Reporting of Breast Cancer Do s and Don ts

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1 Reporting of Breast Cancer Do s and Don ts 7 th SGH Annual Breast Pathology Course Professor Michael Bilous Conjoint Professor Western Sydney University Consultant Pathologist, Australian Clinical Labs, Sydney 1

2 The Role of the Breast Pathologist Information required from pathology: Prognostic and Predictive factors 1. Local Control margin status 2. Extent of Spread lymph nodes 3. Factors to determine optimal therapy

3 The Role of the Breast Pathologist Information required from pathology: Prognostic and Predictive factors 1. Local Control margin status 2. Extent of Spread lymph nodes 3. Factors to determine optimal therapy

4 How is breast cancer pathology determining therapy? An Historical Perspective Early detection of breast cancer via education, awareness and screening programs has meant that most breast cancers in developed nations are detected early i.e. <50mm, without distant metastases.

5 How is breast cancer pathology determining therapy? Adjuvant chemotherapy for early breast cancer has been effective. Patients are selected on the basis of pathology features such as tumour size and grade, and axillary lymph node status.

6 How is breast cancer pathology determining therapy? Additional biological markers such as ER/PR and HER2 have allowed more targeted treatment (endocrine and anti-her2) Personalised Medicine There was no test which will determine whether an individual patient will benefit from chemotherapy. This results in overtreatment of some and undertreatment of others.

7 How is breast cancer pathology determining therapy? The Human Genome Project enabled the identification of genes in breast cancer that could assist in finding those cancers that have an intrinsic poor prognosis for which adjuvant chemotherapy is indicated: The Gene Expression Profile of the cancer. There is increasing interest in these molecular tests that can more accurately predict the response of a cancer to targeted therapy

8 Pathology Reporting of Breast Cancer Traditional Pathology Molecular Markers Gene Expression Tumour size and grade ER, PR, HER2 Oncotype DX Tumour type Ki-67 MammaPrint Margin status EndoPredict Lymph node status Prosigna LVI Breast Cancer Index {TIL s} Mammostrat etc Prognostic Indices Nottingham Prognostic Index Adjuvant! On line St Gallen 2015 (surrogate intrinsic classification)

9 Pathology Reporting of Breast Cancer Traditional Pathology Molecular Markers Gene Expression Tumour size and grade ER, PR, HER2 Oncotype DX Tumour type Ki-67 MammaPrint Margin status EndoPredict Lymph node status Prosigna LVI Breast Cancer Index {TIL s} Mammostrat etc Prognostic Indices Nottingham Prognostic Index Adjuvant! On line St Gallen 2015 (surrogate intrinsic classification)

10 Invasive Carcinoma Traditional Pathology: Size Type Histological Grade Margin Status Lymph Node (Sentinel Node) status Lymphovascular invasion Changes in adjacent breast tissue All reported according to published Guidelines

11 Size Single greatest dimension in mm assessed by microscopy or macroscopically or in three dimensions For invasive carcinoma, include any DCIS within the tumour but - If DCIS extends outside the invasive carcinoma then 2 measurements are required whole tumour and invasive component For small carcinomas a small increase in size equates to a marked change in predicted ALN status

12 Size of invasive ca DCIS Invasive ca Size of whole lesion

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15 >5mm

16 Type of invasive carcinoma Special types pure tubular, invasive cribriform, tubulo-lobular and mucinous have an excellent prognosis: 1500 patients, 10 years follow-up >80% 10 year survival Ellis et al Histopathol 1992;20:

17 Type of invasive carcinoma Invasive lobular has a slightly better prognosis than invasive duct NST but this varies with subtype: Classical 60% 10 year survival Mixed 55% 10 year survival Pleomorphic 40% 10 year survival Tubulo-lobular 80% 10 year survival

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20 Grade For all invasive carcinomas grading assesses growth pattern and cytological features Elston and Ellis system: Tubules 1-3 Nuclear grade 1-3 Mitoses 1-3 Grade 1 (3-5) Grade 2 (6-7) Grade 3 (8-9) This grading system replaced the Bloom and Richardson system devised ~10 years earlier

21 Grade Reproducibility (kappa statistic) tubules 0.64 nuclear grade 0.4 mitoses 0.52 Ellis et al. Human Pathol. 1981;12:3-4

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24 Margin status Breast tissue is irregular and crumbly There is a large surface area to sample Margin Assessment is an Imperfect Science

25 Lymphovascular invasion Correlates with ALN involvement and early recurrence in ALN negative patients Predictor of long term survival and local recurrence after breast conservation and radiation therapy Rosen Pathol Annu 1983;18: Pinder et al Histopathol 1994;24:41-47

26 Lymphovascular invasion Caution Retraction Artefact Always assess LVI at the advancing edge of the carcinoma Look for clues to identify the lymphatics ie. small vein and arteriole +/- nerve Dermal LVI has greater significance than LVI within the breast tissue

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28 Gene Expression Profiles - new breast cancer classification- PAGE 28

29 78 carcinomas, 3 benign tumours, 4 normal breast tumours 476 gene chip selected from previous study of 1753 genes Gene clusters: C HER2 associated genes D Novel set unknown function E Basal epithelium and myoepithelium of breast F Normal breast G Luminal cells of breast (ER+) INTRINSIC MOLECULAR SUBTYPES Perou CM, et al. Nature 2000; 406: ; Sorlie T, et al. Proc Natl Acad Sci USA 2003; 100:

30 Prognosis according to genetic profile note luminal B and C merged Perou CM, et al. Nature 2000; 406: ; Sorlie T, et al. Proc Natl Acad Sci USA 2003; 100:

31 Treatment-oriented Molecular Subtypes of breast cancer What the Medical Oncologist wants to know!

32 How do we best use the cancer specimen to supply this information? By using a molecular test? or By using a combination of traditional pathology and immunohistochemistry?

33 Prognosis according to genetic profile note luminal B and C merged Can we identify INTRINSIC Subtypes by Immunohistochemistry? Perou CM, et al. Nature 2000; 406: ; Sorlie T, et al. Proc Natl Acad Sci USA 2003; 100:

34 Pathology Reporting of Breast Cancer Traditional Pathology Molecular Markers Gene Expression Tumour size and grade ER, PR, HER2 Oncotype DX Tumour type Ki-67 MammaPrint Margin status EndoPredict Lymph node status Prosigna LVI Breast Cancer Index {TIL s} Mammostrat etc Prognostic Indices Nottingham Prognostic Index Adjuvant! On line St Gallen 2015 (surrogate intrinsic classification)

35 Gene profiles and pathology Basic Immunohistochemistry and H&E sections can help identify the main intrinsic cancers (not Luminal A or B) Frequency Luminal A cytokeratin 8/18 positive; 50% ER and/or PR+; HER2 - Luminal B cytokeratin 8/18 positive; 20% ER and/or PR+; HER2 +/- Basal-like ER-,PR-,HER2-, HER1+ 15% and/or cytokeratin 5/6 + HER2 enriched HER2 +, ER- 15% Millikan et al Breast Cancer Res Treat 2007 Schnitt et al Int J Surg Pathol 2010

36 Gene profiles and pathology Basic Immunohistochemistry and H&E sections can help identify the main intrinsic cancers (not Luminal A or B) Frequency Luminal A cytokeratin 8/18 positive; 50% ER and/or PR+; HER2 - Luminal B cytokeratin 8/18 positive; 20% ER and/or PR+; HER2 +/- Basal-like ER-,PR-,HER2-, HER1+ 15% and/or cytokeratin 5/6 + HER2 enriched HER2 +, ER- 15% Millikan et al Breast Cancer Res Treat 2007 Schnitt et al Int J Surg Pathol 2010

37 How can we separate Luminal A and B cancers?

38 St Gallen Consensus May 4 th 2015 Coates AS, et al. Ann Oncol 2015; 00: 1 14.

39 St Gallen Consensus May 4 th 2015 Coates AS, et al. Ann Oncol 2015; 00: 1 14.

40 Prognosis according to genetic profile note luminal B and C merged Ki-67 ASSESSMENT Perou CM, et al. Nature 2000; 406: ; Sorlie T, et al. Proc Natl Acad Sci USA 2003; 100:

41 Dowsett M, et al. J Natl Cancer Inst 2011; 103:

42 Ki-67 assessment Core biopsies/tma or surgical excisions 3+ hpf fields selected at the edge of the carcinoma Include any clear hot spots Staining intensity is not relevant Count at least 500 and preferably 1000 cells Express the result as a percentage Cut points?? Apply only if local practice validated against studies with defined cut offs Dowsett M, et al. J Natl Cancer Inst 2011; 103:

43 Ki-67 and breast cancer Problem for the pathologist What threshold to use? 10%, 13%, 20% 25%...other? St Gallen 2015 Laboratory validation, median value used

44 Variable levels of Ki-67 staining; different score in different areas; take average across the whole section

45 Well fixed Poorly fixed

46 Validating Ki-67 scoring in my laboratory Record the Ki-67 score for all cases in which the ER and PR result is >50%; HER2 negative. 2 years counted Median Ki-67 score is 15% Luminal A-like Ki-67 score <5% Luminal B-like Ki-67 score is >25% Intermediate Ki-67 score is 6 25%

47 Maisonneuve P, et al. Br Cancer Res 2014; 16: R65

48 Prognosis according to genetic profile note luminal B and C merged ER/PR expression Perou CM, et al. Nature 2000; 406: ; Sorlie T, et al. Proc Natl Acad Sci USA 2003; 100:

49 Defining molecular subtypes by quantitative receptor (HR) expression 1557 cases; ER/PR/HER2 centrally tested by IHC and RT-qPCR Intrinsic subtype determined by PAM50 Cheang MCU, et al. The Oncologist 2015;20:

50 Defining molecular subtypes by quantitative receptor (HR) expression 283 IHC HER2-ve tumours; HR<1% 73% basal-like; 17% HER2+ve by PAM50 39 IHC HER2-ve tumours; HR 1-9% 44% luminal A/B; 31% HER2+ve; 18% basal-like by PAM IHC HER2+ve tumours; HR<1% 82% HER2+ve by PAM IHC HER2+ve tumours; HR>10% 54% HER2+ve; 43% luminal A/B by PAM50 Cheang MCU, et al. The Oncologist 2015;20:

51 Defining molecular subtypes by quantitative receptor (HR) expression Conclusion: Significant discordance between IHC defined subsets and intrinsic molecular subtype determined by RT-qPCR For identifying basal-like subtype the optimal cut point for ER was <1% Tumours with borderline HR by IHC are molecularly diverse and may require additional assays Cheang MCU, et al. The Oncologist 2015;20:

52 What limits the use of IHC and gene expression profiling? The quality of the paraffin block Access and Cost IHC, ISH, molecular genetics are highly specific and reproducible testing methodologies using paraffin blocks The techniques fail when pre-analytical protocols are not in place The principles of strict QC and QA should be applied Carlson RW, et al. J Natl Compr Canc Netw 2006; 4:S1 S22; Wolff AC, et al. J Clin Oncol 2007; 25:

53 Quality control and quality assurance? Quality control Achieving and/or maintaining the desired level of quality Inspecting samples at each step and assessing what changes may be needed Internal validation of procedures Performed on each batch Guarantees the accuracy of every test BY THE TESTING LABORATORY Quality assurance Aims to improve overall performance of testing Compares performance with other centres BY AN EXTERNAL LABORATORY

54 Strategies for best test performance, interpretation and reporting (best practice) Standardised and recommended practice Monitor and maintain quality (quality system) Test accuracy and reproducibility

55 Quality Control Starts in the Operating Theatre with handling and fixation

56 Why Is Fixation So Important? Aims of Fixation: To prevent autolysis or decomposition To preserve tissue as near to its original form as possible To protect tissue against subsequent changes during processing & embedding Essentials For Good Fixation: Fresh tissue Proper penetration of fixative Right choice of a correctly prepared fixative

57 Specimen Handling Slicing of Excision Specimens Surgical specimens should be sliced into 5-10mm slices to ensure rapid and even fixation Fixative will not penetrate tissue thicker than 10mm

58 Why 10% Neutral Buffered Formalin? Lots of Reasons! Clinically validated for HER2 testing Use of any other fixative will require validation by the lab FDA kit Requirement; otherwise invalidates test/assay Other fixatives shown to produce inconsistent results Alcohol/acetone decrease quality of IHC/ ISH Bouin s decreases preservation of many anitgens Bouin s causes autofluorescence with ISH

59 Fixative Formulation - 10% NBF (ph 7.0) 10% NBF solution Formalin (40% formaldehyde) Sodium dihydrogen phosphate monohydrate Disodium hydrogen phosphate (anhydrous) Distilled water 100 ml 4.0 g 6.5 g 900 ml Termed 10% NBF but is actually 4% solution of formaldehyde

60 Standardisation Of Fixation Formula for Fixative = Step 1 of standardisation Step 2 = Quality Step 3 = Volume Step 4 = Time

61 Quality Step 2 = Quality - Commercial vs Home Made use fresh solution DO NOT RE-USE Quality

62 Volume Step 3 = Volume - size of tissue being fixed: - minimum 10:1 ratio - fixative to tissue Container Must Be of Adequate Size for the Tissue Image from:

63 Time Step 4 = Time - duration of fixation fixation penetration Published guidelines Wolff et al, J Clin Oncol 2007;26 and October2013 Walker et al, J Clin Pathol 2008;61: Ruschoff et al, Mod Pathol 2012;25: hours minimum for breast biopsies 8 hours minimum for gastric biopsies hours for breast and gastric surgical excisions

64 Summary Do s and Don ts! Ensure proper fixation and handling in the Operating Theatre Slice the specimen on receipt in the Laboratory Use validated protocols and quality control measures at all intralaboratory processes Report traditional pathology features and biomarker results following published guidelines If using Ki-67 ensure that the technique used and threshold are validated Talk to members of the multidisciplinary team formally at meetings and informally to explain: problematic/unusual/unexpected results Don t issue a pathology report if you are uncertain of its accuracy consult with a pathologist with more experience Don t leave preparation of reagents to junior members of staff without supervision Don t leave pathology residents in the grossing room without access to a pathologist for guidance Don t report a difficult case on Friday evening if it can wait until Monday morning!

65 Key Points In addition to standard pathology reporting, oncologists are wanting more information, specifically related to proliferation rate and to intrinsic molecular subtypes, which can predict response to therapy Molecular testing is expensive and may not be accessible to all breast cancer patients Using IHC-determined ER/PR/HER2 and Ki67 can assist in categorising cancers into molecular subtypes Attention to pre-analytical and post-analytical processes is crucial to success with all these tests The correlation between IHC results and molecular subtypes assessed by RT-qPCR is imprecise and results should be applied with caution HER2, human epidermal growth receptor 2; QA, quality assurance procedures; QC, quality control.

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