The examined material included blood cells (BC) and formalin-fixed paraffin embedded tissue (FFPE) collected during standard laboratory procedures. The histological diagnosis of ovarian serous carcinoma and the tumor tissue was evaluated by pathologist.
The patient, a 67-year-old female underwent right-sided mastectomy for breast cancer (NST, G2, T2N0M0) in 2011 and complementary chemotherapy (FEC) in 2012. Furthermore, she suffered hypogastric pain in July 2017 while ascites and tumors of the left and right adnexa were detected in September of the same year . The level of CA125 was 14749 U/ml. The use of standard tests did not reveal the presence of foundational mutations in BRCA1/BRCA2 characteristic of the Polish population.
The patient was treated with chemotherapy: 6 courses of Carboplatin AUC5 and Paclitaxel which brought significant regression of the symptoms. In April 2018, the patient underwent radical surgery i.e. bilateral ovariectomy and hysterectomy. Diagnosis: G2 ovarian serous cancer with metastases to the abdomen. In November 2018, the disease progression was recorded. In January 2019, she was reoperated on and second-line chemotherapy implemented: 6 courses of Carboplatin and Paclitaxel with complete response in imaging and marker tests. BRCA1/2 mutation tests in archival material were ordered. A new pathogenic BRCA1 gene mutation was found using NGS technology. The patient was qualified for maintenance therapy with Olaparib (800 mg daily).
Approval for this study was obtained from the Bioethical Commission in Lublin (approval number 10/2020/KB/VIII). All procedures were performed in accordance with the Declaration of Helsinki (ethical principles for medical research involving human subjects).
DNA extraction and genotyping
Genomic DNA was extracted from the whole blood using commercial kit NucleoSpin Dx Blood (Machery-Nagel, Duren, Germany) according to manufacturer's instructions. Quantity and quality of DNA was determined with the use of BioPhotometer (Eppendorf, Hamburg, Germany). Three mutations of BRCA1 gene: 5382insC (c.5266dupC), 300T>G (c.181T>G), 4153delA (c. 4035delA) were amplified by PCR using the primers and under conditions described by [16,17]. For other mutations: 185delAG (c.68_69delAG; p.Glu23Valfs), 3819delGTAAA (c.3700_3704delGTAAA; (p. Val1234Glnfs), 3875delGTCT (c.3756_3759delGTCT; p.Ser1253Argfs), 3896delT (c.3779delT; p.Leu1260Tyrfs), 4160delAG (c.4041_4042delAG, p.Gly1348Asnfs) and 4184delTCAA (c.4065_4068delTCAA; p.Asn1355Lysfs) primers were designed and reaction conditions were experimentally determined (Table S1).
Primer design was performed using freely available Software Primer [18]. The Taq PCR Master Mix kit (EURx, Gdansk, Poland) was used to prepare the PCR reaction (with 40 – 60 ng DNA) in SimplyAmpTM thermal cycler (Applied Biosystems, ThermoFisher Scientific). PCR products were enzymatically purified using ExoBap kit (EURx, Gdansk, Poland). The sequential PCR was then prepared using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). In the next step, terminators were removed after PCR using ExTerminator (A&A Biotechnology, Gdynia, Poland). After the purification. all samples were placed onto a 96-well plate and sequenced using 3100 Capillary Sequencer (Applied Biosystems, ThermoFisher Scientific). After that, the results were pre-analyzed by ABI3100 Data Collection Software. Then, the data files of the samples were checked by FinchTV1.4 (Geospiza) and compared with reference sequences BRCA1: NM_007294.3.
MLPA assay
BRCA1/2 genomic arrangements were searched by multiplex ligation-dependent probe amplification (MLPA) method [19] according to the manufacturer's instructions (MRC-Holland, Amsterdam, the Netherlands) using P087 SALSA and P090 SALSA MPLA kit. The products of MPLA reactions were 10 times diluted in Hi-DiTM Formamide (Applied Biosystems) and GeneScanTM 500 ROXTM dye Size Standard (Applied Biosystems) was added to each sample (as an internal lane size standard). It enabled automated data analysis. Moreover, it was essential for achieving high run to ensure precision in sizing DNA fragments by electrophoresis. The samples were size-separated by capillary electrophoresis (POP7 polymer, ABI PRISM 3100, Applied Biosystems). Thus, obtained electrophoretograms were analyzed by GeneMarker software (version 2.2.0, SoftGenetics, State College, PA, USA).
Next Generation Sequencing
The amount of cancer cells (evaluated by pathologist) was approximately 100% in the sample of FFPE. DNA was extracted using GeneRead DNA FFPE (QIAGEN, Hilden, Germany). The DNA concentration was determined using Qubit I fluorometer and dsDNA High Sensitivity Assay Kit (InvitrogenTM, ThermoFisher Scientific).
The DNA was diluted to a concentration of 10 ng/l. The libraries were prepared using Devyser BRCA kit (Hagersten, Sweden) according to the manufacturer’s instructions. Complete sequence determination of all coding exons and all adjacent exon-intron boundaries (minimum 20 bp proximal to 5' end and 10 bp distal to 3' end of each exon) were covered. The first, amplicon libraries were generated in one multiplex PCR reaction (PCR1). Next, index addition to PCR1 was performed in PCR2. Sample libraries obtained in PCR2 were pooled and purified in a single tube. The library concentration was quantified using Qubit I fluorometer and dsDNA High-Sensitivity Assay Kit (InvitrogenTM, Thermo Fisher Scientific). The purified library pool was diluted to 0.4 ng/ml concentration and denatured.
Sequencing was performed on MiniSeq (Ilumina, San Diego, USA) using Mid Output Reagent Cartridge 300 cycles (Illumina, San Diego, USA). The Devyser BRCA libraries were sequenced in a paired-end mode (2 x 151 bp). The results of sequencing data files (FASTQ) were reviewed using Amplicon Suite (Smartseq s.r.l).
Data analysis
All mutations were reported following the Human Genome Variation Society guidelines (http://varnomen.hgvs.org/) on the basis of the coding sequences NM_007294.3 for BRCA1 and NM_000059.3 for BRCA2. For the detection of sequence variants of somatic origin, we ensured that each amplicon had at least 1,000 coverage for detection of VAF down to 5%. Mutations were classified according to clinical variants (https://www.ncbi.nlm.nih.gov/clinvar/), mutations in COSMIC database (https://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), variants of unknown significance, literature search and in silico analyses using Varsome (https://varsome.com/). The presence of BRCA1/2 genes mutation was confirmed by Sanger sequencing (Table S1).