Preoperative Mutational Analysis of Circulating Tumor Cells (CTCs) and Plasma-cfDNA Provides Complementary Information for Early Prediction of Relapse: A Pilot Study in Early-Stage Non-Small Cell Lung Cancer.

PURPOSE: We assessed whether preoperativemutational analyses of circulating tumor cells (CTCs) and plasma-cfDNA could be used as minimally invasive biomarkers and as complimentary tools for early prediction of relapse in early-stage non-small -cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Using ddPCR assays, hotspot mutations of BRAF, KRAS, EGFR and PIK3CA were identified in plasma-cfDNA samples and size-based enriched CTCs isolated from the same blood samples of 49 early-stage NSCLC patients before surgery and in a control group of healthy blood donors (n= 22). Direct concordance of the mutational spectrum was further evaluated in 27 patient-matched plasma-cfDNA and CTC-derived DNA in comparison to tissue-derived DNA. RESULTS: The prevalence of detectable mutations of the four tested genes was higher in CTC-derived DNA than in the corresponding plasma-cfDNA (38.8% and 24.5%, respectively).The most commonly mutated gene was PIK3CA, in both CTCs and plasma-cfDNA at baseline and at the time of relapse. Direct comparison of the mutation status of selected drug-responsive genes in CTC-derived DNA, corresponding plasma-cfDNA and paired primary FFPE tissues clearly showed the impact of heterogeneity both within a sample type, as well as between different sample components. The incidence of relapse was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA compared with patients in whom no mutation was detected (p =0.023). Univariate analysis showed a significantly higher risk of progression (HR: 2.716; 95% CI, 1.030-7.165; p =0.043) in patients with detectable mutations in plasma-cfDNA compared with patients with undetectable mutations, whereas the hazard ratio was higher when at least one mutation was detected in CTC-derived DNA or plasma-cfDNA (HR: 3.375; 95% CI, 1.098-10.375; p =0.034). CONCLUSIONS: Simultaneous mutational analyses of plasma-cfDNA and CTC-derived DNA provided complementary molecular information from the same blood sample and greater diversity in genomic information for cancer treatment and prognosis. The detection of specific mutations in ctDNA and CTCs in patients with early-stage NSCLC before surgery was independently associated with disease recurrence, which represents an important stratification factor for future trials.

Evaluation of the impact of tumor HPV status on outcome in patients with locally advanced unresectable head and neck squamous cell carcinoma (HNSCC) receiving cisplatin, 5-fluorouracil with or without docetaxel: a subset analysis of EORTC 24971 study.

BACKGROUND: EORTC 24971 was a phase III trial demonstrating superiority of induction regimen TPF (docetaxel, cisplatin, 5-fluorouracil) over PF (cisplatin/5-fluorouracil), in terms of progression-free (PFS) and overall survival (OS) in locoregionally advanced unresectable head and neck squamous cell carcinomas. We conducted a retrospective analysis of prospectively collected data aiming to evaluate whether only HPV(-) patients (pts) benefit from adding docetaxel to PF, in which case deintensifying induction treatment in HPV(+) pts could be considered. PATIENTS AND METHODS: Pretherapy tumor biopsies (blocks or slides) were assessed for high-risk HPV by p16 immunohistochemistry, PCR and quantitative PCR. HPV-DNA+ and/or p16+ tumors were subjected to in situ hybridization (ISH) and HPV E6 oncogene expression qRT-PCR analysis. Primary and secondary objectives were to evaluate the value of HPV/p16 status as predictive factor of treatment benefit in terms of PFS and OS. The predictive effect was analyzed based on the model used in the primary analysis of the study with the addition of a treatment by marker interaction term and tested at two-sided 5% significance level. RESULTS: Of 358, 119 pts had available tumor samples and 58 of them had oropharyngeal cancer. Median follow-up was 8.7 years. Sixteen of 119 (14%) evaluable samples were p16+ and 20 of 79 (25%) evaluable tumors were HPV-DNA+. 13 of 40 pts (33%) assessed with HPV-DNA ISH and 12 of 28 pts (43%) assessed for HPV E6 mRNA were positive. The preplanned analysis showed no statistical evidence of predictive value of HPV/p16 status for PFS (P = 0.287) or OS (P = 0.118). CONCLUSIONS: The incidence of HPV positivity was low in the subset of EORTC 24971 pts analyzed. In this analysis only powered to detect a large treatment by marker interaction, there was no statistical evidence that treatment effect found overall was different in magnitude in HPV(+) or HPV(-) pts. These results do not justify selection of TPF versus PF according to HPV status.

Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Age-associated inflammation connects RAS-induced senescence to stem cell dysfunction and epidermal malignancy.

Aging is the single biggest risk factor for malignant transformation. Among the most common age-associated malignancies are non-melanoma skin cancers, comprising the most common types of human cancer. Here we show that mutant H-Ras activation in mouse epidermis, a frequent event in cutaneous squamous cell carcinoma (SCC), elicits a differential outcome in aged versus young mice. Whereas H-Ras activation in the young skin results in hyperplasia that is mainly accompanied by rapid hair growth, H-Ras activation in the aged skin results in more dysplasia and gradual progression to in situ SCC. Progression is associated with increased inflammation, pronounced accumulation of immune cells including T cells, macrophages and mast cells as well as excessive cell senescence. We found not only an age-dependent increase in expression of several pro-inflammatory mediators, but also activation of a strong anti-inflammatory response involving enhanced IL4/IL10 expression and immune skewing toward a Th2 response. In addition, we observed an age-dependent increase in the expression of Pdl1, encoding an immune suppressive ligand that promotes cancer immune evasion. Moreover, upon switching off oncogenic H-Ras activity, young but not aged skin regenerates successfully, suggesting a failure of the aged epidermal stem cells to repair damaged tissue. Our findings support an age-dependent link between accumulation of senescent cells, immune infiltration and cancer progression, which may contribute to the increased cancer risk associated with old age.

The DNA damage checkpoint precedes activation of ARF in response to escalating oncogenic stress during tumorigenesis.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16(INK4A), a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

The tumor suppressor gene ARF as a sensor of oxidative stress.

Oxidative stress as a result of either exogenous stimuli or cellular metabolism affects several cellular processes such as proliferation, apoptosis, cell death and senescence. Consequently, it is implicated in the pathogenesis of various human diseases like cancer, diabetes mellitus, atherosclerosis, neurodegenerative diseases and aging. Oxidative stress is implicated in carcinogenesis either by directly provoking DNA damage or through the regulation of intracellular signaling cascades. In both cases the cellular response to oxidative stress is determined by the cellular context. ARF, the alternative protein product of the CDKN2A locus has been recently recognized as a novel sensor of oxidative stress, in a ß-catenin and Hsp70-mediated manner. Since, improved understanding of cellular responses to oxidative stress may facilitate the design of novel antineoplastic regimens, we herein review the mechanisms by which oxidative stress promotes carcinogenesis, focusing on the role of ARF as a sensor of oxidative stress.

Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer.

ZBTB7A (Pokemon) is a member of the POK family of transcriptional repressors. Its main function is the suppression of the p14ARF tumour suppressor gene. Although ZBTB7A expression has been found to be increased in various types of lymphoma, there are no reports dealing with its expression in solid tumours. Given that p14(ARF) inhibits MDM2, the main negative regulator of p53, we hypothesized that overexpression of ZBTB7A could lead indirectly to p53 inactivation. To this end, we examined the status of ZBTB7A and its relationship with tumour kinetics (proliferation and apoptosis) and nodal members of the p53 network in a panel of 83 non-small cell lung carcinomas (NSCLCs). We observed, in the majority of the samples, prominent expression of ZBTB7A in the cancerous areas compared to negligible presence in the adjacent normal tissue elements. Gene amplification (two- to five-fold) was found in 27.7% of the cases, denoting its significance as a mechanism driving ZBTB7A overproduction in NSCLCs. In the remaining non-amplified group of carcinomas, analysis of the mRNA and protein expression patterns suggested that deregulation at the transcriptional and post-translational level accounts for ZBTB7A overexpression. Proliferation was associated with ZBTB7A expression (p = 0.033) but not apoptosis. The association with proliferation was reflected in the positive correlation between ZBTB7A expression and tumour size (p = 0.018). The overexpression of ZBTB7A in both p53 mutant and p53 wild-type cases, implies either a synergistic effect or that ZBTB7A exerts its oncogenic properties independently of the p14(ARF)-MDM2-p53 axis. The concomitant expression of ZBTB7A with p14(ARF) (p = 0.039), instead of the anticipated inverse relation, supports the latter notion. In conclusion, regardless of the pathway followed, the distinct expression of ZBTB7A in cancerous areas and the association with proliferation and tumour size pinpoints a role for this novel cell cycle regulator in the pathogenesis of lung cancer.

Centrosome abnormalities are frequently observed in non-small-cell lung cancer and are associated with aneuploidy and cyclin E overexpression.

Centrosome abnormalities are observed in human cancers and have been associated with aneuploidy, a driving force in tumour progression. However, the exact pathways that tend to cause centrosome abnormalities have not been fully elucidated in human tumours. Using a series of 68 non-small-cell lung carcinomas and an array of in vitro experiments, the relationship between centrosome abnormalities, aneuploidy, and the status of key G1 to S-phase transition cell-cycle molecules, involved in the regulation of centrosome duplication, was investigated. Centrosome amplification and structural abnormalities were common (53%), were strongly related to aneuploidy, and, surprisingly, were even seen in adjacent hyperplastic regions, suggesting the possibility that these are early lesions in lung carcinogenesis. Cyclin E and E2F1 overexpression, but not p53 mutation, was observed to correlate with centrosome abnormalities in vivo (p = 0.029 and p = 0.015, respectively). This was further strengthened by the observation that cyclin E was specifically present in the nucleus and/or cytoplasm of the cells that contained centrosome aberrations. The cytoplasmic cyclin E signal may be attributed, in part, to the presence of truncated low-molecular-weight isoforms of cyclin E. In order to isolate the effect of cyclin E on the appearance of centrosome abnormalities, a U2OS tetracycline-repressible cyclin E cell line that has a normal centrosome profile by default was used. With this system, it was confirmed in vitro that persistent cyclin E overexpression is sufficient to cause the appearance of centrosome abnormalities.