence of GBMs. CONCLUSIONS: Restoration of TAM function through pharmacologic blockage of CAV1 may facilitate more successful immunotherapeutic strategies directed against a variety of solid human tumors infiltrated by TAMs

<i>Ex</i><i>vivo</i> isolation and expression of AXL and CAV1 among myeloid cells in non-inflamed human CNS tissue and GBMs.

<p>(<b>a</b>) GBM tumor specimens or non-inflamed CNS tissue isolated from the anterior temporal tip during epileptic surgery were processed to single cell suspensions and stained with antibodies against CD11b and CD11c to sort infiltrating monocytes/microglia. (<b>b</b>) Quantitative PCR analysis of expression levels of AXL and CAV1 were determined for monocytes/microglia from <i>ex </i><i>vivo</i> non-inflamed human CNS tissue (n=3) and primary GBMs (n=10).</p

Flow schematic summarizing results of GBM-induced genes in co-cultured human myeloid cells.

<p>The number of genes satisfying each selection criteria are summarized, resulting in two gene subsets: those affected by LPS stimulation as well as culture with GBM tumor cells but not non-transformed human astrocytes, exemplified by AXL, and those unaffected by LPS stimulation but affected by GBM tumor cells, exemplified by CAV1. Additional genes within these two gene susbsets included CDCP1, CKS2, STC1, KRT18, and PHLDA2, but their differential expression could not be independently replicated (data not shown).</p

Restoration of myeloid cell function with siRNA inhibition of CAV1 but not AXL.

<p>(<b>a</b>) A representative example of the ability of GBM tumor cells to inhibit monocyte function, exemplified by TNF-alpha secretion after LPS stimulation, and the ability of CAV1 siRNA to reverse this inhibition. (<b>b</b>) Results from four independent experiments examining the ability of CAV1 and AXL siRNA inhibition to reverse GBM-mediated inhibition of TNF-alpha secretion. CAV1 suppression by siRNA significantly reverses GBM-mediated suppression and restores monocyte activity as measured by TNF-alpha secretion (p<0.05).</p

Experimental design and sample processing for microarray analysis of GBM-induced molecules in co-cultured myeloid cells.

<p>(<b>a</b>) ABI1700 cDNA microarrays were used to analyze global changes in gene transcripts using a cutoff in the change of gene expression of > 2 fold. We analyzed the expression profile of 29,362 human genes using RNA samples from unstimulated monocytes (n=8), LPS-stimulated monocytes (n=8), LPS-stimulated monocytes cocultured with glioblastoma cells (n=8), and monocytes cocultured with human astrocytes (n=2). Of these, 14,414 gene expression profiles were used for subsequent analysis, as this set of genes passed all quality control criteria. Local Pooled Error Tests (PLPE package from Bioconductor) were performed comparing gene expression values of monocytes stimulated with LPS in the absence vs. presence of GBM cells and subsequently stimulated monocytes co-cultured with GBM cells vs. stimulated monocytes co-cultured with primary human astrocytes. (<b>b</b>) After 4 hours of culture in 4 experimental conditions, monocytes were isolated from GBM or human astrocytes based on FSC/SSC properties. Purity was re-confirmed in a small fraction of each sample based on CD11b expression.</p