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Background: Activation and regulation of androgen receptor (AR) signaling and the DNA damage response impact the prostate cancer (PCa) treatment modalities of androgen deprivation therapy (ADT) and radiotherapy. Here, we have evaluated a role for human single-strand binding protein 1 (hSSB1/NABP2) in modulation of the cellular response to androgens and ionizing radiation (IR). hSSB1 has defined roles in transcription and maintenance of genome stability, yet little is known about this protein in PCa. Methods: We correlated hSSB1 with measures of genomic instability across available PCa cases from The Cancer Genome Atlas (TCGA). Microarray and subsequent pathway and transcription factor enrichment analysis were performed on LNCaP and DU145 prostate cancer cells. Results: Our data demonstrate that hSSB1 expression in PCa correlates with measures of genomic instability including multigene signatures and genomic scars that are reflective of defects in the repair of DNA double-strand breaks via homologous recombination. In response to IR-induced DNA damage, we demonstrate that hSSB1 regulates cellular pathways that control cell cycle progression and the associated checkpoints. In keeping with a role for hSSB1 in transcription, our analysis revealed that hSSB1 negatively modulates p53 and RNA polymerase II transcription in PCa. Of relevance to PCa pathology, our findings highlight a transcriptional role for hSSB1 in regulating the androgen response. We identified that AR function is predicted to be impacted by hSSB1 depletion, whereby this protein is required to modulate AR gene activity in PCa. Conclusions: Our findings point to a key role for hSSB1 in mediating the cellular response to androgen and DNA damage via modulation of transcription. Exploiting hSSB1 in PCa might yield benefits as a strategy to ensure a durable response to ADT and/or radiotherapy and improved patient outcomes. Refereed/Peer-reviewed

[Introduction] In CheckMate 227, nivolumab plus ipilimumab prolonged overall survival (OS) versus chemotherapy in patients with tumor programmed death-ligand 1 (PD-L1) greater than or equal to 1% (primary end point) or less than 1% (prespecified descriptive analysis). We report results with minimum 4 years' follow-up., [Methods] Adults with previously untreated stage IV or recurrent NSCLC were randomized (1:1:1) to nivolumab plus ipilimumab, nivolumab, or chemotherapy (PD-L1 ≥1%); or to nivolumab plus ipilimumab, nivolumab plus chemotherapy, or chemotherapy (PD-L1, [Results] After 54.8 months' median follow-up, OS remained longer with nivolumab plus ipilimumab versus chemotherapy in patients with PD-L1 greater than or equal to 1% (hazard ratio = 0.76; 95% confidence interval: 0.65–0.90) and PD-L1 less than 1% (0.64; 0.51–0.81); 4-year OS rate with nivolumab plus ipilimumab versus chemotherapy was 29% versus 18% (PD-L1 ≥1%); and 24% versus 10% (PD-L1, [Conclusions] At more than 4 years' minimum follow-up, with all patients off immunotherapy treatment for at least 2 years, first-line nivolumab plus ipilimumab continued to demonstrate durable long-term efficacy in patients with advanced NSCLC. No new safety signals were identified. Immune-mediated AEs occurred early and resolved quickly with guideline-based management. Discontinuation of nivolumab plus ipilimumab due to TRAEs did not have a negative impact on the long-term benefits seen in all randomized patients., This study was supported by Bristol Myers Squibb and Ono Pharmaceutical Company Ltd.

Chemotherapy is used as a standard-of-care against cancers that display high levels of inherent genome instability. Chemotherapy induces DNA damage and intensifies pressure on the DNA repair pathways that can lead to deregulation. There is an urgent clinical need to be able to track the emergence of DNA repair driven chemotherapy resistance and tailor patient staging appropriately. There have been numerous studies into chemoresistance but to date no study has elucidated in detail the roles of the key DNA repair components in resistance associated with the frontline clinical combination of anthracyclines and taxanes together. In this study, we hypothesized that the emergence of chemotherapy resistance in triple negative breast cancer was driven by changes in functional signaling in the DNA repair pathways. We identified that consistent pressure on the non-homologous end joining pathway in the presence of genome instability causes failure of the key kinase DNA-PK, loss of p53 and compensation by p73. In-turn a switch to reliance on the homologous recombination pathway and RAD51 recombinase occurred to repair residual double strand DNA breaks. Further we demonstrate that RAD51 is an actionable target for resensitization to chemotherapy in resistant cells with a matched gene expression profile of resistance highlighted by homologous recombination in clinical samples.

Platinum-based chemotherapy remains the cornerstone of treatment for most non-small cell lung cancer (NSCLC) cases either as maintenance therapy or in combination with immunotherapy. However, resistance remains a primary issue. Our findings point to the possibility of exploiting levels of cell division cycle associated protein-3 (CDCA3) to improve response of NSCLC tumours to therapy. We demonstrate that in patients and in vitro analyses, CDCA3 levels correlate with measures of genome instability and platinum sensitivity, whereby CDCA3high tumours are sensitive to cisplatin and carboplatin. In NSCLC, CDCA3 protein levels are regulated by the ubiquitin ligase APC/C and cofactor Cdh1. Here, we identified that the degradation of CDCA3 is modulated by activity of casein kinase 2 (CK2) which promotes an interaction between CDCA3 and Cdh1. Supporting this, pharmacological inhibition of CK2 with CX-4945 disrupts CDCA3 degradation, elevating CDCA3 levels and increasing sensitivity to platinum agents. We propose that combining CK2 inhibitors with platinum-based chemotherapy could enhance platinum efficacy in CDCA3low NSCLC tumours and benefit patients., Kildey et al find that high levels of mitotic regulator CDCA3 correlates with sensitivity to platinum agents in non-small cell lung cancer patients and cell lines. They show that interfering with CDCA3 degradation through CK2 inhibition enhances CDCA3 levels and increases sensitivity to platinum agents suggesting a therapeutic route.

Simple Summary PIM kinases interact with major oncogenic players, including the PI3K/Akt pathway, and provide an escape mechanism leading to drug resistance. This study examined PIM kinase expression in NSCLC and the potential of PIM1 as a prognostic marker. The effect on cell signaling of novel preclinical PI3K/mTOR/PIM kinase inhibitor IBL-301 was compared to PI3K/mTOR inhibition in vitro and ex vivo. PI3K-mTOR inhibitor sensitive (H1975P) and resistant (H1975GR) cells were compared for altered IL6/STAT3 pathway expression and sensitivity to IBL-301. All three PIM kinases are expressed in NSCLC and PIM1 is a marker of poor prognosis. IBL-301 inhibited c-Myc, the PI3K-Akt and JAK/STAT pathways in vitro and in NSCLC tumor tissue explants. IBL-301 also inhibited secreted pro-inflammatory cytokine MCP-1. PIM kinases were activated in H1975GR cells which were more sensitive to IBL-301 than H1975P cells. A miRNA signature of PI3K-mTOR resistance was validated. Co-targeting PIM kinase and PI3K-mTOR warrants further clinical investigation. Abstract PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.

DNA repair and metabolic pathways are vital to maintain cellular homeostasis in normal human cells. Both of these pathways, however, undergo extensive changes during tumorigenesis, including modifications that promote rapid growth, genetic heterogeneity, and survival. While these two areas of research have remained relatively distinct, there is growing evidence that the pathways are interdependent and intrinsically linked. Therapeutic interventions that target metabolism or DNA repair systems have entered clinical practice in recent years, highlighting the potential of targeting these pathways in cancer. Further exploration of the links between metabolic and DNA repair pathways may open new therapeutic avenues in the future. Here, we discuss the dependence of DNA repair processes upon cellular metabolism; including the production of nucleotides required for repair, the necessity of metabolic pathways for the chromatin remodeling required for DNA repair, and the ways in which metabolism itself can induce and prevent DNA damage. We will also discuss the roles of metabolic proteins in DNA repair and, conversely, how DNA repair proteins can impact upon cell metabolism. Finally, we will discuss how further research may open therapeutic avenues in the treatment of cancer.

Background: Circulating tumour DNA (ctDNA) has emerged as a promising biomarker for monitoring non-small-cell lung cancer (NSCLC). This has enabled the monitoring of clinically actionable mutations over the course of therapy. Case presentation: We present the case of a 74-year-old female who was treated with EGFR inhibitors for NSCLC which later transformed to SCLC. The kinetics of ctDNA was monitored by measuring the EGFR Exon 19 mutant L747–A750 > P and PIK3CA E545K over the course of therapy. Stabilisation of the PIK3CA mutation was found in response to therapy, however there appeared to be increasing levels of the EGFR mutant, potentially reflective of untreated EGFR-driven disease. Conclusion: The key finding described here, revealed by mutational tracking of mutations from the blood, is that clinically actionable mutations are assessable and may demonstrate clinical utility in measuring disease burden, multiple clones and progression non-invasively for lung cancer.

Epidermal growth factor receptor (EGFR) mutations are the most common oncogenic drivers in non-small-cell lung cancer (NSCLC). Significant developments have taken place which highlight the differences in tumor biology that exist between the mutant and wild-type subtypes of NSCLC. Patients with advanced EGFR-mutant NSCLC have a variety of EGFR-targeting agents available proven to treat their disease. This has led to superior patient outcomes when used as a monotherapy over traditional cytotoxic systemic therapy. Attempts at combining EGFR agents with other anticancer systemic treatment options, such as chemotherapy, antiangiogenic agents, and immunotherapy, have shown varied outcomes. Currently, no specific combination stands out to cause a shift away from the use of single-agent EGFR inhibitors in the first-line setting. Similarly, adjuvant EGFR inhibitors, are yet to significantly add to patient overall survival if used at earlier timepoints in the disease course. Liquid biopsy is an evolving technology with potential promise of being incorporated into the management paradigm of this disease. Data are emerging to suggest that this technique may be capable of identifying early resistance mechanisms and consequential disease progression on the basis of the analysis of blood-based circulating tumor cells.

Despite advances in our understanding of the molecular biology of the disease and improved therapeutics, lung cancer remains the most common cause of cancer-related deaths worldwide. Therefore, an unmet need remains for improved treatments, especially in advanced stage disease. Genomic instability is a universal hallmark of all cancers. Many of the most commonly prescribed chemotherapeutics, including platinum-based compounds such as cisplatin, target the characteristic genomic instability of tumors by directly damaging the DNA. Chemotherapies are designed to selectively target rapidly dividing cells, where they cause critical DNA damage and subsequent cell death (1, 2). Despite the initial efficacy of these drugs, the development of chemotherapy resistant tumors remains the primary concern for treatment of all lung cancer patients. The correct functioning of the DNA damage repair machinery is essential to ensure the maintenance of normal cycling cells. Dysregulation of these pathways promotes the accumulation of mutations which increase the potential of malignancy. Following the development of the initial malignancy, the continued disruption of the DNA repair machinery may result in the further progression of metastatic disease. Lung cancer is recognized as one of the most genomically unstable cancers (3). In this review, we present an overview of the DNA damage repair pathways and their contributions to lung cancer disease occurrence and progression. We conclude with an overview of current targeted lung cancer treatments and their evolution toward combination therapies, including chemotherapy with immunotherapies and antibody-drug conjugates and the mechanisms by which they target DNA damage repair pathways.

Tumor tissue biopsy is often limited for non-small cell lung cancer (NSCLC) patients and alternative sources of tumoral information are desirable to determine molecular alterations such as anaplastic lymphoma kinase (ALK) rearrangements. Circulating tumor cells (CTCs) are an appealing component of liquid biopsies, which can be sampled serially over the course of treatment. In this study, we enrolled a cohort of ALK-positive (n = 8) and ALK-negative (n = 12) NSCLC patients, enriched for CTCs using spiral microfluidic technology and performed DNA fluorescent in situ hybridization (FISH) for ALK. CTCs were identified in 12/20 NSCLC patients ranging from 1 to 26 CTCs/7.5 mL blood. Our study revealed that 3D imaging of CTCs for ALK translocations captured a well-defined separation of 3′ and 5′ signals indicative of ALK translocations and overlapping 3′/5′ signal was easily resolved by imaging through the nuclear volume. This study provides proof-of-principle for the use of 3D DNA FISH in the determination of CTC ALK translocations in NSCLC.

PURPOSE CheckMate 568 is an open-label phase II trial that evaluated the efficacy and safety of nivolumab plus low-dose ipilimumab as first-line treatment of advanced/metastatic non–small-cell lung cancer (NSCLC). We assessed the association of efficacy with programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB). PATIENTS AND METHODS Two hundred eighty-eight patients with previously untreated, recurrent stage IIIB/IV NSCLC received nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks. The primary end point was objective response rate (ORR) in patients with 1% or more and less than 1% tumor PD-L1 expression. Efficacy on the basis of TMB (FoundationOne CDx assay) was a secondary end point. RESULTS Of treated patients with tumor available for testing, 252 patients (88%) of 288 were evaluable for PD-L1 expression and 98 patients (82%) of 120 for TMB. ORR was 30% overall and 41% and 15% in patients with 1% or greater and less than 1% tumor PD-L1 expression, respectively. ORR increased with higher TMB, plateauing at 10 or more mutations/megabase (mut/Mb). Regardless of PD-L1 expression, ORRs were higher in patients with TMB of 10 or more mut/Mb (n = 48: PD-L1, ≥ 1%, 48%; PD-L1, < 1%, 47%) versus TMB of fewer than 10 mut/Mb (n = 50: PD-L1, ≥ 1%, 18%; PD-L1, < 1%, 5%), and progression-free survival was longer in patients with TMB of 10 or more mut/Mb versus TMB of fewer than 10 mut/Mb (median, 7.1 v 2.6 months). Grade 3 to 4 treatment-related adverse events occurred in 29% of patients. CONCLUSION Nivolumab plus low-dose ipilimumab was effective and tolerable as a first-line treatment of advanced/metastatic NSCLC. TMB of 10 or more mut/Mb was associated with improved response and prolonged progression-free survival in both tumor PD-L1 expression 1% or greater and less than 1% subgroups and was thus identified as a potentially relevant cutoff in the assessment of TMB as a biomarker for first-line nivolumab plus ipilimumab.

Non-small cell lung cancer (NSCLC) accounts for a large proportion of cancer deaths and is characterized by low treatment response rates and poor overall prognosis. In the absence of specific treatable mutations, cisplatin-based chemotherapy plays an important role in the treatment of this disease. Unfortunately, the development of resistance has become a major therapeutic challenge in the use of this cytotoxic drug. Elucidating the mechanisms underlying this resistance phenotype, may result in the development of novel agents that enhance sensitivity to cisplatin in lung cancer patients. In this study, targeting the cancer stem cell activity of aldehyde dehydrogenase 1 (ALDH1) was investigated as a strategy to overcome chemoresistance in NSCLC. Tumors from NSCLC patients showed an increase in their profile of pluripotent stemness genes. Cisplatin exposure induced the emergence or expansion of an ALDH1-positive subpopulation in cisplatin sensitive and resistant NSCLC cell lines, respectively, further enhancing cisplatin resistance. Using the Aldefluor assay and FACS analysis, ALDH1 subpopulations were isolated and evaluated in terms of stem cell characteristics. Only ALDH1-positive cells exhibited asymmetric division, cisplatin resistance and increased expression of stem cell factors in vitro. Xenograft studies in NOD/SCID mice demonstrated efficient tumorigenesis from low cell numbers of ALDH1-positive and ALDH1-negative subpopulations. Targeting ALDH1 with Diethylaminobenzaldehyde (DEAB) and Disulfiram, significantly re-sensitized resistant lung cancer cells to the cytotoxic effects of cisplatin. Our data demonstrate the existence of a lung CSC population and suggest a role for targeting ALDH1 as a potential therapeutic strategy in re-sensitizing NSCLC cells to the cytotoxic effects of cisplatin.

SASH1 (SAM and SH3 domain-containing protein 1) is a tumor suppressor protein that has roles in key cellular processes including apoptosis and cellular proliferation. As these cellular processes are frequently disrupted in human tumours and little is known about the role of SASH1 in the pathogenesis of the disease, we analysed the prognostic value of SASH1 in non-small cell lung cancers using publicly available datasets. Here, we show that low SASH1 mRNA expression is associated with poor survival in adenocarcinoma. Supporting this, modulation of SASH1 levels in a panel of lung cancer cell lines mediated changes in cellular proliferation and sensitivity to cisplatin. The treatment of lung cancer cells with chloropyramine, a compound that increases SASH1 protein concentrations, reduced cellular proliferation and increased sensitivity to cisplatin in a SASH1-dependent manner. In summary, compounds that increase SASH1 protein levels could represent a novel approach to treat NSCLC and warrant further study.

Immunotherapies have shown long‐lasting and unparalleled responses for cancer patients compared to conventional therapy. However, they seem to only be effective in a subset of patients. Therefore, it has become evident that a greater understanding of the tumor microenvironment (TME) is required to understand the nuances which may be at play for a favorable outcome to therapy. The immune contexture of the TME is an important factor in dictating how well a tumor may respond to immune checkpoint inhibitors. While traditional immunohistochemistry techniques allow for the profiling of cells in the tumor, this is often lost when tumors are analysed using bulk tissue genomic approaches. Moreover, the actual cellular proportions, cellular heterogeneity and deeper spatial distribution are lacking in characterisation. Advances in tissue interrogation technologies have given rise to spatially resolved characterisation of the TME. This review aims to provide an overview of the current methodologies that are used to profile the TME, which may provide insights into the immunopathology associated with a favorable outcome to immunotherapy., In this review, we discuss spatial analysis tools that can be used to characterise the tumor microenvironment. We present a number of technologies that are transforming the field and providing insights into the immunopathology associated with a favorable outcome to immunotherapy.

Identifying and targeting specific oncogenic drivers has become standard of care in the routine management of patients with lung cancer. Research is ongoing to expand the number of drug targets that can offer clinically meaningful outcomes. Rearranged during transfection (RET) fusions are the latest oncogenic driver alterations that show potential as a drug target. RET fusions occur in 1–2% of non-small cell lung cancer (NSCLC) cases. They are more commonly associated with younger age, female gender, non-smokers and Asian ethnicity. The RET kinase is abnormally activated through fusion with a partner protein such as KIF5B, CCDC6 or NCOA4. This leads to downstream intracellular signalling and enhancement of gene transcription and cell proliferation. The effectiveness of multi-kinase inhibitors in RET positive NSCLC has been explored in early phase and retrospective studies. From these studies, the most effective agents identified include cabozantanib and vandetanib. Overall response rates (ORR) vary from 18–47% across studies. In general, these agents have a manageable toxicity profile, although there are a number of off-target toxicities. Similar to the increased activity in ALK rearranged disease, pemetrexed has demonstrated superior response rates in this patient group and should be considered. Selective RET inhibitors, including LOXO-292 and BLU-667, are progressing in clinical trials. LOXO-292 has demonstrated an impressive ORR of 77% in RET positive solid tumours. It is anticipated this agent will be an effective targeted therapeutic option for patients with RET positive lung cancer.

In pre-clinical studies, triple-negative breast cancer (TNBC) cells have demonstrated sensitivity to the multi-targeted kinase inhibitor dasatinib, however, clinical trials with single-agent dasatinib showed limited efficacy in unselected populations of breast cancer, including TNBC. To study potential mechanisms of resistance to dasatinib in TNBC, we established a cell line model of acquired dasatinib resistance (231-DasB). Following an approximately three-month exposure to incrementally increasing concentrations of dasatinib (200 nM to 500 nM) dasatinib, 231-DasB cells were resistant to the agent with a dasatinib IC50 value greater than 5 &mu, M compared to 0.04 &plusmn, 0.001 &micro, M in the parental MDA-MB-231 cells. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970. Treatment of 231-DasB cells with dasatinib did not inhibit phosphorylation of Src kinase. The 231-DasB cells also had significantly increased levels of p-Met compared to the parental MDA-MB-231 cells, as measured by luminex, and resistant cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpdA, with an IC50 value of 1.4 &plusmn, 0.5 &micro, M compared to an IC50 of 6.8 &plusmn, 0.2 &micro, M in the parental MDA-MB-231 cells. Treatment with CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Combined treatment with dasatinib and CpdA significantly inhibited the growth of MDA-MB-231 parental cells and prevented the emergence of dasatinib resistance. If these in vitro findings can be extrapolated to human cancer treatment, combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance and improve response rates to dasatinib treatment in TNBC.

Expression of the SASH1 protein is reduced in a range of human cancers and has been implicated in apoptotic cancer cell death. This study investigated whether increasing SASH1 expression could be a useful therapeutic strategy in breast cancer. Ectopic SASH1 expression increased apoptosis in 7/8 breast cancer cell lines. Subsequent in silico connectivity screening demonstrated that the clinically approved antihistamine drug, chloropyramine, increased SASH1 mRNA levels. Chloropyramine has previously been shown to have anti-tumour activity in breast cancer in part through modulation of FAK signalling, a pathway also regulated by SASH1. This study demonstrated that chloropyramine increased SASH1 protein levels in breast cancer cells. Consistent with this the agent reduced cell confluency in 7/8 cell lines treated irrespective of their ER status but not apoptosis incompetent MCF7 cells. In contrast SASH1 siRNA-transfected breast cancer cells exhibited reduced chloropyramine sensitivity. The prognostic significance of SASH1 expression was also investigated in two breast cancer cohorts. Expression was associated with favourable outcome in ER-positive cases, but only those of low histological grade/proliferative status. Conversely, we found a very strong inverse association in HER2+ disease irrespective of ER status, and in triple-negative, basal-like cases. Overall, the data suggest that SASH1 is prognostic in breast cancer and could have subtype-dependent effects on breast cancer progression. Pharmacologic induction of SASH1 by chloropyramine treatment of breast cancer warrants further preclinical and clinical investigation.

Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) contains a single oligosaccharide/oligonucleotide binding (OB) domain followed by a charged C-terminus and is structurally homologous to the SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus. Recent work has revealed that hSSB1 is critical to homologous recombination and numerous other important biological processes such as the regulation of telomeres, the maintenance of DNA replication forks and oxidative damage repair. Since the ability of hSSB1 to directly interact with single-stranded DNA (ssDNA) is paramount for all of these processes, understanding the molecular details of ssDNA recognition is essential. In this study, we have used solution-state nuclear magnetic resonance in combination with biophysical and functional experiments to structurally analyse ssDNA binding by hSSB1. We reveal that ssDNA recognition in solution is modulated by base-stacking of four key aromatic residues within the OB domain. This DNA binding mode differs significantly from the recently determined crystal structure of the SOSS1 complex containing hSSB1 and ssDNA. Our findings elucidate the detailed molecular mechanism in solution of ssDNA binding by hSSB1, a major player in the maintenance of genomic stability.

Objectives: In non-small cell lung cancers (NSCLC), tumour biopsy can often be an invasive procedure. The development of a non-invasive methodology to study genetic changes via circulating tumour cells (CTCs) is an appealing concept. Whilst CTCs typically remain as rare cells, improvements in epitope-independent CTC isolation techniques has given rise to a greater capture of CTCs. In this cross sectional study, we demonstrate the capture and characterization of NSCLC CTCs for the clinically actionable markers epidermal growth factor receptor (EGFR) alterations, anaplastic lymphoma kinase (ALK) rearrangements and programmed death ligand-1 (PD-L1) expression. The study identified CTCs/CTC clusters in 26/35 Stage IV NSCLC patients, and subsequently characterized the CTCs for EGFR mutation, ALK status and PD-L1 status. This pilot study demonstrates the potential of a non-invasive fluid biopsy to determine clinically relevant biomarkers in NSCLC.

Poster Abstract

The maintenance of genome stability is essential to prevent loss of genetic information and the development of diseases such as cancer. One of the most common forms of damage to the genetic code is the oxidation of DNA by reactive oxygen species (ROS), of which 8-oxo-7,8-dihydro-guanine (8-oxoG) is the most frequent modification. Previous studies have established that human single-stranded DNA-binding protein 1 (hSSB1) is essential for the repair of double-stranded DNA breaks by the process of homologous recombination. Here we show that hSSB1 is also required following oxidative damage. Cells lacking hSSB1 are sensitive to oxidizing agents, have deficient ATM and p53 activation and cannot effectively repair 8-oxoGs. Furthermore, we demonstrate that hSSB1 forms a complex with the human oxo-guanine glycosylase 1 (hOGG1) and is important for hOGG1 localization to the damaged chromatin. In vitro, hSSB1 binds directly to DNA containing 8-oxoguanines and enhances hOGG1 activity. These results underpin the crucial role hSSB1 plays as a guardian of the genome.

Background Afatinib is approved in the US, Europe, and several other regions for first-line treatment for epidermal growth factor receptor mutation-positive (EGFRm+) non-small-cell lung cancer (NSCLC). Patients and Methods Treatment-naive patients with advanced EGFRm+ NSCLC were randomized to afatinib (40 mg/d) versus cisplatin/pemetrexed (LUX-Lung 3 [LL3]) or cisplatin/gemcitabine (LUX-Lung 6 [LL6]), or versus gefitinib (250 mg/d; LUX-Lung 7 [LL7]). We report subgroup analyses according to age, including 65 years or older versus younger than 65 years (preplanned; LL3/LL6) and additional cutoffs up to 75 years and older (exploratory; LL7). Progression-free survival (PFS), overall survival (OS), and adverse events (AEs) were evaluated. Results Among the 134 of 345 (39%) and 86 of 364 (24%) patients aged 65 years and older in LL3 and LL6, median PFS was improved with afatinib versus chemotherapy (LL3: hazard ratio [HR], 0.64 [95% confidence interval (CI), 0.39-1.03]; LL6: HR, 0.16 [95% CI, 0.07-0.39]). Afatinib significantly improved OS versus chemotherapy in elderly patients with Del19+ NSCLC in LL3 (HR, 0.39 [95% CI, 0.19-0.80]). Among the 40 of 319 patients (13%) aged 75 years or older in LL7, median PFS (HR, 0.69 [95% CI, 0.33-1.44]) favored afatinib, consistent with the overall population. Afatinib-associated AEs in older patients were consistent with the overall populations. Conclusions Subgroup analyses of the LL3, LL6, and LL7 trials show that afatinib is an effective and tolerable treatment for patients with EGFRm+ NSCLC, independent of age.

Whilst locoregional control of head and neck cancers (HNCs) has improved over the last four decades, long-term survival has remained largely unchanged. A possible reason for this is that the rate of distant metastasis has not changed. Such disseminated disease is reflected in measurable levels of cancer cells in the blood of HNC patients, referred to as circulating tumour cells (CTCs). Numerous marker-independent techniques have been developed for CTC isolation and detection. Recently, microfluidics-based platforms have come to the fore to avoid molecular bias. In this pilot, proof of concept study, we evaluated the use of the spiral microfluidic chip for CTC enrichment and subsequent detection in HNC patients. CTCs were detected in 13/24 (54%) HNC patients, representing both early to late stages of disease. Importantly, in 7/13 CTC-positive patients, CTC clusters were observed. This is the first study to use spiral microfluidics technology for CTC enrichment in HNC.

Lung cancer is the leading cause of cancer-related deaths. Most patients develop resistance to platinum within several months of treatment. We investigated whether triggering lysosomal membrane permeabilization (LMP) or suppressing autophagy can restore cisplatin susceptibility in lung cancer with acquired chemoresistance. Cisplatin IC50 in A549Pt (parental) and A549cisR (cisplatin resistant) cells was 13 μM and 47 μM, respectively. Following cisplatin exposure, A549cisR cells failed to elicit an apoptotic response. This was manifested by diminished Annexin-V staining, caspase 3 and 9, BAX and BAK activation in resistant but not in parental cells. Chloroquine preferentially promoted LMP in A549cisR cells, revealed by leakage of FITC-dextran into the cytosol as detected by immunofluorescence microscopy. This was confirmed by increased cytosolic cathepsin D signal on Immunoblot. Cell viability of cisplatin-treated A549cisR cells was decreased when co-treated with chloroquine, corresponding to a combination index below 0.8, suggesting synergism between the two drugs. Notably, chloroquine activated the mitochondrial cell death pathway as indicated by increase in caspase 9 activity. Interestingly, inhibition of lysosomal proteases using E64 conferred cytoprotection against cisplatin and chloroquine co-treatment, suggesting that chloroquine-induced cell death occurred in a cathepsin-mediated mechanism. Likewise, blockage of caspases partially rescued A549cisR cells against the cytotoxicity of cisplatin and chloroquine combination. Cisplatin promoted a dose-dependent autophagic flux induction preferentially in A549cisR cells, as evidenced by a surge in LC3-II/α-tubulin following pre-treatment with E64 and increase in p62 degradation. Compared to untreated cells, cisplatin induced an increase in cyto-ID-loaded autophagosomes in A549cisR cells that was further amplified by chloroquine, pointing toward autophagic flux activation by cisplatin. Interestingly, this effect was less pronounced in A549Pt cells. Blocking autophagy by ATG5 depletion using siRNA markedly enhances susceptibility to cisplatin in A549cisR cells. Taken together, our results underscore the utility of targeting lysosomal function in overcoming acquired cisplatin refractoriness in lung cancer.

PURPOSE: Although oral fluoropyrimidine prodrugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. METHOD: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m2) OGT 719. RESULTS: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. CONCLUSION: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.

OA hybrid Introduction: Metastatic spread to the brain is common in patients with non–small cell lung cancer (NSCLC), but these patients are generally excluded from prospective clinical trials. The studies, phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations (LUX-Lung 3) and a randomized, open-label, phase III study of BIBW 2992 versus chemotherapy as first-line treatment for patients with stage IIIB or IV adenocarcinoma of the lung harbouring an EGFR activating mutation (LUX-Lung 6) investigated first-line afatinib versus platinum-based chemotherapy in epidermal growth factor receptor gene (EGFR) mutation-positive patients with NSCLC and included patients with brain metastases; prespecified subgroup analyses are assessed in this article. Methods: For both LUX-Lung 3 and LUX-Lung 6, prespecified subgroup analyses of progression-free survival (PFS), overall survival, and objective response rate were undertaken in patients with asymptomatic brain metastases at baseline (n = 35 and n = 46, respectively). Post hoc analyses of clinical outcomes was undertaken in the combined data set (n = 81). Results: In both studies, there was a trend toward improved PFS with afatinib versus chemotherapy in patients with brain metastases (LUX-Lung 3: 11.1 versus 5.4 months, hazard ratio [HR] = 0.54, p = 0.1378; LUX-Lung 6: 8.2 versus 4.7 months, HR = 0.47, p = 0.1060). The magnitude of PFS improvement with afatinib was similar to that observed in patients without brain metastases. In combined analysis, PFS was significantly improved with afatinib versus with chemotherapy in patients with brain metastases (8.2 versus 5.4 months; HR, 0.50; p = 0.0297). Afatinib significantly improved the objective response rate versus chemotherapy in patients with brain metastases. Safety findings were consistent with previous reports. Conclusions: These findings lend support to the clinical activity of afatinib in EGFR mutation–positive patients with NSCLC and asymptomatic brain metastases.

8073 Background: Afatinib 40 mg/day (oral) is approved for the treatment of pts with advanced EGFRm+ NSCLC. Dose adjustment is recommended according to pre-defined tolerability criteria. We perform...

To complement the existing treatment guidelines for all tumour types, ESMO organises consensus conferences to focus on specific issues in each type of tumour. The 2nd ESMO Consensus Conference on Lung Cancer was held on 11-12 May 2013 in Lugano. A total of 35 experts met to address several questions on non-small-cell lung cancer (NSCLC) in each of four areas: pathology and molecular biomarkers, first-line/second and further lines of treatment in advanced disease, early-stage disease and locally advanced disease. For each question, recommendations were made including reference to the grade of recommendation and level of evidence. This consensus paper focuses on locally advanced disease.