High-resolution melting analysis coupled with next-generation sequencing as a simple tool for the identification of a novel somatic BRCA2 variant: a case report

In a 72-year-old woman with no associated personal or family history of breast and/or ovarian cancers, we identified a novel somatic pathogenic BRCA2 variant (c.18_28delAGAGAGGCCAA, p.Lys6Asnfs*4) using next-generation sequencing (NGS). The variant allele frequency (VAF) was 16%, and Sanger sequencing was unable to identify this variant. Adopting a high-resolution melting analysis strategy coupled with NGS, we successfully highlighted the presence of the c.18_28delAGAGAGGCCAA allele.

sequencing. Furthermore, these types of sources cannot be re-analyzed by NGS because of the small amount of DNA. To avoid considering these variants to be PCR artefacts, it is highly recommended to use alternative methodologies.
In this context, we used high-resolution melting analysis (HRMA) as a simple, cost-effective, rapid and sensitive method to confirm a novel somatic BRCA2 variant (c.18_28delAGAGAGGCCAA, p.Lys6Asnfs*4) that was previously identified by NGS in a patient with high-grade serous ovarian cancer (HGSOC).
The present study involved a 72-year-old woman who presented to an oncologist with a complex right ovarian mass and elevated CA-125 level. Her gynecological history was negative. A transvaginal and transabdominal ultrasound examination revealed a multilocular solid cyst with >10 locules, papillary projections, and irregular surface with a Color Score of 4. Computed tomography of her abdomen and pelvis showed a 10 × 5 cm right ovarian mass and diffuse peritoneal enhancement, consistent with peritoneal carcinomatosis. Ovarian cancer was suspected, and the patient consented to complete surgical staging. She underwent a total abdominal hysterectomy, bilateral salpingo-oophorectomy, partial pelvic peritonectomy, and radical omentectomy 2 months after her initial presentation. The surgery was largely uncomplicated, with no significant hemostasis or coagulation issues, and optimal cytoreduction was achieved. Surgery was followed by six cycles of chemotherapy with paclitaxel and carboplatin. Written informed consent was obtained to allow BRCA testing to be performed after the pathological diagnosis of HGSOC was made.
DNA was extracted from FT HGSOC sections from areas with a minimum neoplastic cellularity of 70% using the MagCore Genomic DNATissue Kit by MagCore HF16 Plus (Diatech Lab Line, Jesi, Italy). The DNA concentration and quality were determined using a Qubit dsDNA HS assay (Thermo Fisher Scientific, Waltham, MA, USA).
BRCA analysis was performed using the Devyser BRCA kit (Devyser, Hägersten, Sweden). Sequencing reactions were carried out on the MiSeq instrument (Illumina, CA, USA). NGS data were processed using the Amplicon Suite software (SmartSeq s.r.l., Novara, Italy) with the parameters of aligning reads to the HG19 reference genome and to generate run metrics, including the depth of sequencing, total read count, and quality. In addition, BRCA large genomic rearrangements were also investigated as previously reported 3,4 .
Sanger sequencing and PCR-HRMA were performed on an ABI 3500 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific) and the LightCycler® 480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland), respectively.
The WT allele is longer by 11 nucleotides compared to c.18_28delAGAGAGGCCAA allele; this DNA size difference allows the allele separation by capillary Sanger sequencing, which was used to confirm the presence of the c.18_28delAGAGAGGCCAA allele, did not reveal this allele (Fig. 1). By contrast, high-resolution melting profiles for the patient showed a specific melting behavior compared to the FT samples (n = 10) that did not carry the c.18_28delAGAGAGGCCAA allele.
Finally, capillary electrophoresis also confirmed the presence of the c.18_28delAGAGAGGCCAA allele, although it was difficult to discriminate between the two alleles (Fig. 2).
The c.18_28delAGAGAGGCCAA variant was considered to be a novel variant because it was absent from Melting profile evaluation of the patient shows a specific melting behavior, as observed in both the normalized and the temperature-shifted and difference plots compared to the FT samples (n = 10) that do not carry this variant. Each experiment is reported in duplicate. The same forward primer used for sequencing was used for PCR-HRMA, while the PCR-HRMA-reverse primer was 5′-TCATTAGGGAGATACATATGGA-3′. The PCR-HRMA primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3) and certified as high molecular-quality products via HPLC purification (Eurofin MWG Operon, Ebersberg, Germany) 300 FT samples as well as two main variant databases: Clin Var (https://www.ncbi.nlm.nih.gov/clinvar/) and Cosmic (http://cancer.sanger.ac.uk/cosmic). This allele was also considered to be pathogenic because of its deleterious impact on the BRCA2 protein sequence.
With the implementation of treatment-focused BRCA testing on patients with somatic BRCA PVs 6 , there is an increasing clinical need for routine BRCA screening on DNA from FFPE and FT tumor samples. In this context, it is recommended to perform cost-effective, complete, and accurate BRCA gene sequencing with a sensitivity, throughput, and sample input that cannot be achieved by Sanger sequencing. In fact, Sanger approaches are not fit for detecting low VAFs, leading researchers to confuse these variants with PCR artefacts, which are indistinguishable from the background sequencing noise, and thus consider them false.
For these reasons, many diagnostic laboratories have adopted NGS technology, which offers the potential for fast, cost-efficient, and comprehensive sequencing-based testing of tumor tissue, enabling the identification of somatic BRCA variants.
HRMA represents a high-throughput, rapid, and inexpensive screening test for germline variants 7,8 , and because of its high sensitivity, it has also proven to be effective at identifying somatic variants 9 .
The aim of this study was to report a combined approach with NGS and HRMA to identify and confirm a novel somatic BRCA2 variant. NGS data demonstrated a VAF of 16%, and the depth and quality of the sequencing led us to suspect that this variant was true. Using HRMA as a confirmatory test allowed us to draw up the final clinical report for this patient.
We suggest that this integrated approach can be used in diagnostic settings to improve the molecular assessment of the somatic BRCA status. In fact, our study provides details regarding a rapid and reliable confirmatory assay for low allele-frequency somatic BRCA variants that would otherwise be difficult to confirm with other molecular methods.
Best-practice guidelines for the analysis of FFPE and FT samples 10 recommend that significant NGS findings must be replicated to ensure the reliability of the results before adopting therapeutic decisions in a clinical context.
This study demonstrates that a combined approach with NGS and HRMA allows for the reliable detection of somatic alterations affecting BRCA genes in FT samples, improving clinical decision-making for the treatment of HGSOC patients.
Finally, epithelial ovarian cancer treatment, which has historically been based on surgery and platinum doublet chemotherapy, is associated with a high risk of relapse and a poor prognosis for recurrent disease. In this landscape, molecular diagnosis of this somatic pathogenic BRCA2 variant made our patient eligible for therapeutic treatments based on poly ADP ribose polymerase, which is a valuable option with promising activity in recurrent ovarian cancer patients and at the different stages of this disease.

HGV Database
The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi. org/10.6084/m9.figshare.hgv.1946