PDF file - 1045K, Figure S1. The comparability of whole exome sequencing for saliva and FFPE AT/RT tissue of the adult patient. The sequencing coverage folds for each chromosome of A. saliva genomic DNA and B. AT/RT genomic DNA.

PDF file - 72K, Table S1. Genetic alterations and protein expression of the SMARCB1 and HMGA2 in AT/RT patients.

PDF file - 382K, Figure S2.The relative whole exome copy number of genomic DNA from FFPE AT/RT tissue (normalized to genomic DNA extracted from saliva).

PDF file - 3492K, Figure S3. Sanger DNA-sequencing for AT/RT tissue specific point mutations including DBF4 (c.794C>G, p.T265S); PMS1 (c.2524C>A, p.L842I); RASSF4 (c.494A>C, p.H165P); Rap2b (c.240G>C, p.L80I); and Tie2 (c.1249C>A, p.P417T) genes. The upper panel for sequencing of genomic DNA extracted from saliva, the lower panel for AT/RT.

PDF file - 224K, Figure S4. Impact of let-7 miRNA and HMGA2 on proliferation and colony formation of G401 cells. A. Let-7 inhibitors up-regulated HMGA2 and promoted cell proliferation, and B. Overexpression HMGA2 increased colony formation of G401 cells, *: p < 0.05, compared with G401 cells transfected with control smRNA.

Intrahepatic cholangiocarcinoma (ICC) is a rare and highly aggressive malignancy. In this study, we identified the presence of gene deletion and missense mutation leading to inactivation or underexpression of liver kinase B1 (LKB1) tumor suppressor and excluded the involvement of LKB1 gene hypermethylation in ICC tissues. Immunohistochemical analysis showed that LKB1 was underexpressed in a portion of 326 ICC tissues compared to their adjacent normal tissues. By statistical analysis underexpression of LKB1 in ICC tissues significantly correlated with poor survival and malignant disease characteristics in ICC patients. Moreover, we showed that knockdown of LKB1 significantly enhanced growth, migration, and invasion of three LKB1-competent ICC cell lines. Global transcriptional profiling analysis identified multiple malignancy-promoting genes, such as HIF-1α, CD24, Talin1, Vinculin, Wnt5, and signaling pathways including Hedgehog, Wnt/β-catenin, and cell adhesion as novel targets of LKB1 underexpression in ICC cells. Furthermore, knockdown of LKB1 gene expression dramatically enhanced Wnt/β-catenin signaling in ICC cells, while an inverse correlation between LKB1 and nuclear β-catenin was observed in ICC tissues. Our findings suggest a novel mechanism for ICC carcinogenesis in which LKB1 underexpression enhances multiple signaling pathways including Wnt/β-catenin to promote disease progression.

Piwi-interacting RNAs (piRNAs) are a distinct group of small noncoding RNAs (sncRNAs) that silence transposable genetic elements to protect genome integrity. Because of their limited expression in gonads and sequence diversity, piRNAs remain the most mysterious class of small RNAs. Studies have shown piRNAs are present in somatic cells and dysregulated in gastric, breast and liver cancers. By deep sequencing 24 frozen benign kidney and clear cell renal cell carcinoma (ccRCC) specimens and using the publically available piRNA database, we found 26,991 piRNAs present in human kidney tissue. Among 920 piRNAs that had at least two copies in one specimen, 19 were differentially expressed in benign kidney and ccRCC tissues, and 46 were associated with metastasis. Among the metastasis-related piRNAs, we found three piRNAs (piR-32051, piR-39894 and piR-43607) to be derived from the same piRNA cluster at chromosome 17. We confirmed the three selected piRNAs not to be miRNAs or miRNA-like sncRNAs. We further validated the aberrant expression of the three piRNAs in a 68-case formalin-fixed and paraffin-embedded (FFPE) ccRCC tissue cohort and showed the upregulation of the three piRNAs to be highly associated with ccRCC metastasis, late clinical stage and poor cancer-specific survival.

Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive pediatric malignancies characterized by biallelic inactivation of the SMARCB1 tumor suppressor gene. We searched for novel genomic aberrations by investigating the copy number and expression alterations of let-7a3/let-7b microRNA (miRNA) and correlated these with expression of high-mobility group AT-hook 2 (HMGA2) oncoprotein, a target of let-7 miRNA family, in 18 AT/RT samples to elucidate potential roles of HMGA2 in the pathogenesis of AT/RT. Experimental Design: Genomic aberrations, let-7a3/let-7b miRNA and HMGA2 expression in AT/RT tissues were identified using quantitative PCR, reverse transcription PCR (RT-PCR), and immunohistochemistry. The impact of let-7b miRNA on HMGA2 expression and the malignant potential of human rhabdoid tumor cell G401 (SMARCB1 / ) were investigated by antisense inhibition and ectopic over-

Abstract Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive pediatric malignancies characterized by biallelic inactivation of the SMARCB1 tumor suppressor gene. We searched for novel genomic aberrations by investigating the copy number and expression alterations of let-7a3/let-7b microRNA (miRNA) and correlated these with expression of high-mobility group AT-hook 2 (HMGA2) oncoprotein, a target of let-7 miRNA family, in 18 AT/RT samples to elucidate potential roles of HMGA2 in the pathogenesis of AT/RT. Experimental Design: Genomic aberrations, let-7a3/let-7b miRNA and HMGA2 expression in AT/RT tissues were identified using quantitative PCR, reverse transcription PCR (RT-PCR), and immunohistochemistry. The impact of let-7b miRNA on HMGA2 expression and the malignant potential of human rhabdoid tumor cell G401 (SMARCB1−/−) were investigated by antisense inhibition and ectopic overexpression studies. Results: The copy number of let-7a3/let-7b miRNA was substantially decreased in 4 of 11 AT/RT samples. A significantly inverse correlation between let-7a3/let-7b miRNA expression and HMGA2 mRNA expression was observed in AT/RT tissues (R = −0.34; P < 0.05). Immunohistochemistry analysis demonstrated that HMGA2 was highly overexpressed in 83.3% (15 of 18) of AT/RT tissues. Restoration of let-7 miRNA or knockdown of HMGA2 expression significantly suppressed proliferation and colony formation, and almost abolished the invasive potential of G401 cells. Conclusion: Reduction of let-7a3/let-7b miRNA may be one of mechanisms leading to overexpression of HMGA2 in AT/RT tissues. HMGA2 oncoprotein plays critical roles in the pathogenesis of AT/RT development; and reconstitution of let-7 miRNA or knockdown of HMGA2 oncoprotein may provide a novel therapeutic strategy for the treatment of patients with AT/RT. Clin Cancer Res; 20(5); 1179–89. ©2014 AACR.

Abstract Background: miRNAs are small regulatory elements involved in regulating gene expression. They have been shown to play a role in tumor initiation, progression, and metastasis in many cancers and their expression is often tissue-specific. Cancers of the large bowel are often grouped together as colorectal cancers; but tumors from different locations require different treatment and have different prognosis and molecular characteristics. We tested the hypothesis that cancers of the colon and rectum have different molecular miRNA profiles that can be used to distinguish between these cancer types. Methods: We collected 40 pretreatment rectal cancer biopsies and 20 pretreatment surgical colon cancer specimens. All samples were formalin fixed and paraffin embedded (FFPE). Tumor cells were microdissected manually under inverted microscopy and total RNA was extracted. A miRNA expression profile for rectal and colon cancer was generated using deep sequencing, and miRNA expression in each cancer type was compared using T-test and multiple testing (Q-bound <0.05) to determine significant miRNA expression changes in rectal versus colon cancer. Results: 143 miRNAs were differentially expressed between rectal and colon cancer tissues (Q-bound <0.05); 106 miRNAs were up-regulated and 37 were down-regulated in rectal cancer compared to colon cancer. Thirteen miRNAs showed a greater than 2-fold increase in expression in rectal cancer compared to colon cancer (mir-1291, mir-1248, mir1973, mir-3653, mir-483-5p, mir-720, mir-3647-3p, mir3687, mir-3607-3p, mir4286, mir-1274b, mir-483-3p, and mir3648). Six miRNAs showed a greater than 1.5-fold decrease in rectal cancer compared to colon cancer (mir-374a, mir-3613-5p, mir-362-3p, mir-340, mir-3, and mir-424). Based on these significant expression differences, the unique miRNA expression profiles of rectal and colon cancer were used to separate these cancers by hierarchical cluster analysis (p<0.0001). Conclusions: We identified a number miRNAs that are differentially expressed between rectal and colon cancer. These tissue-specific miRNA expression profiles may help us understand the biological differences between colon and rectal cancer, which may lead to improved treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3159. doi:1538-7445.AM2012-3159

Chen, Zhenbin MD, PhD; Mu, Bing PhD; Qin, Hanjun PhD; Wang, Jinhui PhD; Gao, Harry PhD; Li, Wanyan BA; Cataldo, Peter MD; Marcet, Jorge MD; Garcia-Aguilar, Julio MD, PhD Author Information

487 Background: miRNAs have been shown to be involved in tumor initiation, progression and metastasis in many cancers including colorectal cancer. However, miRNA profiling specifically in rectal cancer is not well characterized. Our objective was to generate a miRNA expression profile in locally advanced rectal cancer using formalin fixed paraffin embedded (FFPE) biopsy tissue collected from patients with locally advanced rectal cancer. Methods: We collected pre-treatment biopsy tissue and matched normal tissue from 40 rectal cancer patients treated with pre-operative chemoradiation (CRT) and total mesorectal excision (TME). We extracted 50-1000ng of total RNA from FFPE biopsies and optimized small RNA sample preparation for deep-sequencing. We then performed deep sequencing on biopsy and matched normal tissue and compared miRNA expression in biopsy and normal tissues using paired T-test and multiple testing (Q-bound <0.05) to determine significant miRNA expression changes in rectal cancer. Results: 182 miRNAs were differentially expressed in tumor versus normal tissues (Q-bound <0.05); 15 of these miRNAs showed a greater than 2-fold change in expression in tumor tissue; mir-18a, mir-135b, mir-503, mir-584, mir-106b, mir-224, mir-92a, mir-181d were up-regulated and mir-375, mir-378, mir-378c, mir-137, mir-378, mir-147b, mir-30a were down-regulated. miRNA mir-31 showed the highest up-regulation in tumor tissue (16-fold increase) while mir-215 expression decreased 8-fold in tumor compared to normal tissue. Tumor and normal tissues were completely separated by hierarchical cluster analysis based on their distinctive miRNA profiles. Conclusions: We optimized small RNA sample preparation for deep sequencing of miRNAs, an approach which may be useful for quantifying miRNA expression in tissues with limited starting material. We also identified a novel miRNA expression profile in rectal cancer that may be useful as a diagnostic biomarker of disease.

Abstract Introduction: Clear cell renal cell carcinoma (ccRCC) represents the most common renal cancer histology. In the setting of metastatic disease, few patients achieve a durable remission with currently available therapies. Early determination of metastatic potential may help guide therapy and improve clinical outcome. MicroRNA (miRNA) is a group of small non-coding RNAs that regulate gene expression during development and differentiation. miRNA expression is altered in malignant tissue, and signatures based on miRNA expression can aid in diagnosis and prognostication. In this study, we have characterized ccRCC miRNA expression in a 28-sample training cohort using microarray technology. From this, we have developed a 5-miRNA expression signature to predict the risk of metastasis and overall prognosis. This signature has been further validated by an independent 34-sample testing cohort. Study Design: Training and testing cohorts were established, comprised of 28 and 34 ccRCC frozen tissue specimens, respectively. The training cohort included specimens from patients characterized as stage I (T1; n=14) and stage IV (M1; n=14). The testing cohort included specimens from patients with (n=20) and without (n=14) metastatic disease. All cases used for the testing cohort had been followed for at least 5 years if there was no tumor metastasis reported. Total RNAs of these samples were analyzed using Agilent miRNA microarray (probes for 723 human miRNAs, Sanger miRBase 10.1). Results: (1) Differentially expressed miRNAs between metastatic ccRCCs and their non-metastatic counterparts in the training cohort were identified, using the criteria of fold change >1.5 (p value <0.05 by ANOVA). The 28 samples in the cohort were found to form distinct groups with different miRNA expression profiles using hierarchical clustering method. (2) A 5-miRNA ccRCC metastasis-specific signature has been identified using a customized computational method, which was based on logistic regression and Linear Discriminant Analysis classification, by comparing miRNA expression between metastatic and non-metastatic samples. (3) The signature has been successfully validated in the 34-sample testing cohort. With the clinical follow-up information (> 5 years if no metastasis reported), the signature had very high sensitivity (77%) and specificity (100%) when it was used to predict the tumor status of metastasis and metastatic potential. (4) This 5-miRNA signature may supplement widely used prognostic tools in RCC, such as the UCLA Integrated Staging System (UISS). The 5-miRNA signature stratified outcome within groups classified as high, intermediate or low risk by this schema. Conclusions: We have developed a 5-miRNA expression signature to determine ccRCC metastasis and prognosis. With further validation in larger cohorts, the signature may be applied towards early prediction of metastatic potential, and may augment currently available risk stratification tools for RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-275.

Renal cell carcinoma (RCC) is one of the leading causes of cancer mortality. Characterization of microRNA (miRNA) expression of RCC will help disclose new pathogenic pathways in tumourigenesis and progression and may lead to the development of molecular biomarkers and target‐specific therapies for diagnosis, prognostication and treatment. With limitations in test specificity and the ability to detect novel miRNA and other small non‐coding RNAs (smRNAs), microarray and RT–PCR techniques are being replaced by the evolving deep‐sequencing technologies, at least in the discovery phase. Until now, cancer miRNA profiling of human benign and tumour specimen sets, using smRNA deep‐sequencing (smRNA‐seq), has not been reported. Specifically, due to concern over possible poor RNA quality/integrity, formalin‐fixed paraffin‐embedded (FFPE) samples have not been used for such studies. Here, we performed whole‐genome smRNA‐seq analysis using a benign and RCC specimen set and have successfully profiled the miRNA expression. Studies performed on paired frozen and FFPE specimens showed very similar results. Moreover, a comparison study of microarray, deep‐sequencing and RT–PCR methodologies also showed a high correlation among the three technologies. To our knowledge, this is the first study to demonstrate that FFPE specimens can be used reliably for miRNA deep‐sequencing analysis, making future large‐scale clinical cohort/trial‐based studies possible. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction: MicroRNA (miRNA) is a group of small non-coding regulating RNA and has shown altered expression in cancer. With great advantages in test specificity and capability to detect novel (unknown) miRNA and other small RNA targets, the next-generation deep sequencing technology is replacing microarray platforms, at least in the discovery phase. Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney malignancies and one of the leading causes of death. Characterizing miRNA expression of ccRCC will enhance the understanding of its tumorigenesis and progression and will, therefore, lead to the development of cancer-specific molecular therapy. In this study, we analyzed miRNA expression using a human ccRCC cohort by whole genome small RNA deep sequencing. We characterized the miRNA expression in association with CCRCC and further validated the altered miRNA expression in a larger clinical cohort by RT-PCR. A group of tumor associated novel miRNAs have been discovered. Design : (1) Deep sequencing of whole genome small RNA (17-52 nucleotides in size) in a 6-sample frozen CCRCC cohort (3 benign and 3 ccRCC samples) using Illumina system (Solexa) was performed. (2) The miRNA expression of benign kidney and ccRCC samples was quantitatively compared. (3) 8 miRNAs, which were randomly selected from the list of top 20 aberrantly expressed miRNAs detected by deep sequencing, were tested using ABI RT-PCR technology in a 38-sample ccRCC cohort (9 benign kidney and 29 different stage ccRCC samples) for validation. (4) Analysis of unknown small RNAs was performed to find tumor associated novel miRNAs. Results : (1) Using a deep sequencing technology, we detected miRNAs and other small RNAs in frozen human samples of ccRCC cohort and successfully profiled miRNA expression in association with tumor. (2) We validated the altered expression of 8 selected miRNA targets in the 38-sample clinical ccRCC cohort using an RT-PCR method. (3) We discovered a group of novel miRNAs associated with ccRCC. Conclusions : We performed whole genome small RNA deep sequencing of human ccRCC tissue with further RT-PCR validation in a clinical sample cohort. We have characterized miRNA expression and discovered a group of novel miRNAs in association with ccRCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-356.