Intratumoral heterogeneity: IHC and ISH analysis. Tibor Tot, M.D., Ph.D Laboratory Medicine Dalarna Falun, Sweden

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1 Intratumoral heterogeneity: IHC and ISH analysis Tibor Tot, M.D., Ph.D Laboratory Medicine Dalarna Falun, Sweden

2 Breast cancer: a need for multiparameter characterization

3 Size E X T E N T

4 When describing malignant lesions in the breast, the following morphologic parameters should be listed (independent of the used imaging method): the distribution of the lesions (as unifocal, multifocal or diffuse) separately for invasive and in situ lesions, the extent of the disease (representing the whole area including all the invasive, in situ, and intravascular malignant structures), the size of the tumor corresponding to the largest diameter of the lagest individual invasive tumor focus, evidence for intratumoral or intertumoral heterogeneity.

5 Optimal breast cancer pathology manifesto T. Tot 1 ; G. Viale 2 ; E. Rutgers 3 ; E. Bergsten-Nordström 4 ; A. Costa 5

6 Prognostic parameters Radiological/surgical Distant metastasis Lymph node status Tumor size Multifocality Extent Resection margins Oncological Histology grade ER/PR status HER-2 status Proliferation Molecular subtypes

7 Surgery / radiology Radicality Extent Size Multifocality Lymph nodes mm numbers Oncology ER HER2 Ki67 Genetics Molecular % Reproducibility Pathology Histology Benign/malignant Invasive/in situ Tumor type/grade Immunohistochemistry description

8 Radiological pathological correlation is essential in diagnosing breast carcinoma The radiology images are courtesy of Prof Laszlo Tabár, DRs Nadja Lindhe and Mats Ingvarsson

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10 Standardization of assessment Guidelines Registry Population-based data comparison External quality control

11 Breast pathology, Sweden, 2015 WHO blue book AJCC Staging handbook: TNM7 ASCO/CAP guidelines EWGBCSP European guidelines Socialstyrelsens nationella riktlinjer för bröstcancersjukvård 2007 (revised 2014) KVAST Swedish pathology guidelines Nationellt vårdprogram Regionala (RCC) vårdprogram Ackrediterade labs metodbeskrivningar

12 Guidelines Too many Too complicated Too often modified Insufficient with regard to tumor heterogeneity

13 Tumor heterogeneity Intratumoral Intertumoral - multifocal cancers - primary versus metastasis - from case to case

14 Intratumoral heterogeneity, histology level

15 Large-format histology slide showing intratumoral heterogeneity Image from Tot/Tabár/Dean: Practical Breast Pathology, 2nd ed. 2014

16 Histology slide showing intratumoral heterogeneity Image from Tot/Tabár/Dean: Practical Breast Pathology, 2nd ed. 2014

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18 Intratumoral heterogeneity, IHC level

19 ER PR HER2 CK14 CK5,6 E-cad Triple negative basal-like breast cancer showing Intratumoral heterogeneity in CK14 staining.

20 57-year-old woman, 13 mm, grade 2, ER pos PR pos, HER2 neg, Ki67 6%, SN: ITC

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31 Intratumoral heterogeneity, the level of genes

32 Darryl Shibata Heterogeneity and tumor history. Science 336:304-5, 2012 All cells are almost perfect copies of prior cells. Imperfect DNA replication creates random variation, which is the substrate for evolution.

33 Darryl Shibata Heterogeneity and tumor history. Science 336:304-5, 2012 Normally: 1 new mutation per human cell division Accumulation of mutations over time can eventually transform a single cell.

34 Darryl Shibata Heterogeneity and tumor history. Science 336:304-5, 2012 Public mutations are present in all cells of the tumor and appear during the period from the zygote to the first transformed cell. Semiprivate mutations are present in a detectable fraction of the cancer cells (subclone) Private mutations are present in a single or few cancer cells.

35 Darryl Shibata Heterogeneity and tumor history. Science 336:304-5, 2012 The more advanced the tumor, the more numerous different mutations are present. Tumorogenesis can be represented by an ancestral tree that starts from the zygote and ends with present-day cancer cells.

36 Darryl Shibata Heterogeneity and tumor history. Science 336:304-5, 2012 Molecular clock hypothesis Increasing heterogeneity Intratumoral heterogeneity is present whereever one looks.

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38 Hypothetical models explaining intratumor heterogeneity. (A C) Different models of tumor progression can give rise to distinct types of intratumor heterogeneity, exemplified here by the clonal evolution (A), the cancer stem cell (B), and the mutator phenotype (C) models. (D) The different models can result in distinct spatial distributions of subpopulations. Russnes HG et al. Journal of Clinical Investigation

39 Intratumoral heterogeneity Clustered (regional) Scattered ( genetic ) Mosaic pattern

40 Clustered HER2 Intratumoral heterogeneity Scattered HER2 Intratumoral heterogeneity

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42 The painter s palette in Opatija, 2013

43 Intratumoral heterogeneity, HER-2

44 FDA HER2 approved tests Immunohistochemistry In situ hybridisation DNA probe coupled to fluorescence (FISH), chromogenic (CISH) or silver (SISH) Combinations: bright-field dual ISH (CISH + Sish = BDISH or dual hapten, dual color ISH, DDISH Gene-protein assay: tricolor (DISH+IH)

45 Tricolor IH/B-DISH HER2 stainingt

46 Tricolor IH/B-DISH HER2 stainingt

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48 Tricolor brightfield dual in situ hybridization HER2 non-amplified (negative) HER2 amplified (positive)

49 Tricolor brightfield dual in situ hybridization IH 2+, amplified

50 BDISH compares favorable with FISH in IH2+ cases Gao FF, Dabbs DJ, Cooper KL, Bhargava R. Bright-Field HER2 Dual In Situ Hybridization (DISH) Assay vs Fluorescence In Situ Hybridization (FISH): Focused Study of Immunohistochemical 2+ Cases. Am J Clin Pathol Jan;141(1): doi: /AJCP6CXS8OSRHXIR

51 The cases were categorized into positive, equivocal, or negative for HER2 gene amplification using the 2013 American Society of Clinical Oncology/College of American Pathologists criteria. This resulted in 82% agreement (41 of 50) between FISH and GPA. In addition, 25 known IHC 3+ breast carcinomas analyzed by GPA showed protein overexpression and clusters of HER2 gene consistent with unequivocal amplification, and 22 known IHC-negative cases were negative for HER2 gene amplification by GPA. Li Z at al. Am J Clin Pathol March 2015;143:

52 FISH Gold standard Less optimal histology details Takes 3 days Signals fade over time Heterogeneity less evident Assessed by technicians Dual SISH FDA Approved Optimal histology details Takes few hours Signals preserved for archival review Heterogeneity evident Assessed by pathologists Clark HZ, Bhargava R. Bright-field Microscopy for HER2 Gene Assessment. Not Just DISH-ful Thinking? Editorieal, Am J Clin Pathol 2013:139;137-39

53 Falun: Västerås: Tricolor BDISH analysis, Falun, : cases cases Mora: - 28 cases Syria: - 1 cases Total: 894 cases 960 tests

54 Tricolor BDISH analysis, Falun, : Organ Specimen N Breast Core biopsy 133 Sector resection 406 Mastectomy 290 Postmastectomy scar 6 Neoadjuvant therapy 1 Ventricle Gastric biopsy 7 Metastasis Lymph nodes 14 Liver 4 Bones 8 Lungs 2 pleura, peritoneum 5 Ovary, cervix 2 Other Chorion villi, FNAB, unknown 6 Total 884

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56 Problems Technical issues Criteria / interpretation Interobserver/interlaboratory agreement

57 HER2 immunohistochemistry HER2 tricolor BDISH

58 control Artefact: very small HER-2 signals.

59 Extranuclear signals

60 Nuclear dust

61 Diagn Pathol May 30;7:60. doi: / A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections. Nitta H, Kelly BD, Padilla M, Wick N, Brunhoeber P, Bai I, Singh S, Ranger-Moore J, Bieniarz C, Tsuda H, Grogan TM

62 Tricolor BDISH: staining repeated for technical reasons Falun, Total 213 cases Repeated 9% (19/213) Västerås 12% (12/98) Falun 6% (7/115)

63 Tricolor BDISH analysis, Falun, : Antal bedömbara fall 884 Antal obedömbara - tekniskt fel 5 - ej representativt prov 5 Total 894

64 Problems Technical issues Criteria / interpretation Interobserver/interlaboratory agreement

65 Problems: monosomy

66 Problems: polysomi

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70 HER2 IHC och ISH HER2 IHC 0 eller 1+ HER2 IHC 2+ eller 3+ i åtminstone 10% av cellerna i snittet ISH Ingen ISH Kvot < 2 och HER2 < 4 Kvot 2 och HER2 < 4 Kvot 2 och HER2 4 Kvot < 2 och HER2 4 HER2 status Negativ Positiv Courtesy of Dr Dorthe Grabau KVAST 2015

71 HER2 genetic heterogeneity Definitions: CAP Guidelines 2009: 5 50% of the invasive tumor cells are amplified Italian Society of Human Genetics: distinct cell populations are identified e.g.: low vs high ratio UK guidelines identified amplified and nonamplified subpopulation TMA studies: differences between the cores

72 HER2 genetic heterogeneity Prevalence: 5-40% Hanna et al. Modern Pathology 2014

73 HER2 genetic heterogeneity Clustered (regional) Scattered ( genetic )

74 Clustered HER2 Intratumoral heterogeneity Scattered HER2 Intratumoral heterogeneity

75 Seol H et al. Modern Pathology 2012:25,

76 Clustered heterogeneity Scattered haterogeneity

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78 Assessing HER-2 status - details Polysomy HER2 status Parameters Positive Negative Average HER2 gene copy number 4 < 4 Number of cells with >4 HER2 copy number 10% < 10% Average centromer 17 copy number 2 Number of cells with >2 centromere 17 copies Number of assessed cells 10% (minimum level) HER2 copy number /centromere 17 ratio 2 <2 Immunohistochemistry pattern 10% 3+ (2+) 0, 1+

79 Tricolor BDISH analysis, Falun, : primary breast carcinomas (recurrences and tumors asfter neoadjuvant treatment excluded) HER2 positive 18,5%* (152/822) Borderline 2% (18/822) - HER2 negative 79,5% (652/822) Not assessable 9 *non-consecutive series

80 Problems : Gene expression protein expression discordance

81 Tricolor BDISH analysis, Falun, : Unexpected findings in 289 cases 0/1+ IH - 6 amplified (ratio 2 and/or HER2 copy number 4-3 borderline cases (10%+ cells with 4 HER2 copies)

82 Tricolor BDISH analysis, Falun, : Unexpected findings in IH - 1 case with monosomi chr 17

83 Problems Technical issues Criteria / interpretation Interobserver/interlaboratory agreement

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85 Intertumoral heterogeneity

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87 E-cadherin E-cadherin ER ER Ki67 Ki67

88 Case Size (mm) Grade Histology Nielsen * Int. St Gallen 2011 Sotiriou 1. 2n 10/10 2/2 L/L LuB LuA LuB LuA HER LuA 2. 2n 21/3 2/2 D/D LuA LuA LuB LuA LuA LuA 3. 2n 15/10 3/1 D/SP LuA LuA LuA LuA LuB LuA 4. 2n 35/3 2/2 D/D Bas TN Bas Bas Bas Bas 5. 2n 32/2 2/2 L/L LuA LuA LuA LuB LuA LuA 6. 2n 28/10 2/2 D/D TN LuA Bas LuA Bas LuA 7. 2n 30/10 2/2 D/D LuA LuA LuA LuB LuA LuA 8. 3n 21/10/10 3/2/2 PL/L/L HER TN TN HER Bas Bas HER Bas Bas 9. 3n 18/7/3 2/2/2 L/D/L LuA LuA HER LuA LuA HER LuA LuA HER 10. 3n 28/6/5 2/2/2 Mu-D/D/D HER LuA LuA HER LuA LuA HER LuA LuA 11. 4n 20/12/4/3 3/3/3/3 D/D/D/D HER HER TN HER HER HER Bas HER HER HER Basl HER 12. 4n 25/14/12/5 3/3/3/3 Mp/Mp/Mp/D LuB LuA LuA LuA LuB LuB LuB LuB HER LuB LuB LuB 13. 5n 9/4/2/2/2 2/2/2/2/2 D/D/L/D/D LuA LuA LuA LuB LuB LuA LuA LuA LuA 14. >5n 29/5/4/2/1 2/2/1/1 D/D/D/D LuB LuA LuA LuB LuA LuA HER LuA LuA 15. >5n 22/17/5/4/2 2/1/1/1/1 Mu/TL/TL/TL/TL LuA TN LuA LuA LuA Bas LuA LuA LuA Bas LuA LuA 16. >5n 35/14/6/6/4 3/3/3/3/3 Mp/Mp/Mp/Mp/Mp LuA LuA LuA LuA LuB LuA LuA LuA LuB LuB LuB LuB Pekar et al. Cancer, >5n 30/13/8/4/3 2/2/2/2/2 PL/PL/PL/PL/PL LuA LuA LuA LuA LuA LuB LuB LuB LuA LuA LuA LuA 18. >5n 20/9/8/7/5 2/2/2/2/2 D/D/D/D/D LuA PR+ PR+ LuA LuA LuA LuA LuA LuA Bas Bas LuA

89 Variable Univariate analysis Multivariate analysis HR (95% CI) P HR (95% CI) P Age: 50 vs < ( ) ( ) Index tumor size: 15 mm vs. < 15 mm 3.86 ( ) ( ) Number of tumor foci: >2n vs 2n 1.47 ( ) ( ) Histology grade: Grade 3 vs. grade 1, ( ) ( ) Heterogeneous by: histology/grade vs. other 1.07 ( ) ( ) Nielsen vs. other 2.87 ( ) ( ) St. Gallen vs other 0.89 ( ) ( ) Sotiriou vs. other 2.80 ( ) ( ) Pekar et al. Cancer, 2013

90 Pekar et al. Cancer, 2013

91 Intertumoral heterogeneity in multifocal/multicentric breast carcinomas References Mismatches in any features (tumor type, grade, IH) Mismatches in ER PR HER2 Ki67 Middleton et al % (3/14) 0% (0/14) 0% (0/14) 0% (0/14) 14% (2/14) Garimella et al % (2/18) 0% (0/18) 11% (2/18) n.a. n.a. Dawson et al % (14/24) n.a. n.a. * n.a. Choi et al % (24/65) 3% (2/65) 11% (7/65) 6% (4/65) n.a. Pekmezci et al % (13/51) 8% (4/51) 8% (4/51) 2% (1/50) 22% (11/49) Buggi et al % (5-22/113)** 4% (5/113) 16% (18/113) 10% (11/113) 15% (17/113) Boros et al % (11/91) n.a. n.a. n.a. n.a. Boros et al % (71/132) 12% (18/132) 19% (29/132) 16% (24/132) 29% (45/132) Pekar et al % (21-25/110)*** 4% (4/110) 1% (1/110) 6% (7/110) 3% (3/110) Total 27-30% ( /608) 7% (33/503) 12% (61/503) 10% (47/484) 19% (75/404) * Not assessed as predictive marker ** Aggregate % is not given *** % provided in 3 different molecular classification systems

92 36 patients with MFMC ductal cancer of the same grade, ER and HER2 status. 11 carried the same mutations in all lesions, 13 had both common and private mutations 12 had no common mutations Presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. C Desmedt, D Fumagalli, E Pietri C. Sotiriou Uncovering the genomic heterogeneity of multifocal breast cancer J Pathol, 2015 April DOI: /path.4540

93 Heterogeneity, primary versus metastatic

94 Gong Y, Booser DJ, Sneige N: Comparison of HER-2 statusdetermined by fluorescence in situ hybridization in primaryand metastatic breast carcinoma. Cancer 2005, 103: Gancberg D, Di Leo A, Cardoso F, Rouas G, Pedrocchi M, PaesmansM, Verhest A, Bernard- Marty C, Piccart MJ, Larsimont D: Comparison of HER-2 status between primary breast cancer and corresponding distant metastatic sites. Ann Oncol 2002,13: Regitnig P, Schippinger W, Lindbauer M, Samonigg H, Lax SF: Change of HER-2/neu status in a subset of distant metastases from breast carcinomas. J Pathol 2004, 203: Bozzetti C, Personeni N, Nizzoli R, Guazzi A, Flora M, Bassano C, Negri F, Martella E, Naldi N, Franciosi V, et al.: HER-2/neu amplification by fluorescence in situ hybridization in cytologic samples from distant metastatic sites of breast carcinoma. Cancer2003, 99: Tanner M, Jarvinen P, Isola J: Amplification of HER-2/neu and topoisomerase IIα in primary and metastatic breast cancer.cancer Res 2001, 61:

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98 HER2 heterogeneity Intratumoral herterogeneity: up to 40% Intertumoral heterogeneity: up to 16% Metastasis versus primary tumor: up to 22%

99 Conclusions

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101 More than assumptions Tumor heterogeneity Impacts on diagnosis (tumor sampling bias) Impacts on treatment decisions and effect (minute subclones may determine the outcome of the disease being resistent to the applied therapy) Impacts on the outcome

102 Guidelines Too many Too complicated Too often modified Insufficient with regard to tumor heterogeneity Focus on treatable, not on curable

103 Thank You