OVERVIEW OF CIRCULATING TUMOR CELLS
Circulating tumor cells (CTCs) were first identified in 1869 by Ashworth, a resident physician who noted that a 38-year-old man who died with metastatic tumors had similar cells within his blood through simple microscopic examination.1 Despite the explosion in our knowledge of cancer biology in recent years, CTCs have only recently become a regularly utilized tool in clinical research. Despite the availability of an US Food and Drug Administration (FDA)-cleared CTC test in breast, prostate, and colorectal cancers, these tests are currently used largely in a research setting as there are no established guidelines for incorporating CTC enumeration testing into clinical practice.2 The utility of CTC testing will be in its incorporation into predictive biomarker testing, which is currently being explored.2
An important distinction should be made between CTCs and cell-free DNA (cfDNA). CTCs and cfDNA are both types of liquid biopsy, or tumor components detectable in blood. Although CTCs are whole single cells and cell clusters released from tumors into the blood, cfDNA is derived from apoptosis and necrosis of tumor cells. cfDNA may be released early in tumor development and may assess a different tumor compartment than CTCs.3 These techniques may be complementary in assessing molecular alterations and metastatic potential.4 This review will focus on the relevant aspects of CTCs that pathologists should be aware of and how CTC technologies may benefit the analysis of small pathology specimens, with a particular focus on cytology.
OVERVIEW OF TERMINOLOGY AND CIRCULATING TUMOR CELLS PLATFORMS
CTC enumeration refers to simply counting the number of CTCs, whereas CTC enrichment enhances the proportion of CTCs in a specimen as compared with the number of nontumor cells [predominantly white blood cells (WBC)]. Much of the established literature and guidelines relating to CTCs focuses on enumeration, whereas more recent literature looks toward enrichment and characterization of CTCs (Fig. 1). The review by Ferreira et al,5 is recommended for a detailed explanation of the range of CTC technologies and their technical aspects.5 Briefly, immunoaffinity strategies with positive enrichment, such as EpCAM-based enrichment, may concentrate CTCs relative to WBCs. Of these methods, only the CellSearch method is currently FDA-cleared for clinical use for enumeration in the setting of metastatic breast, prostate, and colorectal cancers.6 Note that all other uses of CTC technology are considered investigational from a regulatory perspective. The drawback of positive enrichment is that cells expressing the enrichment target at low levels may be missed, resulting in low CTC capture efficiency.5 Immunoaffinity strategies with negative enrichment may yield higher capture efficiency, but may be less enriched.
Techniques based on physical properties of CTCs, such as deformability, buoyancy, size (by filtration or microfluidic techniques such as inertial focusing) and chemical composition (dielectrophoresis) are less commonly used and must be thoroughly validated, but may provide good capture efficiency and enrichment of CTCs.5 Multiparameter immunofluorescent imaging is usually utilized following enrichment for verification of the enrichment (most frequently identifying cytokeratin-positive, CD45-negative nucleated cells); however, some early systems were based solely upon morphologic detection. In addition, direct imaging of CTCs without enrichment is a possible method when computational methods are used to identify CTCs among the background blood components, with a large number of cells analyzed.5 Isolation or enrichment for subsequent testing may be less straightforward with direct imaging but may be possible using laser microdissection or other techniques. The use of an EpCAM antibody-coated wire inserted into a vein for 30 minutes may provide a potential alternative to drawing blood for CTC enumeration.7 Development of similar devices for clinical use may reduce the concern for iatrogenic anemia due to repeated blood draws in cancer patients. Using a multiparameter fluorescence CTC imaging technique, CTC yield depends both on the type of collection tube used and the time since collection (24 vs. 72 h).8 Further study on preanalytic variables in CTC collection is needed.
PITFALLS OF EpCAM-BASED CIRCULATING TUMOR CELLS TECHNIQUES
Although CellSearch and other EpCAM-based CTC technologies are popular and well-validated, they are likely to be ineffective in cancers with low or variable EpCAM expression. Variation in the expression of EpCAM may account for differences in CTC enumeration between platforms.9 In particular, papillary thyroid carcinoma may produce CTCs with very low or undetectable levels of EpCAM.10 Immunoaffinity systems may increase yield by utilizing multiple antigens concurrently for CTC enrichment. Jackson et al,11 critically review current label-free microfluidic-based CTC enrichment techniques in terms of capture efficiency and enrichment. Of particular interest for enrichment and subsequent study of viable CTCs is the “catch and release” concept, in which CTCs can be isolated and subsequently released by digestion of the affinity agent, cleavage of a specific linker between the affinity agent and the surface of the device or manipulation of polymer coatings.11
Many CTCs bear morphologic similarities to the tumor cells seen in cytologic preparations. The morphologic criteria that have been proposed to identify CTCs include12:
- Anisonucleosis (ratio from one nucleus to another of >0.5)
- Nuclei larger than 3 times the calibrated pore size (pore: 8 μm; nucleus>24 μm)
- Irregular nuclei
- Presence of 3-dimensional sheets
- High nuclear/cytoplasmic ratio
These criteria are particularly used in filtration-based CTC enrichment methods. Endothelial cells have overlapping morphologic features with CTCs, and putative CTCs identified by morphologic criteria alone in renal cell carcinoma patients did not show similar chromosomal aberrations by comparative genomic hybridization array as compared with the primary tumors.13 Morphologically, CTCs by CellSearch criteria should have appropriate nuclear 4’,6-diamidino-2-phenylindole and cytoplasmic keratin immunofluorescent signals (Fig. 2), lack CD45 cytoplasmic staining, show cell-like morphology and meet a minimum size requirement.14 CTC morphology is usually similar among carcinomas of different primary sites (eg, breast, prostate, colorectal, or lung carcinomas). In one study of CTCs in patients with breast, colorectal, and prostate cancers, automated image analysis and manual morphologic assessment achieve similar quantitative results.14
In comparison to cultured prostate cancer cell lines, CTCs from patients with prostate cancer show elongated shapes and increased nuclear: cytoplasmic ratios.15 CTCs from patients with prostate cancer may be significantly smaller than cultured prostate cancer cell lines.16 An in vitro study suggests that minimal artifacts are introduced by CTC processing.17 In lung adenocarcinoma, morphologic details of CTCs were sufficient to distinguish adenocarcinoma from squamous cell carcinoma in ∼60% of patients with histologically confirmed lung carcinomas.18 Cultured tumor cells subjected to CTC processing and enrichment via the CellSearch Profile kit are overall well-preserved with some degradation of nuclear detail in cytologic preparations.17
Although morphology is one mechanism to identify CTCs, immunofluorescent phenotyping, especially when using multiple antigens for characterization, provides greater specificity and is amenable to automation. Therefore, it is the method that most CTC systems use for CTC verification.
CIRCULATING TUMOR CELL CLUSTERS AND BIOLOGICAL MECHANISMS OF METASTASIS
A growing body of evidence indicates that CTC clusters may confer an increased risk of metastasis compared with single CTCs. Experiments in mice implanted with fluorescently labeled breast carcinomas show that clusters are more likely to give rise to metastases than single cells.19 In human studies, survival is decreased in breast carcinoma patients with multiple occurrences of CTC clusters.19 The CellSearch platform does not currently allow for separate enumeration of CTC clusters compared with single CTCs, which may be somewhat of an impediment to research on CTC clusters. Epithelial-to-mesenchymal (EMT) transition and cell-cell adhesion may play a critical role in formation and maintenance of CTC clusters. CTC clusters express increased EMT markers such as Snail, Twist, and the tranforming growth factor-β pathway, as well as the desmosome component plakoglobin.19,20 It has been shown that CTC clusters can traverse capillary-sized spaces in a microfluidic channel as well as capillaries in zebrafish embryos, and that they do so in a single-file arrangement.21 Thus, CTC clusters would not be limited to metastasizing to the first small caliber vessel they reach, but may have the opportunity to continue through the circulation to reach favored sites.
RATIONALE FOR PATHOLOGIST INVOLVEMENT IN CIRCULATING TUMOR CELL ANALYSIS
To assure patient safety and appropriate test utilization, pathologists should be involved in the clinical implementation of CTC testing. Regulating preanalytic and analytic factors in CTC testing is of key importance so that morphologic detail and molecular biomarkers are well-preserved, and the sensitivity and specificity of the test are appropriately balanced.22 We also should take an active role in integration of potentially discordant data from the analysis of CTCs as well as biopsies from primary tumors and metastatic sites.22 The advantages of adopting CTC technology in pathology extend beyond the testing of peripheral blood specimens, as will be discussed at the end of this review.
EVIDENCE FOR UTILITY OF CIRCULATING TUMOR CELLS BY PRIMARY TUMOR SITE
CTCs have been well studied in clinical trials in a variety of cancers. This section summarizes recent literature on CTCs by individual primary tumor site, with a focus on recent systematic reviews and meta-analyses.
CTC enumeration is a well-established prognostic indicator in metastatic and clinically localized breast carcinoma in treatment naive and refractory patients.23 A recent systematic review of biomarkers in breast carcinoma identified 181 articles involving cells within the blood, of which 156 addressed CTCs and the majority (154, 98.7% of all CTC articles) included data on CTC enumeration.24 In total, 29 were observational studies, whereas 2 were clinical trials. Gene and protein expression within CTCs are frequent targets of study. None of the studies performed health economic analyses of CTCs, and this was identified as a major barrier to the adoption of CTCs as a biomarker.24 Many methods to assess estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) status in breast cancer CTCs have been developed; although no standard assay has been established. CTCs may better represent tumor heterogeneity than sampling of a single tumor site and provide a method for continuous, real-time monitoring of tumor in response to treatment.23 More specifically, a systematic review by Wang et al,25 suggests that HER2-positive CTCs may be associated with worse overall survival. In ∼10% to 30% of HER2-negative primary tumors, HER2-positive CTCs were detected.25 The authors suggest that patients with HER2-positive CTCs may benefit from more aggressive or targeted HER2 therapy, even if the primary tumor or sampled metastases are HER2-negative.25 Additional studies will be needed to extend established HER2 guidelines to the potential testing of HER2 in CTCs. Clinical trials to evaluate the efficacy of anti-HER2 therapy in patients with CTCs are ongoing (TREAT-CTC, DETECT III).26,27
CTC enumeration is also a prognostic indicator in prostate carcinoma. Two systematic reviews find that many studies have correlated CTCs with worse overall survival.28,29 This correlation is seen in metastatic and localized disease and in the context of castrate-resistant and hormone-sensitive tumors.30 A recent study shows that gene expression testing of CTCs for a panel of genes associated with seminal vesicle invasion and lymph node metastasis predicts a group with decreased overall survival and progression-free survival, and potential resistance to androgen deprivation therapy.31 A study by Scher et al32 found that the presence of androgen receptor variant 7 (ARv7) expressing CTCs before initiating therapy correlated with worse overall survival and predicted response to taxanes, but not androgen receptor signaling inhibitors. It will be of interest to observe how the potential for CTC-based prediction and monitoring evolves as prostate specific antigen screening becomes less prevalent and as active surveillance of prostate carcinoma increases.
The third primary tumor site for which CTC enumeration has been FDA-cleared is colorectal carcinoma. In a meta-analysis including only the CellSearch method, CTCs have been associated with both worse overall survival and increased risk of liver metastasis.33 Likewise, a meta-analysis of colorectal cancer studies in patients with liver or widespread metastasis showed worse overall and progress-free survival in patients with CTCs.34 Testing for microsatellite instability has been successfully performed in CTCs and suggests that CTCs may develop microsatellite instability when the primary tumor was previously found to microsatellite stable.35
Meta-analyses of both small cell lung carcinoma and non–small cell lung carcinoma (NSCLC) show that the presence of CTC is correlated with reduced overall survival.36–38 In NSCLC, the presence of CTCs is additionally correlated with increased risk of lymph node metastasis.37 CTCs may be particularly useful in monitoring disease progression and resistance to targeted therapies. In a study of NSCLC patients who progressed on anti-EGFR therapy, EGFR T790M mutations were identified in CTCs in 70% of patients, and the combination of testing of CTCs and cfDNA allowed a result in 35% of patients with negative or indeterminate biopsy results.39 Another study performed repeat CTC testing in NSCLC patients over the course of radiation therapy and found that fluctuations in CTC count may be useful in assessing treatment response.40 CTC testing may facilitate early detection of NSCLC. A small prospective study of chronic obstructive pulmonary disease patients at risk for developing NSCLC found that all 5 patients (3% of those screened) who had CTCs at the time of screening developed NSCLC within 4 years of testing.41 Larger trials to confirm these findings are ongoing.
Head and Neck Squamous Cell Carcinoma
CTC-positive patients with head and neck squamous cell carcinoma had an increased risk of disease progression, which was independent of tumor stage and nodal status.42 The human papillomavirus (HPV) status of these tumors was not specified and would be of particular interest given the difference in prognosis and management between HPV-related and non–HPV-related head and neck squamous cell carcinoma. HPV testing has been successful in uterine cervical squamous cell carcinoma CTCs and may be helpful for early diagnosis and in treatment decisions for head and neck squamous cell carcinomas.43
Thyroid carcinoma CTCs have been less straightforward to study due to their potentially low EpCAM expression.10 No systematic reviews or meta-analyses of CTCs in thyroid carcinoma patients were identified upon searching in PubMed or Web of Science. In patients with papillary thyroid carcinoma, the combination of postsurgical serum thyroglobulin surveillance and CTCs detected by EpCAM as well as TSHR expression separated patients with metastatic disease from those without.44 CTCs may also provide a useful substrate for BRAF V600E testing.45 As concern for thyroid carcinoma overtreatment grows, CTCs may provide a minimally invasive way to predict which patients may benefit from additional workup and more aggressive therapy, such as radioactive iodine treatment.
In a meta-analysis of urothelial carcinoma studies, the CTCs correlate with increased clinical stage, suggesting that CTCs may be useful to gauge the adequacy of clinical staging.46 However, overall survival and progression-free survival were reported in too few studies to be analyzed.46 A clinical trial is ongoing to evaluate a telomerase-based CTC assay.47
Of note, melanoma does not typically express EpCAM; therefore, positive selection for melanoma CTCs uses different antigens than those for carcinoma, but CTC methods based on physical properties or negative selection do not require modification. One study utilized melanoma-associated chondroitin proteoglycan for immunomagnetic enrichment of CTCs.48 The detection of CTCs in melanoma correlated with disease stage as well as worse overall survival.49,50 BRAF V600E testing has been accomplished in melanoma CTCs, which could expedite targeted therapy in melanoma.51 However, because BRAF V600E has been identified in a growing number of benign and malignant neoplasms, such results will require appropriate pathologist interpretation and clinical correlation.
Pitfalls in Systematic Reviews/Meta-Analyses of Circulating Tumor Cell Studies
Of note, a number of systematic reviews failed to separate the CTC enumeration/enrichment method (eg, CellSearch profile kit) from specific biomarker detection methods that do not identify individual CTCs (eg, real-time polymerase chain reaction).25,37,52–54 Many older studies utilized nucleic acid-based methods, but it is important to distinguish whether the substrate is truly enriched CTCs through a validated method, nucleated cell fractions of blood or whole blood itself. Preparations may contain varying amounts of CTCs, cfDNA, and background WBC and other blood components, which may complicate quantitative analyses and reduce the sensitivity of sequencing-based methods if the proportion of true CTCs is low.
PRECISION MEDICINE AND OTHER APPLICATIONS OF CIRCULATING TUMOR CELLS BEYOND ENUMERATION
In era of precision/personalized medicine, CTCs represent a minimally invasive window into detection, characterization, and monitoring of cancers. Important therapeutic targets such as EGFR mutation and HER2 overexpression have been detected in CTCs.55 CTCs may also be useful for monitoring expression of these therapeutic targets over time, such as HER2 and estrogen receptor in breast cancer.56 For enrichment methods that do not require fixation of CTCs, culture of these cells may provide options for ex vivo therapeutic predictive testing. Organoids have been successfully grown from CTCs from patients with prostate cancer and small cell lung cancer.57–59 CTCs may also be grown in culture and implanted into mice, for translational research purposes as well as for potential future personalized therapeutic testing.59
POTENTIAL APPLICATIONS IN SCREENING, EARLY DETECTION, AND MINIMALLY INVASIVE DIAGNOSIS
Although circulating epithelial cells (the counterpart of CTCs when the etiology is unknown) are infrequently identified in asymptomatic individuals, they are found often enough to pose a problem. For example, in a study of non–small cell lung cancer patients as compared with healthy controls, a specificity of 94% was achieved.52 Even in selected populations, a false positive rate of 6% is too high to be useful as a screening modality. However, the specificity may be high enough to use as a diagnostic modality in patients with a high clinical suspicion for lung carcinoma, for example, in patients with a radiographically suspicious lung mass.52
A number of other confounding issues will need to be addressed before CTC enumeration and characterization have hope of becoming a viable cancer screening strategy. Currently, CTCs are usually detected in patients with known primary tumors with archival material available for paired molecular analysis.60 In some cancers, clinical guidelines for the interpretation of CTC enumeration data exist.60 CTCs identified in asymptomatic patients will not have the benefit of tissue comparison to verify the relevance of mutations, and established clinical guidelines do not yet exist.60 Extensive clinical and technical validation will be necessary before detection of CTCs can be considered as a screening technique. Potential patient safety, public health, and economic impacts of such screening should be included in any potential consideration of using CTCs for widespread cancer screening.
In patients with known or clinically suspected cancer, CTCs enumeration may provide a mechanism for detecting early recurrence and CTC enrichment could provide a substrate for minimally invasive molecular characterization of the cancer. This could help focus clinical workup as well as guide-targeted therapy. Because of the rarity of these cells and the paucity of nucleic acid, differentiating mutations from sequencing errors is an important concern.61 Single cell picking and sequencing is perhaps the most attractive method for early detection.62
OPPORTUNITIES IN CYTOLOGY
We posit that the cytology laboratory is a natural home for CTC testing. In cytology, we are very familiar with performing high complexity testing on liquid specimens. Cytotechnologists have the technical and morphologic skills to help integrate these technologies into our daily work. Existing workflows connect the cytology laboratory to molecular testing, including HPV testing and molecular testing for solid tumors. CTC technology has the potential to facilitate diagnosis and improve sample conservation of small specimens and potentially rare diagnostic cells. CTC technology could help confirm the identity of rare tumor cells within paucicellular specimens. This testing could be performed as a reflex on excess specimen in indeterminate diagnoses or could potentially be performed on a glass slide, as some CTC technologies incorporate morphologic image interpretation on standard slides. Currently, these techniques have found success in analyzing CSF, which is often both paucicellular and very low volume.63,64 Many other specimen types may benefit from the multilabel testing that CTC testing affords. For example, rare epithelial cells could be distinguished from endothelial and hematopoietic cells on bone fine needle aspiration biopsies utilizing existing CTC labeling methods to evaluate for metastatic carcinoma. A specialized panel of immunolabels could be utilized to distinguish mesothelial cells and histiocytes from tumor cells in body cavity fluid specimens.
CTC technology can be used as an additional mechanism to perform multiparameter immunophenotyping of rare cells seen on routine cytology preparations (smears, liquid-based preparations, cytospins, and cell block) or paraffin-embedded tissue. Instead of expending further specimen to create a cell block and hoping that the diagnostic cells will be abundant enough to appear on multiple levels, the original slide could be reprocessed using 4- to 6-color immunolabeling to further characterize the cells in question. Although the artifacts introduced by previous staining will need to be overcome, CTC technology may be helpful in reprocessing and characterizing these rare cells.
Upfront multiparameter phenotyping, as is frequently utilized in CTC testing, may be helpful to conserve small samples, such as fine needle aspiration or core biopsies in new cancer diagnoses. For example, ALK, ROS1, and PDL1 are currently performed on all lung adenocarcinomas, on serial levels of a core biopsy or cell block specimen. With multiparameter testing, tissue could be conserved for future testing of newly discovered biomarkers, reducing the need for repeat biopsies.
Although it is currently performed predominantly in the research setting, CTC testing can facilitate much of the work that we perform daily in cytology and pathology as we evaluate liquid and tissue specimens. Appropriate incorporation of CTC technology into clinical practice requires us to understand its capabilities and to develop thoughtful, fiscally responsible mechanisms of integrating this powerful testing modality into pathology practice.
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