Charissa Kim

Highly multiplexed targeted DNA sequencing from single nuclei

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48–96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5–6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Systemic sarcoidosis first manifesting in a tattoo in the setting of immune checkpoint inhibition

The use of immune checkpoint inhibitors is revolutionising the treatment of cancer. However, their unique toxicity profile is substantially different from what has been observed with traditional chemotherapy, resulting in a novel learning curve for medical oncologists. Early recognition of these toxicities can make a substantial impact in ameliorating these side effects in the oncological and medical–surgical fields. Here, we present a case of Lofgren syndrome sarcoidosis, which first manifested in a tattoo in a patient with metastatic urothelial cancer on therapy with anti-CTLA-4 (ipilimumab) and anti-PD1 (nivolumab).

Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer

Single cell RNA sequencing has emerged as a powerful tool for resolving transcriptional diversity in tumors, but is limited by throughput, cost and the ability to process archival frozen tissue samples. Here we develop a high-throughput 3′ single-nucleus RNA sequencing approach that combines nanogrid technology, automated imaging, and cell selection to sequence up to ~1800 single nuclei in parallel. We compare the transcriptomes of 485 single nuclei to 424 single cells in a breast cancer cell line, which shows a high concordance (93.34%) in gene levels and abundance. We also analyze 416 nuclei from a frozen breast tumor sample and 380 nuclei from normal breast tissue. These data reveal heterogeneity in cancer cell phenotypes, including angiogenesis, proliferation, and stemness, and a minor subpopulation (19%) with many overexpressed cancer genes. Our studies demonstrate the utility of nanogrid single-nucleus RNA sequencing for studying the transcriptional programs of tumor nuclei in frozen archival tissue samples.Single cell RNA sequencing is a powerful tool for understanding cellular diversity but is limited by cost, throughput and sample preparation. Here the authors use nanogrid technology with integrated imaging to sequence thousands of cancer nuclei in parallel from fresh or frozen tissue.

Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing

Triple-negative breast cancer (TNBC) is an aggressive subtype that frequently develops resistance to chemotherapy. An unresolved question is whether resistance is caused by the selection of rare pre-existing clones or alternatively through the acquisition of new genomic aberrations. To investigate this question, we applied single-cell DNA and RNA sequencing in addition to bulk exome sequencing to profile longitudinal samples from 20 TNBC patients during neoadjuvant chemotherapy (NAC). Deep-exome sequencing identified 10 patients in which NAC led to clonal extinction and 10 patients in which clones persisted after treatment. In 8 patients, we performed a more detailed study using single-cell DNA sequencing to analyze 900 cells and single-cell RNA sequencing to analyze 6,862 cells. Our data showed that resistant genotypes were pre-existing and adaptively selected by NAC, while transcriptional profiles were acquired by reprogramming in response to chemotherapy in TNBC patients.

Influence of Surveillance PET/CT on Detection of Early Recurrence After Definitive Radiation in Stage III Non–small-cell Lung Cancer

Background There are few data to support the use of varying imaging modalities in evaluating recurrence in non–small‐cell lung cancer (NSCLC). We compared the efficacy of surveillance positron emission tomography (PET)/computed tomography (CT) versus CT scans of the chest in detecting recurrences after definitive radiation for NSCLC. Materials and Methods We retrospectively analyzed 200 patients treated between 2000 and 2011 who met the inclusion criteria of stage III NSCLC, completion of definitive radiation treatment, and absence of recurrence within the initial 6 months. These patients were then grouped on the basis of the use of PET/CT imaging during postradiation surveillance. Patients who received ≥ 1 PET/CT scans within 6 months of the end of radiation treatment were placed in the PET group whereas all others were placed in the CT group. We compared survival times from the end of treatment to the date of death or last follow‐up using log rank tests. Multivariate analysis was conducted to identify factors associated with decreased survival. Results In the entire cohort, median event‐free survival (EFS) was 26.7 months, and median overall survival (OS) was 41.2 months. The CT group had a median EFS of 21.4 months versus 29.4 months for the PET group (P = .59). There was no difference in OS between the CT and PET groups (median OS of 41.2 and 41.3 months, respectively; P = .59). There was also no difference in local recurrence‐free survival or distant metastases‐free survival between the CT‐only and PET/CT groups (P = .92 and P = .30, respectively). Similarly, in multivariate analysis, stratification into the PET group was not associated with improved EFS (hazard ratio [HR], 0.90; 95% confidence interval [CI], 0.61‐1.34; P = .60) or OS (HR, 1.2; 95% CI, 0.83‐1.7; P = .34). Conclusions In stage III NSCLC patients treated with definitive radiation and without early recurrence, PET/CT scan surveillance did not result in decreased time to detection of locoregional or distant recurrence or improved survival. Micro‐Abstract To determine the optimal surveillance approach in the setting of locally‐advanced non–small‐cell lung cancer (NSCLC) treated with definitive radiation, we compared 2 commonly used strategies, positron emission tomography (PET)/computed tomography (CT)‐based and CT‐based. There were no differences in baseline clinical or treatment characteristics or any of the survival outcomes examined between the 2 groups. These results suggest that routine PET/CT imaging after definitive radiation treatment for locally‐advanced NSCLC confers no benefit in early detection of recurrence.

Selinexor (KPT-330) demonstrates anti-tumor efficacy in preclinical models of triple-negative breast cancer

BackgroundSelinexor (KPT-330) is an oral agent that has been shown to inhibit the nuclear exporter XPO1. Given the pressing need for novel therapies for triple-negative breast cancer (TNBC), we sought to determine the antitumor effects of selinexor in vitro and in vivo.MethodsTwenty-six breast cancer cell lines of different breast cancer subtypes were treated with selinexor in vitro. Cell proliferation assays were used to measure the half-maximal inhibitory concentration (IC50) and to test the effects in combination with chemotherapy. In vivo efficacy was tested both as a single agent and in combination therapy in TNBC patient-derived xenografts (PDXs).ResultsSelinexor demonstrated growth inhibition in all 14 TNBC cell lines tested; TNBC cell lines were more sensitive to selinexor (median IC50 44 nM, range 11 to 550 nM) than were estrogen receptor (ER)-positive breast cancer cell lines (median IC50 > 1000 nM, range 40 to >1000 nM; P = 0.017). In multiple TNBC cell lines, selinexor was synergistic with paclitaxel, carboplatin, eribulin, and doxorubicin in vitro. Selinexor as a single agent reduced tumor growth in vivo in four of five different TNBC PDX models, with a median tumor growth inhibition ratio (T/C: treatment/control) of 42% (range 31 to 73%) and demonstrated greater antitumor efficacy in combination with paclitaxel or eribulin (average T/C ratios of 27% and 12%, respectively).ConclusionsCollectively, these findings strongly suggest that selinexor is a promising therapeutic agent for TNBC as a single agent and in combination with standard chemotherapy.

Cost Analysis of PET/CT Versus CT as Surveillance for Stage III Non–Small-Cell Lung Cancer After Definitive Radiation Therapy

&NA; We estimated and compared the costs of positron emission tomography (PET)/computed tomography (CT) versus CT for surveillance of patients with stage III non–small‐cell lung cancer and identified patient and provider demographic characteristics associated with preference for PET/CT. PET/CT was associated with higher costs for 18 months post‐treatment, but the difference was borderline statistically significant at 24 months. Consistent with national guidelines, PET/CT surveillance was not cost‐saving and did not provide an economic benefit over CT. Introduction: A previous study showed that use of positron emission tomography (PET)/computed tomography (CT) for surveillance after treatment of non–small‐cell lung cancer (NSCLC) does not yield a detection or survival benefit over the use of chest CT. However, PET/CT remains a common method of follow‐up imaging. Here we estimated and compared the costs of PET/CT versus CT for surveillance of patients with stage III NSCLC and identified patient and provider demographic characteristics associated with preference for use of PET/CT. Patients and Methods: We reviewed 178 patients with stage III NSCLC who had received ≥ 1 PET/CT scan within 6 months of completing radiotherapy (n = 89) or had received CT after radiotherapy (n = 89) from 2000 to 2011. Costs were measured according to Medicare payments converted from institutional billing records. Total and imaging costs were analyzed at 6, 12, 18, and 24 months after the end of treatment. Patient and provider demographic characteristics were also evaluated for potential associations with PET/CT use. Results: Total costs in the PET/CT group were higher during the first 18 months after treatment (P = .002 at 6 months, P = .019 at 12 months, and P = .018 at 18 months) but was marginally significant (P = .05) at 24 months. In univariate analysis of demographic variables, patients who lived in a state different from the treatment center might have been more likely to receive PET/CT (odds ratio [OR], 1.76; P = .051). In multivariate analysis, patients treated in 2007 to 2010 (OR, 29.9; P < .001) or 2003 to 2006 (OR, 11.6; P = .002) were more likely to receive PET/CT than patients treated in 1999 to 2002. In addition, radiation oncologists with > 10 years of experience were more likely to use PET/CT than those with less experience, although this result might be confounded by the small number of providers. Conclusion: Use of PET/CT was associated with higher costs for 18 months after treatment, but the difference was at the borderline of statistical significance at 24 months.

Abstract 418: Adaptive resistance to chemotherapy in triple-negative breast cancer revealed by single cell DNA and RNA sequencing

Triple-negative breast cancer (TNBC) is an aggressive subtype that displays extensive intratumor heterogeneity and frequently (46%) develops resistance to neoadjuvant chemotherapy (NAC). Currently, the genomic basis of chemoresistance remains poorly understood. An important question is whether resistance to chemotherapy is driven by the selection of rare pre-existing subclones with genomic mutations and transcriptional programs that confer resistance to chemotherapy (adaptive resistance) or by the spontaneous induction of new mutations and expression changes that confer a resistant phenotype (acquired resistance). To investigate this question we applied single cell DNA and RNA sequencing methods and deep-exome sequencing to longitudinal time-point samples collected from a cohort of 20 TNBC patients. Deep-exome sequencing of the cohort at three time points revealed a random death model, wherein multiple clones were targeted, as opposed to the selection of specific somatic mutations. In contrast, single cell copy number profiling of ~800 cells from 8 patients identified an adaptive resistance model, wherein minor subclones from the pre-treatment tumors were selected and expanded in response to NAC. Similarly, single cell RNA sequencing of ~8000 cells from 8 patients identified subclones with chemoresistant phenotypes that were selected in response to NAC, resulting in the expansion of the resistant tumor mass. These data suggest that chemoresistance evolves through the selection of copy number changes and expression changes in signaling pathways associated with chemoresistance, rather than point mutations. This adaptive resistance model has important translational implications in clinical diagnostics, by suggesting that resistant clones can be detected in TNBC patients prior to the administration of chemotherapy. Citation Format: Charissa Kim, Ruli Gao, Emi Sei, Rachel Brandt, Nicola Crosetto, Theodoros Foukakis, Nicholas Navin. Adaptive resistance to chemotherapy in triple-negative breast cancer revealed by single cell DNA and RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 418. doi:10.1158/1538-7445.AM2017-418

Abstract 5399: Investigating phenotypic plasticity in breast cancer with high-throughput nanogrid single-nucleus RNA sequencing

Single-cell RNA sequencing (RNA-seq) is a powerful tool for investigating rare tumor subpopulations and resolving intra-tumor heterogeneity, but is low throughput, expensive, and requires fresh tissue samples. To address these limitations, we developed a 5’ high-throughput single-nucleus RNA sequencing (SNRS) approach that uses nanogrid technology to perform single-cell imaging and sequencing of 500-2500 nuclei in parallel. The automated image scanning procedure allowed us to exclude doublets and select live cells with DAPI/PI staining. This approach allows the transcriptomic profiling of frozen tissue samples, in which the cytoplasmic membrane is ruptured in cells, but leaves the nuclear membrane intact. We validated SNRS in a breast cancer cell line (SK-BR-3) and compared the transcriptomes of 500 nuclei to 500 whole cells, which revealed a high concordance in the number of genes expressed as well as their expression levels. We also performed bulk RNA-seq of isolated nuclear and cellular fractions from 5 breast cancer cell lines, which showed a high concordance in genes and expression levels. Differentially expressed genes in the nucleus mainly included lincRNAs, pseudogenes and mitochondria genes, but did not affect most cancer genes and pathway analysis. We further applied SNRS to sequence 500 nuclei from a triple-negative breast cancer patient and identified diverse phenotypes in tumor cells, including variation in cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition. These studies demonstrated the technical feasibility of using a nanogrid platform to perform high-throughput single-cell RNA sequencing and showed that nuclei from cell lines and tumors can be used to study signaling pathways and gene networks that play an important role in tumor progression. Citation Format: Ruli Gao, Charissa Kim, Emi Sei, Jie Yang, Leo Chan, Maithreyan Srinivasan, Hong Zhang, Funda Meric-Bernstam, Nicholas E. Navin. Investigating phenotypic plasticity in breast cancer with high-throughput nanogrid single-nucleus RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5399. doi:10.1158/1538-7445.AM2017-5399

Abstract P3-07-01: Selinexor, a selective inhibitor of nuclear export, demonstrates efficacy in preclinical models of triple negative breast cancer

Background: Approximately 15% of all breast cancers are categorized as triple negative (TNBC) for which the only chemotherapy is known to be effective, yet often fails to achieve remission. Nuclear exporter XPO1 (Exportin1 or CRM1) is a promising target for cancer therapy that mediates the transport of multiple tumor suppressors and cell cycle regulators that have been known to be relevant predictors in the mechanism and severity of TNBC. Given the pressing need for novel therapies for this disease, we sought to determine the antitumor effects of selinexor, a novel inhibitor of nuclear export, on triple negative breast cancers in vitro and in vivo as well as to address its mechanism of action. Methods: 26 breast cancer cell lines of different breast cancer subtypes were treated with selinexor in vitro. Using cell proliferation assays the half maximal inhibitory concentration (IC50) was calculated using isobologram curves after 3 days of treatment; sensitivity was defined as IC50 Results: Selinexor demonstrated growth inhibition in all fourteen TNBC cell lines tested; TNBC cell lines were more sensitive to selinexor (median IC50 44nM, range 11 - 550nM), compared to ER+ cells lines (median IC50 of 13000 nM, range of 40nM - > 1000 nM; P=0.017). Treatment with selinexor decreased expression levels of XPO1, as well as survivin and XIAP, and induced apoptosis. In multiple TNBC cell lines selinexor was synergistic with paclitaxel, carboplatin, eribulin and doxorubicin in vitro (median combination index 0.6, range 0.5-0.8). Selinexor as a single agent reduced tumor growth in vivo in 4 of 5 different TNBC PDX models with a median tumor growth inhibition ratio score (T/C) of 48% (range 34-59%) and demonstrated greater antitumor efficacy in combination with paclitaxel or eribulin with an average T/C score of 27% and 12% respectively. Conclusions: Selinexor is a promising therapeutic agent for triple negative breast cancer and it has potential as a combination agent with standard chemotherapy. Citation Format: Paez Arango N, Evans KW, Zhao M, Yuca E, Scott SM, Janku F, Ueno NT, Tripathy D, Kim C, Naing A, Funda M-B. Selinexor, a selective inhibitor of nuclear export, demonstrates efficacy in preclinical models of triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-01.