bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4: Temporal and spatial topography of cell proliferation in cancer
Authors: Gaglia Giorgio , Kabraji Sheheryar , Argyropoulou Danae , Dai Yang , Bergholz Johann , Coy Shannon , Lin Jia-Ren , Winer Eric P. , Dillon Deborah , Zhao Jean J. , Sorger Peter K. , Santagata Sandro
Published: July 2021

Uncontrolled cell proliferation is a defining feature of malignancy. Current understanding of proliferation, particularly in humans, derives primarily from studying cells growing rapidly in the non-physiological conditions of cell culture. However, tumors are exceedingly complex admixtures of different cell types and subclonal malignant populations comprising proliferative, non-proliferative, and arrested states that are influenced by physical, metabolic, and molecular conditions. Images of single or small sets of protein markers from fixed tissue samples only provide limited and static views into the nature of these complex states. Here we identify proliferation states and develop a quantitative framework to extract cell cycle dynamics from multiplexed, spatially-resolved tissue images of millions of tumor cells from human cancers and genetically engineered tumors in mice. Across spatial scales, tumors display intrinsic regional variability in proliferation patterns and in the coherence of cell cycle markers in high-dimensional space. Cell cycle dynamics and cell cycle coherence are not solely a function of tumor growth and oncogene expression and rapidly adapt following genetic and therapeutic perturbations. Replacing binary metrics with multivariate traits provides a quantitative framework for extracting multidimensional dynamic information from static images that is broadly applicable to the study of temporal processes within the native architecture of human disease tissues.

Citation Format: Giorgio Gaglia, Sheheryar Kabraji, Danae Argyropoulou, Yang Dai, Johann Bergholz, Shannon Coy, Jia-Ren Lin, Eric P. Winer, Deborah Dillon, Jean J. Zhao, Peter K. Sorger, Sandro Santagata. Temporal and spatial topography of cell proliferation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 4.
Abstract 408: Quantification of cell surface HLA-A2 and intracellular Survivin protein levels for tumor blasts and non-blast immune cells in multiple myeloma bone marrow aspirates using a rapid sample preservation methodology followed by 10-color FACS assay
Authors: Hardas Tejaswini , Ra Hyun-Jeong , Sheridan James , Kovalenko Maria , Tribouley Catherine
Published: July 2021

Introduction: Multiple myeloma (MM) is a plasma cell disorder characterized by clonal expansion and accumulation of immature undifferentiated neoplastic cells in the bone marrow (BM). Recent advances in fluorochrome chemistries, flow cytometry (FACS) instrumentation and rapid tissue preservation methodologies have created opportunities for broader adoption of FACS in the clinical management of multiple myeloma patients.

Methods: We have developed a 10 color, 2 tube FACS assay based on recommendations made by the European Myeloma Network for phenotyping plasma cell gammopathies to identify abnormal blasts in multiple myeloma patient bone marrow aspirates. The assay includes essential plasma cell markers CD138, CD38, CD56 and CD19 to differentiate abnormal blasts from reactive plasma cells. Use of a multi epitope fluorochrome conjugated anti-CD38 antibody enables CD38 detection in patients on anti-CD38 therapy.

Results: Quantification of cell surface HLA-A2 and intracellular survivin protein was made possible using fluorochrome conjugated antibodies in combination with bead standards. The SmartTube™ proteomic stabilizer system for rapid preservation was used to freeze BM aspirate specimens for later analysis at locations with appropriate FACS equipment and trained staff. Consistent with previously published literature, patient survivin levels were elevated on abnormal MM blasts in comparison to survivin levels on non-blast immune cells.

Conclusion: We have developed a robust FACS methodology that would be easy to implement in the clinical trial and development setting with minimal training. The assay can consistently identify abnormal blasts in BM samples of multiple myeloma patients and quantify intracellular and cell surface biomarkers related to MM disease or disease treatment responses.

Citation Format: Tejaswini Hardas, Hyun-Jeong Ra, James Sheridan, Maria Kovalenko, Catherine Tribouley. Quantification of cell surface HLA-A2 and intracellular Survivin protein levels for tumor blasts and non-blast immune cells in multiple myeloma bone marrow aspirates using a rapid sample preservation methodology followed by 10-color FACS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 408.
Abstract 416: Toripalimab plus paclitaxel and carboplatin as neoadjuvant therapy in patients with locally advanced resectable esophageal squamous carcinoma: An open-label, single arm phase 2 trial
Authors: He Wenwu , Peng Lin , Bao Yu , Xiao Wenguang , Fang Qiang , Leng Xuefeng , Yan Jiaxin , Mao Tianqin , Shen Xudong , Huang Depei , Zhang Hushan , She Xueke , Han Yongtao
Published: July 2021

Background: Immune checkpoint inhibitors (ICIs) have shown promising results in patients with advanced esophageal squamous carcinoma (ESCC), while their efficacy for locally advanced patients is unclear. We conducted a neoadjuvant immunotherapy trial, investigating the safety and feasibility of toripalimab, a PD-1 inhibitor, combined with paclitaxel and carboplatin in patients with resectable ESCC (ClinicalTrials.gov identifier: NCT04177797).

Methods: Between May 2020 and June 2020, 18 patients with histopathologically diagnosed ESCC, clinically staged as T3N1-3M0, ECOG PS 0, received toripalimab 240 mg IV, D1, Q3W and paclitaxel 135 mg/m2, IV, D1, Q3W and carboplatin AUC 5 mg/mL per min, IV, D1, Q3W for two cycles, followed by McKeown esophagectomy 4-6 weeks later. The primary endpoint were safety and major pathological response (MPR) rate, and the secondary point were pathological complete response (pCR) rate, DFS and OS.

Results: Sixteen patients received surgery with no treatment-related surgical delay, and the R0 resection rate was 87.5% (14/16). Two patients refused surgery due to the clinical assessment as complete response. Among 16 patients undergoing surgery, 3 (16.6%) were pCR and 7 (43.8%) were MPR. Treatment-related adverse events occurred in all patients (100%), and 4 patients (22.2%) experienced grade 3 or higher treatment-related adverse events including neutropenia (2, 12.5%), leukopenia (1, 6.2%), diarrhea (1, 6.2%), fatigue (1, 6.2%). No surgery-related adverse events or treatment-related death occurred.

Conclusions: The combination of toripalimab, paclitaxel and carboplatin for patients with locally advanced resectable ESCC is well tolerated and shows antitumor activity, which might be a potential neoadjuvant therapy regimen.

Citation Format: Wenwu He, Lin Peng, Yu Bao, Wenguang Xiao, Qiang Fang, Xuefeng Leng, Jiaxin Yan, Tianqin Mao, Xudong Shen, Depei Huang, Hushan Zhang, Xueke She, Yongtao Han. Toripalimab plus paclitaxel and carboplatin as neoadjuvant therapy in patients with locally advanced resectable esophageal squamous carcinoma: An open-label, single arm phase 2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 416.
Abstract 421: Characteristics of melanoma diagnosed in patients with underlying HIV infection
Authors: Wei Grace , Sinha Sweta , Messina Jane , Coghill Anna E.
Published: July 2021

Background: Melanoma is diagnosed more frequently in patients living with HIV (PLWH) compared to the general US population and HIV-infected melanoma patients also experience higher cancer-specific mortality. The mechanisms underlying these outcome disparities are poorly understood, and data describing tumor pathology and non-mortality outcomes for melanoma patients with HIV are lacking.

Objective: To describe differences in tumor presentation and disease course among melanoma patients presenting with or without underlying HIV infection in the era of effective HIV therapy.

Methods: We conducted an initial retrospective chart review of 29 HIV+ and 29 HIV- patients, matched as possible to HIV+ patients on age and disease stage, who presented to Moffitt Cancer Center between 2004 and 2019. Data abstracted included patient demographics, tumor pathology, treatment details, and clinical outcomes.

Results: Certain melanoma pathology features differed by HIV status, with a higher prevalence of elevated mitotic rate (78.9% vs 41.2%, p=0.02) and vertical growth phase (90% vs 55%, p=0.02) in melanoma patients with HIV. Nearly all patients underwent lesion excision, but post-treatment complications were greater in PLWH (44.8% vs 24%, p=0.09). Among 16 patients who received systemic therapy after excision (HIV+ 6; HIV- 10), progressive disease was more frequent in PLWH (100% vs 30%, p=0.01), with further suggestion of higher severe side effect rates, defined as events interrupting systemic therapy (66.7% vs 30%; p=0.18). Despite these differences in progression, overall survival rate were similar in PLWH (79.3%) and uninfected patients (82.8%).

Conclusions: These preliminary results suggest that PLWH and melanoma experience a unique disease course. Higher prevalence of post-excision complications and disease progression following systemic therapy in particular warrant further attention as age-related cancers, such as melanoma, are expected to grow in the US HIV population in coming years.

Citation Format: Grace Wei, Sweta Sinha, Jane Messina, Anna E. Coghill. Characteristics of melanoma diagnosed in patients with underlying HIV infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 421.
Abstract 432: Sarcopenia predicts dose-limiting toxicity in metastatic pancreatic cancer treated with gemcitabine plus nab-paclitaxel
Authors: Youn Susie , Chen Angela , Ha Vincent , Chambers Carole , Eurich Dean T. , McCall Michael , Sawyer Michael B.
Published: July 2021

Introduction: Gemcitabine (GEM) plus nab-paclitaxel (nab) has been shown to improve overall survival (OS) compared to GEM monotherapy in patients with metastatic pancreatic cancer. However, GEM/nab is associated with increased toxicity. Our study evaluated whether sarcopenia increased the likelihood of chemotherapy toxicity in pancreatic cancer treated with GEM/nab.

Methods: A retrospective review was performed of all patients who received GEM/nab as first-line therapy for metastatic pancreatic cancer at a northern Alberta cancer institute (Canada) from 2014-2017. Patients were included if a computed tomography (CT) scan of the abdomen and pelvis was performed within 60 days of starting chemotherapy. Skeletal muscle surface area was measured at L3 on baseline CT scans and normalized for height to determine skeletal muscle index (SMI, cm2/m2). Dose-limiting toxicity (DLT) was defined as dose reduction or treatment discontinuation due to toxicity. Optimal stratification was used to establish sex-specific SMI cut-offs with DLT as an outcome.

Results: 152 patients were included in the study. 88 patients (57.8%) were male and median age was 66.5 years (range 34-95). 62 patients (40.8%) experienced DLT. SMI cut-offs were determined as <48.0 cm2/m2 in males and <39.55 cm2/m2 in females. Sarcopenia prevalence using these cut-offs was 54.6%. DLT incidence was significantly higher in sarcopenic versus non-sarcopenic patients (55.4 vs. 23.2% respectively, p<0.001). In multivariate logistic regression accounting for advanced age (>65), sex, BMI category, and performance status (PS), sarcopenia significantly increased the likelihood of DLT (OR 7.21, 95% CI 3.02-17.24, p<0.001). Sarcopenia did not impact OS (HR 1.30, 95% CI 0.94-1.80, p=0.118) or progression-free survival (HR 0.66, 95% CI 0.42-1.03, p=0.071).

Conclusion: In pancreatic cancer treated with GEM/nab, sarcopenic patients are significantly more likely to experience DLT, independent of age, sex, BMI, and PS. These findings could have implications for reduced chemotherapy dosing in sarcopenic patients.

Citation Format: Susie Youn, Angela Chen, Vincent Ha, Carole Chambers, Dean T. Eurich, Michael McCall, Michael B. Sawyer. Sarcopenia predicts dose-limiting toxicity in metastatic pancreatic cancer treated with gemcitabine plus nab-paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 432.
Abstract 457: A comparison of IDH1 R132H [H09] and [IHC132] antibodies on diffuse gliomas
Authors: Figueroa Sara , Vargas Joseph , Ramos Jason
Published: July 2021

Introduction In the 2016 World Health Organization Central Nervous System Tumor Classification, diffuse gliomas were reclassified to include astrocytic tumors (grade II and III), oligodendrogliomas (grade II and III), and grade IV glioblastomas with diffuse astrocytoma being distinct from the diffuse gliomas. This reclassification is due to the abundance of IDH1 mutations in many of these tumors. A much better outcome has been observed in these patients than those without this mutation. IDH1 mutation of the R132H type constitutes more than 90% of all IDH1 and IDH2 mutations. Further, IDH1 IHC is now widely applied for the differential diagnosis of these tumors. There are two distinct IDH1 R132H clones on the market, the H09 and IHC132. Comparing these markers for the IDH1 R132H mutations by IHC can help determine if one is superior for diagnostics.

Materials and Methods IHC was performed on 3-5 um thick formalin-fixed, paraffin-embedded (FFPE) brain tissue arrays containing glioblastoma, astrocytoma, oligoastrocytoma, oligodendroglioma with adjacent normal tissue (US Biomax, Derwood, USA). These slides were deparaffinized, and peroxidase blocked in the usual manner. Slides were then subjected to HIER in a pressure cooker using a citrate-based buffer at 110°C for 15 minutes before being stained on a semi-automated instrument. The slides were stained with rabbit monoclonal IDH1 R132H antibody H09 (Dianova, Hamburg, Germany) and mouse monoclonal IDH1 R132H antibody IHC132 (GenomeMe, Richmond, Canada) at concentrations and protocols predetermined to match staining intensity.

Results The mouse monoclonal anti-IDH1 R132H IHC132 has shown that it is analogous to the current IDH1 R132H [H09]. Upon review of the data, IHC132 marker stains a higher percentage of cases in glioblastoma compared to H09 (11%, n=36) and equal in astrocytoma (73%, n=15), oligoastrocytoma (75%, n=4), oligodendroglioma (100%, n=8). Neither clone stained normal brain tissue per negative controls.

Conclusion Mouse monoclonal anti-IDH1 R132H [IHC132] compared with that of the rabbit monoclonal anti-IDH1 R132H [H09] showed equal sensitivity for astrocytoma, oligodendroglioma, and oligoastrocytomas with higher sensitivity for glioblastoma. The results provide evidence to support a higher percentage of positive staining and equal or greater intensity for clone IHC132 versus H09.

Citation Format: Sara Figueroa, Joseph Vargas, Jason Ramos. A comparison of IDH1 R132H [H09] and [IHC132] antibodies on diffuse gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 457.
Abstract 46: A new class of precision therapeutics that inhibit prostate cancer mediated bone destruction
Authors: Gordon Ryan R. , Pattanayak Abhinandan , Li Wenqi , Bergan Raymond
Published: July 2021

The destructive nature of bone metastases dominates the clinical management of advanced prostate cancer (PCa) and is a common cause of morbidity and mortality. Our current ability to therapeutically target this process is limited. Here we describe the synthesis and functional characterization of the first-in-class Dual Acting Bone Defending agents (DABDs), which couple a small molecule therapeutic that inhibits cell movement to bone trophic bisphosphonates. Upfront in-vitro screens assessed DABD efficacy at inhibiting PCa cell migration and invasion and osteoclast mediated bone destruction. Systemic models of PCa metastasis, inclusive of intracardiac (IC) and intratibial (IT) injection models, were deployed to characterize tolerance and efficacy in-vivo. Finally, in-vitro characterization of DABD molecular mechanism of action was performed, as guided by the existing knowledge base related to the parental compounds. Results demonstrated that DABDs inhibit PCa cell migration and invasion, chemically bind hydroxyappetite, and inhibit osteoclast mediated bone destruction in vitro. Following IC injection of PCa cells, a model where cells are placed into circulation, metastasize to bone, grow and induce bone destruction, emulating the clinical scenario in humans, DABD treatment inhibited circulating PCa cells from forming new bone metastasis and prolonged life in a dose-dependent fashion. In the IT mouse models of established metastatic lesions, DABDs induce reversion of PCa cell growth and disrupt further bone destruction. DABDs at doses 1000 fold higher than those associated with efficacy are well tolerated. At the molecular level, DABDs retain the molecular mechanisms of action of their constituent chemicals. They inhibit farnesyl diphosphate synthase (FDPS), in the mevalonate pathway, leading to decreased prenylation of small GTPases, including the Ras related protein, Rap-1A, as well as inhibit Raf1 activation, inducing apoptosis and impaired cell movement, respectively. DABDs are a new class of agents that inhibit formation of new bone metastasis, revert growth of established metastasis, prolong life, and have a high potential for improving clinical outcome in humans.

Citation Format: Ryan R. Gordon, Abhinandan Pattanayak, Wenqi Li, Raymond Bergan. A new class of precision therapeutics that inhibit prostate cancer mediated bone destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 46.
Abstract 479: mRNA in situ expression of candidate genes for progression risk tissue biomarkers, in premalignant cervical lesions
Authors: Benedetti Ines , Ruiz Felipe , Rodríguez Reinhard
Published: July 2021

Background Comparing the in situ differences in mRNA expression of candidate genes between normal cervical tissue and premalignant lesions, could contribute to detect progression risk biomarkers. Use of RNAscope chromogenic in situ hybridization (CISH) that amplifies specific mRNA signals and detects molecules as points that can be quantified, could help the detection of these biomarkers.

Methods A tissue microarray was constructed from cervical samples (conization and histerectomy). Representative areas of normal cervix, low-grade (LSIL), and high-grade squamous intraepithelial lesions (HSIL) were chosen. RNAscope 2.5HD Red HPV-HR18 assay that detect E6/E7 mRNA for eighteen high-risk (HR) HPV genotypes, and RNAscope 2.5HD Red assays to detect mRNA from: KRT16, TMEM45A and RBP1, were performed. The stainings were scored by two pathologist from 0 to 4 in a semi-quantitative analysis based on the average number of dots per cell; mean score values were compared using GraphPad Prism 7.0. A p<0.05 was considered statistically significant.

Results A total of 390 cervical tissues cores from 169 patients with mean age 36.08 years (SD ±10.48) were included. Mean mRNA expression of HR-HPV E6/E7, TMEM45A and RBP1 were significantly higher in HSIL compared to LSIL and normal cervix squamous epithelium. KRT16 mRNA expression scores in normal cervix squamous epithelium, LSIL, and HSIL, were not significantly different (Table 1).

Conclusions CISH allowed to detect E6/E7 mRNA molecules for eighteen HR-HPV genotypes in routinely processed tissues, and to evaluate the expression of the described candidate genes in their context without destroying the tissue morphology. Identification of differential expression between normal cervical tissue and HSIL supports the possible role of TMEM45A and RBP1 in cervical cancer progression and its potential use as progression risk tissue biomarkers.

Table 1.Biomarkers candidate genes expression profiles in normal cervix, LSIL and HSILCISH scoresMean (±SD)95% CINormal cervixLSILHSILp valueHR-HPV E6/E7 mRNAn=640.4098(±0.7611)0.2149-0.6048n=640.4098 (±0.7611)0.2149-0.6048n=1381.294 (±1.189)1.078-1.51<0.0001*TMEM45A mRNAn=761.105 (± 1.322)0.8031-1.407n=691.232 (±1.477)0.8771-1.587n=2381.514(± 1.491)1.264-1.7650.046*RBP1 mRNAn=660.4242(±0.7658)0.236-0.6125n=740.5135 (±0.9826)0.2859-1.159n=1301.3 (±1.186)1.094-1.506<0.0001*KRT16 mRNAn=840.9524(±1.15)0.7028-1.202n=770.8831 (±1.214)0.6076-1.159n=1571.115 (±1.41)0.8924-1.3370.573*

Citation Format: Ines Benedetti, Felipe Ruiz, Reinhard Rodríguez. mRNA in situ expression of candidate genes for progression risk tissue biomarkers, in premalignant cervical lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 479.
Abstract 497: Human CD73 knock-in mice facilitate evaluation of in vivo efficacy of anti-human CD73 cancer immunotherapies
Authors: Shang Chengzhang , Li Huilin , Zhang Jing , Lin Eugene , Yu Luke (Zhaoxue) , Shen Yuelei
Published: July 2021

In recent years, immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. However, most patients with solid cancers do not respond to the current ICIs against PD-1/PD-L1 or CTLA-4 alone. Stimulating an effective anti-tumor immune response may require countering additional immunosuppressive pathways in the tumor microenvironment (TME). Adenosinergic pathways are critical to maintain normal immune system homeostasis, and tumors could exploit them to avoid immune attack. CD73 (NT5E) is a surface enzyme that catalyzes the final hydrolysis of AMP to adenosine, which impairs immune function through adenosine receptors like A2AR (see our poster on humanized A2AR mice) on immune cells. Some tumors overexpress CD73 driven by hypoxia in the TME, which associates with poor prognosis in some cases. It is also expressed on the surface of multiple immune cells that infiltrate tumors, including T-cells, B-cells, NK cells, macrophages, and myeloid-derived suppressor cells. Several anti-CD73 antibodies have started early stage clinical trials as a treatment for advanced cancers. Combined anti-CD73, anti-PD-1, and anti-CTLA4 therapies synergize to reduce tumor burden and extend survival. Thus, there is a need for pre-clinical models to evaluate the safety and efficacy of CD73-targeted therapeutics before they enter human trials. Biocytogen has generated a human CD73 knock-in (KI) mouse (B-hCD73) and a human CD73-expressing MC38 cell line (MC38-hCD73) for both in vitro functional validation and in vivo efficacy evaluation of CD73-specific antibodies. Human CD73 (hCD73) whole coding sequence was inserted following exon 2 of the mouse Cd73 gene. B-hCD73 mice did not exhibit any immune or developmental abnormalities compared to wild-type mice, but splenic Tregs of B-hCD73 mice and not wild-type expressed hCD73. Anti-hCD73 antibodies bound well only to splenocytes of hCD73 KI mice and also blocked the enzyme activity of CD73. Using our MC38-hCD73 tumor model, we showed robust efficacy of anti-hCD73 antibodies in inhibiting tumor growth in vivo. Furthermore, in PD-1/PD-L1/CD73 triple KI humanized mice implanted with MC38 tumors, combined CD73 and PD-1/PD-L1 blockade showed greater tumor inhibition than monotherapies. Taken together, CD73 humanized mice and their variants are useful tools for in vivo efficacy evaluation of candidate human CD73 antibodies and combination therapies being considered for clinical trials.

Citation Format: Chengzhang Shang, Huilin Li, Jing Zhang, Eugene Lin, Luke (Zhaoxue) Yu, Yuelei Shen. Human CD73 knock-in mice facilitate evaluation of in vivo efficacy of anti-human CD73 cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 497.
Abstract 501: Double-humanized 4-1BB/4-1BBL mouse is an advanced model for liver toxicity and efficacy evaluation of 4-1BB therapeutic antibodies
Authors: Zhao Lei , Wang Linlin , Li Dirui , Hui Li , Lin Qingcong
Published: July 2021

4-1BB is an activation-induced costimulatory TNF receptor family member which is expressed on a variety of cell types such as activated T cells, NK cells, DCs, B cells, monocytes and Neutrophils. 4-1BB/4-1BBL interaction leads to a series of activation effects including increased cytotoxic T lymphocytes (CTL) activity, cytokine induction, prevention of activation-induced cells death (AICD). Agonistic monoclonal antibodies targeting 4-1BB have shown robust anti-tumor activity but has also caused substantial liver toxicity which hampered their clinical development. To better assist the efficacy and toxicity evaluation of anti 4-1BB antibodies, Biocytogen developed a 4-1BB/4-1BBL double humanized knock in mice, which is the second generation of humanized 4-1BB mice. In the double humanized mouse, the exons of mouse 4-1BBL and 4-1BB genes that encode the transmembrane domain and extracellular domain were replaced by human counterpart exons. The expression of knocked in exons were confirmed by both RT-PCR and Flow cytometry. In vivo efficacy study showed that 4-1BB antibodies significantly inhibited tumor growth in mice bearing the colon cancer cell line MC38 and this effect was dose-dependent. The tumor growth inhibition (TGI) reached approximately 70% in the mice group dosed 1mg/kg. More importantly, Toxicity study showed that compared to the single humanized 4-1BB mice, double humanized 4-1BB/4-1BBL mice have higher sensitivity on liver toxicity testing, reflected by sera AST and ALT level as well as liver pathological analysis. These results indicate that the double humanized B-h4-1BB/h4-1BBL mouse model, by mimicking a more human like 4-1BB and 4-1BBL interaction, has been proved to be a more advanced option for preclinical toxicity and efficacy evaluation for 4-1BB therapeutic antibodies compared to single humanized 4-1BB mice.

Citation Format: Lei Zhao, Linlin Wang, Dirui Li, Li Hui, Qingcong Lin. Double-humanized 4-1BB/4-1BBL mouse is an advanced model for liver toxicity and efficacy evaluation of 4-1BB therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 501.
Abstract 514: Specific anti-HER2 host immunity conferred by HER2-targeted antibody drug conjugate therapy and checkpoint blockade
Authors: Qin You , Dobson Hannah E. , Comer Frank I. , Chang Alfred E. , Wicha Max S. , Li Qiao
Published: July 2021

Using HER2-targeted antibody drug conjugates (ADC), we tested the therapeutic efficacy, direct impact on cancer stem cells (CSCs), and potential added benefit when combined with PD-L1 blockade in immunocompetent mouse tumor models. Two murine mammary tumor models were used in this study: D2F2/E2, a cell line resulting from stable transfection of parental D2F2 murine mammary tumor cells with wild-type human HER2, and 4T1-HER2, derived from parental 4T1 by stable transduction with wild-type human HER2. We demonstrate that HER2-targeted ADC treatment induced both cellular and humoral anti-HER2 adaptive immune responses which effectively targeted both bulk tumor and cancer stem cells. We recently investigated induction of host immunity using a tumor re-challenge strategy in tumor free animals subjected to ADC or ADC + anti-PD-L1 therapy. D2F2/E2 tumor-bearing mice were treated with ADC or ADC + anti-PD-L1 to generate tumor free mice. These tumor free animals were re-challenged with the relevant tumor D2F2/E2 or irrelevant tumor WT 4T1 respectively. While all the tumor free animals re-challenged with WT 4T1 showed aggressive tumor growth, ADC-treated tumor free animals re-challenged with D2F2/E2 demonstrated significantly (p<0.0001) slower tumor growth compared with the age-matched naive mice, and this protection was significantly (p=0.0078) enhanced in ADC + anti-PD-L1-treated tumor free animals, resulting in completely rejection of the re-challenge and thus documenting immunological memory formation. B cell activation was obvious by the significant upregulation of Ki67 and GL7 CD86 on both CD45+CD19+ tumor-infiltration lymphocytes (TILs) and splenocytes. B cell differentiation was evident by the upregulation of GL7 on both TILs and splenocytes, as well as the expression of CD138 on splenotytes. We found that IgG1 was the predominant isotype in treated mice vs. IgG2a, IgG2b, IgG3, and ADC + anti-PD-L1 dual treatment drastically increased the amount of IgG1 in serum. In addition, ADC+ anti-PD-L1 dual treatment significantly increased the frequency of CD8+IFNγ+ cells in TILs. Both ADC alone (p=0.0004) and ADC+anti-PD-L1 dual treatment (p=0.01) significantly decreased CD4+FoxP3+ regulatory T cells. These results provide evidence that HER2-targeted ADC + anti-PD-L1 immunotherapy-induced B cell activation and differentiation along with the modulation of T cell subsets could confer host anti-HER2 immunity.

Citation Format: You Qin, Hannah E. Dobson, Frank I. Comer, Alfred E. Chang, Max S. Wicha, Qiao Li. Specific anti-HER2 host immunity conferred by HER2-targeted antibody drug conjugate therapy and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 514.
Abstract 544: Urine as a non-invasive alternative to blood for germline and somatic mutation detection in hepatocellular carcinoma
Authors: Kim Amy , Lin Selena , Cui Yixiao , Gade Terence , Shieh Fwu-Shan , Chao Max , Shieh John , Jain Surbhi , Cheng Jonathan , Hamilton James , Hann Hie-Won L. , Goryunov Dmitry , Su Ying-Hsiu
Published: July 2021

BACKGROUND: Cell-free DNA (cfDNA) from blood has become a promising analyte for cancer diagnostics. However, because blood contains high concentrations of proteins and enzyme inhibitors, plasma cfDNA quality can be variable. Urinary cfDNA has been shown to contain mutations associated with non-genitourologic cancers including hepatocellular carcinoma (HCC). In this study, we evaluate urine as a noninvasive alternative to blood-based liquid biopsy for both germline and ctDNA genotyping in HCC.

METHOD: Using qPCR, whole-genome sequencing (WGS), and targeted NGS, DNA isolated from blood or urine of patients with HCC was evaluated and analyzed for overall genome coverage, HCC hotspot coverage, and germline or somatic mutation concordance. Targeted NGS of plasma and urine cfDNA was also performed for detection of somatic variants.

RESULTS: Similar to plasma cfDNA, urine cfDNA showed a major mononucleosomal species of 165-170 bp, with dinucleosomal species also discernable in most healthy individuals and patients with HCC. Overall genome coverage breadth was similar between urine and plasma cfDNA, with higher fraction of covered cancer-associated mutation hotspots in urine cfDNA. Quantitative PCR analyses of HCC-associated mutations (TP53, CTNNB1, and hTERT) in 102 patients with HCC, revealed 78% overall concordance between plasma and urine. Targeted NGS of HCC-associated gene regions in additional 15 patients showed an 97% overall position-level concordance between plasma and urine cfDNA. Furthermore, urine outperformed plasma in both the sensitivity and specificity of detection of CTNNB1 and hTERT mutations.

CONCLUSION urine DNA can potentially be used as a completely noninvasive diagnostic biomarker for HCC.

Citation Format: Amy Kim, Selena Lin, Yixiao Cui, Terence Gade, Fwu-Shan Shieh, Max Chao, John Shieh, Surbhi Jain, Jonathan Cheng, James Hamilton, Hie-Won L. Hann, Dmitry Goryunov, Ying-Hsiu Su. Urine as a non-invasive alternative to blood for germline and somatic mutation detection in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 544.
Abstract 578: Comparative analysis of cell free DNA with whole-exome sequencing (cfWES) and application to personalized minimal residual disease (MRD) monitoring
Authors: Wang Amy Xiaohong , Zhao Zhixin , Dai Chao , Zhou Kemin , Jia Shidong , Du Pan , Luo Shujun
Published: July 2021

Liquid biopsy has become increasingly important in cancer diagnosis, personalized medicine, and disease progression monitoring. Conventional liquid biopsy relies on cancer gene panels which often contain fewer than 500 genes. Despite the revolutionary impact it has brought to the cancer research and cancer patient care, gene panels often miss many key mutations involved in cancer development and drug response. To fully capture the genetic variations embedded in cfDNA, we have developed Predicine-cfWES assay, which expands the features of our existing 152-gene PredicineCARE and 600-gene PredicineATLAS NGS panel with additional coverage of coding regions of the entire human genome. With 2500 x sequencing depth, Predicine-cfWES assay detects common cancer variants at sensitivity of 1% allele frequency and precisely measures copy number variations, such as loss of tumor suppressor genes (PTEN, BRCA2, p53) and gain of oncogenic driver genes (AR, Myc, ERBB2). Variants detected by Predicine-cfWES assay was confirmed by PredicineCARE panel based NGS assay and further used in tumor-agnostic personalized MRD assay. In conclusion, we have developed a comprehensive liquid biopsy solution to profile cfDNA with broad coverage of ~20,000 genes in the whole exome and higher sensitivity in a focused set of clinically actionable cancer variants for more personalized MRD monitoring in cancer patients.

Citation Format: Amy Xiaohong Wang, Zhixin Zhao, Chao Dai, Kemin Zhou, Shidong Jia, Pan Du, Shujun Luo. Comparative analysis of cell free DNA with whole-exome sequencing (cfWES) and application to personalized minimal residual disease (MRD) monitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 578.
Abstract 581: Prognostic implication of circulating tumor-derived KRAS mutation in pancreatic ductal adenocarcinoma
Authors: Kim Min Kyeong , Lee Dong-Eun , Kim Yun Hee , Park Sang Jae , Woo Sang Myung , Kong Sun-Young
Published: July 2021

Circulating tumor DNA (ctDNA) has been evaluated as potential biomarkers for prognosis in pancreatic adenocarcinoma(PDAC) including our previous study, which proved KRAS mutation as a prognostic marker through the quantitative analysis. Then we investigated in a larger samples to validate the results of previous studies. Total of 213 PDAC patients has been enrolled in this study and the median age was 66 years old and 120 males (56.1%). Plasma was separated by established centrifugation method and ctDNA was extracted from 2.0 mL plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen Cat# 61504, Hilden, Germany) or a Chemagic cfDNA 2K kit (PerkinElmer, Waltham, Massachusetts, U.S.). Extracted ctDNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). The detection of KRAS mutation was performed using QX200 Droplet Digital PCR System (Biorad, USA) by KRAS screening multiplex droplet digital PCR kit which covers seven common mutation sites (Biorad, USA) and compared with that in 93 matched formalin-fixed paraffin-embedded (FFPE) tissues. Mutant concentration and fractional abundance were analyzed by QuantaSoft software (Biorad, USA). The KRAS mutation rates in tissue and ctDNA were 98.9% and 82.8%, respectively, and the concordance rate between them was 78 of 93 samples (83.9%). Univariable analyses demonstrated that KRAS fraction (Hazard ratio (HR), 1.59; 95% CI, 1.02-2.49; P = 0.040) were significant factors for PFS, whereas KRAS mutation concentration (HR, 1.69; 95% CI, 1.03-2.77; P = 0.038) and KRAS fraction (HR, 1.88; 95% CI, 1.17-3.02; P = 0.010) for OS. This study proved the usefulness of the KRAS mutation of ctDNA as a prognostic marker in PDAC. (This study was supported by National cancer center, Korea, Grant no. 1910192)

Citation Format: Min Kyeong Kim, Dong-Eun Lee, Yun Hee Kim, Sang Jae Park, Sang Myung Woo, Sun-Young Kong. Prognostic implication of circulating tumor-derived KRAS mutation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 581.
Abstract 584: Circulating tumor cells as prognostic biomarkers in glioblastoma
Authors: Bark Juliana Müller , Kulasinghe Arutha , Leo Paul , Hartel Gunter , Warkiani Majid E. , Jeffree Rosalind L. , Chua Benjamin , Day Bryan W. , Punyadeera Chamindie
Published: July 2021

Background: Glioblastoma (GBM) is the most common and aggressive type of tumor arising from the central nervous system. GBM remains an incurable disease despite current advancement in therapies, with survival rates of approximately 15 months. Recent literature has highlighted that GBM can release tumoral content which crosses the blood-brain barrier (BBB) and as a consequence can be detected in patients' blood, such as circulating tumor cells (CTCs). CTCs carry tumor information and have shown promise as prognostic and predictive biomarkers in other cancer types. Currently, the prognostic utility of CTCs in GBM is not well understood.

Methods: Blood samples from GBM patients (n=18) attending a tertiary referral hospital were collected before and after surgery to investigate the prognostic role of CTCs. CTCs were isolated using spiral microfluidic technology and characterized using immunofluorescence for GFAP, cell surface vimentin and CD45, in addition to fluorescence in situ hybridization (FISH) to detect EGFR amplification. CTC counts of patients with poor outcomes (recurrence or death) versus patients with good outcomes (stable disease and no recurrence) were compared using a negative binomial distribution.

Results: CTCs were found in 12 out of 18 patients (66.7%), 8/18 before surgery and 10/17 after surgery. CTC clusters were found in 2 patients. CTCs counts before surgery were significantly higher for patients with poor clinical outcome (p=0.0396).

Conclusion: Our preliminary data support the use of this spiral technology to isolate CTCs. CTCs and CTC clusters can be used as prognostic biomarkers for managing GBM patients.

Citation Format: Juliana Müller Bark, Arutha Kulasinghe, Paul Leo, Gunter Hartel, Majid E. Warkiani, Rosalind L. Jeffree, Benjamin Chua, Bryan W. Day, Chamindie Punyadeera. Circulating tumor cells as prognostic biomarkers in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 584.
Abstract 597: Comprehensive analysis of blood-based biomarkers to immunotherapy using circulating tumor cells, peripheral blood cells and circulating tumor DNA in advanced non-small cell lung cancer
Authors: Park Cheol-Kyu , Cho Hyun-Ju , Oh In-Jae , Kim Young-Chul
Published: July 2021

Background: This study aimed to investigate the feasibility of liquid biopsy using circulating tumor cells (CTCs), peripheral blood cells (PBCs) and circulating tumor DNA (ctDNA) as predictive blood-based biomarkers to immunotherapy in advanced non-small cell lung cancer (NSCLC).

Methods: This study included patients with advanced NSCLC receiving pembrolizumab or atezolizumab from July 2019 to June 2020. Blood was collected in K2-EDTA tube before each administration from cycle 1 to 4 (C1-4), and PBC counts (absolute neutrophil count[ANC], neutrophil-to-lymphocyte ratio[NLR], derived NLR[dNLR], platelet-to-lymphocyte ratio[PLR]) were calculated at each cycle. CTCs were enriched by using CD-PRIMETM system, the antibody-independent size-based isolation method, and identified by single positive cells (EpCAM/CK+CD45-) in BioViewCCBSTM system. Cell-free DNA was purified and extracted from plasma at baseline and C4 or end-of-treatment.

Results: Among 83 response-evaluable patients, objective response rate was 19% and durable clinical benefit (DCB) were 34%. Baseline (C1) CTC, PBC count and ctDNA amount did not show a significant difference according to best response and DCB. However, patients with lower NLR, dNLR, PLR and ctDNA level at C1 showed significant benefit in progression-free survival (PFS) and overall survival (OS) (p<0.05 for all). Patients with decreased CTC at C2 compared to C1 showed benefit in median PFS (6.2 vs 2.3, p=0.078) and OS (not reached [NR] vs 6.8, p=0.021) than those with increased CTC count. Low dNLR (≤2.0) at C1 and decreased CTC at C2 compared to C1 were the independently significant factors for survival.

Conclusion: Comprehensive analysis of CTC, PBC count and ctDNA amount at baseline and their changes during treatment could be potential biomarkers to predict the survival benefit and may help risk-group stratification in patients with advanced NSCLC who received anti-PD-1/PD-L1 immunotherapy.

Citation Format: Cheol-Kyu Park, Hyun-Ju Cho, In-Jae Oh, Young-Chul Kim. Comprehensive analysis of blood-based biomarkers to immunotherapy using circulating tumor cells, peripheral blood cells and circulating tumor DNA in advanced non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 597.
Abstract 647: CXCL17 as a prognostic biomarker for aggressive cutaneous squamous cell carcinoma
Authors: Fermin Janmaris Marin , Alam Maksudul MD , Gu Xin , Asarkar Ameya , Nathan Cherie-Ann , Khandelwal Alok R.
Published: July 2021

Cutaneous squamous cell cancer (cSCC) is one of the most rapidly increasing cancers in the USA, striking 200,000 Americans annually. cSCC is most often observed in the sun-exposed areas such as the head and neck region. Although most patients have an improved prognosis after surgical treatment, a subset carries an increased risk of local recurrence, nodal metastasis, and disease-specific death. CXCL17 (C-X-C Motif Chemokine Ligand 17) is the latest chemokine family member, and our preliminary studies identified a causative role of CXCL17 in skin tumorigenesis. Accordingly, we aimed to investigate the correlation between CXCL17 and clinicopathological factors associated with recurrence, metastasis, and disease-specific death in cSCC. A retrospective chart review was conducted to identify patients with cSCC at Louisiana State University Health Sciences Center Shreveport (LSUHSC-S) and Overton Brooks Veterans Affairs Medical Center. Tissue samples were immunostained for human CXCL17/VCC-1. Risk factors such as the depth of invasion, perineural invasion, tumor diameter, location, and differentiation were obtained from chart review/clinical databases and correlated with the expression of CXCL17. Among 42 subjects included in the analysis, the Histo-score of CXCL17 expression in cytoplasm and nucleus was higher in the malignant group compared to the normal skin group (p=0.01 and p=0.03, respectively). Interestingly, the Histo-score of CXCL17 expression was associated with perineural invasion (p=0.01) suggesting a role for CXCL17 in disease aggressiveness. Our results indicate a positive correlation between CXCL17 and cSCC aggressiveness. Increased expression of CXCL17 was associated with perineural invasion, a principal risk factor for recurrence, metastasis, and disease-specific death in cSCC patients. These results suggest that CXCL17 plays a role as a prognostic marker and warrants further exploration as a potential target for treatment in aggressive cSCC.

Citation Format: Janmaris Marin Fermin, Maksudul MD Alam, Xin Gu, Ameya Asarkar, Cherie-Ann Nathan, Alok R. Khandelwal. CXCL17 as a prognostic biomarker for aggressive cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 647.
Abstract 664: Development and validation of a glycolysis-related gene signature to predict overall survival in lung adenocarcinoma patients
Authors: Wang Shengguang , Bei Zhang
Published: July 2021

Background: Genomic expression profiles have enabled the classification of lung adenocarcinoma (LUAD) into molecular sub-types and provide prognostic information, which have implications for the personalized treatment of LUAD beyond clinical and pathological features.

Methods: Gene-expression profiling was performed on 459 patients in TCGA-LUAD(I-IIIa) for which RNA sequencing data and clinical covariates were available. A single-sample gene set enrichment analysis (ssGSEA) algorithm were used to quantified the levels of various hallmarks of cancer. And LASSO Cox regression model was used to screen robust prognostic biomarkers to identify the best set of survival-associated gene signatures in LUAD. Statistical analyses were performed using R version 3.4.4.

Results: We identified glycolysis as the primary risk factor for survival(HR=0.55 [0.38-0.8], P<0.001)among various hallmarks of cancer in TCGA-LUAD. And glycolysis ssGESA scores were significantly elevated in patients who died during follow up (P<0.001). Kaplan-Meier analysis showed that patients with high ssGSEA scores of glycolysis exhibited worse OS ( HR=1.9,P= 0.001).Adjusted for stage,age,gender, our signature was an independent risk factor for overall survival in TCGA cohorts (HR=1.67[1.19-2.30],P= 0.003). We then apply the glycolysis -related gene signature to classify patients into the high risk group and the low risk group with the cutoff of median. In external independent cohorts, similar results were observed. In the validation cohort GEO31210, the multivariate Cox regression analysis showed that glycolysis -related gene signature exhibited an independent risk prediction for overall survival in 226 patients with stage I-II LUAD (HR=2.5 [1.11-5.5], P =0.025).

Conclusions: By analyzing the gene-expression data with bioinformation approach, we developed and validated a risk prediction model with glycolysis -related expression scores in early stage LUAD, which have the potential to identify patients who could have better overall survival. Furthmore, our study confirmed that tumor glycolysis has wide-ranging effects, would become an attractive therapeutic target.

Citation Format: Shengguang Wang, Zhang Bei. Development and validation of a glycolysis-related gene signature to predict overall survival in lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 664.