Effects of KEAP1 Silencing on NRF2 and NOTCH Pathways in SCLC Cell Lines.

The KEAP1/NRF2 pathway is a master regulator of several redox-sensitive genes implicated in the resistance of tumor cells against therapeutic drugs. The dysfunction of the KEAP1/NRF2 system has been correlated with neoplastic patients' outcomes and responses to conventional therapies. In lung tumors, the growth and the progression of cancer cells may also involve the intersection between the molecular NRF2/KEAP1 axis and other pathways, including NOTCH, with implications for antioxidant protection, survival of cancer cells, and drug resistance to therapies. At present, the data concerning the mechanism of aberrant NRF2/NOTCH crosstalk as well as its genetic and epigenetic basis in SCLC are incomplete. To better clarify this point and elucidate the contribution of NRF2/NOTCH crosstalk deregulation in tumorigenesis of SCLC, we investigated genetic and epigenetic dysfunctions of the KEAP1 gene in a subset of SCLC cell lines. Moreover, we assessed its impact on SCLC cells' response to conventional chemotherapies (etoposide, cisplatin, and their combination) and NOTCH inhibitor treatments using DAPT, a γ-secretase inhibitor (GSI). We demonstrated that the KEAP1/NRF2 axis is epigenetically controlled in SCLC cell lines and that silencing of KEAP1 by siRNA induced the upregulation of NRF2 with a consequent increase in SCLC cells' chemoresistance under cisplatin and etoposide treatment. Moreover, KEAP1 modulation also interfered with NOTCH1, HES1, and DLL3 transcription. Our preliminary data provide new insights about the downstream effects of KEAP1 dysfunction on NRF2 and NOTCH deregulation in this type of tumor and corroborate the hypothesis of a cooperation of these two pathways in the tumorigenesis of SCLC.

Assessment of bone turnover markers and DXA parameters to predict bone metastasis progression during zoledronate treatment: a single-center experience.

Bone metastases (BM) are a serious cancer complication, potentially causing substantial morbidity. Among the clinical issues related to BM, there is the lack of specific tools for early diagnosis and prognosis. We explored whether combining bone turnover markers (BTM) with dual-energy X-ray absorptiometry (DXA) assessment could identify early BM progression and risk of skeletal-related events (SREs) during zoledronate treatment. Before the initiation of zoledronate (T0) and after six months of treatment (T1), serum levels of five BTM were measured, and patients (N = 47) underwent DXA evaluation. Standard radiological imaging was performed to assess bone tumor response to medical anti-cancer treatment. High tumor burden in bone correlated with higher serum CTX (p = 0.007) and NTX (p = 0.005) at baseline. Low concentrations of OPG at T0 predicted BM progression with a sensitivity and specificity of 63% and 77%, respectively, when a cutoff of 5.2 pmol/l was used; such a predictive meaning was stronger in patients with lytic BM (sensitivity: 88%, specificity: 80%; p = 0.0006). As for the risk of SREs, we observed an association between low baseline OC (p = 0.04) and OPG (p = 0.08) and the onset of any-time SREs, whereas an increase in OPG over time was associated with reduced risk of on-study events (p = 0.03). Moreover, a statistically significant correlation emerged between low baseline lumbar T-score and femur BMD and on-study SREs (p < 0.001 in both instances). These findings suggest that addition of DXA to BTM dosage could help stratifying the risk of SREs at the time of BM diagnosis but does not enhance our capability of detecting bone progression, during zoledronate treatment.

Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing.

DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA aberrant methylation events occur simultaneously in a group of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Due to this reason, methylation profiles have been suggested as promising cancer biomarkers. Here, we designed and performed a first step of validation of a novel targeted next generation sequencing (NGS) panel for methylation analysis, which can simultaneously evaluate the methylation levels at CpG sites of multiple cancer-related genes. The OPERA_MET-A methylation panel was designed using the Ion AmpliSeq™ technology to amplify 155 regions with 125-175 bp mean length and covers a total of 1107 CpGs of 18 cancer-related genes. The performance of the panel was assessed by running commercially available fully methylated and unmethylated control human genomic DNA (gDNA) samples and a variable mixture of them. The libraries were run on Ion Torrent platform and the sequencing output was analyzed using the "methylation_analysis" plugin. DNA methylation calls on both Watson (W) and Crick (C) strands and methylated:unmethylated ratio for each CpG site were obtained. Cell lines, fresh frozen and formalin-fixed paraffin-embedded (FFPE) lung cancer tissues were tested. The OPERA_MET-A panel allows to run a minimum of 6 samples/530 chip to reach an observed mean target depth ≥2,500X (W and C strands) and an average number of mapped reads >750,000/sample. The conversion efficiency, determined by spiking-in unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed percentage of global methylation for all CpGs was >95% and <5% for fully methylated and unmethylated gDNA samples, respectively, and the observed results for the variable mixtures were in agreement with what was expected. Methylation-specific NGS analysis represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. Moreover, it offers the opportunity to construct a more robust algorithm for disease prediction in cancer patients having a low quantity of biological material available.

Mixed Pulmonary Adenocarcinoma and Atypical Carcinoid: A Report of Two Cases of a Non-codified Entity With Biological Profile.

Pulmonary carcinoids combined with a non-neuroendocrine component have rarely been described, and this histological subtype is not included as a specific entity in the current World Health Organization classification of pulmonary neoplasms. Here, we described the molecular and histological features of two rare cases of mixed lung neoplasms, composed of atypical carcinoid and adenocarcinoma. The targeted next-generation sequencing analysis covering single nucleotide variations, copy number variations, and transcript fusions in a total of 161 cancer genes of the two different tumor components shows a similar molecular profile of shared and private gene mutations. These findings suggest their monoclonal origin from a transformed stem/progenitor tumor cell, which acquires a divergent differentiation during its development and progression and accumulates novel, specific mutations.

Application of "omics" sciences to the prediction of bone metastases from breast cancer: State of the art.

Breast cancer (BC) is the most frequent malignancy and the first cause of cancer-related death in women. The majority of patients with advanced BC develop skeletal metastases which may ultimately lead to serious complications, termed skeletal-related events, that often dramatically impact on quality of life and survival. Therefore, the identification of biomarkers able to stratify BC patient risk to develop bone metastases (BM) is fundamental to define personalized diagnostic and therapeutic strategies, possibly at the earliest stages of the disease. In this regard, the advent of "omics" sciences boosted the investigation of several putative biomarkers of BC osteotropism, including deregulated genes, proteins and microRNAs. The present review revisits the current knowledge on BM development in BC and the most recent studies exploring potential BM-predicting biomarkers, based on the application of omics sciences to the study of primary breast malignancies.