Spatial Intratumoral Heterogeneity Expression of PD-L1 Antigen in Head and Neck Squamous Cell Carcinoma.

INTRODUCTION: Immune-checkpoint inhibitors have demonstrated a significant survival benefit in metastatic and non-resectable head and neck squamous cell carcinoma (HNSCC). Patients with a combined positivity score (CPS) of 20 and higher benefit the most from therapy. Inaccurate definition of the CPS category might lead to the incorrect stratification of patients to immunotherapy. This study's main aim was to investigate programmed death-ligand 1 (PD-L1) antigen expression in HNSCC in diverse clinical situations and histological settings. MATERIALS AND METHODS: This is a prospective cohort study conducted in a tertiary referral medical center. Tissues were investigated for PD-L1 expression using the FDA-approved 22C3 immunohistochemistry assay (Dako). We analyzed potential associations between the CPS category and meaningful demographic, clinical, and outcome metrics. Furthermore, we investigated morphologically separate sites for CPS scores in whole surgical tissue specimens and matched preoperative biopsies. RESULTS: We analyzed 36 patients, of whom 26 had oral cavity SCC and 10 had laryngeal SCC. The overall, disease-specific, and progression-free survival of the HNSCC group of patients were not associated with the CPS category (p = 0.45, p = 0.31, and p = 0.88, respectively). There was a significant (18%, 95% CI 0.65-0.9) inconsistency between the CPS category determined in biopsies versus whole carcinoma analyses. We also found an uneven distribution of whole-tumor CPS attributed to spatial carcinoma invasiveness, tumor differentiation, and inflammatory cell infiltration heterogeneity. DISCUSSION AND CONCLUSIONS: Our data suggest that careful selection of tumor area for CPS analysis is important. PD-L1 antigen expression, clinically represented by CPS, may be up- or down-categorized in different clinical and pathological circumstances. The high whole-tissue CPS category scatter may clinically result in potential treatment modifications. We argue that CPS analysis requires not only adequacy (at least 100 viable tumor cells), but also correct representation of the tumor microenvironment.

Histological and immunohistochemical characterization of 17 gonadal tumours in koi carp (Cyprinus carpio koi).

Reports on abdominal tumours in koi carp are scarce and most are from the gonads. Their histological diagnosis is challenging due to the occurrence of mixed populations of neoplastic cells and the few availability of cross-reactive antibodies in fish tissues. The present study aims to provide a histopathological characterization of seventeen gonadal tumours, enriched by a wide antibody panel (vimentin, CD117, placental alkaline phosphatase-PLAP, AE1/AE3 cytokeratin, E-cadherin, proliferating cell nuclear antigen-PCNA, müllerian-inhibiting substance-MIS, GATA4 and Inhibin-α) applied on whole and tissue microarray (TMA) sections. Abdominal enlargement was associated with tumours filling 30%-80% of the abdominal cavity; frequently, the gonads had been completely replaced by neoplastic tissue. Twelve cases were characterized as sex cord-stromal tumours (SCSTs), three as germ cell tumours (GCTs), one as mixed germ cell sex cord-stromal tumour (MGCSCST) and one as carcinoma. By immunohistochemistry, PLAP enabled confirmation of GCTs, ovarian carcinoma and the objective identification of a further cell component in 8 out of the 12 SCSTs that were reclassified as mixed tumours. The use of an immunohistochemical panel can help in refining the histological diagnosis, but the morphological diagnosis still represents the main tool for the characterization of these tumours in koi carp.

Expression of Aldehyde Dehydrogenase 1A1 (ALDH1A1) as a Prognostic Biomarker in Colorectal Cancer Using Immunohistochemistry.

BACKGROUND The expression of aldehyde dehydrogenase 1A1 (ALDH1A1) is increased in several human tumors, including colorectal carcinoma (CRC). The aim of this study was to compare the expression ALDH1A1 in CRC tumor tissue compared with non-tumor adjacent tissue (NAT), using immunohistochemistry (IHC), and to determine whether the expression of the ALDH1A1 protein was associated with prognostic factors in CRC. MATERIAL AND METHODS Formalin-fixed paraffin-embedded (FFPE) tissue from 424 patients diagnosed with CRC, and 196 matched NATs were used to prepare tissue microarrays (TMAs). IHC was performed using an immunoperoxidase method with a primary polyclonal rabbit anti-ALDH1A1 antibody. The IHC scores by light microscopy were the staining intensity (scored from 0-3) multiplied by the percentage area of positive immunostaining within the visual field (scored from 0-4). Associations between tumor expression levels of ALDH1A1 and patient clinicopathological characteristics, including tumor grade, size, and TNM stage at surgery were analyzed. RESULTS ALDH1A1 protein expression was significantly increased in CRC tissues compared with matched NATs. In patients with CRC, increased expression of the ALDH1A1 protein was significantly associated with the presence of lymph node metastasis: 64.28% in N0 cases; 75.49% in N1 cases; and 82.14% in N2 cases, (P=0.002). Univariate and multivariate analysis showed that ALDH1A1 expression was an independent prognostic marker for CRC (P<0.001). CONCLUSIONS Using IHC, the expression of the ALDH1A1 protein in CRC tissues was significantly associated with the presence of lymph node metastases and might be a potential prognostic marker in patients with CRC.

Downregulation of Drp1, a fission regulator, is associated with human lung and colon cancers.

Dynamin-related protein 1 (Drp1), a dynamin-related GTPase, is a key regulator of mitochondrial fission. Although recent studies have shown that Drp1 plays important roles in various important cellular processes, such as maintaining proper mitochondrial function, apoptosis and necrosis, the potential involvement of Drp1 in cancer development has not been fully addressed. To explore the role of Drp1 in cancer, we examined Drp1 levels in various human cancer tissues. Tissue array analysis showed that the level of Drp1 was decreased significantly in malignant colon and lung cancer tissues, whereas no change in Drp1 was observed in breast and prostate tumors. Pairwise comparisons of cancer tissue and adjacent normal tissue from colon and lung cancer patients further confirmed decreases in Drp1 expression of 75% in colon cancer patients and 78% in lung cancer patients. Moreover, Drp1 levels were decreased further with advanced grade in both colon and lung cancers, suggesting that loss of Drp1 is associated with the progression of human lung and colon cancer. Consistent with this observation, knockdown of Drp1 increased cellular migration activity in human lung cancer cells and tumor formation in a xenograft tumor model. Taken together, these results suggest that the loss of Drp1 expression could contribute to the development of human lung and colon cancers.

Inverse Correlation between Methylation and Expression of the Delta-like Ligand 1 Gene in Gastric Cancer.

Notch signaling is a candidate pathway that transmits environmental information into the cell and interferes with the epigenome of gastric cancer. This study aimed to explore if the Notch pathway was abnormally regulated during gastric tumorigenesis. To achieve the goal, Delta-like ligand 1 (DLL1) gene expression, Notch upstream signal, promoter methylation and its correlation with DLL1 expression were examined by methylation-specific polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in cultured gastric cancer cell lines or gastric cancer patient samples. Immunostainings and tissue arrays (n = 40) were used to confirm the DLL1 expression was down-regulated in cancer cells. Transient or stable Notch1 active domain (NICD)-overexpression suppressed proliferation of the gastric cells but the in vivo tumor growth was enhanced. The results of abnormal DLL1 methylation and expression observed in early gastric lesions and in gastric cancers may be relevant to the pathogenesis of gastric cancer.

CCR4 frameshift mutation identifies a distinct group of adult T cell leukaemia/lymphoma with poor prognosis.

Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next-generation sequencing-based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell-surface CCR4 positivity. Semi-quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild-type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology.

Prostate Cancer Expression Profiles of Cytoplasmic ERß1 and Nuclear ERß2 are Associated with Poor Outcomes following Radical Prostatectomy.

PURPOSE: Existing data regarding the expression of estrogen receptors (ERs) and prostate cancer outcomes have been limited. We evaluated the relationship of expression profiles of ERß subtypes and the ER GPR30 (G-protein-coupled receptor-30) with patient factors at diagnosis and outcomes following radical prostatectomy. MATERIALS AND METHODS: Tissue microarrays constructed using samples from 566 men with long-term clinical followup were analyzed by immunohistochemistry targeting ERß1, ERß2, ERß5 and GPR30. An experienced pathologist scored receptor distribution and staining intensity. Tumor staining characteristics were evaluated for associations with patient characteristics, recurrence-free survival and prostate cancer specific mortality following radical prostatectomy. RESULTS: Prostate cancer cells had unique receptor subtype staining patterns. ERß1 demonstrated predominantly nuclear localization while ERß2, ERß5 and GPR30 were predominantly cytoplasmic. After controlling for patient factors intense cytoplasmic ERß1 staining was independently associated with time to recurrence (HR 1.7, 95% CI 1.1-2.6, p = 0.01) and prostate cancer specific mortality (HR 6.6, 95% CI 1.8-24.9, p = 0.01). Intense nuclear ERß2 staining was similarly independently associated with prostate cancer specific mortality (HR 3.9, 95% CI 1.1-13.4, p = 0.03). Patients with cytoplasmic ERß1 and nuclear ERß2 co-staining had significantly worse 15-year prostate cancer specific mortality than patients with expression of only cytoplasmic ERß1, only nuclear ERß2 and neither ER (16.4%, 4.3%, 0.0% and 2.0 %, respectively, p = 0.001). CONCLUSIONS: Increased cytoplasmic ERß1 and nuclear ERß2 expression is associated with worse cancer specific outcomes following radical prostatectomy. These findings suggest that tumor ERß1 and ERß2 staining patterns provide prognostic information on patients treated with radical prostatectomy.

A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer.

BACKGROUND: Loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision-making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study. METHODS: We used recently developed probes optimized for sensitivity and specificity in a four-color FISH deletion assay to study the Canary Retrospective multicenter Prostate Cancer Tissue Microarray (TMA). This TMA was constructed specifically for biomarker validation from radical prostatectomy specimens, and is accompanied by detailed clinical information with long-term follow-up. RESULTS: In 612 prostate cancers, the overall rate of PTEN deletion was 112 (18.3%). Hemizygous PTEN losses were present in 55/612 (9.0%) of cancers, whereas homozygous PTEN deletion was observed in 57/612 (9.3%) of tumors. Significant associations were found between PTEN status and pathologic stage (P < 0.0001), seminal vesicle invasion (P = 0.0008), extracapsular extension (P < 0.0001), and Gleason score (P = 0.0002). In logistic regression analysis of clinical and pathological variables, PTEN deletion was significantly associated with extracapsular extension, seminal vesicle involvement, and higher Gleason score. In the 406 patients in which clinical information was available, PTEN homozygous (P = 0.009) deletion was associated with worse post-operative recurrence-free survival (number of events = 189), pre-operative prostate specific antigen (PSA) (P < 0.001), and pathologic stage (P = 0.03). CONCLUSION: PTEN status assessed by FISH is an independent predictor for recurrence-free survival in multivariate models, as were seminal vesicle invasion, extracapsular extension, and Gleason score, and preoperative PSA. Furthermore, these data demonstrate that the assay can be readily introduced at first diagnosis in a cost effective manner analogous to the use of FISH for analysis of HER2/neu status in breast cancer. Combined with published research beginning 17 years ago, both the data and tools now exist to implement a PTEN assay in the clinic.

Histochemical analysis of testis specific gene 13 in human normal and malignant tissues.

Testis-specific gene 13 (TSGA13) is abundantly expressed in testis. As previous studies of TSGA13 expression pattern have all been based on mRNA analysis, it is imperative to investigate its actual protein expression. Here, we first examined TSGA13 gene tree and protein homology among species, and found that TSGA13 is relatively well conserved. Next, we detected its protein expression in normal human tissues as well as in a limited number of malignant tumors by immunohistochemistry (IHC). It was demonstrated that, in addition to testis, high expression of TSGA13 could also be observed in multiple normal tissues, including stomach, larynx, spleen, bladder, tonsil, liver and thyroid. Notably, most types of human carcinoma tissues displayed reduced expression of TSGA13 rather than their adjacent normal tissues except glioblastoma and lung cancer. Hence, the data from the current study strongly suggest the association between TSGA13 and tumor malignancy.

A CD44 specific peptide developed by phage display for targeting gastric cancer.

OBJECTIVE: To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. RESULTS: A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells. CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (-8.6 kcal/mol). CONCLUSIONS: The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.

Osteopontin expression is an independent factor for poor survival in oral squamous cell carcinoma: a computer-assisted analysis on TMA sections.

INTRODUCTION: Osteopontin (OPN) is non-collagenous extracellular matrix protein involved in various physiological and pathological events, including tumor progression. The aim of this study was to analyze the expression of OPN in normal oral mucosa and oral squamous cell carcinoma (OSCC) and to assess its prognostic significance. METHODS: The expression of OPN was immunohistochemicaly analyzed in 86 OSCC and compared with clinicopathological variable such as tumor size, nodal stage, WHO clinical stage, Ki-67 proliferation index, and patients' outcome. OPN mRNA was analyzed using quantitative real-time PCR and compared with protein OPN expression and clinical outcome in 18 OSCC samples. RESULTS: The expression of OPN protein was found in OSCC tumor cells (t-OPN) and various stromal cells (s-OPN). High level of t-OPN expression was associated with higher nodal stage (P = 0.045), higher WHO clinical stage (P = 0.033), and poor clinical outcome (P = 0.022). In multivariate analysis, t-OPN emerged as an adverse independent factor for survival (P = 0.049). Although correlated with t-OPN (P = 0.005), s-OPN was not significantly associated with clinical parameters, including patients' outcome. Also, there was no association between OPN and clinical parameters at the mRNA level. CONCLUSION: OPN is upregulated in tumor and stromal OSCC cells. Tumor cell-derived OPN is involved in tumor progression and can independently predict the clinical outcome. Stromal-derived OPN probably has a different function compared with OPN secreted from tumor cells.

Expression of DDB1 is associated with subtypes of epithelial ovarian cancer and predicts clinical outcomes.

BACKGROUND: Ovarian cancer is the most lethal gynaecological malignancy. Damage specific DNA-binding protein 1 (DDB1) functions in nucleotide-excision repair and has been reported to be involved in cancer development. In this study, we aimed to determine the expression levels of DDB1 and their association with the clinical outcomes of patients with ovarian cancer. METHODS: Tissue arrays were performed on 54 epithelial ovarian cancer (EOC) samples. Immunohistochemistry was performed to determine DDB1 expression. DDB1 expression levels among different EOC subtypes were analysed via one-way analysis of variance using SPSS Statistics 19.0. Correlation between DDB1 expression and chemotherapy course/progression-free survival (PFS) of patients was determined via Kaplan-Meier survival analysis using GraphPad Prism 5. Moreover, knockdown of DDB1 in ovarian cancer cells ES2 and OVCAR3 was used to preliminarily validate the role of DDB1. RESULTS: DDB1 was detected in the cytoplasm, especially in the nucleus, of all subtypes of EOC. However, DDB1 expression levels were significantly different between clear cell carcinoma and low-grade serous carcinoma (P = 0.022) and clear cell carcinoma and endometrioid cancer (P = 0.016). In addition, DDB1 expression was not significantly correlated with chemotherapy course (P = 0.433) or PFS (P = 0.566). High expression levels of DDB1 were correlated with significantly worse overall survival (P = 0.017) in patients with EOC. In addition, DDB1 knockdown in ovarian cancer cells decreased their proliferation in vitro. CONCLUSION: Our results revealed that DDB1 expression is heterogeneous in ovarian cancer, suggesting its use as a potential biomarker for poor survival in ovarian cancer.

CopA3 peptide inhibits MDM2-p53 complex stability in colorectal cancers and activates p53 mediated cell death machinery.

The diverse expression patterns of the tumor suppressor p53 in cancer cells reflect the regulatory efficiency of multiple cellular pathways. By contrast, many human tumors are reported to develop in the presence of wild-type p53. Recently, several oncogene inhibitors have been used clinically to suppress tumor development by functionally reactivating other oncoproteins. On the other hand, p53 reactivation therapies have not been well established, as few of the p53-MDM2 complex inhibitors such as Nutlin-3 induces mutation in p53 gene upon prolonged usage. Therefore, in this study CopA3, a 9-mer dimeric D-type peptide with anticancer activity against the human colorectal cancer cells, was used to explore the efficacy of p53 reactivation in-vitro and in-vivo. The anticancer activity of CopA3 was more selective towards the wild-type p53 expressing cells than the p53 deficient or mutant colorectal cancer cells. In response to this, this study investigated the signaling pathway in vitro and validated its anti-tumor activity in-vivo. The protein-peptide interaction and molecular docking efficiently provided insight into the specific binding affinity of CopA3 to the p53-binding pocket of the MDM2 protein, which efficiently blocked the p53 and MDM2 interaction. CopA3 plays a crucial role in the binding with MDM2 and enhanced the nuclear translocation of the p53 protein, which sequentially activated the downstream targets to trigger the autophagic mediated cell death machinery through the JNK/Beclin-1 mediated pathway. Collectively, CopA3 affected the MDM2-p53 interaction, which suppressed tumor development. This study may provide a novel inhibitor candidate for the MDM2-p53 complex, which could ultimately suppress the growth of colorectal cancer cells without being cytotoxic to the healthy neighboring cells present around the tumor microenvironment.

Downregulated miRNAs associated with auditory deafferentation and compensatory neural plastic changes following single-sided deafness in the inferior colliculi of rats.

BACKGROUND AND AIMS: Deafferentation and compensatory neural plastic changes in the inferior colliculus (IC) have been suggested following single-sided deafness (SSD). We explored related miRNA changes in the IC of SSD rats using miRNA microarray analyses. METHODS: Eight-week-old rats were divided into control and SSD rats (n = 8 for each group). SSD rats underwent right-side cochlear ablation surgery, with the IC harvested two weeks post-surgery. miRNA microarray analysis was performed using GeneChip miRNA 4.0, microarray (Affymetrix Inc.). miRNAs whose expression levels differed between SSD and control rats with a fold-change ≥ 1.5 and P < 0.05 were examined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Target genes of differentially expressed miRNAs were predicted using TargetScan software. The pathways related to predicted target genes were analyzed. mRNA levels of predicted target genes were estimated using qRT-PCR. RESULTS: The expression of miR-15b-5p, miR-202-5p, and miR-212-3p was lower in the contralateral (left) IC of SSD rats than that of control rats. In SSD rats, miRNA expression levels in the contralateral IC were 0.45-, 0.25-, and 0.50-fold lower for miR-15b-5p, miR-202-5p, and miR-212-3p, respectively (P < 0.05). The expression of predicted target genes (Spred1, Rasa1, Lsm11, and Srsf1) was higher in the contralateral IC of SSD rats than in control rats. The targets were predicted to be related with cleavage of growing transcripts in the termination region, mitogen-activated protein kinase family signaling cascades, RAF/AMP kinase cascade, regulation of RAS by GTPase activating proteins (GAPs), and RNA polymerase II transcription termination. For ipsilateral ICs, miR-425-3p, miR-199a-5p, and miR-134-3p showed lower expressions in SSD rats than in control rats, which were 0.55-, 0.61-, and 0.69-fold lower, respectively (P < 0.05). The expression of predicted target genes (Atp2b2, Grin2b, Foxp1, Ztbt20, Zfp91, and Strn) was higher in the ipsilateral IC of SSD rats; the regulation of synaptic plasticity, cAMP signaling pathway, metal ion binding, and calcium ion transport can be associated with these target genes. CONCLUSION: Adult rats with unilateral auditory deprivation showed miRNA changes in the IC. The contralateral IC showed decreased miRNA expression predicted to be related to MAPK and RAS signaling, whereas the ipsilateral IC revealed decreased miRNA expression predicted to be associated with synaptic plasticity and calcium ion transport.

Decreased expression of transcription factor Homeobox A11 and its potential target genes in bladder cancer.

Bladder cancer (BC) ranks as the ninth most commonly diagnosed cancer worldwide. The presence of a transcription factor (TF) has been uncovered as a significant contributor to the pathophysiological changes of cancers. In present study, we elucidated the expression and clinical significance of Homeobox A11 (HOXA11) in BC for the first time, and originally investigated HOXA11 as a TF. Employing in-house immunohistochemistry (IHC), we incorporated 137 BC and 34 non-BC cases to detect the expression of HOXA11 protein in BC tissues. HOXA11-related RNA-sequencing (RNA-seq) expression and RNA microarrays were collected from public databases, the "sva" and "limma" R packages were implemented to integrate and normalize the RNA-seq data and microarrays separately. Integration expression was carried out to further evaluate the HOXA11 expression by utilizing the standard mean difference (SMD). The expression level of HOXA11 in various BC cell lines was also evaluated. We further systematically analyzed the downstream target genes of HOXA11 in BC by utilizing Chromatin Immunoprecipitation Sequencing (ChIP-seq) profiles, differentially expressed genes (DEGs), and HOXA11-related genes. Modification of histone marks on the promoter region of target genes were also discovered by histone ChIP-seq data. Results of the IHC and RNA-seq revealed the protein and mRNA expression of HOXA11 was significantly decreased in BC tissues compared to non-BC tissues (2.98 ± 1.48 vs. 8.23 ± 2.64; 6.87 ± 1.54 vs. 8.38 ± 1.42). Five platforms significantly revealed the down-regulation of HOXA11 expression in BC (GPL96, GPL570, GPL6102, GPL6884, and GPL13497). A similar decreased trend was discovered in BC tissues in expression integration with the incorporated SMD reaching -0.843 (-1.362 ~ -0.325, p = 0.001) and -1.051 (-1.674 ~ -0.428, p = 0.001). Expression of HOXA11 was down-regulated in most of the BC cell lines. COL1A1 was considered as a final HOXA11 target gene and positively related to HOXA11 with the correlation coefficient as 0.584 (95% CI: 0.371-0.739, p < 0.001). HOXA11 regulates COL1A1 expression in BC via H3K27ac modification. The expression of COL1A1 was down-regulated with the SMD reached -0.312 (p < 0.001). In conclusion, HOXA11 expression is markedly decreased and might promote the transcription of COL1A1 to inhibit BC.

Novel resin tissue array system reduces sample preparation time, labour and reagent costs in bone tissue histology.

Resin histology plays an essential role in the analysis of hard tissues, such as bone and teeth, as well as in the context of metallic implant analysis. However, the techniques of resin embedding, followed by ground sectioning, are very costly due to significantly increased reagent cost and labour time when compared to the conventional paraffin histology approach. In the present study, a novel resin array system was developed to increase the affordability of a project analysing rat femur tissues containing metallic or polymeric implants. The resin array system enabled the simultaneous embedding of the femur samples in groups of eight samples compared to the conventional resin method where samples are processed individually. The ground sections produced with the resin array system allowed uniform ROI selection, ground section thickness, staining consistency, and histological analysis with Goldner's trichrome stain, offering a substantial opportunity for reproducible immunohistochemistry which is unable to be achieved when processing samples embedded individually. The application of this novel resin array system significantly reduced resource usage when compared to doing the same analysis on individual samples. A reduction of approximately 40% was achieved for both total labour time and total reagent cost through the use of the array system compared with individual embedding. This novel resin array system has widespread applicability to many bone, hard tissue, and metallic implant studies, offering substantial conservation of research funds and increased accessibility to advanced techniques for commercial partners due to more cost-effective sample preparation and more accurate, reproducible data.

A Dynamic Transcription Factor Signature Along the Colorectal Adenoma-Carcinoma Sequence in Patients With Co-Occurrent Adenoma and Carcinoma.

BACKGROUND: Colorectal carcinoma (CRC) often arises from benign adenoma after a stepwise accumulation of genetic alterations. Here, we profiled the dynamic landscapes of transcription factors (TFs) in the mucosa-adenoma-carcinoma progression sequence. METHODS: The transcriptome data of co-occurrent adenoma, carcinoma, and normal mucosa samples were obtained from GSE117606. Identification of differentially expressed TFs (DE-TFs) and subsequent function annotation were conducted in R software. Expression patterns of DE-TFs were clustered by Short Time-series Expression Miner software. Thereafter, modular co-expression analysis, Kaplan-Meier survival analysis, mutation profiling, and gene set enrichment analysis were conducted to investigate TF dynamics in colorectal tumorigenesis. Finally, tissue microarrays, including 51 tumors, 32 adenomas, and 53 normal tissues, were employed to examine the expression of significant candidates by immunohistochemistry staining. RESULTS: Compared to normal tissues, 20 (in adenoma samples) and 29 (in tumor samples) DE-TFs were identified. During the disease course, 28 expression patterns for DE-TFs and four co-expression modules were clustered. Notably, six DE-TFs, DACH1, GTF2IRD1, MEIS2, NR3C2, SOX9, and SPIB, were identified as having a dynamic signature along the colorectal adenoma-carcinoma sequence. The dynamic signature was of significance in GO enrichment, prognosis, and co-expression analysis. Among the 6-TF signature, the roles of GTF2IRD1, SPIB and NR3C2 in CRC progression are unclear. Immunohistochemistry validation showed that GTF2IRD1 enhanced significantly throughout the mucosa-adenoma-carcinoma sequence, while SPIB and NR3C2 kept decreasing in stroma during the disease course. CONCLUSIONS: Our study provided a dynamic 6-TF signature throughout the course of colorectal mucosa-adenoma-carcinoma. These findings deepened the understanding of colorectal cancer pathogenesis.

YKL-40 protein expression in human tumor samples and human tumor cell line xenografts: implications for its use in tumor models.

BACKGROUND: YKL-40, also known as non-enzymatic chitinase-3 like-protein-1 (CHI3L1), is a glycoprotein expressed and secreted mainly by inflammatory cells and tumor cells. Accordingly, several studies demonstrated elevated YKL-40 serum levels in cancer patients and found YKL-40 to be correlated with a poor prognosis and disease severity in some tumor entities. YKL-40 was suggested to be involved in angiogenesis and extracellular matrix remodeling. As yet, however, its precise biological function remains elusive. METHODS: As YKL-40 protein expression has only been investigated in few malignancies, we employed immunohistochemical detection in a large multi-tumor tissue microarray consisting of 2,310 samples from 72 different tumor entities. In addition, YKL-40 protein expression was determined in primary mouse xenograft tumors derived from human cancer cell lines. RESULTS: YKL-40 could be detected in almost all cancer entities and was differently expressed depending on tumor stage and subtype (e.g., thyroid cancer, colorectal cancer, gastric cancer and ovarian cancer). While YKL-40 was absent in in vitro grown human cancer cell lines, YKL-40 expression was upregulated in xenograft tumor tissues in vivo. CONCLUSIONS: These data provide new insights into YKL-40 expression at the protein level in various tumor entities and its regulation in tumor models. Our data suggest that upregulation of YKL-40 expression is a common feature in vivo and is finely regulated by tumor cell-microenvironment interactions.

Thioredoxin Interacting Protein (TXNIP) Is Differentially Expressed in Human Tumor Samples but Is Absent in Human Tumor Cell Line Xenografts: Implications for Its Use as an Immunosurveillance Marker.

Thioredoxin interacting protein (TXNIP) is a metabolic protein critically involved in redox homeostasis and has been proposed as a tumor suppressor gene in a variety of malignancies. Accordingly, TXNIP is downregulated in breast, bladder, and gastric cancer and in tumor transplant models TXNIP overexpression inhibits growth and metastasis. As TXNIP protein expression has only been investigated in few malignancies, we employed immunohistochemical detection in a large multi-tumor tissue microarray consisting of 2,824 samples from 94 different tumor entities. In general, TXNIP protein was present only in a small proportion of primary tumor samples and in these cases was differently expressed depending on tumor stage and subtype (e.g., renal cell carcinoma, thyroid cancer, breast cancer, and ductal pancreatic cancer). Further, TXNIP protein expression was determined in primary mouse xenograft tumors derived from human cancer cell lines and was immunohistochemically absent in all xenograft tumors investigated. Intriguingly, TXNIP expression became gradually lower in the proximity of the primary tumor tissue and was absent in leukocytes directly adjacent to tumor tissue. In conclusion, these findings suggest that TXNIP downregulation is as a common feature in human tumor xenograft models and that intra-tumoral leukocytes down-regulate TXNIP. Hence TXNIP expression might be used to monitor the functional state of tumor-infiltrating leukocytes in tissue sections.

Global gene expression profile of periodontal ligament cells submitted to mechanical loading: A systematic review.

OBJECTIVE: To evaluate the evidence reporting gene expression array data of human in vitro cultured periodontal ligament cells (PDLCs) submitted to static mechanical loading compared to a control group. DESIGN: Systematic searches were performed in MEDLINE/PubMed, Scopus, Web of Science, Virtual Health Library, The Cochrane Library and the System for Information on Grey Literature in Europe up to June 2019. A narrative synthesis was performed to summarize differentially expressed genes (DEGs). These were grouped according to the culture method (2D or 3D), force type (compression or tension) and observation time. Additionally, gene ontology (GO) analysis was performed using the Database for Annotation Visualization and Integrated Discovery. The risk of bias (RoB) and certainty of evidence (CoE) were assessed using a modified CONSORT checklist and the GRADE tool, respectively. RESULTS: Of eight studies included (all rated as having moderate RoB), only two provided the complete list of DEGs and four studies performed GO, gene network or pathways analysis. "Cell proliferation", "cell-cell signaling", "response to hypoxia and to mechanical stimulus" were among the significantly enriched biological processes in 3D-cultured compressed PDLCs (moderate CoE); while "collagen catabolic process", "extracellular matrix organization" and "cell proliferation" were associated with DEGs of 3D-cultured PDLCs submitted to tension (very low CoE). Biological processes significantly enriched in 2D-cultured PDLCs under compression were "extracellular matrix organization", "canonical glycolysis" and "glycolytic process" (very low CoE). CONCLUSION: Genes such as NR4A2, NR4A3, NAMPT, PGK1, and REDD1 are suggested as novel biomarkers for orthodontic tooth movement. Limited amount of evidence on the complete gene expression profile and the high heterogeneity in methodologies make it impossible to obtain definite conclusions. New studies following standardized and well-designed in vitro model and reporting complete gene expression datasets are needed.