Comparison between photoacoustic mammography images of breasts cancers and histological staining patterns of CD31 and carbonic anhydrase IX

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1 Comparison between photoacoustic mammography images of breasts cancers and histological staining patterns of CD31 and carbonic anhydrase IX Poster No.: C-1893 Congress: ECR 2013 Type: Scientific Exhibit Authors: I. Yamaga, Y. asao, M. Torii, E. Fakharejahani, M. Takada, S. Kanao, M. Kataoka, T. Shiina, M. Toi; Kyoto/JP Keywords: DOI: Cancer, Laser, Ultrasound, Mammography, Vascular, Breast /ecr2013/C-1893 Any information contained in this pdf file is automatically generated from digital material submitted to EPOS by third parties in the form of scientific presentations. References to any names, marks, products, or services of third parties or hypertext links to thirdparty sites or information are provided solely as a convenience to you and do not in any way constitute or imply ECR's endorsement, sponsorship or recommendation of the third party, information, product or service. ECR is not responsible for the content of these pages and does not make any representations regarding the content or accuracy of material in this file. As per copyright regulations, any unauthorised use of the material or parts thereof as well as commercial reproduction or multiple distribution by any traditional or electronically based reproduction/publication method ist strictly prohibited. You agree to defend, indemnify, and hold ECR harmless from and against any and all claims, damages, costs, and expenses, including attorneys' fees, arising from or related to your use of these pages. Please note: Links to movies, ppt slideshows and any other multimedia files are not available in the pdf version of presentations. Page 1 of 16

2 Purpose Photoacoustic tomography using near-infrared light can describe hemoglobin distribution and oxygenation state inside tissue non-invasively with high spatial resolution. We have already reported our initial study with photoacoustic mammography (PAM). Breast cancer images of PAM were compared with those of magnetic resonance imaging[1]. In this report, we compared PAM images with CD31 and carbonic anhydrase (CA) IX staining patterns. CD31 is one of the markers for endothelial cell of blood vessel, and CAIX is a membraneassociated CA, strongly induced by hypoxia[2,3]. The purpose in this report is to examine the correlation between vessel density, hypoxia and PAM images. Methods and Materials A prototype machine of a dual illuminated mode photoacoustic tomography system (Canon Inc., Tokyo, Japan) was used for PAM[1]. Fig. 1 on page 3 shows a photograph of the PAM and a schematic of the measurement. 38 patients diagnosed with breast cancer were assessed with PAM before surgery from August 2010 to March 2012 in Kyoto university hospital. In these cases, we report 4 patients whose PAM images and histological stainings were comparable. Four surgical resection specimens with invasive ductal carcinoma were analyzed. Tissues were fixed in 10% formalin and embedded in paraffin wax. Five-micrometre consecutive sections were used for the haematoxylin and eosin (HE) staining and for the immunohistochemistry (IHC). Number and density of blood vessels in tumor were quantified by CD31 IHC staining. Taking digital image of IHC section, CD31 positive cells were extracted by thresholding whole image pixels (comuptational process called "binarization"). CD31 positive structure were automatically detected after optimizing the threshold utilizing "Image-Pro Plus ver.6.3 ( Media Cybernetics, Inc.)". Counts of stained vessels were mapped for each tumor. CAIX IHC stainings were categorized positive versus negative. Page 2 of 16

3 Images for this section: Page 3 of 16

4 Page 4 of 16

5 Fig. 1: a) A photograph of the PAM. b) A Schematic of the PAM measurement: A patient lies in prone position on the bed with her breast placed in the hole. The breast is mildly compressed in craniocaudal direction between holding plates. Page 5 of 16

6 Results Four cases are presented in. All cases were diagnosed as invasive ductal carcinoma. PAM signals were significant at the tumor parts in 3 patients out of 4 (case1,2,3: Fig. 2 on page 6, Fig. 3 on page 8, Fig. 4 on page 10). In these cases the numbers of tumor vessels were more than that of normal parts. PAM signal of the other patient was not significant and interestingly the number of tumor vessels was not significantly increased compared to normal parts (case4: Fig. 5 on page 12). However, blood supplying vessels to the tumor could be detected in this case. Out of the 3 patients who had significant signals of PAM at the tumor sites, CAIX was positive in 2 cases (case1,2: Fig. 2 on page 6, Fig. 3 on page 8). Their oxygen saturation (SO 2 ) were 53.8, 68.6% at the tumor cites, and 90.6, 88.1% at the same depth in the contralateral normal breast, respectively. In the other case out of 3, CAIX was negative (case3: Fig. 4 on page 10). SO 2 was 93.5% in the cancer part and 93.2% in the contralateral breast. For the case that PAM signal was not detected in the tumor, SO 2 from vessels around the tumor was calculated 79.0% (case4: Fig. 5 on page 12). It was lower than 84.9% at the same depth in the contralateral normal breast. CAIX was positive at the tumor. Images for this section: Page 6 of 16

7 Fig. 2: Case1. Yellow dotted lines show tumor site. a) PAM image: Signals were detected at the tumor. b) PAM Oxygen saturation image: SO2 of tumor part was calculated as 53.8%, and SO2 of the same depth in the contralateral normal breast was 90.6%. c) HE stain d) CD31 counting map: Number of vessels was increased at the tumor site. Page 7 of 16

8 e) CAIX IHC stain: positive PAM: Photoacoustic mammography, SO2:Oxygen saturation HE: Haematoxylin and eosin, CA: Carbonic anhydrase, IHC: Immunohistochemistry Page 8 of 16

9 Page 9 of 16

10 Fig. 3: Case2. Yellow dotted lines show tumor site. a) PAM image: Signals were detected at the tumor. b) PAM SO2 image: SO2 was 93.5% at the tumor, and 93.2% at the same depth in the contralateral normal breast. c) HE stain d) CD31 counting map: Number of vessels was increased at the tumor site. d) CAIX IHC stain: Negative PAM: Photoacoustic mammography, SO2:Oxygen saturation HE: Haematoxylin and eosin, CA: Carbonic anhydrase, IHC: Immunohistochemistry Page 10 of 16

11 Page 11 of 16

12 Fig. 4: Case3. Yellow dotted circle s show tumor site. a) PAM image: Signals were detected at the tumor. b) PAM SO2 image: SO2 were 68.6% at lower half of the tumor, and 88.1% at the same depth in the contralateral normal breast. b) HE stain c) CD31 counting map: Number of vessels was increased at the tumor site. d) CA IX IHC stain: Positive at ductal carcinoma in situ. PAM: Photoacoustic mammography, SO2:Oxygen saturation HE: Haematoxylin and eosin, CA: Carbonic anhydrase, IHC: Immunohistochemistry Page 12 of 16

13 Page 13 of 16

14 Fig. 5: Case4. Yellow dotted lines show tumor site. a) PAM image: No signal was detected at the tumor. Signals from vessels around the tumor were detected. b) PAM SO2 image: SO2 were 79.0% at the tumor, and 84.9% at the same depth in the contralateral normal breast. c) HE stain d) CD31 counting map: Number of vessels was not significantly increased at the tumor. e) CA IX IHC stain: Positive at the tumor. PAM: Photoacoustic mammography, SO2:Oxygen saturation HE: Haematoxylin and eosin, CA: Carbonic anhydrase, IHC: Immunohistochemistry Page 14 of 16

15 Conclusion We compared PAM images with CD31 and CAIX staining patterns. This preliminary analysis shows that PAM signal might be more significantly visible in the cases where the tumor is more angiogenic. Further investigation is required to confirm correlation of PAM signals with vessel density and hypoxia. References 1)Kitai T, Torii M, Sugie T, Kanao S, Mikami Y, Shiina T, Toi. Photoacoustic mammography: initial clinical results. Breast Cancer 2012; Apr 7. [Epub ahead of print] 2)Charles C. Wykoff, Nigel J. P. Beasley, Peter H. atson, Kevin J. Turner, Jaromir Pastorek, Amen Sibtain, George D. Wilson, Helen Turley, Kate L. Talks, Patrick H. Maxwell,Christopher W. Pugh, Peter J. Ratcliffe2, andadrian L. Harris. Hypoxia-inducible Expression of Tumor-associated Carbonic Anhydrases. Cancer Res 2000; 60: )Chiche J, Ilc K, Laferrière J, Trottier E, Dayan F, Mazure NM, Brahimi-Horn MC, Pouysségur J. Hypoxia-inducible carbonic anhydrase IX and XII promote tumor cell growth by counteracting acidosis through the regulation of the intracellular ph. Cancer Res ; 69(1): Personal Information Iku Yamaga 1, Yasufumi Asao 1, Masae Torii 1, Elham fakharejahani 1, Masahiro Takada 1, Shotaro Kanao 2, Masako Kataoka 2, Tsuyoshi Shiina 3 and Masakazu Toi 1 1. Department of Breast Surgery, Graduate School of Medicine, Kyoto University 2. Department of Diagnostic Imaging and Nuclear Medicine, Graduate School of Medicine, Kyoto University 3. Department of Human Health Science, Graduate School of Medicine, Kyoto University yamaga@kuhp.kyoto-u.ac.jp Special thanks to Dr. Toshiyuki Kitai (Deparment of Surgery, Kishiwada City Hospital), Dr. Tomoharu Sugie ( Department of Breast Surgery, Graduate School of Medicine, Kyoto Page 15 of 16

16 University ),and Dr.Yoshiki Mikami (Department of Diagnostic Pathology, Graduate School of Medicine, Kyoto University) Page 16 of 16

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