MicroRNA-27a-3p targets FoxO signalling to induce tumour-like phenotypes in bile duct cells.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of transcriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour. METHODS: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High-throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts. RESULTS: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth. CONCLUSIONS: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA. IMPACT AND IMPLICATIONS: Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic and non-genetic alterations, but the functional roles of these non-genetic events remain poorly understood. By unveiling global miRNA upregulation in patient tumours and their functional ability to increase proliferation of cholangiocytes, these small non-coding RNAs are implicated as critical non-genetic alterations promoting biliary tumour initiation. These findings identify possible mechanisms for transcriptome rewiring during transformation, with potential implications for patient stratification.

Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells.

Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system.

LncRNA-OIS1 regulates DPP4 activation to modulate senescence induced by RAS.

Oncogene-induced senescence (OIS), provoked in response to oncogenic activation, is considered an important tumor suppressor mechanism. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nt without a protein-coding capacity. Functional studies showed that deregulated lncRNA expression promote tumorigenesis and metastasis and that lncRNAs may exhibit tumor-suppressive and oncogenic function. Here, we first identified lncRNAs that were differentially expressed between senescent and non-senescent human fibroblast cells. Using RNA interference, we performed a loss-function screen targeting the differentially expressed lncRNAs, and identified lncRNA-OIS1 (lncRNA#32, AC008063.3 or ENSG00000233397) as a lncRNA required for OIS. Knockdown of lncRNA-OIS1 triggered bypass of senescence, higher proliferation rate, lower abundance of the cell-cycle inhibitor CDKN1A and high expression of cell-cycle-associated genes. Subcellular inspection of lncRNA-OIS1 indicated nuclear and cytosolic localization in both normal culture conditions as well as following oncogene induction. Interestingly, silencing lncRNA-OIS1 diminished the senescent-associated induction of a nearby gene (Dipeptidyl Peptidase 4, DPP4) with established role in tumor suppression. Intriguingly, similar to lncRNA-OIS1, silencing DPP4 caused senescence bypass, and ectopic expression of DPP4 in lncRNA-OIS1 knockdown cells restored the senescent phenotype. Thus, our data indicate that lncRNA-OIS1 links oncogenic induction and senescence with the activation of the tumor suppressor DPP4.

MicroRNA Biomarkers in IBD-Differential Diagnosis and Prediction of Colitis-Associated Cancer.

Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC). These are chronic autoimmune diseases of unknown etiology affecting the gastrointestinal tract. The IBD population includes a heterogeneous group of patients with varying disease courses requiring personalized treatment protocols. The complexity of the disease often delays the diagnosis and the initiation of appropriate treatments. In a subset of patients, IBD leads to colitis-associated cancer (CAC). MicroRNAs are single-stranded regulatory noncoding RNAs of 18 to 22 nucleotides with putative roles in the pathogenesis of IBD and colorectal cancer. They have been explored as biomarkers and therapeutic targets. Both tissue-derived and circulating microRNAs have emerged as promising biomarkers in the differential diagnosis and in the prognosis of disease severity of IBD as well as predictive biomarkers in drug resistance. In addition, knowledge of the cellular localization of differentially expressed microRNAs is a prerequisite for deciphering the biological role of these important epigenetic regulators and the cellular localization may even contribute to an alternative repertoire of biomarkers. In this review, we discuss findings based on RT-qPCR, microarray profiling, next generation sequencing and in situ hybridization of microRNA biomarkers identified in the circulation and in tissue biopsies.

Co-Detection of miR-21 and TNF-α mRNA in Budding Cancer Cells in Colorectal Cancer.

MicroRNA-21 (miR-21) is upregulated in many cancers including colon cancers and is a prognostic indicator of recurrence and poor prognosis. In colon cancers, miR-21 is highly expressed in stromal fibroblastic cells and more weakly in a subset of cancer cells, particularly budding cancer cells. Exploration of the expression of inflammatory markers in colon cancers revealed tumor necrosis factor alpha (TNF-α) mRNA expression at the invasive front of colon cancers. Surprisingly, a majority of the TNF-α mRNA expressing cells were found to be cancer cells and not inflammatory cells. Because miR-21 is positively involved in cell survival and TNF-α promotes necrosis, we found it interesting to analyze the presence of miR-21 in areas of TNF-α mRNA expression at the invasive front of colon cancers. For this purpose, we developed an automated procedure for the co-staining of miR-21, TNF-α mRNA and the cancer cell marker cytokeratin based on analysis of frozen colon cancer tissue samples (n = 4) with evident cancer cell budding. In all four cases, TNF-α mRNA was seen in a small subset of cancer cells at the invasive front. Evaluation of miR-21 and TNF-α mRNA expression was performed on digital slides obtained by confocal slide scanning microscopy. Both co-expression and lack of co-expression with miR-21 in the budding cancer cells was noted, suggesting non-correlated expression. miR-21 was more often seen in cancer cells than TNF-α mRNA. In conclusion, we report that miR-21 is not linked to expression of the pro-inflammatory cytokine TNF-α mRNA, but that miR-21 and TNF-α both take part in the cancer expansion at the invasive front of colon cancers. We hypothesize that miR-21 may protect both fibroblasts and cancer cells from cell death directed by TNF-α paracrine and autocrine activity.

Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.

Mucosal microRNAs relate to age and severity of disease in ulcerative colitis.

Despite significant evidence that the expression of several microRNAs (miRNAs) impacts disease activity in patients with ulcerative colitis (UC), it remains unknown if the more severe disease phenotype seen in pediatric onset UC can be explained by an altered miRNA expression. In this study, we assessed the relationship between miRNA expression, age, and disease severity in pediatric and adult patients with UC. Using RT-qPCR, we analyzed the expression of miR-21, miR-31, miR-126, miR-142 and miR-155 in paraffin embedded rectum biopsies from 30 pediatric and 30 adult-onset UC patients. We found that lesions from adult patients had significantly higher expression levels of miR-21 compared to pediatric patients and that the expression levels of miR-31 (all patients) and miR-155 (pediatric patients only) correlated inversely with histological assessed disease severity. Using in situ hybridization followed by image analysis, the expression level estimates of miR-21 and miR-126 correlated with histological assessed disease severity. In conclusion, we found that the expression of miRNAs depends on the age of the patient and/or the severity of the disease, suggesting that miRNAs may contribute to the regulation of inflammation in UC and could be useful biomarkers in the surveillance of disease severity.

Staphylococcus aureus Induces Signal Transducer and Activator of Transcription 5‒Dependent miR-155 Expression in Cutaneous T-Cell Lymphoma.

Staphylococcal enterotoxins are believed to fuel disease activity in cutaneous T-cell lymphoma. Recent data support this by showing that antibiotics inhibit malignant T cells in skin lesions in mycosis fungoides and Sézary syndrome, the most common forms of cutaneous T-cell lymphoma. Yet, it remains incompletely characterized how staphylococcal enterotoxins fuel disease activity. In this study, we show that staphylococcal enterotoxins induce the expression of the oncogenic microRNA miR-155 in primary malignant T cells. Thus, staphylococcal enterotoxins and Staphyloccocus aureus isolates from lesional skin of patients induce miR-155 expression at least partly through the IL-2Rg‒Jak‒signal transducer and activator of transcription 5 pathway, and the effect is augmented by the presence of nonmalignant T cells. Importantly, mycosis fungoides lesions harbor S. aureus, express Y-phosphorylated signal transducer and activator of transcription 5, and display enhanced miR-155 expression, when compared with nonlesional and healthy skin. Preliminary data show that aggressive antibiotic therapy is associated with decreased Y-phosphorylated signal transducer and activator of transcription 5 and miR-155 expression in lesional skin in two patients with Sézary syndrome. In conclusion, we show that S. aureus and its enterotoxins induce enhanced expression of oncogenic miR-155, providing mechanistic insight into the role of S. aureus in cutaneous T-cell lymphoma. Our findings support that environmental stimuli such as bacteria can fuel disease progression in cutaneous T-cell lymphoma.

Estrogen Receptor-α Quantification in Breast Cancer: Concordance Between Immunohistochemical Assays and mRNA-In Situ Hybridization for ESR1 Gene.

Immunohistochemical (IHC) quantification of estrogen receptor-α (ER) is used for assessment of treatment regimen in breast cancer. Different ER IHC assays may produce diverging results, because of different antibody clones, protocols, and stainer platforms. Objective tissue-based techniques to assess sensitivity and specificity of IHC assays are therefore needed. We tested the usability of ER mRNA-in situ hybridization (mRNA-ISH) in comparison with assays based on clones SP1 and 6F11. We selected 56 archival specimens according to their reported ER IHC positivity, representing a wide spectrum from negative to strongly positive cases. The specimens were used to prepare 4 TMAs with 112 cores. Serial sections of each TMA were stained for ER and pan-cytokeratin (PCK) by IHC and ESR1 (ER gene) by mRNA-ISH. Digital image analysis (DIA) was used to determine ER IHC H-score. ESR1 mRNA-ISH was scored both manually and by DIA. DIA showed a nonlinear correlation between IHC and ESR1 mRNA-ISH with R-values of 0.80 and 0.78 for the ER antibody clones SP1 and 6F11, respectively. Comparison of manual mRNA-ISH scoring categories and SP1 and 6F11 IHC H-scores showed a highly significant relationship (P<0.001). In conclusion, the study showed good correlation between mRNA-ISH and IHC, suggesting that mRNA-ISH can be a valuable tool in the assessment of the sensitivity and specificity of ER IHC assays.

Selecting optimal spectral bands for improved detection of autofluorescent biomarkers in multiphoton microscopy.

SIGNIFICANCE: In multiphoton microscopy, two-photon excited fluorescence (TPEF) spectra carry valuable information on morphological and functional biological features. For measuring these biomarkers, separation of different parts of the fluorescence spectrum into channels is typically achieved by the use of optical band pass filters. However, spectra from different biomarkers can be unknown or overlapping, creating a crosstalk in between the channels. Previously, establishing these channels relied on prior knowledge or heuristic testing. AIM: The presented method aims to provide spectral bands with optimal separation between groups of specimens expressing different biomarkers. APPROACH: We have developed a system capable of resolving TPEF with high spectral resolution for the characterization of biomarkers. In addition, an algorithm is created to simulate and optimize optical band pass filters for fluorescence detection channels. To demonstrate the potential improvements in cell and tissue classification using these optimized channels, we recorded spectrally resolved images of cancerous (HT29) and normal epithelial colon cells (FHC), cultivated in 2D layers and in 3D to form spheroids. To provide an example of an application, we relate the results with the widely used redox ratio. RESULTS: We show that in the case of two detection channels, our system and algorithm enable the selection of optimized band pass filters without the need of knowing involved fluorophores. An improvement of 31,5% in separating different 2D cell cultures is achieved, compared to using established spectral bands that assume NAD(P)H and FAD as main contributors of autofluorescence. The compromise is a reduced SNR in the images. CONCLUSIONS: We show that the presented method has the ability to improve imaging contrast and can be used to tailor a given label-free optical imaging system using optical band pass filters targeting a specific biomarker or application.

Altered expression of the urokinase receptor homologue, C4.4A, in invasive areas of human esophageal squamous cell carcinoma.

C4.4A is a glycolipid-anchored membrane protein with structural homology to the urokinase-type plasminogen activator receptor (uPAR). Although C4.4A was identified as a metastasis-associated protein little is known about its actual expression and possible function in malignant disease. In the present study, we have therefore analyzed the expression of C4.4A in 14 esophageal squamous cell carcinomas (ESCC). Normal squamous esophageal epithelium shows a strong cell surface associated C4.4A expression in the suprabasal layers, whereas basal cells are negative. Upon transition to dysplasia and carcinoma in situ the expression of C4.4A is abruptly and coordinately weakened. Double immunofluorescence staining of normal and dysplastic tissue showed that C4.4A colocalizes with the epithelial cell surface marker E-cadherin in the suprabasal cells and has a complementary expression pattern compared to the proliferation marker Ki-67. A prominent, but frequently intracellular, C4.4A expression reappeared in tumor cells located at the invasive front and local lymph node metastases. Because C4.4A was reported previously to be a putative laminin-5 (LN5) ligand, and both proteins are expressed by invasive tumor cells, we analyzed the possible coexpression of C4.4A and the gamma 2-chain of LN5 (LN5-gamma 2). Although these proteins are indeed expressed by either neighboring cancer cells or in a few cases even coexpressed by the same cells in the tumor front and metastases, we found no evidence for a general colocalization in the extracellular compartment by confocal microscopy. In conclusion, C4.4A is expressed during invasion and metastasis of human ESCC and may thus provide a new histological marker in this disease.

Crohn's disease but not chronic ulcerative colitis induces the expression of PAI-1 in enteric neurons.

OBJECTIVES: Chronic inflammation of the intestinal wall is the common characteristic of Crohn's disease and ulcerative colitis; disorders, which in some cases can be difficult to distinguish. The inflammation also affects the local neuronal plexuses of the enteric nervous system. It is known that plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) are upregulated in neurons after experimental peripheral nerve injury and have been linked to nerve regeneration. METHODS: The expression of PAI-1 and uPAR in neuronal cells in lesions of the gastrointestinal tract was analyzed by immunohistochemical techniques. RESULTS: PAI-1 was found in a subset of neurons primarily located in the submucosal plexus of the small and large intestine in 24 of 28 cases (86%) with Crohn's disease, but in none of 17 cases with chronic ulcerative colitis and other severe inflammatory conditions in the intestinal wall. The PAI-1 was seen in the perikarya of the neurons and a few proximal axons, whereas nerves were negative. uPAR was seen in nerves in all types of lesion varying from 21% to 88% of the cases, most frequent in colon adenocarcinomas. No uPAR-positive nerves were detected in normal colon. CONCLUSIONS: PAI-1-positive neurons in inflammatory bowel disease are linked to chronic inflammation in Crohn's disease, implying PAI-1 as a potential parameter for the differential diagnosis between Crohn's disease and ulcerative colitis. The findings also suggest that PAI-1 in neurons is related to pain and that both PAI-1 and uPAR are involved in neuronal repair in the inflamed tissue.

Tumor cell-derived and macrophage-derived cathepsin B promotes progression and lung metastasis of mammary cancer.

Proteolysis in close vicinity of tumor cells is a hallmark of cancer invasion and metastasis. We show here that mouse mammary tumor virus-polyoma middle T antigen (PyMT) transgenic mice deficient for the cysteine protease cathepsin B (CTSB) exhibited a significantly delayed onset and reduced growth rate of mammary cancers compared with wild-type PyMT mice. Lung metastasis volumes were significantly reduced in PyMT;ctsb(+/-), an effect that was not further enhanced in PyMT;ctsb(-/-) mice. Furthermore, lung colonization studies of PyMT cells with different CTSB genotypes injected into congenic wild-type mice and in vitro Matrigel invasion assays confirmed a specific role for tumor-derived CTSB in invasion and metastasis. Interestingly, cell surface labeling of cysteine cathepsins by the active site probe DCG-04 detected up-regulation of cathepsin X on PyMT;ctsb(-/-) cells. Treatment of cells with a neutralizing anti-cathepsin X antibody significantly reduced Matrigel invasion of PyMT;ctsb(-/-) cells but did not affect invasion of PyMT;ctsb(+/+) or PyMT;ctsb(+/-) cells, indicating a compensatory function of cathepsin X in CTSB-deficient tumor cells. Finally, an adoptive transfer model, in which ctsb(+/+), ctsb(+/-), and ctsb(-/-) recipient mice were challenged with PyMT;ctsb(+/+) cells, was used to address the role of stroma-derived CTSB in lung metastasis formation. Notably, ctsb(-/-) mice showed reduced number and volume of lung colonies, and infiltrating macrophages showed a strongly up-regulated expression of CTSB within metastatic cell populations. These results indicate that both cancer cell-derived and stroma cell-derived (i.e., macrophages) CTSB plays an important role in tumor progression and metastasis.

Quantification of microRNA-21 and microRNA-125b in melanoma tissue.

Although microRNAs (miRNAs) have emerged as potent mediators of melanoma development and progression, a precise understanding of their oncogenic role remains unclear. In this study, we analysed formalin-fixed and paraffin-embedded tissues from two separate melanoma cohorts and from a series of benign melanocytic nevi. Using three different quantification methods [array analysis, quantitative PCR (qPCR) and in-situ hybridization (ISH) quantified by digital image analysis], we found considerable miRNA dysregulation in tumours. Using array analysis, samples mainly clustered according to their biological group (benign vs. malignant) and 77 miRNAs differed significantly between nevi and melanoma samples. Increase of miR-21 and miR-142, and decrease of miR-125b, miR-211, miR-101 and miR-513c in the melanomas were verified in both cohorts using qPCR, whereas the decrease of miR-205 observed with array analysis could not be confirmed using qPCR. ISH with digital quantification showed expression of miR-21 and miR-125b in the melanocytic lesions. miR-21 ISH was increased in melanomas, whereas quantification of miR-125b showed uniform ISH expression across nevi and melanomas. Our results support the important involvement of different miRNAs in melanoma biology and may serve as solid basics for further miRNA investigations in melanoma formalin-fixed and paraffin-embedded tissue. In particular, there is increased expression of miR-21 in melanomas compared with benign nevi.

Metastasis of transgenic breast cancer in plasminogen activator inhibitor-1 gene-deficient mice.

The plasminogen activator inhibitor-1 (PAI-1) blocks the activation of plasmin(ogen), an extracellular protease vital to cancer invasion. PAI-1 is like the corresponding plasminogen activator uPA (urokinase-type plasminogen activator) consistently expressed in human breast cancer. Paradoxically, high levels of PAI-1 as well as uPA are equally associated with poor prognosis in cancer patients. PAI-1 is thought to play a vital role for the controlled extracellular proteolysis during tumor neovascularization. We have studied the effect of PAI-1 deficiency in a transgenic mouse model of metastasizing breast cancer. In these tumors, the expression pattern of uPA and PAI-1 resembles that of human ductal breast cancer and plasminogen is required for efficient metastasis. In a cohort of 63 transgenic mice that were either PAI-1-deficient or wild-type sibling controls, primary tumor growth and vascular density were unaffected by PAI-1 status. PAI-1 deficiency also did not significantly affect the lung metastatic burden. These results agree with the virtual lack of spontaneous phenotype in PAI-1-deficient mice and humans and may reflect that the plasminogen activation reaction is not rate limiting for tumor vascularization and metastasis, or that there is a functional redundancy between PAI-1 and other inhibitors of the uPA/plasmin system, masking the effect of PAI-1 deficiency.

Structural analysis and tissue localization of human C4.4A: a protein homologue of the urokinase receptor.

C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor (uPAR), was originally identified as a metastasis-associated membrane protein, but little is known about its structural and functional properties. Therefore, we expressed, purified and characterized a soluble truncated form of human C4.4A, and used this protein to produce specific polyclonal anti-C4.4A antibodies. By immunohistochemistry we observed a pronounced surface staining for C4.4A in suprabasal keratinocytes of chronic human wounds and found C4.4A expression markedly upregulated in migrating keratinocytes during re-epithelisation of incisional skin wounds. Phorbol-ester-induced hyperplasia of mouse skin is also accompanied by a significant induction of C4.4A expression in the multilayered, suprabasal keratinocytes. C4.4A contains two Ly-6 (leucocyte antigen 6)/uPAR/alpha-neurotoxin modules. Our recombinant human C4.4A is extensively modified by post-translational glycosylation, which include 5-6 N-linked carbohydrates primarily located in or close to its second Ly-6/uPAR/alpha-neurotoxin module and approximately 15 O-linked carbohydrates clustered in a Ser/Thr/Pro-rich region at the C-terminus. A highly protease-sensitive region (Tyr200-Arg204) is located between these two clusters of N- and O-linked carbohydrates. The natural, glycolipid-anchored C4.4A from amnion membranes of human term placenta exhibits similar properties. Using recombinant, soluble C4.4A or MCF 7 cells, which express significant amounts of GPI-anchored C4.4A, we find no evidence for an interaction between C4.4A and uPA, a property suggested previously for rat C4.4A. Collectively these data indicate that C4.4A, although being a structural homologue of uPAR, is unlikely to have a functional overlap with uPAR.

Differential expression of miR-155 and miR-21 in tumor and stroma cells in diffuse large B-cell lymphoma.

OncomiRs miR-21 and miR-155 have been linked to lymphomagenesis, but information on their implication in diffuse large B-cell lymphoma (DLBCL) is limited. Here, we used locked nucleic acid-based in situ hybridization (ISH) detection techniques on formalin-fixed paraffin-embedded DLBCL tissue samples to identify miR-155 and miR-21 at the cellular level in 56 patients diagnosed with DLBCL, and compared them to miR array data. miR-155 was observed in tumor cells in 19/56 (33.9%) of the samples evaluated by ISH. miR-21 was localized to the stromal compartment in 41/56 (73.2%). A subset of these, 16/56 (28.6%), also showed labeling in tumor cells. When comparing ISH-scores and miR array data, miR-155 in tumor cells, identified by ISH, was associated with miR-155 expression in miR array data (P=0.030). Equally, miR-21 expression by miR array data were highly associated with miR-21 ISH-scores in the stromal cells (P=0.002), whereas no association between miR array data and ISH of miR-21 in tumor cells was observed (P=0.673). We found no association of miR-155 and miR-21 with overall survival or germinal center B-cell-like (GCB) versus non-GCB-like subtypes of DLBCL. In conclusion, miR-ISH added to the biological interpretation of miR expression in DLBCL compared with miR array data, but miR-155 and miR-21 ISH did not add prognostic information in this series.

Detection of plasminogen activators in oral cancer by laser capture microdissection combined with zymography.

Plasminogen activation is believed to be critical to the progression of oral squamous cell carcinoma by facilitating matrix degradation during invasion and metastasis, and high levels of urokinase plasminogen activator (uPA) and plasminogen activator (PA) inhibitor-1 (PAI-1) in tumors predict poor disease outcome. We describe the development of a novel method for studying PA in oral cancer that combines the sensitivity and specificity of zymography with the spatial resolution of immunohistochemistry. Laser capture microdissection (LCM) was combined with plasminogen-casein zymography to analyze uPA, tissue PA (tPA), uPA-PAI-1 complexes, and tPA-PAI-1 complexes in 11 tumors and adjacent non-malignant epithelium from squamous cell carcinomas of the tongue, floor of mouth, larynx, and vocal cord. uPA was detectable in all tumor samples analyzed, uPA-PAI-1 complexes in three samples, and tPA in nine. PA was detectable in as little as 0.5 microg protein lysate from microdissected tumors. In all specimens, uPA expression was highly increased in tumor tissue compared to adjacent non-malignant tissue. In conclusion, LCM combined with zymography may be excellently suited for analyzing the prognostic significance and causal involvement of the plasminogen activation system in oral cancer.

Efficient identification of miRNAs for classification of tumor origin.

Carcinomas of unknown primary origin constitute 3% to 5% of all newly diagnosed metastatic cancers, with the primary source difficult to classify with current histological methods. Effective cancer treatment depends on early and accurate identification of the tumor; patients with metastases of unknown origin have poor prognosis and short survival. Because miRNA expression is highly tissue specific, the miRNA profile of a metastasis may be used to identify its origin. We therefore evaluated the potential of miRNA profiling to identify the primary tumor of known metastases. Two hundred eight formalin-fixed, paraffin-embedded samples, representing 15 different histologies, were profiled on a locked nucleic acid-enhanced microarray platform, which allows for highly sensitive and specific detection of miRNA. On the basis of these data, we developed and cross-validated a novel classification algorithm, least absolute shrinkage and selection operator, which had an overall accuracy of 85% (CI, 79%-89%). When the classifier was applied on an independent test set of 48 metastases, the primary site was correctly identified in 42 cases (88% accuracy; CI, 75%-94%). Our findings suggest that miRNA expression profiling on paraffin tissue can efficiently predict the primary origin of a tumor and may provide pathologists with a molecular diagnostic tool that can improve their capability to correctly identify the origin of hitherto unidentifiable metastatic tumors and, eventually, enable tailored therapy.

Tissue microRNAs as predictors of outcome in patients with metastatic colorectal cancer treated with first line Capecitabine and Oxaliplatin with or without Bevacizumab.

PURPOSE: We tested the hypothesis that expression of microRNAs (miRNAs) in cancer tissue can predict effectiveness of bevacizumab added to capecitabine and oxaliplatin (CAPEOX) in patients with metastatic colorectal cancer (mCRC). EXPERIMENTAL DESIGN: Patients with mCRC treated with first line CAPEOX and bevacizumab (CAPEOXBEV): screening (n = 212) and validation (n = 121) cohorts, or CAPEOX alone: control cohort (n = 127), were identified retrospectively and archival primary tumor samples were collected. Expression of 754 miRNAs was analyzed in the screening cohort using polymerase chain reaction (PCR) arrays and expression levels were related to time to disease progression (TTP) and overall survival (OS). Significant miRNAs from the screening study were analyzed in all three cohorts using custom PCR arrays. In situ hybridization (ISH) was done for selected miRNAs. RESULTS: In the screening study, 26 miRNAs were significantly correlated with outcome in multivariate analyses. Twenty-two miRNAs were selected for further study. Higher miR-664-3p expression and lower miR-455-5p expression were predictive of improved outcome in the CAPEOXBEV cohorts and showed a significant interaction with bevacizumab effectiveness. The effects were strongest for OS. Both miRNAs showed high expression in stromal cells. Higher expression of miR-196b-5p and miR-592 predicted improved outcome regardless of bevacizumab treatment, with similar effect estimates in all three cohorts. CONCLUSIONS: We have identified potentially predictive miRNAs for bevacizumab effectiveness and additional miRNAs that could be related to chemotherapy effectiveness or prognosis in patients with mCRC. Our findings need further validation in large cohorts, preferably from completed randomized trials.