Ultra-deep mutational landscape in chronic lymphocytic leukemia uncovers dynamics of resistance to targeted therapies.

BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved the outcomes of patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients we detected variants that expanded and reached significant cancer cell fractions (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S; PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment, including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while the patients were receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1,000-fold (from a CCF of 0.02% to 35%), and the CCF in p.T474I in patient R002 increased from 0.03% to 4.2% (more than 100-fold). Our data illuminate the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.

Stochastic model for cell population dynamics quantifies homeostasis in colonic crypts and its disruption in early tumorigenesis.

The questions of how healthy colonic crypts maintain their size, and how homeostasis is disrupted by driver mutations, are central to understanding colorectal tumorigenesis. We propose a three-type stochastic branching process, which accounts for stem, transit-amplifying (TA) and fully differentiated (FD) cells, to model the dynamics of cell populations residing in colonic crypts. Our model is simple in its formulation, allowing us to estimate all but one of the model parameters from the literature. Fitting the single remaining parameter, we find that model results agree well with data from healthy human colonic crypts, capturing the considerable variance in population sizes observed experimentally. Importantly, our model predicts a steady-state population in healthy colonic crypts for relevant parameter values. We show that APC and KRAS mutations, the most significant early alterations leading to colorectal cancer, result in increased steady-state populations in mutated crypts, in agreement with experimental results. Finally, our model predicts a simple condition for unbounded growth of cells in a crypt, corresponding to colorectal malignancy. This is predicted to occur when the division rate of TA cells exceeds their differentiation rate, with implications for therapeutic cancer prevention strategies.

Clonal interactions in cancer: Integrating quantitative models with experimental and clinical data.

Tumors consist of different genotypically distinct subpopulations-or subclones-of cells. These subclones can influence neighboring clones in a process called "clonal interaction." Conventionally, research on driver mutations in cancer has focused on their cell-autonomous effects that lead to an increase in fitness of the cells containing the driver. Recently, with the advent of improved experimental and computational technologies for investigating tumor heterogeneity and clonal dynamics, new studies have shown the importance of clonal interactions in cancer initiation, progression, and metastasis. In this review we provide an overview of clonal interactions in cancer, discussing key discoveries from a diverse range of approaches to cancer biology research. We discuss common types of clonal interactions, such as cooperation and competition, its mechanisms, and the overall effect on tumorigenesis, with important implications for tumor heterogeneity, resistance to treatment, and tumor suppression. Quantitative models-in coordination with cell culture and animal model experiments-have played a vital role in investigating the nature of clonal interactions and the complex clonal dynamics they generate. We present mathematical and computational models that can be used to represent clonal interactions and provide examples of the roles they have played in identifying and quantifying the strength of clonal interactions in experimental systems. Clonal interactions have proved difficult to observe in clinical data; however, several very recent quantitative approaches enable their detection. We conclude by discussing ways in which researchers can further integrate quantitative methods with experimental and clinical data to elucidate the critical-and often surprising-roles of clonal interactions in human cancers.

Waiting times in a branching process model of colorectal cancer initiation.

We study a multi-stage model for the development of colorectal cancer from initially healthy tissue. The model incorporates a complex sequence of driver gene alterations, some of which result in immediate growth advantage, while others have initially neutral effects. We derive analytic estimates for the sizes of premalignant subpopulations, and use these results to compute the waiting times to premalignant and malignant genotypes. This work contributes to the quantitative understanding of colorectal tumor evolution and the lifetime risk of colorectal cancer.

In Vivo Modeling of CLL Transformation to Richter Syndrome Reveals Convergent Evolutionary Paths and Therapeutic Vulnerabilities.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.

Inferring parameters of cancer evolution in chronic lymphocytic leukemia.

As a cancer develops, its cells accrue new mutations, resulting in a heterogeneous, complex genomic profile. We make use of this heterogeneity to derive simple, analytic estimates of parameters driving carcinogenesis and reconstruct the timeline of selective events following initiation of an individual cancer, where two longitudinal samples are available for sequencing. Using stochastic computer simulations of cancer growth, we show that we can accurately estimate mutation rate, time before and after a driver event occurred, and growth rates of both initiated cancer cells and subsequently appearing subclones. We demonstrate that in order to obtain accurate estimates of mutation rate and timing of events, observed mutation counts should be corrected to account for clonal mutations that occurred after the founding of the tumor, as well as sequencing coverage. Chronic lymphocytic leukemia (CLL), which often does not require treatment for years after diagnosis, presents an optimal system to study the untreated, natural evolution of cancer cell populations. When we apply our methodology to reconstruct the individual evolutionary histories of CLL patients, we find that the parental leukemic clone typically appears within the first fifteen years of life.

A mechanistic mathematical model of initiation and malignant transformation in sporadic vestibular schwannoma.

BACKGROUND: A vestibular schwannoma (VS) is a relatively rare, benign tumour of the eighth cranial nerve, often involving alterations to the gene NF2. Previous mathematical models of schwannoma incidence have not attempted to account for alterations in specific genes, and could not distinguish between nonsense mutations and loss of heterozygosity (LOH). METHODS: Here, we present a mechanistic approach to modelling initiation and malignant transformation in schwannoma. Each parameter is associated with a specific gene or mechanism operative in Schwann cells, and can be determined by combining incidence data with empirical frequencies of pathogenic variants and LOH. RESULTS: This results in new estimates for the base-pair mutation rate u = 4.48 × 10-10 and the rate of LOH = 2.03 × 10-6/yr in Schwann cells. In addition to new parameter estimates, we extend the approach to estimate the risk of both spontaneous and radiation-induced malignant transformation. DISCUSSION: We conclude that radiotherapy is likely to have a negligible excess risk of malignancy for sporadic VS, with a possible exception of rapidly growing tumours.

Quantification of the Selective Advantage of Driver Mutations Is Dependent on the Underlying Model and Stage of Tumor Evolution.

Measuring the selective fitness advantages provided by driver mutations has the potential to facilitate a precise quantitative understanding of cancer evolution. However, accurately measuring the selective advantage of driver mutations has remained a challenge in the field. Early studies reported small selective advantages of drivers, on the order of 1%, whereas newer studies report much larger selective advantages, as high as 1,200%. In this article, we argue that the calculated selective advantages of cancer drivers are dependent on the underlying mathematical model and stage of cancer evolution and that comparisons of numerical values of selective advantage without regard for the underlying model and stage can lead to spurious conclusions.

Modeling CAR T-Cell Therapy with Patient Preconditioning.

The Federal Drug Administration approved the first Chimeric Antigen Receptor T-cell (CAR T-cell) therapies for the treatment of several blood cancers in 2017, and efforts are underway to broaden CAR T technology to address other cancer types. Standard treatment protocols incorporate a preconditioning regimen of lymphodepleting chemotherapy prior to CAR T-cell infusion. However, the connection between preconditioning regimens and patient outcomes is still not fully understood. Optimizing patient preconditioning plans and reducing the CAR T-cell dose necessary for achieving remission could make therapy safer. In this paper, we test treatment regimens consisting of sequential administration of chemotherapy and CAR T-cell therapy on a system of differential equations that models the tumor-immune interaction. We use numerical simulations of treatment plans from within the scope of current medical practice to assess the effect of preconditioning plans on the success of CAR T-cell therapy. Model results affirm clinical observations that preconditioning can be crucial for most patients, not just to reduce side effects, but to even achieve remission at all. We demonstrate that preconditioning plans using the same CAR T-cell dose and the same total concentration of chemotherapy can lead to different patient outcomes due to different delivery schedules. Results from sensitivity analysis of the model parameters suggest that making small improvements in the effectiveness of CAR T-cells in attacking cancer cells will significantly reduce the minimum dose required for successful treatment. Our modeling framework represents a starting point for evaluating the efficacy of patient preconditioning in the context of CAR T-cell therapy.

Mathematical model of colorectal cancer initiation.

Quantifying evolutionary dynamics of cancer initiation and progression can provide insights into more effective strategies of early detection and treatment. Here we develop a mathematical model of colorectal cancer initiation through inactivation of two tumor suppressor genes and activation of one oncogene, accounting for the well-known path to colorectal cancer through loss of tumor suppressors APC and TP53 and gain of the KRAS oncogene. In the model, we allow mutations to occur in any order, leading to a complex network of premalignant mutational genotypes on the way to colorectal cancer. We parameterize the model using experimentally measured parameter values, many of them only recently available, and compare its predictions to epidemiological data on colorectal cancer incidence. We find that the reported lifetime risk of colorectal cancer can be recovered using a mathematical model of colorectal cancer initiation together with experimentally measured mutation rates in colorectal tissues and proliferation rates of premalignant lesions. We demonstrate that the order of driver events in colorectal cancer is determined primarily by the fitness effects that they provide, rather than their mutation rates. Our results imply that there may not be significant immune suppression of untreated benign and malignant colorectal lesions.

Delineating the evolutionary dynamics of cancer from theory to reality.

Uncovering and quantifying the laws of the evolutionary dynamics of cancer, in particular in the context of specific genetic lesions and in individual patients, has the potential to revolutionize precision oncology. Recent technological advances in the study of human cancer have increased access to in vivo human data and have thereby facilitated the confirmation or refutation of existing theoretical models. In this Perspective, we discuss recent work at the intersection of quantitative mathematical models of cancer evolution and patient data that provides insights into different stages of tumor evolution, including premalignant and malignant progression and response to therapy.

On measuring selection in cancer from subclonal mutation frequencies.

Recently available cancer sequencing data have revealed a complex view of the cancer genome containing a multitude of mutations, including drivers responsible for cancer progression and neutral passengers. Measuring selection in cancer and distinguishing drivers from passengers have important implications for development of novel treatment strategies. It has recently been argued that a third of cancers are evolving neutrally, as their mutational frequency spectrum follows a 1/f power law expected from neutral evolution in a particular intermediate frequency range. We study a stochastic model of cancer evolution and derive a formula for the probability distribution of the cancer cell frequency of a subclonal driver, demonstrating that driver frequency is biased towards 0 and 1. We show that it is difficult to capture a driver mutation at an intermediate frequency, and thus the calling of neutrality due to a lack of such driver will significantly overestimate the number of neutrally evolving tumors. Our approach provides quantification of the validity of the 1/f statistic across the entire range of relevant parameter values. We also show that our conclusions remain valid for non-exponential models: spatial 3d model and sigmoidal growth, relevant for early- and late stages of cancer growth.

Growth dynamics in naturally progressing chronic lymphocytic leukaemia.

How the genomic features of a patient's cancer relate to individual disease kinetics remains poorly understood. Here we used the indolent growth dynamics of chronic lymphocytic leukaemia (CLL) to analyse the growth rates and corresponding genomic patterns of leukaemia cells from 107 patients with CLL, spanning decades-long disease courses. We found that CLL commonly demonstrates not only exponential expansion but also logistic growth, which is sigmoidal and reaches a certain steady-state level. Each growth pattern was associated with marked differences in genetic composition, the pace of disease progression and the extent of clonal evolution. In a subset of patients, whose serial samples underwent next-generation sequencing, we found that dynamic changes in the disease course of CLL were shaped by the genetic events that were already present in the early slow-growing stages. Finally, by analysing the growth rates of subclones compared with their parental clones, we quantified the growth advantage conferred by putative CLL drivers in vivo.

Dormant tumour cells, their niches and the influence of immunity.

Despite increased focus on the clinical relevance of dormant metastatic disease, our understanding of dormant niches, mechanisms underlying emergence from dormancy, and the immune system's role in this phenomenon, remains in its infancy. Here, we discuss key work that has shaped our current understanding of these topics. Because tumour dormancy provides a unique therapeutic window to prevent metastatic disease, we discuss on-going clinical trials and weigh the potential for immunotherapy to eradicate dormant disease.

The evolutionary landscape of chronic lymphocytic leukemia treated with ibrutinib targeted therapy.

Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts (change >0.1 in clonal cancer cell fraction, Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones.

Reconstructing metastatic seeding patterns of human cancers.

Reconstructing the evolutionary history of metastases is critical for understanding their basic biological principles and has profound clinical implications. Genome-wide sequencing data has enabled modern phylogenomic methods to accurately dissect subclones and their phylogenies from noisy and impure bulk tumour samples at unprecedented depth. However, existing methods are not designed to infer metastatic seeding patterns. Here we develop a tool, called Treeomics, to reconstruct the phylogeny of metastases and map subclones to their anatomic locations. Treeomics infers comprehensive seeding patterns for pancreatic, ovarian, and prostate cancers. Moreover, Treeomics correctly disambiguates true seeding patterns from sequencing artifacts; 7% of variants were misclassified by conventional statistical methods. These artifacts can skew phylogenies by creating illusory tumour heterogeneity among distinct samples. In silico benchmarking on simulated tumour phylogenies across a wide range of sample purities (15-95%) and sequencing depths (25-800 × ) demonstrates the accuracy of Treeomics compared with existing methods.

Limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer.

The extent of heterogeneity among driver gene mutations present in naturally occurring metastases-that is, treatment-naive metastatic disease-is largely unknown. To address this issue, we carried out 60× whole-genome sequencing of 26 metastases from four patients with pancreatic cancer. We found that identical mutations in known driver genes were present in every metastatic lesion for each patient studied. Passenger gene mutations, which do not have known or predicted functional consequences, accounted for all intratumoral heterogeneity. Even with respect to these passenger mutations, our analysis suggests that the genetic similarity among the founding cells of metastases was higher than that expected for any two cells randomly taken from a normal tissue. The uniformity of known driver gene mutations among metastases in the same patient has critical and encouraging implications for the success of future targeted therapies in advanced-stage disease.

Tumour and immune cell dynamics explain the PSA bounce after prostate cancer brachytherapy.

BACKGROUND: Interstitial brachytherapy for localised prostate cancer may be followed by transient increases in prostate-specific antigen (PSA) that resolve without therapy. Such PSA bounces may be associated with an improved outcome but often cause alarm in the patient and physician, and have defied explanation. METHODS: We developed a mathematical model to capture the interactions between the tumour, radiation and anti-tumour immune response. The model was fitted to data from a large cohort of patients treated exclusively with interstitial brachytherapy. Immunohistological analysis for T-cell infiltration within the same tumours was also performed. RESULTS: Our minimal model captures well the dynamics of the tumour after therapy, and suggests that a strong anti-tumour immune response coupled with the therapeutic effect of radiation on the tumour is responsible for the PSA bounce. Patients who experience a PSA bounce had a higher density of CD3 and CD8 cells within the tumour that likely contribute to the PSA bounce and the overall better outcomes observed. CONCLUSIONS: Our observations provide a novel and unifying explanation for the PSA bounce in patients with early prostate cancer and also have implications for the use of immune-based therapies in such patients to improve outcomes.

Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition.

Resistance to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.