Sam M. Janes

Spatial and temporal diversity in genomic instability processes defines lung cancer evolution

Spatial and temporal dissection of the genomic changes occurring during the evolution of human non–small cell lung cancer (NSCLC) may help elucidate the basis for its dismal prognosis. We sequenced 25 spatially distinct regions from seven operable NSCLCs and found evidence of branched evolution, with driver mutations arising before and after subclonal diversification. There was pronounced intratumor heterogeneity in copy number alterations, translocations, and mutations associated with APOBEC cytidine deaminase activity. Despite maintained carcinogen exposure, tumors from smokers showed a relative decrease in smoking-related mutations over time, accompanied by an increase in APOBEC-associated mutations. In tumors from former smokers, genome-doubling occurred within a smoking-signature context before subclonal diversification, which suggested that a long period of tumor latency had preceded clinical detection. The regionally separated driver mutations, coupled with the relentless and heterogeneous nature of the genome instability processes, are likely to confound treatment success in NSCLC. Different regions of a human lung tumor harbor different mutations, possibly explaining why the disease is so tough to treat. [Also see Perspective by Govindan] Space, time, and the lung cancer genome Lung cancer poses a formidable challenge to clinical oncologists. It is often detected at a late stage, and most therapies work for only a short time before the tumors resume their relentless growth. Two independent analyses of the human lung cancer genome may help explain why this disease is so resilient (see the Perspective by Govindan). Rather than take a single “snapshot” of the cancer genome, de Bruin et al. and Zhang et al. identified genomic alterations in spatially distinct regions of single lung tumors and used this information to infer the tumors evolutionary history. Each tumor showed tremendous spatial and temporal diversity in its mutational profiles. Thus, the efficacy of drugs may be short-lived because they destroy only a portion of the tumor. Science, this issue p. 251, p. 256; see also p. 169

Mesenchymal Stem Cell Delivery of TRAIL Can Eliminate Metastatic Cancer

Cancer is a leading cause of mortality throughout the world and new treatments are urgently needed. Recent studies suggest that bone marrow-derived mesenchymal stem cells (MSC) home to and incorporate within tumor tissue. We hypothesized that MSCs engineered to produce and deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a transmembrane protein that causes selective apoptosis of tumor cells, would home to and kill cancer cells in a lung metastatic cancer model. Human MSCs were transduced with TRAIL and the IRES-eGFP reporter gene under the control of a tetracycline promoter using a lentiviral vector. Transduced and activated MSCs caused lung (A549), breast (MDAMB231), squamous (H357), and cervical (Hela) cancer cell apoptosis and death in coculture experiments. Subcutaneous xenograft experiments confirmed that directly delivered TRAIL-expressing MSCs were able to significantly reduce tumor growth [0.12 cm(3) (0.04-0.21) versus 0.66 cm(3) (0.21-1.11); P < 0.001]. We then found, using a pulmonary metastasis model, systemically delivered MSCs localized to lung metastases and the controlled local delivery of TRAIL completely cleared the metastatic disease in 38% of mice compared with 0% of controls (P < 0.05). This is the first study to show a significant reduction in metastatic tumor burden with frequent eradication of metastases using inducible TRAIL-expressing MSCs. This has a wide potential therapeutic role, which includes the treatment of both primary tumors and their metastases, possibly as an adjuvant therapy in clearing micrometastatic disease following primary tumor resection.

Stem-cell-based, tissue engineered tracheal replacement in a child: A 2-year follow-up study

BACKGROUND Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor β to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

Tracking the Evolution of Non–Small-Cell Lung Cancer

BACKGROUND Among patients with non‐small‐cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early‐stage NSCLC. METHODS In this prospective cohort study, we performed multiregion whole‐exome sequencing on 100 early‐stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence‐free survival. RESULTS We observed widespread intratumor heterogeneity for both somatic copy‐number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy‐number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy‐number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10‐4), which remained significant in multivariate analysis. CONCLUSIONS Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601.)

Murine but not human mesenchymal stem cells generate osteosarcoma-like lesions in the lung.

Murine mesenchymal stem cells are capable of differentiation into multiple cell types both in vitro and in vivo and may be good candidates to use as cell therapy for diseased or damaged organs. We have previously reported a method of enriching a population of murine MSCs that demonstrated a diverse differentiation potential both in vitro and in vivo. In this study, we show that this enriched population of murine mesenchymal stem cells embolize within lung capillaries following systemic injection and then rapidly expand within, and invade into, the lung parenchyma, forming tumor nodules. These lesions rarely contain cells bearing the immunohistochemical characteristics of lung epithelium, but they do show the characteristics of immature bone and cartilage that resembles exuberant fracture callus or well‐differentiated osteosarcoma. Our findings indicate that murine mesenchymal stem cells can behave in a manner similar to tumor cells, with dysregulated growth and aberrant differentiation within the alveolar microenvironment after four passages. We demonstrate that unlike human MSCs, MSCs from different mouse strains can acquire chromosomal abnormalities after only a few in vitro passages. Moreover, other parameters, such as mouse strain used, might also play a role in the induction of these tumors. These findings might be clinically relevant for future stem cell therapy studies.

Magnetic Resonance Imaging of Mesenchymal Stem Cells Homing to Pulmonary Metastases Using Biocompatible Magnetic Nanoparticles

The ability of mesenchymal stem cells (MSC) to specifically home to tumors has suggested their potential use as a delivery vehicle for cancer therapeutics. MSC integration into tumors has been shown in animal models using histopathologic techniques after animal sacrifice. Tracking the delivery and engraftment of MSCs into human tumors will need in vivo imaging techniques. We hypothesized that labeling MSCs with iron oxide nanoparticles would enable in vivo tracking with magnetic resonance imaging (MRI). Human MSCs were labeled in vitro with superparamagnetic iron oxide nanoparticles, with no effect on differentiation potential, proliferation, survival, or migration of the cells. In initial experiments, we showed that as few as 1,000 MSCs carrying iron oxide nanoparticles can be detected by MRI one month after their coinjection with breast cancer cells that formed subcutaneous tumors. Subsequently, we show that i.v.- injected iron-labeled MSCs could be tracked in vivo to multiple lung metastases using MRI, observations that were confirmed histologically. This is the first study to use MRI to track MSCs to lung metastases in vivo. This technique has the potential to show MSC integration into human tumors, allowing early-phase clinical studies examining MSC homing in patients with metastatic tumors.

A general mechanism for intracellular toxicity of metal-containing nanoparticles

We demonstrate a general mechanism for the toxicity induced by metal-containing NPs, named “lysosome-enhanced Trojan horse effect”, which provides design rules to engineer safer NPs.

Suitability of endobronchial ultrasound-guided transbronchial needle aspiration specimens for subtyping and genotyping of non-small cell lung cancer: a multicenter study of 774 patients.

RATIONALE The current management of advanced non-small cell lung cancer (NSCLC) requires differentiation between squamous and nonsquamous subtypes as well as epidermal growth factor receptor (EGFR) mutation status. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis and staging of lung cancer. However, it is unclear whether cytology specimens obtained with EBUS-TBNA are suitable for the subclassification and genotyping of NSCLC. OBJECTIVES To determine whether cytology specimens obtained from EBUS-TBNA in routine practice are suitable for phenotyping and genotyping of NSCLC. METHODS Cytological diagnoses from EBUS-TBNA were recorded from 774 patients with known or suspected lung cancer across five centers in the United Kingdom between 2009 and 2011. MEASUREMENTS AND MAIN RESULTS The proportion of patients with a final diagnosis by EBUS-TBNA in whom subtype was classified was 77% (95% confidence interval [CI], 73-80). The rate of NSCLC not otherwise specified (NSCLC-NOS) was significantly reduced in patients who underwent immunohistochemistry (adjusted odds ratio, 0.50; 95% CI, 0.28-0.82; P = 0.016). EGFR mutation analysis was possible in 107 (90%) of the 119 patients in whom mutation analysis was requested. The sensitivity, negative predictive value, and diagnostic accuracy of EBUS-TBNA in patients with NSCLC were 88% (95% CI, 86-91), 72% (95% CI, 66-77), and 91% (95% CI, 89-93), respectively. CONCLUSIONS This large, multicenter, pragmatic study demonstrates that cytology samples obtained from EBUS-TBNA in routine practice are suitable for subtyping of NSCLC and EGFR mutation analysis and that the use of immunohistochemistry reduces the rate of NSCLC-NOS.

New roles for integrins in squamous-cell carcinoma

Although integrins are known to mediate invasion and metastasis, recent studies reveal new ways in which they contribute to squamous-cell carcinoma. Integrin mutation or upregulation can expand the tumour stem-cell compartment by inhibiting differentiation or apoptosis. Integrins that are expressed by differentiated cells can stimulate or inhibit the proliferation of neighbouring tumour stem cells. These findings provide a mechanistic basis for the well-established links between altered integrin expression and tumour prognosis.

Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes

Introduction The human genome is peppered with mobile repetitive elements called long interspersed nuclear element–1 (L1) retrotransposons. Propagating through RNA and cDNA intermediates, these molecular parasites copy and insert themselves throughout the genome, with potentially disruptive effects on neighboring genes or regulatory sequences. In the germ line, unique sequence downstream of L1 elements can also be retrotransposed if transcription continues beyond the repeat, a process known as 3′ transduction. There has been growing interest in retrotransposition and 3′ transduction as a possible source of somatic mutations during tumorigenesis. The activity of individual L1 elements fluctuates during tumor evolution. In a lung tumor, hundreds of 3′ transductions arose from a small number of active L1 source elements (colored circles on outer rim of circle). As the tumor evolved from the preinvasive common ancestor to invasive cancer, individual elements exhibited variable activity over time. Rationale To explore whether 3′ transductions are frequent in cancer, we developed a bioinformatic algorithm for identifying somatically acquired retrotranspositions in cancer genomes. We applied our algorithm to 290 cancer samples from 244 patients across 12 tumor types. The unique downstream sequence mobilized with 3′ transductions effectively fingerprints the L1 source element, providing insights into the activity of individual L1 loci across the genome. Results Across the 290 samples, we identified 2756 somatic L1 retrotranspositions. Tumors from 53% of patients had at least one such event, with colorectal and lung cancers being most frequently affected (93% and 75% of patients, respectively). Somatic 3′ transductions comprised 24% of events, half of which represented mobilizations of unique sequence alone, without any accompanying L1 sequence. Overall, 95% of 3′ transductions identified derived from only 72 germline L1 source elements, with as few as four loci accounting for 50% of events. In a given sample, the same source element could generate 50 or more somatic transductions, scattered extensively across the genome. About 5% of somatic transductions arose from L1 source elements that were themselves somatic retrotranspositions. In three of the cases in which we sequenced more than one sample from a patient’s tumor, we were able to place 3′ transductions on the phylogenetic tree. We found that the activity of individual source elements fluctuated during tumor evolution, with different subclones exhibiting much variability in which elements were “on” and which were “off.” The ability to identify the individual L1 source elements active in a given tumor enabled us to study the promoter methylation of those elements specifically. We found that 3′ transduction activity in a patient’s tumor was always associated with hypomethylation of that element. Overall, 2.3% of transductions distributed exons or entire genes to other sites in the genome, and many more mobilized deoxyribonuclease I (DNAse-I) hypersensitive sites or transcription factor binding sites identified by the ENCODE project. Occasionally, somatic L1 insertions inserted near coding sequence and redistributed these exons elsewhere in the genome. However, we found no general effects of retrotranspositions on transcription levels of genes at the insertion points and no evidence for aberrant RNA species resulting from somatically acquired transposable elements. Indeed, as with germline retrotranspositions, somatic insertions exhibited a strong enrichment in heterochromatic, gene-poor regions of the genome. Conclusion Somatic 3′ transduction occurs frequently in human tumors, and in some cases transduction events can scatter exons, genes, and regulatory elements widely across the genome. Dissemination of these sequences appears to be due to a small number of highly active L1 elements, whose activity can wax and wane during tumor evolution. The majority of the retrotransposition events are likely to be harmless “passenger” mutations. Hitchhiking through the tumor genome Retrotransposons are DNA repeat sequences that are constantly on the move. By poaching certain cellular enzymes, they copy and insert themselves at new sites in the genome. Sometimes they carry along adjacent DNA sequences, a process called 3′ transduction. Tubio et al. found that 3′ transduction is a common event in human tumors. Because this process can scatter genes and regulatory sequences across the genome, it may represent yet another mechanism by which tumor cells acquire new mutations that help them survive and grow. Science, this issue p. 10.1126/science.1251343 Tumor genomes are peppered with mobile repeat sequences that carry along adjacent DNA when they insert into new genomic sites. Long interspersed nuclear element–1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3′ transduction. Because 3′ transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3′ transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3′ transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3′ transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome.