Anders Valind

Intratumoral genome diversity parallels progression and predicts outcome in pediatric cancer

Genetic differences among neoplastic cells within the same tumour have been proposed to drive cancer progression and treatment failure. Whether data on intratumoral diversity can be used to predict clinical outcome remains unclear. We here address this issue by quantifying genetic intratumoral diversity in a set of chemotherapy-treated childhood tumours. By analysis of multiple tumour samples from seven patients we demonstrate intratumoral diversity in all patients analysed after chemotherapy, typically presenting as multiple clones within a single millimetre-sized tumour sample (microdiversity). We show that microdiversity often acts as the foundation for further genome evolution in metastases. In addition, we find that microdiversity predicts poor cancer-specific survival (60%; P=0.009), independent of other risk factors, in a cohort of 44 patients with chemotherapy-treated childhood kidney cancer. Survival was 100% for patients lacking microdiversity. Thus, intratumoral genetic diversity is common in childhood cancers after chemotherapy and may be an important factor behind treatment failure.

Whole chromosome gain does not in itself confer cancer-like chromosomal instability

Significance Aneuploidy, denoting cells with an abnormal number of chromosomes, is a common phenomenon in cancer. Another common finding in cancer cells is chromosomal instability, a condition in which cells change their chromosomal content at a high rate. It is clear that chromosomal instability can lead to aneuploidy, but whether the opposite is true has been much debated in the field of cancer biology. The concept that aneuploidy automatically triggers chromosomal instability has been propagated in the scientific literature in recent years. Here, we show that aneuploidy does not, on its own, lead to chromosomal instability, even when cells acquire chromosome alterations typical of cancer. This has important implications for understanding the role of aneuploidy in cancer development. Constitutional aneuploidy is typically caused by a single-event meiotic or early mitotic error. In contrast, somatic aneuploidy, found mainly in neoplastic tissue, is attributed to continuous chromosomal instability. More debated as a cause of aneuploidy is aneuploidy itself; that is, whether aneuploidy per se causes chromosomal instability, for example, in patients with inborn aneuploidy. We have addressed this issue by quantifying the level of somatic mosaicism, a proxy marker of chromosomal instability, in patients with constitutional aneuploidy by precise background-filtered dual-color FISH. In contrast to previous studies that used less precise methods, we find that constitutional trisomy, even for large chromosomes that are often trisomic in cancer, does not confer a significantly elevated rate of somatic chromosomal mosaicism in individual cases. Constitutional triploidy was associated with an increased level of somatic mosaicism, but this consisted mostly of reversion from trisomy to disomy and did not correspond to a proportionally elevated level of chromosome mis-segregation in triploids, indicating that the observed mosaicism resulted from a specific accumulation of cells with a hypotriploid chromosome number. In no case did the rate of somatic mosaicism in constitutional aneuploidy exceed that of “chromosomally stable” cancer cells. Our findings show that even though constitutional aneuploidy was in some cases associated with low-level somatic mosaicism, it was insufficient to generate the cancer-like levels expected if aneuploidy single-handedly triggered cancer-like chromosomal instability.

BCOR internal tandem duplication and YWHAE–NUTM2B/E fusion are mutually exclusive events in clear cell sarcoma of the kidney

Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal tumor. Two recurrent genetic aberrations have been described in CCSK. One is a fusion of YWHAE and NUTM2B/E, the other is an internal tandem duplication (ITD) in the BCOR gene. Here it is shown that YWHAE–NUTM2B/E fusion and the BCOR ITD are mutually exclusive events and activated different downstream signaling systems. This has important diagnostic implications and opens up for further mechanistic studies of CCSK pathogenesis.

Activation of human telomerase reverse transcriptase through gene fusion in clear cell sarcoma of the kidney.

Clear cell sarcoma of the kidney (CCSK) is a rare tumor type affecting infants and young children. Most CCSKs display few genomic aberrations, and no general underlying mechanism for tumor initiation has yet been identified, although a YWHAE-NUTM2B/NUTM2E fusion gene has been observed in a minority of cases. We performed RNA-sequencing of 22 CCSKs to investigate the presence of additional fusion transcripts. The presence of the YWHAE-NUTM2B/NUTM2E fusion was confirmed in two cases. In addition, a novel IRX2-TERT fusion transcript was identified in one case. SNP-array analyses revealed the underlying event to be an interstitial deletion in the short arm of chromosome 5 (5p15.33). TERT was dramatically upregulated under the influence of the IRX2 promoter. In line with TERT expression being driven by active IRX2 regulatory elements, we found a high expression of IRX2 in CCSKs irrespective of fusion gene status. IRX2 was also expressed in human fetal kidney - the presumed tissue of origin for CCSK. We conclude that in addition to promoter mutations and epigenetic events, TERT can also be activated in tumors via formation of fusion transcripts.

Elevated Tolerance to Aneuploidy in Cancer Cells: Estimating the Fitness Effects of Chromosome Number Alterations by In Silico Modelling of Somatic Genome Evolution

An unbalanced chromosome number (aneuploidy) is present in most malignant tumours and has been attributed to mitotic mis-segregation of chromosomes. However, recent studies have shown a relatively high rate of chromosomal mis-segregation also in non-neoplastic human cells, while the frequency of aneuploid cells remains low throughout life in most normal tissues. This implies that newly formed aneuploid cells are subject to negative selection in healthy tissues and that attenuation of this selection could contribute to aneuploidy in cancer. To test this, we modelled cellular growth as discrete time branching processes, during which chromosome gains and losses were generated and their host cells subjected to selection pressures of various magnitudes. We then assessed experimentally the frequency of chromosomal mis-segregation as well as the prevalence of aneuploid cells in human non-neoplastic cells and in cancer cells. Integrating these data into our models allowed estimation of the fitness reduction resulting from a single chromosome copy number change to an average of ≈30% in normal cells. In comparison, cancer cells showed an average fitness reduction of only 6% (p = 0.0008), indicative of aneuploidy tolerance. Simulations based on the combined presence of chromosomal mis-segregation and aneuploidy tolerance reproduced distributions of chromosome aberrations in >400 cancer cases with higher fidelity than models based on chromosomal mis-segregation alone. Reverse engineering of aneuploid cancer cell development in silico predicted that aneuploidy intolerance is a stronger limiting factor for clonal expansion of aneuploid cells than chromosomal mis-segregation rate. In conclusion, our findings indicate that not only an elevated chromosomal mis-segregation rate, but also a generalised tolerance to novel chromosomal imbalances contribute to the genomic landscape of human tumours.

Reply to Duesberg: Stability of peritriploid and triploid states in neoplastic and nonneoplastic cells

We thank P. H. Duesberg for his interesting comments (1) on our recently published report that aneuploidy in itself does not cause chromosomal instability (2). The topic of aneuploidy in cancer cells has been surprisingly little debated publicly, despite the fact that it is a prevalent phenomenon that remains enigmatic from many viewpoints. We welcome this opportunity to discuss our data further, with a particular focus on chromosomal instability at triploid and near-triploid levels.

Reply to Heng: Inborn aneuploidy and chromosomal instability

It is with great interest that we read the letter by Heng (1), who kindly brought up the interesting topic of nonclonal-chromosome aberrations (NCCAs) in the context of our recent report on the relationship between inborn aneuploidy and chromosome instability (CIN). We fully agree with Heng that NCCAs are better proxies for CIN than clonal-chromosome aberrations (CCAs). In fact, we used the frequency of NCCA as proxy to CIN in our study (2). We also agree that it might well be that aneuploidy causes other forms of genomic instability, but our focus lay on whether aneuploidy per se is a cause of CIN. Measuring instability at the level of single nuclear variants or small segmental changes in single cells is still very much a technical challenge. As pointed out in our report, as well as by Heng in his letter (1), our study does not exclude that certain combinations of CCA may trigger further NCCAs/CIN in specific biological contexts. It is still possible that some trisomies or aneuploidies have a more profound effect on the CIN-phenotype than others. However, this would not be in line with recent work showing that there seems to be a rather uniform cellular response to whole chromosome gains (3). Furthermore, our report included cells from patients with constitutional trisomy 8, a type of aneuploidy common in hematological as well as solid neoplasms. We also included cells with multiple trisomies. Neither trisomy 8 nor such double trisomies are compatible with live birth.

Confined trisomy 8 mosaicism of meiotic origin: A rare cause of aneuploidy in childhood cancer

Whether chromosome abnormalities observed in tumor cells may in some cases reflect low‐grade somatic mosaicism for anomalies present already at zygote formation, rather than acquired somatic mutations, has for long remained a speculation. We here report a patient with Wilms tumor, where constitutional somatic mosaicism of trisomy 8 was detected in a previously healthy 2 ½‐year‐old boy. Single Nucleotide Polymorphism (SNP) array analysis of tumor tissue revealed a complex distribution of allele frequencies for chromosome 8 that could not be explained solely by mitotic events. Combined analysis of allele frequencies, chromosome banding, and fluorescence in situ hybridization revealed that the majority of tumor cells contained four copies of chromosome 8, with three distinct haplotypes at a 2:1:1 ratio. Because the patient had not been subject to organ transplantation, these findings indicated that the tumor karyotype evolved from a cell with trisomy 8 of meiotic origin, with subsequent somatic gain of one additional chromosome copy. Haplotype analysis was consistent with trisomy 8 through nondisjunction at meiosis I. Matched normal renal tissue or peripheral blood did not contain detectable amounts of cells with trisomy 8, consistent with the complete lack of mosaic trisomy 8 syndrome features in the patient. This case provides proof of principle for the hypothesis that tumor genotypes may in rare cases reflect meiotic rather than mitotic events, also in patients lacking syndromic features.

Convergent evolution of 11p allelic loss in multifocal Wilms tumors arising in WT1 mutation carriers

Wilms tumors in patients with constitutional WT1 mutations are examples of Knudsons tumor suppressor paradigm, with somatic inactivation of the second allele occurring through 11p loss of heterozygosity. The time point of this second hit has remained unknown. We analyzed seven Wilms tumors from two patients with constitutional WT1 mutations by whole exome sequencing and genomic array. All tumors exhibited wild type WT1 loss through uniparental isodisomy. Each tumor had a unique genomic breakpoint in 11p, typically accompanied by a private activating mutation of CTNNB1. Hence, convergent evolution rather than field carcinogenesis underlies multifocal tumors in WT1 mutation carriers.

Four evolutionary trajectories underlie genetic intratumoral variation in childhood cancer

A major challenge to personalized oncology is that driver mutations vary among cancer cells inhabiting the same tumor. Whether this reflects principally disparate patterns of Darwinian evolution in different tumor regions has remained unexplored1–5. We mapped the prevalence of genetically distinct clones over 250 regions in 54 childhood cancers. This showed that primary tumors can simultaneously follow up to four evolutionary trajectories over different anatomic areas. The most common pattern consists of subclones with very few mutations confined to a single tumor region. The second most common is a stable coexistence, over vast areas, of clones characterized by changes in chromosome numbers. This is contrasted by a third, less frequent, pattern where a clone with driver mutations or structural chromosome rearrangements emerges through a clonal sweep to dominate an anatomical region. The fourth and rarest pattern is the local emergence of a myriad of clones with TP53 inactivation. Death from disease was limited to tumors exhibiting the two last, most dynamic patterns.Multiregional analysis in 54 childhood cancers highlights four evolutionary patterns of intratumoral variation. Multiple patterns are often found in the same tumor, suggesting that tumors follow different evolutionary strategies concurrently.