bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 672: Baseline serum albumin as a prognostic factor in patients with stage I and II colon adenocarcinoma
Authors: Onate-Ocana Luis F.
Published: July 2017

Background: Patients with stage I or II colon carcinoma (CC) have a significant risk of recurrence after adequately performed curative resection. Adjuvant chemotherapy has not improved recurrence or survival in such patients. In this study, a prognostic model in patients with stage I or II CC is presented.

Methods: Consecutive cases with CC treated at a cancer center in Mexico City from January 2008 to December 2014, with diagnosis of adenocarcinoma by colonoscopy and biopsy were included in this cohort. Patients were treated according to standard guidelines. Follow-up continued until June 2016; the Kaplan-Meier method and the Cox model were used to analyze the association of prognostic factors and overall survival (OS).

Results: 1,259 cases of CC were treated; only 496 patients have stage I and II CC and were included in the database: 234 women (47.2%) and 262 males (52.8%) and their mean age was 59.9 years (SD 14.9); 96, 339, 31 and 30 cases were pTNM stages I, IIa, IIb and IIc, respectively. Multivariate analysis of prognostic factors is depicted below. Predicted survival functions using this model, defined 4 risk groups that showed better prognostic value than simple TNM stages I-IIc.

Conlusion: BSA is an independent, significant, simple, cheap and widely available prognostic factor in patients with CC. BSA and other clinical factors add significant prognostic information to TNM classification. Feasibly, it can be used to develop multivariate prognostic models with clinical impact defining the use of adjuvant chemotherapy. The use of histopathological and immunohistochemical markers could improve the prognostic information of this model.

Multivariate análisis of prognostic factorsBetaSEpHR95% CIMale gender0.3550.1660.0321.4261.031-1.973Hemoglobin-0.0880.0380.020.9160.85-0.986Platelet count-0.0020.0010.0010.9980.996-0.999Neutrophil count0.0820.0280.0041.0851.027-1.147Albumin0.7820.2280.0012.1851.397-3.417Albumin/Globulin-1.6240.4880.0010.1970.076-0.513BMI-0.0420.0220.0510.9580.918-1.0TNM 0/I0.0541TNM IIa0.5540.2550.031.741.056-2.869TNM Iib0.7340.4040.0692.0830.944-4.597TNM IIc0.950.3670.012.5861.26-5.305Chemotherapy-0.7650.2<0.00010.4650.315-0.688R0<0.00011R10.2670.4750.5741.3060.515-3.311R20.9360.3480.0072.5511.29-5.044No Resection1.9360.288<0.00016.9323.944-12.18Platelet/Lymphocute ratio0.0010.0010.081.0011.0-1.002

Citation Format: Luis F. Onate-Ocana. Baseline serum albumin as a prognostic factor in patients with stage I and II colon adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 672. doi:10.1158/1538-7445.AM2017-672
Abstract 679: Serine/threonine kinase inhibitors on improving symptoms of neuroendocrine tumor
Authors: Liu I-Hua , Ding Yusian , Liou Chiung-Wen , Chang Jia-Ming
Published: July 2017

Recently, neuroendocrine tumors (NETs) are increased and mostly occurred in gastrointestinal tract and lung in the worldwide. It secretes functional hormones and non-functional neural transmitters to affect patients unwell so called carcinoid syndrome. In clinically, surgical removal and somatostatin treatment are used to ease the syndrome by NETs. A new strategy for advanced NETs is used by targeting mTOR signaling and neovascularization. In 2011, Everolimus, an mTOR inhibitor, was approved for the treatment of progressive NETs of pancreatic origin in patients with unresectable, locally advanced or metastatic disease, but safety and effectiveness of everolimus have not been established. In this study, low dose of mTOR inhibitor was investigated for capability in modifying the carcinoid syndrome. In an NCI-H727 NET xenograft model, the level of chromogranin A (CgA) was determined in accordance with tumor volume. With multiplexing the serum hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin were lowered in tumor-bearing mice, but they were rebound when animals treated with Everolimus. Another mTOR inhibitor INK 128 had no apparent tumor inhibition in the same model with doses ranged from 0.25-1 mg/kg. There was no change in the level of CgA in INK128-treated tumor-bearing mice but alpha-MSH level was restored compared with normal mice at a dose-dependent manner. In conclusion, we discovered that low dose of mTOR inhibitor could modify the carcinoid syndrome, where alpha-MSH and beta-endorphin may serve as biomarkers for prognoses of mTOR treatment.

Citation Format: I-Hua Liu, Yusian Ding, Chiung-Wen Liou, Jia-Ming Chang. Serine/threonine kinase inhibitors on improving symptoms of neuroendocrine tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 679. doi:10.1158/1538-7445.AM2017-679
Abstract 68: Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models
Authors: King Emily M. , Mazor Ronit , Pastan Ira
Published: July 2017

Recombinant immunotoxins (rITs) are composed of a tumor antigen-targeting antibody fragment fused to a portion of Pseudomonas exotoxin A. rITs have been effective in clinical trials for patients with hematologic malignancies. The CD22 targeting rIT Moxetumomab Pasudotox has achieved overall response rates of 86% and complete remission rates of 46% in patients with relapsed-refractory Hairy Cell Leukemia. However, the therapeutic efficacy of rITs against solid tumors is limited by their immunogenicity in immune-competent patients. In clinical trials to treat mesothelioma patients with SS1P, a rIT targeting mesothelin, 90% of patients developed neutralizing antibodies against SS1P after one cycle of treatment. When immunosuppressive chemotherapy and SS1P were combined, more cycles of rIT could be given and several patients with advanced chemo-refractory mesothelioma had striking tumor regressions. This implicates high therapeutic potential for rITs against solid tumors once immunogenicity is surmounted.

Methotrexate (MTX) is a folate antagonist which interferes with purine biosynthesis, and is used to treat osteosarcomas and other cancers. MTX also interferes with T cell responses and is used to treat autoimmune diseases. Based on its immunosuppressive properties, Joly et al. demonstrated that low-dose MTX prevented the formation of ADAs against recombinant human alglucosidase alfa in mice in an antigen-specific manner. We hypothesized that MTX would similarly prevent the formation of ADAs against rITs in an antigen-specific manner. To test our hypotheses, mice were treated with the mesothelin-targeting rIT RG7787 with or without MTX given 0, 24, and 48 hours after RG7787 treatment. Serum was collected and anti-RG7787 ADAs were measured by direct ELISA. We found that six doses of RG7787 combined with low dose MTX (1 mg/kg) inhibited the formation of ADAs against RG7787. This inhibition was sustained through six challenges with RG7787 without additional MTX. Further, we found that immunization with RG7787 plus MTX induced RG7787-specific tolerance, and had no effect on the ADA response against a second protein, ovalbumin.

We conclude that combination of MTX and RG7787 is effective at preventing primary immune responses in a durable, antigen-specific manner. We propose to combine this agent in immune-competent cancer patients receiving rIT therapy to prevent rIT immunogenicity.

Citation Format: Emily M. King, Ronit Mazor, Ira Pastan. Methotrexate prevents primary immune responses against recombinant immunotoxin in murine models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2017-68
Abstract 681: Methylomics profiling reveals epigenetically silenced HS3ST2 sensitize IL6 signaling and confers poor prognosis of ovarian cancer
Authors: Huang Rui-Lan , Chan Hsiang-Ju , Chen Lin-Yu , Hsu Yaw-Wen , Chao Tai-Kuang , Su Po-Hsuan , Weng Yu-Chun , Yuan Chiou-Chung , Lai Hung-Cheng
Published: July 2017

The attempts of target therapy in ovarian cancer therapy mostly fail. There is a need of biomarkers for better patient stratification in clinical trials. Epigenetics is a driving force for cancer development and may serve as a molecular marker of disease. The present study was to analyze the clinical relevance of methylation phenotypes and figure out the mechanism in ovarian cancer. We integrated the analysis using public dataset of epigenomics and transcriptomics, and our hospital-based methylomic database and tissue arrays of patients diagnosed with epithelial ovarian cancers (EOC). Bioinformatics results were verified and validated in cell lines and clinical samples. From 25 genes, we identified even genes, ADRA1A, CD248, HS3ST2, NEFH, IGSF21, POU4F2, and TWIST1, of which hypermethylation was significantly related to the poor overall survival (OS). The association of NEFH and HS3ST2 hypermethylation and poor 5-year OS was validated in our independent dataset and TCGA database. The NEFH/HS3ST2 hypermethylated signature was an independent risk factor for 5-year OS (adjusted Hazard ratio = 2.93, 95% confidence interval = 1.25 to 6.85, P = 0.013). In vitro experiment re-expressing HS3ST2 repressed migration and invasion, and suppressed IL-6 signaling pathways. The combination of hypermethylation of HS3ST2 and high expression of IL-6 confers the worse outcome. Our data suggested that methylation of NEFH/HS3ST2 constituted a poor prognostic signature of ovarian cancer. The sulfation status of proteoglycan modified by HS3ST2 implicates an intrinsic sensitivity of ovarian cancer cells to IL-6, which shed a new light on the application of HS3ST2 as a biomarker for personalized medicine of targeting IL-6.

Note: This abstract was not presented at the meeting.

Citation Format: Rui-Lan Huang, Hsiang-Ju Chan, Lin-Yu Chen, Yaw-Wen Hsu, Tai-Kuang Chao, Po-Hsuan Su, Yu-Chun Weng, Chiou-Chung Yuan, Hung-Cheng Lai. Methylomics profiling reveals epigenetically silenced HS3ST2 sensitize IL6 signaling and confers poor prognosis of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 681. doi:10.1158/1538-7445.AM2017-681
Abstract 690: Identifying the genetic basis of stage 4S neuroblastoma
Authors: Vaksman Zalman , McDaniel Lee D. , Diamond Maura , Maris John M. , Diskin Sharon J.
Published: July 2017

Introduction: The 5 year mortality rate for stage 4 metastatic neuroblastoma (NBL) is ~50%; however, the stage 4S subset of NBL is typically classified as low-risk with tumors exhibiting spontaneous regression. To date, the underlying genetic mechanisms influencing this distinct phenotype remain unknown.

Method: To identify genetic determinants specific to the 4S phenotype, we performed genome-wide association study (GWAS) analyses. Blood DNA samples from 6,195 NBL patients and 11,384 non-NBL controls were genotyped using Illumina SNP arrays. European ancestry subjects were selected for inclusion in a discovery [stage 4S (n=148), stage 4 (n=996), non-NBL controls (n=9,923)] or replication cohort [stage 4S (n=76), stage 4 (n=608), non-NBL controls (n=1,461)]. Following imputation using 1KG Phase 3, we conducted case:case comparisons including stage 4S vs. 4 and stage 4S vs. 1, followed by comparisons of NBL cases in each group to matched non-NBL controls. Variants uniquely associated with 4S NBL were defined as those meeting the following criteria: (1) stage 4S vs. 4 meta P < 5.0 x 10-5 and P < 0.005 in the discovery cohort (2) 4S vs. non-NBL control P < 0.05 and (3) either not significant (P > 0.05) or odds ratio in the opposite direction in 4 vs. non-NBL controls.

Results: 4S-specific variants that met our criteria spanned multiple regions with several harboring genes important to neuronal function including 9q34 (SARDH/DBH; rs2502746), 1p32.2 (KCNH1; rs12122529), 1p34 (KCNQ4; chr 1:41317634), 7q34 (CNTNAP2; rs79403134) and 2q24.1 (KCNJ3; rs78829625). At the 9q34 locus, rs2502746 showed further evidence as an expression quantitative trait locus (eQTL) for Dopamine Beta-Hydroxylase Antisense RNA1( DBH-AS1 P = 1.9 x 10-5 sun-exposed skin) and Sarcosine Dehydrogenase SARDH expression (P = 2.5 x 10-6 whole blood). Other 4S-specific loci observed include 3q27.3 (rs113207181), 8p21.3 (rs12548388), and 15q12 (rs62189512). Notably, none of the previously identified NBL susceptibility loci were found to be associated with 4S NBL in the 4S vs. non-NBL control comparison, suggesting a unique genetic basis for this enigmatic subset.

Discussion: Although a unifying mechanism underlying 4S NBL has yet to be identified, our results suggest a potential role for ion-channel and neuronal function genes. Ongoing efforts include investigating the role these genes and associated pathways play in NBL, including an integrative genomic, epigenomic and transcriptome analysis of stage 4 and 4S NBL.

Citation Format: Zalman Vaksman, Lee D. McDaniel, Maura Diamond, John M. Maris, Sharon J. Diskin. Identifying the genetic basis of stage 4S neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 690. doi:10.1158/1538-7445.AM2017-690
Abstract 695: Targeted nanoparticle therapy for poor prognosis Ewing’s sarcoma
Authors: Kang HyungGyoo , Nagy Jon , Samulak Julia , Triche Timothy
Published: July 2017

Targeted nanoparticles have shown the potential to deliver anticancer drugs to cancer cells selectively and thus to overcome unexpected cytotoxicity and limited efficacy of chemotherapy caused by non-selective delivery to normal cells. However, the optimal formulation of targeted nanoparticles for actual translational applications has not been established. By refining early formulations and after extensive testing, we have created an optimized targeted delivery system, HPLNs (Hybrid Polymerized Liposomal Nanoparticles) and demonstrated that this new, targeted nanoparticle technology overcomes many of these deficiencies. This technology has tremendous potential for treating virtually any type of cancer. The goal of our present study was to determine the efficacy and toxicity of this novel targeted nanoparticle in the treatment of Ewing’s Sarcoma, a rare and highly lethal cancer of children and young adults. The efficacy of this targeted delivery system was compared to 1) administration of the free drug and 2) the drug in untargeted nanoparticle form. Our studies have shown that HPLN-Dox targeted with human antiCD99 antibodies can successfully inhibit the growth of Ewing’s Sarcoma in murine models without any detectable off-target toxicity. The deliverable for our study was to demonstrate that the effectiveness of doxorubicin in the treatment of recurrent Ewing’s sarcoma could be dramatically improved when encapsulated in human antiCD99 antibody targeted HPLNs. At the same time, toxic side effects normally seen with free drug was not detectable. Success with human anti-CD99 antibodies targeted HPLNs containing Doxorubicin was then easily adapted to other tumor types (Leukemia, Prostate or Pancreatic cancers) and other payloads such as irinotecan, EWS-FLI1 specific ASOs, siRNA and CRISPR/Cas9 targeting the ETS domain of the chimeric EWS-ets gene found in all Ewing sarcoma. In conclusion, our study showed a potential of targeted HPLN as a selective delivery vehicle of cytotoxic payloads to cancers and this technology is then easily adapted to other tumor types and virtually any small molecule therapeutic.

Citation Format: HyungGyoo Kang, Jon Nagy, Julia Samulak, Timothy Triche. Targeted nanoparticle therapy for poor prognosis Ewing’s sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 695. doi:10.1158/1538-7445.AM2017-695
Abstract 711: Mechanistic implications of COL1A1 as a prostate cancer biomarker
Authors: Jedinak Andrej , Vuichoud Camille , El-Hayek Andrew , Kaplan Katherine , Savage Jason , Prophet Sarah , Feldman Adam S. , Camphausen Kevin A. , Loughlin Kevin R. , Moses Marsha A.
Published: July 2017

Prostate cancer (PCa) is the second most frequently diagnosed form of male cancer and shares similar symptoms with BPH (Benign Prostate Hyperplasia), a disease characterized by prostate enlargement. Elevated levels of prostate-specific antigen (PSA) can be observed with either benign or malignant growth of the prostate and therefore cannot effectively discriminate between these two prostate diseases. Currently, a test that sensitively and accurately distinguishes between BPH and localized prostate cancer does not exist creating an urgent need for novel biomarkers that can successfully distinguish between these two prostate diseases. The goal of this study was to identify and validate non-invasive urinary biomarkers that distinguish between BPH and PCa. Our previous proteomic study identified elevated levels of several proteins, including EGF (epidermal growth factor), HE-4 (human epididymis protein 4), COL1A1 (collagen, type I, alpha 1) and other proteins in the urine of PCa patients compared to urine samples from patients with BPH. In this current study we have analyzed and validated the presence of EGF, HE-4 and COL1A1 by enzyme-linked immunosorbent assay (ELISA). Our ELISA experiments revealed that COL1A1 was significantly (P < 0.002) elevated in the urine of patients diagnosed with early or localized PCa vs. BPH. In vitro experiments performed on seven different prostate cell lines identified cells from the tumor microenvironment that secrete the highest levels of COL1A1. Expression of COL1A1 by these cells was confirmed by immunohistochemistry (IHC) using prostate tissue microarrays (TMA). In addition, protein array experiments identified elevated levels of several proteases in the urine of PCa patients, including MMP-9, uPA, ADAM-TS1 and several cathepsins. Substrate gel electrophoresis (zymography) revealed elevated activity of both MMP-9 and MMP-2 in urine from PCa patients. These data suggest that MMP-9 and MMP-2 may participate in the cleavage of collagen type 1, resulting in elevated levels of COL1A1 in urine of PCa patients. We are currently testing this hypothesis. In summary, COL1A1 may represent a novel non-invasive urinary biomarker that can effectively discriminate between BPH vs. localized PCa. (Supported by The Ellison Foundation)

Citation Format: Andrej Jedinak, Camille Vuichoud, Andrew El-Hayek, Katherine Kaplan, Jason Savage, Sarah Prophet, Adam S. Feldman, Kevin A. Camphausen, Kevin R. Loughlin, Marsha A. Moses. Mechanistic implications of COL1A1 as a prostate cancer biomarker [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 711. doi:10.1158/1538-7445.AM2017-711
Abstract 723: Improved detection of cancer specific serum exosomal aspartyl (asparaginyl) beta hydroxylase (HAAH)
Authors: Semenuk Mark A. , Cifuentes Anokhi S. , Ghanbari Eleanor R. , Lebowitz Michael S. , Ghanbari Hossein A.
Published: July 2017

By means of an ELISA, we have found the pan-cancer biomarker HAAH to be closely associated with serum exosomes primarily in cancers of the breast, lung, colon, and prostate. This new understanding of the HAAH target has led to a dramatically improved serological detection assay, and in turn diagnostic reagent kit. In keeping with the multivalent particulate nature of exosomes, appropriately reformatting the ELISA assay has yielded a simplified so-called simultaneous-homologous format. Currently in this format, a single monoclonal anti-HAAH antibody (FB50) serves both as microplate capture and as a biotinylated detector. All reagents, including peroxidase-streptavidin, are co-incubated in the FB50- coated microplate simultaneously with serum samples, in the absence of intervening sequential steps.

This considerably shorter exosome-enabled ELISA format requires half the time and yields generally on average a two-fold or higher increase in detection of HAAH. This format was evaluated with over 100 banked serum samples from several early stage cancers. While increasing the sensitivity for HAAH in cancer samples, the improved detection and performance has not caused any undesirable increases in false positives among more than 30 normal serum samples from healthy subjects. The reagent kit components include recombinant HAAH calibrators, positive /negative controls, vialed detection reagents and a Mylar packaged FB50 pre-coated microplate. The simplified reagent kit format has yielded close inter-operator and day to day trending consistency with a limit of detection (LOD) of 3 ng/mL.

Recognizing the exosomal nature of the HAAH target has resulted in some changes in blood sample procurement, sample processing, and sample shipping. Such field testing of the HAAH reagent kit was recently done with serum samples from 48 high risk or mildly symptomatic volunteers with general concerns about cancer. Upon testing these samples 9 had an HAAH level greater than 3.0 ng/ mL (range 4.2 to 116.7) and 39 had less than 3.0 ng/ mL. Based upon the post-analysis diagnostic determination, there were two false negatives and one false positive, hence an overall accuracy of 93.8%.

Citation Format: Mark A. Semenuk, Anokhi S. Cifuentes, Eleanor R. Ghanbari, Michael S. Lebowitz, Hossein A. Ghanbari. Improved detection of cancer specific serum exosomal aspartyl (asparaginyl) beta hydroxylase (HAAH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 723. doi:10.1158/1538-7445.AM2017-723
Abstract 730: High sensitive detecting procedure of circulating repetitive RNA as novel early marker of pancreatic cancer
Authors: Kishikawa Takahiro , Otsuka Motoyuki , Koike Kazuhiko
Published: July 2017

Pancreatic ductal adenocarcinoma (Pdac) is one of the most intractable malignancies due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. It is difficult to correctly quantitate repeat arrays because its repetitive nature makes it difficult to establish appropriate primers that amplify a single product using simple PCR procedures. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues. HSATII RNA core sequence levels in sera were significantly higher in Pdac patients compared with non-cancer patients (median copy number: 14.75 and 3.17 per µl in the training set and 17.35 and 2.9 in the validation set, respectively). In addition, patients with intraductal papillary mucinous neoplasm (IPMN), a precancerous lesion of Pdac, could also be efficiently detected. This method can be routinely applied to screen patients with Pdac and high-risk patients, facilitating the development of preventive medicine for this disease.

Citation Format: Takahiro Kishikawa, Motoyuki Otsuka, Kazuhiko Koike. High sensitive detecting procedure of circulating repetitive RNA as novel early marker of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 730. doi:10.1158/1538-7445.AM2017-730
Abstract 737: Signficant DLX4 expression in inflammatory breast cancer tumors from African American patients
Authors: Jeong Jaehong , Naab Tammey , Ongkeko Martin , Makambi Kepher , Khan Farhan A. , Blancato Jan
Published: July 2017

Purpose: To examine protein expression characteristics of Distal-less homeobox4 (DLX4) in inflammatory breast cancer (IBC) cases from an African-American population to determine if a) DLX4 over-expression occurs in IBC vs. normal mammary tissue and b) over-expression is associated with clinico-pathologic features such as ER, PR, HER2.

Experimental Design: Twenty nine blocks of formalin-fixed paraffin-embedded (FFPE) tissues from well-characterized human IBC cases were used for immuno-histochemical staining (IHC). Tumor tissues were from African American subjects seen at Howard University Hospital. Normal breast tissues from 30 mammoplasties were controls. IHC results were assigned intensity and percentage scores based upon intensity of positive staining and % of stained cells. Percentage scores were assigned as 0, 1 (0%-25%), 2 (26%-50%), 3 (51%-75%) or 4 (76%-100%) and intensity scores were assigned 0, 1+, 2+ or 3+. For the analysis of the IHC, a percentage score of 3 or 4 was considered high and an intensity score of 2+ or 3+ was categorized as high. Chi-square and Fisher’s exact tests were used for analysis.

Results: The staining pattern for the categorized cohort in which 82.8% (24 out of 29) of IBC cases showed high percentages of positive cells staining for DLX4 protein, while 40.0% (12 out of 30) normal breast tissues demonstrated DLX4 expression (P=0.001). In terms of staining intensity, 75.9% (22 out of 29) of IBC cases showed a high level of intensity, compared to 20.0% (6 out of 30) of normal breast tissues (P<0.001). In terms of the association between breast cancer characteristics and IHC staining, intensity of DLX4 was higher in HER2- (87.0%, 20 out of 23) than HER2+ (33.3%, 2 out of 6; P=0.018). Other associations were not statistically significant in this pilot study.

Conclusions: DLX4 expression is significantly higher in this pilot study of IBC cases from AA patients than in normal breast tissue cases. In addition, HER2- is associated with high intensity of DLX4 expression in IBC.

Associations DLX4 protein expression (IHC) In IBC and normal breast tissuesLowHighP-valuePercent positivityn (%)n (%)Controls (n=30)18 (60.0)12 (40.0)0.001IBCs (n=29)5 (17.2)24 (82.8)Staining intensityn (%)n (%)Controls (n=30)24 (80.0)6 (20.0)<0.001IBCs (n=29)7 (24.1)22 (75.9)

Citation Format: Jaehong Jeong, Tammey Naab, Martin Ongkeko, Kepher Makambi, Farhan A. Khan, Jan Blancato. Signficant DLX4 expression in inflammatory breast cancer tumors from African American patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 737. doi:10.1158/1538-7445.AM2017-737
Abstract 746: TaqMan Rare Mutation Assays targeting the TERT promoter region
Authors: Laig Marion , Varma Kamini , Venkatesh Vidya
Published: July 2017

The enzyme Telomerase maintains telomeres at the ends of chromosomes. The Telomerase Reverse Transcriptase (TERT) gene codes for the enzyme’s catalytic domain and is not expressed in normal somatic cells. As a consequence, normal cells acquire senescence by shortening of their telomeres during cell division and eventually undergo apoptosis. In contrast to normal somatic cells, expression of TERT is reinstated in cancer cells causing escape from senescence and apoptosis by maintaining the telomeres. It has recently been shown that mutations in the TERT promoter region play a key role in regulating and reinstating TERT expression. 90% of cancers carry a mutation in the TERT promoter region. Mutations like C228T and C250T create new binding site for the E26 transformation-specific (ETS) transcription factor that regulates TERT expression. Experimental evidence showed that the ETS factor GA-binding protein, alpha subunit (GABPA) binds to the de novo ETS motif and activates TERT transcription in cancer cells.

We undertook a project designing TaqMan Rare Mutation assays addressing mutations in the TERT promoter. These assays are TaqMan SNP Genotyping assays that are optimized for use in digital PCR with the Applied Biosystems QuantStudio 3D. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. This will enable easy and sensitive detection of TERT promoter mutations in cancer research samples. These assays are suitable for detection in liquid biopsy applications with cell free DNA (cfDNA).

Assay design proved to be challenging due to high GC content and repetitive elements in this region of the TERT gene and required varying both the assay design and experimental cycling conditions. Template for wet-lab testing was synthetic plasmid carrying the mutation spiked into wild-type genomic DNA and wild-type genomic DNA control.

The result was a TaqMan Rare Mutation assay detecting the C228T mutation in the TERT promoter region. We showed that designing TaqMan Rare Mutation Assays for digital PCR is feasible for the challenging TERT promoter region. Work addressing additional mutations in this region is ongoing.

For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Marion Laig, Kamini Varma, Vidya Venkatesh. TaqMan Rare Mutation Assays targeting the TERT promoter region [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2017-746
Abstract 747: An improved fixative for histomorphology, recovery of nuclic acids and proteins
Authors: Hewitt Stephen M. , Chung Joon-Yong , Perry Candice , Ylaya Kris , Smith William M-A , Star Robert A.
Published: July 2017

Neutral Buffered Formalin (NBF) has been the primary diagnostic fixative for histopathology for nearly a century. The limitations of NBF are well known, especially with reference to the analysis of RNA and proteins. Alternative fixatives have been proposed, but not been widely adopted. Recently, the US Environmental Protection Agency (EPA) classified formaldehyde as a carcinogen, further tilting the balance away from the continued use of NBF. We recently described a fixative based on 70% ethanol and phosphate buffered saline, called BE70. Using BE70 as the base fixative, we have developed a modular fixative in which different chemicals can be supplemented for “fit for purpose” applications. The addition of guanidinium salts to BE70 , termed BE70G results in a fixative in which both proteins and nucleic acids are improved in quantity and quality after extraction from the paraffin block. The guanidinium replaces non-freezable water, and prevents hydrolysis and oxidation of the biomolecules. Histomorphology is not disrupted at lower concentrations, with only alterations of hematoxylin intensity at higher concentrations. Studies are underway to evaluate the stability of RNA and protein in the tissue block as well as on cut sections. This fixative, BE70G has the potential to address a number of challenges facing the development of precision biomarkers, while preserving histomorphology, and improving the safety of pathology staff. Other formulations are currently being evaluated that may support applications for cytology and electron microscopy. The goal is a well characterized fixative, in which modifications to the chemistry are defined and can be applied to obtain the optimal pathology specimen for both histomorphologic and molecular analysis.

Citation Format: Stephen M. Hewitt, Joon-Yong Chung, Candice Perry, Kris Ylaya, William M-A Smith, Robert A. Star. An improved fixative for histomorphology, recovery of nuclic acids and proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 747. doi:10.1158/1538-7445.AM2017-747
Abstract 76: Synthetic lethal targeting of BRCA mutant tumors with antibody linked pyrrolobenzodiazepine dimers
Authors: Zhong Haihong , Tammali Ravinder , Chen Cui , Fazenbaker Christine , Maureen Kennedy , Monks Noel , Harper Jay , Herbst Ronald , Tice Dave
Published: July 2017

Pyrrolbenzodiazepine dimers (PBDs) are amongst the most potent DNA alkylating agents, with activity against a broad spectrum of tumors. PBDs form cross-links within the minor groove of DNA causing double strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair (HRR) of DSB. Cells defective in BRCA1 or BRCA2 are known to be sensitive to DNA interstrand crosslinks. Accordingly, it is possible that PBD-based ADCs will have enhanced killing of cells (synthetic lethality) in which HR processes are defective by inactivation of BRCA1 or BRCA2 genes in breast, ovarian and other cancers. To determine anti-tumor activity of PBD dimers, we have used MEDI0641, PBD-dimer conjugated to anti-5T4 antibody, against BRCA wild type and mutant xenograft tumor models. MEDI0641 was >3-fold more potent in BRCA1 or BRCA2 mutant models than in wild-type xenografts. Similar observations were seen in 25 patient-derived xenograft (PDX) models (19 breast and 6 ovarian) bearing mutations in BRCA1 or BRCA2 (blinded to 5T4 expression) treated with MEDI0641. Out of a total of 25 PDX models, 17 models had tumor regression with a single administration of MEDI0641 at 0.3 mg/kg (response rate = 68%), and 14 models showed response to 0.1 mg/kg of MEDI0641 (response rate = 56%). In BRCA wild-type PDX models, a higher dose of 1 mg/kg was required to achieve full anti-tumor efficacy. Retrospective analysis of 5T4 expression in PDX tumors demonstrated no correlation between efficacy and target expression in BRCA mutant PDX models. To further delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knock-down of both BRCA1 and BRCA2 in the DNA repair wild type HeLa cells. Knockdown of BRCA genes sensitized Hela cells to PBD payload and MEDI0641 in vitro. Anti-tumor activity of MEDI0641 was further examined in isogenic BRCA2 knockout xenograft models. Genetic deletion of BRCA2 markedly increased anti-tumor activity of MEDI0641. In conclusion, PBD based ADCs may have improved therapeutic window in cancer patients with somatic BRCA mutations.

Citation Format: Haihong Zhong, Ravinder Tammali, Cui Chen, Christine Fazenbaker, Kennedy Maureen, Noel Monks, Jay Harper, Ronald Herbst, Dave Tice. Synthetic lethal targeting of BRCA mutant tumors with antibody linked pyrrolobenzodiazepine dimers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 76. doi:10.1158/1538-7445.AM2017-76
Abstract 760: Detection of EGFR T790M mutation by ddPCR in untreated NSCLC patients: Correlation with clinical outcome
Authors: Ganzinelli Monica , Rulli Eliana , Tamborini Elena , Busico Adele , Russo Giuseppe Lo , Corrao Giulia , Vitali Milena , Garassino Marina Chiara , Broggini Massimo , Marabese Mirko
Published: July 2017

Background: Activating EGFR mutations are associated with response to first generation tyrosine kinase inhibitors (TKI) that significantly increased PFS and OS in NSCLC patients. However, almost invariably, patients relapse while in treatment with TKI, mostly for the presence of EGFR T790M mutation. New second and third generation EGFR inhibitors active against T790M are now under development with very promising results. To date it is not known if the presence of T790M (and its amount) at diagnosis influences clinical outcome of patients with activating EGFR mutation.

Methods: We analysed 50 cases with NSCLC. 48 patients harbour TKI sensitizing mutation in either exon 19 (N=24) or 21 (N=19). 2 EGFR wt patients (blinded to the investigator) were analysed in parallel. These patients were screened by ddPCR for the presence of T790M mutation using Biorad QX200 Droplet Digital PCR System. We used rough DNA extract from FFPE sections after deparaffinization and proteinase K digestion, as available in pathology unit for daily diagnosis.

Results: 12 samples failed to amplify, maybe due to the poor quality of DNA. 13 samples produced results, but were excluded for the analysis because of too low number of calls retrieved. T790M mutation was detected in a significant proportion of remaining cases. Among the latter, 4 patients were sampled after TKI treatment and here the percentage of T790M were higher compared to those patients not treated [0.65% - 28% vs 0.019% - 1.65%, respectively]. One out four of these patients was not detected by routinely method of sequencing (Sanger or RT-PCR). T790M positive samples harboured mutations affecting exon 19 or 21 in equal manner.

Conclusions: ddPCR has proven to be a sensitive method to detect T790M mutation, although the quality of DNA could affect the results. The system has been challenged for specificity an sensitivity and the results proved to be clinically relevant. The correlation between the T790M positivity and clinical outcome will be available and we plan to define a ddPCR threshold value which can help in selecting those patients who are likely to have early recurrence under first generation TKI and that could therefore be directly shifted to second or third generation inhibitors.

Citation Format: Monica Ganzinelli, Eliana Rulli, Elena Tamborini, Adele Busico, Giuseppe Lo Russo, Giulia Corrao, Milena Vitali, Marina Chiara Garassino, Massimo Broggini, Mirko Marabese. Detection of EGFR T790M mutation by ddPCR in untreated NSCLC patients: Correlation with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 760. doi:10.1158/1538-7445.AM2017-760
Abstract 762: A concordance study of the ArcherDX RevealTM ctDNA 28 NGS panel and Biocept’s Target SelectorTM mutation assay using ctDNA collected in Biocept CEE-sureTM blood collection tubes
Authors: Poole Jason C. , Kudlow Brian , Stefanelli Jill , Mishkin Skyler , Pham Anh , Chen Jeff , Singh Veena M. , Stahl Josh , Arnold Lyle J.
Published: July 2017

Biocept’s Target Selector technology is a targeted hotspot mutation panel designed to enrich for mutant targets in a large excess of WT DNA. The test is specific to small regions of interest, is highly sensitive, validated down to 7 mutant copies in a background of 14,000 WT at >98% sensitivity. The ArcherDX Reveal ctDNA test is a 28 gene NGS panel that targets key oncogene activating mutations, drug resistance mutations, in addition to full coverage of TP53. The Reveal ctDNA assay utilizes Anchored Multiplex PCR to enrich, tag, and efficiently capture short ctDNA fragments. Both technologies are designed specifically for use with plasma associated ctDNA. We undertook to evaluate the feasibility of using the ArcherDX Reveal ctDNA 28 NGS panel with the DNA extracted from plasma collected with Biocept’s patented blood collection tube which has been validated at room temperature for 4 days for circulating tumor cells and 8 days for ctDNA. The ability to build complex, targeted libraries, free of chemically induced mutations is of major importance for the detection of the vanishingly rare mutations that can be present in the plasma of cancer patients. We found that high quality NGS libraries were produced from plasma collected and stored in the Biocept blood tube, indicating that little damage occurred to the DNA during preservation. In addition, the two methods were found to be highly concordant and complementary when using both mutation positive and negative patient samples. The Target-Selector assay was the more sensitive, however, the ArcherDX panel revealed several additional mutations not targeted by the Biocept Target Selector assay.

Citation Format: Jason C. Poole, Brian Kudlow, Jill Stefanelli, Skyler Mishkin, Anh Pham, Jeff Chen, Veena M. Singh, Josh Stahl, Lyle J. Arnold. A concordance study of the ArcherDX RevealTM ctDNA 28 NGS panel and Biocept’s Target SelectorTM mutation assay using ctDNA collected in Biocept CEE-sureTM blood collection tubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 762. doi:10.1158/1538-7445.AM2017-762
Abstract 769: Matched-pair comparisons of chemotherapy plus bevacizumab versus chemotherapy alone as third-line treatment or beyond for advanced non-small cell lung cancer
Authors: Hu Binbin , Lu You , Zhou Xiaojuan , Li Qian , Xiang Mengmeng
Published: July 2017

Background: The addition of bevacizumab to chemotherapy has proven efficacy as first-line treatment for advanced NSCLC. Whether this combination still be efficient as salvage treatment for NSCLC patients who have not received bevacizumab previously remains unclear. This retrospective matched-pair study was designed to compare the efficacy and safety of chemotherapy plus bevacizumab with chemotherapy alone as third-line treatment or beyond for NSCLC patients.

Methods: Patients with stage IV NSCLC who had received single-agent chemotherapy plus bevacizumab after failure of at least two prior regimens were enrolled in this study. Each of them was matched with two patients who had received chemotherapy alone (1:2) by age (within ±5 years), sex, smoking history, histology, EGFR mutation status and number of prior regimens. Tumor response rate, survival and toxicity analyses were performed.

Results: Between January 2011 and June 2016, a total of 40 patients who had received chemotherapy plus bevacizumab (chemotherapy-bevacizumab group) were matched with 80 patients who had received chemotherapy alone (chemotherapy-alone group) as third-line treatment or beyond. Objective response rate (ORR) and Disease control rate (DCR) were improved for chemotherapy-bevacizumab compared with chemotherapy alone (ORR, 22.5% v 6.2%, respectively, p= .014; DCR, 62.5% v 40.0%, respectively, p= .032). Progression-free survival was prolonged for chemotherapy-bevacizumab compared with chemotherapy alone (median, 3.9 v 2.5 months, respectively; HR, 0.64; 95%CI, 0.43 to 0.97; p = .032). Incidence of severe (≥ grade 3) adverse events was low and similar across groups.

Conclusion: The combination of chemotherapy and bevacizumab can be an efficient alternative salvage treatment for NSCLC patients who have not received bevacizumab previously. But taking this combination as first-line treatment for advanced NSCLC still should be a priority.

Note: This abstract was not presented at the meeting.

Citation Format: Binbin Hu, You Lu, Xiaojuan Zhou, Qian Li, Mengmeng Xiang. Matched-pair comparisons of chemotherapy plus bevacizumab versus chemotherapy alone as third-line treatment or beyond for advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 769. doi:10.1158/1538-7445.AM2017-769
Abstract 788: Less is more: macrophage depletion via CSF1/CSF1R pathway improves anti-VEGF therapy after adaptive resistance
Authors: Lyons Yasmin A. , Pradeep Sunila , Hansen Jean M. , Wagner Michael J. , Dood Robert L. , Wu Sherry Y. , Previs Rebecca A. , Hu Wei , Coleman Robert L. , Sood Anil K.
Published: July 2017

Objectives: Anti-angiogenesis therapy shows clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance typically occurs. Thus, approaches to overcome such resistance are needed. Here, we systematically assessed immune cell populations enriched during adaptive resistance and identify novel therapeutic avenues.

Methods: A series of in vitro and in vivo (immune competent and nude mice) experiments were carried out. Animals were treated with anti-VEGF antibody continuously until resistance emerged, at which point full immune profiling was performed. Based on these results, efficacy of AC708 (CSF1R inhibitor to target tumor-associated macrophages) was tested in the adaptive-resistance models. F4/80 antibody was used as a macrophage marker.

Results: On the basis of full immune profiling, we detected significantly increased macrophage infiltration in tumors with anti-VEGF antibody resistance compared to tumors from sensitive mice (p<0.0001). Given the dominant role of CSF1R in macrophage function and overexpression of CSF1R in HGSC, we added AC708 following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with AC708 after anti-VEGF antibody resistance demonstrated complete response upon completion of the experiment, while those that did not receive AC708 still had abundant tumor. To mimic treatment with the AURELIA regimen, we next treated mice with anti-VEGF antibody and paclitaxel until resistance emerged, and then AC708 was added. The addition of AC708 restored response to anti-angiogenesis therapy, resulting in 82% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone (p < 0.0001), and a substantial decrease in macrophages (p<0.0004).

Conclusions: The addition of CSf1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects. To confirm these findings, a clinical trial at our institution is nearing activation. The REDIRECT (RandomizEd Induction DIscontinuation TRial of EmaCTuzumab) trial will randomize patients to continue weekly paclitaxel and biweekly bevacizumab with or without a CSF1R inhibitor (emactuzumab) following an induction phase.

Citation Format: Yasmin A. Lyons, Sunila Pradeep, Jean M. Hansen, Michael J. Wagner, Robert L. Dood, Sherry Y. Wu, Rebecca A. Previs, Wei Hu, Robert L. Coleman, Anil K. Sood. Less is more: macrophage depletion via CSF1/CSF1R pathway improves anti-VEGF therapy after adaptive resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 788. doi:10.1158/1538-7445.AM2017-788