Patient-derived models recapitulate heterogeneity of molecular signatures and drug response in pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. Here we report establishment of 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulate histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and include rare subgroups not well-represented by existing models. We deploy 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predict variable in vivo response to PI3K/mTOR and MEK pathway inhibitors. These unique new models and an online interactive data portal for exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.

An in vitro model of tumor heterogeneity resolves genetic, epigenetic, and stochastic sources of cell state variability.

Tumor heterogeneity is a primary cause of treatment failure and acquired resistance in cancer patients. Even in cancers driven by a single mutated oncogene, variability in response to targeted therapies is well known. The existence of additional genomic alterations among tumor cells can only partially explain this variability. As such, nongenetic factors are increasingly seen as critical contributors to tumor relapse and acquired resistance in cancer. Here, we show that both genetic and nongenetic factors contribute to targeted drug response variability in an experimental model of tumor heterogeneity. We observe significant variability to epidermal growth factor receptor (EGFR) inhibition among and within multiple versions and clonal sublines of PC9, a commonly used EGFR mutant nonsmall cell lung cancer (NSCLC) cell line. We resolve genetic, epigenetic, and stochastic components of this variability using a theoretical framework in which distinct genetic states give rise to multiple epigenetic "basins of attraction," across which cells can transition driven by stochastic noise. Using mutational impact analysis, single-cell differential gene expression, and correlations among Gene Ontology (GO) terms to connect genomics to transcriptomics, we establish a baseline for genetic differences driving drug response variability among PC9 cell line versions. Applying the same approach to clonal sublines, we conclude that drug response variability in all but one of the sublines is due to epigenetic differences; in the other, it is due to genetic alterations. Finally, using a clonal drug response assay together with stochastic simulations, we attribute subclonal drug response variability within sublines to stochastic cell fate decisions and confirm that one subline likely contains genetic resistance mutations that emerged in the absence of drug treatment.

Evolutionary dynamics at the tumor edge reveal metabolic imaging biomarkers.

Human cancers are biologically and morphologically heterogeneous. A variety of clonal populations emerge within these neoplasms and their interaction leads to complex spatiotemporal dynamics during tumor growth. We studied the reshaping of metabolic activity in human cancers by means of continuous and discrete mathematical models and matched the results to positron emission tomography (PET) imaging data. Our models revealed that the location of increasingly active proliferative cellular spots progressively drifted from the center of the tumor to the periphery, as a result of the competition between gradually more aggressive phenotypes. This computational finding led to the development of a metric, normalized distance from 18F-fluorodeoxyglucose (18F-FDG) hotspot to centroid (NHOC), based on the separation from the location of the activity (proliferation) hotspot to the tumor centroid. The NHOC metric can be computed for patients using 18F-FDG PET-computed tomography (PET/CT) images where the voxel of maximum uptake (standardized uptake value [SUV]max) is taken as the activity hotspot. Two datasets of 18F-FDG PET/CT images were collected, one from 61 breast cancer patients and another from 161 non-small-cell lung cancer patients. In both cohorts, survival analyses were carried out for the NHOC and for other classical PET/CT-based biomarkers, finding that the former had a high prognostic value, outperforming the latter. In summary, our work offers additional insights into the evolutionary mechanisms behind tumor progression, provides a different PET/CT-based biomarker, and reveals that an activity hotspot closer to the tumor periphery is associated to a worst patient outcome.

Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy.

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.

Clonal Evolution and Heterogeneity of Osimertinib Acquired Resistance Mechanisms in EGFR Mutant Lung Cancer.

Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as EGFR C797S. MET amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, PD-L1, KRAS amplification, and ESR1-AKAP12, MKRN1-BRAF fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies.

Impact of tumor heterogeneity and tissue sampling for genetic mutation testing: a systematic review and post hoc analysis.

OBJECTIVE: The objective of the study was to identify guidelines to assist systematic reviewers or clinical researchers in identifying sampling bias due to tumor heterogeneity (TH) in solid cancers assayed for somatic mutations. We also assessed current reporting standards to determine the impact of TH on sample bias. STUDY DESIGN AND SETTING: We conducted a systematic review searching 13 databases (to January 2019) to identify guidelines. A post hoc analysis was performed using 12 prostate tumor somatic mutation data sets from a previous systematic review to assess reporting on TH. RESULTS: Searches identified 2,085 records. No formal guidelines were identified. Forty publications contained incidental recommendations across five major themes: using multiple tumor samples (n = 29), sample purity thresholds (n = 14), using specific sequencing methods (n = 8), using liquid biopsies (n = 4), and microdissection (n = 4). In post hoc analyses, 50% (6 of 12) clearly reported pathology methods. Forty-two percent (5 of 12) did not report pathology results. Forty-two percent (5 of 12) confirmed the pathology of the sample by direct diagnosis rather than inference. Forty-two percent (5 of 12) used multiple samples per patient. Fifty-eight percent (7 of 12) reported on tumor purity (reported ranges 10% to 100%). CONCLUSIONS: As precision medicine progresses to the clinic, guidelines are required to help evidence-based decision makers understand how TH may impact sample bias. Authors need to clearly report pathology methods and results and tumor purity methods and results.

Single-cell RNA sequencing reveals a heterogeneous response to Glucocorticoids in breast cancer cells.

Steroid hormone receptors such as the Glucocorticoid Receptor (GR) mediate transcriptional responses to hormones and are frequently targeted in the treatment of human diseases. Experiments using bulk populations of cells have provided a detailed picture of the global transcriptional hormone response but are unable to interrogate cell-to-cell transcriptional heterogeneity. To examine the glucocorticoid response in individual cells, we performed single cell RNA sequencing (scRNAseq) in a human breast cancer cell line. The transcriptional response to hormone was robustly detected in individual cells and scRNAseq provided additional statistical power to identify over 100 GR-regulated genes that were not detected in bulk RNAseq. scRNAseq revealed striking cell-to-cell variability in the hormone response. On average, individual hormone-treated cells showed a response at only 30% of the total set of GR target genes. Understanding the basis of this heterogeneity will be critical for the development of more precise models of steroid hormone signaling.

Understanding the Cause and Consequence of Tumor Heterogeneity.

Tumor heterogeneity is a large conundrum in cancer medicine, making most therapeutic interventions palliative rather than curative. Here we discuss the implications of how molecularly targeted therapies in solid malignancies that promote limited cancer cell death may in fact make tumors more heterogeneous, increase aggressive phenotypes, and thus worsen patient outcomes.

Ribosomal heterogeneity - A new inroad for pharmacological innovation.

The paradigm of ribosome usage in protein translation has shifted from a stance proposed as scientists began to unpick the genetic code that each mRNA was partnered by its own, unique ribosome to a rapid reversal of this view that ribosomes are completely interchangeable and simply recruited to mRNAs from a completely homogenous cellular pool. Evidence that the ribosomal proteome, ribosomal gene transcriptome and ribosome protein and RNA modifications differ between cells and tissues points to the fact that ribosomes are heterogeneous in their composition and have a degree of specialisation in their function. It has also been posited that the tissue-specificity of ribosome diseases provides an indication of functional ribosome heterogeneity, but there are substantial caveats to this interpretation. Only now have proteomic technologies developed to a level enabling accurate stoichiometric comparison of the abundance of specific ribosomal proteins in actively translating ribosomes and to measure protein in non-denatured ribosomes. This poises the field for the provocation that ribosome heterogeneity offers a novel and powerful inroad for the pharmacological targeting of disease. Such ribosome-targeted treatments may extend beyond specific ribosomopathies through strategies such as targeting features of ribosomes that are unique to diseased cells, particularly cancer cells, or to activated immune cells, as well as augmenting the action of other drugs through weakening the production of new proteins in target tissues. We may also be able to harness the potential power in ribosome diversity and specialism to better tune synthetic biology for the production of pharmaceutical proteins.

Chromatin mapping and single-cell immune profiling define the temporal dynamics of ibrutinib response in CLL.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib provides effective treatment for patients with chronic lymphocytic leukemia (CLL), despite extensive heterogeneity in this disease. To define the underlining regulatory dynamics, we analyze high-resolution time courses of ibrutinib treatment in patients with CLL, combining immune-phenotyping, single-cell transcriptome profiling, and chromatin mapping. We identify a consistent regulatory program starting with a sharp decrease of NF-κB binding in CLL cells, which is followed by reduced activity of lineage-defining transcription factors, erosion of CLL cell identity, and acquisition of a quiescence-like gene signature. We observe patient-to-patient variation in the speed of execution of this program, which we exploit to predict patient-specific dynamics in the response to ibrutinib based on the pre-treatment patient samples. In aggregate, our study describes time-dependent cellular, molecular, and regulatory effects for therapeutic inhibition of B cell receptor signaling in CLL, and it establishes a broadly applicable method for epigenome/transcriptome-based treatment monitoring.

Characterizing smoking-induced transcriptional heterogeneity in the human bronchial epithelium at single-cell resolution.

The human bronchial epithelium is composed of multiple distinct cell types that cooperate to defend against environmental insults. While studies have shown that smoking alters bronchial epithelial function and morphology, its precise effects on specific cell types and overall tissue composition are unclear. We used single-cell RNA sequencing to profile bronchial epithelial cells from six never and six current smokers. Unsupervised analyses led to the characterization of a set of toxin metabolism genes that localized to smoker ciliated cells, tissue remodeling associated with a loss of club cells and extensive goblet cell hyperplasia, and a previously unidentified peri-goblet epithelial subpopulation in smokers who expressed a marker of bronchial premalignant lesions. Our data demonstrate that smoke exposure drives a complex landscape of cellular alterations that may prime the human bronchial epithelium for disease.

Heterogeneity and dynamics of active Kras-induced dysplastic lineages from mouse corpus stomach.

Dysplasia is considered a key transition state between pre-cancer and cancer in gastric carcinogenesis. However, the cellular or phenotypic heterogeneity and mechanisms of dysplasia progression have not been elucidated. We have established metaplastic and dysplastic organoid lines, derived from Mist1-Kras(G12D) mouse stomach corpus and studied distinct cellular behaviors and characteristics of metaplastic and dysplastic organoids. We also examined functional roles for Kras activation in dysplasia progression using Selumetinib, a MEK inhibitor, which is a downstream mediator of Kras signaling. Here, we report that dysplastic organoids die or show altered cellular behaviors and diminished aggressive behavior in response to MEK inhibition. However, the organoids surviving after MEK inhibition maintain cellular heterogeneity. Two dysplastic stem cell (DSC) populations are also identified in dysplastic cells, which exhibited different clonogenic potentials. Therefore, Kras activation controls cellular dynamics and progression to dysplasia, and DSCs might contribute to cellular heterogeneity in dysplastic cell lineages.

Association between the UGT1A1*28 allele and hyperbilirubinemia in HIV-positive patients receiving atazanavir: a meta-analysis.

Objectives The uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1)*28 allele in HIV-positive patients receiving atazanavir (ATV) might be associated with the risk of hyperbilirubinemia. Owing to mixed and inconclusive results, a meta-analysis was conducted to systematically summarize and clarify this association.Methods Based on a comprehensive search of PubMed, Embase and Web of Science databases, studies investigating the association between UGT1A1 alleles and hyperbilirubinemia was retrieved. We evaluated the strength of this relationship using odds ratios (ORs) with 95% confidence intervals (CIs). Sensitivity analysis was performed by removing each study one at a time and calculating the pooled ORs of the remaining studies to test the robustness of the meta-analysis results. The Q statistic and the I2 index statistic were used to assess heterogeneity. Publication bias was evaluated using Orwin's fail-safe N test.Results A total of six individual studies were included in this meta-analysis. A significantly increased risk of hyperbilirubinemia was observed in HIV-positive patients receiving ATV with the UGT1A1*1/*28 or UGT1A1*28/*28 genotype, and the risk was higher with the UGT1A1*28/*28 genotype than with the UGT1A1*1/*28 genotype. (UGT1A1*28/*28 versus UGT1A1*1/*28: OR = 3.69, 95%CI = 1.82-7.49; UGT1A1*1/*28 versus UGT1A1*1/*1: OR = 3.50, 95%CI = 1.35-9.08; UGT1A1*28/*28 versus UGT1A1*1/*1: OR = 10.07, 95%CI = 4.39-23.10). All of the pooled ORs were not significantly affected by the remaining studies and different modeling methods, indicating robust results.Conclusions This meta-analysis suggests that the UGT1A1*28 allele represents a biomarker for an increased risk of hyperbilirubinemia in HIV-positive patients receiving ATV.

Dissecting heterogeneity in malignant pleural mesothelioma through histo-molecular gradients for clinical applications.

Malignant pleural mesothelioma (MPM) is recognized as heterogeneous based both on histology and molecular profiling. Histology addresses inter-tumor and intra-tumor heterogeneity in MPM and describes three major types: epithelioid, sarcomatoid and biphasic, a combination of the former two types. Molecular profiling studies have not addressed intra-tumor heterogeneity in MPM to date. Here, we use a deconvolution approach and show that molecular gradients shed new light on the intra-tumor heterogeneity of MPM, leading to a reconsideration of MPM molecular classifications. We show that each tumor can be decomposed as a combination of epithelioid-like and sarcomatoid-like components whose proportions are highly associated with the prognosis. Moreover, we show that this more subtle way of characterizing MPM heterogeneity provides a better understanding of the underlying oncogenic pathways and the related epigenetic regulation and immune and stromal contexts. We discuss the implications of these findings for guiding therapeutic strategies, particularly immunotherapies and targeted therapies.

Genetic heterogeneity of cytogenetically normal AML with mutations of CEBPA.

Biallelic mutations of the CCAAT/enhancer binding protein α (CEBPA) gene define a distinct genetic entity of acute myeloid leukemia (AML) with favorable prognosis. The presence of GATA2 and CSF3R mutations that are specifically associated with this subgroup but not mutated in all samples suggests a genetic heterogeneity of biCEBPA-mutated AML. We characterized the mutational landscape of CEBPA-mutated cytogenetically normal AML by targeted amplicon resequencing. We analyzed 48 biallelically mutated CEBPA (biCEBPA), 32 monoallelically mutated CEBPA (moCEBPA), and 287 wild-type CEBPA (wtCEBPA) patient samples from German AML Cooperative Group studies or registry. Targeted sequencing of 42 genes revealed that moCEBPA patients had significantly more additional mutations and additional mutated genes than biCEBPA patients. Within the group of biCEBPA patients, we identified 2 genetic subgroups defined by the presence or absence of mutations in chromatin/DNA modifiers (C), cohesin complex (C), and splicing (S) genes: biCEBPA CCSpos (25/48 [52%]) and biCEBPA CCSneg (23/48 [48%]). Equivalent subgroups were identified in 51 biCEBPA patients from the Cancer Genome Project. Patients in the biCEBPA CCSpos group were significantly older and had poorer overall survival and lower complete remission rates following intensive chemotherapy regimens compared with patients in the biCEBPA CCSneg group. Patients with available remission samples from the biCEBPA CCSpos group cleared the biCEBPA mutations, but most had persisting CCS mutations in complete remission, suggesting the presence of a preleukemic clone. In conclusion, CCS mutations define a distinct biological subgroup of biCEBPA AML that might refine prognostic classification of AML. This trial was registered at www.clinicaltrials.gov as #NCT00266136 and NCT01382147.

Studying cellular heterogeneity and drug sensitivity in colorectal cancer using organoid technology.

Intra-tumor heterogeneity (genotypic and functional diversity among cancer cells within the same tumor) represents one of the key challenges in cancer medicine. As heterogeneity of cancer cells constitutes an important parameter in the development of therapy resistance, an accurate assessment of intra-tumor heterogeneity is essential for the prediction of drug resistance and development of effective treatment. In this review, we evaluate primary patient derived-tumor organoid technology as a new tool for colorectal cancer research and treatment. Furthermore, we discuss organoid use to understand intra-tumor heterogeneity, both in terms of mutational diversification and of diversification in drug sensitivity. Finally, we address the exciting recent results that show that tumor organoid technology is highly predictive for drug response in metastatic colorectal cancer.

Mechanisms of acquired resistance to rapalogs in metastatic renal cell carcinoma.

The mechanistic target of rapamycin (mTOR) is an established therapeutic target in renal cell carcinoma (RCC). Mechanisms of secondary resistance to rapalog therapy in RCC have not been studied previously. We identified six patients with metastatic RCC who initially responded to mTOR inhibitor therapy and then progressed, and had pre-treatment and post-treatment tumor samples available for analysis. We performed deep whole exome sequencing on the paired tumor samples and a blood sample. Sequence data was analyzed using Mutect, CapSeg, Absolute, and Phylogic to identify mutations, copy number changes, and their changes over time. We also performed in vitro functional assays on PBRM1 in RCC cell lines. Five patients had clear cell and one had chromophobe RCC. 434 somatic mutations in 416 genes were identified in the 12 tumor samples. 201 (46%) of mutations were clonal in both samples while 129 (30%) were acquired in the post-treatment samples. Tumor heterogeneity or sampling issues are likely to account for some mutations that were acquired in the post-treatment samples. Three samples had mutations in TSC1; one in PTEN; and none in MTOR. PBRM1 was the only gene in which mutations were acquired in more than one post-treatment sample. We examined the effect of PBRM1 loss in multiple RCC cell lines, and could not identify any effect on rapalog sensitivity in in vitro culture assays. We conclude that mTOR pathway gene mutations did not contribute to rapalog resistance development in these six patients with advanced RCC. Furthermore, mechanisms of resistance to rapalogs in RCC remain unclear and our results suggest that PBRM1 loss may contribute to sensitivity through complex transcriptional effects.

Tumor-stroma interactions differentially alter drug sensitivity based on the origin of stromal cells.

Due to tumor heterogeneity, most believe that effective treatments should be tailored to the features of an individual tumor or tumor subclass. It is still unclear, however, what information should be considered for optimal disease stratification, and most prior work focuses on tumor genomics. Here, we focus on the tumor microenvironment. Using a large-scale coculture assay optimized to measure drug-induced cell death, we identify tumor-stroma interactions that modulate drug sensitivity. Our data show that the chemo-insensitivity typically associated with aggressive subtypes of breast cancer is not observed if these cells are grown in 2D or 3D monoculture, but is manifested when these cells are cocultured with stromal cells, such as fibroblasts. Furthermore, we find that fibroblasts influence drug responses in two distinct and divergent manners, associated with the tissue from which the fibroblasts were harvested. These divergent phenotypes occur regardless of the drug tested and result from modulation of apoptotic priming within tumor cells. Our study highlights unexpected diversity in tumor-stroma interactions, and we reveal new principles that dictate how fibroblasts alter tumor drug responses.

Update on the Treatment of Early-Stage Triple-Negative Breast Cancer.

OPINION STATEMENT: Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancers and is associated with poor long-term outcomes compared to other breast cancer subtypes. Currently, chemotherapy remains the main modality of treatment for early-stage TNBC, as there is no approved targeted therapy for this subtype. The biologic heterogeneity of TNBC has hindered the development and evaluation of novel agents, but recent advancements in subclassifying TNBC have paved the way for further investigation of more effective systemic therapies, including cytotoxic and targeted agents. TNBC is enriched for germline BRCA mutation and for somatic deficiencies in homologous recombination DNA repair, the so-called "BRCAness" phenotype. Together, germline BRCA mutations and BRCAness are promising biomarkers of susceptibility to DNA-damaging therapy. Various investigational approaches are consequently being investigated in early-stage TNBC, including immune checkpoint inhibitors, platinum compounds, PI3K pathway inhibitors, and androgen receptor inhibitors. Due to the biological diversity found within TNBC, patient selection based on molecular biomarkers could aid the design of early-phase clinical trials, ultimately accelerating the clinical application of effective new agents. TNBC is an aggressive breast cancer subtype, for which multiple targeted approaches will likely be required for patient outcomes to be substantially improved.

Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors.

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.