INVESTIGATING INVASION IN DUCTAL CARCINOMA IN SITU WITH TOPOGRAPHICAL SINGLE CELL GENOME SEQUENCING

Synchronous Ductal Carcinoma in situ (DCIS-IDC) is an early stage breast cancer invasion in which it is possible to delineate genomic evolution during invasion because of the presence of both in situ and invasive regions within the same sample. While laser capture microdissection studies of DCIS-IDC examined the relationship between the paired in situ (DCIS) and invasive (IDC) regions, these studies were either confounded by bulk tissue or limited to a small set of genes or markers. To overcome these challenges, we developed Topographic Single Cell Sequencing (TSCS), which combines laser-catapulting with single cell DNA sequencing to measure genomic copy number profiles from single tumor cells while preserving their spatial context. We applied TSCS to sequence 1,293 single cells from 10 synchronous DCIS patients. We also applied deep-exome sequencing to the in situ, invasive and normal tissues for the DCIS-IDC patients. Previous bulk tissue studies had produced several conflicting models of tumor evolution. Our data support a multiclonal invasion model, in which genome evolution occurs within the ducts and gives rise to multiple subclones that escape the ducts into the adjacent tissues to establish the invasive carcinomas. In summary, we have developed a novel method for single cell DNA sequencing, which preserves spatial context, and applied this method to understand clonal evolution during the transition between carcinoma in situ to invasive ductal carcinoma

Punctuated copy number evolution and clonal stasis in triple-negative breast cancer

Aneuploidy is a hallmark of breast cancer; however, our knowledge of how these complex rearrangements evolve during tumorigenesis is limited. In this study we developed a highly-multiplexed single-nucleus-sequencing (HM-SNS) method to investigate DNA copy number evolution in triple-negative breast cancer (TNBC) patients. We applied this method to sequence 1000 single cells from 12 TNBC patients and identified 1-3 major clonal subpopulations in each tumor, with large genetic distances from the ancestral diploid cells. Mathematical modeling suggests that these data are unlikely to be explained by the gradual accumulation of copy number aberrations (CNAs) over extended periods of time. We also identified a minor (~7%) subpopulation of non-clonal cells that were classified as: 1) metastable tumor cells, 2) pseudo-diploid cells, and 3) chromazemic cells. Collectively, these data challenge the paradigm of gradual copy number evolution by showing that the majority of CNAs are acquired early in tumor evolution, in short punctuated bursts, followed by stable clonal expansions that form the tumor mass. These data have important implications for understanding the evolutionary dynamics of tumor growth and the diagnosis and treatment of TNBC patients

Genomic analysis defines clonal relationships of ductal carcinoma in situ and recurrent invasive breast cancer.

Ductal carcinoma in situ (DCIS) is the most common form of preinvasive breast cancer and, despite treatment, a small fraction (5-10%) of DCIS patients develop subsequent invasive disease. A fundamental biologic question is whether the invasive disease arises from tumor cells in the initial DCIS or represents new unrelated disease. To address this question, we performed genomic analyses on the initial DCIS lesion and paired invasive recurrent tumors in 95 patients together with single-cell DNA sequencing in a subset of cases. Our data show that in 75% of cases the invasive recurrence was clonally related to the initial DCIS, suggesting that tumor cells were not eliminated during the initial treatment. Surprisingly, however, 18% were clonally unrelated to the DCIS, representing new independent lineages and 7% of cases were ambiguous. This knowledge is essential for accurate risk evaluation of DCIS, treatment de-escalation strategies and the identification of predictive biomarkers

Comprehensive molecular characterization of human colon and rectal cancer

To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome sequencing. In total, 16% of colorectal carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase ε (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression.National Institutes of Health (U.S.) (Grant U24CA143799)National Institutes of Health (U.S.) (Grant U24CA143835)National Institutes of Health (U.S.) (Grant U24CA143840)National Institutes of Health (U.S.) (Grant U24CA143843)National Institutes of Health (U.S.) (Grant U24CA143845)National Institutes of Health (U.S.) (Grant U24CA143848)National Institutes of Health (U.S.) (Grant U24CA143858)National Institutes of Health (U.S.) (Grant U24CA143866)National Institutes of Health (U.S.) (Grant U24CA143867)National Institutes of Health (U.S.) (Grant U24CA143882)National Institutes of Health (U.S.) (Grant U24CA143883)National Institutes of Health (U.S.) (Grant U24CA144025)National Institutes of Health (U.S.) (Grant U54HG003067)National Institutes of Health (U.S.) (Grant U54HG003079)National Institutes of Health (U.S.) (Grant U54HG003273

Integrated genomic characterization of endometrial carcinoma

We performed an integrated genomic, transcriptomic and proteomic characterization of 373 endometrial carcinomas using array- and sequencing-based technologies. Uterine serous tumours and ~25% of high-grade endometrioid tumours had extensive copy number alterations, few DNA methylation changes, low oestrogen receptor/progesterone receptor levels, and frequent TP53 mutations. Most endometrioid tumours had few copy number alterations or TP53 mutations, but frequent mutations in PTEN, CTNNB1, PIK3CA, ARID1A and KRAS and novel mutations in the SWI/SNF chromatin remodelling complex gene ARID5B. A subset of endometrioid tumours that we identified had a markedly increased transversion mutation frequency and newly identified hotspot mutations in POLE. Our results classified endometrial cancers into four categories: POLE ultramutated, microsatellite instability hypermutated, copy-number low, and copy-number high. Uterine serous carcinomas share genomic features with ovarian serous and basal-like breast carcinomas. We demonstrated that the genomic features of endometrial carcinomas permit a reclassification that may affect post-surgical adjuvant treatment for women with aggressive tumours.National Institutes of Health (U.S.) (Grant 5U24CA143799-04)National Institutes of Health (U.S.) (Grant 5U24CA143835-04)National Institutes of Health (U.S.) (Grant 5U24CA143840-04)National Institutes of Health (U.S.) (Grant 5U24CA143843-04)National Institutes of Health (U.S.) (Grant 5U24CA143845-04)National Institutes of Health (U.S.) (Grant 5U24CA143848-04)National Institutes of Health (U.S.) (Grant 5U24CA143858-04)National Institutes of Health (U.S.) (Grant 5U24CA143866-04)National Institutes of Health (U.S.) (Grant 5U24CA143867-04)National Institutes of Health (U.S.) (Grant 5U24CA143882-04)National Institutes of Health (U.S.) (Grant 5U24CA143883-04)National Institutes of Health (U.S.) (Grant 5U24CA144025-04)National Institutes of Health (U.S.) (Grant U54HG003067-11)National Institutes of Health (U.S.) (Grant U54HG003079-10)National Institutes of Health (U.S.) (Grant U54HG003273-10

Comprehensive genomic characterization of squamous cell lung cancers

Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers.National Institutes of Health (U.S.) (Grant U24 CA126561)National Institutes of Health (U.S.) (Grant U24 CA126551)National Institutes of Health (U.S.) (Grant U24 CA126554)National Institutes of Health (U.S.) (Grant U24 CA126543)National Institutes of Health (U.S.) (Grant U24 CA126546)National Institutes of Health (U.S.) (Grant U24 CA126563)National Institutes of Health (U.S.) (Grant U24 CA126544)National Institutes of Health (U.S.) (Grant U24 CA143845)National Institutes of Health (U.S.) (Grant U24 CA143858)National Institutes of Health (U.S.) (Grant U24 CA144025)National Institutes of Health (U.S.) (Grant U24 CA143882)National Institutes of Health (U.S.) (Grant U24 CA143866)National Institutes of Health (U.S.) (Grant U24 CA143867)National Institutes of Health (U.S.) (Grant U24 CA143848)National Institutes of Health (U.S.) (Grant U24 CA143840)National Institutes of Health (U.S.) (Grant U24 CA143835)National Institutes of Health (U.S.) (Grant U24 CA143799)National Institutes of Health (U.S.) (Grant U24 CA143883)National Institutes of Health (U.S.) (Grant U24 CA143843)National Institutes of Health (U.S.) (Grant U54 HG003067)National Institutes of Health (U.S.) (Grant U54 HG003079)National Institutes of Health (U.S.) (Grant U54 HG003273