Search

The discovery of high-risk breast cancer susceptibility genes, such as Breast cancer associated gene 1 (BRCA1) and Breast cancer associated gene 2 (BRCA2) has led to accurate identification of individuals for risk management and targeted therapy. The rapid decline in sequencing costs has tremendously increased the number of individuals who are undergoing genetic testing world-wide. However, given the significant differences in population-specific variants, interpreting the results of these tests can be challenging especially for novel genetic variants in understudied populations. Here we report the characterization of novel variants in the Malaysian and Singaporean population that consist of different ethnic groups (Malays, Chinese, Indian, and other indigenous groups). We have evaluated the functional significance of 14 BRCA2 variants of uncertain clinical significance by using multiple in silico prediction tools and examined their frequency in a cohort of 7840 breast cancer cases and 7928 healthy controls. In addition, we have used a mouse embryonic stem cell (mESC)-based functional assay to assess the impact of these variants on BRCA2 function. We found these variants to be functionally indistinguishable from wild-type BRCA2. These variants could fully rescue the lethality of Brca2-null mESCs and exhibited no sensitivity to six different DNA damaging agents including a poly ADP ribose polymerase inhibitor. Our findings strongly suggest that all 14 evaluated variants are functionally neutral. Our findings should be valuable in risk assessment of individuals carrying these variants.

The thymus is critical for the development of mainstream T cell mediated adaptive immunity. A key stromal component is the thymic epithelium, which can be broadly divided into cortical and medullary thymic epithelial cells (cTEC and mTEC). Developing thymocytes migrate through and interact with specialised TEC subsets in a co dependent manner, known as cross talk. Despite its immunological importance, the thymus paradoxically involutes with age and is proposed to contribute to the clinical symptoms of immunosenescence. Thymic epithelium, is unique as it begins to atrophy soon after birth but, under appropriate stimuli, is able to regenerate partially following damage, at least in the young to middle aged. This probably involves resident thymic epithelial progenitor cells (TEPC), but to date they have not been identified in the adult. Investigations into thymic development, maintenance in the adult and the capacity to regenerate are critical for developing clinically relevant strategies to treat immune degenerative conditions. Such studies will also serve as important proof of concept contributions to regenerative medicine in general. The Forkhead box transcription factor n1 (Foxn1) is the best characterised and earliest known TEC regulator, marking bipotent TEPC in the embryo and required for the maintenance of TEC and their sub lineages. In this project, we generated Foxn1eGFP/w knock in reporter mouse embryonic stem cell (mESC) lines in two strains (C57BL/6 and 129/Sv) and a corresponding C57BL/6 mouse line, to assist in the study of TEC development and maintenance at all life stages, and the role of Foxn1 in these processes. The Foxn1eGFP/w mouse described in this thesis is currently the only fluorescent reporter knock-in mouse model for Foxn1. In the Foxn1eGFP allele, eGFP is under the transcriptional control of the endogenous Foxn1 promoter and regulatory elements, ensuring accurate reporter expression compared to other transgenic mouse models. Its main advantage over existing Foxn1LacZ/+ reporter mice is the ability to identify, isolate and characterise viable eGFP+ cells, which are exclusively in epithelial cells of the thymus and skin. The generated young Foxn1eGFP/w heterozygotes were phenotypically normal and indistinguishable from Foxn1w/w mice with normal fur development, thymic cellularity, TEC and T cell proportions and numbers. Foxn1eGFP/eGFP mice phenocopied conventional BALB/c Foxn1nu/nu (nude) mice, with thymic dysgenesis, hairlessness and T cell defects. eGFP expression reflected reported Foxn1 expression throughout ontogeny. There was a very close correlation between Foxn1 and eGFP at the protein and mRNA levels. The majority of TEC in the young and middle aged Foxn1eGFP/w mice are eGFP+ confirming previous reports that the majority of TEC arise from a Foxn1+ lineage. eGFP- TEC in the adult could also represent immature TEC that express eGFP and Foxn1 mRNA and are still in the process of translating eGFP and Foxn1 protein or they could be apoptosing TEC. Early studies in our Foxn1eGFP/w mice show a possible reduction in Foxn1 and eGFP mRNA in aged but not middle aged TEC, compared to young levels. Collectively, the Foxn1eGFP/w colony reflects the reported Foxn1LacZ/+ colony with the added benefit of isolating and culturing viable eGFP+ TEC in vitro. Mild haploinsufficiency as previously documented in Foxn1nu/+ heterozygotes, was not observed in young Foxn1eGFP/w steady state thymii. Furthermore, Foxn1eGFP/w thymii, despite having reduced Foxn1 levels, responded in a similar manner to wildtype thymii in preliminary studies of damage induced thymic regeneration and castration induced thymic hypertrophy. The Foxn1eGFP/w reporter mouse colony is a useful tool in thymic epithelial research at all life stages - from the technical isolation of early embryonic or Foxn1eGFP/eGFP “nude” thymii, characterising adult and aged TEPC, establishing in vitro TEC cultures and determining the precise roles for Foxn1 in TEPC and TEC maintenance and ageing.

BACKGROUND: Genetic testing for BRCA1 and BRCA2 is offered typically to selected women based on age of onset and family history of cancer. However, current internationally accepted genetic testing referral guidelines are built mostly on data from cancer genetics clinics in women of European descent. To evaluate the appropriateness of such guidelines in Asians, we have determined the prevalence of germ line variants in an unselected cohort of Asian patients with breast cancer and healthy controls. METHODS: Germ line DNA from a hospital-based study of 2575 unselected patients with breast cancer and 2809 healthy controls were subjected to amplicon-based targeted sequencing of exonic and proximal splice site junction regions of BRCA1 and BRCA2 using the Fluidigm Access Array system, with sequencing conducted on a Illumina HiSeq2500 platform. Variant calling was performed with GATK UnifiedGenotyper and were validated by Sanger sequencing. RESULTS: Fifty-five (2.1%) BRCA1 and 66 (2.6%) BRCA2 deleterious mutations were identified among patients with breast cancer and five (0.18%) BRCA1 and six (0.21%) BRCA2 mutations among controls. One thousand one hundred and eighty-six (46%) patients and 97 (80%) carriers fulfilled the National Comprehensive Cancer Network guidelines for genetic testing. CONCLUSION: Five per cent of unselected Asian patients with breast cancer carry deleterious variants in BRCA1 or BRCA2. While current referral guidelines identified the majority of carriers, one in two patients would be referred for genetic services. Given that such services are largely unavailable in majority of low-resource settings in Asia, our study highlights the need for more efficient guidelines to identify at-risk individuals in Asia.

Funder: National University Cancer Institute, Singapore; FundRef: http://dx.doi.org/10.13039/501100011105, Background: Rare protein-truncating variants (PTVs) in partner and localiser of BRCA2 (PALB2) confer increased risk to breast cancer, but relatively few studies have reported the prevalence in South-East Asian populations. Here, we describe the prevalence of rare variants in PALB2 in a population-based study of 7840 breast cancer cases and 7928 healthy Chinese, Malay and Indian women from Malaysia and Singapore, and describe the functional impact of germline missense variants identified in this population. Methods: Mutation testing was performed on germline DNA (n=15 768) using targeted sequencing panels. The functional impact of missense variants was tested in mouse embryonic stem cell based functional assays. Results: PTVs in PALB2 were found in 0.73% of breast cancer patients and 0.14% of healthy individuals (OR=5.44; 95% CI 2.85 to 10.39, p

1026 Background: A germline deletion in the APOBEC3B cytosine deaminase gene [A3Bdel] occurs more frequently in Asian women (45% heterozygous deletion (hetD) and 15% homozygous deletion (homD)) compared to in Caucasian women (15% hetD and 4% homD). Carriers are more likely to develop breast cancer, and cancers in carriers are more likely to have a hypermutator phenotype (with C > T transitions) and to be immune-enriched. In this clinical trial, we aim to evaluate whether the immune activation increases response to checkpoint immunotherapy. Methods: In this open label, single arm Phase II study of single agent pembrolizumab in metastatic HER2-receptor negative breast cancer patients with germline deletion in A3B, 40 evaluable subjects who have received > = 1 but < = 3 lines of therapy in a metastatic setting will be enrolled and given pembrolizumab 200 mg intravenously (IV) every 3 weeks (Q3W) for up to 35 administrations (2 years), with Objective Response Rate (ORR) using RECIST 1.1 as the primary study endpoint. The study applies a Simon two-stage design, where if at least 3 out of 22 evaluable patients achieve CR/PR in stage I, the study will proceed to stage II. Results: To date, 84 breast cancer patients were screened for germline A3Bdel, of whom 46 (54.8%) were heterozygous carriers and 12 (14.3%) were homozygous carriers. The study enrolled 22 female A3Bdel carriers with a median age of 59.4 years (range: 32.1, 82.9 years) between September 2020 and September 2021 for stage I analysis. On average, patients received 2 prior lines of chemotherapy in a metastatic setting [6 with 1, 8 with 2 and 8 with 3 lines of prior chemotherapy]. Complete response (CR) was observed in one patient, while partial response (PR) was observed in 4 patients, with an ORR of 22.7% (5 over 22 subjects) in stage I, meeting the pre-defined criteria to proceed to stage II. Notably, the patient with complete response had received 2 prior lines of chemotherapy, whereas of the patients with partial response, 1 had received 1 prior line and 3 had received 3 prior lines of chemotherapy in a metastatic setting. As the observed ORR was greater than the value of r1 (13.6%), the study has met the statistical criteria to proceed to the stage II enrolment with an additional 18 patients required to complete the entire study. Conclusions: Single agent pembrolizumab demonstrates promising efficacy in germline A3Bdel carriers, who constitute almost two-thirds of Asian patients. Clinical trial information: NCT03989089.

BACKGROUND: Rare protein-truncating variants (PTVs) in partner and localiser of BRCA2 (PALB2) confer increased risk to breast cancer, but relatively few studies have reported the prevalence in South-East Asian populations. Here, we describe the prevalence of rare variants in PALB2 in a population-based study of 7840 breast cancer cases and 7928 healthy Chinese, Malay and Indian women from Malaysia and Singapore, and describe the functional impact of germline missense variants identified in this population. METHODS: Mutation testing was performed on germline DNA (n=15 768) using targeted sequencing panels. The functional impact of missense variants was tested in mouse embryonic stem cell based functional assays. RESULTS: PTVs in PALB2 were found in 0.73% of breast cancer patients and 0.14% of healthy individuals (OR=5.44; 95% CI 2.85 to 10.39, p