Fine-Needle Aspiration Is Suitable for Breast Cancer BRCA Molecular Assessment: A Case Report

Abstract

Breast cancer is the most common cause of cancer-related deaths in the female population worldwide. To the best of our knowledge, breast cancer (BRCA)1/2 gene mutations have not been described yet on breast cancer cytological specimens. Here we describe the case of a 38-year old woman with a family and personal history for breast cancer, who underwent a fine needle aspiration (FNA) procedure for a novel 30 mm lesion located in the external quadrants of the contralateral (left) breast. Cytological findings and ancillary immunostaining confirmed the diagnosis of a triple negative NST carcinoma. BRCA1/2 molecular assessment was carried out on DNA extracted from cytological (November 2020), biopsy (December 2014) and surgical resection (July 2015) specimens, as well as on the resection of a benign fibroadenoma, by using a next generation sequencing approach. Molecular analysis showed a pathogenic BRCA1 insertion (c.5266dupC; p.Q1756PfsTer74) in the cytological specimen (allelic fraction 92.0%), biopsy (allelic fraction 84.2%), surgical resection (allelic fraction 87.8%) and fibroadenoma (58.9%), demonstrating a germinal BRCA mutated status.

1. Introduction

Breast cancer represents the most common cause of cancer-related deaths in the female population worldwide, with a dramatically increasing numbers [1]. Despite the improvements in the screening and therapeutic management of breast cancer patients, the advanced stages of disease are still difficult to treat [2]. Beyond standard systemic therapies, an additional arrow in the quiver of oncologists is represented by poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) [3]. To date, two PARPi monotherapies, olaparib and talazoparib have, in the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA), obtained approval for advanced stages germline breast cancer (BRCA) 1/2 genes-mutated, human epidermal growth factor receptor 2 (HER2)-negative, in breast cancer patients [4,5,6,7]. In advanced stages of the disease, a minimal invasive fine needle aspiration (FNA) approach for primitive or metastatic lesions plays a key role in the morphological and molecular evaluation of different solid tumors, including breast cancer [8,9,10]. Nowadays, next generation sequencing (NGS) represents a valid approach for BRCA tumor testing in breast cancer patients [11]. However, BRCA1/2 mutations on cytological specimens have not been described yet. Here we report a case harboring a BRCA1 mutation, identified through an NGS approach in both cytological and histological specimens.

2. Case Report

In November 2014, a 38-year old woman with a family history for breast cancer underwent a fine needle aspiration (FNA) procedure at the University of Naples “Federico II” on a right, under-areolar, breast lesion of 33 mm of diameter. The US-guided procedure was performed by a trained interventional cytopathologist, as previously reported [9]. Smears were readily fixed in 95% ethanol and subsequently stained with Papanicolaou. An additional pass was taken and rinsed in formalin to ensure a cell block (CB) preparation. The direct smears showed a necrotic background with occasional histiocytes, and numerous solid groups of epithelial cells feature striking nuclear abnormalities. The immunocytochemical (ICC) analysis performed on CB showed negativity for estrogen (ER clone SP1, Ventana, Oro Valley, AZ, USA), progesterone (PR clone 1E2, Ventana) receptors and Human Epidermal Growth Factor Receptor 2 (HER2, Dako test, Agilent Technologies, Santa Clara, CA, USA). Ki67 (clone 30-9, Ventana) was expressed by 40% of the cells. A diagnosis of high grade, triple negative, breast carcinoma of no special type (NST) was carried out. Subsequently, the histological evaluation performed on both a core biopsy (December 2014) and surgical resection (July 2015), confirmed the evidence of an invasive, high grade, triple negative, breast NST carcinoma. Concurrently, a benign fibroadenoma was also resected. The patient was then administered with sequential chemotherapy, which included a combination of anthracyclines and taxanes. During the follow-up, in November 2020, a second FNA was requested upon the evidence of a novel 30 mm lesion located in the external quadrants of the contralateral (left) breast. The cytological and ICC features overlapped with that of the FNA from the primary lesion. Thus, a metastasis from the previous triple negative NST carcinoma was diagnosed. Due to the lack of receptor expressions on ICC, the aggressive clinical course and a family history for breast cancer, the patient underwent a molecular analysis for BRCA 1/2 alterations. To this end, next generation sequencing (NGS) analysis was carried out on DNA extracted from cytological smear (November 2020), biopsy (December 2014) and surgical resection (July 2015) specimens. To evaluate the presence of the genomic alteration at the germinal level, DNA extracted from the concurrent benign lesion (fibroadenoma) was also tested. The analysis was carried out by adopting a commercially available NGS 2-pool panel (Oncomine™ BRCA Research Assay, Thermo Fisher Scientific, Waltham, MA, USA) able to cover 100% of the coding sequences in BRCA1/2 genes, including all coding splice site and acceptor sites, with an average of 64 bp extension into adjoining introns on Ion Torrent S5 (Thermo Fisher Scientific). Data inspection was automatically carried out using Ion Reporter Torrent Suite version 5.18.0.1 with a dedicated analysis workflow optimized for somatic annotation of BRCA alterations. In detail, a minimum depth of 500×, a quality score ≥ 20 and an allele mutation frequency for ≥5% were required to successfully identify BRCA1/2 mutations. In addition, BAM files were visually inspected with the Golden Helix Genome Browser v.2.0.7 (Bozeman, MT, USA). Variant annotation was performed according to Human Genome Variation Society nomenclature. Variants were classified as pathogenic or likely pathogenic (collectively termed pathogenic) according to the American College of Medical Genetics and Genomics (ACMG) recommendations with the inspection of Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) and ClinVar (NCBI) databases. Molecular analysis showed a pathogenic BRCA1 insertion (c.5266dupC; p.Q1756PfsTer74) in the cytological specimen (allelic fraction 92.0%, number of mapped reads: 1934441, uniformity of amplicon coverage: 7003), biopsy (allelic fraction 84.2%, number of mapped reads: 1177566, uniformity of amplicon coverage: 5369), surgical resection (allelic fraction 87.8%, number of mapped reads: 1229612, uniformity of amplicon coverage: 4016) and fibroadenoma (58.9%, number of mapped reads: 9027266, uniformity of amplicon coverage: 5065), demonstrating a germinal BRCA mutated status (Figure 1). No other clinically relevant alterations were found. Thus, since the BRCA mutation was also retrieved as germinal alteration in the fibroadenoma, the patient was referred to the oncologist for the PARPi therapy.

3. Discussion

Here we showed the technical feasibility of the BRCA1/2 molecular assessment on FNA from a metastatic breast lesion. Given its cost-effectiveness and tolerability, FNA for breast solid lesions is still employed to guide clinicians and surgeons for the best treatment approach [12]. To date, the introduction of the Yokohama System for Reporting Breast FNA by the International Academy of Cytology has improved the diagnostic value of this minimal invasive approach by standardizing the morphological report [13]. From a molecular point of view, cytological samples represent a suitable starting material to carry out NGS molecular analysis for predictive purposes and to refine the risk of malignancy of indeterminate cytological diagnosis [14,15]. Less is known about the feasibility of NGS analysis on breast cytological samples for BRCA1/2 genomic assessment. To date, the analysis of BRCA1/2 genes has acquired relevant clinical importance due to the FDA and EMA approval of two PARPi monotherapies, olaparib and talazoparib, for advanced stage, germline BRCA1/2 genes-mutated, HER2-negative, breast cancer patients [4,5,6,7]. In this setting, the absence of surgical resection or of tissue biopsy specimens due to the high invasiveness, risks and discomfort for patients, may be associated with a clinical bias for treatment decision making. Conversely, the minimal invasive FNA may be a valid solution to overcome these limitations. In addition, cytological smears do not suffer from the issue related to formalin fixation that impact on the quality of extracted nucleic acids in histological samples [16]. However, in the present case, the histological material from the first occurrence of the cancer was available and we were able to verify the presence of BRCA1 mutation on both the primary and benign lesion, thus confirming the germline origin of the BRCA1 mutation. It has been widely demonstrated that among patients with HER2-negative metastatic breast cancer harboring a germline BRCA mutation, olaparib or talazoparib monotherapy determine a significant clinical outcome respect to standard therapies [17,18]. In the OlympiA clinical trial high-risk, HER2-negative early breast cancer and germline BRCA1/2 pathogenic or likely pathogenic variants, received adjuvant olaparib with the evidence of a significant improvement in progression free survival respect to placebo group [19]. Another possible clinical application of PARPi is the association with immunotherapy in advanced stage, triple negative breast cancer patients. Vinayak et al. [20] and Domchek et al. [21] highlighted that the association of niraparib plus pembrolizumab and olaparib and durvalumab showed promising antitumour activity and safety.

The main limitations of this study lie in the absence of control samples to check the reproducibility of NGS experiments and the absence of blood sample-derived DNA to confirm the germinal origin of the detected alteration. However, further studies by our group are ongoing to evaluate the role of our NGS approach for the identification of BRCA1/2 gene alterations in diagnostic routine practice. However, we speculate that the detected alteration has a germline origin due to identification in either malignant (allelic fraction of about 100%) or benign (allelic fraction of about 50%) lesions.

In conclusion, to the best of our knowledge, this is the first report describing the NGS assessment of BRCA1/2 on cytological samples obtained from breast cancer, confirming the suitability of minimal invasive FNA as a valuable biospecimen for molecular-based predictive tests. Further studies are needed to confirm the suitability of cytology specimens for detecting BRCA1/2 molecular alterations by using NGS.