CD34 as a marker of angiogenesis in breast cancer A study of 74 cases

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 751 IJAMSCR |Volume 5 | Issue 3 | July-Sep – 2017 www.ijamscr.com Review article Medical research CD34 as a marker of angiogenesis in breast cancer: A study of 74 cases with brief review of literature 1 Azka Anees Khan 2 Kiran Alam 2 Hasan Harris 1 MD Pathology at JN Medical College AMU Aligarh 2 Professor Department of Pathology J. N. Medical College AMU Aligarh 2 Professor Department of Surgery J. N. Medical College AMU Aligarh Corresponding Author: Azka Anees Khan Email id: azka.asquare.aneesgmail.com ABSTRACT CD34 was studied as a marker of angiogenesis in breast cancer. 74 cases of IDC were studied for this purpose. The mean microvessel density grade and the mean microvessel count per 400X field were determined for each case. Further these 2 parameters were compared between node negative and node positive cases in order to determine whether increased angiogenesis would increase the chances of metastasis to lymph nodes. It was observed that cases with lymph node metastasis showed increased angiogenesis with a mean microvessel count and microvessel density grade of 45.6 and 2.91 respectively. In comparison cases with absence of lymph node metastasis showed less angiogenesis and the mean microvessel count and microvessel density grade was 16.15 and 1.05 respectively. On statistical analysis the difference between both the microvessel count and microvessel density grade for the two groups came out to be highly significant p0.001. Hence increased angiogenesis was seen to lead to increased chances of metastasis to lymph nodes in our study. Keywords: CD34 Angiogenesis Breast Breast cancer. INTRODUCTION There is considerable experimental evidence to prove that tumor growth is dependent on angiogenesis 1. Thus after a new tumor has attained a small size of few millimeters in diameter about 10 6 cells further expansion of the tumor cell population requires the generation of new vessels. These new vessels also increase the opportunity of tumor cells to enter the blood circulation 2. The first quantitative evidence that intensity of angiogenesis in a human tumor could predict the probability of metastasis was reported for cutaneous melanoma 3. Later many studies demonstrated the same in breast carcinoma 4 5 6 7 8. Different studies have used different markers to highlight blood vessels in breast carcinoma in order to measure angiogenesis. The different antibodies that can be used are factor VIII anti CD31 and anti CD34. Martin et al 1997 conducted a Meta- analysis and showed that anti-CD34 gave the most significant results of the three antibodies for ISSN:2347-6567 International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 752 examining angiogenesis in cases of breast carcinoma 9. CD34 has been widely used as a marker to assist in the identification and isolation of hematopoietic stem cells HSCs and progenitors in preparation for bone-marrow transplantation more recently it has been employed as a marker to help identify other tissue-specific stem cells including muscle satellite cells and epidermal precursors. Notably however the function of CD34 and its family members has not yet been definitively determined although several roles have been ascribed to the proteins. CD34 is a transmembrane highly glycosylated protein expressed by hematopoietic stem/progenitor cells HPSCs 10 endothelial cells 11 and mesenchymal cells at several different sites including breast 12. It is thought to be involved in the modulation of cell adhesion and signal transduction 10. Known tissue and cell types where CD34 is distributed include 13  Multipotent precursors: HSCs multipotent haemopoeitic progenitors  Mast cells  Eosinophils  Muscle satellite cells  Hair follicle stem cells  Vascular endothelia  Fibrocytes  Neurons +/- REVIEW OF LITERATURE Angiogenesis or neovascularization consists in the formation of new blood vessels from the endothelium of the existing vasculature. When a new tumor reaches the size of 1–2 mm its ulterior growth requires the induction of new blood vessels which may consequently lead to the development of metastases via the penetration of malignant cells into the circulation. Vascular endothelial growth factor VEGF platelet-derived endothelial cell growth factor and basic fibroblast growth factor produced by tumor and stromal cells are potent inducers of the angiogenic switch. However angiogenesis is necessary but not sufficient to the development of metastases. Angiogenic activity is heterogeneous within a given tumor type. Concerning the relationship between angiogenesis and clinical outcome breast cancer has been the most studied tumor. For ˃10 years microvessel density MVD a surrogate marker of tumoral angiogenesis has been proposed to identify patients at high risk of recurrence more precisely than classical indicators. The identification of such patients at an early stage of their disease would be of great interest allowing for a more appropriate and effective treatment by adjuvant chemotherapy or by specific antiangiogenic drugs of patients at higher risk and possibly predicting the activity of these latter drugs. Microvessel density assessment is the most commonly used technique to quantify Intratumoral angiogenesis in breast cancer. It was first developed by Weidner et al. in 1991 and uses panendothelial immunohistochemical staining of blood microvessels. CD34 is a known endothelial cell marker and is usually used for calculating microvessel density. The first step in Weidner’s approach is the identification by light microscopy of the area of highest neovessel density the so called “hot spot” by scanning the whole tumoral section at low power. Then individual microvessels are counted at a higher power X200 field in an adequate area e.g. 0.74 mm 2 per field using X20 objective lens and X10 ocular. Any stained endothelial cell or clusters separate from adjacent vessels are counted as a single microvessel even in the absence of vessel lumen. Each single count is expressed as the highest number of microvessels identified at the hot spot 4. Some authors use Chalkley count or computerized image analysis systems both aimed to minimize the subjectivity in the quantification of MVD. The Chalkley count consists of applying a 25-point eyepiece graticule on several hot spots usually 3. The graticule is oriented to allow the maximum number of points to hit on or within the areas of stained microvessel profiles Chalkley grid area: 0.196mm 2 14. Later in the year 1994 R. K. Vartanian and Weidner published a study titled” Correlation of intratumoral endothelial cell proliferation with microvessel density tumor angiogenesis and tumor cell proliferation in breast carcinoma” 15. This study aimed to establish how intratumoral microvessel density correlates with tumor cell and intratumoral endothelial cell proliferation. The results of this study indicated no correlation between tumor cell proliferation determined by

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 753 tumor cell Ki67 labelling index and endothelial cell proliferation determined by endothelial cell Ki67 labelling index. CD34 was also used to label microvessesls. Endothelial cell Ki67 labelling index did not correlate with intratumoral microvessel density or mitotic figure index nor did intratumoral microvessel density correlate with tumor cell Ki67 labelling index or mitotic figure index. The authors concluded that intratumoral microvessel density endothelial cell proliferation and tumor cell proliferation may be regulated by separate mechanisms. Hence it was becoming clear at this point that angiogenesis as determined by microvessel density grade and microvessel count may be a definite prognostic factor in women with breast cancer. However the logical conclusion that increased tumor cell proliferation would directly lead to increased endothelial cell proliferation higher microvessel counts and increased angiogenesis was disproven by this study. In the year 1995 Siitonen et al conducted a study called “Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas”. They compared the different antibodies used to stain microvessels and the different quantitative methods used to measure angiogenesis. Anti-CD34 and anti- VWF showed better staining performances than anti-CD31 although the staining results with different antibodies were comparable. Two different methods of microvessel quantitation the highest microvessel count and percentage microvessel area were evaluated and also showed significant correlation. However they found that neither highest microvessel counts nor microvessel area measurements quantitated with anti-CD34 or anti-VWF immunohistochemistry were able to discriminate between favourable and unfavourable outcome patients. They challenged the earlier view saying that further evidence was still needed on tumor angiogenesis immunohistochemistry before it could be adopted as a prognostic marker in routine clinical practice 16. In 1996 Heimann et al again proved the prognostic significance of angiogenesis in breast cancer by determining microvessel count MVC after highlighting microvessels by CD34. They correlated angiogenesis with clinical outcome in a series of patients with axillary lymph node-negative breast cancer who received no adjuvant therapy and who were followed for a long period of time. They found that angiogenesis as measured by MVC is a reliable independent prognostic marker of long- term survival in patients with node-negative breast cancer 17. In 1998 concerns were raised by Hansen et al about the observer variability of methods for determining microvessel density. The microvessel endothelium was stained immunohistochemically by antibodies against CD34. The investigated methods included Chalkley counting estimation of intratumoral microvessel density MVD by one hot-spot MVD by the mean value of three hot- spots and the highest value of MVD in three hot- spots. Each of forty tumors was measured with all methods twice by the same observer and once by another observer. They found that the Chalkley and MVD methods had moderate reproducibility and the Chalkley method had low variation due to observers alone. They concluded that the Chalkley method has less observer variation and may be superior from a methodologic point of view 18. A new antibody was brought into focus in 1999 when Kumar et al assessed the microvessel density IMD in 106 breast carcinomas using a pan- endothelial marker CD34 and a new mAb to CD105 which preferentially reacts with endothelial cell in angiogenic tissues. IMD values for CD105 expression showed a statistically significant correlation with overall P 0.0029 and disease- free survival P 0.0362. In contrast blood vessel counts using a pan-endothelial marker CD34 did not correlate with overall or disease-free survival P 0.2912 and P 0.3153 respectively. Multivariate analysis confirmed that IMD values using CD105 were an independent prognostic factor. The authors concluded that the ability to quantitatively distinguish between tumor neovascularization and preexisting vessels may be important in the assessment of tumor angiogenesis but requires confirmation in a greater number of patients with a longer follow-up 19. In 2000 Hansen et al conducted a study “Vascular grading of angiogenesis: prognostic significance in breast cancer” and again concluded that the angiogenesis determined by vascular grading after staining for CD34 has independent prognostic value of clinical relevance for patients with breast cancer 20. In 2004 Uzzan et al performed a meta-analysis of all 87 published studies linking intratumoral

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 754 microvessel density MVD reflecting angiogenesis to relapse-free survival RFS and overall survival OS. They found that MVD was a significant although weak prognostic factor in women with breast cancer 21. In 2011 Mikalsen et al sought to decrease variability associated with different methods of microvessel quantification by introducing the method of automated vessel identification in CD34 immunohistochemical sections. They found this method to be highly reliable and recommended its use on colour photographs of staining for CD34 to quantify angiogenesis 22. To summarize many observational studies either prospective or retrospective studies have concluded that MVD is a prognostic factor in invasive breast cancer but others reached the opposite conclusion. There have been concerns over the type of antibodies used and the method used for quantification. Therefore although there is a consensus among most scholars that angiogenesis is an important factor determining outcome in women with breast cancers the lack of standardization and high observer variability associated with different methods preclude its current clinical use as a prognostic factor. MATERIALS AND METHODS The present study was carried out on 74 patients of infiltrating ductal carcinoma attending the OPD/IPD of Department of Surgery and on the histopathological specimens received in the Department of Pathology J. N. Medical College AMU Aligarh. The study was conducted over a period of 2 years from July 2012 to June 2014. A detailed clinical history and examination along with routine investigations were carried out in each case. Post-surgical specimens included mastectomies and a few biopsies. Gross examination of all the specimens was performed and sections were taken from the representative areas. They were processed by an automatic tissue processor Histokinette. Blocks were prepared in paraffin wax with the help of Paraffin Embedding Station. Sections were cut at 4-5 µm thickness with the help of rotary microtome. All the cases were stained by Haematoxylin and Eosin and examined microscopically. Further CD34 immunostaining was done on all the cases. Immuno-histochemistry for CD34 was performed on paraffin embedded tissue sections using the kit Thermo Scientific CD34 Clone QBEnd/10. The antibody provided is prediluted and ready to use. Tissue was first microwaved in citrate buffer at pH- 6.0 95ºC 10 min for antigen retrieval. This was followed by peroxide block protein block and incubation in primary antibody for 20 minutes at room temperature. Further incubation with HRP polymer and DAB was done. Finally counterstaining with haematoxylin was done and dried mounted slides were examined microscopically. Staining for CD34 highlighted all the blood vessels. The method used for microvessel counting was modified from Weidner et al. 1991. Tumors were frequently heterogeneous in their vessel density however the area of highest neovascularization was found by scanning the tumor section at low power 40X and 100X and identifying the areas of invasive carcinoma with the highest number of discrete microvessels staining for CD34. After the area of highest neovascularization was identified it was subjectively graded on a scale of +1 to +4. This was called the microvessel density grade .Then the individual microvessels were counted on a 400x microscopic field i.e. 40X objective lens and 10X ocular lens. This was called the microvessel count. Any brown staining endothelial cell or endothelial cell cluster that was clearly separate from adjacent microvessels tumor cells and other connective tissue elements was considered a single countable microvessel. Vessel lumina were not necessary for a structure to be defined as a microvessel 4. Hence a value for microvessel density grade and microvessel count was determined for each case. This data was analysed statistically to determine if there was a significant difference in the values for node positive and node negative patients. The test of significance used was student`s t test. A p value of less than 0.05 was considered as significant. OBSERVATIONS It was observed that cases with lymph node metastasis showed increased angiogenesis with higher values of microvessel count and microvessel density grade Fig.1. In comparison cases with

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 755 absence of lymph node metastasis showed less angiogenesis and low microvessel count and microvessel density grade Fig. 2 Microvessel density grade microvessel count tumor grade tumor size and age of patients were compared between the node negative and node positive group. Difference in age and tumor grade was not found to be significant while the rest of the parameters were significantly different in the 2 groups as described below: Patients with breast carcinoma showing lymph node metastasis had a mean age of 48.68 while those without metastasis had the corresponding value of 48.08. No significant difference was found to be present between the two groups on statistical analysis p 0.05. The mean tumor grade was 2.17 in patients with lymph node metastasis while it was 2 in patients without metastasis. The difference in the tumor grade between the two groups was not found to be statistically significant. Microvessel density grade microvessel count and tumor size were found to be significantly different between node positive and node negative patients. As shown in table 1 the mean microvessel density grade was 2.91 in node positive cases with a median of 3. In comparison the value in node negative patients was 1.05 with a median of 1. Statistical analysis revealed the difference between the 2 groups to be significant p 0.001. Also in patients with metastasis to lymph nodes the mean microvessel count was 45.6 per 400X field. In those without metastasis the corresponding value was 16.15. Statistical analysis revealed the difference between these 2 groups also to be highly significant p 0.001. The mean tumor size was 6.54 cm. in cases with lymph node metastasis in comparison to 4.63 cm. in those without metastasis. Statistical analysis revealed the difference in tumor size between the two groups to be statistically significant p0.001. Hence it was observed that cases with lymph node metastasis had increased angiogenesis seen in the form of high microvessel density grade and microvessel count and higher mean tumor size. Table 1: Clinico-histological characteristics CD34 expression in 74 breast cancer cases with or without lymph node metastasis. Values expressed as mean ± standard deviation median range. CHARACTERISTIC METASTASIS – n39 METASTASIS+ n35 P VALUE Microvessel density grade 1.05 ± 0.22 1 1-2 2.91 ± 0.56 3 1-4 0.001significant Microvessel count per 400x field 16.15 ± 3.29 16 10-22 45.6 ± 9.35 49 25-65 0.001significant Tumor grade 2 ± 0.73 21-3 2.17 ± 0.62 2 1-3 0.146notsignificant Tumor size cm. 4.63 ± 2.38 4 1-12 6.54 ± 3.07 6 1-16.5 0.003significant Age yrs 48.08 ± 11.61 50 25-70 48.68 ± 11.29 48 32-80 0.42not significant

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 756 Fig. 1: A case of infiltrating ductal carcinoma with DCIS showing high microvessel densityCD3440X Fig. 2: A case of infiltrating ductal carcinoma with DCIS showing low microvessel density CD3440X DISCUSSION Our findings were similar to those of Weidner et al 1991 who showed a significant correlation between the density of microvessels in histologic sections of invasive breast carcinoma and the occurrence of metastases. However the marker used by Weidner to highlight the microvessels was factor VIII instead of CD34. A significant correlation was also found between the size of IDC and lymph node metastasis. The mean tumor size was 6.54cm. for

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 757 cases with lymph node metastasis in comparison to 4.63cm. in those without metastasis. Statistical analysis revealed the difference in tumor size between the two groups to be statistically significant p0.001. It is already well known that the measured gross size represented by the largest diameter of a mammary carcinoma is one of the most significant prognostic variables. Numerous studies have shown that survival decreases with increasing tumor size and that there is a coincidental rise in the frequency of axillary zodal metastases 5678. Hence our findings are consistent with different researchers earlier. Although there has been much research in this field with newer antibodies and automated procedures introduced to detect angiogenesis in breast cancers we sought to find out whether the oldest and simplest method introduced by Weidner et al still hold true. As suggested by our findings it is obvious that this method showed a significant correlation between angiogenesis and lymph node metastasis. However since this study was retrospective and did not involve follow up of the patients we cannot comment on the effect of angiogenesis on patient outcome. The role of CD34 as a marker of angiogenesis in breast cancer is still undecided. There are many studies which have proven that estimating angiogenesis via CD34 microvessel staining is a good method to predict patient outcome. However there are also few with conflicting views on the topic. We are of the view that CD34 can definitely be used to measure angiogenesis in breast cancer and in turn can be utilized as a predictor of lymph node metastasis. CONCLUSION CD34 can be used for determining angiogenesis in breast cancer. Increased angiogenesis was seen to lead to increased chances of metastasis to lymph nodes in our study. REFERENCES 1. Folkman J. What is the evidence that tumors are angiogenesis dependent J Natl Cancer Inst 8219904 -6. 2. Liotta L Kleinerman J Saidel G. Quantitative relationships of intravascular tumor cells tumor ve ssels and pulmonary metastasis following tumor implantation. Cancer Res 34 1974 997- 1004. 3. Srivastava A Laidler P Davies RP Horgan K Hughes LE. The prognostic significance of tumor vascuarity in intermediate thickness skin melanoma: a quantitative histological study. Am J Pathol 133 1988 419-423. 4. Weidner N Semple JP Welch WR Folkman J. Tumor angiogenesis and metastasis– correlation in invasive breast carcinoma.N Engl J Med 324 1991 1– 8. 5. Yoshimoto M Sakamoto G Ohashi Y. Time dependency of the influence of prognostic factors on relapse in breast cancer. 72 1993 2993–3001. 6. Adair F Berg J Joubert L et al. Long term follow-up of breast cancer patients. The 30-year report. Cancer 33 1974 1145–1150. 7. Say CC Donegan WL. Invasive carcinoma of the breast: Prognostic significance of tumor size and involved axillary lymph nodes. Cancer 34 1974 468–471. 8. Smart CR Myers MH Gloecker LA. Implications for SEER data on breast cancer management. Cancer 41 1978 787–789. 9. L Martin B Green C Renshaw D Lowe P Rudland SJ Leinsterl and J Winstanley. Examining the technique of angiogenesis assessment in invasive breast cancer. British Journal of Cancer 768 1997 1046-1054 10. Van de Rijn M Rouse RV. CD34. A review. Appl Immunohistochem 2 1994 71–80 11. Fina L Molgaard HV Robertson D et al. Expression of the CD34 gene in vascular endothelial cells. Blood 12 1990 2417–26. 12. Yamazaki K Eyden BP. Ultrastructural and immunohistochemical observations on intralobular fibroblasts of human breast with observations on the CD34 antigen. J Submicrosc Cytol Pathol 27 1995 309–323. 13. Julie S. Nielsen Kelly M. McNagny .Novel functions of the CD34 family. Journal of Cell Science 121 2008 3683-3692.

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Azka A K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-53 2017 751-758 758 14. Chalkley HW. Method for the quantitative morphologic analysis of tissues. J. Natl. Cancer Inst 4 1943 47–53. 15. Vartanian RK Weidner N. Correlation of intratumoral endothelial cell proliferation with microvessel density tumor angiogenesis and tumor cell proliferation in breast carcinoma. The American Journal of Pathology. 1446 1994 1188-1194. 16. Siitonen SM Haapasalo HK Rantala IS Helin HJ Isola JJ . Comparison of different immunohistochemical methods in the assessment of angiogenesis: lack of prognostic value in a group of 77 selected node-negative breast carcinomas. Mod Pathol 7 1995 745-752. 17. Ruth Heimann Donald Ferguson Claire Powers Wendy M. Recant Ralph R. Weichselbaum Samuel Hellman. Angiogenesis as a Predictor of Long-term Survival for Patients With Node-Negative Breast Cancer JNCI: Journal of the National Cancer Institute 8823 1996 1764–1769. 18. Hansen S Grabau DA Rose C Bak M Sørensen FB. Angiogenesis in breast cancer: a comparative study of the observer variability of methods for determining microvessel density. Lab Invest 7812 1998 1563 - 1573. 19. Kumar Shant et al. Breast Carcinoma. Cancer Research 1999 856-861. 20. Hansen S Grabau DA Sørensen FB Bak M Vach W Rose C. Vascular grading of angiogenesis: prognostic significance in breast cancer. British Journal of Cancer. 822 2000 339-347. 21. Uzzan Bernard et al .Microvessel Density as a Prognostic Factor in Women with Breast Cancer. Cancer Research 2004 2941-2955. 22. Lars Tore Gyland Mikalsen Hari Prasad Dhakal Oyvind S. Bruland Jahn M. Nesland Dag Rune Olsen. Quantification of Angiogenesis in Breast Cancer by Automated Vessel Identification in CD34 Immunohistochemical Sections. Anticancer Research December 3112 2011 4053-4060. How to cite this article: Azka Anees Khan Kiran Alam Hasan Harris. CD34 as a marker of angiogenesis in breast cancer: A study of 74 cases with brief review of literature. Int J of Allied Med Sci and Clin Res 2017 53: 751-758. Source of Support: Nil. Conflict of Interest: None declared.

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