Andrea Marion Marquard

Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data

We describe our algorithm and software for determining copy number profiles from tumor genome sequencing data, and find that it compares favorably to existing algorithms for the same purpose.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes

Identification of the peptides recognized by individual T cells is important for understanding and treating immune-related diseases. Current cytometry-based approaches are limited to the simultaneous screening of 10–100 distinct T-cell specificities in one sample. Here we use peptide–major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer. Barcode-labeled pMHC multimers enable the combination of functional T-cell analysis with large-scale epitope recognition profiling for the characterization of T-cell recognition in various diseases, including in small clinical samples.

Pan-cancer analysis of genomic scar signatures associated with homologous recombination deficiency suggests novel indications for existing cancer drugs.

BackgroundOvarian and triple-negative breast cancers with BRCA1 or BRCA2 loss are highly sensitive to treatment with PARP inhibitors and platinum-based cytotoxic agents and show an accumulation of genomic scars in the form of gross DNA copy number aberrations. Cancers without BRCA1 or BRCA2 loss but with accumulation of similar genomic scars also show increased sensitivity to platinum-based chemotherapy. Therefore, reliable biomarkers to identify DNA repair-deficient cancers prior to treatment may be useful for directing patients to platinum chemotherapy and possibly PARP inhibitors. Recently, three SNP array-based signatures of chromosomal instability were published that each quantitate a distinct type of genomic scar considered likely to be caused by improper DNA repair. They measure telomeric allelic imbalance (named NtAI), large scale transition (named LST), and loss of heterozygosity (named HRD-LOH), and it is suggested that these signatures may act as biomarkers for the state of DNA repair deficiency in a given cancer.ResultsWe explored the pan-cancer distribution of scores of the three signatures utilizing a panel of 5371 tumors representing 15 cancer types from The Cancer Genome Atlas, and found a good correlation between scores of the three signatures (Spearman’s ρ 0.73–0.87). In addition we found that cancer types ordinarily receiving platinum as standard of care have higher median scores of all three signatures. Interestingly, we also found that smaller subpopulations of high-scoring tumors exist in most cancer types, including those for which platinum chemotherapy is not standard therapy.ConclusionsWithin several cancer types that are not ordinarily treated with platinum chemotherapy, we identified tumors with high levels of the three genomic biomarkers. These tumors represent identifiable subtypes of patients which may be strong candidates for clinical trials with PARP inhibitors or platinum-based chemotherapeutic regimens.

Prediction of neoepitopes from murine sequencing data

We recently published the tool MuPeXI, the mutant peptide extractor and informer, enabling neoepitope prediction from tumor sequencing data [1]. MuPeXI is originally designed for variant calls obtained from sequencing data of human origin but increasing interest to determine neoepitopes in murine models have encouraged us to update and test MuPeXI for mouse compatibility. The murine-compatible MuPeXI is now available as a command line tool (https :// githu b.com/ambj/MuPeX I) together with a mouse-specific web server (http://www.cbs.dtu.dk/servi ces/MuPeX I-mouse /). Despite the interest for determining neoepitopes from preclinical mouse models, only few tools for neoepitope prediction have been designed and evaluated to allow neoepitope prediction from data of murine origin. To fulfill this need, we optimized MuPeXI to enable identification of murine neopeptides. MuPeXI is now compatible with the genetic reference of mus musculus, as well as the two commonly used mouse strains, BALBc and C57BL/6. To test the NGS pipeline and optimize MuPeXI, we evaluate the neoepitope landscape in the CT26 tumor cell line, which has been extensively used in mode-of-action studies in syngeneic mouse tumor models [2], and proven especially valuable as an experimental model for immune therapy interventions. We used sequencing data from Castle et al. [2] and Mosely et al. [3], including both the CT26 cell line (CL) and the CT26 tumors grown in vivo on BALBc mice (TU). The NGS analysis pipeline was followed as suggested by Genome Analysis Tool Kit (GATK), best practice guidelines, using the same tools as in the original MuPeXI paper [1]. References were downloaded from Ensembl’s mouse genome assemble, further detail can be found in the MuPeXI user manual (https :// githu b.com/ambj/MuPeX I/blob/maste r/doc/MuPeX I_User_ Manua l.md#refer ences ). In the analysis we incorporated the new binding predictor netH2pan into MuPeXI. NetH2pan is trained solely on mouse-binding affinity and eluted ligand data [4], thereby providing the most suitable H2-binding predictions for the neopeptides extracted from somatic variant of murine sequencing data by MuPeXI. The mutational landscape was compared for the three samples tested, and although the total number of mutations identified in the three samples were high, it was in accordance with the original papers. The analysis revealed that only a fraction of the mutations, 7898 (~ 26% of the total) was identified in all 3 samples (Fig. 1a). The number of nonsynonymous mutations (NSmut) did not vary substantially compared to the total number of mutations identified, i.e., Castle: 5994–3.8%, Mosely CL: 4921–3.1%, Mosely TU: 4793–3.0% (Fig. 1b). Besides missense variant (MV) mutations, frame shifts, and indels (FI) were also identified, but present to a lesser degree (MV: 15349–9.6%, FI: 359–0.2%). To identify how many of the NSmuts lead to potential neoepitopes, the updated murine-compatible MuPeXI software was used with the relevant BALBc references. We identified 79,958 (61% of all) neopeptides shared among the two tumor samples obtained from two different studies and the original CT26 cell line sample (Fig. 1c). Of these, 4399 (3.4%) had an eluted ligand percentile rank score (%Rank EL) below 2 and were considered binding peptides in all 3 samples (Fig. 1c, blue). A total of 7034 (5.4%) peptides were identified as binders and therefore potential neoepitopes out of the total 124,467 unique neopeptides extracted from all 3 samples. Of the binding peptides, 61 potential minimal neopeptides matched previously described long peptides shown to evoke immune responses [5]. The potential neoepitopes originate from various types of NSmuts, including Anne-Mette Bjerregaard and Thomas Kainamura Pedersen contributed equally.

Quantification of within-sample genetic heterogeneity from SNP-array data

Intra-tumour genetic heterogeneity (ITH) fosters drug resistance and is a critical hurdle to clinical treatment. ITH can be well-measured using multi-region sampling but this is costly and challenging to implement. There is therefore a need for tools to estimate ITH in individual samples, using standard genomic data such as SNP-arrays, that could be implemented routinely. We designed two novel scores S and R, respectively based on the Shannon diversity index and Ripley’s L statistic of spatial homogeneity, to quantify ITH in single SNP-array samples. We created in-silico and in-vitro mixtures of tumour clones, in which diversity was known for benchmarking purposes. We found significant but highly-variable associations of our scores with diversity in-silico (p < 0.001) and moderate associations in–vitro (p = 0.015 and p = 0.085). Our scores were also correlated to previous ITH estimates from sequencing data but heterogeneity in the fraction of tumour cells present across samples hampered accurate quantification. The prognostic potential of both scores was moderate but significantly predictive of survival in several tumour types (corrected p = 0.03). Our work thus shows how individual SNP-arrays reveal intra-sample clonal diversity with moderate accuracy.

Next-generation detection of antigen-responsive T cells using DNA barcode-labeled peptide-major histocompatibility complex I multimers

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.Changes in microbiome, mucosal immunity and intestinal integrity have been associated with the onset of Type 1 Diabetes (T1D) in children. Toll-like Receptors (TLR) have been associated all three factors. The role of TLR and their effects on microbiome in autoimmunity were studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. Physical parameters of the intestines were compared; ileal weight was reduced in NOD.Tlr1-/-mice. Similarly, by histology, these mice had reduced villus length and width. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA), in two cohorts, 18 months apart. Analysis of caecal 16S sequences clearly resolved the mouse lines and there were significant differences in beta diversity between the strains, with individual bacterial species contributing greatly to the differences in the microbiota of individual TLR-deficient strains. To test the relationship between microbiome and T1D, all strains were re-derived onto the parental NOD/Lt line and the incidence of T1D re-assessed within two generations. All rederived lines expressed an incidence of disease similar to that of the parental line. TLR deficiencies are associated with changes in microbiome; changes of microbiome are associated with T1D; the effects of TLR deficiencies on T1D appear to be mediated by their effects on gut flora.Intestinal TCRb+CD4-CD8b-CD8a+ (CD8aa) IELs alleviate T cell induced colitis and have been suggested to play a role in virus infection and cancer. Their thymic development has been elucidated to some extent, as IEL precursors (IELp) are known to be CD4-CD8-CD5+TCRb+, but is not yet fully understood. Within the thymus, mature IELp were identified based on their expression of CD122 and MHC class I. Two major phenotypic subsets exist within this mature thymic IELp population: a PD1+Tbet- population that preferentially expresses a4b7, and a PD1-Tbet+ population with preferential CD103 expression. These two populations were also distinct in their Valpha repertoire. The PD1+a4b7+ population contains clones that are strongly self-reactive as judged by Nur77GFP and their dramatic increase in Bim deficient mice, while the PD1-Tbet+ population did not show these characteristics. Both gave rise to CD8aa IELs upon adoptive transfer into RAG-/- recipients. However intrathymic labeling revealed that PD1+a4b7+ IELp were the major thymic emigrating population, and emigration was S1P1-dependent. In contrast, PD1-Tbet+ IELp expressed CXCR3, were retained, and accumulated in the thymus with age. Preliminary immunofluorescence data furthermore indicate differential thymic cortico-medullary localization of the IELp subtypes. These experiments more precisely define the behavior of IEL precursors.

Abstract 2822: A pan-cancer analysis of inferred homologous recombination deficiency identifies potential platinum benefit in novel subtypes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Personalized medicine in cancer aims to improve treatment outcome, by exploiting the molecular alterations of the individual tumor to inform therapeutic decisions. Ovarian and triple-negative breast cancers with defects in homologous recombination (HR) DNA repair are highly sensitive to treatment with platinum-based DNA-damaging agents. Reliable biomarkers to identify HR-deficient cancers prior to the initial treatment may be used to stratify patients for platinum chemotherapy. Extensive genome damage caused by deficient HR is readily observed as high frequencies of allelic imbalance and loss of heterozygosity in cancers with loss of either of the tumor suppressor genes BRCA1 or BRCA2, but is also common in ovarian and triple-negative breast cancers with no BRCA1/2 mutations, indicating HR loss due to alternative mechanisms. Recently, three independent methods were published that each quantitate the state of HR deficiency in a given cancer, by summarizing different types of DNA aberrations that are likely to be caused by improper DNA repair. Here we compare the three scores, named NtAI (1), LST (2), and HRD (3), utilizing a panel of 4400 patients representing 13 cancer types from The Cancer Genome Atlas. We found that the three scores are highly correlated with each other, suggesting they measure the effect of similar types of DNA damage. We found a strong association with overall survival only in ovarian cancer, which is consistent with frequent BRCA-related HR deficiency reported for this type of cancer. Next, we compared the distribution of the scores across cancer types, and found that those types ordinarily receiving platinum as standard of care have the highest median scores. Importantly, in most types not generally given platinum chemotherapy we also found small sub-populations of high scoring tumors, which may represent subtypes with a previously overlooked potential to respond to platinum agents. Lastly, we used RNAseq to identify genes whose expression is associated with high DNA aberration scores. We compared the 100 genes most highly correlated with each score and found a shared set of 53 genes; these were enriched for genes involved in cell cycle progression, mitosis and chromosome segregation. This suggests that replication stress, perhaps combined with or induced by HR deficiency, could play a role in the generation of the measured DNA aberrations. Overall, our results demonstrate that the three methods measure correlated aberration patterns possibly generated through replication stress, and that they show prognostic potential in patients who receive platinum chemotherapy. In addition, we identify subsets of patients suffering from cancers not presently receiving platinum chemotherapy, who may benefit from it. 1) Birkbak NJ, et al. (2012) Cancer Discov 2: 366-375 2) Popova T, et al. (2012) Cancer Res 72: 5454-5462 3) Abkevich V, et al. (2012) Br J Cancer 107: 1776-1782 Citation Format: Andrea M. Marquard, Aron C. Eklund, Zhigang C. Wang, Andrea L. Richardson, Zoltan Szallasi, Nicolai J. Birkbak. A pan-cancer analysis of inferred homologous recombination deficiency identifies potential platinum benefit in novel subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2822. doi:10.1158/1538-7445.AM2014-2822