1. The Cavitary” Type of Angiogenesis in Gastric and Breast Cancer. Clinical and Morphological Aspects Dr. Marina Senchukova, Associate Professor, Department of Oncology, Orenburg State Medical Academy, Russia 7th Global Summit on Cancer therapy

2. At 1971 year J. Folkman formulated his celebrated scientific concept that the tumor growth and metastasis depend on angiogenesis and lymphangiogenesis triggered by chemical signals from tumor cells. An angiogenesis activity correlated with the depth of tumor invasion, the presence of metastases in regional lymph nodes (RLN) and the prognosis of the disease (Ding S. et al, 2006; Ma J. et al, 2007; Lazar D. et al, 2008; Poon RT et al, 2003; Wang YD et al, 2007). The tumor vessels are heterogeneous in origin, morphology as well as in the clinical significance and in their sensitivity to the anti-angiogenic therapy (Dvorak et al, 1995; Nagy et al, 2012).

3. The “cavitary” type of angiogenesis I step - The formation of “cavitary” structures in tumor stroma or the adjacent gastric mucosa; II step - their lining by endothelial cells; III step - their merger into the blood vessels of the organ. * Senchukova, M. & Kiselevsky, M.V. (2014). The “Cavitary” type of angiogenesis by gastric cancer. Morphological characteristics and prognostic value, J Cancer, 5 (5), 311 – 319. * Senchukova M, Ryabov A, Karmakova T, Tomchuk O, Stadnikov A (2015) The Morphological Features of “Cavitary” Type Angiogenesis in Diffuse and Intestinal Types of Gastric Cancer and Its Relationship with Tumor-Infiltrating Immune Cells. BJMMR 7(4): 272-284. * Senchukova M, Nikitenko N, Tomchuk O, Zaitsev N, Stadnikov A. (2015). Different Types of Tumor Vessels in Breast Cancer. Morphology and Clinical Value. Springer plus 4; 512.

4. Characteristics of the patients with gastric cancer 73 patients with gastric cancer The average age was 61.2±9,3 years (from 34 to 78 years) The median – 61 years. 59 patients with T1-T2 stages of ductal invasive carcinomas. The average age was 58,1±10.1 years (from 35 to 75 years). The median – 57 years. The patients with decompensation of chronic diseases, acute infection pathology, severe allergic processes were not included in the study as well as the ones who received corticosteroids, antihistamines, non-steroidal anti- inflammatory drugs and neoadjuvant chemotherapy radiotherapy.

5. Clinicopathologic characteristics of gastric carcinoma cases Clinicopathologic variables Number of cases (n) Percent (%) ______________________________________________________________________ Gender Male4358.9 Female3041.1 Location of tumor Upper third1419.2 Middle third1824.7 Lower third3953.4 Total cancer22.7 Lauren classification Intestinal type4156.2 Diffuse type3243.8 Differentiation Well (G1)2736.9 Moderate (G2)1419.3 Poorly (G3-G4)912.3 Signet ring cell carcinoma2331.5 Nodal status (N) pN04359.9 pN1912.3 pN22128.8 Tumor status (T) pT11621.9 pT21824.7 pT33649.3 pT434.1 Stage (TNM) T1-2N0M03446.6 T3N0M0912.3 T3-4N1M0912.3 T3-4N2M02128.8

6. Surgery Subtotal distal resection – 56 patients (76.7%) Subtotal proximal resection – 10 patients (13.7%) Gastrectomy – 7 patients (9.5%) D2 lymphadenectomy - all patients With D3 elements – 38 patients (52.0%).

7. Clinicopathologic characteristics of breast carcinoma cases Clinicopathologic variables Number of cases (n) Percent (%) _________________________________________________________________________________ Age <501118.6 >504881.4 Tumor status (T) pT12135.6 pT23864.4 Nodal status (N) pN03050.8 pN11525.4 pN211.7 pN31322.1 Number of lymph nodes 03050.8 1-32033.9 4-6610.2 >635.1 Tumour grade G1711.9 G24271.2 G31016.9 ER status Negative 2949.1 Positive 3050. PR status Negative 3864.4 Positive 2135.6 HER2/neu status Negative 4881.4 Positive 1118.6

8. Surgery Modified radical mastectomy - 45 patients (76,2%) Breast-conserving surgery – 14 patients (23,8%), all with adequate lymph node dissection (at least 10 - 12 nodes were examined).

9. METHODS The specimens of gastric mucosa and tumor were stained with Mayer’s hematoxylin and eosin and by van Gieson. We used the follow antibodies for IGH:   anti-CD4;   anti-CD-8;   anti-CD20;   anti-CD68;   anti-CD34 The visualization system has included DAB and Hematoxylin counterstaining. For negative control sections, primary antibody was replaced with phosphate-buffered saline and processed in the same manner.

10. METHODS It was evaluated: The number of “cavitary” structures type-1 and atypical dilated vessels. These vessels were estimated by the visual analog way using x100 magnification (none, single – no more than two in the field of view, and multiple – more than two in the field of view). The presence of “cavitary” structures type-2 The density of CD4, CD8, CD20 lymphocytes and CD68 macrophages (on the relative area unit equal to 0.42 x 0.28 mm²). Microvessels density was assessed using antibodies to CD34, in accordance with the international consensus on the methodology and criteria for quantitative evaluation of angiogenesis in human solid tumors [Vermeulen PB]. The severity of polymorphonuclear cell infiltration (PCI) was calculated as density of cells on area unit equal to 0.42 x 0.28 mm²; The obtained data were compared with the clinical and morphological characteristics of gastric and breast cancer.

11. Statistical methods Spearman’s rank correlation or gamma correlation ( for the evaluation of the correlations between different data ); Chi-square test ( it was carried out to analyze the difference of distribution among the categorized data ). The survival was analyzed by the Kaplan-Meier method. Log-rank test ( it was used to compare survival curves between subgroups of patients ). Cox's proportional hazards model (it was used for multivariate analysis of prognostic factors). Odds ratio (OR) and a 95% confidence interval (CI) (this methods were used for the estimated the association between the 3-year survival and different types of vessels). A value of P< 0.05 was considered statistically significant.

12. The “cavitary” type of angiogenesis I step - The formation of “cavitary” structures in tumor stroma or the adjacent gastric mucosa; II step - their lining by endothelial cells; III step - their merger into the blood vessels of the organ.

13. There are two main types of the formation of the “cavitary” structures. first type The first type is associated with the abruption of layers of epithelial cells from their underlying foundation and their desquamation into the lumen of the “obliterated” cancerous glands.

14. The first type of “cavitary” structures (CS type-1) formation A - H&E stain (x400); B - H&E stain (x100) The abruption of tumor cells from their underlying foundation and their desquamation into the lumen of the cancerous glands in breast (A) and gastric (B) cancer. This step of angiogenesis corresponds to the previously described the “Retraction Artifact” (Acs G et al, 2007; Acs G et al 2012).

15. The main sign of “cavitary” angiogenesis of type-1 partial endothelial lining number of protuberances The main sign of “cavitary” angiogenesis of type-1 is the presence of CS with partial endothelial lining. The cytoplasm of cells lining of the CS type-1 has an uneven surface with a number of protuberances CS type-1 with partial endothelial lining (black arrows); CS type-1 without endothelial lining (green arrows); Blood vessel with tumor emboli in the lumen (red arrow); Staining with CD34 antibodies, x400.

16. The features of “cavitary” angiogenesis of type-1 in gastric and breast cancer CS type-1 without endothelial lining (black arrows); CS type-1 with partial endothelial lining (green arrows); (red arrow); Dilated vessel with tumor emboli in the lumen (red arrow); Staining with CD34 antibodies, Fig A – samples of gastric cancer, x100; Fig B – samples of breast cancer, x400.

17. The features of “cavitary” angiogenesis of type-1 in gastric and breast cancer Fig. A shows the dilated blood vessels with tumor emboli in the lumen in the samples of gastric cancer, x400; Fig. B shows the atypical dilated vessel with the marked atypia of the lining endothelial cells in the samples of breast cancer, x 400; Staining with CD34 antibodies.

18. The features of atypical dilated vessels in breast cancer Atypical dilated vessel (red arrows): Fig. A - with tumor emboli in the lumen; Fig. B – with free (unrelated with wall of vessel CD34 positive cells and tumor emboli in the lumen, CS type-1 without endothelial lining (black arrows); Staining with CD34 antibodies, x 400.

19. The second type of “cavitary” angiogenesis in gastric cancer. This type angiogenesis are associated with a characteristic structure of tumor stroma presented by loose fine-fibered connective tissue with a distinctive cellular structure (CS type-2). Fig. A shows a characteristic cellular structure (CS type-2) of the connective tissue in the stroma bordering upon the tumor tissue; Fig. B shows the CS type-2 (red arrows) and cavitary vessels type-2 (black arrows). H&E stain, x400.

20. The second type of “cavitary” angiogenesis in breast cancer Fig. A - B show a characteristic cellular structure and CS type-2 in the peritumoral stroma, H&E stain, x400.

21. The “cavitary” vessels type-2 with endothelial lining in gastric and breast cancer Fig. A shows the CS type-2 in gastric cancer; Fig. B shows the CS type-2 in breast caner, Staining with CD34 antibodies, x 400. A

22. The clinical significance of “cavitary” type of angiogenesis on the examples of gastric and breast cancer

23. THE “CAVITARY” STRUCTURES TYPE-1 IN GASTRIC CANCER The presence of “cavitary” structures type-1 in tumor stroma correlated with: the tumor sizes (ρ=0,316, p=0,006); histological type (gamma=0,344, p=0,01); Grade (gamma=0,318, p=0,005); Tumor Stage (gamma=0,549, p<0,00001); Nodal Stage (gamma=0,520, p=0,00003); 3-year overall survival rate (gamma=-0,778, p<0,00001) 3-year Relapse-free survival rate (gamma=-0,766, p<0,00001).

24. The tumor sizes according to the number of “cavitary” structures type-1 in tumor stroma Median test (p=0,02)

25. The frequency of CS type-1 depending on the histology type of gastric cancer X 2 =3,42, p=0,18

26. The frequency of CS type-1 depending on the Grade of gastric cancer X 2 =9,64, p=0,14

27. The frequency of CS type-1 depending on the Tumor Stage (T) of gastric cancer X 2 =17,48, p=0,008

28. The frequency of CS type-1 depending on the Nodal Stage (N) of gastric cancer X 2 =11,69, p=0,01

29. Survival of the patients depending on the number of “cavitary” structures type-1 A - The curves of 3-year relapse-free surviving (p=0,00001, Log-Rank Test) B - The curves of 3-year overall surviving (p=0,0012, Log-Rank Test ) AB

30. With or without the multiple “cavitary” structures type-1, the 3-year overall survival was 52.7% and 93.9% respectively (p=0,0013, OR=15,0, 95% CI=2,96 – 76,31), and relapse-free survival - 32.4% and 87.7% respectively (t=0,0001, OR=14,93, 95%CI=4,34-51,38). The multivariate Cox proportional hazard regression analysis indicated that TNM stage (p=0,003), nodal stage (p=0,013), the number of “cavitary” structures type-1 (p=0,005) were significantly independent prognostic factors in patients with GC.

31. THE “CAVITARY” STRUCTURES TYPE-1 IN BREAST CANCER The presence of “cavitary” structures type-1 with partial endothelial lining correlated with: the number of atypical dilated cappilaries (gamma=0,753, p<0,00001); ER status (gamma=-0,493, p=0,004); PR status (gamma=-0,627, p=0,001); the presence of tumor emboli in the vessels (gamma=0,515, p=0,004); the presence of free CD34 positive cells in the lumen of tumor vessels (gamma=0,673, p=0,001). :

32. THE FREQUENCY OF CS TYPE-1 DEPENDING ON THE NUMBER OF ATYPICAL DILATED VESSELS X 2 =19,73, p=0,0006

33. THE FREQUENCY OF CS TYPE-1 DEPENDING ON THE ESTROGEN RECEPTOR STATUS X 2 =4,66, p=0,03

34. THE FREQUENCY OF CS TYPE-1 DEPENDING ON THE PROGESTERONE RECEPTOR STATUS X 2 =11,69, p=0,01

35. THE NUMBER OF CS TYPE-1 DEPENDING ON THE PRESENCE OF LYMPHOVASCULAR INVASION X 2 =2,01, p=0,16

36. THE CAVITARY STRUCTURES TYPE-2 IN GASTRIC CANCER In gastric cancer the presence of “cavitary” structures type-2 correlated only with the histological type of tumor (gamma=0,403, p=0,008). By the diffuse and intestinal type of GC they were revealed in 55,9% and 35,0% cases respectively (р=0,07).

37. THE CAVITARY STRUCTURES TYPE-2 IN BREAST CANCER The number of CS type-2 correlated with: Her2/new status (gamma=0,680, p=0,0002) the presence of lymphovascular invasion (gamma=- 0,441, p=0,01). The CS type-2 were observed in 100,0% and 56,3% cases in the positive and negative Her2/new status (χ 2 =9,69, р=0,008). The tumor emboli in vessels were revealed in 43,7%, 69,2% and 75,0% cases in the absence of CS type-2, single and multiple ones (χ 2 =3,71, р=0,16).

38. The density of CD68 macrophages in gastric mucosa according to the number of “cavitary” vessels type-1 Kruskal-Wallis ANOVA by Ranks, p=0,057

39. The density of CD20 B-lymphocytes in tumor stroma according to the presence of CS type-2 Mann-Whitney U Test, p=0,035

40. The presence of CS type-2 according to the presence of focal CD20-cell infiltrates at the boundary of gastric mucosa and tumor X 2 =10,64, p=0,001

41. Factors that may be associated with “cavitary” angiogenesis type-1 The disorder of the adhesive properties of tumour cells; The inflammatory changes in the tumour stroma and the adjacent GM. The formation of “cavitary” structures type-1 and type-2 is likely related to the different classes of tumor-infiltrating immune cells (type-1 – with CD68 macrophages, type-2 – with CD20 B- lymphocytes; The focal disruptions in the tumor capsule associated with increased immune cell infiltration (Man Y.G., 2010; Man Y.G. et al, 2013). The increase the vascular permeability that may influence the process of stroma “retraction” and promote the formation of a fibrin matrix and a migration of endothelial cells (Nagy J.A. et al, 2012).

42. Thank for your attention