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2007 Nature Publishing Group http://www.nature.com/naturebiotechnology

The terminology of teratocarcinomas and teratomas

To the editor: In their paper in the January issue entitled Isolation of amniotic stem cell lines with potential for therapy, De Coppi et al.1 claim that embryonic stem (ES) cells grow as teratocarcinomas when implanted in vivo. This disagrees both with standard histopathological classification of germ cell neoplasms and with terminology previously used in the field to define ES cellderived tissues. Historically, the term teratocarcinoma has been used by pathologists to specifically define a malignant testicular germ cell tumor that is composed of teratoma and embryonal carcinoma. This neoplasm is associated with a malignant phenotype2,3, including the potential for distant metastasis4, local invasion and marked cytogenetic defects, as well as serial transplantability in experimental systems57. Teratocarcinomas are malignant tumors and may cause death due to invasion and metastasis if left untreated. A more correct term for the experimental tissue masses formed by human ES cells is mature teratomas. Mature teratomas and ES cellderived teratoma-like masses contain differentiated derivatives of all three embryonic germ layers and differ from teratocarcinomas in that they are considered benign2,6,8, do not invade adjacent tissues or metastasize to distant organs and typically do not reoccur after surgical removal9,10. Owing to their developmental potency, ES cell derived masses that form in immunodeficient animals are referred to as teratomas by most other authors in the field11. That said, to a purist, neither term is correct, as both teratocarcinomas and teratomas are naturally occurring neoplasms that arise as a result of specific, acquired genetic and epigenetic alterations. In contrast, experimental human ES cellbased lumps result from disorganized but normal tissue generation and are a manifestation of a failed attempt, resulting from ectopic implantation, to form an embryo by ES cells that are genetically normal. Referring to the ES cellderived tissue masses as teratocarcinomas (neoplastic tumors) is not only ontologically inaccurate: in view of the heated debate surrounding the conduct of human ES cell research, failing to make this important distinction could also have political implications.
M William Lensch1 & Tan A Ince2
1Division of Hematology/Oncology, Childrens Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. 2Harvard Medical School, Department of Pathology, Brigham and Womens Hospital, 75 Francis Street, Boston, Massachusetts 02115, USA. e-mail: Mathew.Lensch@childrens.harvard.edu or tince@partners.org

1. De Coppi, P. et al. Nat. Biotechnol. 25, 100106 (2007). 2. Martin, G.R. & Evans, M.J. Cell 2, 163172 (1974). 3. Cooper, M. Med. Humanit. 30, 1222 (2004). 4. Andrews, P.W. Phil. Trans. R. Soc. Lond. B Biol. Sci. 357, 405417 (2002). 5. Kleinsmith, L.J. & Pierce, G.B. Jr. Cancer Res. 24, 15441551 (1964). 6. Martin, G.R. Cell 5, 229243 (1975). 7. Zwaka, T.P. & Thomson, J.A. Development 132, 227233 (2005). 8. Isaacs, H. Jr. J. Pediatr. Surg. 39, 10031013 (2004). 9. Lopes, M.A., Pereira, C.M., da Cruz Perez, D.E., Vargas, P.A. & de Almeida, O.P. Oral. Surg. Oral. Med. Oral. Pathol. Oral. Radiol. Endod. 100, 598602 (2005). 10. Heerema-McKenney, A., Harrison, M.R., Bratton, B., Farrell, J. & Zaloudek, C. Am. J. Surg. Pathol. 29, 2938 (2005). 11. Brivanlou, A.H. et al. Science 300, 913916 (2003).

Anthony Atala and Mark Furth respond: We used the term teratocarcinoma in our paper because it accurately reflects observations and language from some of the original reports on ES cells that we cited. For instance, in her seminal paper from 1981, Martin1 describes a method...for isolating and establishing pluripotent cell lines with

the properties of teratocarcinoma stem cells directly from normal early mouse embryos in vitro. She characterizes the tumor type formed after injection of ES cells into athymic mice as follows: In most cases, a typical teratocarcinoma, containing derivatives of all three primary germ layers, formed within 6 weeks. Furthermore, she documents the presence of undifferentiated stem cells within tumors formed from ES cell clones. This may help to explain Martins choice of the term teratocarcinoma, rather than teratoma, as it was one of the key criteria used by original workers in the field, including Stevens2. Evans and Kaufman3 also use the term teratocarcinomas to refer to tumors formed by mouse ES cells and ectopically implanted embryos. Moreover, they retain this terminology when reporting the efficient formation of germline chimeras from ES cells (embryo-derived teratocarcinoma cell lines) but not EC cells4. In 1995, Thomson et al.5, like us, used the term teratocarcinomanot teratomawhen citing the prior mouse ES cell literature and observed that when injected into severe combined immunodeficient mice, R278.5 [primate ES] cells consistently differentiate into derivatives of all three embryonic germ layers. Three years later, Thomson and coworkers chose to refer to tumors formed after injection of human ES cells into immune deficient mice as teratomas6; however, a survey of the subsequent literature reveals that even among leaders in the field, the distinction between the terms seems subtle at best. For instance, Andrews and colleagues7 use the term teratoma to describe tumors formed by most human ES cells, but report the production of malignant teratocarcinoma tumors by culture adapted human ES cells that, nonetheless, retain the ability to generate differentiated cell types corresponding to the three germ layers7. Andrews8 has also, on occasion, described tumors formed by cancer-derived human embryonal carcinoma (EC) cells as teratomas rather than teratocarcinomas8. Elsewhere, Solter9 refers to a human ES cellderived tumor as a teratocarcinoma. In contrast, Schuldiner

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et al.10 use teratoma for tumors formed by human ES cells. However, they report that ES cell lines consistently can be cultured from these tumors, demonstrating that stem cells are still presenta criterion that could justify the alternative terminology, as employed by earlier workers14. In a more extreme example, Erdo et al.11 report the formation of highly malignant teratocarcinomas by mouse ES cells injected into mouse brain. They support the nomenclature by noting that tumor growth was highly invasive and micrometastases outside the tumor were repeatedly detected11. What is clear, regardless of the terminology used, is that potential tumorigenicity must be evaluated directly before the clinical application of any stem cellembryonic, amniotic or adultin regenerative medicine. The more important issue remains assuring the safety of patients.
1. Martin, G.R. Proc. Natl. Acad. Sci. USA 78, 76347638 (1981). 2. Stevens, L.C. Results Probl. Cell. Differ. 11, 265274 (1980). 3. Evans, M.J. & Kaufman, M.H. Nature 292, 154156 (1981). 4. Bradley, A., Evans, M., Kaufman, M.H. & Robertson, E. Nature 309, 255256 (1984). 5. Thomson, J.A. et al. Proc. Natl. Acad. Sci. USA 92, 78447848 (1995). 6. Thomson, J.A. et al. Science 282, 11451147 (1998). 7. Andrews, P.W. et al. Biochem. Soc. Trans. 33, 15261530 (2005). 8. Andrews, P.W. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 357, 405417 (2002). 9. Solter, D. Nat. Rev. Genet. 7, 319327 (2006). 10. Schuldiner, M., Itskovitz-Eldor, J. & Benvenisty, N. Stem Cells 21, 257265 (2003). 11. Erdo, F. et al. J. Cereb Blood Flow Metab. 23, 780785 (2003).

To the editor: We appreciate the opportunity to respond to preprint copies of correspondence from Lensch and Ince and the response from Atala and Furth. From our point of view, most of the problems and controversies mentioned in this correspondence have arisen from the inconsistent usage of the terms teratoma and teratocarcinoma by many scientists working in this field. Thus, we encourage the editors of Nature Biotechnology to standardize the terminology, at least for human embryonic stem (ES) cellderived xenografts . In our view, the term teratocarcinoma should be used only for malignant tumors, which in this context are malignant by virtue of the continued presence of stem cellsthe embryonal carcinoma (EC) cells1. EC cells, as suggested by almost all studies on mouseand human-derived cells, are the malignant equivalents or cognates of ES cells2. Any pathologist trained to identify human EC

cells should be able to distinguish malignant teratocarcinomas from benign teratomas, which are defined as tumors composed only of somatic tissues and devoid of EC cells. In an experimental setting, the malignancy of a tumor due to the presence of morphologically identifiable EC cells can be tested by retransplantation to a new host. The distinction between teratomas and teratocarcinomas is crucial, especially for the future usage of ES cells in human medicine. Using xenografting as an essential preclinical safety control, one could predict that the ES cell lines that form only teratomas are benign or safe for human usage, whereas the cell lines that produce teratocarcinomas are malignant and not safe for injection into humans3. Thus, we would discourage the indiscriminate usage of terms teratoma and teratocarcinoma, even though in the past some eminent scientists have used those term interchangeably and even as synonyms. Previous imprecision is, in our opinion, not a valid justification for future use of a confusing terminology. A minor but not insurmountable problem pertains to the usage of teratocarcinoma for tumors produced from human ES cells. Even though the term teratocarcinoma has been used as a synonym for malignant teratoma in mice for more than four decades, most leaders in human pathology have consistently refused to accept it. In the recent consensus book on human testicular tumors compiled by the experts of the World Health Organization (WHO; Geneva, Switzerland)4, the term teratocarcinoma is mentioned only in passing for the animal model of human germ cell tumors. Because the work on human ES cells is, in a sense, a continuation of the experiments first performed on mouse ES cells and teratocarcinoma-derived EC cells, we feel that the term teratocarcinoma will be more readily accepted by laboratory researchers than diagnostic pathologists. At least it is less cumbersome than the WHO-recommended term mixed embryonal carcinoma and teratoma or indeed the British classification malignant teratoma intermediate4.
Ivan Damjanov1 & Peter W Andrews2
1Department of Pathology, The University of Kansas School of Medicine, Kansas City, Kansas 66160, USA. 2Centre for Stem Cell Biology and Department of Biomedical Science, The University of Sheffield, Sheffield S10 2TN, UK. e-mail: idamjano@kumc.edu or p.w.andrews@shef. ac.uk

4. Eble, J.N., Sauter, G., Epstein, J.I. & Sesterhen, I.A. (eds). in World Health Organization Classification of Tumours, Pathology & Genetics: Tumours of the Urinary System and Male Genital Organs 217278 (IARC Press, Lyon, France, 2004).

1. Damjanov, I. Int. J. Dev. Biol. 37, 3946 (1993). 2. Andrews, P.W. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 405417 (2002). 3. Andrews, P.W. et al. Biochem. Trans. 33, 15251529 (2005).

Nature Biotechnology responds: A survey of the literature indicates no consensus on how to distinguish the terms teratoma and teratocarcinoma. The term teratocarcinoma is used as a synonym for human tumors clinically known as teratoma with embryonal carcinoma (according to the World Health Organization) or teratoma intermediate (according to the British classification of germ cell tumors). Some pathologists include these tumors in the group of testicular nonseminomas (also known as nonseminomatous germ cell tumors (NSGCT)) or use imprecise terms, such as malignant teratoma. The somatic tissues in teratocarcinoma may be fully differentiated (equivalent to adult tissue) or only partially differentiated (corresponding to immature tissues in fetal organs). On the basis of the above exchange and after expert consultation, Nature Biotechnology will adopt the term teratocarcinoma to describe malignant tumors comprising both somatic tissues and undifferentiated malignant stem cells, identifiable as EC cells. EC cells are malignant equivalents of ES cells. Human EC cells should be identifiable microscopically according to the pathologic and immunohistochemical criteria used to identify human EC cells in malignant germ cell tumors of the ovary or testis or extragonadal sites. In an experimental setting, the malignancy of a tumor due to the presence of morphologically identifiable undifferentiated EC cells may be defined by their ability to form a new tumor after transplantation to a new host. We will apply the term teratoma only to tumors composed of normal, benign somatic tissue and their immature (fetal) precursors derived from more than one of the three embryonic germ layers (ectoderm, mesoderm and endoderm). Teratomas comprising nonproliferating somatic tissue may be further labeled as benign, mature or fully differentiated. Teratomas composed of immature, proliferating fetal-like tissues may be labeled immature. It should be noted that almost all tumors produced in immunosuppressed mice from xenografted human ES cells have proven to be teratomas. Some data suggest that teratocarcinomas may occasionally be produced from human ES cells upon xenografting, but these tumors have not been fully documented.

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