Germline and Somatic Neurofibromatosis Type 1 Aberrations in Breast Cancer

Title: Germline and Somatic Neurofibromatosis Type 1 Aberrations in Breast Cancer Overview: These studies were initiated after seeing a series of women with Neurofibromatosis Type 1 (NF1) and breast cancer (BC) at National Cancer Centre Singapore (NCCS) from 2006 to 2009. Neurofibromatosis type 1 (NF1) is a relatively common tumour predisposition syndrome related to germline aberrations of NF1, a tumour suppressor gene. NF1 is usually a clinical diagnosis as individuals with NF1 typically develop multiple neurofibromas which can be cosmetically disfiguring, in addition to other features such as café-au-lait spots, skin tags and Lisch nodules. These patients under my care had aggressive HER2-positive breast cancers that did not seem to respond to standard systemic therapies as well as in individuals without NF1 syndrome. Individuals with NF1, an autosomal dominant genetic disorder, are known to be at increased risk of developing various tumours, such as malignant peripheral nerve sheath tumour (MPNST), phaeochromocytoma, glioma, and rhabdomyosarcoma. In 2007, the first study which reported an increased risk of breast cancer in women with NF1 was published. Since then, there have been a number of other epidemiological studies with the consistent finding that women with NF1 are have a three- to eight-fold increased risk of breast cancer, especially for women aged less than 50 years. Data on the characteristics of BC in NF1 patients is currently still limited. Our group was the first to discover the higher frequency of HER2-positive, hormone receptor negative and grade 3 breast cancers in women with NF1 compared to breast cancers in women without NF1. We have also performed genomic profiling of these NF1-associated breast cancers. Over the course of my candidature, large-scale exome or genome sequencing studies led by various groups such as The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and METABRIC, have revealed somatic NF1 aberrations in different sporadic tumours from individuals in the absence of a clinical diagnosis of NF1. These somatic NF1 alterations appear to be associated with resistance to standard therapy and adverse outcomes, similar to the breast cancers in women with clinical NF1 syndrome. Improved understanding of the implications of NF1 aberrations is critical for the development of novel therapeutic strategies. In Asia, women with breast cancer are on average younger than in Western populations, resulting in higer rates of poor prognosis breast cancers in premenopausal women. Since somatic NF1 mutations in BC are associated with poor prognosis, we also aimed to explore the potential role of NF1 and neurofibromin in the sporadic BCs from patients without NF1. This included immunohistochemical staining of tissue micrroarrays, and targeted gene sequencing (with NF1 in the gene panel). Structure of Thesis and Research Questions: Chapter 1: Systematic Review: This literature review focused on the germline NF1 disorder, the biology of the NF1 gene and neurofibromin, tumours associated with NF1 as well as sporadic tumours harbouring somatic NF1 aberrations in individuals without NF1 disorder. This review identified an important role of NF1 in carcinogenesis as well as the challenges of detecting NF1 aberrations and deficiency or dysfunction of the encoded protein neurofibromin. It also highlights the need to pursue further research, especially in the area of therapeutic strategies for individuals with germline NF1 syndrome and for sporadic tumours with somatic NF1 aberrations. Chapter 2: Whole exome sequencing of multiple tumours from an NF1 patient: This paper describes the exome sequencing of BC, MPNST, and neurofibroma from a patient with NF1. Apart from the germline NF1 mutation, we demonstrated independent somatic NF1 mutations in all three tumors. Each tumor had a distinct genomic profile with mutually exclusive aberrations in different genes. Although second-hit NF1 mutation may be critical in tumorigenesis, different additional mutations are required to drive the formation of different tumors. Chapter 3: Comprehensive case series of BCs in women with NF1 with molecular insights into its aggressive phenotype: The aim was to elucidate the clinical, pathological and molecular characteristics of NF1-associated BCs at National Cancer Centre Singapore. There was a higher frequency of grade 3, oestrogen receptor (ER) negative and human epidermal growth factor receptor 2 (HER2) positive tumours among NF1 patients with inferior overall survival compared to non-NF1 BCs. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all NF1-associated BC specimens, but without any discernable consistent pattern in the intensity or extent of staining. It appears that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes. Chapter 4: Immunohistochemical expression of neurofibromin in sporadic breast cancers: From the initial discovery cohort of 314 sporadic breast cancers of all subtypes, tumours with both nuclear and cytoplasmic expression of neurofibromin seemed to have better outcomes, especially in the triple negative subset. However, there was no correlation between expression of neurofibromin and survival outcomes in a larger validation cohort of triple negative breast cancers. Chapter 5: Elucidating therapeutic molecular targets in premenopausal Asian women with recurrent breast cancers: Targeted sequencing was performed on a separate cohort of premenopausal poor prognosis BCs. The most prevalent alterations included TP53 (65%). PIK3CA (32%), GATA3 (29%), ERBB2 (27%), MYC (25%) and KMT2C (21%). The frequency of NF1 mutations was 2%. Detecting changes in dosage of the NF1 gene in formalin-fixed paraffin-embedded specimens was not feasible. Chapter 6: Conclusion and future directions: The final chapter summarises the findings of these studies and highlights future directions that are clinically relevant.Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 201

Improving Indel Detection Specificity of the Ion Torrent PGM Benchtop Sequencer

10.1371/journal.pone.0045798PLoS ONE79

Replication and Meta-analysis of the Association between BDNF Val66Met Polymorphism and Cognitive Impairment in Patients Receiving Chemotherapy.

Cancer-related cognitive impairment (CRCI) adversely affects cancer patients. We had previously demonstrated that the BDNF Val66Met genetic polymorphism is associated with lower odds of subjective CRCI in the multitasking and verbal ability domains among breast cancer patients receiving chemotherapy. To further assess our previous findings, we evaluated the association of BDNF Val66Met polymorphism with subjective and objective CRCI in a temporally separate cohort of patients and pooled findings from both the original (n = 145) and current (n = 193) cohorts in a meta-analysis. Subjective CRCI was assessed using FACT-Cog. Objective CRCI was evaluated using computerized neuropsychological tests. Genotyping was carried out using Sanger sequencing. The association of BDNF Val66Met genotypes and CRCI was examined with logistic regression. A fixed-effect meta-analysis was conducted using the inverse variance method. In the meta-analysis (n = 338), significantly lower odds of CRCI were associated with Met allele carriers based on the global FACT-Cog score (OR = 0.52, 95% CI 0.29-0.94). Furthermore, Met allele carriers were at lower odds of developing impairment in the domains of memory (OR = 0.34, 95% CI: 0.17-0.70), multitasking (OR = 0.33, 95% CI: 0.18-0.59), and verbal ability (OR = 0.46, 95% CI: 0.24-0.88). Consistent with the previous study, lower odds of subjective CRCI among patients with the BDNF Met allele was observed after adjusting for potential confounders in the multitasking (OR = 0.30, 95% CI: 0.14-0.67) domain. In conclusion, carriers of the BDNF Met allele were protected against global subjective CRCI, particularly in the domains of memory, multitasking, and verbal ability. Our findings further contribute to the understanding of CRCI pathophysiology

Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background: Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Methodology/Principal Findings: Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. Conclusions/Significance: We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.Singapore-MIT Alliance for Research and Technolog

Joint effects of known type 2 diabetes susceptibility loci in genome-wide association study of Singapore Chinese: The Singapore Chinese health study

Background: Genome-wide association studies (GWAS) have identified genetic factors in type 2 diabetes (T2D), mostly among individuals of European ancestry. We tested whether previously identified T2D-associated single nucleotide polymorphisms (SNPs) replicate and whether SNPs in regions near known T2D SNPs were associated with T2D within the Singapore Chinese Health Study. Methods: 2338 cases and 2339 T2D controls from the Singapore Chinese Health Study were genotyped for 507,509 SNPs. Imputation extended the genotyped SNPs to 7,514,461 with high estimated certainty (r2>0.8). Replication of known index SNP associations in T2D was attempted. Risk scores were computed as the sum of index risk alleles. SNPs in regions ±100 kb around each index were tested for associations with T2D in conditional fine-mapping analysis. Results: Of 69 index SNPs, 20 were genotyped directly and genotypes at 35 others were well imputed. Among the 55 SNPs with data, disease associations were replicated (at p<0.05) for 15 SNPs, while 32 more were directionally consistent with previous reports. Risk score was a significant predictor with a 2.03 fold higher risk CI (1.69-2.44) of T2D comparing the highest to lowest quintile of risk allele burden (p = 5.72×10-14). Two improved SNPs around index rs10923931 and 5 new candidate SNPs around indices rs10965250 and rs1111875 passed simple Bonferroni corrections for significance in conditional analysis. Nonetheless, only a small fraction (2.3% on the disease liability scale) of T2D burden in Singapore is explained by these SNPs. Conclusions: While diabetes risk in Singapore Chinese involves genetic variants, most disease risk remains unexplained. Further genetic work is ongoing in the Singapore Chinese population to identify unique common variants not already seen in earlier studies. However rapid increases in T2D risk have occurred in recent decades in this population, indicating that dynamic environmental influences and possibly gene by environment interactions complicate the genetic architecture of this disease. © 2014 Chen et al

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000–3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons—Leu844, Cys845, Ala846, Leu847, and Gly848—located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844–848 exists and will be valuable in the management and genetic counseling of a significant number of individuals

Ribociclib as First-Line Therapy for HR-Positive, Advanced Breast Cancer

The inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) could potentially overcome or delay resistance to endocrine therapy in advanced breast cancer that is positive for hormone receptor (HR) and negative for human epidermal growth factor receptor 2 (HER2). In this randomized, placebo-controlled, phase 3 trial, we evaluated the efficacy and safety of the selective CDK4/6 inhibitor ribociclib combined with letrozole for first-line treatment in 668 postmenopausal women with HR-positive, HER2-negative recurrent or metastatic breast cancer who had not received previous systemic therapy for advanced disease. We randomly assigned the patients to receive either ribociclib (600 mg per day on a 3-weeks-on, 1-week-off schedule) plus letrozole (2.5 mg per day) or placebo plus letrozole. The primary end point was investigator-assessed progression-free survival. Secondary end points included overall survival, overall response rate, and safety. A preplanned interim analysis was performed on January 29, 2016, after 243 patients had disease progression or died. Prespecified criteria for superiority required a hazard ratio of 0.56 or less with P The duration of progression-free survival was significantly longer in the ribociclib group than in the placebo group (hazard ratio, 0.56; 95% CI, 0.43 to 0.72; P=3.29×10-6 for superiority). The median duration of follow-up was 15.3 months. After 18 months, the progression-free survival rate was 63.0% (95% confidence interval [CI], 54.6 to 70.3) in the ribociclib group and 42.2% (95% CI, 34.8 to 49.5) in the placebo group. In patients with measurable disease at baseline, the overall response rate was 52.7% and 37.1%, respectively (P Among patients receiving initial systemic treatment for HR-positive, HER2-negative advanced breast cancer, the duration of progression-free survival was significantly longer among those receiving ribociclib plus letrozole than among those receiving placebo plus letrozole, with a higher rate of myelosuppression in the ribociclib group. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT01958021 .)

Cabbage and fermented vegetables : From death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe, or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage have been associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin-converting enzyme 2 (ACE2). As a result of SARS-CoV-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT(1)R) axis associated with oxidative stress. This leads to insulin resistance as well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block in particular the AT(1)R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are: kimchi in Korea, westernized foods, and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects, helpful in mitigating COVID-19 severity.Peer reviewe