Ricky Thakrar

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.)

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

RATIONALE Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

Genetically modified mesenchymal stromal cells in cancer therapy

The cell therapy industry has grown rapidly over the past 3 decades, and multiple clinical trials have been performed to date covering a wide range of diseases. The most frequently used cell is mesenchymal stromal cells (MSCs), which have been used largely for their anti-inflammatory actions and in situations of tissue repair and although they have demonstrated a good safety profile, their therapeutic efficacy has been limited. In addition to these characteristics MSCs are being used for their homing and engraftment properties and have been genetically modified to enable targeted delivery of a variety of therapeutic agents in both malignant and nonmalignant conditions. This review discusses the science and technology behind genetically modified MSC therapy in malignant disease and how potential problems have been overcome to enable their use in two novel clinical trials in metastatic gastrointestinal and lung cancer.

Combined cell-gene therapy for lung cancer: rationale, challenges and prospects

Lung cancer is the leading cause of cancer-related deaths worldwide, with a 5-year survival of approximately 6%.[1] Around 80% of these are of non-small-cell (NSCLC) histological type for which surgical resection or radical chemoradiotherapy offers the best prospect of cure. The vast majority of cases however are diagnosed at an advanced stage, and therapy options are limited. Although, cisplatin-based chemotherapy trials in this group show a clear survival benefit over supportive care alone, the actual increase in median survival is small (4 vs. 5.5 months).[2] The landscape of treating stage IV disease changed around a decade ago with the discovery of the epidermal growth factor receptor (EGFR) mutations.[3] These were found in lung adenocarcinoma and were associated with a favorable response to EGFR tyrosine kinase inhibitors. Whilst new targeted therapies have been introduced in routine clinical practice, they are only suitable for a low percentage of patients and outside of this outlook has changed little.[3] A better understanding of genetic changes in NSCLC has pushed the development of novel treatments for oncogenic drivers or actionable mutations as identified by preclinical evidence. However, only a small number of patients will benefit. Novel therapies are needed and we have developed a cell and gene therapy for the treatment of metastatic lung cancer. In this article, we discuss some hurdles we face as we take our product into man in our recently funded phase I/II randomized trial, TACTICAL (Targeted stem cells expressing TRAIL as a therapy for lung cancer).

Optimized isolation and expansion of human airway epithelial basal cells from endobronchial biopsy samples

Autologous airway epithelial cells have been used in clinical tissue‐engineered airway transplantation procedures with a view to assisting mucosal regeneration and restoring mucociliary escalator function. However, limited time is available for epithelial cell expansion due to the urgent nature of these interventions and slow epithelial regeneration has been observed in patients. Human airway epithelial cells can be expanded from small biopsies or brushings taken during bronchoscopy procedures, but the optimal mode of tissue acquisition from patients has not been investigated. Here, we compared endobronchial brushing and endobronchial biopsy samples in terms of their cell number and their ability to initiate basal epithelial stem cell cultures. We found that direct co‐culture of samples with 3T3‐J2 feeder cells in culture medium containing a Rho‐associated protein kinase inhibitor, Y‐27632, led to the selective expansion of greater numbers of basal epithelial stem cells during the critical early stages of culture than traditional techniques. Additionally, we established the benefit of initiating cell cultures from cell suspensions, either using brushing samples or through enzymatic digestion of biopsies, over explant culture. Primary epithelial cell cultures were initiated from endobronchial biopsy samples that had been cryopreserved before the initiation of cell cultures, suggesting that cryopreservation could eliminate the requirement for close proximity between the clinical facility in which biopsy samples are taken and the specialist laboratory in which epithelial cells are cultured. Overall, our results suggest ways to expedite epithelial cell preparation in future airway cell therapy or bioengineered airway transplantation procedures.

S9 The role of LRIG1-dependent EGFR signalling in airway homoeostasis and squamous cell lung cancer development

Background Aberrations of EGFR signalling drive cancer development. In squamous cell lung cancer (SqCLC), EGFR is overexpressed. LRIG1 is a negative regulator of EGFR and patient pre-invasive SqCLC samples show LRIG1 loss, suggesting involvement in early disease pathogenesis. In skin and gut homeostasis, LRIG1 regulates stem cells. In the upper airway, basal cells act as stem cells and are the putative origin of SqCLC. We hypothesise LRIG1 has a key role in airway homeostasis and its loss promotes pre-invasive SqCLC development. Methods Lrig1 EGFP-ires-CreERT2 mice were used to delineate airway LRIG1 expression. Flow sorted LRIG1-positive and -negative murine basal cells were used in 2D and 3D colony-forming, spheroid and proliferation assays. A murine SqCLC model was set up through application of N-Nitrosotris-(2-chloroethyl)urea (NTCU). Pre-invasive lesions and tumour development were compared between wild-type (WT), heterozygous and LRIG1-knockout (KO) animals. Human basal cells obtained from bronchoscopy were sorted according to LRIG1 expression and used directly in colony-forming assays or maintained in primary culture to assess the effect of shRNA knockdown of LRIG1. LRIG1-knockdown cells were assessed in colony-forming and proliferation assays, and differentiation and invasion were assessed using organotypic models. Results LRIG1 is expressed by 40% of airway basal cells. LRIG1-expressing murine basal cells exhibit increased colony-forming capacity (p = 0.0286), spheroid formation (p = 0.0043) and proliferation (p = 0.0043) compared with LRIG1-negative cells. Similarly, LRIG1-expressing human airway basal cells isolated from endobronchial brush biopsy samples exhibit increased colony-forming capacity (p = 0.0469). Topical application of NTCU to mice recapitulates the development of human pre-invasive and SCLC lesions after 23 weeks. Results show lesions in LRIG1-KO mice to be larger than those of WT animals. Knock down of LRIG1 in cultured human airway basal cells alters cell phenotype, leading to an increased colony-forming efficiency and greater proliferation at cell confluence. Conclusions LRIG1 has an important role in stem cell homeostasis of the human and murine airway epithelium. Loss of LRIG1 promotes pre-cancerous lesion development in a murine SqCLC mouse model and behaviour of human epithelial cells in culture, indicating a potential target for chemoprevention of SqCLC in humans.

S42 Transbronchial cryobiopsies in the diagnosis of Interstitial Lung Diseases- first UK experience

Introduction Despite radiological advancements histology is often needed in the diagnosis of Interstitial Lung Disease (ILD). In order to obtain lung biopsies of adequate size and quality patients traditionally undergo a surgical lung biopsy associated with a mean hospital stay of 3.5 days and a complication rate of up to 28%.1 We are the first UK centre to have set up a minimally invasive, day case transbronchial cryobiopsy service in the diagnosis of ILDs. Aims To establish a transbronchial cryobiopsy service for ILDs and assess complication rates and diagnostic yield. Methods Patients were selected following discussion at the Interstitial Lung Disease Multidisciplinary Team meeting. Only patients in whom significant diagnostic doubt remained after thorough clinic-radiological work up were considered. All procedures were performed with the patient self-ventilating under deep sedation with propofol using a flexible bronchoscope and 2.4 mm cryoprobe (ERBE). All but one procedure was performed under fluoroscopy guidance. All but the first two cases were performed following intubation with an uncuffed ET tube (Bronchoflex, Rusch). We introduced prophylactic IV tranexamic acid 1 g as premedication and endobronchial adrenaline routinely as part of our protocol after the first 5 cases. 1–5 samples were taken per procedure from several sub-segments of one lobe pre-selected by CT imaging. Results 14 procedures were carried out on 13 patients. 9 patients were male. Mean age 63, mean predicted TLCO 50%; mean number of biopsies 2.7; mean aggregate biopsy size 624 mm3. Complications: 2 pneumothoraces requiring chest drain insertion; 1 case of moderate bleeding and 1 case of severe bleeding managed with endobronchial adrenaline and suction only. All cases were performed as outpatient day cases with patients discharged home 2– 4 h after the procedure except the 2 patients requiring chest drains.Abstract S42 Table 1 Patient characteristics Case Sex Age Number of biopsies Aggregate biopsy size in mm3 Diagnosis 1 M 66 3 1800 Normal lung parenchyma 2 M 75 2 118 UIP 3 F 60 3 410 NSIP 4 M 66 1 125 Interstitial fibrosis 5 M 64 1 60 HP 6 M 44 1 80 NSIP 7 F 73 4 147 HP 8 M 74 5 1200 CLL 9 M 68 2 1120 Interstitial fibrosis 10 M 41 4 810 Organising Pneumonia 11 M 59 3 168 Interstitial fibrosis 12 F 57 4 2000 UIP 13 M 64 1 75 Non-diagnostic 14 F 67 2 pending pending Conclusions Our transbronchial cryobiopsy service is comparable in complication rates and diagnostic yield with larger case series from established centres outside the UK. Transbronchial cryobiopsy provides potential for minimally-invasive acquisition of sizeable lung biopsies. Further research is needed to establish whether transbronchial cryobiopsy should replace surgical lung biopsy in selected cases. Reference 1 Morris D, Zamvar V. The efficacy of video-assisted thoracoscopic surgery lung biopsies in patients with interstitial lung disease: a retrospective study of 66 patients. J Cardiothorac Surg. 2014;9:45

S108 MIF as the key regulator for mesenchymal stem cells homing to tumours by 3D and in vivo lung metastasis models

Mesenchymal stromal cells (MSCs) are inherently tumour-homing and can be isolated, expanded and transduced, making them viable candidates for cell therapy. This tumour-tropism has been used to deliver anti-cancer therapies to various tumour models in several organs. In a previous study we have shown that MIF is the key director of MSC migration and infiltration towards tumour cells. We have shown this major role for MIF (mainly via CXCR4), using in vitro migration and invasion assays, in presence of different receptor inhibitors and achieving a drastic decrease in both processes using MIF inhibitor. Importantly we show that knock down of either CXCR4 or MIF abrogates MSC homing to tumours in an in vivo pulmonary metastasis model, confirming the in vitro 2D and 3D assays. In this study we define the mechanism behind MIF stimulation of MSC homing to tumours. We show that MIF upregulates other cytokines involved in chemotaxis, such as IL6, IL8 and CCL2 and upregulates MIF as well, amplifying the initial trigger and generating a positive feedback loop. However when inhibiting those cytokines individually, we never achieved a decrease in migration as drastic as for MIF inhibition. This suggests that the up-regulation of this set of cytokines would lead to chemoattraction of leucocytes to the site of the tumour, which was observed in a 3D model. Therefore, MIF trigger is amplified by its own upregulation in MSCs via a positive feedback loop, confirming again our previous findings and its key role as a regulator of MSC homing to tumours. This improved understanding of MSC tumour tropism will further enable development of novel cellular therapies for cancers.