A survey of chromosomal instability measures across mechanistic models.

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, six-centromere FISH, bulk transcriptomics, and single-cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples significantly correlated (R = 0.72; P < 0.001) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also significantly correlate (R = 0.76; P < 0.001) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, scDNAseq detects CIN with high sensitivity, and significantly correlates with imaging methods (R = 0.82; P < 0.001). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate the comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division. This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles.

Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741.

HPV16 E6 induces chromosomal instability due to polar chromosomes caused by E6AP-dependent degradation of the mitotic kinesin CENP-E.

Chromosome segregation during mitosis is highly regulated to ensure production of genetically identical progeny. Recurrent mitotic errors cause chromosomal instability (CIN), a hallmark of tumors. The E6 and E7 oncoproteins of high-risk human papillomavirus (HPV), which causes cervical, anal, and head and neck cancers (HNC), cause mitotic defects consistent with CIN in models of anogenital cancers, but this has not been studied in the context of HNC. Here, we show that HPV16 induces a specific type of CIN in patient HNC tumors, patient-derived xenografts, and cell lines, which is due to defects in chromosome congression. These defects are specifically induced by the HPV16 oncogene E6 rather than E7. We show that HPV16 E6 expression causes degradation of the mitotic kinesin CENP-E, whose depletion produces chromosomes that are chronically misaligned near spindle poles (polar chromosomes) and fail to congress. Though the canonical oncogenic role of E6 is the degradation of the tumor suppressor p53, CENP-E degradation and polar chromosomes occur independently of p53. Instead, E6 directs CENP-E degradation in a proteasome-dependent manner via the E6-associated ubiquitin protein ligase E6AP/UBE3A. This study reveals a mechanism by which HPV induces CIN, which may impact HPV-mediated tumor initiation, progression, and therapeutic response.

High nuclear TPX2 expression correlates with TP53 mutation and poor clinical behavior in a large breast cancer cohort, but is not an independent predictor of chromosomal instability.

BACKGROUND: Targeting Protein for Xenopus Kinesin Like Protein 2 (TPX2) is a microtubule associated protein that functions in mitotic spindle assembly. TPX2 also localizes to the nucleus where it functions in DNA damage repair during S-phase. We and others have previously shown that TPX2 RNA levels are strongly associated with chromosomal instability (CIN) in breast and other cancers, and TPX2 RNA levels have been demonstrated to correlate with aggressive behavior and poor clinical outcome across a range of solid malignancies, including breast cancer. METHODS: We perform TPX2 IHC on a cohort of 253 primary breast cancers and adopt a clinically amenable scoring system to separate tumors into low, intermediate, or high TPX2 expression. We then correlate TPX2 expression against diverse pathologic parameters and important measures of clinical outcome, including disease-specific and overall survival. We link TPX2 expression to TP53 mutation and evaluate whether TPX2 is an independent predictor of chromosomal instability (CIN). RESULTS: We find that TPX2 nuclear expression strongly correlates with high grade morphology, elevated clinical stage, negative ER and PR status, and both disease-specific and overall survival. We also show that increased TPX2 nuclear expression correlates with elevated ploidy, supernumerary centrosomes, and TP53 mutation. TPX2 nuclear expression correlates with CIN via univariate analyses but is not independently predictive when compared to ploidy, Ki67, TP53 mutational status, centrosome number, and patient age. CONCLUSIONS: Our findings demonstrate a strong correlation between TPX2 nuclear expression and aggressive tumor behavior, and show that TPX2 overexpression frequently occurs in the setting of TP53 mutation and elevated ploidy. However, TPX2 expression is not an independent predictor of CIN where it fails to outperform existing clinical and pathologic metrics.

Chromosomal instability upregulates interferon in acute myeloid leukemia.

Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis-segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis-segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.

IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation.

Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and ß family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin ß family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response.

Centrosome amplification induces high grade features and is prognostic of worse outcomes in breast cancer.

BACKGROUND: Centrosome amplification (CA) has been reported in nearly all types of human cancer and is associated with deleterious clinical factors such as higher grade and stage. However, previous reports have not shown how CA affects cellular differentiation and clinical outcomes in breast cancer. METHODS: We analyzed centrosomes by immunofluorescence and compared to ploidy and chromosomal instability (CIN) as assessed by 6-chromosome FISH in a cohort of 362 breast cancers with median clinical follow-up of 8.4 years. Centrosomes were recognized by immunofluorescence using antibodies for pericentriolar material (PCM; pericentrin) and centrioles (polyglutamylated tubulin). CA was experimentally induced in cell culture by overexpression of polo-like kinase 4 (PLK4). RESULTS: CA is associated with reduced all-cause and breast cancer-specific overall survival and recurrence-free survival. CA correlates strongly with high-risk subtypes (e.g. triple negative) and higher stage and grade, and the prognostic nature of CA can be explained largely by these factors. A strong correlation between CA and high tumor ploidy demonstrates that chromosome and centrosome doubling often occur in concert. CA is proposed to be a method of inducing CIN via aberrant mitotic cell divisions; consonant with this, we observed a strong correlation between CA and CIN in breast cancers. However, some CA tumors had low levels of CIN, indicating that protective mechanisms are at play, such as centrosome clustering during mitosis. Intriguingly, some high-risk tumors have more acentriolar centrosomes, suggesting PCM fragmentation as another mechanism of CA. In vitro induction of CA in two non-transformed human cell lines (MCF10A and RPE) demonstrated that CA induces a de-differentiated cellular state and features of high-grade malignancy, supporting the idea that CA intrinsically causes high-grade tumors. CONCLUSIONS: CA is associated with deleterious clinical factors and outcomes in breast cancer. Cell doubling events are the most prevalent causes of CA in cancer, although PCM fragmentation may be a secondary cause. CA promotes high-risk breast cancer in part by inducing high-grade features. These findings highlight the importance of centrosome aberrations in the biology of human breast cancer.

Chromosome missegregation rate predicts whether aneuploidy will promote or suppress tumors.

Aneuploidy, a chromosome content other than a multiple of the haploid number, is a common feature of cancer cells. Whole chromosomal aneuploidy accompanying ongoing chromosomal instability in mice resulting from reduced levels of the centromere-linked motor protein CENP-E has been reported to increase the incidence of spleen and lung tumors, but to suppress tumors in three other contexts. Exacerbating chromosome missegregation in CENP-E(+/-) mice by reducing levels of another mitotic checkpoint component, Mad2, is now shown to result in elevated cell death and decreased tumor formation compared with reduction of either protein alone. Furthermore, we determine that the additional contexts in which increased whole-chromosome missegregation resulting from reduced CENP-E suppresses tumor formation have a preexisting, elevated basal level of chromosome missegregation that is exacerbated by reduction of CENP-E. Tumors arising from primary causes that do not generate chromosomal instability, including loss of the INK4a tumor suppressor and microsatellite instability from reduction of the DNA mismatch repair protein MLH1, are unaffected by CENP-E-dependent chromosome missegregation. These findings support a model in which low rates of chromosome missegregation can promote tumorigenesis, whereas missegregation of high numbers of chromosomes leads to cell death and tumor suppression.

Interphase cytofission maintains genomic integrity of human cells after failed cytokinesis.

In cell division, cytokinesis is tightly coupled with mitosis to maintain genomic integrity. Failed cytokinesis in humans can result in tetraploid cells that can become aneuploid and promote cancer. However, the likelihood of aneuploidy and cancer after a failed cytokinesis event is unknown. Here we evaluated cell fate after failed cytokinesis. We interrupted cytokinesis by brief chemical treatments in cell populations of human epithelial lines. Surprisingly, up to 50% of the resulting binucleate cells generated colonies. In RPE1 cells, 90% of colonies obtained from binucleate founders had a karyotype that matched the parental cell type. Time-lapse videomicroscopy demonstrated that binucleate cells are delayed in the first growth phase of the cell cycle (G1) and undergo interphase cellular fission (cytofission) that distributes nuclei into separate daughters. The fission is not compatible with delayed cytokinesis because events occur in the absence of polymerized microtubules and without canonical components of the cytokinetic machinery. However, the cytofission can be interrupted by inhibiting function of actin or myosin II. Fission events occur in both two- and three-dimensional culture. Our data demonstrate that cytofission can preserve genomic integrity after failed cytokinesis. Thus, traction-mediated cytofission, originally observed in Dictyostelium, is relevant to human biology--where it seems to be an evolutionarily conserved mechanism that can preserve genomic integrity.

2n or not 2n: Aneuploidy, polyploidy and chromosomal instability in primary and tumor cells.

Mitotic defects leading to aneuploidy have been recognized as a hallmark of tumor cells for over 100 years. Current data indicate that ∼85% of human cancers have missegregated chromosomes to become aneuploid. Some maintain a stable aneuploid karyotype, while others consistently missegregate chromosomes over multiple divisions due to chromosomal instability (CIN). Both aneuploidy and CIN serve as markers of poor prognosis in diverse human cancers. Despite this, aneuploidy is generally incompatible with viability during development, and some aneuploid karyotypes cause a proliferative disadvantage in somatic cells. In vivo, the intentional introduction of aneuploidy can promote tumors, suppress them, or do neither. Here, we summarize current knowledge of the effects of aneuploidy and CIN on proliferation and cell death in nontransformed cells, as well as on tumor promotion, suppression, and prognosis.

Aneuploidy acts both oncogenically and as a tumor suppressor.

An abnormal chromosome number, aneuploidy, is a common characteristic of tumor cells. Boveri proposed nearly 100 years ago that aneuploidy causes tumorigenesis, but this has remained untested due to the difficulty of selectively generating aneuploidy. Cells and mice with reduced levels of the mitosis-specific, centromere-linked motor protein CENP-E are now shown to develop aneuploidy and chromosomal instability in vitro and in vivo. An increased rate of aneuploidy does drive an elevated level of spontaneous lymphomas and lung tumors in aged animals. Remarkably, however, in examples of chemically or genetically induced tumor formation, an increased rate of aneuploidy is a more effective inhibitor than initiator of tumorigenesis. These findings reveal a role of aneuploidy and chromosomal instability in preventing tumorigenesis.

Up-regulation of the mitotic checkpoint component Mad1 causes chromosomal instability and resistance to microtubule poisons.

The mitotic checkpoint is the major cell cycle checkpoint acting during mitosis to prevent aneuploidy and chromosomal instability, which are hallmarks of tumor cells. Reduced expression of the mitotic checkpoint component Mad1 causes aneuploidy and promotes tumors in mice [Iwanaga Y, et al. (2007) Cancer Res 67:160-166]. However, the prevalence and consequences of Mad1 overexpression are currently unclear. Here we show that Mad1 is frequently overexpressed in human cancers and that Mad1 up-regulation is a marker of poor prognosis. Overexpression of Mad1 causes aneuploidy and chromosomal instability through weakening mitotic checkpoint signaling caused by mislocalization of the Mad1 binding partner Mad2. Cells overexpressing Mad1 are resistant to microtubule poisons, including currently used chemotherapeutic agents. These results suggest that levels of Mad1 must be tightly regulated to prevent aneuploidy and transformation and that Mad1 up-regulation may promote tumors and cause resistance to current therapies.

On the road to cancer: aneuploidy and the mitotic checkpoint.

Abnormal chromosome content - also known as aneuploidy - is the most common characteristic of human solid tumours. It has therefore been proposed that aneuploidy contributes to, or even drives, tumour development. The mitotic checkpoint guards against chromosome mis-segregation by delaying cell-cycle progression through mitosis until all chromosomes have successfully made spindle-microtubule attachments. Defects in the mitotic checkpoint generate aneuploidy and might facilitate tumorigenesis, but more severe disabling of checkpoint signalling is a possible anticancer strategy.

CENP-E inhibition induces chromosomal instability and synergizes with diverse microtubule-targeting agents in breast cancer.

Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are only effective in approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers revealed that they only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. While maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles dramatically reduces CIN and confers resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin CENP-E overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to anti-microtubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E.

Does aneuploidy cause cancer?

Aneuploidy has been recognized as a common characteristic of cancer cells for >100 years. Aneuploidy frequently results from errors of the mitotic checkpoint, the major cell cycle control mechanism that acts to prevent chromosome missegregation. The mitotic checkpoint is often compromised in human tumors, although not as a result of germline mutations in genes encoding checkpoint proteins. Less obviously, aneuploidy of whole chromosomes rapidly results from mutations in genes encoding several tumor suppressors and DNA mismatch repair proteins, suggesting cooperation between mechanisms of tumorigenesis that were previously thought to act independently. Cumulatively, the current evidence suggests that aneuploidy promotes tumorigenesis, at least at low frequency, but a definitive test has not yet been reported.

Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death.

Disrupted passage through mitosis often leads to chromosome missegregation and the production of aneuploid progeny. Aneuploidy has long been recognized as a frequent characteristic of cancer cells and a possible cause of tumorigenesis. Drugs that target mitotic spindle assembly are frequently used to treat various types of human tumors. These lead to chronic mitotic arrest from sustained activation of the mitotic checkpoint. Here, we review the linkage between the mitotic checkpoint, aneuploidy, adaptation from mitotic arrest, and antimitotic drug-induced cell death.

A survey of CIN measures across mechanistic models.

Chromosomal instability (CIN) is the persistent reshuffling of cancer karyotypes via chromosome mis-segregation during cell division. In cancer, CIN exists at varying levels that have differential effects on tumor progression. However, mis-segregation rates remain challenging to assess in human cancer despite an array of available measures. We evaluated measures of CIN by comparing quantitative methods using specific, inducible phenotypic CIN models of chromosome bridges, pseudobipolar spindles, multipolar spindles, and polar chromosomes. For each, we measured CIN fixed and timelapse fluorescence microscopy, chromosome spreads, 6-centromere FISH, bulk transcriptomics, and single cell DNA sequencing (scDNAseq). As expected, microscopy of tumor cells in live and fixed samples correlated well (R=0.77; p<0.01) and sensitively detect CIN. Cytogenetics approaches include chromosome spreads and 6-centromere FISH, which also correlate well (R=0.77; p<0.01) but had limited sensitivity for lower rates of CIN. Bulk genomic DNA signatures and bulk transcriptomic scores, CIN70 and HET70, did not detect CIN. By contrast, single-cell DNA sequencing (scDNAseq) detects CIN with high sensitivity, and correlates very well with imaging methods (R=0.83; p<0.01). In summary, single-cell methods such as imaging, cytogenetics, and scDNAseq can measure CIN, with the latter being the most comprehensive method accessible to clinical samples. To facilitate comparison of CIN rates between phenotypes and methods, we propose a standardized unit of CIN: Mis-segregations per Diploid Division (MDD). This systematic analysis of common CIN measures highlights the superiority of single-cell methods and provides guidance for measuring CIN in the clinical setting.

Misaligned Chromosomes are a Major Source of Chromosomal Instability in Breast Cancer.

Chromosomal instability (CIN), the persistent reshuffling of chromosomes during mitosis, is a hallmark of human cancers that contributes to tumor heterogeneity and has been implicated in driving metastasis and altering responses to therapy. Though multiple mechanisms can produce CIN, lagging chromosomes generated from abnormal merotelic attachments are the major cause of CIN in a variety of cell lines, and are expected to predominate in cancer. Here, we quantify CIN in breast cancer using a tumor microarray, matched primary and metastatic samples, and patient-derived organoids from primary breast cancer. Surprisingly, misaligned chromosomes are more common than lagging chromosomes and represent a major source of CIN in primary and metastatic tumors. This feature of breast cancers is conserved in a majority of breast cancer cell lines. Importantly, though a portion of misaligned chromosomes align before anaphase onset, the fraction that remain represents the largest source of CIN in these cells. Metastatic breast cancers exhibit higher rates of CIN than matched primary cancers, primarily due to increases in misaligned chromosomes. Whether CIN causes immune activation or evasion is controversial. We find that misaligned chromosomes result in immune-activating micronuclei substantially less frequently than lagging and bridge chromosomes and that breast cancers with greater frequencies of lagging chromosomes and chromosome bridges recruit more stromal tumor-infiltrating lymphocytes. These data indicate misaligned chromosomes represent a major mechanism of CIN in breast cancer and provide support for differential immunostimulatory effects of specific types of CIN. Significance: We surveyed the single-cell landscape of mitotic defects that generate CIN in primary and metastatic breast cancer and relevant models. Misaligned chromosomes predominate, and are less immunostimulatory than other chromosome segregation errors.

Cell biology: nondisjunction, aneuploidy and tetraploidy.

One simple, widely accepted mechanism for generating an aberrant chromosome number, or aneuploidy, is through nondisjunction--a chromosome distribution error that occurs during mitosis when both copies of a duplicated chromosome are deposited into one daughter cell and none into the other. Shi and King challenge this view, concluding that nondisjunction does not yield aneuploid cells directly, but instead gives rise to tetraploid cells that may subsequently become aneuploid through further division. Here we show that the direct result of chromosome nondisjunction is gain or loss of a single chromosome, which results in near-diploid aneuploidy, not tetraploidy. We suggest that chromatin trapped in the cytokinetic cleavage furrow is the more likely reason for furrow regression and tetraploidization.

Quantifying chromosomal instability from intratumoral karyotype diversity using agent-based modeling and Bayesian inference.

Chromosomal instability (CIN)-persistent chromosome gain or loss through abnormal mitotic segregation-is a hallmark of cancer that drives aneuploidy. Intrinsic chromosome mis-segregation rate, a measure of CIN, can inform prognosis and is a promising biomarker for response to anti-microtubule agents. However, existing methodologies to measure this rate are labor intensive, indirect, and confounded by selection against aneuploid cells, which reduces observable diversity. We developed a framework to measure CIN, accounting for karyotype selection, using simulations with various levels of CIN and models of selection. To identify the model parameters that best fit karyotype data from single-cell sequencing, we used approximate Bayesian computation to infer mis-segregation rates and karyotype selection. Experimental validation confirmed the extensive chromosome mis-segregation rates caused by the chemotherapy paclitaxel (18.5 ± 0.5/division). Extending this approach to clinical samples revealed that inferred rates fell within direct observations of cancer cell lines. This work provides the necessary framework to quantify CIN in human tumors and develop it as a predictive biomarker.

DNA contains all the information that cells need to function. The DNA inside cells is housed in structures called chromosomes, and most healthy human cells contain 23 pairs. When a cell divides, all chromosomes are copied so that each new cell gets a complete set. However, sometimes the process of separating chromosomes is faulty, and new cells may get incorrect numbers of chromosomes during cell division. Cancer cells frequently exhibit this behavior, which is called chromosomal instability', or CIN. Chromosomal instability affects many cancer cells with varying severity. In cancers with high chromosomal instability, the number of chromosomes may change almost every time the cells divide. These cancers are often the most aggressive and difficult to treat. Scientists can estimate chromosomal instability by counting differences in the number of chromosomes across many cells. However, many cells that are missing chromosomes die, resulting in inaccurate measures of chromosomal instability. To find a solution to this problem, Lynch et al. counted chromosomes in human cells with different levels of chromosomal instability and created a computer model to work out the relationship between chromosomal instability and chromosome number. The model could account for both living and dead cells, which gave more accurate results. Lynch et al. then confirmed the accuracy of their approach by using it on a group of cells treated with a chemotherapy drug that causes a known level of chromosomal instability. They also used existing data from breast and bowel cancer, which revealed that levels of chromosomal instability varied between one mistake per three to twenty cell divisions. Lower levels of chromosomal instability can be linked to a better prognosis for cancer patients, but it currently cannot be measured reliably. These results may help to reveal the causes of chromosomal instability and the role it has in cancer. If this method is successfully applied to patient samples, it could also improve our ability to predict how each cancer will progress and may lead to better treatments.