bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2304: Biologically distinct mammary tumors develop from Trp53 null tissue transplanted to juvenile versus adult gland.
Authors: Nguyen David H. , Mao Jian-Hua , Barcellos-Hoff Mary Helen
Published: August 2014

The biology of the organism is a key regulator of breast cancer development. Our previous work showed that irradiating the host accelerated the development of carcinomas from unirradiated Trp53 null mammary fragments transplanted into the inguinal fat pads previously divested of endogenous epithelia (Nguyen et al., Cancer Cell, 2011). In this report, we investigated the age of transplantation by comparing carcinogenesis in Trp53 null mammary outgrowths that were transplanted during pubertal, 5 week-old mice compared to 10 week-old adult hosts. Tumors arising over the course of a year from Trp53 null tissue transplanted into adult hosts (AH) (n=26), were compared to tumors arising following transplantation in juvenile hosts (JH) (n=55). The rate of tumor occurrence was not significantly different as a function of the transplantation age but once detected, JH tumors grew several times faster. JH tumors were more perfused, as indicated by 2-fold larger blood vessels (p=0.015), and exhibited a 2-fold higher mitotic index (p=0.009) compared to AH tumors. Tumors transplanted at either age were predominantly ER+ (80% JH vs. 60% AH), but more tumor cells were ER immunoreactive as measured by the Allred score in JH tumors compared to AH tumors (p=0.0001). Microarray analysis of 10 JH and 6 AH tumors identified 119 genes that were modulated by at least 1.5-fold in JH tumors. Main biological processes enriched in JH tumors were organ morphology, proliferation, and lipid metabolism. The resulting profile revealed a luminal transcriptional program in JH tumors that included expression of Foxa1 and Areg; Foxa1 protein expression in JH tumors was confirmed by immunohistochemistry. The distinct biology of tumors arising from tissue undergoing morphogenesis in the context of puberty suggests that: (1) the window of susceptibility for breast cancer involves host biology during puberty; and (2) that the tumor intrinsic subtype is not necessarily epithelial autonomous, but can be influenced by the host environment. Supported by DOD-BCRP and DOE Low Dose Radiation Program.

Citation Format: David H. Nguyen, Jian-Hua Mao, Mary Helen Barcellos-Hoff. Biologically distinct mammary tumors develop from Trp53 null tissue transplanted to juvenile versus adult gland. [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 2304. doi:10.1158/1538-7445.AM2013-2304
Abstract 2309: Identification and targeting of small noncoding RNAs implicated in tumor angiogenesis.
Authors: Mellick Albert S. , Plummer Prue N. , Mittal Vivek
Published: August 2014

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) contribute to angiogenesis-mediated tumor growth. In previous studies[1], we showed that EPCs can be tracked from the BM to sites throughout the body, including the BM compartment of the tumor stroma. We also demonstrated that the Inhibitor of DNA binding 1 (ID1) proximal promoter can be used to silence genes in EPCs, through directed delivery of short hairpin RNA inhibition. Here we report the elucidation of a novel small RNA pathway regulated by miR-10b, required for regulation of EPC function and tumor angiogenesis. We demonstrate that this pathway is required for tumor vascular biology and through in situ hybridization (ISH) that it is preferentially up-regulated in the vasculature of aggressive late stage Infiltrating Ductal Carcinomas, and not in more contained Ductal Carcinomas. Such analysis reveals the clinical implications of in vivo targeting of miRNAs, such as miR-10b in cancer therapeutics, and reveals the need for cell specific localisation of miRNA expression in understanding tumor malignancy.[2]

1. Mellick AS, Plummer P, Nolan D, et al. Using Transcription Factor Id1 to Selectively Target Endothelial Progenitor Cells Offers Novel Strategies to Inhibit Tumor Angiogenesis and Growth. Cancer Res. 70:7273-82, 2010.

2. Plummer PN, Freeman R, Taft R, Vider J, Sax M, Umer BA, Gao D, Johns CA, Mattick JS, Wilton SD, Ferro V, McMillan NA, Swarbrick A, Mittal V, Mellick AS. MicroRNAs regulate tumor angiogenesis modulated by endothelial progenitor cells. Cancer Res. 2012 Jul 25. [Epub ahead of print].

Citation Format: Albert S. Mellick, Prue N. Plummer, Vivek Mittal. Identification and targeting of small noncoding RNAs implicated in tumor angiogenesis. [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 2309. doi:10.1158/1538-7445.AM2013-2309
Abstract 2315: Reversible disruption of mSWI/SNF-like BAF complexes by the SS18-SSX oncogenic fusion in synovial sarcoma.
Authors: Kadoch Cigall , Crabtree Gerald R.
Published: August 2014

Recent exon sequencing studies have revealed frequent mutations in subunits of SWI/SNF or BAF complexes and implicated them as tumor suppressors. Indeed, we estimate that they are mutated in 15.8 percent of sequenced human cancers. Interestingly, the subunits most commonly mutated are not required for chromatin remodeling in vitro and thus may contribute to chromatin regulation in a novel way. One difficultly in understanding the role of these complexes in cancer is distinguishing primary driving mutations from passenger mutations. To investigate the underlying mechanism we studied human synovial sarcoma (SS), in which transformation results from a remarkably precise and uniform translocation of exactly 78 amino acids of SSX to the SS18 protein. In a proteomic study we discovered that SS18 is a dedicated, non-exchangeable subunit of BAF complexes. We demonstrate that the hallmark SS18-SSX fusion incorporates into BAF complexes, evicting both the wild-type SS18 protein and the tumor suppressor, hSNF5 (BAF47). The altered complex binds the Sox2 locus, reversing polycomb-mediated repression and results in Sox2 activation. Sox2 is uniformly expressed in SS tumors and is essential for their proliferation. Increasing the concentration of wild-type SS18 leads to reassembly of the wild-type complex, retargeting of BAF complexes away from the Sox2 locus, polycomb-mediated repression of Sox2 and cessation of proliferation. This mechanism of transformation depends on only two amino acids of SSX and hence provides a potential foundation for therapeutic intervention.

Citation Format: Cigall Kadoch, Gerald R. Crabtree. Reversible disruption of mSWI/SNF-like BAF complexes by the SS18-SSX oncogenic fusion in synovial sarcoma. [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 2315. doi:10.1158/1538-7445.AM2013-2315
Abstract 2320: Oncogenic K-ras and loss of Smad4 mediate invasion by activating a NF-kappaB/EGFR axis that induces expression of MMP9/uPA in human pancreas progenitor cells.
Authors: Bera Alakesh , Zhao Shujie , Cao Lin , Freeman James W.
Published: August 2014

Pancreatic ductal adenocarcinoma (PDAC) is an extremely fatal and highly metastatic in nature. Several pathways and mutations contribute to the development and pathogenesis of PDAC. Activating K-ras mutations and inactivating mutations of Smad4 are two common genetic alterations that occur in the development and progression of PDAC. To further study the role of these two mutations in the pathogenesis of PDAC, immortalized human pancreas nestin postive cells (HPNE) were genetically modified by expressing oncogenic K-ras (GD12) and /or by shRNA knock down of Smad4. Present studies show that these two mutations caused increase in expression of EGFR and erbB2 growth factor receptors and increased expression and nuclear translocation of NF-κB. The increase in EGFR expression and nuclear translocation of NF-κB was associated with development of an invasive phenotype as measured by matrigel assays.

Our data also demonstrated that MMP9 and uPA were substantially up regulated in HPNE cells that express K-ras or along with loss of Smad4 activities. MMP-9 and uPA are two extracellular matrix (ECM)-degrading proteinases known to play important roles in driving invasion and metastasis. Zymography studies confirm the expression and enzymatic activities of uPA and MMP-9 in the progenitor cells. The mechanism underlying this uPA/MMP9 induction is linked with NF-κB activation. Immuno-fluorescence, Western blot and chromatin immuno-precipitation (ChIP) analysis confirm the nuclear translocation of NF-κB RelA (p65) sub-unit, by induction of K-ras. Following signaling cascade, EGFR activated by the K-ras mediated NF-κB nuclear localization and further activation induced by loss of Smad4. Our results also suggest that the EGFR pathway as a critical mediator connecting Ras to NF-κB in proteolytic enzyme secretion as well as cancer invasion.

In conclusion, the present study reveals a new insight in pancreatic progenitor cell invasion which includes signal transduction of K-ras activity and Smad4 inactivation in regulation of NF-κB/EGFR axis to induce expression of uPA and MMP9. Therefore, induction of K-ras and loss of Smad4 could play important roles in PDAC invasion through NF-κB/EGFR activation and subsequent induced expression of MMP9 and uPA, and might be suitable molecular candidates for the development of new therapeutic strategies for pancreatic cancer.

Citation Format: Alakesh Bera, Shujie Zhao, Lin Cao, James W. Freeman. Oncogenic K-ras and loss of Smad4 mediate invasion by activating a NF-kappaB/EGFR axis that induces expression of MMP9/uPA in human pancreas progenitor 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 2320. doi:10.1158/1538-7445.AM2013-2320
Abstract 2322: The KIAA1549:BRAF fusion gene regulates mTOR signaling and gliomagenesis in a cell type- and brain region-specific manner.
Authors: Kaul Aparna , Chen Yi-Hsien , Emnett Ryan J. , Dahiya Sonika , Gutmann David
Published: August 2014

The two most common genetic alterations in low-grade pediatric brain tumors (glioma) are NF1 gene inactivation and tandem duplications involving the BRAF kinase gene. In this study, we sought to define the role of KIAA1549:BRAF fusion (fusion BRAF; f-BRAF) gene in glioma development. We demonstrate that f-BRAF regulates cerebellar NSC proliferation by activating the mTOR pathway downstream of MAPK. Also, f-BRAF expression in cerebellar neuroglial progenitors is sufficient to induce glioma-like lesions in mice. Interestingly, f-BRAF expression does not increase the proliferation of NSCs derived from other brain regions, nor does it increase primary astrocyte proliferation. These brain region- and cell type-specific growth promoting effects of f-BRAF are mediated by tuberin/Rheb-dependent modulation of mTOR, leading to S6-kinase activation and p27 degradation. Finally, we demonstrate that mTOR activation is also observed in fusion BRAF-associated human sporadic pilocytic astrocytomas. Our findings highlight the importance of spatial, cellular and molecular heterogeneity in brain tumor development, and provide compelling preclinical evidence for the use of mTOR pathway inhibitors for both sporadic and familial low-grade gliomas.

Citation Format: Aparna Kaul, Yi-Hsien Chen, Ryan J. Emnett, Sonika Dahiya, David Gutmann. The KIAA1549:BRAF fusion gene regulates mTOR signaling and gliomagenesis in a cell type- and brain region-specific manner. [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 2322. doi:10.1158/1538-7445.AM2013-2322
Abstract 233: Therapeutic efficacy of cancer stem cell-based vaccine in an adjuvant setting.
Authors: Lu Lin , Tao Huimin , Egenti Martin , Wicha Max S. , Chang Alfred E. , Li Qiao
Published: August 2014

So far, the majority of cancer stem cell (CSC) studies have been confined to human tumors inoculated into severely immunosuppressed hosts (e.g. SCID mice), where adaptive immune responses are absent, and such hosts are therefore not suitable for the immunologic evaluation of CSCs. The lack of immunocompetent syngeneic animal tumor models where stem cells can be isolated has been the primary obstacle to evaluating the immunogenicity of CSCs. We recently reported the identification of CSC-enriched populations using ALDEFLUOR/ALDH as a marker in murine melanoma D5 and squamous cell cancer SCC7, and evaluated their immunogenicity in two genetically distinct syngeneic immunocompetent hosts. Enriched CSCs are immunogenic and significantly more effective as an antigen source in inducing protective anti-tumor immunity than unsorted tumor cells or purified non-CSCs. We further found that selective targeting of CSCs by CSC-primed antibodies and T cells represents the mechanisms involved in CSC vaccine-conferred protective immunity. If a CSC vaccine is to be clinically relevant, it needs to be evaluated in a therapeutic model. In this study, we examined two models for this purpose. The first model involves the treatment of micrometastatic disease. We initiated vaccination 24 hrs after s.c. inoculation of D5 tumor cells in the syngeneic immunocompetent host (B6 mice), and repeated the vaccination one week later. Comparison was made among the group with PBS-injected controls; mice vaccinated with dendritic cells (DC) pulsed with the lysate of ALDHhigh D5 cells (CSC-TPDC) vs. ALDHlow D5 cells (ALDHlow -TPDC). We found that CSC-TPDC inhibited the tumor growth more than ALDHlow-TPDC and PBS controls, and significantly prolonged the survival of the tumor-bearing mice. The second model involves the treatment of established tumors using CSC-TPDC vaccine as an additional strategy to radiation therapy (RT). Day 7 D5 sc tumors were treated with localized RT with repeat treatments on D8. Vaccine therapy commenced on D8. This combination therapy was repeated twice with one week apart. CSC-TPDC plus RT mediated D5 tumor regression more than ALDHlow-TPDC + RT, RT alone and PBS controls, and prolonged the survival of the tumor-bearing mice. Importantly, we observed metastasis of the D5 tumors to the lung in all the groups except for the one treated with CSC-TPDC plus RT. The findings may help develop novel approaches for cancer treatment by utilizing an autologous cancer stem cell-based vaccine to selectively and immunologically target cancer stem cells.

Citation Format: Lin Lu, Huimin Tao, Martin Egenti, Max S. Wicha, Alfred E. Chang, Qiao Li. Therapeutic efficacy of cancer stem cell-based vaccine in an adjuvant setting. [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 233. doi:10.1158/1538-7445.AM2013-233
Abstract 2332: Oncogenic MYD88 mutants require Toll-like receptors.
Authors: Lim Kian-Huat , Barton Gregory M. , Staudt Louis M.
Published: August 2014

Oncogenic mutations affecting the signaling adapter MyD88 are present in 39% of activated B-cell-like (ABC) diffuse large B cell lymphomas (DLBCLs), with the most recurrent MyD88 mutant, L265P, present in 29% of cases. MYD88 mutations constitutively activate multiple signaling cascades that promote the survival and proliferation of ABC DLBCLs, including the NF-B, type-1 interferon, p38 and JAK/STAT pathways. It is unknown whether MyD88 mutants act autonomously or require a receptor to engage these downstream pathways. Here we show that the MyD88 L265P oncoprotein binds constitutively to two members of the Toll-like receptor family, TLR7 and TLR9, thereby amplifying signals that emanate from these receptors. Knockdown of TLR7 or TLR9 potently suppressed NF-B activity in ABC DLBCL cell lines and promoted apoptosis. Depletion of known proteins essential for TLR7 and TLR9 trafficking and signaling, including UNC93B1, PRAT4A or CD14, was lethal for ABC DLBCL lines. Similarly, pharmacologic inhibition of TLR7 and TLR9 function in lysosomal compartments using the cathepsin inhibitor zFA-fmk or hydroxychloroquine diminished MyD88 signaling and reduced survival of ABC DLBCL lines. TLR7 or TLR9 mutants that are defective in ligand binding failed to promote the survival of ABC DLBCL lines following knockdown of endogenous TLR7 or TLR9 expression in ABC DLBCL lines. Moreover, an inhibitory oligonucleotide capable of suppressing TLR9 responses in normal B cells blocked NF-B signaling and survival of ABC DLBCL lines. These new insights into the oncogenic mechanisms of MyD88 mutants provide a rationale for targeting of TLR7 and TLR9 signaling for the therapy of ABC DLBCL.

Citation Format: Kian-Huat Lim, Gregory M. Barton, Louis M. Staudt. Oncogenic MYD88 mutants require Toll-like receptors. [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 2332. doi:10.1158/1538-7445.AM2013-2332
Abstract 2340: Preferential targeting of KRAS mutant or BRAF mutant tumors using MEK inhibitors with distinct mechanisms of action.
Authors: Hatzivassiliou Georgia , Haling Jacob , Chen Huifen , Song Kyung , Luoh Shiuh-Ming , Price Steve , Heald Robert , Zak Mark , Merchant Mark , Hoeflich Klaus , Friedman Lori , Malek Shiva , Belvin Marcia P.
Published: August 2014

KRAS and BRAF mutant cancers represent an area of high unmet medical need in oncology. Multiple allosteric MEK inhibitors are being tested in clinical trials in BRAF mutant and KRAS mutant cancers, where to date they have demonstrated impressive single agent activity in BRAF mutant melanoma, but not in KRAS mutant disease. Active, phosphorylated MEK is elevated in BRAFV600E mutant melanoma tumor models compared to KRAS mutant tumor models, in line with evidence that MAPK pathway regulation differs depending on the upstream oncogenic lesion. However, the precise differences in MEK activation by mutant KRAS or BRAF and how to create MEK inhibitors to block that activation are not known. In this study we describe two clinical, allosteric MEK inhibitors, GDC-0973 and GDC-0623, that have complementary efficacy profiles, with one showing superior efficacy in BRAF mutant models and the other showing superior efficacy in KRAS mutant models. Using structural information and targeted mutagenesis, we demonstrate that although both inhibitors effectively block MEK kinase activity and downstream ERK activation, their unique binding modes result in differential effects on active versus inactive MEK and MEK interaction with RAF that translates into unique efficacy profiles in BRAFV600E versus KRAS mutant tumors. GDC-0973 has already shown promising efficacy as a single agent in BRAFV600E mutant melanoma, and both inhibitors are currently being tested in KRAS mutant tumors in the clinic. Our study highlights that subtle differences in the binding mode of kinase inhibitors can have a large impact on inhibitor/target interactions and efficacy through allosteric mechanisms. These differences may impact the therapeutic index of MEK inhibitors in either BRAF mutant or KRAS mutant cancers.

Citation Format: Georgia Hatzivassiliou, Jacob Haling, Huifen Chen, Kyung Song, Shiuh-Ming Luoh, Steve Price, Robert Heald, Mark Zak, Mark Merchant, Klaus Hoeflich, Lori Friedman, Shiva Malek, Marcia P. Belvin. Preferential targeting of KRAS mutant or BRAF mutant tumors using MEK inhibitors with distinct mechanisms of action. [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 2340. doi:10.1158/1538-7445.AM2013-2340
Abstract 2345: Combined inhibition of protein kinase C and MEK is a rational therapeutic approach for melanomas with GNAQ or GNA11 mutations.
Authors: Chen Xu , Wu Qiuxia , Bastian Boris C.
Published: August 2014

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults. There currently is no effective treatment for metastatic UM. UM differs from cutaneous melanoma in that it lacks BRAF, NRAS, or KIT mutations but instead harbors mutations in the Gαq family members GNAQ and GNA11 in over 80 to 90% of cases. Understanding the downstream effector pathways of these oncoproteins is of considerable interest to identify opportunities for targeted therapy. Using 293T cells, immortalized mouse and human melanocytes, we find that GNAQQ209L or GNA11Q209L induce activation of the protein kinase C (PKC) and MAPK pathways. ShRNA mediated knock-down of mutant GNAQ abrogates both PKC and MAPK activation in human melanoma cell lines or model systems. Using two different PKC inhibitors we found selective extinction of PKC and MAPK signalling and induction of G1 arrest in panels of melanoma cell lines carrying GNAQ or GNA11 mutations, whereas no such effect was observed in melanoma cell lines with other mutations. MAPK activation occurred downstream of activated PKC in cells with GNAQ or GNA11 mutations. In contrast to PKC inhibitors, which selectively inhibited the growth of melanoma cell lines with GNAQ or GNA11 mutations, the MEK inhibitor PD0325901 inhibited melanoma cell lines irrespective of their GNAQ/11 mutation status. In vivo the PKC inhibitor AEB071 slowed the growth of tumors in an allograft model of GNAQQ209L transduced melanocytes, but did not induce tumor shrinkage. Analysis of tumor lysates identified suppression of PKC signaling but not of MAP-kinase pathway signaling under chronic PKC inhibition. The rebound of MAP-kinase activation could be overcome using a combination of AEB071 with the MEK inhibitor PD0325901, which showed strong synergy in halting proliferation and in inducing apoptosis. Our data suggest that PKC is a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and indicate that a combination therapy with MEK inhibitor is superior to treatment with either approach alone.

Citation Format: Xu Chen, Qiuxia Wu, Boris C. Bastian. Combined inhibition of protein kinase C and MEK is a rational therapeutic approach for melanomas with GNAQ or GNA11 mutations. [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 2345. doi:10.1158/1538-7445.AM2013-2345
Abstract 2350: Discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design.
Authors: Lee Taekyu , Friberg Anders , Vigil Dominico , Zhao Bin , Daniels R. Nathan , Burke Jason P. , Garcia-Barrantes Pedro M. , Camper DeMarco , Chauder Brian A. , Olejniczak Edward T. , Fesik Stephen W.
Published: August 2014

Myeloid cell leukemia 1 (Mcl-1), a member of the Bcl-2 family of proteins, is overexpressed and amplified in various cancers and promotes the aberrant survival of tumor cells that otherwise would undergo apoptosis. Overexpression of Mcl-1 is also a resistance mechanism that prevents tumor cells from being effectively treated by existing chemotherapies. Thus, inhibition of Mcl-1 is a promising therapeutic strategy for the treatment of cancer. However, Mcl-1 exerts its effects through protein-protein interactions and is thought to be very challenging to target with small molecules. Here we describe the discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. From an NMR-based screen of a large fragment library, over 130 hits were identified. Two distinct chemical hit series were found that bind to two different sites on Mcl-1 as revealed by NMR-derived model structures. Members of the two fragment classes were merged together to produce compounds that bind to Mcl-1 with greater than two orders of magnitude improved binding affinity. Structures of these compounds when complexed to Mcl-1 were obtained by X-ray crystallography and provide detailed information about the molecular recognition involved in small-molecule binding to Mcl-1. Lead compounds exhibited a dissociation constant of <100 nM, with selectivity for Mcl-1 over Bcl-xL and Bcl-2. These compounds represent useful starting points for the discovery of clinically useful Mcl-1 inhibitors for the treatment of a wide variety of cancers.

Citation Format: Taekyu Lee, Anders Friberg, Dominico Vigil, Bin Zhao, R. Nathan Daniels, Jason P. Burke, Pedro M. Garcia-Barrantes, DeMarco Camper, Brian A. Chauder, Edward T. Olejniczak, Stephen W. Fesik. Discovery of potent and selective Mcl-1 inhibitors using fragment-based methods and structure-based design. [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 2350. doi:10.1158/1538-7445.AM2013-2350
Abstract 2355: Telomere length of tumor tissues and survival in patients with early stage non-small cell lung cancer .
Authors: Song Ji Woong , Jeon Hyo-Sung , Lee Eung Bae , Lee Shin Yup , Park Jae Yong
Published: August 2014

Telomere shortening leads to genomic instability that drives oncogenesis through the activation of telomerase and the generation of other mutations necessary for tumor progression. This study was conducted to determine the impact of telomere shortening on the survival of patients with early stage non-small cell lung cancer (NSCLC).

Relative telomere length in tumor tissues was measured by quantitative polymerase chain reaction in 164 patients with surgically-resected NSCLC. The association between telomere length and overall survival (OS) and disease-free survival (DFS) was analyzed.

When the patients were categorized into quartiles based on telomere length, those patients with the 1st quartile (shortest) of telomere length had a significantly worse OS and DFS compared to patients with the 2nd to the 4th quartiles of telomere length (adjusted hazard ratio for OS =

2.67, 95% confidence interval = 1.50-4.75, P = 0.001; and adjusted hazard ratio for

DFS = 1.92, 95% confidence interval = 1.17-3.14, P = 0.01). An association between telomere length and survival outcome was more pronounced in squamous cell carcinomas than adenocarcinomas (P-value of test for homogeneity for OS and DFS = 0.05 and 0.02, respectively).

Telomere length of tumor tissues is an independent prognostic factor in patients with surgically resected early stage NSCLC.

Citation Format: Ji Woong Song, Hyo-Sung Jeon, Eung Bae Lee, Shin Yup Lee, Jae Yong Park. Telomere length of tumor tissues and survival in patients with early stage 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 2355. doi:10.1158/1538-7445.AM2013-2355
Abstract 2359: Characterization of a lung cancer growth factor, LASEP3, as a serological and prognostic biomarker and a therapeutic target.
Authors: Takano Atsushi , Nakamura Yusuke , Daigo Yataro
Published: August 2014

To identify novel biomarkers or therapeutic targets for lung cancers, we have been screening genes encoding transmembrane/secretory proteins that are up-regulated in lung cancers, using cDNA microarrays. During this process, we identified a secreted protein, LASEP3 (lung cancer-associated serum protein 3) as a candidate.

Immunohistochemical analysis showed that strong positive staining of LASEP3 was observed in 198 (54.8%) of 361 NSCLCs (non-small cell lung cancer).

High level of LASEP3 expression was associated with poor prognosis of NSCLC patients. (P=0.0183 by log-rank test). Serum LASEP3 levels were higher in NSCLC patients than in healthy volunteers. The proportion of serum LASEP3-positive cases was 160 (61.8%) of 259 NSCLCs, while 6 (5.5%) of 109 healthy volunteers were falsely diagnosed. Moreover serum LASEP3 levels were significantly higher in breast and colon cancer patients than in healthy volunteers.

A combined assay using both LASEP3 and CEA increased sensitivity because 79.5% of the NSCLC patients were then diagnosed as positive, whereas only 7.3% of 109 healthy volunteers were falsely diagnosed as positive. In addition, the use of both LASEP3 and proGRP increased sensitivity because 78.3% of the lung SCLC patients were diagnosed as positive, whereas only 6.6% of 121 healthy volunteers were falsely diagnosed. LASEP3 protein was overexpressed in breast, cervical and colon cancer tissues. Furthermore, treatment of lung cancer cells with

LASEP3-siRNAs suppressed its expression and suppressed cell growth and invasion.

To clarify the mechanism of tumor suppression by siRNAs against LASEP3, we performed flow cytometric analysis of the tumor cells transfected with these siRNAs. We found a significant increase of the cells at the G0/G1 phase. These data suggest that LASEP3 is a diagnostic and prognostic biomarker and therapeutic target for lung and various type of human cancer.

Citation Format: Atsushi Takano, Yusuke Nakamura, Yataro Daigo. Characterization of a lung cancer growth factor, LASEP3, as a serological and prognostic biomarker and a therapeutic target. [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 2359. doi:10.1158/1538-7445.AM2013-2359

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2364: Long non-coding RNA HOTAIR is expressed in small cell lung cancer and relevant to cellular proliferation, invasiveness and clinical relapse - analyses based on tumor tissues and cell lines.
Authors: Ono Hiroshi , Miyauchi Eisaku , Motoi Noriko , Ushijima Masaru , Matsuura Masaaki , Okumura Sakae , Nishio Makoto , Hirose Tetsuro , Itou Satoru , Fujiwara Mutsunori , Inase Naohiko , Ishikawa Yuichi
Published: August 2014

Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis, showing early relapse after initial fair response to chemotherapy. A long non-coding RNA, Hox transcript antisense intergenic RNA (HOTAIR) has been reported to be involved in breast and colon cancer metastasis. Here we revealed, using 35 surgical SCLC cases, that HOTAIR was expressed highly in pure, rather than combined, SCLC (p=0.012) and that a subset of SCLC with its higher expression tended to relapse earlier (p=0.086). Also, knock down of HOTAIR by siRNA transfection in an SCLC cell line, SBC-3, led to the reduced proliferation activity and invasiveness, due to changes of expression of cell adhesion-related genes. These findings suggest that the HOTAIR expression changes gene expression in relation to cell adhesion and subsequently alters proliferation activity and invasiveness of tumor cells, resulting in early relapse of SCLC. Our results imply that HOTAIR could be a prognostic biomarker and a therapeutic target of SCLC.

Citation Format: Hiroshi Ono, Eisaku Miyauchi, Noriko Motoi, Masaru Ushijima, Masaaki Matsuura, Sakae Okumura, Makoto Nishio, Tetsuro Hirose, Satoru Itou, Mutsunori Fujiwara, Naohiko Inase, Yuichi Ishikawa. Long non-coding RNA HOTAIR is expressed in small cell lung cancer and relevant to cellular proliferation, invasiveness and clinical relapse - analyses based on tumor tissues and cell lines. [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 2364. doi:10.1158/1538-7445.AM2013-2364
Abstract 2369: Pharmacogenomic assessment of outcomes of pemetrexed-treated patients with adenocarcinoma of the lung.
Authors: Jung Minkyu , Lee Chul Ho , Park Hyung Soon , Lee Ji Hyun , Kim Dae Joon , Lim Sun Min , Kim Joo Hang , Cho Byoung Chul
Published: August 2014

Purpose: The main objective of this study was to evaluate the association between polymorphisms of the target genes of pemetrexed and clinical outcomes in non-small cell lung cancer (NSCLC) patients treated with pemetrexed. Materials and Methods: We assessed polymorphisms at 8 sites in 4 genes (thymidylate synthase (TS), dihydrofolate reductase (DHFR) [1610, 680, 317, intron 1], methylenetetrahydrofolate reductase (MTHFR) [677, 1298], glycinamide ribonucleotide formyl transferase (GARFT) [2255]) associated with pemetrexed metabolism using polymerase chain reaction, gene scanning, and restriction fragment length polymorphism analysis in 90 patients with adenocarcinoma of the lung. Results: Survival was significantly longer with pemetrexed in patients with TS 3RGCC/3RGCC or 3RGGC/3RGGC compared with the other groups (PFS; 5.2 months vs. 3.7 months, p=0.03: OS; 31.8 months vs. 18.5 months, p=0.001). Patients with DHFR 680CC experienced fatigue more frequently (50% vs. 8.6%, p=0.008). Polymorphisms of MTHFR and GARFT were not significantly associated with clinical outcomes with of pemetrexed. Conclusions: The TS genotype was associated with survival and one DHFR polymorphism was associated with fatigue in NSCLC patients treated with pemetrexed. Further large prospective studies are required to identify other biomarkers that affect patients being treated with pemetrexed for adenocarcinoma of the lung.

Citation Format: Minkyu Jung, Chul Ho Lee, Hyung Soon Park, Ji Hyun Lee, Dae Joon Kim, Sun Min Lim, Joo Hang Kim, Byoung Chul Cho. Pharmacogenomic assessment of outcomes of pemetrexed-treated patients with adenocarcinoma of the lung. [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 2369. doi:10.1158/1538-7445.AM2013-2369

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2380: Reduced CD44 and OTP gene expression are strong indicators of poor prognosis in pulmonary carcinoids.
Authors: Swarts Dorian RA , Henfling Mieke , van Neste Leander , van Suylen Robert J. , Dingemans Anne-Marie , Dinjens Winand NM , Haesevoets Annick , Rudelius Martina , Thunnissen Erik , Volante Marco , van Criekinge Wim , van Engeland Manon , Ramaekers Frans CS , Speel Ernst-Jan M.
Published: August 2014

Purpose: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a favorable prognosis. However, there is a risk for late recurrence and/or distant metastasis. Because histological classification in typical and atypical (AC) carcinoids is difficult and its reliability to predict disease outcome varies, we evaluated three genes as potential prognostic markers, i.e., OTP, CD44 and RET.

Methods: These genes were analyzed in 56 frozen carcinoids by quantitative RT-PCR. RET was further studied by methylation and mutation analysis. Immunohistochemistry for CD44 and OTP protein expression was performed on 292 carcinoids.

Results: Low mRNA expression levels of CD44 (p=1.3e-5) and OTP (p=0.012), and high levels of RET (p=0.0025), were strongly associated with a low 20-year survival of carcinoid patients. High RET expression was not related to promoter hypomethylation or gene mutations. A direct link between gene expression and protein levels was confirmed for CD44 and OTP, but not for RET. Within all carcinoids as well as ACs, absence of CD44 protein was significantly associated with low 20-year survival (p=8.6e-5 and p=0.00015, respectively). The absence of nuclear OTP followed by complete loss of expression was also significantly associated with unfavorable disease outcome in all carcinoids (p=2.9e-5). Multivariate analysis revealed that CD44 and OTP immunostaining combined with histopathology is the optimal indicator for patient outcome.

Conclusions: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We therefore argue for implementation of these markers in routine diagnostics in addition to histopathology to improve subclassification of pulmonary carcinoids into prognostically relevant categories.

Citation Format: Dorian RA Swarts, Mieke Henfling, Leander van Neste, Robert J. van Suylen, Anne-Marie Dingemans, Winand NM Dinjens, Annick Haesevoets, Martina Rudelius, Erik Thunnissen, Marco Volante, Wim van Criekinge, Manon van Engeland, Frans CS Ramaekers, Ernst-Jan M. Speel. Reduced CD44 and OTP gene expression are strong indicators of poor prognosis in pulmonary carcinoids. [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 2380. doi:10.1158/1538-7445.AM2013-2380
Abstract 2389: Positive or negative clinical implications of PI3K/Akt/mTOR pathway activation for predicting patient response to targeted therapy using multiplexed protein imaging of tumor tissue.
Authors: Derrien-Colemyn Alexandrine , Park Soon , Gillespie John , Lebowitz Michael , Tunon Peter
Published: August 2014

Background: In this ongoing clinical exploratory study, FFPE tissue sections are probed for protein biomarkers along the PI3K/Akt/mTOR pathway. A panel of assays was developed using Layered Immunohistochemistry (L-IHC). L-IHC allows for concurrent measurement of 6-8 proteins (total and/or phosphorylation sites) using a single tissue section. The goal was to discover applications of the technique based on complementary rationales about the implications of pathway activation. Those were: 1) in a scenario where the test indicates drug target activation, analysis for a positive response to an mTOR-targeted tyrosine kinase inhibitor (everolimus and/or temsirolimus) in kidney cancer; or 2) analysis for predicting no response to a HER2-targeted drug (trastuzumab) in breast cancer, assuming a scenario where the test would indicate activation of a functional by-pass pathway.

Methods: FFPE pre-treatment tissue samples from patients (pts) with advanced renal cell carcinoma (aRCC) treated with temsirolimus and/or everolimus, and trastuzumab treated HER2-positive breast cancer pts were studied. In L-IHC tissue-transfer layers, the intensity of specific protein biomarker signals in the cancer regions is visually scored. The expression levels are then summed and correlated with the patient's clinical outcome status.

Results: In the trastuzumab study, four biomarkers were determined to correlate with patient response. A total of 32 responders and 13 non-responders have been analyzed to date. The sum score for a 4-biomarker panel discriminated between responders and non-responders. The test correctly identified 28/32 responders and 10/13 non-responders for a sensitivity of 87%, specificity of 76%, and an accuracy of 81%.

In the mTOR inhibitor study, 35 RCC cases were analyzed, and a composite score based on 6 biomarkers was found to correlate to patient response. 11/14 OR/SD pts (sensitivity 79%) and 18/21 PD pts (specificity 86%).

Conclusions: These results indicate that multiplexed protein analysis of tumor tissue is capable of providing scenario-specific information, which could help guide targeted therapy with either a positive or a negative clinical significance.

Funding source: Supported in part by NCI Grant 5R44CA123994-06 and by 20/20 GeneSystems.

Citation Format: Alexandrine Derrien-Colemyn, Soon Park, John Gillespie, Michael Lebowitz, Peter Tunon. Positive or negative clinical implications of PI3K/Akt/mTOR pathway activation for predicting patient response to targeted therapy using multiplexed protein imaging of tumor tissue. [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 2389. doi:10.1158/1538-7445.AM2013-2389
Abstract 2396: High EGFR gene copy number predicts poor outcome in triple-negative breast cancer.
Authors: Park Heae Surng , Jang Min Hye , Kim Eun Joo , Kim Hyun Jeong , Lee Hee Jin , Park So Yeon
Published: August 2014

INTRODUCTION: Epidermal growth factor receptor (EGFR) is frequently expressed in triple-negative breast cancer and is emerging as a new therapeutic target. However, the rate of EGFR gene copy number alteration and mutation has been reported to be quite variable and their prognostic significance is poorly defined in triple-negative breast cancer.

METHOD: We examined EGFR protein expression, gene copy number alteration and mutation in 150 cases of tripe-negative breast cancer and correlated their findings with clinical outcome of the patients. EGFR protein expression was evaluated by immunohistochemistry using whole tissue sections and EGFR copy number alteration was assessed by dual-color fluorescence in situ hybridization using tissue microarrays. EGFR mutation analysis was performed by polymerase chain reaction of exon 18 to 21 of EGFR gene and direct sequencing of PCR products.

RESULT: EGFR protein was expressed in 117 of 150 cases (78.0%). High EGFR gene copy number was found in 33.3% of the cases (amplification in 2.0% and high polysomy in 31.3%). Five EGFR mutations were detected in 4 of 150 cases (2.7%) with G719A in exon 18 (n=1), V786M in exon 20 (n=1) and L858R in exon 21 (n=3). One case had two mutations (G719A and L858R). High EGFR copy number, but not EGFR mutation, was correlated with EGFR protein over-expression. In survival analyses, high EGFR copy number gain was found to be a poor prognostic factor for relapse-free survival (p=0.046), especially for metastasis-free survival (p=0.016).

CONCLUSION: EGFR mutation is found to be a rare event in triple-negative breast cancer. However, high EGFR copy number is relatively frequent and is associated with poor clinical outcome in triple-negative breast cancer, suggesting that evaluation of EGFR gene copy number is beneficial for the prediction of outcome in patients with triple-negative breast cancer and selection of the patients for anti-EGFR targeted therapy.

Citation Format: Heae Surng Park, Min Hye Jang, Eun Joo Kim, Hyun Jeong Kim, Hee Jin Lee, So Yeon Park. High EGFR gene copy number predicts poor outcome in triple-negative 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 2396. doi:10.1158/1538-7445.AM2013-2396
Abstract 2399: The analytic performance of a TaqMelt PCR assay for the detection of KRAS mutations in formalin-fixed paraffin-embedded tissue (FFPET) samples of Non-Small Cell Lung Cancer (NSCLC).
Authors: Lee Sung C. , Cao Jianli , Adhikary Kiran , Corona Lilia , Lee Nitta , Li Jingchuan , May Theresa , Wu Yiqiao , Brophy Victoria
Published: August 2014

Mutations in the Kras gene that lead to its constitutive activation are quite common in many tumor types including NSCLC. We describe the analytic performance of a TaqMelt PCR assay (cobas® KRAS Mutation Test) that detects 19 mutations in codons 12, 13 and 61 which generates test results within 8 hours. Using a panel of DNA blends from 18 FFPET samples of NSCLC, tested at 4 genomic DNA input levels, the correct mutation call was observed at 5% mutant alleles from the prescribed 50 ng of gDNA/PCR down to 12.5 ng of gDNA/PCR. A separate panel of 194 FFPET samples of NSCLC was tested with the PCR test and compared to Sanger sequencing. There were 15 discordant samples, 9 PCR (+), Sanger (-) and 6 PCR (-), Sanger (+) samples, that were further analyzed using a quantitative 454 sequencing method. All Sanger discordant samples except 1 PCR (+), Sanger (-) sample yielded 454 results that agreed with the PCR result. The PPA, NPA, and overall agreement with sequencing (Sanger resolved by 454) are 100.0%, 98.9% and 99.5%, respectively.

Sanger/454 Sequencing MUT WT Total MUT 99 1 100 cobas® KRAS Mutation Test WT 0 94 94 Total 99 95 194* *Includes 1 result for double mutations by Sanger and excludes 1 invalid result by PCR

The same panel of 194 specimens tested with another lot of the PCR test gave similar performance. When a panel of 8 FFPET samples of NSCLC (including 3 near the limit of analytical sensitivity) was repeatedly tested by 2 operators, using multiple instruments and reagent lots, a correct result was observed for 100% of tests. PCR test results were not affected by repeated freezing and thawing of the DNA sample or test reagents, or by addition of hemoglobin or triglycerides. These studies show that this PCR test is a robust and reproducible assay that 1) detects 19 mutations in codons 12, 13 and 61, 2) requires a small amount of DNA (which can generally be obtained from 1x5 μm section), and 3) is more sensitive and specific than Sanger sequencing.

Citation Format: Sung C. Lee, Jianli Cao, Kiran Adhikary, Lilia Corona, Nitta Lee, Jingchuan Li, Theresa May, Yiqiao Wu, Victoria Brophy. The analytic performance of a TaqMelt PCR assay for the detection of KRAS mutations in formalin-fixed paraffin-embedded tissue (FFPET) samples of Non-Small Cell Lung Cancer (NSCLC). [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 2399. doi:10.1158/1538-7445.AM2013-2399
Abstract 240: Therapeutic targeting of EMP2 reduces breast cancer stem cells.
Authors: Wadehra Madhuri , Kiyohara Meagan , Ashki Negin
Published: August 2014

There is increasing evidence that tumor-initiating cancer stem cells (CSCs) contribute to tumor metastasis and therapeutic resistance. In breast cancer, metastatic CSCs are defined as CD44+/CD24- and ALDH+, and in this study, we define another marker epithelial membrane protein-2 (EMP2) as a novel target for this population of cells. EMP2 is an oncogene whose expression has been shown to correlate with tumor progression and survival in a number of human cancers including triple negative breast, ovarian, and endometrial tumors. In breast cancer, EMP2 is expressed on the majority of invasive tumors examined, and in particular 70% of triple negative breast tumors showed surface expression of this marker. Clinical data also revealed higher levels of EMP2 in metastatic lesions compared to primary tumors.

In this study, we show new evidence that EMP2 is highly expressed in CSCs. New data suggests that EMP2 upregulates the expression of HIF-1α, CD44, and ALDH and directly contributes to the metastatic potential of the tumor cell. High levels of EMP2 promote mammosphere formation and increased tumor forming potential. In tumors created from these cells, high levels of ALDH+ expression were also observed. In contrast, reduction in EMP2 decreased CD44 expression and ALDH activity both in vitro and in vivo, with the net consequence of poorly vascularized and slow growing tumors.

We have recently developed a novel IgG1 monoclonal antibody to EMP2 and have started testing its therapeutic efficacy. In vitro treatment with EMP2 IgG1 reduced HIF-1α, CD44, and ALDH levels, ultimately leading to caspase mediated apoptosis. Treatment of breast cancer cells with EMP2 IgG1 reduced tumor load in both subcutaneous and metastatic models of breast cancer with a significant improvement in survival. Histological examination of the tumors revealed significant necrosis and elimination of ALDH+ CSCs. These results suggest that targeting EMP2 may reduce CSCs and represent an attractive target for further therapeutic development.

Citation Format: Madhuri Wadehra, Meagan Kiyohara, Negin Ashki. Therapeutic targeting of EMP2 reduces breast cancer stem 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 240. doi:10.1158/1538-7445.AM2013-240