bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4485: VGLL3 is a novel outlier in African American prostate tumors and its expression is associated with a tumor suppressor phenotype in prostate cancer
Authors: Kang Hongjun
Published: August 2018

Higher incidence, progression and mortality of prostate cancer (PCa) have been noted in African American (AA) men compared with Caucasian (CA) men. In fact AA race is an independent predictor of biochemical recurrence for all stage of PCa (Dr. Schaeffer's data analysis, n=1634 of AA patients). The etiology of PCa racial disparities is likely multi-factorial with increasing support of biologic contributors to these differences. By analyzing large cohort of PCa, we have demonstrated that AA PCa exhibits a unique spectrum of molecular subtypes. For example, AA men presented with PCa have a significant decrease in fusion proteins such as ERG, ETS and SPINK, with an enrichment of fusion unknown (FU; triple negative) cases. Given the increased mortality of PCa in AA populations and the enrichment of FU cases among AA tumors, there is an urgent need to define and explore individual outlier as race-based modifiers of disease aggressiveness. By utilizing a large multi-institutional cohort of prostate cancer cases of mixed racial composition, we have identified novel outliers enriched in AA among FU group by COPA outlier analysis.VGLL3 is one of the top outliers enriched in AA. VGLL3 positive cases are associated with low stage (less LNI, p=0.001; SVI, p<0.001; and EPE, p=0.009) and low grade disease (less Gleason >7, p=0.012) of higher protective phenotype of adverse cancer grade and stage. This preliminary work on VGLL3 expression suggests it may be associated with a tumor suppressor-like phenotype with reductions in expression with increasingly aggressive PCa. Studies of VGLL3 in cancer are limited and the role of VGLL3 in prostate has not been explored at all. The goal of this study is to investigate the role of VGLL3 as a novel outlier in AA prostate cancer that modulates disease aggressiveness. We found that mRNA expression levels of VGLL3 were lower in prostate cancer epithelial cells than in normal epithelial cells or stromal cells. By establishing and characterizing PCa models overexpressing VGLL3 in vitro and in vivo, our data demonstrate that VGLL3 significantly reduced migration and invasion of PCa cells and inhibited primary tumor cell growth. Our preliminary data also indicate VGLL3 inhibits PCa metastasis. Furthermore, VGLL3 seems to negatively affect both hippo and AR activity in PCa cells and these might be the mechanisms that VGLL3 inhibits tumor cell growth. These data is consistent with our clinical data suggesting tumor suppressive role of VGLL3 in advanced PCa.

Citation Format: Hongjun Kang. VGLL3 is a novel outlier in African American prostate tumors and its expression is associated with a tumor suppressor phenotype in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4485.
Abstract 4487: Gene expression changes elicited by crosstalk between adipocytes and breast cancer cells
Authors: Lovejoy Leann A. , Costantino Nicholas S. , Shriver Craig D. , Iida Geogre , Ellsworth Rachel E.
Published: August 2018

Background: No longer considered simply an inert energy depot, adipose in the breast is increasingly recognized as an active endocrine tissue. Given the increasing rates of obesity in the US, choice of breast conserving surgery and use of autologous fat grafting, it is critical to understand how signals from adipocytes affect breast tumor cells and vice versa.

Methods: Human subcutaneous preadipocytes were co-cultured for 3 days. Triple negative cell lines MDA-MB-231, SUM-149 and HCC-38 were then added and grown for a total of 10 days, with exposure to mature adipocytes for 7 days. Adipocytes and tumors cells were lysed from the plate and total RNA was isolated. Libraries were prepared for RNAseq and sequenced using a HiSeq2000. Differentially expressed genes were detected using the edge R package in R (v 3.3.3).

Results: Co-culturing breast tumor cells demonstrated 110 differentially expressed genes compared to unexposed tumor cells including 6-fold lower expression of CLCA2 and 9-fold higher expression ZBTB16 in exposed tumor cells compared to controls. Pathway analysis demonstrated upregulation of 23 pathways such in the FoxO signaling and longevity regulating pathways and downregulation of tryptophan metabolism. In co-cultured adipocytes, 89 genes, including 9-fold higher expression of WNT7B and 13-fold lower expression of ADIPOQ. Pathways such as ECM-receptor interaction were upregulated and fat digestion and absorption were downregulated in co-cultured adipocytes.

Conclusions: Exposure of adipocytes to tumor cells alters pathways involved in lipolysis and fatty acid metabolism in the adipocytes which may serve as a fuel source for tumor cells. In conjunction, exposure to adipocytes alters gene expression profiles in tumor cells associated with proliferation, migration and metastasis. These data demonstrate that proximity of breast tumor cells to adipocytes elicit significant gene expression changes in both cell types which synergistically promote tumorigenesis. The views expressed in this abstract are those of the author and do not reflect the official policy of the Department of Army, Navy, Air Force, Department of Defense, or U.S. Government. The identification of specific products, scientific instrumentation, or organization is considered an integral part of the scientific endeavor and does not constitute endorsement or implied endorsement on the part of the author, DoD, or any component agency.

Citation Format: Leann A. Lovejoy, Nicholas S. Costantino, Craig D. Shriver, Geogre Iida, Rachel E. Ellsworth. Gene expression changes elicited by crosstalk between adipocytes and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4487.
Abstract 4488: Osteosarcoma cells interact with bronchial cells in lung to drive production of pro-metastatic cytokines
Authors: Hinckley John M. , Gross Amy , Roberts Ryan
Published: August 2018

The survival rate for patients diagnosed with osteosarcoma (OS) drops from 70% to an abysmal 20% when diagnosed with lung metastasis. We have shown that metastatic colonization of lungs by OS cells depends on the production of IL6 and CXCL8 by tumor cells, which seems to increase within the metastatic niche of the lung. Identification of key tumor-host interactions that drive this phenomenon will facilitate the development of novel therapies aimed at prevention of metastases in patients diagnosed with OS. We hypothesized that OS cells interact with lung tissues in ways that establish paracrine signal amplification loops, where IL6 and CXCL8 stimulate lung-resident cells to produce secondary signaling molecules that, in turn, further stimulate tumor cell production of IL6 and CXCL8 in a feed-forward mechanism. Co-culture experiments have identified strong interactions between OS cells and primary cultures of bronchial epithelial cells and lung smooth muscle cells. Exposure of OS cells to conditioned supernatants from the primary cell cultures show that these interactions result from exchange of soluble mediators secreted by each cell type. Proteomic profiling of bronchial cell supernatants following incubation with OS conditioned media identified a number of intriguing candidate mediators, which might facilitate the growth and survival of OS cells within the microenviroment of the lung. Some of those same candidate mediators trigger expression of IL6 and CXCL8 in OS cell cultures. These results suggest a paracrine loop mechanism, where OS cells trigger a response from lung stromal cells that promotes survival and proliferation of the OS cells, establishing a favorable niche for the development of metastasis.

Citation Format: John M. Hinckley, Amy Gross, Ryan Roberts. Osteosarcoma cells interact with bronchial cells in lung to drive production of pro-metastatic cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4488.
Abstract 4492: Aggressive breast cancer cells reduce CCL5 and Nodal expression in response to a human adipose-derived stromal cell microenvironment
Authors: Berrebi Daniel , Yadav Abhishek , Lundstrom Eric , Werwie Nicole , Muttillo Dominic , Seftor Richard E. , Seftor Elisabeth A. , Vona-Davis Linda
Published: August 2018

Clinical studies indicate that obesity is correlated with poor outcomes in breast cancer patients. Obese individuals have a high proportion of adipose tissue, which is abundant in stem and progenitor cells. Human adipose-derived stromal cells (hADSCs) secrete several cytokines including CCL5, an inflammatory chemokine shown to increase tumor metastatic ability in vitro. We previously identified that CCL5 is secreted into the microenvironment when hADSCs are co-cultured with breast tumor cells. When the clinical relevance of CCL5 was assessed using cBioPortal, an open-source genomics database, we found that CCL5 mRNA overexpression was correlated with increased overall survival in breast cancer patients, which counters what we and others have observed in vitro. In this study, we compared the expression of CCL5 and Nodal, a potent embryonic morphogen re-expressed by aggressive cancer cells, in either hADSC or MDA-MB-231 breast cancer cells in response to three-dimensional collagen matrices conditioned by hADSCs or breast cancer cells. After preconditioning a three-dimensional collagen matrix with either hADSCs or MDA-MB-231 cells (and following removal of the cells), the denuded three-dimensional matrices were reseeded with the opposite cell type. We found that the expression of both CCL5 and Nodal was significantly reduced in the breast cancer cells plated on the hADSC-preconditioned matrix. However, hADSCs did not express Nodal or alter CCL5 protein expression when grown on a MDA-MB-231-preconditioned matrix. MDA-MB-231 cells plated onto a hADSC-preconditioned matrix appeared more mesenchymal and stem-like, while the adipose stem cells placed into a tumor-preconditioned matrix were induced into a more aggressive cancer cell phenotype by engaging in vasculogenic mimicry. Furthermore, we found there was no change in cell proliferation of any cells grown under these different conditions. These observations suggest a relationship between hADSCs and the breast tumor microenvironment where human adipose-derived stem cells appear to inhibit the expression of CCL5 and Nodal by the aggressive tumor cells without altering tumor cell proliferation.

Citation Format: Daniel Berrebi, Abhishek Yadav, Eric Lundstrom, Nicole Werwie, Dominic Muttillo, Richard E. Seftor, Elisabeth A. Seftor, Linda Vona-Davis. Aggressive breast cancer cells reduce CCL5 and Nodal expression in response to a human adipose-derived stromal cell microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4492.
Abstract 4503: The paracrine role of p53 in skin
Authors: Terzian Tamara , Sobhani Nema , Mylavarapu Rohan , Kharbili Manale El , Parker Andrew , Little Colleen , Krauspe Ethan , Box Neil
Published: August 2018

Exposure to ultraviolet radiation (UVR) in sunlight activates both defensive and repair pathways in the skin. As the major cell type affected by UVR, keratinocytes coordinate critical aspects of the skin response. Molecularly, the transcription factor p53 is induced by UVR in keratinocytes leading to their cell death by apoptosis (peeling). p53 also stimulates the expression of paracrine melanocyte growth factors involved in tanning defined as increase in melanocyte proliferation and melanin synthesis. Here, we investigate the less defined role of p53 in tanning. For this, we use a mouse model that has high p53 levels in keratinocytes and presents with elevated melanocyte number and melanin production. Interestingly, loss of p53 in keratinocytes completely abrogates the hyperpigmentation phenotype. These data demonstrate in a powerful way the paracrine role of p53 expressed in keratinocytes on melanocyte behavior and function. Moreover, it provides an excellent tool to examine molecular and cellular changes that accompany the melanocyte response to sunburn. Sunburn increases melanoma risk in epidemiological studies. To elucidate the role of the p53 response in melanoma, we treated mice with “sunburning” doses of UVB and followed melanocyte proliferation and the expression of keratinocyte-induced melanocyte growth factors. We present our latest findings reinforcing the signs of cross-talk between keratinocytes and melanocytes which support a paracrine role for p53 expressed in keratinocyte on promoting melanocyte proliferation through the release of paracrine factors.

Citation Format: Tamara Terzian, Nema Sobhani, Rohan Mylavarapu, Manale El Kharbili, Andrew Parker, Colleen Little, Ethan Krauspe, Neil Box. The paracrine role of p53 in skin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4503.
Abstract 4518: Prevalence of high-grade prostatic adenocarcinoma in a black African population
Authors: Nnakenyi Emeka F. , Nnakenyi Ifeyinwa D. , Okafor Okechukwu C. , Ugwumba Fred O.
Published: August 2018

INTRODUCTION:

Prostate cancer is the second most common type of cancer in men worldwide. Despite this, screening is currently disputed because studies in the western world have shown that more men die with the disease than from it. Black men have been documented to have a greater prevalence of prostate cancer which runs an aggressive course. Therefore, we aimed to determine the prevalence of high grade prostatic adenocarcinoma among Nigerian men.

METHOD:

This was a retrospective study of Nigerian men attending the urology clinic at University of Nigeria Teaching Hospital, Enugu, south-east Nigeria. Data on prostate histology were retrieved from the histopathology laboratory records for the last 5 years as thus: age, prostate diagnosis, gleason score. Histologic slides from core-needle biopsies had been reviewed by two Pathologists independently. If adenocarcinoma was diagnosed, Gleason system was used to grade the tumors, and a consensus was reached by both Pathologists. The data were analyzed with SPSS statistical software.

RESULTS:

Three hundred and forty-five (345) requests for histology on prostate biopsies were received, of which 118 (34.2%) were diagnosed with prostatic adenocarcinoma (PCa) at a mean age(sd) of 69(8.4) years. Minimum and maximum gleason scoring observed were 4 and 10.

Gleason systemNo. of prostatic adenocarcinoma (%)Gleason score 2 – 6 (well differentiated PCa with excellent prognosis)22 (18.6%)Gleason score 7 (3+4 moderately differentiated PCa)19 (16.1%)Gleason score 7 (4+3 moderate to poorly differentiated PCa)11 (9.4%)Gleason score 8-10 (poorly differentiated with aggressive biologies)66 (55.9%)

CONCLUSION:

A high prevalence of high grade PCa was observed in this study, which are less likely to be cured. Therefore, early detection is crucial to prevent mortality. This may justify the need for prostate cancer screening in our African environment.

Citation Format: Emeka F. Nnakenyi, Ifeyinwa D. Nnakenyi, Okechukwu C. Okafor, Fred O. Ugwumba. Prevalence of high-grade prostatic adenocarcinoma in a black African population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4518.
Abstract 4524: Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification
Authors: Agersborg Sally , Mixon Christopher , Nguyen Thanh , Aithal Sramila , Blocker Forrest , Weiss Lawrence , Gasparini Robert , Jiang Shiping , Chen Wayne , Hess Gregory , Albitar Maher
Published: August 2018

PURPOSE

While HER2 testing is well established in directing appropriate treatment for breast cancer, a small percentage of cases show equivocal results by IHC and FISH. Alternative probes may be used in equivocal cases. We present a single community-based institution's experience in further evaluating these cases.

PATIENTS AND METHODS

Between 2014-2016, 4255 samples were submitted for HER2 amplification testing by alternative probes, TP53, RAI1, and RARA. Of patients tested by FISH, 505/3908 (12.9%) also had IHC data.

RESULTS

Most (73.9%) FISH equivocal cases remained equivocal after IHC testing. Duplicate testing by IHC using a different paraffin block resulted in poor reproducibility as 44% of IHC negative became equivocal and 40% of IHC equivocal became negative. Of the equivocal cases by IHC, 52.3% became positive by alternative FISH. However, 50.5% of cases equivocal by conventional FISH were classified as HER2 amplified by alternative probes. Most cases were positive by more than one probe; 78% of positive cases were positive by RAI1 and 73.9% by TP53. There was a significant difference between IHC and FISH alternative testing (P<0.0001) among the equivocal cases by conventional FISH testing, 44% of IHC negative cases became positive while 36% of the positive IHC cases became negative by alternative FISH testing. Available data showed that 41% of patients were treated with palbociclib and were positive by alternative FISH.

CONCLUSION

The prevalence of double HER2 equivocal cases and the discrepancy between IHC and alternative FISH testing suggest that FISH alternative testing using both RAI1 and TP53 probes is necessary for conclusive classification. Due to the significant difference between IHC repeat testing, reflexing on IHC alone in this group of cases may not be reproducible. Because almost half of FISH equivocal cases converted to HER2 amplified upon alternative testing, clinical studies to determine benefit of anti-HER2 therapy in these patients is urgently needed.

Alternative FISHIHC Score 0 NegativeIHC Score 1 NegativeIHC Score 2 EquivocalIHC Score 3 PositiveNo%No%No%No%Negative1463.65454.517847.7436.4Positive836.44545.519552.3763.6Total22100.099100.0373100.011100.0

Citation Format: Sally Agersborg, Christopher Mixon, Thanh Nguyen, Sramila Aithal, Forrest Blocker, Lawrence Weiss, Robert Gasparini, Shiping Jiang, Wayne Chen, Gregory Hess, Maher Albitar. Immunohistochemistry and alternative FISH testing in breast cancer with HER2 equivocal amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4524.
Abstract 4540: Development of well characterized breast, lung, and brain cancer copy number variation reference materials
Authors: Lowe Dana Ruminski , Dickens Jessica , Huang Catherine , Konigshofer Yves , Anekella Bharathi
Published: August 2018

Introduction: Copy number variation (CNV), including gene amplification and deletion, can be a key driver of oncogenesis. Pathogenic CNVs are often associated with unfavorable prognosis and drug resistance, therefore detection of these types of genetic changes will be important for personalized treatment. Many tumor profiling workflows can detect CNVs in addition to somatic mutations, but well-characterized reference materials for CNVs are not widely available, which makes it difficult to assess accuracy and sensitivity. CNV content of cell lines can be variable and may change with passages, the genomic background may not be diploid, and germline and somatic variants may not be well characterized. Remnant patient samples often lack characterization and the volumes needed for such characterization and larger validation studies. Therefore, we developed and evaluated Seraseq CNV reference materials to serve as accuracy, precision, and limit-of-detection (LOD) controls for tumor profiling assay development and validation. By providing a consistent source of purified DNA with stable CNV content, these reference materials allow for repeatable assessment of clinical diagnostic assays targeting CNVs. Methods: Seraseq CNV reference materials were prepared by mixing genomic DNA with additional copies of target genes from the well-characterized cell line GM24385. Copies of ERBB2, FGFR3, and MYC were combined with genomic DNA to simulate CNVs found in breast cancer, while sequences for EGFR, MET, and MYCN were used to mimic lung and brain cancer CNVs. Three levels of amplification were targeted: +3, +6, and +12 additional copies. The copy number of each gene was precisely quantified using digital PCR and confirmed on several NGS-based assays internally and externally. Results & Conclusions: Testing revealed CNVs of each target gene were close to the expected copy number. Good correlation was observed between digital PCR data and NGS data generated internally with the Archer VariantPlex Solid Tumor assay. In addition, internal and external NGS data showed correlation and broad assay compatibility. While CNVs observed clinically can be upwards of 50 additional copies and more easily detectable, LOD controls such as those described here are imperative in evaluating assay performance. Collectively, the data demonstrate the utility of biosynthetic Seraseq CNV reference materials, which serve as superior alternatives to DNA from cell lines or patient-derived material.

Citation Format: Dana Ruminski Lowe, Jessica Dickens, Catherine Huang, Yves Konigshofer, Bharathi Anekella. Development of well characterized breast, lung, and brain cancer copy number variation reference materials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4540.
Abstract 4543: Combined analysis of cell-free DNA methylation in plasma for early detection of NSCLC
Authors: Cao Li , Wang Hao , Dong Xu , Zhong Li
Published: August 2018

Introduction: Detection of cell-free DNA (cfDNA) methylations in plasma samples may provide diagnostic information for non-small lung cancer (NSCLC). Here, we evaluated 5 preselected genes for methylation analysis from patient and control plasma samples.

Method: Plasma samples were collected from 91 patients who were diagnosed with primary NSCLC with pathological or cytological confirmation. All the cases did not received any surgery or chemotherapy treatments when blood samples were collected. Meanwhile, 58 cases of healthy blood samples were collected as the control group. Methylation specific PCR (methylation specific-PCR, MS-PCR) was used to detect RUNX3, RASSF1A, 3-OST-2, DAPK, PTPRO genes methylation. Data was analyzed individually as well as jointly for the optimal diagnostic accuracy.

Results: The DNA methylation rates for the 5 genes RUNX3, RASSF1A, 3-OST-2, DAPK, PTPRO were 47.25% (43/91), 51.65% (47/91), 27.47% (25/91), 36.26% (33/91), and 41.76% (38/91) respectively in patient plasma samples. In the normal control group, only PTPRO gene methylation was detected with the methylation rate of 5.2% (3/58), and the rest of 4 genes were not detected. Methylation of RUNX3 gene was found to have higher frequencies in adenocarcinoma than other cell types of NSCLC (P < 0.05). DAPK gene methylation was found to be associated with the stages of lung cancer, and methylation rate was higher in stage III/IV than in stage I/II (P < 0.05). Combined analysis of the 5 genes methylation rates was achieved the sensitivity 86.81% (79/91) and the specificity 79.31% (46/58).

Conclusion: The combined DNA methylation analysis of RUNX3, RASSF1A, 3-OST-2, DAPK, and PTPRO genes with MS-PCR achieved a satisfied sensitivity and specificity in NSCLC diagnosis.

Citation Format: Li Cao, Hao Wang, Xu Dong, Li Zhong. Combined analysis of cell-free DNA methylation in plasma for early detection of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4543.
Abstract 4551: Thyroid hormone induces PD-L1 expression in oral cancer cells
Authors: Chin Yu-Tang , Su Kwan-Wei , Lim Yee-Tang , Shih Ya-Jung , Chen Yi-Ru , Lee Sheng-Yang , Davis Paul J. , Lin Hung-Yun , Fu Earl
Published: August 2018

Oral cancer is a fatal disease, which accounts for the fourth highest incidence of malignancy in males and the seventh highest in the general population of Taiwan. Oral cancer is increasing in Taiwan. About 95% of oral cancer in Taiwan is oral squamous cell carcinoma (OSCC). With the development of cancer molecular biology and immunology, targeted therapy for immune checkpoints of programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has shown enormous development prospects for treatment of head and neck cancer. The PD-1/PD-L1 checkpoint is a critical regulator of activated T cell-cancer cell interactions which defend tumor cells against immune surveillance. Thyroid hormone induces PD-L1 expression in human oral cancer cells. Human oral cancer OEC-M1 and SCC-25 cells were treated with different concentrations of T4 (10-8 to 10-6M) for 24 h and cells were harvested and total RNA was extracted. qPCR of PD-L1 revealed that PD-L1 mRNA was significantly induced by thyroid hormone on a concentration-dependent basis. Parallel studies were conducted to study the effect of thyroid hormone on PD-L1 protein accumulation. Cancer cells were treated with different concentrations of T4 for 24 h. Total proteins were extracted and western blot analysis of PD-L1 was conducted. Thyroid hormone-activated ERK1/2 and STAT3 were companied with PD-L1 expression. Inhibition of ERK1/2 and consequently STAT3 activation also blocked PD-L1 induced by thyroid hormone. Knockdown of PD-L1 expression by siRNA also inhibits thyroid hormone-induced proliferation of oral cancer cells which indicated that PD-L1 expression is involved in thyroid hormone-induced cancer growth via an ERK1/2-STAT3 signal transduction pathway in oral cancer cells.

Citation Format: Yu-Tang Chin, Kwan-Wei Su, Yee-Tang Lim, Ya-Jung Shih, Yi-Ru Chen, Sheng-Yang Lee, Paul J. Davis, Hung-Yun Lin, Earl Fu. Thyroid hormone induces PD-L1 expression in oral cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4551.
Abstract 4552: Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox
Authors: Fiedler Ulrike , Reichen Christian , Taylor Joanna , Schildknecht Patricia , Barsin Sophie , Metz Clara , Schlegel Anja , Fontaine Simon , Villemagne Denis , Ahlskog Julia , Kaufmann Yvonne , Link Alexander , Rigamonti Nicolo , Hepp Julia , Stumpp Michael T.
Published: August 2018

During the last years, immune-modulating drugs became an important cornerstone in the treatment of cancer patients. In particular, the PD1/PDL-1 and CTLA-4 antagonists revolutionized the field. However, only a limited number of patients benefit from these antagonistic molecules and more combination therapies are on the way to increase the number of patients benefiting from these novel therapies. Among the combinations, drugs that are T-cell or myeloid cell agonists belonging to the TNFR-superfamily show first promising clinical results. However, systemic immune activation bears the risk of severe side effects that will not allow using these powerful drugs at an effective dose. We have developed a new class of DARPin molecules that enable tumor-restricted immune cell activation of TNFR-superfamily agonists in the tumor only, thereby preventing systemic immune-activation. We generated DARPin molecules that bind with high affinity to TNFR-superfamily members (CD134, CD137 and CD40) and DARPin molecules that bind to tumor-specific antigens such as HER2 and EGFR or targets restricted to the tumor stroma compartment like FAP and extra-domain B of fibronectin (ED-B). Using these building blocks from our DARPin toolbox, we constructed a variety of multi-specific molecules consisting of a TNFR-superfamily receptor targeting DARPin molecule and a tumor-localizing DARPin molecule. In reporter cell assays the multi-specific DARPin molecules activate the respective TNF-superfamily receptor only in the presence of a cell expressing the tumor-localizer; e.g. the FAP-CD134 molecule activates CD134 only in the presence of a stromal cell expressing FAP and not in its absence. Moreover, activation of CD134 was dependent on the density of FAP expression on cells, showing that CD134 only becomes activated if a certain level of the tumor-localizing target is expressed. These finding were confirmed in experiments with primary immune cells where we see immune-cell activation only upon binding to the tumor-localizing target. This could be shown for multiple combinations (>8) and supports the concept that multi-specific DARPin molecules are powerful drug-candidates allowing tumor-restricted immune cell activation.

Citation Format: Ulrike Fiedler, Christian Reichen, Joanna Taylor, Patricia Schildknecht, Sophie Barsin, Clara Metz, Anja Schlegel, Simon Fontaine, Denis Villemagne, Julia Ahlskog, Yvonne Kaufmann, Alexander Link, Nicolo Rigamonti, Julia Hepp, Michael T. Stumpp. Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4552.
Abstract 4556: Phagocytosis response of macrophage to CD47 expression of tumor cells in anti-CD47 therapy
Authors: Yang Huajun , Li Zhongliang , Wang Phillip , Tang Beibei , Ma Qinyun , Xing Frank , Shi Qian
Published: August 2018

Macrophage as an important surveillance system is escaped by human cancer cells expressing CD47. To reinstate macrophage functionality in clearing tumor cells targeting CD47 and thereby blocking phagocytosis inhibitory CD47-SIRPalpha interaction has been an important strategy in immune-oncology therapy. We profiled a large panel of tumor cell lines, Crown's proprietary PDX models and clinical lung cancer tumors for CD47 expression. Although it has been reported as a ubiquitously expressed immune-escape marker in cancer cells, the expression levels of CD47 vary among different tumor cell lines and models when the cells were examined by flow cytometry. It is of interest that a portion of tumor cells in fact do not present CD47. To tackle the cause for the loss of CD47 expression, whole-exome sequencing was performed to examine the intactness of the gene in the CD47 loss models. The results will be presented. We next established a robust and reproducible in vitro assay platform to support anti-CD47 drug research. We applied a macrophage differentiation system using isolated CD14+ monocytes as starting material, and were able to achieve >80% of M1 and M2 populations, respectively, through the 6-day differentiation. Differentiated macrophage was co-cultured with target tumor cells labeled for flow cytometry analysis. In the presence of blocking anti-CD47 antibody BRICK126, steady and specific phagocytosis effect was observed when target tumor cells express high level of CD47 markers. BRIC126 antibody blocks the CD47-SIRPalpha interaction. In contrast, aCD274 antibody did not yield more than background phagocytosis effect in the same assay. To prove the CD47-specific effect, we constructed isogenic cell models from a couple of parental cell models in that CD47 gene is knocked out in one sub-cell line and overexpressed in another sub-cell line. The isogenic systems were then subject to the CD47 biology assay and more profound changes in phagocytosis responses were observed. Furthermore, the in vitro findings were validated in in vivo setting.

Citation Format: Huajun Yang, Zhongliang Li, Phillip Wang, Beibei Tang, Qinyun Ma, Frank Xing, Qian Shi. Phagocytosis response of macrophage to CD47 expression of tumor cells in anti-CD47 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4556.
Abstract 4583: Combined analysis of circulating tumor cells, fecal occult blood test and serum carcinoembryonic antigen for early detection and monitoring recurrence of patients with colorectal cancer
Authors: Hung Wei-Shan , Tsai Wen-Sy , Wang Tzu-Min , Hsu Hsueh-Ling , Liu Hsuan , Yang Chia-Yu , Tseng Ching-Ping , Cheng Ju-Chien
Published: August 2018

Current methods for clinical early detection and disease monitoring of patients with colorectal cancer (CRC) are immunochemical fecal occult blood test (iFOBT) and serum levels of carcinoembryonic antigen (CEA), which are known with low sensitivity. There is an unmet need to seek for additional molecular tools for early detection and disease monitoring of patients with CRC. Whether enumeration of circulating tumor cells (CTCs) supplements current clinical tests and facilitates early detection and monitoring disease progression was investigated. A total of 109 pre-operative patients with CRC (including early and advanced stages) and 65 non-cancerous controls were enrolled in this study. CTCs were enriched from the peripheral blood of these individuals by using the PowerMag negative selection system. Immunofluorescence staining of epithelial cell adhesion molecule (EpCAM), and podoplanin (PDPN, a marker related to poor cancer progression) was performed to define the two distinct subtypes of CTCs. Heterogeneous CTCs expressing PDPN or EpCAM are defined in the peripheral blood of patients with CRC. Regardless clinical stages in patients, the number of both EpCAM+-CTCs and PDPN+-CTCs explicitly differentiated non-cancerous controls and patients with CRC, and were also correlated with clinicopathological diagnosis. Receiver operating characteristic analysis demonstrated that the sensitivity and specificity of EpCAM+-CTCs was 86.32% and 78.46%, respectively, when the cutoff value was 23 EpCAM+-CTCs/mL. Using 7 PDPN+-CTCs/mL as the cutoff value, the sensitivity and specificity of PDPN+-CTCs was 77.98% and 75.38%, respectively. 84.2% and 80.3% of patients with serum CEA < 5 ng/mL were found to have higher EpCAM+-CTCs and PDPN+-CTCs counts, respectively. In addition, simultaneous measurements of iFOBT and CTCs could compromise the false-positive rate of iFOBT, while measurements of CEA and CTCs are applicable to monitor cancer recurrence after surgery. This study implicates that CTC testing supplements the current clinical methods to facilitate early detection and disease monitoring in CRC.

Citation Format: Wei-Shan Hung, Wen-Sy Tsai, Tzu-Min Wang, Hsueh-Ling Hsu, Hsuan Liu, Chia-Yu Yang, Ching-Ping Tseng, Ju-Chien Cheng. Combined analysis of circulating tumor cells, fecal occult blood test and serum carcinoembryonic antigen for early detection and monitoring recurrence of patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4583.
Abstract 4586: Evaluation of available blood collection tubes for use in stabilizing concentrations of extracellular vesicles / exosomes and associated cell-free RNA
Authors: George Nicholas
Published: August 2018

Introduction: Liquid Biopsy has revolutionized the detection and the monitoring of cancer disease states. In addition to the widely utilized circulating cell-free DNA (ccfDNA), cell-free RNAs (cfRNA) and the extracellular vesicles (EV's) / exosomes they are stored in, is drawing significant interest in cancer diagnostics. Analysis of disease-specific EV's / exosomes or cfRNA requires that collected samples maintain draw-time concentrations of these analytes in order to remain relevant. Blood components, such as platelets, red, and white blood cells, are unstable when stored in normal blood collection tubes (BCT's), thus leading to increases of EVs and associated cfRNA concentration. We undertook an analysis of currently available commercial BCT's to determine whether any of these sufficiently stabilize EV / exosome and cfRNA concentration. Materials and Methods: Healthy consenting adult donors were drawn into blood collection tubes per approved protocol. Samples were either processed immediately (Day-0) or allowed to incubate at room temperature for the indicated times. Cleared plasma was isolated and subsequently frozen at -80oC until use. Assay of extracellular vesicle concentration utilized the Nanosight-300 (Malvern Instruments) per manufactures protocol. Isolation of cell-free RNA utilized the Circulating Nucleic Acid Isolation Kit (Qiagen) per manufacturer's protocol with the exception of the lysis step; this was extended from 30 minutes to 60 minutes. Digital PCR (ddPCR) utilized the Bio-Rad workflow (QX200 Droplet Generator and Reader). Conclusion: Stabilization of EV's and cfRNA was assayed for blood samples drawn into a variety of Streck nucleic acid stabilizing BCT's, non-Streck nucleic acid stabilizing BCT's, and a traditional K3EDTA BCT. A majority of the assayed tubes led to time-dependent increases in EV concentration with the most robust increases occurring between Day-3 and Day-7. Time-dependent increases in cfRNA concentration mirrored the increases observed in EV's, and based on ddPCR analysis; the majority of this increase was likely the result of erythrocyte membrane shedding. One type of Streck BCT was found to stabilize both EV and cfRNA concentration as a function of blood storage time. Incorporation of this tube into the Liquid Biopsy workflow will maximize assay target sensitivity by preventing the deleterious effects of excess cfRNA from non-specific release of EV's / exosomes.

Citation Format: Nicholas George. Evaluation of available blood collection tubes for use in stabilizing concentrations of extracellular vesicles / exosomes and associated cell-free RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4586.
Abstract 4587: ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer
Authors: Giannopoulou Lydia , Mastoraki Sofia , Strati Areti , Chebouti Issam , Pavlakis Kitty , Kasimir-Bauer Sabine , Lianidou Evi
Published: August 2018

Background: The estrogen receptor is highly expressed in epithelial ovarian cancer and represents the main target for endocrine therapy. The ESR1 gene is frequently methylated in many types of gynecological malignancies and previous studies have shown an inverse correlation between ESR1 methylation and gene expression. Few studies attempted to investigate the role of ESR1 methylation in ovarian cancer so far, and the clinical significance of ESR1 methylation status is not as yet clear.

Methods: The ESR1 methylation status was examined in primary tumors and corresponding circulating tumor DNA (ctDNA) samples of patients with high-grade serous ovarian cancer. For the detection of methylation we applied a novel highly specific and sensitive real-time methylation specific PCR (real-time MSP) assay. Two groups of primary tumor samples were recruited (training group, n=66 and validation group, n=61), along with the corresponding plasma samples (n=58) for the validation group. ESR1 methylation was also analyzed in a small group of 16 normal fallopian tube samples.

Results: ESR1 was found methylated in both groups of primary tumor samples and in the corresponding plasma samples of the validation group. More specifically, ESR1 methylation was detected in 32/66 (48.5%) and 17/61 (27.9%) primary tumor samples of the training and the validation group, respectively, and in 23/58 (39.7%) corresponding plasma samples. A significant agreement between ESR1 methylation in primary tumors and paired ctDNA was observed in 40/56 (71.4%) samples of the validation group (P=0.004, k=0.360). Almost all normal fallopian tube samples (15/16) were found methylated. Interestingly, the presence of ESR1 methylation in the primary tumor samples of the validation group was nearly significantly correlated (P=0.057) with a better overall survival.

Conclusions: We detected for the first time ESR1 methylation in ctDNA of patients with high-grade serous ovarian cancer. The agreement between ESR1 methylation in the primary tumors and paired ctDNA is statistically significant. Our results indicate a potential correlation between ESR1 methylation and better overall survival in high-grade serous ovarian cancer patients.

Citation Format: Lydia Giannopoulou, Sofia Mastoraki, Areti Strati, Issam Chebouti, Kitty Pavlakis, Sabine Kasimir-Bauer, Evi Lianidou. ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4587.
Abstract 459: Therapy-induced senescence is reversible in vitro and in vivo
Authors: Saleh Tareq , Alotaibi Moureq R. , Kyte Sarah L. , Cudjoe Emmanuel K. , Kawale Ajinkya , Xu Jingwen , Elsayed Zeinab , Landry Joseph W. , Henderson Scott C. , Yakovlev Vasily , Elmore Lynne W. , Gewirtz David A.
Published: August 2018

While cellular senescence has long been recognized as an irreversible form of growth arrest, evidence in the literature has suggested that subpopulations of senescent tumor cells may retain proliferative capacity. To directly address this question, H460 non-small cell lung cancer cells induced into senescence by exposure to etoposide, and enriched based on β-galactosidase staining and size, were shown to recover reproductive capacity, which was accompanied by resolution of the DNA-damage-response (downregulation of p53 and p21Waf1/Cip1 induction), attenuation of the senescence-associated secretory phenotype (SASP) as well as downregulation of miRNA34 expression and an increase in c-myc. The senescence-enriched lung cancer cells were also capable of tumor growth upon transplantation into immunodeficient mice. Re-emergence from etoposide-induced senescence was also observed using HCT116 colon cancer cells, as evidenced by concomitant downregulation of a senescence-associated reporter (BTG1-RFP). Collectively, these findings indicate that therapy-induced senescence (TIS) may ultimately be a transient process in that at least a subpopulation of tumor cells can resume proliferation. We propose that some forms of tumor dormancy that lead to disease recurrence may reflect cells that enter into and ultimately escape from senescence.

Citation Format: Tareq Saleh, Moureq R. Alotaibi, Sarah L. Kyte, Emmanuel K. Cudjoe, Ajinkya Kawale, Jingwen Xu, Zeinab Elsayed, Joseph W. Landry, Scott C. Henderson, Vasily Yakovlev, Lynne W. Elmore, David A. Gewirtz. Therapy-induced senescence is reversible in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 459.
Abstract 4599: Detecting MSI phenotype in circulating blood DNA
Authors: Stern Marc-Henri , Silveira Amanda B. , Bieche Ivan , Melaabi Samia , Cabel Luc , Buecher Bruno , Pierga Jean-Yves , Bidard Francois-Clement , Proudhon Charlotte
Published: August 2018

Background: Microsatellite instability (MSI) is a hypermutator phenotype occurring in gastrointestinal, endometrial and colorectal tumors, and more rarely, in urinary tract, ovarian, breast, prostate, lung, head and neck, liver and glioblastoma tumors. The diagnosis of MSI phenotype has recently emerged as the first pan-tumor biomarker likely to predict clinical benefit from immune-checkpoint blockade therapy, making its precise identification primordial for treatment decisions and disease monitoring. Current diagnosis of MSI is performed by multiplex PCR of five microsatellite markers, followed by capillary electrophoresis to detect shifts in allele size. An important limitation of this method is its low sensitivity preventing its use when tumor content is below 10%.

Methods: We designed ddPCR assays examining 3 microsatellites. Analytical performance was assessed in vitro. Clinical performance was evaluated in a series of FFPE and body fluid DNA samples obtained from patients with Stage IV colorectal or endometrial carcinomas, previously classified as MSI-H or MSS using the standard pentaplex PCR method. Mutant allele frequencies (MAF) quantified with the ddPCR MSI assay were also compared with the MAFs reported by ddPCR assays targeting specific BRAF or PIK3CA mutations.

Results: The ddPCR assays for the 3 microsatellites were found to have high specificity and reached a limit of detection of <0.1%. A perfect concordance with the MSI status determined by the pentaplex assay was observed in the 50 tumor samples tested (25 MSI-H and 25 MSS). Importantly, the ddPCR assays diagnosed the MSI phenotype in the plasma of all patients with MSI-H tumors, without false positives in MSS cases. Furthermore, an excellent correlation was observed in the 6 FFPE (R2:0.81) and 8 body fluid (R2:0.99) MSI-H samples were the MAFs measured by our assay could be compared with the ones obtained by ddPCR assays targeting specific mutations in BRAF or PIK3CA genes.

Conclusion: This technique allows a cost-effective, sensitive and large scale screening of the MSI phenotype, as well as the follow-up of MSI patients using liquid biopsies.

Citation Format: Marc-Henri Stern, Amanda B. Silveira, Ivan Bieche, Samia Melaabi, Luc Cabel, Bruno Buecher, Jean-Yves Pierga, Francois-Clement Bidard, Charlotte Proudhon. Detecting MSI phenotype in circulating blood DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4599.