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.

Identification of novel biomarkers with potential for diagnosis and prognosis of gastric cancer: a bioinformatics approach

INTRODUCTION: Gastric cancer (GC) is the fifth most diagnosed neoplasia and the third leading cause of cancer-related deaths. A substantial number of patients exhibit an advanced GC stage once diagnosed. Therefore, the search for biomarkers contributes to the improvement and development of therapies. OBJECTIVE: This study aimed to identify potential GC biomarkers making use of in silico tools. METHODS: Gastric tissue microarray data available in Gene Expression Omnibus and The Cancer Genome Atlas Program was extracted. We applied statistical tests in the search for differentially expressed genes between tumoral and non-tumoral adjacent tissue samples. The selected genes were submitted to an in-house tool for analyses of functional enrichment, survival rate, histological and molecular classifications, and clinical follow-up data. A decision tree analysis was performed to evaluate the predictive power of the potential biomarkers. RESULTS: In total, 39 differentially expressed genes were found, mostly involved in extracellular structure organization, extracellular matrix organization, and angiogenesis. The genes SLC7A8, LY6E, and SIDT2 showed potential as diagnostic biomarkers considering the differential expression results coupled with the high predictive power of the decision tree models. Moreover, GC samples showed lower SLC7A8 and SIDT2 expression, whereas LY6E was higher. SIDT2 demonstrated a potential prognostic role for the diffuse type of GC, given the higher patient survival rate for lower gene expression. CONCLUSION: Our study outlines novel biomarkers for GC that may have a key role in tumor progression. Nevertheless, complementary in vitro analyses are still needed to further support their potential.

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.

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.

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.

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.

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.

Neuregulin 1 enhances cell adhesion molecule L1 like expression levels and promotes malignancy in human glioma.

Neural cell adhesion molecular L1-like protein (CHL1) is a member of the cell adhesion molecule L1 family and serves an important role in the development and progression of tumors. The cytokine neuregulin 1 (NRG1) has been indicated in the tumorigenesis and promotion of metastasis through the modulation of L1. However, the roles of NRG1 in regulating CHL1 in glioma have not been elucidated. The present study investigated the protein expression levels and roles of CHL1 and the possible correlation between NRG1 and CHL1 protein expression levels in human gliomas, both in vivo and in vitro. Using immunohistochemistry coupled with a human glioma tissue microarray, it was demonstrated that the percentage of CHL1-positive areas was the highest in grade II glioma tissues. Using immunofluorescence staining, a positive correlation was identified between the expression levels of CHL1 and proliferating cell nuclear antigen. In addition, CHL1 downregulation also resulted in increased senescence of U-87 MG human glioblastoma cells. In vitro, administration of NRG1α induced a significant increase in CHL1 protein expression levels in human glioma SHG-44 and U251 cells and in human glioblastoma U-87 MG cells, whereas NRG1ß failed to increase CHL1 expression levels in U251 cells. These findings were further confirmed by the downregulation of NRG1 expression levels using small interfering RNA treatment, which resulted in the reduction of CHL1 protein expression levels in U-87 MG cells. These data indicate that NRG1 can regulate CHL1 protein expression levels in gliomas, that it is correlated with malignancy, and that NRG1 may contribute to malignancy by upregulating CHL1 protein expression levels in glioma/glioblastoma cells.

Expedited large-volume 3-D SEM workflows for comparative microanatomical imaging.

To-date, different electron microscopy (EM) approaches are available (e.g., TEM, SEM, STEM, CLEM, etc.) to collect three-dimensional (3-D) information in tissues and cells from the microscale up to the nanometer scale. However, an abundant amount of possibilities and methodologies exist to reconstruct volumes of biological matter. In this topical paper we outline two specimen manipulation workflows for the generation of 3-D scanning electron microscopy (3-D SEM) data by means of serial-block face/focussed ion beam SEM and array tomography (AT). We applied these commonly used workflows to rodent and zebrafish liver as model experimental systems. In doing so, we outline the specific steps of each procedure and discuss in-depth the strengths vs. limitations for each of the respective 3-D SEM specimen manipulation workflows.

A molecular approach combined with American Thyroid Association classification better stratifies recurrence risk of classic histology papillary thyroid cancer.

BACKGROUND: Prognosis among patients with differentiated thyroid cancer is widely variable. Better understanding of biologic subtypes is necessary to stratify patients and improve outcomes. METHODS: In patients diagnosed with classic histology papillary thyroid cancer treated from 1973 to 2009, BRAF V600E mutation status was determined on surgical tumor specimens by restriction fragment length polymorphism analysis. A tissue microarray (TMA) was constructed from tumor specimens in triplicate and stained by immunohistochemistry for RET, phospho-MEK, MAPK(dpERK), PPARγ, and phospho-AKT(pAKT). Stained slides were scored independently and blindly by two investigators and compared to tumor and patient characteristics and outcomes. RESULTS: A total of 231 patients had archived formalin-fixed, paraffin-embedded tumor tissue available and were included on the TMA. Mean age at diagnosis was 44 years (range 6-82 years); proportion of patients with female sex was (72%); 2015 American Thyroid Association (ATA) risk stratification was low (26%), intermediate (32%), and high (42%). BRAF V600E mutation was found in 74% of specimens, and IHC was scored as positive for RET (61%), MAPK (dpERK) (14%), PPARγ (27%), and pAKT (39%). Positive RET staining was associated with a lower risk of recurrence (HR = 0.46, 95% CI 0.22-0.96). No other molecular biomarkers were independent predictors of recurrence on univariable analysis. On RPA, patients with RET-negative and either MAPK(dpERK)-positive or pAKT-positive tumors were identified to have a high risk of recurrence (HR = 5.4, 95%CI 2.5-11.7). This profile remained associated with recurrence in a multivariable model including ATA risk stratification (HR = 2.8, 95% CI 1.3-6.0). CONCLUSION: Characterization of molecular pathways involved in cPTC tumorigenesis may add further risk stratification for recurrence beyond the 2015 ATA risk categories alone.

[Tissue arrays: Two simple and economical methods for manual construction.] / Matrices tisulares: presentación de dos métodos sencillos y económicos de elaboración manual.

OBJECTIVES: Tissue array (TA) technologyis widely used as a method for the in situ investigation oftissue markers in cancer studies. A limitation of this techniqueis the high price of tissue arrayers. We describetwo easy and non-expensive manual methods, that haveproduced small and medium format arrays. MATERIAL AND METHODS: 16 TAs were manuallyconstructed from conventional paraffin blocks using twodifferent techniques. For the first method, a 16G Tru-Cutneedle whose bevel edge had been cut, was used tomake the holes in the donor blocks (80 cases) and thereceptor ones (resulting in 2 TAs each one with 55 casesand two with 25 cases). In the second technique, a 4mm-diameter punch for cutaneous biopsies was appliedto the donor blocks (obtaining 210 cylinders from 108blocks) and to the receptor ones (12 TAs). Hematoxylin-eosin, immunohistochemical and in situ hybridizationstains were performed on sections from these TAs. RESULTS: The tissue loss rate in the sections obtainedfrom the TAs constructed with the first method was26.5%, but as two cylinders were included from eachcase, at least one of them was retained. There was notany loss of tissue in the sections from the TAs constructedwith the second method. The results of all of the stainsperformed were successful. CONCLUSIONS: These two manual methods of elaborationof TAs result rather simple and they are economical.The tissue loss rate is significant in the first methodbut it can be compensated embedding more than onecylinder from each donor block. There was not anyproblem in the sectioning of the TAs constructed with thesecond method.

OBJETIVOS: La tecnología de matricestisulares (MTs) se ha implantado como método de trabajohabitual en la investigación de marcadores tisularesrelacionados con el cáncer. Su inconveniente esla necesidad de contar con un dispositivo especial deprecio elevado. Presentamos dos métodos manuales,económicos, que han sido válidos para construir MTsde pequeño y mediano formato.MATERIAL Y MÉTODOS: Se han elaborado 16 MTs deforma manual a partir de bloques convencionales deparafina, mediante dos técnicas diferentes. En la primerase utilizó una aguja Tru-Cut 16G a la que se cortó el bisel, para realizar los orificios en los bloques donantes(80 casos) y en los receptores (resultando dos MTs de55 casos y dos de 25 casos). Para la segunda técnicase utilizó un dispositivo "punch" para biopsias cutáneas,de 4 mm de diámetro, que se aplicó a los bloques donantes(obteniendo 210 cilindros de 108 bloques) y alos receptores (12 MTs). En las secciones de las MTs obtenidasse realizaron tinciones de hematoxilina-eosina,inmunohistoquímica (IHQ) e hibridación in situ. RESULTADOS: La tasa de pérdida de material en las seccionesobtenidas con el primer método fue del 26,5%,pero al haberse incluído dos cilindros de cada caso, almenos uno de ellos se conservó. En las MTs obtenidascon el método de punch biopsia no hubo pérdidas detejido. Los resultados de todas las tinciones realizadasfueron óptimos. CONCLUSIONES: Estos dos métodos manuales de elaboraciónde MTs resultan relativamente sencillos y soneconómicos. La tasa de pérdida de tejido es sólo significativaen el primero de los métodos pero se puedecompensar incluyendo varios cilindros de cada bloquedonante. En el segundo método no han existido problemasdestacables en la fase de microtomía.

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.

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.

Sialyl Lewisx expression at the invasive front as a predictive marker of liver recurrence in stage II colorectal cancer.

Sialyl Lewisx (SLX) is a carbohydrate ligand for endothelial selectin that participates in cell adhesion, proliferation and scattering. It plays an important role in cancer cell adhesion to vascular endothelial cells, leading to hematogenous metastasis. The prognostic significance of SLX expression level at the invasive front in patients with stage II colorectal cancer (CRC) was examined. A total of 209 patients with stage II CRC curatively resected between 1997 and 2000 were enrolled. The preoperative serum SLX levels measured by radioimmunoassay and SLX immunoexpression levels at the invasive front, and at the non-invasive frontal region determined by tissue microarray were analyzed. SLX expression at the invasive front was positively associated with tumor invasion depth (P=0.007) and tumor budding grade (P=0.038). Disease-free survival curves differed between the high and low SLX-expression groups (5-year survival rates, 77.0 and 89.7%, respectively; P=0.036). Liver cancer recurrence was more frequent in the high-expression group than in the low-expression group (15.9 and 2.4%; P=0.002). Multivariate analysis revealed that its expression (hazard ratio, 5.26; P=0.015) and venous invasion (hazard ratio, 4.14; P=0.040) were independent predictive markers of liver cancer recurrence. Neither the preoperative serum SLX level nor SLX expression at the non-invasive frontal region showed any association with histopathological features or disease-free survival. SLX expression level at the invasive front is a promising marker for identifying patients with stage II CRC with a high risk of liver cancer recurrence.

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.