Monitor Tumor Burden Research Articles

Abstract 2929: Comparative analysis of circulating human tumor DNA and bioluminescent imaging in monitoring tumor burden and therapeutic response of ionizing radiation in intratibial human prostate tumor xenografts

Abstract Purpose/Objective: Pre-clinical models of prostate cancer (PCa) metastases provide critical insight into tumor biology and therapeutic response. Current models require sophisticated reporter transgene models and/or expensive tools to noninvasively monitor tumors in metastatic sites, such as bone. Recent developments in the measurement of circulating tumor DNA (ctDNA) provide an alternative method to noninvasively assess tumor burden at a low cost. Here we present a comparative analysis of human PCa xenograft tumor growth and therapeutic response by simultaneous measurement of ctDNA and bioluminescence. Materials and methods: Male athymic nude mice were used for intra-tibial (n=10) modeling. LNCaP-CMV-Luc cells were implanted in the tibial medullary canal. Tumor burden was evaluated at pre and post-IR (focal exposure 6 Gy on day 28 post-implantation) time-points by in vivo bioluminescent imaging and quantitative real-time polymerase chain reaction (qPCR) for human LINE1/Alu DNA. qPCR was performed on plasma-derived DNA consecutively on the same day as bioluminescence. Results: Human genomic DNA quantification was sensitive to 0.1 pg and linear by hAlu (R2=0.98, for 0.1pg-150ng) and LINE DNA (R2=0.99, for 0.1pg-150ng) qPCR. hAlu DNA qPCR produces a much lower Ct value when compared to LINE1; possibly due to its higher copies in the human genome (Ct value 28 versus 22/0.1 pg). Following tumor inoculation, ctDNA level and tumor bioluminescence were strongly and significantly correlated over time (LINE1, r=0.65 and Alu, r=0.59, p<0.002). Following radiation, we observed an immediate (day 32) but transient decrease in the levels of ctDNA and bioluminescence, followed by a gradual increase due to tumor relapse. The correlation between ctDNA level and bioluminescence was not maintained, but responses followed a similar trend. Conclusions: In the present study, we report that the volume of human PCa xenografts can be accurately measured in the intra-tibial compartments of athymic nude mice by qPCR of circulating human Alu and LINE1 elements. Plasma-derived ctDNA levels highly correlated with tumor bioluminescence over time. Tumor directed radiation therapy exhibited slightly differential responses in ctDNA level and tumor bioluminescence, but overall both measurements followed a similar trend. Citation Format: Alok Mishra, Kenji Zennami, Esteban Velarde, Jonathan B. Coulter, Srinivasan Yegnasubramanian, Shawn E. Lupold, Theodore L. DeWeese. Comparative analysis of circulating human tumor DNA and bioluminescent imaging in monitoring tumor burden and therapeutic response of ionizing radiation in intratibial human prostate tumor xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2929.

Abstract 3008: Characterization of a panel of orthotopic bioluminescent patient-derived models for drug discovery

Abstract Background: Cancer is a complicated disease featuring geographic, genetic and immunological diversity. The tumor microenvironment (TME) is an essential part of tumor development, influencing tumor growth, metastasis and also therapeutic response. Patient-derived xenografts (PDX) and PDX-derived organoids (PDXO) are widely-used preclinically for drug evaluation with the prominent advantage of largely maintaining the histo- and molecular- pathology of the original tumor. Over decades of studies using PDX models, we found limitations in monitoring the orthotopic and metastatic pattern of cancer. With the development of gene engineering technologies (particularly for luciferase labeling), we now present the transduction of patient-derived models with firefly luciferase, to enable real-time monitoring of tumor burden in mice following orthotopic engraftment of either PDX tumor cells or PDXO in vivo. Methods: A panel of PDX cell slurry or PDXO cultures were transduced with lentiviral vectors carrying both the firefly luciferase gene as well as GFP. Mixed pools or GFP-sorted cells were used for in vivo validation. Bioluminescent PDX or PDXO were orthotopically and subcutaneously engrafted into immunodeficient mice. Real-time tumor growth and metastasis were monitored using the IVIS® Spectrum In Vivo Imaging System. Results: We have established a series of luciferase-labeled PDXO from different cancer types, including liver, ovarian, pancreatic and lung cancer and characterized their growth following orthotopic implantation. We have also established a series of luciferase-labeled PDX models from non-epithelial origin, including brain tumors, osteosarcoma and lymphomas. All of the models were confirmed with GFP signaling in vitro and showed significant luciferase signaling and growth in vivo. A mixed pool of bioluminescent PDXs or PDXOs presented heterogenous luciferase signals in tumors, while single clones or cell sorting by GFP or selected passage in vivo improved the consistency of the bioluminescent signal. Conclusions: PDX models are widely used as preclinical tools for clinical predictivity, however their application in orthotopic/metastatic modeling has been challenging due to the limitation in labelling PDXs for real-time monitoring. With the development of gene engineering technology in vitro and bioluminescent imaging (BLI) in vivo, we have developed and characterized a large panel of bioluminescent PDX and PDXO models from a range of different cancer types for real-time monitoring of orthotopic engrafted tumors in an organ-specific location to represent the TME, and the potential metastatic spread. Citation Format: Jessie Jingjing Wang, Jun Zhou, Ling Chen, Rongyun Zhou, Xiaoxi Xu, Henry Q. X. Li, Davy Xuesong Ouyang. Characterization of a panel of orthotopic bioluminescent patient-derived models for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3008.

Detection of MYD88 L265P Mutation By Next Generation Deep Sequencing in Peripheral Blood Mononuclear Cells of Waldenstrom Macroguloblinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Introduction: Whole genome sequencing has recently revealed MYD88 L265P somatic mutations in Waldenstrom macroguloblinemia (WM) tumor cells. Allele-specific polymerase chain reaction (AS-PCR) using bone marrow (BM) aspirate and peripheral blood (PB) is the most frequently used technique to detect the specific mutation. In search for a non-invasive, simple, and more sensitive detection of MYD88 L265P mutation, we performed next generation deep sequencing (NGS) technique using unselected peripheral blood mononuclear cells (PBMCs) collected from WM and IgM monoclonal gammopathy of undetermined significance (IgM MGUS) patients. The main purpose of this study is to assess the clinical implication of MYD88 L265P mutation burden detected by NGS technique using unselected PBMCs.Patients and methods: The study was approved from the Institutional Review Board. After informed consent was obtained in accordance with the Declaration of Helsinki, PBMCs were collected from 38 patients with WM and 20 patients with IgM MGUS. The median time from diagnosis to PBMC collection was 2.2 years (range, 0-25 years). Twenty-nine (76.3%) of WM patients were previously treated. The overall response (CR+PR+MR) rate at the time of analysis was 75.8% (22/29). Genomic DNA was extracted from unselected PBMCs and 300 ng of DNA was used for NGS analysis. Variation rates of MYD88 at the nucleotide position corresponding to L265P were measured with an Illumina MiSeq DNA sequencer by molecular barcoding technology essentially as previously reported (Nature Protocols 12, 664:2017). PCR products were sequenced on a MiSeq DNA sequencer with a Reagent kit v2. for 300 cycles. Serial dilution assessment was carried out to investigate the sensitivity of the method by analyzing the DNA of BM sample from a MYD88 mutation positive patient diluted in DNA from a healthy control.Results: Dilution assessment demonstrated that NGS technique was detectable to a sensitivity of 0.02%, ×5 higher than previously reported sensitivity by AS-PCR. MYD88 L265P mutation was detected from 6/9 (66.7%), 8/29 (27.6%), and 11/20 (55%) in patients with untreated WM, previously treated WM, and IgM MGUS, respectively. Among the patients who were positive forMYD88 L265P mutations, the median percentage of mutant allele relative to WT was 3.0% (0.14-32.3%), 0.24% (0.02-33.8%), and 0.55% (0.06-2.85%), in untreated WM, previously treated WM, and IgM MGUS, respectively. Patients with Untreated WM and those with lymphadenopathy showed a trend toward higher mutant allele burden in PBMCs, but the difference was not statistically significant. Among 26 previously treated WM patients, those with at least minimal response showed significantly lower detection rate of MYD88 L265P mutation (13.6 vs. 75.0%, P=0.02) and mutant allele burden (median, 0.00% vs. 0.23%, P=0.01), compared to patients with less than minimal response.Conclusion: This study is the first to report NGS analysis of MYD88 L265P mutation using unselected PBMCs of WM and IgM MGUS patients. The sensitivity for MYD88 L265P mutation in unselected PBMCs from IgM MGUS patients was comparable with that detected by AS-PCR using CD19-selected PB cells (Leukemia, 2014;28:1698-704). Although its specificity is in need for further investigations, NGS technique successfully detected the mutation carried by minimal tumor cells included in unselected PBMCs with high sensitivity as AS-PCR using CD19-selected PB cells. The mutation detection rate of previously treated WM patients was lower than that of untreated patients in our study and previously reported cohort; however, this in turn may potentially indicate that NGS detection of MYD88 L265P mutation in PBMC be clinically utilized in assessing treatment response by monitoring tumor burden during patients' clinical course. DisclosuresNo relevant conflicts of interest to declare.

OSU-2S, a Novel PKC Activator, Mediates PKC Dependent Cell Death, Differentiation and Cell Cycle Arrest in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a near incurable disease with 5-year survival rates of 10% in older patients. Poor tolerance to chemotherapy, chemo resistance and high rate of relapse warrants less toxic and more effective regimens in AML. AML leads to a block in normal myeloid differentiation, leading to an accumulation of undifferentiated blasts. Thus, therapies directed towards promoting these cells to differentiate re-establishes the ability of AML cells to undergo cell death, reducing the leukemic population. Such differentiation therapy with all-trans retinoic acid (ATRA) has been successful in acute promyelocytic leukemia (APL), but not in other AML subtypes resistant to ATRA, thus presenting a promising and unexploited avenue for targeted therapies. There is accumulating evidence that Protein Kinase C (PKC) activation induces myeloid differentiation. However, since most PKC activators are either tumor promoters or have inconclusive activity, their potential for use in AML has yet to be realized. OSU-2S is a non-immunosuppressive derivative of FTY720 (a sphingosine analogue with anti-tumorigenic properties) and has previously been reported to directly activate PKC to mediate cytotoxicity, showing promising in vitro and in vivo activity against a number of leukemias and lymphomas, and other malignancies such as hepatocellular carcinoma. This impelled us to evaluate the activity of OSU-2S in causing cell death and differentiation in AML. We found that OSU-2S resulted in increased activation of PKC in HL-60 cells. In addition to effecting significant dose-dependent cytotoxicity against AML cell lines HL-60, MV4-11 and MOLM-13 [mean decrease in viability with OSU-2S in HL-60: 85±7% (p=0.08), MV4-11: 74±20% (p=0.02), MOLM-13: 60±25% (p=0.04)], and in primary samples from leukemic patients, including high risk FLT3-ITD mutated AMLs (65±8% decrease in viability with OSU-2S, p<0.0001, n=13, dose trend p<0.0001), OSU-2S caused a significant upregulation of CD11b, a cell-surface marker associated with myeloid maturation, in HL-60 and primary AML samples [(25±5% and 14±7% increase in CD11b+ cells with OSU-2S vs vehicle control in HL-60 (p =0.0126) and primary AML (p=0.043) cells respectively]. In addition, OSU-2S treated HL-60 and primary AML cells exhibited morphological features indicative of differentiation, as evidenced by lower nuclear to cytoplasmic ratio, and highly vacuolated and granulated cytoplasm. The observed differentiation activity prompted us to test if OSU-2S affected the leukemia-initiating cell population. Colony forming assays showed OSU-2S significantly reduced the number of colony forming units-leukemic (CFU-L) in primary AML blasts (52±13% decrease in CFU-L in response to OSU-2S vs vehicle control; p=0.018, n=4). OSU-2S mediated cytotoxicity was significantly abrogated in the presence of PKC inhibitor Bisindolylmaleimide (BIS) in HL-60 cell line [39±8% decrease in OSU-2S induced cytotoxicity in the presence of BIS vs control (p=0.008), n=3] as well as in primary AML cells [41±16% decrease in OSU-2S induced cytotoxicity (p=0.02), n=6], indicating that OSU-2S mediates its cytotoxic effects in AML via PKC activation.To further gain insight into the mechanism of cell death and differentiation effected by OSU-2S, we performed RNA-Sequencing analysis of total RNA isolated from MV4-11 cells treated with OSU-2S. Ingenuity Pathway Analysis on these transcripts showed significant changes in gene expression affecting cell cycle regulation and proliferation (p=10-13). Cell cycle analysis of MV4-11, HL-60 and U-937 cells by PI staining followed by flow cytometry confirmed a decrease in cells in G2/M phase of the cell cycle associated with increased cells in S but not Go/G1 phase (69.63% decrease in G2/M phase cells with OSU-2S vs vehicle control, p=0.04). Current ongoing studies in cell line-derived xenografted mouse models of AML indicate favorable therapeutic effect of OSU-2S in providing survival advantage in AML. Tumor burden monitoring by bioluminescent imaging shows reduction in tumor burden following OSU-2S treatment. Our results show promising pre-clinical efficacy of OSU-2S against AML, and warrants further studies to establish clinical efficacy as a potential therapeutic agent. Ongoing studies are also directed towards studying the involvement and role of PKC and other target molecules in the cell death and differentiation effected by OSU-2S. DisclosuresWalker:Gilead: Research Funding. Mims:Novartis: Honoraria. Chen:The Ohio State University: Patents & Royalties: OSU-2S. Byrd:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; Janssen: Research Funding; Genentech: Research Funding; The Ohio State University: Patents & Royalties: OSU-2S. Vasu:Boehringer-Ingelheim: Research Funding; Stemline Therapeutics: Research Funding.

Targeting T-Cell Malignancies with Chimeric Antigen Receptors Using Two Structurally Different Antigen Binding Domains Against CD5

Patients with relapsed T-cell acute lymphoblastic leukemia (T-ALL) have extremely poor survival when treated with chemotherapy alone. Allogeneic hematopoietic stem cell transplantation (HSCT) offers a chance of cure for these patients, but attaining clinical remission in order to undergo HSCT remains the biggest therapeutic challenge. Chimeric antigen receptor (CAR) T-cell therapy has been used with great success in relapsed/refractory B-ALL patients. The same approach, however, is difficult in targeting T-cell disease given the lack of a T-lymphoblast specific surface antigen. The use of natural killer (NK) cells as the effector cell provides a potential solution. We are testing two different CAR structures directed against CD5, a pan T-cell marker; one consisting of a more traditional immunoglobulin (Ig) based single chain variable fragment (scFv), while the other comprising of a variable lymphocyte receptor (VLR) as the antigen recognition domain. VLRs are single chain crescent shaped proteins that represent the functional unit of the adaptive immune system in jawless vertebrates. Our previous work has shown that a VLR can be used as an alternative for CAR-based antigen recognition. Here, we directly compare a VLR to its scFv counterpart in a CAR setting. The single chain structure of a VLR allows for the rapid creation of novel CARs, compared to the corresponding use of a scFv-CAR, in which the variable heavy and light sequences need further engineering for adapting to CAR technologies. Thus, our hypothesis was that natural killer cells engineered to express an anti-CD5 CAR can specifically be used to target T-cell malignancies, with the VLR-CAR being equal or superior to scFv-based cassettes. We constructed two second generation CARs using the VLR or scFv sequence targeting CD5 as our antigen recognition domain. Both CD5-CAR constructs showed similar activity when tested in CD5-positive Jurkat cells. For our cytotoxicity studies, we used the IL-2 dependent natural killer cell line NK-92. To generate uniform populations of CAR-expressing NK cells we transduced NK-92 cells with a dual lentiviral construct expressing the CD5-CAR and green fluorescent protein (GFP), and then selected for the positively transduced cells using flow sorting for GFP. In vitro cytotoxicity studies showed significantly increased killing using the CD5-CAR expressing NK-92 cells compared to unmodified NK-92 cells (p<0.05), for both VLR and scFv modified cells. We then tested the two CD5-CARs in a T-cell leukemia mouse model. NOD scid gamma (NSG) mice were intravenously injected with luciferase expressing Jurkat leukemia cells to establish the T-cell leukemia xenograft model. Four doses of NK-92 cells were injected over a two week period starting on day 7 post-tumor injection. Bioluminescence imaging was used to monitor tumor burden. The CD5-scFv-CAR NK-92 treated mice group had a significant survival advantage over the other groups (p<0.01). Interestingly, the CD5-VLR-CAR NK-92 treated mice did not have a significant advantage over naïve NK-92 or saline treated groups. Given the superior in vivo cytotoxicity demonstrated in our studies, we plan to use the CD5-scFV-CAR going forward as we transition to primary cells. Overall, these studies provide the foundation to move these studies into preclinical testing as a treatment for relapsed T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive molecular profiling of advanced cancers in a real-world setting using an ultrasensitive amplicon-based next-generation sequencing (NGS) liquid biopsy assay.

3062 Background: Molecular profiling of circulating tumor DNA (ctDNA) by NGS has demonstrated the value of liquid biopsy in informing treatment decisions and monitoring disease progression in cancer patients. Here we report results from the real-world clinical application of an ultrasensitive amplicon-based NGS liquid biopsy assay for advanced cancers. Methods: Plasma cell-free DNA (cfDNA) from 1,338 consecutive samples (51.3% lung, 15.6% breast, 8.4% colorectal, 26.8% from 18 other cancer types; 86.3% of cancer samples were metastatic) underwent real-world testing in a Singapore-based, CAP-accredited, CLIA-certified laboratory from Jan 2018 – Nov 2020. Genomic alterations were analyzed using an amplicon-based NGS assay that detects alterations in 80 cancer-related genes, microsatellite instability (MSI) and cancer-causing viruses, with previously validated cfDNA detection limits of 0.1% variant allele frequency (VAF) for SNPs and indels, 5% tumor fraction, and 2 IU/mL plasma, respectively. Results: ctDNA was successfully detected in 70.0% of cancer samples (76.3% from metastatic and 24.7% from localized tumors) for a total of 977 unique variants. Across ctDNA-positive samples the median VAF was 1.4%, and 8.5% of all reported variants had VAFs between 0.01 – 0.09%. The most frequently altered genes were TP53 (48.8%), EGFR (36.9%), KRAS (17.4%), PIK3CA (15.3%), and APC (9.5%). In ctDNA-positive lung cancer, 74.6% of samples (66.5% EGFR+) harbored ≥1 actionable target, with 29.9% of patients treated with a first- or second-generation EGFR tyrosine kinase inhibitor harboring an EGFR T790M resistance mutation. Among breast cancers, ESR1 or PIK3CA alterations predicting drug resistance/response were detected in 44.2% of HR+ cases. In colorectal cancer, alterations implicated in anti-EGFR resistance ( NRAS, KRAS, BRAF, ERBB2) were found in 50.0% of samples. Profiling of cfDNA also enabled cancer monitoring, even among cancers with low genetic biomarker prevalence. 50% of nasopharyngeal cancer samples were Epstein-Barr virus-positive while 35.7% of liver cancer samples were Hepatitis B virus-positive, enabling an additional yield of 14.3% and 7.1% respectively, over ctDNA detection. cfDNA profiling of cancers of unknown primary origins informed potential tumor origins via the detection of tumor type-specific alterations such as mutations in AR, ESR1, GNAS, EGFR genes, viral biomarkers, and MSI. Conclusions: The use of a comprehensive pan-cancer liquid biopsy panel with rational inclusion of actionable and driver genes resulted in detection of ctDNA in the vast majority of clinical cases processed routinely. These findings support the utility of ultrasensitive amplicon-based NGS liquid biopsy assays in probing cancer drug sensitivity/resistance, tumor burden monitoring, and molecular characterization of tumors.

Liquid Biopsy Targeting Monocarboxylate Transporter 1 on the Surface Membrane of Tumor-Derived Extracellular Vesicles from Synovial Sarcoma.

Simple SummarySynovial sarcoma (SS) is associated with a high risk of recurrence and poor prognosis, and no biomarker useful in monitoring tumor burden exists. We identified monocarboxylate transporter 1 (MCT1) expressed in extracellular vesicles (EVs) derived from synovial sarcoma as a potential such marker. Circulating levels of MCT1+CD9+ EVs were significantly correlated with tumor volume in a SS mouse model. Serum levels of MCT1+CD9+ EVs reflected tumor burden and treatment response in SS patients. Patients with MCT1 expression on the plasma membrane have significantly worse overall survival than those with nuclear expression. Silencing of MCT1 reduced the malignant phenotype including cellular viability, migration, and invasion of SS cells. MCT1 may thus be a promising novel target for liquid biopsies and a novel therapeutic target.The lack of noninvasive biomarkers that can be used for tumor monitoring is a major problem for soft-tissue sarcomas. Here we describe a sensitive analytical technique for tumor monitoring by detecting circulating extracellular vesicles (EVs) of patients with synovial sarcoma (SS). The proteomic analysis of purified EVs from SYO-1, HS-SY-II, and YaFuSS identified 199 common proteins. DAVID GO analysis identified monocarboxylate transporter 1 (MCT1) as a surface marker of SS-derived EVs, which was also highly expressed in SS patient-derived EVs compared with healthy individuals. MCT1+CD9+ EVs were also detected from SS-bearing mice and their expression levels were significantly correlated with tumor volume (p = 0.003). Furthermore, serum levels of MCT1+CD9+ EVs reflected tumor burden in SS patients. Immunohistochemistry revealed that MCT1 was positive in 96.7% of SS specimens and its expression on the cytoplasm/plasma membrane was significantly associated with worse overall survival (p = 0.002). Silencing of MCT1 reduced the cellular viability, and migration and invasion capability of SS cells. This work describes a new liquid biopsy technique to sensitively monitor SS using circulating MCT1+CD9+ EVs and indicates the therapeutic potential of MCT1 in SS.

Exploring the concepts and practices of advanced breast cancer treatment: a narrative review.

ObjectiveTo explore the concepts and practices of advanced breast cancer treatment.BackgroundMetastatic breast cancer (MBC) has become a chronic disease, with a median overall survival (OS) of around 3 years and a 5-year survival rate of about 25%. OS are strongly associated with the best available care, which consists of not only application of guidelines, but also multidisciplinary specialized care, the most efficacious medicines, and so on. Advanced breast cancer (ABC) Guidelines are the most important and authoritative guidelines for MBC.MethodsIn this review, we demonstrate the history and evolution of the global ABC Guidelines. Since 2015, Chinese multidisciplinary experts have drafted guidelines for clinical diagnosis and treatment of MBC. All of these ABC guidelines describe specialized therapeutic principles for different subtypes MBC in detail. Encouragingly, we have found that some special subtypes are hopeful of being cured, such as HER-2 positive patients with low tumor burden or HR-positive (HR+) MBC with non-visceral metastasis. In our opinion, the definition of cure of MBC is that MBC patients achieve CR and remain for more than five years after systemic treatment, including those with local therapy. Consequently, we also have conducted some researches and meaningful explorations in different subtypes of MBC. In HER2 positive MBC, our study revealed that regular HER2 circulating extracellular domain (ECD) assay can provide the real-time monitoring of tumor burden and prediction of poor outcome, and may present an important opportunity to reassess HER2 status. In HR+ MBC, we suggested that hormone therapy (HT) maintenance is the priority choice for HR+/HER2− MBC after first-line combined chemotherapy. Besides, our real-world study revealed that fulvestrant combined with ovarian suppression was an active option for premenopausal HR+/HER2- MBC. And also, we observed that everolimus (low-dose) combined with hormone therapy was still effective for HR+/HER2− MBC. For mTNBC patients, we found that THA and endostatin exhibited potential efficacy and was well tolerated in pretreated patients.ConclusionsOur concepts and practices will contribute to the design of relevant clinical research and accumulation of evidence, and cure of MBC is promising.

Imaging of neuroendocrine tumors: A pictorial review of the clinical value of different imaging modalities.

Neuroendocrine tumors (NETs) are multifaceted tumors occurring in a variety of organs and often present as metastatic at the time of diagnosis. Accurate staging is the most significant factor in therapy planning, but it remains a challenge. Imaging is established as the cornerstone for disease detection/diagnosis, staging, and follow up. To accurately assess and monitor tumor burden in patients with NETs, various imaging techniques have been developed and optimized. Current recommendations for the imaging of patients with NETs include a combination of both morphologic (or anatomic) and molecular imaging, but a final choice can be puzzling for clinicians. Recognizing that there is no uniform sequence consensus on the "best" imaging test, and the heterogeneity of technologicavailability at different centers, we hope to provide a pictorial review of the different imaging techniques and their role and utility in management of patients with NETs, aimed to provide a practical guide for all clinicians.

The Role of Exosomes in Breast Cancer Diagnosis.

The importance of molecular re-characterization of metastatic disease with the purpose of monitoring tumor evolution has been acknowledged in numerous clinical guidelines for the management of advanced malignancies. In this context, an attractive alternative to overcome the limitations of repeated tissue sampling is represented by the analysis of peripheral blood samples as a ‘liquid biopsy’. In recent years, liquid biopsies have been studied for the early diagnosis of cancer, the monitoring of tumor burden, tumor heterogeneity and the emergence of molecular resistance, along with the detection of minimal residual disease. Interestingly, liquid biopsy consents the analysis of circulating tumor cells, circulating tumor DNA and extracellular vesicles (EVs). In particular, EVs play a crucial role in cell communication, carrying transmembrane and nonmembrane proteins, as well as metabolites, lipids and nucleic acids. Of all EVs, exosomes mirror the biological fingerprints of the parental cells from which they originate, and therefore, are considered one of the most promising predictors of early cancer diagnosis and treatment response. The present review discusses current knowledge on the possible applications of exosomes in breast cancer (BC) diagnosis, with a focus on patients at higher risk.

Frequent post-operative monitoring of colorectal cancer using individualised ctDNA validated by multiregional molecular profiling

BackgroundCirculating tumour DNA (ctDNA) is known as a tumour-specific personalised biomarker, but the mutation-selection criteria from heterogeneous tumours remain a challenge.MethodsWe conducted multiregional sequencing of 42 specimens from 14 colorectal tumours of 12 patients, including two double-cancer cases, to identify mutational heterogeneity to develop personalised ctDNA assays using 175 plasma samples.Results“Founder” mutations, defined as a mutation that is present in all regions of the tumour in a binary manner (i.e., present or absent), were identified in 12/14 tumours. In contrast, “truncal” mutations, which are the first mutation that occurs prior to the divergence of branches in the phylogenetic tree using variant allele frequency (VAF) as continuous variables, were identified in 12/14 tumours. Two tumours without founder and truncal mutations were hypermutators. Most founder and truncal mutations exhibited higher VAFs than “non-founder” and “branch” mutations, resulting in a high chance to be detected in ctDNA. In post-operative long-term observation for 10/12 patients, early relapse prediction, treatment efficacy and non-relapse corroboration were achievable from frequent ctDNA monitoring.ConclusionsA single biopsy is sufficient to develop custom dPCR probes for monitoring tumour burden in most CRC patients. However, it may not be effective for those with hypermutated tumours.

Time to focus on circulating nucleic acids for diagnosis and monitoring of gliomas: A systematic review of their role as biomarkers.

Gliomas are diffusely growing tumours arising from progenitors within the central nervous system. They encompass a range of different molecular types and subtypes, many of which have a well-defined profile of driver mutations, copy number changes and DNA methylation patterns. A majority of gliomas will require surgical intervention to relieve raised intracranial pressure and reduce tumour burden. A proportion of tumours, however, are located in neurologically sensitive areas and a biopsy poses a significant risk of a deficit. A majority of gliomas recur after surgery, and monitoring tumour burden of the recurrence is currently achieved by imaging. However, most imaging modalities have limitations in assessing tumour burden and infiltration into adjacent brain, and sometimes imaging is unable to discriminate between tumour recurrence and pseudo-progression. Liquid biopsies, obtained from body fluids such as cerebrospinal fluid or blood, contain circulating nucleic acids or extracellular vesicles containing tumour-derived components. The studies for this systematic review were selected according to PRISMA criteria, and suggest that the detection of circulating tumour-derived nucleic acids holds great promises as biomarker to aid diagnosis and prognostication by monitoring tumour progression, and thus can be considered a pathway towards personalized medicine.

Serial Circulating Tumor DNA in Predicting and Monitoring the Effect of Neoadjuvant Chemoradiotherapy in Patients with Rectal Cancer: A Prospective Multicenter Study.

We investigated the value of circulating tumor DNA (ctDNA) in predicting tumor response to neoadjuvant chemoradiotherapy (nCRT), monitoring tumor burden, and prognosing survival in patients with locally advanced rectal cancer (LARC). This prospective multicenter trial recruited 106 patients with LARC for treatment with nCRT followed by surgery. Serial ctDNAs were analyzed by next-generation sequencing at four timepoints: at baseline, during nCRT, presurgery, and postsurgery. In total, 1,098 mutations were identified in tumor tissues of the 104 patients being analyzed (median, seven mutations/patient). ctDNA was detected in 75%, 15.6%, 10.5%, and 6.7% of cases at the four timepoints, respectively. None of the 29 patients with pathologic complete response (ypCR) had preoperative ctDNA detected. The preoperative ctDNA-positive rate was significantly lower in the well-responded patients with pathologic tumor regression grade of ypCAP 0-1 than ypCAP 2-3 group (P < 0.001), lower in ypCR than non-ypCR group (P = 0.02), and lower in pathologic T stage (ypT) 0-2 than ypT 3-4 group (P = 0.002). With a median follow-up of 18.8 months, 13 patients (12.5%) experienced distant metastasis. ctDNA positivity at all four timepoints was associated with a shorter metastasis-free survival (MFS; P < 0.05). Multivariate analyses showed that the median variant allele frequency (VAF) of mutations in baseline ctDNA was a strong independent predictor of MFS (HR, 1.27; P < 0.001). We show that ctDNA is a real-time monitoring indicator that can accurately reflect the tumor burden. The median VAF of baseline ctDNA is a strong independent predictor of MFS.

Frequent Tumor Burden Monitoring of Esophageal Squamous Cell Carcinoma With Circulating Tumor DNA Using Individually Designed Digital Polymerase Chain Reaction

Frequent Tumor Burden Monitoring of Esophageal Squamous Cell Carcinoma With Circulating Tumor DNA Using Individually Designed Digital Polymerase Chain Reaction

Prognostic implications of preoperative versus postoperative circulating tumor DNA in surgically resected lung cancer patients: a pilot study.

BackgroundRecent studies of advanced lung cancer patients have shown that circulating tumor DNA (ctDNA) analysis is useful for molecular profiling, monitoring tumor burden, and predicting therapeutic efficacies and disease progression. However, the usefulness of ctDNA analysis in surgically resected lung cancers is unclear.MethodsThis study included 20 lung cancer patients with clinical stage IIA–IIIA disease. Preoperative and postoperative (3–12 days) plasma samples were collected for ctDNA analysis. Cancer personalized profiling by deep sequencing, which can detect mutations in 197 cancer-related genes, was used for ctDNA detection. The cohort consisted of 18 men and 2 women with a median age of 69 (range, 37–88) years. Sixteen patients (80%) had a history of smoking. Histologically, there were four squamous cell carcinomas, 13 adenocarcinomas, two adenosquamous cell carcinomas, and one small cell carcinoma.ResultsAt the time of data analysis, the 20 patients had been monitored for a median follow-up of 12 months. Eight patients (40%) were positive for preoperative ctDNA, and this was significantly correlated with tumor size (≥5 vs. <5 cm, P=0.018). Four patients (20%) were positive for postoperative ctDNA, and this was significantly correlated with histological grade (3 vs. 1 or 2, P=0.032). Postoperative positivity for ctDNA also predicted shorter recurrence-free survival (RFS) (P=0.015), while pre- and post-operative carcinoembryonic antigen levels (P=0.150 and P=0.533, respectively) and preoperative positivity for ctDNA (P=0.132) were not correlated with RFS.ConclusionsDetecting ctDNA postoperatively was a poor prognostic factor in surgically resected lung cancer patients that may suggest there is minimal residual disease (MRD).

Prognostic implications of preoperative versus postoperative circulating tumor DNA in surgically resected lung cancer patients: a pilot study

Background: Recent studies of advanced lung cancer patients have shown that circulating tumor DNA (ctDNA) analysis is useful for molecular profiling, monitoring tumor burden, and predicting therapeutic efficacies and disease progression. However, the usefulness of ctDNA analysis in surgically resected lung cancers is unclear.

Longitudinal multi-gene panel assessment of circulating tumor DNA revealed tumor burden and molecular characteristics along treatment course of non-small cell lung cancer.

BackgroundMost studies associating circulating tumor DNA (ctDNA) with outcome in lung cancer treatment were either cross-sectional or, if longitudinal, only analyzed a limited number of genes. This study evaluated the potential of utilizing ctDNA profiled by a panel of common cancer genes to monitor tumor burden and to reveal molecular characteristics of tumor along treatment course.MethodsTwenty Chinese non-small cell lung cancer (NSCLC) patients with serial plasma samples collected (I) before starting on either first- or second-line treatment, (II) at stable disease on treatment, and (III) upon disease progression, were analyzed for mutations in ctDNA using the PGDx 64-gene panel. Paired statistics compared mutation profiles between any two of the three time points.ResultsProportions with detectable ctDNA decreased from 65% at baseline to 35% at stable disease and rose to 80% at progression (P=0.012, between stable disease and progression); median ctDNA levels (mutated fragments per mL) were 7.8, 0, and 24.7 at the three time points, respectively (P=0.013 between baseline and progression; P=0.007 between stable disease and progression). Although plasma epidermal growth factor receptor (EGFR) mutations were commonly detected, 15% of patients had mutations other than EGFR detected during progression, such as various types of TP53 mutations.ConclusionsctDNA profiling in serial blood samples reflected tumor burden over time, and a multi-gene panel was more sensitive in indicating lung cancer progression on treatment than a single gene approach. The detection of additional oncogenic mutations or their disappearance suggested evolution of tumor heterogeneity along treatment course.

Locally invasive, castrate-resistant prostate cancer in a Pten/Trp53 double knockout mouse model of prostate cancer monitored with non-invasive bioluminescent imaging.

Here we have improved an existing mouse model of prostate cancer based on prostate-specific deletion of Pten and Trp53 by incorporating a Cre-activatable luciferase reporter. By coupling the deletion of those genes to the activation of a luciferase reporter, we were able to monitor tumor burden non-invasively over time. We show that, consistent with previous reports, deletion of both Pten and Trp53 on a C57BL/6 background accelerates tumor growth and results in both the loss of androgen receptor expression and castrate resistant tumors as compared with loss of Pten alone. Loss of Trp53 results in the development of sarcomatoid histology and the expression of markers of epithelial-to-mesenchymal transition Zeb1 and vimentin, with kinetics and penetrance dependent on whether one or both alleles of Trp53 were deleted. Homozygous deletion of Trp53 and Pten resulted in uniformly lethal disease by 25 weeks. While we were able to detect locally invasive disease in the peritoneal cavity in aggressive tumors from the double knockout mice, we were unable to detect lymphatic or hematogenous metastatic disease in lymph nodes or at distant sites.

Allelic Imbalance Analysis in Liquid Biopsy to Monitor Locally Advanced Esophageal Cancer Patients During Treatment.

Esophageal cancer (EC) is a highly aggressive tumor, and the current monitoring procedures are partially inadequate to evaluate treatment efficacy. The aim of this study was to investigate whether allelic imbalance analysis in liquid biopsy could be used as an additional tool to monitor tumor burden in EC patients. For this purpose, circulating cell-free DNA (cfDNA) from 52 patients with a locally advanced EC, which underwent neoadjuvant treatment and resection, was analyzed. Data from four representative longitudinally followed patients are also reported. Furthermore, 17 DNAs from formalin-fixed paraffin-embedded (FFPE) tumor samples were analyzed and compared to time-matched cfDNAs. To look for allelic imbalance, which is the main genetic alteration in both EC histotypes, we used a panel of five microsatellites (MSs) and three single-nucleotide polymorphisms (SNPs) near genes described as frequently altered. The Fisher exact and Mann-Whitney U tests were used to analyze categorical and continuous data, respectively. The correlation coefficient between cfDNA and FFPE-DNA was calculated with the Pearson's correlation test. We found that the selected tumor-related alterations are present in cfDNA of both adenocarcinoma (EADC) and squamous cell carcinoma (ESCC) with similar frequencies. The only exception were the MSs, one downstream and one upstream, of SMAD4 of which the loss was only observed in EADC (26 vs. 0%, P = 0.018). More interestingly, longitudinal studies disclosed that in patients with disease progression, tumor-related alterations were present in cfDNA before overt clinical or instrumental signs of relapse. In conclusion, our data indicate that the evaluation of tumor-related gene allelic imbalance in cfDNA might be a useful tool to complement the current monitoring procedures for EC patients and to guide their management.

Abstract 3390: Predictive and prognostic value of circulating tumor DNA (ctDNA) compared to circulating tumor cells (CTC) in a prospective cohort of metastatic breast cancer patients: The UCBG COMET trial

Abstract Background: In metastatic breast cancer (MBC), monitoring circulating tumor DNA (ctDNA) can detect mutation associated with resistance to treatment and its variations reflect changes in tumor burden. We prospectively monitored CTC and ctDNA during first line chemotherapy for MBC. Methods: The French cohort COMET is a prospective study including first line HER2- negative pts receiving weekly paclitaxel and bevacizumab. Blood samples were obtained at baseline (BL) and before the second cycle of chemotherapy (C2). ctDNA was analyzed by targeted resequencing (custom panel 220kb) SNV (28 genes + 8 promoters and CNA (18 genes). Results: For ctDNA, out of 196 pts analyzed, 147 had at least one somatic mutation (SNV) detected in plasma (75%). Despite no complete overlap, 24 pts (12%) had no CTC nor ctDNA detected at baseline. Including Copy Number Variation analysis (CNV), the number of patients without detectable ctDNA at baseline could be reduced to 18 (9%). The average number of mutations per pt was 2.4 (range 1 to 9). Median Allelic Frequency (MAF) was 9.1%. Most commonly mutated genes were TP53 and GATA3 with 14% and 7% of all mutations, respectively. TP53 mutations were detected in 31 % of patients and were associated with a shorter progression-free and overall survival. PI3KCA mutations were detected in 23.2% of the pts, were correlated with presence of bone metastasis and had no prognostic value. ESR1 was mutated in 10.6% of the pts, restricted to the ER+ subgroup and had no impact on overall survival. At baseline, CTC and ctDNA levels were correlated (r = 0.40, p &amp;lt; 0.0001). CTC detection was correlated with TP53 mutations detection but not with mutations PI3KCA or ESR1. Only 68 pts (36%) had detectable ctDNA at C2 with a MAF of 2% (0.3%-40%). 52.8% of patients positive for ctDNA at D1C1 became negative at D1C2 and only one negative at baseline became positive at C2. Correlation rate with CTC was the same after one cycle of chemotherapy (r = 0.40). Median follow-up was 53 months and median OS was 32 months. At multivariate analysis, triple negative status, detectable ctDNA at C2, CTC ≥5 at C2 and grade 3 on primary tumor were independent prognostic for OS. Conclusion: Early decrease of CTC and/or ctDNA after one cycle of chemotherapy are independent predictive markers of favorable outcome. Compared to CTC, ctDNA allows monitoring of tumor burden during chemotherapy and specific detection of targetable mutations as PI3KCA, HER2 or BRCA. Citation Format: Jean-Yves Pierga, Amanda Silveira, Elodie Girard, Veronique Lorgis, Marie-Laure Tanguy, Benoit Albaud, Olivier Tredan, Coraline Dubot, Caroline Hego, William Jacot, Anthony Goncalves, Marc Debled, Christelle Levy, Jean-Marc Ferrero, Christelle Jouannaud, Marie-Ange Mouret-Reynier, Florence Dalenc, Sylvain Baulande, Jerome Lemonnier, Frederique Berger, Francois-Clement Bidard, Charlotte Proudhon. Predictive and prognostic value of circulating tumor DNA (ctDNA) compared to circulating tumor cells (CTC) in a prospective cohort of metastatic breast cancer patients: The UCBG COMET trial [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 3390.