Analysis of Circulating Tumor DNA Predicts Outcomes of Short Course Consolidation Immunotherapy in Unresectable Stage III Non-Small Cell Lung Cancer.

INTRODUCTION: The current standard of care for patients with inoperable stage III non-small cell lung cancer (NSCLC) includes chemoradiotherapy (CRT) followed by one year of checkpoint inhibitor (CPI) therapy. However, the optimal duration of consolidation CPI remains unknown. Here, we characterized the relationship between circulating tumor DNA (ctDNA) minimal residual disease (MRD) and clinical outcomes of unresectable locally advanced NSCLC patients treated on a phase 2 trial of short course consolidation immunotherapy after CRT, with the goal of testing if ctDNA may be able to identify patients who do not require a full year of treatment. PATIENTS AND METHODS: Plasma samples for ctDNA analysis were collected from patients on the BTCRC LUN 16-081 trial after completion of CRT, prior to C2D1 of CPI (i.e. 1 month after treatment start), and at the end of up to 6 months of treatment. Tumor-informed ctDNA MRD analysis was performed using CAPP-Seq. Levels of ctDNA at each time point were correlated with clinical outcomes. RESULTS: Detection of ctDNA predicted significantly inferior progression-free survival (PFS) after completion of CRT (24-month 29% vs 65%, P = 0.0048), prior to C2D1 of CPI (24-month 0% vs 72%, P < 0.0001) and at the end of CPI (24-month 15% vs 67%, P = 0.0011). Additionally, patients with decreasing or undetectable ctDNA levels after one cycle of CPI had improved outcomes compared to patients with increasing ctDNA levels (24-month PFS 72% vs 0%, P < 0.0001). Progression of disease occurred within <12 months of starting CPI in all patients with increasing ctDNA levels at C2D1. CONCLUSION: Detection of ctDNA before, during, or after 6 months of consolidation CPI is strongly associated with inferior outcomes. Our findings suggest that analysis of ctDNA MRD may enable personalizing the duration of consolidation immunotherapy treatment.

Quantification of cerebrospinal fluid tumor DNA in lung cancer patients with suspected leptomeningeal carcinomatosis.

Cerebrospinal fluid tumor-derived DNA (CSF-tDNA) analysis is a promising approach for monitoring the neoplastic processes of the central nervous system. We applied a lung cancer-specific sequencing panel (CAPP-Seq) to 81 CSF, blood, and tissue samples from 24 lung cancer patients who underwent lumbar puncture (LP) for suspected leptomeningeal disease (LMD). A subset of the cohort (N = 12) participated in a prospective trial of osimertinib for refractory LMD in which serial LPs were performed before and during treatment. CSF-tDNA variant allele fractions (VAFs) were significantly higher than plasma circulating tumor DNA (ctDNA) VAFs (median CSF-tDNA, 32.7%; median plasma ctDNA, 1.8%; P < 0.0001). Concentrations of tumor DNA in CSF and plasma were positively correlated (Spearman's ρ, 0.45; P = 0.03). For LMD diagnosis, cytology was 81.8% sensitive and CSF-tDNA was 91.7% sensitive. CSF-tDNA was also strongly prognostic for overall survival (HR = 7.1; P = 0.02). Among patients with progression on targeted therapy, resistance mutations, such as EGFR T790M and MET amplification, were common in peripheral blood but were rare in time-matched CSF, indicating differences in resistance mechanisms based on the anatomic compartment. In the osimertinib cohort, patients with CNS progression had increased CSF-tDNA VAFs at follow-up LP. Post-osimertinib CSF-tDNA VAF was strongly prognostic for CNS progression (HR = 6.2, P = 0.009). Detection of CSF-tDNA in lung cancer patients with suspected LMD is feasible and may have clinical utility. CSF-tDNA improves the sensitivity of LMD diagnosis, enables improved prognostication, and drives therapeutic strategies that account for spatial heterogeneity in resistance mechanisms.

Evaluation of the diagnostic accuracy of exome sequencing and its impact on diagnostic thinking for patients with rare disease in a publicly funded health care system: A prospective cohort study.

PURPOSE: To evaluate the diagnostic utility of publicly funded clinical exome sequencing (ES) for patients with suspected rare genetic diseases. METHODS: We prospectively enrolled 297 probands who met eligibility criteria and received ES across 5 sites in Ontario, Canada, and extracted data from medical records and clinician surveys. Using the Fryback and Thornbury Efficacy Framework, we assessed diagnostic accuracy by examining laboratory interpretation of results and assessed diagnostic thinking by examining the clinical interpretation of results and whether clinical-molecular diagnoses would have been achieved via alternative hypothetical molecular tests. RESULTS: Laboratories reported 105 molecular diagnoses and 165 uncertain results in known and novel genes. Of these, clinicians interpreted 102 of 105 (97%) molecular diagnoses and 6 of 165 (4%) uncertain results as clinical-molecular diagnoses. The 108 clinical-molecular diagnoses were in 104 families (35% diagnostic yield). Each eligibility criteria resulted in diagnostic yields of 30% to 40%, and higher yields were achieved when >2 eligibility criteria were met (up to 45%). Hypothetical tests would have identified 61% of clinical-molecular diagnoses. CONCLUSION: We demonstrate robustness in eligibility criteria and high clinical validity of laboratory results from ES testing. The importance of ES was highlighted by the potential 40% of patients that would have gone undiagnosed without this test.

NFE2L2 Mutations Enhance Radioresistance in Head and Neck Cancer by Modulating Intratumoral Myeloid Cells.

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC-attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations. SIGNIFICANCE: NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

Induction EGFR tyrosine kinase inhibitors prior to definitive chemoradiotherapy in unresectable stage III EGFR-mutated non-small cell lung cancer.

INTRODUCTION: Increasing evidence suggests that consolidation durvalumab confers limited benefits for patients with stage III EGFR-mutated NSCLC. Induction or maintenance EGFR tyrosine kinase inhibitors (TKIs) added to concurrent chemoradiotherapy (CRT) may optimize definitive treatment, but there are limited data supporting an induction TKI strategy. METHODS: We evaluated the efficacy and safety of induction EGFR TKIs administered before concurrent CRT in a retrospective series of patients with unresectable locally advanced EGFR-mutated NSCLC. Circulating tumor DNA (ctDNA) analysis was performed on a patient subset using CAPP-seq and correlated with outcomes. RESULTS: Of six patients, three received erlotinib and three osimertinib as induction therapy before CRT. Induction TKIs were administered for a median of 2.5 months. The objective response rate after induction TKI was 83%. One patient had a complete response to induction erlotinib and continued erlotinib for 4 years until local progression, which was treated with CRT. Two patients completed maintenance erlotinib after CRT, and another received consolidation durvalumab. After a median follow-up of 20.5 months, only one patient developed disease recurrence, with rising ctDNA coinciding with recurrence. ctDNA remained undetectable in patients without recurrence, or low-level in a patient receiving maintenance erlotinib. Adverse events were mild and expected, and none developed pneumonitis. CONCLUSION: Induction EGFR TKI before CRT may achieve high disease control rates with promising signs of durability in patients with locally advanced EGFR-mutated NSCLC. ctDNA analysis after CRT can correlate well with clinical outcomes. Prospective studies are needed to define the role of induction EGFR TKIs in this setting.

CD8+ Tumor-Infiltrating Lymphocyte Abundance Is a Positive Prognostic Indicator in Nasopharyngeal Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are immune cell populations found within tumors, critical in the antigen-specific host immune response. In this study, we aimed to elucidate the prognostic significance of CD3+, CD4+, and CD8+ TILs in nasopharyngeal cancer (NPC). EXPERIMENTAL DESIGN: Immune cell infiltration was quantified in NPC samples (n = 50) using RNA-sequencing (RNA-seq) data based on rearranged T-cell receptor (TCR) reads and the Estimation of Stromal and Immune cells in malignant tumors using expression data (ESTIMATE) immune score tool. The differential abundances of TIL subset populations were also characterized through IHC staining of formalin-fixed, paraffin-embedded samples from a training cohort (n = 35), which was a subset of the RNA-seq cohort (n = 50). RESULTS: In the RNA-seq cohort, patients with higher rearranged TCR reads experienced superior 5- and 10-year overall survival (OS; P < 0.001), and disease-free survival (DFS; P < 0.001). Similarly, patients with higher ESTIMATE immune scores experienced superior 5- and 10-year OS (P = 0.024) and DFS (P = 0.007). In the training cohort, high abundances of CD8+ TILs were significantly associated with improved 5- and 10-year OS (P = 0.003) and DFS (P = 0.005). These findings were corroborated in an independent validation cohort (n = 84), and combined analysis of the training and validation cohorts [n = 119 (35+84)], which further demonstrated improved 5- and 10-year survival in terms of locoregional control (P < 0.001) and distant metastasis (P = 0.03). CONCLUSIONS: Taken together, our study highlights the prognostic value of CD8+ TILs in NPC, and the potential of future investigations into cellular-based immunotherapies employing CD8+ lymphocytes.

Genomic Profiling of Bronchoalveolar Lavage Fluid in Lung Cancer.

Genomic profiling of bronchoalveolar lavage (BAL) samples may be useful for tumor profiling and diagnosis in the clinic. Here, we compared tumor-derived mutations detected in BAL samples from subjects with non-small cell lung cancer (NSCLC) to those detected in matched plasma samples. Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) was used to genotype DNA purified from BAL, plasma, and tumor samples from patients with NSCLC. The characteristics of cell-free DNA (cfDNA) isolated from BAL fluid were first characterized to optimize the technical approach. Somatic mutations identified in tumor were then compared with those identified in BAL and plasma, and the potential of BAL cfDNA analysis to distinguish lung cancer patients from risk-matched controls was explored. In total, 200 biofluid and tumor samples from 38 cases and 21 controls undergoing BAL for lung cancer evaluation were profiled. More tumor variants were identified in BAL cfDNA than plasma cfDNA in all stages (P < 0.001) and in stage I to II disease only. Four of 21 controls harbored low levels of cancer-associated driver mutations in BAL cfDNA [mean variant allele frequency (VAF) = 0.5%], suggesting the presence of somatic mutations in nonmalignant airway cells. Finally, using a Random Forest model with leave-one-out cross-validation, an exploratory BAL genomic classifier identified lung cancer with 69% sensitivity and 100% specificity in this cohort and detected more cancers than BAL cytology. Detecting tumor-derived mutations by targeted sequencing of BAL cfDNA is technically feasible and appears to be more sensitive than plasma profiling. Further studies are required to define optimal diagnostic applications and clinical utility. SIGNIFICANCE: Hybrid-capture, targeted deep sequencing of lung cancer mutational burden in cell-free BAL fluid identifies more tumor-derived mutations with increased allele frequencies compared with plasma cell-free DNA. See related commentary by Rolfo et al., p. 2826.

Standardization of rounds on a general paediatric ward: Implementation of a checklist to improve efficiency, quality of rounds, and family satisfaction.

Objective: The purpose of this study was to develop a standardized rounding tool for use on the general paediatric ward and to determine if its use can improve quality of rounds as well as patient and parent satisfaction. Methodology: The study used a pre- and post-intervention prospective observational methodology. The intervention consisted of the implementation of a Checklist Rounding Tool (CRT) entitled the 'Paediatric Inpatient Rounding Checklist (PIRC)' which outlined items deemed essential to discuss during rounds for all patients admitted to the paediatric ward. The PIRC was created by the research team after reviewing the literature and it was peer reviewed by a panel of expert paediatricians. Performance on rounds based on discussion of checklist items as well as patient and parent satisfaction were evaluated by an external observer both pre- and post-PIRC implementation. Results: Four of the five less frequently addressed checklist items were discussed significantly more post-intervention. The Rounds Quality Score was significantly improved after checklist implementation, the pre- and post-intervention scores being 8.24 and 9.61/10, respectively (P-value <0.001). Patient and parent satisfaction were rated higher with the use of the checklist. There was no difference in the duration of rounds between the pre- and post-implementation phases. Conclusion: In summary, utilization of a standardized rounding tool on an inpatient paediatric ward led to improvement in quality of rounds as well as patient and parent satisfaction.

Inferring gene expression from cell-free DNA fragmentation profiles.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.

Noninvasive Early Identification of Therapeutic Benefit from Immune Checkpoint Inhibition.

Although treatment of non-small cell lung cancer (NSCLC) with immune checkpoint inhibitors (ICIs) can produce remarkably durable responses, most patients develop early disease progression. Furthermore, initial response assessment by conventional imaging is often unable to identify which patients will achieve durable clinical benefit (DCB). Here, we demonstrate that pre-treatment circulating tumor DNA (ctDNA) and peripheral CD8 T cell levels are independently associated with DCB. We further show that ctDNA dynamics after a single infusion can aid in identification of patients who will achieve DCB. Integrating these determinants, we developed and validated an entirely noninvasive multiparameter assay (DIREct-On, Durable Immunotherapy Response Estimation by immune profiling and ctDNA-On-treatment) that robustly predicts which patients will achieve DCB with higher accuracy than any individual feature. Taken together, these results demonstrate that integrated ctDNA and circulating immune cell profiling can provide accurate, noninvasive, and early forecasting of ultimate outcomes for NSCLC patients receiving ICIs.

A human lung tumor microenvironment interactome identifies clinically relevant cell-type cross-talk.

BACKGROUND: Tumors comprise a complex microenvironment of interacting malignant and stromal cell types. Much of our understanding of the tumor microenvironment comes from in vitro studies isolating the interactions between malignant cells and a single stromal cell type, often along a single pathway. RESULT: To develop a deeper understanding of the interactions between cells within human lung tumors, we perform RNA-seq profiling of flow-sorted malignant cells, endothelial cells, immune cells, fibroblasts, and bulk cells from freshly resected human primary non-small-cell lung tumors. We map the cell-specific differential expression of prognostically associated secreted factors and cell surface genes, and computationally reconstruct cross-talk between these cell types to generate a novel resource called the Lung Tumor Microenvironment Interactome (LTMI). Using this resource, we identify and validate a prognostically unfavorable influence of Gremlin-1 production by fibroblasts on proliferation of malignant lung adenocarcinoma cells. We also find a prognostically favorable association between infiltration of mast cells and less aggressive tumor cell behavior. CONCLUSION: These results illustrate the utility of the LTMI as a resource for generating hypotheses concerning tumor-microenvironment interactions that may have prognostic and therapeutic relevance.

Integrating genomic features for non-invasive early lung cancer detection.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality1,2; however, a small minority of eligible individuals undergo such screening in the United States3,4. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq)5, a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.

Circulating Tumor DNA Analysis to Assess Risk of Progression after Long-term Response to PD-(L)1 Blockade in NSCLC.

PURPOSE: Treatment with PD-(L)1 blockade can produce remarkably durable responses in patients with non-small cell lung cancer (NSCLC). However, a significant fraction of long-term responders ultimately progress and predictors of late progression are unknown. We hypothesized that circulating tumor DNA (ctDNA) analysis of long-term responders to PD-(L)1 blockade may differentiate those who will achieve ongoing benefit from those at risk of eventual progression. EXPERIMENTAL DESIGN: In patients with advanced NSCLC achieving long-term benefit from PD-(L)1 blockade (progression-free survival ≥ 12 months), plasma was collected at a surveillance timepoint late during/after treatment to interrogate ctDNA by Cancer Personalized Profiling by Deep Sequencing. Tumor tissue was available for 24 patients and was profiled by whole-exome sequencing (n = 18) or by targeted sequencing (n = 6). RESULTS: Thirty-one patients with NSCLC with long-term benefit to PD-(L)1 blockade were identified, and ctDNA was analyzed in surveillance blood samples collected at a median of 26.7 months after initiation of therapy. Nine patients also had baseline plasma samples available, and all had detectable ctDNA prior to therapy initiation. At the surveillance timepoint, 27 patients had undetectable ctDNA and 25 (93%) have remained progression-free; in contrast, all 4 patients with detectable ctDNA eventually progressed [Fisher P < 0.0001; positive predictive value = 1, 95% confidence interval (CI), 0.51-1; negative predictive value = 0.93 (95% CI, 0.80-0.99)]. CONCLUSIONS: ctDNA analysis can noninvasively identify minimal residual disease in patients with long-term responses to PD-(L)1 blockade and predict the risk of eventual progression. If validated, ctDNA surveillance may facilitate personalization of the duration of immune checkpoint blockade and enable early intervention in patients at high risk for progression.

Circulating Tumor DNA Dynamics Predict Benefit from Consolidation Immunotherapy in Locally Advanced Non-Small Cell Lung Cancer.

Circulating tumor DNA (ctDNA) molecular residual disease (MRD) following curative-intent treatment strongly predicts recurrence in multiple tumor types, but whether further treatment can improve outcomes in patients with MRD remains unclear. We applied CAPP-Seq ctDNA analysis to 218 samples from 65 patients receiving chemoradiation therapy (CRT) for locally advanced NSCLC, including 28 patients receiving consolidation immune checkpoint inhibition (CICI). Patients with undetectable ctDNA after CRT had excellent outcomes whether or not they received CICI. Among such patients, one died from CICI-related pneumonitis, highlighting the potential utility of only treating patients with MRD. In contrast, patients with MRD after CRT who received CICI had significantly better outcomes than patients who did not receive CICI. Furthermore, the ctDNA response pattern early during CICI identified patients responding to consolidation therapy. Our results suggest that CICI improves outcomes for NSCLC patients with MRD and that ctDNA analysis may facilitate personalization of consolidation therapy.

Circulating Tumor DNA Analysis for Detection of Minimal Residual Disease After Chemoradiotherapy for Localized Esophageal Cancer.

BACKGROUND & AIMS: Biomarkers are needed to risk stratify after chemoradiotherapy for localized esophageal cancer. These could improve identification of patients at risk for cancer progression and selection of additional therapy. METHODS: We performed deep sequencing (CAncer Personalized Profiling by deep Sequencing, [CAPP-Seq]) analyses of plasma cell-free DNA collected from 45 patients before and after chemoradiotherapy for esophageal cancer, as well as DNA from leukocytes and fixed esophageal tumor biopsy samples collected during esophagogastroduodenoscopy. Patients were treated from May 2010 through October 2015; 23 patients subsequently underwent esophagectomy, and 22 did not undergo surgery. We also sequenced DNA from blood samples from 40 healthy control individuals. We analyzed 802 regions of 607 genes for single-nucleotide variants previously associated with esophageal adenocarcinoma or squamous cell carcinoma. Patients underwent imaging analyses 6-8 weeks after chemoradiotherapy and were followed for 5 years. Our primary aim was to determine whether detection of circulating tumor DNA (ctDNA) after chemoradiotherapy is associated with risk of tumor progression (growth of local, regional, or distant tumors, detected by imaging or biopsy). RESULTS: The median proportion of tumor-derived DNA in total cell-free DNA before treatment was 0.07%, indicating that ultrasensitive assays are needed for quantification and analysis of ctDNA from localized esophageal tumors. Detection of ctDNA after chemoradiotherapy was associated with tumor progression (hazard ratio, 18.7; P < .0001), formation of distant metastases (hazard ratio, 32.1; P < .0001), and shorter disease-specific survival times (hazard ratio, 23.1; P < .0001). A higher proportion of patients with tumor progression had new mutations detected in plasma samples collected after chemoradiotherapy than patients without progression (P = .03). Detection of ctDNA after chemoradiotherapy preceded radiographic evidence of tumor progression by an average of 2.8 months. Among patients who received chemoradiotherapy without surgery, combined ctDNA and metabolic imaging analysis predicted progression in 100% of patients with tumor progression, compared with 71% for only ctDNA detection and 57% for only metabolic imaging analysis (P < .001 for comparison of either technique to combined analysis). CONCLUSIONS: In an analysis of cell-free DNA in blood samples from patients who underwent chemoradiotherapy for esophageal cancer, detection of ctDNA was associated with tumor progression, metastasis, and disease-specific survival. Analysis of ctDNA might be used to identify patients at highest risk for tumor progression.

Functional significance of U2AF1 S34F mutations in lung adenocarcinomas.

The functional role of U2AF1 mutations in lung adenocarcinomas (LUADs) remains incompletely understood. Here, we report a significant co-occurrence of U2AF1 S34F mutations with ROS1 translocations in LUADs. To characterize this interaction, we profiled effects of S34F on the transcriptome-wide distribution of RNA binding and alternative splicing in cells harboring the ROS1 translocation. Compared to its wild-type counterpart, U2AF1 S34F preferentially binds and modulates splicing of introns containing CAG trinucleotides at their 3' splice junctions. The presence of S34F caused a shift in cross-linking at 3' splice sites, which was significantly associated with alternative splicing of skipped exons. U2AF1 S34F induced expression of genes involved in the epithelial-mesenchymal transition (EMT) and increased tumor cell invasion. Finally, S34F increased splicing of the long over the short SLC34A2-ROS1 isoform, which was also associated with enhanced invasiveness. Taken together, our results suggest a mechanistic interaction between mutant U2AF1 and ROS1 in LUAD.

Reprogramming the immunological microenvironment through radiation and targeting Axl.

Increasing evidence suggests that ionizing radiation therapy (RT) in combination with checkpoint immunotherapy is highly effective in treating a subset of cancers. To better understand the limited responses to this combination we analysed the genetic, microenvironmental, and immune factors in tumours derived from a transgenic breast cancer model. We identified two tumours with similar growth characteristics but different RT responses primarily due to an antitumour immune response. The combination of RT and checkpoint immunotherapy resulted in cures in the responsive but not the unresponsive tumours. Profiling the tumours revealed that the Axl receptor tyrosine kinase is overexpressed in the unresponsive tumours, and Axl knockout resulted in slower growth and increased radiosensitivity. These changes were associated with a CD8+ T-cell response, which was improved in combination with checkpoint immunotherapy. These results suggest a novel role for Axl in suppressing antigen presentation through MHCI, and enhancing cytokine release, which promotes a suppressive myeloid microenvironment.

Prognostic microRNAs modulate the RHO adhesion pathway: A potential therapeutic target in undifferentiated pleomorphic sarcomas.

A common and aggressive subtype of soft-tissue sarcoma, undifferentiated pleomorphic sarcoma (UPS) was examined to determine the role of micro-RNAs (miRNAs) in modulating distant metastasis. Following histopathologic review, 110 fresh frozen clinically annotated UPS samples were divided into two independent cohorts for Training (42 patients), and Validation (68 patients) analyses. Global miRNA profiling on the Training Set and functional analysis in vitro suggested that miRNA-138 and its downstream RHO-ROCK cell adhesion pathway was a convergent target of miRNAs associated with the development of metastasis. A six-miRNA signature set prognostic of distant metastasis-free survival (DMFS) was developed from Training Set miRNA expression values. Using the six-miRNA signature, patients were successfully categorized into high- and low-risk groups for DMFS in an independent Validation Set, with a hazard ratio (HR) of 2.25 (p = 0.048). After adjusting for other known prognostic variables such as age, gender, tumor grade, size, depth, and treatment with radiotherapy, the six-miRNA signature retained prognostic value with a HR of 3.46 (p < 0.001). A prognostic miRNA biomarker for clinical validation was thus identified along with a functional pathway that modulates UPS metastatic phenotype.

Chromosomal instability as a prognostic marker in cervical cancer.

BACKGROUND: Cervical cancer is the third most common cancer in women globally, and despite treatment, distant metastasis and nodal recurrence will still develop in approximately 30% of patients. The ability to predict which patients are likely to experience distant relapse would allow clinicians to better tailor treatment. Previous studies have investigated the role of chromosomal instability (CIN) in cancer, which can promote tumour initiation and growth; a hallmark of human malignancies. In this study, we sought to examine the published CIN70 gene signature in a cohort of cervical cancer patients treated at the Princess Margaret (PM) Cancer Centre and an independent cohort of The Cancer Genome Atlas (TCGA) cervical cancer patients, to determine if this CIN signature associated with patient outcome. METHODS: Cervical cancer samples were collected from 79 patients, treated between 2000-2007 at the PM, prior to undergoing curative chemo-radiation. Total RNA was extracted from each patient sample and analyzed using the GeneChip Human Genome U133 Plus 2.0 array (Affymetrix). RESULTS: High CIN70 scores were significantly related to increased chromosomal alterations in TCGA cervical cancer patients, including a higher percentage of genome altered and a higher number of copy number alterations. In addition, this same CIN70 signature was shown to be predictive of para-aortic nodal relapse in the PM Cancer Centre cohort. CONCLUSIONS: These findings demonstrate that chromosomal instability plays an important role in cervical cancer, and is significantly associated with patient outcome. For the first time, this CIN70 gene signature provided prognostic value for patients with cervical cancer.

Developing a prognostic micro-RNA signature for human cervical carcinoma.

Cervical cancer remains the third most frequently diagnosed and fourth leading cause of cancer death in women worldwide. We sought to develop a micro-RNA signature that was prognostic for disease-free survival, which could potentially allow tailoring of treatment for cervical cancer patients. A candidate prognostic 9-micro-RNA signature set was identified in the training set of 79 frozen specimens. However, three different approaches to validate this signature in an independent cohort of 87 patients with formalin-fixed paraffin-embedded (FFPE) specimens, were unsuccessful. There are several challenges and considerations associated with developing a prognostic micro-RNA signature for cervical cancer, namely: tumour heterogeneity, lack of concordance between frozen and FFPE specimens, and platform selection for global micro-RNA expression profiling in this disease. Our observations provide an important cautionary tale for future miRNA signature studies for cervical cancer, which can also be potentially applicable to miRNA profiling studies involving other types of human malignancies.