We believe that we are the first to demonstrate that the previously reported breast cancer-associated change seen in XRD analysis of hair samples is of lipid origin, in particular phospholipids.
To demonstrate that a similar feature to that reported for XRD analysis of hair from breast cancer patients could be added to hair samples ex vivo, we applied olive oil to hair samples that did not demonstrate the breast cancer ring, and reported the presence of a ring in those samples subsequently. We used olive oil because it is the vegetable oil that is most similar to human fatty acids.24
Interestingly, olive oil contains significant amounts of phospholipids, including phosphatidylinositol.25
We were able to remove the ring from a majority of samples using a range of organic solvents known to solubilise lipids. We were also able to enhance the XRD intensity of the ring in several of the breast cancer positive samples using a chemical known to bind to lipids. Whilst not every sample reacted the same to the treatments, we speculate that this was due to the variation in the ability of the solvents and chemicals to penetrate the hair fibre, which could be attributed to the individual differences in the cuticle density packing.
In attempting to characterise the lipid(s) associated with breast cancer, we used mass spectrometry on hair samples that had been extensively extracted using a cryo-mill, to overcome the individual extractability variation. This showed that there is significantly more phosphatidylcholine present in the hairs of breast cancer patients when compared to controls. From this data we propose that the tumour in the breast produces and releases phospholipids, probably from the tumour cell membrane, which enter the circulation and are incorporated into the matrix of the hair fibre resulting in an alteration in the pattern represented by a circular feature when the hair is subjected to XRD.
There are several lines of evidence which support this proposal and suggest that lipogenesis is closely linked to tumorigenesis. BRCA1 (breast cancer susceptibility gene 1) was the first susceptibility gene linked to breast and ovarian cancer.26
It is frequently lost in breast cancer. In 1999, James also reported that women who tested positive for a mutation of the BRCA1 gene also showed the alteration in their hair XRD pattern.11
A study by Moreau et al demonstrated that BRCA1 negatively regulates lipogenesis through binding to phosphorylated and inactive form of acetyl coenzyme A carboxylase, P-ACCA.27
Thus there is a link between loss of BRCA1 and increased lipogenesis.
A number of other groups have also suggested that lipogenesis is closely linked to tumorigenesis in breast cancer. Chajes et al examined lipids in breast cancer tissues in comparison to normal tissue from the same patients and found that mechanisms specifically related to malignant transformation and tumour progression influence the membrane fatty-acid profile of breast carcinoma.28
A mouse study showed similar effects. Only mammary tumour tissues showed a drastic increase in the total phospholipid content (P
< 0.0001) associated with a significant upregulation of phosphatidylethanolamine, phosphatidylcholine (PC), and sphingomyelin (P
Breast cancer has been associated with increased levels of PC in several studies which have suggested it can serve as a biomarker of breast cancer reflecting upregulation of specific choline transporters and choline kinase genes.30
The level of PC in human breast cancer cells has been reported to be 10-fold higher than in normal mammary epithelial cells.31
In addition to being upregulated in breast cancer tissues and in cultured breast cancer cell membranes, it has been reported that lipids are also elevated in serum. In 1971 Feldman and Carter reported the association between serum lipids and breast cancer.32
More recently, Alexopoulos et al found that there was a significant difference in serum phospholipid content between stage-IV breast cancer patients and disease-free individuals. The most significant differences in lipid profiles among disease-free and cancer subjects were attributed to three phosphocholine species and to three unidentified fatty acid species.33
Serum lipids elevated in breast cancer decrease significantly after treatment,34
in a parallel fashion to the reported disappearance of the ring following mastectomy and chemotherapy.15
There are no reports which have looked at whether elevated levels of phosphocholine in serum are also seen in hair from the same patient.
The results presented in this study are from a small sample size designed as a pilot study to establish the nature of the circular breast cancer-associated XRD feature and formulate an idea of the likely lipids involved. We can conclude that phosphatidylcholine, and in particular sub-species thereof, is the most likely candidate, however, the possible association of other phospholipid molecules needs to be investigated and an understanding of the relationship between lipid shedding and the cancer process needs to be elucidated in a larger sample size. Furthermore, it is possible that hair from patients with other cancers may also exhibit incorporation of cancer-associated phospholipids. Therefore in future studies on breast cancer it would be necessary to rule out the presence of other cancers in the patients from which the hair samples have been collected.
Further characterization of the phospholipids associated with breast cancer could be used to develop a novel sensitive and specific diagnostic screening test for breast cancer, based on hair initially, and potentially extendable to other biological samples.