The prognostic and potentially immunomodulatory role of cartilage oligomeric matrix protein in patients with gastric and esophageal adenocarcinoma.

BACKGROUND: Cartilage oligomeric matrix protein (COMP) is a novel regulator of the tumor microenvironment. Studies in colon cancer and pancreatobiliary adenocarcinoma have revealed COMP expression to be associated with decreased infiltration of immune cells in the tumor microenvironment. Herein, the expression of COMP was investigated in gastric and esophageal adenocarcinoma with particular reference to its the relationship with the immune microenvironment. METHODS: COMP expression was evaluated in tissue microarrays representing primary tumors from 159 patients with chemo- and radiotherapy naïve esophageal and gastric adenocarcinoma and 67 matched samples of lymph node metastases using immunohistochemistry. Additionally, collagen fibers were stained with Sirius Red and evaluated with the FIJI macro TWOMBLI algorithm. RESULTS: The expression of COMP in cancer cells in the entire cohort was associated with shorter overall survival (OS) (p = 0.013) and recurrence-free survival (RFS) (p = 0.029), while COMP expression in the stroma was correlated with shorter RFS (p = 0.042). Similar correlations were found for patients with gastric adenocarcinoma, whereas COMP expression was not prognostic in esophageal adenocarcinoma. Further, in the entire cohort, the expression of COMP in the stroma was correlated with exclusion of different populations of immune cells (CD8+, CD3+, FoxP3+, CD20+) from the tumor microenvironment. Finally, higher density and alignment of collagen fibers were correlated with the expression of COMP in the stroma. CONCLUSIONS: Expression of COMP in gastric and esophageal adenocarcinoma was correlated with shorter OS and RFS. A reduced number of immune cells infiltrated the tumor microenvironment when COMP expression was detected. This phenomenon could be attributed to the denser collagen deposits, a hallmark of tumor fibrosis observed in COMP-expressing tumors.

Stromal cartilage oligomeric matrix protein as a tumorigenic driver in ovarian cancer via Notch3 signaling and epithelial-to-mesenchymal transition.

BACKGROUND: Cartilage oligomeric matrix protein (COMP), an extracellular matrix glycoprotein, is vital in preserving cartilage integrity. Further, its overexpression is associated with the aggressiveness of several types of solid cancers. This study investigated COMP's role in ovarian cancer, exploring clinicopathological links and mechanistic insights. METHODS: To study the association of COMP expression in cancer cells and stroma with clinicopathological features of ovarian tumor patients, we analyzed an epithelial ovarian tumor cohort by immunohistochemical analysis. Subsequently, to study the functional mechanisms played by COMP, an in vivo xenograft mouse model and several molecular biology techniques such as transwell migration and invasion assay, tumorsphere formation assay, proximity ligation assay, and RT-qPCR array were performed. RESULTS: Based on immunohistochemical analysis of epithelial ovarian tumor tissues, COMP expression in the stroma, but not in cancer cells, was linked to worse overall survival (OS) of ovarian cancer patients. A xenograft mouse model showed that carcinoma-associated fibroblasts (CAFs) expressing COMP stimulate the growth and metastasis of ovarian tumors through the secretion of COMP. The expression of COMP was upregulated in CAFs stimulated with TGF-ß. Functionally, secreted COMP by CAFs enhanced the migratory capacity of ovarian cancer cells. Mechanistically, COMP activated the Notch3 receptor by enhancing the Notch3-Jagged1 interaction. The dependency of the COMP effect on Notch was confirmed when the migration and tumorsphere formation of COMP-treated ovarian cancer cells were inhibited upon incubation with Notch inhibitors. Moreover, COMP treatment induced epithelial-to-mesenchymal transition and upregulation of active ß-catenin in ovarian cancer cells. CONCLUSION: This study suggests that COMP secretion by CAFs drives ovarian cancer progression through the induction of the Notch pathway and epithelial-to-mesenchymal transition.

Expression of Cartilage Oligomeric Matrix Protein in colorectal cancer is an adverse prognostic factor and correlates negatively with infiltrating immune cells and PD-L1 expression.

Introduction: Cartilage Oligomeric Matrix Protein (COMP) is an oncogenic protein that has been associated with a decrease in infiltrating T-cells in periampullary adenocarcinoma. This study aimed to investigate whether this is also the case for colorectal cancer (CRC) and to evaluate the relationship between COMP expression and clinopathological features. Methods: Immunohistochemistry was used to determine the expression levels of COMP in tumor cells and stroma in primary tumors from a cohort of 537 CRC patients. The expression of immune cell markers, including CD3+, CD8+, FoxP3+, CD68+, CD56+, CD163+, and PD-L1, was evaluated previously. Tumor fibrosis was assessed by Sirius Red staining and evaluation of collagen fiber organization. Results: COMP expression correlated positively with TNM-stage and grade of differentiation. Patients with CRC expressing high levels of COMP had significantly shorter OS than those with low COMP expression (p<0.0001), and fewer infiltrating T-cells were detected in tumors with high COMP expression. Additionally, a negative correlation was identified between the expression of COMP and PD-L1 on both tumor cells and immune cells. Cox regression analysis showed that tumors expressing high levels of COMP had significantly shorter OS, independent of all evaluated immune cell markers. Tumor fibrosis was correlated with high expression of COMP in the stroma (p<0.0001), and tumors with high levels of COMP expression and denser fibrosis displayed more sparse immune cell infiltration. Discussion: The results suggest that COMP expression in CRC may exert an immune regulatory effect by increasing dense fibrosis and decreasing immune cell infiltration. These findings support the notion that COMP is an important factor in the development and progression of CRC.

Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner.

BACKGROUND: Sushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor. METHODS: In a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method. RESULTS: We discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells. CONCLUSIONS: In this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane.

Expression of cartilage oligomeric matrix protein in periampullary adenocarcinoma is associated with pancreatobiliary-type morphology, higher levels of fibrosis and immune cell exclusion.

Cartilage oligomeric matrix protein (COMP) is an emerging regulator of tumor progression. The aim of this study was to evaluate the expression of COMP in periampullary adenocarcinoma with respect to prognostic value for survival and relapse, levels of fibrosis and infiltrating immune cells. COMP expression was evaluated using immunohistochemistry in primary tumors and subsets of paired lymph node metastases in tissue microarrays including 175 patients with periampullary adenocarcinoma. Collagen content was assessed with Sirius Red-Fast Green staining. High COMP levels were detected in cancer cells and in stroma, in 46% and 57% of the patients, respectively. High COMP expression was strongly associated with more aggressive pancreatobiliary-type (PB-type) compared to intestinal-type tumors (p < .0001). Importantly, high expression of COMP correlated with the exclusion of cytotoxic T-cells from the cancer cell compartment of the tumors, particularly in PB-type tumors. Higher levels of fibrosis measured by the density of collagen fibers correlated with high COMP levels in both cancer cells and stroma. This in turn could lead to exclusion of cytotoxic T-cells from accessing the cancer cells, a recognized immunotherapy resistance mechanism. Targeting COMP could therefore be considered as a novel therapeutic strategy in PB-type periampullary adenocarcinoma.

High Levels of Expression of Cartilage Oligomeric Matrix Protein in Lymph Node Metastases in Breast Cancer Are Associated with Reduced Survival.

Cartilage oligomeric matrix protein (COMP) is a regulator of the extracellular matrix and is expressed primarily in the cartilage. Recently, COMP expression was also documented in breast cancer patients both in sera and tumor biopsies, in both of which it could serve as an independent prognostic marker. This study aimed to assess COMP as a potential biomarker in the group of metastatic breast cancer patients. Levels of COMP were measured by ELISA in serum samples of 141 metastatic breast cancer patients. Biopsies from primary tumors, synchronous lymph node metastases, and distant metastases were stained for COMP expression. The levels of serum COMP were higher in patients with ER- and HER2-positive tumors when compared to triple-negative tumors and correlated with the presence of bone and lung metastases, circulating tumor cell count, and clusters. Most of the primary tumors expressing COMP (70%) retained the expression also in the lymph node metastases, which correlated with visceral metastases and reduced survival. In conclusion, COMP appears as a valuable biomarker in metastatic breast cancer patients indicating a more severe stage of the disease. Serum COMP levels were associated with specific types of metastases in patients with metastatic breast cancer emphasizing that further studies are warranted to elucidate its potential role as a monitoring marker.

Complement inhibitor CSMD1 modulates epidermal growth factor receptor oncogenic signaling and sensitizes breast cancer cells to chemotherapy.

BACKGROUND: Human CUB and Sushi multiple domains 1 (CSMD1) is a large membrane-bound tumor suppressor in breast cancer. The current study aimed to elucidate the molecular mechanism underlying the effect of CSMD1 in highly invasive triple negative breast cancer (TNBC). METHODS: We examined the antitumor action of CSMD1 in three TNBC cell lines overexpressing CSMD1, MDA-MB-231, BT-20 and MDA-MB-486, in vitro using scanning electron microscopy, proteome array, qRT-PCR, immunoblotting, proximity ligation assay, ELISA, co-immunoprecipitation, immunofluorescence, tumorsphere formation assays and flow cytometric analysis. The mRNA expression pattern and clinical relevance of CSMD1 were evaluated in 3520 breast cancers from a modern population-based cohort. RESULTS: CSMD1-expressing cells had distinct morphology, with reduced deposition of extracellular matrix components. We found altered expression of several cancer-related molecules, as well as diminished expression of signaling receptors including Epidermal Growth Factor Receptor (EGFR), in CSMD1-expressing cells compared to control cells. A direct interaction of CSMD1 and EGFR was identified, with the EGF-EGFR induced signaling cascade impeded in the presence of CSMD1. Accordingly, we detected increased  ubiquitination levels of EGFR upon activation in CSMD1-expressing cells, as well as increased degradation kinetics and chemosensitivity. Accordingly, CSMD1 expression rendered tumorspheres pretreated with gefitinib more sensitive to chemotherapy. In addition, higher mRNA levels of CSMD1 tend to be associated with better outcome of triple negative breast cancer patients treated with chemotherapy. CONCLUSIONS: Our results indicate that CSMD1 cross-talks with the EGFR endosomal trafficking cascade in a way that renders highly invasive breast cancer cells sensitive to chemotherapy. Our study unravels one possible underlying molecular mechanism of CSMD1 tumor suppressor function and may provide novel avenues for design of better treatment.

High Levels of Cartilage Oligomeric Matrix Protein in the Serum of Breast Cancer Patients Can Serve as an Independent Prognostic Marker.

Background: Cartilage oligomeric matrix protein (COMP) is a pentameric cartilage protein also expressed in breast cancer tumors. A high expression of COMP evaluated by immunohistochemical staining is as an independent prognostic marker associated with poor patients' prognosis. Methods: Herein, levels of COMP were analyzed using an IVD approved ELISA in serum samples from 233 well-characterized breast cancer patients; 176 with metastatic breast cancer; and 57 in an early stage of the disease. Results: The metastatic patients had double the concentration of serum COMP compared with those with early breast cancer. High levels of COMP in sera of metastatic patients were associated with the histological subtype (p = 0.025) and estrogen receptor positivity (p = 0.019) at the time of breast cancer diagnosis. Further, correlation was observed between the serum levels of COMP and the presence of liver (p = 0.010) or bone (p = 0.010) metastases in this population. Most importantly, elevated serum levels of COMP appear to serve as an independent prognostic marker of survival as assessed by Cox proportional hazard regression analysis (p = 0.001) for the metastatic patients. Among metastatic patients treated with taxanes (Docetaxel-Paclitaxel) as part of their first metastatic line (n = 25), those with high levels of serum COMP detected in the metastatic stage of the disease had a shorter median survival (0.2 years) compared with those with low levels of serum COMP (1.1 years) (p = 0.001). Conclusions: Taken together, the serum levels of COMP are elevated in the metastatic patients and may be a potential novel biomarker for the evaluation of the prognosis in this population.

Cartilage Oligomeric Matrix Protein initiates cancer stem cells through activation of Jagged1-Notch3 signaling.

Cancer stem cell populations are important for the initiation, progression and metastasis of tumors. The mechanisms governing cancer stem cell control are only partially understood, but activation of the Notch3 pathway plays a crucial role in the maintenance of breast cancer stem cells. Expression of Cartilage Oligomeric Matrix Protein (COMP) in breast cancer cells is correlated with poor survival and higher recurrence rates in patients. In this study, we provide in vivo and in vitro evidence that COMP expression increases the proportion of cancer stem cells in breast cancer. Thus, MDA-MB-231 and BT-20 cells expressing COMP formed larger tumorspheres in vivo and in vitro and displayed higher ALDH-activity than cells lacking COMP. Additionally, BT-20 COMP-expressing cells displayed higher expression of CD133 compared with the control cells. Furthermore, among the different Notch receptors, Notch3 is specifically activated in COMP-expressing cells. Mechanistically, activation of Notch3 is mediated by secreted, polymeric COMP, which interacts with both Notch3 and its ligand Jagged1, bridging the receptor and ligand together, enhancing Notch3-specific signaling. COMP-dependent Notch3 activation also leads to cross-talk with ß-Catenin and AKT pathways. Using the model of MMTV-PyMT mouse breast tumorigenesis, we observed a decrease in the size of tumors and the amount of cancer stem cells as well as reduced Notch3 activation, in COMP knockout mice in comparison to wild type mice. In conclusion, we reveal a novel molecular mechanism whereby COMP regulates the cancer stem cell population through increasing the interaction between Notch3 and Jagged1, leading to increased activation of Notch3 signaling.

Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis.

Cartilage oligomeric matrix protein (COMP) was recently implicated in the progression of breast cancer. Immunostaining of 342 prostate cancer specimens in tissue microarrays showed that COMP expression is not breast cancer-specific but also occurs in prostate cancer. The expression of COMP in prostate cancer cells correlated with a more aggressive disease with faster recurrence. Subcutaneous xenografts in immunodeficient mice showed that the prostate cancer cell line DU145 overexpressing COMP formed larger tumors in vivo as compared to mock-transfected cells. Purified COMP bound to and enhanced the invasion of DU145 cells in vitro in an integrin-dependent manner. In addition, intracellular COMP expression interfered with cellular metabolism by causing a decreased level of oxidative phosphorylation with a concurrent upregulation of lactate production (Warburg effect). Further, expression of COMP protected cells from induction of apoptosis via several pathways. The effect of COMP on metabolism and apoptosis induction was dependent on the ability of COMP to disrupt intracellular Ca2+ signalling by preventing Ca2+ release from the endoplasmic reticulum. In conclusion, COMP is a potent driver of the progression of prostate cancer, acting in an anti-apoptotic fashion by interfering with the Ca2+ homeostasis of cancer cells.

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

Helicobacter pylori CagA protein induces factors involved in the epithelial to mesenchymal transition (EMT) in infected gastric epithelial cells in an EPIYA- phosphorylation-dependent manner.

As a result of Helicobacter pylori adhesion to gastric epithelial cells, the bacterial effector cytotoxin-associated gene A (CagA) is translocated intracellularly, and after hierarchical tyrosine phosphorylation on multiple EPIYA motifs, de-regulates cellular polarity and contributes to induction of an elongation and scattering phenotype that resembles the epithelial to mesenchymal transition (EMT). Stromelysin-1/matrix metalloproteinase-3 (MMP-3) has been reported to induce a sequence of molecular alterations leading to stable EMT transition and carcinogenesis in epithelial cells. To identify the putative role of CagA protein in MMP-3 induction, we exploited an experimental H. pylori infection system in gastric epithelial cell lines. We utilized isogenic mutants expressing CagA protein with variable numbers of EPIYA and phosphorylation-deficient EPIFA motifs, as well as cagA knockout and translocation-deficient cagE knockout strains. Increased levels of MMP-3 transcriptional activation were demonstrated by quantitative real time-PCR for strains with more than two terminal EPIYA phosphorylation motifs in CagA. MMP-3 expression in total cell lysates and the corresponding culture supernatants was associated with CagA expression and translocation and was dependent on CagA phosphorylation. A CagA EPIYA phosphorylation-dependent increase in gelatinase and caseinolytic activity was also detected in culture supernatants by zymography. A significant increase in the transcriptional activity of the mesenchymal markers Vimentin, Snail and ZEB1 and the stem cell marker CD44 was observed in the case of CagA containing phosphorylation-functional EPIYA motifs. Our data suggest that CagA protein induces EMT through EPIYA phosphorylation-dependent up-regulation of MMP-3. Moreover, no significant increase in EMT and stem cell markers was observed following infection with H. pylori strains that cannot effectively translocate CagA protein.

Presence of terminal EPIYA phosphorylation motifs in Helicobacter pylori CagA contributes to IL-8 secretion, irrespective of the number of repeats.

CagA protein contributes to pro-inflammatory responses during H. pylori infection, following its intracellular delivery to gastric epithelial cells. Here, we report for the first time in an isogenic background, on the subtle role of CagA phosphorylation on terminal EPIYA-C motifs in the transcriptional activation and expression of IL-8. We utilized isogenic H. pylori mutants of P12 reference strain, expressing CagA with varying number of EPIYA-C motifs and the corresponding phosphorylation defective EPIFA-C motifs while preserving intact the CM multimerization motifs. These mutants had been previously closely scrutinized in terms of type IV secretion system functionality, CagA translocation and its subsequent phosphorylation. Following infection of gastric epithelial cell lines, transcriptional activation of IL-8 gene and secreted IL-8 levels were found to be strictly dependent upon the functionality of the EPIYA-C phosphorylation motifs, as EPIFA-C phosphorylation-deficient CagA expression failed to induce full IL-8 transcriptional activity. Interestingly, levels of IL-8 gene activation and of secreted IL-8 were the same, irrespective of the number of EPIYA-C terminal repeats. We monitored IkBα phosphorylation and confirmed CagA involvement in NF-kB activation. Furthermore, we observed that presence of EPIYA-C functional phosphorylation motifs contributed to NF-kB activation. NF-kB upstream signaling events, such as early ERK1/2 and AKT activation were confirmed to be independent of EPIYA-C phosphorylation. On the contrary, use of TAK1 specific inhibitor 5Z-7-Oxozeaenol resulted in complete arrest of IL-8 secretion, in a dose-dependent manner, irrespective of CagA status. H. pylori-infected TAK1(-/-) mouse embryonic fibroblasts (MEFs) failed to induce NF-kB activity, unlike the respective control MEFs. CagA and TAK1 were found to immunoprecipitate together, irrespective of CagA EPIYA-C status, thus confirming earlier reports of TAK1 and CagA protein interaction. Our data suggest that CagA may potentially interfere with TAK1 activity during NF-kB activation for IL-8 induction in early H. pylori infection.

A mutagenesis method for the addition and deletion of highly repetitive DNA regions: the paradigm of EPIYA motifs in the cagA gene of Helicobacter pylori.

BACKGROUND: CagA protein of Western origin Helicobacter pylori isolates contains at its carboxyl-terminal end repeating types of EPIYA motifs, depending on the surrounding sequence, which dictate hierarchic tyrosine phosphorylation. To produce, in an isogenic background, mutant strains expressing CagA protein with variable numbers of EPIYA-C terminal motifs, we have adopted a mutagenesis assay using a megaprimer approach. MATERIALS AND METHODS: The H. pylori P12 reference strain containing two terminal EPIYA-C motifs was utilized. Initially, we cloned, full-length cagA gene, next to the Campylobacter jejuni kanamycin-resistance cassette, followed by the 1200-bp region located immediately after cagA gene (metacagA region). Then, we generated a megaprimer consisting of three consecutive copies of the EPIYA-C coding sequence of cagA gene, followed by the 140-bp region of the cagA genomic sequence present immediately after the second EPIYA-C repeat. We utilized these two products to perform a QuikChange mutagenesis assay and were able to obtain all desired combinations of EPIYA-C motifs, followed by Kan(r) cassette and metacagA region. These constructions were used to perform natural transformation of the P12 parental strain, by directional homologous recombination. RESULTS: We produced isogenic H. pylori strains that express CagA with variable number of EPIYA-C motifs (AB, ABC, ABCCC) and their phosphorylation-deficient counterparts. They exhibited similar growth characteristics to the parental strain, adhered equally well to gastric cells and successfully translocated CagA, following pilus induction. CONCLUSIONS: Our method can be used in other cases where highly repetitive sequences need to be reproduced.

Expansion of European vacA and cagA alleles to East-Asian Helicobacter pylori strains in Cambodia.

Helicobacter pylori infection is associated with gastric cancer (GC). The highest incidence rates have been described in Asia, but regional variations exist that do not match the distribution of infection prevalence rates. The aim of the study was to examine the possible contribution of H. pylori virulence factors to geographic differences in the incidence of GC across East and Southeast Asia. We studied 66 isolates from Cambodian patients that had previously been assigned to two genetic populations based on sequences of seven housekeeping genes, namely hpEurope (n = 34, 51.5%) and hpEastAsia, subpopulation hspEAsia (n = 32, 48.5%). These strains were characterized with respect to vacA polymorphism and cagA status by PCR, and the CagA C-terminal region was sequenced. We also sequenced the complete cagA gene from 10 hpEurope and 10 hspEAsia strains chosen at random. The cagA gene was present in 92.4% of the 66 isolates and was mainly of Western type (n = 36, 59.0%). hspEAsia strains carrying East-Asian CagA and the m1-type vacA allele (15.2%) were less frequent among the 66 Cambodian isolates than reported in East Asian countries, a finding that might partly explain the intermediate incidence of GC in Cambodia, and by extension, in Southeast Asia (except for Vietnam). The observed high prevalence of s1a alleles (34.4%) and Western CagA (28.1%) among hspEAsia strains indicates frequent introgression of European vacA and cagA alleles into East Asian H. pylori strains. This expansion might have severe consequences for individual disease outcome.

CagA and VacA polymorphisms are associated with distinct pathological features in Helicobacter pylori-infected adults with peptic ulcer and non-peptic ulcer disease.

Polymorphic variability in Helicobacter pylori factors CagA and VacA contributes to bacterial virulence. The presence of one CagA EPIYA-C site is an independent risk factor for gastroduodenal ulceration (odds ratio [OR], 4.647; 95% confidence interval [CI], 2.037 to 10.602), while the presence of the vacA i1 allele is a risk factor for increased activity (OR, 5.310; 95% CI, 2.295 to 12.287) and severity of gastritis (OR, 3.862; 95% CI, 1.728 to 8.632).