The portfolio of adjuvant systemic treatment of breast cancer nowadays contains novel anti-hormonal and chemotherapeutic drugs, immunotherapeutic approaches and small molecules that are only effective in a limited number of patients and are often associated with high costs and significant side effects. Therefore, a personalised approach based on individual tumour biomarkers is required to arrive at the optimal balance between effectiveness on the one hand, and costs and side effects on the other. The aim of this paper is to provide an overview of the molecular biomarkers and associated molecular tests that are currently relevant in pathology of invasive breast cancer.
Breast cancer, pathology, molecular biomarkers
Natalie D ter Hoeve, Cathy B Moelans, Willemijne AME Schrijver, Wendy de Leng and Paul J van Diest have nothing to disclose in relation to this article. This study involves a review of the literature and did not involve any studies with human or animal subjects performed by any of the authors. No funding was received for the publication of this article.
Authorship: All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship of this manuscript, take responsibility for the integrity of the work as a whole, and have given final approval to the version to be published.
This article is published under the Creative Commons Attribution Noncommercial License, which permits any non-commercial use, distribution, adaptation and reproduction provided the original author(s) and source are given appropriate credit.
October 12, 2016 Accepted:
February 13, 2017 Published Online:
June 14, 2017
Paul J van Diest, Department of Pathology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht. E: email@example.com
Adjuvant systemic treatment of breast cancer is moving away from the limited portfolio of traditional hormonal drugs and chemotherapy, towards a gamma of novel anti-hormonal and chemotherapeutic drugs, immunotherapeutic approaches and small molecules. All these therapeutic approaches are unfortunately effective in a limited number of patients and are often associated with high costs and significant side effects. Therefore, the traditional “one size fits all” approach can no longer be upheld, and a personalised approach based on individual tumour biomarkers is required to arrive at the optimal balance between effectiveness on the one hand and costs and side effects on the other.
Over recent years, progress in molecular techniques has made it possible to analyse formalin fixed paraffin embedded (FFPE) tumour material of larger cohorts of patients. This has incentivised many translational studies relating molecular tumour biomarkers to diagnosis, prognosis and/or response to therapy, yielding many relevant molecular biomarkers that have rather quickly made it to clinical pathology practice. The aim of this paper is to provide an overview of the molecular biomarkers and associated molecular tests that are currently relevant in pathology of invasive breast cancer.
Hereditary breast cancers
About 5–10% of breast cancer cases are due to a hereditary predisposition.1 In most of these cases, mutations are found in well-characterised, medium to high-risk genes, such as BRCA1, BRCA2, CHEK2, TP53, PALB2, or BRIP1,2 of which BRCA1 and BRCA2 are the most important ones. Promotor hypermethylation of BRCA1 and BRCA2 seems to be very infrequent in BRCA1/2 germline mutation related breast cancers, and significantly more frequent in sporadic cancers [data unpublished, submitted for publication]. BRCA1/2 promoter methylation testing, pointing to sporadic cancers when present, may grow out to be clinically useful once the diagnostically optimal CpG islands have been identified.3
Copy number analysis by array comparative genomic hybridisation (CGH) showed frequently occurring gains of 3q, 7p, 8q 10p, 12p, 16p and 17q, and loss of 2q, 3p, 4p, 4q, 5q, 12q, 16p and 18q in BRCA1 germline mutation related cancers. BRCA2 related breast cancers show more frequently gains of 8q, 17q22–q24 and 20q13, and loss of 8p, 6q, 11q and 13q compared to BRCA1 related cancers.4,5 A multiplex ligation-dependent probe amplification (MLPA) kit® (MRC Holland, Amsterdam, The Netherlands) for copy number testing pointing to BRCA1 related cancers is commercially available.
Molecular (intrinsic) typing
Several gene expression studies have revealed the existence of five molecular subtypes of breast cancer: a “basal-like” subgroup with low oestrogen receptor (ER)/progesterone receptor(PR)/ human epidermal growth factor receptor 2 (HER2) and expression of basal cytokeratins; a subgroup mainly driven by HER2 amplification and overexpression while being ER/PR low; a luminal A group with high ER/PR and low HER2; and a luminal B group that is also ER/PR high but with additional HER2 overexpression and/ or high proliferation.6,7 A further “normal breast like” group is likely the result of absence of tumour in the frozen pieces that were analysed without morphological control by a pathologist. These different classes have varying clinical behaviour and have led to a new way of thinking about classification of breast cancer going beyond morphology. Nevertheless, good correlations exist between these intrinsic subtypes and morphology. The basal-like group contains high grade ductal medullary and metaplastic cancers as well as the low grade salivary gland type cancers (adenoid cystic cancers [AdCC], acinic cell cancers, myoepithelial cancers). The HER2 group contains poorly differentiated HER2 overexpressing ductal and apocrine cancers. The luminal A group contains mainly low grade ductal, lobular, ductulolobular, tubular, cribriform, mucinous and micropapillary cancers. Several intrinsic typing tests are commercially available (PAM50®, NanoString, Washington, US; Blueprint®, Agendia, Amsterdam, The Netherlands). However, these intrinsic subtypes have little added value to type, grade and expression of ER/PR/HER2, and the reproducibility of intrinsic subtyping based on gene expression has proven to be low across datasets and technology platforms. Besides, these tests are not available for local testing in pathology labs, are time consuming and expensive. Therefore, a more practical approach is probably to use an immunohistochemical surrogate as depicted in Table 1.
PAM50 and Blueprint are two commercially available gene expression test for intrinsic subtyping (from centrally at Nanostring and Agendia, respectively). The PAM50 signature employs 50 genes and can be applied on FFPE material.8 Blueprint contains 80 genes and can be applied on FFPE material as well.
Adenoid cystic carcinoma
While being a frequent cancer type in the minor and major salivary glands with frequent perineural invasion and poor prognosis, AdCC is a rare cancer type in the breast, accounting for 0.1–1% of breast cancers, with infrequent perineural invasion and indolent clinical behaviour despite their triple negative (ER/PR/HER2) state.9 These carcinomas often display the recurrent chromosomal translocation t(6;9) (q22e23;p23e24), which generates oncogenic fusion transcripts involving the two transcription factor genes MYB and NFIB. In the t(6;9) (q22eq23;p23ep24), the exon 14 of MYB is fused to the final coding exons of NFIB, usually due to breakpoints in MYB intron 14 and intron 8 in NFIB. The fusion results in loss of the 3’-end of MYB, including several conserved binding sites for microRNAs that regulate MYB expression negatively. More recently, also recurring t(8;9) and t(8;14) translocations have been described fusing the MYBL1 gene to the NFIB and RAD51B genes, respectively.10,11 Due to the characteristic histological features of AdCC, translocation assays may not be often necessary in diagnostic practice, but may be assessed by fluorescent in situ hybridisation (FISH) kits or reverse transcription polymerase chain reaction (RT-PCR) in difficult cases.
Secretory cancer is a rare breast cancer type that occurs at all ages, even at quite young age. Recently, it was shown that secretory cancer is characterised by a balanced chromosomal translocation t(12;15) (p13;q25), which leads to the formation of an oncogenic ETV6-NTRK3 fusion gene encoding a chimeric tyrosine kinase, also demonstrated in paediatric mesenchymal cancers.12 Translocations may be assessed by FISH or RT-PCR in difficult cases.
1. Economopoulou P, Dimitriadis G, Psyrri A, Beyond BRCA: new hereditary breast cancer susceptibility genes,Cancer Treat Rev, 2015;41:1–8.
2. Vos S, Van der Groep P, Van der Wall E, Van Diest PJ, Hereditary Breast Cancer Syndromes: Molecular Pathogenesis and Diagnostics. eLS: New Jersey, US, John Wiley & Sons, Ltd; 2001;1-16.
3. Vos S, Moelans CB, van Diest PJ, BRCA promoter methylation in sporadic versus BRCA germline mutation-related breast cancers, Breast Cancer Res, 2017: in press.
4. Wessels LF, van Welsem T, Hart AA, et al., Molecular classification of breast carcinomas by comparative genomic hybridization: a specific somatic genetic profile for BRCA1 tumors, Cancer Res, 2002;62:7110–17.
5. Joosse SA, Brandwijk KI, Devilee P, et al., Prediction of BRCA2- association in hereditary breast carcinomas using array-CGH, Breast Cancer Res Treat, 2012;132:379–89.
6. Perou CM, Sorlie T, Eisen MB, et al., Molecular portraits of human breast tumours, Nature, 2000;406:747–52.
7. Sorlie T, Perou CM, Tibshirani R, et al., Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications, Proc Natl Acad Sci U S A, 2001;98:10869–74.
8. Ellis MJ, Suman VJ, Hoog J, et al., Randomized phase II neoadjuvant comparison between letrozole, anastrozole, and exemestane for postmenopausal women with estrogen receptor-rich stage 2 to 3 breast cancer: clinical and biomarker outcomes and predictive value of the baseline PAM50-based intrinsic subtype-ACOSOG Z1031, J Clin Oncol, 2011;29:2342–9.
9. Marchio C, Weigelt B, Reis-Filho JS, Adenoid cystic carcinomas of the breast and salivary glands (or ‘The strange case of Dr Jekyll and Mr Hyde’ of exocrine gland carcinomas), J Clin Pathol, 2010;63:220–8.
10. Brayer KJ, Frerich CA, Kang H, Ness SA, Recurrent fusions in MYB and MYBL1 define a common, transcription factor-driven oncogenic pathway in salivary gland adenoid cystic carcinoma, Cancer Discov, 2016;6:176–87.
11. Persson M, Andren Y, Mark J, et al., Recurrent fusion of MYB and NFIB transcription factor genes in carcinomas of the breast and head and neck, Proc Natl Acad Sci U S A, 2009;106:18740–4.
12. Tognon C, Knezevich SR, Huntsman D, et al., Expression of the ETV6-NTRK3 gene fusion as a primary event in human secretory breast carcinoma, Cancer Cell, 2002;2:367–76.
13. Filipits M, Rudas M, Jakesz R, et al., A new molecular predictor of distant recurrence in ER-positive, HER2-negative breast cancer adds independent information to conventional clinical risk factors, Clin Cancer Res, 2011;17:6012–20.
14. Sapino A, Roepman P, Linn SC, et al., MammaPrint molecular diagnostics on formalin-fixed, paraffin-embedded tissue, J Mol Diagn, 2014;16:190–7.
15. Paik S, Shak S, Tang G, et al., A multigene assay to predict recurrence of tamoxifen-treated, node-negative breast cancer, N Engl J Med, 2004;351:2817–26.
16. Gyanchandani R, Lin Y, Lin HM, et al., Intratumor Heterogeneity Affects Gene Expression Profile Test Prognostic Risk Stratification in Early Breast Cancer, Clin Cancer Res, 2016;22:5362-9.
17. Eden P, Ritz C, Rose C, et al., “Good Old” clinical markers have similar power in breast cancer prognosis as microarray gene expression profilers, Eur J Cancer, 2004;40:1837–41.
18. Cuzick J, Dowsett M, Pineda S, et al., Prognostic value of a combined estrogen receptor, progesterone receptor, Ki-67, and human epidermal growth factor receptor 2 immunohistochemical score and comparison with the Genomic Health recurrence score in early breast cancer, J Clin Oncol, 2011;29:4273–8.
19. Dowsett M, Sestak I, Lopez-Knowles E, et al., Comparison of PAM50 risk of recurrence score with oncotype DX and IHC4 for predicting risk of distant recurrence after endocrine therapy, J Clin Oncol, 2013;31:2783–90.
20. Dowsett M, Nielsen TO, A’Hern R, et al., Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group, J Natl Cancer Inst, 2011;103:1656–64.
21. Roepman P, Horlings HM, Krijgsman O, et al., Microarray-based determination of estrogen receptor, progesterone receptor, and HER2 receptor status in breast cancer, Clin Cancer Res, 2009;15:7003–11.
22. Robinson DR, Wu YM, Vats P, et al., Activating ESR1 mutations in hormone-resistant metastatic breast cancer, Nat Genet, 2013;45:1446–51.
23. Toy W, Shen Y, Won H, et al., ESR1 ligand-binding domain mutations in hormone-resistant breast cancer, Nat Genet, 2013;45:1439–45.
24. Roodi N, Bailey LR, Kao WY, et al., Estrogen receptor gene analysis in estrogen receptor-positive and receptornegative primary breast cancer, J Natl Cancer Inst, 199515;87:446–51.
25. Jeselsohn R, Buchwalter G, De Angelis C, et al., ESR1 mutations-a mechanism for acquired endocrine resistance in breast cancer, Nat Rev Clin Oncol, 2015;12:573–83.
26. Merenbakh-Lamin K, Ben-Baruch N, Yeheskel A, et al., D538G mutation in estrogen receptor-alpha: A novel mechanism for acquired endocrine resistance in breast cancer, Cancer Res, 2013;73:6856–64.
27. Sefrioui D, Perdrix A, Sarafan-Vasseur N, et al., Short report: Monitoring ESR1 mutations by circulating tumor DNA in aromatase inhibitor resistant metastatic breast cancer, Int J Cancer, 2015;137:2513–9.
28. Schiavon G, Hrebien S, Garcia-Murillas I, et al., Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer, Sci Transl Med, 2015;7:313ra182.
29. Angus L, Beije N, Jager A, et al., ESR1 mutations: Moving towards guiding treatment decision-making in metastatic breast cancer patients, Cancer Treat Rev, 2017;52:33–40.
30. Babyshkina N, Vtorushin S, Zavyalova M, et al., The distribution pattern of ER alpha expression, ESR1 genetic variation and expression of growth factor receptors: association with breast cancer prognosis in Russian patients treated with adjuvant tamoxifen, Clin Exp Med, 2016; [Epub ahead of print].
31. Moelans CB, Holst F, Hellwinkel O, et al., ESR1 amplification in breast cancer by optimized RNase FISH: frequent but low-level and heterogeneous, PLoS One, 2013;8:e84189.
32. Lapidus RG, Ferguson AT, Ottaviano YL, et al., Methylation of estrogen and progesterone receptor gene 5’ CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors, Clin Cancer Res, 1996;2:805–10.
33. Ferguson AT, Lapidus RG, Baylin SB, Davidson NE, Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression, Cancer Res, 1995;55:2279–83.
34. Moelans CB, de Weger RA, Van der Wall E, van Diest PJ, Current technologies for HER2 testing in breast cancer, Crit Rev Oncol Hematol, 2011;80:380–92.
35. Lim TH, Lim AS, Thike AA, et al., Implications of the updated 2013 American Society of Clinical Oncology/College of American Pathologists guideline recommendations on human epidermal growth factor receptor 2 gene testing using immunohistochemistry and fluorescence in situ hybridization for breast cancer, Arch Pathol Lab Med, 2016;140:140–7.
36. Moelans CB, de Weger RA, Ezendam C, van Diest PJ, HER-2/ neu amplification testing in breast cancer by Multiplex Ligationdependent Probe Amplification: influence of manual- and laser microdissection, BMC Cancer, 2009;9:4.
37. Singh K, Tantravahi U, Lomme MM, et al., Updated 2013 College of American Pathologists/American Society of Clinical Oncology (CAP/ASCO) guideline recommendations for human epidermal growth factor receptor 2 (HER2) fluorescent in situ hybridization (FISH) testing increase HER2 positive and HER2 equivocal breast cancer cases; retrospective study of HER2 FISH results of 836 invasive breast cancers, Breast Cancer Res Treat, 2016;157:405–11.
38. Moelans CB, Reis-Filho JS, van Diest PJ, Implications of rarity of chromosome 17 polysomy in breast cancer, Lancet Oncol, 2011;12:1087–9.
39. Moelans CB, de Weger RA, van Diest PJ, Absence of chromosome 17 polysomy in breast cancer: analysis by CEP17 chromogenic in situ hybridization and multiplex ligationdependent probe amplification, Breast Cancer Res Treat, 2010;120:1–7.
40. Weigelt B, Reis-Filho JS, Activating mutations in HER2: neu opportunities and neu challenges, Cancer Discov, 2013;3:145–7.
41. Bose R, Kavuri SM, Searleman AC, et al., Activating HER2 mutations in HER2 gene amplification negative breast cancer, Cancer Discov, 2013;3:224–37.
42. Dave B, Migliaccio I, Gutierrez MC, et al., Loss of phosphatase and tensin homolog or phosphoinositol-3 kinase activation and response to trastuzumab or lapatinib in human epidermal growth factor receptor 2-overexpressing locally advanced breast cancers, J Clin Oncol, 2011;29:166–73.
43. Wang L, Zhang Q, Zhang J, et al., PI3K pathway activation results in low efficacy of both trastuzumab and lapatinib, BMC Cancer, 2011;11:248.
44. Ma CX, Crowder RJ, Ellis MJ, Importance of PI3-kinase pathway in response/resistance to aromatase inhibitors, Steroids, 2011;76:750–2.
45. Vollebergh MA, Lips EH, Nederlof PM, et al., An aCGH classifier derived from BRCA1-mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients, Ann Oncol, 2011;22:1561–70.
46. Lips EH, Laddach N, Savola SP, et al., Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness, Breast Cancer Res, 2011;13:R107.
47. Ibragimova I, Cairns P, Assays for hypermethylation of the BRCA1 gene promoter in tumor cells to predict sensitivity to PARP-inhibitor therapy, Methods Mol Biol, 2011;780:277–91.
48. Hoefnagel LD, Moelans CB, Meijer SL, et al., Prognostic value of estrogen receptor alpha and progesterone receptor conversion in distant breast cancer metastases, Cancer, 2012;118:4929–35.
49. Bertucci F, Finetti P, Guille A, et al., Comparative genomic analysis of primary tumors and metastases in breast cancer, Oncotarget, 2016;7:27208–19.
50. Beelen K, Hoefnagel LD, Opdam M, et al., PI3K/AKT/mTOR pathway activation in primary and corresponding metastatic breast tumors after adjuvant endocrine therapy, Int J Cancer, 2014;135:1257–63.
51. Cizkova M, Dujaric ME, Lehmann-Che J, et al., Outcome impact of PIK3CA mutations in HER2-positive breast cancer patients treated with trastuzumab, Br J Cancer, 2013;108:1807–9.
52. de Leng WW, Gadellaa-van Hooijdonk CG, Barendregt-Smouter FA, et al., Targeted next generation sequencing as a reliable diagnostic assay for the detection of somatic mutations in tumours using minimal DNA amounts from formalin fixed paraffin embedded material, PLoS One, 2016;11:e0149405.
53. Schouten PC, Dackus GM, Marchetti S, et al., A phase I followed by a randomized phase II trial of two cycles carboplatinolaparib followed by olaparib monotherapy versus capecitabine in BRCA1- or BRCA2-mutated HER2-negative advanced breast cancer as first line treatment (REVIVAL): study protocol for a randomized controlled trial, Trials, 2016;17:293.
54. Hoogstraat M, Hinrichs JW, Besselink NJ, et al., Simultaneous detection of clinically relevant mutations and amplifications for routine cancer pathology, J Mol Diagn, 2015;17:10–18.
55. Olsson E, Winter C, George A, et al., Serial monitoring of circulating tumor DNA in patients with primary breast cancer for detection of occult metastatic disease, EMBO Mol Med, 2015;7:1034–47.
56. Rothe F, Laes JF, Lambrechts D, Smeets D, Vincent D, Maetens M, et al. Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer. Ann Oncol, 2014 Oct;25(10):1959–1965.
57. Ignatiadis M, Rack B, Rothe F, Riethdorf S, Decraene C, Bonnefoi H, et al. Liquid biopsy-based clinical research in early breast cancer: The EORTC 90091-10093 Treat CTC trial. Eur J Cancer 2016 Aug;63:97–104.