An evaluation of clinical fellow programmes in an acute teaching hospital trust.

BACKGROUND/AIMS: Clinical fellows support the hospital workforce while gaining experience in different specialities, research, leadership and teaching. The authors aimed to assess the impact of clinical fellow programmes in an acute teaching hospital trust. METHODS: An anonymous electronic service evaluation was sent to clinical fellows to investigate their views on whether the programme had improved patient safety, doctors' clinical performance, training and wellbeing. Thematic analysis was used to analyse the free-text responses. RESULTS: A total of 95 out of 144 clinical fellows responded to the evaluation survey. The clinical fellows believed that the programme had improved patient safety, clinical performance (time to manage acute patients), foundation and internal medicine training, undergraduate teaching and junior doctors' wellbeing. Four similar themes emerged from the free-text responses: career development, patient safety, training and doctors' wellbeing. CONCLUSIONS: Clinical fellow programmes may improve patient safety, clinical performance, training, undergraduate education and doctors' wellbeing.

The effects of coagulation factors and their inhibitors on proliferation and migration in colorectal cancer.

BACKGROUND/AIM: Clotting factors promote cancer development. We investigated if coagulation proteins promote proliferation and migration in colorectal cancer (CRC) cell lines and whether their direct inhibitors can attenuate these effects. MATERIALS AND METHODS: DLD-1 and SW620 cells were treated with tissue factor (0, 50, 100 and 500 pg/mL ± 10 µg/mL 10H10 [anti-tissue factor antibody]), thrombin (0.0, 0.1, 1.0 and 10.0 U/mL ± 0.5 µM dabigatran [thrombin inhibitor]) and Factor Xa, FXa (0.0, 0.1, 1.0 and 10.0 U/mL ± 100 ng/mL rivaroxaban [FXa inhibitor]) and their effects on proliferation and migration were quantified using the PrestoBlue® and transwell migration assays, respectively. RESULTS: Thrombin increased proliferation from 48 h treatment compared to its control (48 h 6.57 ± 1.36 u vs. 2.42 ± 0.13 u, p = 0.001, 72 h 9.50 ± 1.54 u vs. 4.50 ± 0.47 u, p = 0.004 and 96 h 10.77 ± 1.72 u vs. 5.57 ± 0.25 u, p = 0.008). This increase in proliferation was attenuated by dabigatran at 72 h (2.23 ± 0.16 u vs. 3.26 ± 0.43 u, p = 0.04). Tissue factor (0 pg/mL 20.7 ± 1.6 cells/view vs. 50 pg/mL 32.4 ± 1.9 cells/view, p = 0.0002), FXa (0.0 U/mL 8.9 ± 1.1 cells/view vs. 10.0 U/mL 17.7 ± 1.7 cells/view, p < 0.0001) and thrombin (0.0 U/mL 8.9 ± 1.3 cells/view vs. 10.0 U/mL 20.2 ± 2.0 cells/view, p < 0.0001) all increased migration compared to their controls. However, their direct inhibitors did not attenuate these increases. CONCLUSION: Thrombin, FXa and TF all increase migration in CRC in vitro. Thrombin induced increase in proliferation is abrogated by dabigatran. Dabigatran may have potential as an anti-cancer therapy in CRC.

Breast cancer cells mediate endothelial cell activation, promoting von Willebrand factor release, tumor adhesion, and transendothelial migration.

BACKGROUND: Breast cancer results in a three- to four-fold increased risk of venous thromboembolism (VTE), which is associated with reduced patient survival. Despite this, the mechanisms underpinning breast cancer-associated thrombosis remain poorly defined. Tumor cells can trigger endothelial cell (EC) activation resulting in increased von Willebrand factor (VWF) secretion. Importantly, elevated plasma VWF levels constitute an independent biomarker for VTE risk. Moreover, in a model of melanoma, treatment with low molecular weight heparin (LMWH) negatively regulated VWF secretion and attenuated tumor metastasis. OBJECTIVE: To investigate the role of VWF in breast cancer metastasis and examine the effect of LMWH in modulating EC activation and breast tumor transmigration. METHODS: von Willebrand factor levels were measured by ELISA. Primary ECs were used to assess tumor-induced activation, angiogenesis, tumor adhesion, and transendothelial migration. RESULTS AND CONCLUSION: Patients with metastatic breast cancer have markedly elevated plasma VWF:Ag levels that also correlate with poorer survival. MDA-MB-231 and MCF-7 breast cancer cells induce secretion of VWF, angiopoietin-2, and osteoprotegerin from ECs, which is further enhanced by the presence of platelets. Vascular endothelial growth factor-A (VEGF-A) plays an important role in modulating breast cancer-induced VWF release. Moreover, VEGF-A from breast tumor cells also contributes to a pro-angiogenic effect on ECs. VWF multimers secreted from ECs, in response to tumor-VEGF-A, mediate adhesion of breast tumor cells along the endothelium. LMWH inhibits VWF-breast tumor adhesion and transendothelial migration. Our findings highlight the significant crosstalk between tumor cells and the endothelium including increased VWF secretion which may contribute to tumor metastasis.

Neoantigen vaccine-induced CD4 T cells confer protective immunity in a mouse model of multiple myeloma through activation of CD8 T cells against non-vaccine, tumor-associated antigens.

BACKGROUND: Cancer-associated neoantigens (neoAg) derived from tumor genomic sequencing and predictive algorithms for mutated peptides are a promising basis for therapeutic vaccines under investigation. Although these are generally designed to bind major histocompatibility complex class I and induce CD8 cytolytic T lymphocyte (CTL) activity, results from preclinical and clinical studies demonstrate that the majority of neoAg vaccines efficiently induce CD4 T helper (Th) responses but not CTL. Despite this, these vaccines have demonstrated clinical efficacy. Therefore, understanding the mechanisms of CD4 + T cell-mediated tumor protection is critical to optimizing this immunotherapeutic strategy. METHODS: We investigated this phenomenon in the mineral oil-induced plasmacytoma (MOPC).315.BM (MOPC315) mouse model of multiple myeloma, a malignancy of plasma cells. MOPC315 cells express in their lambda chain a unique tumor-specific neoAg, an idiotypic (Id) peptide. We generated a vaccine formulated with this Id peptide fused to a heat shock protein HSC70 binding (HSB) motif co-delivered with poly (I:C). The immunogenicity of the Id-vaccine was measured in splenocytes by ELISpot. Mice were challenged with MOPC315 cells and antitumor immunity was assessed by co-incubating splenocytes and bone marrow mononuclear cells derived from vaccinated mice and controls, with the Id antigen and irradiated MOPC315 cells. The frequency of activated CD4 and CD8 T cells and their phenotype were characterized by flow cytometry. RESULTS: Id-vaccine efficiently induced antigen-specific CD4 Th activity and antitumor immunity, protecting mice from MOPC315 tumor growth. CD4 cytolytic activity was not detected under these conditions. Polyfunctional CD8 T cells homed to the bone marrow microenvironment of protected mice and preferentially expanded only when restimulated ex vivo with both Id peptide and MOPC315 cells. Protective activity was abrogated by depletion of either CD4 or CD8 lymphocytes. CONCLUSION: These results demonstrate that Id-HSB +poly (I:C) vaccine protects against MOPC315 growth by priming Id-specific CD4 Th cells that confer protection against tumor but are not directly cytotoxic. These data indicate that activation of CD8 CTL against MOPC315-associated antigens not present in the vaccine is one of the major mechanisms of tumor immunity.

Current status of use of high throughput nucleotide sequencing in rheumatology.

OBJECTIVE: Here, we assess the usage of high throughput sequencing (HTS) in rheumatic research and the availability of public HTS data of rheumatic samples. METHODS: We performed a semiautomated literature review on PubMed, consisting of an R-script and manual curation as well as a manual search on the Sequence Read Archive for public available HTS data. RESULTS: Of the 699 identified articles, rheumatoid arthritis (n=182 publications, 26%), systemic lupus erythematous (n=161, 23%) and osteoarthritis (n=152, 22%) are among the rheumatic diseases with the most reported use of HTS assays. The most represented assay is RNA-Seq (n=457, 65%) for the identification of biomarkers in blood or synovial tissue. We also find, that the quality of accompanying clinical characterisation of the sequenced patients differs dramatically and we propose a minimal set of clinical data necessary to accompany rheumatological-relevant HTS data. CONCLUSION: HTS allows the analysis of a broad spectrum of molecular features in many samples at the same time. It offers enormous potential in novel personalised diagnosis and treatment strategies for patients with rheumatic diseases. Being established in cancer research and in the field of Mendelian diseases, rheumatic diseases are about to become the third disease domain for HTS, especially the RNA-Seq assay. However, we need to start a discussion about reporting of clinical characterisation accompany rheumatological-relevant HTS data to make clinical meaningful use of this data.

Rivaroxaban compared to no treatment in ER-negative stage I-III early breast cancer patients (the TIP Trial): study protocol for a phase II preoperative window-of-opportunity study design randomised controlled trial.

BACKGROUND: Breast cancer patients are at a four-fold increased risk of developing a venous thromboembolism (VTE), a major cause of death in this group. Conversely, coagulation factors promote tumour growth and metastasis. This has been evidenced in preclinical models, with an inhibitory effect of anticoagulants on cancer growth through proliferative, angiogenic, apoptotic, cancer stem cell and metastatic processes. The extrinsic clotting pathway is also more upregulated in patients in the relatively poorer prognosis oestrogen receptor (ER)-negative breast cancer subgroup, with increased tumour stromal expression of the coagulation factors Tissue Factor and thrombin. Rivaroxaban (Xarelto®, Bayer AG, Leverkusen, Germany) is a direct oral anticoagulant (DOAC). It is a Factor Xa inhibitor that is routinely prescribed for the prevention of stroke in non-valvular atrial fibrillation and for both VTE prophylaxis and treatment. This trial will assess the anti-proliferative and other anti-cancer progression mechanisms of Rivaroxaban in ER-negative early breast cancer patients. METHODS: This UK-based preoperative window-of-opportunity phase II randomised control trial will randomise 88 treatment-naïve early breast cancer patients to receive 20 mg OD Rivaroxaban treatment for 11 to 17 days or no treatment. Treatment will be stopped 24 h (range 18-36 h) prior to surgery or repeat core biopsy. All patients will be followed up for 2 weeks following surgery or repeat core biopsy. The primary endpoint is change in tumour Ki67. Secondary outcome measures include tumour markers of apoptosis and angiogenesis, extrinsic clotting pathway activation and systemic markers of metastasis, tumour load and coagulation. DISCUSSION: Laboratory evidence supports an anti-cancer role for anticoagulants; however, this has failed to translate into survival benefit when trialled in patients with metastatic disease or poor prognosis cancers, such as lung cancer. Subgroup analysis supported a potential survival benefit in better prognosis advanced disease patients. This is the first study to investigate the anti-cancer effects of anticoagulants in early breast cancer. TRIAL REGISTRATION: UK National Research Ethics Service (NRES) approval 15/NW/0406, MHRA Clinical Trials Authorisation 48380/0003/001-0001. The sponsor is Manchester University NHS Foundation Trust, and the trial is co-ordinated by Cancer Research UK Liverpool Cancer Trials Unit (LCTU). EudraCT 2014-004909-33 , registered 27 July 2015. ISRCTN14785273 .

Breast cancer stromal clotting activation (Tissue Factor and thrombin): A pre-invasive phenomena that is prognostic in invasion.

BACKGROUND: Tumor stroma, of which fibroblasts are the most abundant cell, resembles a non-healing wound, where a procoagulant environment creates a permissive milieu for cancer growth. We aimed to determine if tumor expression of coagulation factors (procoagulant phenotype), and systemic hypercoagulability, occur at the preinvasive (ductal carcinoma in situ; DCIS) stage and correlate with breast cancer subtype, disease-free survival (DFS), and overall survival (OS). METHODS: In a prospective cohort of early breast cancer (DCIS, n = 76; invasive, n = 248) tumor, normal breast and plasma were examined. Fibroblast and epithelial expression of Tissue Factor (TF), thrombin, PAR1, PAR2, and plasma thrombin-antithrombin (TAT) and D-dimer were correlated with clinicopathological data, and 5-year survival. RESULTS: Fibroblast expression of TF, thrombin, and PAR1 was increased in DCIS and invasive cancer compared to normal breast fibroblasts (P ≤ .003, all). Fibroblast TF, thrombin, PAR1, and PAR2 was increased in cancers with high Ki67, high grade, ER- (vs ER+), and HER2+ (vs HER2-) (all P < .05). On univariate analysis, fibroblast TF expression was inversely associated with DFS (P = .04) and OS (P = .02). D-dimer was higher in node positive (507 (CI: 411-625) ng/mL, n = 68) vs negative patients (428 (CI: 387-472) ng/mL, n = 171, P = .004) and inversely associated with OS (P = .047). On multivariate analysis, plasma TAT was associated with reduced OS (HR 3.26, CI 1.16-3.1, P = .02), with a high plasma TAT (≥3.2 ng/mL) associated with > 3-fold mortality risk compared to low TAT. CONCLUSION: This demonstrates procoagulant phenotypic changes occur in fibroblasts at the preinvasive stage. Fibroblast procoagulant phenotype is associated with aggressive breast cancer subtypes and reduced survival. Coagulation may be a therapeutic target in breast cancer.

Results of XPAND II: A Multicenter, Prospective, Continued-Access Clinical Trial Using the AeroForm Tissue Expander for Two-Stage Breast Reconstruction.

BACKGROUND: XPAND II was a prospective, multicenter, single-arm, open-label, continued-access study designed to confirm the results from the XPAND study, a multicenter, prospective, randomized study for breast reconstruction. The AeroForm device received clearance from the U.S. Food and Drug Administration in December 2016 based on the results of the pivotal XPAND trial, which compared the AeroForm to saline expanders. METHODS: Fifty women were treated in the XPAND II study and implanted with the AeroForm device (86 devices). The study endpoint was successful completion of the second-stage surgery, and secondary endpoints were days to complete expansion and reconstruction, and patient/physician satisfaction. Following implantation, women were administered 10-cc doses of carbon dioxide at home up to three times daily. When adequate expansion was achieved, the expanders were exchanged for standard breast implants. RESULTS: The primary endpoint (successful exchange to standard breast implant, precluding non-device-related failures) is 100 percent. All-cause interim success is 95 percent, with three subjects (four breasts) failing primary exchange because of non-device-related reasons. Median time to complete expansion was 21 days (range, 5 to 117 days). Median time to complete the reconstruction was 112 days (range, 55 to 329 days). Ninety-six percent of the subjects were very or moderately satisfied with the AeroForm expansion process. CONCLUSIONS: Results of the XPAND II continued access study confirm and improve on previous results from the randomized trial (XPAND). These results validate that the AeroForm patient-controlled, needle-free carbon dioxide tissue expander is safe and effective for two-stage breast reconstruction. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

TCR Fingerprinting and Off-Target Peptide Identification.

Adoptive T cell therapy using patient T cells redirected to recognize tumor-specific antigens by expressing genetically engineered high-affinity T-cell receptors (TCRs) has therapeutic potential for melanoma and other solid tumors. Clinical trials implementing genetically modified TCRs in melanoma patients have raised concerns regarding off-target toxicities resulting in lethal destruction of healthy tissue, highlighting the urgency of assessing which off-target peptides can be recognized by a TCR. As a model system we used the clinically efficacious NY-ESO-1-specific TCR C259, which recognizes the peptide epitope SLLMWITQC presented by HLA-A*02:01. We investigated which amino acids at each position enable a TCR interaction by sequentially replacing every amino acid position outside of anchor positions 2 and 9 with all 19 possible alternative amino acids, resulting in 134 peptides (133 altered peptides plus epitope peptide). Each peptide was individually evaluated using three different in vitro assays: binding of the NY-ESOc259 TCR to the peptide, peptide-dependent activation of TCR-expressing cells, and killing of peptide-presenting target cells. To represent the TCR recognition kernel, we defined Position Weight Matrices (PWMs) for each assay by assigning normalized measurements to each of the 20 amino acids in each position. To predict potential off-target peptides, we applied a novel algorithm projecting the PWM-defined kernel into the human proteome, scoring NY-ESOc259 TCR recognition of 336,921 predicted human HLA-A*02:01 binding 9-mer peptides. Of the 12 peptides with high predicted score, we confirmed 7 (including NY-ESO-1 antigen SLLMWITQC) strongly activate human primary NY-ESOc259-expressing T cells. These off-target peptides include peptides with up to 7 amino acid changes (of 9 possible), which could not be predicted using the recognition motif as determined by alanine scans. Thus, this replacement scan assay determines the "TCR fingerprint" and, when coupled with the algorithm applied to the database of human 9-mer peptides binding to HLA-A*02:01, enables the identification of potential off-target antigens and the tissues where they are expressed. This platform enables both screening of multiple TCRs to identify the best candidate for clinical development and identification of TCR-specific cross-reactive peptide recognition and constitutes an improved methodology for the identification of potential off-target peptides presented on MHC class I molecules.

Correction to: Angiosarcoma patients treated with immune checkpoint inhibitors: a case series of seven patients from a single institution.

Following publication of the original article [1], the authors have reported that the following sentence "While of the same IgG1 class as ipilimumab, preclinical data suggests this molecule may have enhanced activity against T regulatory cells".

Angiosarcoma patients treated with immune checkpoint inhibitors: a case series of seven patients from a single institution.

BACKGROUND: Angiosarcoma is an uncommon endothelial malignancy and a highly aggressive soft tissue sarcoma. Due to its infiltrative nature, successful management of localized angiosarcoma is often challenging. Systemic chemotherapy is used in the metastatic setting and occasionally in patients with high-risk localized disease in neoadjuvant or adjuvant settings. However, responses tend to be short-lived and most patients succumb to metastatic disease. Novel therapies are needed for patients with angiosarcomas. METHODS: We performed a retrospective analysis of patients with locally advanced or metastatic angiosarcoma, who were treated with checkpoint inhibitors at our institution. We collected their clinical information and outcome measurements. In one patient with achieved complete response, we analyzed circulating and infiltrating T cells within peripheral blood and tumor tissue. RESULTS: We have treated seven angiosarcoma (AS) patients with checkpoint inhibitors either in the context of clinical trials or off label [Pembrolizumab + Axitinib (NCT02636725; n = 1), AGEN1884, a CTLA-4 inhibitor (NCT02694822; n = 2), Pembrolizumab (n = 4)]. Five patients had cutaneous angiosarcoma, one primary breast angiosarcoma and one radiation-associated breast angiosarcoma. At 12 weeks, 5/7 patients (71%) had partial response of their lesions either on imaging and/or clinical exam and two (29%) had progressive disease. 6/7 patients are alive to date and, thus far, 3/7 patients (43%) have progressed (median 3.4 months)- one achieved partial response after pembrolizumab was switched to ongoing Nivolumab/Ipilimumab, one died of progressive disease at 31 weeks (primary breast angiosarcoma) and one was placed on pazopanib. One patient had a complete response (CR) following extended treatment with monotherapy AGEN1884. No patient experienced any ≥ grade 2 toxicities. CONCLUSIONS: This case series underscores the value of targeted immunotherapy in treating angiosarcoma. It also identifies genetic heterogeneity of cutaneous angiosarcomas and discusses specific genetic findings that may explain reported benefits from immunotherapy.

Mutation-Derived Neoantigens for Cancer Immunotherapy.

Mutation-derived neoantigens distinguish tumor from normal cells. T cells can sense the HLA-presented mutations, recognize tumor cells as non-self and destroy them. Therapeutically, immunotherapy antibodies can increase the virulence of the immune system by increasing T-cell cytotoxicity targeted toward neoantigens. Neoantigen vaccines act through antigen-presenting cells, such as dendritic cells, to activate patient-endogenous T cells that recognize vaccine-encoded mutations. Infusion of mutation-targeting T cells by adoptive cell therapy (ACT) directly increases the number and frequency of cytotoxic T cells recognizing and killing tumor cells. At the same time, publicly-funded consortia have profiled tumor genomes across many indications, identifying mutations in each tumor. For example, we find basal and HER2 positive tumors contain more mutated proteins and more TP53 mutations than luminal A/B breast tumors. HPV negative tumors have more mutated proteins than HPV positive head and neck tumors and in agreement with the hypothesis that HPV activity interferes with p53 activity, only 14% of the HPV positive mutations have TP53 mutations vs. 86% of the HPV negative tumors. Lung adenocarcinomas in smokers have over four times more mutated proteins relative to those in never smokers (median 248 vs. 61, respectively). With an eye toward immunotherapy applications, we review the spectrum of mutations in multiple indications, show variations in indication sub-types, and examine intra- and inter-indication prevalence of re-occurring mutation neoantigens that could be used for warehouse vaccines and ACT.

Bioinformatic methods for cancer neoantigen prediction.

Tumor cells accumulate aberrations not present in normal cells, leading to presentation of neoantigens on MHC molecules on their surface. These non-self neoantigens distinguish tumor cells from normal cells to the immune system and are thus targets for cancer immunotherapy. The rapid development of molecular profiling platforms, such as next-generation sequencing, has enabled the generation of large datasets characterizing tumor cells. The simultaneous development of algorithms has enabled rapid and accurate processing of these data. Bioinformatic software tools encoding the algorithms can be strung together in a workflow to identify neoantigens. Here, with a focus on high-throughput sequencing, we review state-of-the art bioinformatic tools along with the steps and challenges involved in neoantigen identification and recognition.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Further, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

Publisher Correction: Actively personalized vaccination trial for newly diagnosed glioblastoma.

The additional author support information was erroneously omitted from the Supplementary Information. This has been corrected online.

Actively personalized vaccination trial for newly diagnosed glioblastoma.

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30-50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens-that is, both unmutated antigens and neoepitopes-may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-L-lysine carboxymethylcellulose) and granulocyte-macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.

Responsiveness to anti-PD-1 and anti-CTLA-4 immune checkpoint blockade in SB28 and GL261 mouse glioma models.

Immune checkpoint blockade (ICB) is currently evaluated in patients with glioblastoma (GBM), based on encouraging clinical data in other cancers, and results from studies with the methylcholanthrene-induced GL261 mouse glioma. In this paper, we describe a novel model faithfully recapitulating some key human GBM characteristics, including low mutational load, a factor reported as a prognostic indicator of ICB response. Consistent with this observation, SB28 is completely resistant to ICB, contrasting with treatment sensitivity of the more highly mutated GL261. Moreover, SB28 shows features of a poorly immunogenic tumor, with low MHC-I expression and modest CD8+ T-cell infiltration, suggesting that it may present similar challenges for immunotherapy as human GBM. Based on these key features for immune reactivity, SB28 may represent a treatment-resistant malignancy likely to mirror responses of many human tumors. We therefore propose that SB28 is a particularly suitable model for optimization of GBM immunotherapy.

Identification of Tumor Antigens Among the HLA Peptidomes of Glioblastoma Tumors and Plasma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor with poor prognosis to most patients. Immunotherapy of GBM is a potentially beneficial treatment option, whose optimal implementation may depend on familiarity with tumor specific antigens, presented as HLA peptides by the GBM cells. Furthermore, early detection of GBM, such as by a routine blood test, may improve survival, even with the current treatment modalities. This study includes large-scale analyses of the HLA peptidome (immunopeptidome) of the plasma-soluble HLA molecules (sHLA) of 142 plasma samples, and the membranal HLA of GBM tumors of 10 of these patients' tumor samples. Tumor samples were fresh-frozen immediately after surgery and the plasma samples were collected before, and at multiple visits after surgery. In total, this HLA peptidome analysis involved 52 different HLA allotypes and resulted in the identification of more than 35,000 different HLA peptides. Strong correlations were observed in the signal intensities and in the repertoires of identified peptides between the tumors and plasma-soluble HLA peptidomes of the individual patients, whereas low correlations were observed between these HLA peptidomes and the tumors' proteomes. HLA peptides derived from Cancer/Testis Antigens (CTAs) were selected based on their presence among the HLA peptidomes of the patients and absence of expression of their source genes from any healthy and essential human tissues, except from immune-privileged sites. Additionally, peptides were selected as potential biomarkers if their levels in the plasma-sHLA peptidome were significantly reduced after the removal of tumor mass. The CTAs identified among the analyzed HLA peptidomes provide new opportunities for personalized immunotherapy and for early diagnosis of GBM.

In Silico Typing of Classical and Non-classical HLA Alleles from Standard RNA-Seq Reads.

Next-Generation Sequencing (NGS) enables the rapid generation of billions of short nucleic acid sequence fragments (i.e., "sequencing reads"). Especially, the adoption of gene expression profiling using whole transcriptome sequencing (i.e., "RNA-Seq") has been rapid. Here, we describe an in silico method, seq2HLA, that takes standard RNA-Seq reads as input and determines a sample's (classical and non-classical) HLA class I and class II types as well as HLA expression. We demonstrate the application of seq2HLA using publicly available RNA-Seq data from the Burkitt's lymphoma cell line DAUDI and the choriocarcinoma cell line JEG-3.