BRIEF COMMUNICATION

Experimental Confirmation of a Distinctive Diffraction Pattern in Hair From Women With Breast Cancer

Peter Meyer, Veronica J. James

Affiliations of authors: P. Meyer, Department of Dermatology, University of Tuebingen and Human Resources Inc., Tuebingen, Germany; V. J. James, Research School of Chemistry, Australian National University, Canberra.

Correspondence to: Veronica J. James, Ph.D., Research School of Chemistry, Australian National University, Canberra ACT, Australia 0200 (e-mail: vjs{at}bigpond.com).

Since distinctive changes in the fiber diffraction of hair from women with breast cancer were reported by James et al. (1), several groups (24) have reported apparently conflicting results. This brief communication presents, to our knowledge, the first comparison of two double-blind studies using samples from the same patients. The first experiment replicated exactly the procedures by Wilk et al. (5)1 and by James et al. (6)1 and showed distinctive changes in the fiber-diffraction patterns from all patients with breast cancer. The second experiment (3) used a different protocol and did not show these distinctive changes, indicating the need to observe the protocol as described by James et al. (1).



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Fig. 1. (a) Diffraction pattern taken from the hair of a control subject. The arrows indicate the very strong 7th, 19th, and 38th orders of the 47-nm lattice along the hair's axis (James' protocol). (b) An intensity diffraction pattern taken from the hair of a patient with breast cancer. Arrow indicates the weak additional ring (protocol of James et al.). (c) Diffraction pattern of a hair using an alternate method showing the spurious ring and virtually no keratin pattern. Briefly, the final meridional pattern is a composite of six superimposed lattices, with spacings of 62.6, 47, 19.8, 27.3, 12.5, and 7.6 nm. The relevant section of this meridional pattern has eight normal peaks superimposed on each other and, in the case of breast cancer, a ring is superimposed on this array. The determination as to whether or not this weak ring is present requires great care. The equatorial pattern is also a complicated helix-within-helix structure giving rise to a series of "spots." These spots originate from cylindrical Bessel functions relating to the cross-sectional packing. By use of a Bessel function analysis, it is possible to determine the diameters and center-to-center spacings of the {alpha}-keratin helices, the tetramers, and the intermediate filaments of the hair fiber. The equatorial pattern also includes 11 Bragg orders of the phospholipid bilayer membrane. Being off axis can also cause problems if the sample is in any way disoriented, because this results in extended meridionals, in particular, the split reflection near the 10th order of the 47-nm lattice. In some cases, this split reflection can almost meet up with the disordered membrane arc resulting in an egg-shaped "circle."

 
A synchrotron fiber-diffraction pattern of normal human hair obtained by use of the protocol of James et al. is shown in Fig. 1, AGo.2 This diffraction pattern is not apparent in the diffraction patterns from normal hair reported by other groups (24). There are several important features visible in diffraction patterns of hair. These features include the meridional arcs arising from long-range periodicity of intermediate filaments along the length of the hair and the equatorial spots arising from the hexagonal packing of the filaments across the hair. These features must be visible in all hair-diffraction patterns (for control subjects and patients with breast cancer) if the changes associated with breast cancer reported by James et al. (1) are to be detected reliably.

For any hair-diffraction image to be valid for this analysis, the 7th, 19th, and 38th meridional reflections of the 47-nm lattice must be clearly visible, with at least some of the weaker meridionals just discernible above the background. For samples from patients with breast cancer, James et al. (1) showed that a ring or rings of comparatively low intensity superimpose on the normal diffraction pattern for hair (Fig. 1, BGo). The samples were taken at the outpatient clinic of the Institute of Human Genetics, University of Heidelberg, Germany. The control samples were from healthy cancer-free patients. The breast cancer samples were from women who had a familial background of breast cancer. Patients 1 and 6 showed the most frequent mutation (5382insC) in the BRCA1 gene. All of these women underwent surgical treatment. Individual follow-up chemotherapeutic treatments are noted in Table 1Go.


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Table 1. Results of fiber-diffraction studies by use of the protocol of James et al. (1)*
 
In a properly mounted sample, the intensity of the first-order ring is invariably much less than that of the 7th meridional reflection of the 47-nm lattice. These rings are often not discernible until after the background has been fitted with a boxcar average and removed from the original image (5). Their presence is confirmed by intensity plots along both axes and at some suitable off-axis location. The changes in the diffraction patterns are further verified by use of a second-order derivative plot of data smoothed previously by the application of best-fit Legendre polynomials (6). All peaks are then confirmed by manually scanning the original data on a computer screen with a mouse.

The main difficulty encountered in the second experiment (2), which has also been experienced by other groups attempting to reproduce the results of James et al., has been the presence of a very intense ring (Fig. 1, CGo) that is sometimes accompanied by much weaker second- and third-order arcs or rings. These rings have intensities that exceed even the 7th meridional intensities and tend to swamp the true pattern. Such rings have no relationship to the ring reported for patients with breast cancer but simply confirm macroscopic disorder in the arrangement of the cylindrical arrays, probably as a result of poorly selected, badly aligned, or improperly tensioned multistranded samples.

In addition to the question of relative intensity, the ring that reflects breast cancer can be distinguished from other rings by its radius. The first reported radius of the breast cancer ring (1) was calculated from the third order in patterns taken on the Australian National Beamline Facility diffractometer at the Photon Factory, Tsukuba, Japan. From data collected at higher resolution with the BioCAT Facility at the Advanced Photon Source, at Argonne National Laboratory (Argonne, IL), a powerful electron synchrotron that has a specific beamline for biomolecular measurements, the first order of this ring can be resolved from the membrane arc and has a radius of 4.7 ± 0.1 nm (mean ± standard deviation). In contrast, the heavy ring arising from macroscopic disorder has a radius of 4.5 ± 0.2 nm (mean ± standard deviation). A summary of the experimental results for this study by use of the technique of James et al. is given in Table 1Go.

These results demonstrated a clear association between a change in the diffraction pattern of hair and the presence of breast cancer, whereas results from the second experiment did not (2). Given that both experiments were performed independently on the same blinded set of samples, this work shows that careful attention in sample alignment and mounting is critical, as is a suitable scattering configuration optimized for a high signal-to-noise ratio.

NOTES

1 Experimental details are also provided on the Australian Nuclear Science and Technology Organisation (ANSTO) website (www.ansto.gov.au/natfac/asrp9.htm). Back

2 The three diffraction patterns shown in Fig. 1Go have the same orientation as the patterns shown in Meyer et al. (2) but are rotated 90° from the patterns shown in James et al. (1). Back

Supported by the Access to the Major Research Facilities Program, which is funded by the Commonwealth of Australia; by Human Resources Inc., Tuebingen, Germany, which enabled the collection of the hair samples and the relevant personal details on the patients; and by contract W-31–109-ENG-38 from the BioCAT Facility at the Advanced Photon Source, which is supported by the U. S. Department of Energy, Basic Energy Sciences, Office of Science. BioCAT is supported by Public Health Service grant RR08630 from the National Center for Research Resources, National Institutes of Health, Department of Health and Human Services.

We thank the technical staff of BioCAT for their assistance in setting up the equipment and Colin Gageler for his assistance with the data analysis.

REFERENCES

1 James V, Kearsley J, Irving T, Amemiya Y, Cookson D. Using hair to screen for breast cancer [letter]. Nature 1999;398:33–4.[Medline]

2 Meyer P, Goergl R, Botz JW, Fratzl P. Breast cancer screening using small-angle X-ray scattering analysis of human hair. J Natl Cancer Inst 2000;92:1092–3.[Free Full Text]

3 Howell A, Grossmann JG, Cheung KC, Kanbi L, Evans DG, Hasnain SS. Can hair be used to screen for breast cancer? [letter]. J Med Genet 2000;37:297–8.[Free Full Text]

4 Briki F, Busson B, Salicru B, Esteve F, Doucet J. Breast-cancer diagnosis using hair [letter]. Nature 1999;400:226.[Medline]

5 Wilk KE, James VJ, Amemiya Y. The intermediate filament structure of human hair. Biophys Biochim Acta 1995;1245:392–6.

6 James VJ, Wilk KE, McConnell JF, Baranov EP, Amemiya Y. Intermediate filament structure of {alpha}-keratin in baboon hair. Int J Biol Macromol 1995;17:99–104.[Medline]

Manuscript received September 28, 2000; revised March 15, 2001; accepted March 27, 2001.


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