In-depth Molecular Profiling Research Articles

Dissecting the Natural Patterns of Progression and Senescence in Pediatric Low-Grade Glioma: From Cellular Mechanisms to Clinical Implications.

Pediatric low-grade gliomas (PLGGs) comprise a heterogeneous set of low-grade glial and glioneuronal tumors, collectively representing the most frequent CNS tumors of childhood and adolescence. Despite excellent overall survival rates, the chronic nature of the disease bears a high risk of long-term disease- and therapy-related morbidity in affected patients. Recent in-depth molecular profiling and studies of the genetic landscape of PLGGs led to the discovery of the paramount role of frequent upregulation of RAS/MAPK and mTOR signaling in tumorigenesis and progression of these tumors. Beyond, the subsequent unveiling of RAS/MAPK-driven oncogene-induced senescence in these tumors may shape the understanding of the molecular mechanisms determining the versatile progression patterns of PLGGs, potentially providing a promising target for novel therapies. Recent in vitro and in vivo studies moreover indicate a strong dependence of PLGG formation and growth on the tumor microenvironment. In this work, we provide an overview of the current understanding of the multilayered cellular mechanisms and clinical factors determining the natural progression patterns and the characteristic biological behavior of these tumors, aiming to provide a foundation for advanced stratification for the management of these tumors within a multimodal treatment approach.

Advancing drug development by leveraging microdissection for proteogenomic analysis of histological sections.

e14660 Background: Combining laser capture microdissection (LCM) with proteogenomics in cancer research offers a targeted way to way to explore the intricate interactions between the tumor microenvironment and tumor cells proper. For immuno-oncology (IO) research, this is especially important, since improvements in the predictability of patient response to IO-based drugs is greatly needed. An improved understanding of the spatial relationships involving tumor cells, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: From fresh frozen tissue, LCM is used to isolate and obtain distinct histological cell types. Nucleic acids and proteins are extracted from captured cells for in-depth multi-modality molecular profiling assays. This includes multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing the analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, DNA sequencing can be performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. RNA-seq may be performed for gene expression. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. Our novel proteogenomics approach is focused around a customized enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: A proteogenomic approach is reported that is utilizing amount limited tissue quantities from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

Abstract 730: Leveraging microdissection for proteogenomic analysis of histological sections to advance drug development

Abstract Background: Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the tumor microenvironment. This is especially important for immuno-oncology (IO) research where improvements in the predictability of patient response to IO-based drugs is sorely needed. An improved understanding of the spatial relationships involving tumor cells proper, stromal cells, blood supply and immune cell interactions may help to inform drug development and improve the indicators of success for IO-based drug regimens. Methods: LCM is used to isolate and obtain distinct histological cell types from fresh frozen tissue. Once cells have been captured, nucleic acids and proteins are extracted for in-depth multi-modality molecular profiling assays involving multiple genomic modalities and a customized solution-based mass spectrometry proteomics approach for samples or specimens with a limited amount of tissue. Results: Optimizing analysis of minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, and methylation profiles. Regarding genomic-based studies, commercial kits are often utilized for these steps due to their general availability, rapid innovation, and the robust performance of these products. However, this kit-based adaptive approach is not true for proteomics. There remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of our proteogenomics approach is focused around an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. This includes an in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Conclusions: Reported here is a proteogenomic approach that is leveraging from microdissected fresh frozen tissue. The methodology may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume. Citation Format: Ik Jae Shin, Michael Tangrea, Michael R. Emmert-Buck, Donald J. Johann. Leveraging microdissection for proteogenomic analysis of histological sections to advance drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 730.

A Microdissection Protocol for Proteogenomic Analysis of Histological Sections to Advance Drug Development.

Combining proteogenomics with laser capture microdissection (LCM) in cancer research offers a targeted way to explore the intricate interactions between tumor cells and the different microenvironment components. This is especially important for immuno-oncology (IO) research where improvements in the predictability of IO-based drugs are sorely needed, and depends on a better understanding of the spatial relationships involving the tumor, blood supply, and immune cell interactions, in the context of their associated microenvironments. LCM is used to isolate and obtain distinct histological cell types, which may be routinely performed on complex and heterogeneous solid tumor specimens. Once cells have been captured, nucleic acids and proteins may be extracted for in-depth multimodality molecular profiling assays. Optimizing the minute tissue quantities from LCM captured cells is challenging. Following the isolation of nucleic acids, RNA-seq may be performed for gene expression and DNA sequencing performed for the discovery and analysis of actionable mutations, copy number variation, methylation profiles, etc. However, there remains a need for highly sensitive proteomic methods targeting small-sized samples. A significant part of this protocol is an enhanced liquid chromatography mass spectrometry (LC-MS) analysis of micro-scale and/or nano-scale tissue sections. This is achieved with a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) approach developed for LC-MS analysis of fresh-frozen tissue specimens obtained via LCM. Included is a detailed in-gel digestion method adjusted and specifically designed to maximize the proteome coverage from amount-limited LCM samples to better facilitate in-depth molecular profiling. Described is a proteogenomic approach leveraged from microdissected fresh frozen tissue. The protocols may also be applicable to other types of specimens having limited nucleic acids, protein quantity, and/or sample volume.

In-depth molecular profiling of an intronic GNAO1 mutant as the basis for personalized high-throughput drug screening

The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants. In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery. We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gαo. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracellular localization, Gβγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged. This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this analysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gαo. This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.

Mutational Impacts on the N and C Terminal Domains of the MUC5B Protein: A Transcriptomics and Structural Biology Study.

Cholangiocarcinoma (CCA) involves various epithelial tumors historically linked with poor prognosis because of its aggressive sickness course, delayed diagnosis, and limited efficacy of typical chemotherapy in its advanced stages. In-depth molecular profiling has exposed a varied scenery of genomic alterations as CCA's oncogenic drivers. Previous studies have mainly focused on commonly occurring TP53 and KRAS alterations, but there is limited research conducted to explore other vital genes involved in CCA. We retrieved data from The Cancer Genome Atlas (TCGA) to hunt for additional CCA targets and plotted a mutational landscape, identifying key genes and their frequently expressed variants. Next, we performed a survival analysis for all of the top genes to shortlist the ones with better significance. Among those genes, we observed that MUC5B has the most significant p-value of 0.0061. Finally, we chose two missense mutations at different positions in the vicinity of MUC5B N and C terminal domains. These mutations were further subjected to molecular dynamics (MD) simulation, which revealed noticeable impacts on the protein structure. Our study not only reveals one of the highly mutated genes with enhanced significance in CCA but also gives insights into the influence of its variants. We believe these findings are a good asset for understanding CCA from genomics and structural biology perspectives.

Robust quantitation of gangliosides and sulfatides in human brain using UHPLC-MRM-MS: Method development and application in Alzheimer's disease

Gangliosides (GAs) and sulfatides (STs) are acidic glycosphingolipids that are particularly abundant in the nervous system and are closely related to aging and neurodegenerative disorders. To explore their roles in brain diseases, in-depth molecular profiling, including structural variations of sphingoid backbone, fatty acyl group, and sugar chain of GAs and STs was performed. A total of 210 GAs and 38 STs were characterized in the inferior frontal gyrus (IFG) of human brain, with 90 GAs discovered in brain tissues for the first time. Influential MS parameters for detecting GAs and STs in multiple reaction monitoring (MRM) mode were systematically examined and optimized to minimize in-source fragmentation, resulting in remarkable signal intensity enhancement for GAs and STs, especially for polysialylated species. To eliminate analytical variations, isotopic interference-free internal standards were prepared by simple and fast reduction reaction. The final established method facilitated the simultaneous quantitation of 184 GAs and 30 STs from 25 subtypes, which represents the highest number of GAs quantitated among all quantitation methods recorded in literature so far. The method was further validated and applied to reveal the aberrant change of GAs and STs in the IFG of 12 Alzheimer's disease (AD) patients. Four GAs exhibited high classification capacity for AD (AUC ≥0.80) and were thereby considered the most promising signatures for AD. These findings suggested the close correlation between GAs and the pathogenesis of AD, highlighting the achievements of our robust method for investigating the roles of GAs and STs in various physiological states and diseases.

A patient with two gliomas with independent oligodendroglioma and glioblastoma biology proved by DNA-methylation profiling: a case report and review of the literature

Here, we report on a patient presenting with two histopathologically distinct gliomas. At the age of 42, the patient underwent initial resection of a right temporal oligodendroglioma IDH mutated 1p/19q co-deleted WHO Grade II followed by adjuvant radiochemotherapy with temozolomide. 15 months after initial diagnosis, the patient showed right hemispheric tumor progression and an additional new left frontal contrast enhancement in the subsequent imaging. A re-resection of the right-sided tumor and resection of the left frontal tumor were conducted. Neuropathological work-up showed recurrence of the right-sided oligodendroglioma with features of an anaplastic oligodendroglioma WHO Grade III, but a glioblastoma WHO grade IV for the left frontal lesion. In depth molecular profiling revealed two independent brain tumors with distinct molecular profiles of anaplastic oligodendroglioma IDH mutated 1p/19q co-deleted WHO Grade III and glioblastoma IDH wildtype WHO grade IV. This unique and rare case of a patient with two independent brain tumors revealed by in-depth molecular work-up and epigenomic profiling emphasizes the importance of integrated work-up of brain tumors including methylome profiling for advanced patient care.

An Observational Study on the Molecular Profiling of Primary Melanomas Reveals a Progression Dependence on Mitochondrial Activation.

Simple SummaryMalignant melanoma is the deadliest among the skin cancers. In the advanced stage, it shows extremely poor prognosis. The incidence and mortality of melanoma has been continuously increasing worldwide, primarily among the fair-skinned populations. To date, the best indicator for progression is the tumor thickness (Breslow). In this sense, there is a fundamental need to discover early indicators of disease progression. We believe that in-depth molecular profiling at the multiomic level of primary melanomas may find a signature that predicts disease progression toward metastasis. Here we report an observational study of a small primary melanoma cohort, in which in-depth molecular profiling was performed at the levels of protein expression, phosphorylation, and lysine acetylation. Our results provided evidence for dysregulation of pathways and biological processes at all molecular levels evaluated, in primary tumors from patients that ultimately developed a metastatic disease. Moreover, we provided evidence at the molecular level of the sex-related differences in the melanoma disease presentation.Melanoma in advanced stages is one of the most aggressive tumors and the deadliest of skin cancers. To date, the histopathological staging focuses on tumor thickness, and clinical staging is a major estimate of the clinical behavior of primary melanoma. Here we report on an observational study with in-depth molecular profiling at the protein level including post-translational modifications (PTMs) on eleven primary tumors from melanoma patients. Global proteomics, phosphoproteomics, and acetylomics were performed on each sample. We observed an up-regulation of key mitochondrial functions, including the mitochondrial translation machinery and the down-regulation of structural proteins involved in cell adhesion, the cytoskeleton organization, and epidermis development, which dictates the progression of the disease. Additionally, the PTM level pathways related to RNA processing and transport, as well as chromatin organization, were dysregulated in relation to the progression of melanoma. Most of the pathways dysregulated in this cohort were enriched in genes differentially expressed at the transcript level when similar groups are compared or metastasis to primary melanomas. At the genome level, we found significant differences in the mutation profiles between metastatic and primary melanomas. Our findings also highlighted sex-related differences in the molecular profiles. Remarkably, primary melanomas in women showed higher levels of antigen processing and presentation, and activation of the immune system response. Our results provide novel insights, relevant for developing personalized precision treatments for melanoma patients.

Systematic analysis of migration factors by MigExpress identifies essential cell migration control genes in non-small cell lung cancer.

Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking—in part due to the difficulty in assessing migration using high‐throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the ‘identification of Migration control genes by differential Expression’. MigExpress exploits the combination of in‐depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration‐associated genes by their differential expression in slow‐ versus fast‐migrating cells. We applied MigExpress to investigate non‐small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration.

Multi-omics phenotyping of the gut-liver axis reveals metabolic perturbations from a low-dose pesticide mixture in rats

Health effects of pesticides are not always accurately detected using the current battery of regulatory toxicity tests. We compared standard histopathology and serum biochemistry measures and multi-omics analyses in a subchronic toxicity test of a mixture of six pesticides frequently detected in foodstuffs (azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole) in Sprague-Dawley rats. Analysis of water and feed consumption, body weight, histopathology and serum biochemistry showed little effect. Contrastingly, serum and caecum metabolomics revealed that nicotinamide and tryptophan metabolism were affected, which suggested activation of an oxidative stress response. This was not reflected by gut microbial community composition changes evaluated by shotgun metagenomics. Transcriptomics of the liver showed that 257 genes had their expression changed. Gene functions affected included the regulation of response to steroid hormones and the activation of stress response pathways. Genome-wide DNA methylation analysis of the same liver samples showed that 4,255 CpG sites were differentially methylated. Overall, we demonstrated that in-depth molecular profiling in laboratory animals exposed to low concentrations of pesticides allows the detection of metabolic perturbations that would remain undetected by standard regulatory biochemical measures and which could thus improve the predictability of health risks from exposure to chemical pollutants.

Therapeutic Interleukin-6 Trans-signaling Inhibition by Olamkicept (sgp130Fc) in Patients With Active Inflammatory Bowel Disease

A large unmet therapeutic need exists in inflammatory bowel disease (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL6/IL6 receptor (IL6R) blockade is limited by profound immunosuppression. Evidence has emerged that chronic proinflammatory activity of IL6 is mainly mediated by trans-signaling via a complex of IL6 bound to soluble IL6R engaging the gp130 co-receptor without the need for membrane-bound IL6R. We have developed a decoy protein, sgp130Fc, that exclusively blocks IL6 proinflammatory trans-signaling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression. We present a 12-week, open-label, prospective phase 2a trial (FUTURE) in 16 patients with active IBD treated with the trans-signaling inhibitor olamkicept (sgp130Fc) to assess the molecular mechanisms, safety, and effectiveness of IL6 trans-signaling blockade invivo. We performed in-depth molecular profiling at various timepoints before and after therapy induction to identify the mechanism of action of olamkicept. Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-tumor necrosis factor (infliximab) or anti-integrin (vedolizumab) therapies was identified. Our data suggest that blockade of IL6 trans-signaling holds great promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy for IBD. (EudraCT no., Nu 2016-000205-36).

Nutrient recycle from algae hydrothermal liquefaction aqueous phase through a novel selective remediation approach

Algae have received increasing interest in the past several decades as a biofuel feedstock source. However, sustainable nutrient supply has presented algal biofuels with a major obstacle in the value chain. At a scale where algal biofuels would meet a significant portion of transportation fuel needs, the demand for nutrients, specifically nitrogen and phosphorus, would exceed current global agricultural production. One downstream conversion pathway, hydrothermal liquefaction (HTL), produces bio-crude oils from wet algal biomass with a waste aqueous phase (HTL-AP), containing a significant amount of carbon and nitrogen. While this stream is rich in organic content and nutrients, it also contains toxic components, which include heterocyclic nitrogen compounds and phenolic compounds. Thus, the recyclability and potential toxicity of HTL-AP need to be studied in detail. The feasibility of utilizing nutrients available in HTL-AP was experimentally determined for Chlorella vulgaris and Desmodesmus armatus monocultures. Our work focused on determining the tolerance of these algae species toward HTL-AP toxicity through varying dilutions. Nitrogen replacement in the growth media was varied from a low of 18% to a high of 141% across both species. The most notable of these results show that addition of a 100× dilution (35% nitrogen replacement) of untreated HTL-AP decreased growth in C. vulgaris by 47 ± 7% with respect to a control medium. Adsorption treatments, including activated carbon and various resins, were introduced to remediate the HTL-AP toxic effects. Treatment of the HTL-AP portion with an ion-exchange resin, Dowex 50WX8, supported C. vulgaris growth at a 100× dilution (35% nitrogen replacement) with no statistical change compared to the control. An in-depth molecular profiling demonstrated for the first time the selective removal of high‑nitrogen containing components by resin treatment. This work provides a foundation for studying the toxic components of HTL-AP and possible mechanisms by which treatments can remove these components.

Sepsis in the era of data-driven medicine: personalizing risks, diagnoses, treatments and prognoses.

Sepsis is a series of clinical syndromes caused by the immunological response to infection. The clinical evidence for sepsis could typically attribute to bacterial infection or bacterial endotoxins, but infections due to viruses, fungi or parasites could also lead to sepsis. Regardless of the etiology, rapid clinical deterioration, prolonged stay in intensive care units and high risk for mortality correlate with the incidence of sepsis. Despite its prevalence and morbidity, improvement in sepsis outcomes has remained limited. In this comprehensive review, we summarize the current landscape of risk estimation, diagnosis, treatment and prognosis strategies in the setting of sepsis and discuss future challenges. We argue that the advent of modern technologies such as in-depth molecular profiling, biomedical big data and machine intelligence methods will augment the treatment and prevention of sepsis. The volume, variety, veracity and velocity of heterogeneous data generated as part of healthcare delivery and recent advances in biotechnology-driven therapeutics and companion diagnostics may provide a new wave of approaches to identify the most at-risk sepsis patients and reduce the symptom burden in patients within shorter turnaround times. Developing novel therapies by leveraging modern drug discovery strategies including computational drug repositioning, cell and gene-therapy, clustered regularly interspaced short palindromic repeats -based genetic editing systems, immunotherapy, microbiome restoration, nanomaterial-based therapy and phage therapy may help to develop treatments to target sepsis. We also provide empirical evidence for potential new sepsis targets including FER and STARD3NL. Implementing data-driven methods that use real-time collection and analysis of clinical variables to trace, track and treat sepsis-related adverse outcomes will be key. Understanding the root and route of sepsis and its comorbid conditions that complicate treatment outcomes and lead to organ dysfunction may help to facilitate identification of most at-risk patients and prevent further deterioration. To conclude, leveraging the advances in precision medicine, biomedical data science and translational bioinformatics approaches may help to develop better strategies to diagnose and treat sepsis in the next decade.

Fibroblast growth factor receptor status and response to immune checkpoint inhibition in metastatic urothelial cancer.

458 Background: Fibroblast growth factor receptor (FGFR) inhibitors are a promising new targeted therapy for patients with metastatic urothelial cancer (UC) and FGFR alterations. FGFR-altered tumors are more likely to be of the luminal molecular subtype, which is less immune infiltrated and may be less likely to respond to immune checkpoint inhibitors (ICP). Methods: Metastatic UC patients at the University of North Carolina who underwent targeted exon sequencing (any CLIA-certified platform) and were treated with ICP since 2014 were identified. Patients with any FGFR alteration were compared to patients without alterations (including mutations, fusions, and amplifications in FGFR1-4). Overall response rates (ORR) to ICP were assessed by a radiologist (K.M.) per RECIST 1.1 and compared between FGFR-altered and unaltered tumors using Fisher’s exact tests. Patients who died prior to radiologic assessment were considered non-responders. Results: 66 patients (median age 70, 65% male, 76% white, 21% black) were identified. Most patients (74%) had received prior platinum-based chemotherapy, and 13% had received 2 or more prior lines of therapy. At the time of initiation of ICP, 32% of patients had a hemoglobin < 10, 33% had liver metastases, and 72% had a performance status > 0. Fifteen (22%) patients had FGFR alterations. The ORR for all patients was 15%, with ORR of 13% in FGFR-altered patients compared with 16% in unaltered patients (p = 1.0). No patients (0/9, 0%) with known pathogenic mutations in FGFR3 responded to ICP compared to 10/57 (18%) of patients without these alterations (p = 0.33). 46% of FGFR-altered patients who stopped ICP due to progression received subsequent therapy. Conclusions: Response rates to ICP are low and there was no difference in ORR between FGFR-altered and unaltered patients. While no patient with pathogenic FGFR3 mutations responded to ICP in our cohort, this difference did not reach statistical significance. Given low response rates overall, some FGFR-altered patients may benefit from treatment with FGFR inhibitors prior to ICP. Analysis of larger cohorts of patients as well as patients from clinical trials and more in-depth molecular profiling may add further clarity.

Development and characterisation of a panel of phosphatidylinositide 3-kinase \u2013 mammalian target of rapamycin inhibitor resistant lung cancer cell lines

The PI3K-mTOR pathway is involved in regulating all hallmarks of cancer, and is often dysregulated in NSCLC, making it an attractive therapeutic target in this setting. Acquired resistance to PI3K-mTOR inhibition is a major hurdle to overcome in the success of PI3K-mTOR targeted agents. H460, A549, and H1975 resistant cells were generated by prolonged treatment in culture with Apitolisib (GDC-0980), a dual PI3K-mTOR inhibitor over a period of several months, from age-matched parent cells. Resistance was deemed to have developed when a log fold difference in IC50 had been achieved. Resistant cell lines also exhibited resistance to another widely investigated PI3K-mTOR dual inhibitor; Dactolisib (BEZ235). Cell lines were characterised at the level of mRNA (expression array profiling expression of >150 genes), miRNA (expression array profiling of 2100 miRNAs), protein (bottoms-up label-free mass spectrometry) and phosphoprotein (expression array profiling of 84 phospho/total proteins). Key alterations were validated by qPCR and Western blot. H1975 cells were initially most sensitive to Apitolisib (GDC-0980), but developed resistance more quickly than the other cell lines, perhaps due to increased selective pressure from the impressive initial effect. In-depth molecular profiling suggested epithelial-mesenchymal transition (EMT) may play a role in resistance to PI3K-mTOR dual inhibition in NSCLC.

Protocol for the Analysis of Laser Capture Microdissected Fresh-Frozen Tissue Homogenates by Silver-Stained 1D SDS-PAGE.

The heterogeneity present in solid tumors adds significant difficulty to scientific analysis and improved understanding. Fundamentally, solid tumor formation consists of cancer cells proper along with stromal elements. The burgeoning malignant process is dependent upon modified stromal elements. Collectively, the stroma forms an essential microenvironment, which is indispensable for the survival and growth of the malignant neoplasm. This cellular heterogeneity makes molecular profiling of solid tumors via mass spectrometry (MS)-based proteomics a daunting task. Laser capture microdissection (LCM) is commonly used to obtain distinct histological cell types (e.g., tumor parenchymal cells, stromal cells) from tumor tissue and attempt to address the tumor heterogeneity interference with downstream liquid chromatography (LC) MS analysis. To provide optimal LC-MS analysis of micro-scale and/or nano-scale tissue sections, we modified and optimized a silver-stained one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) protocol for the LC-MS analysis of LCM-procured fresh-frozen tissue specimens. Presented is a detailed in-gel digestion protocol adjusted specifically to maximize the proteome coverage of amount-limited LCM samples, and facilitate in-depth molecular profiling. Following LCM, targeted tissue sections are further fractionated using silver-stained 1D-SDS-PAGE to resolve and visualize tissue proteins prior to in-gel digestion and subsequent LC-MS analysis.

Circulating and disseminated tumor cells: diagnostic tools and therapeutic targets in motion.

Enumeration of circulating tumor cells (CTCs) in peripheral blood with the gold standard CellSearchTM has proven prognostic value for tumor recurrence and progression of metastatic disease. Therefore, the further molecular characterization of isolated CTCs might have clinical relevance as liquid biopsy for therapeutic decision-making and to monitor disease progression. The direct analysis of systemic cancer appears particularly important in view of the known disparity in expression of therapeutic targets as well as epithelial-to-mesenchymal transition (EMT)-based heterogeneity between primary and systemic tumor cells, which all substantially complicate monitoring and therapeutic targeting at present. Since CTCs are the potential precursor cells of metastasis, their in-depth molecular profiling should also provide a useful resource for target discovery. The present review will discuss the use of systemically spread cancer cells as liquid biopsy and focus on potential target antigens.

In-Depth Proteomics Identifies a Role for Autophagy in Controlling Reactive Oxygen Species Mediated Endothelial Permeability

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells.

In-Depth Molecular Profiling of Multiple Myeloma in African Americans

Introduction: Multiple Myeloma (MM) is a complex malignancy of plasma cells well-described hyperdyploidy, and immunoglobulin gene rearrangements. To promote rapid advances in the field, Multiple Myeloma Research Foundation (MMRF) initiative is an intensive and comprehensive longitudinal study (CoMMpass) designed to create a rich discovery ecosystem to through in-depth clinical and molecular profiling to understand the molecular perturbations of the disease in the context of therapy. The large study population empowered us to stratify mutational landscapes among different ethnicities to influence on broader disparities in tumor dynamics.Methods: Clinical data, tumor/normal sample collection, and mutational landscape analysis described in this abstract are derived from MMRF CoMMpass IA7 release that is composed of self-identified 93 African American (AA) and 377 European American (EA). The post-processing and primary analysis was done on baseline samples only. Whole exome sequencing was analyzed for the detection of somatic events. Secondary analysis was performed using MutSigCV (Mutation Significance) algorithm and GISTIC (The Genomic Identification of Significant Targets in Cancer) to determine the significance of coding mutations and copy number events.Results: Our preliminary comparison analysis of CoMMpass IA7 data demonstrated that overall there was no statistical difference (p=0.5973) in nonsilent mutation burden between the two stratified groups, AA (μ=63.9 mutations/patient) vs EA (μ=73.7 mutations/patient). However, we have observed several notable differences. The most notable population difference (p<0.0001) was TP53 mutation occurrence, which was more common in EA 5.6% (21/377) compared AA 2.1% (2/93).Furthermore, MutSig analysis also revealed a novel candidate PTCHD3 (p = 7.07E-06, q = 3.33E-02) with 6% occurrence in AA compared to only 0.04% in EA. Furthermore, we looked at mutation occurrence difference of at least 5 fold between the two stratified populations. We have identified several additional genes that have higher mutation frequencies in tumors from AA patients including: ANKRD26, BCL7A, BRWD3. Interestingly, majority is implicated in epigenetic regulatory mechanisms.Moreover, despite the complex karyotype in MM translocations, our analysis demonstrated a lower frequency of 14q32 translocations in tumors from AA patients (10%) compared to tumors from EA patients (37%). These data would independently validate our previously reported differences in 14q32 breakpoints between these two populations.Conclusion: Data from this comprehensive diverse multi-institutional longitudinal study has afforded us the opportunity to study population differences among MM patients in an unprecedented way. Taking advantage of high quality, high-resolution whole exome and whole-genome data has allowed us to identify potential differences in the genomic and molecular landscape of MM from AA and EA patients. These data may help us further understand the incidence and outcomes among patients from these populations. As CoMMpass continues to mature these datasets will be retested that may result in some of these preliminary reported events to either level off, or increase in power. DisclosuresMulligan:Millennium Pharmaceuticals, Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Lonial:Janssen: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Onyx: Consultancy, Research Funding. Keats:Translational Genomic Research Institute: Employment.