Expression Profiling of Circulating Tumor Cells

Institution: University of Southern California
Investigator(s): Julie Lang, M.D. -
Award Cycle: 2013 (Cycle 19) Grant #: 19IB-0112 Award: $244,591
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2013)

Non-technical overview of the research topic and relevance to breast cancer:
The spread of cancer to distant sites, known as metastasis, is responsible for virtually all breast cancer related deaths. Detection of circulating tumor cells (CTCs) has been demonstrated to be prognostic in both metastatic and non-metastatic breast cancer. Our study uses a highly innovative method to isolate pure CTCs, allowing for detailed molecular descriptions of these rare cells. Our research group has recently discovered a method for the isolation and characterization of CTCs in breast cancer. These rare cancer cells in the blood (CTCs) appear to be precursors of metastatic disease. We wish to identify and target them before they have advanced to the point that they may be found through imaging or physical examination. Cancers are highly variable - although sophisticated technology exists to classify primary tumors, primary tumor biology does not reveal the entire story as to why some patients recur and others do not. This is likely because measurable indications of disease, known as biomarkers, of the non-aggressive disease within a bulk tumor dilute important markers for aggressive disease.

The question(s) or central hypotheses of the research:
We hypothesize that molecular profiling (identifying molecular patterns or signatures) of CTCs may be a clinically useful biomarker to predict response to neoadjuvant chemotherapy in Stage II-III breast cancer. Neoadjuvant chemotherapy (NC) is defined as chemotherapy given prior to surgery.

The general methodology:
Our CTC isolation strategy involves separating rare cancer cells from millions of red and white cells found in blood. We propose to study whether sequencing the molecular patterns of CTCs from non-metastatic breast cancer patients may provide clinically relevant information that could potentially help guide patient treatment. Sequencing involves collecting all the available genetic information that describes which genes are turned on or off in cells. We propose to conduct a prospective study enrolling 120 Stage II-III newly diagnosed breast cancer patients to screen for CTCs. Our innovative method allows us to separate pure cancer cells from peripheral blood, asking patients to undergo a minimally invasive blood draw of 2 small tubes (20 milliliters) of blood. Our primary objective is to determine if CTC profiling can predict for complete tumor eradication after neoadjuvant chemotherapy. This is determined by comparing CTC signatures to pathology results at the time of definitive cancer surgery. We will also look for evidence of stem cell markers or markers indicating that CTCs may or may not be actively dividing cells. We will use two independent technologies to verify our CTC signature results are correct.

Innovative elements of the project:
To our knowledge, this is the first such method permitting successful capture of CTCs suitable for profiling pure CTCs across many thousands of genes (the whole genome) without bias from contaminating white cells. We will carry out a series of studies in order to better understand the relationship of CTCs to their primary tumors and understand whether CTCs may be used as a surrogate for metastasis. Our study uses a highly innovative method to isolate pure CTCs, allowing for detailed molecular characterization of these rare cells. Such profiling allows us to learn which genes are active in CTCs. We will test several important hypotheses to gain a better understanding of the biology of CTCs. This proposal will have a potential impact on the eradication of breast cancer by determining if CTC gene profiling may play a useful clinical role in predicting for response to NC. This type of approach has tremendous potential for personalized medicine, allowing for a variety of molecular approaches never before possible. My laboratory seeks to move the field beyond counting circulating tumor cells to a more detailed molecular understanding of how circulating tumor cells fit into the process of metastasis and how to target these cells before clinically detectable metastases occur. If successful, detailed molecular profiling of patientsí CTCs may potentially benefit all stages of breast cancer patients as an opportunity to assess in real time the current state of a patientís tumor biology and explore rational therapeutic strategies and novel targets for treatment.




Final Report (2015)

Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer (BC), yet few studies have examined their molecular biology in non-metastatic BC. We have previously reported a method for the isolation and gene expression profiling of pure CTCs that permits gene expression profiling without background subtraction of leukocytes. We hypothesized that transcriptional profiling of CTCs prior to therapy may predict for pathologic complete response (pCR) to neoadjuvant chemotherapy (NC) in stage II-III breast cancer.

Currently we are enrolling patients to a prospective, observational clinical study in which CTCs are enumerated and captured from 10- 20ml of peripheral blood (PB) via immunomagnetic enrichment based on EpCAM followed by fluorescence-activated cell sorting (IEFACS). CTCs and tumors are profiled with RNA Sequencing (RNA-Seq) via Illumina HiSeq (primary predictor); NanoString PAM50 and quantitative real-time polymerase chain reaction will be used as validation studies.

To date, we have isolated CTCs from 29/33 patients (88%). No CTCs were found in 23 healthy controls. The median number of CTCs isolated was 7 (range 0-65). Currently 16/33 patients had NC and 17 had no NC; 11/13 patients had CTCs isolated and 6/10 patients had a pCR. We have performed RNA-Seq on 20 patientís CTCs, their corresponding peripheral blood, and 5 primary tumors. RNA-Seq results from those 20 patients showed a total of 253 differently expressed genes (fold change of at least 2, false-discovery rate adjusted p<0.001) between CTCs and PB. Only 6 genes were found to be in common between CTCs and peripheral blood. A gene set enrichment analysis on the first 17 CTC samples revealed up-regulation of cancer related pathways (p<0.001). RNA-Seq results from primary tumors showed 230 genes differentially expressed among CTCs vs primary tumor samples p<0.001; fold change =2.

RNA Seq of rare CTCs is feasible in stage II-III breast cancer even without background subtraction of leukocytes using IE/FACS, and shows evidence of oncogenes and tumor suppressor genes. RNA Seq of 20 patientsí CTCs shows clear differentiation between CTCs and PB. RNA Seq and validation studies for additional CTC and additional tumor samples are currently in progress. This pilot study demonstrates that gene expression profiling of CTCs with RNA Seq is a feasible, may predict response to therapy, and may identify potentially actionable targets for therapy.




Conference Abstract (2016)

Molecular Profiling of Circulating Tumor Cells In Non-metastatic Breast Cancer

Dany Barrak1,2,Victoria Forte1,2, Weizhu Zhu1,2, Alexander Ring1,2, Vasu Punj3, Debu Tripathy4, Min Yu2,4, Tania Porras1, Akshara Raghavendra1, Gloria Yang1, Julie E. Lang1
1 Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
2 Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA
3 Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA
4 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
5 Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA

Background: Circulating tumor cells are prognostic in all stages of breast cancer (BC), yet few studies have examined their molecular biology in non-metastatic BC. We have previously reported a method for isolation and gene expression profiling of pure CTCs that permits gene expression profiling without background subtraction of leukocytes. We hypothesized that transcriptional profiling of CTCs prior to therapy may predict for pathologic complete response (pCR) to neoadjuvant chemotherapy (NC) in Stage II-III breast cancer.

Methods: We are currently enrolling patients to a prospective, observational clinical study in which CTCs are enumerated and captured from 10-20 mL peripheral blood (PB) via immunomagnetic enrichment based on EpCAM followed by fluorescence-activated cell sorting (IE-FACS). CTCs and tumors are profiled with RNA Seq via the Illumina HiSeq (primary predictor); NanoString PAM50 and real-time polymerase chain reaction will be used as validation studies.

Results: To date, we have isolated CTCs from 29/33 patients (88%). No CTCs were found in 23 healthy controls. The median number of CTCs isolated was 7 (range 0-65). We will analyze our primary endpoint when n=20 and n=40 NC treated patients. Currently 12/33 patients had NC and 21 had no NC; 10/12 patients had CTCs isolated and 5/10 patients had a pCR.

RNA Seq of the first 17 patients CTCs shows clear differentiation between CTCs and PB with 253 differentially expressed genes with a fold-change of at least 2 (false-discovery rate adjusted p<0.001). A gene set enrichment analysis of the 17 CTC samples demonstrated up-regulation of cancer related pathways (p<0.001). RNA Seq and validation studies of additional CTC and tumor samples is currently in progress

Conclusion: RNA Seq of rare CTCs is feasible in Stage II-III breast cancer and shows evidence of oncogenes and tumor suppressor genes with differential expression.



Expression profiling of circulating tumor cells in metastatic breast cancer.
Periodical:Breast Cancer Research and Treatment
Index Medicus: Breast Cancer Res Treat
Authors: Lang JE, Scott JH, Wolf DM, Novak P Punj V et al.
Yr: 2015 Vol: 149 Nbr: 1 Abs: Pg:121-131

EpCAM Based Capture Detects and Recovers Circulating Tumor Cells From All Subtypes of Breast Cancer Except Claudin-low. doi: 10.18632/oncotarget.5977
Periodical:Oncotarget
Index Medicus:
Authors: A Ring, N Mineyev, W Zhu, E Park, C Lomas, V Punj, M Yu, D Barrak, V Forte, et al.
Yr: 2015 Vol: 6 Nbr: 42 Abs: Pg:44623-34