The life history of 21 breast cancers.

Cancer evolves dynamically as clonal expansions supersede one another driven by shifting selective pressures, mutational processes, and disrupted cancer genes. These processes mark the genome, such that a cancer's life history is encrypted in the somatic mutations present. We developed algorithms to decipher this narrative and applied them to 21 breast cancers. Mutational processes evolve across a cancer's lifespan, with many emerging late but contributing extensive genetic variation. Subclonal diversification is prominent, and most mutations are found in just a fraction of tumor cells. Every tumor has a dominant subclonal lineage, representing more than 50% of tumor cells. Minimal expansion of these subclones occurs until many hundreds to thousands of mutations have accumulated, implying the existence of long-lived, quiescent cell lineages capable of substantial proliferation upon acquisition of enabling genomic changes. Expansion of the dominant subclone to an appreciable mass may therefore represent the final rate-limiting step in a breast cancer's development, triggering diagnosis.

Mutational processes molding the genomes of 21 breast cancers.

All cancers carry somatic mutations. The patterns of mutation in cancer genomes reflect the DNA damage and repair processes to which cancer cells and their precursors have been exposed. To explore these mechanisms further, we generated catalogs of somatic mutation from 21 breast cancers and applied mathematical methods to extract mutational signatures of the underlying processes. Multiple distinct single- and double-nucleotide substitution signatures were discernible. Cancers with BRCA1 or BRCA2 mutations exhibited a characteristic combination of substitution mutation signatures and a distinctive profile of deletions. Complex relationships between somatic mutation prevalence and transcription were detected. A remarkable phenomenon of localized hypermutation, termed "kataegis," was observed. Regions of kataegis differed between cancers but usually colocalized with somatic rearrangements. Base substitutions in these regions were almost exclusively of cytosine at TpC dinucleotides. The mechanisms underlying most of these mutational signatures are unknown. However, a role for the APOBEC family of cytidine deaminases is proposed.

Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Targeted sequencing in DLBCL, molecular subtypes, and outcomes: a Haematological Malignancy Research Network report.

Based on the profile of genetic alterations occurring in tumor samples from selected diffuse large B-cell lymphoma (DLBCL) patients, 2 recent whole-exome sequencing studies proposed partially overlapping classification systems. Using clustering techniques applied to targeted sequencing data derived from a large unselected population-based patient cohort with full clinical follow-up (n = 928), we investigated whether molecular subtypes can be robustly identified using methods potentially applicable in routine clinical practice. DNA extracted from DLBCL tumors diagnosed in patients residing in a catchment population of ∼4 million (14 centers) were sequenced with a targeted 293-gene hematological-malignancy panel. Bernoulli mixture-model clustering was applied and the resulting subtypes analyzed in relation to their clinical characteristics and outcomes. Five molecular subtypes were resolved, termed MYD88, BCL2, SOCS1/SGK1, TET2/SGK1, and NOTCH2, along with an unclassified group. The subtypes characterized by genetic alterations of BCL2, NOTCH2, and MYD88 recapitulated recent studies showing good, intermediate, and poor prognosis, respectively. The SOCS1/SGK1 subtype showed biological overlap with primary mediastinal B-cell lymphoma and conferred excellent prognosis. Although not identified as a distinct cluster, NOTCH1 mutation was associated with poor prognosis. The impact of TP53 mutation varied with genomic subtypes, conferring no effect in the NOTCH2 subtype and poor prognosis in the MYD88 subtype. Our findings confirm the existence of molecular subtypes of DLBCL, providing evidence that genomic tests have prognostic significance in non-selected DLBCL patients. The identification of both good and poor risk subtypes in patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) clearly show the clinical value of the approach, confirming the need for a consensus classification.

NRG1 fusions in breast cancer.

BACKGROUND: NRG1 gene fusions may be clinically actionable, since cancers carrying the fusion transcripts can be sensitive to tyrosine kinase inhibitors. The NRG1 gene encodes ligands for the HER2(ERBB2)-ERBB3 heterodimeric receptor tyrosine kinase, and the gene fusions are thought to lead to autocrine stimulation of the receptor. The NRG1 fusion expressed in the breast cancer cell line MDA-MB-175 serves as a model example of such fusions, showing the proposed autocrine loop and exceptional drug sensitivity. However, its structure has not been properly characterised, its oncogenic activity has not been fully explained, and there is limited data on such fusions in breast cancer. METHODS: We analysed genomic rearrangements and transcripts of NRG1 in MDA-MB-175 and a panel of 571 breast cancers. RESULTS: We found that the MDA-MB-175 fusion-originally reported as a DOC4(TENM4)-NRG1 fusion, lacking the cytoplasmic tail of NRG1-is in reality a double fusion, PPP6R3-TENM4-NRG1, producing multiple transcripts, some of which include the cytoplasmic tail. We hypothesise that many NRG1 fusions may be oncogenic not for lacking the cytoplasmic domain but because they do not encode NRG1's nuclear-localised form. The fusion in MDA-MB-175 is the result of a very complex genomic rearrangement, which we partially characterised, that creates additional expressed gene fusions, RSF1-TENM4, TPCN2-RSF1, and MRPL48-GAB2. We searched for NRG1 rearrangements in 571 breast cancers subjected to genome sequencing and transcriptome sequencing and found four cases (0.7%) with fusions, WRN-NRG1, FAM91A1-NRG1, ARHGEF39-NRG1, and ZNF704-NRG1, all splicing into NRG1 at the same exon as in MDA-MB-175. However, the WRN-NRG1 and ARHGEF39-NRG1 fusions were out of frame. We identified rearrangements of NRG1 in many more (8% of) cases that seemed more likely to inactivate than to create activating fusions, or whose outcome could not be predicted because they were complex, or both. This is not surprising because NRG1 can be pro-apoptotic and is inactivated in some breast cancers. CONCLUSIONS: Our results highlight the complexity of rearrangements of NRG1 in breast cancers and confirm that some do not activate but inactivate. Careful interpretation of NRG1 rearrangements will therefore be necessary for appropriate patient management.

Defining the clinical genomic landscape for real-world precision oncology.

Through the delivery of large international projects including ICGC and TCGA, knowledge of cancer genomics is reaching saturation point. Enabling this to improve patient outcomes now requires embedding comprehensive genomic profiling into routine oncology practice. Towards this goal, this study defined the biologically and clinically relevant genomic features of adult cancer through detailed curation and analysis of large genomic datasets, accumulated literature and biomarker-driven therapeutics in clinic and development. The characteristics and prevalence of these features were then interrogated in 2348 whole genome sequences, covering 21 solid tumour types, generated by the PCAWG project. This analysis highlights the predominant contribution of copy number alterations and identifies a critical role for disruptive structural variants in the inactivation of clinically important tumour suppressor genes, including PTEN and RB1, which are not currently captured by diagnostic assays. This study defines a set of essential genomic features for the characterisation of common adult cancers.

C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency.

Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage-fusion-bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling "chromoanasynthesis," a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease.

Spatial and temporal heterogeneity in high-grade serous ovarian cancer: a phylogenetic analysis.

BACKGROUND: The major clinical challenge in the treatment of high-grade serous ovarian cancer (HGSOC) is the development of progressive resistance to platinum-based chemotherapy. The objective of this study was to determine whether intra-tumour genetic heterogeneity resulting from clonal evolution and the emergence of subclonal tumour populations in HGSOC was associated with the development of resistant disease. METHODS AND FINDINGS: Evolutionary inference and phylogenetic quantification of heterogeneity was performed using the MEDICC algorithm on high-resolution whole genome copy number profiles and selected genome-wide sequencing of 135 spatially and temporally separated samples from 14 patients with HGSOC who received platinum-based chemotherapy. Samples were obtained from the clinical CTCR-OV03/04 studies, and patients were enrolled between 20 July 2007 and 22 October 2009. Median follow-up of the cohort was 31 mo (interquartile range 22-46 mo), censored after 26 October 2013. Outcome measures were overall survival (OS) and progression-free survival (PFS). There were marked differences in the degree of clonal expansion (CE) between patients (median 0.74, interquartile range 0.66-1.15), and dichotimization by median CE showed worse survival in CE-high cases (PFS 12.7 versus 10.1 mo, p = 0.009; OS 42.6 versus 23.5 mo, p = 0.003). Bootstrap analysis with resampling showed that the 95% confidence intervals for the hazard ratios for PFS and OS in the CE-high group were greater than 1.0. These data support a relationship between heterogeneity and survival but do not precisely determine its effect size. Relapsed tissue was available for two patients in the CE-high group, and phylogenetic analysis showed that the prevalent clonal population at clinical recurrence arose from early divergence events. A subclonal population marked by a NF1 deletion showed a progressive increase in tumour allele fraction during chemotherapy. CONCLUSIONS: This study demonstrates that quantitative measures of intra-tumour heterogeneity may have predictive value for survival after chemotherapy treatment in HGSOC. Subclonal tumour populations are present in pre-treatment biopsies in HGSOC and can undergo expansion during chemotherapy, causing clinical relapse.

Single-cell paired-end genome sequencing reveals structural variation per cell cycle.

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis.

The role of tandem duplicator phenotype in tumour evolution in high-grade serous ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC) is characterized by genomic instability, ubiquitous TP53 loss, and frequent development of platinum resistance. Loss of homologous recombination (HR) is a mutator phenotype present in 50% of HGSOCs and confers hypersensitivity to platinum treatment. We asked which other mutator phenotypes are present in HGSOC and how they drive the emergence of platinum resistance. We performed whole-genome paired-end sequencing on a model of two HGSOC cases, each consisting of a pair of cell lines established before and after clinical resistance emerged, to describe their structural variants (SVs) and to infer their ancestral genomes as the SVs present within each pair. The first case (PEO1/PEO4), with HR deficiency, acquired translocations and small deletions through its early evolution, but a revertant BRCA2 mutation restoring HR function in the resistant lineage re-stabilized its genome and reduced platinum sensitivity. The second case (PEO14/PEO23) had 216 tandem duplications and did not show evidence of HR or mismatch repair deficiency. By comparing the cell lines to the tissues from which they originated, we showed that the tandem duplicator mutator phenotype arose early in progression in vivo and persisted throughout evolution in vivo and in vitro, which may have enabled continual evolution. From the analysis of SNP array data from 454 HGSOC cases in The Cancer Genome Atlas series, we estimate that 12.8% of cases show patterns of aberrations similar to the tandem duplicator, and this phenotype is mutually exclusive with BRCA1/2 carrier mutations.

Tandem duplication of chromosomal segments is common in ovarian and breast cancer genomes.

The application of paired-end next generation sequencing approaches has made it possible to systematically characterize rearrangements of the cancer genome to base-pair level. Utilizing this approach, we report the first detailed analysis of ovarian cancer rearrangements, comparing high-grade serous and clear cell cancers, and these histotypes with other solid cancers. Somatic rearrangements were systematically characterized in eight high-grade serous and five clear cell ovarian cancer genomes and we report here the identification of > 600 somatic rearrangements. Recurrent rearrangements of the transcriptional regulator gene, TSHZ3, were found in three of eight serous cases. Comparison to breast, pancreatic and prostate cancer genomes revealed that a subset of ovarian cancers share a marked tandem duplication phenotype with triple-negative breast cancers. The tandem duplication phenotype was not linked to BRCA1/2 mutation, suggesting that other common mechanisms or carcinogenic exposures are operative. High-grade serous cancers arising in women with germline BRCA1 or BRCA2 mutation showed a high frequency of small chromosomal deletions. These findings indicate that BRCA1/2 germline mutation may contribute to widespread structural change and that other undefined mechanism(s), which are potentially shared with triple-negative breast cancer, promote tandem chromosomal duplications that sculpt the ovarian cancer genome.

Clinically significant copy number alterations and complex rearrangements of MYB and NFIB in head and neck adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) of the head and neck is a malignant tumor with poor long-term prognosis. Besides the recently identified MYB-NFIB fusion oncogene generated by a t(6;9) translocation, little is known about other genetic alterations in ACC. Using high-resolution, array-based comparative genomic hybridization, and massively paired-end sequencing, we explored genomic alterations in 40 frozen ACCs. Eighty-six percent of the tumors expressed MYB-NFIB fusion transcripts and 97% overexpressed MYB mRNA, indicating that MYB activation is a hallmark of ACC. Thirty-five recurrent copy number alterations (CNAs) were detected, including losses involving 12q, 6q, 9p, 11q, 14q, 1p, and 5q and gains involving 1q, 9p, and 22q. Grade III tumors had on average a significantly higher number of CNAs/tumor compared to Grade I and II tumors (P = 0.007). Losses of 1p, 6q, and 15q were associated with high-grade tumors, whereas losses of 14q were exclusively seen in Grade I tumors. The t(6;9) rearrangements were associated with a complex pattern of breakpoints, deletions, insertions, inversions, and for 9p also gains. Analyses of fusion-negative ACCs using high-resolution arrays and massively paired-end sequencing revealed that MYB may also be deregulated by other mechanisms in addition to gene fusion. Our studies also identified several down-regulated candidate tumor suppressor genes (CTNNBIP1, CASP9, PRDM2, and SFN) in 1p36.33-p35.3 that may be of clinical significance in high-grade tumors. Further, studies of these and other potential target genes may lead to the identification of novel driver genes in ACC.

Structural analysis of the genome of breast cancer cell line ZR-75-30 identifies twelve expressed fusion genes.

BACKGROUND: It has recently emerged that common epithelial cancers such as breast cancers have fusion genes like those in leukaemias. In a representative breast cancer cell line, ZR-75-30, we searched for fusion genes, by analysing genome rearrangements. RESULTS: We first analysed rearrangements of the ZR-75-30 genome, to around 10kb resolution, by molecular cytogenetic approaches, combining array painting and array CGH. We then compared this map with genomic junctions determined by paired-end sequencing. Most of the breakpoints found by array painting and array CGH were identified in the paired end sequencing-55% of the unamplified breakpoints and 97% of the amplified breakpoints (as these are represented by more sequence reads). From this analysis we identified 9 expressed fusion genes: APPBP2-PHF20L1, BCAS3-HOXB9, COL14A1-SKAP1, TAOK1-PCGF2, TIAM1-NRIP1, TIMM23-ARHGAP32, TRPS1-LASP1, USP32-CCDC49 and ZMYM4-OPRD1. We also determined the genomic junctions of a further three expressed fusion genes that had been described by others, BCAS3-ERBB2, DDX5-DEPDC6/DEPTOR and PLEC1-ENPP2. Of this total of 12 expressed fusion genes, 9 were in the coamplification. Due to the sensitivity of the technologies used, we estimate these 12 fusion genes to be around two-thirds of the true total. Many of the fusions seem likely to be driver mutations. For example, PHF20L1, BCAS3, TAOK1, PCGF2, and TRPS1 are fused in other breast cancers. HOXB9 and PHF20L1 are members of gene families that are fused in other neoplasms. Several of the other genes are relevant to cancer-in addition to ERBB2, SKAP1 is an adaptor for Src, DEPTOR regulates the mTOR pathway and NRIP1 is an estrogen-receptor coregulator. CONCLUSIONS: This is the first structural analysis of a breast cancer genome that combines classical molecular cytogenetic approaches with sequencing. Paired-end sequencing was able to detect almost all breakpoints, where there was adequate read depth. It supports the view that gene breakage and gene fusion are important classes of mutation in breast cancer, with a typical breast cancer expressing many fusion genes.

Evolution of platinum resistance in high-grade serous ovarian cancer.

High-grade serous ovarian cancers account for most ovarian-cancer mortality. Although this disease initially responds well to platinum-based chemotherapy, relapse and progression to chemotherapy resistance are frequently seen. Time to relapse after first-line therapy is a predictor of response to secondary platinum treatment: more than 12 months is associated with high chance of a secondary response, whereas relapses within 6 months generally indicate platinum resistance. In this Personal View we discuss whether patterns of response, relapse, and the development of drug resistance in high-grade serous ovarian cancers are related to distinct underlying molecular and cellular biological characteristics. In particular, we propose that rapid relapse with platinum-resistant disease is due to minor subpopulations of intrinsically resistant cancer cells at presentation.

Molecular subclusters of follicular lymphoma: a report from the United Kingdom's Haematological Malignancy Research Network.

Follicular lymphoma (FL) is morphologically and clinically diverse, with mutations in epigenetic regulators alongside t(14;18) identified as disease-initiating events. Identification of additional mutational entities confirms this cancer's heterogeneity, but whether mutational data can be resolved into mechanistically distinct subsets remains an open question. Targeted sequencing was applied to an unselected population-based FL cohort (n = 548) with full clinical follow-up (n = 538), which included 96 diffuse large B-cell lymphoma (DLBCL) transformations. We investigated whether molecular subclusters of FL can be identified and whether mutational data provide predictive information relating to transformation. DNA extracted from FL samples was sequenced with a 293-gene panel representing genes frequently mutated in DLBCL and FL. Three clusters were resolved using mutational data alone, independent of translocation status: FL_aSHM, with high burden of aberrant somatic hypermutation (aSHM) targets; FL_STAT6, with high STAT6 & CREBBP mutation and low aSHM; and FL_Com, with the absence of features of other subtypes and enriched KMT2D mutation. Analysis of mutation signatures demonstrated differential enrichment of predicted mutation signatures between subgroups and a dominant preference in the FL_aSHM subgroup for G(C>T)T and G(C>T)C transitions consistent with previously defined aSHM-like patterns. Of transformed cases with paired samples, 17 of 26 had evidence of branching evolution. Poorer overall survival (OS) in the aSHM group (P = .04) was associated with older age; however, overall tumor genetics provided limited information to predict individual patient risk. Our approach identifies 3 molecular subclusters of FL linked to differences in underlying mechanistic pathways. These clusters, which may be further resolved by the inclusion of translocation status and wider mutation profiles, have implications for understanding pathogenesis as well as improving treatment strategies in the future.

Epithelial-to-Mesenchymal Transition Supports Ovarian Carcinosarcoma Tumorigenesis and Confers Sensitivity to Microtubule Targeting with Eribulin.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. SIGNIFICANCE: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Genomic characterization of hepatoid tumors: context matters.

Hepatoid tumors (HT) are rare neoplasms morphologically resembling hepatocellular carcinoma, which arise in several organs other than the liver. A comprehensive molecular profile of this group of neoplasms is still lacking. Genomic characterization of 19 HTs from different organs (three colon HTs, four esophagogastric HTs, four biliary HTs, six genitourinary HTs, two lung HTs) was performed using a multigene next-generation sequencing panel. NGS unraveled a composite molecular profile of HT. Their genetic alterations were clearly clustered by tumor site: (i) colorectal HT displayed microsatellite instability, high tumor mutational burden, mutations in ARID1A/B genes and NCOA4-RET gene fusion (2/3 cases); (ii) gastric HT had TP53 mutations (2/4); (iii) biliary HT displayed loss of CDKN2A (3/4) and loss of chromosome 18 (2/4); (iv) genital HT showed gain of chromosome 12 (3/6); (v) lung HT had STK11 somatic mutations (2/2). The only commonly mutated gene occurring in HT of different sites was TP53 (8/19 cases: colon 2, esophagogastric 2, biliary 2, genital 1, lungs 1). This study shows that most genetic alterations of HT were clustered by site, indicating that context matters. The novel potential targets for HT precision oncology are also clustered based on the anatomic origin. This study shed light on the biology of these rare cancers and may have important consequences for treatment decisions and clinical trial selection for HT patients.

Genetic modification of primary human B cells to model high-grade lymphoma.

Sequencing studies of diffuse large B cell lymphoma (DLBCL) have identified hundreds of recurrently altered genes. However, it remains largely unknown whether and how these mutations may contribute to lymphomagenesis, either individually or in combination. Existing strategies to address this problem predominantly utilize cell lines, which are limited by their initial characteristics and subsequent adaptions to prolonged in vitro culture. Here, we describe a co-culture system that enables the ex vivo expansion and viral transduction of primary human germinal center B cells. Incorporation of CRISPR/Cas9 technology enables high-throughput functional interrogation of genes recurrently mutated in DLBCL. Using a backbone of BCL2 with either BCL6 or MYC, we identify co-operating genetic alterations that promote growth or even full transformation into synthetically engineered DLBCL models. The resulting tumors can be expanded and sequentially transplanted in vivo, providing a scalable platform to test putative cancer genes and to create mutation-directed, bespoke lymphoma models.

High-resolution array CGH clarifies events occurring on 8p in carcinogenesis.

BACKGROUND: Rearrangement of the short arm of chromosome 8 (8p) is very common in epithelial cancers such as breast cancer. Usually there is an unbalanced translocation breakpoint in 8p12 (29.7 Mb - 38.5 Mb) with loss of distal 8p, sometimes with proximal amplification of 8p11-12. Rearrangements in 8p11-12 have been investigated using high-resolution array CGH, but the first 30 Mb of 8p are less well characterised, although this region contains several proposed tumour suppressor genes. METHODS: We analysed the whole of 8p by array CGH at tiling-path BAC resolution in 32 breast and six pancreatic cancer cell lines. Regions of recurrent rearrangement distal to 8p12 were further characterised, using regional fosmid arrays. FISH, and quantitative RT-PCR on over 60 breast tumours validated the existence of similar events in primary material. RESULTS: We confirmed that 8p is usually lost up to at least 30 Mb, but a few lines showed focal loss or copy number steps within this region. Three regions showed rearrangements common to at least two cases: two regions of recurrent loss and one region of amplification. Loss within 8p23.3 (0 Mb - 2.2 Mb) was found in six cell lines. Of the genes always affected, ARHGEF10 showed a point mutation of the remaining normal copies in the DU4475 cell line. Deletions within 12.7 Mb - 19.1 Mb in 8p22, in two cases, affected TUSC3. A novel amplicon was found within 8p21.3 (19.1 Mb - 23.4 Mb) in two lines and one of 98 tumours. CONCLUSION: The pattern of rearrangements seen on 8p may be a consequence of the high density of potential targets on this chromosome arm, and ARHGEF10 may be a new candidate tumour suppressor gene.