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PDF file - 1001KB, S1. Transformation of NIH3T3 cells expressing CD74-NRG1, EZR-ERBB4, or TRIM24-BRAF cDNAs. S2. Electropherogram of Sanger sequencing of cDNAs of novel fusion transcripts. S3. Schematic diagrams of genomic organization for NRG1, ERBB4, BRAF, and RET rearrangements. S4. Break-apart fluorescence in situ hybridization (FISH) for the NRG1 fusion. S5. Representative histological images obtained from fusion-positive IMAs.

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Clinicopathological characteristics of study populations.

Supplemental Figure 1. Nomogram to predict 5-year survival rates for stage IA (A) or stage IB (B) lung adenocarcinoma separately. Each clinical variable (4-gene score, sex and age) is assigned a point value. The sum of those points can then be used to estimate probability of survival for 5-years.

Supplementary Figure Legends

Purpose: To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur.Experimental Design: From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products.Results: We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use.Conclusions: Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs. Clin Cancer Res; 20(12); 3087–93. ©2014 AACR.

Supplemental Table 1. Datasets considered for inclusion in the meta-analysis and the rationale for inclusion or exclusion.

Supplemental Table 2. Selected probes for each platform

PDF file, 165K, Little overlap between prediction algorithms.

Table S3: List of 1820 differentially expressed genes between short and long survival groups

Supplemental Table 3. Univariable and Multivariable Cox regression of a linear score for the 4-coding gene classifier in the combined cohorta of Stage I, adenocarcinoma patients

Purpose: The dismal outcome of esophageal cancer patients highlights the need for novel prognostic biomarkers, such as microRNAs (miRNA). Although recent studies have established the role of miRNAs in esophageal carcinoma, a comprehensive multicenter study investigating different histologic types, including squamous cell carcinoma (SCC) and adenocarcinoma with or without Barrett's, is still lacking.Experimental Design: miRNA expression was measured in cancerous and adjacent noncancerous tissue pairs collected from 100 adenocarcinoma and 70 SCC patients enrolled at four clinical centers from the United States, Canada, and Japan. Microarray-based expression was measured in a subset of samples in two cohorts and was validated in all available samples.Results: In adenocarcinoma patients, miR-21, miR-223, miR-192, and miR-194 expression was elevated, whereas miR-203 expression was reduced in cancerous compared with noncancerous tissue. In SCC patients, we found elevated miR-21 and reduced miR-375 expression levels in cancerous compared with noncancerous tissue. When comparing cancerous tissue expression between adenocarcinoma and SCC patients, miR-194 and miR-375 were elevated in adenocarcinoma patients. Significantly, elevated miR-21 expression in noncancerous tissue of SCC patients and reduced levels of miR-375 in cancerous tissue of adenocarcinoma patients with Barrett's were strongly associated with worse prognosis. Associations with prognosis were independent of tumor stage or nodal status, cohort type, and chemoradiation therapy.Conclusions: Our multicenter-based results highlight miRNAs involved in major histologic types of esophageal carcinoma and uncover significant associations with prognosis. Elucidating miRNAs relevant to esophageal carcinogenesis is potentially clinically useful for developing prognostic biomarkers and identifying novel drug targets and therapies. (Clin Cancer Res 2009;15(19):6192–200)

XLSX file, 64K, Clinical data.

XLS file, 36K, Target Prediction.

XLS file, 56K, miRNAs that are differentially expressed between rectal cancer tissue and normal adjacent mucosa.

Purpose: To examine the clinical utility of intratumor microRNAs (miRNA) as a biomarker for predicting responses to platinum-based doublet chemotherapy in patients with recurring lung adenocarcinoma (LADC).Experimental Design: The expression of miRNAs was examined in LADC tissues surgically resected from patients treated with platinum-based doublet chemotherapy at the time of LADC recurrence. Microarray-based screening of 904 miRNAs followed by quantitative reverse transcription-PCR–based verification in 40 test cohort samples, including 16 (40.0%) responders, was performed to identify miRNAs that are differentially expressed in chemotherapy responders and nonresponders. Differential expression was confirmed in a validation cohort (n = 63 samples), including 18 (28.6%) responders. An miRNA signature that predicted responses to platinum-based doublet chemotherapy was identified and its accuracy was examined by principal component and support vector machine analyses. Genotype data for the TP53-Arg72Pro polymorphism, which is associated with responses to platinum-based doublet chemotherapy, were subsequently incorporated into the prediction analysis.Results: A signature comprising three miRNAs (miR1290, miR196b, and miR135a*) enabled the prediction of a chemotherapeutic response (rather than progression-free and overall survival) with high accuracy in both the test and validation cohorts (82.5% and 77.8%). Examination of the latter was performed using miRNAs extracted from archived formalin-fixed paraffin-embedded tissues. Combining this miRNA signature with the TP53-Arg72Pro polymorphism genotype marginally improved the predictive power.Conclusion: The three-miRNA signature in surgically resected primary LADC tissues may by clinically useful for predicting responsiveness to platinum-based doublet chemotherapy in patients with LADC recurrence. Clin Cancer Res; 20(18); 4784–93. ©2014 AACR.

PDF file, 164K, Lack of correlation between array and qRT-PCR data for two miRNAs.

Purpose: Inflammatory genes and microRNAs have roles in colon carcinogenesis; therefore, they may provide useful biomarkers for colon cancer. This study examines the potential clinical utility of an inflammatory gene expression signature as a prognostic biomarker for colon cancer in addition to previously examined miR-21 expression.Experimental Design: Quantitative reverse transcriptase-PCR. was used to measure the expression of 23 inflammatory genes in colon adenocarcinomas and adjacent noncancerous tissues from 196 patients. These data were used to develop models for cancer-specific mortality on a training cohort (n = 57), and this model was tested in both a test (n = 56) and a validation (n = 83) cohort. Expression data for miR-21 were available for these patients and were compared and combined with inflammatory gene expression.Results: PRG1, IL-10, CD68, IL-23a, and IL-12a expression in noncancerous tissue, and PRG1, ANXA1, IL-23a, IL-17a, FOXP3, and HLA-DRA expression in tumor tissues were associated with poor prognosis based on Cox regression (|Z-score| >1.5) and were used to generate the inflammatory risk score (IRS). IRS was associated with cancer-specific mortality in the training, test (P = 0.01), and validation (P = 0.02) cohorts. This association was strong for stage II cases (P = 0.002). Expression of miR-21 was associated with IL-6, IL-8, IL-10, IL-12a, and NOS2a, providing evidence that the function of this microRNA and these inflammatory genes are linked. Both IRS and miR-21 expression were independently associated with cancer-specific mortality, including stage II patients alone.Conclusion: IRS and miR-21 expression are independent predictors of colon cancer prognosis and may provide a clinically useful tool to identify high-risk patients. (Clin Cancer Res 2009;15(18):5878–87)

Supplementary Data from Association of Inflammation-Related and microRNA Gene Expression with Cancer-Specific Mortality of Colon Adenocarcinoma

PDF file, 248K, miRNA networks identified by IPA based on miRNAs that are differentially regulated between tumor and mucosa.

PDF file, 208K, Identfication of miR-652*.

Supplementary Data. This excel file contains all of the data on stage I lung adenocarcinoma that was used for this manuscript. The second worksheet contains a description of the columns

Supplementary methods: Describes additional details of generation of stable NOSTRIN overexpressing cells, western blotting, measurement of Nitric Oxide production, Xcelligence real-time migration assay and Luciferase Reporter Assay; Supplementary figure legends

Correlation of microarray data with quantitative RT-PCR data for 12-miRNAs expression in the test cohort.

Purpose: miRNAs play a prominent role in a variety of physiologic and pathologic biologic processes, including cancer. For rectal cancers, only limited data are available on miRNA expression profiles, whereas the underlying genomic and transcriptomic aberrations have been firmly established. We therefore, aimed to comprehensively map the miRNA expression patterns of this disease.Experimental Design: Tumor biopsies and corresponding matched mucosa samples were prospectively collected from 57 patients with locally advanced rectal cancers. Total RNA was extracted, and tumor and mucosa miRNA expression profiles were subsequently established for all patients. The expression of selected miRNAs was validated using semi-quantitative real-time PCR.Results: Forty-nine miRNAs were significantly differentially expressed (log2-fold difference >0.5 and P < 0.001) between rectal cancer and normal rectal mucosa. The predicted targets for these miRNAs were enriched for the following pathways: Wnt, TGF-beta, mTOR, insulin, mitogen-activated protein kinase, and ErbB signaling. Thirteen of these 49 miRNAs seem to be rectal cancer-specific, and have not been previously reported for colon cancers: miR-492, miR-542-5p, miR-584, miR-483-5p, miR-144, miR-2110, miR-652, miR-375, miR-147b, miR-148a, miR-190, miR-26a/b, and miR-338-3p. Of clinical impact, miR-135b expression correlated significantly with disease-free and cancer-specific survival in an independent multicenter cohort of 116 patients.Conclusion: This comprehensive analysis of the rectal cancer miRNAome uncovered novel miRNAs and pathways associated with rectal cancer. This information contributes to a detailed view of this disease. Moreover, the identification and validation of miR-135b may help to identify novel molecular targets and pathways for therapeutic exploitation. Clin Cancer Res; 18(18); 4919–30. ©2012 AACR.

Supplementary Data from MicroRNA Expression in Squamous Cell Carcinoma and Adenocarcinoma of the Esophagus: Associations with Survival

Table S4: List of 201 differentially expressed genes (>1.5 fold) that were significantly associated with survival. Table S5: List of 66 inflammatory genes associated with survival. Table S6: List of 35 inflammatory genes associated with survival by Kaplan-Meier analysis. Table S7: Univariable and Multivariable Cox-Regression Analysis

Supplemental Table 4. Nine cohorts of stage I, lung squamous cell carcinoma patients

Purpose: Esophageal cancer is one of the most aggressive and deadly forms of cancer; highlighting the need to identify biomarkers for early detection and prognostic classification. Our recent studies have identified inflammatory gene and microRNA signatures derived from tumor and nontumor tissues as prognostic biomarkers of hepatocellular, lung, and colorectal adenocarcinoma. Here, we examine the relationship between expression of these inflammatory genes and micro RNA (miRNA) expression in esophageal adenocarcinoma and patient survival.Experimental Design: We measured the expression of 23 inflammation-associated genes in tumors and adjacent normal tissues from 93 patients (58 Barrett's and 35 Sporadic adenocarcinomas) by quantitative reverse transcription-polymerase chain reaction. These data were used to build an inflammatory risk model, based on multivariate Cox regression, to predict survival in a training cohort (n = 47). We then determined whether this model could predict survival in a cohort of 46 patients. Expression data for miRNA-375 were available for these patients and was combined with inflammatory gene expression.Results: IFN-γ, IL-1α, IL-8, IL-21, IL-23, and proteoglycan expression in tumor and nontumor samples were each associated with poor prognosis based on Cox regression [(Z-score)>1.5] and therefore were used to generate an inflammatory risk score (IRS). Patients with a high IRS had poor prognosis compared with those with a low IRS in the training (P = 0.002) and test (P = 0.012) cohorts. This association was stronger in the group with Barrett's history. When combining with miRNA-375, the combined IRS/miR signature was an improved prognostic classifier than either one alone.Conclusion: Transcriptional profiling of inflammation-associated genes and miRNA expression in resected esophageal Barrett's-associated adenocarcinoma tissues may have clinical utility as predictors of prognosis. Clin Cancer Res; 16(23); 5824–34. ©2010 AACR.

XLSX file - 22KB, Supplementary Table S1. RNA sequencing of 32 IMAs. Supplementary Table S2. PCR Primers used for RT-PCR screening.

XLS file, 32K, Target sequences for the miRNA PCR reactions.

Figure S1: Representative pictures of immunohistochemical staining, showing NOSTRIN expression largely in the tumor cells in the patient with long survival. Figure S2: NOSTRIN overexpression sensitizes the Panc1 pancreatic cancer cells to gemcitabine. Figure S3: (a) A negative correlation existed between miR-222 and NOSTRIN expression in tumors from PDAC cases (N=37). (b) miR-222 expression does not show any significant association with survival. (ns=non significant).

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Supplementary Appendix 1 from MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

PDF file - 10.6MB, Mapping the CAV1 interaction to the START domain in the C-terminus of DLC1. The DLC1-Del (929-957) mutant is active in RhoGAP activity in transiently transfected H1299 cells and stably transfected H358 cells

PDF file - 1MB, Expression of CAV1 or the combination of low CAV1 with low DLC1 do not affect the prognostic significance of lung cancer patients in Director's challenge cohort

Supplementary Figure 1 from MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

Supplementary Methods, Figures 1-8, Tables 1-4 from Tumor Suppressor miR-22 Determines p53-Dependent Cellular Fate through Post-transcriptional Regulation of p21

Supplementary Tables 1-9 from MicroRNA Expression and Clinical Outcomes in Patients Treated with Adjuvant Chemotherapy after Complete Resection of Non–Small Cell Lung Carcinoma

PDF file - 836K, Supplemental Figure 1: Overview of the study design for the identification of candidate coding genes, development and validation of the four coding gene classifier using five independent datasets. Supplemental Figure 2: Technical validation of the microarray experiments showing Pearson�s correlation between microarray expression values and qRT-PCR expression values for each gene across all tumors that were examined.Supplemental Figure 3: Kaplan-Meier survival curves according to tertiles of the four coding gene classifier in AJCC 6th edition stage I-II lung adenocarcinoma from five independent cohorts, including the Japan (n=199, relapse-free survival), US/Norway (n=92, cancer specific mortality), Directors� (n=378, overall survival), Bhattacharjee (n=100, overall survival) and Tomida (n=92, overall survival) cohorts. P-values were estimated by log-rank test for trend. Supplemental Figure 4: For survival analysis, miR-21 expression values as measured by nCounter miRNA assays were normalized to the average of the 5 most highly expressed microRNAs in tumor tissue, excluding miR-21 itself. Supplemental Figure 5: In the Japan cohort, the four coding gene classifier and the non-coding miR-21 classifier are each significantly associated with relapse-free survival.Supplemental Figure 6: In the US/Norway cohort, the four coding gene classifier and the non-coding miR-21 classifier are each significantly associated with relapse-free survival. Supplemental Figure 7: Estimation of miR-21 copies per cell in lung adenocarcinoma tissue by PCR-based absolute quantification. Supplemental Figure 8: Total RNA content per lung tumor cell was estimated at approximately 20pg per cell based on a series of total RNA extraction from serially diluted cultured lung adenocarcinoma cell lines A549 and NCI-H23. Supplemental Figure 9: Prognostic significance of BRCA1 expression in multiple cancer types. Supplemental Table 1. Characteristics of study populations of patients from public database. Supplemental Table 2. Univariate Cox regression analysis in the Japan cohort using microarray data (AJCC TNM 6th, Stage I, n=148). Supplemental Table 3. Univariate Cox Regression analysis of the expression of six genes.

This study determined whether expression levels of a panel of biologically relevant microRNAs can be used as prognostic or predictive biomarkers in patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected non–small cell lung carcinoma (NSCLC). Expression of miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a was determined by quantitative real-time PCR in formalin-fixed paraffin-embedded tumor specimens from 639 IALT patients. The prognostic and predictive values of microRNA expression for survival were studied using a Cox model, which included every factor used in the stratified randomization, clinicopathologic prognostic factors, and other factors statistically related to microRNA expression. Investigation of the expression pattern of microRNAs in situ was performed. We also analyzed the association of TP53 mutation status and miR-34a/b/c expression, epidermal growth factor receptor and KRAS mutation status, and miR-21 and Let-7a expression. Finally, the association of p16 and miR-29b expression was assessed. Overall, no significant association was found between any of the tested microRNAs and survival, with the exception of miR-21 for which a deleterious prognostic effect of lowered expression was suggested. Otherwise, no single or combinatorial microRNA expression profile predicted response to adjuvant cisplatin-based chemotherapy. Together, our results indicate that the microRNA expression patterns examined were neither predictive nor prognostic in a large patient cohort with radically resected NSCLC, randomized to receive adjuvant cisplatin-based chemotherapy versus follow-up only. Cancer Res; 70(21); 8288–98. ©2010 AACR.

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PDF file - 4MB, siRNA-mediated reduction of CAV1, DLC1, or both CAV1 and DLC1 increases the migration of H1299 cells

On March 29, 2018, the FDA granted accelerated approval for blinatumomab (Blincyto; Amgen, Inc.) for the treatment of adults and children with B-cell precursor acute lymphoblastic leukemia (BCP ALL) in first or second complete remission with minimal residual disease (MRD) greater than or equal to 0.1%. Blinatumomab is a CD3xCD19 bispecific antibody approved previously for the treatment of relapsed or refractory BCP ALL. The basis for this accelerated approval was a single-arm trial. For the 86 patients in first and second complete remission with MRD ≥ 0.1%, conversion to MRD < 0.01% was achieved after one cycle of blinatumomab by 85.2% [95% confidence interval (CI): 73.8%, 93.0%] and 72.0% (95% CI: 50.6%, 87.9%), respectively, and the estimated median hematologic relapse-free survivals (RFS) were 35.2 months (95% CI: 0.4–53.5) and 12.3 months (95% CI: 0.7–42.3), respectively. Hematologic RFS was considered substantial independent of whether patients underwent subsequent allogeneic stem cell transplantation. The safety profile for blinatumomab was established in prior studies, and no new safety signals were observed in the new population. Cytokine release syndrome and neurotoxicity remain significant risks. The FDA is requiring confirmation of clinical benefit in a randomized trial.

Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.