NGS Analysis of Liquid Biopsy (LB) and Formalin-Fixed Paraffin-Embedded (FFPE) Melanoma Samples Using Oncomine™ Pan-Cancer Cell-Free Assay.

Next-generation sequencing (NGS) in liquid biopsies may contribute to the diagnosis, monitoring, and personalized therapy of cancer through the real-time detection of a tumor's genetic profile. There are a few NGS platforms offering high-sensitivity sequencing of cell-free DNA (cfDNA) samples. The aim of this study was to evaluate the Ion AmpliSeq HD Technology for targeted sequencing of tumor and liquid biopsy samples from patients with fourth-stage melanoma. Sequencing of 30 samples (FFPE tumor and liquid biopsy) derived from 14 patients using the Oncomine™ Pan-Cancer Cell-Free Assay was performed. The analysis revealed high concordance between the qPCR and NGS results of the BRAF mutation in FFPE samples (91%), as well as between the FFPE and liquid biopsy samples (91%). The plasma-tumor concordance of the non-BRAF mutations was 28%. A total of 17 pathogenic variants in 14 genes (from 52-gene panel), including TP53, CTNNB1, CCND1, MET, MAP2K1, and GNAS, were identified, with the CTNNB1S45F variant being the most frequent. A positive correlation between the LDH level and cfDNA concentration as well as negative correlation between the LDH level and time to progression was confirmed in a 22-patient cohort. The analysis showed both the potential and limitations of liquid biopsy genetic profiling using HD technology and the Ion Torrent platform.

Genetic Profiling of Advanced Melanoma: Candidate Mutations for Predicting Sensitivity and Resistance to Targeted Therapy.

BACKGROUND: Molecularly targeted therapy has revolutionized the treatment of advanced melanoma. However, despite its high efficiency, a majority of patients experience relapse within 1 year of treatment because of acquired resistance, and approximately 10-25% patients gain no benefit from these agents owing to intrinsic resistance. This is mainly caused by the genetic heterogeneity of melanoma cells. OBJECTIVE: We aimed to validate the predictive significance of selected genes in advanced melanoma patients before treatment with BRAF/MEK inhibitors. PATIENTS AND METHODS: Archival DNA derived from 37 formalin-fixed paraffin-embedded pre-treatment advanced melanoma samples of patients treated with targeted therapy was used for next-generation sequencing analysis using the Ion Torrent platform. The AmpliSeq Custom Panel comprised coding sequences or hot spots of 23 melanoma genes: ATM, BRAF, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, IDH1, KIT, KRAS, MAP3K8, MAP2K1, MAP2K2, MITF, MYC, NF1, NRAS, PAX5, PIK3R1, PTEN, RAC1, and RB1. The sequences were evaluated for genomic alterations and further validated using Sanger sequencing. RESULTS: Our analysis revealed non-BRAF genetic alterations in 28 out of 37 samples (75.7%). Genetic changes were identified in PTEN, CDK4, CDKN2A, CTNNB1, EGFR, HOXD8, HRAS, KIT, MAP2K1, MAP2K2, MITF, MYC, NF1, PAX5, RAC1, and RB1. Fifteen known pathogenic mutations (single nucleotide variants or indels) and 11 variants of unknown significance were detected. Statistical analysis revealed an association between the presence of pathogenic mutations and time to progression during treatment with combination therapy. CONCLUSIONS: Pathogenic mutations identified by gene panel sequencing have potential predictive value for targeted therapy of melanoma and are worth further validation in a larger series of cases. The role of some known mutations (e.g. CDK4R24, PTEN c.801 + 1G > A, CTNNB1S45F) as well as variants of unknown significance identified in this study (e.g. MITFR316K, KITG498S) in the generation of resistance to BRAF/MEK inhibitors should be further investigated.

BRCA2 genemutation, c.2808_2811delACAA (p.Ala938Profs), in malebreastcancer - clinicopathologicalanalysisbased on a case report.

Male breast cancer (MBC) is a rare disease that occurs in ~0.2% of all neoplasms among men. The risk of developing MBC is higher in men with a BRCA2 genetic mutation (7%). The aim of this study was to evaluate the association between c.2808_2811del ACAA (p.Ala938Profs) BRCA2 mutation in MBC and clinicopathological factors. A 75-year-old patient was admitted to the Genetic Outpatient Clinic with a diagnosis of right breast cancer and with a family history of cancer (two daughters who were diagnosed with breast cancer at ages 46 and 38 years). Postoperative histopathological examination revealed tumor type pT2 N1a Mx, NST NG-3 G-3, Ki-67 (75%), HER2 (+), ER (+++), PR (+++), invasive carcinoma and luminal B subtype. The complete coding sequences of the BRCA1 and BRCA2 genes were analyzed for genomic DNA material using next generation sequencing on the Ion Torrent platform. The c.2808_2811delACAA (p.Ala938Profs) mutation was observed in the BRCA2 gene. The presence of the c.2808_2811delACAA (p.Ala938Profs) mutation in the BRCA2 gene was confirmed using the Sanger method. The same BRCA2 gene mutation was reported in one of the patient's daughters. The detected mutation causes the frameshift mutation, resulting in the creation of an additional translation termination codon and the synthesis of an abnormal protein. This mutation was associated with a later age of disease, a higher histological degree, a higher mitotic index, a positive steroid receptor status and the luminal B subtype HER2- breast cancer.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Lomianki/Kielpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Cell-cycle gene expression analysis using real time PCR in locally advanced squamous-cell head and neck cancer.

PURPOSE: The analysis of gene expression, especially those involved in cell cycle control, can help to discover mechanisms determining the outcome of radiation treatment. The main purpose of this study was to examine the expression level of genes responsible for cell cycle regulation in samples of the head and neck cancer, obtained during surgery. METHODS: Postsurgical samples of SCC of head and neck region were collected. Over 80 genes were analysed using cell cycle quantitative real-time RT-PCR Array method. Presence of 14 high-risk HPV types DNA in frozen or paraffin-embedded tumour pathological samples was also assessed. To correlate gene expression with selected pathological features and clinical outcome we used different hierarchical clustering method. RESULTS: Hierarchical clustering demonstrated the association between gene expression within certain clusters and gender, tumour site, T stage, N stage, grade, pathological subtype or tumour recurrence. CONCLUSIONS: Despite some limitations we were able to identify gene clusters that allowed to classify patients according to selected clinical features and occurrence of tumour recurrence. The results of the analysis also confirm that the incidence of HPV infection among the patients from Upper Silesia is relatively low, whereas HPV negative tumours, likely associated with smoking, appeared dominant.

Assessment of the total cfDNA and HPV16/18 detection in plasma samples of head and neck squamous cell carcinoma patients.

OBJECTIVES: The advantages of the circulating cell-free DNA (cfDNA) methodology are quick results and the possibility of repeated analysis. The main aim of our study was to establish the relationship of the total cfDNA with patients' clinical characteristics and circulating HPV DNA detection in the blood of patients with head and neck squamous cell carcinoma (HNSCC). METHODS: The cfDNA level of 200 HNSCC patients in plasma was quantified using TaqMan-based TERT amplification. TaqMan technology was also used for HPV16/18 detection. Additionally, mutations in KRAS and EGFR were investigated. RESULTS: A higher level (p=0.011) of the total cfDNA was found in patients with oropharyngeal squamous cell carcinoma (OPSCC) (9.60 ± 6.23 ng/ml) in comparison with other HNSCC (7.67 ± 4.44 ng/ml). The level of cfDNA in patients with clinical N2-N3 disease (9.28 ± 6.34 ng/ml) was (p=0.015) higher than in patients with a clinical N0-N1 disease (7.50 ± 3.69 ng/ml). It was also higher in patients with stage IV (9.16 ± 6.04 ng/ml) compared with stages I-III of cancer (7.26 ± 3.63 ng/ml) (p=0.011). Analysis of HPV16/18 in plasma revealed that 14% of patients were HPV-positive, the majority of whom had the type HPV16 (96.4%). CfDNA level was comparable in HPV-positive and HPV-negative HNSCC patients, as well in the OPSCC subgroup. Somatic mutations in EGFR and KRAS were not found. CONCLUSIONS: A high level of cfDNA is specific for patients with OPSCC. HPV detection in cfDNA does not depend on the cfDNA concentration. Our results prove the diagnostic potential of plasma-based HPV cfDNA tests for the early detection and monitoring of HPV-positive HNSCC.

Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells.

BACKGROUND: During functional studies on the rat stress-inducible Hspa1b (hsp70.1) gene we noticed that some liposome-based DNA carriers, which are used for transfection, induce its promoter activity. This observation concerned commercial liposome formulations (LA), Lipofectin and Lipofectamine 2000. This work was aimed to understand better the mechanism of this phenomenon and its potential biological and practical consequences. RESULTS: We found that a reporter gene driven by Hspa1b promoter is activated both in the case of transient transfections and in the stably transfected cells treated with LA. Using several deletion clones containing different fragments of Hspa1b promoter, we found that the regulatory elements responsible for most efficient LA-driven inducibility were located between nucleotides -269 and +85, relative to the transcription start site. Further studies showed that the induction mechanism was independent of the classical HSE-HSF interaction that is responsible for gene activation during heat stress. Using DNA microarrays we also detected significant activation of the endogenous Hspa1b gene in cells treated with Lipofectamine 2000. Several other stress genes were also induced, along with numerous genes involved in cellular metabolism, cell cycle control and pro-apoptotic pathways. CONCLUSIONS: Our observations suggest that i) some cationic liposomes may not be suitable for functional studies on hsp promoters, ii) lipofection may cause unintended changes in global gene expression in the transfected cells.

Stress proteins HSP27 and HSP70i predict survival in non-small cell lung carcinoma.

BACKGROUND: The expression pattern of stress (heat shock) proteins (HSPs) in cancer cells is frequently different from that observed in normal cells; most often some stress-inducible HSPs are constitutively and highly expressed. The objective of this study was to determine the prognostic significance of stress proteins HSP70i and HSP27 in non-small cell lung carcinoma (NSCLC). MATERIALS AND METHODS: An immunohistochemical procedure that enables unambiguous detection of HSP70i protein was used. RESULTS: Strong HSP70i staining showed a survival advantage, although multivariate analysis did not confirm this result. There was an evident correlation between HSP27 overexpression and survival of patients and the results were confirmed by multivariate analysis: 70% of patients with HSP27-negative tumors died within one year after the surgery. CONCLUSION: Our data suggest that HSP27 and HSP70i positivity may represent a favorable prognostic factor in NSCLC.

Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line.

Targeting of the Hsp function in tumor cells is currently being assessed as potential anticancer therapy. An improved understanding of the molecular signals that trigger or attenuate the stress protein response is essential for advances to be made in this field. The present study provides evidence that the membrane fluidizer benzyl alcohol (BA), a documented nondenaturant, acts as a chaperone inducer in B16(F10) melanoma cells. It is demonstrated that this effect relies basically on heat shock transcription factor 1 (HSF1) activation. Under the conditions tested, the BA-induced Hsp response involves the up-regulation of a subset of hsp genes. It is shown that the same level of membrane fluidization (estimated in the core membrane region) attained with the closely analogous phenethyl alcohol (PhA) does not generate a stress protein signal. BA, at a concentration that activates heat shock genes, exerts a profound effect on the melting of raft-like cholesterol-sphingomyelin domains in vitro, whereas PhA, at a concentration equipotent with BA in membrane fluidization, has no such effect. Furthermore, through the in vivo labeling of melanoma cells with a fluorescein labeled probe that inserts into the cholesterol-rich membrane domains [fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol)], we found that, similarly to heat stress per se, BA, but not PhA, initiates profound alterations in the plasma membrane microdomain structure. We suggest that, apart from membrane hyperfluidization in the deep hydrophobic region, a distinct reorganization of cholesterol-rich microdomains may also be required for the generation and transmission of stress signals to activate hsp genes.

Oligo-3-hydroxybutyrates as potential carriers for drug delivery.

In the present paper we describe the synthesis and toxicity studies of well-defined tailor made oligo-[R,S]-3-hydroxybutyrates (OHBs). The results indicate potential applicability of these nano-polymers as drug delivery carriers. Several OHBs of number average molecular weight (M(n)) ranging from 800 to 2400 have been synthesized and tested on transformed hamster V79 fibroblasts and murine melanoma B16(F10) cells using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) based drug resistance and clonogenic survival assays. We show that 96-h incubation of cells with 1-9 microg/ml of OHBs did not affect cell viability. Incubation of OHBs with rat hepatoma FTO-2B cells stably transfected with chloramphenicol acetyltransferase (CAT) gene ligated to heat-inducible hsp70i gene promoter demonstrated that OHBs did not induce cellular stress response. Finally, we demonstrate that doxorubicin conjugated with OHB is effectively taken up by murine melanoma B16(F10) cells in vitro and localizes in the cytoplasm. These data show for the first time that tailor-made biodegradable and biocompatible oligomers of 3-hydroxybutyric acid can be taken into consideration as effective, non-toxic vectors for delivery of drugs in a conjugated form.

Structure of gene flanking regions and functional analysis of sequences upstream of the rat hsp70.1 stress gene.

We present structural and comparative analysis of the flanking regions of the rat hsp70.1 stress gene. Several repetitive sequences, microsatellites and short interspersed repetitive elements (SINEs) were found, as well as a significant gap in the 3' UTR, as compared to the orthologous mouse gene. We also show that the complex microsatellite region composed of partially overlapping inverted repeat and long homopurine-homopyrimidine sequence, which is localized 1.8 kbp upstream of the transcription start site, is capable to adopt non-B DNA structures (an H-DNA and a cruciform structure) in vitro. Functional analysis performed with the use of various fragments of the 5'end flanking regions ligated to the chloramphenicol acetyltransferase (CAT) reporter gene revealed a crucial role of cooperation between heat shock element (HSE) regulatory sequences, while none of the three HSEs alone is able to drive efficient heat induced transcription of the reporter gene. We also found that the microsatellite region does not influence transcription by itself, however, it abolishes the effect of the adjacent putative silencing element. To our knowledge, this is a first extensive structural and functional analysis of the promoter region of the mammalian heat inducible hsp70i gene localized distally to the hsp70-related spermatid-specific gene in the major histocompatibility complex III.

HSP70 overexpression increases resistance of V79 cells to cytotoxicity of airborne pollutants, but does not protect the mitotic spindle against damage caused by airborne toxins.

Exposure of Chinese hamster V79 cells to extracts of airborne pollutants induced formation of multipolar or incomplete mitotic spindles. To find out whether overexpression of the HSP70 chaperone protein could protect spindles against airborne toxins we constructed V79 cells stably transfected with an expression vector containing rat heat-inducible hsp70.1 gene under the control of a constitutive CMV promoter. When cells were incubated with extracts of airborne pollutants (5-20 microg/ml) no protective effect of the HSP70 protein against mitotic spindle damage was observed. Moreover, at 20 microg/ml of extracts of airborne toxins the frequency of mitotic malformations was even higher in HSP70-overexpressing cells than in control ones. Extracts of airborne pollutants of 50 microg/ml blocked the formation of mitotic figures both in control and HSP70-overexpressing cells and led to destruction of cell nuclei. However, the HSP70-overproducing cells exhibited higher survival rates when exposed to heat shock and airborne toxins than the control ones, as determined by MTT assay. This suggests that HSP70 overexpression-a frequent feature of cancer cells-should be considered as a factor facilitating survival of cells with damaged mitotic spindles and aberrantly segregated chromosomes.