Molecular Diagnostics in Breast Cancer Routine Practice

European Oncology & Haematology, 2017;13(1):74–9 DOI:


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.
Keywords: Breast cancer, pathology, molecular biomarkers
Disclosure: 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.
Received: October 12, 2016 Accepted February 13, 2017 Published Online: June 14, 2017
Correspondence: Paul J van Diest, Department of Pathology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht. E:
Open Access: 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.

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.

Diagnostic markers
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
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.

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Keywords: Breast cancer, pathology, molecular biomarkers