bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4144: Improvement of tumor cell transduction with lentiviral vectors using chemical and cell targeting approaches.
Authors: Höfig Ines , Atkinson Michael J. , Thirion Christian , Anastasov Natasa
Published: August 2014

Although lentiviral transduction methods are widely used in cancer research their application is frequently hindered by the low transduction efficiency seen for a number of different cell types, in particular primary cells. We have previously reported that a mixture of poloxamer synperonic F108 and polybrene combines reduced toxicity and increased lentiviral gene delivery into a range of different primary cell cultures (1). We now report further improvement in efficiency through a novel combination of the poloxamer-based adjuvant with a modified lentiviral envelope. The envelope was rendered cell specific by fusing monoclonal antibody fragments directed against specific cell-surface epitopes with the viral attachment glycoprotein VSV-G.

For specific cellular targeting we inserted chimeric antiCD30 or antiEGFR antibody-VSV-G fusion proteins into the lentiviral genome. The combination of VSV-G-fused antibody fragments against specific tumor cell antigens and the improved adjuvant resulted in a 3-fold higher transduction rate using a GFP reporter containing lentivirus in CD30-positive lymphoma and EGFR-positive epithelial breast cancer cells. The combination of optimized transduction enhancer molecules and retargeted lentiviral vectors enabled quantitative transduction of previously hard-to-transduce cell types. When mixtures of antigen-negative and antigen-positive tumor cells were selected the modified lentivirus particles showed a preference for infecting antigen-positive cells by a factor 4.

These results demonstrate that improvements in both chemical charge protecting adjuvants and membrane targeting can lead to increased transduction efficiency without a loss of specificity. The combination of chemical adjuvant and envelope design can lead to the optimization of lentiviral transductions in large-scale systems.

*1. Höfig I, Atkinson MJ, Mall S, Krackhardt AM, Thirion C, Anastasov N. Poloxamer synperonic F108 improves cellular transduction with lentiviral vectors. J Gene Med. 2012 Aug;14(8):549-60.

Citation Format: Ines Höfig, Michael J. Atkinson, Christian Thirion, Natasa Anastasov. Improvement of tumor cell transduction with lentiviral vectors using chemical and cell targeting approaches. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4144. doi:10.1158/1538-7445.AM2013-4144
Abstract 4148: Sensitive detection of tumor nucleic acids in plasma by mutation-enriched next generation sequencing.
Authors: Marziali Andre
Published: August 2014

We have demonstrated a novel somatic mutation enrichment methodology that allows DNA sequencing to work beyond its usual limit of detection to accurately profile solid tumors by detecting their mutation signature in plasma, even when the tumor DNA is present in abundances below 0.01%.

Next Generation DNA Sequencing (NGS) is becoming the new standard for mutational profiling of tumor tissue, due to its flexibility, speed, and decreasing cost. While generally exceptional in performance, NGS suffers from a sequencing error rate of 0.1% - 1%, largely due to amplification induced artifacts in its workflow. While this does not constitute a significant problem in application of NGS to sequencing of most tumour tissues, it makes NGS impractical as a method to search for low abundance mutation signatures in plasma samples.

Numerous publications have shown the presence of tumor signatures in the cell-free DNA (cfDNA) circulating in plasma, but concordance between the tumor signature and the plasma signature has been limited. This is likely due to limitations in the detection technologies used to search for cfDNA in plasma. To maximize concordance between plasma and tissue, it will be essential that sensitivities reaching 0.01% and below (as little as a single tumor mutant allele per sample) be achieved, and ideally that multiple mutational hot spots be analyzed to maximize the chance of detection. Current technologies are incapable of such sensitivity over a large number of mutation loci.

We have developed a novel electrophoretic method that can enrich nucleic acid samples over 1,000,000-fold for up to 100 somatic mutations, enabling reliable profiling of samples containing as little as 0.01% mutant. By enriching nucleic acid samples for specific targets prior to amplification and sequencing, we enable the use of NGS in plasma-based mutation detection and profiling.

We present data demonstrating highly sensitive multiplexed mutation detection in plasma samples, demonstrating 0.01% sensitivity. We also report on concordance studies between mutation incidence in tissue and matched plasma in human samples.

Citation Format: Andre Marziali. Sensitive detection of tumor nucleic acids in plasma by mutation-enriched next generation sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4148. doi:10.1158/1538-7445.AM2013-4148
Abstract 4154: Rapid, highly multiplexed somatic mutation analysis of FFPE tissues by deep amplicon sequencing on MiSeq®.
Authors: Upsall Elizabeth , Chang Katherine , Lewis Ian , Tian April , Iyer Anita , Shen Richard , Lin Charles
Published: August 2014

DNA extraction from formalin-fixed, paraffin-embedded (FFPE) tumor tissues typically results in low quantities of poor-quality, damaged DNA, which present challenges for sequencing. Here we present a fast, streamlined sample-to-answer workflow using probe design software and sample preparation methods optimized to overcome these challenges. DesignStudio® allows for the targeting of up to 1,536 user-defined genomic regions and supports amplicon sizes (150- and 175-bp) ideal for characterizing genomic hot-spot regions or sequencing of whole exons with FFPE DNA. The TruSeq® Custom Amplicon (TSCA) workflow delivers high target enrichment specificity (>80% of reads on target), robust target multiplexing (up to 1,536 amplicons per reaction), and scalable sample indexing (up to 96 libraries) that supports simultaneous sequencing using the MiSeq. We have optimized the DNA extraction and hybridization processes to achieve higher DNA yields and more uniform target representation, respectively. In addition, the improved TSCA assay and software analysis tools significantly reduce false-positive variant calls. In conclusion, we have developed an integrated solution for highly accurate and reproducible detection of somatic variants implicated in cancer biology.

Citation Format: Elizabeth Upsall, Katherine Chang, Ian Lewis, April Tian, Anita Iyer, Richard Shen, Charles Lin. Rapid, highly multiplexed somatic mutation analysis of FFPE tissues by deep amplicon sequencing on MiSeq®. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4154. doi:10.1158/1538-7445.AM2013-4154
Abstract 4155: Personalized chemosensitivity profiling of cancers isolated from genetically-engineered mice.
Authors: Kamiyama Hirohiko , Rauenzahn Sherri , Shim Joong Sup , Liu Jun O. , Maitra Anirban , Eshleman James R.
Published: August 2014

BACKGROUNDHigh-throughput functional chemosensitivity testing is a potentially effective way to customize personalized chemotherapy treatment. This testing requires low-passage patient-specific cancer cell lines, however, generating such cell lines from patients’ tumors is difficult, due in large part, to contaminating fibroblasts that often overgrow the malignant cells.

METHODSWe constructed a series of hypoxanthine phosphoribosyl transferase (hprt) null immunodeficient mice. These include nude hprt-null, SCID hprt-null and NOD-SCID hprt-null mice. During growth of xenografted human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Tumors from patients were implanted and expanded in these mice. Explanted tumors were then grown in tissue culture flasks with selection media (hypoxanthine, aminopterin, thymidine; HAT media) to eliminate the mouse biochemically-defective fibroblasts and produce pure human cancer cell lines. We screened one cell line against the Johns Hopkins Drug Library containing ∼3,100 drugs. Selected potent drugs were used for in vivo xenograft tumor inhibition studies.

RESULTSWe successfully established several pancreatic cancer cell lines from patients using the hprt null immunodeficient mice. We performed chemosensitivity profiling on one of the pancreatic cancer cell lines using 3,100 drugs. From the profiling, we selected potent drugs, which are FDA-approved but are not currently used for pancreatic cancer. We chose two drugs that demonstrated greater in vitro chemosensitivity compared to control cell lines. The two drugs were tested against mouse xenografts where both drugs demonstrated superior in vivo efficacy to the sensitive cell line tumors compared to control tumors.

CONCLUSIONSChemotherapy for cancer may be personalized using low passage cell lines derived from patients through biochemically selectable mice. Further tests are needed to determine whether in vitro chemosensitivity testing of low-passage patient-specific cancer cell lines can predict clinical patient response.

Citation Format: Hirohiko Kamiyama, Sherri Rauenzahn, Joong Sup Shim, Jun O. Liu, Anirban Maitra, James R. Eshleman. Personalized chemosensitivity profiling of cancers isolated from genetically-engineered mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4155. doi:10.1158/1538-7445.AM2013-4155
Abstract 4158: MicroRNA-mediated regulation of Bmi1 expression in gastric and colon cancer cells.
Authors: Sugihara Hidetaka , Ishimoto Takatsugu , Watanabe Masayuki , Baba Hideo
Published: August 2014

Background: MicroRNAs (miRs) target the 3’-untranslated region (3’-UTR) of mRNA and induce translational suppression or mRNA degradation. Bmi1 is a member of the polycomb-repressive complex 1 (PRC1) that has an essential role in maintaining chromatin silencing, and is involved in the self-renewal of neuronal, haematopoietic and intestinal cells through repression of the INK4A-ARF locus. The regulatory mechanism of Bmi1 in cancer cells is still unknown. The aim of this study is to clarify the miR-mediated regulatory mechanism of Bmi1 expression in gastric and colon cancer cells.

Methods and Results: We searched for miRNAs that might regulate Bmi1 using miRBase and selected 64 miRNAs as a candidate. In these miRNAs, we focused miR-30e-3p that was known to be down-regulated in colon, breast and esophageal cancer cells. First, we examined the expression of miR-30e-3p in matched cancer tissue and non-tumor tissue using formalin-fixed paraffin-embedded(FFPE) samples of gastric and colon cancer. We confirmed the expression of miR-30e-3p was significantly lower in cancer tissue than in non-tumor tissue. Next, we measured the expression of miR-30e-3p and Bmi1 by real time PCR and western blotting using gastric and colon cancer cell lines. The endogenous expression of Bmi1 and miR-30e-3p showed inverse correlation in each cell lines. Then we examined the effect of overexpression or down regulation of miR-30e-3p on the expression of Bmi1. The suppression of miR-30e-3p increased Bmi1 expression, and the overexpression of miR-30e-3p decreased Bmi1 expression. Conclusion: Our current study revealed that the expression of Bmi1 was affected by miR-30e-3p in gastric and colon cancer. Mir-30e-3p may be a therapeutic target for the treatment of gastric and colon cancer.

Citation Format: Hidetaka Sugihara, Takatsugu Ishimoto, Masayuki Watanabe, Hideo Baba. MicroRNA-mediated regulation of Bmi1 expression in gastric and colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4158. doi:10.1158/1538-7445.AM2013-4158
Abstract 4163: Oncogenic function of miR-196s promote cell invasion through suppression of NME4 to regulate JNK-TIMP1-MMP signaling pathway in head and neck cancer.
Authors: Cheng Ann-Joy , Lu Ya-Ching , Chang Joseph T.
Published: August 2014

Dysregulation of microRNA (miRNA) is associated with malignant transformation, while elucidating the roles remains an ongoing process. In this study, we characterize miR-196 functions in head and neck cancer (HNC). We found that neither miR-196a or miR-196b has an effect on cell growth, only miR-196a plays a role in protection cells from radiation damage, while both miR-196a and miR-196b actively promotes cell migration (1.7- to 2.7-fold) and invasion (2.1- to 2.3-fold), which were supported by the altered expressions of fibronectin and N-cadherin. Mechanistically, miR-196 achieves these functions by targeting and inhibiting the anti-metastatic family member NME4, which is further elucidated as a tumor suppressor to repress JNK protein phosphorylation, leading to TIMP1 inhibition and MMP1/9 activation. Furthermore, both miR-196a and miR-196b was significantly over-expressed in the cancer tissues compared to the normal counterparts, with approximately 60 and 10 fold elevations respectively in average. Over-expression of miR-196 was correlated to lymph node metastasis and poor survival. In summary, our findings define miR-196 as a crucial oncomir in HNC which acts at least in part, through NME4-pJNK-TIMP-MMP signaling pathway to promote cell invasion and cellular transformation.

Citation Format: Ann-Joy Cheng, Ya-Ching Lu, Joseph T. Chang. Oncogenic function of miR-196s promote cell invasion through suppression of NME4 to regulate JNK-TIMP1-MMP signaling pathway in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4163. doi:10.1158/1538-7445.AM2013-4163
Abstract 4178: The role of miR-1 in RAS mutation prostate cancer metastasis.
Authors: Liu Yen-Nien , Yin Juan Juan , Barrett Ben , Sheppard-Tillman Heather , Casey Orla , Kelly Kathy
Published: August 2014

Several mechanisms have been suggested to impact prostate cancer progression, including mutation of Ras, either genetically or epigenetically during prostate cancer metastasis. MicroRNAs (miRs) are involved in embryonic development and prostate cancer metastasis. MiR-1 expression has been found to be downregulated in prostate cancer clinical cases relative to normal prostate tissue. It is not clear how miR-1 expression relates to Ras mutation in prostate cancer metastasis. Identifying the regulatory mechanisms of miR-1 in relation to Ras signaling will define roles of the miR-1 regulatory pathway in prostate metastatic processes. To investigate the contribution of this miRNA in Ras signaling pathway, we are using lentiviral expression systems to enhance miR-1 expression in a metastatic human prostate cancer cell line. Following confirmation of miR-1 over-expression, we have found that the metastatic prostate cancer cell line DU145 with RasV12G37 mutant displayed reduced metastasis and tumor growth ability in vivo, and decreased expression of a miR-1 target gene and Ras-related gene expression in vitro. We hypothesize that upon activation of Ras signaling genes, reduced expression of miR-1 is required for prostate cancer metastasis. Our results will provide evidence to show how miR-1 regulates Ras signaling genes in prostate cancer progression.

Citation Format: Yen-Nien Liu, Juan Juan Yin, Ben Barrett, Heather Sheppard-Tillman, Orla Casey, Kathy Kelly. The role of miR-1 in RAS mutation prostate cancer metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4178. doi:10.1158/1538-7445.AM2013-4178
Abstract 4183: MicroRNA-7 targeting PSME3 inhibits the growth of non-small cell lung cancer.
Authors: Chu Yiwei
Published: August 2014

MicroRNAs (miRNAs) are a family of small non-coding RNAs that function as important regulators of multiple target genes and participate in diverse biological roles in carcinogenesis..In this study, we found that the expression of miR-7 was downregulated not only in cell lines, but also in human NSCLC tissues compared to the matched adjacent tissues, which exhibited positive correlation with tumor volume.Restoration of its expression through miR-7 mimics in NSCLC cell lines including A549 and H1299 cells inhibited cell proliferation, colony formation, and cell cycle progression in vitro. More importantly, the tumorigenicity in nude mice was reduced after administration of miR-7 in vivo, strongly indicating that miR-7 is a main tumor suppressor miRNA in NSCLC. In advance, through a series of analysis derived from bioinformatics prediction, western blot and luciferase assay, we identified a novel target of miR-7, PSME3 (a proteasome activator) to be enrolled in the regulation with tumor. Expression of PSME3 in NSCLC tumor samples was significantly upregulated compared to adjacent non-tumor tissues, exhibiting a strong inverse relation with that of miR-7. In addition, knockdown of PSME3 gene induced similar effects to overexpression of miR-7 in NSCLC cells. All these findings strongly imply that the overexpression of PSME3 resulted from miR-7 downexpression in NSCLC plays an important role in promoting cancer cell progress, and consequently results in NSCLC growth. Thus, strategies targeting PSME3 and/or miR-7 may be the promising molecular therapy in NSCLC treatment.

Citation Format: Yiwei Chu. MicroRNA-7 targeting PSME3 inhibits the growth of non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4183. doi:10.1158/1538-7445.AM2013-4183
Abstract 4185: miR-129 is a potential tumor suppressor in colorectal cancer by regulating apoptosis and modulating fluropyrimidine based chemosensitivity.
Authors: Karaayvaz Mihriban , Zhai Haiyan , Ju Jingfang
Published: August 2014

Resistance to fluoropyrimidine based chemotherapy is the major reason for the failure of treating advanced colorectal cancer. The lack of ability for tumor cells to undergo apoptosis after genotoxic stress is the key contributor to such intrinsic mechanism. Mounting evidence has demonstrated that non-coding microRNAs (miRNAs) are crucial regulators of gene expression, in particular, under acute stress. However, there is still very limited knowledge about the role of miRNAs in apoptosis. In this study, we discovered a novel mechanism mediated by miR-129 directly inducing apoptosis by suppressing a key anti-apoptotic protein Bcl-2. Ectopic expression of miR-129 promotes apoptosis, inhibits cell proliferation and causes G2-cell-cycle arrest in colon cancer cells in vitro. The intrinsic apoptotic pathway triggered by miR-129 is activated by cleaving caspase-9 and caspase-3. The expression of miR-129 is very significantly down-regulated in colorectal cancer tissue specimens compared with the paired normal control samples (p<0.00001). More importantly, we demonstrated that miR-129 functions synergistically with 5-fluorouracil (5-FU) in vitro and in vivo to enhance 5-FU cytotoxicity. These results suggest that miR-129 is a major regulator of apoptosis in colorectal cancer. Therefore, miR-129 may be a potential tumor suppressor in colorectal cancer and a novel candidate for developing miR-129 based therapeutic strategies.

Citation Format: Mihriban Karaayvaz, Haiyan Zhai, Jingfang Ju. miR-129 is a potential tumor suppressor in colorectal cancer by regulating apoptosis and modulating fluropyrimidine based chemosensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4185. doi:10.1158/1538-7445.AM2013-4185
Abstract 419: A comparison between the OV100 and Maestro fluorescence imaging systems for noninvasive detection of metastasis.
Authors: Zhang Yong , Zhang Nan , Hoffman Robert M. , Zhao Ming
Published: August 2014

The OV100 (Olympus Corp.), and Maestro (Perkin-Elmer Inc.), were compared for imaging metastasis in orthotopic lung, liver, pancreas and colon nude mouse tumor models expressing RFP. Non-invasive images of tumor-bearing mice indicated that the Maestro systems outperformed the OV100 for macro imaging. Tumors could be detected earlier with the Maestro system than with the OV100. By separating skin and food autofluorescence, the Maestro provides a high-contrast image. The Maestro can produce a composite image of five unmixed components and detects tumors deeper than the OV100. The Maestro can also detect more color signals simultaneously. However, the OV100 has higher resolution and even a single cell can be imaged, for example via a skin-flap. The results of this study suggest that the two instruments are complimentary.

Citation Format: Yong Zhang, Nan Zhang, Robert M. Hoffman, Ming Zhao. A comparison between the OV100 and Maestro fluorescence imaging systems for noninvasive detection of metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 419. doi:10.1158/1538-7445.AM2013-419
Abstract 4195: Identification of dysregulated miRs and its target genes in head and neck cancer.
Authors: Kuo Yi-Zih , Huang Yun-Chu , Chen Yuh-Ling , Lo Hung-I , Tsai Sen-Tien , Chiang Chi-Wu , Wu Li-Wha
Published: August 2014

Oral cancer is a subtype of head and neck cancer, the eighth common cancer type in the world. MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading target messenger RNA or repressing their translation. The deregulation of certain microRNAs has been associated with the progression and metastasis of various cancer types. A total of 133 miRs were dysregulated in oral cancer cells when compared with normal oral keratinocytes by using microRNA microarray hybridization. Among them, miR-22, often silenced in breast cancer, colon cancer, and multiple myeloma, is downregulated and least known in oral carcinogenesis. In this study, we confirmed the down-regulation of miR-22 in the majority of oral cell lines and 70 % of the tested clinical specimens, as measured by real-time PCR. Both gain-of-function and loss-of-function experiments showed that increased miR-22 expression significantly reduced oral cancer cell migration and invasion, whereas decreased miR-22 expression dramatically enhanced cell migration and invasion. Snail, a transcription factor that promotes cell invasion and tumor metastasis, was predicted to be a target of miR-22. The prediction was validated by the inverse correlation of miR-22 and snail protein, and the luciferase reporter assay bearing 3’- translated region derived from snail. Silencing snail expression by using shRNA recapitulated the anti-metastatic function of miR-22, whereas restored snail expression attenuated the function of miR-22 in oral cancer cells. Together, these results suggest that miR-22 may act as a tumor suppressor through decreasing snail expression in oral cancer and thus be used as a therapeutic target to block oral cancer metastasis.

Citation Format: Yi-Zih Kuo, Yun-Chu Huang, Yuh-Ling Chen, Hung-I Lo, Sen-Tien Tsai, Chi-Wu Chiang, Li-Wha Wu. Identification of dysregulated miRs and its target genes in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4195. doi:10.1158/1538-7445.AM2013-4195
Abstract 4202: Oncogenic microRNA-31 as prognostic potential biomarker in histologically negative surgical margins of head and neck cancer patients.
Authors: Horst Maria Aderuza , Carvalho Ana Carolina , Maia Danielle Campelo , Aymberé Bello Juliana , Kowalski Luiz Paulo , Carvalho Andre Lopes , Vettore Andre Luiz
Published: August 2014

The development of locoregional recurrence is the main reason for treatment failure in head and neck squamous cell carcinomas (HNSCC) and the remaining of tumor cells in surgical margins is associated with recurrence. Surgical margins are considered negative based on histologic assessment of the pathological specimen. However, this method lacks sensitivity in identifying cells that already started malignant transformation but have not yet developed a pathologic phenotype. We investigated the usefulness of assessing the expression of microRNA-31 for the detection of molecular alterations in histologically negative surgical margins (HNSM) and determine the correlation of these tumor-related alterations with clinical and prognostic parameters. MicroRNAs are endogenously expressed non-coding RNAs that have important biological and pathological functions. The microRNA-31 was found markedly upregulated in different malignancies, including HNSCC. The microRNA-31 expression was determined by qRT-PCR in HNSCC (n=19), HNSM (n=37) and normal mucosa (n=10). According to our results, 76% of tumor samples and 30% of the HNSM specimens were positive for microRNA-31 overexpression. Moreover, the overexpression of this microRNA in the HNSM was associated with perineural invasion (p < 0.05 Fisher's exact tests) and showed a tendency to correlates with poor disease-free survival (p < 0,09 log-rank).

These findings demonstrate that the overexpression of microRNA-31 in histologically negative surgical margins is a frequent event. The use of qRT-PCR may be an useful tool in detecting actually negative HNSCC surgical margins and the overexpression of microRNA-31 in these margins could be helpful in the identification of patients with worst outcome.

Supported by Fapesp (Grant 2011/01919-2).

Citation Format: Maria Aderuza Horst, Ana Carolina Carvalho, Danielle Campelo Maia, Juliana Aymberé Bello, Luiz Paulo Kowalski, Andre Lopes Carvalho, Andre Luiz Vettore. Oncogenic microRNA-31 as prognostic potential biomarker in histologically negative surgical margins of head and neck cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2013-4202
Abstract 4206: Impact of nuclear telomere architecture in the transition of myelodysplastic syndromes to acute myeloid leukemias.
Authors: Gadji Macoura , de Oliveira Fábio Morato , Mai Sabine
Published: August 2014

Myelodysplastic Syndromes (MDS) are a group of disorders characterized by cytopenias, with a propensity for evolution into Acute Myeloid Leukemias (AML). This transformation is driven by genomic instability and epigenetic events. However, how genomic instability occurs in this disease remains unknown. Telomere dysfunction might be the generator of genomic instability leading to cytopenias and disease progression. Despite several studies demonstrating the role of telomere dysfunction in the occurrence of hematopoietic malignancies, little is known about their role in the evolution of MDS to AML/MDS. Our preliminary data allowed us to define three-dimensional nuclear telomeric profiles on the basis of telomere numbers, telomeric aggregates, telomere signal intensities, nuclear volumes, and nuclear telomere distribution. Using these parameters, we blindly subdivided the MDS patients into nine subgroups and the AML patients into six subgroups. We showed distinct telomeric profiles specific to patients with MDS, AML, and suggested for the first time a chronological and evolutionary process of telomere dysfunction in both diseases and the transformation of MDS to AML. To validate the clinical significance of 3D telomere profiling of MDS-patients and AML-patients, we are studying new patient cohorts and patients progressing from MDS to AML over time. Such a longitudinal study will allow for precise 3D telomeric profiling during disease progression. Since transformation of MDS to AML can occur over a long period of time, we are also studying disease progression from MDS to AML using a mouse model (C57BL/6-Tg(Vava1-NUP98/HOXD13)G2Apla/J). These mice will transform their MDS-disease to AML disease in a time period of in between 4-14 months. Therefore, we are following the mice monthly up to 14 months. Our study will allow us to gain an understanding of the 3D telomeric signatures that predict and accompany the transition of MDS to AML. In addition, we will define the molecular profiles at disease transition to improve, once validated, treatment strategies for patients with MDS/AML.

Citation Format: Macoura Gadji, Fábio Morato de Oliveira, Sabine Mai. Impact of nuclear telomere architecture in the transition of myelodysplastic syndromes to acute myeloid leukemias. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4206. doi:10.1158/1538-7445.AM2013-4206
Abstract 4210: An osteopontin promoter polymorphism is associated with aggressiveness in breast cancer.
Authors: Weber Georg F. , Ramchandani Divya
Published: August 2014

Background: Like cancer initiation, cancer progression and metastasis are genetically programmed. However, unlike oncogenes metastasis genes are deregulated in cancer by aberrant expression or splicing. We have previously described splicing of the metastasis gene osteopontin in cancers. Here we have analyzed polymorphic sites in the osteopontin promoter as potential contributors to aberrant expression in breast cancers.

Methods: This study comprised 241 breast cancer specimens, for 111 of which DNA from normal surrounding tissue was available, and 65 healthy breast samples. One probe set tested the polymorphic promoter sites -66, -443, -1748 and -1776. For comparison, we also investigated non-synonymous DNA sequence variations in the coding region positions 305, 367, 794 and 1025.

Results: The polymorphic sites in positions -66 and 305 were not in Hardy-Weinberg equilibrium and hence were not included in further analyses. The polymorphic site in position -443 of the promoter was associated with tumor grade. Expectedly, there was no association of the promoter SNPs with tumor stage or with in situ carcinoma versus cancer, as stage and early transformation are determined by the sampling time more than by tumor genetics. In a subset of samples, osteopontin expression levels had previously been obtained. The allelic distribution in positions -443 and -1748 was distinct between high and low expressors, confirming the importance of promoter SNPs. These two sites also form a haplotype. Osteopontin expression has been associated with breast cancer progression, regardless of the histologic subtype of the cancer. Remarkably, the polymorphic site at -443, but not -1748 or -1776, showed differences between ER positive and ER negative breast cancers and between PR positive and PR negative breast cancers, but there was no association with HER2 status. In five cases, the genotype of the tumor was different from the genotype of the host, implying the possibility of somatic mutations in the osteopontin promoter that may affect expression. The polymorphic sites in positions 367, 794, and 1025 showed one homozygous genotype for all specimens in this study.

Conclusion: Our results show that the osteopontin promoter SNPs -443 (rs11730582) and -1748 (rs2728127) are important for gene expression and tumor aggressiveness.

Citation Format: Georg F. Weber, Divya Ramchandani. An osteopontin promoter polymorphism is associated with aggressiveness in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4210. doi:10.1158/1538-7445.AM2013-4210
Abstract 422: Early growth response 1 is required for radiation-induced apoptosis in normal tissues.
Authors: Zhao Yi D. , Jacobs Keith M. , Karvas Rowan M. , Hallahan Dennis E. , Thotala Dinesh
Published: August 2014

Although radiation therapy is a mainstay of cancer treatments, it can have deleterious effects on normal tissues, leading to poor quality of life for cancer survivors. Radiation can cause depletion of stem cells in normal tissues that can result in late sequelae, including poor learning, cognitive disorders, intestinal malabsorption, infertility, and skin injuries. Our laboratory has shown that low doses of radiation can induce apoptosis selectively in undifferentiated normal stem cells compared to adjacent differentiated cells in multiple niches including the brain and intestine. To elucidate the molecular basis underlying stem cell sensitivity to radiation, we performed discovery research to detect genomic differences between stem cells and differentiated cells. Among the candidates, the Early growth response 1 (Egr1) gene was expressed at high levels in stem cells, but not in isogenic differentiated cells. Egr1 is a zinc-finger transcription factor that is rapidly induced in response to radiation and activates the transcription of pro-apoptotic genes. Since cancer survivors can suffer from cognitive difficulties and intestinal malabsorption, we investigated the role of Egr1 in neuronal and intestinal cell death following irradiation. Knockdown of Egr1 using shRNA increased clonogenic survival in irradiated HT-22 hippocampal neuronal cells and IEC-6 intestinal epithelial cells. This increased radioprotection was largely due to reduced apoptosis in Egr1 knockdown cells. Additionally, suppression of Egr1 promoter binding by mithramycin A and chromomycin A3 also conferred radioprotection to HT-22 and IEC-6 cells. Mouse models further supported Egr1’s role in radiation-induced apoptosis. Egr1 null mice had significantly fewer apoptotic cells in the hippocampus and small intestinal crypts following irradiation, as compared to their wild-type littermates. Finally, we examined mechanisms by which Egr1 regulates radiation-induced apoptosis. Irradiation of HT-22 and IEC-6 cells led to a G2/M cell cycle arrest, which was abrogated by the knockdown of Egr1. Also, knockdown of Egr1 by shRNA and inhibition of Egr1 promoter binding both attenuated radiation induction of the pro-apoptotic factors p53, Bim, and Bax, and upregulated the anti-apoptotic protein Bcl-2. Thus, this study establishes a crucial role for Egr1 in the radiation-induced apoptosis of hippocampal and intestinal tissues. Egr1 could be a potential molecular target to minimize the normal tissue complications associated with radiation therapy.

Citation Format: Yi D. Zhao, Keith M. Jacobs, Rowan M. Karvas, Dennis E. Hallahan, Dinesh Thotala. Early growth response 1 is required for radiation-induced apoptosis in normal tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 422. doi:10.1158/1538-7445.AM2013-422
Abstract 4222: Ion AmpliSeq Panel for the DNA sequencing of BRCA1 and BRCA2 genes on Ion Torrent PGM.
Authors: Costa Jose L. , Mensenkamp Arjen , Rico Alain , Liu Guoying , Petraroli Rosella , Ligtenberg Marjolijn , Machado Jose Carlos
Published: August 2014

The development of massive parallel sequencing (MPS) has revolutionized the field of genomics and created new avenues for basic research. The introduction of the benchtop sequencers enables the development of methodologies for genetic screening that are faster, more comprehensive and more cost-effective than conventional Sanger sequencing. In this study we describe a collaborative effort for the validation of a strategy for the screening of the breast cancer susceptibility genes- BRCA1 and BRCA2, making use of the most recent advances on Ion AmpliSeq™ multiplex PCR technology combined with Ion PGM™ System.

Strict criteria were set to the design of the amplification primers: 1) 100% coverage of all coding exons and exon-intron boundaries; 2) amplicons covering exons must be overlapping; 3) primers must not overlap; 4) no SNPs in the last five nucleotides of primer; and 5) maximum of three non-validated SNPs per primer. Constitutional genomic DNA of 50 BRCA1 or BRCA2 mutated cases are used. The cases are divided in two groups. The first 20 cases are used to test and optimize the methodology and are selected based on the presence of mutations in or within close proximity to homopolymer regions. The additional 30 cases are used for an independent and blind evaluation and power estimation of the new methodology. All samples are studied using both “gold standard” Sanger sequencing and Ion PGM System. For data-analysis various software packages are evaluated.

In the first 19 cases all SNPs and mutations, identified by Sanger sequencing were detected with our Ion AmpliSeq™ based approach. The disease-causing mutations identified included missense, nonsense and indel mutations. The number false positive results were always less than five per sample and reproducible. This work demonstrates the potential for mutational screening of BRCA1 and BRCA2 using the Ion AmpliSeq™ technology combined with the Ion Torrent PGM.

Citation Format: Jose L. Costa, Arjen Mensenkamp, Alain Rico, Guoying Liu, Rosella Petraroli, Marjolijn Ligtenberg, Jose Carlos Machado. Ion AmpliSeq Panel for the DNA sequencing of BRCA1 and BRCA2 genes on Ion Torrent PGM. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4222. doi:10.1158/1538-7445.AM2013-4222
Abstract 4223: Combining the CEE-selector assay with sequencing for sensitive mutation detection in cancer.
Authors: Alexiadis Vassilios , Watanaskul Tim , Arnold Lyle
Published: August 2014

We previously described the utility of the CEE-Selector assay for the sensitive detection of the EGFR T790M mutation in plasma samples from lung cancer patients. The assay combined the CEE-Selector assay with melt curve analysis of the mutant PCR product, followed by Sanger sequencing to verify the presence of the mutation. We showed that the assay selectively amplified the mutant sequence compared to wild type by >100,000 fold. We have now expanded the CEE-Selector assay to include additional mutations relevant to patient care (KRAS, BRAF V600E, EGFR L858R). We are presenting data of allele/mutation-specific as well as “footprint” versions of the CEE-Selector assay. The “footprint” version of the assay provides for the interrogation of “hot spot” regions of the genome where multiple cancer relevant mutations are located within a small region, as in the case of KRAS codons 12 and 13 where the CEE-Selector assay is sensitive to all known mutations. Combining the “footprint” version of the CEE-Selector assay with next-generation sequencing, allows us to simultaneously detect, with extremely high sensitivity, a broad array of mutations of interest. Additionally, when used with barcoding, samples from multiple patients can be interrogated simultaneously. The combination of the CEE-Selector assay with next-generation sequencing increases the sensitivity of sequencing by a factor of >1000 and allows for simultaneous sensitive detection of multiple mutations from complex samples, like plasma, where the mutations may be very rare. With the increases in sensitivity made possible using the CEE-Selector assay, the limiting factor for the sensitive detection of mutations/alleles is determined fundamentally by the ability to obtain at least a single copy of the mutant allele within the sample under interrogation.

Citation Format: Vassilios Alexiadis, Tim Watanaskul, Lyle Arnold. Combining the CEE-selector assay with sequencing for sensitive mutation detection in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4223. doi:10.1158/1538-7445.AM2013-4223
Abstract 4224: A quick and easy way of assembling TALE/TALEN modules for genetic modifications in mammalian cells .
Authors: Zhang Juan , Mo Yin-Yuan
Published: August 2014

The transcription activator-like effectors (TALEs) are a family of virulence factors produced by plant bacterial pathogens, and they are able to regulate expression of specific host genes by binding to the corresponding promoter regions in a sequence-specific manner. The transcription activator-like effector nuclease (TALEN) is made by fusing a nuclease to the DNA binding modules of TALE, which would allow for generation of DNA deletions, knockouts or knockins. Since their discovery, genes from different organisms have been successfully targeted using designer TALEs through assembling sequence specific modules, which is a critical step to make TALE/TALEN vectors. However, current methods of assembling DNA binding modules, such as the golden-gate ligation, are still time consuming and labor intensive. Moreover, they are also technically challenging. In the present study, we adopted a single step approach to assemble TALE modules. For proof of principle, we used p53 binding site (Tgacttgcatgtacatgcc) derived from the lncRNA-RoR promoter for proof of principle. First, we designed four gBlock DNA oligos which carried 30 bp overlapping between two adjacent oligos. Then, we linked them together by PCR and subsequently directly cloned into an expression vector which carries two flanking TALE sequences. To determine the function of this construct, we generated a reporter vector carrying this p53 binding site and mCherry and showed that this newly made construct was able to activate the mCherry reporter. Experiments are underway to test if the p53 specific TALE is able to activate the endogenous lncRNA-RoR.

Citation Format: Juan Zhang, Yin-Yuan Mo. A quick and easy way of assembling TALE/TALEN modules for genetic modifications in mammalian cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2013-4224
Abstract 4227: Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE).
Authors: Richardson Katherine A. , Wu Grant , Lin Rui , Shi Yanggu , Eastlake Phil , Legendre Benjamin
Published: August 2014

Transgenomic, Inc. has developed a size-based analysis for the detection of all EGFR Exon 19 deletions (including complex mutation) greater than 6 base pairs (ACE™ Kit EGFR Exon 19). Currently there are over 70 known EGFR Exon 19 deletions in the region important for response of non-small cell lung cancer (NSCLC) to thymidine kinase inhibitors (TKIs). The majority of these deletions are rare, but the patients may benefit from TKI treatment if the deletion is present. In most current testing paradigms, a NSCLC patient with one of these rare mutations would be considered EGFR mutation negative and not receive TKI therapy. We have designed an easy, size-based PCR assay to detect any of the deletions present in any sample after a single PCR reaction. The assay is based on the separation of PCR fragments of different sizes using microcapillary electrophyoresis (MCE). Transgenomic's WAVE® MCE platform can discriminate the deletions from the wild-type sequence in EGFR Exon 19, however the exact nature of the mutation cannot be determined. If the exact nature of the mutation is required, the PCR fragment can be sequenced.

To confirm that the platform can detect common and rare mutations, plasmids containing various EGFR Exon 19 deletions were synthesized, mixed with EGFR wild-type plasmids and analyzed on the WAVE® MCE platform. The Limit of detection for the plasmid mixing experiments is between 0.5 and 1% for all plasmid constructs tested. These plasmids include 6, 9, 15, 21, 24, and 25 bp deletions . As all samples tested have some wild-type sequence present, 6 base pair deletions cannot be resolved from the wild-type fragment. Several of the deletion constructs contain those not detected by any allele-specific kit, however patients with these deletions, as determined by Sanger sequence analysis, have benefitted from TKI therapy. In addition cell line DNA and DNA isolated from FFPE sections have been analyzed for the detection of EGFR Exon 19 deletions and similar limits of detection (0.5-1.0%) were seen.

This simple, size-based analysis for EGFR Exon deletions will result in NSCLC patients with both common and rare EGFR Exon 19 deletions being able to receive the treatment option best suited for their molecular profile.

Citation Format: Katherine A. Richardson, Grant Wu, Rui Lin, Yanggu Shi, Phil Eastlake, Benjamin Legendre. Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4227. doi:10.1158/1538-7445.AM2013-4227