bluuebunny/crossref_metadata_2025 huggingface dataset:
ESR1 Mutations and Overall Survival on Fulvestrant versus Exemestane in Advanced Hormone Receptor–Positive Breast Cancer: A Combined Analysis of the Phase III SoFEA and EFECT Trials
Authors: Turner Nicholas C. , Swift Claire , Kilburn Lucy , Fribbens Charlotte , Beaney Matthew , Garcia-Murillas Isaac , Budzar Aman U. , Robertson John F.R. , Gradishar William , Piccart Martine , Schiavon Gaia , Bliss Judith M. , Dowsett Mitch , Johnston Stephen R.D. , Chia Stephen K.
Published: June 2020

Purpose:

ESR1 mutations are acquired frequently in hormone receptor–positive metastatic breast cancer after prior aromatase inhibitors. We assessed the clinical utility of baseline ESR1 circulating tumor DNA (ctDNA) analysis in the two phase III randomized trials of fulvestrant versus exemestane.

Experimental Design:

The phase III EFECT and SoFEA trials randomized patients with hormone receptor–positive metastatic breast cancer who had progressed on prior nonsteroidal aromatase inhibitor therapy, between fulvestrant 250 mg and exemestane. Baseline serum samples from 227 patients in EFECT, and baseline plasma from 161 patients in SoFEA, were analyzed for ESR1 mutations by digital PCR. The primary objectives were to assess the impact of ESR1 mutation status on progression-free (PFS) and overall survival (OS) in a combined analysis of both studies.

Results:

ESR1 mutations were detected in 30% (151/383) baseline samples. In patients with ESR1 mutation detected, PFS was 2.4 months [95% confidence interval (CI), 2.0–2.6] on exemestane and 3.9 months (95% CI, 3.0–6.0) on fulvestrant [hazard ratio (HR), 0.59; 95% CI, 0.39–0.89; P = 0.01). In patients without ESR1 mutations detected, PFS was 4.8 months (95% CI, 3.7–6.2) on exemestane and 4.1 months (95% CI, 3.6–5.5) on fulvestrant (HR, 1.05; 95% CI, 0.81–1.37; P = 0.69). There was an interaction between ESR1 mutation and treatment (P = 0.02). Patients with ESR1 mutation detected had 1-year OS of 62% (95% CI, 45%–75%) on exemestane and 80% (95% CI, 68%–87%) on fulvestrant (P = 0.04; restricted mean survival analysis). Patients without ESR1 mutations detected had 1-year OS of 79% (95% CI, 71%–85%) on exemestane and 81% (95% CI, 74%–87%) on fulvestrant (P = 0.69).

Conclusions:

Detection of ESR1 mutations in baseline ctDNA is associated with inferior PFS and OS in patients treated with exemestane versus fulvestrant.
Predictive Value of Molecular Subtypes in Premenopausal Women with Hormone Receptor–positive Early Breast Cancer: Results from the ABCSG Trial 5
Authors: Bago-Horvath Zsuzsanna , Rudas Margaretha , Singer Christian F. , Greil Richard , Balic Marija , Lax Sigurd F. , Kwasny Werner , Hulla Wolfgang , Gnant Michael , Filipits Martin
Published: June 2020

Purpose:

To assess the predictive value of molecular breast cancer subtypes in premenopausal patients with hormone receptor–positive early breast cancer who received adjuvant endocrine treatment or chemotherapy.

Experimental Design:

Molecular breast cancer subtypes were centrally assessed on whole tumor sections by IHC in patients of the Austrian Breast and Colorectal Cancer Study Group Trial 5 who had received either 5 years of tamoxifen/3 years of goserelin or six cycles of cyclophosphamide, methotrexate, and fluorouracil (CMF). Luminal A disease was defined as Ki67 <20% and luminal B as Ki67 ≥20%. The luminal B/HER2-positive subtype displayed 3+ HER2-IHC or amplification by ISH. Recurrence-free survival (RFS) and overall survival (OS) were analyzed using Cox models adjusted for clinical and pathologic factors.

Results:

185 (38%), 244 (50%), and 59 (12%) of 488 tumors were classified as luminal A, luminal B/HER2-negative and luminal B/HER2-positive, respectively. Luminal B subtypes were associated with poor outcome. Patients with luminal B tumors had a significantly shorter RFS [adjusted HR for recurrence: 2.22; 95% confidence interval (CI), 1.41–3.49; P = 0.001] and OS (adjusted HR for death: 3.51; 95% CI, 1.80–6.87; P < 0.001). No interaction between molecular subtypes and treatment was observed (test for interaction: P = 0.84 for RFS; P = 0.69 for OS).

Conclusions:

Determination of molecular subtypes by IHC is an independent prognostic factor for recurrence and death in premenopausal women with early-stage, hormone receptor–positive breast cancer but is not predictive for outcome of adjuvant treatment with tamoxifen/goserelin or CMF.

See related commentary by Hunter et al., p. 5543
Statins Reduce Intratumor Cholesterol Affecting Adrenocortical Cancer Growth
Authors: Trotta Francesca , Avena Paola , Chimento Adele , Rago Vittoria , De Luca Arianna , Sculco Sara , Nocito Marta C. , Malivindi Rocco , Fallo Francesco , Pezzani Raffaele , Pilon Catia , Lasorsa Francesco M. , Barile Simona N. , Palmieri Luigi , Lerario Antonio M. , Pezzi Vincenzo , Casaburi Ivan , Sirianni Rosa
Published: June 2020

Mitotane causes hypercholesterolemia in patients with adrenocortical carcinoma (ACC). We suppose that cholesterol increases within the tumor and can be used to activate proliferative pathways. In this study, we used statins to decrease intratumor cholesterol and investigated the effects on ACC growth related to estrogen receptor α (ERα) action at the nuclear and mitochondrial levels. We first used microarray to investigate mitotane effect on genes involved in cholesterol homeostasis and evaluated their relationship with patients' survival in ACC TCGA. We then blocked cholesterol synthesis with simvastatin and determined the effects on H295R cell proliferation, estradiol production, and ERα activity in vitro and in xenograft tumors. We found that mitotane increases intratumor cholesterol content and expression of genes involved in cholesterol homeostasis, among them INSIG, whose expression affects patients' survival. Treatment of H295R cells with simvastatin to block cholesterol synthesis decreased cellular cholesterol content, and this affected cell viability. Simvastatin reduced estradiol production and decreased nuclear and mitochondrial ERα function. A mitochondrial target of ERα, the respiratory complex IV (COXIV), was reduced after simvastatin treatment, which profoundly affected mitochondrial respiration activating apoptosis. Additionally, simvastatin reduced tumor volume and weight of grafted H295R cells, intratumor cholesterol content, Ki-67 and ERα, COXIV expression and activity and increase terminal deoxynucleotidyl transferase dUTP nick end labeling–positive cells. Collectively, these data demonstrate that a reduction in intratumor cholesterol content prevents estradiol production and inhibits mitochondrial respiratory chain–inducing apoptosis in ACC cells. Inhibition of mitochondrial respiration by simvastatin represents a novel strategy to counteract ACC growth.
A Novel Approach to Safer Glucocorticoid Receptor–Targeted Anti-lymphoma Therapy via REDD1 (Regulated in Development and DNA Damage 1) Inhibition
Authors: Lesovaya Ekaterina A. , Savinkova Alena V. , Morozova Olga V. , Lylova Evgeniya S. , Zhidkova Ekaterina M. , Kulikov Evgeny P. , Kirsanov Kirill I. , Klopot Anna , Baida Gleb , Yakubovskaya Marianna G. , Gordon Leo I. , Readhead Ben , Dudley Joel T. , Budunova Irina
Published: June 2020

Glucocorticoids are widely used for therapy of hematologic malignancies. Unfortunately, chronic treatment with glucocorticoids commonly leads to adverse effects including skin and muscle atrophy and osteoporosis. We found recently that REDD1 (regulated in development and DNA damage 1) plays central role in steroid atrophy. Here, we tested whether REDD1 suppression makes glucocorticoid-based therapy of blood cancer safer. Unexpectedly, approximately 50% of top putative REDD1 inhibitors selected by bioinformatics screening of Library of Integrated Network-Based Cellular Signatures database (LINCS) were PI3K/Akt/mTOR inhibitors. We selected Wortmannin, LY294002, and AZD8055 for our studies and showed that they blocked basal and glucocorticoid-induced REDD1 expression. Moreover, all PI3K/mTOR/Akt inhibitors modified glucocorticoid receptor function shifting it toward therapeutically important transrepression. PI3K/Akt/mTOR inhibitors enhanced anti-lymphoma effects of Dexamethasone in vitro and in vivo, in lymphoma xenograft model. The therapeutic effects of PI3K inhibitor+Dexamethasone combinations ranged from cooperative to synergistic, especially in case of LY294002 and Rapamycin, used as a previously characterized reference REDD1 inhibitor. We found that coadministration of LY294002 or Rapamycin with Dexamethasone protected skin against Dexamethasone-induced atrophy, and normalized RANKL/OPG ratio indicating a reduction of Dexamethasone-induced osteoporosis. Together, our results provide foundation for further development of safer and more effective glucocorticoid-based combination therapy of hematologic malignancies using PI3K/Akt/mTOR inhibitors.
Abstract A06: Multiplatform analysis of early-stage cancer signatures in blood
Authors: Li Bingsi , Wang Chenyang , Xu Jiayue , Fang Shuai , Qiu Fujun , Su Jing , Chu Huiling , Han-Zhang Han , Mao Xinru , Liu Hao , Liu Xianling , Zhang Wei , Zhao Heng , Zhang Zhihong
Published: June 2020

Background: Detection of cancer in its early stages is likely to be the most effective way to improve clinical outcome. In patients with cancer, a portion of cell-free DNA (cfDNA) in the blood stream is tumor derived, providing an opportunity to analyze the cancer genome in real time noninvasively. MERMAID is a retrospective multicenter case-control study to investigate early tumor signatures in blood using different platforms.

Methods: The study was conducted among 452 surgery-resectable patients with lung cancer (N=180), colorectal cancer (N=210), liver cancer (N=62), and 290 age-/sex- matched non-cancer controls. Patients who were recognized to have anemia, autoimmune diseases, treated with neoadjuvant therapy were excluded from the study. The non-cancer controls were recruited with the criteria of showing no clinical symptoms or history of cancer at time of administration. The participants were divided into subgroups and analyzed by ultra-deep mutation sequencing (HS-UMI), droplet digital PCR (ddPCR), and deep methylation sequencing (ELSA-seq) singly or in combination.

Results: Overall, the highest accuracy was achieved using ELSA-seq and will be reported in full. Highly similar classification results were obtained in training and test sets, with the area under the curve (AUC) value ranging from 0.90-0.97. The specificity for each cancer type ranged from 96-99%, and the average sensitivities with 95CI were lung cancer (61%, 53-70%), colorectal cancer (82%, 76-87%), and hepatocellular cancer (91%, 80-96%). Moreover, incorporation of somatic variants and epigenetic alterations improved the overall accuracy.

Conclusions: This study highlighted the potential of machine learning-aided deep methylation sequencing as a sensitive ctDNA profiling approach for early cancer detection. Further investigation in large-scale clinical studies is ongoing.

Citation Format: Bingsi Li, Chenyang Wang, Jiayue Xu, Shuai Fang, Fujun Qiu, Jing Su, Huiling Chu, Han Han-Zhang, Xinru Mao, Hao Liu, Xianling Liu, Wei Zhang, Heng Zhao, Zhihong Zhang. Multiplatform analysis of early-stage cancer signatures in blood [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A06.
Abstract A17: Pan-solid tumor comparison of variant detection in paired liquid and tissue biopsies
Authors: Assaf Zoe June F. , Sivakumar Smruthy , Jin Dexter X. , Maund Sophia L. , Lyalina Svetlana , Walia Guneet , Sokol Ethan S. , Trabucco Sally E.
Published: June 2020

Comprehensive genomic profiling (CGP) of circulating tumor DNA (ctDNA) provides an opportunity to noninvasively monitor a patient’s tumor burden via liquid biopsy. Liquid biopsies can assess a patient’s mutational landscape through time and provide information on treatment response and relapse. It has become increasingly common to have paired genomic data analysis between liquid and tissue biopsies from the same patient. However, the impact of clinical, temporal, and biologic factors on percentage of positive agreement (PPA) of variant detection between these biopsies is unclear. For our study, we leveraged two databases containing CGP data from paired liquid and tissue biopsy specimens tested by Foundation Medicine (FMI): the FMI database (>1700 paired samples) and the Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB). The CGDB is a subset of the FMI database linked with the Flatiron Health nationwide de-identified EHR-derived database, which includes demographic, treatment, and clinical outcomes information (>500 paired samples). We report pan-solid tumor and per-indication prevalence and PPA for variants commonly assayed in both liquid and tissue tests. We found that PPA depended on the tumor DNA concentration in plasma and tissue biopsies, as well as the amount of time between tests. We also investigated the effect of treatments administered in the time between liquid and tissue tests on the detected variants and observed some well-described resistance alterations at a higher frequency in liquid samples, including a higher prevalence of ESR1 point mutations in breast cancer, more EGFR T790M alterations in lung cancer, and frequent KRAS Q61H alterations in colorectal cancer. Liquid biopsies can also contain DNA shed from multiple metastatic sites. We observed evidence of this in the form of polyclonal resistance alterations, which may also account for differences in PPA over time. Our findings indicate that while PPA is generally high between samples, it may be influenced by factors such as intervening therapies, resistance, therapy efficacy, and polyclonality of liquid samples.

Citation Format: Zoe June F. Assaf, Smruthy Sivakumar, Dexter X. Jin, Sophia L. Maund, Svetlana Lyalina, Guneet Walia, Ethan S. Sokol, Sally E. Trabucco. Pan-solid tumor comparison of variant detection in paired liquid and tissue biopsies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A17.
Abstract A18: Plasma circulating tumor DNA is scarce and confounded by clonal hematopoiesis in metastatic renal cell carcinoma
Authors: Bacon Jack V. W. , Annala Matti , Soleimani Maryam , Lavoie Jean-Michel , So Alan I. , Gleave Martin E. , Fazli Ladan , Chi Kim N. , Kollmannsberger Christian K. , Wyatt Alexander W. , Nappi Lucia
Published: June 2020

Despite the recent implementation of novel targeted therapies, the 5-year survival rate for metastatic renal cell carcinoma (mRCC) remains dismal. Prognostic and predictive biomarkers are urgently required to avoid unnecessary patient morbidity and financial toxicity. Exploratory studies leveraging archival primary tumor tissue are considered suboptimal in this setting due to the considerable heterogeneity of metastatic lesions. Circulating tumor DNA (ctDNA) permits the noninvasive characterization of metastatic cancers using a simple blood draw. In this study, we sought to establish the utility of a ctDNA-based assay as a tool to profile the somatic genome of patients with mRCC. We collected whole blood from 55 progressing mRCC patients. All patients were systemic therapy naïve at the time of sample collection. Plasma cell-free DNA (cfDNA) and matched leukocyte DNA were subjected to targeted sequencing across 981 cancer-associated genes. Matched tumor tissue from 14 patients was also analyzed. The median cfDNA sequencing depth for this cohort was 938x. 33% of patients had evidence for RCC-derived ctDNA above 1% of total cfDNA; this was significantly lower than prostate or bladder cancer patients analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no association with clinical variables or cfDNA yield. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential (CHIP), was detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. Patients with ctDNA above 1% had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of CHIP but not ctDNA had an intermediate prognosis compared to ctDNA-positive and ctDNA-negative patients. CfDNA sequencing enables characterization of the somatic RCC genome in only a minority of metastatic RCC patients. Due to low ctDNA abundance and presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC.

Citation Format: Jack V. W. Bacon, Matti Annala, Maryam Soleimani, Jean-Michel Lavoie, Alan I. So, Martin E. Gleave, Ladan Fazli, Kim N. Chi, Christian K. Kollmannsberger, Alexander W. Wyatt, Lucia Nappi. Plasma circulating tumor DNA is scarce and confounded by clonal hematopoiesis in metastatic renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A18.
Abstract A37: Early change in circulating tumor DNA as a potential predictor of response to chemotherapy in patients with metastatic colorectal cancer
Authors: Zembutsu Hitoshi , Osumi Hiroki , Shinozaki Eiji , Yamaguchi Kensei
Published: June 2020

The impact of the change in ctDNA levels after chemotherapy on the clinical outcomes of patients with metastatic colorectal cancer (mCRC) remains unclear. The present study evaluated the clinical implications of the early change in ctDNA levels as a predictor of objective response and the clinical outcome in mCRC patients who received chemotherapy. We investigated the effects of after/before ratio of ctDNA levels 2 and 8 weeks after initiation of second-line chemotherapy on objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). ctDNA was detected using amplicon-based deep sequencing with a molecular barcode that includes >240 hotspot mutations in 14 colon cancer-related genes. Patients with lower ctDNA level (≤ 50%) 8 weeks after initiation of chemotherapy compared to baseline showed significantly longer PFS and OS than the patients with higher (> 50%) ctDNA level in multivariate analysis. In patients achieving partial response or stable disease, the after/before ratio of ctDNA level 8 weeks after initiation of chemotherapy was significantly lower than those in patients with progressive disease. The present study suggests that an early change in the ctDNA level might serve as a biomarker to predict the chemotherapeutic efficacy and clinical outcomes in patients with mCRC.

Citation Format: Hitoshi Zembutsu, Hiroki Osumi, Eiji Shinozaki, Kensei Yamaguchi. Early change in circulating tumor DNA as a potential predictor of response to chemotherapy in patients with metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A37.
Abstract A52: Role of circulating tumor DNA (ctDNA) from liquid biopsy in early-stage NSCLC resected lung tumor investigation (LIBERTI)
Authors: Morgensztern Daniel , Green Emma , King Jennifer C. , Sable-Hunt Alicia , Erwin Richard , Moore Amy , Addario Tony , Morris Clive , Govindan Ramaswamy
Published: June 2020

The detection of ctDNA after cancer surgery with curative intent may indicate the presence of minimal residual disease and a higher risk for relapse. The Addario Lung Cancer Medical Institute (ALCMI) has launched a multicenter study to evaluate the role of ctDNA in patients with completely resected stage II to III non-small cell lung cancer (NSCLC) using a highly sensitive assay developed by Inivata. This molecular residual disease (MRD) personalized assay is used to detect tumor-specific variants in plasma DNA from patients with NSCLC. The study will enroll 500 patients across three cohorts, with the initial ctDNA collection prior to neoadjuvant therapy (cohort 1), prior to surgery (cohort 2), or between the surgery and initiation of adjuvant chemotherapy (cohort 3). We will conduct an initial evaluation of ctDNA results after enrollment of 100 eligible patients. All patients will be followed for survival and recurrence for a maximum of 60 months from the date of surgery. The primary objective is to correlate the presence of ctDNA following complete surgical resection with disease-free survival.

Citation Format: Daniel Morgensztern, Emma Green, Jennifer C. King, Alicia Sable-Hunt, Richard Erwin, Amy Moore, Tony Addario, Clive Morris, Ramaswamy Govindan. Role of circulating tumor DNA (ctDNA) from liquid biopsy in early-stage NSCLC resected lung tumor investigation (LIBERTI) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A52.
Abstract B10: Feasibility of bronchial washing fluid-based approach to early-stage lung cancer diagnosis
Authors: Ryu Jeong Seon , Lee Myoung Kyu , Lee Seung Jae
Published: June 2020

Circulating tumor DNA has few applications for detecting early-stage lung cancers. Bronchial washing (BW) is minimally invasive, and BW fluids can contain tumor DNA. Thus, analysis of BW fluids could be used for precision medicine. Forty-eight paired specimens (primary tumor tissue, normal tissue, BW supernatant, and BW precipitate) from twelve patients with early-stage non-small cell lung cancer were analyzed using ultradeep next-generation sequencing with a custom panel containing 113 genes. In primary tumors, 130 missense mutations/indels (5–16 per patient) and 20 driver mutations (0–3) were found; 105 (80.8%) and 97 (74.6%) of these missense mutations/indels were identified in BW supernatants and precipitates, respectively, and 19 (95.0%) of the driver mutations in both BW supernatants and precipitates. Concordance between EGFR mutations in primary tumors, BW supernatants, and BW precipitates was 100%. The median allele frequencies (AFs) for missense mutations/indels in primary tumors, BW supernatants, and BW precipitates were 13.0%, 0.3%, and 0.1%, respectively. The AFs of missense mutations/indels in BW supernatants correlated with those in primary tumors and in BW precipitates (P<0.001). In conclusion, this study provides evidence that the BW fluids, especially supernatants, can be an alternative to overcome the limitation that clinical utility of ctDNA is often not reliable in early-stage NSCLC.

Citation Format: Jeong Seon Ryu, Myoung Kyu Lee, Seung Jae Lee. Feasibility of bronchial washing fluid-based approach to early-stage lung cancer diagnosis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B10.
Abstract B15: Circulating tumor cells express tissue specific antigens in multiple cancers
Authors: Akolkar Dadasaheb , Patil Revati , Patil Darshana , Fulmali Pradip , Fulmali Pooja , Patel Shoeb , Devhare Pradip , Ainwale Akshay , Datta Vineet , Sims Cynthe , Srinivasan Ajay , Datar Rajan
Published: June 2020

Definitive diagnosis of solid organ cancers is based on histopathologic analysis of tumor tissue obtained by invasive biopsies. However, invasive biopsies are associated with procedural expenses, sequelae, and risks. Though immunocytochemistry (ICC) profiling of circulating tumor cells (CTCs) has been previously attempted for diagnosis in some cancers, they have been unviable due to limited yields of CTCs. We used a novel approach for enrichment of sufficient viable CTCs that permit meaningful ICC profiling for diagnosis of cancers in suspected cases. We obtained 15 mL of venous blood draw from 965 patients with confirmed diagnosis of various solid organ cancers. CTCs were enriched from PBMCs fraction using an epigenetically activated enrichment process that is lethal towards normal (nonmalignant) cells but confers survival privilege on apoptosis-resistant CTCs. Harvested CTCs were confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of these CTCs was performed with organ-specific marker for various organs such as lungs (TTF-1 or Napsin), breast (GCDFP15 or GATA3), ovarian (CA125 or WT-1), pancreatic (CK19 or CA19-9), cervical (P63), liver (Hep Par-1 or Glypican), colorectal (CK20 or CDX-2), head and neck (P63), kidney (Pax-8 or RCC), and prostate (PSMA or AMACR). CTCs (EpCAM+, pan-CK+) could be obtained from 94.4% of all samples. Organ-specific markers were positive in 100% of lung cancer, 79.3% of breast cancers, 70.0% of ovarian cancers, 93.3% of pancreatic cancers, 100% of cervical cancers, 61% of hepatocellular carcinomas, 92.6% ofcColorectal cancers, 98.8% of head and neck cancers, 100% of renal cancers, and 100% of prostate cancers. Our results show that deep ICC profiling of CTCs can determine tissue of origin in solid organ cancers noninvasively to substitute conventional procedures dependent on tissue extraction.

Citation Format: Dadasaheb Akolkar, Revati Patil, Darshana Patil, Pradip Fulmali, Pooja Fulmali, Shoeb Patel, Pradip Devhare, Akshay Ainwale, Vineet Datta, Cynthe Sims, Ajay Srinivasan, Rajan Datar. Circulating tumor cells express tissue specific antigens in multiple cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B15.
Abstract B26: A new way of fabricating high-porosity parylene membranes for high-throughput capturing of viable circulating and exfoliated tumor cells from large-volume bodily fluids
Authors: Gu Jian , Rabadi Inad , Zenhausern Frederic , Carpentieri David , Wang Jue
Published: June 2020

Liquid biopsy is a critical component in cancer precision medicine. The ability to capture rare cancer cells from liquid samples, such as circulating tumor cells (CTCs) from peripheral blood as well as exfoliated tumor cells (ETCs) from other body fluids, has shown great promises in cancer detection and prognosis, as well as understanding of cancer biology at different stages. Different ways have been developed for rare cancer cell capture, including biomarker-based immunocapture and capture through other physical processes, such as size, mechanical or dielectric properties, hydrodynamic fluids, etc. However, the throughputs of these techniques are limited to 0.2-2 mL/min. Recently, a high-porosity parylene membrane has been shown to be able to process sample at a fluid rate of > 100 mL/min, which would greatly extend the capability of CTC and ETC-based liquid biopsy. The high porosity of the membrane can also dramatically reduce the shear stress the tumor cells encountered during capture, which has been shown to be critical to maintain cell viability. However, the current parylene membrane fabrication involves deep Si reactive ion etching, which is an expensive process and not widely available. The release of the parylene membrane can also be challenging. We report here a new method to fabricate the parylene membrane using simple photolithography and reactive ion etching techniques. The fabricated parylene membranes and their capability of processing large volume sample for viable cell capture will be presented.

Citation Format: Jian Gu, Inad Rabadi, Frederic Zenhausern, David Carpentieri, Jue Wang. A new way of fabricating high-porosity parylene membranes for high-throughput capturing of viable circulating and exfoliated tumor cells from large-volume bodily fluids [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B26.
Abstract B37: Analysis of non-small cell lung cancer (NSCLC) circulating biomarkers for monitoring early response to radiation therapy
Authors: Purcell Emma , Niu Zeqi , Grzesik Madeline , Owen Sarah , Fairbairn Heather , Jolly Shruti , Nagrath Sunitha
Published: June 2020

Approximately 20% of non-small cell lung cancer (NSCLC) patients will be diagnosed with stage 3 cancer, with a predicted survival rate between 20-30%. Stage 3 NSCLC patients commonly undergo chemoradiation treatment, with imaging scans being the standard of care for determining tumor control or progression both during and after radiation treatment. Imaging technologies can only show progression after it has occurred, making treatment challenging. We suggest that the molecular characterization of biomarkers found in the bloodstream can identify or predict radiation treatment efficacy before progression becomes visible on a scan. In this study, patients undergo a response-driven adaptive radiation therapy (RDART) protocol: four weeks of chemoradiation followed by a set of imaging scans before an additional 2 weeks of adapted, personalized radiation with chemotherapy. In this work, we provide combined analysis of both circulating tumor cells (CTCs), a rare cell found in a patient’s peripheral blood, and exosomes, nanovesicles excreted from cells, for use in assessing radiation efficacy. Our initial cohort is 30 patients, and we aimed to collect six timepoints: one pretreatment, two during radiation treatment, and three after radiation treatment. In this study, we look at the changes in several characteristics: CTC counts, PD-L1 expression on CTCs, as well as gene expression changes of both CTCs and exosomes using Affymetrix microarrays. CTCs were isolated using a graphene oxide (GO)-based microfluidic device and were found to be present in all pretreatment samples and most following samples. Initial transcriptome profiling of 5 patients using microarrays highlighted clustered gene expression differences for both CTCs and exosomes when comparing pretreatment samples with those collected during radiation.

Citation Format: Emma Purcell, Zeqi Niu, Madeline Grzesik, Sarah Owen, Heather Fairbairn, Shruti Jolly, Sunitha Nagrath. Analysis of non-small cell lung cancer (NSCLC) circulating biomarkers for monitoring early response to radiation therapy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B37.
Abstract B44: An optimized method of multimodal mRNA and gDNA isolation from low biomass input
Authors: van de Flierdt Jens , Sprenger-Haussels Markus , Otte Michael , Hauch Siegfried
Published: June 2020

Background: In cell-based applications in liquid biopsy research (circulating tumor cells [CTC], fetal cells, endothelial cells), often transcriptomes get analyzed. Here and in single-cell research there is a requirement to also investigate genomic variants. Therefore, a multimodal sample preparation for both gDNA and RNA (preferably mRNA) is increasingly needed in the market. There are several combined RNA/DNA preparation solutions already available with the following caveats: Optimizing DNA yield and quality is often achieved under the compromise of accepting losses in RNA yield and/or quality or vice versa. This may be acceptable for larger sample inputs but is critical for low-biomass samples. The goal of this project, therefore, was to develop an optimized AllPrep solution for low-biomass samples that provides both highest mRNA and gDNA quality and yield without any compromises.

Material and Methods: As starting material for the mRNA purification from low-biomass input samples, the AdnaTest EMT 2/StemCell Select/Detect procedure was used. The procedure has already been tested widely to get highest yields of mRNA from as few as 2 cells spiked into 5 mL of blood. Cells captured with immunomagnetic beads as provided in the AdnaTest CTC-Select procedure were lysed using the AdnaTest Lysis/Binding Buffer and mRNA was purified using Oligo(dT)25 beads. After mRNA depletion cell lysates still contained gDNA, which can be isolated using magnetic affinity binding beads in an optimized workflow. mRNA recovery was demonstrated by reverse transcription and subsequent endpoint PCR. gDNA was analyzed using PCR and pyrosequencing as downstream applications.

Results: It has been shown that the optimized workflow combination allowed the isolation of mRNA and gDNA from as few as 5 tumor cells spiked into 5 mL of blood. Tumor cell-related gene expression profiles can be analyzed after reverse transcription of mRNA and multiplex PCR. In parallel, the recovered gDNA was successfully used in PCR assays as well as for detection of known gene variants by means of pyrosequencing. Furthermore, gel electrophoretic analysis of the gDNA eluates showed good gDNA integrity with mean molecular weight values of >60 kbp.

Citation Format: Jens van de Flierdt, Markus Sprenger-Haussels, Michael Otte, Siegfried Hauch. An optimized method of multimodal mRNA and gDNA isolation from low biomass input [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B44.
Abstract B48: Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA
Authors: Van Paemel Ruben , De Koker Andries , Vandeputte Charlotte , Van Zogchel Lieke , Lammens Tim , Laureys Geneviève , Vandesompele Jo , Schleiermacher Gudrun , Chicard Matthieu , Van Roy Nadine , Vicha Ales , Tytgat Godelieve A. M. , Callewaert Nico , De Wilde Bram , De Preter Katleen
Published: June 2020

In the clinical management of pediatric solid tumors, histologic examination of tumor tissue obtained by an invasive biopsy remains the gold standard to establish a conclusive pathologic diagnosis. However, correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. The DNA methylation pattern of a tumor is known to correlate with the histopathologic diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. It has previously been shown that this methylation pattern can be detected in the cell-free circulating DNA isolated from blood plasma of cancer patients. Here, we provide proof of concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. Using computational deconvolution of the DNA methylation profiles, we were able to obtain the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free circulating DNA. We could demonstrate that the majority of misclassifications were associated with bad sample quality, evidenced by an increase of the test accuracy to 94.3% when only including good-quality samples. Misclassifications were not associated with the extent of the disease. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Our findings show the potential of the approach to tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner. Given the fact that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in these difficult cases. A preprint of the study manuscript is already available at bioRxiv: https://doi.org/10.1101/795047.

Citation Format: Ruben Van Paemel, Andries De Koker, Charlotte Vandeputte, Lieke Van Zogchel, Tim Lammens, Geneviève Laureys, Jo Vandesompele, Gudrun Schleiermacher, Matthieu Chicard, Nadine Van Roy, Ales Vicha, Godelieve A. M. Tytgat, Nico Callewaert, Bram De Wilde, Katleen De Preter. Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B48.
Abstract B58: Detection of ESR1 gene fusions in breast cancer cell-derived exosomal RNA
Authors: Liu Tiantong , Hooda Jagmohan , Ludwig Nils , Whiteside Theresa , Atkinson Jennifer M. , Oesterreich Steffi , Lee Adrian
Published: June 2020

Background: Estrogen receptor-positive (ER+) breast cancer is often intractable due to endocrine therapy resistance. Although ER basepair hotspot mutations have been well characterized in ~20-30% of endocrine-resistant disease, accumulating evidence from our group and others suggests that other genetic alterations such as ER fusions (e.g., ESR1-DAB2) could also play a role. As an important component of liquid biopsies, exosomes have recently gained much attention in the field of clinical oncology. It is well established that some species of RNA, typically microRNA and mRNA, are packaged into these small membrane-enclosed structures, which could potentially serve as a noninvasive biomarker to constantly and comprehensively monitor tumor progression and tumor cell evolution. We tested whether ESR1 fusions could be detected in exosomal RNA (exoRNA) produced by breast cancer cell lines.

Methods: Multiple methods were used to characterize exosomes isolated from cell culture media: 1) Nanoparticle tracking analysis (NTA) confirmed concentration and size of exosomes, 2) immunoblotting identified protein markers that are enriched or lacking in exosomes, 3) transmission electron microscopy validated the size and shape of exosomes. During exoRNA extraction, DNA contamination was removed by DNase treatment. Fusion-specific regions in exoRNA were amplified by standard PCR.

Results: We used breast cancer cell lines stably infected with cDNA encoding ESR1 fusions ESR1-DAB2 and ESR1-SOX9. NTA shows that our isolated exosomes had a size ranging from 30-150 nm, with the peak at 80 nm. Immunoblotting showed TSG101 and CD81 as positive markers of our exosomes, while Grp94 was negative. By RT-PCR, we identified a fusion-specific region amplified for both ESR1-SOX9 and ESR1-LPP from exoRNA for the fusion cell lines.

Conclusions: Collectively, these data indicate that multiple ESR1 fusions are detectable in breast cancer cell line-derived exoRNA.

Citation Format: Tiantong Liu, Jagmohan Hooda, Nils Ludwig, Theresa Whiteside, Jennifer M. Atkinson, Steffi Oesterreich, Adrian Lee. Detection of ESR1 gene fusions in breast cancer cell-derived exosomal RNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B58.
Abstract IA08: Use of liquid biopsy to facilitate drug development in early-stage disease—FDA perspective
Authors: Beaver Julia A.
Published: June 2020

Background: With advances in precision oncology, liquid biopsies have shown promise as a minimally invasive means to potentially diagnose cancer, detect resistance mutations and monitor tumor evolution, predict relapse, provide prognosis, and guide treatment decisions. One unique setting for the use of liquid biopsy to facilitate drug development is in early-stage disease where there is the potential for use in patient selection, prognostication, and drug activity demonstration.

Discussion: From the regulatory and clinical trial perspective, liquid biopsies can be used as prognostic biomarkers to identify the likelihood of disease recurrence in early-stage cancer. For example, circulating cell-free tumor DNA (ctDNA) in an adjuvant cancer setting affords the potential to select a high-risk population that could be utilized as a stratification factor, or selection of an enriched high-risk population in which specific drug development could be pursued. Use of liquid biopsy as a putative surrogate endpoint or efficacy-response biomarker, in this setting, would require a large body of evidence demonstrating biologic plausibility, demonstration of studies of prognostic value of the surrogate endpoint for the clinical outcome, and evidence from clinical trials (or meta-analysis) that the treatment effects on the biomarker correlate to the long-term outcome. Another use of liquid biopsy as an efficacy-response biomarker could allow for go or no-go decision making earlier on in drug development, which could also be applied in the adjuvant setting.

Conclusions: Liquid biopsies will continue to play a role in the clinical management of malignancies and have the potential to support drug approval and become more broadly incorporated and utilized in early-stage clinical trials.

Citation Format: Julia A. Beaver. Use of liquid biopsy to facilitate drug development in early-stage disease—FDA perspective [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA08.
Abstract IA20: Circulating tumor cells: Biology and clinical applications as liquid biopsy
Authors: Pantel Klaus
Published: June 2020

The analysis of circulating tumor cells (CTCs) in blood may provide clinically relevant information as “liquid biopsy” and provide new insights into tumor biology (Alix-Panabieres, Pantel, Nature Rev Cancer 2014; Keller, Pantel, Nature Rev Cancer 2019). Various technologies have been developed over the past 10 years, which include label-dependent assays based on the expression of tumor-associated cell surface antigens and label-independent assays based on physical properties of tumors cells distinct from the surrounding leukocytes (Pantel, Alix-Panabieres, Nature Rev Cancer 2019). After detection, CTCs can be further analyzed at the DNA, RNA, and protein level to obtain global information on tumor biology and targets relevant to cancer therapy (Pantel, Alix-Panabieres, Nature Rev Clin Oncol 2019). Liquid biopsy analyses with validated platforms provide reliable information on prognosis and may serve to identify therapeutic targets or mechanisms of resistance on metastatic cells. Metastatic cells might have unique characteristics that can differ from the bulk of cancer cells in the primary tumor currently used for stratification of patients to systemic therapy. Moreover, monitoring of blood samples before, during, and after systemic therapy (e.g., chemotherapy or targeted therapy) provides unique information for the future clinical management of the individual cancer patient and serves as surrogate marker for response to therapy (Alix-Panabieres, Pantel, Cancer Discovery 2016; Bardelli, Pantel, Cancer Cell 2017).

Citation Format: Klaus Pantel. Circulating tumor cells: Biology and clinical applications as liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA20.
Abstract IA21: Exploiting the biology of exosomes for diagnosis and treatment of cancer
Authors: Kalluri Raghu
Published: June 2020

Humans circulate trillions of exosomes at all times. Exosomes are extracellular vesicles with a size range of 40-150 nm and a lipid bilayer membrane and are released by all cell types. Normal exosomes contain DNA, RNA, and proteins. Some proteins such as CD9, CD63, and flotillin are often detected on the surface of exosomes and serve as markers. Exosomes likely remove excess and/or unnecessary constituents from the cells, functioning like garbage bags. Although their precise physiologic role remains largely unknown, is it suggested that exosomes may mediate specific cell-cell communication and activate signaling pathways in cells they fuse or interact with. Exosomes are detected in the tumor microenvironment, and emerging evidence suggests that they play a role in facilitating tumorigenesis by regulating angiogenesis, immunity, and metastasis. Circulating exosomes could be used as liquid biopsies and noninvasive biomarkers to potentially inform on early detection and diagnosis of cancer patients. Exosomes can be used for the treatment of cancer. This lecture will highlight some of the recent advances in the area of exosomes’ biology and their utility in the diagnosis and treatment of cancer. Additionally, the use of exosomes in personalized therapy for cancer patients will be discussed.

Citation Format: Raghu Kalluri. Exploiting the biology of exosomes for diagnosis and treatment of cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA21.
Abstract IA23: Phospho-RNA-seq: A liquid biopsy approach for cell-free mRNA/lncRNA profiling
Authors: Giraldez Maria D. , Spengler Ryan M. , Etheridge Alton , Goicochea Annika J. , Tuck Missy , Choi Sung W. , Galas David J. , Tewari Muneesh
Published: June 2020

Measuring disease biomarkers in biofluids to detect and monitor pathophysiologic events in tissues has enormous appeal. While detecting specific DNA sequences in blood plasma has been successful in informing about the presence of cancers, viruses, and a range of pathogens, this approach does not provide gene expression information, which could reveal more detailed information about disease activity. Measuring cell-free RNA in plasma would provide such information and has the potential to be more sensitive, given that one DNA molecule in a cell gives rise often to thousands or more RNA molecules.

However, with the exception of microRNAs, broad sequencing of cell-free messenger RNA (mRNA) profiles in plasma has been generally challenging. We discovered that a major barrier to cell-free RNA sequencing from plasma is that the large majority of mRNAs are both fragmented and have modified phosphorylation states at their 5’ and 3’ ends that make them invisible to standard small RNA-seq methods. We present a modified RNA-seq methodology, called phospho-RNA-seq, which revealed thousands of mRNA in blood plasma. The key to this approach is (i) the incorporation of T4-polynucleotide kinase treatment to change the phosphorylation states at the RNA ends, and (ii) a stringent bioinformatics analysis pipeline to reduce false positive alignments.

Phospho-RNA-seq identified cohorts of gene transcripts in plasma, including ones expressed in a tissue-specific manner. As proof-of-concept validation of the approach for biomarker identification, we used phospho-RNA-seq to longitudinally profile plasma specimens collected from patients undergoing hematopoietic stem cell transplantation. We detected bone marrow-enriched and liver-enriched transcript sets in plasma, which tracked with bone marrow recovery and hepatic injury, respectively. By providing expanded access to the transcriptome in plasma and potentially other biofluids, phospho-RNA-seq enables the discovery of new cell-free RNA biomarker signatures for a wide variety of liquid biopsy applications.

Citation Format: Maria D. Giraldez, Ryan M. Spengler, Alton Etheridge, Annika J. Goicochea, Missy Tuck, Sung W. Choi, David J. Galas, Muneesh Tewari. Phospho-RNA-seq: A liquid biopsy approach for cell-free mRNA/lncRNA profiling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA23.