Analysis Of Tumor Tissue Research Articles

EXTH-30. PHARMACOKINETICS- AND PHARMACODYNAMICS-BASED EVALUATION OF QUISINOSTAT AS A RADIOSENSITIZER IN PRECLINICAL MODELS OF GBM

Abstract Glioblastoma (GBM) is characterized by an aberrant yet druggable epigenetic landscape. Class I HDACs mediate chromatin compaction and are frequently overexpressed in GBM. Hence, there has been considerable interest in HDAC inhibitors (HDACi) for the treatment of malignant brain tumors. However, to date almost all HDACi tested clinically have failed to provide significant therapeutic benefit to primary and recurrent GBM patients. These HDACi are broad-spectrum with poor or unknown pharmacokinetic profiles and narrow therapeutic window. Isoform specificity for HDACi is important given that not all HDAC enzymes are equally expressed in GBM. Recently, we uncovered the functional importance of HDAC1 in therapy-resistant glioma stem cells, where we found that its expression increases with brain tumor grade and is correlated with decreased survival. While no class I HDAC isoform-specific inhibitors are currently available, the second-generation HDACi quisinostat harbors high specificity for HDAC1. In this study, we assessed the pharmacokinetic, pharmacodynamic and radiation-sensitizing properties of quisinostat in preclinical models of human GBM. In vitro analyses conducted in patient-derived glioma stem cell (GSC) lines revealed that quisinostat exhibited potent growth inhibition, induced global histone hyperacetylation, elevated DNA damage, cell death and cell cycle arrest. To determine the efficacy and drug distribution profile of quisinostat in vivo, athymic mice with orthotopic or flank tumors were treated with an optimized treatment schedule. At various timepoints during treatment, plasma and brain/tumor tissue were collected to measure total and unbound drug levels by liquid chromatography tandem mass spectrometry (LC-MS/MS), coupled with transcriptomic and pharmacodynamic analyses of tumor tissues. We demonstrate quisinostat is a brain-penetrant molecule that reduces tumor burden in flank and orthotopic models of GBM, and significantly extends survival when administered with radiation. Together, these results provide a rationale for developing quisinostat as a potential combination therapy with radiation for the treatment of GBM.

INNV-28. A PILOT STUDY EVALUATING THE FEASIBILITY OF USING AN ULTRA-FAST DIGITAL CONFOCAL MICROSCOPY SCANNER FOR REAL-TIME INTRA-OPERATIVE BRAIN TUMOUR DIAGNOSIS

Abstract INTRODUCTION Real-time intra-operative brain tumour tissue analysis in the operating room (OR) can shorten turnaround times and facilitate repeated sampling; the latter can improve diagnostic accuracy and guide resection to maximise extent. We evaluated the feasibility of using an ultra-fast confocal microscopy scanner (Histolog®, SamanTree Medical SA) in the OR for real-time intra-operative brain tumour tissue diagnosis. This study is the first use of Histolog® on primary human brain tissue globally. METHODS An observational pilot study with varying brain tumour types was conducted in a tertiary UK brain tumour centre. Multiple, freshly excised tissue samples were stained with Acridine Orange, and images captured within 60 seconds on Histolog® in the OR in parallel to current diagnostic pathways. A Consultant Neuropathologist performed a blinded correspondence assessment between Histolog® and current gold standard histopathology. Qualitative logging of diagnostic features was performed. RESULTS Cases studied (n=12) included glioma, metastasis, meningioma, schwannoma, and pituitary adenoma. Concordance between Histolog® and traditional (frozen section) intra-operative histopathological diagnosis was 83% (10/12 cases). One Histolog® case demonstrated superior diagnostic accuracy to frozen section. Histolog® images demonstrated specific diagnostic features such as clusters of cohesive epithelioid cells (metastatic carcinoma), sheet-like variably cellular and pleomorphic cells (gliomas), diffuse sheet-like monomorphic round nuclei (pituitary adenoma), elongated spindle cells (schwannoma), and nodular architecture and oval nuclei (meningioma). DISCUSSION Ultra-fast confocal microscopy scanning with Histolog® is feasible in the OR and Histolog® image resolution is adequate to identify histopathological features necessary for intra-operative brain tumour diagnosis. Training and clinico-radiological details are important in minimising discordance. A larger study with greater numbers and tumour types will now be performed to define an optimal scanning protocol and determine the strengths and limitations of the technology. Future studies should investigate the effect of real-time margin zone analysis on extending resection.

Classic and New Markers in Diagnostics and Classification of Breast Cancer.

Breast cancer remains the most frequently diagnosed form of female's cancer, and in recent years it has become the most common cause of cancer death in women worldwide. Like many other tumours, breast cancer is a histologically and biologically heterogeneous disease. In recent years, considerable progress has been made in diagnosis, subtyping, and complex treatment of breast cancer with the aim of providing best suited tumour-specific personalized therapy. Traditional methods for breast cancer diagnosis include mammography, MRI, biopsy and histological analysis of tumour tissue in order to determine classical markers such as estrogen and progesterone receptors (ER, PR), cytokeratins (CK5/6, CK14, C19), proliferation index (Ki67) and human epidermal growth factor type 2 receptor (HER2). In recent years, these methods have been supplemented by modern molecular methodologies such as next-generation sequencing, microRNA, in situ hybridization, and RT-qPCR to identify novel molecular biomarkers. MicroRNAs (miR-10b, miR-125b, miR145, miR-21, miR-155, mir-30, let-7, miR-25-3p), altered DNA methylation and mutations of specific genes (p16, BRCA1, RASSF1A, APC, GSTP1), circular RNA (hsa_circ_0072309, hsa_circRNA_0001785), circulating DNA and tumour cells, altered levels of specific proteins (apolipoprotein C-I), lipids, gene polymorphisms or nanoparticle enhanced imaging, all these are promising diagnostic and prognostic tools to disclose any specific features from the multifaceted nature of breast cancer to prepare best suited individualized therapy.

Novel plasma exosome biomarkers for prostate cancer progression in co-morbid metabolic disease

Comorbid Type 2 diabetes (T2D), a metabolic complication of obesity, associates with worse cancer outcomes for prostate, breast, head and neck, colorectal and several other solid tumors. However, the molecular mechanisms remain poorly understood. Emerging evidence shows that exosomes carry miRNAs in blood that encode the metabolic status of originating tissues and deliver their cargo to target tissues to modulate expression of critical genes. Exosomal communication potentially connects abnormal metabolism to cancer progression. Here, we hypothesized that T2D plasma exosomes induce epithelial-mesenchymal transition (EMT) and immune checkpoints in prostate cancer cells. We demonstrate that plasma exosomes from subjects with T2D induce EMT features in prostate cancer cells and upregulate the checkpoint genes CD274 and CD155. We demonstrate that specific exosomal miRNAs that are differentially abundant in plasma of T2D adults compared to nondiabetic controls (miR374a-5p, miR-93-5p and let-7b-3p) are delivered to cancer cells, thereby regulating critical target genes. We build on our previous reports showing BRD4 controls migration and dissemination of castration-resistant prostate cancer, and transcription of key EMT genes, to show that T2D exosomes require BRD4 to drive EMT and immune ligand expression. We validate our findings with gene set enrichment analysis of human prostate tumor tissue in TGCA genomic data. These results suggest novel, non-invasive approaches to evaluate and potentially block progression of prostate and other cancers in patients with comorbid T2D.

A Sampling of Highlights from the Literature.

Macrophages can be reprogrammed to promote antitumor responses (by Noah Smith via Wikimedia Commons).Multiple factors contribute to the suppression that occurs in the tumor microenvironment (TME) and this could potentially be alleviated through various mechanisms. Chen et al. find that high potassium in the TME inhibits antitumor responses from tumor-associated macrophages (TAM) via alteration of their metabolism. Blocking this metabolic switch enables TAMs to mount effective antitumor responses. Chryplewicz et al. find that TAMs in glioma are reprogrammed to a proinflammatory phenotype, along with other antitumor remodeling of the TME and tumor vasculature, after treatment with VEGF inhibitors and imipramine (tricyclic anti-depressant). These studies provide insight into multiple mechanisms of TAM suppression and potential targets that can be used to boost antitumor responses.Chen S, …, Wang D. Cell Metab 2022 September 13. DOI:10.1016/j.cmet.2022.08.016.Chryplewicz A, …, Hanahan D. Cancer Cell 2022 September 15;40:1111–27.E9.Dietary intake can affect microbiota and immunity (by the NCI via Wikimedia Commons).Dietary intake can affect antitumor immunity and response to immunotherapy. In a prospective study, Simpson et al. evaluated dietary patterns and microbial signatures of different patient populations and how these factors related to response to neoadjuvant immune checkpoint inhibition. Distinct differences between response groups were observed not only in microbial composition but also in immune-related adverse events. Specific diets, as well as certain microbial compositions, were associated with better treatment outcomes. The data highlight the crossroads of cancer-intrinsic and -extrinsic factors with patient lifestyle, and how these factors together can shape responses to treatment.Simpson RC, …, Long GV. Nat Med 2022 September 12. DOI:10.1038/s41591-022-01965-2.Immunotherapy can overcome resistance to HER2-targeted therapy (from IrinaPav via Wikimedia Commons).HER2-specific antibodies such as trastuzumab are used to treat HER2+ breast cancer, but not all patients respond and some that do ultimately relapse. Using orthogonal approaches, including analysis of tumor tissue and transcriptomic data from trastuzumab-treated patients with HER2+ breast cancer and a fully humanized immunocompetent model of HER2+ breast cancer, Rivas et al. find that a population of TGFβ-activated cancer-associated fibroblasts (CAF) and low IL2 activity associate with trastuzumab resistance. Targeting IL2 to fibroblast activation protein-α (FAP)–expressing cells restores tumor responsiveness to trastuzumab by enhancing NK cell–mediated antibody-dependent cytotoxicity, highlighting the therapeutic potential of targeting stromal components in the tumor microenvironment.Rivas EI, …, Calon A. Nat Commun 2022 September 9;13:5310.A Treg subpopulation of CAR T cells drives therapeutic resistance (from NIH via Flickr).Using single-cell profiling methods, two groups have identified subpopulations of CD19-targeting CAR T cells with T regulatory (Treg)–cell phenotypes associated with poor response in patients with B-cell lymphoma. Good et al. find a higher proportion of circulating CD4+Helios– Treg CAR T cells in patients with progressive disease who received axicabtagene ciloleucel (axi-cel), highlighting their potential as a biomarker prognostic for progression at day 7 post infusion. Haradhvala et al. similarly identify CAR+ Treg cells in blood and infusion products for patients nonresponsive to either axi-cel or tisagenlecleucel, and validate their role in mouse models of CAR T-cell treatment. The studies provide new insight into therapeutic resistance to CAR T-cell therapy.Good Z, …, Mackall CL. Nat Med 2022 September 12;28:1860–71.Haradhvala NJ, …, Maus MV. Nat Med 2022 September 12;28:1848–59.Covalent K-Ras(G12C) inhibitor provides a new target for immunotherapy (from Rawpixel).MHC class I presentation of tumor-specific peptides derived from intracellular proteins provides a target for anticancer therapy. Zhang et al. have identified a new class of therapeutically targetable MHC class I epitopes: covalently modified intracellular proteins. K-Ras(G12C) covalently conjugated to the K-Ras(G12C)-specific inhibitor ARS1620 can undergo antigen processing and presentation on MHC class I. ARS1620-specific antibodies can recognize MHC class I–restricted ARS1620-peptides. Incorporating such an antibody into a bispecific T-cell engager (BiTE) induces T-cell responses against K-Ras(G12C) tumor cells, suggesting a new strategy for cancer immunotherapy.Zhang Z, …, Craik CS. Cancer Cell 2022 September 12;40:1060–69.e7.NK cells are key component of the antitumor immune response (from NIAID via Wikimedia Commons).Understanding the factors that regulate the maturation and function of natural killer (NK) cells is important for understanding the antitumor immune response. Imianowski et al. show that the transcriptional regulator BACH2 is expressed in developing and mature NK cells and is a negative regulator of NK-cell maturation and function, keeping NK cells quiescent under conditions of IL15 stimulation. In mice lacking BACH2 in NK cells, the NK cells show increased cytotoxicity and enhanced ability to limit metastasis to the lungs in two preclinical models. The data provide new avenues for potential therapeutic modulation of NK cell–mediated antitumor immunity.Imianowski CJ, …, Roychoudhuri R. J Exp Med 2022 September 30;219:e20211476.

Increased FOXJ1 protein expression is associated with improved overall survival in high-grade serous ovarian carcinoma: an Ovarian Tumor Tissue Analysis Consortium Study

BackgroundRecently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC.MethodsTwo prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis.ResultsConsistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67–0.91, p < 0.0001). GMNN protein expression was not significantly associated with overall HSGC patient survival. However, HGSCs with >35% GMNN expression showed a trend for better outcomes, though this was not significant.ConclusionWe provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

188P Spatial transcriptomic analysis of tumor tissue in ovarian cancer patients treated with neoadjuvant chemotherapy

Ovarian cancer (OC) is one of the most common gynecological cancers, with the worst prognosis and the highest mortality rate out of gynecological diseases. Despite a good response to the first line of standard neoadjuvant chemotherapy (NAC), the relapses in patients with sporadic ovarian cancer are detected within a short period of time in 70% of cases. The most relevant task is to identify the key features of tumor tissue of non-responders and to find out how these features can affect the development and outcome of OC.

Low Dose Fractionated Radiotherapy Alters Tumor Blood Vessel Morphology, Increases CD3+CD8+ T Cell Infiltration to the Tumor, and Sensitizes Mesothelioma to Immune Checkpoint Blockade

<h3>Purpose/Objective(s)</h3> Radiotherapy is widely used to palliate symptomatic lesions in patients with mesothelioma. However, radiotherapy alone does not improve patient survival. There is currently little published data on radiotherapy and immunotherapy combination in mesothelioma. Here, we aim to optimize a clinically relevant radiotherapy and immunotherapy combination by pre-conditioning tumor microenvironment using low dose radiotherapy for effective immunotherapy responses using mouse model of mesothelioma. <h3>Materials/Methods</h3> Mice were implanted in the flank with 1 × 10^5 murine AB1-HA mesothelioma cells and radiotherapy treatment commenced on day 10 once tumors were approximately 20-30 mm². Treatment was delivered using an X-RAD SmART small animal image-guided precision irradiator at 225 kV X-RAY (HVL 1mm Cu) with a dose rate of 3 Gy/min. Animals were treated with either 0 Gy (sham-irradiation), or as single or multiple doses of either 2 Gy or 6 Gy over the course of five days. Tumors were harvested two weeks following radiotherapy to assess vessel morphology, T cell infiltration by immunohistochemistry and gene expression using RNA-sequencing. In subsequent experiments, the combination of immunotherapy (anti-PD-1 and anti-CTLA-4) with radiotherapy (2 Gy × 5) were administered concurrently and after radiotherapy as one day and six days apart after the final dose. <h3>Results</h3> Radiotherapy did not change the blood vessel density, pericyte coverage and the basement of the blood vessels (collagen-IV) between sham- and radiotherapy treated groups, suggesting the prescribed radiotherapy doses did not damage the blood vessels. Interestingly, the group receiving radiotherapy dose of 2 Gy × 5 had their vessel length and diameter reduced compared with sham-radiotherapy group and these features are similar to normal vessel characteristics. Analysis of tumor tissues revealed significant influx of CD3⁺CD8⁺ T cells following radiotherapy done of 2 Gy × 5 compared with sham radiotherapy group. Gene expression analysis demonstrated inflammatory response gene sets such as type I interferon and tumor necrotic factor-alpha were upregulated in group receiving radiotherapy of 2 Gy × 5, compared with sham radiotherapy group indicating tumor microenvironment may have been an inflamed "hot tumor" following radiotherapy. The combination of anti-PD-1 monotherapy with radiotherapy cured 50 % of the tumor bearing mice. This low cure rate was increased to 100% once dual immunotherapy (anti-PD-1/anti-CTLA-4) was administered concurrently with radiotherapy. All cured mice rejected secondary tumor rechallenge, suggesting cured mice may have developed immunological memory. <h3>Conclusion</h3> Our preliminary finding suggests radiotherapy dose of 2 Gy × 5 was optimal to normalize the tumor blood vessels, increased T cell infiltration, and induced transcriptomic changes associated with inflammatory responses, which may sensitize mesothelioma to immunotherapy. The combination of anti-CTLA-4/anti-PD-1 and radiotherapy were required to achieve 100% cure rate.

Laser microdissection for the analysis of molecular heterogeneity in colorectal cancer

Oncogenic driver mutations in RAS/RAF oncogenes are frequent in colorectal cancer (CRC). The presence of different subclones within asingle tumor can lead to treatment failure in anti-EGFR/epidermal growth factor receptor-directed antibody therapies. The identification of different subclones and their mutational profiles within asingle tumor and the identification of morphologically distinct tumor areas might help to unravel novel aspects of tumor biology and therapy resistance. The aim of this study was to identify intratumoral heterogeneity in CRC by using laser microdissection (LMD) in comparison to the routinely used method. We hereby applied LMD to identify and investigate tumor heterogeneity in CRC. We established LMD and purified DNA from several morphologically distinct tumor areas (n = 13) in CRCs from 2patients and compared the results from routine testing to our newly established LMD approach. LMD enabled the comparative analysis of small tumor areas by cutting histologically selected elements under microscopic control using alaser beam. In some cases, potential low-level mutations (PLLM) could not be detected using the routine method since they were masked by high-level mutations (HLM). The application of LMD enabled the identification of concomitant PLLM in NRAS and BRAF genes in the identical patient sample. LMD improved spatial resolution in the molecular analysis of CRC tumor tissue compared to routine methods. Our results confirmed the presence of molecular heterogeneity in CRC. This should be kept in mind when interpreting sequencing results, since low frequency mutations can have an impact on the effectiveness of targeted therapy.

Scaffold-mediated switching of lymphoma metabolism in culture

BackgroundDiffuse large B cell lymphoma (DLBCL) is an aggressive subtype of non-Hodgkin lymphoma (NHL) and accounts for about a third of all NHL cases. A significant proportion (~40%) of treated DLBCL patients develop refractory or relapsed disease due to drug resistance which can be attributed to metabolomic and genetic variations amongst diverse DLBCL subtypes. An assay platform that reproduces metabolic patterns of DLBCL in vivo could serve as a useful model for DLBCL.MethodsThis report investigated metabolic functions in 2D and 3D cell cultures using parental and drug-resistant DLBCL cell lines as compared to patient biopsy tissue.ResultsA 3D culture model controlled the proliferation of parental and drug-resistant DLBCL cell lines, SUDHL-10, SUDHL-10 RR (rituximab resistant), and SUDHL-10 OR (obinutuzumab resistant), as well as retained differential sensitivity to CHOP. The results from metabolic profiling and isotope tracer studies with d-glucose-13C6 indicated metabolic switching in 3D culture when compared with a 2D environment. Analysis of DLBCL patient tumor tissue revealed that the metabolic changes in 3D grown cells were shifted towards that of clinical specimens.Conclusion3D culture restrained DLBCL cell line growth and modulated metabolic pathways that trend towards the biological characteristics of patient tumors. Counter-intuitively, this research thereby contends that 3D matrices can be a tool to control tumor function towards a slower growing and metabolically dormant state that better reflects in vivo tumor physiology.

Gene-Expression Profiling of Mucinous Ovarian Tumors and Comparison with Upper and Lower Gastrointestinal Tumors Identifies Markers Associated with Adverse Outcomes.

Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features. Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55). Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04-7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04-1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01-1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%). An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.

The Immunocytokine FAP-IL-2v Enhances Anti-Neuroblastoma Efficacy of the Anti-GD2 Antibody Dinutuximab Beta.

Simple SummarySince IL-2 co-treatment did not show any therapeutic benefit in the GD2-directed treatment of high-risk neuroblastoma (NB) but strongly induced regulatory T cells (Treg), we investigated here the immunocytokine FAP-IL-2v stimulating NK and cytotoxic T cells without induction of Treg. We first detected FAP on NB- and myeloid-derived suppressor cells (MDCS) in tumor tissue and showed a tumor-cell-dependent enhancement in FAP expression by fibroblasts. Treatment of leukocytes with FAP-IL-2v increased ADCC mediated by the anti-GD2 antibody dinutuximab beta (DB) against NB cells. We next evaluated the antitumor efficacy of a combinatorial immunotherapy by applying DB and FAP-IL-2v and observed strongly reduced tumor growth and improved survival in experimental mice. Analysis of tumor tissue revealed increased NK and cytotoxic T cell numbers and reduced Treg compared to controls. Our data show that FAP-IL-2v is a potent immunocytokine that augments the efficacy of DB against NB, providing a promising alternative to IL-2.Treatment of high-risk neuroblastoma (NB) patients with the anti-GD2 antibody (Ab) dinutuximab beta (DB) improves survival by 15%. Ab-dependent cellular cytotoxicity (ADCC) is the major mechanism of action and is primarily mediated by NK cells. Since IL-2 co-treatment did not show a therapeutic benefit but strongly induced Treg, we investigated here a DB-based immunotherapy combined with the immunocytokine FAP-IL-2v, which comprises a fibroblast activation protein α (FAP)-specific Ab linked to a mutated IL-2 variant (IL-2v) with abolished binding to the high-affinity IL-2 receptor, thus stimulating NK cells without induction of Treg. Effects of FAP-IL-2v on NK cells, Treg and ADCC mediated by DB, as well as FAP expression in NB, were investigated by flow cytometry, calcein-AM-based cytotoxicity assay and RT-PCR analysis. Moreover, the impact of soluble factors released from tumor cells on FAP expression by primary fibroblasts was assessed. Finally, a combined immunotherapy with DB and FAP-IL-2v was evaluated using a resistant syngeneic murine NB model. Incubation of leukocytes with FAP-IL-2v enhanced DB-specific ADCC without induction of Treg. FAP expression on NB cells and myeloid-derived suppressor cells (MDCS) in tumor tissue was identified. A tumor-cell-dependent enhancement in FAP expression by primary fibroblasts was demonstrated. Combination with DB and FAP-IL-2v resulted in reduced tumor growth and improved survival. Analysis of tumor tissue revealed increased NK and cytotoxic T cell numbers and reduced Treg compared to controls. Our data show that FAP-IL-2v is a potent immunocytokine that augments the efficacy of DB against NB, providing a promising alternative to IL-2.

134P Whole transcriptome RNA-Seq analysis of formalin-fixed and paraffin-embedded tumour tissue

Pure uterine sarcomas are rare tumours which account for less than 3 % of all uterine malignancies. Knowledge about their molecular background is limited. Identification of new molecular pattern and new molecular markers could help to better stratify these tumours in terms of their diagnostic or therapeutic options. Whole transcriptome RNA-Seq is a robust tool which could reveal novel gene fusions, aberrant splicing events, or gene expression. However, optimization of library preparation from archive specimens (FFPE) is crucial for a retrospective study, which is necessary due to the rarity of these tumours.

Design and characterizing of robust probes for enhanced mass spectrometry imaging and spatially resolved metabolomics

As for the emerging and cut edge spatially resolved metabolomics, mass spectrometry imaging (MSI) is a powerful tool that can map thousands of metabolites from bio-tissue sections without chemical labels. However, the stability, sensitivity and spatial resolution of MSI are always limited by the performance of its ionization probe. Herein, two types of probes (fine probe (P-100) and large probe (P-200)) were designed and characterized to perform air-flow assisted desorption electrospray ionization (AFA-DESI) MSI analysis for spatially resolved metabolomics. It was determined that the spray introduced by P-100 was homogenous and stable under the spray solvent at a flow rate of 5-10 µL/min, while P-200 can endure a high flow rate of up to 10-30 µL/min. Moreover, the MSI images were acquired by AFA-DESI-MSI with P-100 from rat brain tissue section and with P-200 from whole-body tissue section of mouse, and these results presented unambiguous tissue structure with the distribution information of numerous metabolites. Furthermore, the spatially resolved metabolomic analysis of tumor tissue was successfully realized to discover the tumor associated biomarkers. As the key parts of AFA-DESI-MSI system, it has been demonstrated that the designed probs have excellent performance for spatially resolved metabolomics, and it will further promote its application in life science, and drug research and development.

Innovations in the Diagnosis and Surgical Management of Low-Grade Gliomas

Low-grade gliomas are a broad category of tumors that can manifest at different stages of life. As a group, their prognosis has historically been considered to be favorable, and surgery is a mainstay of treatment. Advances in the molecular characterization of individual lesions has led to newer classification systems, a better understanding of the biological behavior of different neoplasms, and the identification of previously unrecognized entities. New prospective genetic and molecular data will help delineate better treatment paradigms and will continue to change the taxonomy of central nervous system tumors in the coming years. Advances in the field of radiomics will help predict the molecular profile of a particular tumor through noninvasive testing. Similarly, more precise methods of intraoperative tumor tissue analysis will aid surgical planning. Improved surgical outcomes propelled by novel surgical techniques and intraoperative adjuncts and emerging forms of medical treatment in the field of immunotherapy have enriched the management of these lesions. We review the contemporary management and innovations in the treatment of low-grade gliomas.

Characteristics of homologous recombination repair pathway genes mutation in ovarian cancers.

Few studies have investigated the characteristics of non-BRCA homologous recombination repair (HRR) pathway somatic mutations, and the impact of these mutations on efficacy of treatment in ovarian cancer patients is not clear. Therefore, we conducted this study to analyze the frequency and spectrum of somatic mutations in HRR pathway genes in patients with ovarian cancer and to examine the relationships between somatic mutations in HRR pathway genes and their effects on the efficacy of platinum-based chemotherapy. We performed targeted sequencing of 688 genes related to the occurrence, development, treatment, and prognosis of solid tumors. Somatic mutations were identified by paired analysis of tumor tissue and germline DNA in blood cells. A total of 38 patients with ovarian cancer were included in the study, and 35 (92.1%) patients were diagnosed with high-grade serous carcinoma. All patients exhibited somatic mutations in the tumor tissue samples. The commonly mutated genes were TP53 (73.7%), BRCA2 (55.3%), NF1 (52.6%), BRCA1 (47.4%), and CDH1 (47.4%). Overall, 71.1% of the patients exhibited mutation in at least one HRR pathway gene. The most frequently altered HRR genes were BRCA2 (55.3%), followed by BRCA1 (47.4%), ATM (44.7%), BARD1 (42.1%), and CHEK1 (36.8%). The median progression-free survival (PFS) in patients with HRR pathway mutation was 36.0 months compared with 13.6 months in patients with no HRR pathway mutation (hazard ratio [HR], 0.25; 95% confidence interval [CI], 0.08-0.77; p = 0.016). Patients harboring BRCA1/2 and/or CDK12 mutations displayed a longer PFS (median, 36.0 months) compared with patients with no BRCA1/2 or CDK12 mutation (median, 13.6 months; HR, 0.21; 95% CI, 0.07-0.61; p = 0.004). In multivariate analysis Cox proportional hazards models, after adjustment for tumor stage at diagnosis and histology of initial diagnosis, patients with HRR pathway mutation had a longer PFS than patients with HRR wild-type genes (p = 0.006). HRR pathway somatic mutations are common in Chinese patients with ovarian cancer. HRR pathway somatic mutations were associated with improved sensitivity to platinum-based chemotherapy. Large-scale prospective studies are needed to verify our findings.

Tumor purity adjusted beta values improve biological interpretability of high-dimensional DNA methylation data.

A common issue affecting DNA methylation analysis in tumor tissue is the presence of a substantial amount of non-tumor methylation signal derived from the surrounding microenvironment. Although approaches for quantifying and correcting for the infiltration component have been proposed previously, we believe these have not fully addressed the issue in a comprehensive and universally applicable way. We present a multi-population framework for adjusting DNA methylation beta values on the Illumina 450/850K platform using generic purity estimates to account for non-tumor signal. Our approach also provides an indirect estimate of the aggregate methylation state of the surrounding normal tissue. Using whole exome sequencing derived purity estimates and Illumina 450K methylation array data generated by The Cancer Genome Atlas project (TCGA), we provide a demonstration of this framework in breast cancer illustrating the effect of beta correction on the aggregate methylation beta value distribution, clustering accuracy, and global methylation profiles.

Tumor-derived exosomes deliver the tumor suppressor miR-3591-3p to induce M2 macrophage polarization and promote glioma progression

Exosomes can selectively secrete harmful metabolic substances from cells to maintain cellular homeostasis, and complex crosstalk occurs between exosomes and tumor-associated macrophages (TAMs) in the glioma immune microenvironment. However, the precise mechanisms by which these exosome-encapsulated cargos create an immunosuppressive microenvironment remain unclear. Herein, we investigated the effect of glioma-derived exosomes (GDEs) on macrophage polarization and glioma progression. We performed sequencing analysis of cerebrospinal fluid (CSF) and tumor tissues from glioma patients to identify functional microRNAs (miRNAs). High levels of miR-3591-3p were found in CSF and GDEs but not in normal brain tissue or glial cells. Functionally, GDEs and miR-3591-3p significantly induced M2 macrophage polarization and increased the secretion of IL10 and TGFβ1, which in turn promoted glioma invasion and migration. Moreover, miR-3591-3p overexpression in glioma cell lines resulted in G2/M arrest and markedly increased apoptosis. Mechanistically, miR-3591-3p can directly target CBLB and MAPK1 in macrophages and glioma cells, respectively, and further activate the JAK2/PI3K/AKT/mTOR, JAK2/STAT3, and MAPK signaling pathways. In vivo experiments confirmed that macrophages lentivirally transduced with miR-3591-3p can significantly promote glioma progression. Thus, our study demonstrates that tumor-suppressive miR-3591-3p in glioma cells can be secreted via exosomes and target TAMs to induce the formation of an immunosuppressive microenvironment. Collectively, these findings provide new insights into the role of glioma exosomal miRNAs in mediating the establishment of an immunosuppressive tumor microenvironment and show that miR-3591-3p may be a valuable biomarker and that blocking the encapsulation of miR-3591-3p into exosomes may become a novel immunotherapeutic strategy for glioma.

MA12.04 SAKK 16/14: CD8 T Cell Positioning Correlates with Survivalin Stage IIIA(N2) NSCLC After Neoadjuvant Immunotherapy

Spatial distribution of immune cells within the tumor immune microenvironment (TIME) influences response to immunotherapy in many malignancies, but its role in neoadjuvant immunotherapy in non-small cell lung cancer (NSCLC) is not clearly established. To address this knowledge gap, we performed digital pathology analysis of tumor tissue obtained from patients with stage IIIA(N2) NSCLC undergoing neoadjuvant chemotherapy with three cycles of cisplatin/docetaxel followed by treatment with the PD-L1 antibody durvalumab in the single-arm phase II trial SAKK 16/14.

490P Safety and efficacy of tusamitamab ravtansine in patients with colorectal or gastric cancer expressing carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5)

In the first-in-human study of tusamitamab ravtansine (NCT02187848), an anti-CEACAM5 antibody-drug conjugate, 100 mg/m2 every 2 weeks (Q2W) showed antitumor activity in heavily pretreated patients with nonsquamous non-small cell lung cancer with high CEACAM5 expression. In the dose-escalation phase, 2 of 18 patients with colorectal cancer (CRC) and 1 of 7 with gastric cancer (GC) had confirmed partial responses (PR). Here, we report results from the CRC and GC dose-expansion cohorts. Patients were ≥ 18 years with refractory CRC (all comers) or GC. In the GC cohort, inclusion was restricted to those with high CEACAM5 expression (≥ 2+ intensity in ≥ 50% of tumor cells, as assessed by immunohistochemical analysis of tumor tissue). All patients received tusamitamab ravtansine 100 mg/m2 Q2W. The primary efficacy endpoint was overall objective response. Time to progression (TTP) and treatment-emergent adverse events (AEs) were also assessed. As of November 19, 2020, 46 patients with CRC and 16 with GC were treated. In the CRC cohort, the median age was 62 years (range, 36–77; 32.6% ≥ 65 years) and 84.1% of patients had high CEACAM5 expression. In the GC cohort, the median age was 59 years (range, 35–77; 37.6% ≥ 65 years). Patients in the CRC and GC cohorts had a median of 4 and 3 prior regimens. In the CRC cohort, the best overall response (BOR) was stable disease (SD) in 12 patients (26.1%) and a median TTP of 1.8 months (95% confidence interval [CI], 1.64–1.91). One patient had an unconfirmed PR. In the GC cohort, the BOR was SD in 6 patients (37.5%) and a median TTP of 1.7 months (95% CI, 1.18-2.60). Treatment-related AEs occurred in 34 (73.9%) and 9 (56.3%) patients in the CRC and GC cohorts. Corneal AEs (keratitis and keratopathy) occurred in 13 (28.3%) and 2 (12.5%) patients in the CRC and GC cohorts, leading to treatment modification in 5 (10.9%) patients and 1 (6.3%) patient. Grade ≥ 3 corneal AEs occurred in 2 patients with CRC and none with GC. Corneal toxicity was manageable with dose delay or modification. Tusamitamab ravtansine was well tolerated in patients with CRC or GC.