“Thyroid Cancer” Cell Line Misidentification: A Time for Proactive Change

Cell lines isolated from human cancers that survive in long-term culture conditions are commonly used to help determine the functional relevance of cancer-related markers, identify and define key molecules in cancer cell biology, and develop and test new treatments in vitro and in vivo as preclinical models. Data derived from these cell lines have led to important scientific advances. Because of their low cost, ease of use, and the reproducibility of results between experiments and laboratories, continuous cell lines are often used for years. They are frequently shared by investigators, and allow for collaboration and confirmation of data. From a preclinical testing standpoint, results from experiments with stable cancer cell lines have been used for preclinical modeling for nearly all cancers. In 1985, the U.S. National Cancer Institute created a panel consisting of 60 human cancer cell lines derived from several tumor types for rapid and comparable screening of compounds using in vitro and in vivo methods (1). The ability of these results to predict response in clinical trials has been variable, with some differences between different histological subtypes of tumors (2). Because thyroid cancer cell lines are not included in this panel, there has been a reliance on investigator-maintained cell lines for similar studies in this disease.

Alternative cell culture systems, such as primary cultures from human cancers, have also been used by investigators. Advantages of this cell culture model include the low likelihood of cellular changes resulting from long-term survival in cell culture and the more direct relationship between the tumor tissue and the biology of the cells in an individual patient (1,2). However, these cells are difficult to obtain and have limitations related to reproducibility between laboratories due to the short-term nature of cell survival. Finally, cells and tissue slices derived from nonhuman sources have been used to define thyroid cell signaling and biological functions of thyroid cancer-related oncogenes in the context of a differentiated thyroid cell. The advantages and disadvantages of these systems have been reviewed (3).

It is well known that “stable” cancer cell lines have been selected for their ability to survive and proliferate in vitro. The clones that grow best after isolation may no longer reflect the biology of the majority of cells in the primary tumors from which they are derived (4,5). Furthermore, secondary selection can occur in cell culture, thereby allowing subclones to emerge with different characteristics than the parental cell line over time. To detect cellular changes in the cells in culture, investigators have traditionally stored early passage cells in liquid nitrogen for future use, observed cellular physical characteristics and biological behaviors, and periodically evaluated cell lines for residual tissue-specific gene expression or protein function when possible using a variety of methods.

The advent of genetic evaluation of cell lines to determine their unique gene profile (DNA “fingerprinting”) has allowed for a new level of evidence to characterize the identity and stability of a particular cell line (6). Using these methods, the frequency of cell line cross contamination has been alarmingly common, with percentages of misidentification as high as 36% in some studies (6,7,8). Until recently, this type of analysis had not been applied to thyroid cancer cell lines. However, several cell lines thought to be unique now have been reported to be genetically similar to other thyroid cancer cell lines (9,10,11). In the current issue of The Journal of Clinical Endocrinology and Metabolism, Schweppe et al. (12) applied a comprehensive genetic approach to evaluating a large panel of commonly used human thyroid cancer cell lines and have carefully established the extent of this problem in thyroid cancer cell culture systems.

In their manuscript, Schweppe et al. (12) subjected thyroid cancer cell lines to genetic analysis, including the standard short tandem repeat profiling in all cases, and in some particularly commonly used cell lines, single nucleotide polymorphism array analysis. The study included cell lines from different laboratories, and the investigators carefully compared the results between cell lines and also between their findings to those in cell line repository databases.

There are several important observations of this study. First, many of the reportedly unique thyroid cancer cell lines are in fact not genetically unique. Second, several of these groups of cell lines that are nonunique are genetically identical to nonthyroid cancer cell lines whose development predated the thyroid cancer cell line original reports, suggesting they are not of thyroid lineage. Third, there are panels of unique cancer cell lines that either have thyroid-specific gene expression (presumed bona fide thyroid cancer cell lines) or have no thyroid-specific gene expression (presumed anaplastic thyroid cancer cell lines). Fourth, the authors demonstrate that several subclones exist for many of these cell lines. Overall, these findings make this carefully performed study a landmark that will reshape the landscape for molecular thyroid cancer research.

Although it is tempting to speculate when or how these events occurred, it is difficult to recreate the past. Several of these original cell lines have been characterized as having evidence of thyroid-specific gene expression by Northern analysis and TSH receptor binding by several groups (13,14,15). The use of RT-PCR to detect gene expression may lead to false-positive results depending on the precise method used and the number of amplification cycles in the reaction, as noted by Schweppe et al. (12). Thus, it is not certain precisely when the cross contamination occurred. In addition, some of the cell lines are poorly differentiated or anaplastic and, therefore, have low or no evidence of thyroid-specific gene expression. The finding of distinct subclones for some of the lines makes it clear that not all of the cell lines carrying a particular name are identical (12). With regard to previous published work including misidentified cell lines, the erratum from Haugen et al. in this issue of the Journal represents an admirable approach for others to consider.

Moving forward, it is imperative that thyroid cancer researchers confront this issue in a proactive manner. It seems unlikely that all work in long-term cancer cell lines will disappear for the reasons noted previously, and the continued use of permanent cell lines may be appropriate if approached with care. By characterizing a panel of unique cell lines with varying degrees of thyroid-specific gene expression, Schweppe et al. (12) have provided a new starting point for thyroid cancer researchers. The availability of DNA “fingerprints” for these cell lines provides a solid method for confirming and maintaining the identity of each cell line.

With these data, thyroid cancer researchers are now prepared to join in the “call for action” proposed for all cell culture researchers (16). Because of potential differences between different clones of the same named cell lines, it would be prudent for researchers to establish if the cell lines in their laboratories are either thyroid or unique by comparing them with the data in this manuscript. Short tandem repeat analysis as performed in this study is inexpensive and commercially available, making this a reasonable level of evidence for individual investigators to achieve. For cell lines not included in the manuscript, it is important that similar approaches be taken to confirm uniqueness and identity. Data from nonthyroid cancer cell lines should not be published as deriving from thyroid cancer cells. Work in which redundant thyroid cancer cell lines are used will need to be reinterpreted. Standards for new cell lines should be created. In addition to reporting detailed information regarding the source of the cell line, the thyroid-specific gene expression profiles, and biological characteristics, publicly available DNA footprints from the primary tumor and cell line should also be provided. Moreover, it seems reasonable that all cell lines should be submitted to a cell line repository to allow investigators to obtain cell lines that have been confirmed to maintain their DNA fingerprint with confidence. Alternatively, thyroid cancer investigators could choose to create their own central cell line repository that would maintain the cell lines and confirm their genetic integrity over time. In this manner, thyroid cancer researchers can move forward with confidence in the identity of the cell lines, taking care to monitor their stocks over time and to avoid extension of interpretation beyond the systems being studied.