Case report
The proband investigated in the current study is a 54-year-old Chinese female clinically diagnosed with ovarian cancer who belongs to a non-consanguineous Chinese family. The proband's elder sister passed away at the age of 46 years as a result of ovarian cancer. The age and cause of mortality for the proband's parents are unknown (Fig. 1). The proband had been diagnosed with stage III right ovarian cancer, along with liver metastasis. The top edge of the liver exhibited a nodule with mild leukoaraiosis. The magnetic resonance imaging (MRI) scan demonstrated no metastasis around the brain.
The biopsy sections (thickness, 3–4 µm) were paraffin embedded, fixed with 4% neutral buffer formaldehyde solution, at room temperature (25°C) for 6–48 h. Pathological biopsy was performed at the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Hematoxylin and eosin staining of tumor tissue was performed by optical microscopy (magnification, ×460) and microscopy images were obtained (Fig. 2A-C). Paraffin-embedded slices were stained with IHC markers (Ventana Benchmark Ultra automatic immunohistochemical staining platform, primary antibodies: ER: Anti-estrogen receptor (SP1) rabbit monoclonal primary antibody, Roche, catalog number: 790–4325; PR: Anti-progesterone receptor (1E2) rabbit monoclonal primary antibody, Roche, catalog number: 790–4296; P53: Anti-P53 mouse monoclonal primary antibody, Roche, catalog number: 800–2912; Ki-67: Anti-Ki-67 (30–9) rabbit monoclonal primary antibody, Roche, catalog number: 790–4286; CEA: Anti-CEA (COL-1) mouse monoclonal primary antibody, From: MXB Biotechnologies, catalog number: Kit-0008; CA125: Anti-CA125 (TA347) mouse monoclonal primary antibody, MXB Biotechnologies, catalog number: MAB-0007; WT1: Anti-WT1 (MX012) mouse monoclonal primary antibody, MXB Biotechnologies, catalog number: MAB-0678; PAX-8: Anti-PAX-8 rabbit polyclonal primary antibody, MXB Biotechnologies, catalog number: RAB-0657; PAX-2: MXB Biotechnologies, catalog number: RAB-0648. Secondary antibodies: MaxVision DAB, From: MXB Biotechnologies, catalog number: KIT-5220. All antibodies were purchased in their ready-to-use format so dilutions weren't required. IHC experimental procedure according to the manufacturer's protocols), the percentage of positive staining was calculated by eye. Immunohistochemistry (IHC) for estrogen receptors demonstrated a high (70%) expression level in cancer cells (Fig. 2D). IHC for progesterone receptors revealed a moderate (30%) expression level in cancer cells (Fig. 2E). IHC for p53 demonstrated zero expression level of p53 in cancer cells (Fig. 2F). IHC for Ki67 revealed a moderate (30%) expression level in cancer cells (Fig. 2G). Staining for cancer antigen 125 demonstrated a high expression level (Fig. 2H). IHC for Wilms tumor protein (WT1) revealed a moderate expression level in cancer cells (Fig. 2I). IHC for paired box gene PAX-2, PAX-8 and CEA was also performed, revealing zero, positive and positive expression, respectively (Fig. 2J-L). An MRI scan was performed, which demonstrated enhancement of the pelvic tumor (Fig. 3A), enhancement of hepatic metastasis (Fig. 3B,C), and thickening of the omentum majus and tumor tissue (Fig. 3D).
The patient's treatment regimen consisted of TC scheme (paclitaxel and carboplatin: Paclitaxel liposome, 240 mg/day 1+ carboplatin, 0.5 g/day 1, intravenous injection) chemotherapy for two cycles. Following treatment, the patient showed an improvement, and a gynecological surgeon contacted the patient's family.
Prior to the experiment, written informed consent was obtained from each patient. Peripheral blood samples of the patients were collected and genomic DNA was extracted. Targeted high-throughput sequencing (DNA polymerase is T4 DNA POLYMERASE, supplier is ENZYMATICS; Qiagen, Inc., Valencia, CA, USA) was performed with a panel of a 21 genes (BRCA1, BRCA2, CHEK2, PALB2, BRIP1, TP53, PTEN, STK11, CDH1, ATM, BARD1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PMS1, PMS2, RAD50 and RAD51C). Roche NimbleGen's (Roche Applied Science, Madison, MI, USA) custom Sequence Capture Human array was used to designed to capture targeted sequence, according to the manufacturer's protocols. A target area of 172,959 bp, a target area coverage of 99.49% and a target area average depth of 586.13×. Samples were sequenced simultaneously on Illumina HiSeq 2500 Analyzers (Illumina Inc., San Diego, CA, USA) for 90 cycles according to the manufacturer's protocols. Targeted high-throughput sequencing identified a novel heterozygous mutation, c.3195_3196insA, in BRCA2, which causes a frameshift mutation that generates a premature stop codon and results in the formation of a truncated BRCA2 protein. This mutation was confirmed by Sanger sequencing (Fig. 1R). The mutation was absent in the Human Gene Mutation database (www.hgmd.cf.ac.uk/) and the Online Mendelian Inheritance in Man database (www.omim.org). In addition, the mutation could not be identified in our in-house databases, which contains ~30,000 samples from Chinese Han individuals.
Since the proband's elder sister passed away at an age of 46 years due to ovarian cancer, it can be hypothesized that she carried the same mutation in BRCA2. In addition, the proband's father or mother may carry the same mutation. However, as the proband's elder sister and parents had passed away, it was not possible to investigate this. The proband's children did not volunteer genetic testing. Therefore, only the following suggestions can be made. Firstly, it could be hypothesized that the proband and her sister carried the same mutation on the BRCA2 gene, which was inherited from their father or mother. By contrast, it could be hypothesized that the mutation carried by the proband is a de novo mutation. In this case, her parents and sister would not have carried the same mutation. With the knowledge that the proband's sister passed away due to ovarian cancer, we speculate that the first hypothesis is more likely. The Ethical Committee of The Fourth Hospital of Hebei Medical University (Shijiazhuang, China) approved the current study protocol, which was in compliance with the Declaration of Helsinki.
Discussion
A c.3195_3196insA frameshift mutation in exon 10 of the BRCA2 gene, which has not previously been reported in the BIC database, was detected in a 54 year-old female Chinese patient diagnosed with ovarian cancer. Not all genetic variants are harmful; BRCA1/2 variations can be classified into the five following categories: ‘Pathogenic’, ‘likely pathogenic’, ‘uncertain significance’, ‘likely benign’ and ‘benign’, according to American College of Medical Genetics’ guidelines (8). ‘Pathogenic’ or ‘likely pathogenic’ indicates that a mutation was identified in a specific gene, which increases the lifetime risk of developing certain cancer types, but does not indicate that cancer will definitely develop. Germline BRCA1/2 deleterious mutations lead to an increased risk of early onset hereditary breast and ovarian cancer (HBOC)-associated tumors, including breast, ovarian and pancreatic cancer in females, and to a lesser extent, breast and prostate cancer in males (9,10).
Genetic sequencing techniques have developed radically during the past few decades; therefore, novel testing techniques have been widely used in the medical sector (11–15). For example, the enrichment step, a preparation technique used prior to next-generation sequencing (NGS), can significantly improve the accuracy and efficiency of medical screening (16). This technique was used in the current study to identify the largest possible number of mutations that may be associated with HBOC. Due to technology limitations, there are still many challenges in detecting copy-number variants by NGS (17), certain patients with a negative NGS testing result may also require multiplex ligation-dependent probe application, particularly patients with a family history of cancer.
BRCA1 and BRCA2 were not the only genes analyzed in the current study; multiple gene analysis was performed. Numerous genes should be involved in sequencing tests as the mutational spectra of these genes can be accurately detected with NGS technology, which provides additional data that can support clinical diagnoses. However, involving multiple genes in sequencing tests may lead to the detection of a higher number of variants of uncertain significance (VUS) (18). Clinical recommendations may not be standardized for the presence of VUS mutations in certain cancer susceptibility genes, which may complicate results. These complex results are why suggestions from medical geneticists or certified genetic counselors are important. Since a number of NGS-based techniques are used in genetic testing and different genetic companies perform different techniques, customers should have access to the sensitivity and specificity of any results, as well as comparisons of various techniques (19). In our opinion, positive results should be validated using standard Sanger sequencing, which should also be available to relatives who are concerned about the risk of cancer.