The clonal architecture and tumour microenvironment of breast cancers are shaped by neoadjuvant chemotherapy

Neoadjuvant chemotherapy has become standard practice in patients with high-risk early breast cancer as it improves rates of breast conservation surgery and enables prediction of recurrence and survival by using response to treatment as a surrogate. Previous studies have focused on generating molecular datasets to develop prediction models of response, though little is known on how tumours and their microenvironments are modulated by neoadjuvant chemotherapy. The thesis aims at molecularly characterising tumour changes during neoadjuvant chemotherapy in a cohort of 168 patients. Serial tumour samples at diagnosis, and, when available, midway through chemotherapy and on completion of treatment were profiled by shallow whole genome sequencing, deep exome sequencing and transcriptome sequencing, resulting in the generation of an unprecedented genomics dataset with tumours in situ while patients received chemotherapy. Molecular predictors of response to chemotherapy were inferred from the diagnostic biopsy. Several novel observations were made, including previously undescribed associations between copy number alterations, mutational genotypes, neoantigen load, HLA genotypes and intra-tumoural heterogeneity with chemosensitivity. Possible mechanisms of chemoresistance included LOH at the MHC Class I locus, decreased expression of MHC Class I and II genes and drug influx molecules, as well as increased expression of drug efflux pumps. A complex relationship between proliferation, tumour microenvironment composition (TME) and response to treatment was explored by deconvoluting bulk RNAseq data and performing digital pathology orthogonal validation. Clonal and microenvironment dynamic changes induced by/associated with chemotherapy were then modelled. Two types of genomic responses were identified, one in which the clonal composition was stable throughout treatment and another where clonal emergence and/or extinction was evident. Validation by multi-region deep sequencing confirmed the dynamics of the clonal landscape. Clonal emergence was shown to be associated with higher proliferation and decreased immune infiltrate, with an increase in genomic instability and homologous recombination deficiency during treatment. The immune TME composition and activity mirrored response to treatment, with cytolytic activity and innate and adaptive immune infiltrates linearly correlating with the degree of residual disease remaining after chemotherapy. Finally, the circulating tumour DNA (ctDNA) genomic landscape was explored by using shallow whole genome sequencing and targeted sequencing of plasma DNA. Tumour mutations detected on exome sequencing were also detected in ctDNA in plasma, supporting the use of liquid biopsies as a biomarker for monitoring response to therapy and detection of minimal residual disease.Wellcome Trus

Identification and characterisation of germline-associated genes as potential human cancer biomarkers

It is increasingly apparent that cancer cells exhibit major intraclonal and interclonal heterogeneity that impacts on disease behaviour, response to treatments as well as clinical outcomes for patients. Understanding this diversity is critical to gain insights that ultimately influence treatment decisions in the era of personalised medicine. There is an increasing need to identify unique and relevant diagnostic and prognostic protein biomarkers to allow better stratification. Identification of aberrant gene expression patterns is one way in which additional insights into cancer causation can be discovered and can also inform novel therapeutic targets. In this study we sought to focus on a group of proteins that are predominantly present in the germline but not in somatic cells. It is now well established that cancer cells exhibit aberrant expression of germline factors. Cancer testis antigens (CTAs) are one such family of proteins that exhibit increased presence in a variety of cancers and are also potentially immunogenic. As meiotic cell division is restricted to germline cells such as the testis, we hypothesised that identification and characterisation of genes that govern this process may yield additional clinically useful biomarkers that are also relevant to disease biology and behaviour. With the aid of a pre-existing computational and bioinformatics approach, we first identified a subset of genes, some of which were likely to be involved in meiosis, and interrogated normal and cancerous cells to ascertain their expression pattern. This allowed us to identify a distinct cohort of candidate genes that appeared to be predominantly expressed in the germline and cancer. Immunohistochemistry using normal and (predominantly colorectal) cancer tissues was performed for further evaluation. Significantly, we identified two novel proteins (C20orf201 and TEX19) as putative meiosis-associated proteins that may have enhanced presence in cancer and the potential to be immunogenic. In summary, this research into germline restricted genes in cancer vs normal tissues has identified two previously uncharacterised proteins that are likely to be relevant for the biology, behaviour or therapy of some cancers and has also identified a cohort of further genes that warrant further scientific exploration

Next Generation-Targeted Amplicon Sequencing (NG-TAS): an optimised protocol and computational pipeline for cost-effective profiling of circulating tumour DNA

Cancer; Computational pipeline; Deep sequencingCàncer; Segmentació computacional; SeqüenciacióCáncer; Segmentación computacional; SecuenciaciónCirculating tumour DNA (ctDNA) detection and monitoring have enormous potential clinical utility in oncology. We describe here a fast, flexible and cost-effective method to profile multiple genes simultaneously in low input cell-free DNA (cfDNA): Next Generation-Targeted Amplicon Sequencing (NG-TAS). We designed a panel of 377 amplicons spanning 20 cancer genes and tested the NG-TAS pipeline using cell-free DNA from two HapMap lymphoblastoid cell lines. NG-TAS consistently detected mutations in cfDNA when mutation allele fraction was > 1%. We applied NG-TAS to a clinical cohort of metastatic breast cancer patients, demonstrating its potential in monitoring the disease. The computational pipeline is available at https://github.com/cclab-brca/NGTAS_pipelin

ARL3 mutations cause Joubert syndrome by disrupting ciliary protein composition

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTSrelated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128 (hg 19)), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP Ribosylation Factor-Like GTPase 3, ARL3, is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localisation in cilia. We propose that ARL3 provides a potential hub in the network of encoded ciliopathy genes, whereby perturbation of ARL3 results in the mislocalisation of multiple ciliary proteins due to abnormal displacement of lipidated protein cargo

Intersect-then-combine approach: improving the performance of somatic variant calling in whole exome sequencing data using multiple aligners and callers.

Bioinformatic analysis of genomic sequencing data to identify somatic mutations in cancer samples is far from achieving the required robustness and standardisation. In this study we generated a whole exome sequencing benchmark dataset using the platinum genome sample NA12878 and developed an intersect-then-combine (ITC) approach to increase the accuracy in calling single nucleotide variants (SNVs) and indels in tumour-normal pairs. We evaluated the effect of alignment, base quality recalibration, mutation caller and filtering on sensitivity and false positive rate. The ITC approach increased the sensitivity up to 17.1%, without increasing the false positive rate per megabase (FPR/Mb) and its validity was confirmed in a set of clinical samples

Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers.

Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling

O direito tradicional hindu: análise de um sistema jurídico integral

TCC(graduação) - Universidade Federal de Santa Catarina. Centro de Ciências Jurídicas. Direito.O eixo teórico desta pesquisa está na análise dos elementos jurídicos que se pode extrair das fontes da tradição hindu, sobretudo através de suas escrituras de escopo legal, conhecidas como Smritis, da qual a mais conhecida no ocidente foram As Leis de Manu. Esta cultura jurídica foi tomada em sua singularidade, e não como tela de projeção para os conceitos do direito ocidental. Destinou-se, principalmente, muitos capítulos a análise das principais categorias teóricas e os fundamentos do raciocínio indiano em temas como organização social, administração da justiça e certos tópicos de direito material, como os 18 títulos legais ou vyavaharapadas. Deu-se um destaque ao sistema de varnas e ashramas, que é tido pelos próprios tratados de direito hindu, os dharmashastras, como o principal eixo normativo da sociedade védica. Por fim, também se trabalhou com as concepções hindus de processo, sobretudo na figura do vyavahara, espécie de procedimento legal, e mesmo alguns elementos punitivos da justiça tradicional hindu, como danda e prayascitta, as punições e penitências. Deste modo, desenhou-se um quadro axiológico e topográfico do direito na tradição hindu, privilegiando uma visão de sistema sobre as especificidades da hermenêutica oriental, sem insistir tanto em casuísticas de direito material

The Genomic and Immune Landscapes of Lethal Metastatic Breast Cancer

TCR repertoire; Breast cancer; Clade mutationsRepertori TCR; Càncer de mama; Mutacions cladeRepertorio TCR; Cáncer de mama; Mutaciones cladoThe detailed molecular characterization of lethal cancers is a prerequisite to understanding resistance to therapy and escape from cancer immunoediting. We performed extensive multi-platform profiling of multi-regional metastases in autopsies from 10 patients with therapy-resistant breast cancer. The integrated genomic and immune landscapes show that metastases propagate and evolve as communities of clones, reveal their predicted neo-antigen landscapes, and show that they can accumulate HLA loss of heterozygosity (LOH). The data further identify variable tumor microenvironments and reveal, through analyses of T cell receptor repertoires, that adaptive immune responses appear to co-evolve with the metastatic genomes. These findings reveal in fine detail the landscapes of lethal metastatic breast cancer

The temporal mutational and immune tumour microenvironment remodelling of HER2-negative primary breast cancers.

The biology of breast cancer response to neoadjuvant therapy is underrepresented in the literature and provides a window-of-opportunity to explore the genomic and microenvironment modulation of tumours exposed to therapy. Here, we characterised the mutational, gene expression, pathway enrichment and tumour-infiltrating lymphocytes (TILs) dynamics across different timepoints of 35 HER2-negative primary breast cancer patients receiving neoadjuvant eribulin therapy (SOLTI-1007 NEOERIBULIN-NCT01669252). Whole-exome data (N = 88 samples) generated mutational profiles and candidate neoantigens and were analysed along with RNA-Nanostring 545-gene expression (N = 96 samples) and stromal TILs (N = 105 samples). Tumour mutation burden varied across patients at baseline but not across the sampling timepoints for each patient. Mutational signatures were not always conserved across tumours. There was a trend towards higher odds of response and less hazard to relapse when the percentage of subclonal mutations was low, suggesting that more homogenous tumours might have better responses to neoadjuvant therapy. Few driver mutations (5.1%) generated putative neoantigens. Mutation and neoantigen load were positively correlated (R2 = 0.94, p = 2 = 0.16, p = 0.02). An enrichment in pathways linked to immune infiltration and reduced programmed cell death expression were seen after 12 weeks of eribulin in good responders. VEGF was downregulated over time in the good responder group and FABP5, an inductor of epithelial mesenchymal transition (EMT), was upregulated in cases that recurred (p < 0.05). Mutational heterogeneity, subclonal architecture and the improvement of immune microenvironment along with remodelling of hypoxia and EMT may influence the response to neoadjuvant treatment