A “homeopathic” bit of breast cancer “science,” or: Who knew alcohol was so toxic?

Homeopaths are irritating.

They’re irritating for a number of reasons. One is their magical thinking, and, make no mistake, their thinking is nothing but pure magic, sympathetic magic to be precise. That’s all that the principle of “like cures like” really is at its heart. Normally, that principle states that “like produces like,” but homeopathy reverses that principle by saying that, while like produces like at normal concentrations, like reverses like at high dilutions. Somehow the magical process of shaking the remedy very hard between each dilution step (called “succussion”) imbues it with the property of curing what it would normally cause. Of course, scientists know that the whole “shake and dilute” process is bunk, given that a typical 30C homeopathic dilution dilutes a substance to the point where it is incredibly unlikely that even a single molecule remains.

This brings us to the second magical principle upon which homeopathy rests: The law of contagion, which states that things that have once been in contact with each other continue to act on each other at a distance after the physical contact has been severed. The perfect embodiment of this is the claimed “memory of water,” which is how homeopaths get around the inconvient fact that most of their remedies contain not a single molecule of the original remedy. Somehow the water “remembers” the contact with the remedy and magically forgets the contact it’s had with all the other things that water has been contact with, like poo, pee, chemicals, foodstuffs, and everything else. It’s not as if waterfalls and various other natural processes don’t “succuss” the water quite nicely.

What makes homeopaths particularly irritating is that they believe so highly in their magic that they all too often abuse science to try to “prove” that it works. Lately, homeopaths have been throwing a new study in my face as “evidence” that their woo works:

Frenkel M, Mishra BM, Sen S, Yang P, Pawlus A, Vence L, Leblanc A, Cohen L, Banerji P, Banerji P. (2010). Cytotoxic effects of ultra-diluted remedies on breast cancer cells. Int J Oncol. 2010 Feb;36(2):395-403.

Let’s looks at the abstract:

The use of ultra-diluted natural products in the management of disease and treatment of cancer has generated a lot of interest and controversy. We conducted an in vitro study to determine if products prescribed by a clinic in India have any effect on breast cancer cell lines. We studied four ultra-diluted remedies (Carcinosin, Phytolacca, Conium and Thuja) against two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and a cell line derived from immortalized normal human mammary epithelial cells (HMLE). The remedies exerted preferential cytotoxic effects against the two breast cancer cell lines, causing cell cycle delay/arrest and apoptosis. These effects were accompanied by altered expression of the cell cycle regulatory proteins, including downregulation of phosphorylated Rb and upregulation of the CDK inhibitor p27, which were likely responsible for the cell cycle delay/arrest as well as induction of the apoptotic cascade that manifested in the activation of caspase 7 and cleavage of PARP in the treated cells. The findings demonstrate biological activity of these natural products when presented at ultra-diluted doses. Further in-depth studies with additional cell lines and animal models are warranted to explore the clinical applicability of these agents.

Basically, it’s a test of homeopathic remedies against breast cancer cell lines in a dish that claims differential toxicity aginast breast cancer cell lines compared to normal breast cells and claim that homeopathic remedies did just as well as cytotoxic chemotherapy. For three weeks, I have been assiduously ignoring them because I thought that Dr. Rachie’s takedown of the study was excellent (not to mention that neither my university library nor those of several of my friends apparently carry a digital subscription to the journal–the best that anyone could send me was from a library loan, which was FAXED, and the figures come out as not that clear), but yesterday was the last straw. I don’t know what it was, but finally I had had enough. Dr. Rachie did good, but I’m a breast cancer researcher, and I have worked with the cell lines used in the paper. It’s time for a heapin’ helpin’ of not-so-Respectful Insolence. It’s also time for me to make a note never to submit a paper to the International Journal of Oncology, whose editorial and peer review standards are clearly lacking.

Dr. Rachie hit upon one huge flaw in the study, and I can’t help but mention it as well. There isn’t a single mention of statistics to show that the differences described are significant. In fact, there is a rather disturbing lack of proper quantification of results throughout the paper, in particular with image-driven data, such as assays for DNA breakage by FISH. For these analyses, all the authors show are pictures of “representative” cells, but they didn’t bother to analyze large numbers of cells to see if the qualitative results that show up on the panels they chose to print are real, if they hold up to statistical analysis. It’s very easy to be fooled, even unintentionally, if you don’t look at large numbers of cells. The same is true of the flow cytometry data, as Dr. Rachie also points out.

Let’s step back a minute, now. This study is based on homeopathic remedies of the P. Banerji Homeopathic Research Foundation (homeopatic research: an oxymoron if I’ve ever heard one!), which claims to be able to cure a lot of cancers that “conventional therapy” can’t. Indeed, they even submitted a “best case” series to the NIH in which, out of 941 patients with breast cancer, the PBHRF claimed that in 19% of patients, tumors regressed and that 21% of tumors were stable. One thing I noted is that the reference cited for this claim is not a peer-reviewed article. It’s merely cited as:

Banerji, P. P. Banerji Homeopathic Research Foundation (PBHRF) in Kolkata, India, 2008.

Gee, I wish I could cite claims like that in my papers!

Of course, the claim above is meaningless without knowing a lot more about the patients. For instance, as I’ve pointed out before, as many as 20% of mammographically detected breast cancers might spontaneously regress. I’ve also pointed out that the natural history of breast cancer is highly variable, from relatively rapid growth and decline to a slow and indolent course. Indeed, a subset of women with untreated breast cancer can survive as long as 10 years or more, and this data comes from before the age of mammography, which means that these were at the very least palpable tumors. Indeed, most tumors detected at the end of the 19th century and the beginning of the 20th century (which is when this data was collected) were stage II and III. These days, the vast majority of breast cancers are detected by mammography and are stages 0 (noninvasive–or preinvasive–ductal carcinoma in situ) or stage I. Without stage-specific survival data, with stages documented by pathology, imaging studies, and clinical data, a claim like the one made above is uninterpretable and, in essence, meaningless.

The remedies, however, are Carcinosin, 30C; Conium maculatum, 3C; Phytolacca decandra, 200C and Thuja occidentalis, 30C. At this point I can’t help but note a couple of things. First, a 3C dilution of conium muculatum is only a 106 or one to one million dilution, which means that there might well be something active left in this particular remedy. The second point I’d like to make is just to express awe at a 200C homeopathic dilution, which would represent a dilution of 10400, or 10367 or so (what’s an order of magnitude between friends?) greater than Avagadro’s number. This is a mind-boggling number, not to mention a mind-bogglingly tedious process.

There’s one other aspect to consider. The diluent was not water. Rather, it was 87% “extra neutral alcohol,” and this alcohol was used as the control. So far, fair enough. At least they used a solvent-only control, which is more than I can say for a lot of homeopathic “researchers.” They then treated the cells with different “doses” (although how one can speak of a dose of a nonexistent remedy, I don’t know) of the remedies or solvent. The cells treated included MCF-7 cells (which are estrogen receptor-positive, or ER(+), cells and a very commonly used cell line in breast cancer research; I use them myself) and MDA-MB-231 cells (an ER(-), progesterone receptor-negative, HER2/neu-negative cell line, which I’ve also used in cell culture and xenograft experiments). The former is relatively slow-growing and somewhat tricky to grow in xenografts, although easy to grow in cell culture, and the latter is a model of a particularly nasty form of breast cancer known as “triple negative” breast cancer.. Control cells are an immortalized human mammary epithelial cell line, HMLE, which are supposed to represent “normal” mammary epithelial cells.

I think the entire problem with the paper can be summed up with this figure, which shows the effect of these remedies on cell viability:

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Notice how each remedy and the controls are graphed in little bar graphs for each cell type. This makes it very difficult to compare directly effects of each remedy at each dose compared to the solvent control. Really. Click on the picture and see if you can easily tell whether the differences shown look significant. You can’t. Moreover, no statistics are provided to tell you whether the differences might be significant. Worse, however, look at the solvent-only control groups. Do you see what I see? The solvent is killing the cells, up to 50% of them! Not only that, but the normal control cells appear to be more resistant to the effect of the solvent, with very little effect seen. In fact, by the lack of a clear dose-response curve for the solvent-only control, with the measurement fluctuating around a point slightly lower than that seen with no solvent added, you can get an idea of the variability of this assay from well to well, which is quite a bit. The cynic in me thinks that the data were graphed this way intentionally, to obscure just how weak it is, but the angel in me will attribute it to gross incompetence.

If I had reviewed this paper, I would have insisted for the figure above that all the data for each cell line be graphed on a single graph, with a dose-response curve clearly indicated for each and error bars, along with proper statistical analysis for a dose-response effect showing that there were significant differences between the effect of the homeopathic remedies at each dose compared to the solvent control. All that the above figure shows is that adding 10 μl/ml of 87% alcohol has a significant effect on cell viability for the cancer cell lines. They have not shown in any way that I can tell that any of their remedies have a significant effect on cell viability compared to the alcohol solvent alone. As I’ve said time and time again, anyone can kill cells in a dish with alcohol or bleach or any number of other nonspecific solvents. That does not mean that this study shows that these homeopathic remedies are as “effective as paclitaxel” (a commonly used chemotherapeutic agent in breast cancer) against these cells. All it proves is that the solvent at the doses used can nonspecifically kill cells and that perhaps the normal cells are more resistant to the alcohol.

Speaking of the solvent, there’s something very fishy in this passage from the paper:

All four remedies had very similar HPLC chromatograms to each other, with only trace amounts of limited number of peaks. They were not significantly distinct from the solvent and they lacked the distinct peak seen in the solvent.

The chromatograms are not shown, but note the last part: They lacked the distinct peak seen in the solvent. What on earth does this mean? One guess is that the solvent peak was wider and less distinct in the homeopathic remedies, but another is that it was gone. Which is it? Who knows? As I said before, the chromatograms weren’t shown. In any case, if these remedies are truly homeopathic, then why are the solvent peaks different?

I seriously have to wonder about this solvent. The maximum dose used (10 μl/ml) is in essence a 1:100 dilution of alcohol, making the alcohol content in the media less than 1%. The lowest dose (1.25 μl/ml), is close to a 1:1000 dilution. Alcohol alone should not be that toxic to these cells. At least, in our hands it isn’t. We’ve used drugs diluted in alcohol at similar concentrations to the lowest dose of solvent used in this study (1.25 μl/ml), and we don’t see 35% cell kill in MDA-MB-231 or MCF-7 cells due to solvent effects alone. Something odd is going on here.

A journalist acquaintance of mine once asked me for examples of massive failures in the peer review process. I can’t think of a better one than this paper. It tests a remedy so highly implausible as to be safely considered, for all practical intents and purposes, impossible barring some truly extraordinary evidence coming to light, evidence sufficient to overthrow long-established science in multiple disciplines. Its statistical analysis is nonexistent, and its quantification dodgy in many places. All of this means that its conclusions do not flow from its data and are not supported by its data. The only conclusion that is supported by the data is that the solvent in which the homeopathic remedies had been diluted is toxic to MCF-7 and MDA-MB-231 cells.

Finally, I can accept that perhaps a 3C homeopathic dilution might have an effect on cells. There could be an actual drug remaining there. However, 30C and 200C homeopathic dilutions leave nothing behind, and there is nothing in this paper to show that there is an effect above and beyond solvent effects from either of these remedies. Come to think of it, there’s nothing in this paper to show that the 3C homeopathic dilution really has any effect above and beyond solvent toxicity effects. Put it all together, and this is a EPIC FAIL on the part of the peer reviewers and the editors.

You know what’s more irritating than homeopaths? Peer reviewers and editors who let dreck like this see print, giving homeopaths something to irritate me with.