NTRK and ALK rearrangements in malignant pleural mesothelioma, pulmonary neuroendocrine tumours and non-small cell lung cancer.

OBJECTIVES: Gene rearrangements involving NTRK1, NTRK2, NTRK3, ROS1 and ALK have been identified in many types of cancer, including non-small cell lung cancer (NSCLC). Data in malignant pleural mesothelioma (MPM), lung neuroendocrine tumors (NETs) and small-cell lung cancer (SCLC) are lacking. Given the activity of NTRK, ROS-1 and ALK inhibitors in tumors harboring gene fusions, we sought to explore such rearrangements in these less common tumors in addition to NSCLC. METHODS: Archival tumor tissue from patients with MPM, lung NETs, SCLC and NSCLC were used to create tissue microarrays. Immunohistochemistry (IHC) was performed using a cocktail of antibodies against TRK, ROS1 and ALK. IHC positive samples underwent RNA sequencing using the ArcherDX FusionPlex CTL diagnostic assay. Clinical data were obtained through retrospective chart review. RESULTS: We performed IHC on 1116 samples: 335 MPMs, 522 NSCLCs, 105 SCLCs and 154 lung NETs. There were 23 IHC positive cases (2.1%) including eight MPMs (2.4%), eight NETs (5.2%), five SCLC (4.8%) and two NSCLC (0.4%). The following fusions were detected: one MPM with an NTRK ex10-TPM3 ex8, another MPM with an ALK ex20-EML4ex13, one lung intermediate-grade NET (atypical carcinoid) with an ALK ex20-EML4 ex6/intron6, and two NSCLCs with an ALK ex20-EML4 ex6/intron6 rearrangement. None of the patients received targeted treatment. CONCLUSIONS: To our knowledge, we report for the first time NTRK and ALK rearrangements in a small subset of MPM. An ALK rearrangement was also detected in lung intermediate-grade NET (or atypical carcinoid). Our data suggest that IHC could be a useful screening test in such patients to ensure that all therapeutic strategies including targeted therapy are utilized.

Downregulation of CEACAM6 gene expression in laryngeal squamous cell carcinoma is an effect of DNA hypermethylation and correlates with disease progression.

We have turned our attention to CEACAM6 gene, already described as deregulated in various types of cancer. By using the expression microarrays performed on the set of 16 laryngeal squamous cell carcinoma (LSCC) samples: 11 cell lines and 5 primary tumors we have shown downregulation of CEACAM6 gene as compared to non cancer controls from head and neck region. CEACAM6 gene downregulation, further confirmed by quantitative PCR on 25 LSCC cell lines, was observed in cell lines derived from recurrent tumors in comparison to controls. A significant gene downregulation was observed in cell lines derived from advanced, high grade tumors in comparison to controls. Intrigued by the recurrent transcriptional loss of CEACAM6 we searched for the mechanism potentially responsible for its downregulation and hence we analyzed DNA copy number changes (a-CGH), promoter DNA methylation status and occurrence of gene mutations (in silico). Neither the analysis of gene copy number, nor the mutation screen has shown recurrent deletions or mutations, that could contribute to the observed downregulation of the gene. However, by using bisulfite pyrosequencing, we have shown DNA hypermethylation (mean DNA methylation > 78%) of CEACAM6 promoter region in 9/25 (36%) LSCC cell lines. Importantly, the 5-aza-2-deoxycytidine-induced inhibition of DNA methylation resulted in restoration of CEACAM6 expression in the two LSCC cell lines on mRNA level. In summary, we have shown that recurrent downregulation of CEACAM6 in LSCC is dependent on the gene's promoter DNA methylation and is observed predominantly in large, poorly differentiated tumors and recurrences.

DNA Methylation Profiling of Breast Cancer Cell Lines along the Epithelial Mesenchymal Spectrum-Implications for the Choice of Circulating Tumour DNA Methylation Markers.

(1) Background: Epithelial⁻mesenchymal plasticity (EMP) is a dynamic process whereby epithelial carcinoma cells reversibly acquire morphological and invasive characteristics typical of mesenchymal cells. Identifying the methylation differences between epithelial and mesenchymal states may assist in the identification of optimal DNA methylation biomarkers for the blood-based monitoring of cancer. (2) Methods: Methylation-sensitive high-resolution melting (MS-HRM) was used to examine the promoter methylation status of a panel of established and novel markers in a range of breast cancer cell lines spanning the epithelial⁻mesenchymal spectrum. Pyrosequencing was used to validate the MS-HRM results. (3) Results: VIM, DKK3, and CRABP1 were methylated in the majority of epithelial breast cancer cell lines, while methylation of GRHL2, MIR200C, and CDH1 was restricted to mesenchymal cell lines. Some markers that have been used to assess minimal residual disease such as AKR1B1 and APC methylation proved to be specific for epithelial breast cell lines. However, RASSF1A, RARß, TWIST1, and SFRP2 methylation was seen in both epithelial and mesenchymal cell lines, supporting their suitability for a multimarker panel. (4) Conclusions: Profiling DNA methylation shows a distinction between epithelial and mesenchymal phenotypes. Understanding how DNA methylation varies between epithelial and mesenchymal phenotypes may lead to more rational selection of methylation-based biomarkers for circulating tumour DNA analysis.

Recurrent transcriptional loss of the PCDH17 tumor suppressor in laryngeal squamous cell carcinoma is partially mediated by aberrant promoter DNA methylation.

Protocadherins are cell-cell adhesion molecules encoded by a large family of genes. Recent reports demonstrate recurrent silencing of protocadherin genes in tumors and provide strong arguments for their tumor supresor functionality. Loss of protocadherins may contribute to cancer development not only by altering cell-cell adhesion, that is a hallmark of cancer, but also by enhancing proliferation and epithelial mesenchymal transition of cells via deregulation of the WNT signaling pathway. In this study we have further corroborated our previous findings on the involvement of PCDH17 in laryngeal squamous cell carcinoma (LSCC). We used bisulfite pyrosequencing to analyze a cohort of primary LSCC tumors for alterations in PCDH17 promoter DNA methylation as an alternative gene inactivation mechanism to the homozygous deletions reported earlier. Moreover, we analyzed primary LSCC samples by immunohistochemistry for PCDH17 protein loss. We identified recurrent elevation of PCDH17 promoter DNA methylation in 32/81 (40%) primary tumors (P < 0.001) and therein hypermethylation of 12 (15%) cases in contrast to no tumor controls (n = 24) that were all unmethylated. Importantly, DNA demethylation by decitabine has restored low level PCDH17 expression in LSCC cell lines. In conclusion, we provide a mechanistic explanation of recurrently observed PCDH17 silencing in LSCC by demonstrating the role of promoter methylation in this process. In light of these findings and recent reports showing that PCDH17 methylation is detectable in serum of cancer patients we suggest that testing PCDH17 DNA methylation might serve as a potential biomarker in LSCC.

HSD11B2, RUNX3, and LINE-1 Methylation in Placental DNA of Hypertensive Disorders of Pregnancy Patients.

Hypertensive Disorders of Pregnancy (HDsP) remain leading causes of maternal and perinatal morbidity and mortality. Growing evidence suggests the involvement of epigenetic factors, such as gene-specific and global DNA methylation changes, both in the etiology and as an effect of HDsP. In this study, we investigated the potential association between placental DNA methylation status in selected CpGs of HSD11B2 cortisol level controlling gene, RUNX3 tumor suppressor gene, and long interspersed nucleotide element-1 (LINE-1) repetitive elements and HDsP-preeclampsia (PE), gestational hypertension (GH), and chronic hypertension (CH). Methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ) were used to analyze placental DNA methylation. Plasma and urine cortisol and cortisone levels were measured using high performance liquid chromatography with fluorescence detection (HPLC-FLD), whereas serum progesterone level was determined by electrochemiluminescence immunoassay. The mean percentage of HSD11B2, RUNX3, and LINE-1 methylation was not altered in the placentas of patients with HDsP, as compared to the controls. However, among patients from PE, GH, and CH groups, several significant correlations were observed between the methylation status of HSD11B2, RUNX3, or LINE-1 and children's birth weight, gestational age at delivery, mother's age, and body mass index as well as hormones levels. These results indicate lack of association between methylation status of HSD11B2, RUNX3, or LINE-1 repetitive elements and HDsP. However, association of these parameters with some clinical variables may suggest the role of placental DNA methylation in fetal development and should be further explored.

Combined deletion and DNA methylation result in silencing of FAM107A gene in laryngeal tumors.

Larynx squamous cell carcinoma (LSCC) is characterized by complex genotypes, with numerous abnormalities in various genes. Despite the progress in diagnosis and treatment of this disease, 5-year survival rates remain unsatisfactory. Therefore, the extended studies are conducted, with the aim to find genes, potentially implicated in this cancer. In this study, we focus on the FAM107A (3p14.3) gene, since we found its significantly reduced expression in LSCC by microarray profiling (Affymetrix U133 Plus 2.0 array). By RT-PCR we have confirmed complete FAM107A downregulation in laryngeal cancer cell lines (15/15) and primary tumors (21/21) and this finding was further supported by FAM107A protein immunohistochemistry (15/15). We further demonstrate that a combined two hit mechanism including loss of 3p and hypermethylation of FAM107A promoter region (in 9/15 cell lines (p < 0.0001) and in 15/21 primary tumors (p < 0.0001)) prevails in the gene transcriptional loss. As a proof of principle, we show that Decitabine - a hypomethylating agent - restores FAM107A expression (5 to 6 fold increase) in the UT-SCC-29 cell line, characterized by high DNA methylation. Therefore, we report the recurrent inactivation of FAM107A in LSCC, what may suggest that the gene is a promising tumor suppressor candidate involved in LSCC development.

Recurrent epigenetic silencing of the PTPRD tumor suppressor in laryngeal squamous cell carcinoma.

Cellular processes like differentiation, mitotic cycle, and cell growth are regulated by tyrosine kinases with known oncogenic potential and tyrosine phosphatases that downmodulate the first. Therefore, tyrosine phosphatases are recurrent targets of gene alterations in human carcinomas. We and others suggested recently a tumor suppressor function of the PTPRD tyrosine phosphatase and reported homozygous deletions of the PTPRD locus in laryngeal squamous cell carcinoma. In this study, we investigated other gene-inactivating mechanisms potentially targeting PTPRD, including loss-of-function mutations and also epigenetic alterations like promoter DNA hypermethylation. We sequenced the PTPRD gene in eight laryngeal squamous cell carcinoma cell lines but did not identify any inactivating mutations. In contrast, by bisulfite pyrosequencing of the gene promoter region, we identified significantly higher levels of methylation (p = 0.001 and p = 0.0002, respectively) in 9/14 (64%) laryngeal squamous cell carcinoma cell lines and 37/79 (47%) of primary laryngeal squamous cell carcinoma tumors as compared to normal epithelium of the upper aerodigestive tract. There was also a strong correlation (p = 0.0001) between methylation and transcriptional silencing for the PTPRD gene observed in a cohort of 497 head and neck tumors from The Cancer Genome Atlas dataset suggesting that DNA methylation is the main mechanism of PTPRD silencing in these tumors. In summary, our data provide further evidence of the high incidence of PTPRD inactivation in laryngeal squamous cell carcinoma. We suggest that deletions and loss-of-function mutations are responsible for PTPRD loss only in a fraction of cases, whereas DNA methylation is the dominating mechanism of PTPRD inactivation.

Exposure to volatile anaesthetics is not followed by a massive induction of single-strand DNA breaks in operation theatre personnel.

Volatile anaesthetics such as halothane, isoflurane and others were expected to produce a health challenge for operation room personnel because of prolonged occupational exposure to anaesthetic gases. To estimate a molecular background of adverse health effects, a cohort of 100 exposed individuals was studied by the single-cell gene electrophoresis (comet assay) test. DNA lesions in lymphocytes of the exposed group did not differ significantly compared with non-exposed blood donors. Then, the exposed group was further divided according to job position. A highest level of DNA lesions was established in nurses but without significant difference compared with other groups. When a time period of exposure was taken into account, a tendency to cumulate DNA lesions was found only in the group of anaesthesiologists. A very weak genotoxic effect established in this study is discussed in relation to DNA repair, adaptative response and potential self-elimination of sensitive individuals.

Global miRNA Expression Profiling Identifies miR-1290 as Novel Potential oncomiR in Laryngeal Carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is the most common group among head and neck cancers. LSCC is characterized by a high incidence in Europe. With the aim of better understanding its genetic background we performed global miRNA expression profiling of LSCC cell lines and primary specimens. By this approach we identified a cohort of 33 upregulated and 9 downregulated miRNA genes in LSCC as compared to epithelial no tumor controls. RESULTS: Within this group we identified overexpression of the novel miR-1290 gene not reported in the context of LSCC before. Using a combined bioinformatical approach in connection with functional analysis we delineated two putative target genes of miR-1290 namely ITPR2 and MAF which are significantly downregulated in LSCC. They are interesting candidates for tumor suppressor genes as they are implicated in apoptosis and other processes deregulated in cancer. CONCLUSION: Taken together, we propose miR-1290 as the new oncomiR involved in LSCC pathogenesis. Additionally, we suggest that the oncogenic potential of miR-1290 might be expressed by the involvement in downregulation of its target genes MAF and ITPR2.

Diversified expression of aryl hydrocarbon receptor dependent genes in human laryngeal squamous cell carcinoma cell lines treated with ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study the effect of administration of ß-naphthoflavone (BNF), potent AhR ligand, on the expression of AhR, AhRR, CYP1A1, CYP1A2, CYP1B1, NQO1, GSTA1, ALDH3A1 and UGT1A genes encoding the enzymes controlled by AhR were examined in thirteen laryngeal tumor cell lines and in HepaRG cell line. The analyzed cell lines were derived from patients with squamous laryngeal cancer, with history of cigarette smoking and without signs of human papillomavirus types 16 and 18 infection in investigated cells. Quantitative real-time RT-PCR analysis revealed huge interindividual differences in expression of genes from AhR regulatory network. Our results strongly suggest predominant effect of DNA methylation on induction of CYP1A1 expression by AhR ligands as well. Our results indicate that differentiated HepaRG cell line appeared to be very good substitute for human liver in studies on xenobiotic metabolism by AhR regulated enzymes.

microRNAs are important players in head and neck carcinoma: a review.

The results of treatment of head and neck tumors remain poor for decades. It means that after surgery, chemotherapy is not a proper choice, as tumors of this region are relatively resistant to cytotoxic drugs. A little progress was noted only for radiotherapy outcome. Consequently, clinicians and researchers' expectations are focused on targeted therapy, where microRNAs (miRNAs, miRs) seem to be the most promising target. After the year 2000, miRNAs became new players on the scene of cancer science. Since then, extensive investigations have been performed with a hope of finding a new prognostic and diagnostic tool and bridging them with a bright new way of understanding the basis of molecular carcinogenesis. miRNAs display astonishing specificity and thus are associated with pathoclinical parameters of the disease. After more than a decade of ongoing studies, in this review we attempt to summarize the current knowledge of miRNAs in malignancies arising in head and neck sites and with a majority of squamous cells of the epithelium.

Heterogeneity of 11q13 region rearrangements in laryngeal squamous cell carcinoma analyzed by microarray platforms and fluorescence in situ hybridization.

We reinvestigated rearrangements occurring in region q13 of chromosome 11 aiming to: (i) describe heterogeneity of the observed structural alterations, (ii) estimate amplicon size and (iii) identify of oncogenes involved in laryngeal cancer progression as potential targets for therapy. The study included 17 cell lines derived from laryngeal cancers and 34 specimens from primary laryngeal tumors. The region 11q13 was analyzed by fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and gene expression microarray. Next, quantitative real time PCR was used for chosen genes to confirm results from aCGH and gene expression microarray. The observed pattern of aberrations allows to distinguish three ways, in which gain and amplification involving 11q13 region may occur: formation of a homogeneously staining region; breakpoints in/near 11q13, which lead to the three to sevenfold increase of the copy number of 11q13 region; the presence of additional copies of the whole chromosome 11. The minimal altered region of gain and/or amplification was limited to ~1.8 Mb (chr.11:69,395,184-71,209,568) and comprised mostly 11q13.3 band which contain 12 genes. Five, out of these genes (CCND1, ORAOV1, FADD, PPFIA1, CTTN) had higher expression levels in comparison to healthy controls. Apart from CCND1 gene, which has an established role in pathogenesis of head and neck cancers, CTTN, ORAOV1 and FADD genes appear to be oncogene-candidates in laryngeal cancers, while a function of PPFIA1 requires further studies.

Simple technique for RNA purification from mouse inner ear hair cells.

Obtaining a good quality of RNA from small population of cells remain an issue. Isolation for a special anatomic location such as inner ear placed in the temporal bone become a challenge, especially in terms of time needed for isolation of living tissue from the bone, which is a key factor to preserve the RNA. Due to limited accessibility to the technologies such as laser dissection, we present a simplified procedure for isolation of good quality of RNA from the inner ear for further studies.

Loss of protein expression and recurrent DNA hypermethylation of the GNG7 gene in squamous cell carcinoma of the head and neck.

Although down-regulation of GNG7 in cancer was reported before, its role in carcinogenesis is poorly understood. It belongs to a family of large G-proteins that may be involved in cell-contact-induced growth arrest and function in tumor suppression. In the present study, we stained immunohistochemically 188 tumors derived from larynx or floor of the mouth for GNG7 protein and confronted it with clinicopathologic data. Moreover, we performed bisulfite pyrosequencing to analyze GNG7 promoter methylation. We identified recurrent loss of GNG7 protein expression in 68/188 (36%) cases and promoter hypermethylation in (42/98; 43%) primary tumors, predominantly in young patients (p < 0.001). Loss of GNG7 expression correlated with hypermethylation of GNG7 promoter region (p < 0.001). Moreover, loss of GNG7 protein expression correlated with tumor size (p = 0.012) and lack of cervical metastasis (p = 0.02) whereas sustained expression correlated with keratinization (p = 0.008). Taken together, loss of GNG7 protein expression is a frequent event in head and neck cancer. Moreover, our data suggest that hypermethylation of the promoter region of GNG7 is probably the mechanism of the observed inactivation.

Pyrosequencing-based DNA methylation profiling of Fanconi anemia/BRCA pathway genes in laryngeal squamous cell carcinoma.

Fanconi anemia (FA) associated genes [FANCA, -B, -C, FANCD1(BRCA2), -D2, -E, -F, -G, -I, -L, -M, FANCN (PALB2), FANCJ(BRIP1) and FA-linked BRCA1] encode proteins of DNA damage response pathways mutated in FA patients. FA is characterized by congenital malformations, chromosomal instability and high cancer susceptibility. FA patients have a 500-700 times higher risk of head and neck squamous cell carcinoma (HNSCC) compared to the non-FA population. As DNA methylation comprises one of the known gene inactivation mechanisms in cancer we have investigated the methylation status of 13 FA and one FA-linked gene in order to assess the role of FA in sporadic laryngeal squamous cell carcinoma (LSCC) tumor samples. Thirteen laryngeal squamous carcinoma cell lines (UT-SCC) and 64 primary laryngeal carcinoma cases were analyzed by bisulfite pyrosequencing. DNA from buccal swabs of 10 healthy volunteers was used as a control group. Promoter regions of FANCA, BRCA1 and BRCA2 displayed recurrent alterations in the methylation levels in cancer samples as compared to buccal swabs controls. For FANCA, hypomethylation was observed in 11/13 cell lines (p<0.0003) and all 64 primary larynx samples (p<0.001) compared to buccal swabs. For BRCA1, 4/13 cell lines (p=0.04) and 3/58 primary laryngeal cases (p=0.22) showed hypomethylation. In BRCA2, all 13 cell lines (p<0.0001) 4/63 primary LSCC (p<0.01) showed hypermethylation as compared to controls. In conclusion, we show recurrent alterations of DNA methylation levels in three Fanconi anemia genes which might contribute to the pathogenesis of LSCC.

High resolution ArrayCGH and expression profiling identifies PTPRD and PCDH17/PCH68 as tumor suppressor gene candidates in laryngeal squamous cell carcinoma.

Many classical tumor suppressor genes (TSG) were identified by delineation of bi-allelic losses called homozygous deletions. To identify systematically homozygous deletions in laryngeal squamous cell carcinoma (LSCC) and to unravel novel putative tumor suppressor genes, we screened 10 LSCC cell lines using high resolution array comparative genomic hybridization (arrayCGH) and array based expression analysis. ArrayCGH identified altogether 113 regions harboring protein coding genes that showed strong reduction in copy number indicating a potential homozygous deletion. Out of the 113 candidate regions, 22 novel homozygous deletions that affected the coding sequences of 15 genes were confirmed by multiplexPCR. Three genes were homozygously lost in two cell lines: PCDH17/PCH68, PRR20, and PTPRD. For the 15 homozygously deleted genes, four showed statistically significant downregulation of expression in LSCC cell lines as compared with normal human laryngeal controls. These were ATG7 (1/10 cell line), ZMYND11 (BS69) (1/10 cell line), PCDH17/PCH68 (9/10 cell lines), and PTPRD (7/10 cell lines). Quantitative real-time PCR was used to confirm the downregulation of the candidate genes in 10 expression array-studied cell lines and an additional cohort of cell lines; statistical significant downregulation of PCDH17/PCH68 and PTPRD was observed. In line with this also Western blot analyses demonstrated a complete absence of the PCDH17 and PTPRD proteins. Thus, expression profiling confirmed recurrent alterations of two genes identified primarily by delineation of homozygous deletions. These were PCDH17/PCH68, the protocadherin gene, and the STAT3 inhibiting receptor protein tyrosine phosphatase gene PTPRD. These genes are good candidates for novel TSG in LSCC.

[An attempt to rationalize adverse health effects following tobacco smoking]. / Próba racjonalizacji zagrozenia dla zdrowia w nastepstwie palenia tytoniu.

Statistics concerning tobacco smoking confronted with cancer epidemiology indicates that only a minority of smokers develops terminal cancer. To much extent it could be explained by genetic polymorphism responsible for a variable risk to develop cancer and its further progression. Then, a comparative analysis of the data concerning cancer deaths regarding a decline of smoking in developed countries unravels other factors previously not considered to represent carcinogenic agents. Human papilloma virus (HPV) could serve as an example of such agent exerting adverse health effects once attributed only to tobacco.

Polymorphisms in base-excision repair and nucleotide-excision repair genes in relation to lung cancer risk.

Polymorphisms in DNA repair genes may be associated with differences in DNA repair capacity, thereby influencing the individual susceptibility to smoking-related cancer. We investigated the association of 10 base-excision and nucleotide-excision repair gene polymorphisms (XRCC1 -77 T/C, Arg194Trp, Arg280His and Arg399Gln; APE1 Asp148Glu; OGG1 Ser326Cys; XPA -4 G/A; XPC PAT; XPD Asp312Asn and Lys751Gln) with lung cancer risk in Caucasians. Genotypes were determined by PCR-RFLP and PCR-single base extension assays in 110 lung cancer patients and 110 age- and sex-matched controls, and the results were analyzed using logistic regression adjusted for relevant covariates. A significant association between the APE1 Asp148Glu polymorphism and lung cancer risk was found, with adjusted odds ratios (OR) of 3.38 (p=0.001) for the Asp/Glu genotype and 2.39 (p=0.038) for the Glu/Glu genotype. Gene-smoking interaction analyses revealed a statistically significant interaction between cumulative cigarette smoking and the XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms: these polymorphisms were significantly associated with lung cancer in nonsmokers and light smokers (<25 PY; OR=4.92, p=0.021 for XRCC1 399 Gln/Gln; OR=3.62, p=0.049 for XPD 751 Gln/Gln), but not in heavy smokers (> or =25 PY; OR=0.68, p=0.566 for XRCC1 399 Gln/Gln; OR=0.46, p=0.295 for XPD 751 Gln/Gln). Both the XRCC1 Arg194Trp and Arg280His as well as the OGG1 Ser326Cys heterozygous genotypes were associated with a significantly reduced risk for lung cancer (OR=0.32, p=0.024; OR=0.25, p=0.028; OR=0.51, p=0.033, respectively). No associations with lung cancer risk were found for the XRCC1 -77 T/C, the XPA -4 G/A and the XPC PAT polymorphisms. In conclusion, the APE1 Asp148Glu polymorphism is highly predictive for lung cancer, and cumulative cigarette smoking modifies the associations between the XRCC1 Arg399Gln and the XPD Lys751Gln polymorphisms and lung cancer risk.