bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 675: The interaction between hedgehog and EMT pathway in non-small cell lung cancer and its prognostic implication
Authors: An Ho Jung , Kim Tae-Jung , Sa Young Jo , Kang Chang Suk
Published: July 2017

Introduction: The Hedgehog (Hh) signaling plays essential role in transcription regulation and embryonic development. In many cancers including lung cancer, it is related to carcinogenesis and poor prognosis. This signaling also mediates epithelial to mesenchymal transition (EMT) by paracrine mechanism in various diseases. We aim to study the interaction between Hedgehog and EMT pathway and its clinical implication in non-small cell lung cancer (NSCLC).

Method: Total 271 cases of NSCLC who received surgical resection in Yeouido St. Mary’s Hospital between 1997 and 2011 were included. For immunohistochemical analysis of Hh and EMT related proteins, 4um sections were sliced from tissue microarray. Hh signaling proteins including Sonig hedgehog (Shh), Patched (Ptch), Smoothened (Smo), Glioma-associated oncogene (Gli-1) and Suppressor of fused (Sufu), and EMT associated molecules including E-cadherin and vimentin were analyzed. Staining intensity (IS) was estimated as follows 0 (no staining), +1 (weak), +2 (distinct), +3 (strong). Intensity score (IS) ≥1 was defined as positive. Medical record was retrospectively reviewed. The study was approved by the institutional review board the hospital.

Result: The median age was 63 (18-84) years, and 65.3% were male. Squamous cell carcinoma was diagnosed in 113 (41.7%), adenocarcinoma in 153 (56.5%), and mixed type in 153 (56.5%). Stage 1 and 2 were 46.9% and 30.6%, respectively, and 8 cases had distant metastasis. The Hh proteins showed positive correlation each other. For EMT, the expressions of Hh protein was significantly correlated with tumoral and stromal positivity of vimentin, and inversely with positivity of E-cadherin (P = 0.002 and P = 0.028, respectively). The expression of Hh proteins were significantly associated with lymph node metastasis along with large tumor size (P = 0.021 and P = 0.035, respectively). In survival analysis, expressions of Shh and Gli1 were independent predictor of disease free survival and cancer-specific survival (P = 0.007 and P = 0.025, respectively).

Conclusion: Expression of Hh protein is associated with epithelial-mesenchymal transition and associated with poor prognosis in NSCLC.

Citation Format: Ho Jung An, Tae-Jung Kim, Young Jo Sa, Chang Suk Kang. The interaction between hedgehog and EMT pathway in non-small cell lung cancer and its prognostic implication [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 675. doi:10.1158/1538-7445.AM2017-675
Abstract 676: Nuclear expression of insulin receptor (InsR) adds prognostic information in primary breast cancer patients with high BMI
Authors: Björner Sofie , Rosendahl Ann H. , Simonsson Maria , Markkula Andrea , Jirström Karin , Borgquist Signe , Rose Carsten , Ingvar Christian , Jernström Helena
Published: July 2017

Aim and introduction The aim of this study was to elucidate the prognostic importance of nuclear expression of the insulin receptor (nucInsR) in primary breast cancer. The InsR is important for the metabolism, for the insulin-like growth factor (IGF) signaling network and is implicated in breast cancer. Insulin and InsR are interlinked with obesity and body mass index (BMI), which are also associated with breast cancer. The InsR is present both in the membrane and cytoplasm and can additionally translocate to the nucleus. The role of nucInsR is not well understood and the prognostic importance is unclear.

Methods The tumor-specific expression of nucInsR was evaluated by immunohistochemistry on invasive breast cancer tissue microarrays from 984 primary breast cancer patients included in a population-based cohort in Sweden. Patients were followed for up to 11 years, the median follow-up for patients still at risk was five years. nucInsR in relation to prognosis was analyzed using Cox regression, adjusted for age, invasive tumor size, axillary lymph node involvement, histological grade, estrogen receptor (ER) status, BMI, tumor storage time, and treatment.

Results Status for nucInsR was available for 900 patients (91.5%). nucInsRpos staining was present in 214 patients (23.8%) and was associated with lower age (P=0.004), and lower waist-to-hip ratio (P=0.006), but not with BMI, ER or PR status. During follow-up, 107 patients had a first breast cancer event. nucInsR had no prognostic significant impact among all patients. However, there was a significant effect modification of BMI on nucInsR regarding event-free survival (Pinteraction=0.006). Patients with nucInsRpos-expressing tumors and higher BMI (≥25 kg/m2) had lower risk for any breast cancer event, adjusted HR (0.49; 95% CI 0.26-0.93), while patients with nucInsRpos-expressing tumors and normal BMI (<25 kg/m2) were associated with a non-significant higher risk of events, adjusted HR (1.72; 95% CI 0.93-3.17).

Conclusions These results indicate that nucInsR status may add prognostic information in primary breast cancer depending on BMI.

Citation Format: Sofie Björner, Ann H. Rosendahl, Maria Simonsson, Andrea Markkula, Karin Jirström, Signe Borgquist, Carsten Rose, Christian Ingvar, Helena Jernström. Nuclear expression of insulin receptor (InsR) adds prognostic information in primary breast cancer patients with high BMI [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 676. doi:10.1158/1538-7445.AM2017-676
Abstract 677: Progranulin/GP88 tumor tissue expression further stratifies survival outcomes of patients classified by their Nottingham Prognostic Index
Authors: Serrero Ginette , Hawkins Douglas , Ioffe Olga , Bejarano Pablo , Yue Binbin
Published: July 2017

Background: The Nottingham Prognostic Index (NPI) combines nodal status, tumor size and histological grade and is used to provide predictive value information on survival for patients with primary breast cancer. Attempts to improve the performance of NPI have been carried out by inclusion of other factors, such as biomarker expression as well as addition of other structural features such as vascular invasion. In the present study, we investigated whether expression of the survival factor GP88 (progranulin), known to be overexpressed in breast cancer, would improve NPI’s predictive value. GP88/Progranulin is an 88 kDa autocrine growth and survival factor that is overexpressed in estrogen receptor positive (ER+) invasive ductal carcinoma (IDC) in association with resistance to anti-estrogens and aromatase inhibitor while the cells remain ER+. Previous training and validation pathological studies totaling 600 cases of ER+ IDC had demonstrated that GP88/Progranulin tissue expression was an independent prognostic marker od recurrence and mortality for early stage ER+ IDC.

Methods: We examined by immunohistochemistry the GP88 tumor tissue expression of 508 formalin fixed paraffin embedded cases of ER+ IDC with known clinical outcomes and for which NPI had been determined. GP88 IHC expression was scored by two board certified pathologists and classified into two score groups of GP88 < 3+ (0, 1+, 2+) and GP88 = 3+. The correlation between GP88 scoring, NPI and disease-free (DFS) and overall survival outcomes (OS) was then examined by Kaplan Meier analysis, Cox proportional Hazard ratio and Pearson’s X2 test.

Results: Patients were categorized into three NPI groups (< 3.4, 3.4-5.4, >5.4). Kaplan-Meier survival graphs of cases categorized by their NPI scores and GP88 expression showed that for each NPI subgroup, patients having tumors with a high GP88 expression (GP88 IHC score of 3+) had a worse DFS than patients within the same NPI subgroup with low GP88 expression (GP88 IHC score < 3+). When adjusted for NPI, high GP88 score was highly significantly associated with recurrence. Its hazard ratio is 3.30 (95% CI 2.12 to 5.14).

Conclusions: These data suggest that the determination GP88 tumor expression at time of diagnosis for early stage breast cancer patients could provide additional survival information than the ones provided by NPI alone that maybe useful for improving the risk management of patients.

Citation Format: Ginette Serrero, Douglas Hawkins, Olga Ioffe, Pablo Bejarano, Binbin Yue. Progranulin/GP88 tumor tissue expression further stratifies survival outcomes of patients classified by their Nottingham Prognostic Index [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 677. doi:10.1158/1538-7445.AM2017-677
Abstract 678: A strategy to overcome the interfering problem of tissue embedding OCT compound in activity based multiplex profiling of tyrosine kinase substrates
Authors: Arni Stephan , Caviezel Claudio , Weder Walter , Hillinger Sven
Published: July 2017

Introduction: The analysis of clinically relevant human tissue preserved in optimal cutting temperature (OCT) medium with activity based proteomic approaches are promising for the discovery of novel druggable disease biomarkers for the diagnosis, prognosis and prediction of response to therapeutic interventions. Nonetheless, and for many different proteomic approaches, there are important signal interferences observed in the presence of the OCT compound.

Methods: We tested activity based multiplex profiling tyrosine kinase substrates in a large batch of neoplastic and non-neoplastic lung resection specimen embedded with or without OCT. We observed significant differences in overall phosphorylation levels and searched for reasons explaining such a large effect.

Results: We ruled out the implication of either short versus long storage time after sample extraction or of nonhomogeneous batch processing of samples. We documented that the clear downward shift in overall phosphorylation levels coincided with the introduction of OCT as an improved embedding medium for resection specimen. For all the kinomes extracted, we developed a corrective procedure where a median centering was performed on the values of each peptide, separately for the with or without OCT samples.

Conclusions: We applied corrective filtering of data to the multiplex profiling approach of well characterised tyrosine kinase substrates obtained in lung resection specimen embedded with or without OCT. With the OCT correction parameters applied, the quantitation of molecular prognosis signature based on tyrosine kinase activity differences found in lung adenocarcinoma resection specimens may result in the identification of novel targets for future anti-lung cancer therapies.

Citation Format: Stephan Arni, Claudio Caviezel, Walter Weder, Sven Hillinger. A strategy to overcome the interfering problem of tissue embedding OCT compound in activity based multiplex profiling of tyrosine kinase substrates [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 678. doi:10.1158/1538-7445.AM2017-678
Abstract 689: Immune checkpoint targeting to improve immunotherapy in neuroblastoma
Authors: Ortiz-Ruiz Myrna L.
Published: July 2017

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Current treatments accounts only for at the most 40% of survival rate in high-risk NB patients. Hence, there is a need to develop new strategies that disrupt immunosuppressive signals in the tumor microenvironment in order to achieve robust anti-tumor immune responses. Co-inhibitory signals through receptors such as PD-1 can lead to the inactivation of T cells. On the other hand, co-stimulatory signals through receptors such as 41BB can enhance T cell activity against tumors. These receptors can serve as tools to develop strategies to optimize and enhance T cell anti-tumor functions. In this study, we used 9464D NB tumor mouse model to evaluate antibody treatment as a possible immunotherapy strategy. We further examine the infiltration and function of T cells after targeting checkpoints in 9464D NB tumor bearing mice. Blockade of various receptors in mice bearing 9464D tumor showed that anti-PD1 and anti-41BB delayed tumor growth. Combination therapy with anti-PD1 and anti-41BB antibodies significantly delayed tumor growth compared to single antibody treatment. This combination therapy led to a higher infiltration of CD8+ cells within the tumor. Splenocytes from 9464D tumor bearing mice treated with anti-PD1 and anti-41BB are tumor specific. In conclusion, our findings intend to design the best complement therapy to improve survival of patients with advanced NB. Our data demonstrates that tumor reactive T cells in NB tumors may express different checkpoint receptors including PD1 and 41BB. 9464D tumor bearing mice that received a combination of anti-PD1 and anti-41BB antibodies have smaller tumor size compared to single antibody therapy. Future direction includes examine T cells activation after combination therapy of BMT and immunecheckpoint targeting. Also we want to test adoptive transfer of T cells in tumor bearing mice after BMT. Further we want to measure the infiltration of other immune cells, such as myeloid cells and Tregs, in the tumor environment and possible target immune suppressive populations to see the effects in tumor growth.

Citation Format: Myrna L. Ortiz-Ruiz. Immune checkpoint targeting to improve immunotherapy in 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 689. doi:10.1158/1538-7445.AM2017-689
Abstract 692: Pappalysin-1 is a suitable target for T cell receptor transgenic T cells to kill Ewing sarcoma in vivo and in vitro
Authors: Thiel Uwe , Kirschner Andreas , Thiede Melanie , Grünewald Thomas GP , Rubio Rebeca Alba , Richter Günther , Kirchner Thomas , Busch Dirk , Sorensen Poul , Burdach Stefan
Published: July 2017

Pregnancy-associated plasma protein-A (PAPPA), also known as pappalysin, is a member of the insulin like growth factor (IGF) family. PAPPA acts as a protease, cleaving IGF inhibitors, i.e. IGF binding proteins (IGFBPs), thereby releasing IGFs from IGFBPs. The insulin/IGF-axis is involved in cancer in general and in Ewing sarcoma (ES) in particular. ES is a highly malignant bone tumor characterized by early metastatic spread. PAPPA stimulates normal bone growth, and is also associated with various cancers. In particular, PAPPA is overexpressed and required for proliferation in ES. We isolated HLA-A*02:01+/peptide restricted T cells from A*02:01- healthy donors directed against PAPPA, generated by priming with A*02:01+ PAPPA peptide-loaded dendritic cells. After T cell receptor (TCR) identification, retrovirally TCR transduced CD8+ T cells were assessed for their in vitro specificity and in vivo efficacy in human ES bearing Rag2-/-γc-/- mice. Engraftment in mice and tumor infiltration of TCR transgenic T cells in the mice was evaluated. The TCR transgenic T cell clone PAPPA-2G6 demonstrated specific reactivity towards HLA-A*02:01+/PAPPA+ ES cell lines. We furthermore detected circulating TCR transgenic T cells in the blood in Rag2-/-γc-/- mice and in vivo engraftment of the in bone marrow. Tumor growth in mice with xenografted ES was significantly reduced after treatment with PAPPA-2G6 TCR transgenic T cells compared to controls, and tumors from treated mice revealed tumor infiltrating PAPPA-2G6 TCR transgenic T cells. In summary, we demonstrate that PAPPA is a promising target for TCR based immunotherapy of ES. We demonstrate that TCR transgenic T cells recognize this target, home to the tumor, and causes tumor regression in a preclinical mouse model.

Citation Format: Uwe Thiel, Andreas Kirschner, Melanie Thiede, Thomas GP Grünewald, Rebeca Alba Rubio, Günther Richter, Thomas Kirchner, Dirk Busch, Poul Sorensen, Stefan Burdach. Pappalysin-1 is a suitable target for T cell receptor transgenic T cells to kill Ewing sarcoma in vivo and in vitro [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 692. doi:10.1158/1538-7445.AM2017-692
Abstract 7: Ceftriaxone, an FDA-approved cephalosporin antibiotic, suppresses lung cancer growth by targeting Aurora B
Authors: Li Xiang , Li Haitao , Li Shengqing , Zhu Feng , Kim Dong Joon , Xie Hua , Li Yan , Nadas Janos , Oi Naomi , Zykova Tatyana , Yu Dong Hoon , Lee Mee-Hyun , Kim Myoung Ok , Wang Lei , Ma Wei Ya , Lubet Ronald A. , Bode Ann M. , Dong Ziming , Dong Zigang
Published: July 2017

Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential ‘off’ target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.

Citation Format: Xiang Li, Haitao Li, Shengqing Li, Feng Zhu, Dong Joon Kim, Hua Xie, Yan Li, Janos Nadas, Naomi Oi, Tatyana Zykova, Dong Hoon Yu, Mee-Hyun Lee, Myoung Ok Kim, Lei Wang, Wei Ya Ma, Ronald A. Lubet, Ann M. Bode, Ziming Dong, Zigang Dong. Ceftriaxone, an FDA-approved cephalosporin antibiotic, suppresses lung cancer growth by targeting Aurora B [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 7. doi:10.1158/1538-7445.AM2017-7
Abstract 700: Characterization of the mode of action of Fenretinide treatment in alveolar rhabdomyosarcoma cells
Authors: Brack Eva , Wachtel Marco , Schaefer Beat W.
Published: July 2017

Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by a specific chromosomal translocation encoding the oncogenic transcription factor PAX3-FOXO1. As aRMS cells are addicted to the tumor-specific fusion protein, it may serve as an ideal therapeutic target. Previously, we have identified from a large drug library screen the compound Fenretinide (retinoic acid p-hydroxyanilide), which is already in clinical use, to affect both PAX3-FOXO1 expression as well as aRMS cell viability. The aim of this study was therefore to characterize the mode of action of Fenretinide in more detail. First, we were able to show that Fenretinide induced the generation of reactive oxygen species (ROS) in mitochondria. A more detailed characterization revealed that the Fenretinide-induced ROS derived from an interaction of Fenretinide around complex II of the mitochondrial respiratory chain, leading to the production of superoxides. ROS scavenging as well as complexing of iron ions completely abolished cell death.

To identify the mode of cell death involved, we then used a range of pharmacological inhibitors of specific cell death pathways including Z-vad (pan -caspase inhibitor), Necrostatin-1 (necroptosis pathway inhibitor (RIP-1 kinase inhibitor)), 3-Methyadenine (3-MA) (autophagy pathway inhibitor (phosphatidylinositol 3-kinase inhibitor)) and Ferrostatin (ferroptosis pathway inhibitor) during Fenretinide treatment. Surprisingly, none of these inhibitors alone was able to prevent cell death and even different combinations were not sufficient to completely inhibit cell death. CRISPR/Cas9 mediated depletion of key players in the apoptotic and necroptotic pathway (Bak, Bax and RIPK1) confirmed the pharmacological data. We therefore conclude that other, less characterized cell death pathways or a combination of several pathways including apoptosis and necroptosis might be crucial. Interestingly, electron microscopic examination of cells pointed towards an excessive accumulation of vacuoles to be characteristic.

Taken together, our data show that Fenretinide shows high potential for the treatment of aRMS, inducing several forms of cell death mediated through the production of ROS. These properties open the search for additional compounds acting in a combinatorial manner.

Citation Format: Eva Brack, Marco Wachtel, Beat W. Schaefer. Characterization of the mode of action of Fenretinide treatment in alveolar rhabdomyosarcoma cells [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 700. doi:10.1158/1538-7445.AM2017-700
Abstract 706: Combination ionizing radiation and oncolytic Seneca Valley virus injection enhances tumor cell killing of pediatric gliomas
Authors: Du Yuchen
Published: July 2017

Background and Purpose: Radiation therapy is the traditional and only therapy that offers benefits to patients with diffuse intrinsic pontine gliomas (DIPG) which are highly aggressive pediatric central nervous system tumors without known curative therapies. However, unfortunately, efficacy of radiation therapy is transient. Seneca Valley virus-001(SVV-001) is a novel non-pathogenic oncolytic piconavirus that can be systemically administered and pass through brain blood barrier (BBB). Phase I clinical trial of SVV has proven its efficacy in adults patients with cancers with neuroendocrine features and in pediatric patients with non-central nerve system (CNS) tumors. We sought to examine if combining ionizing radiation with an oncolytic virus SVV-001 would lead to synergistically enhanced tumor cell killing and significantly improve therapeutic efficacy in vivo in DIPG patient tumor-derived intra-brain stem orthotopic xenograft mouse models (PDOX).

Methods: By infecting PDOX derived tumor cells with green fluorescent protein (GFP)-coupled SVV (SVV-GFP) or SVV-001, in vitro virus infection and anti-tumor activity of SVV were examined by cell viability assay, Western Blotting for apoptosis and autophagy-related protein and immunohistochemistry (IHC) staining or Flow cytometry for cell mitochondria content. In vivo therapeutic efficacy was evaluated by systemic administration of SVV into PDOX derived DIPG xenografts alone or in combination with irradiation.

Results: We first had established a novel panel of nine PDOX derived orthotopic xenograft mouse models of DIPG. Our data confirmed that SVV can infect and efficiently kill DIPG tumor cells in vitro by inducing apoptosis and autophagy, leading to improved animal survival in a subset of DIPG xenograft models. We found that the lack of mitochondria in the xenograft tumor cells in vivo impaired the intracellular replication of SVV-001 and subsequently compromised its oncolytic cell killing of tumor cells in vivo. Radiation induced elevated mitochondria biogenesis in tumor cells therefore boosted SVV oncolysis of tumor cells. Moreover, combining fractionated radiation with single i.v.injection of SVV led to significant improvement of animal survival in a subset of DIPG models.

Conclusion: SVV-001 killed DIPG xenograft cells in vitro. Radiation activated mitochondria content of tumor cells in vivo, which restored impaired SVV oncolytic function.Combining radiation with single systemic administration of SVV synergistically enhanced animal survival in a subset of DIPG xenograft models.

Citation Format: Yuchen Du. Combination ionizing radiation and oncolytic Seneca Valley virus injection enhances tumor cell killing of pediatric gliomas [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 706. doi:10.1158/1538-7445.AM2017-706
Abstract 71: Bystander activity and in vivo efficacy of a folate receptor α (FRα)-targeting antibody-drug conjugate with a novel peptide linker
Authors: Qiu Qifeng , Wu Rui , Lanieri Leanne , Maloney Erin , Skaletskaya Anna , Jin Shan , Wang Lintao , Ab Olga , Ponte Joe , Setiady Yulius , Widdison Wayne , Keating Thomas , Chari Ravi , Gregory Richard , Hong Erica
Published: July 2017

Folate receptor α (FRα) is an antigen that is overexpressed on the cell surface of solid tumors including ovarian cancer. The differential expression on cancer cells makes FRα an attractive target for antibody-drug conjugates (ADCs), and an ADC targeting FRα, Mirvetuximab soravtansine, has demonstrated promising activity and safety profiles in the clinic. Here, we employed a new linker (NL) to enhance the bystander activity of ADCs, which is the ability of ADCs to generate cell-permeable catabolites that can diffuse into and kill proximal cancer cells with little or no target expression. With the goal of improving the potency of anti-FRα ADC in solid tumors with heterogeneous FRα expression, we constructed the M9346A-NL-DM. M9346A-NL-DM is a conjugate of the tubulin-disrupting maytansinoid (DM), linked via a novel linker to M9346A, a humanized antibody that binds FRα with high affinity. After cellular uptake and catabolism of the ADC, the new linker undergoes lysosomal cleavage followed by self-immolation to generate free DM that can readily penetrate neighboring cancer cells. Correspondingly, M9346A-NL-DM showed enhanced bystander cytotoxic activity against proximal antigen-negative cells in vitro. In the xenograft tumor models in vivo, M9346A-NL-DM demonstrated enhanced efficacy against tumors with heterogeneous expression of FRα. Interestingly, M9346A-NL-DM also showed improved antitumor activity against a tumor model with homogeneous expression of FRα, possibly due to better tumor penetration of the cell-permeable catabolite. In summary, M9346A-NL-DM is a novel ADC with enhanced bystander activity and antitumor activity that can target tumors with heterogeneous expression of FRα.

Citation Format: Qifeng Qiu, Rui Wu, Leanne Lanieri, Erin Maloney, Anna Skaletskaya, Shan Jin, Lintao Wang, Olga Ab, Joe Ponte, Yulius Setiady, Wayne Widdison, Thomas Keating, Ravi Chari, Richard Gregory, Erica Hong. Bystander activity and in vivo efficacy of a folate receptor α (FRα)-targeting antibody-drug conjugate with a novel peptide linker [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 71. doi:10.1158/1538-7445.AM2017-71
Abstract 720: Systematic discovery and evaluation of tumor-associated antigens (TAAs) as biomarkers in cancer immunodiagnosis
Authors: Shi Jianxiang , Zhang Lu , Wang Peng , Ye Hua , Dai Liping , Li Pei , Luo Chenglin , Song Chuhua , Wang Kaijuan , Wang Xiao , Ouyang Jun , Ji Zhenyu , Zhang Jianying
Published: July 2017

Proteins aberrantly over-expressed in tissues from nineteen types of cancers have been identified from the Human Protein Atlas (HPA) database. Among these proteins, some were overexpressed in several types of cancers while certain proteins were found only overexpressed in certain type of cancers. It is demonstrated that aberrantly overexpressed proteins might shed into the blood and trigger the immune system to produce autoantibodies during the process of oncogenesis. Autoantibodies are more stable and often have higher titer compared to autoantigens. Therefore, we hypothesize that autoantibodies against these aberrantly overexpressed proteins may have the potential to serve as diagnostic biomarkers in certain type of cancers. In current study, we have selected 11 proteins, including EDNRB, KRT4, PLAT, MSLN, WFDC2, F5, FOXA1, AURKC, STC1, CAB39L, and SFTPA1, to further evaluate the diagnostic values of their corresponding serum autoantibodies in five types of cancers, including hepatocellular carcinoma, lung cancer, gastric cancer, breast cancer and ovarian cancer. Sera from 470 patients (94 per cancer type), 94 hepatitis patients and 192 age- and gender-matched normal controls were used in current study. Serum autoantibodies were detected by ELISA. Receiver operating curve (ROC) analysis was used to evaluate the diagnostic value of serum autoantibodies against these potential TAAs. The data indicates that several anti-TAA autoantibodies can be found to have diagnostic value for certain types of cancers. Autoantibodies against PLAT can distinguish breast and ovarian cancer while autoantibodies against SFTPA1 and CAB39L can distinguish cancers both in male and female. However, none of these serum anti-TAAs was found to be specific to a single type of cancer. In summary, HPA database has made systematic discovery of cancer biomarkers possible. Whether these in silicon biomarkers can be used to distinguish normal and cancer still needs to be evaluated in more serum samples from normal controls and cancer patients.

Note: This abstract was not presented at the meeting.

Citation Format: Jianxiang Shi, Lu Zhang, Peng Wang, Hua Ye, Liping Dai, Pei Li, Chenglin Luo, Chuhua Song, Kaijuan Wang, Xiao Wang, Jun Ouyang, Zhenyu Ji, Jianying Zhang. Systematic discovery and evaluation of tumor-associated antigens (TAAs) as biomarkers in cancer immunodiagnosis [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 720. doi:10.1158/1538-7445.AM2017-720
Abstract 725: Feasibility and evaluation of exosomal RNAs as novel diagnostic biomarkers for high grade serous epithelial ovarian cancer
Authors: Prendergast Emily N. , Lin Xianzhi , Corona Rosario I. , Hazelett Dennis J. , Karlan Beth Y. , Lawrenson Kate
Published: July 2017

Introduction: High-grade serous ovarian cancer (HGSOC) survival has been essentially unchanged over the last ten years despite new therapeutic strategies. In order to make a large-scale impact on overall survival, early detection strategies are imperative. Exosomes are cell-derived vesicles derived from multivesicular bodies or the plasma membrane that carry a variety of biomolecules, including protein, DNA, and RNA. Exosomal contents represent a novel and underexplored source of tumor biomarkers, in particular for HGSOC. This study aimed to first evaluate approaches to exosomal RNA purification and to then compare the exosomal RNA profiles from serum of patients with benign ovarian disease and HGSOC to determine if unique profiles exist, and to identify biomarkers useful for early detection.

Methods: Cell free serum was obtained from patients in each of the following categories: HGSOC with early (n=24) or late stage (n=24) disease, benign ovarian masses (n=24) and healthy age-matched controls (n=24). We compared ultracentrifugation and a commercial precipitation solution to enrich and isolate exosomes. Exosomal enrichment was confirmed by both transmission electron microscopy (TEM) and western blot using anti-CD63 antibodies. RNA was extracted for each and deep sequencing analysis was performed for 12 candidates within each group. A second cohort of 48 samples will be used for validation of the best performing candidate biomarkers from the discovery cohort via RT-qPCR.

Results: Enrichment of exosomes was confirmed via qualitative assessment using TEM and Western blot analysis. RNA quality was superior when RNA isolations were performed using the QIAgen RNeasy kit compared to the AllPrep kit. Mean RNA yield for each group ranged from 10.3-28.6ng/µl and RNA yield and quality was independent of sample age. RNAseq analyses are ongoing to identify biomarkers specific to HGSOC that can be detected at the earliest stages. The top differentially RNAs for the ovarian cancer groups will be validated in an independent cohort of specimens. A panel will then be generated in combination with CA125 to determine the clinical utility of an integrated -omics approach in early stage detection of HGSOC.

Conclusions: Efficient isolation of exosomes and RNA can be achieved from archived specimens >10 years old. Evaluation of exosomal RNA in HGSOC provides a novel method for biomarker discovery.

Citation Format: Emily N. Prendergast, Xianzhi Lin, Rosario I. Corona, Dennis J. Hazelett, Beth Y. Karlan, Kate Lawrenson. Feasibility and evaluation of exosomal RNAs as novel diagnostic biomarkers for high grade serous epithelial 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 725. doi:10.1158/1538-7445.AM2017-725
Abstract 733: Detection of HCC-derived major HBV integration junctions in urine and their implications for driver identification
Authors: Lin Selena Y. , Steffen Jamin D. , Su Yih-Ping , Jain Surbhi , Chang Ting-Tsung , Song Wei , Su Ying-Hsiu
Published: July 2017

Chronic Hepatitis B Virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC), a leading cause of cancer mortality worldwide. Integration of HBV DNA into the host genome occurs during the course of chronic infection. Integrated HBV DNA is observed in most of HBV-associated HCC. The integration junctions derived from the original tumor cell become abundant (referred to as major integration junctions) in the infected liver because of clonal expansion during tumor development. Detection of circulating DNA containing these tumor-derived integration junctions may therefore be useful for cancer detection. We have adapted a method known as Primer Extension Capture (PEC) to enrich integrated HBV DNA for next-generation sequencing (NGS). Our initial studies using this approach to enrich the HBV DR1-2 region (a common site for HBV integrated breakpoints) identified major integration junctions from HBV-HCC tissue samples and matched urine. Further analysis revealed that most recurrently targeted integrations from these HCC tumors have previously reported involvement in cancer. This suggested that identification of recurrently targeted genes is applicable for driver identification. Interestingly, we show how HBV targets the TERT promoter in a localized region even though no two TERT junctions examined are identical. We have further developed this PEC to enrich for the entire HBV genome and applied it to (i) liver tissue DNA from 20 matched HCC and adjacent non-HCC samples, and (ii) DNA from urine of 20 hepatitis, 20 cirrhosis and 20 HCC patients. The HBV enriched libraries were sequenced by NGS and the integration events were analyzed using the in-house developed software. The complexity of HBV junction sites in HCC and non-HCC tissue and urine derived circulating DNA is reported. Our approach has potential to be used for liquid biopsies to study the complexities of HBV integrations in chronic HBV infection and carcinogenesis, and to identify HCC-related DNA modifications for early detection and disease management.

Citation Format: Selena Y. Lin, Jamin D. Steffen, Yih-Ping Su, Surbhi Jain, Ting-Tsung Chang, Wei Song, Ying-Hsiu Su. Detection of HCC-derived major HBV integration junctions in urine and their implications for driver identification [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 733. doi:10.1158/1538-7445.AM2017-733
Abstract 745: Interleukin-13 conjugated quantum dots to identify glioma initiating cells and exosomes
Authors: Madhankumar Achuthamangalam B. , Mrowczynski Oliver , Zacharia Brad , Glantz Michael , Xu Lichong , Webb Becky , Siedlecki Christopher , Mintz Akiva , Connor James R.
Published: July 2017

High grade brain tumors like glioblastoma multiforme (GBM) and certain other brain metastatic cancers possess high tumor invasive and infiltrating properties making them harder to detect. Identification of cancer stem cells and exosomes secreted by them in the biofluids like cerebrospinal fluid (CSF) will be an ideal way of detecting the presence of residual cancer after therapy. In the current approach, a ligand conjugated quantum dot (QD) was utilized to specifically label the cancer stem cells and exosomes to identify the presence of IL13Rα2 a tumor invasive marker. Human glioma initiating cells were cultured in adherent form and were exposed to interleukin-13 conjugated quantum dots (IL13QD), which binds the glioma initiating cells expressing IL13Rα2 receptor. Similarly the exosomes isolated from cancer stem cell condition media and CSF was briefly incubated with IL13QD, before analyzing their complexing pattern. Exosomes that were isolated from glioma stem cells and patients CSF were demonstrated to express IL13Rα2 receptor. Fluorescent microscopy, transmission electron microscopy and flow cytometry were performed on the exosomes after complexing with IL13QD. IL13QD was demonstrated to specifically bind glioma stem cells both in the monolayer and spheroid culture. The binding affinity of the exosomes to the quantum dots were quantitatively and qualitatively confirmed by atomic force microscopy (AFM). The morphology and size of exosomes before and after complexing with exosomes were confirmed by electron microscopy and AFM. The density plot from the flow cytometry experiments with IL13QD-exosomes complex indicates a possibility to identify the tumor associated exosomes. Our experiment infers that tumor targeted quantum dots can selectively bind with glioma stem cells and extracellular vesicles (exosomes) that are secreted by cancer cells. This binding profile could be utilized to identify the expression of certain tumor promoting receptors and antigens on the surface of exosomes present in the CSF and cancer stem cells from the glioma patients. Experiments are in progress to identify the tumor initiating cells in an orthotopic mouse glioma tumor model after intravenous administration of wild type IL13 and certain high affinity mutated IL13 conjugated quantum dots, by non-invasive way.

Citation Format: Achuthamangalam B. Madhankumar, Oliver Mrowczynski, Brad Zacharia, Michael Glantz, Lichong Xu, Becky Webb, Christopher Siedlecki, Akiva Mintz, James R. Connor. Interleukin-13 conjugated quantum dots to identify glioma initiating cells and exosomes [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 745. doi:10.1158/1538-7445.AM2017-745
Abstract 749: Novel ALK specific mRNA in situ hybridization assay for non-small cell lung carcinoma
Authors: Hirai Noriko , Sasaki Takaaki , Ohsaki Yoshinobu
Published: July 2017

A recent technical advance in the mRNA in situ hybridization (mRNA-ISH) assay provides simultaneous signal amplification and background suppression with a unique probe design to achieve single molecule visualization. We assessed the utility of the mRNA-ISH assay as a diagnostic tool to detect anaplastic lymphoma receptor tyrosine kinase (ALK) mRNA in non-small cell lung carcinoma. We compared the mRNA-ISH assay with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). The study included 279 surgically resected lung adenocarcinomas and 44 transbronchial biopsied (TBB) adenocarcinomas. mRNA-ISH was conducted using the RNAscope 2.0 system which includes pre-designed detection probes for the tyrosine kinase domain of ALK mRNA. IHC was conducted on all of the 323 samples using ALK-specific antibodies. mRNA-ISH was performed on 279 surgical samples and 6 TBB samples. Break-apart FISH was used to assess cases that were mRNA-ISH or IHC positive. ALK protein expression was detected in 11/279 (3.9%) specimens. ALK mRNA was also detected by mRNA-ISH in these cases, and 9 of the 11 specimens (81%), were also positive by ALK FISH. Using the IHC results as a reference, the sensitivity and specificity of mRNA-ISH was 100%. In the TBB cohort, ALK protein expression was observed in 3/44 (6.8%) of specimens, in which ALK mRNA expression was also detected. ALK mRNA-ISH data were highly correlated with the IHC data, and ALK mRNA-ISH was able to identify every FISH-positive sample. We conclude that mRNA-ISH could play an alternative or complementary role in ALK genetic diagnosis in NSCLC.

Citation Format: Noriko Hirai, Takaaki Sasaki, Yoshinobu Ohsaki. Novel ALK specific mRNA in situ hybridization assay for non-small cell lung carcinoma [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 749. doi:10.1158/1538-7445.AM2017-749
Abstract 751: Characterization of STAT3 activation in human prostate cancer
Authors: Velasco Marco A. De , Hatanaka Yuji , Kura Yurie , Ando Naomi , Sakai Kazuko , Sugimoto Koichi , Nozawa Masahiro , Yoshimura Kazuhiro , Yoshikawa Kazuhiro , Nishio Kazuto , Uemura Hirotsugu
Published: July 2017

Signal transducers and activators of transcription (STATs) were initially associated with cytokine signal transduction pathways but are now also recognized as key modulators of key survival processes in in various cancers. Activation of STAT3 occurs by the binding of various cytokines to its receptors leading to the activation of the JAK/ STAT3 signaling pathway. Interleukin-6 (IL-6) has been implicated in regulating growth of various malignant tumors. Activated IL-6 has also been shown to be elevated in the sera from patients with metastatic prostate cancer, and persistent activation of STAT3 is a common feature. To better characterize the potential role of JAK/STAT3 as a therapeutic target for advanced prostate cancer, we examined expression patterns of activated STAT3 in 111 cases of localized prostate cancer from patients who underwent radical prostatectomy. Association studies were conducted with clinicopathological characteristics and biochemical recurrence. We also analyzed the expression of IL-6/KAK/STAT3 genes from RNA-sequencing data form the Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data set and its association to long-term outcomes. Immunohistochemical analysis revealed that 11.7%, 42.3% and 45.9% of patients had high, moderate or low/negative expression of phosphorylated-STAT3 (Tyr705). Cancerous glands tended to have higher STAT3 expression compared to paired normal adjacent tissue, P=0.088. No correlations were observed between STAT3 expression and clinicopathological parameters. Patients with moderate-high p-STAT3 expression tended to experience shorter times to biochemical recurrence (median time, 15 months) compared to patients with low/negative STAT3 activity, (median time to recurrence not reached, P=0.064). Cluster analysis suggested a trend for decreased disease-free survival for patients with high IL-6/KAK/STAT3 gene expression signature. Our data suggests an association between STAT3 signaling and prostate cancer recurrence and provides clinical evidence to support JAK/STAT3 as a potential therapeutic target for patients with advanced prostate cancer.

Citation Format: Marco A. De Velasco, Yuji Hatanaka, Yurie Kura, Naomi Ando, Kazuko Sakai, Koichi Sugimoto, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Characterization of STAT3 activation in human prostate 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 751. doi:10.1158/1538-7445.AM2017-751
Abstract 753: Human epidermal growth receptor-2 immunohistochemistry in gastric cancer
Authors: Duduyemi Babatunde M. , Afihene Mary Y. , Asiamah Emmanuel A.
Published: July 2017

Introduction: Gastric cancer is the second most common GI cancers and third leading cause of cancer-related deaths worldwide. In Ghana, gastric cancer contributes significantly to cancer morbidity and mortality. The recent development on the benefit of targeted therapy in the management of gastric cancer using HER-2 monoclonal antibody in combination with chemotherapy has greatly improved the overall survival of patients. This study therefore aims at determining the frequency of Her-2 over-expression in gastric carcinomas in Kumasi, Ghana and comparing with other prognostic factors.

Methodology: Demographic data and histological diagnosis were retrieved from the surgical day book of the Department of Pathology, Komfo Anokye Teaching Hospital, Kumasi. The slides were retrieved and reviewed to confirm diagnosis, grading and classification of gastric cancer. Immunohistochemistry was done with anti-HER-2 antibody to confirm HER-2 over-expression. The data was analysed with SPSS version 17. Results: Of the 99 cases of gastric cancer seen over the 8 year period, there were 57 males and 42 females, adenocarcinoma (91), GIST (5) and Non Hodgskin Lymphoma (3). The age range of the study population was 8-90 years with modal age group in the 6th decade. There were more biopsies (63) than resections (36). Of the adenocarcinomas, 45 were poorly differentiated, 38 moderately differentiated and 8 well differentiated. Diffuse type is most common with 47 cases followed by intestinal 41cases and mixed type 3 cases. Three of the 4 patients under the age of 30 years had lymphoma. HER-2 over-expression was seen in 14 out of the 47 tested and all were intestinal type.

Conclusion: HER -2 over-expression was seen a small cohort of our patients with gastric cancer especially those with intestinal type.

Citation Format: Babatunde M. Duduyemi, Mary Y. Afihene, Emmanuel A. Asiamah. Human epidermal growth receptor-2 immunohistochemistry in gastric 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 753. doi:10.1158/1538-7445.AM2017-753