Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Characterizing Evolutionary Dynamics Reveals Strategies to Exhaust the Spectrum of Subclonal Resistance in EGFR-Mutant Lung Cancer.

The emergence of resistance to targeted therapies restrains their efficacy. The development of rationally guided drug combinations could overcome this currently insurmountable clinical challenge. However, our limited understanding of the trajectories that drive the outgrowth of resistant clones in cancer cell populations precludes design of drug combinations to forestall resistance. Here, we propose an iterative treatment strategy coupled with genomic profiling and genome-wide CRISPR activation screening to systematically extract and define preexisting resistant subpopulations in an EGFR-driven lung cancer cell line. Integrating these modalities identifies several resistance mechanisms, including activation of YAP/TAZ signaling by WWTR1 amplification, and estimates the associated cellular fitness for mathematical population modeling. These observations led to the development of a combination therapy that eradicated resistant clones in large cancer cell line populations by exhausting the spectrum of genomic resistance mechanisms. However, a small fraction of cancer cells was able to enter a reversible nonproliferative state of drug tolerance. This subpopulation exhibited mesenchymal properties, NRF2 target gene expression, and sensitivity to ferroptotic cell death. Exploiting this induced collateral sensitivity by GPX4 inhibition clears drug-tolerant populations and leads to tumor cell eradication. Overall, this experimental in vitro data and theoretical modeling demonstrate why targeted mono- and dual therapies will likely fail in sufficiently large cancer cell populations to limit long-term efficacy. Our approach is not tied to a particular driver mechanism and can be used to systematically assess and ideally exhaust the resistance landscape for different cancer types to rationally design combination therapies. SIGNIFICANCE: Unraveling the trajectories of preexisting resistant and drug-tolerant persister cells facilitates the rational design of multidrug combination or sequential therapies, presenting an approach to explore for treating EGFR-mutant lung cancer.

A multi-targeted investigation of Deepwater Horizon crude oil exposure impacts on the marine teleost stress axis.

The toxicity of the polycyclic aromatic hydrocarbons (PAHs) in Deepwater Horizon (DWH) oil is well-established, but a knowledge gap exists regarding how this combination of PAHs affects the vertebrate stress axis. We hypothesized that (1) marine vertebrates exposed to DWH PAHs experience stress axis impairment, and co-exposure to an additional chronic stressor may exacerbate these effects, (2) serotonin (5-hydroxytryptamine; 5-HT) may act as a secondary cortisol secretagogue in DWH PAH-exposed fish to compensate for impairment, and (3) the mechanism of stress axis impairment may involve downregulation of cyclic adenosine monophosphate (cAMP; as proxy for melanocortin 2 receptor (MC2R) functionality), total cholesterol, and/or mRNA expression of CYP1A and steroidogenic proteins StAR, P450scc, and 11ß-h at the level of the kidney. We found that in vivo plasma cortisol and plasma adrenocorticotropic hormone (ACTH) concentrations in Gulf toadfish exposed to an environmentally relevant DWH PAH concentration (ΣPAH50= 4.6 ± 1.6 µg/L) for 7 days were not significantly different from controls, whether fish were chronically stressed or not. However, the rate of cortisol secretion by isolated kidneys after acute stimulation with ACTH was significantly lower in PAH-exposed toadfish compared to clean seawater (SW) controls. 5-HT does not appear to be acting as a secondary cortisol secretagogue, rather, PAH-exposed + stressed toadfish exhibited significantly lower plasma 5-HT concentrations than clean SW + stressed fish as well as a reduced sensitivity to 5-HT at the level of the kidney. There was a tendency for kidney cAMP concentrations to be lower in PAH-exposed fish (p = 0.069); however, mRNA expression of steroidogenic proteins between control and PAH-exposed toadfish were not significantly different and a significant elevation in total cholesterol concentration in PAH-exposed toadfish compared to controls was measured. Future work is needed to establish whether the slower cortisol secretion rate by isolated kidneys of PAH-exposed fish is detrimental, to determine the potential role of other secretagogues in compensating for the impaired kidney interrenal cell function, and to determine whether there is a reduction in MC2R mRNA expression or an impairment in the function of steroidogenic proteins.

Genomic ALK alterations in primary and relapsed neuroblastoma.

BACKGROUND: Genomic alterations of the anaplastic lymphoma kinase gene (ALK) occur recurrently in neuroblastoma, a pediatric malignancy of the sympathetic nervous system. However, information on their development over time has remained sparse. METHODS: ALK alterations were assessed in neuroblastomas at diagnosis and/or relapse from a total of 943 patients, covering all stages of disease. Longitudinal information on diagnostic and relapsed samples from individual patients was available in 101 and 102 cases for mutation and amplification status, respectively. RESULTS: At diagnosis, ALK point mutations occurred in 10.5% of all cases, with highest frequencies in stage 4 patients <18 months. At relapse, ALK alteration frequency increased by 70%, both in high-risk and non-high-risk cases. The increase was most likely due to de novo mutations, frequently leading to R1275Q substitutions, which are sensitive to pharmacological ALK inhibition. By contrast, the frequency of ALK amplifications did not change over the course of the disease. ALK amplifications, but not mutations, were associated with poor patient outcome. CONCLUSIONS: The considerably increased frequency of ALK mutations at relapse and their high prevalence in young stage 4 patients suggest surveying the genomic ALK status regularly in these patient cohorts, and to evaluate ALK-targeted treatment also in intermediate-risk patients.

Chromothripsis followed by circular recombination drives oncogene amplification in human cancer.

The mechanisms behind the evolution of complex genomic amplifications in cancer have remained largely unclear. Using whole-genome sequencing data of the pediatric tumor neuroblastoma, we here identified a type of amplification, termed 'seismic amplification', that is characterized by multiple rearrangements and discontinuous copy number levels. Overall, seismic amplifications occurred in 9.9% (274 of 2,756) of cases across 38 cancer types, and were associated with massively increased copy numbers and elevated oncogene expression. Reconstruction of the development of seismic amplification showed a stepwise evolution, starting with a chromothripsis event, followed by formation of circular extrachromosomal DNA that subsequently underwent repetitive rounds of circular recombination. The resulting amplicons persisted as extrachromosomal DNA circles or had reintegrated into the genome in overt tumors. Together, our data indicate that the sequential occurrence of chromothripsis and circular recombination drives oncogene amplification and overexpression in a substantial fraction of human malignancies.

Variant profiling of colorectal adenomas from three patients of two families with MSH3-related adenomatous polyposis.

The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21-42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis.

Exposure and Recovery from Environmentally Relevant Levels of Waterborne Polycyclic Aromatic Hydrocarbons from Deepwater Horizon Oil: Effects on the Gulf Toadfish Stress Axis.

There is evidence that the combination of polycyclic aromatic hydrocarbons (PAHs) released in the Deepwater Horizon oil spill impairs the glucocorticoid stress response of vertebrates in the Gulf of Mexico, but the mechanisms are unclear. We hypothesized that inhibition of cortisol release may be due to 1) overstimulation of the hypothalamic-pituitary-inter-renal (HPI) axis, or 2) an inhibition of cortisol biosynthesis through PAH activation of the aryl hydrocarbon receptor (AhR). Using a flow-through system, Gulf toadfish (Opsanus beta) were continuously exposed to control conditions or one of 3 environmentally relevant concentrations of PAHs from Deepwater Horizon oil (∑PAH50 = 0-3 µg L-1 ) for up to 7 d. One group of toadfish was then exposed to a recovery period for up to 7 d. No changes in corticotrophin-releasing factor mRNA expression, adrenocorticotropic hormone (ACTH), or pituitary mass suggested that overstimulation of the HPI axis was not a factor. The AhR activation was measured by an elevation of cytochrome P4501A1 (CYP1A) mRNA expression within the HPI axis in fish exposed to high PAH concentrations; however, CYP1A was no longer induced after 3 d of recovery in any of the tissues. At 7 d of recovery, there was an impairment of cortisol release in response to an additional simulated predator chase that does not appear to be due to changes in the mRNA expression of the kidney steroidogenic pathway proteins steroidogenic acute regulatory protein, cytochrome P450 side chain cleavage, and 11ß-hydroxylase. Future analyses are needed to determine whether the stress response impairment is due to cholesterol availability and/or down-regulation of the melanocortin 2 receptor. Environ Toxicol Chem 2021;40:1062-1074. © 2020 SETAC.

Exposure and Recovery of the Gulf Toadfish (Opsanus beta) to Weathered Deepwater Horizon Slick Oil: Impacts on Liver and Blood Endpoints.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be responsible for a variety of deleterious effects on organisms. These adverse outcomes are relatively well studied, but at concentrations rarely found in the environment. Among the documented effects of sublethal acute PAH exposure are reductions in osmoregulatory capacity and immune function, and changes in the function of critical metabolic organs such as the liver. Gulf toadfish (Opsanus beta) were exposed to control seawater (0.006 µg tPAH50 /L) or water accommodated fractions of Deepwater Horizon spill oil diluted to 3 flow-through exposure regimes (0.009, 0.059, and 2.82 µg tPAH50 /L) for 7 d, with a recovery period of equal duration. We hypothesized that these chronic exposures would induce the aryl hydrocarbon receptor (AhR)-mediated pathways and result in significant impacts on markers of osmoregulatory, immune, and metabolic function. We further hypothesized that measurable reversal of these impacts would be observed during the recovery period. Our results indicate that activation of cytochrome P 450 (CYP)1A1 was achieved during exposure and reversed during the recovery phase. The only significant deviations from controls measured were a reduction in plasma glucose in fish exposed to medium and high levels of PAH after 7 d of exposure and a reduction in plasma osmolality fish exposed to high levels of PAHs after 7 d of recovery, when CYP1A1 messenger (m)RNA levels had returned to control levels. Our study illustrates a disconnect between the activation of CYP1A1 in response to environmentally realistic PAHs concentrations and several physiological endpoints and supports the idea that the AhR might not be associated with mediating osmoregulatory, immune, and metabolic changes in Gulf toadfish. Environ Toxicol Chem 2021;40:1075-1086. © 2020 SETAC.

CaMuS: simultaneous fitting and de novo imputation of cancer mutational signature.

The identification of the mutational processes operating in tumour cells has implications for cancer diagnosis and therapy. These processes leave mutational patterns on the cancer genomes, which are referred to as mutational signatures. Recently, 81 mutational signatures have been inferred using computational algorithms on sequencing data of 23,879 samples. However, these published signatures may not always offer a comprehensive view on the biological processes underlying tumour types that are not included or underrepresented in the reference studies. To circumvent this problem, we designed CaMuS (Cancer Mutational Signatures) to construct de novo signatures while simultaneously fitting publicly available mutational signatures. Furthermore, we propose to estimate signature similarity by comparing probability distributions using the Hellinger distance. We applied CaMuS to infer signatures of mutational processes in poorly studied cancer types. We used whole genome sequencing data of 56 neuroblastoma, thus providing evidence for the versatility of CaMuS. Using simulated data, we compared the performance of CaMuS to sigfit, a recently developed algorithm with comparable inference functionalities. CaMuS and sigfit reconstructed the simulated datasets with similar accuracy; however two main features may argue for CaMuS over sigfit: (i) superior computational performance and (ii) a reliable parameter selection method to avoid spurious signatures.

Integrative Analysis of Pleomorphic Dermal Sarcomas Reveals Fibroblastic Differentiation and Susceptibility to Immunotherapy.

PURPOSE: Pleomorphic dermal sarcoma (PDS) is a rare malignant cutaneous tumor with an unknown cell of origin. Locally defined tumors can be treated by curative excisions, whereas advanced stages of the disease are difficult to treat, using standard regimens. EXPERIMENTAL DESIGN: We performed whole-exome sequencing on a cohort of 28 individuals and corresponding transcriptomic analysis on 21 patients, as well as quantitative IHC image analysis on 27 patients. RESULTS: PDS exhibits a universally high mutational load (42.7 mutations/mega base) with an inflamed, immunogenic tumor microenvironment. Three cases of PDS showed response to immune checkpoint blockade. Local mutation rate variation together with mRNA expression data demonstrate that PDS form a distinct entity, with PDGFRB as a lineage marker. In addition, we found that PDS is of mesenchymal, fibroblastic differentiation. CONCLUSIONS: PDS is of fibroblastic differentiation and exhibits a strong susceptibility to immunotherapy, including a high mutational burden and an inflamed tumor microenvironment.

Do reproductive hormones control Gulf toadfish pulsatile urea excretion?

Gulf toadfish (Opsanus beta) can excrete the majority of their nitrogenous waste as urea in distinct pulses across their gill. Urea pulses are controlled by cortisol and serotonin (5-HT) and are believed to contain chemical signals that may communicate reproductive and/or social status. The objectives of this study were to determine if reproductive hormones are involved in controlling pulsatile urea excretion, and if toadfish respond to prostaglandins as a chemical signal. Specifically, 11-ketotestosterone (11-KT), estradiol (E2), and the teleost pheromone prostaglandin E2 (PGE2) were investigated. Castration during breeding season did not affect pulsatile urea excretion but serial injections of 11-KT outside of breeding season did result in a 48% reduction in urea pulse size in fish of both sexes. Injections of E2 and PGE2, on the other hand, did not alter urea excretion patterns. Toadfish also did not pulse urea in response to waterborne exposure of PGE2 suggesting that this compound does not serve as a toadfish pheromone alone. Toadfish have significantly higher plasma 5-HT during breeding season compared to the months following breeding season. Future research should focus on the composition of the chemical signal in toadfish and the potential importance of seasonal changes in plasma 5-HT in toadfish pulsatile urea excretion and teleost reproduction in general.

A mechanistic classification of clinical phenotypes in neuroblastoma.

Neuroblastoma is a pediatric tumor of the sympathetic nervous system. Its clinical course ranges from spontaneous tumor regression to fatal progression. To investigate the molecular features of the divergent tumor subtypes, we performed genome sequencing on 416 pretreatment neuroblastomas and assessed telomere maintenance mechanisms in 208 of these tumors. We found that patients whose tumors lacked telomere maintenance mechanisms had an excellent prognosis, whereas the prognosis of patients whose tumors harbored telomere maintenance mechanisms was substantially worse. Survival rates were lowest for neuroblastoma patients whose tumors harbored telomere maintenance mechanisms in combination with RAS and/or p53 pathway mutations. Spontaneous tumor regression occurred both in the presence and absence of these mutations in patients with telomere maintenance-negative tumors. On the basis of these data, we propose a mechanistic classification of neuroblastoma that may benefit the clinical management of patients.

Clonal dynamics towards the development of venetoclax resistance in chronic lymphocytic leukemia.

Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.

Extrinsic nerves are not involved in branchial 5-HT dynamics or pulsatile urea excretion in Gulf toadfish, Opsanus beta.

Gulf toadfish (Opsanus beta) can switch from continuously excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses. Previous studies have shown that the neurotransmitter serotonin (5-HT) is involved in controlling this process, but it is unknown if 5-HT availability is under central nervous control or if the 5-HT signal originates from a peripheral source. Following up on a previous study, cranial nerves IX (glossopharyngeal) and X (vagus) were sectioned to further characterize their role in controlling pulsatile urea excretion and 5-HT release within the gill. In contrast to an earlier study, nerve sectioning did not result in a change in urea pulse frequency. Total urea excretion, average pulse size, total nitrogen excretion, and percent ureotely were reduced the first day post-surgery in nerve-sectioned fish but recovered by 72h post-surgery. Nerve sectioning also had no effect on toadfish urea transporter (tUT), 5-HT transporter (SERT), or 5-HT2A receptor mRNA expression or 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) abundance in the gill, all of which were found consistently across the three gill arches except 5-HIAA, which was undetectable in the first gill arch. Our findings indicate that the central nervous system does not directly control pulsatile urea excretion or local changes in gill 5-HT and 5-HIAA abundance.

cDNA Library Enrichment of Full Length Transcripts for SMRT Long Read Sequencing.

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci.