Germline and Somatic Neurofibromatosis Type 1 Aberrations in Breast Cancer

Abstract

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.