Circulating Tumor Cells

Isolation and Analysis

Z. Hugh Fan, Mark F. Vitha, Z. Hugh Fan

• Publisher: Wiley
• Year: 2016
• ISBN:
  9781119244547
• Edition: 1
• Topic: Physical Sciences
• Subtopic: Analytic Chemistry

Introduces the reader to Circulating Tumor Cells (CTCs), their isolation method and analysis, and commercially available platforms

• Presents the historical perspective and the overview of the field of circulating tumor cells (CTCs)
• Discusses the state-of-art methods for CTC isolation, ranging from the macro- to micro-scale, from positive concentration to negative depletion, and from biological-property-enabled to physical-property-based approaches
• Details commercially available CTC platforms
• Describes post-isolation analysis and clinical translation
• Provides a glossary of scientific terms related to CTCs

Part I
Introduction

Chapter 1
Circulating Tumor Cells and Historic Perspectives

Jonathan W. Uhr

Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA

This introduction reviews the history of research on circulating tumor cells (CTCs) with brief reviews of the present opportunities and challenges using CTCs for diagnostic and treatment decisions.

1.1 Early Studies on Cancer Dormancy Led to the Development of a Sensitive Assay for CTCs (1970–1998)

Prior to my involvement in the development of the first Food and Drug Administration (FDA)-approved capture device for CTCs, our laboratory's research was involved with investigations of the signaling pathways in B lymphocytes. We were studying the mechanisms underlying induction of replication, differentiation, apoptosis, and cell cycle arrest when an important event took place at Stanford University that affected our future plans. In 1980, Slavin and Strober [1] isolated the first murine B-cell lymphoma that spontaneously arose in a BALB/c mouse and allowed us to propagate the tumor. We found that an antibody to the tumor immunoglobulin [anti-idiotype (Id)] injected into the tumor-bearing mice could induce a state of cancer dormancy [2] by its ability to induce cell cycle arrest and apoptosis (antibodies are immunoglobulins; each antibody has a unique antigenicity, so it can stimulate production of an antibody to it, that is, an antibody to an antibody called an anti-idiotypic antibody). Dormancy could last up to 2 years. The population of dormant lymphoma cells in the spleen was stable for the 210 days of observation, although a subpopulation of tumor cells were replicating; loss of dormancy occurred at a steady rate during the 2 years of observation; and, about 90% of the lymphoma cells that were now replicating remained Id+ [3–6]. However, the majority of such antibodies had undergone minor changes that made them Id+ variants with decreased or no susceptibility to anti-Id-mediated induction of dormancy. With time, these lymphoma cells regained their full malignant potential; as few as three of these cells transferred progressive tumor growth to syngeneic recipients [6]. Thus, anti-Id suppresses the malignant phenotype, observed in control mice that do not receive anti-Id, by signal transduction mechanisms that override the genetic lesions that cause neoplasia. In searching for potential signaling molecules, alterations in Syk, Lyn, and HS1 were suggested by either the loss of an epitope recognized by a monoclonal antibody or the loss of functional kinase activity [5]. Significant advances in characterizing cancer dormancy followed from studies by Meltzer [7], Stewart [8], and Demichelli [9].

At the same time, the Stevensons reported impressive results of anti-Id therapy of murine B-cell tumors [10, 11], and Levy obtained groundbreaking results in treating patients with B-cell lymphomas with anti-Id serum [12, 13]. Prior conventional treatment could induce long-term remission, but dormant lymphoma cells were not eliminated and virtually all patients eventually died of the disease. Levy treated such patients with anti-Id and the vast majority went into long-term remissions even though tumor cells remained [14]. As with the B-cell tumor lymphoma 1 (BCL1) mouse tumor model, the clinical studies also indicated that the antitumor effect of anti-Id and several other B-cell reactive antibodies related to their ability to act as agonists rather than conventional effector antibodies. Levy's results initiated a burst of research into immunological methods for cancer treatment, which continue to the present day. There are currently over 30 immunologically based drugs in clinical trials to treat patients with various cancers.

These observations in human B-cell lymphoma and in the BCL1 mouse tumor model – that long-lasting tumor dormancy can be induced by antibodies to the tumor immunoglobulin in the face of persisting tumor cells, a portion of which are replicating in the BCL1 tumor – gave us the impetus to study clinical dormancy. We were interested in analyzing those tumor types in which metastases can develop many years after primary tumor removal even though the patient has appeared clinically disease-free. Breast cancer was an excellent example because of the prevalence of the disease and because recurrences occur at a steady rate from 7 to 20 years after mastectomy in patients who appear well [9, 15]. To study the tumor cell dynamics in clinically healthy humans, we needed a relatively noninvasive procedure, namely, a very sensitive blood test to determine whether CTCs were present in a proportion of these patients. However, the methods under development were insensitive, could not be quantified, frequently gave false positives, and were impractical for the challenge.

The history of CTCs began in 1869, when Ashworth [16] described cells in the blood that appeared similar to those observed in the tumor at autopsy. In the mid-twentieth century, there were many claims that CTCs as determined by cytology were commonly seen in cancer patients. However, further studies indicated that hematopoietic cells, particularly megakaryocytes, were responsible for almost all of these results [17] and such studies were then abandoned. However, there were positive exceptions. Drye et al. [18] had convincing evidence that the presence of cancer cells in peripheral blood of 17 patients related to their clinical progress. Engell [19] claimed to have found cancer cells in venous blood draining the tumor as well as in peripheral blood.

Beginning in the 1970s, experimental models were developed to study the events that led to metastases [20, 21]. Metastases had been generally regarded as a late event in the development of epithelial tumors. However, the poor prognosis of patients with clinically localized lung cancer suggested that micrometastases may have taken place before the primary tumor was diagnosed. Pantel et al. [22, 23] searched for tumor cells in the bone marrow by immunohistochemistry in patients with breast, gastrointestinal tract, or non-small-cell lung carcinomas with and without evidence of metastases. In the majority of lung cancer patients without metastases, cytokeratin-positive cells were detected at significant concentrations, whereas they were rarely found in controls. The authors concluded that early dissemination of isolated tumor cells is a frequent occurrence in non-small-cell lung carcinomas; in breast and gastrointestinal carcinomas, the majority of these disseminated cells in the bone marrow were in a dormant state. In a series of pioneering experiments, Folkman et al. [24, 25] defined a critical role for angiogenesis in the metastatic cascade. Liotta et al. [26] studied mechanisms of metastases and focused on the role of angiogenesis. The primary tumor must first develop an adequate vascular supply. This is achieved by balancing angiogenesis-promoting and -inhibiting factors released by tumor cells, inflammatory cells, and extracellular matrices. Although there were problems with the methods for quantification of angiogenesis, the results indicated that increased angiogenesis within the primary tumor resulted in a worse prognosis.

Using a fibrosarcoma model, Liotta also quantified some of the major processes that occur following transplantation of tumor into the leg muscle of a mouse and the subsequent rapid development of pulmonary metastases [27]. He showed that by about Day 4, a vascular network first appeared in the peripheral regions of the tumor and grew throughout the tumor mass by Day 10. Following intratumoral perfusion (using a solution of human hemoglobin, calf serum, Eagle's medium, amino acids, glucose, and insulin), fibrosarcoma tumor cells were detectable in the draining venous blood vessels by Day 5. The number of these cells in the blood stream, both as single cells and tumor clumps (2–30 tumor cells, comprising about ...

Table of contents

• Cover
• Title Page
• Copyright
• Table of Contents
• List of Contributors
• Foreword
• Preface
• Part I: Introduction
• Part II: Isolation Methods
• Part III: Post-Isolation Analysis and Clinical Translation
• Part IV: Commercialization
• Part V: Glossary
• Index
• End User License Agreement