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1.  Histological Transformation and Progression in Follicular Lymphoma: A Clonal Evolution Study 
PLoS Medicine  2016;13(12):e1002197.
Follicular lymphoma (FL) is an indolent, yet incurable B cell malignancy. A subset of patients experience an increased mortality rate driven by two distinct clinical end points: histological transformation and early progression after immunochemotherapy. The nature of tumor clonal dynamics leading to these clinical end points is poorly understood, and previously determined genetic alterations do not explain the majority of transformed cases or accurately predict early progressive disease. We contend that detailed knowledge of the expansion patterns of specific cell populations plus their associated mutations would provide insight into therapeutic strategies and disease biology over the time course of FL clinical histories.
Methods and Findings
Using a combination of whole genome sequencing, targeted deep sequencing, and digital droplet PCR on matched diagnostic and relapse specimens, we deciphered the constituent clonal populations in 15 transformation cases and 6 progression cases, and measured the change in clonal population abundance over time. We observed widely divergent patterns of clonal dynamics in transformed cases relative to progressed cases. Transformation specimens were generally composed of clones that were rare or absent in diagnostic specimens, consistent with dramatic clonal expansions that came to dominate the transformation specimens. This pattern was independent of time to transformation and treatment modality. By contrast, early progression specimens were composed of clones that were already present in the diagnostic specimens and exhibited only moderate clonal dynamics, even in the presence of immunochemotherapy. Analysis of somatic mutations impacting 94 genes was undertaken in an extension cohort consisting of 395 samples from 277 patients in order to decipher disrupted biology in the two clinical end points. We found 12 genes that were more commonly mutated in transformed samples than in the preceding FL tumors, including TP53, B2M, CCND3, GNA13, S1PR2, and P2RY8. Moreover, ten genes were more commonly mutated in diagnostic specimens of patients with early progression, including TP53, BTG1, MKI67, and XBP1.
Our results illuminate contrasting modes of evolution shaping the clinical histories of transformation and progression. They have implications for interpretation of evolutionary dynamics in the context of treatment-induced selective pressures, and indicate that transformation and progression will require different clinical management strategies.
Sohrab Shah and colleagues explore the evolutionary histories that shape clinical and transformation dynamics in follicular lymphoma
Author Summary
Why Was This Study Done?
Follicular lymphoma (FL) is a largely incurable malignancy in which early progression and transformation have consistently been linked to lymphoma-related mortality.
We contended that detailed characterization of clonal dynamics would reveal fundamental biological properties with implications for future patient management strategies relating to both transformation and progression.
We also sought to identify recurrent gene mutations associated with transformation and/or early progression in a large patient cohort.
What Did the Researchers Do and Find?
Using whole genome sequencing, deep allelic sampling by amplicon sequencing, and digital droplet PCR, we found dramatic clonal expansions in transformed disease, whereby dominant clones in transformation samples emerged from extremely low prevalence clones or from clones that were not detected in the diagnostic samples.
The dynamics of disease progression during treatment in the absence of transformation showed markedly different characteristics, with much of the clonal architecture preserved from diagnostic to relapse specimens.
Targeted capture-based sequencing in a large extension cohort then established genetic variants associated with transformation and early progression in the broader patient population.
What Do These Findings Mean?
Taken together, our findings illuminate previously undescribed patterns of clonal expansion underpinning FL clinical histories suggesting that contrasting management strategies will be necessary across the FL patient population.
We uncovered novel associations of gene mutations with early progression that could inform future prognostic assay development.
PMCID: PMC5154502  PMID: 27959929
2.  Divergent clonal selection dominates medulloblastoma at recurrence 
Morrissy, A. Sorana | Garzia, Livia | Shih, David J. H. | Zuyderduyn, Scott | Huang, Xi | Skowron, Patryk | Remke, Marc | Cavalli, Florence M. G. | Ramaswamy, Vijay | Lindsay, Patricia E. | Jelveh, Salomeh | Donovan, Laura K. | Wang, Xin | Luu, Betty | Zayne, Kory | Li, Yisu | Mayoh, Chelsea | Thiessen, Nina | Mercier, Eloi | Mungall, Karen L. | Ma, Yusanne | Tse, Kane | Zeng, Thomas | Shumansky, Karey | Roth, Andrew J. L. | Shah, Sohrab | Farooq, Hamza | Kijima, Noriyuki | Holgado, Borja L. | Lee, John J. Y. | Matan-Lithwick, Stuart | Liu, Jessica | Mack, Stephen C. | Manno, Alex | Michealraj, K. A. | Nor, Carolina | Peacock, John | Qin, Lei | Reimand, Juri | Rolider, Adi | Thompson, Yuan Y. | Wu, Xiaochong | Pugh, Trevor | Ally, Adrian | Bilenky, Mikhail | Butterfield, Yaron S. N. | Carlsen, Rebecca | Cheng, Young | Chuah, Eric | Corbett, Richard D. | Dhalla, Noreen | He, An | Lee, Darlene | Li, Haiyan I. | Long, William | Mayo, Michael | Plettner, Patrick | Qian, Jenny Q. | Schein, Jacqueline E. | Tam, Angela | Wong, Tina | Birol, Inanc | Zhao, Yongjun | Faria, Claudia C. | Pimentel, José | Nunes, Sofia | Shalaby, Tarek | Grotzer, Michael | Pollack, Ian F. | Hamilton, Ronald L. | Li, Xiao-Nan | Bendel, Anne E. | Fults, Daniel W. | Walter, Andrew W. | Kumabe, Toshihiro | Tominaga, Teiji | Collins, V. Peter | Cho, Yoon-Jae | Hoffman, Caitlin | Lyden, David | Wisoff, Jeffrey H. | Garvin, James H. | Stearns, Duncan S. | Massimi, Luca | Schüller, Ulrich | Sterba, Jaroslav | Zitterbart, Karel | Puget, Stephanie | Ayrault, Olivier | Dunn, Sandra E. | Tirapelli, Daniela P. C. | Carlotti, Carlos G. | Wheeler, Helen | Hallahan, Andrew R. | Ingram, Wendy | MacDonald, Tobey J. | Olson, Jeffrey J. | Van Meir, Erwin G. | Lee, Ji-Yeoun | Wang, Kyu-Chang | Kim, Seung-Ki | Cho, Byung-Kyu | Pietsch, Torsten | Fleischhack, Gudrun | Tippelt, Stephan | Ra, Young Shin | Bailey, Simon | Lindsey, Janet C. | Clifford, Steven C. | Eberhart, Charles G. | Cooper, Michael K. | Packer, Roger J. | Massimino, Maura | Garre, Maria Luisa | Bartels, Ute | Tabori, Uri | Hawkins, Cynthia E. | Dirks, Peter | Bouffet, Eric | Rutka, James T. | Wechsler-Reya, Robert J. | Weiss, William A. | Collier, Lara S. | Dupuy, Adam J. | Korshunov, Andrey | Jones, David T. W. | Kool, Marcel | Northcott, Paul A. | Pfister, Stefan M. | Largaespada, David A. | Mungall, Andrew J. | Moore, Richard A. | Jabado, Nada | Bader, Gary D. | Jones, Steven J. M. | Malkin, David | Marra, Marco A. | Taylor, Michael D.
Nature  2016;529(7586):351-357.
The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon–driven, functional genomic mouse model of medulloblastoma with ‘humanized’ in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (<5%). Whole-genome sequencing of 33 pairs of human diagnostic and post-therapy medulloblastomas demonstrated substantial genetic divergence of the dominant clone after therapy (<12% diagnostic events were retained at recurrence). In both mice and humans, the dominant clone at recurrence arose through clonal selection of a pre-existing minor clone present at diagnosis. Targeted therapy is unlikely to be effective in the absence of the target, therefore our results offer a simple, proximal, and remediable explanation for the failure of prior clinical trials of targeted therapy.
PMCID: PMC4936195  PMID: 26760213
3.  Dynamics of genomic clones in breast cancer patient xenografts at single cell resolution 
Nature  2014;518(7539):422-426.
Human cancers, including breast cancers, are comprised of clones differing in mutation content. Clones evolve dynamically in space and time following principles of Darwinian evolution1,2, underpinning important emergent features such as drug resistance and metastasis3–7. Human breast cancer xenoengraftment is used as a means of capturing and studying tumour biology, and breast tumour xenografts are generally assumed to be reasonable models of the originating tumours8–10. However the consequences and reproducibility of engraftment and propagation on the genomic clonal architecture of tumours has not been systematically examined at single cell resolution. Here we show by both deep genome and single cell sequencing methods, the clonal dynamics of initial engraftment and subsequent serial propagation of primary and metastatic human breast cancers in immunodeficient mice. In all 15 cases examined, clonal selection on engraftment was observed in both primary and metastatic breast tumours, varying in degree from extreme selective engraftment of minor (<5% of starting population) clones to moderate, polyclonal engraftment. Furthermore, ongoing clonal dynamics during serial passaging is a feature of tumours experiencing modest initial selection. Through single cell sequencing, we show that major mutation clusters estimated from tumour population sequencing relate predictably to the most abundant clonal genotypes, even in clonally complex and rapidly evolving cases. Finally, we show that similar clonal expansion patterns can emerge in independent grafts of the same starting tumour population, indicating that genomic aberrations can be reproducible determinants of evolutionary trajectories. Our results show that measurement of genomically defined clonal population dynamics will be highly informative for functional studies utilizing patient-derived breast cancer xenoengraftment.
PMCID: PMC4864027  PMID: 25470049 CAMSID: cams5567
4.  Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer 
Nature Communications  2015;6:8760.
Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.
Individual tumours are heterogeneous with regards to genetic mutations. In this study, the authors use sequencing to follow multiple tumour and plasma samples over 3 years from a breast cancer patient and show mutations detected in the plasma samples could partially reproduce the clonal nature of the primary tumour.
PMCID: PMC4659935  PMID: 26530965
5.  Associations of interleukin-1 gene cluster polymorphisms with C-reactive protein concentration and lung function decline in smoking-induced chronic obstructive pulmonary disease 
Objective: We reported association of haplotypes formed by IL-1b (IL1B)-511C/T (rs16944) and a variable number of tandem repeats (rs2234663) in intron 3 of IL-1 receptor antagonist (IL1RN) with rate of lung function decline in smoking-induced COPD. The aim of current study was to further investigate this association. Methods: We genotyped an additional 19 polymorphisms in IL1 cluster (including IL1A, IL1B and IL1RN) in non-Hispanic whites who had the fastest (n = 268) and the slowest (n = 292) decline of FEV1% predicted in the same study. We also analyzed the association of all 21 polymorphisms with serum CRP levels. Results: None of 21 polymorphisms showed significant association with rate of decline of lung function or CRP levels after adjusting for multiple comparisons. Before adjusting for multiple comparisons, only IL1RN_19327 (rs315949) showed significant association with lung function decline (P = 0.03, additive model). The frequencies of genotypes containing the IL1RN_19327A allele were 71.9% and 62.2%, respectively in the fast and slow decline groups (P = 0.02, odds ratio = 1.6, 95% confidence interval = 1.1-2.3); the IL1B_5200 (rs1143633) and rs2234663 in IL1RN were associated with serum CRP levels (P=0.04 and 0.03, respectively). Conclusions: No single marker was significantly associated with either rate of lung function decline or serum CRP levels.
PMCID: PMC4680456  PMID: 26722511
Chronic obstructive pulmonary disease (COPD); forced expiratory volume in one second (FEV1); genetic polymorphism; IL1 gene cluster; lung function decline; C-reactive protein
6.  Small cell carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and somatic mutations in SMARCA4 
Nature genetics  2014;46(5):427-429.
Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 69% (9/13) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.
PMCID: PMC4332808  PMID: 24658001
7.  The clonal and mutational evolution spectrum of primary triple negative breast cancers 
Nature  2012;486(7403):10.1038/nature10933.
Primary triple negative breast cancers (TNBC) represent approximately 16% of all breast cancers1 and are a tumour type defined by exclusion, for which comprehensive landscapes of somatic mutation have not been determined. Here we show in 104 early TNBC cases, that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some exhibiting only a handful of somatic aberrations in a few pathways, whereas others contain hundreds of somatic events and multiple pathways implicated. Integration with matched whole transcriptome sequence data revealed that only ~36% of mutations are expressed. By examining single nucleotide variant (SNV) allelic abundance derived from deep re-sequencing (median >20,000 fold) measurements in 2414 somatic mutations, we determine for the first time in an epithelial tumour, the relative abundance of clonal genotypes among cases in the population. We show that TNBC vary widely and continuously in their clonal frequencies at the time of diagnosis, with basal subtype TNBC2,3 exhibiting more variation than non-basal TNBC. Although p53 and PIK3CA/PTEN somatic mutations appear clonally dominant compared with other pathways, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal and cell shape/motility proteins occurred at lower clonal frequencies, suggesting they occurred later during tumour progression. Taken together our results show that future attempts to dissect the biology and therapeutic responses of TNBC will require the determination of individual tumour clonal genotypes.
PMCID: PMC3863681  PMID: 22495314
9.  Correction: Type-Specific Cell Line Models for Type-Specific Ovarian Cancer Research 
PLoS ONE  2013;8(9):10.1371/annotation/ffcaf179-872f-470b-8bb6-f06d8ba6d03a.
PMCID: PMC3792146  PMID: 24116245
10.  Type-Specific Cell Line Models for Type-Specific Ovarian Cancer Research 
PLoS ONE  2013;8(9):e72162.
Ovarian carcinomas consist of at least five distinct diseases: high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous. Biomarker and molecular characterization may represent a more biologically relevant basis for grouping and treating this family of tumors, rather than site of origin. Molecular characteristics have become the new standard for clinical pathology, however development of tailored type-specific therapies is hampered by a failure of basic research to recognize that model systems used to study these diseases must also be stratified. Unrelated model systems do offer value for study of biochemical processes but specific cellular context needs to be applied to assess relevant therapeutic strategies.
We have focused on the identification of clear cell carcinoma cell line models. A panel of 32 “ovarian cancer” cell lines has been classified into histotypes using a combination of mutation profiles, IHC mutation-surrogates, and a validated immunohistochemical model. All cell lines were identity verified using STR analysis.
Many described ovarian clear cell lines have characteristic mutations (including ARID1A and PIK3CA) and an overall molecular/immuno-profile typical of primary tumors. Mutations in TP53 were present in the majority of high-grade serous cell lines. Advanced genomic analysis of bona-fide clear cell carcinoma cell lines also support copy number changes in typical biomarkers such at MET and HNF1B and a lack of any recurrent expressed re-arrangements.
Conclusions: As with primary ovarian tumors, mutation status of cancer genes like ARID1A and TP53 and a general immuno-profile serve well for establishing histotype of ovarian cancer cell We describe specific biomarkers and molecular features to re-classify generic “ovarian carcinoma” cell lines into type specific categories. Our data supports the use of prototype clear cell lines, such as TOV21G and JHOC-5, and questions the use of SKOV3 and A2780 as models of high-grade serous carcinoma.
PMCID: PMC3762837  PMID: 24023729
11.  Effect of heme oxygenase-1 polymorphisms on lung function and gene expression 
BMC Medical Genetics  2011;12:117.
Oxidative stress induced by smoking is considered to be important in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Heme oxygenase-1 (HMOX1) is an essential enzyme in heme catabolism that is induced by oxidative stress and may play a protective role as an antioxidant in the lung. We determined whether HMOX1 polymorphisms were associated with lung function in COPD patients and whether the variants had functional effects.
We genotyped five single nucleotide polymorphisms (SNPs) in the HMOX1 gene in Caucasians who had the fastest (n = 278) and the slowest (n = 304) decline of FEV1 % predicted, selected from smokers in the NHLBI Lung Health Study. These SNPs were also studied in Caucasians with the lowest (n = 535) or the highest (n = 533) baseline lung function. Reporter genes were constructed containing three HMOX1 promoter polymorphisms and the effect of these polymorphisms on H2O2 and hemin-stimulated gene expression was determined. The effect of the HMOX1 rs2071749 SNP on gene expression in alveolar macrophages was investigated.
We found a nominal association (p = 0.015) between one intronic HMOX1 SNP (rs2071749) and lung function decline but this did not survive correction for multiple comparisons. This SNP was in perfect linkage disequilibrium with rs3761439, located in the promoter of HMOX1. We tested rs3761439 and two other putatively functional polymorphisms (rs2071746 and the (GT)n polymorphism) in reporter gene assays but no significant effects on gene expression were found. There was also no effect of rs2071749 on HMOX1 gene expression in alveolar macrophages.
We found no association of the five HMOX1 tag SNPs with lung function decline and no evidence that the three promoter polymorphisms affected the regulation of the HMOX1 gene.
PMCID: PMC3180266  PMID: 21902835
Heme oxygenase; polymorphism; chronic obstructive pulmonary disease
12.  Associations of IL6 polymorphisms with lung function decline and COPD 
Thorax  2009;64(8):698-704.
Interleukin-6 (IL6) is a pleiotropic pro-inflammatory and immunomodulatory cytokine which likely plays an important role in the pathogenesis of COPD. There is a functional single nucleotide polymorphism (SNP), −174G/C, in the promoter region of IL6. We hypothesized that IL6 SNPs influence susceptibility for impaired lung function and COPD in smokers.
Seven and 5 SNPs in IL6 were genotyped in two nested case-control samples derived from the Lung Health Study (LHS) based on phenotypes of rate of decline of forced expiratory volume in one second (FEV1) over 5 years and baseline FEV1 at the beginning of the LHS. Serum IL6 concentrations were measured for all subjects. A partially overlapping panel of 9 IL6 SNPs was genotyped in 389 COPD cases from the National Emphysema Treatment Trial (NETT) and 420 controls from the Normative Aging Study (NAS).
In the LHS, three IL6 SNPs were associated with FEV1 decline (0.023 ≤ P ≤ 0.041 in additive models). Among them the IL6_−174C allele was associated with rapid decline of lung function. The association was more significant in a genotype-based analysis (P = 0.006). In the NETT-NAS study, IL6_−174G/C and four other IL6 SNPs, all of which are in linkage disequilibrium with IL6_−174G/C, were associated with susceptibility to COPD (0.01 ≤ P ≤ 0.04 in additive genetic models).
Our results suggest that the IL6_−174G/C SNP is associated with rapid decline of FEV1 and susceptibility to COPD in smokers.
PMCID: PMC2859187  PMID: 19359268
genetic polymorphism; IL6; forced expiratory volume in one second (FEV1); lung function; chronic obstructive pulmonary disease (COPD)
13.  Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling 
The Journal of Pathology  2013;231(1):21-34.
High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment-resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2–91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole-genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug-resistance mechanisms.
PMCID: PMC3864404  PMID: 23780408
high-grade serous ovarian cancer; intratumoural heterogeneity; clonal evolution

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