bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4621: Validation and calibration of next-generation sequencing to identify Epstein-Barr Virus-positive gastric cancer
Authors: Camargo M. Constanza , Bowlby Reanne , Chu Andy , Pedamallu Chandra S. , Thorsson Vesteinn , Elmore Sandra , Mungall Andrew , Bass Adam , Gulley Margaret L. , Rabkin Charles S.
Published: August 2015

Background: Gastric adenocarcinoma was recently classified by a large genomic study into four molecular subtypes, including one defined by Epstein-Barr virus (EBV)-positivity. EBV infection in tumors is conventionally assessed by in situ hybridization (ISH), but detection of viral nucleic acids by next-generation sequencing represents a potential alternative.

Methods: Normalized EBV read counts were determined by whole genome, whole exome, mRNA and miRNA sequencing for 295 fresh-frozen gastric tumor samples. Formalin-fixed, paraffin-embedded tissue sections were retrieved for ISH confirmation of 13 high-EBV and 11 low-EBV cases selected at random.

Results: By each sequencing method, numbers of EBV reads were bimodally distributed across tumors, with a minority having much higher counts. In pairwise comparisons, individual samples were either consistently high or consistently low by all genomic methods for which data were available. Empiric cut-offs based on molecular data of 1000 normalized reads for whole genome, 100 for exome, 4 for mRNA and 5000 for miRNA had perfect concordance identifying 26 (9%) tumors as EBV-positive. One sample tested by ISH lacked tumor cells. For the 23 tumors with EBV status determined by both approaches, EBV-positivity or -negativity by molecular testing was confirmed by EBER-ISH in all but one case (kappa = 0.91). The exception was a microsatellite instability-type cancer EBV-negative by both mRNA and miRNA sequencing, with equivocally positive ISH interpretation.

Conclusions: EBV-positive gastric tumors may be accurately identified by quantifying viral sequences in genomic data. Simultaneous analyses of human and viral DNA, mRNA and miRNA could streamline tumor profiling for clinical care and research.

Citation Format: M. Constanza Camargo, Reanne Bowlby, Andy Chu, Chandra S. Pedamallu, Vesteinn Thorsson, Sandra Elmore, Andrew Mungall, Adam Bass, Margaret L. Gulley, Charles S. Rabkin. Validation and calibration of next-generation sequencing to identify Epstein-Barr Virus-positive gastric cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4621. doi:10.1158/1538-7445.AM2015-4621
Abstract 4628: Analysis of melanoma GWAS data suggests specific risk loci influencing age of onset of melanoma
Authors: Cymerman Rachel M. , Bigio Benedetta , Seybold Martin P. , Polsky David , Wittkowski Knut M.
Published: August 2015

Introduction/Purpose: The average age of diagnosis of melanoma is younger than that of other major cancers. In addition, melanoma is the second most common cancer among individuals in their 20s. Analyzing GWAS data by ‘wide-locus region’ (i.e. analyzing multiple nearby single nucleotide polymorphisms (SNPs) together) increases statistical power compared to single-SNP analyses. This approach has uncovered new associations in GWAS data for diseases other than melanoma. By applying wide-locus analysis to GWAS data from melanoma cases, we aimed to discover common genetic risk variants related to age of melanoma onset.

Procedures: We re-analyzed data from the 1977 melanoma cases of European ancestry made available by the MD Anderson Cancer Center study (accession: phs000187.v1.p1). Patients were genotyped on the Illumina OMNI1-Quad chip. Biostatistical analysis was performed from a window moving across the whole genome using u-statistics for multivariate, genetically structured wide-locus data to derive genetic risk scores to determine if any gene regions were associated with age of onset in melanoma patients. This statistical approach resulted in a genome-wide significance level of p<10−5.64.

Results: Common variants in several genes were associated with melanoma age-of-onset at the level of genome-wide significance. The most significant functional risk loci were located at DLEU1 (p<10−6.75), NRP1 (p<10−6.66), PTPN11P (p<10−6.49) and BAT1/ATP6/NFKBIL (p<10−6.41). Of note, none of these risk loci have been shown to be associated with risk of developing melanoma overall, or longevity in prior GWAS. The NRP1 and PTPN11P genes have been linked to the RAS pathway, an important driver of melanoma pathogenesis.

Conclusions: Our analysis revealed novel associations between several common genetic variants and earlier age of melanoma diagnosis. Some of these loci interact with the RAS pathway. Additional studies of common genetic variants and age of cancer diagnosis using this approach may reveal similar findings in other melanoma cohorts and possibly other cancer types.

Citation Format: Rachel M. Cymerman, Benedetta Bigio, Martin P. Seybold, David Polsky, Knut M. Wittkowski. Analysis of melanoma GWAS data suggests specific risk loci influencing age of onset of melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4628. doi:10.1158/1538-7445.AM2015-4628
Abstract 463: Successful generation of tumor infiltrating lymphocytes from human non-small cell lung cancer specimens
Authors: Kim Kyoung-Hee , Kargl Julia , Lee Sylvia , Houghton McGarry
Published: August 2015

Tumor infiltrating lymphocytes (TILs) are naturally occurring cancer-fighting T cells found within malignant tumors. After decades of research, patient-derived TIL populations have been successfully re-infused into melanoma subjects, and have produced impressive treatment effects in a subset of cases. To date, the successful generation and usage of TIL populations for non-small cell lung cancer (NSCLC) patients has not been reported. Generating TILs from NSCLC tissue presents several challenges, as immune cell populations residing within the lungs are constantly exposed to numerous inhaled antigens and other particulate matter.

Here, we report the successful generation of TILs from three NSCLC specimens from subjects undergoing surgical resection for curative intent. Six distinct lung cancer tissue fragments were cultured in the presence of IL-2 for each of the three cases. Each fragment yielded a TIL population, although their proliferative rates differed substantially. Each TIL population was subjected to flow cytometry to identify the cellular subtypes, and these results were compared to the lymphocyte subset content measured in the original in vivo tumor specimen. Preliminary findings suggest that the ability to generate CD8+ enriched TILs is dependent upon the CD8+ content present in vivo. Low CD8+ content was associated with an abundance of either Tregs or NK cells. Greater than 70% of the CD8+ populations generated displayed an effector phenotype (CCR7-CD45RA+), regardless of the overall cellular composition of the specimen. However, these cells displayed heterogeneous levels of exhaustion markers (PD1, Tim3), which inversely correlated with proliferative rates.

Our results show that TIL populations can be successfully generated from primary human NSCLC specimens. Although the relative cellular content differed for each tissue fragment, we were able to generate at least one CD8+ effector population with low levels of exhaustion for each case. In vitro and in vivo studies to determine the tumor killing capacity of these TIL populations are currently underway.

Note: This abstract was not presented at the meeting.

Citation Format: Kyoung-Hee Kim, Julia Kargl, Sylvia Lee, McGarry Houghton. Successful generation of tumor infiltrating lymphocytes from human non-small cell lung cancer specimens. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 463. doi:10.1158/1538-7445.AM2015-463
Abstract 4630: Heterogeneity revealed through meta-analysis might link geographical differences with nasopharyngeal carcinoma incidence in Han Chinese
Authors: Su Wen-Hui , Chiu Chi-Cking , Shugart Yin Yao
Published: August 2015

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy highly prevalent in Southern China with varying incidence rates among Han Chinese in different geographic regions. Recently, three independent genome-wide association studies (GWAS) confirmed that the HLA-A was the main risk gene of NPC. However, studies from regions with different incidence rates led to contradictory claims regarding whether HLA-A was the sole risk gene and the association of rs29232 was independent from the HLA-A effect in chromosome 6p21.3 region. To explore whether the differences were caused by genetic factors underlying the varying incidence rates by region in NPC incidence, we performed a meta-analysis by selecting 5 single-nucleotide polymorphisms (SNPs) in chromosome 6p21.3 from 3 published GWAS studies and 3 published case-control studies from Southern China (Taiwan, Guangdong and Guangxi) involving 7 828 NPC patients and 8 813 healthy controls from Han Chinese. The rs29232 SNP exhibited a moderate level of heterogeneity (I2 = 43%) that disappeared (I2 = 0% and 4%) after stratifying the studies according to moderate- and high-incidence NPC regions. The estimated odds ratio (OR) of rs29232 (OR: 1.70, 95% confidence interval [CI]: 1.47-1.95) was higher in the moderate-incidence region than in the high-incidence region (OR: 1.42, 95% CI: 1.30-1.56). The heterogeneity revealed by meta-analysis of rs29232 might be associated with differences in NPC incidence in Han Chinese between geographic regions. Therefore, we postulate that moderate heterogeneity in rs29232 might contribute to the varying incidence rates between regions due to a genetic cause in NPC incidence. The higher OR of rs29232 and the fact that it was independent of the HLA-A effect in the moderate-incidence population suggested that rs29232 might have greater relevance to NPC in a moderate-incidence population than to a high-incidence population. To our knowledge, this is the first study to demonstrate that genetic factors might involve in the varying disease incidence rates by region. This further suggests that the disease incidence rate should be considered as a potential confounding factor when conducting large, cross-region meta-analysis in NPC.

Citation Format: Wen-Hui Su, Chi-Cking Chiu, Yin Yao Shugart. Heterogeneity revealed through meta-analysis might link geographical differences with nasopharyngeal carcinoma incidence in Han Chinese. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4630. doi:10.1158/1538-7445.AM2015-4630
Abstract 4632: Poor survival associated with NUAK1 overexpression in serous ovarian cancer may be explained by chemotherapy resistance
Authors: Phippen Neil T. , Bateman Nicholas W. , Wang Guisong , Hamilton Chad A. , Maxwell George L. , Darcy Kathleen M. , Conrads Thomas P.
Published: August 2015

Objectives: The purpose of this study was to identify and validate novel gene transcripts associated with poor prognosis ovarian cancer.

Methods: Clinical, pathologic, outcomes and transcriptomics data for 22,277 probesets were downloaded for each of 1,183 eligible patients representing six datasets. Cohorts 1 (n = 638) and 2 (n = 545) were used for discovery and validation, respectively. Associations with survival were evaluated using univariate Cox modeling with Wald test and Kaplan-Meier method with log rank test. Odds ratios were evaluated using the Mantel-Haenszel test. NUAK1 expression in ovarian cancer cell lines was examined using immunoblotting.

Results: There were 293 (1.3%) probesets whose expression was associated with survival in serous ovarian cancer patients in cohort 1 that passed false discovery correction, and 19 of these, including NUAK1, validated in cohort 2 (q<0.05). Women with high versus low transcript expression of NUAK1 had worse survival (hazard ratio = 1.4, 95% confidence interval = 1.2-1.64) and increased odds of being diagnosed at advanced age with late stage disease in addition to experiencing suboptimal debulking and earlier disease progression. Higher levels of NUAK1 protein were observed in a panel of chemoresistant versus chemosensitive ovarian cancer cells lines.

Conclusions: Overexpression of NUAK1, also known as AMPK-related protein kinase 5 (ARK5) and a survival factor in nutrient starved cells with low oxygen tension, was identified and validated to predict poor prognosis in a cohort of 1,183 serous ovarian cancer patients, and to be associated with chemoresistance using in vitro models of platinum and taxane resistance in human ovarian cancer.

Citation Format: Neil T. Phippen, Nicholas W. Bateman, Guisong Wang, Chad A. Hamilton, George L. Maxwell, Kathleen M. Darcy, Thomas P. Conrads. Poor survival associated with NUAK1 overexpression in serous ovarian cancer may be explained by chemotherapy resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4632. doi:10.1158/1538-7445.AM2015-4632
Abstract 4646: Cumin extract prevents estrogen-associated breast cancer in ACI rats
Authors: Aqil Farrukh , Jeyabalan Jeyaprakash , Munagala Radha , Gupta Ramesh C.
Published: August 2015

Spices are highly chemoprotective because of their antioxidant, antiproliferative, and anti-inflammatory activities. We have previously demonstrated significant chemoprotective effects of cumin seed powder (7.5%, w/w) against estrogen-induced mammary tumorigenesis in ACI rats by inhibiting cell proliferation as well as cellular and molecular markers. Here we report the inhibition of E2-mediated mammary tumorigenesis by cumin extract (1%, w/w) that is equivalent to the dietary dose in our previous study. Three extractions of cumin seeds with 80% aqueous ethanol followed by evaporation provided 13% of total dried extract (w/w). Groups of female ACI rats were given either AIN-93M diet or diet supplemented with cumin extract and then treated with subcutaneous 17ß-estradiol silastic implants (1.2 cm; 9 mg). Two additional groups were maintained on control or experimental diet. Rats received diet and water ad libitum. Five-six animals from each group were euthanized three weeks after the E2 treatment to determine the effect of interventions on early biomarkers. The remaining animals (n = 20-22) were euthanized when estrogen-treated group on control diet reached over 90% tumor incidence.

Diet consumption and body weight gain was found to be largely unaffected by the cumin extract, suggesting that it was well tolerated. The first tumor incidence in control group was after 92 days, this was significantly delayed by 35 days in cumin extract treated group. Experimental diet significantly offset estrogen-related growth of pituitary prolactinomas as early as after three weeks (p = 0.019), the effect was more pronounced at tumor time point (p < 0.0001). At the end of the study (25 wks), the tumor incidence was found to be 96% in control group whereas only 45% animals had tumors in the intervention group. Significant reduction in average tumor volume (474 vs. 75 mm3) and tumor multiplicity (4.1 vs. 0.8 tumors/animal) was also observed by cumin extract compared to control. The mechanistic studies including effect on the plasma prolactin levels, and the cell proliferation marker, PCNA, estrogen-associated expression of the ERα and cyclin D1 in the mammary tissues are being evaluated. Together, our data show that cumin extract can significantly delay and prevent estrogen-mediated mammary tumorigenesis. This data further support our ultimate goal to develop these spice bioactives as clinically translatable chemopreventives and therapeutics against breast cancer. (Supported by CA-125152, Helmsley Fund, and Agnes Brown Duggan Endowment).

Citation Format: Farrukh Aqil, Jeyaprakash Jeyabalan, Radha Munagala, Ramesh C. Gupta. Cumin extract prevents estrogen-associated breast cancer in ACI rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4646. doi:10.1158/1538-7445.AM2015-4646
Abstract 4651: Urolithin A (UA) inhibits both androgen receptor and AKT signaling and prevents the progression of castration resistant prostate cancer
Authors: Vadhanam Manicka V. , Das Trinath P. , Suman Suman , Annamalai Lakshmanan , Damodaran Chendil
Published: August 2015

Current therapeutic regimens are insufficient for treating advanced and metastatic prostate cancer. Compelling evidence suggest existence of concomitant aberrations in both activation of Androgen Receptor (AR) and Protein Kinase-B (AKT) signaling pathway in advanced castration resistant prostate cancer (CRPC). Hence, simultaneous inhibition of both AR and AKT appears to be an effective strategy for the prevention and treatment of CRPC.

We are interested in identifying small molecules from natural plant origin, which targets both AR and AKT signaling for the prevention and treatment of CRPC. Ellagic acid/ellagitannins, abundantly present in the pomegranate and berries, are actively metabolized by the intestinal microflora to Urolithin-A (UA). Oral administration of UA has been equivocally shown to be highly bioavailable in the prostate gland, and is non-toxic.

In our in vitro studies, UA treatment inhibited the growth of both AR-expressing (LNCaP, C42B) and AR-negative (PC-3) prostate cancer cell lines. The molecular analyses suggested that UA transcriptionally regulated AR function and its downstream events in these cell lines. In addition, dihydrotestosterone (DHT) induced AR signaling was attenuated by UA.

While dissecting the effect of UA on AKT signaling, we found UA inhibited pAKT (Ser 473) expression and its pro-survival target genes. Phenotypic analysis confirmed UA reduces the motility and invasive potential of prostate cancer cells by inhibiting the expression of genes that are implicated in the epithelial-to-mesenchymal transition (EMT) (e.g., β-catenin, vimentin, Snail, and Slug).

We also demonstrated the in vivo efficacy of UA against CRPC. Oral administration of UA (50 mg/kg body weight) inhibited prostate tumor growth in xenografts from PC-3 and C4-2B cells. Moreover, histopathological evaluation of the xenografts revealed a marked reduction in the tumor cell density, loss of anisocytosis and anisokaryosis, and lower mitosis in animals that received UA as compared to the control group. Studies are underway to analyze the plasma and tissue levels of UA and our ongoing immunohistochemistry studies may validate the in vitro findings and confirms whether AKT/AR are the target of UA.

Together, these results suggest that targeting and thus, overriding both AR and AKT mediated pro-survival signaling could be an effective strategy for preventing the progression of CRPC.

Citation Format: Manicka V. Vadhanam, Trinath P. Das, Suman Suman, Lakshmanan Annamalai, Chendil Damodaran. Urolithin A (UA) inhibits both androgen receptor and AKT signaling and prevents the progression of castration resistant prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4651. doi:10.1158/1538-7445.AM2015-4651
Abstract 4669: Druggable oncogene fusions in lung invasive mucinous adenocarcinoma
Authors: Nakaoku Takashi , Tsuta Koji , Ichikawa Hitoshi , Shiraishi Kouya , Sakamoto Hiromi , Enari Masato , Furuta Koh , Shimada Yoko , Ogiwara Hideaki , Watanabe Shun-ichi , Nokihara Hiroshi , Yasuda Kazuki , Hiramoto Masaki , Nammo Takao , Kim Young Hak , Mishima Michiaki , Yokota Jun , Yoshida Teruhiko , Kohno Takashi
Published: August 2015

Invasive mucinous adenocarcinoma (IMA) of lung, a subtype of lung adenocarcinoma, is known to be strongly associated with KRAS mutation. Genetic aberration is unknown in IMA without KRAS mutation.

From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations, we conducted whole-transcriptome sequencing on 32 IMAs, including 27 cases without KRAS mutation. We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1 (NRG1 fusions: 6/90), EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET.

The CD74-NRG1 and SLC3A2-NRG1 fusion proteins contained the transmembrane domain of CD74 and SLC3A2 and retained the EGF-like domain of the NRG1 protein (NRG1 III-β3 form). Membrane-tethered EGF-like domain might activate juxtacrine signaling through HER2:HER3 receptors. To reveal the mechanism of the oncogenicity, exposing EFM-19 cells which express HER2 and HER3 to conditioned media from H1299 cells expressing exogenous CD74-NRG1 fusion protein resulted in phosphorylation of HER2 and HER3, and downstream mitogen-activated kinase and AKT. The phosphorylation was suppressed by lapatinib and afatinib.

When EZR-ERBB4 or TRIM24-BRAF cDNA was exogenously expressed in NIH3T3 cells, ERBB4 or downstream mediator of BRAF was phosphorylated under serum-free condition. The expression of fusion genes induced anchorage-independent growth. The phosphorylation and growth were suppressed by the kinase inhibitors targeting fusion-induced signaling.

These results show that the NRG1, ERBB4 and BRAF fusions are novel driver mutations and potential targets. About 15% (13/90; 14.4%) of IMAs harbor druggable aberrations, taken together a small fraction of known driver aberrations.

Citation Format: Takashi Nakaoku, Koji Tsuta, Hitoshi Ichikawa, Kouya Shiraishi, Hiromi Sakamoto, Masato Enari, Koh Furuta, Yoko Shimada, Hideaki Ogiwara, Shun-ichi Watanabe, Hiroshi Nokihara, Kazuki Yasuda, Masaki Hiramoto, Takao Nammo, Young Hak Kim, Michiaki Mishima, Jun Yokota, Teruhiko Yoshida, Takashi Kohno. Druggable oncogene fusions in lung invasive mucinous adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4669. doi:10.1158/1538-7445.AM2015-4669
Abstract 4691: KRAS mutation specific alkylating pyrrole-imidazole polyamide (KR12) suppresses mutant KRAS expression and inhibits tumor growth by showing accumulation in KRAS mutant xenografts
Authors: Inoue Takahiro , Hiraoka Kiriko , Suzuki Yusei , Yoda Hiroyuki , Watanabe Takayoshi , Takatori Atsushi , Koshikawa Nobuko , Ozaki Toshinori , Nagase Hiroki
Published: August 2015

Constitutive active mutations of KRAS are detected in 35-40% of human colon cancers, and almost all of them are the constitutive active missense mutations at codon 12 (80%) or codon 13 (20%). Consistent with these observations, the presence of KRAS mutations has been shown to be associated with malignant properties of tumors as well as a poor clinical outcome of the patients bearing these tumors. Unfortunately, yet no effective anti-cancer drug(s) specifically targeting KRAS mutations have been developed. Hence, we synthesized an alkylating agent conjugated with the Pyrrole-Imidazole polyamide (KR12: PI-polyamide-seco-CBI), which recognized KRAS G12D or G12V mutations at codon12. We have previously found that KR12 has anti-tumor effects in vitro and in vivo. However, it still remains elusive whether KR12 exerts its selective toxicity towards colon cancer cells by penetrating into the tumor tissues and inhibiting the expression of mutant KRAS gene in mouse model of human cancer. To address this issue, we decided to examine the distribution of KR12 using FITC labeled PI polyamide. In vivo imaging of tumor-bearing mice after single intravenous administration demonstrated that the highest fluorescence intensity was seen in the tumor sites 24 hours after injection with showing nuclear localization. Quantitative RT-PCR revealed that KR12 decreased the mutated KRAS expression in tumor tissues obtained from KRAS-heterozygous-mutated-LS180-xenografted mice (G12D heterozygous mutation). Since KR12 showed long lasting accumulation in xenografts we compared the effect of single and multiple administration of KR12. Once a week injection for five to eight weeks resulted in significant suppression of tumor growth in homozygous mutant SW480 (G12V homozygous mutation) xenografts. Surprisingly, both single and multiple treatments of KR12 induced massive tumor volume reduction without affecting body weight gain. These data suggest that KR12 accumulation in colon xenograft tumor tissues may emphasize drug local effect and minimize systemic adverse effect.

Citation Format: Takahiro Inoue, Kiriko Hiraoka, Yusei Suzuki, Hiroyuki Yoda, Takayoshi Watanabe, Atsushi Takatori, Nobuko Koshikawa, Toshinori Ozaki, Hiroki Nagase. KRAS mutation specific alkylating pyrrole-imidazole polyamide (KR12) suppresses mutant KRAS expression and inhibits tumor growth by showing accumulation in KRAS mutant xenografts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4691. doi:10.1158/1538-7445.AM2015-4691
Abstract 47: KRAS-mutation dependent effect of zoledronic acid in human NSCLC preclinical models
Authors: Kenessey István , Kói Krisztina , Cserepes Mihály , Dobos Judit , Hegedűs Balázs , Tóvári József , Tímár József
Published: August 2015

In non-small cell lung cancer (NSCLC) KRAS-mutant status is a negative prognostic and predictive factor of both classic and target based therapy. In human malignancies bisphosphonate drugs are applied to treat or prevent formation of bone metastases. Nitrogen-containing bisphosphonates inhibit the posttranslational modification of small G-proteins (e.g. Ras, Rac, Rho) and thus may affect proliferation and migration. In our preclinical work, we investigated the effect of an aminobisphosphonate compound (zoledronic acid) on mutant and wild type KRAS-expressing human NSCLC cell lines.

In vitro cell viability was measured by MTT assay in H358, LCLC-103H, H1650 and H1975 cells. Apoptotic cell death was quantified by flow cytometric detection of sub-G1-fraction in propidium-iodide stained cultures. Furthermore, we investigated the in vivo effect of zoledronic acid in a SCID mouse subcutaneous xenograft model. The extracted tumors were examined by traditional histological methods and the proportion of proliferating tumor cells was determined by Ki67 labeling.

KRAS-mutant H358 human NSCLC cell line showed relative resistance to zoledronic acid on proliferation, while human lung cancer cell lines carried wild type KRAS proved to be sensitive. Zoledronic acid improved the cytostatic effect of traditional anti-cancer agent cisplatin. Zoledronic acid did not induce significant apoptosis in NSCLC cell lines in vitro, rather the blockade of the cell cycle appeared. In in vivo subcutaneous animal model, zoledronic acid significantly reduced the weight of LCLC-103H xenograft tumor harboring wild type KRAS, furthermore, the treatment decreased the number of Ki67-labeled tumor. Zoledronic acid induced the expression of VEGF in LCLC-103H cells and stimulated in vivo angiogenesis.

We concluded that in human NSCLC cells the antiproliferative effect of zoledronic acid dependended on KRAS-status (wild-type or mutant). The inhibitory effect of zoledronic manifested through the in vitro and in vivo blockade of cell cycle. The in vivo angiogenic effect of zoledronic acid supported cytostatic effect of cisplatin. Our preclinical investigation suggests that patients with wild type KRAS-expressing NSCLC could potentially benefit from aminobisphosphonate therapy.

Citation Format: István Kenessey, Krisztina Kói, Mihály Cserepes, Judit Dobos, Balázs Hegedűs, József Tóvári, József Tímár. KRAS-mutation dependent effect of zoledronic acid in human NSCLC preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 47. doi:10.1158/1538-7445.AM2015-47
Abstract 4708: Mammalian glutamine metabolism controls circadian rhythm through regulation of reactive oxygen species
Authors: Altman Brian J. , Stine Zachary E. , Hsieh Annie L. , Deberardinis Ralph J. , Dang Chi V.
Published: August 2015

Circadian rhythms are twenty-four hour physiologic cycles present in all eukaryotes that control a variety of organismal processes, including metabolism, but the role of metabolism in control of circadian rhythms is still not well understood. Peripheral clocks such as those present in the liver control metabolic pathways such as glucose metabolism and respiration as well as amino acid metabolism. It has been recently demonstrated that the availability of the metabolite NAD (nicotinamide adenine dinucleotide) can feed back to control circadian rhythm. There is much interest in targeting glutamine metabolism in cancer, but it is still not fully understood how inhibition of glutamine metabolism affects normal cell physiology, including circadian rhythm. Here we show using the commonly-used circadian model U2OS osteosarcoma cells and other cell lines that glutamine withdrawal blocked proper circadian oscillation of gene expression. Glutamine withdrawal led to distinct and dramatic changes in circadian gene expression in several cell lines with highly different tissue origins, which could be rescued by addition of the cell permeable TCA-intermediate α-ketoglutarate. However, cells withdrawn from glutamine did not show signs of metabolic stress or impairment of the mTOR pathway. While alterations to histone modifications possibly stemming from impairment of αKG-dependent enzymes were observed, these did not explain the observed alterations in circadian rhythm. Rather, RNA-seq analysis of genetic changes after glutamine withdrawal and α-ketoglutarate rescue revealed strong induction of several genetic pathways associated with reactive-oxygen species (ROS) induction, particularly those resulting from chemotherapy or photodynamic therapy of cancer. Further supporting the importance of ROS in regulation of circadian rhythm, addition of cell permeable antioxidants rescued the disruption of circadian oscillation in the absence of glutamine. Finally, inhibiting expression of the key ROS-defense catalase phenocopied circadian rhythm disruption observed after glutamine withdrawal. Together, these data suggest that glutamine availability and metabolism are critical to support circadian rhythm and gene expression through modulation of intracellular ROS, and furthermore that cancer treatments that lead to induction of ROS could affect normal cellular circadian rhythm.

We thank the following funding: NIH F32CA180370, NIH F32CA174148, NIH R01CA051497, LLS 610614

Citation Format: Brian J. Altman, Zachary E, Stine, Annie L. Hsieh, Ralph J. Deberardinis, Chi V. Dang. Mammalian glutamine metabolism controls circadian rhythm through regulation of reactive oxygen species. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4708. doi:10.1158/1538-7445.AM2015-4708
Abstract 4726: Gene expression signatures of isolated circulating tumor cells from metastatic breast cancer patients reveal presence of breast cancer stem cells with EMT or MET phenotypes
Authors: Fouladdel Shamileh , Yoon Hyeun Joong , Lin Eric , Kim Tae Hyun , Deol Yadwinder S. , Luther Tahra K. , Clouthier Shawn G. , Jiang Hui , Burness Monika L. , Nagrath Sunitha , Azizi Ebrahim , Wicha Max S.
Published: August 2015

Breast cancer stem cells are responsible for tumor recurrence, metastasis and drug resistance. One of the important steps in metastasis is entry of tumor cells into the blood circulation. These circulating tumor cells (CTCs) may serve as markers of cancer progression and as a “liquid biopsy” to provide information on tumor biology at single cell resolution. Current methods of CTC capture are not fully inclusive to isolate all phenotypic variations of cancer stem cells (CSCs) among the CTCs. Using newly developed microfluidic methods to isolate CTCs from blood samples of metastatic breast cancer patients, we assessed gene expression profiles in these cells. The gene expression signatures of isolated CTCs were determined using a highly sensitive microfluidic-based 96-plex RT-qPCR method. Analyzed multiplex RT-qPCR data revealed distinct gene expression patterns among CTCs isolated from different patients. Cancer stem cell markers such as ALDH1a1, ALDH1a2 and ALDH1a3 as well as CD44 and CD24 genes were differentially expressed in isolated CTCs. In addition, markers of the epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) phenotypes such as Vimentin, EpCAM, HER2, CDH1, CDH2, TGFb1 and cytokeratins were also expressed variably in the CTC samples. Furthermore, MMP9, TIMP1, CD90, CD14, SPARC, BRAF, KRAS, PI3K and ERCC1 genes which play a role in cancer initiation, promotion, metastasis and drug resistance were also differentially expressed in the isolated CTCs. Overall, these studies further our understanding into the heterogeneity of CTCs and CSCs as well as providing a potential tool for real-time monitoring of cancer patients on clinical trials.

Citation Format: Shamileh Fouladdel, Hyeun Joong Yoon, Eric Lin, Tae Hyun Kim, Yadwinder S. Deol, Tahra K. Luther, Shawn G. Clouthier, Hui Jiang, Monika L. Burness, Sunitha Nagrath, Ebrahim Azizi, Max S. Wicha. Gene expression signatures of isolated circulating tumor cells from metastatic breast cancer patients reveal presence of breast cancer stem cells with EMT or MET phenotypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4726. doi:10.1158/1538-7445.AM2015-4726
Abstract 4730: Molecular analysis of the pediatric cancer fibrolamellar hepatocellular carcinoma
Authors: Simon Elana P. , Honeyman Joshua N. , Darcy David G. , Rosenberg Brad R. , Lim Iris I. , Murphy Jennifer M. , Farber Ben , Lalazar Gadi , Freije Catherine , La Quaglia Michael P. , Simon Sanford M.
Published: August 2015

Genomic analysis of the pediatric cancer fibrolamellar hepatocellular carcinoma

Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver tumor that usually occurs in adolescents and young adults. Originally considered a variant of hepatocellular carcinoma (HCC), FLHCC is characterized by hepatocytes with deeply eosinophilic, granular cytoplasm interspersed with fibrous bands, without signs of cirrhosis as in HCC. Since little is known of its molecular pathogenesis, we performed RNA-seq and whole genome sequencing of FLHCC paired with normal liver from the same patient.

Our results demonstrated that the majority of the DNA is unremarkable with few recurrent mutations or structural variants such as amplifications or inversions The major finding is a heterogeneous deletion of ∼400 kB in chromosome 19 which produces a chimeric transcript and a fully functional protein, a fusion between the heat shock protein DNAJB1 and the catalytic subunit of protein kinase A (PRKACA). Our initial observation of this chimera in 15 out of 15 patients has now been expanded to 116 out of 116 patients. Differential expression analysis of RNA-seq revealed many changes in the gene expression in FLHCC as compared to its paired normal liver. These changes were highly consistent from patient to patient, suggesting a unifying pathogenesis. The RNA-seq from our FLHCC samples had little in common with HCC samples from the TCGA or published reports of other cancers. An analysis of protein expression correlated well with the RNA-seq results. Some observed changes can directly be linked to increased activity of PRKACA. Other changes involve pathways known to participate in the initiation and progression of other cancers, which, along with an increase of PRKACA activity, represent targets for therapeutic intervention. Several of these targets are now being explored in cellular models, genetic, and PDX mouse models, as well as clinical trials.

Citation Format: Elana P. Simon, Joshua N. Honeyman, David G. Darcy, Brad R. Rosenberg, Iris I. Lim, Jennifer M. Murphy, Ben Farber, Gadi Lalazar, Catherine Freije, Michael P. La Quaglia, Sanford M. Simon. Molecular analysis of the pediatric cancer fibrolamellar hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4730. doi:10.1158/1538-7445.AM2015-4730
Abstract 4738: Maximize recovery of quality nucleic acid from formalin-fixed paraffin-embedded tissue samples using a novel, flexible purification technology
Authors: Wieczorek Doug , Hook Brad , Vincent Eric , Schagat Trista
Published: August 2015

Formalin-fixed paraffin-embedded (FFPE) tissue samples are a valuable source of genetic information for gene expression and clinical research. Extraction of nucleic acid (NA) from FFPE tissues is a challenge because the fixation process results in cross-linking between proteins and nucleic acid, as well as between different strands of DNA or RNA molecules. Recent advances in sample preparation enables access to the valuable information contained within these difficult samples. We use a novel purification technology that does not rely on use of any harsh organic solvents and concentrates the NA for low volume elution on a specially designed spin column, thus maximizing concentration. Using this technology, we examined several aspects of the purification protocol for flexibility and adaptability: effect of de-crosslinking time on the quality and length of nucleic acids released from diverse FFPE tissues, effect of overnight storage between lysis and extraction steps, and whether the technology could be adapted to extract both DNA and RNA from the same FFPE tissue sample. Yield and quality of isolated nucleic acids were examined by absorbance, fluorescent binding dye, and real-time qPCR and

RT-qPCR. We demonstrate that amplification-based quantitation gave the best indication of the quality of NA recovered. We found that a short decrosslinking time gave significant yields, but only with longer decrosslinking, times were amplifiable yields maximized for many of the tissue types tested. Preprocessing conditions were determined that allow release of both DNA and RNA for subsequent capture and elution in separate spin columns, enabling recovery of high quality DNA and RNA from the same sample in separate tubes.

In this study, we demonstrate that de-crosslinking conditions can affect the recovery of amplifiable nucleic acid from FFPE samples and that RNA and DNA can be recovered from the same FFPE sample using an optimized protocol.

Citation Format: Doug Wieczorek, Brad Hook, Eric Vincent, Trista Schagat. Maximize recovery of quality nucleic acid from formalin-fixed paraffin-embedded tissue samples using a novel, flexible purification technology. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4738. doi:10.1158/1538-7445.AM2015-4738
Abstract 474: Regulation of epithelial plasticity of cancer cells by tumor-associated macrophages
Authors: LEE CHIH-CHAN , Yang Muh-Hwa
Published: August 2015

Epithelial plasticity of cancer cells is considered to be critical for the metastatic process of cancer cells. Epithelial-mesenchymal transition (EMT) has been proved to associate with cancer metastatic dissemination from primary tumor site to distal metastatic organ. In contrast, accumulated evidence showed that the reversal process of EMT, i.e., mesenchymal-epithelial transition (MET), is critical for metastatic colonization. However, the mechanism for controlling the epithelial plasticity during metastatic process is unclear. Tumor-associated macrophages (TAMs) are the most abundant host immune cells in tumor microenvironments. TAMs have been shown to modulate cancer progression through regulating cancer cell growth and metastasis, and remodeling extracellular matrix. However, it is still unclear about the roles of TAMs in regulating epithelial plasticity during cancer metastasis. Here, we demonstrate the distinct characteristics of that the TAMs in primary and metastatic tumors. The TAMs in metastatic tumors carry a M2-like phenotype; in contrast, the TAMs in primary tumors are composed of heterogeneous population containing both M1-like and M2-like cells. Furthermore, we polarize human monocytes into M1-like and M2-like macrophages and treat cancer cells with M1 and M2 macrophages conditioned medium. We find that different macrophage subtypes have a distinct effect in modulating epithelial plasticity of cancer cells. Interestingly, polarized macrophages regulate cancer cell morphological changes through epigenetic regulation. Therefore, we propose that TAMs in tumor microenvironment may polarize into different subtypes and facilitate cancer metastatic dissemination. These findings have implications for designing alternative strategies aimed at preventing and inhibiting cancer metastasis.

Citation Format: CHIH-CHAN LEE, Muh-Hwa Yang. Regulation of epithelial plasticity of cancer cells by tumor-associated macrophages. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 474. doi:10.1158/1538-7445.AM2015-474
Abstract 4750: Transcriptome sequencing of the Reed-Sternberg cells of classical Hodgkin lymphoma
Authors: Reichel Jonathan , Brody Joshua , Elemento Olivier , Rabadan Raul , Cesarman Ethel
Published: August 2015

Introduction: Most genomic studies of classical Hodgkin lymphoma (cHL) have been confined to cell lines due to the difficulty of isolating sparsely distributed Hodgkin and Reed-Sternberg (HRS) cells from reactive background tissue. One approach to evaluate primary cases has been to microdissect HRS cells from fresh-frozen tissue biopsies, which has been used for gene expression profiling and array comparative genomic hybridization to assess copy number alterations. However, laser capture microdissection is technically challenging, does not provide a pure tumor cell population, and yields very small amounts of degraded nucleic acids that may not be adequate for whole-transcriptome level RNA sequencing.

Methods: We used a flow cytometric cell isolation method, which has enabled rapid isolation of thousands of viable HRS cells from primary cHL tumors (Fromm, et al., Am J Clin Pathol 126, 2006). Here we combined flow cytometry using CD64, CD95, CD30, CD5, CD20, CD15, CD40, and CD45, with the Arcturus PicoPure RNA Isolation kit to isolate high quality RNA from viable HRS cells and intratumor B cells from nine primary cases of CHL respectively. We applied the Clontech SMARTer Ultra Low Input RNA Kit followed by Kapa Library Preparation kit to generate what is to our knowledge the first full transcriptome RNA sequence data from primary cases of cHL. Four cell lines were sequenced using the same methodology. We performed differential expression and gene fusion analyses, and conducted a search for activated pathways.

Results: We report the expression signature of purified primary HRS cells. PCA analysis showed that these segregate from HRS cell lines and primary B cells. We confirmed at the RNA level overexpression of relevant amplified genes for which inhibitors are available (AURKA and EZH2), and underexpression of transcripts encoded by genes previously found to be deleted (for example ATM). Pathway analysis revealed extensive downregulation of antigen presentation, consistent with our previous exome sequence analysis showing mutations in beta-2 microglobulin as the most common genetic alteration. Results of the mutational and transcriptional landscape of the HRS cells will be presented.

Conclusions: Transcriptome-level analysis of flow-sorted HRS cells allows for expanded study of CHL pathogenesis, new target identification based on altered signaling pathways and potentially individualized approaches to CHL therapy.

Citation Format: Jonathan Reichel, Joshua Brody, Olivier Elemento, Raul Rabadan, Ethel Cesarman. Transcriptome sequencing of the Reed-Sternberg cells of classical Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4750. doi:10.1158/1538-7445.AM2015-4750
Abstract 4751: Simple modifications in Ion Torrent Variant Caller (TVC) parameters improve somatic variant detection from circulating tumor cells and cell free DNA to 1% allele frequency
Authors: Davidson Colin , Sun Yongming , Davies Christopher , Parikh Chaitali , Tom Warren , Hyland Fiona , Scafe Charles , Dhingra Dalia
Published: August 2015

By increasing quality-associated parameters and lowering sensitivity thresholds, a significant improvement in low frequency allele detection was achieved. The approach described was optimized using a dilution series of synthetic spike-ins (the AcroMetrix® Oncology Hotspot Control), and a titrated range of tumor cell lines in a normal background. The optimized variant calling approach was then applied to the analysis of somatic variants in circulating tumor cell (CTC) samples and associated cell-free DNA (cfDNA) using next-generation sequencing (NGS). Libraries were produced using the Ion AmpliSeq™ Cancer Hotspot Panel v2 for coverage of 50 cancer-associated genes and 2,800 COSMIC mutations. Using the above samples, the improved somatic variant detection workflow demonstrated analytical sensitivity down to 1% allele ratio in the observed sequence reads.

Citation Format: Colin Davidson, Yongming Sun, Christopher Davies, Chaitali Parikh, Warren Tom, Fiona Hyland, Charles Scafe, Dalia Dhingra. Simple modifications in Ion Torrent Variant Caller (TVC) parameters improve somatic variant detection from circulating tumor cells and cell free DNA to 1% allele frequency. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2015-4751