A case of mistaken identity

A cell line used in more than 650 published breast cancer studies may not be a breast cancer cell line at all

By | September 16, 2008

Some breast cancer researchers may be studying the wrong type of cancer. A growing body of evidence suggests a cell line that's been a cornerstone of metastatic breast cancer research over the last 25 years is in fact derived from melanoma cells. This case of mistaken identity is causing concern among researchers working with the cells, reviewers, and cell suppliers, with some already reclassifying the cells as melanoma. It began with a Nature Genetics paper in 2000. Reading it, James Rae, then a doctoral student at Georgetown University, thought the National Cancer Institute had made an error. The paper examined gene expression in 60 human cancer cell lines at the NCI, including one Rae was using -- MDA-MB-435, the most widely used model for metastatic breast cancer. The first figure in the paper, a colored tree diagram, showed the 435 cell line in pink, but it was not grouped with the other pink breast cancer lines. It lay sandwiched between brown melanoma cell lines. Two years later, scientists at AstraZeneca investigated the incongruence further. Using RT-PCR and immunohistochemistry, they compared expression of breast and melanoma genes in 435 and nine other breast cancer and melanoma cell lines. The 435 cell lines expressed none of the genes characteristic of breast cancer cells, but did express several genes commonly expressed by melanocytes -- a finding that prompted the study's authors to recommend that 435 not be used as a primary line in breast cancer research. By then Rae was a professor at the University of Michigan Medical School, and he decided to track down the mix-up to see just how many 435 lines looked like melanoma. In 2004, he and colleagues at Michigan reported that all lines of 435 they tested from various repositories -- including the cells' original source, the MD Anderson Cancer Center in Texas -- expressed melanoma-specific genes. "It was all the same cell line, and it was probably melanoma," said Rae. Not all data pointed in the same direction. That same year, researchers at Anderson reported that 435 cells express breast-specific markers and can be induced to secrete milk lipids, a characteristic of well-established breast cancer cell lines. The Anderson authors concluded that 435 was a breast cancer model that simply showed heterogeneity, and its melanoma-like phenotype was probably the result of dedifferentiation due to genetic instability. But Rae wasn't convinced. In 2006, he and colleagues from Georgetown examined 435 cell lines using karyotype analysis, comparative genomic hybridization, single nucleotide polymorphism data, and gene expression studies. They reached one definite conclusion, which they published in Breast Cancer Research and Treatment: The true origin of MDA-MB-435 cells is a melanoma cell line called M14. A 2005 Nature paper from researchers at the Dana-Farber Cancer Institute in Boston had reached the same conclusion, also using SNP array analysis: 435 is derived from the same individual as melanoma cell line M14. Today, Rae has no doubts about the claim: "I'm absolutely confident to say this is a melanoma," he said. What's more, Rae's 2006 paper, as well as NCI DNA fingerprinting of samples of 435, suggest that the misidentification of 435 occurred before 1982, meaning that 25 years of breast cancer research on 435 may be based on an incorrect model system. Although the cells phenotypically resemble breast cancer cells, causing cancer and metastasizing when injected into mouse breast tissue, the melanocytic origin of the cells means that the molecular profile of the tumors are potentially very different. Breast cancer hypotheses based on studies of the cell line may be incorrect, and treatments developed from it may ultimately be ineffective. Even Janet Price, an associate professor at Anderson who distributed the cell line in the 1990s, now doubts its identity. "We thought it was a breast cancer," said Price, "but it does have certain characteristics of melanoma." It's impossible to say where it came from without tracing it back to the original patient, said Price. She believes 435 can still be used as a valid model for metastatic cancer, but no longer advises people to use it as a model for breast cancer. "Colleagues will probably be disappointed that I'm not saying what I used to," she said. "I suggest they find other cell lines if they can." Since Rae's 2006 paper, cell line distributors have taken action. The NCI and the Berkeley Lab, a breast cancer cell line repository, have reclassified the cell line to melanoma. ATCC, a leading cell line distributor, stopped selling the line in March, and is testing the cell line's identity, according to Brian Douglass, product manager for cell biology at the company. Misidentification of cell lines is not rare; a short tandem repeat analysis of 100 human cell lines last year found 18 of the lines were incorrectly designated. But with 435, misidentification may be especially problematic. Because of the cell line's unrivaled metastatic ability in mice, more than 650 studies using 435 as a breast cancer model have been published (including more than 60 so far this year). Despite growing doubts about its identity, with nothing to replace the model line, researchers have been unwilling to let it go. "There are people vested in using the cell line because they have grants involved," said Rae. Alison Allan, an oncologist at the London Health Sciences Center in Ontario, recently attended the Joint Metastasis Research Society-AACR Conference on Metastasis. "A good 40% of the presentations used [435] in studies," said Allan. "Nobody even questioned that they weren't breast. They're carrying on as usual." Allan uses 435 cells to study metastasis in breast cancer, but, she said, "It certainly makes me nervous to use them." Although she isn't convinced it's a melanoma line -- "There are 500 papers that say it's breast and five that say it's melanoma" -- reviewers have now begun questioning its use as a breast cancer model. She now adds a caveat statement to methods sections of papers, citing the debate over the origins of the cell line. At the same time, some of her work in progress is already invested in 435, and she wants to continue to use that data. But, Allan is concerned with keeping her cancer models as clinically relevant as possible, and she is currently testing several new cell lines that could provide alternative metastatic models. Researchers have enough problems extending the relevance of their studies to different subtypes of breast cancer, she said. "Then, when you introduce the possibility it's not even a breast cancer, and you look at molecular mechanisms and different proteins we think might affect metastsasis, metastasis of melanoma might be controlled by a whole of different set of proteins." Researchers who stick with 435 as a breast cancer model don't just jeopardize their own scientific results, said Rae; there are rumors of young PIs being turned down for publishing and grant funding based on incorrect use of the line. What's more, Rae believes researchers turning a blind eye to the controversy are preventing the field from directing effort toward finding a replacement to 435. "It's an overall shame," he said. But Allan noted that aside from her own interest, many of her colleagues at the recent metastasis conference spoke informally about wanting alternative models. "Everyone is in the same boat," she said. "Everyone was saying, 'If you find one, let me know.'" Not all researchers are as concerned. Selvarangan Ponnazhagan, a pathologist at the University of Alabama at Birmingham, injects the cells into bone, where they cause osteolytic bone lesions characteristic of strongly metastatic breast cancer. When reviewers of a paper he published earlier this year raised the issue of the cell line's identity, he responded that he didn't want to mention the controversy in the paper, stating that 435 is a well-known breast cancer cell line, and that it behaves as such in bone lesions. "I'm truly not very interested in finding out what cell line it is," he said. "This is an ideal model [for what I'm studying]." Rae believes all researchers will eventually come around and stop using 435 once a new model is found. And although he is adamant against using 435 in breast cancer studies, Rae, who is a breast cancer researcher himself, agrees it remains a valuable system for studying metastases. He hopes it can be a "boon" for melanoma research, providing years of extensive literature on M14 to the melanoma community. "The efforts spent over the years on studying MDA-MB-435 have not been wasted," he wrote in the conclusion of his 2006 analysis of 435. "The many studies published using MDA-MB-435 as a model for breast cancer could now conceivably be reinterpreted as studies using M14 as a model for melanoma." Megan Scudellari mail@the-scientist.com Correction (September 19): A previous version of this story referred to Janet Price as the original distributor of the MDA-MB-435 cell line. According to officials at MD Anderson, the line was originally distributed by Relda Cailleau in 1984, with Janet Price taking over the role in the 1990s. The Scientist regrets the error.

Comments

Avatar of: anonymous poster

anonymous poster

Posts: 1

September 16, 2008

This is an issue that gets raised from time to time... including commentaries to Nature articles, see..New Scientist 03 October 2007 "Stem cells recruited to help cancers grow" by Colin Barras.\nRae's maverick observations highlight the success of more modern methods for comprehensive cell characterization. MDA-MB-435 cells can provide excellent information regarding metastasis. Ironically, perhaps some of the fundamental molecular mechanisms are indeed shared by some metastatic breast cancers. However it would be prudent to place less emphasis on the direct relevance of this cell line to breast cancer....."accidents happen".
Avatar of: john toeppen

john toeppen

Posts: 52

September 16, 2008

Questioning all of the assumptions, all of the answers, and all of the questions all of the time is good science. Hard work and ingenuity are simply not enough. There is a tendency to presume that the works of others are correct and should be trusted, especially if the view is commonly held. Consensus can mistaken, and the consequences severe. We would all do well to learn a larger lesson from this.
Avatar of: anonymous poster

anonymous poster

Posts: 1

September 16, 2008

293 is a similar example. It is widely quoted as a human embryonic kidney cell line while Shaw et al. (FASEB J. 2002) showed that it is neuronal in character.
Avatar of: anonymous poster

anonymous poster

Posts: 85

September 16, 2008

Let me get this straight. The cell line has been shown to express genes generally associated with melanoma cells, and to NOT express genes generally associated with mammary tissue. Furthermore, genetic analysis has indicated that the line comes from the same human individual as a known established melanoma cell line. Presumably that individual did not also happen to have breast cancer. BUT -- because so much wonderful data has already been collected using this cell line, we are continuing to spend US taxpayer dollars to support continued research on this cell line in the context of breast cancer, and we will continue to do so until someone comes up with another cell line? \n\nDuh? What's wrong with this picture? \n\n
Avatar of: anonymous poster

anonymous poster

Posts: 1

September 16, 2008

although it is highly likely to be very wrong to define these cells as "breast cancer" all may not be loss; during embryogenesis embryonic mammary buds evolve from skin...if cancer mimics development then this cell line may open up new avenues to compare melanoma and breast cancers...
Avatar of: Li Chen

Li Chen

Posts: 10

September 16, 2008

Who is a real New Yorker? \nThe one you meet on Wall Street, or the one who just walk off a plane from New York in Narita Airport?\nSo, what is our definition of breast cell line?\nIn addition, a breast cancer cell, just like a terrorist to New York, could be totally different from conventional breast cells. \nTo me, Rae's studies indicated that we need more cell lines for breast cancer research, but it is too early to jump into the conclusion that 435 is a mistaken identity. \n
Avatar of: anonymous poster

anonymous poster

Posts: 3

September 16, 2008

Some scientist appear to choose to ask whether it can be definitively said that MDA-MB-435≠Breast Cancer. Would they be equally comfortable in trying to question whether MDA-MB-435=M14? Obfuscating over whether it is breast origin or not simply avoids the difficulty of two supposedly different cell lines being identical. The history of the cell line is suspect... \n\nHmmm, should we also begin to doubt paternity tests?
Avatar of: anonymous poster

anonymous poster

Posts: 1

September 17, 2008

Selvarangan Ponnazhagan,. "I'm truly not very interested in finding out what cell line it is," he said. "This is an ideal model [for what I'm studying.\n\nIs it just me, or does anyone else find this comment appalling? It's extraordinarily bad science! If you don't know what your injecting how can you possibly know it's a good model?
Avatar of: Cecily Bishop

Cecily Bishop

Posts: 9

September 17, 2008

While this article is a good example of how modern techniques can be used to characterize a MODEL - there is another important issue some are missing.\nHow many passages has this line undergone? Established cell lines often mutate in culture and drift away from their original genotype/phenotype.\nIt would be interesting to know this data before drawing too many conclusions.
Avatar of: RISTO PENTTINEN

RISTO PENTTINEN

Posts: 1

September 17, 2008

A great story, important to very many scientists. Thank you for publishing this comment.
Avatar of: Roland Nardone

Roland Nardone

Posts: 2

September 17, 2008

The article refers to a serious, widespread problem. Fifteen to 30% of cell lines used for biomedical research are cross-contaminated or misidentified. A current initiative to correct the problem has been underway for three years with some success. However too many scientists and policy makers are indifferent. The initiative, its progress, and how a solution can be achieved is described in "Curbing rampant cross-contamination and mis-identification of cell lines." BioTechniques, Sept. 2008. volume 45: 221-227.
Avatar of: anonymous poster

anonymous poster

Posts: 3

September 19, 2008

Alison Allan-"There are 500 papers that say it's breast and five that say it's melanoma".\nThis is a rather disingenuous and infuriating argument for the continued use of this line. I suspect the 500 papers report them as 'breast' because that is what they were told when they bought them from NCI, ATCC... or received them second hand from other researchers (a sadly common affair). On the other hand the 5 papers stepping out of line are based on real research into the origins/genotype of the line.\nAlmost a save by adding the caveat to her papers-sounds like everyone should be doing the same. The new 'best breast' line is gonna be a while because no-one will want to stick their necks out in support of one!

September 22, 2008

I find it appalling how many "anonymous posters" there are. Good science discussions happen in the open, and anonymity has no place here. If you want to make a comment, please have the decency to sign with your name.
Avatar of: Jacob Schor

Jacob Schor

Posts: 2

September 22, 2008

So shall we run out and start treating melanoma with aromatase inhibitors?\nHow much of the research on this cell line might be turned around and now be applied to treat skin cancers? What a bizarre thought!
Avatar of: Adrian Gonzalez

Adrian Gonzalez

Posts: 2

September 24, 2008

?The sciences do not try to explain, they hardly even try to interpret. They mainly make models. By a model is meant a mathematical construct which, with the addition of certain verbal interpretations, describes observed phenomena. The justification of such a mathematical construct is solely and precisely that it is expected to work.? Johann Von Neumann\n\nJohann Von Neumann, the 20th century mathematical genius was correct about models. Thank you James Rae for asking questions.
Avatar of: Ken Dev

Ken Dev

Posts: 7

October 21, 2008

Your author says "Rae, who is a breast cancer researcher himself, agrees it remains a valuable system for studying metastases." Wouldn't it be better to choose a more appropriate cell line from now on, rather than a mistaken cell line, to study metastases, specifically for breast cancer, now that it seems clear that there has been a mix-up somewhere nearly 25 years ago? Also, various comments following this article show that the last word has not be spoken on this 'mistaken identity.' I don't know whether aromatase inhibitors or other compounds that are used to treat metastatic breast cancers have ever been used to treat metastatic melanoma even in animal models. It would certainly be interesting to find out what happens. There must be some commonalities between various metastatic cancers, although the primary tumors may have different origins.
Avatar of: Ingrid Shafer

Ingrid Shafer

Posts: 1

October 21, 2008

I was struck by Rae's comment that "There are people vested in using the cell line because they have grants involved." This demonstrates the urgency of developing ways of assessing conflicts of interest and insisting on the ethical conduct of research.\n
Avatar of: Colin Smith

Colin Smith

Posts: 1

October 23, 2008

An anonymous poster made the following comment: "although it is highly likely to be very wrong to define these cells as "breast cancer" all may not be loss; during embryogenesis embryonic mammary buds evolve from skin...if cancer mimics development then this cell line may open up new avenues to compare melanoma and breast cancers..."\nThe embryonic mammary buds may evolve from the skin, however melanocyte/melanoma evolve from the neural tube and thus have a different embryonic lineage\n
Avatar of: Ellen Hunt

Ellen Hunt

Posts: 199

October 23, 2008

"500 papers that say it's breast and five that say it's melanoma" - If that isn't a classic of idiot science, I don't know what is. With blither like that from the mouths of highly placed researchers, we scientists wonder why the public doesn't trust or believe us anymore? \n\nCecily Bishop - Your comment is a good and obvious question to ask. This concern was addressed by surveying a all the sources of this cell line, as stated in the article.

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