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Nonrigid forms of motion are commonplace in everyday life. Given previously documented age-related deteriorations in various tasks involving motion (discriminating speed, identifying motion direction, etc.), an experiment was conducted to evaluate the potential effect of age upon the visual ability to detect rigid and nonrigid object motion. Thirty younger and older observers participated in the experiment (mean ages were 19.9 and 75.8 years, respectively). As has been done multiple times in the past, the individual motions of object vertices were manipulated to simulate either rigid motion (rotation in depth, with or without precession) or two different types of nonrigid motion (also rotation in depth with or without precession, but with added object deformation). In confirmation of previous research, there were large effects of nonrigid motion type and precession upon the ability to differentiate between rigid and nonrigid object motion. There was also a large effect of age, such that the discrimination performance of the younger observers was 49.6% higher than that exhibited by the older observers. In this first ever study of aging and nonrigid object motion perception, we thus find that aging is associated with a substantial impairment in the ability to visually perceive object nonrigidity., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Introduction: Acute leukemias (AL) are the main types of cancer in children worldwide. In Mexico, they represent one of the main causes of death in children under 20 years of age. Most of the studies on the incidence of AL in Mexico have been developed in the urban context of Greater Mexico City and no previous studies have been conducted in the central-south of the country through a population-based study. The aim of the present work was to identify the general and specific incidence rates of pediatric AL in three states of the south-central region of Mexico considered as some of the marginalized populations of Mexico (Puebla, Tlaxcala, and Oaxaca)., Methods: A population-based study was conducted. Children aged less than 20 years, resident in these states, and newly diagnosed with AL in public/private hospitals during the period 2021-2022 were identified. Crude incidence rates (cIR), standardized incidence rates (ASIRw), and incidence rates by state subregions (ASIRsr) were calculated. Rates were calculated using the direct and indirect method and reported per million children under 20 years of age. In addition, specific rates were calculated by age group, sex, leukemia subtype, and immunophenotype., Results: A total of 388 cases with AL were registered. In the three states, the ASIRw for AL was 51.5 cases per million (0-14 years); in Puebla, it was 53.2, Tlaxcala 54.7, and Oaxaca de 47.7. In the age group between 0-19 years, the ASIRw were 44.3, 46.4, 48.2, and 49.6, in Puebla, Tlaxcala, and Oaxaca, respectively. B-cell acute lymphoblastic leukemia was the most common subtype across the three states., Conclusion: The incidence of childhood AL in the central-south region of Mexico is within the range of rates reported in other populations of Latin American origin. Two incidence peaks were identified for lymphoblastic and myeloid leukemias. In addition, differences in the incidence of the disease were observed among state subregions which could be attributed to social factors linked to the ethnic origin of the inhabitants. Nonetheless, this hypothesis requires further investigation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that authors JN-E, RP and JM-A were all Topic Editors and were members of Frontiers at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Flores-Lujano, Allende-López, Duarte-Rodríguez, Alarcón-Ruiz, López-Carrillo, Shamah-Levy, Cebrián, Baños-Lara, Casique-Aguirre, Elizarrarás-Rivas, López-Aquino, Garrido-Hernández, Olvera-Caraza, Terán-Cerqueda, Martínez-José, Aristil-Chery, Alvarez-Rodríguez, Herrera-Olivares, Ruíz-Arguelles, Chavez-Aguilar, Márquez-Toledo, Cano-Cuapio, Luna-Silva, Martínez-Martell, Ramirez-Ramirez, Merino-Pasaye, Galván-Díaz, Medina-Sanson, Gutiérrez-Rivera, Martín-Trejo, Rodriguez-Cedeño, Bekker-Méndez, Romero-Tlalolini, Cruz-Maza, Juárez-Avendaño, Pérez-Tapia, Rodríguez-Espinosa, Suárez-Aguirre, Herrera-Quezada, Hernández-Díaz, Galván-González, Mata-Rocha, Olivares-Sosa, Rosas-Vargas, Jiménez-Morales, Cárdenas-González, Álvarez-Buylla Roces, Duque-Molina, Pelayo, Mejía-Aranguré and Núñez-Enriquez.)

Many previous studies have investigated visual distance perception, especially for small to moderate distances. Few experiments, however, have evaluated the perception of large distances (e.g., 100 m or more). The studies that have been conducted have found conflicting results (diametrically opposite conclusions). In the current experiment, the functions relating actual and perceived distance were obtained for sixteen adult observers using the method of equal appearing intervals. These functions relating perceived and actual distance were obtained for outdoor viewing in a typical University environment-the experiment was conducted along a sidewalk adjacent to a typical street where campus buildings, trees, street signs, etc., were visible. The overall results indicated perceptual compression of distances in depth so that the stimulus distance intervals appeared significantly shorter than the actual (physical) distance intervals. It is important to note, however, that there were sizeable individual differences-the judgments of half of the observers were relatively accurate, whereas the judgments of the remaining half were inaccurate to varying degrees. The results of the experiment demonstrate that there is no single function that describes how human observers visually perceive large distance intervals in outdoor environments., (© 2024. The Author(s).)

Objective: Most of the work in terms of liquid biopsies in patients with solid tumors is focused on circulating tumor DNA (ctDNA). Our aim was to evaluate the feasibility of using circulating tumor cells (CTCs) in peripheral blood samples from patients with advanced or metastatic gastrointestinal (GI) cancers., Methods: In this prospective study, blood samples were collected from each patient in 2 AccuCyte ® blood collection tubes and each tube underwent CTC analysis performed utilizing the RareCyte ® platform. The results from both tubes were averaged and a total of 150 draws were done, with 281 unique reported results. The cadence of sampling was based on convenience sampling and piggybacked onto days of actual clinical follow-ups and treatment visits. The CTC results were correlated with patient- and tumor-related variables., Results: Data from a total of 59 unique patients were included in this study. Patients had a median age of 58 years, with males representing 69% of the study population. More than 57% had received treatment prior to taking blood samples. The type of GI malignancy varied, with more than half the patients having colorectal cancer (CRC, 54%) followed by esophageal/gastric cancer (17%). The least common cancer was cholangiocarcinoma (9%). The greatest number of CTCs were found in patients with colorectal cancer (Mean: 15.8 per 7.5 ml; Median: 7.5 per 7.5 ml). In comparison, patients with pancreatic cancer (PC) had considerably fewer CTCs (Mean: 4.2 per 7.5 ml; Median: 3 per 7.5 ml). Additionally, we found that patients receiving treatment had significantly fewer CTCs than patients who were not receiving treatment (Median 2.7 versus 0.7). CTC numbers showed noteworthy disparities between patients with responding/stable disease in comparison to those with untreated/progressive disease (Median of 2.7 versus 0). When CTCs were present, biomarker analyses of the four markers human epidermal growth factor receptor 2 (HER2)/programmed death-ligand 1 (PD-L1)/Kiel 67 (Ki-67)/epidermal growth factor receptor (EGFR) was feasible. Single cell sequencing confirmed the tumor of origin., Conclusion: Our study is one of the first prospective real-time studies evaluating CTCs in patients with GI malignancies. While ctDNA-based analyses are more common in clinical trials and practice, CTC analysis provides complementary information from a liquid biopsy perspective that is of value and worthy of continued research., Competing Interests: Author PK reports grants paid to the institution by Merck, Agenus Bio, Novartis, Advanced Accelerator Applications, Tersera, and Boston Scientific; a consultancy and advisory board relationship with Elicio scientific advisory board member/shares/stock ownership; consultancy/advisory board fees from Guardant Health, Natera, Foundation Medicine, Illumina, BostonGene, Merck/MSD Oncology, Tempus, Bayer, Lilly, Delcath Systems, IPBA, QED Therapeutics, Boston Healthcare Associates, Servier, Taiho Oncology, Exact Sciences, Daiichi Sankyo/AstraZeneca, Eisai, Saga Diagnostics, Neogenomics, Do More Diagnostics AS, and Seattle Genetics; consulting fees paid to the institution by Taiho Pharmaceutical and Ipsen; receiving travel support from AstraZeneca for presentation of an investigator-initiated trial. Authors LC, AR, and TG are employees of RareCyte. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Malkawi, Lutfi, Afghan, Shah, Costandy, Ramirez, George, Toor, Salem and Kasi.)

Introduction: The reliable and accurate detection of rare circulating tumor cells (CTCs) from cancer patient blood samples promises advantages in both research and clinical applications. Numerous CTC detection methods have been explored that rely on either the physical properties of CTCs such as density, size, charge, and/or their antigen expression profiles. Multiple factors can influence CTC recovery including blood processing method and time to processing. This study aimed to examine the accuracy and sensitivity of an enrichment-free method of isolating leukocytes (AccuCyte ® system) followed by immunofluorescence staining and high-resolution imaging (CyteFinder ® instrument) to detect CTCs., Method: Healthy human blood samples, spiked with cancer cells from cancer cell lines, as well as blood samples obtained from 4 subjects diagnosed with cancer (2 pancreatic, 1 thyroid, and 1 small cell lung) were processed using the AccuCyte-CyteFinder system to assess recovery rate, accuracy, and reliability over a range of processing times., Results: The AccuCyte-CyteFinder system was highly accurate (95.0%) at identifying cancer cells in spiked-in samples (in 7.5 mL of blood), even at low spiked-in numbers of 5 cells with high sensitivity (90%). The AccuCyte-CyteFinder recovery rate (90.9%) was significantly higher compared to recovery rates obtained by density gradient centrifugation (20.0%) and red blood cell lysis (52.0%). Reliable and comparable recovery was observed in spiked-in samples and in clinical blood samples processed up to 72 hours post-collection. Reviewer analysis of images from spiked-in and clinical samples resulted in high concordance (R-squared value of 0.998 and 0.984 respectively)., Discussion: The AccuCyte-CyteFinder system is as an accurate, sensitive, and clinically practical method to detect and enumerate cancer cells. This system addresses some of the practical logistical challenges in incorporating CTCs as part of routine clinical care. This could facilitate the clinical use of CTCs in guiding precision, personalized medicine., Competing Interests: Authors AR, LC, RH, BG are employees of RareCyte, Inc. Author JR reports advisory roles in The Gene Technology Technical Advisory Committee, Office of the Gene Technology Regulator, Australian Government. Author JR also reports honoraria, speaker fees or advisory roles for GSK, Takeda, Gilead, Cynata, Pfizer, Spark, Novartis, Celgene, bluebird bio, Shire, Avrobio; stocks in Genea; consultant role for Rarecyte stocks in lieu. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yeo, Kao, Gupta, Wahlroos, Bastian, Strauss, Klemm, Shrestha, Ramirez, Costandy, Huston, Gardner, Grimison, Clark and Rasko.)

Background: Reliable biomarkers that can be serially monitored to predict treatment response to immune checkpoint inhibitors (ICIs) are still an unmet need. Here, we present a multiplex immunofluorescence (IF) assay that simultaneously detects circulating tumor cells (CTCs) and assesses CTC expression of programmed death ligand-1 (PD-L1) and interferon regulatory factor 1 (IRF-1) as a candidate biomarker related to ICI use., Objective: To assess the potential of CTC PD-L1 and IRF-1 expression as candidate biomarkers for patients with advanced epithelial solid tumors receiving ICIs., Patients and Methods: We tested the IF CTC assay in a pilot study of 28 patients with advanced solid tumors who were starting ICI. Blood for CTC evaluation was obtained prior to starting ICI, after a single cycle of therapy, and at the time of radiographic assessment or treatment discontinuation., Results: At baseline, patients with 0-1 CTCs had longer progression-free survival (PFS) compared to patients with ≥ 2 CTCs (4.3 vs 1.3 months, p = 0.01). The presence of any PD-L1+ CTCs after a single dose of ICI portended shorter PFS compared to patients with no CTCs or PD-L1- CTCs (1.2 vs 4.2 months, p = 0.02); the presence of any PD-L1+ or IRF-1+ CTCs at time of imaging assessment or treatment discontinuation also was associated with shorter PFS (1.9 vs 5.5 months, p < 0.01; 1.6 vs 4.7 months, p = 0.05). CTC PD-L1 and IRF-1 expression did not correlate with tumor tissue PD-L1 or IRF-1 expression. Strong IRF-1 expression in tumor tissue was associated with durable (≥ 1 year) radiographic response (p = 0.02)., Conclusions: Based on these results, CTC PD-L1 and IRF-1 expression is of interest in identifying ICI resistance and warrants further study., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The practice of medicine has steadily employed less invasive methods to obtain information derived from the tumor to guide clinical management of patients. Liquid biopsy-the sampling of blood-is a non-invasive method for generating information previously only available from tissue biopsies of the tumor mass. Analysis of fragmented circulating tumor DNA in the plasma is clinically used to identify actionable mutations and detect residual or recurrent disease. Plasma analysis cannot, however, assess cancer phenotypes, including the expression of drug targets and protein biomarkers. Circulating tumor cells (CTCs) are intact cancer cells that have entered the blood that have the potential for distant metastasis. While enumeration of CTCs is prognostic of outcome, recently developed technology allows for the interrogation of protein biomarkers on CTCs that could be predictive of response. Furthermore, since CTCs contain intact whole cancer genomes, isolating viable CTCs detected during therapy could provide a rational approach to assessing mutational profiles of resistance. Identification, characterization and molecular analysis of CTCs together will advance the capacity of liquid biopsy to meet the requirements of twenty-first century medicine., Competing Interests: EK, AR, LC, NE and TG: employees of RareCyte, Inc. PK: Consulting/Advisory Board for AstraZeneca/Daiichi Sankyo, Axiom Healthcare Strategies, Bayer, Delcath Systems, Foundation Medicine, Incyte, Ipsen, IPBA, Lilly, Merck, MSD Oncology, Natera, Servier, Taiho Oncology, and Tempus; Research/Trial funding: BTG/Boston Scientific, Tersera, Merck. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kaldjian, Ramirez, Costandy, Ericson, Malkawi, George and Kasi.)

Purpose: Patients with metastatic triple-negative breast cancer (mTNBC) have poor outcomes. The Intensive Trial of Omics in Cancer (ITOMIC) sought to determine the feasibility and potential efficacy of informing treatment decisions through multiple biopsies of mTNBC deposits longitudinally over time, accompanied by analysis using a distributed network of experts., Methods: Thirty-one subjects were enrolled and 432 postenrollment biopsies performed (clinical and study-directed) of which 332 were study-directed. Molecular profiling included whole-genome sequencing or whole-exome sequencing, cancer-associated gene panel sequencing, RNA-sequencing, and immunohistochemistry. To afford time for analysis, subjects were initially treated with cisplatin (19 subjects), or another treatment they had not received previously. The results were discussed at a multi-institutional ITOMIC Tumor Board, and a report transmitted to the subject's oncologist who arrived at the final treatment decision in conjunction with the subject. Assistance was provided to access treatments that were predicted to be effective., Results: Multiple biopsies in single settings and over time were safe, and comprehensive analysis was feasible. Two subjects were found to have lung cancer, one had carcinoma of unknown primary site, tumor samples from three subjects were estrogen receptor-positive and from two others, human epidermal growth factor receptor 2-positive. Two subjects withdrew. Thirty-four of 112 recommended treatments were accessed using approved drugs, clinical trials, and single-patient investigational new drugs. After excluding the three subjects with nonbreast cancers and the two subjects who withdrew, 22 of 26 subjects (84.6%) received at least one ITOMIC Tumor Board-recommended treatment., Conclusion: Further exploration of this approach in patients with mTNBC is merited., Competing Interests: Kimberly A. BurtonEmployment: All4Cure, Just Biotherapeutics (I), Northwest Medical SpecialtiesLeadership: Just Biotherapeutics (I)Stock and Other Ownership Interests: Just Biotherapeutics (I)Other Relationship: NantWorks Eric Quentin KonnickHonoraria: Ventana Medical Systems, Roche, Clinical Care Options, Medscape, National Comprehensive Cancer NetworkConsulting or Advisory Role: River West Meeting AssociatesTravel, Accommodations, Expenses: Ventana Medical Systems, Roche, Clinical Care Options LLC, Medscape, National Comprehensive Cancer Network, River West Meeting Associates Sibel BlauEmployment: Northwest Medical SpecialtiesLeadership: Northwest Medical Specialties, Quality Cancer Care AllianceStock and Other Ownership Interests: Northwest Medical Specialties, All4Cure (I)Honoraria: Cardinal Health, Novartis, Puma Biotechnology, American Journal of Managed CareResearch Funding: Northwest Medical SpecialtiesExpert Testimony: Northwest Medical SpecialtiesTravel, Accommodations, Expenses: Northwest Medical Specialties, Quality Cancer Care AllianceOther Relationship: Northwest Medical Specialties, All4Cure (I), Quality Cancer Care Alliance Michael O. DorschnerEmployment: Guardant HealthStock and Other Ownership Interests: Guardant Health Arturo B. RamirezEmployment: RareCytePatents, Royalties, Other Intellectual Property: I hold six patents for my work at RareCyte Stephanie ParkerEmployment: IQvia Colin C. PritchardConsulting or Advisory Role: AstraZeneca, Sana BiotechnologyResearch Funding: Color Genomics (I) Christopher SzetoEmployment: NantHealth, Zai Lab, ImmunityBioStock and Other Ownership Interests: NantWorks, Zai LabPatents, Royalties, Other Intellectual Property: Listed as inventor or coinventor on several patents and patent applications filed with NantOmics; listed as inventor or coinventor on several patents and applications filed with ImmunityBio Patricia SpilmanEmployment: ImmunityBio IncStock and Other Ownership Interests: ImmunityBio IncResearch Funding: ImmunityBio Inc Vijayakrishna K. GadiStock and Other Ownership Interests: Sengine Precision Medicine, Novilla, 3rdEyeBio, Phoenix Molecular Designs, New Equilibrium BiosciencesConsulting or Advisory Role: Seattle Genetics, Puma Biotechnology, Novartis, SanofiSpeakers' Bureau: Seattle Genetics, Hologics, Genentech/Roche, Puma BiotechnologyResearch Funding: Genentech/Roche (Inst), SignalOne Bio, AgendiaTravel, Accommodations, Expenses: Seattle Genetics, Genentech/Roche, Puma BiotechnologyOpen Payments Link: https://openpaymentsdata.cms.gov/physician/2511 Roy RonenOther Relationship: Data4Cure Jackie StilwellEmployment: Umoja Biopharma, Seattle GeneticsStock and Other Ownership Interests: Seattle Genetics Eric KaldjianEmployment: RareCyte IncStock and Other Ownership Interests: RareCyte Inc Janusz DutkowskiPatents, Royalties, Other Intellectual Property: Patents pending on methods and systems for data analysis including biomedical data analysis (Inst)Other Relationship: Data4Cure Inc Stephen Charles BenzEmployment: NantWorks, ImmunityBio, NantHealthStock and Other Ownership Interests: Novartis, Celgene, NantWorks, NantHealth, Pfizer, Regeneron, Bluebird BioPatents, Royalties, Other Intellectual Property: Patents issued and pending Shahrooz RabizadehEmployment: ImmunityBio, Sagittarius BioLeadership: NantBioScience Inc, NantWorks, ImmunityBio, Sagittarius BioStock and Other Ownership Interests: NantHealth, ImmunityBio, Sagittarius BioPatents, Royalties, Other Intellectual Property: Made inventions that resulted in IP for NantBioScience and NantOmics; made inventions that resulted in IP for ImmunityBio; made inventions that resulted in IP for Sagittarius Bio Patrick Soon-ShiongEmployment: ImmunityBio, NantWorks, NantHealthLeadership: ImmunityBio, NantWorks, NantHealthStock and Other Ownership Interests: ImmunityBio, NantWorks, NantHealthPatents, Royalties, Other Intellectual Property: NantWorks and Affiliates, NantKwest, ImmunityBio C. Anthony BlauEmployment: All4CureLeadership: All4CureStock and Other Ownership Interests: All4CureConsulting or Advisory Role: GlaxoSmithKline (Inst)Travel, Accommodations, Expenses: Novartis (I)No other potential conflicts of interest were reported.

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths worldwide. Liquid biopsy by a blood draw to detect circulating tumor cells (CTCs) is a tool for molecular profiling of cancer using single-cell and next-generation sequencing (NGS) technologies. The aim of the study was to identify somatic variants in single CTCs isolated from NSCLC patients by targeted NGS. Thirty-one subjects (20 NSCLC patients, 11 smokers without cancer) were enrolled for blood draws (7.5 mL). CTCs were identified by immunofluorescence, individually retrieved, and DNA-extracted. Targeted NGS was performed to detect somatic variants (single-nucleotide variants (SNVs) and insertions/deletions (Indels)) across 65 oncogenes and tumor suppressor genes. Cancer-associated variants were classified using OncoKB database. NSCLC patients had significantly higher CTC counts than control smokers ( p = 0.0132; Mann-Whitney test). Analyzing 23 CTCs and 13 white blood cells across seven patients revealed a total of 644 somatic variants that occurred in all CTCs within the same subject, ranging from 1 to 137 per patient. The highest number of variants detected in ≥1 CTC within a patient was 441. A total of 18/65 (27.7%) genes were highly mutated. Mutations with oncogenic impact were identified in functional domains of seven oncogenes/tumor suppressor genes ( NF1, PTCH1, TP53, SMARCB1, SMAD4, KRAS, and ERBB2 ). Single CTC-targeted NGS detects heterogeneous and shared mutational signatures within and between NSCLC patients. CTC single-cell genomics have potential for integration in NSCLC precision oncology.

Background: Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®-CyteFinder® system., Methods: CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation., Results: The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method., Conclusions: Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte-CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression.

Circulating tumor cells (CTCs) can reliably be identified in cancer patients and are associated with clinical outcome. Next-generation "liquid biopsy" technologies will expand CTC diagnostic investigation to include phenotypic characterization and single-cell molecular analysis. We describe here a rare cell analysis platform designed to comprehensively collect and identify CTCs, enable multi-parameter assessment of individual CTCs, and retrieve single cells for molecular analysis. The platform has the following four integrated components: 1) density-based separation of the CTC-containing blood fraction and sample deposition onto microscope slides; 2) automated multiparameter fluorescence staining; 3) image scanning, analysis, and review; and 4) mechanical CTC retrieval. The open platform utilizes six fluorescence channels, of which four channels are used to identify CTC and two channels are available for investigational biomarkers; a prototype assay that allows three investigational biomarker channels has been developed. Single-cell retrieval from fixed slides is compatible with whole genome amplification methods for genomic analysis. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry., (© 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.)

The CytePicker module built into the RareCyte CyteFinder instrument allows researchers to easily retrieve individual cells from microscope slides for genomic analyses, including array CGH, targeted sequencing, and next-generation sequencing. Here, we describe the semiautomated retrieval of CTCs from the blood processed by AccuCyte (see Chapter 13) and amplification of genomic DNA so that molecular analysis can be performed.

The RareCyte platform addresses important technology limitations of current circulating tumor cell (CTC) collection methods, and expands CTC interrogation to include advanced phenotypic characterization and single-cell molecular analysis. In this respect, it represents the "next generation" of cell-based liquid biopsy technologies. In order to identify and analyze CTCs, RareCyte has developed an integrated sample preparation, imaging and individual cell retrieval process. The first step in the process, AccuCyte ® , allows the separation, collection, and transfer to a slide the nucleated cell fraction of the blood that contains CTCs. Separation and collection are based on cell density-rather than size or surface molecular expression-and are performed within a closed system, without wash or lysis steps, enabling high CTC recovery. Here, we describe our technique for nucleated cell collection from a blood sample, and the spreading of these nucleated cells onto glass slides permitting immunofluorescent staining, cell identification, and individual cell picking described in subsequent chapters. In addition to collection of rare cells such as CTCs, AccuCyte also collects cells of the circulating immune system onto archivable slides as well as plasma from the same sample.

Ultrasound imaging is used for detecting and characterizing breast lesions. A state of the art imaging method is the contrast source inversion (CSI), which solves the full wave nonlinear inverse problem. However, when the measurements are acquired in noisy environments, CSI can diverge from the correct solution after several iterations. Problems associated with noisy data were originally solved by including total variation (TV) regularization. Unfortunately, for very noisy data, TV regularization alone is not sufficient. In this work, compressed sensing ideas are used to regularize the inversion process by restricting the solution of the CSI method to be sparse in a transformation domain. The proposed method estimates the contrast source and contrast function by minimizing the mean squared error between the measured and modeled data. An extra penalty term is added to measure sparsity in the transformation domain. A second method that combines sparsity of the contrast source and minimal TV in the contrast function is also presented. The proposed methods are tested on noise-free and noisy synthetic data sets representing a scan of a cancerous breast. Numerical experiments show that, for measurements contaminated with 1% noise, the sparsity constrained CSI improves the normalized mean squared error of the reconstructed speed-of-sound profiles up to 36% in comparison with traditional CSI. Also, for measurements contaminated with 5% noise, the proposed methods improve the quality of the reconstruction up to 70% in comparison with the traditional CSI method. Experimental results also show that the methods remain convergent to the correct speed-of-sound profile as the number of iterations increases.

Accelerating cancer research is expected to require new types of clinical trials. This report describes the Intensive Trial of OMics in Cancer (ITOMIC) and a participant with triple-negative breast cancer metastatic to bone, who had markedly elevated circulating tumor cells (CTCs) that were monitored 48 times over 9 months. A total of 32 researchers from 14 institutions were engaged in the patient's evaluation; 20 researchers had no prior involvement in patient care and 18 were recruited specifically for this patient. Whole-exome sequencing of 3 bone marrow samples demonstrated a novel ROS1 variant that was estimated to be present in most or all tumor cells. After an initial response to cisplatin, a hypothesis of crizotinib sensitivity was disproven. Leukapheresis followed by partial CTC enrichment allowed for the development of a differential high-throughput drug screen and demonstrated sensitivity to investigational BH3-mimetic inhibitors of BCL-2 that could not be tested in the patient because requests to the pharmaceutical sponsors were denied. The number and size of CTC clusters correlated with clinical status and eventually death. Focusing the expertise of a distributed network of investigators on an intensively monitored patient with cancer can generate high-resolution views of the natural history of cancer and suggest new opportunities for therapy. Optimization requires access to investigational drugs., (Copyright © 2016 by the National Comprehensive Cancer Network.)

Background: Circulating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. There is great interest in using CTCs to monitor response to therapies, to identify clinically actionable biomarkers, and to provide a non-invasive window on the molecular state of a tumor. Here we characterize the performance of the AccuCyte®--CyteFinder® system, a comprehensive, reproducible and highly sensitive platform for collecting, identifying and retrieving individual CTCs from microscopic slides for molecular analysis after automated immunofluorescence staining for epithelial markers., Methods: All experiments employed a density-based cell separation apparatus (AccuCyte) to separate nucleated cells from the blood and transfer them to microscopic slides. After staining, the slides were imaged using a digital scanning microscope (CyteFinder). Precisely counted model CTCs (mCTCs) from four cancer cell lines were spiked into whole blood to determine recovery rates. Individual mCTCs were removed from slides using a single-cell retrieval device (CytePicker™) for whole genome amplification and subsequent analysis by PCR and Sanger sequencing, whole exome sequencing, or array-based comparative genomic hybridization. Clinical CTCs were evaluated in blood samples from patients with different cancers in comparison with the CellSearch® system., Results: AccuCyte--CyteFinder presented high-resolution images that allowed identification of mCTCs by morphologic and phenotypic features. Spike-in mCTC recoveries were between 90 and 91%. More than 80% of single-digit spike-in mCTCs were identified and even a single cell in 7.5 mL could be found. Analysis of single SKBR3 mCTCs identified presence of a known TP53 mutation by both PCR and whole exome sequencing, and confirmed the reported karyotype of this cell line. Patient sample CTC counts matched or exceeded CellSearch CTC counts in a small feasibility cohort., Conclusion: The AccuCyte--CyteFinder system is a comprehensive and sensitive platform for identification and characterization of CTCs that has been applied to the assessment of CTCs in cancer patient samples as well as the isolation of single cells for genomic analysis. It thus enables accurate non-invasive monitoring of CTCs and evolving cancer biology for personalized, molecularly-guided cancer treatment.

Triple-negative breast cancer is a particularly aggressive and lethal breast cancer subtype that is more likely to be interval-detected rather than screen-detected. The purpose of this study is to discover and initially validate novel early detection biomarkers for triple-negative breast cancer using preclinical samples. Plasma samples collected up to 17 months before diagnosis from 28 triple-negative cases and 28 matched controls from the Women's Health Initiative Observational Study were equally divided into a training set and a test set and interrogated by a customized antibody array. Data were available on 889 antibodies; in the training set, statistically significant differences in case versus control signals were observed for 93 (10.5 %) antibodies at p < 0.05. Of these 93 candidates, 29 were confirmed in the test set at p < 0.05. Areas under the curve for these candidates ranged from 0.58 to 0.79. With specificity set at 98 %, sensitivity ranged from 4 to 68 % with 20 candidates having a sensitivity ≥ 20 % and 6 having a sensitivity ≥ 40 %. In an analysis of KEGG gene sets, the pyrimidine metabolism gene set was upregulated in cases compared to controls (p = 0.004 in the testing set) and the JAK/Stat signaling pathway gene set was downregulated (p = 0.003 in the testing set). Numerous potential early detection biomarkers specific to triple-negative breast cancer in multiple pathways were identified. Further research is required to followup on promising candidates in larger sample sizes and to better understand their potential biologic importance as our understanding of the etiology of triple-negative breast cancer continues to grow.

The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.

Here we demonstrate the utility of high-density antibody microarrays for ovarian cancer biomarker discovery. This report describes the technology and how it can be optimized for hypothesis-generating and testing experiments. Our previous results validated the high density antibody array technology platform, the current work expands on it utilizing a second generation array that we tested with a larger set of ovarian case and control serum samples. We then describe our strategies and methods for result validation, including Western immunoblots to confirm antibody specificity. By comparing and combining the current results with our previous study, we solidified the case that the markers found could be used for ovarian cancer diagnosis using this technology. These results set the stage for further validation of these potential biomarkers and the use of this technology in future biomarker discovery studies.

Background: Strategies to discover circulating protein markers of ovarian cancer are urgently needed. We developed a novel technology that permits us to isolate recombinant antibodies directed against the potential serum biomarkers, to facilitate the further development of affinity reagents necessary to construct diagnostic tests., Methods: This study presents a novel discovery approach based on serum immunoprecipitation with cancer-specific in vivo biotinylated recombinant antibodies (biobodies) derived from differentially selected yeast-display scFv, and analysis of the eluted serum proteins by electrophoresis and/or mass spectrometry., Results: Using this strategy we identified catabolic fragments of complement factors, EMILIN2, Von Willebrand factor and phosphatidylethanolamine-binding protein 1 (PEBP1 or RKIP) in patient sera. To our knowledge, this is the first report of a soluble form of PEBP1 in human. Independent evidence for ovarian cancer-specific expression of PEBP1 in patient sera was found by ELISA assays and antibody arrays with anti-PEBP1 antibodies. PEBP1 was detected in 29 out of 30 ascites samples and discriminated ovarian cancer sera from controls (p = 0.02). Finally, we confirmed by western blots the presence of a 21-23 kDa fragment corresponding to the expected size of PEBP1 but we also showed additional bands of 38 kDa and 50-52 kDa in various tissues and cell lines., Conclusion: We conclude that the novel strategy described here allows the identification of candidate biomarkers that can be variants of normally expressed proteins or that display cancer-specific post-translational modifications.

Perhaps the greatest barrier to translation of serum biomarker discoveries is the inability to evaluate putative biomarkers in high throughput validation studies. Here we report on the development, production, and implementation of a high-density antibody microarray used to evaluate large numbers of candidate ovarian cancer serum biomarkers. The platform was shown to be useful for evaluation of individual antibodies for comparative analysis, such as with disease classification, and biomarker validation and discovery. We demonstrate its performance by showing that known tumor markers behave as expected. We also identify several promising biomarkers from a candidate list and generate hypotheses to support new discovery studies.