An 80-year-old woman presented to our hospital with mild headache in November 2015. She had a history of hypertension, diabetes mellitus, and hyperlipidaemia. She was febrile (38.6°C) but did not appear septic. Abdominal examination revealed mild splenomegaly but no hepatomegaly and there were no focal neurological signs or suggestions of other organ involvement. A full blood count showed leukocytes 124.0 × 10 9 /L (neutrophils 120.3 × 10 9 /L, lymphocytes 2.5 × 10 9 /L, monocytes 1.2 × 10 9 /L); haemoglobin 7.8 g/dL, mean corpuscular volume 89.1 fL; and platelets 384 × 10 9 /L. The blood film showed marked neutrophilia, occasional myelocytes, and absolute basophilia but no blasts. The neutrophils showed toxic granules and were not dysplastic (Fig 1). Plain radiographs of the chest, kidney, ureter, and urinary bladder did not reveal any abnormalities. Bacterial cultures of the blood and urine did not reveal any septic foci. Because of the marked neutrophilia, the patient was initially treated for bacterial sepsis with empirical intravenous amoxicillin with clavulanic acid. Subsequent ultrasonography of the abdomen Hong Kong Med J 2019;25:248-50 https://doi.

Background: Hereditary disposition accounts for 10-15% in breast cancers and 20-25% in ovarian cancers, in which 5-10% of women have genomic alteration in breast cancer predisposition genes, BRCA1 and BRCA2, while the rest are likely due to less penetrant genes. In specific ethnicities such as Ashkenazi Jewish, three founder mutations have been identified which covers 95 % of all the BRCA mutations identified in this race. These genes are screened prior to the gold standard Sanger Sequencing in order to reduce cost. Sanger Sequencing, however, still has the limitation on the necessity of laborious processing and results interpretation. Moreover, it limits the number of genes that can be analyzed in one setting. With the use of next-generation sequencing (NGS), identification of hereditary breast and ovarian cancer (HBOC) syndrome associated genes, other than BRCA, can be sequenced at the same time but yet a faster turnover time. This allows more timely targeted risk-reducing strategies and interventions to be implemented for mutation positive carriers and their family members. Methods: In this study cohort, 948 high-risk breast/ovarian patients who met the HBOC selection criteria were recruited for mutation screening by our NGS pipeline. With the inclusion of 90 Sanger-validated known mutation cases, the performance of the NGS pipeline were proven to be comparable to Sanger sequencing. PTEN and TP53, other than BRCA1 and BRCA2, a 4 gene sequencing panel were included in the mutation screening for high-risk patients. Results: The prevalence of BRCA1/BRCA2 germline mutations was 7.28% in our Chinese cohort and 47.8% of the mutation were recurrent mutations. Based on this finding, we further adopted a new workflow by screening the recurrent mutations including founder mutations from Chinese cohort prior to NGS for those who tested negative. In a testing cohort of 343 cases, the recurrent mutation pick-up rate was 3.5%, this implicated a more cost-effective method for mutation screening in the clinical setting. Moreover, the frequencies of PTEN and TP53 were 0.21% and 0.53% respectively in our population with breast and ovarian cases. Conclusion: Taken together, our data demonstrated a strategic upfront screening for recurrent mutations in Chinese population which is highly applicable in most of the diagnostic laboratories. Multi-gene sequencing using the NGS technology will be the upcoming strategies for mutation screening for HBOC patients. Citation Format: Kwong A, Shin VY, Au CH, Law FB, Ho DN, Ip BK, Wong AT, Lau SS, To RM, Choy G, Ford JM, Ma ES, Chan TL. Evaluation on the mutation screening by next-generation sequencing in hereditary breast and ovarian cancer: Implementation of recurrent mutation panel. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-09-20.

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Introduction: Breast cancer is the most common malignancy and 3rd leading cause of deaths among the female population in Hong Kong. Since the establishment of The Hong Kong Hereditary Breast Cancer Family Registry in 2007, 1344 patients with breast and/or ovarian cancer who met the selection criteria were recruited for genetic testing in Hong Kong. Since 2011 we started to employ next-generation DNA sequencing (NGS) to expedite the analysis workflow and expand the panel of genes for sequencing. Aim: To evaluate the workflow of NGS in mutation screening of BRCA1, BRCA2, TP53 and PTEN genes, and compared with the sequence data obtained by Sanger sequencing. Methods: We sequenced BRCA1, BRCA2, TP53 and PTEN genes in peripheral blood samples of 410 patients, 53 positive controls and 107 healthy local individuals using 454 GS Junior System. Generation of barcoded amplicon libraries was streamlined by microfluidic PCR using Fluidigm Access Array System. Sequencing data were analyzed by an in-house developed fully automatic bioinformatics pipeline, which mainly consists of GS Amplicon Variant Analyzer, SAMtools and Ensembl Variant Effect Predictor. All putative mutations identified were validated by Sanger sequencing. Furthermore, the frequency of BRCA1, BRCA2 and PTEN missense variants of unknown significance (VUS) identified in the cohort were compared among 107 healthy local individuals and 1000 Genomes project samples. The VUS were also subjected to a panel of in silico prediction methods including PolyPhen and SIFT. Results: Among 410 patients, there were 7 in BRCA1, 6 in BRCA2 and 1 in TP53 mutations found, including 1 novel recurrent BRCA2 (c.7007G>T) and 1 novel founder BRCA2 (c.5164_5165delAG) mutations. Based on multiple criteria, 12 in BRCA1, 12 in BRCA2 and 1 in PTEN VUS could be prioritized for further investigation. The bioinformatics pipeline was extensively evaluated with Sanger-validated controls. The evaluation determined minimum sequencing coverage needed in this sequencing platform for accurate analysis. The pipeline accuracy was demonstrated by successful detecting mutations from 53 positive controls, including single nucleotide variants, insertions and deletions in different sequence context. Conclusion: BRCA1, BRCA2, TP53 and PTEN mutation screening of 410 patients were expedited by high-throughput DNA sequencing. This method could detect 14 positive cases, including recurrent mutations, in a shorter period of time when compared with Sanger full gene sequencing. High-risk patients who are negative for the gene panel may need further investigation other than screening for BRCA1/2. The in-house developed bioinformatics pipeline was validated to detect various types of mutations and potentially become a conventional platform for genetic screening. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-07-03.

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