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Purpose:Imaging mass cytometry (IMC) uses metal-conjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this high-throughput platform to perform an 18-plex assessment of HER2 ICD/ECD, cytotoxic T-cell infiltration and other structural and signaling proteins in a cohort of patients treated with trastuzumab to discover associations with trastuzumab benefit.Experimental Design:An antibody panel for detection of 18 targets (pan-cytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, β-catenin, HER3, MET, EGFR, ERK 1–2, MEK 1–2, PTEN, PI3K p110 α, Akt, mTOR, Ki67, and Histone H3) was used with a selection of trastuzumab-treated patients from the Hellenic Cooperative Oncology Group 10/05 trial (n = 180), and identified a case–control series.Results:Patients that recurred after adjuvant treatment with trastuzumab trended toward a decreased fraction of HER2 ECD pixels over threshold compared with cases without recurrence (P = 0.057). After exclusion of the lowest HER2 expressers, 5-year recurrence events were associated with reduced total extracellular domain (ECD)/intracellular domain (ICD) ratio intensity in tumor (P = 0.044). These observations are consistent with our previous work using quantitative immunofluorescence, but represent the proof on identical cell content. We also describe the association of the ECD of HER2 with CD8 T-cell infiltration on the same slide.Conclusions:The proximity of CD8 cells as a function of the expression of the ECD of HER2 provides further evidence for the role of the immune system in the mechanism of action of trastuzumab.

Circulating tumor cells (CTCs) represent a unique population of cells that can be used to investigate the mechanistic underpinnings of metastasis. Unfortunately, current technologies designed for the isolation and capture of CTCs are inefficient. Existing literature for in vitro CTC cultures report low (6&minus, 20%) success rates. Here, we describe a new method for the isolation and culture of CTCs. Once optimized, we employed the method on 12 individual metastatic breast cancer patients and successfully established CTC cultures from all 12 samples. We demonstrate that cells propagated were of breast and epithelial origin. RNA-sequencing and pathway analysis demonstrated that CTC cultures were distinct from cells obtained from healthy donors. Finally, we observed that CTC cultures that were associated with CD45+ leukocytes demonstrated higher viability. The presence of CD45+ leukocytes significantly enhanced culture survival and suggests a re-evaluation of the methods for CTC isolation and propagation. Routine access to CTCs is a valuable resource for identifying genetic and molecular markers of metastasis, personalizing the treatment of metastatic cancer patients and developing new therapeutics to selectively target metastatic cells.

Introduction: Recent studies have shown that the molecular heterogeneity of HER2 intracellular (ICD) and extracellular (ECD) domains, as well as overall immune infiltration, are associated with response to adjuvant trastuzumab. Traditional strategies for in situ measurement in the tumor microenvironment allow the combination of up to 6 targets, limiting our capability for in-depth interrogation of tissues. Imaging Mass Cytometry (IMC) uses metal-conjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this high-throughput multiplexing platform to perform an 18-plex assessment of HER2 ICD/ECD, cytotoxic T cell infiltration and other structural and signaling proteins in a cohort of patients treated with trastuzumab. Methods: An antibody panel for detection of 18 targets (Pancytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, beta-catenin, HER3, MET, EGFR, ERK 1-2, MEK 1-2, PTEN, PI3K p110 alpha, Akt, mTOR, Ki67 and Histone H3) was conjugated to unique metals for detection in an IMC instrument (Fluidigm). All assays were objectively standardized and validated using quantitative immunofluoresce (QIF). Finally, the IMC technique was validated against HER2 single marker assays by QIF. We used a collection of trastuzumab-treated patients from the HeCOG 10/05 trial (n=180), and identified a case:control series using 5-year recurrence events (n=19), which were matched to controls (n=41) by age and TNM stage. Formalin-fixed, paraffin embedded tissues in tissue microarray format were ablated in the IMC attachment to the CyTOF flow cytometer for simultaneous detection of markers. Image visualization was conducted using MCD Viewer (Fluidigm). Statistical analyses were performed using a range of platforms. Results: Patients that recurred after adjuvant treatment with trastuzumab showed a decreased fraction of HER2 ECD pixels over threshold in a compartment determined by CK and HER2 ICD compared to cases without recurrence (p=0.057). After exclusion of the lowest HER2 expressers (that would have fallen below the threshold for positive by current HER2 assays), 5-year recurrence events where associated with reduced total ECD/ ICD ratio intensity in tumor (p=0.044). Patients below the median for total ECD/ICD ratio showed a trend for decreased benefit from trastuzumab (p=0.066). Levels of cytotoxic T cell infiltration, depicted by total CD8 intensity, were lower in patients with recurrences (p=0.05). Conclusion: Objective measurement of highly multiplexed protein targets in routine, fixed breast cancer tissues shows that a decreased ratio of HER2 ECD/ ICD is associated with 5-year recurrence after trastuzumab treatment. This observation is consistent with our previous work using QIF but represents the first time this has been done on identical cell content (on a single tissue section). Additionally, on the same section we found that lower levels of overall cytotoxic T cell infiltration were associated with worse outcome. Further analysis of the multiplexed data, including both correlative and distance-based analyses are underway. Citation Format: Carvajal-Hausdorf DE, Stanton KP, Patsenker J, Villarroel-Espindola F, Esch A, Montgomery RR, Psyrri A, Kalogeras KT, Kotoula V, Fountzilas G, Schalper KA, Kluger Y, Rimm DL. Multiplexed (18-Plex) measurement of protein targets in trastuzumab-treated patients using imaging mass cytometry [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-18.

In the United States, an estimated 280,000 people will be diagnosed with invasive breast cancer, which has a 5-year survival rate of 27% and 22% in females and males, respectively. Despite the clinical burden of metastasis, the underlying machinery of metastasis remains unclear. This is largely due to a lack of available technologies that can robustly expand patient-derived metastatic cell populations for study ex vivo. One such population is circulating tumor cells (CTCs), which are cells that escape from the primary tumor, intravasate through blood vessel endothelium, and circulate throughout the body. Eventually, CTCs will extravasate at the metastatic site and become the seeds for metastatic colonization. Based on their role in metastasis, CTCs yield great promise for identifying key mechanistic drivers of metastasis. Unfortunately, CTCs exist in low levels within the blood (typically < 100 cells/10mL of blood). Previous methods for culturing CTCs ex vivo have reported low success rates (~ 20%), and often depend on the presence of a high number of CTCs for success. To better interrogate the mechanisms of metastasis, robust methods to propagate CTCs for study are needed. Recently, our lab developed a method using an unbiased selection criterion for isolating and propagating CTCs derived from metastatic breast cancer, yielding 100% success in twelve distinct individuals. This method, based on a density gradient centrifugation, extracts not only CTCs from blood plasma, but also includes potential cancer-associated neutrophils and macrophages. All cultures under our conditions demonstrated growth for > 30 days and were confirmed to have an epithelial and breast phenotype. Notably, 6/12 samples grew longer and were associated with increased CD45 expression using qRT-PCR. We have also recently demonstrated further success in culturing CTCs derived from a variety of other cancers, including colorectal, lung, and pancreatic cancers. This study therefore not only defines a novel technology for expanding CTCs, but also suggests a deficiency in modern isolation methods, which rely on CD45 exclusion, that is limiting culture success in previous methods. Citation Format: Jerry Xiao, Joshua D. Kassner, Kelly Stanton, Richard Schlegel, Paula R. Pohlmann, Seema Agarwal. A robust method for expanding patient-derived circulating tumor cells ex vivo [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 585.

Up to 90% of cancer-associated deaths are due to metastasis. Nonetheless, the mechanisms of metastasis are poorly understood, largely because of a lack of technologies to routinely and robustly isolate, propagate, and culture metastatic cells. Current cellular models for metastasis are hindered by a lack of scalability, a high cost per experiment, and the length of experiments. In this proof-of-principle study, we injected an invasive triple-negative breast cancer cell line, MDA-MB-231, into zebrafish embryos. Cells were monitored for invasion from the yolk sac into the zebrafish circulatory system, representing a cell population analogous to metastatic, circulating tumor cells. Within 5 days, cells had migrated from the primary injection site to the tail. Tails showing migration of cells were excised, digested, and the labeled human-cancer cells were propagated. Re-injection of propagated cells resulted in serial generations (F1 and F2). The time required for migration decreased with each succeeding generation; with the latest (F2) generation migrating to the tail within 24 hours. In addition to being more invasive, cells that migrated to the tail displayed a more mesenchymal phenotype compared to parental cells. Consistent with this finding, RNA-seq analysis showed an upregulation of genes involved in an epithelial to mesenchymal transition. Moreover, the highest differentially expressed genes have been independently associated with metastasis. Finally, in silico analyses demonstrated that the upregulated genes were clinically significant. Together these data provide proof-of-principle that we have established a scalable, rapid, and robust method that can provide routine access to invasive metastatic cells. The platform combines in vivo and in vitro components that can be used to isolate, enrich, and propagate metastatic cells from individual patients, and has the potential to advance basic and translational research in tumor metastasis. This cellular model relies solely on cell behavior and is a non-biased method that can be widely applied to all tumor types as it does not require specific biomarkers. In addition to providing a tool for advancing our understanding of metastases, the technology has the potential to be used in screening for drugs that specifically target metastatic cells; a critical unmet need in the treatment of human cancers. Citation Format: Jerry Xiao, Alex Lekan, Kelly Stanton, Eric Glasgow, Seema Agarwal. A scalable, non-biased screen for isolating functionally metastatic cell populations [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 6129.

Purpose: Imaging mass cytometry (IMC) uses metal-conjugated antibodies to provide multidimensional, objective measurement of protein targets. We used this high-throughput platform to perform an 18-plex assessment of HER2 ICD/ECD, cytotoxic T-cell infiltration and other structural and signaling proteins in a cohort of patients treated with trastuzumab to discover associations with trastuzumab benefit. Experimental Design: An antibody panel for detection of 18 targets (pan-cytokeratin, HER2 ICD, HER2 ECD, CD8, vimentin, cytokeratin 7, β-catenin, HER3, MET, EGFR, ERK 1–2, MEK 1–2, PTEN, PI3K p110 α, Akt, mTOR, Ki67, and Histone H3) was used with a selection of trastuzumab-treated patients from the Hellenic Cooperative Oncology Group 10/05 trial (n = 180), and identified a case–control series. Results: Patients that recurred after adjuvant treatment with trastuzumab trended toward a decreased fraction of HER2 ECD pixels over threshold compared with cases without recurrence (P = 0.057). After exclusion of the lowest HER2 expressers, 5-year recurrence events were associated with reduced total extracellular domain (ECD)/intracellular domain (ICD) ratio intensity in tumor (P = 0.044). These observations are consistent with our previous work using quantitative immunofluorescence, but represent the proof on identical cell content. We also describe the association of the ECD of HER2 with CD8 T-cell infiltration on the same slide. Conclusions: The proximity of CD8 cells as a function of the expression of the ECD of HER2 provides further evidence for the role of the immune system in the mechanism of action of trastuzumab.

Recent years have witnessed intense development of randomized methods for low-rank approximation. These methods target principal component analysis and the calculation of truncated singular value decompositions. The present article presents an essentially black-box, foolproof implementation for Mathworks’ MATLAB, a popular software platform for numerical computation. As illustrated via several tests, the randomized algorithms for low-rank approximation outperform or at least match the classical deterministic techniques (such as Lanczos iterations run to convergence) in basically all respects: accuracy, computational efficiency (both speed and memory usage), ease-of-use, parallelizability, and reliability. However, the classical procedures remain the methods of choice for estimating spectral norms and are far superior for calculating the least singular values and corresponding singular vectors (or singular subspaces).

With the advent of next generation high-throughput DNA sequencing technologies, omics experiments have become the mainstay for studying diverse biological effects on a genome wide scale. ChIP-seq is the omics technique that enables genome wide localization of transcription factor binding or epigenetic modification events. Since the inception of ChIP-seq in 2007, many methods have been developed to infer ChIP target binding loci from the resultant reads after mapping them to a reference genome. However, interpreting these data has proven challenging, and as such these algorithms have several shortcomings, including susceptibility to false positives due to artifactual peaks, poor localization of binding sites, and the requirement for a total DNA input control which increases the cost of performing these experiments. We present Ritornello, a new approach with roots in digital signal processing (DSP) that addresses all of these problems. We show that Ritornello generally performs equally or better than the peak callers tested and recommended by the ENCODE consortium, but in contrast, Ritornello does not require a matched total DNA input control to avoid false positives, effectively decreasing the sequencing cost to perform ChIP-seq.

With the advent of next generation high-throughput DNA sequencing technologies, omics experiments have become the mainstay for studying diverse biological effects on a genome wide scale. Chromatin immunoprecipitation (ChIP-seq) is the omics technique that enables genome wide localization of transcription factor (TF) binding or epigenetic modification events. Since the inception of ChIP-seq in 2007, many methods have been developed to infer ChIP-target binding loci from the resultant reads after mapping them to a reference genome. However, interpreting these data has proven challenging, and as such these algorithms have several shortcomings, including susceptibility to false positives due to artifactual peaks, poor localization of binding sites and the requirement for a total DNA input control which increases the cost of performing these experiments. We present Ritornello, a new approach for finding TF-binding sites in ChIP-seq, with roots in digital signal processing that addresses all of these problems. We show that Ritornello generally performs equally or better than the peak callers tested and recommended by the ENCODE consortium, but in contrast, Ritornello does not require a matched total DNA input control to avoid false positives, effectively decreasing the sequencing cost to perform ChIP-seq. Ritornello is freely available at https://github.com/KlugerLab/Ritornello.

Researchers generating new genome-wide data in an exploratory sequencing study can gain biological insights by comparing their data with well-annotated data sets possessing similar genomic patterns. Data compression techniques are needed for efficient comparisons of a new genomic experiment with large repositories of publicly available profiles. Furthermore, data representations that allow comparisons of genomic signals from different platforms and across species enhance our ability to leverage these large repositories. Here, we present a signal processing approach that characterizes protein–chromatin interaction patterns at length scales of several kilobases. This allows us to efficiently compare numerous chromatin-immunoprecipitation sequencing (ChIP-seq) data sets consisting of many types of DNA-binding proteins collected from a variety of cells, conditions and organisms. Importantly, these interaction patterns broadly reflect the biological properties of the binding events. To generate these profiles, termed Arpeggio profiles, we applied harmonic deconvolution techniques to the autocorrelation profiles of the ChIP-seq signals. We used 806 publicly available ChIP-seq experiments and showed that Arpeggio profiles with similar spectral densities shared biological properties. Arpeggio profiles of ChIP-seq data sets revealed characteristics that are not easily detected by standard peak finders. They also allowed us to relate sequencing data sets from different genomes, experimental platforms and protocols. Arpeggio is freely available at http://sourceforge.net/p/arpeggio/wiki/Home/.

Introduction: For decades, scientists have attempted to develop models for growing and studying primary tumors outside of the human body. Even today, one of the greatest challenges in cancer research has been generation of stable cancer cell lines from primary tumors. Most primary cell cultures, regardless of the method used to generate them, suffer from limited lifespan due to cellular senescence. Cultured cells transformed with SV40 virus large T antigen or over-expression of hTERT has been limited by genomic variation and other changes that vary with the passage number. Recently, a new approach was described by the Schlegel group at Georgetown (Liu et al, AJP 2012) called “conditionally reprogrammed cells” (CRCs). They showed these cells (derived from primary tumors or normal tissue) could be grown indefinitely under defined conditions without using any extrinsic genetic immortalization technique. These cells have been shown to maintain the karyotype similar to the tissue of origin even after prolonged passaging. Here we show that we can obtain confirmatory results at a separate institution and further show that in lung cancer, the CRC lines maintain histotype specific characteristics. Method: The CRC technique requires mouse irradiated J2 cells as feeder cells and a ROCK inhibitor to rapidly grow and expand patient's epithelial cells. We have grown cultures from six tissue types including lung, breast, pancreas, kidney, colon, and salivary gland. All tissue samples were collected by Yale University Pathology Tissue Services and processed in our lab for generating CRC lines. We have characterized lung CRC lines for morphology and immunophenotyping using H&E stains and histological markers including CK7, p63 and TTF1. We have also performed gene expression profiling using Illumina bead arrays on selected lung and pancreatic samples. Results: We have successfully established CRC lines from all six tissue types. Our success rate exceeds 70% for lung and breast tumor samples while other tissues show somewhat lower rates. Even traditionally challenging cells to culture, like pancreatic and salivary gland tumors have been successfully cultured. Preliminary morphological and histological analysis on lung CRC lines show that features of lung adenocarcinoma (ADCA) and lung squamous cell carcinoma (SQCC) are maintained in CRC lines. Gene expression profiling shows that lung CRC lines from SQCC samples clustered together and are separate from ADCA. Profiles from a pancreatic CRC line recapitulated the profile of the corresponding frozen tissue. Conclusion: This novel CRC technology represents a new approach to culturing cells from tumors and normal cells, including tumors that have been historically challenging to grow. The resulting cells appear highly similar to the tumors from which they were derived. Future studies are needed to further characterize the CRC system and to test its value for assessment of personalized drug sensitivity. Citation Format: Seema Agarwal, Joanna Hu, Kelly Stanton, Kurt Schalper, Yuval Kluger, Elizabeth Zarrella, Xuefeng Liu, Richard Schlegel, David L. Rimm. Next generation cell line models: conditionally reprogrammed cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1569. doi:10.1158/1538-7445.AM2013-1569