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Supplemental Figure 5, online only: Copy Number Alteration (CNA) profiles from Case 11, 21, and 25 demonstrating change in amplitude of the alterations in longitudinal AH samples taken over time. Case 11 showed a progressive gain of 6p and was eventually enucleated whereas Cases 21 and 25 showed a decrease in the amplitude of alterations and these eyes were salvaged.

Supplemental Figure 4, online only: Initial Copy Number Alteration (CNA) profile from eyes that were successfully salvaged with treatment

Supplemental Figure 3, online only: Initial Copy Number Alteration (CNA) profile from eyes that required enucleation + indicates that in Case 11 the second AH sample (which had good alignment and demonstrates gain of 6p) is shown instead of the initial sample (see also Table 1).

Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal MYCN amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes (P = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes (P = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8–55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes (P = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH (P = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy.Implications: The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. Mol Cancer Res; 16(11); 1701–12. ©2018 AACR.

Supplemental Figure 2, online only: Hierarchical clustering matrix based on the genomic instability of the Copy Number Alteration (CNA) profiles of all the AH samples included in this analysis. Samples are listed by Case number_# based on the order of AH sampling (e.g. 1, 2, 3). Samples from eyes that were enucleated are indicated by the red bar adjacent the dentogram, those from eyes that were salvaged are indicated in blue.

Supplemental Figure 1, online only: a positive correlation was seen between the age at diagnosis and genomic instability which is defined as the overall size of the copy number variation found in the initial AH samples (p=

Supplementary Figure from A Randomized Trial of Fulvestrant, Everolimus, and Anastrozole for the Front-line Treatment of Patients with Advanced Hormone Receptor–positive Breast Cancer, SWOG S1222

Purpose:Metastatic hormone receptor (HR)-positive, HER2-negative breast cancer is an important cause of cancer mortality. Endocrine treatment with or without additional targeted therapies has been the mainstay of treatment. This trial was designed to evaluate the combination of fulvestrant plus everolimus versus fulvestrant, everolimus, and anastrozole compared with fulvestrant alone in the first-line treatment of advanced HR-positive, HER2-negative breast cancer.Patients and Methods:This randomized placebo-controlled trial included postmenopausal women with HR-positive, HER2-negative advanced breast cancer who had received no prior systemic therapy for metastatic disease. Participants were randomized to one of three treatment arms and the primary outcome was progression-free survival (PFS), comparing combinations of fulvestrant and everolimus with or without anastrozole with fulvestrant alone. Circulating tumor cells (CTC), as measured with two different methods, and circulating tumor DNA (ctDNA) were evaluated serially prior to treatment and the beginning of the second cycle of therapy.Results:Due in part to changes in clinical practice, the study was closed after accruing only 37 participants. There was no evidence that everolimus-containing combination treatment improved PFS or overall survival relative to fulvestrant alone. When modeled continuously, an association was observed of baseline CTC and ctDNA with poorer survival.Conclusions:Although power of the study was limited, the findings were unable to support the routine use of everolimus combination endocrine therapy in the first-line treatment of advanced hormone-sensitive breast cancer. Prognostic impact of baseline ctDNA and copy-number variations in CTC was demonstrated.

Purpose: Metastatic hormone receptor (HR)-positive, HER2-negative breast cancer is an important cause of cancer mortality. Endocrine treatment with or without additional targeted therapies has been the mainstay of treatment. This trial was designed to evaluate the combination of fulvestrant plus everolimus versus fulvestrant, everolimus, and anastrozole compared with fulvestrant alone in the first-line treatment of advanced HR-positive, HER2-negative breast cancer. Patients and Methods: This randomized placebo-controlled trial included postmenopausal women with HR-positive, HER2-negative advanced breast cancer who had received no prior systemic therapy for metastatic disease. Participants were randomized to one of three treatment arms and the primary outcome was progression-free survival (PFS), comparing combinations of fulvestrant and everolimus with or without anastrozole with fulvestrant alone. Circulating tumor cells (CTC), as measured with two different methods, and circulating tumor DNA (ctDNA) were evaluated serially prior to treatment and the beginning of the second cycle of therapy. Results: Due in part to changes in clinical practice, the study was closed after accruing only 37 participants. There was no evidence that everolimus-containing combination treatment improved PFS or overall survival relative to fulvestrant alone. When modeled continuously, an association was observed of baseline CTC and ctDNA with poorer survival. Conclusions: Although power of the study was limited, the findings were unable to support the routine use of everolimus combination endocrine therapy in the first-line treatment of advanced hormone-sensitive breast cancer. Prognostic impact of baseline ctDNA and copy-number variations in CTC was demonstrated.

The liquid biopsy has the potential to improve current clinical practice in oncology by providing real-time personalized information about a patient’s disease status and response to treatment. In this study, we evaluated 161 peripheral blood (PB) samples that were collected around surgical resection from 47 metastatic colorectal cancer (mCRC) patients using the High-Definition Single Cell Assay (HDSCA) workflow. In conjunction with the standard circulating tumor cell (CTC) enumeration, cellular morphology and kinetics between time-points of collection were considered in the survival analysis. CTCs, CTC-Apoptotic, and CTC clusters were found to indicate poor survival with an increase in cell count from pre-resection to post-resection. This study demonstrates that CTC subcategorization based on morphological differences leads to nuanced results between the subtypes, emphasizing the heterogeneity within the CTC classification. Furthermore, we show that factoring in the time-point of each blood collection is critical, both for its static enumeration and for the change in cell populations between draws. By integrating morphology and time-based analysis alongside standard CTC enumeration, liquid biopsy platforms can provide greater insight into the pathophysiology of mCRC by highlighting the complexity of the disease across a patient’s treatment.

Tumor-derived cell-free DNA (cfDNA) has biomarker potential; therefore, this study aimed to identify cfDNA in the aqueous humor (AH) of retinoblastoma eyes and correlate somatic chromosomal copy-number alterations (SCNA) with clinical outcomes, specifically eye salvage. AH was extracted via paracentesis during intravitreal injection of chemotherapy or enucleation. Shallow whole-genome sequencing was performed using isolated cfDNA to assess for highly recurrent SCNAs in retinoblastoma including gain of 1q, 2p, 6p, loss of 13q, 16q, and focal MYCN amplification. Sixty-three clinical specimens of AH from 29 eyes of 26 patients were evaluated; 13 eyes were enucleated and 16 were salvaged (e.g., saved). The presence of detectable SCNAs was 92% in enucleated eyes versus 38% in salvaged eyes (P = 0.006). Gain of chromosome 6p was the most common SCNA found in 77% of enucleated eyes, compared with 25% of salvaged eyes (P = 0.0092), and associated with a 10-fold increased odds of enucleation (OR, 10; 95% CI, 1.8–55.6). The median amplitude of 6p gain was 1.47 in enucleated versus 1.07 in salvaged eyes (P = 0.001). The presence of AH SCNAs was correlated retrospectively with eye salvage. The probability of ocular salvage was higher in eyes without detectable SCNAs in the AH (P = 0.0028), specifically 6p gain. This is the first study to correlate clinical outcomes with SCNAs in the AH from retinoblastoma eyes, as such these findings indicate that 6p gain in the aqueous humor is a potential prognostic biomarker for poor clinical response to therapy. Implications: The correlation of clinical outcomes and SCNAs in the AH identified in the current study requires prospective studies to validate these finding before SCNAs, like 6p gain, can be used to predict clinical outcomes at diagnosis. Mol Cancer Res; 16(11); 1701–12. ©2018 AACR.

Currently, clinical characterization of metastatic breast cancer is based on tissue samples taken at time of diagnosis. However, tissue biopsies are invasive and tumors are continuously evolving, which indicates the need for minimally invasive longitudinal assessment of the tumor. Blood-based liquid biopsies provide minimal invasive means for serial sampling over the course of treatment and the opportunity to adjust therapies based on molecular markers. Here, we aim to identify cellular changes that occur in breast cancer over the lifespan of an affected patient through single-cell proteomic and genomic analysis of longitudinally sampled solid and liquid biopsies. Three solid and 17 liquid biopsies from peripheral blood of an ER+/HER2− metastatic breast cancer patient collected over 4 years and eight treatment regimens were analyzed for morphology, protein expression, copy-number alterations, and single-nucleotide variations. Analysis of 563 single morphometrically similar circulating tumor cells (CTCs) and 13 cell-free DNA (cfDNA) samples along with biopsies of the primary and metastatic tumor revealed progressive genomic evolution away from the primary tumor profiles, along with changes in ER expression and the appearance of resistance mutations. Both the abundance and the genomic alterations of CTCs and cfDNA were highly correlated and consistent with genomic alterations in the tissue samples. We demonstrate that genomic evolution and acquisition of drug resistance can be detected in real time and at single-cell resolution through liquid biopsy analytes and highlight the utility of liquid biopsies to guide treatment decisions.

Tumor heterogeneity is prevalent in both treatment-naive and end-stage metastatic castration-resistant prostate cancer (PCa), and may contribute to the broad range of clinical presentation, treatment response, and disease progression. To characterize molecular heterogeneity associated with de novo metastatic PCa, multiplatform single cell profiling was performed using high definition single cell analysis (HD-SCA). HD-SCA enabled morphoproteomic and morphogenomic profiling of single cells from touch preparations of tissue cores (prostate and bone marrow biopsies) as well as liquid samples (peripheral blood and bone marrow aspirate). Morphology, nuclear features, copy number alterations, and protein expression were analyzed. Tumor cells isolated from prostate tissue touch preparation (PTTP) and bone marrow touch preparation (BMTP) as well as metastatic tumor cells (MTCs) isolated from bone marrow aspirate were characterized by morphology and cytokeratin expression. Although peripheral blood was examined, circulating tumor cells were not definitively observed. Targeted proteomics of PTTP, BMTP, and MTCs revealed cell lineage and luminal prostate epithelial differentiation associated with PCa, including co-expression of EpCAM, PSA, and PSMA. Androgen receptor expression was highest in MTCs. Hallmark PCa copy number alterations, including PTEN and ETV6 deletions and NCOA2 amplification, were observed in cells within the primary tumor and bone marrow biopsy samples. Genomic landscape of MTCs revealed to be a mix of both primary and bone metastatic tissue. This multiplatform analysis of single cells reveals several clonal origins of metastatic PCa in a newly diagnosed, untreated patient with polymetastatic disease. This case demonstrates that real-time molecular profiling of cells collected through prostate and bone marrow biopsies is feasible and has the potential to elucidate the origin and evolution of metastatic tumor cells. Altogether, biological and genomic data obtained through longitudinal biopsies can be used to reveal the properties of PCa and can impact clinical management.

Liquid biopsy allows assessment of multiple analytes, providing temporal information with potential for improving understanding of cancer evolution and clinical management of patients. Although liquid biopsies are intensely investigated for prediction and response monitoring, preanalytic variables are of primary concern for clinical implementation, including categories of collection method and sample storage. Herein, an integrated high-density single-cell assay workflow for morphometric and genomic analysis of the liquid biopsy is used to characterize the effects of preanalytical variation and reproducibility of data from a breast cancer cohort. Following prior work quantifying performance of commonly used blood collection tubes, this study completes the analysis of four time points to assay (24, 48, 72, and 96 hours), demonstrating precision up to 48 hours after collection for assay sensitivity, highly reproducible rare cell enumeration, morphometric characterization, and high efficiency and capacity for single-cell genomic analysis. For the cell-free analysis, both freezing and use of fresh plasma produced similar quality and quantity of cell-free DNA for sequencing. The genomic analysis (copy number variation and single-nucleotide variation) described herein is broadly applicable to liquid biopsy platforms capable of isolating cell-free and cell-based DNA. Morphometric parameters and genomic signatures of individual circulating tumor cells were evaluated in relation to patient clinical response, providing preliminary evidence of clinical validity as a potential biomarker aiding clinical diagnostics or monitoring progression.

The isotopic composition of iron, zinc, copper, and cadmium (δ(56)Fe, δ(66)Zn, δ(65)Cu, and δ(114)Cd) are novel and promising tools to study the metabolism and homeostasis of trace metals in the human body. Serum δ(65)Cu has been proposed as a potential tool for diagnosis of cancer in liquid biopsy, and other metals may have similar utility. However, accurate analysis of trace metal isotopes is challenging because of the difficulties in purifying the metals from biological samples. Here we developed a simple and rapid method for sequential purification of Cu, Fe, Zn, and Cd from a single blood plasma sample. By using a combination of 11 M acetic acid and 4 M HCl in the first steps of column chemistry on AG-MP1 resin, we dramatically improve the separation of Cu from matrix elements compared to previous methods which use concentrated HCl alone. Our new method achieves full recovery of Cu, Fe, Zn, and Cd to prevent column-induced isotope fractionation effects, and effectively separates analytes from the matrix in order to reduce polyatomic interferences during isotope analysis. Our methods were verified by the analysis of isotope standards, a whole blood reference material, and a preliminary sample set including five plasma samples from healthy individuals and five plasma samples from cancer patients. This new method simplifies preparation of blood samples for metal isotope analysis, accelerating multi-isotope approaches to medical studies and contributing to our understanding of the cycling of Fe, Zn, Cu, and Cd in the human body.

Background: Retinoblastoma (RB) was one of the first tumors to demonstrate a genetic basis to the development of cancer. However, unlike other cancers, RB cannot be directly biopsied due to the high risk of extraocular cancer spread. Therefore, unless the eye is enucleated, tumor tissue is not evaluated for genetic and genomic changes and these alterations are not used to inform diagnosis or prognosis for this disease. However, in a 2017 publication in JAMA Ophthalmology, we demonstrated that tumor-derived cell-free DNA can be extracted from the aqueous humor (AH) of RB eyes, which is safe to remove even with active intraocular disease. The purpose of this current study (Berry JL, Xu L et al. Molec Canc Res 2018) was to identify somatic chromosomal copy number alterations (SCNA) in tumor-derived cell-free DNA in the AH of RB eyes and to correlate with clinical outcomes particularly tumor relapse requiring enucleation. Methods: AH was extracted via paracentesis from RB eyes during intravitreal injection of chemotherapy or post enucleation. Shallow whole genome sequencing was performed to assess for cell-free tumor DNA fractions and highly-recurrent SCNAs in RB which include gain of 1q, 2p, 6p and loss of 13q and 16q. Globe salvage was recorded. Results: 26 patients were included; 3 patients had both eyes included for 29 eyes. From these, 63 samples of AH were analyzed; 5 post-enucleations and 58 during intravitreal chemotherapy injection. Ultimately 13 eyes required enucleation and 16 eyes were salvaged. Follow-up ranged from 8-43 months (median 17 months). The presence of any detectable SCNA was 92% in enucleated eyes versus 38% in salvaged eyes (p=0.006). 6p gain was the most common SCNA found in 77% of enucleated eyes versus 25% of salvaged eyes (p=0.0092). 6p gain was associated with a ten-fold increased odds of enucleation (OR=10, 95% CI:1.8-55.6). The mean amplitude of 6p gain was 1.47 in enucleated eyes versus 1.07 in salvaged eyes (p=0.001). The probability of ocular survival was higher in eyes without detectable SCNAs in the AH (p=0.0028). Serum testing was positive for a germline mutation in 17 eyes of 14 patients with the following incidence of RB SCNAs 1q (35%); 2p (18%); 6p (47%); 13q (18%) and 16q (35%) versus 12 eyes of 12 patients without germline disease with 1q (33%); 2p (8%); 6p (50%); 13q (17%) and 16q (33%) (p=0.71). Conclusions: This is the first study to show that clinical outcomes correlate with highly-recurrent SCNAs in the AH from RB eyes. This study suggests that the AH can reliably serve as a surrogate to tumor biopsy and improves upon current clinical staging to predict tumor response to therapy and the ability to salvage the eye. Unlike previous studies that suggest a greater incidence of RB SCNAs in patients with non-germline disease, this was not seen in this evaluation which may be because all previous work was done on tumor from enucleated eyes instead of salvaged eyes. Citation Format: Liya Xu, Jesse L. Berry, Irsan Kooi, A Linn Murphree, Rishvanth K. Prabakar, Mark W. Reid, Kevin Stachelek, Bao Han A. Le, Lisa Welter, Rima Jubran, Thomas C. Lee, Jonathan W. Kim, Peter Kuhn, David Cobrinik, James B. Hicks. Genomic cfDNA analysis of aqueous humor in retinoblastoma (RB) predicts eye salvage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2877.

Metastatic breast cancer patients have a high risk of progression and face poor prognosis overall, with about one third (34%) surviving 5 years or more. In rare instances (2-4% of cases) patients with metastatic breast cancer have ERBB2 (HER2) activating mutations, but are HER2 negative (non-amplified. Neratinib is a potent, irreversible inhibitor that binds HER2 and inhibits downstream signaling. We used the previously validated high-definition single cell analysis (HD-SCA) workflow to investigate the clinical significance of circulating tumor cells (HD-CTCs) in HER2-mutant, HER2 non-amplified, post-menopausal metastatic breast cancer patients starting neratinib and fulvestrant combinational therapy. Genomic analysis of the cell-free DNA (cfDNA) and single cell HD-CTCs was conducted to monitor tumor evolution and identify potential mutational variants unique to the patient’s clinical response. A subset of 5 patients presented here were from the MutHER (NCT01670877) and/or SUMMIT (NCT01953926) trials. Patients had an average of 5.4 lines of therapy before enrollment, variable hormone receptor status, and ERBB2 mutations at diagnosis and during treatment. HD-CTC enumeration alone was not sufficient to predict clinical response. Interestingly, treatment pressure was shown to lead to an observable change in HD-CTC morphology and genomic instability, suggesting these parameters may inform prognosis. Single cell copy number variation (CNV) analysis indicated that the persistence or development of a clonal population of HD-CTCs during treatment was associated with a worse response. Hierarchical clustering analysis of the single cells across all patients and timepoints identified distinct aberrant regions shared among patients, comprised of 26 genes that are similarly affected and may be related to drug resistance. Additionally at the cfDNA level, we identified new mutations in ERBB2, PIK3CA, and TP53 that arose due to treatment pressure in a patient with poor response, further informing us on the dynamics of the cancer genome over the course of therapy. The data presented in this small cohort study demonstrates the feasibility of real-time molecular profiling of the cellular and acellular fractions of the liquid biopsy using the HD-SCA platform. Additional insight is warranted to determine the potential use of morphometric and genomic analysis as a prognostic tool to advance personalized oncology. Citation Format: Stephanie Nicole Shishido, Rahul Masson, Lisa Welter, Liya Xu, Anishka D'Souza, Darcy Spicer, James Hicks, Janice Lu, Peter Kuhn. Morphologic and genomic characterization of circulating tumor cells in patients with ERBB2 mutant HER2 non-amplified metastatic breast cancer treated with neratinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4459.

Background: Copy number variation analysis has been shown as a valuable tool to infer clonal evolution of tumor cells. While bulk genomic analysis of FFPE tissue can provide information about the characteristics of a primary tumor or a metastatic nodule, these biopsies are rarely repeated due to their invasive nature. Liquid biopsies on the other hand, provide minimally invasive access to tumor derived cells and DNA and can therefore be used to monitor genomic changes of the tumor longitudinally and in real time. Here we evaluate the kinetics of liquid biopsy biomarkers and monitor genomic changes in single circulating tumor cells (CTCs) and cell-free DNA (cfDNA) from 17 longitudinal blood draws collected from a metastatic breast cancer patient over seven treatment regimes. FFPE tissue from the primary tumor and two metastatic sites in liver and bone are available for comparative analysis. Methods: We used the high definition – single cell assay (HD-SCA) platform to detect and enumerate CTCs of the blood of one metastatic breast cancer patient followed for 4 years. CTCs were characterized morphometrically and single CTCs of each draw were sequenced for copy number variation (CNV). cfDNA was extracted from plasma and tissue samples of the primary breast, bone and liver metastasis were micro-dissected. Extracted DNA from tissue samples and plasma underwent CNV analysis and were compared with CNV data of CTCs. Results: CTCs were identified in 14/17 draws and varied from 0 to >3000 cells/ml, spiking at two different times concordant with clinical progression. Changes in cell free tumor DNA fraction (ctDNA) correlated strongly with CTC count. Comparative CNV analysis of CTCs, cfDNA and FFPE tissue confirmed the tumorous origin of both cells and cfDNA. Single cell CNV analysis found four genomically related sub-clones in the patient's blood, three of which were present at time of enrollment, while the fourth clone emerged 3.5 years later concordant with the decline in the patient's health. Genomic copy number losses included tumor suppressor genes such as ARID1A, FOXP1, ATM, BRCA2, RB1, CDH11 and CDH1. Genomic gains were limited but included the proto-oncogenes BCL9, ABL2 and MDM4. It is striking that the sub-clonal structure persisted nearly unchanged for 3.5 years throughout various hormone and cytotoxic treatments, indicating high genomic stability. Despite their genomic differences, cells from the different clones are indistinguishable by immunofluorescence or morphometric analysis. CNV profiles of cfDNA reflect the most abundant CTC clone at each time-point. Conclusion: Using the HD-SCA assay cellular (CTC), and soluble (cfDNA) fractions of liquid biopsies can be compared at the molecular level over a long time course, and verified by comparison with tissue biopsy. This work confirms that liquid biopsies can be used to access the molecular state of a patient's cancer in near real-time and provide insight into the response to treatment years after the original characterization of the primary tissue. Here we show in a single patient example the power of single CTC analysis and highlight how CNV analysis of liquid biopsies could serve as minimally invasive tool to monitor tumor evolution longitudinally. Citation Format: Welter L, Xu L, McKinley D, Dago AE, Restrepo-Vassalli S, Rodriguez Lee M, Kolatkar A, Nieva J, Hicks J, Kuhn P. Tracing clonal evolution of circulating tumor cells and cell-free DNA in a metastatic breast cancer patient [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-09.

Tumor heterogeneity is prevalent in both treatment-naïve and end-stage metastatic castration-resistant prostate cancer (PCa), and may contribute to the broad range of clinical presentation, treatment response, and disease progression. To characterize molecular heterogeneity associated with de novo metastatic PCa, multiplatform single cell profiling was performed using High Definition Single Cell Analysis (HD-SCA). HD-SCA enabled morphoproteomic and morphogenomic profiling of single cells from touch preparations of tissue cores (prostate and bone marrow biopsies) as well as liquid samples (peripheral blood and bone marrow aspirate). Morphology, nuclear features, copy number alterations, and protein expression were analyzed. Tumor cells isolated from prostate tissue touch preparation (PTTP) and bone marrow touch preparation (BMTP) as well as metastatic tumor cells (MTCs) isolated from bone marrow aspirate were characterized by morphology and cytokeratin expression. Although peripheral blood was examined, circulating tumor cells were not definitively observed. Targeted proteomics of PTTP, BMTP, and MTCs revealed cell lineage and luminal prostate epithelial differentiation associated with PCa, including co-expression of EpCAM, PSA, and PSMA. Androgen receptor expression was highest in MTCs. Hallmark PCa copy number alterations, including PTEN and ETV6 deletions and NCOA2 amplification, were observed in cells within the primary tumor and bone marrow biopsy samples. Genomic landscape of MTCs revealed to be a mix of both primary and bone metastatic tissue. This multiplatform analysis of single cells reveals several clonal origins of metastatic PCa in a newly diagnosed, untreated patient with polymetastatic disease. This case demonstrates that real-time molecular profiling of cells collected through prostate and bone marrow biopsies is feasible and has the potential to elucidate the origin and evolution of metastatic tumor cells. Altogether, biological and genomic data obtained through longitudinal biopsies can be used to reveal the properties of PCa and can impact clinical management. Citation Format: Paymaneh D. Malihi, Michael Morikado, Lisa Welter, Anders Carlsson, Carmen Ruiz Velasco, Anand Kolatkar, Mariam Rodriguez-Lee, James Hicks, Peter Kuhn, Sandy T. Liu, Eric T. Miller, Radu M. Cadaneanu, Beatrice S. Knudsen, Michael S. Lewis, Isla P. Garraway. Clonal diversity revealed by morphoproteomic and copy number profiles of single prostate cancer cells at diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1576.

Background: It is established that tumors evolve over time due to treatment pressure or physiological constraints. Tissue biopsies give a one-time window into the characteristics of a primary tumor or metastatic nodule. Their main pitfall is procedure risk, pain, and cost, which is why they are rarely repeated. Liquid biopsies, however, are performed with minimal discomfort and risk, providing an easier way to monitor disease. Circulating tumor cells (CTCs) and cell-free DNA (cfDNA) have proven to be of high prognostic and diagnostic value. Here we evaluate 17 longitudinal blood draws collected from a metastatic breast cancer patient over 4 years and assess the power of single-cell analysis relative to analysis of tissue biopsies and cfDNA. Methods: CTCs were enumerated using the high-definition single-cell assay (HD-SCA) and single CTCs were isolated by micromanipulation. DNA of single cells underwent whole genome amplification and Illumina library construction for copy number variation (CNV) analysis. Genomic DNA from microdissected FFPE tissue and cfDNA extracted from plasma were converted into Illumina sequencing libraries for CNV analysis. CA27.29 levels were reported by the clinic. Results: Levels of CTCs and circulating tumor DNA (ctDNA) fractions were positively correlated with CA27.29. CNV analysis of single tumor cells at time of enrollment revealed 3 morphometrically indistinguishable clones that persisted during the 4-year follow-up. Genomic analysis of primary breast tissue and bone metastasis detected at time of diagnosis matched CNV data of the evolutionary first clone, while a later liver metastasis was comprised solely of clone 2. Remarkably, CTCs in clone 2 were identified years before discovery of the liver metastasis. To date, we have not detected a metastatic site harboring clone 3. We found that CNV profiles from cfDNA represented the aggregate of CTC clones. Conclusion: Longitudinal blood sampling enabled tracing of tumor evolution robustly and less invasively than solid tumor biopsies. Metastasis associated clones were identified years before measureable disease, providing an opportunity for earlier interventions. While plasma-based assays detected the presence of tumor-derived DNA, the single-cell high-content resolution of the HD-SCA workflow was required to deconvolute 3 genomically distinct clones present in this patient. We propose that enumeration of CTCs and analysis of ctDNA could be used as additional clinical markers to monitor disease recurrence, adding valuable morphogenomic information about the primary tumor and metastatic sites. Additionally, we are currently analyzing the results of single nucleotide variation analysis of single CTCs, cfDNA and FFPE tissue from this patient. Citation Format: Lisa Welter, Liya Xu, Dillon McKinley, Sara Restrepo-Vassalli, Angel Dago, Mariam Rodriguez Lee, Anand Kolatkar, James Hicks, Jorge Nieva, Peter Kuhn. Characterization of disease evolution in sequential sampled metastatic breast cancer using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2963.

The liquid biopsy allows assessment of multiple biological analytes over time to provide dynamic temporal information with the potential for improving clinical management and guiding treatment decisions. While the promise of liquid biopsies for prediction and response monitoring are intensely investigated, the pre-analytic variables are of primary concern for its implementation in diagnostic clinical medicine, including such categories as collection method, shipping conditions, and sample storage. Here we utilize an integrated high-definition single cell analysis (HD-SCA) workflow for genomic analysis of rare cells and cfDNA from the liquid biopsy to characterize the effects of pre-analytical variation and reproducibility of data analysis from the same cohort of patients. The results presented here confirm consistent rare cell enumeration and morphometric characterization between 24h and 48h time to assay (TTA), with a high efficiency and capacity for both copy number variation (CNV) and single nucleotide variation (SNV) analysis at the single cell level. Additionally, the freezing process neither diminishes or increases the DNA quantity, nor does it affect the DNA quality for CNV or SNV analysis. The integration of genomic information is imperative to reveal essential disease-implicated mutational profiles, but effective molecular diagnostic tests require reproducible coverage over a broad dynamic range. The performance of the HD-SCA platform quantified here may be utilized as a guide for implementation into patient care and/or research biorepository processes. Citation Format: Stephanie Nicole Shishido, Mariam Rodriguez_Lee, Anand Kolatkar, Liya Xu, Sara Restrepo-Vassalli, Lisa Welter, Anders Carlsson, Emily Greenspan, Shelley Hwang, Kathryn Waitman, Jorge Nieva, Kelley Bethel, James Hicks, Peter Kuhn. Integration of genomic analysis and assessment of pre-analytic variables in the HD-SCA workflow: A technical validation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4591.

e12571 Background: Chemotherapy-induced cognitive impairment, also known as 'chemobrain', is widely recognized as a frequent adverse effect of chemotherapy, occurs in 10-40% of all cancer patients. Those cancer survivors suffer from poor concentration, memory, abstract reasoning, and motor dysfunction. The etiology is unclear. In our study, we analyzed the metabolite panels in breast cancer patients with vs. without chemotherapy trying to identify metabolic mediators of neurologic injury. Methods: We obtained plasma sample from 18 breast cancer patients, 9 received chemotherapy prior to blood drawn; while the other 9 had no systemic therapy. The plasma samples were sent for mass spectroscopy. Each metabolites level was normalized, and the two groups were compared in each metabolite by t-test with statistical significance corrected for multiple comparisons using the Holm-Sidak method. Results: We identified 57 amines and their metabolites; 106 carbohydrate related metabolites, and 228 lipid molecules. While amino acid, and carbohydrate did not show significant difference, phosphatidylcholine level in the chemotherapy treated group demonstrated lower level in the patients received chemotherapy. Among 59 phosphatidylcholine identified, 6 variants were significantly lower in the chemo group compare with non chemo group. Additionally the sum of all phosphatidylcholine variants was diminished in the chemotherapy treated patients compared with untreated controls. No other differences in plasma lipid levels were identified. Conclusions: Phosphatidylcholine is a major component of cell membranes and lipid rafts which are critical elements in nerve conduction. It also plays a role as the precursor to the neurotransmitter acetylcholine. The finding that phosphatidylcholine is significantly different between chemotherapy treated vs. the no chemotherapy group raises the possibility that lipid metabolism contributes to chemotherapy-induced cognitive impairment and further experiments are planned to explore this hypothesis.

e12554 Background: ERBB2 mutations in the absence of gene amplification are rare, with an incidence of 2-4%. Neratinib is a HER2/EGFR tyrosine kinase inhibitor being evaluated for use in ERBB2 mutated breast cancer. Neratinib has been found to have clinical activity on heavily pre-treated ERBB2 mutant breast cancer patients. We are evaluating the response and genomic profiles of 3 postmenopausal patients with metastatic ERBB2 mutant/non-amplified breast cancer receiving neratinib and fulvestrant NCT01953926, NCT01670877. Methods: Samples were collected at different points during treatment and CTCs were identified. Other representative cells were tracked but not classified as CTCs. CD45-CK+ cells with cytoplasmic and/or nuclear apoptosis were defined as CTC-Apoptotic. CD45- cells expressing little to no CK but otherwise meeting morphological criteria for CTCs are CTC-LowCK. CD45-CK+ cells with small nuclear size are CTC-SmallCK. Single CTCs will be analyzed using whole genome copy number variation to determine chromosomal alterations. Results: The patients had an average of 4.7 lines of therapy for metastatic disease before neratinib. Two had stable disease and one had progression. The patient who progressed had a rise in the number of CTCs from 37 to 52 cells/ml and drop in apoptotic cells from 5 to 0 cells/ml. Conclusions: The high CTC count of the patient who progressed may suggest more aggressive disease. The drop in the apoptotic cell count may correlate with a failure to respond to therapy. Samples have been sent for copy number variation profiling. The goal is to identify any genomic amplifications or deletions associated with clinical response and progression after targeted therapy. We hope to demonstrate the timeframe of tumor evolution in response to therapy and provide a framework for the use of fluid biopsies to monitor disease progression. [Table: see text]

The use of ARMS (amplification refractory mutation system) PCR coupled with RFLP (restriction fragment length polymorphism) analysis has been used to identify a unique genetic marker on the Ambico oral vaccine strain. This method was also used to characterize the genetic profiles of a number of other TGEV strains. This procedure takes advantage of the nucleotide differences between the Ambico strain, and the Miller and Purdue strains. Within the S gene there are three nucleotide differences between the Ambico strain and the published Purdue sequence. There are additional nucleotide dif-ferences in the structural and non-structural gene sequences, but we have chosen to focus on the differences contained within the S gene. The Ambico strain has a closer sequence homology to the Purdue strain than to the Miller strain. The Ambico and Purdue strains contain a six nucleotide deletion at position 1122 that is not present in the Miller published sequence or the ISU-1 strain of PRCV (based on our PCR experiments). We have designed a 5’ oligo whose sequence is homologous to a region located 80 nucleotides upstream of the TGEV and PRCV S gene initiation codons to be used in conjunction with either of two 3’ oligos whose sequences are identical with the exception of the last six nucleotides of their 3’ ends. When utilized with the appropriate PCR conditions, these oligos can differentiate between PRCV, Miller and Purdue prototype virus strains. These PCR products were then subjected to RFLP analysis using four separate restriction enzymes (BstE II, Alw26 I, Dra III, or MspA\ I). We have used this procedure to analyze six TGEV vaccine strains, intestinal derived virulent viruses, cell cultured viruses at different cell passage numbers, and field isolates of TGEV or PRCV.

Porcine respiratory coronavirus(PRCV) is a new variant of TGE with an altered pathogenesis. PRCV multiplies mainly in tonsilar tissues and the respiratory tract. There are no enteric symptoms and in experimentally infected pigs, even the respiratory tract infection is usually asymptomatic. PRCV is spread aerogenically through herds and the significance of PRCV as a pathogen in swine has yet to be determined. Despite the differences in pathogenesis and tissue tropism, the behavior of TGEV and PRCV are closely related antigenically. PRCV induces an antibody response in pigs that cannot be distinguished from TGEV-infected pigs by conventional serological assays. PRCV sensitized animals are not protected from TGEV challenge nor is the milk antibody provided to nursing piglets completely effective in prevention of TGEV infections; thus PRCV is not a good vaccine candidate for TGEV infections. PRCV subclinical infections have led to several reported cases of enzootic TGEV in herds that had been diagnosed as TGEV immune strictly on the basis of serum neutralizing titers which were later found to be due to exposure to PRCV. Vaccination studies conducted with the Ambico, oral modified live TGEV vaccine have led to some startling new results: (1)Use of Ambico TGEV modified live vaccine has been shown to provide complete protection against subsequent PRCV challenge and (2) the effectiveness of TGEV vaccination is actually enhanced by previous exposure to PRCV (3) Weanling pigs which have passively acquired circulating TGEV neutralizing antibodies are protected from subsequent PRCV infections.

Leader RNA is found at the 5′ end of the mouse hepatitis virus (MHV) genomic RNA, at the 5′ end of the seven viral mRNAs, and free in the cell. Leader RNA is synthesized by a transcriptional activity separate from the activities that synthesize both (-) sense genomic length RNA and the virus mRNAs1. It is believed to function as a primer, binding to complementary intergenic sites, on (-) sense template, situated 5′ of each of the initiation sites for viral mRNAs2. Using monoclonal antibodies specific for the nucleocapsid (N) protein, immunoprecipitations of RNA/protein complexes from infected cells indicate that the N protein is complexed to: 1) genomic RNA; Z) viral mRNAs; and 3) even free leader containing RNA fragments as small as 60 nucleotides in length3. Northwestern blot analysis showed that the viral N protein exhibits RNA binding activity that is specific for viral leader containing RNA when expressed in the (+) sense4,5. However, this system has several limitations. First, in denaturing conditions, RNA/protein interactions which require the interaction of multiple protein subunits cannot be studied, and second, it is not possible to quantify relative affinities and binding characteristics.

According to established notion, one of the major angiogenesis-inducing factors, pro-matrix metalloproteinase-9 (proMMP-9), is supplied to the tumor microenvironment by tumor-associated macrophages (TAMs). Accumulated evidence, however, indicates that tumor-associated neutrophils (TANs) are also critically important for proMMP-9 delivery, especially at early stages of tumor development. To clarify how much angiogenic proMMP-9 is actually contributed by TAMs and TANs, we quantitatively evaluated TAMs and TANs from different tumor types, including human xenografts and syngeneic murine tumors grown in wild-type and Mmp9-knockout mice. Whereas host MMP-9 competence was required for full angiogenic potential of both normal and tumor-associated leukocytes, direct comparisons of neutrophils versus macrophages and TANs versus TAMs demonstrated that macrophages and TAMs secrete 40- to 50-fold less proMMP-9 than the same numbers of neutrophils or TANs. Correspondingly, the levels of MMP-9–mediated in vivo angiogenesis induced by neutrophils and TANs substantially exceeded those induced by macrophages and TAMs. MMP-9–delivering TANs were also required for development of metastasis-supporting intratumoral vasculature, characterized by ≥11-μm size lumens and partial coverage with stabilizing pericytes. Importantly, MMP-9–producing TAMs exhibit M2-skewed phenotype but do not express tissue inhibitor of metalloproteinases-1 (TIMP-1), a novel characteristic allowing them to secrete TIMP-1–free, neutrophil-like MMP-9 zymogen unencumbered by its natural inhibitor. Together, our findings support the notion whereby TANs, capable of immediate release of their pre-stored cargo, are the major contributors of highly angiogenic MMP-9, whereas tumor-influxing precursors of macrophages require time to differentiate, polarize into M2-skewed TAMs, shut down their TIMP-1 expression, and only then, initiate relatively low-level production of TIMP-free MMP-9 zymogen.

Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. Our accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article we report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. We have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.