Breast tissue diagnosis by Raman spectroscopy

B. Broek-Puska*, J.Surmacki*, J. Jaboska**, R. Kordek**, H. Abramczyk*

*Laboratory of Laser Molecular Spectroscopy, Institute of Applied Radiation Chemistry, Technical University of d, Faculty of Chemistry, Poland **Department of Oncology, Medical University of d, Poland

GOAL
Some substances are produced by the organism in response to the cancers presence. They are called tumor markers. Tumor markers are molecules occurring in blood or tissue that are associated with cancer and whose measurement or identification is useful in patient diagnosis or clinical management. The ideal marker would be a blood test for cancer in which a positive result would occur only in patients with malignancy, one that would correlate with stage and response to treatment and that was easily and reproducibly measured. No tumor marker now available has met this ideal.

We believe that optical methods including Raman spectroscopy will provide such a marker. Possibly it will help to find the hallmarks of cancer.

Why ?
immediate in vivo diagnosis reduction the number of biopsies

combination of biochemical and histopathological diagnosis provides more information because pathology is intimately related to biochemistry
method has a potential to remove human interpretation non-invasive,non-ionizing method that probes with chemical specificity vibrations and fluorescence (not just structure) extremaly high spatial resolution (optical imaging)

Tissue Preparation

carcinoma mammae
20000

fibroadenoma mammae

20000

514nm, 100mW, cancer

26000

10000

0 15000

24000

Intensity (arbitrary units)

22000

10000
20000

maligant tissue

18000

5000
16000

14000

0
12000

500

1000

1500

2000

2500

3000
-1

3500

4000

Raman shift in wavennumbers [cm ]

normal tissue
514nm, normal tissue
35000
5000 8000

30000
0 6000

25000

100mW
4000

20000

15000

2000

10000

25mW
0

5000

500

1000

1500

2000

2500

3000
-1

3500

4000

Raman shift in wavennumbers [cm ]

Human speciemens obtained from surgery. Upon removal during the operation, the ex vivo sample is devided by a doctor into two parts, one goes to our lab the second goes to the pathology examination The ex vivo samples are neither frozen in liquid nitrogen for storage nor fixed in formalin, the fresh tissue is measured immeditely after delivering from the hospital The samples for pathology measurements are passively thawed at room temperature and kept moist with PBS fixed in formalin Cut through the marked locations into 5-m-thick sections, and stained with eosin

Intensity (arbitrary units)

Measurements Parameters
The laser excitation is 514 nm, the spot is d=500 m in diameter Light diffusion in the tissue results in a spot of v1mm Integration time 0.5 s Spectral resolution 2 and 8 cm -1

The laser excitation power: 25 mW, 100mW

Patients Statistics
0014 KT - normal tissue 4000

Intensity [counts/s]

1100 spectra from 100 patients

3000

2000

fibroadenomas infiltrating carcinoma infiltrating ductal carcinoma (IDC) infiltrating lobular carcinoma (ILC) IDC+ILC multifocal carcinoma carcinoma microinvasive intracystic papillary carcinoma (noninvasive) carcinoma mucinosum carcinoma intraductal microinvasive benign dysplasia dysplasia benign dysplasia (cystes, apocrinal metaplasia, adenosis) ductal-lobular hyperplasia cystic mastopathy
0 0
514 541 572 607 647

1000

25mW

1000 2000 3000 4000 5000 6000 7000 8000 -1 relative wavenumber [cm ]
691 743 802 872 [nm]

0014 KT - carcinoma lobulare infiltrans mammae

4000

Intensity [counts/s]

3000

2000

1000

25mW

1000 2000 3000 4000 5000 6000 7000 8000 -1 relative wavenumber [cm ]
541 572 607 647 691 743 802 872 [nm]

514

Comparison between the Raman spectra for normal and malignant tissue (infiltrating ductal carcinoma (IDC))

0006 KT - normal tissue 15000 12500 10000 7500 5000 2500

25mW

15000 12500
Intensity [counts/s]

0006 KT - carcinoma ductale infiltrans G2

Intensity [counts/s]

10000 7500 5000 2500

25mW

0 0
514

1000 2000 3000 4000 5000 6000 7000 8000 -1 relative wavenumber [cm ]
541 572 607 647 691 743 802 872 [nm]

0 0
514

1000 2000 3000 4000 5000 6000 7000 8000 -1 relative wavenumber [cm ]
541 572 607 647 691 743 802 872 [nm]

Quenching of autofluorescence by low temperature Raman spectroscopy

normal tissue - patient nr 90

2500 2000
intensity (cts/s)

K90T33 - 250K K90T34 - 200K K90T35 - 170K K90T36 - 150K K90T37 - 110K K90T38 - 77K

cancer tissue - patient nr 90

600 500
intensity 9cts/s)

K90T14 - 250K K90T15 - 200K K90T16 - 170K K90T17 - 150K K90T18 - 110K K90T19 - 77K

1500 1000 500 0 -500 0 500 1000 1500 2000 2500 3000 3500 4000
wavenumber (cm-1)

400 300 200 100 0 -100 0 500 1000 1500 2000 2500 3000 3500 4000
wavenumber (cm-1)

Comparison between the Raman spectra for normal and malignant tissue (carcinoma mucinosum )

5000 4500 4000

K14T2 (carcinoma mucinosum mammae sinistri) K14T2 (normal tissue)

Title Y Axis intensity [cts/s]

3500 3000 2500 2000 1500 1000 500 0 -500 0 2000 4000 6000 8000

X Axis [1/cm] Title wavenumber

Comparison between the Raman spectra for normal and benign tumour tissue (fibroadenoma)

K31T5 (normal tissue) K31T8 (Fibroadenoma mamae)

1800 1600 1400

Title Y Axis intensity [cts/s]

1200 1000 800 600 400 200 0 0 2000 4000 6000 8000

wavenumber [1/cm]

X Axis Title

PRINCIPAL COMPONENT ANALYSIS (PCA)

PLS_Toolbox Version 4.0 for use with MATLAB
Barry M. Wise Neal B. Gallagher Rasmus Bro Jeremy M. Shaver Willem Windig R. Scott Koch

Eigenvector Research, Inc. 3905 West Eaglerock Drive Wenatchee, WA 98801

helpdesk@eigenvector.com www.eigenvector.com

PCA score plot mean center, SNV, 1-st derivative

Low autofluorescence
0.4 0.2 w w Samples/Scores Plot of HY w c ww cw hh c cc w cw cc c wh c cc c c h h cc h h h c w hw c w c c c 60 hc h h h c h h h h 30 w h h h h 40 hh h w h h

Scores on PC 2 (7.70%)

0 -0.2 -0.4 -0.6 -0.8 -1 -1.2

50

Normal tissue

Malignant tissue
c h

Benign tissue

20

10 c -0.5 0 0.5 1 1.5 2

-1.4 -1 High autofluorescence Decluttered

Scores on PC 1 (66.25% )
Intensive Raman peaks

No Raman peaks

12

PCA loading plot mean center, SNV, 1-st derivative

Variables/Loadings Plot for HY 0.2 0.15 C-C and C=C stretching carotenoids C-H streching lipids 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 -0.1 0.2 -0.15 -0.2 0.1 500 1000 1500 2000 2500 wavenumber (cm-1) 3000 3500 0

Loadings on PC 1 (66.25%)

0.1 0.05 0 -0.05

CONCLUSIONS
The results clearly illustrates the ability of Raman spectroscopy to accurately diagnose breast cancer and demonstrates how the diagnostic scheme can be adjusted to obtain the desired degree of sensitivity and specificity (88%,72%) The normal tissue has a characteristic bands: C-C (1110 cm-1) and C=C (1520 cm-1) stretching bands of carotenoids and at 2840-2900 cm-1 for the C-H symmetric and asymmetric bands of lipids (fat) which are not visible in the malignant tissue and in benign tumor tissue. Moreover, the fluorescence is much higher in the malignant tissue.

We belive that in a very near future a good quality Raman signal will be obtained with the optical fibers coupled with a biopsy needle and incorporated into Raman spectrometer for breast tissue measurements in vivo.