Multiple Tissue Samples Research Articles

Somatic Platelet Derived Growth Factor Receptor Beta Activating Variants in Fusiform Cerebral Aneurysms

Abstract INTRODUCTION The role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-yr-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of unrevealing genetic evaluations for known connective-tissue disorders, and served as a somatic mosaic for discovery of novel mutations involved in aneurysm pathogenesis. METHODS Deoxyribonucleic acid (DNA) was extracted from multiple vascular and perivascular tissue samples obtained during surgery on the index patient. Initial variant discovery was carried out via paired-sample exome sequencing between blood and diseased area fibroblasts. A single novel variant was discovered in the platelet derived growth factor receptor beta (PDGFRB), and mutations in this gene were tested in exome sequencing of aneurysm walls from 6 patients with fusiform intracranial aneurysm and 38 patients with saccular aneurysm. RESULTS Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T > C [GRCh37/hg19]; c.1685A > G) change within PDGFRB, a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting postzygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, P < .001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive autophosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated nonligand-dependent autophosphorylation, responsive to the kinase inhibitor sunitinib. CONCLUSION Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors.

A real-time ex vivo model (eIBUB) for optimizing intraperitoneal drug delivery as an alternative to living animal models.

BackgroundOptimization of intraperitoneal drug delivery systems requires functional models. We proposed the Inverted Bovine Urinary Bladder Model (IBUB), but IBUB does not allow repeated measurements over time and there is a significant biological variability between organs.MethodsA further development of IBUB is presented, based on the physical principle of communicating vessels. Fresh bovine bladders were inverted so that the peritoneum lines up the inner surface. The IBUB and a second vessel were then interconnected under the same CO2 pressure and placed on two scales. The therapeutic solution (Doxorubicin 2.7 mg and Cisplatin 13.5 mg) was delivered via an aerosolizer. All experiments were in triplicate and blinded to the origin of samples, measurements in a GLP-certified laboratory.ResultsThe enhanced IBUB (eIBUB) model allows measurements of tissue drug concentration, depth of tissue penetration and spatial distribution. The homogeneous morphology of the peritoneum enables standardized, multiple tissue sampling. eIBUB minimizes biological variability between different bladders and eliminates the bias caused by the liquid collecting at the bottom of the model. Concentration of doxorubicin in the eIBUB (mean ± STDV: 18.5 ± 22.6 ng/mg) were comparable to clinical peritoneal biopsies (19.2 ± 38.6 ng/mg), as was depth of drug penetration (eIBUB: mean (min-max) 433 (381–486) µm, clinical ~ 500 µm).ConclusionsThe eIBUB model is a simple and powerful ex vivo model for optimizing intraperitoneal drug delivery and represents an attractive alternative to animal models. Results obtained are similar to those obtained in the human patient.

What Is the Optimal Number of Samples for Culture in Patients Undergoing Surgery for Foot and Ankle Infections?

The optimal number of samples for culture in patients undergoing surgery for foot and ankle infections is unknown. We recommend that multiple tissue samples be taken. Consensus. Agree: 100%, Disagree: 0%, Abstain: 0% (Unanimous, Strongest Consensus).

Abstract 4555: Characterization of the tumor microenvironment with an automated 10-plex IHC panel using UltiMapper™ I/O assays

Abstract Background: In recent years, identifying and characterizing biomarkers within the tumor microenvironment (TME) has become a major focus for immune-oncology research. The growing field of immuno-oncology necessitates the identification of more biomarkers that help characterize the TME, and development of complimentary processes to rapidly analyze precious tissue samples. Ultivue® has developed UltiMapper™ assays that provide numerous advantages over alternative multiplexed IHC approaches, including high multiplexing in situ, sample preservation, streamlined workflows with staining completed in the same day, and easy integration into any histology lab. In this study we demonstrate a 10-plex immuno-oncology assay in FFPE tumor tissues by combining markers that profile the T-cell infiltrate, antigen presenting cells (APC), and checkpoint expression along the PD-L1/PD-1 axis. This 10-plex is achieved with a single antibody staining step for a fast workflow that is independent of staining order. The combination of 10 different biomarkers allows for characterization of the TME and investigation of PD-L1 expression on tumor and antigen presenting cells on a single slide. Methods: A 10-plex immunofluorescence assay was carried out using the UltiMapper approach on multiple deidentified FFPE tissue samples, including lung, melanoma, colon, and breast. Each sample was stained for the following 10 markers: CK or SOX10, PD-L1, PD-1, CD3, CD8, CD11c, CD20, CD68, CD163, and MHCII. Staining was performed in a single step using the Leica® Bond® Rx autostainer. Whole slide imaging was performed on various tissue scanners including the Zeiss® Axio Scan.Z1 slide scanner, and image analysis was performed using HALO® software from Indica Labs. Results: Cells were characterized and phenotyped based on their expression of specific biomarkers and the abundance of PD-L1 on macrophages, dendritic cells, B cell, cytotoxic T-cells, and tumor cells as measured. PD-L1 expression was observed on both immune and tumor cells with a range of different expression levels. Spatial analysis was employed to measure the distances between immune cells with different phenotypes and tumor cells to further characterize tumors as hot or cold. Conclusions: The multiplexed UltiMapper IHC approach allows for fast and comprehensive analysis of tumor samples necessary to research complex cancer biology. This approach achieves high quality multiplexed data on various tissue types and preserves precious tissue samples. The ability to interrogate a 10-plex I/O panel on a single slide with whole slide imaging presents a significant leap forward for IHC analysis, providing a faster and more thorough approach to characterize the TME and analyze PD-L1 expression on antigen presenting cells and tumor cells. Citation Format: Heike Boisvert, Alexis Wong, Gourab Chatterjee, Abdul Mohammed, Douglas Wood, Mael Manesse. Characterization of the tumor microenvironment with an automated 10-plex IHC panel using UltiMapper™ I/O assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4555.

Clonal diversity of Cutibacterium acnes (formerly Propionibacterium acnes) in prosthetic joint infections

Prosthetic joint infections (PJIs) are rare but feared complications following joint replacement surgery. Cutibacterium acnes is a skin commensal that is best known for its role in acne vulgaris but can also cause invasive infections such as PJIs. Some phylotypes might be associated with specific diseases, and recently, a plasmid was detected that might harbour important virulence genes. In this study, we characterized C. acnes isolates from 63 patients with PJIs (n = 140 isolates) and from the skin of 56 healthy individuals (n = 56 isolates), using molecular methods to determine the phylotype and investigate the presence of the plasmid. Single-locus sequence typing and a polymerase chain reaction designed to detect the plasmid were performed on all 196 isolates. No statistically significant differences in sequence types were seen between the two study groups indicating that the C. acnes that causes PJIs originates from the patients own normal skin microbiota. Of the 27 patients with multiple tissue samples, 19 displayed the same sequence types among all their samples. Single-locus sequence typing identified different genotypes among consecutive C. acnes isolates from four patients with recurrent infections. The plasmid was found among 17 isolates distributed in both groups, indicating that it might not be a marker for virulence regarding PJIs. Patients presenting multiple sequence types in tissue samples may represent contamination or a true polyclonal infection due to C. acnes.

High incidence of early periprosthetic joint infection following total hip arthroplasty with concomitant or previous hardware removal

Hardware removal preceding total hip arthroplasty may increase the risk of prosthetic joint infection (PJI). Whether hardware removal and total hip arthroplasty (THA) should be performed in a single- or two-stage procedure remains controversial. In this comparative retrospective study, the incidence of PJI following either single- or two-stage THA with hardware removal was assessed in a consecutive series. All patients that underwent THA preceded by hardware removal from January 2006 until March 2018 were retrospectively reviewed and checked for the occurrence of early PJI. Recognized risk factors for PJI at the time of surgery were evaluated and the incidence of early PJI was compared between one- and two-stage THA regarding hardware removal. 145 patients underwent THA and hardware removal (52 two-stage surgery and 93 single-stage surgery). There were no significant differences between both groups regarding pre-operative hemoglobulin levels, time interval between internal fixation and THA, antibiotic-loaded-cement use, BMI and ASA classification. Overall the incidence of early PJI was 6.9%. The incidence of PJI was 8.6% in the single-stage group versus 3.8% in the two-stage group (P = 0.234). Irrespective of single- or two-stage procedures, a high incidence of PJI was encountered. Despite non-significance, a trend towards a higher proportion of patients developing PJI after single-stage surgery was encountered. We recommend a two-stage surgical procedure regarding hardware removal and THA in patients that are expected to tolerate this surgical strategy. When considering a one-stage procedure, it should be preceded by a thorough pre-operative workup including joint aspiration and serum determination of inflammatory parameters. Multiple tissue samples should be obtained during hardware removal in either one- or two-stage procedures since the risk for development of PJI is relevant.

Bortezomib Augments Natural Killer Cell Targeting of Stem-Like Tumor Cells.

Tumor cells harboring stem-like/cancer stem cell (CSC) properties have been identified and isolated from numerous hematological and solid malignancies. These stem-like tumor cells can persist following conventional cytoreductive therapies, such as chemotherapy and radiotherapy, thereby repopulating the tumor and seeding relapse and/or metastasis. We have previously shown that natural killer (NK) cells preferentially target stem-like tumor cells via non- major histocompatibility complex (MHC) restricted mechanisms. Here, we demonstrated that the proteasome inhibitor, bortezomib, augments NK cell targeting of stem cell-like tumor cells against multiple solid human tumor-derived cancer lines and primary tissue samples. Mechanistically, this was mediated by the upregulation of cell surface NK ligands MHC class I chain-related protein A and B (MICA and MICB) on aldehyde dehydrogenases (ALDH)-positive CSCs. The increased expression of MICA and MICB on CSC targets thereby enhanced NK cell mediated killing in vitro and ex vivo from both human primary tumor and patient-derived xenograft samples. In vivo, the combination of bortezomib and allogeneic NK cell adoptive transfer in immunodeficient mice led to increased elimination of CSCs as well as tumor growth delay of orthotopic glioblastoma tumors. Taken together, our data support the combination bortezomib and NK transfer as a strategy for both CSC targeting and potentially improved outcomes in clinical cancer patients.

Metabolomic prostate cancer fields in HRMAS MRS-profiled histologically benign tissue vary with cancer status and distance from cancer.

In this article, we review the state of the field of high resolution magic angle spinning MRS (HRMAS MRS)-based cancer metabolomics since its beginning in 2004; discuss the concept of cancer metabolomic fields, where metabolomic profiles measured from histologically benign tissues reflect patient cancer status; and report our HRMAS MRS metabolomic results, which characterize metabolomic fields in prostatectomy-removed cancerous prostates. Three-dimensional mapping of cancer lesions throughout each prostate enabled multiple benign tissue samples per organ to be classified based on distance from and extent of the closest cancer lesion as well as the Gleason score (GS) of the entire prostate. Cross-validated partial least squares-discriminant analysis separations were achieved between cancer and benign tissue, and between cancer tissue from prostates with high (≥4+3) and low (≤3+4) GS. Metabolomic field effects enabled histologically benign tissue adjacent to cancer to distinguish the GS and extent of the cancer lesion itself. Benign samples close to either low GS cancer or extensive cancer lesions could be distinguished from those far from cancer. Furthermore, a successfully cross-validated multivariate model for three benign tissue groups with varying distances from cancer lesions within one prostate indicates the scale of prostate cancer metabolomic fields. While these findings could, at present, be potentially useful in the prostate cancer clinic for analysis of biopsy or surgical specimens to complement current diagnostics, the confirmation of metabolomic fields should encourage further examination of cancer fields and can also enhance understanding of the metabolomic characteristics of cancer in myriad organ systems. Our results together with the success of HRMAS MRS-based cancer metabolomics presented in our literature review demonstrate the potential of cancer metabolomics to provide supplementary information for cancer diagnosis, staging, and patient prognostication.

A protocol for making and sectioning multiple embedded Swiss-rolls in a gelatin matrix

The appropriate methodological approach for intestinal preparation enables researchers to create representative histological and immunostaining images which validate their biochemical data. The Swiss-roll technique was first introduced by Reilly and Kirsner in 1965. Later, Moolenbeek and Ruitenberg described a detailed procedure to longitudinally study the rodent intestine in 1981 [1]. In this publication, our slightly different approach for co-embedding four different Swiss-rolls in a gelatin block provides a full-length overview of cross-sectional colons on a single slide. This protocol allows for longitudinal histologic examination of multiple tissue samples on a single slide simultaneously. In this method, antigenicity is retained for immunohistology. In addition, the accessibility of samples during the prolonged hardening time required of the gelatin matrix allows the tissue samples to be adjusted/re-adjusted to provide the desired orientation and spatial arrangement for ideal cross sections with similar planes of section and optimum space utilization for slide mounting. Although not the focus of this protocol, the room temperature stability of the gelatin matrix and the ability to contain numerous tissue samples in a block allows the flexibility of performing thicker sections for free-floating tissue staining and ease of mounting a single gelatin sheet rather than individual tissue sections. This is a convenient approach for allowing precise preparation of multi-tissue blocks and simultaneous sectioning, staining, and slide mounting of tissue for subsequent comparisons.•A single gelatin block is prepared by simultaneously embedding at least four different intestinal Swiss-rolls.•The tissue orientation can be adjusted for each sample as desired which facilitates the comparison of different colon samples on a single gelatin section.•The gelatin sections containing tissue samples are stable at least overnight at room temperature for staining.

Microbiome-Derived Lipopolysaccharide (LPS) Selectively Inhibits Neurofilament Light Chain (NF-L) Gene Expression in Human Neuronal-Glial (HNG) Cells in Primary Culture.

The remarkable co-localization of highly pro-inflammatory lipopolysaccharide (LPS) with sporadic Alzheimer’s disease (AD)-affected neuronal nuclei suggests that there may be some novel pathogenic contribution of this heat stable neurotoxin to neuronal activity and neuron-specific gene expression. In this communication we show for the first time: (i) the association and envelopment of sporadic AD neuronal nuclei with LPS in multiple AD neocortical tissue samples; and (ii) a selective repression in the output of neuron-specific neurofilament light (NF-L) chain messenger RNA (mRNA), perhaps as a consequence of this association. The down-regulation of NF-L mRNA and protein is a characteristic attribute of AD brain and accompanies neuronal atrophy and an associated loss of neuronal architecture with synaptic deficits. To study this phenomenon further, human neuronal-glial (HNG) cells in primary culture were incubated with LPS, and DNA arrays, Northern, Western, and ELISA analyses were used to quantify transcription patterns for the three member neuron-specific intermediate filament-gene family NF-H, NF-M, and NF-L. As in sporadic AD limbic-regions, down-regulated transcription products for the NF-L intermediate filament protein was significant. These results support our novel hypothesis: (i) that internally sourced, microbiome-derived neurotoxins such as LPS contribute to a progressive disruption in the read-out of neuron-specific genetic-information; (ii) that the presence of LPS-enveloped neuronal nuclei is associated with a down-regulation in NF-L expression, a key neuron-specific cytoskeletal component; and (iii) this may have a bearing on progressive neuronal atrophy, loss of synaptic-contact and disruption of neuronal architecture, all of which are characteristic pathological features of sporadic-AD brain. This is the first report that provides evidence for a neuron-specific effect of a human GI-tract microbiome-derived neurotoxin on decreased NF-L abundance in both sporadic AD temporal lobe neocortex in vivo and in LPS-stressed HNG cells in vitro.

High prevalence of bacteria in clinically aseptic non-unions of the tibia and the femur in tissue biopsies

There are several hints that bacterial colonization might be an often overseen cause of non-union. Modern procedures like PCR have been reported to diagnose bacterial colonization with a high degree of accuracy. While PCR is not ubiquitously available, we hypothesize that biopsies from the non-union site are comparable to PCR results reported in the literature. Retrospective analysis of microbiological results of biopsies from non-unions (femoral or tibial, history of revision surgery, and/or open fracture) with stable osteosynthesis, no clinical signs of local infection were analysed. CRP and leucocyte count were taken on admission. Multiple tissue samples (soft tissue and bone) were from the non-union (1-4cm incision). Samples were cultivated for 2 weeks and tested following EUCAST protocols using VITEK® 2. 11 tibia- and 7 femur non-union (44 ± 23.9years), 11 open fractures (1 I°, 6 II°, 4 III° Gustillo Anderson), 0-5 revisions, and 4.1 (± 1.8) tissue samples were taken 8.5 (± 1.7) months after trauma. Cultures were positive in 8/18 (44,4%) (3/18 Propionibacterium acnes, 1/18 S. capitis, and 4/18 S. epidermidis). There was neither a correlation between number of biopsies taken and positive culture results (Pearson R: - 0.0503, R2 0.0025), nor between positive culture results and leucocytes counts (Pearson R: - 0.0245, R2 0.0006) or CRP concentration (Pearson R: 0.2823, R2 0.0797). The results confirm that the presence of bacteria in cases with no clinical signs of infection is a relevant issue. The prevalence of bacteria reported here is comparable that reported from cohorts tested with PCR or sonication. In most cases, there was only one positive biopsy, raising the question whether a contamination has been detected. Thus, to better understand the problem, it is necessary to gather more knowledge regarding the sensitivities and specificities of the different diagnostic procedures.

DNA methylation age is accelerated in alcohol dependence

Alcohol dependence (ALC) is a chronic, relapsing disorder that increases the burden of chronic disease and significantly contributes to numerous premature deaths each year. Previous research suggests that chronic, heavy alcohol consumption is associated with differential DNA methylation patterns. In addition, DNA methylation levels at certain CpG sites have been correlated with age. We used an epigenetic clock to investigate the potential role of excessive alcohol consumption in epigenetic aging. We explored this question in five independent cohorts, including DNA methylation data derived from datasets from blood (n = 129, n = 329), liver (n = 92, n = 49), and postmortem prefrontal cortex (n = 46). One blood dataset and one liver tissue dataset of individuals with ALC exhibited positive age acceleration (p < 0.0001 and p = 0.0069, respectively), whereas the other blood and liver tissue datasets both exhibited trends of positive age acceleration that were not significant (p = 0.83 and p = 0.57, respectively). Prefrontal cortex tissue exhibited a trend of negative age acceleration (p = 0.19). These results suggest that excessive alcohol consumption may be associated with epigenetic aging in a tissue-specific manner and warrants further investigation using multiple tissue samples from the same individuals.

Functional Characterization of the Osteoarthritis Genetic Risk Residing at ALDH1A2 Identifies rs12915901 as a Key Target Variant

ObjectiveTo identify the functional single‐nucleotide polymorphisms (SNPs) and mechanisms conferring increased risk of hand osteoarthritis (OA) at the ALDH1A2 locus, which is a retinoic acid regulatory gene.MethodsTissue samples from 247 patients with knee, hip, or hand OA who had undergone joint surgery were included. RNA‐sequencing analysis was used to investigate differential expression of ALDH1A2 and other retinoic acid signaling pathway genes in cartilage. Expression of ALDH1A2 in joint tissues obtained from multiple sites was quantified using quantitative reverse transcription–polymerase chain reaction. Allelic expression imbalance (AEI) was measured by pyrosequencing. The consequences of ALDH1A2 depletion by RNA interference were assessed in primary human chondrocytes. In silico and in vitro analyses were used to pinpoint which, among 62 highly correlated SNPs, could account for the association at the locus.Results ALDH1A2 expression was observed across multiple joint tissue samples, including osteochondral tissue from the hand. The expression of ALDH1A2 and of several retinoic acid signaling genes was different in diseased cartilage compared to non‐diseased cartilage, with ALDH1A2 showing lower levels in OA cartilage. Experimental depletion of ALDH1A2 resulted in changes in the expression levels of a number of chondrogenic markers, including SOX9. In addition, reduced expression of the OA risk–conferring allele was witnessed in a number of joint tissues, with the strongest effect in cartilage. The intronic SNP rs12915901 recapitulated the AEI observed in patient tissues, while the Ets transcription factors were identified as potential mediators of this effect.ConclusionThe ALDH1A2 locus seems to increase the risk of hand OA through decreased expression of ALDH1A2 in joint tissues, with the effect dependent on rs12915901. These findings indicate a mechanism that may now be targeted to modulate OA risk.

Demonstration of P-selectin expression and potential function in human corneal epithelial cells

In response to an unexpected observation of apparent localisation by immunocytochemistry, we have investigated the potential expression and function of P-selectin (CD62P) in human corneal epithelial cells. The SV40 immortalised cell line, HCE-T (validated by STR profiling), along with multiple donor corneal-limbal tissue samples, were examined for P-selectin expression using a combination of immunocytochemistry, Western blotting, RT-PCR and immunohistochemistry. Potential expression of the major ligand for P-selectin (P-selectin glycoprotein ligand-1; PSGL-1; CD162) was also examined by immunocytochemistry and RT-PCR. A selective inhibitor of P-selectin-PSGL-1 binding (KF38789) was subsequently tested for effects on HCE-T cells using a cell culture gap-closure assay. HCE-T cells as well as primary epithelial cultures derived from donor corneal-limbal tissue, displayed positive immunostaining for P-selectin. Staining was particularly evident at cell-cell boundaries and at the outer edge of expanding epithelial islands. P-selectin expression was confirmed by Western blotting and RT-PCR (validated by product sequencing), as well as by immunohistochemistry performed on serial sections of corneal-limbal tissue stained for P-selectin, keratin 3 and p63. PSGL-1 was detected by RT-PCR and immunocytochemistry in both corneal epithelial cells as well as human limbal fibroblasts (HLF). KF38789 (5 μM) significantly reduced closure of a 500-μm gap between confluent sheets of HCE-T cells over an 8-hr period (by ∼40%, p < 0.01; paired two-tailed T test), but had no effect on culture gap-closure by either HLF or murine 3T3 fibroblasts. These results provide evidence of P-selectin expression in human corneal epithelial cells and suggest a potential role for this glycoprotein in facilitating the net movement of confluent sheets of human corneal epithelial cells.

Intra-tumor molecular heterogeneity in breast cancer: definitions of measures and association with distant recurrence-free survival.

The purpose of the study was to define quantitative measures of intra-tumor heterogeneity in breast cancer based on histopathology data gathered from multiple samples on individual patients and determine their association with distant recurrence-free survival (DRFS). We collected data from 971 invasive breast cancers, from 1st January 2000 to 23rd March 2014, that underwent repeat tumor sampling at our institution. We defined and calculated 31 measures of intra-tumor heterogeneity including ER, PR, and HER2 immunohistochemistry (IHC), proliferation, EGFR IHC, grade, and histology. For each heterogeneity measure, Cox proportional hazards models were used to determine whether patients with heterogeneous disease had different distant recurrence-free survival (DRFS) than those with homogeneous disease. The presence of heterogeneity in ER percentage staining was prognostic of reduced DRFS with a hazard ratio of 4.26 (95% CI 2.22-8.18, p < 0.00002). It remained significant after controlling for the ER status itself (p < 0.00062) and for patients that had chemotherapy (p < 0.00032). Most of the heterogeneity measures did not show any association with DRFS despite the considerable sample size. Intra-tumor heterogeneity of ER receptor status may be a predictor of patient DRFS. Histopathologic data from multiple tissue samples may offer a view of tumor heterogeneity and assess recurrence risk.

Abstract 5661: A multi modal mass spectrometry imaging strategy to profile the metabolic hallmarks of colorectal cancer

Abstract New methods are required to image the metabolic hallmarks of cancerous tissues and correlate this information with patient tumour pathology, cancer phenotype and treatment outcome. Mass spectrometry imaging (MSI) offers a powerful suite of techniques for imaging metabolites, drugs and proteins in cells and tissues. Here we present a multi-modal MSI strategy and use it to profile PDX, GEMM and biopsy samples. Human and murine colon and small intestine samples were sectioned onto glass slides and stored at -80°C until use. MALDI-MSI (Waters and Bruker) was carried out with pixel sizes between 10 and 50 microns, DESI (Waters) at 20-50 microns and SIMS (IONTOF) at approximately 200 nm per pixel. Data were converted from proprietary format to imzML and analysed in MatLab (2015b, MathWorks) via the SpectralAnalysis software package using non-negative matrix factorisation (NMF) and principal component analysis (PCA). Further reduction and segmentation was achieved using stacked autoencoders and t-distributed stochastic neighbour embedding (t-SNE). To assign the peaks detected to molecular identities, peaks picked from mean spectra were matched to the human metabolome database (HMDB) using custom Matlab scripts. Possible adducts were queried against the database masses, and filtered based on comparison of expected and observed mass, and correlation between the images of the monoisotopic mass and 13C isotope image. Preliminary data show the successful preparation and analysis of multiple GEMM and human tissue samples across several sites by MALDI-MSI, DESI-MSI and SIMS imaging. Converting data to imzML and combining into large, single multi-file images enabled multivariate analysis (MVA) to be performed simultaneously within the same comparison. Of these MVA methods, mass spectrometry imaging (MSI) in combination with t-SNE has been shown to differentiate tumour subpopulations linked with patient outcomes. Current t-SNE methods suffer from three main limitations as compared to other MVA methods; poor computational scaling to large datasets, inability to add new data into the embedding, and inability to return to spectral contributions. By combining t-SNE performed on a subset of data and learning the embedding via deep learning, almost limitless size datasets can be analysed by t-SNE, new data can be incorporated into the embedding, and the spectral contributions towards the three dimensional space can be determined. Database matching on the data from these GEMM samples to the HMDB reveals over 1000 possible molecular assignments, primarily structural lipids, within 10 ppm mass accuracy and with isotope image correlations above 0.6. From these methods, comparisons can be made between the unique molecules detected in different tissues, as well as the relative changes in intensity of those detected between different tissues. Citation Format: Josephine Bunch, Rory T. Steven, Adam J. Taylor, Spencer A. Thomas, Alan M. Race, Alex Dexter, Gregory Hamm, Nicole Strittmatter, Rasmus Havelund, Renata F. Soares, Andrew D. Campbell, Owen J. Sansom, Richard J. Goodwin, Zoltan Takats. A multi modal mass spectrometry imaging strategy to profile the metabolic hallmarks of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5661.

Canine Gastrointestinal Spindle Cell Tumors Efficiently Diagnosed by Tissue Microarray-Based Immunohistochemistry.

Tissue microarray (TMA) is a time- and cost-saving technique allowing the simultaneous immunohistochemical evaluation of multiple tissue samples. The aim of this study was to assess the efficacy of TMA at classifying canine gastrointestinal spindle cell tumors as gastrointestinal stromal tumor (GIST), smooth muscle tumor (SMT), and non-GIST/non-SMT based on the expression of α-smooth muscle actin (α-SMA), desmin, and CD117. Thirty-four cases were investigated on TMAs, sampling 2 cores each. Immunohistochemistry was performed on TMAs and full sections, and the results were compared. Comparing full sections, TMA specificity and sensitivity were 100% and 93.8%, respectively, for α-SMA; 100% and 80.8% for desmin; and 100% and 100% for CD117. TMA allowed the identification of 6 of 6 GISTs, 25 of 26 SMTs, and 2 of 2 non-GIST/non-SMTs. One SMT was misdiagnosed as non-GIST/non-SMT. Based on these results, TMA-based immunohistochemistry is efficient at diagnosing canine gastrointestinal spindle cell tumors and might be applied on large caseloads in a research setting.

Evaluation of [89Zr]trastuzumab-PET/CT in differentiating HER2-positive from HER2-negative breast cancer.

To evaluate whether tumor uptake of [89Zr]trastuzumab can distinguish HER2-positive from HER2-negative breast cancer. Women with HER2-positive (n=34) and HER2-negative (n=16) breast cancer underwent PET/CT 5±2days following [89Zr]trastuzumab administration. HER2 status was determined based on immunohistochemistry and/or fluorescence in situ hybridization of primary or metastatic/recurrent tumor. Tumor [89Zr]trastuzumab uptake was assessed qualitatively and semiquantitatively as maximum standardized uptake value (SUVmax), and correlated with HER2 status. Additionally, intrapatient heterogeneity of [89Zr]trastuzumab uptake was evaluated. On a per-patient basis, [89Zr]trastuzumab-PET/CT was positive in 30/34 (88.2%) HER2-positive and negative in 15/16 (93.7%) HER2-negative patients. Considering all lesions, the SUVmax was not significantly different in patients with HER2-positive versus HER2-negative disease (p=0.06). The same was true of when only hepatic lesions were evaluated (p=0.42). However, after excluding hepatic lesions, tumor SUVmax was significantly higher in HER2-positive compared to HER2-negative patients (p=0.003). A cutoff SUVmax of 3.2, determined by ROC analysis, demonstrated positive-predictive value of 83.3% (95% CI 65.3%, 94.4%), sensitivity of 75.8% (57.7%, 88.9%), negative-predictive value of 50% (24.7%, 75.3%), and specificity of 61.5% (95% 31.6%, 86.1%) for differentiating HER2-positive from HER2-negative lesions. There was intrapatient heterogeneity of [89Zr]trastuzumab uptake in 20% of patients with multiple lesions. [89Zr]trastuzumab has the potential to characterize the HER2 status of the complete tumor burden in patients with breast cancer, thus obviating repeat or multiple tissue sampling to assess intrapatient heterogeneity of HER2 status.

CDK13 RNA Over-Editing Mediated by ADAR1 Associates with Poor Prognosis of Hepatocellular Carcinoma Patients

Background/Aims: Aberrant RNA editing, mediated by adenosine deaminases acting on RNA (ADAR), serves as a post-transcriptional event participating in tumorigenesis and prognosis. However, the RNA editing profiles during HCC progression and their clinical correlations remain unclear. Methods: Multiple tissue samples were collected from an advanced HCC patient. RNA-seq was performed to obtain the RNA editing profiles for each sample. Two RNA editing sites from CDK13 were further validated in 60 HCC patients; and their potential regulatory mechanisms were investigated. Results: In-depth analysis of the RNA-seq data revealed a significant number of editing sites (632-816) in coding regions for each tissue sample, showing branched evolution during tumorigenesis and metastasis. Two editing sites (Q103R and K96R) in CDK13 showed significant over-editing in tumor, and these phenomenon were validated in 60 HCC patients. Furthermore, the clinicopathological analysis revealed that these CDK13 over-editing sites were positively associated with TNM, PVTT and poor prognosis. In addition, the editing level of these sites were significantly correlated with the expression of ADAR1. Loss of function assays further proved that these CDK13 over-editing sites were mediated by ADAR1 in HCC cells. Conclusions: CDK13 RNA over-editing sites mediated by ADAR1 may serve as novel cancer driver events in HCC progression.

Genomic mosaicism in paternal sperm and multiple parental tissues in a Dravet syndrome cohort

Genomic mosaicism in parental gametes and peripheral tissues is an important consideration for genetic counseling. We studied a Chinese cohort affected by a severe epileptic disorder, Dravet syndrome (DS). There were 56 fathers who donated semen and 15 parents who donated multiple peripheral tissue samples. We used an ultra-sensitive quantification method, micro-droplet digital PCR (mDDPCR), to detect parental mosaicism of the proband’s pathogenic mutation in SCN1A, the causal gene of DS in 112 families. Ten of the 56 paternal sperm samples were found to exhibit mosaicism of the proband’s mutations, with mutant allelic fractions (MAFs) ranging from 0.03% to 39.04%. MAFs in the mosaic fathers’ sperm were significantly higher than those in their blood (p = 0.00098), even after conditional probability correction (p’ = 0.033). In three mosaic fathers, ultra-low fractions of mosaicism (MAF < 1%) were detected in the sperm samples. In 44 of 45 cases, mosaicism was also observed in other parental peripheral tissues. Hierarchical clustering showed that MAFs measured in the paternal sperm, hair follicles and urine samples were clustered closest together. Milder epileptic phenotypes were more likely to be observed in mosaic parents (p = 3.006e-06). Our study provides new insights for genetic counseling.