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PDF file - 56K, Supplementary Table S3 Read count, filtering and number of peaks.

PDF file - 101K, Supplementary Figure S5 Venn diagram, depicting the level of overlap of genes between 'proximity' and 'functionality' genes.

PDF file - 189K, Supplementary Figure S1 Overlap of binding events for the three markers in all tumors.

PDF file - 46K, Supplementary Table S2 Clinicopathological characteristics of the aromatase inhibitor cohort.

PDF file - 100K, Supplementary Figure S2 Overlap of peaks from the same ChIP-seq condition between tumors.

PDF file - 129K, Supplementary Table S4 Differential bound regions for ERalpha, H3K4me3 or H3K27me3 in 'poor' and 'good' outcome patients.

PDF file - 198K, Supplementary Figure S6 Differential response to aromatase inhibitors, using ChiA-PET associated genes and genes overlapping between proximity and ChIA-PET data.

PDF file - 192K, Supplementary Figure S4 Clustering heatmap analysis for ERalpha, H3K4me3 and H3K27me3.

PDF file - 35K, Supplementary Table S6 Gene lists of the ChIP-seq based classifiers for outcome after aromatase inhibitor treatment.

PDF file - 69K, Supplementary Table S7 Gene Ontology (GO) pathway enrichment for genes proximal to differentially enriched ERalpha, H3K4me3 and H3K27me3 binding events.

PDF file - 39K, Supplementary Table S5 QPCR primer sequences for 'good outcome' and 'poor outcome' enriched peaks for ERalpha, H3K4me3 and H3K27me3.

PDF file - 46K, Supplementary Table S8 Gene Ontology (GO) pathway overlap between ChIP-seq identified classifiers and pathways identified by Ellis et al., 2012 Nature.

PDF file - 55K, Supplementary Table S1 Patient and tumor characteristics of the discovery cohort.

PDF file - 43K, Supplementary Table S9 Univariate and multivariate analyses for TTP after treatment with first-line aromatase inhibitors.

PDF file - 110K, Supplementary Figure S3 Principal Component Analysis for all three ChIP-seq conditions.

PDF file - 169K, Supplementary Material and Methods Information on tumor samples, motif analysis, heatmaps, and genomic distributions of binding events, mRNA expression analysis, survival analysis and supplementary references.

SummaryAgeing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we developed a novel in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis, rather than increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remained constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity.

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

Discovering biomarkers of drug response and finding powerful drug combinations can support the reuse of previously abandoned cancer drugs in the clinic. Indisulam is an abandoned drug that acts as a molecular glue, inducing degradation of splicing factor RBM39 through interaction with CRL4DCAF15. Here, we performed genetic and compound screens to uncover factors mediating indisulam sensitivity and resistance. First, a dropout CRISPR screen identified SRPK1 loss as a synthetic lethal interaction with indisulam that can be exploited therapeutically by the SRPK1 inhibitor SPHINX31. Moreover, a CRISPR resistance screen identified components of the degradation complex that mediate resistance to indisulam: DCAF15, DDA1, and CAND1. Lastly, we show that cancer cells readily acquire spontaneous resistance to indisulam. Upon acquiring indisulam resistance, pancreatic cancer (Panc10.05) cells still degrade RBM39 and are vulnerable to BCL-xL inhibition. The better understanding of the factors that influence the response to indisulam can assist rational reuse of this drug in the clinic.

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

Discovering biomarkers of drug response and finding powerful drug combinations can support the reuse of previously abandoned cancer drugs in the clinic. Indisulam is an abandoned drug that acts as a molecular glue, inducing degradation of splicing factor RBM39 through interaction with CRL4DCAF15. Here, we performed genetic and compound screens to uncover factors mediating indisulam sensitivity and resistance. First, a dropout CRISPR screen identified SRPK1 loss as a synthetic lethal interaction with indisulam that can be exploited therapeutically by the SRPK1 inhibitor SPHINX31. Moreover, a CRISPR resistance screen identified components of the degradation complex that mediate resistance to indisulam: DCAF15, DDA1, and CAND1. Last, we show that cancer cells readily acquire spontaneous resistance to indisulam. Upon acquiring indisulam resistance, pancreatic cancer (Panc10.05) cells still degrade RBM39 and are vulnerable to BCL-xL inhibition. The better understanding of the factors that influence the response to indisulam can assist rational reuse of this drug in the clinic.

Background Chromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice. Methods To investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1−/−;TP53−/− mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination. Results The enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment. Conclusions Our study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.

Background Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary. Results We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations. Conclusions XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR. Electronic supplementary material The online version of this article (10.1186/s12859-018-2353-5) contains supplementary material, which is available to authorized users.

Contains fulltext : 193611.pdf (Publisher’s version ) (Open Access) Mutations in Fanconi Anemia or Homologous Recombination (FA/HR) genes can cause DNA repair defects and could therefore impact cancer treatment response and patient outcome. Their functional impact and clinical relevance in head and neck squamous cell carcinoma (HNSCC) is unknown. We therefore questioned whether functional FA/HR defects occurred in HNSCC and whether they are associated with FA/HR variants. We assayed a panel of 29 patient-derived HNSCC cell lines and found that a considerable fraction is hypersensitive to the crosslinker Mitomycin C and PARP inhibitors, a functional measure of FA/HR defects. DNA sequencing showed that these hypersensitivities are associated with the presence of bi-allelic rare germline and somatic FA/HR gene variants. We next questioned whether such variants are associated with prognosis and treatment response in HNSCC patients. DNA sequencing of 77 advanced stage HNSCC tumors revealed a 19% incidence of such variants. Importantly, these variants were associated with a poor prognosis (p = 0.027; HR = 2.6, 1.1-6.0) but favorable response to high cumulative cisplatin dose. We show how an integrated in vitro functional repair and genomic analysis can improve the prognostic value of genetic biomarkers. We conclude that repair defects are marked and frequent in HNSCC and are associated with clinical outcome.

Mutational load varies widely between malignancies and has been used as a proxy for the immunological foreignness of human cancers. However, without well-defined reference points it is difficult to determine which human tumors can be considered sufficiently foreign to the T-cell-based immune system. We established a neo-antigen prediction pipeline that processes single nucleotide variants, indels and gene fusion events and established its precision in identifying T-cell-recognized antigens. We used this pipeline to benchmark immunological foreignness of human cancers against pathogens for which T-cell control has been established. We demonstrate that up to 50% of tumors, spanning 25 sites of origin, are more foreign than these pathogen benchmarks. In addition, we demonstrate that the neo-antigen repertoire of treatment-naive tumors is not detectably influenced by immune editing. These data suggest that immunotherapeutic strategies that enhance activity of the endogenous T-cell compartment may be of value for a large fraction of human cancers.

Poly(ADP-ribose) polymerase inhibition (PARPi) is a promising new therapeutic approach for the treatment of cancers that show homologous recombination deficiency (HRD). Despite the success of PARPi in targeting HRD in tumors that lack the tumor suppressor function of BRCA1 or BRCA2, drug resistance poses a major obstacle. We developed three-dimensional cancer organoids derived from genetically engineered mouse models (GEMMs) for BRCA1- and BRCA2-deficient cancers. Unlike conventional cell lines or mammospheres, organoid cultures can be efficiently derived and rapidly expanded in vitro. Orthotopically transplanted organoids give rise to mammary tumors that recapitulate the epithelial morphology and preserve the drug response of the original tumor. Notably, GEMM-tumor-derived organoids can be easily genetically modified, making them a powerful tool for genetic studies of tumor biology and drug resistance.

Tumor-specific neo-antigens that arise as a consequence of mutations(1,2) are thought to be important for the therapeutic efficacy of cancer immunotherapies(3-5). Accumulating evidence suggests that neo-antigens may be commonly recognized by intratumoral CD8(+) T cells(3-7), but it is unclear whether neoantigen-specific CD4(+) T cells also frequently reside within human tumors. In view of the accepted role of tumor-specific CD4(+) T-cell responses in tumor control(8-10), we addressed whether neo-antigen-specific CD4(+) T-cell reactivity is a common property in human melanoma

Over the past years we have learned that the T-cell-based immune system frequently responds to the neoantigens that arise as a consequence of the accumulated DNA damage causing the malignant transformation. Furthermore, recognition of neoantigens appears an important driver of the clinical activity of both T-cell checkpoint blockade and adoptive T-cell therapy as cancer immunotherapies. From the efforts dissecting the neoantigen-specific T-cell response it has become clear that only a very minor fraction of the accumulated mutations is recognized by the immune system, and the challenge to unravel the neoantigen-specific T-cell response lies in identifying which neoantigens are more likely to be true T-cell epitopes. We have analyzed neoantigen-specific T-cell reactivity in 12 melanoma patients using an in silico epitope prediction pipeline based on RNA expression, predicted HLA binding affinity, proteasomal processing and self-similarity to predict potential neoepitopes. We screened for T-cell recognition of 7000 epitopes from these 12 patients (average ~550 epitopes per patient, range: 96-1902) using our pMHC multimer combinatorial encoding technology and found 19 epitopes to be recognized by T-cells (hits) and 6981 to be “non-hits.” Based on these data we have examined the properties of T-cell recognized neoantigens. An intriguing observation is an enrichment within T-cell recognized epitopes of epitopes with the mutation positioned within the last 4 amino acids (C-terminal end of the peptide) compared to the screened set of epitopes. Fifteen out of 19 hits (approximately 80%) harbored a mutation within the last 4 amino acids of the peptide, whereas within the full set of screen epitopes it is 43%. While it is currently unclear what the reason is for this, this could reflect a biologic importance in T-cell recognition of the C-terminal part of the epitope. Furthermore, RNA expression and predicted binding affinity to HLA are important informative parameters for selecting T-cell recognized epitopes. A striking observation is that predicted binding affinity not only correlates with likelihood of observing a T-cell response but also the magnitude of this T-cell response, suggesting a hierarchy within neoantigens, and that not all neoantigens are of equal immunologic quality. In summary, our findings indicate that T-cell recognized neoantigens may differ from the neoantigen pool not recognized. In particular regarding position of the mutation with the epitope, RNA abundance and predicted HLA binding affinity. Importantly, our data reveal a hierarchy within neoantigens comparable to immunodominance known from viral infections. This hierarchy appears to depend mostly on binding affinity. These observations are likely to be highly relevant when selecting neoantigens for therapeutic manipulation such as vaccines. Citation Format: Pia Kvistborg, Marit M. van Buuren, Daisy Philips, Nienke van Rooij, Arno Velds, Sam Behjati, Marlous van den Braber, Mireille Toebes, Lorenzo Fanchi, Maarten Slagter, Marie Stentoft Svane, Patrick Hwu, Joost van den Berg, Michael Stratton, Christian Blank, John B.A.G. Haanen, Can Kesmir, Ton N.M. Schumacher. Properties of T-cell-recognized neoantigens [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B022.

The evidence for T-cell–mediated regression of human cancers such as non–small-cell lung carcinoma, renal cell carcinoma, and—in particular—melanoma after immunotherapy is strong. Anti-CTLA4 (ipilimumab) treatment has been approved for treatment of meta-static melanoma,1 and antibody-mediated blockade of PD-1, a second inhibitory receptor on T cells, has shown highly encouraging results in early clinical trials.2,3 Although the clinical activity of these treatments is apparent, it is still unknown which T-cell reactivities are involved in immunotherapy-induced cancer regression.4 T-cell reactivity against nonmutated tumor-associated self-antigens has been analyzed in patients treated with ipilimumab or with autologous tumor-infiltrating T cells, but the magnitude of the T-cell responses observed has been relatively modest.5,6 In part on the basis of such data, recognition of patient-specific mutant epitopes (hereafter referred to as neoantigens) has been suggested to be a potentially important component.7 A potential involvement of mutated epitopes in T-cell control would also fit well with the observation that the mutation load in sun-exposed melanomas is particularly high.8-10 Intriguingly, on the basis of animal model data, it has recently been suggested that (therapy-induced) analysis of T-cell reactivity against patient-specific neoantigens may be feasible through exploitation of cancer genome data.11,12 However, human data have thus far been lacking. Here we report a case of a patient with stage IV melanoma who exhibited a clinical response to ipilimumab treatment. Cancer exome–guided analysis of T-cell reactivity in this patient revealed reactivity against two neoantigens, including a dominant T-cell response against a mutant epitope of the ATR (ataxia telangiectasia and Rad3 related) gene product that increased strongly after ipilimumab treatment. These data provide the first demonstration (to our knowledge) of cancer exome–guided analysis to dissect the effects of melanoma immunotherapy.

Aromatase inhibitors are the major first-line treatment of estrogen receptor–positive breast cancer, but resistance to treatment is common. To date, no biomarkers have been validated clinically to guide subsequent therapy in these patients. In this study, we mapped the genome-wide chromatin-binding profiles of estrogen receptor α (ERα), along with the epigenetic modifications H3K4me3 and H3K27me3, that are responsible for determining gene transcription (n = 12). Differential binding patterns of ERα, H3K4me3, and H3K27me3 were enriched between patients with good or poor outcomes after aromatase inhibition. ERα and H3K27me3 patterns were validated in an additional independent set of breast cancer cases (n = 10). We coupled these patterns to array-based proximal gene expression and progression-free survival data derived from a further independent cohort of 72 aromatase inhibitor–treated patients. Through this approach, we determined that the ERα and H3K27me3 profiles predicted the treatment outcomes for first-line aromatase inhibitors. In contrast, the H3K4me3 pattern identified was not similarly informative. The classification potential of these genes was only partially preserved in a cohort of 101 patients who received first-line tamoxifen treatment, suggesting some treatment selectivity in patient classification. Cancer Res; 73(22); 6632–41. ©2013 AACR.

SummaryPosttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.

Chromosomal translocations are a hallmark of cancer. Unraveling the molecular mechanism of these rare genetic events requires a clear distinction between correlative and causative risk-determinants, where technical and analytical issues can be excluded. To meet this goal, we performed in-depth analyses of publicly available genome-wide datasets. In contrast to several recent reports, we demonstrate that chromosomal translocation risk is causally unrelated to promoter stalling (Spt5), transcriptional activity, or off-targeting activity of the activation-induced cytidine deaminase. Rather, an open chromatin configuration, which is not promoter-specific, explained the elevated translocation risk of promoter regions. Furthermore, the fact that gene size directly correlates with the translocation risk in mice and human cancers further demonstrated the general irrelevance of promoter-specific activities. Interestingly, a subset of translocations observed in cancer patients likely initiates from double-strand breaks induced by an access-independent process. Together, these unexpected and novel insights are fundamental in understanding the origin of chromosome translocations and, consequently, cancer.

Knockdown of the scaffolding protein Mek binding protein 1 (Mp1) in mouse embryonic stem cells suppresses differentiation but not proliferation, and more invasive human germ cell tumors express lower amounts of Mp1., Despite intense investigation of intrinsic and extrinsic factors that regulate pluripotency, the process of initial fate commitment of embryonic stem (ES) cells is still poorly understood. We used a genome-wide short hairpin RNA screen in mouse ES cells to identify genes that are essential for initiation of differentiation. Knockdown of the scaffolding protein Mek binding protein 1 (Mp1, also known as Lamtor3 or Map2k1ip1) stimulated self-renewal of ES cells, blocked differentiation, and promoted proliferation. Fibroblast growth factor 4 (FGF4) signaling is required for initial fate commitment of ES cells. Knockdown of Mp1 inhibited FGF4-induced differentiation but did not alter FGF4-driven proliferation. This uncoupling of differentiation and proliferation was also observed when oncogenic Ras isoforms were overexpressed in ES cells. Knockdown of Mp1 redirected FGF4 signaling from differentiation toward pluripotency and up-regulated the pluripotency-related genes Esrrb, Rex1, Tcl1, and Sox2. We also found that human germ cell tumors (GCTs) express low amounts of Mp1 in the invasive embryonic carcinoma and seminoma histologies and higher amounts of Mp1 in the noninvasive carcinoma in situ precursor and differentiated components. Knockdown of Mp1 in invasive GCT cells resulted in resistance to differentiation, thereby showing a functional role for Mp1 both in normal differentiation of ES cells and in germ cell cancer.

Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analysis of their RNA and DNA profiles is challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin can result both in the generation of false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. Therefore, we developed the open-source R-package XenofilteR, which separates mouse from human sequence reads based on the number of discordant base pairs between each read and the reference genomes. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human whole genome and whole exome DNA sequence data. This analysis revealed that XenofilteR removes >99.9% of sequence reads of mouse origin while retaining sequence reads of human origin. The filtering allowed for mutation analysis of PDX samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic mutations present in the original tumor. These findings were further validated in breast cancer and melanoma PDX samples, confirming the retrieval of accurate variant allele frequencies and somatic mutations. In conclusion, XenofilteR accurately dissects sequence reads from mouse and human origin in PDX sequence data, thereby outperforming currently available tools. Citation Format: Oscar Krijgsman, Roelof JC Kluin, Kristel Kemper, Thomas Kuilman, Julian R. de Ruiter, Vivek Iyer, Josep V. Forment, Paulien Cornelissen-Steijger, Iris de Rink, Petra ter Brugge, Ji-Ying Song, Sjoerd Klarenbeek, Ultan McDermott, Jos Jonkers, Arno Velds, David J. Adams, Daniel S. Peeper. XenofilteR: Computational dissection of mouse and human reads in PDX and xenograft sequence data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1041.

OBJECTIVE To determine the value of gene-expression profiling as a predictor of the status of the regional nodes in patients with penile carcinoma. PATIENTS AND METHODS Tumour samples of 56 patients with penile squamous cell carcinoma were analysed for the gene expression on 35 k oligoarrays; 32 were from patients with histopathologically confirmed lymph node metastases and 24 from those with no lymph node involvement. The 56 patients were divided into a training and validation set. For the training set, 15 patients with histopathologically confirmed nodal metastases and 15 without were selected. The validation set consisted of the remaining 26 patients, containing 17 node-positive and nine with no nodal metastases. RESULTS A 44-probe classifier had the best performance within the training set; this classifier correctly assigned 29 of 30 specimens in the training set to the two outcome groups. In the validation set of 26 tumours, the classifier correctly assigned 14 of the 26 (54%) specimens to the two outcome groups. Of the 17 specimens with histologically confirmed nodal involvement, 12 were classified as node-positive and five as node-negative, resulting in a sensitivity of 71%. Of the nine specimens from node-negative patients, two were correctly classified as node-negative and seven as node positive, resulting in a specificity of 22%. CONCLUSIONS In this series, gene expression profiling did not produce a useful classifier to predict nodal involvement in patients with penile carcinoma.

T cells, as well as other cell types, are composed of phenotypically and functionally distinct subsets. However, for many of these populations it is unclear whether they develop from common or separate progenitors. To address such issues, we developed a novel approach, termed cellular barcoding, that allows the dissection of lineage relationships. We demonstrate that the labeling of cells with unique identifiers coupled to a microarray-based detection system can be used to analyze family relationships between the progeny of such cells. To exemplify the potential of this technique, we studied migration patterns of families of antigen-specific CD8+ T cells in vivo. We demonstrate that progeny of individual T cells rapidly seed independent lymph nodes and that antigen-specific CD8+ T cells present at different effector sites are largely derived from a common pool of precursors. These data show how locally primed T cells disperse and provide a technology for kinship analysis with wider utility.

BRCA1 is an important protein in the repair of DNA double strand breaks (DSBs), which are induced by alkylating chemotherapy. A BRCA1-like DNA copy number signature derived from tumors with a BRCA1 mutation is indicative for impaired BRCA1 function and associated with good outcome after high dose (HD) and tandem HD DSB inducing chemotherapy. We investigated whether BRCA1-like status was a predictive biomarker in the WSG AM 01 trial. WSG AM 01 randomized high-risk breast cancer patients to induction (2× epirubicin-cyclophosphamide) followed by tandem HD chemotherapy with epirubicin, cyclophosphamide and thiotepa versus dose dense chemotherapy (4× epirubicin-cyclophospamide followed by 3× cyclophosphamide-methotrexate-5-fluorouracil). We generated copy number profiles for 143 tumors and classified them as being BRCA1-like or non-BRCA1-like. Twenty-six out of 143 patients were BRCA1-like. BRCA1-like status was associated with high grade and triple negative tumors. With regard to event-free-survival, the primary endpoint of the trial, patients with a BRCA1-like tumor had a hazard rate of 0.2, 95% confidence interval (CI): 0.07-0.63, p = 0.006. In the interaction analysis, the combination of BRCA1-like status and HD chemotherapy had a hazard rate of 0.19, 95% CI: 0.067-0.54, p = 0.003. Similar results were observed for overall survival. These findings suggest that BRCA1-like status is a predictor for benefit of tandem HD chemotherapy with epirubicin-thiotepa-cyclophosphamide.

Background Next generation sequencing (NGS) of amplified DNA is a powerful tool to describe genetic heterogeneity within cell populations that can both be used to investigate the clonal structure of cell populations and to perform genetic lineage tracing. For applications in which both abundant and rare sequences are biologically relevant, the relatively high error rate of NGS techniques complicates data analysis, as it is difficult to distinguish rare true sequences from spurious sequences that are generated by PCR or sequencing errors. This issue, for instance, applies to cellular barcoding strategies that aim to follow the amount and type of offspring of single cells, by supplying these with unique heritable DNA tags. Results Here, we use genetic barcoding data from the Illumina HiSeq platform to show that straightforward read threshold-based filtering of data is typically insufficient to filter out spurious barcodes. Importantly, we demonstrate that specific sequencing errors occur at an approximately constant rate across different samples that are sequenced in parallel. We exploit this observation by developing a novel approach to filter out spurious sequences. Conclusions Application of our new method demonstrates its value in the identification of true sequences amongst spurious sequences in biological data sets. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-0999-4) contains supplementary material, which is available to authorized users.