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Objectives Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. Methods In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. Results Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. Conclusions This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology. [ABSTRACT FROM AUTHOR]

Most multigene mutation tests require tissue specimens. However, cytological specimens are easily obtained in the clinical practice and provide high‐quality DNA and RNA. We aimed to establish a test that utilizes cytological specimens and performed a multi‐institutional study to investigate the performance of MINtS, a test based on next‐generation sequencing. A standard procedure for specimen isolation was defined. The specimens were considered suitable for the test if >100 ng DNA and >50 ng RNA could be extracted from them. In total, 500 specimens from 19 institutions were investigated. MINtS detected druggable mutations in 63% (136 of 222) of adenocarcinomas. Discordant results between MINtS and the companion diagnostics were observed in 14 of 310 specimens for the EGFR gene, and 6 of 339 specimens for the ALK fusion genes. Confirmation by other companion diagnostics for the EGFR mutations or the clinical response to an ALK inhibitor all supported the results obtained by MINtS. MINtS along with the isolation procedure presented in the current study will be a platform to establish multigene mutation tests that utilize cytological specimens. UMIN000040415. [ABSTRACT FROM AUTHOR]

Introduction: Metabolite stability is critical for tissue metabolomics. However, changes in metabolites in tissues over time from the operating room to the laboratory remain underexplored. Objectives: In this study, we evaluated the effect of postoperative freezing delay time on the stability of metabolites in normal and oral squamous cell carcinoma (OSCC) tissues. Methods: Tumor and paired normal tissues from five OSCC patients were collected after surgical resection, and samples was sequentially quenched in liquid nitrogen at 30, 40, 50, 60, 70, 80, 90 and 120 min (80 samples). Untargeted metabolic analysis by liquid chromatography–mass spectrometry/mass spectrometry in positive and negative ion modes was used to identify metabolic changes associated with delayed freezing time. The trends of metabolite changes at 30–120 and 30–60 min of delayed freezing were analyzed. Results: 190 metabolites in 36 chemical classes were detected. After delayed freezing for 120 min, approximately 20% of the metabolites changed significantly in normal and tumor tissues, and differences in the metabolites were found in normal and tumor tissues. After a delay of 60 min, 29 metabolites had changed significantly in normal tissues, and 84 metabolites had changed significantly in tumor tissues. In addition, we constructed three tissue freezing schemes based on the observed variation trends in the metabolites. Conclusion: Delayed freezing of tissue samples has a certain impact on the stability of metabolites. For metabolites with significant changes, we suggest that the freezing time of tissues be reasonably selected according to the freezing schemes and the actual clinical situation. [ABSTRACT FROM AUTHOR]

Objective: In approximately 80% of the patients with advanced non-small cell lung cancer (NSCLC), the only samples available are cytological material or a limited amount of tissue specimens. Therefore, we aimed to explore the effect of the Idylla™ system for rapid evaluation of epidermal growth factor receptor ( EGFR ) mutation status in formalin-fixed and paraffin-embedded (FFPE) samples of NSCLC patients., Methods: A total of 221 archived FFPE NSCLC tissue specimens were included in this study. Idylla™ system and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were used to detect the EGFR mutation status. The Idylla™ system results and ARMS-PCR results were compared. When results were discordant, the Idylla™ system would be retested, and a third analysis method was performed to confirm test results when possible., Results: Among the 220 valid results of the Idylla™ system, the Idylla™ identified EGFR mutations in 109 cases with an incidence of mutation of 49.55%. The Idylla™ assay results were in complete agreement with the results of the reference methods for 207 cases, yielding an overall accordance of 94.09% (207/220, 95% CI, 90.11%-96.82%). Consequently, the overall concordance with routine reference methods, including further analysis, was found to be 96.81% (213/220, 95% CI: 93.28%-98.60%), with a negative percentage agreement of 97.3% (108/111, 95% CI: 91.72%-99.30%) and a positive percentage agreement of 96.33% (105/109, 95% CI: 90.32%-98.81%)., Conclusion: The Idylla™ system is a rapid and effective method that enables sensitive and reliable detection of EGFR mutations in FFPE samples of NSCLC patients, with minimal molecular expertise or infrastructure., (© 2024 by the Association of Clinical Scientists, Inc.)

Introduction  Fixation is the critical step in the preservation of tissues in diagnostic pathology. The formalin is an economical and excellent fixative with the inherent property of adequate fixation. The well-established side effects of formalin include mucosal irritation, upper respiratory diseases, and corrosive injury to the gastrointestinal tract. In addition, substantial evidence exists regarding the potential role of formaldehyde as a human carcinogen. The carcinogenic and toxic effects of formalin encourage searching for alternative fixatives for tissue fixation. However, "the formalin dogma" has severely hampered the search for alternative fixatives for many years. Material and Methods  Ninety tissues of liver and skeletal muscle obtained during autopsies were immersed in adequate amounts of the following fixatives: formalin (10%), methyl alcohol (70%), and acetone (100%). The comparison among the three was made based on time for fixation, preservation of tissue architecture, cell borders, cytoplasm, nuclear contours, chromatin texture, and uniformity of staining. Results  The tissue preserved in formalin undergoes rapid fixation compared with alcohol and acetone. The tissue architecture, cell border characteristics of alcohol and acetone was found satisfactory compared with formalin. The cytoplasm and nuclear contour were superior with the formalin. The chromatin texture and uniformity of staining were similar with all the three fixatives. Conclusion  The formalin is considered superior to most of the parameters, whereas both methyl alcohol and acetone showed nearly equivalent scores. Hence, owing to the potential human health hazards and carcinogenicity of formalin, no rational reasons hamper the complete substitution of formalin with alternative fixatives such as alcohol and acetone in diagnostic pathology and medical research. [ABSTRACT FROM AUTHOR]

Purpose: Projects evaluating the effects of radiation, within the National Institutes of Quantum and Radiological Science and Technology (QST), National Institute of Radiological Sciences (NIRS), have focused on risk analyses for life shortening and cancer prevalence using laboratory animals. Genetic and epigenetic alterations in radiation-induced tumors have been also analyzed with the aim of better understanding mechanisms of radiation carcinogenesis. As well as the economic and practical limitations of repeating such large-scale experiments, ethical considerations make it vital that we store and share the pathological data and samples of the animal experiments for future use. We are now constructing such an archive called the Japan-Storehouse of Animal Radiobiology Experiments (J-SHARE). Methods: J-SHARE records include information such as detailed experimental protocols, necropsy records and photographs of organs at necropsy. For each animal organs and tumor tissues are dissected, and parts are stored as frozen samples at −80 °C. Samples fixed with formalin are also embedded in paraffin blocks for histopathological analyses. Digital copies of stained tissues are being systematically saved using a virtual slide system linked to original records by barcodes. Embedded and frozen tissues are available for molecular analysis. Conclusion: Similar archive systems for radiation biology have also been under construction in the USA and Europe, the Northwestern University Radiation Archive (NURA), and STORE at the BfS, respectively. The J-SHARE will be linked with the sister-archives and made available for collaborative research to institutions and universities all over the world. [ABSTRACT FROM AUTHOR]

Background: Diabetes mellitus is a chronic lifelong disorder distinguishes oneself by chronic hyperglycemia and caused damage of organs. Bad control of diabetes leads to skin infections and non-healing foot ulcers which common in clinical practice. The access to the hospital and mutilation will increase and conversely result in long-term economic, physical, social, and mental disability to the patient. Methodology: This study is designed to identified bacteria from diabetic foot ulcer (DFU) patients by tissue specimen and investigates the sensitivity of these bacteria to Nigella sativa (NS) oil, other essential oils, and compounds. Results: Staphylococcus epidermidis 50% was recorded the highest occurrence in patients followed by Klebsiella pneumoniae 20%, Staphylococcus aureus, and Escherichia coli 13.3%. Pseudomonas aeruginosa 3.3% was recorded as the lowest occurrence in patients. Polymicrobial growth cultures were present in 6 (100%) of patients and appeared as S. aureus with E. coli and K. pneumoniae in 2 patient’s 33.3%, S. epidermidis with E. coli in 2 patient’s 33.3%, S. epidermidis with P. aeruginosa in 1 patient 16.6%, and S. aureus with K. pneumoniae in 1 patient 16.6%. (NS) oil has antibacterial activity against S. aureus and S. epidermidis. Almond oil, silver sulfadiazine, and hydrogen peroxide 3% were the only showed antibacterial activity. Conclusion: Tissue specimen is a good method for bacterial isolation in DFU. (NS) oil, almond oil, and some compounds have antibacterial properties. [ABSTRACT FROM AUTHOR]

Background: Bulk tumor tissue samples are used for generating gene expression profiles in most research studies, making it difficult to decipher the stroma-cancer crosstalk networks. In the present study, we describe the use of microdissected transcriptome profiles for the identification of cancer-stroma crosstalk networks with prognostic value, which presents a unique opportunity for developing new treatment strategies for ovarian cancer.Methods: Transcriptome profiles from microdissected ovarian cancer-associated fibroblasts (CAFs) and ovarian cancer cells from patients with high-grade serous ovarian cancer (n = 70) were used as input data for the computational systems biology program CCCExplorer to uncover crosstalk networks between various cell types within the tumor microenvironment. The crosstalk analysis results were subsequently used for discovery of new indications for old drugs in ovarian cancer by computational ranking of candidate agents. Survival analysis was performed on ovarian tumor-bearing Dicer/Pten double-knockout mice treated with calcitriol, a US Food and Drug Administration-approved agent that suppresses the Smad signaling cascade, or vehicle control (9-11 mice per group). All statistical tests were two-sided.Results: Activation of TGF-β-dependent and TGF-β-independent Smad signaling was identified in a particular subtype of CAFs and was associated with poor patient survival (patients with higher levels of Smad-regulated gene expression by CAFs: median overall survival = 15 months, 95% confidence interval [CI] = 12.7 to 17.3 months; vs patients with lower levels of Smad-regulated gene expression: median overall survival = 26 months, 95% CI = 15.9 to 36.1 months, P = .02). In addition, the activated Smad signaling identified in CAFs was found to be targeted by repositioning calcitriol. Calcitriol suppressed Smad signaling in CAFs, inhibited tumor progression in mice, and prolonged the median survival duration of ovarian cancer-bearing mice from 36 to 48 weeks (P = .04).Conclusions: Our findings suggest the feasibility of using novel multicellular systems biology modeling to identify and repurpose known drugs targeting cancer-stroma crosstalk networks, potentially leading to faster and more effective cures for cancers. [ABSTRACT FROM AUTHOR]

Background: Liposuction is one of the most performed cosmetic surgery procedures. In a previously reported study, gold-nanoparticle (GNP) laser-assisted liposuction (NanoLipo) was shown to improve procedure parameters and outcomes in a porcine model.Objectives: An ex vivo human liposuction model was developed to assess the ease, efficacy, and outcomes of NanoLipo, and to further explore its mechanism of action in facilitating liposuction.Methods: NanoLipo was compared to a control without GNPs in sets of fresh, nonperfused, anatomically symmetric, matched tissue specimens from 12 patients. A subset of three experiments was performed under single-blinded conditions. Intraoperative assessments included lipoaspirate volume, percentage of free oil, ease of removal, and temperature rise. Specimens were palpated, visualized for evenness, and graded with and without skin. Postoperative assessment included viability staining of the lipoaspirate and remaining tissues. Microcomputed tomography was used to assess the distribution of infused GNPs within the tissues.Results: NanoLipo consistently removed more adipose tissue with more liberated triglycerides compared to control. NanoLipo specimens were smoother, thinner, and had fewer and smaller irregularities. Infused solutions preferentially distributed between fibrous membranes and fat pearls. After NanoLipo, selective structural-tissue disruptions, indicated by loss of metabolic activity, were observed. Thus, NanoLipo likely creates a bimodal mechanism of action whereby fat lobules are dislodged from surrounding fibro-connective tissue, while lipolysis is simultaneously induced.Conclusions: NanoLipo showed many advantages compared to control under blinded and nonblinded conditions. This technology may be promising in facilitating fat removal.Level Of Evidence5: [ABSTRACT FROM AUTHOR]

Background: Hypochlorous acid (HOCl) demonstrates rapid and broad antimicrobial activity against planktonic and biofilm phenotype bacteria in vitro.Objectives: To identify the protein content present in breast pockets in vivo and calculate the estimated active concentration of HOCl, (PhaseOne, Integrated Healing Technologies, Franklin, TN) following HOCl-protein interactions.Methods: Fluid samples were collected prior to implant insertion in 18 consecutive patients, representing 36 pocket samples, with all cases being bilateral primary breast augmentations. Samples were evaluated by an independent CLIA approved laboratory for albumin and total protein concentration in g/dL. Results were compared to HOCl solution concentration and protein binding potential to determine availability of free HOCl.Results: The mean tissue sample concentration (right and left breast) was 31.6 mg/dL which translates to 0.0001 mmol per 20 cc of interstitial fluid. Mean total protein levels (right and left breast) were 62.3 mg/dL or 0.000187 mmol per 20 cc interstitial fluid. Based upon potential stoichiometric neutralization of HOCl by proteins in either a 1:1 or 3:1 ratio, using 115 cc of HOCl solution (per breast) at a concentration of 250 ppm/mL or 0.025% HOCl or = 0.48 mmol HOCl/dL, there would be 2950 times the amount of active HOCl at a 1:1 reaction ratio, or 983 times more HOCl assuming a 3:1 reaction ratio. Based on the range of identified levels of protein in individual surgical pockets in the study, there is an estimated 242 to 12,500 times more HOCl molecules than protein at a 3:1 molar ratio of binding or reactive protein.Conclusions: n estimated range of 983-2950 times more HOCl molecules are present during irrigation with 230 cc of PhaseOne® (115 cc for each breast) than available protein. This supports the antimicrobial and anti-biofilm activity as described in previous in vitro studies when using PhaseOne® as part of pocket irrigation.Level of Evidence5: [ABSTRACT FROM AUTHOR]

Poor clinical responses to checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies in melanoma have recently been associated with acquired IFNγ resistance that protects tumor cells from the antiproliferative and pro-apoptotic cytokine activity. IFNγ-resistant melanoma cells very often lack functional expression of the IFNγ signaling pathway gene JAK2 due to gene deletions or inactivating gene mutations. Analyzing melanoma cell lines (n = 46, applying next-generation targeted sequencing and single nucleotide polymorphism arrays) as well as available genomic data sets from The Cancer Genome Atlas (TCGA) tumor tissue samples (cutaneous melanoma n = 367, lung squamous cell carcinoma n = 501, bladder urothelial carcinoma n = 408, breast invasive carcinoma n = 768, colorectal adenocarcinoma n = 257), we demonstrate that the frequent chromosomal losses of the tumor suppressor CDKN2A in melanoma and other tumor entities enhance the susceptibility to IFNγ resistance by concomitant deletion of the JAK2 gene (odds ratio = 223.17, 95% confidence interval = 66.91 to 1487.38, two-sided P = 7.6×10-46). Tumors with JAK2 mutations or homozygous JAK2 deletions demonstrate allelic losses covering both CDKN2A and JAK2. This suggests that patients with tumor chromosomal CDKN2A losses are susceptible to developing immunotherapy resistance and should be screened for JAK2 deficiency prior to and under immune checkpoint blocking therapy. [ABSTRACT FROM AUTHOR]

With the development of high‐speed, high‐resolution whole slide histology digital scanners, glass slides of tissue specimen can now be digitised at high magnification to create the whole slide image. Quantitative image analysis tools are then desirable to help the pathologist for their routine examination. Epidermis area is a very important observation area for the cancer diagnosis. Therefore, in order to build up a computer‐aided diagnosis system, segmentation of the epidermis area is often the very first and crucial step. An improved computer‐aided epidermis segmentation technique for the whole slide skin histopathological image is proposed in this study. The proposed technique first obtains an initial segmentation result with the help of global thresholding and shape analysis. A template matching method, with adaptive template intensity value, is then applied. Finally, a threshold is calculated based on the probability density function of the response value image. Experimental results show that the proposed technique overcomes the limitation of the existing technique and provides superior performance, with sensitivity of 95.68%, specificity of 99.41% and precision of 93.13%. The performance of the proposed technique is satisfactory for future clinical use. [ABSTRACT FROM AUTHOR]

We describe a homicide case involving Aconitum tuberous roots, in which formalin-fixed solid tissues were analyzed for diester-type Aconitum alkaloids and their hydrolysates by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The specimens were taken from a cadaver 12 days after death, and preserved in formalin solution for 5 years. Polymer-based solid-phase extraction was employed for sample cleanup. This procedure allowed detection of 0.10 ng/g for each of the spiked alkaloids from the formalin-fixed liver by quadrupole LC-MS-MS, and 10 pg/g for benzoylaconine and benzoylmesaconine by ion-trap LC-MS-MS. Sub-parts-per-billion levels of benzoylaconine and benzoylmesaconine could be detected from the formalin-fixed liver, kidney, and lung specimens. [ABSTRACT FROM AUTHOR]

Background Molecular diagnostic tests can identify bacterial and fungal pathogens from clinical samples. Nucleic acid detection tests include 16S and 18S rRNA gene PCR (16/18S PCR) and plasma next-generation sequencing (NGS). Other assays (fungal galactomannan and 1,3-β- d -glucan) detect structural factors. Our objective was to assess the utilization, yield, and impact of molecular diagnostics in pediatric patients who had samples sent for 16/18S PCR. Methods Sterile site fluid or tissue specimens were collected as part of standard care at Lurie Children's Hospital, cultured, and sent to Northwestern Memorial Hospital for 16/18S PCR as clinically indicated. Medical records were reviewed for diagnostics, antibiotics, and clinical course. Results From 1/2016–8/2018, 236 samples were sent for 16 and/or 18S PCR from 183 patients. 83% had a concurrent ID consult. 16S PCR was done on 215 samples, 42 (20%) were positive, and 36 yielded species identification (Table 1). Antibacterial agents were administered prior to specimen collection in 73% and did not affect likelihood of positive 16S PCR. 18S PCR was sent on 163 samples; 12 (7.4%) were positive (Table 2) of which 10 were from immunocompromised hosts. 40% of patients were on antifungals prior to sample acquisition. 16/18S PCR impacted antimicrobial decision-making in 70 cases (30%). A pathogenic fungus was detected by PCR but not culture in 2 cases. Time to positivity of fungal culture was 1–15 days. Fungal culture was positive in 5 cases with-negative 18S PCR. Seventeen patients had positive serum 1,3-ß-D-glucan and/or galactomannan: 3 of which had positive 18S PCR, 5 with fungal growth, 5 presumed infection based on imaging, 1 Nocardia , and 3 noninfectious etiology. Plasma NGS was sent on 45 cases, was positive in 34, and affected clinical management in 10. Conclusion 16S PCR can identify bacterial pathogens in the setting of negative culture and impact clinical care. Abscess, bronchial/pleural fluid, and brain/organ tissue were high yield specimens. 18S PCR can provide expeditious fungal identification in cases of suspected invasive disease, but fungal culture and serum molecular testing increase diagnostic yield. No single fungal test is comprehensive. Plasma NGS had relatively high yield and clinical impact in selected patients. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to explore the differential expression profiles of microRNAs (miRNAs) in paraffin-embedded acute aortic dissection (AAD) tissues to find potential biomarkers for this disease., Methods: A total of 92 paraffin-embedded tissue specimens were collected from 92 patients with AAD who underwent surgical replacement. Among these specimens, 54 had partial normal aortic segments (smooth intima surface, non-atherosclerotic lesions) in proximal crevasse of aorta. Samples of these segments were taken 1 cm away from aortic lesions as the control group, after eliminating the tunica adventitia tissues. miRNA expression profiles were obtained by miRNA microarray analysis. Differentially expressed miRNAs were found by comparing the AAD group with the control group and were verified by fluorescence real-time quantitative polymerase chain reaction and by fluorescence in situ hybridization., Results: A total of 71 differentially expressed miRNAs were detected. Twenty-two were up-regulated and 49 were down-regulated. Four up-regulated miRNAs (hsa-miR-636, hsa-miR-142-3p, hsa-miR-425-3p, hsa-miR-191-3p) were selected for validation by real-time fluorescence quantitative polymerase chain reaction and fluorescence in situ hybridization. In the fluorescence real-time quantitative polymerase chain reaction analysis, only hsa-miR-636 showed a statistically significant difference in the AAD versus control comparison (3.3-fold, P = 0.012). The fluorescence in situ hybridization validation showed that the expression level of hsa-miR-636 was significantly increased in the AAD versus control comparison (P < 0.001), with average optical densities of 61.29 ± 16.83 in the AAD group and 9.30 ± 3.98 in the control group., Conclusions: Hsa-miR-636 is involved in the pathogenesis of AAD and may be a potential biomarker for this disease., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Background Molecular and biological characterization of nasopharyngeal cancer (NPC) is scarce. Clinical trials of emerging treatment such as immunotherapy for NPC in endemic area is lacking. Currently the most extensively studied immune related molecule that potentially become the target for treatment is PD-1/PD-L1. PD-L1 profile of NPC patients in Indonesia has never been studied before. This study aimed to elucidate PD-L1 profile from NPC specimens in Indonesia. Methods Nasopharyngeal biopsy specimen was taken for PD-L1 protein examination with enzyme-linked immunosorbent assay (ELISA) method. Tissue specimen was homogenized and then ELISA test was conducted using ABCAM AB214565 kit. All protocols from the manufacturer kit was followed. Total protein concentration from homogenized tissue specimen was obtained through Bradford protein assay method. Then, PD-L1 concentration per mg protein of tissue specimen was calculated. Results Twenty-four nasopharyngeal biopsy specimens was obtained. The average age of all patients was 45 years old. Sixteen (66.67%) out of 24 specimens were confirmed histologically to be undifferentiated nasopharyngeal carcinoma. The rest, 8 other specimens have normal histology. Average PD-L1 concentration from NPC specimens was 2,186.67 ± 1,399.32 pg/mg protein (Range: 461.76 to 4,805.85 pg/mg protein). Average PD-L1 concentration from non NPC specimens was 612.43 ± 314.04 pg/mg protein (Range: 326.87 to 1,155.19 pg/mg protein). Conclusions From this preliminary result, PD-L1 protein (both in tumor cells and in immune cells) from NPC specimens has been shown to be elevated compared to normal nasopharyngeal epithelium. This finding indicate that further work is warranted to test anti PD-L1 drug for NPC patients that has failed all the standard treatment. Furthermore, our upcoming work would be exploring relationship between PD-L1, EBV, and tumor microenvironment from our NPC specimens. Legal entity responsible for the study The authors. Funding University of Indonesia. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background Treatment of non-small cell lung cancer (NSCLC) has undergone a paradigm shift in the last decade with the advent of targeted therapy and immunotherapy. Anaplastic Lymphoma Kinase (ALK) rearrangement and Programmed Death-Ligand 1 (PD-L1) expression are important predictive biomarkers for response to targeted therapy and immunotherapy respectively. How these two biomarkers interplay with each other is yet to be determined. Various studies have tried to establish a relationship between PD-L1 expression in tumors and treatment outcomes in ALK rearranged NSCLC, with conflicting results. There is a paucity of data regarding this relationship from the Indian subcontinent. Methods PD-L1 expression was assessed in tissue samples of 58 patients with advanced ALK rearranged NSCLC treated between 2015 and 2018. PD-L1 expression was measured by immunohistochemistry using VENTANA PD-L1 (SP263) antibody on formalin-fixed paraffin-embedded specimens. The epidemiological profile of patients and the relationship between progression free survival (PFS) on small molecule ALK inhibitor and PD-L1 expression were noted. Results The median age was 47 years (range 25-72 years) and there were 25 males (50%). Most of the patients were never smokers (88%). Thirty-seven (63.7%) of tumors expressed PD-L1 (≥1%) and 13 (22.4%) tumors had a PD-L1 expression of ≥ 50%. All of the patients received crizotinib as first line ALK inhibitor. Of the 44 patients evaluable for treatment response there was no significant difference in PFS between PDL1 negative cohort (median PFS - 13.8 months, CI 6.2-21.3 months) and PDL1 positive cohort (median PFS 10.9 months, CI 6.5-15.2 months, P-0.81). No association was seen between PD-L1 expression and PFS when PD-L1 expression levels were stratified by median or quartiles. In the subgroup analysis, PFS was not significantly different between different patterns of PDL1 expression or intensity of staining. Conclusions In contrast to previous studies, we found a higher incidence of PD-L1 expression in patients with ALK rearranged advanced NSCLC. PFS on ALK inhibitors did not differ based on PD-L1 expression. However, this was a retrospective study in a limited number of patients. More prospective studies are needed to confirm our findings. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background To study of immunohistochemical markers of proliferation and apoptosis are important prognostic indicators for predicting the clinical course of tumours and the development of therapeutic tactics. Methods The p53 suppressor gene, the bcl-2 oncogene and the Ki-67 antigen are responsible for DNA repair, cell division and apoptosis. In all patients, tumour material was obtained using biopsies and operations. In tumour tissue samples fixed by neutral formalin with standard wiring and embedded in paraffin, tumour markers of apoptosis and proliferation were determined - p53, bcl-2, Ki – 67. A positive result was the presence of specific brown staining of the nuclei in the detection of the Ki-67 antigen and the p53 gene suppressor and the cytoplasm in the detection of the expression of bCl-2. Results Cases where the nucleus or cytoplasm was not stained by reaction with antibodies to p53, Ki-67 and bcl-2 or the number of positive cells was less than 10%, the reaction was considered negative (0). Weak (1+) reaction was considered when staining 10-30% of cells; medium reaction (2+) - when staining 30-50% of cells; strong reaction (3+) - intense staining from 70 or more cells. We carried out a comparative assessment of the effectiveness of treatment of patients depending on the type of therapy and the phenotype of tumour cells. Among patients who received systemic chemotherapy, the full effect was observed in 13% of patients. With negative and weakly positive reactions to the mutant p53 gene and Ki 67 among them was 70.6%, and with moderate and strong expression of bcl-2 (80%). Patients with a phenotype of tumour cells with a negative or weakly positive IHC response to the mutant p53 gene (58.5% of patients), weakly positive expression of Ki 67 (70.6% of patients), and high expression of bcl-2 (46.4 % of patients). In 37.4% of patients from this group, the effect of therapy was negative, most of them had tumour cells with high expression of the mutant p53 and Ki 67 gene, and no expression of bcl-2. Conclusions It was established that with positive expression of p53 + Ki-67 + and negative bcl-2 parameters, patients had an unfavourable prognosis. Legal entity responsible for the study Ronc. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background Gastrointestinal stromal tumor (GIST) is a type of mesenchymal tumor and has KIT mutations in 80–90% of the cases. The development of tyrosine kinase inhibitors have greatly improved patient outcomes. We have previously described association between lower pfetin (KCTD12) expression and poor prognosis in the GIST patients (Ref 1. Suenara Y et al. Clin Cancer Res2008). In this study, to investigate the protein profiles of kit expression, we performed comprehensive protein expression analysis using the gastric tumor cell lines and surgical specimens in GIST. Methods We performed KITgene knockdown using siRNA inthe GIST cell line (T1 cell line) and generated the protein expression profiling data by i-TRAQ. Based on this i-TRAQ protein profiling data from the T1 cell line, we referenced our previously published protein expression analysis databases associated with GIST prognosis in the patient samples (Ref 1.) and compared the protein profile of KIT-associated proteins (T1 cell line) and prognosis-associated protein (patient samples). Results In our study, the i-TRAQ profiling method detected expression of approximately 1500–2000 proteins. In KITknockdown cells, 125 proteins were identified to have statistically different expression (p < 0.05). Based on our previously published GIST prognosis protein profile database (using 17 patient tissue samples), 25 proteins were identified to be statistically significant (p < 0.05). Upon comparing the prognosis database with theKIT knockdown database, 6 proteins were identified as common proteins.In particular, the heat shock protein (HSP) 90-beta, which has been previously shown to contribute to the growth of cancer cells, was identified as a critical protein in the biological behavior of GIST. Conclusions HSP 90-beta is currently considered as a therapeutic target for HSP 90 inhibitors against imatinib-resistant GIST. Our data suggests that the differentiation and cell growth of GIST may be enhanced by the identified proteins, which are in turn induced by KIT. We believe that our established protein profile data will help in improving the understanding of malignant behaviors in GIST. Clinical trial identification HSP 90-betaplay an important role in the tumor progression of GISTs. Legal entity responsible for the study Juntendo University. Funding This study was supported by Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number #19K22694, #19H03789 and #15KK0353 to Y.S. ; #17K08730 to T.S.; #17K10987to K.K.; #19K16753 to K.A.; #18K15329 to T.O. , # 18K16634 to Y.K.) and Leading Advanced Projects for Medical Innovation (LEAP) (Grant Number # JP18am0001009) and the Practical Research for Innovative Cancer Control (Grant Number # JP18ck0106252) from the Japan Agency for Medical Research and Development. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background There is an expanding list of therapeutically relevant biomarkers for NSCLC and effective molecular profiling is paramount. We sought to comprehensively and prospectively evaluate the molecular epidemiology of NSCLC in Asia and ascertain the extent of access to relevant therapies. The study is being conducted by the Asian Thoracic Oncology Research Group (ATORG) – an academic co-operative lung cancer trials group in Asia. Methods ATORG-001 is a prospective, multi-centre study of NSCLC patients (pts) throughout Asia. Eligible pts must have good performance status (ECOG ≤2), adequate organ function and ≤3 prior lines of cytotoxic chemotherapy. Archival tissue specimens are sent to a central laboratory in Singapore (Singapore General Hospital) for profiling, consisting of NGS with the Oncomine Focus Assay (52 gene panel including fusions) and PD-L1 immunohistochemistry. Results are returned to investigators in a clinically relevant timeframe. Baseline demographics, pre-existing molecular profile and prior treatment details are collected. Treatment and pt outcomes are followed for 2 years. Recruitment commenced in Jan 2019 in 3 sites (National Cancer Centre Singapore, Chinese University of Hong Kong, University Malaya Medical Centre). We report an interim analysis for the first 27 of a planned 500 pts. Results As of 01 Jul 2019, 27 pts have enrolled with median age 67 yrs (range 46-79), 59% male, 81% adenocarcinoma, 52% stage IV, 37% non-smokers, median lines of prior therapy 1 (range 0-6) and 85% with no known oncogenic driver. Pre-existing mutation status was known in 89% for EGFR and 63% for ALK. Median turnaround time from tissue acquisition to profiling results was 22 days (range 15-74). New alterations were most common in KRAS (19%), MYC (15%), CDK4 (7%) and ERBB2 (7%). Potentially actionable new alterations were found in 7 (26%) pts, including KRAS G12C mutation (7%), BRAF G469A mutation (4%), ERBB2 exon 20 insertion (4%), ERBB2 amplification (4%), FGFR2 amplification (4%) and MET exon 14 skipping mutation (4%). Conclusions Enrolment is ongoing with additional sites and countries planned to comprehensively evaluate the molecular epidemiology of NSCLC in Asia. Legal entity responsible for the study Asian Thoracic Oncology Research Group. Funding Asian Thoracic Oncology Research Group, Novartis. Disclosure A.C. Tan: Travel / Accommodation / Expenses: ASLAN Pharmaceuticals. All other authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background In this study, we examined the TMB landscape of 5,660 pan-cancer cases in Chinese population, using NGS panel. We established cancer-specific and histology-specific biological pathways associated with the TMB status. In addition, as a proof on concept, an unsupervised algorithm was conducted using stepwise logistic regression to generate TMB-predicting signatures from both lung adenocarcinoma and lung squamous cell carcinoma. Methods Patients: 5,660 Chinese cancer patients across 11 cancer types Panel: Targeted sequencing was performed on tissue samples using a panel consisting of 295 or 520 cancer-related genes, spanning 1.4 and 1.6Mb of human genome, respectively. An average sequencing depth of 1,000X and 10,000x were achieved for tissue and plasma samples, respectively. Tumor mutation burden: calculated as the ratio of mutation count to the size of coding region of the panel, excluding CNV, fusions, large genomic rearrangements and mutations occurring on the kinase domain of EGFR and ALK. Results Across the 11 cancer types included in the analysis, lung squamous cell carcinoma had the highest average TMB, whereas sarcoma has the lowest TMB. High microsatellite instability, DNA damage response deficiency, and homologous recombination repair deficiency indicated significantly higher TMB.The independent predictive power for TMB 26 biological pathways was tested in 11 cancer types. Mismatch repair, DNA damage response, homologous recombination repair, and PI3K-AKT signaling pathway were most commonly correlated with high-TMB. In contrast, ERBB signaling pathway, and adhesion-related pathways were most commonly correlated with low-TMB. Moreover, we developed a 23- and 16-gene signature for TMB prediction for LUAD and LUSC, respectively, with 12 genes shared by both signatures, indicating a histology- specific mechanism for driving high- TMB in lung cancer. Conclusions The findings extended the knowledge of the underlying biological mechanisms for high TMB and might be helpful for developing more precise and accessible TMB assessment panels and algorithms in more cancer types. Legal entity responsible for the study The authors. Funding Burning Rock Biotech. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background It is now clear that the immune system has a substantial effect on the progression of colon cancer. Typically, an immune response defined by a polarized Th1 phenotype, characterized by expression of CXCR3/CCR5 chemokine-receptor ligands, activation of interferon stimulated genes and production of cytotoxic molecules by effector immune cells, has been associated with immune-mediated tumor rejection. We have previously introduced a gene signature, called Immunology Constant of Rejection (ICR), that reflects these immune components. This signature was able to differentiate quite well the patients with an active immune environment and improved survival vs those who did not display this phenotype. This phenomenon although expected based on other immune infiltration studies could not be observed in the TCGA colon cancer cohort, likely due to the per protocol exclusion of samples with low purity. To gain more insight in the underlying mechanism of cancer tissue rejection by the immune system, we build an extensive data repository from high quality colon cancer samples unbiased for tumor purity. Methods RNA and DNA were isolated from fresh frozen tissue samples of a cohort of 366 colon cancer patients collected over the last decade at the University of Leiden, Netherlands. Tissue sections flanking the corresponding samples were hematoxylin and eosin stained. RNA-seq (HiSeq4000) data was obtained using HISAT2 alignment and quantile normalization of the GC corrected raw counts. Whole Exome Sequencing (>100X) for our cohort for normal and cancer tissue respectively. T-cell repertoire profiling of 150 tumors was achieved using Adaptive immunoSEQ. Tumor immune phenotype classification was done using unsupervised consensus clustering based on the expression of ICR genes. Results Our preliminary data supports a positive impact of ICR gene expression in our colon cancer cohort: patients in ICR High cluster had a significantly improved survival compared to other clusters. Conclusions This newly generated immune centric NGS dataset, generated in Qatar, will is used to elucidate the genetic determinants of immune phenotype in colon cancer and the relationship of the immune phenotype, the tumors genetics the TCR diversity. Legal entity responsible for the study Sidra Medicine. Funding Qatar National Research Fund. Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background Main challenges in thyroid cancer management are low sensitivity of preoperative differential diagnostics and absence of sufficient prognostic markers for choosing of therapeutic strategy, including decision regarding radioiodine therapy. The study aims evaluation of clinical utility of analyses of comprehensive set of driver and secondary mutations with targeted high-throughput sequencing. Methods Point mutations in 52 genes, CNVs at 1q, 9q, 17q, 22q, and fusions of 66 genes were analyzed with targeted high-throughput sequencing. The analyzed mutations included well known driving and putatively driving mutations selected from TCGA project and literature, and secondary mutations with known or putative prognostic significance. Enrichment of regions of interest was performed with AmpliSeq technology. Fusions were detected by the presence of chimeric transcripts. Sequencing was performed on Illumina NextSeq (for point mutations and CNVs) or MiSeq (for fusions). Samples included 64 confirmed cancer samples (41 samples with Bethesda IV-V cytology, and 23 samples - Bethesda VI cytology), 16 confirmed benign samples, 12 normal tissue samples. Results Mutations detected in carcinomas included, in addition to BRAF and RASs: CDKN2B, ZFHX3, PDGFRA, KIT, NTRK3, ECM2, TERT, KDR, KMT2A, TP53, RNF213, APC, CHEK2, PTEN, PPM1D, ATR, CDKN2A, CTNNB1, ARID1B, KMT2C, ALK, DICER1, fusions of NTRK3, NTRK1, PPARG and RET, and CNVs 22q-del и 9q-del. The detected mutations allow to characterize all studied cancer cases. In benign nodules mutations in NRAS, TSC2, KDR, EIF1AX, ARID1B, TSHR, ZFHX3, GRIN3A, DICER1, HRAS, CHEK2 were found. Conclusions Tests limited to BRAF, K/N/HRAS, RET-PTC1/3, PAX-PPARG mutations have low diagnostic value. The developed panel allows analyzing an expanded range of mutations, including recently described rearrangements and CNV. Design of the panel, initial validation and sequencing of cancer samples were funded by Foundation for Assistance to Small Innovative Enterprises in Science and Technology (№ 442ГС2/9119 - С3-40846); sequencing of benign samples was supported by the grant of the Russian Science Foundation (Agreement 19-75-00053). Legal entity responsible for the study The authors. Funding Foundation for Assistance to Small Innovative Enterprises in Science and Technology (442ГC2/9119 - C3-40846); Russian Science Foundation (Agreement 19-75-00053). Disclosure All authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis and high mortality worldwide. Despite the emergence of new therapeutic approaches for the treatment of inoperable PDAC, reliable prognostic/predictive biomarkers are lacking. PDAC harbours mutations in the KRAS gene in 80-90% of patients. The aim of the present study is the detection and quantification of RAS mutations in the ctDNA of patients with inoperable PDAC by BEAMing digital polymerase chain reaction (PCR), in order to (a) evaluate the correlation with tissue RAS mutation status in the tumour, (b) study the baseline prognostic significance and (c) study the predictive significance of ctDNA RAS mutation kinetics for the prediction of response to systemic/neoadjuvant therapy. Methods Plasma samples of patients with locally advanced or metastatic inoperable PDAC were collected at diagnosis and at 4-6 weeks after treatment initiation and were tested for 34 mutations in the KRAS and NRAS genes by BEAMing digital polymerase chain reaction (PCR). This method has a particularly high sensitivity and can accurately quantify mutated DNA as the Mutant Allele Fraction (MAF). To test the efficacy and validity of ctDNA for the detection of RAS mutations, RAS mutations will also be tested for on paired tumour tissue samples of the patients under study by PCR. Results So far, 38 plasma samples from 20 patients were available for analysis. At baseline, mutant ctDNA pertaining to exon 2 codon 12 of the KRAS gene was detected in all patients' samples and, additionally, to exon 3 codon 61 of the NRAS gene in one of them. Comparison of plasma and tissue results revealed a high agreement of the RAS mutational profile in plasma ctDNA and tissue tumour DNA. Kinetics of the RAS mutational load during therapy and its prognostic or predictive value will be analysed with further cohort expansion. Conclusions This work highlights the potential role of liquid biopsies in the management of PDAC and, more specifically, will yield preliminary data on the role of RAS ctDNA profiling as a predictive and prognostic marker. Legal entity responsible for the study Department of Medical Oncology, University General Hospital of Ioannina. Funding Hellenic Society of Medical Oncology (HeSMO) and Society for Study of Clonal Heterogeneity of Neoplasia (EMEKEN). Disclosure G. Pentheroudakis: Research grant / Funding (institution), Support material was provided for this project: Sysmex Inostics. All other authors have declared no conflicts of interest. [ABSTRACT FROM AUTHOR]

Background Precision medicine based on driver oncogenes is now developed for non-small cell lung cancer. Recent approval of next-generation sequencing (NGS)-based companion diagnostics has increased the need for the use of high-quality specimens for the diagnosis. Methods From August 2015 to May 2018, 82 patients who were enrolled in a nation-wide genome screening, LC-SCRUM-Japan, from our institution were examined for the success rates of genetic analysis according to sample types. A NGS analysis, Oncomine Comprehensive Assay, was performed using DNA and RNA extracted from lung cancer specimens. Results The success rates of DNA and RNA analyses were 100% (82/82 cases) and 78% (64/82), respectively. Of the 82 samples analyzed, 80 (98%) were tissues and two (2%) were pleural effusions. The success rates of RNA analysis in the 80 tissue samples were 45/60 (73%) in bronchoscopic byopsy, 10/10(100%) in operation, 5/6(83%) in Percutaneous biopsy and 3/4(75%) in others. Of the 60 bronchoscopic samples, the success rates were 72% (18/25) in EBUS-TBNA, 88% (7/8) in EBUS-GS (large diameter), 69% (11/16) in EBUS-GS (small diameter) and 82% (9/11) in direct vision biopsy. In the 82 cases analyzed, a total of 52 actionable gene alterations (18 KRAS mut, 6 EGFR mut, 6 MET amp/fus, 5 RET fus, 5 ALK fus, 4 PIK3CA mut, 2 ERBB2 mut, 2 ROS1 fus, 2 FGFR1 amp, 1 BRAF mut, 1 FGFR3 fus) were detected in 48 cases (59%). Twelve of the 48 (15%) were registered in ongoing clinical trials of targeted drugs. Conclusions All the samples were available for DNA analysis of the NGS. The success rates of RNA analysis were lower in samples obtained from EBUS-TBNA and EBUS-GS (small diameter), suggesting that the success rates depend on the sample size. [ABSTRACT FROM AUTHOR]

Clinical research involves the study of people, data or human tissue samples in order to understand health and disease while seeking new and better ways to detect, diagnose, treat and prevent disease. Current global statements support patient and public involvement (also known as PPI or patient engagement) in a range of research activities, including clinical research. These activities include helping to develop research questions, working on advisory groups and/or disseminating research findings. During the dialogues or focus groups, we can combine benefit and risk assessments by professionals with benefit and risk assumptions by lay people; the best way to select participants, the bearable levels of risks and burdens as well as desirable methods for informing participants of the side effects and possible direct benefits. Japan Agency for Medical Research and Development (AMED) began to introduce PPI in 2017, the first comprehensive policy for the country. However, there is no data regarding attitudes about of researchers with PPI in Japan. We conducted cross-sectional online surveys with AMED-funded principal investigators in order to know the current views from researchers about PPI. We would like to discuss benefits of PPI and issues to be solved from our survey results. [ABSTRACT FROM AUTHOR]

Background GEP, a first in class novel triazaacenaphthylene bacterial topoisomerase inhibitor, inhibits bacterial replication and has in vitro activity against key pathogens implicated in a range of infections, including drug-resistant strains of E. coli associated with acute cystitis. Methods PK and PD studies were conducted in murine (male CD-1 mice) thigh and kidney infections. The administered doses ranged from 1 to 200 mg/kg SC every 6 hours starting 1-hour post-infection. Infected tissues were evaluated for bacterial burden at 24-h post-infection (baseline controls at 1-hour post-infection). Plasma and tissue samples (kidney or thigh homogenates) were collected at 15, 30, 60, 120, 240 and 360 minutes. A population PK (PopPK) model was built in NONMEM using plasma exposures. Efficacy was determined against E. coli ALL, 997577, ATCC25922, IR5 and NCTC13441 (MICs of 1 to 4 µg/mL) in thigh-infected neutropenic (I-) mice and against E. coli ALL in kidney-infected immunocompetent (I+) and I- mice. The PopPK model was used to determine GEP exposures associated with efficacy. PK-PD analyses were conducted using Phoenix WinNonLin 6.3 (Pharsight). The change in log10 colony-forming units (CFU) from baseline were correlated with free drug (f) AUC:MIC using an inhibitory model from the Phoenix library, and model parameter values for each isolate were used to calculate the plasma fAUC:MIC associated with stasis, 1- or 2-log10 reductions in CFU. Results Plasma PK data were best fit by a 1-compartment IV model with first-order elimination and were similar in I+ vs. I- and thigh- vs. kidney-infected mice. The AUC0-6 of GEP in kidney was approximately 4- to 5-fold higher than in plasma while the AUC0-6 in thigh was approximately half of plasma. In the I- thigh model, median plasma fAUC:MIC ratios for stasis, 1- or 2-log10 reductions in CFU were 11, 16, and 25 (ranges 3–17, 4–25 and 7–40), respectively. Efficacy vs. E. coli ALL was similar in I- mice infected in thigh or kidney. In I+ mice, the PK-PD target was reduced by half. Conclusion Median plasma fAUC:MIC targets ranged from 11 to 25. Higher drug levels in kidney vs. plasma or thigh did not translate into improved efficacy in pyelonephritis vs. thigh-infection models. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

Background Actinomyces sp. are associated with numerous clinical diseases in humans; however, there are few case reports of necrotizing soft-tissue infections (NSTIs) involving these organisms. Their role in NSTIs has not been well described. At our medical center, we noted an increase in Actinomyces sp. isolated from the tissue specimens of patients with NSTIs, prompting further evaluation. Methods Microbiology databases were utilized to identify patients with clinical cultures growing Actinomyces sp. from January 2008 to December 2018. Adult patients admitted to the R Adams Cowley Shock Trauma Center with a diagnosis of NSTI were included for analysis. Results Nine patients were identified meeting inclusion criteria, the first in February 2018–none prior. Organisms isolated from culture included Actinomyces turicensis (n = 3), Actinomyces europeaus (n = 1), and five organisms identified only as Actinomyces species. 89% of patients had additional co-pathogens identified in their tissue cultures. Eight patients had NSTIs of the lower extremity (n = 5) and/or the genitourinary area (n = 6), and one had chronic decubitus ulcers. Comorbidities included diabetes mellitus (77%), chronic kidney disease (33%). 44% patients were in septic shock at presentation. Surgical debridement was performed in all patients. Eight patients were discharged on amoxicillin, with a mean treatment duration of 75 days (range 31–90). One patient was treated with ampicillin–sulbactam. Readmission rate at 90 days was 37%; only one was related to the index infection. One death occurred during the index hospitalization, secondary to NSTI. No patients experienced adverse drug reactions during therapy. Conclusion We describe one of the largest case series to date of Actinomyces sp. associated with NSTI. The startling appearance of Actinomyces sp. at our institution directly followed the implementation of matrix-assisted laser desorption/ionization time of flight mass spectrometry in January 2018. Actinomyces sp. may act as co-pathogens contributing to the severity of NSTIs, augmenting the virulence of other organisms. As more advanced technology is used in laboratories to identify these organisms, further study is needed to determine pathogenicity and appropriate treatment. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

Histopathological examination is considered as gold standard procedure for arriving at a final diagnosis of various lesions of the human body. However, it is limited by a number of alterations of normal morphologic and cytological features that occur as a result of presence of artifacts. These artifacts may occur during surgical removal, fixation, tissue processing, embedding and microtomy and staining and mounting procedures. They can even lead to complete uselessness of the tissue. It is therefore essential to identify the commonly occurring artifacts during histopathological interpretations of tissue sections. This article reviews the common artifacts encountered during slide examination alongside the remedial measures which can be undertaken to differentiate between an artifact and tissue constituent. [ABSTRACT FROM AUTHOR]