1. Updates on Breast Diseases: What clinicians need to know from pathologists Preah Bat Norodom Sihanouk Hopsital, 22 April 2009 Monirath Hav, MD, Ph.D. fellow (VLIR project) Pathology Department, Ghent University Hospital Ghent University, Belgium

2. Benign breast lesions Richard J et al. The New England Journal of Medicine. Volume 353:275-285 (July 2005)

4. Benign breast lesions: standard pathology report 1. Histologic type + type of proliferation 2. Maximum diameter 3. Nuclear grade (for DCIS only) 4. Resection margin (for DCIS & pleomorphic LCIS only) 5. Presence/absence of micro-invasion (for DCIS only) 6. Areas of involvement (unifocal, multifocal, multicentric)

5. VAN NUYS Prognostic Index for the management of DCIS Size (measured on histology exam) Score 1: size < or = 1.5 cm Score 2: size 1.6 – 4 cm Score 3: size > or = 4.1 cm Nuclear grade Score 1: DCIS nuclear grade 1 Score 2: DCIS nuclear grade 2 Score 3: DCIS nuclear grade 3 Surgical margin Score 1: tumor-free margin < or = 1 cm Score 2: tumor-free margin 0.1 – 0.9 cm Score 3: tumor-free margin < 0.1 cm Age of patient Score 1: > 60 y.o Score 2: 40 – 60 y.o Score 3: < 40 y.o Management Score 4 – 6 : lumpectomy Score 7 – 9 : lumpectomy + radiation Th. Score 10 – 12 : mastectomy Silverstein MJ, Lagios MD, Craig PH, et al. Cancer 77(11): 2267-2274, 1996

6. Malignant lesions

7. 1.Secretory/Juvenile carcinoma (<0.15%) 2.Tubular carcinoma (<2%)- so low recurrence that some centers consider adjuvant th. unnecessary. 3.Invasive cribriform carcinoma (0.8-3.5%) 4.Metaplastic carcinoma (<1%) 5.Invasive papillary carcinoma (1-2%) 6.Mucinous carcinoma (~2%) 7.Neuroendocrine carcinoma (2-5%) 8.Medullary carcinoma (1-7%) 9.Invasive lobular carcinoma (5-15%) 10.Invasive ductal carcinoma (75%)

8. Invasive carcinoma – standard pathology report

9. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

10. Histologic type: different prognosis Darius Dian et al. Arch Gynecol Obstet (2009) 279:23–28

11. Histologic type Gives pathologists and clinicians the ideas of: 1. Tumours aggressiveness 2. Patients overall prognosis 3. Tumours origin (i.e. basal-like + family history of breast CA highly suggestive for hereditary origin of BRCA1 mutation * ) 4. Response to chemotherapy (i.e. basal-like 45% pCR after neoadjuvant therapy using anthracycline and taxane ** ) * Turner NC & Reis-Filho JS (2006). Oncogene 25:5846–5853 * * Rouzier R et al. (2005). Clin Cancer Res 11:5678–585

12. Basal-like?

13. Features of basal-like breast CA Histology: Solid growth pattern High nuclear grade < 5% DCIS Lympho-vascular invasion Central scar Pushing border Marked lymphocytic infiltrates Immunohistochemical profile: CK5 + or CK14 + or CK17 + or EGFR + Mamatha Chivukula Appl Immunohistochem Mol Morphol Volume 16, Number 5, October 2008

14. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

15. Modifed Bloom-Richardson grade Tubule Formation score 1: >75% of tumor has tubules score 2: 10%-75% of tumor has tubules score 3: <10% tubule formation Nuclear Size score 1: tumor nuclei similar to normal duct cell nuclei (2-3÷ rbc) score 2: intermediate size nuclei score 3: very large nuclei, usually vesicular with prominent nucleoli Mitotic Count (per 10 hpf with 40÷ objective and field area of 0.196 mm 2 ) score 1: 0-7 mitoses score 2: 8-14 mitoses score 3: 15 or more mitoses rbc, red blood cells; hpf, high power field From Robbins P, Pinder S, de Klerk N, et al. Histological grading of breast carcinomas: A study of interobserver agreement. Hum Pathol 1995;26:873-879, with permission.

16. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

17. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

18. Ki-67 index -Ki-67 recurrence rate ; overall survival (1) -Ki-67 < 10% no benefit from chemotherapy (2) -Ki-67 > 25% sensitive to chemotherapy (2) -Ki-67 between 10 to 25%? other factors (Bloom-richardson grade, TNM stage, resection margin etc) (2) (1) E de Azambuja et al. British Journal of Cancer (2007) 96, 1504-1513 (2) Frédérique Spyratos et al. Cancer 2002 Apr 15;94(8):2151-9

19. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

20. Sebastian F et al. Ann Surg. 2004 August; 240(2): 306–312.

21. How about peri-neural invasion? No study has yet proven its independent prognostic significance Present in ~10% of high-grade tumours

22. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

23. Carter D et al. Am J Surg Pathol 1978;2:39–46 Prognostic value of Tumor necrosis & Tumor border

24. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

26. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border

27. Survival analysis: DCIS in invasive breast CA Rosemary R. Millis et al. Breast Cancer Research and Treatment 84: 197–198, 2004.

28. 1. Histologic type 2. Histologic grade (Bloom-Richardson) 3. TNM (size, node, distant metastasis) 4. Ki-67 index 5. Lympho-vascular invasion 8. Status of resection margins 9. ER, PR, HER2/neu status 10. In situ components, if present 6. Necrosis 7. Tumour border HER2/neu Estogen receptor

29. Overview on ER, PR, HER2 status in breast cancer HER2/neu overexpressed in 25 – 30%

30. ER, PR, HER2 status (cont) Molecular sub-types of breast CA: Luminal A (ER/PR +, HER2 -) Luminal B (ER/PR +, HER2 +) HER2 sub-type (ER/PR -, HER2 +) Basal-like (ER -, PR -, HER2 -) Perou CM, Sorlie T, Eisen MB et al (2000). Nature 406:747–752

31. Hiroo Nakajima et al. World J Surg (2008) 32:2477–2482 Prognosis of each sub-type of breast CA

32. ER, PR, HER2 status (cont) Therapeutic implication : Luminal A (ER/PR +, HER2 -) Hormonal therapy Luminal B (ER/PR +, HER2 +) Hormonal therapy? + anti-HER2 HER2 sub-type (ER/PR -, HER2 +) anti-HER2 Basal-like (ER -, PR -, HER2 -) No benefit from either therapy

33. Quickscore for ER-PR IHC Staining intensity - Negative (no staining of any nuclei at high magnification)= 0 - Weak (only visible at high magnification) = 1 - Moderate (readily visible at low magnification) = 2 - Strong (strikingly positive at low magnification) = 3 Proportion of positive cells (nuclei) - 0% = 0 - <1% = 1 - 1–10% = 2 - 11–33% = 3 - 34–66% = 4 - 67–100% = 5 Quickscore: 0 8

34. Quickscore : What should be the cut off? Harvey JM et al. J Clin Oncol. 1999 May;17(5):1474-81.

35. Quickscore in ER, PR IHC Score 0 : no response to endocrine treatment Score 2 - 3 : 20% response to endocrine treatment Score 4 - 6 : 50% response to endocrine treatment Score 7 - 8 : 75% response to endocrine treatment

36. But many labs use the 10% cut off rule!

37. HER2/neu Immunohistochemistry

38. What is known about HER2 and response to Trastuzumab? Guido Sauter et al J Clin Oncol 29. 2009 by American Society of Clinical Oncology

39. Mass R et al. Clinical Breast Cancer 6:240-246, 2005 HER2 gene amplication detected by In Situ Hybridization is superior to HER2 protein overexpression detected by IHC in predicting Response to Trastuzumab.

40. Does HER2 over- expression defined by IHC predict response to Trastuzumab? YES! If not false-positive Poor fixation Artifact Antigen retrieval techniques Inexperience interpreter

41. Correlation between HER2 FISH and IHC FISH result IHC score 0 1+ 2+3+Total Amplified4.5%3.27% 8.6% 83.6% 244 cases Not amplified49.5%23.74% 17.22% 9.53% 598 cases Guido Sauter et al J Clin Oncol 29. 2009 by American Society of Clinical Oncology

42. How about HER2 status and response to Tamoxifen?

43. De Laurentiis M et al. Clin Cancer Res. 2005 Jul 1;11(13):4741-8 HER2 overexpression is correlated with resistance to Tamoxifen in metastastic breast cancers ER, PR IHC tests are no longer important in metastatic setting

44. Does HER2 overexpression predict resistance to Tamoxifen in early breast cancers? Controversial studies: no conclusion yet

45. Should we trust all these studies? Why dont we conduct studies on our own population?

46. Standard pathology report for benign breast lesions: Histologic type of lesion + type of proliferation Diameter Areas of involvement (unifocal, multifocal, multicentric) Nuclear grade and growth pattern (for carcinoma in situ) Presence/absence of micro-invasion (for carcinoma in situ) Status of resection margin (for carcinoma in situ > 2mm safe)

47. Sample of a standard report Conclusion: 1.Lumpectomy: Atypical Ductal Hyperplasia (Proliferative lesion with atypia) 2.Nuclear grade: 3 3.Growth pattern: solid type 4.Areas of involvement: multifocal (3 foci) 5.Overall size: 0.8 cm 6.Microinvasion: absent 7.Resection margins: not involved / negative (6 mm)

48. Standard pathology report for invasive breast carcinoma 1.Histologic type 2.Histologic grade (Bloom-Richardson) 3.TNM (size, extension, node, distant meta.) 4.Ki-67 index 5.Lympho-vascular/perineural invasion 6.Status of resection margin (> 1 mm safe) 7.ER, PR, HER2/neu status 8.In situ component, if present

49. Sample of a standard report Conclusion: Tumorectomy – left breast : Invasive component: 1.Type: Invasive ductal adenocarcinoma 2.Poorly differentiated, Bloom score 8 3.Maximal diameter : 1.8 cm 4.Lymphovascular invasion: present 5.Resection margins: minimally safe (3 mm from dorsal margin) 6.Left axillary lymph nodes: 5 lymph nodes found, 2 lymph nodes invaded by carcinoma (2/5) 7.Ki-67 index : approximately 30% of the tumor 8.Receptor status: ER negative (quickscore 0) PR negative (quickscore 2) HER2/neu score 2+ TNM (6 th edition, 2002) : pT1c pN1a p Mx In situ component : absent

50. References and suggested readings 1.Richard J et al. Benign Breast Disorders. The New England Journal of Medicine. Volume 353:275-285 (July 2005) 2.Turner NC & Reis-Filho JS (2006). Basal-like breast cancer and the BRCA1 phenotype. Oncogene 25:5846–5853 3.Rouzier R et al. (2005). Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 11:5678–585 4.Mamatha Chivukula. Evaluation of Morphologic Features to Identify Basal-like Phenotype on Core Needle Biopsies of Breast. Appl Immunohistochem Mol Morphol Volume 16, Number 5, October 2008 5.E de Azambuja et al. Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12 155 patients. British Journal of Cancer (2007) 96, 1504-1513 6.Frédérique Spyratos et al. Correlation between MIB-1 and Other Proliferation Markers: Clinical Implications of the MIB-1 Cutoff Value. Cancer 2002 Apr 15;94(8):2151-9 7.Perou CM, Sorlie T, Eisen MB et al (2000). Molecular portraits of human breast tumors. Nature 406:747– 752 8.Hiroo Nakajima et al. Prognosis of Japanese Breast Cancer Based on Hormone Receptor and HER2 Expression Determined by Immunohistochemical Staining. World J Surg (2008) 32:2477–2482 9.Sebastian F et al. Prognostic Value of Lymphangiogenesis and Lymphovascular Invasion in Invasive Breast Cancer. Ann Surg. 2004 August; 240(2): 306–312. 10.Rosemary R. Millis et al. Ductal in situ component and prognosis in invasive mammary carcinoma. Breast Cancer Research and Treatment 84: 197–198, 2004.