bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3024: Relationship between venetoclax concentrations and undetectable MRD in patients with chronic lymphocytic leukemia
Authors: Gopalakrishnan Sathej , Menon Rajeev , Chyla Brenda , Samineni Divya , Jacobson Amanda , Kim Su Young , Mensing Sven , Salem Ahmed Hamed
Published: September 2020

Minimal residual disease (MRD) is an important clinical endpoint in chronic lymphocytic leukemia (CLL) because it correlates with progression free survival and overall survival. The objective of this work is to investigate the impact of venetoclax (VEN) exposures on undetectable MRD rates in blood and bone marrow (BM) in relapsed/refractory (R/R) CLL patients receiving VEN monotherapy or combination therapy with rituximab (RTX).

Data from a total of 708 patients from four VEN monotherapy studies and one combination Phase 1b study (VEN + 6-9 cycles of RTX) in R/R CLL patients were utilized to perform this analysis. The median number of prior therapies in these studies was 3, 42% of the patients had 17p chromosomal deletion and 28% had prior B-Cell Receptor inhibitor (BCRi) therapy. Blood and BM MRD assessments were available for 70% and 38%, respectively, of patients included in the analysis. Absence of an MRD measurement was assumed as detectable MRD for the purposes of this analysis.

The average concentration (Cavg) of VEN was computed for each patient by fitting a population pharmacokinetic model to the observed VEN concentration data. The model was used to calculate area under the curve and compute Cavg accounting for VEN dose ramp-ups, reductions and interruptions. The relationships between venetoclax Cavg and undetectable MRD rates were assessed by using quartile plots. Logistic regression analyses were also conducted to quantify the exposure-response relationship.

With VEN monotherapy, the undetectable MRD rates in blood and BM increased with the increase in VEN exposures. With VEN + RTX combination treatment, the undetectable MRD rates in blood and BM showed an increase with increasing VEN average concentration, but the benefit saturated with limited increase at exposures beyond 400mg.

Logistic regression analyses indicated statistically significant (p<0.01) relationships between VEN average concentrations and undetectable MRD rates in both blood and BM.

These results demonstrate that higher venetoclax concentrations are associated with higher undetectable MRD rates in both blood and BM. When VEN is combined with RTX, an increase in VEN dose beyond 400mg is unlikely to result in higher undetectable MRD rates.

Citation Format: Sathej Gopalakrishnan, Rajeev Menon, Brenda Chyla, Divya Samineni, Amanda Jacobson, Su Young Kim, Sven Mensing, Ahmed Hamed Salem. Relationship between venetoclax concentrations and undetectable MRD in patients with chronic lymphocytic leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3024.
Abstract 3039: Essential role of IRAK4/TPL2 signaling axis in MAPK activation by oncogenic RAS and genotoxic stress
Authors: Dodhiawala Paarth B. , Khurana Namrata , Zhang Daoxiang , Cheng Yi , Li Lin , Seehra Kuljeet , Jiang Hongmei , Grierson Patrick M. , Wang-Gillam Andrea , Ruzinova Marianna B. , Lim Kian-Huat
Published: September 2020

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival of 9% and effective treatment options remain elusive. Oncogenic mutations of KRAS occur in >95% of PDACs and are well-established as the bona fide driver event. However, inhibition of KRAS oncoprotein or its downstream signaling cascades remains unsuccessful in PDAC patients. Furthermore, parallel survival pathways including constitutive activation of the NF-κB pathway poses an additional therapeutic barrier. Previous work from our lab showed that Interleukin-1 Receptor associated kinase 4 (IRAK4) is a major driver of NF-kB cascade in PDAC. Here, through an unbiased reverse phase protein array screen and RNA sequencing, we discovered IRAK4 controls MAPK activity downstream of KRAS. Ablation of IRAK4 completely abolishes RAS-induced transformation in human and murine cell lines. Mechanistically, we implicate a KRAS-driven IL-1β signaling loop that activates IRAK4 and uncover MAP3K8 (or TPL2/COT) as the kinase through which IRAK4 activates MEK and ERK. Suppression of TPL2 abrogates KRAS-driven MEK-ERK activity and transformed growth of PDAC cell lines. In addition, TPL2 inhibition suppresses p105/p50 NF-kB activation, a valuable phenomenon that distinguishes TPL2 inhibition from MEK inhibition. We find TPL2 inhibition synergizes with chemotherapy to suppress growth of PDAC cell lines in vitro and patient-derived xenograft tumor model in vivo. Analyses of PDAC tissue microarray showed TPL2 expression to be marginally associated with poor prognosis. Additionally, we are the first to characterize gain-of-function point mutations in TPL2 which hyperactivate MAPK and NF-kB, in part by preventing TPL2 protein degradation. Together, our study broadens the understanding of the oncogenic RAS signaling network and reveals IRAK4 and TPL2 as novel practical therapeutic targets in RAS-driven cancers.

Citation Format: Paarth B. Dodhiawala, Namrata Khurana, Daoxiang Zhang, Yi Cheng, Lin Li, Kuljeet Seehra, Hongmei Jiang, Patrick M. Grierson, Andrea Wang-Gillam, Marianna B. Ruzinova, Kian-Huat Lim. Essential role of IRAK4/TPL2 signaling axis in MAPK activation by oncogenic RAS and genotoxic stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3039.
Abstract 3044: The novel bi-specific antibody CKD-702 is a potential agent for NSCLC patients with aberrant cMET and EGFR signaling
Authors: park kyu jin , Jeon Eun-Ju , Choi Ji-Hye , Lee Kwan-Woo , Lee Kyung-Woo , Park Gun-Woo , Hong Hae-Jin , Hwang In-Chang , Moon Seung-Kee , Koh Yeo-Wook
Published: September 2020

Development of drug resistance is one of the main causes of poor prognosis and therapeutic failure in NSCLC. Mutual synergism between cMET and EGFR is known to promote acquired drug resistance. In NSCLC, cMET gene amplification is reported in 2-4% previously untreated patients and in 5-20% of patients with EGFR mutation and acquired resistance to EGFR TKIs. Because of this crosstalk between cMET and EGFR pathways, dual inhibition of these targets holds promise as a treatment for NSCLC patients, especially those with acquired cMET activation.

CKD-702 is a bispecific antibody designed to dually target cMET and EGFR. It is tetravalent IgG1-like bispecific antibody, in which single-chain variable fragment specific to EGFR is genetically fused to the c-terminus of a conventional IgG1 of a cMET. The antibody binds to extracellular domains of cMET and EGFR with high affinity and inhibits signaling by blocking ligand binding. CKD-702 also induces internalization and degradation of both receptors.

There are various types of cMET activation in NSCLC patients, including point mutations and amplification. An emerging aberrant cMET activation is through a splice site mutation of cMET that leads to skipping of exon 14, inducing prolonged signaling and oncogenic activity. In this study, we carried out genetic analysis of cMET activation in cancer cell line as well as PDX model with or without EGFR mutation, and the efficacy of CKD-702 in each model for NSCLC was determined. CKD-702 showed complete tumor regression as monotherapy in tumor models that have primary/secondary activating EGFR mutations and cMET amplification. Also, CKD-702 resulted in complete tumor regression in models where cMET is activated by either skipping of exon 14 or amplification. Interestingly, unlike other previously developed EGFR-targeting agents, CKD-702 showed limited skin rash in GLP toxicity study.

In summary, CKD-702 is confirmed to inhibit tumors with activated cMET pathway such as cMET amplification and exon 14 skipping as well as with EGFR-activating mutations. This profile of preclinical data supports the clinical trial of CKD-702 as monotherapy in selected lung cancer patients with aberrant cMET and EGFR signaling.

A Phase I study of CKD-702 is planned to initiate in 2020.

Citation Format: kyu jin park, Eun-Ju Jeon, Ji-Hye Choi, Kwan-Woo Lee, Kyung-Woo Lee, Gun-Woo Park, Hae-Jin Hong, In-Chang Hwang, Seung-Kee Moon, Yeo-Wook Koh. The novel bi-specific antibody CKD-702 is a potential agent for NSCLC patients with aberrant cMET and EGFR signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3044.
Abstract 3052: Targeting AXL in PIK3R2-amplified ovarian cancer
Authors: Rao Ling , Mak Victor C. , Zhou Yuan , Zhang Dong , Li Xinran , Lu Yiling , Mills Gordon B. , Cheung Lydia W.
Published: September 2020

The p85 regulatory subunit and its associated p110 catalytic subunit are components of the class IA PI3K that mediates the downstream signal of receptor tyrosine kinases. PIK3R2 (encoding p85β, one of the p85 isoforms) is commonly amplified in ovarian cancer patients. Importantly, high PIK3R2 expression significantly correlated with worse patient survival. Here, we showed that p85β promoted tumorigenic properties of ovarian cancer cells. The protein expression of a receptor tyrosine kinase AXL was positively regulated by p85β. AXL is a member of the TAM receptor family. The regulation was specific to AXL because the other two members (Tyro3 and MERTK) were not altered by p85β. Drug sensitivity assays suggested that inhibition of AXL reduced oncogenic activities of p85β in vitro and in vivo. Together, we propose p85β as a potential therapeutic target in ovarian cancer and therapeutic blockade of AXL signaling may be an effective strategy for ovarian cancer with PIK3R2 amplification.

Citation Format: Ling Rao, Victor C. Mak, Yuan Zhou, Dong Zhang, Xinran Li, Yiling Lu, Gordon B. Mills, Lydia W. Cheung. Targeting AXL in PIK3R2-amplified ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3052.
Abstract 3054: Targeting TGF-β signaling for obesity/insulin resistance-associated hepatocellular carcinoma
Authors: Chen Jian , Su Xiaoping , Rojas Andres , Bresalier Robert S , Stroehlein John R. , Yeung Sai-ching
Published: September 2020

Background: Recently, obesity, insulin resistance/type 2 diabetes mellitus and fatty liver disease, i.e., key components of the metabolic syndrome, have been recognized as risk factors for hepatocellular carcinoma (HCC). Obesity and dysmetabolism serve as drivers of oncogenesis in the setting of abnormal hepatic morphology and physiology, and hepatic steatosis may provide the promoting microenvironment for the development of HCC. Dysregulated signaling in the transforming growth factor (TGF)-β pathway plays a central role in immunomodulation, inflammation, and fibrogenesis in the HCC microenvironment. We used a carcinogen Diethylnitrosamine (DEN)-induced HCC mouse model in genetically obese mice [heterozygous Agouti yellow (Ay/a)] and haplo-knock out of Smad3 (a kinase downstream of type II receptor for TGFβ to study the detailed mechanism by which Smad3-dependent TGF-β signaling regulates obesity-associated dysmetabolism in HCC, and to determine whether inhibition of TGF-β signaling could inhibit obesity-associated HCC.

Methods: Ay/a mice in C57B/L genetic background were bred with Smad3 knockout mouse (Smad3+/−). Obese and insulin-resistant Ay/a mice were treated with DEN (i.p. 25mg/kg) at 14 days of age to induce HCC. Ay/a mice and DEN-induce HCC mice were treated with TGF-β receptor 2 inhibitor (EW7919, 20 mg/kg orally 3 times/week for 8 weeks). We performed RNA sequencing analysis for 40 mouse liver samples from the groups of mice above.

Results: 1. Haplodeficiency of Smad3 significantly decreased obesity Ay/a mouse weight and suppressed fatty liver development. 2. Treatment of TGF-β receptor 2 inhibitor to Ay mice successfully blocked weight gain in Ay/a mice. 3. DEN-induced hepatocarcinogenesis in Ay/a mice occurred significantly earlier than in lean (a/a) C57B/L mice. 4. Inhibition of TGF-β signaling by Smad3 haplodeficiency significantly upregulated lipid catabolic processes and downregulated cytokine-induce leukocyte migration in the fatty livers of Ay/a mice. 5. Inhibition of TGF-β signaling by genetic or pharmacologic inhibitors significantly suppressed HCC development in Ay/a mice.

Conclusion: We demonstrated a specific cancer promoting role of TGF-β in metabolic reprogramming in liver cancer cells, highlighting the TGF-β signaling pathway as a potential therapeutic target in obesity/insulin resistance-associated HCC.

Citation Format: Jian Chen, Xiaoping Su, Andres Rojas, Robert S Bresalier, John R. Stroehlein, Sai-ching Yeung. Targeting TGF-β signaling for obesity/insulin resistance-associated hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3054.
Abstract 3068: The glycoprotein inhibitors overcome the resistance of the topoisomerase inhibitors in small cell lung cancer
Authors: Noro Rintaro , Omori Miwako , Fukuizumi Aya , Matsuda kuniko , Hirao Mariko , Takahashi Satoshi , Takano Natsuki , Nakamichi Shinji , Sugano Teppei , Miyanaga Akihiko , Minegishi Yuji , Seike Masahiro , Kubota Kaoru , Gemma Akihiko
Published: September 2020

Small cell lung cancer (SCLC) is a highly aggressive disease with median survival of <2 years. Most patients respond initially to the topoisomerase inhibitors, resistance rapidly emerges. The purpose is overcoming the resistance of the topoisomerase inhibitors in SCLCs. To identify the key factors for the resistance of the topoisomerase I (SN38) or II (VP-16) inhibitors in SCLCs, we analyzed MTS cell proliferation assay. We identified 8 sensitive cell lines and 2 resistant cell lines. We established 4 resistant cell lines by continuous exposure to increasing concentrations of SN-38 or VP-16 stepwise.

RNA sequencing was performed using the established cell lines to discover novel therapeutic targets associated with the resistance to topoisomerase inhibitors. We found that whenever the drug efflux protein ATP-binding cassette transporter B1 (ABCB1) was associated the with resistance of VP-16, ATP-binding cassette super-family G member 2 (ABCG2) was associated with the resistance of SN-38. The silencing of ABCG2 or ABCB1 in every resistant cell line, could induce synergistic apoptosis of every cell line. Elacridar and tariquidar which are glycoprotein inhibitors, recovered the sensitivity of SN38 or VP-16 in every resistant line, increased expression of signaling proteins associated with DNA damage and cell-cycle checkpoints, and promoted G2/M arrest and apoptosis. Furthermore, we are going to analyze the expressions of ABCG2 and ABCB1 in serum before and after the treatment of VP-16 or CPT-11. These results suggested the glycoprotein inhibitors, which phase I study is ongoing, could be a promising therapeutic strategy for the purpose of preventing the resistance of topoisomerase inhibitors in SCLCs.

Citation Format: Rintaro Noro, Miwako Omori, Aya Fukuizumi, kuniko Matsuda, Mariko Hirao, Satoshi Takahashi, Natsuki Takano, Shinji Nakamichi, Teppei Sugano, Akihiko Miyanaga, Yuji Minegishi, Masahiro Seike, Kaoru Kubota, Akihiko Gemma. The glycoprotein inhibitors overcome the resistance of the topoisomerase inhibitors in small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3068.
Abstract 307: KSR1-dependent modulation of the translational landscape in Ras-driven colorectal cancer
Authors: Vieira Heidi , Rao Chaitra , Black Adrian R. , Southekal Siddesh , Mizutani Tomohiro , Guda Babu , Clevers Hans , Black Jennifer D. , Lewis Robert E.
Published: September 2020

KRAS is mutated and activated in an estimated 40% of colon cancers but has proven difficult to target directly due to the lack of drug-binding pockets on the surface of Ras. Thus, identifying effectors that transmit signals from oncogenic Ras is a critical step in targeting vulnerabilities in the tumors of patients with colorectal cancer (CRC). Kinase Suppressor of Ras 1 (KSR1) is a molecular scaffold that coordinates the interaction of effectors in the Raf/MEK/ERK signaling cascade downstream of Ras. KSR1 is necessary for oncogenic transformation of cells expressing mutated Ras but is dispensable for normal cell growth making KSR1 an attractive therapeutic target for Ras-driven colon cancers. In CRC cells, KSR1 and ERK mediate Ras-dependent effects on protein translation, notably driving cap-dependent and cap-independent translation of the Myc oncogene. Depletion of KSR1 or treatment with ERK inhibitor (SCH772984) leads to a decrease in phophorylated 4EBP1 and a decrease in PDCD4, which collectively impair protein translation, notably leading to a decrease in Myc translation. Patient-derived CRC organoids with deletion of APC, p53, and SMAD4, and an activating G12D mutation in KRAS treated with ERK inhibitor show a marked decrease in Myc protein expression, coincident with changes in PDCD4 and 4EBP1 that impair protein translation. These observations indicate that the KSR1-dependent ERK activation observed in CRC cell lines is present in pre-clinical colon tumoroid models. Genome-wide polysome profiling in CRC cell lines HCT116 and HCT15 depleted of KSR1 identified mRNAs that were preferentially translated in a KSR1 and ERK-dependent manner. Several of these mRNAs were previously predicted by our Functional Signature Ontology (FUSION) screen to be critical to CRC viability but dispensable for normal cell growth. GSEA identified functional classes of mRNAs whose translation is KSR1-dependent, including mediators of oncogenic signaling in pathways that regulate mTOR, MAPK, WNT and JAK-STAT. Our data suggest that an essential component of oncogenic Ras-induced and KSR1-dependent signaling is the preferential translation of mRNAs supporting the transformed phenotype of CRC cells.

Citation Format: Heidi Vieira, Chaitra Rao, Adrian R. Black, Siddesh Southekal, Tomohiro Mizutani, Babu Guda, Hans Clevers, Jennifer D. Black, Robert E. Lewis. KSR1-dependent modulation of the translational landscape in Ras-driven colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 307.
Abstract 3083: Selective inhibition of transcription factor BRN2 as a treatment strategy for Small Cell Prostate Cancer
Authors: Thaper Daksh , Munuganti Ravi , Aguda Adeleke , Kim Soojin , Ku Shungyu , Kumar Sahil , Vahid Sepideh , Nouruzi Shaghayegh , Sivak Olena , Ganguli Dwaiyapan , Morrissey Colm , Corey Eva , Beltran Himisha , Zoubeidi Amina
Published: September 2020

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges. In particular, a subset of patients who relapse following ARPI therapy their dependence on AR signaling and emerge with neuroendocrine features. These tumors, termed treatment induced small-cell prostate cancer (t-SCPC) or neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapies. Previously, our group identified the neural transcription factor BRN2 as a major clinically relevant driver of SCPC and now report that targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC.Methods/Results: In silico screening of small molecules was conducted on a model of BRN2 which was later validated with the first-in-field crystal structure of BRN2 DNA binding domain. On the basis of the model, several small molecules were identified that showed direct binding to BRN2 and inhibited its activity. Pharmacokinetic studies measured stability and bioavailability of med-chem optimized lead compound (BRN2i) that significantly reduced tumor growth in multiple xenograft models with no measurable side-effects. In silico modeling predicted a 7Å “closing” in the DBD once it was bound to BRN2i, this shift translated to reduced interaction with DNA by chromatin fractionation and ChIP-seq, thus confirming the mode of action for BRN2i is through loss of DNA binding. Loss of BRN2 binding reduced cell proliferation in tSCPC cell line 42DENZR, de novo SCPC cell line NCI-H660 and NEPC organoids as well as downregulated several known targets like EZH2, ASCL1, SOX2 and PEG10. Down-regulation of these target genes was also measured in the xenograft tumors, confirming on target effect in vivo. Moreover, the specificity of BRN2i was validated with CRISPR/Cas9 mediated knockout of BRN2 with downstream mRNA expression and phenotypic changes. Conclusion: The described work aims to lay the pre-clinical foundation for the integration of BRN2 targeted therapies into the treatment landscape to improve survival for patients suffering from small-cell neuroendocrine prostate cancer.

Citation Format: Daksh Thaper, Ravi Munuganti, Adeleke Aguda, Soojin Kim, Shungyu Ku, Sahil Kumar, Sepideh Vahid, Shaghayegh Nouruzi, Olena Sivak, Dwaiyapan Ganguli, Colm Morrissey, Eva Corey, Himisha Beltran, Amina Zoubeidi. Selective inhibition of transcription factor BRN2 as a treatment strategy for Small Cell Prostate Cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3083.
Abstract 3098: Urine-based molecular biomarker signatures for bladder cancer
Authors: Zhao Zhixin , Chang Amy , Xiang Binggang , Xie Feng , Wang Xiaohong , Zhou Kemin , Yu Jianjun , Du Pan , Jia Shidong
Published: September 2020

Introduction: By 2020 the estimated incidence of genitourinary (GU) cancers (prostate, bladder, and kidney) will be over 2 million worldwide and responsible for ∼800 000 deaths. Current diagnosis and monitoring methods of GU cancer patients are often invasive and/or lack sensitivity and specificity. The tremendous success in cell-free DNA (cfDNA) blood test provides a strong rationale for using body fluids such as blood and urine as minimally invasive “liquid biopsies” to identify DNA-based molecular biomarkers in urologic malignancies. Urinary cfDNA as a liquid biopsy holds greater potential for a more sensitive alternative to blood biopsies-based tests for clinical use in GU cancers. Urinary biomarker test offers advantages including the potential for more convenient testing, monitoring, and home use.

Methods: Analytic validation was conducted using urine cfDNA samples containing different SNPs with different titrations. Urine samples were collected from two different groups, including treatment-naïve patients with pathology-confirmed bladder cancer (BLCA) and healthy donors. Both tumor and germline tissues were collected from the BLCA patients.

Results: We report the development, validation, and clinical application of the urine-based next-generation sequencing for integrated genomic cfDNA profiling of bladder cancers, including genomic alterations, tumor mutation burden (TMB) and microsatellite instability (MSI). The analytic sensitivity goes down to 0.5% with 100% analytical specificity. High concordance for mutations between tumor tissue and urinary cfDNA suggests a higher sensitivity alternative for the non-invasive early detection of BLCA. In addition to the NGS-based test, we also developed a Bio-Rad digital PCR assay for orthogonal confirmation of the detected biomarkers such as FGFR3 hotspot mutations.

Conclusions: Our work has demonstrated the promise of using urinary cfDNA for detecting genomic alterations at the early stage of BLCA for biomarker detections.

Citation Format: Zhixin Zhao, Amy Chang, Binggang Xiang, Feng Xie, Xiaohong Wang, Kemin Zhou, Jianjun Yu, Pan Du, Shidong Jia. Urine-based molecular biomarker signatures for bladder cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3098.
Abstract 3099: Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients
Authors: Ottestad Anine Larsen , Emdal Elisabeth F. , Wahl Sissel G. , Grønberg Bjørn Henning , Dai Hong Yan
Published: September 2020

Purpose

Circulating tumor DNA (ctDNA) can potentially be used to monitor cancer treatment response. In this study, we developed and used a sensitive and selective ultra-deep next-generation sequencing (NGS) approach for detection of ctDNA in lung cancer patients.

Method

Most studies use fixed gene panels for detection of ctDNA by NGS. Deep sequencing of large target regions is expensive and generates a myriad of variants, which are difficult to interpret. For the purpose of using ctDNA for monitoring, we try to overcome these challenges by sequencing only selected regions where mutations were previously found in the tumor. This method is more sensitive and cost-efficient.

In this study, we included about 80 lung cancer patients. Tumor DNA was sequenced with a large gene panel and somatic mutations were identified in each tumor. We then defined the target region according to the tumor mutations and selected primers for constructing NGS libraries. Since the coverage region is small it enables ultra-deep sequencing of several samples simultaneously.

We evaluated the lower limit of detection for this method by sequencing a series of artificially constructed samples with known mutant allele frequencies (MAFs). DNA from peripheral blood leucocytes was sequenced in parallel with plasma DNA to exclude variants from clonal hematopoiesis.

Preliminary results

Using 40 ng as input DNA, which contains approximately 12,000 haploid genomes, we detected mutations down to 0.02% MAF. This corresponds to about 6 mutated copies per mL plasma. Currently, we are applying this method on patient plasma DNA samples.

Conclusion

We have developed a sensitive method for ctDNA detection, and we will present the results of the lung cancer patient samples.

Citation Format: Anine Larsen Ottestad, Elisabeth F. Emdal, Sissel G. Wahl, Bjørn Henning Grønberg, Hong Yan Dai. Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3099.
Abstract 3108: Combined cfDNA and cfRNA NGS testing for cancer patient care
Authors: Huang Yong , Zhang Yue , Xiang Binggang , Zhao Zhixin , Wang Xiaohong , Xie Feng , Moktari Reza , Zhou Kemin , Du Pan , Jia Shidong , Yu Jianjun
Published: September 2020

Background: NGS based liquid biopsy has its unique advantage in cancer patient care/monitoring as it allows repeated testing of a spectrum of target sites at multiple time points. However, cfDNA assays usually can only cover focused genomic regions. In order to monitor dynamic fusion and alternative splicing variants, we developed and validated a cfRNA assay which can be used in combination with cfDNA assays to get clear snapshots of mutation landscapes in cancer patients. Here we introduce our application of the combined cfDNA and cfRNA approach to cancer patient care.

Method: A single tube of blood is taken from cancer patients, which is used to extract both cfDNA and cfRNA. Predicine proprietary cfDNA and cfRNA NGS assays and analysis pipelines are used to detect SNV/INDEL, CNV as well as fusion and alternative RNA splicing variants from cancer samples, collected at multiple time points from patients during or after treatment.

Results: The cfDNA+cfRNA combined approach allows the cross-validation of results from both methods, which greatly elevated the confidence level of our testing results. For example, a case in which EML4-ALK fusion was detected at cfDNA level but only with fairly low allele frequency was confidently confirmed, as EML4-ALK fusion was also detected at cfRNA level. On the other hand, the cfDNA+cfRNA combined approach allows us to study the broad mutation landscape from both cfDNA and cfRNA levels. For example, for a cohort of prostate samples, the changes of AR alternative splicing forms and the cfDNA mutation landscape can be observed from the same tube of blood samples. In general, the cfDNA+cfRNA combined approach provides a more complete molecular biomarker profiles and extract more information that can be used to instruct cancer patient care.

Citation Format: Yong Huang, Yue Zhang, Binggang Xiang, Zhixin Zhao, Xiaohong Wang, Feng Xie, Reza Moktari, Kemin Zhou, Pan Du, Shidong Jia, Jianjun Yu. Combined cfDNA and cfRNA NGS testing for cancer patient care [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3108.
Abstract 3115: A single institution experience with droplet digital polymerase chain reaction (dd-PCR) liquid biopsy (LB) for therapeutic decision in advanced solid tumors
Authors: Murad Andre Marcio , Casali-da-Rocha José Claudio , Carneiro Juliana Garcia
Published: September 2020

Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of cell-free circulating free nucleic acid (DNA and RNA) due to its high sensitivity, low cost, and fast reading, since it dispenses bioinformatics, making it an appropriate alternative to new generation sequencing (NGS) for the detection of biomarkers to guide molecularly targeted cancer therapy. The assay covers main actionable hotspot alterations across many actionable genes: EGFR(mutations), ALK (fusion, mutations), ROS1(fusion), BRAF(mutations), KRAS (mutations), NRAS(mutations), PIK3CA (mutations), ERBB2(CNV- copy number variation), ESR1(mutations), KIT(CNV) and PDGFRA(CNV). Therefore, our genes can be used in panels, both in therapeutically applied genotyping and in detecting molecular responses as well as in secondary resistance to tumors such as NSCLC, breast, cervix. rectal, GIST, melanoma and pancreas. METHODS: dd-PCR was performed using the QX200 system (BIO-RAD®, Hercules). The extraction of DNA and RNA was done using the magnetic beads technique of Thermofisher's MagMAX Kit. All samples were tested in duplicate. 54 metastatic cancer patients were tested so far: NSCLC - 14 (26%), breast - 11 (20%), colorectal - 11 (20%), melanoma - 9(17%), pancreas - 4(7%), ovarian - 3(6%) and salivary glands - 2 (4%). RESULTS: Significant genomic alterations were detected in 19 (35%) patients: 5 (9%) mutations in the KRAS G12V gene (all in colorectal cancer), 5 (9%) ERBB2 amplification (breast cancer), 2 (3.65%) EGFR L858R mutations (NSCLC), 2 (3.5%) EGFR del19 mutations (NSLC), 2 (3.5%) EGFR T790M mutations (NSCLC), 2 (3.5%) BRAF-V600E mutations (colon and melanoma), 1 (1.8%) ALK-EML4 fusion (NSCLC). The MAF (Mutant Allele Fraction) ranged from 0.9% to 24%. In all cases, the results were decisive for the indication or change of a targeted therapy. Median turnaround time was 36 hours and the average cost of the panels was around 500 USD (median of 4 genes per panel). CONCLUSION: Our results suggest that dd-PCR is a highly sensitive method and could be used for a routine laboratory detection of the important genomic variations to determine the targeted therapy in patients with varied advanced solid tumors.

Citation Format: Andre Marcio Murad, José Claudio Casali-da-Rocha, Juliana Garcia Carneiro. A single institution experience with droplet digital polymerase chain reaction (dd-PCR) liquid biopsy (LB) for therapeutic decision in advanced solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3115.
Abstract 3125: Disturbance of CALR mutation profile in MPNs by NGS and Real-Time PCR
Authors: Rencuzogullari Cagla , Bozdogan Sevcan Tug , Guvenc Birol , Bisgin Atil
Published: September 2020

Myeloproliferative neoplasms (MPNs) are clonal disorders which are driven by one of three driver mutations that cause activation of JAK/STAT signaling. MPNs have classically been divided into three main conditions; essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). CALR mutations act as a driver mutation early in the pathogenesis of these disorders and the mutations in exon 9 of CALR gene occur in approximately 30% of patients with MPNs. Thus, in this study CALR mutation profiling had been performed by real time PCR then the next generation sequencing (NGS) to identify the differential genetic profile of Turkish population in MPNs. Genomic DNA was isolated from peripheral blood samples. A real time PCR test was used to detect c.1092_1143del variant, c.1154_1155insTTGTC variant and other 34 minor variants (Ipsogen CALR RGQ PCR Kit). Then the minor variants and possible other variants were identified via NGS (GeneReader NGS System). CALR mutations were detected in 34/128 of the patients. Among the 34 patients with CALR mutations, 15 of them had at least one mutation and 19 had at two mutations. 22 of 34 patients who were classified as in minor variants by real time PCR, were then studied via NGS to specify the mutations. The detected sub-variants are shown at Table 1. Interestingly, five mutations that are unique and not included in the real time PCR were identified by sequencing. Identification of the driver mutations are important for disease diagnosis and progress. Though, our study shows the disturbance of the mutations in Turkey together with the redundance of real time PCR and NGS data.

Identified CALR mutations by NGSVariant DetectedTotal Number of Patientsc.1099_1150delCTTAAGG10c.1142_1144delAGG7c.1100_1101delTTinsAC1c.1154_1155insTTGTC6c.1124A>G1c.1103_1136del AGGAGGA1

Citation Format: Cagla Rencuzogullari, Sevcan Tug Bozdogan, Birol Guvenc, Atil Bisgin. Disturbance of CALR mutation profile in MPNs by NGS and Real-Time PCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3125.
Abstract 314: Micropatterning supported method for cancer spheroid assays under flow and high throughput CAR-T cell potency assay with single cell resolution
Authors: Horn Elias , Balles Miriam , Fischbeck Angelika J. , Schwarz Jan , Thyssen Dane A. , Drell David W. , Noessner Elfriede , Zantl Roman
Published: September 2020

Tumor spheroids, organoids, and cell culture models demand defined cell-cell and cell-matrix interactions, ideally with specific cell attachment on adhesion sites for multicellular spheroids or single cells. Our bioinert based micropatterning technology allows for the creation of adhesion spots while the surrounding passivated area is biologically inert, not interfering with the assay.We use this micro-patterning of adhesion ligands on highly passivated surfaces to generate homogenously distributed spheroids within a microfluidic channel system which can be perfused. Spheroids can either be directly generated by a simple injection of a large number of cells and a self-sorting process or by the addition of pre-formed spheroids, which will bind to the patterned adhesion sites. Perfusion of the spheroid chip enables the homogenous supply of all spheroids with nutrients and oxygen, and also the application of a defined shear stress, metabolite analysis together with toxicological screenings or even co-culture of multiple spheroid types. Applying a flow increases the overall spheroid growth, but condenses the cell aggregate, as well as influences the drug triggered protein expression response towards a more in vivo-like reaction.We also use this technique to generate arrays of homogenously distributed single cancer cells to evaluate cytotoxic T cell activity on a single cell level. By optical analysis combined with machine-learning based image processing of cell / spheroid arrays we obtained efficient label-free analysis of each cancer cell - T cell interaction over time.

Citation Format: Elias Horn, Miriam Balles, Angelika J. Fischbeck, Jan Schwarz, Dane A. Thyssen, David W. Drell, Elfriede Noessner, Roman Zantl. Micropatterning supported method for cancer spheroid assays under flow and high throughput CAR-T cell potency assay with single cell resolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 314.
Abstract 3171: Biomarkers of responses to immunotherapy in advanced hepatocellular carcinoma
Authors: Ting Vox Z. , Chew Valerie , Wong Carmen C. , Yau Thomas C.
Published: September 2020

Biomarkers from the peripheral blood may serve as a useful tool to identify who will likely benefit from immunotherapy in patients with advanced hepatocellular carcinoma; hence, oncologists might be able to monitor treatment responses regularly via blood tests in addition to routine radiological assessment. To investigate immune profiles of patients with advanced HCC after exposure to immunotherapy by analysing circulating T cells from peripheral blood, our study included stage IV HCC patients (n = 14) seen at the Department of Medical Oncology, Queen Mary Hospitals. Nine patients received PD-1/PD-L1 inhibitor immunotherapy at the discretion of their clinicians, and five treatment-naïve patients were included as a control group. A control group of healthy subjects was also included in the study. Samples from patients who received PD-1/PD-L1 inhibitor therapy were collected after the treatment response was confirmed by radiological assessment. T cell profiles and exhaustion markers were compared along with treatment response as determined by radiological assessment.

A total of 14 HCC patients were enrolled in the study: 4 patients showed a partial response to PD-1/PD-L1 inhibitor treatment, 5 patients progressed during PD-1/PD-L1 inhibitor treatment, and 5 patients were not treated with PD-1/PD-L1 inhibitors. Five buffy coat samples from healthy subjects were included as controls. Decreases in the percentage of regulatory T cells (0.8% of T cells in responders, p<0.0001) and in the ratio of effector memory T cells to regulatory T cells (EMT/Treg) were noted in patients who responded to PD-1/PD-L1 inhibitor therapy compared to those who did not, and thus may serve as biomarkers for immunotherapy responses in HCC. Similarly, we observed decreases in Tim-3 (2.09%, p<0.0001) and OX-40 (6.12%, p<0.0001) expression between responders and non-responders, and thus expression levels of these factors could potentially serve as response predictors. An increase in the proportion of CD4 CD8 double-positive (DP) T cells in peripheral blood was observed in immunotherapy responders (16.63%) versus non-responders (4.03%, p<0.05). Biomarkers in circulating T cells allow the investigation of immune profiles after treatment with immunotherapy using non-invasive approach from peripheral blood. Coupled with conventional surveillance strategies, we have identified biomarkers may serve as predictors for HCC patients who will likely benefit from immunotherapy.

Citation Format: Vox Z. Ting, Valerie Chew, Carmen C. Wong, Thomas C. Yau. Biomarkers of responses to immunotherapy in advanced hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3171.
Abstract 3192: Quanticell, a novel histopathology technology for visualization and quantitation of membrane PSMA demonstrated in cell lines and clinical tissue
Authors: Dimant Hemi , Gandhi Ankit , Maker Micheal , Markman Adam , Reinhardt Jeanette , Collins Stefan , Hesterman Jacob , Krueger Joseph , Bloom Kenneth , Bailis Julie , Juan Gloria
Published: September 2020

Prostate specific membrane antigen (PSMA), being both a biomarker and a therapeutic target, is important in prostate cancer (PC) diagnosis and treatment. Imaging and pathology are two of the most widely used approaches for PSMA detection in the diagnosis of PC. However, these techniques lack sensitivity for accurate assessment of low levels of PSMA expression and lack the capabilities to properly characterize heterogeneous PSMA expression. When using anti-PSMA imaging ligands, radiotherapies or immunotherapies, these drawbacks may ultimately prevent proper diagnosis and response prediction and could deny anti-PSMA treatment to qualified patients that have low PSMA expression. Better, more sensitive detection methods are needed for improved PSMA-based diagnosis and prediction of response. Quanticell is a novel histopathology technology utilizing fluorescent nanoparticles that are compatible with standard immunostaining practices. Combined with a dedicated software, Quanticell provides increased sensitivity and quantification and a wide dynamic range for improved detection and characterization of a broad range of target expression. A pilot study was conducted to demonstrate this technology on PSMA-expressing cell lines and clinical samples, and to evaluate its utility for discovery research and clinical studies. An anti-PSMA Quanticell protocol was developed and optimized on several PSMA-expressing cell lines, focusing on the lowest expressing sample. A dual-staining protocol for PSMA and a membrane marker was further developed to allow for detection and analysis of membrane-specific PSMA. Staining of negative and positive controls demonstrate high specificity to PSMA with high signal-to-noise that is not affected with increased primary concentration. When compared to DAB (diaminobenzidine) staining, we observed increased sensitivity in a low PSMA-expressing cell line, VCAP, indicated by increased detection of percent PSMA-positive cells. Additionally, using Quanticell, we demonstrate increased dynamic range in high expressing PSMA clinical samples compared to DAB staining, which is subject to saturation.

Citation Format: Hemi Dimant, Ankit Gandhi, Micheal Maker, Adam Markman, Jeanette Reinhardt, Stefan Collins, Jacob Hesterman, Joseph Krueger, Kenneth Bloom, Julie Bailis, Gloria Juan. Quanticell, a novel histopathology technology for visualization and quantitation of membrane PSMA demonstrated in cell lines and clinical tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3192.
Abstract 3197: Serum CEA change predicts tumor regression and prognosis in locally advanced rectal cancer
Authors: Cai Zerong , Huang Lingyu , Lan Ping , Wu Xiaojian
Published: September 2020

Objective: To investigate the value of carcinoembryonic antigen (CEA) change in predicting pathological tumor regression and outcome for locally advanced rectal cancer (LARC) patients received neoadjuvant therapy.

Material and methods: LARC patients who received neoadjuvant therapy and radical tumor resection were retrospectively collected. The predictive value of baseline CEA, post-treatment CEA and post/pre-treatment CEA ratio for tumor regression grade (TRG), overall survival and diseases free survival were estimated by logistic regression or cox proportional hazard regression.

Results: 700 LARC patients were enrolled in the study with 475 (67.9%) cases of male patients and median age of 56 (20-87). Of the all patients enrolled, multivariate analysis showed post-treatment CEA (OR=1.142, 95% CI: 1.073-1.215, P<0.001) was an independent predictor for TRG.

284 patients with elevated baseline CEA were analyzed. Among them, 168 patients (59.2%) have normalized CEA. Multivariate analysis showed post-treatment CEA (OR=1.068, 95% CI: 1.007-1.133, P=0.027), CEA ratio (OR=3.463, 95% CI: 1.269-9.446, P=0.015) were independent predictors for TRG(Table 1).

Of the all patients enrolled, multivariate analysis showed low preoperative albumin level (HR=0.919, 95% CI: 0.886-0.952, P<0.001), tumor deposit (HR=3.226, 95% CI: 1.470-7.077, P=0.003), nerve infiltration (HR=3.602, 95% CI: 1.736-7.476, P=0.001), high post-treatment CEA (HR=1.029, 95% CI: 1.010-1.049, P=0.003) were independent unfavored predictors for overall survival of LARC patients.

700 patients were divided into 3 groups: A group with normal baseline CEA (n=416,59.4%); B group with normalized CEA (n=168,24.0%); C group with elevated baseline and post-treatment CEA (n=116,16.6%). The overall survival and disease free survival of group B were comparable to that of group A (P=0.588,0.053) and significantly better than that of group C (B vs. C: P=0.010, 0.003; A vs. C: P=0.007, <0.001).

Conclusion: 1. Post-treatment CEA is an independent predictor for TRG of LARC patients and higher post-treatment CEA precdicted poor overall survival.

2. Post/pre-treatment CEA ratio is an independent predictor for TRG of LARC patients with elevated baseline CEA.

Citation Format: Zerong Cai, Lingyu Huang, Ping Lan, Xiaojian Wu. Serum CEA change predicts tumor regression and prognosis in locally advanced rectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3197.