Rigorous benchmarking of T-cell receptor repertoire profiling methods for cancer RNA sequencing.

The ability to identify and track T-cell receptor (TCR) sequences from patient samples is becoming central to the field of cancer research and immunotherapy. Tracking genetically engineered T cells expressing TCRs that target specific tumor antigens is important to determine the persistence of these cells and quantify tumor responses. The available high-throughput method to profile TCR repertoires is generally referred to as TCR sequencing (TCR-Seq). However, the available TCR-Seq data are limited compared with RNA sequencing (RNA-Seq). In this paper, we have benchmarked the ability of RNA-Seq-based methods to profile TCR repertoires by examining 19 bulk RNA-Seq samples across 4 cancer cohorts including both T-cell-rich and T-cell-poor tissue types. We have performed a comprehensive evaluation of the existing RNA-Seq-based repertoire profiling methods using targeted TCR-Seq as the gold standard. We also highlighted scenarios under which the RNA-Seq approach is suitable and can provide comparable accuracy to the TCR-Seq approach. Our results show that RNA-Seq-based methods are able to effectively capture the clonotypes and estimate the diversity of TCR repertoires, as well as provide relative frequencies of clonotypes in T-cell-rich tissues and low-diversity repertoires. However, RNA-Seq-based TCR profiling methods have limited power in T-cell-poor tissues, especially in highly diverse repertoires of T-cell-poor tissues. The results of our benchmarking provide an additional appealing argument to incorporate RNA-Seq into the immune repertoire screening of cancer patients as it offers broader knowledge into the transcriptomic changes that exceed the limited information provided by TCR-Seq.

Clinical characteristics and prognostic characterization of endometrial carcinoma: a comparative analysis of molecular typing protocols.

BACKGROUND: Endometrial carcinoma (EC) is one of the most common gynecological malignancies in China and globally, accounting for the fourth-prevalent cancer in women. Although numerous studies have confirmed prognostic value of The Cancer Genome Atlas (TCGA) molecular subgroups, it is unclear how they are combined with histological features. The main objective of this study was to compare ProMisE and TCGA classification for the rapid and accurate prediction of prognosis within EC patients, together with the provision of a revised strategy for individualized diagnosis and treatment of patients. METHODS: Within this study, 70 patients with EC from Beijing Tsinghua Changgeng Hospital (affiliated to Tsinghua University) were retrospectively examined between July 2015 and December 2021. Samples were processed for determination of clinical markers, together with ProMisE and TCGA classification. RESULTS: Comparative analysis across four TCGA types (POLE, Low-CN, High-CN, and MSI-H) and age, was statistically significant (χ²= 7.000, p = 0.029). There was no significant difference observed among the four TCGA types and FIGO stage, vascular invasion and depth of invasion, or lymph node metastasis and tumor area. There was no significant association between the expression of Vimentin, Ki-67, PTEN, MSH2, PAX-8, ß-catenin, CD10, ER, PR, P16, MLH1, and PMS2 with the four TCGA types. In addition, p63 expression (χ²= 11.09, p = 0.029) and p53 expression (χ²= 11.585, p = 0.005) were statistically significant. Numerous models demonstrated that patients with POLE mutations and low-CN had higher progression free survival (PFS) and overall survival (OS), whereas those with high-CN had lowest values. The log-rank test revealed that the survival rate of PR-positive and ER-positive patients was significantly higher (p < 0.001). CONCLUSION: Overall, these results can be of additional benefit for clinical applications, in comparison to the ProMisE classification method. In addition, PR, ER, vascular infiltration, hyperlipidemia and atherosclerosis were found to be the key factors affecting EC prognosis.

Deletion of CD36 exhibits limited impact on normal hematopoiesis and the leukemia microenvironment.

BACKGROUND: CD36 has been identified as a potential therapeutic target both in leukemic cells and in the tumor immune microenvironment. In acute myeloid leukemia (AML), we found that APOC2 acts with CD36 to promote leukemia growth by activating the LYN-ERK signaling. CD36 also plays a role in lipid metabolism of cancer associated T-cells leading to impaired cytotoxic CD8+ T-cell and enhanced Treg cell function. To establish CD36 as a viable therapeutic target in AML, we investigated whether targeting CD36 has any detrimental impact on normal hematopoietic cells. METHODS: Differential expression data of CD36 during human and mouse normal hematopoiesis were examined and compared. Cd36 knockout (Cd36-KO) mice were evaluated for blood analysis, hematopoietic stem cells and progenitors (HSPCs) function and phenotype analyses, and T cells in vitro expansion and phenotypes in comparison with wild type (WT) mice. In addition, MLL-PTD/FLT3-ITD leukemic cells were engrafted into Cd36-KO and WT mice, and leukemia burden was compared between groups. RESULTS: RNA-Seq data showed that Cd36 expression was low in HSPCs and increased as cells matured. Phenotypic analysis revealed limited changes in blood count except for a slight yet significantly lower red blood cell count and hemoglobin and hematocrit levels in Cd36-KO mice compared with WT mice (P < 0.05). In vitro cell proliferation assays of splenocytes and HSPCs from Cd36-KO mice showed a similar pattern of expansion to that of cells from WT mice. Characterization of HSPCs showed similar percentages of the different progenitor cell populations between Cd36-KO with WT mice. However, Cd36-KO mice exhibited ~ 40% reduction of the number of colonies developed from HSPCs cells compared with WT mice (P < 0.001). Cd36-KO and WT mice presented comparably healthy BM transplant in non-competitive models and developed similar leukemia burden. CONCLUSIONS: Although the loss of Cd36 affects the hematopoietic stem cell and erythropoiesis, limited detrimental overall impact was observed on normal Hematopoietic and leukemic microenvironments. Altogether, considering the limited impact on normal hematopoiesis, therapeutic approaches to target CD36 in cancer are unlikely to result in toxicity to normal blood cells.

Clinicopathological characteristics of endometrial carcinoma with different molecular subtypes and their correlation with lymph node metastasis.

Endometrial carcinoma (EC) is one of the three major malignancies of the female reproductive organs. With intense research of tumor molecular mechanisms and development of precision medicine in recent years, the traditional pathomorphological classification fails to meet the needs of clinical diagnosis and treatment for EC. This study aims to analyze the correlation of different Proactive Molecular Risk Classifier for Endometrial Cancer molecular subtypes with lymph node metastasis (LNM) and other clinical features in EC. 120 treatment-naive EC patients with surgery were enrolled in this study. The molecular subtypes of these patients were classified as follows by Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) molecular subtyping: mismatch repair deficiency (MMRd) in 22 cases (18.33%), polymerase epsilon exonuclease domain mutation (POLE EDM) in 2 cases (1.67%), p53 wild-type (p53-wt) in 64 cases (53.33%), and p53 abnormal (p53-abn) in 32 cases (26.67%). The clinicopathological features of 120 patients were retrospectively analyzed. Statistical significance was identified among the four molecular subtypes in terms of histological classification, International Federation of Gynecology and Obstetrics (FIGO) staging, pathological grading, and LNM. Among the enrolled cases, 26 had LNM and 94 had no lymph node involvement. According to the multivariate Logistic regression analysis, p53 wt (P=0.008, OR=0.078, 95% CI: 0.012-0.510) was a protective factor for LNM in EC patients, while poorly differentiated histology (P=0.001, OR=15.137, 95% CI: 3.013-76.044) was a risk factor. ProMisE classification system, being more objective and reproducible, can provide an important reference for preoperative decision-making. The patients with p53 wt by ProMisE had a low risk of LNM in preoperative diagnostic curettage specimens, while there was a higher risk of LNM among the patients with poorly differentiated EC.

Enhanced T cell activation and cytotoxicity against AML via targeted anti-CD99 nanoparticle treatment.

CD99 is a transmembrane protein overexpressed in Acute Myeloid Leukemia (AML), presenting a potential novel therapeutic target. Our group has previously developed anti-CD99-A192 (α-CD99-A192), comprising of single chain variable fragment (scFv) and elastin-like polypeptides (ELPs), and reported promising anti-leukemic activity in AML preclinical models. Treatment with α-CD99-A192 induced apoptosis in AML cell lines and prolonged survival in AML xenograft models. Considering CD99's expression and role in T cell activation, in the current study, we propose that α-CD99-A192 plays a dual function, i.e., targeting leukemic cells and activating T cells. This manuscript reports the effects of α-CD99-A192 on T cells in the context of AML. α-CD99-A192 treatment enhances T cell proliferation and activation and increases the release of pro-inflammatory cytokines along with increased aggregation of T cells, which culminates in heightened cytotoxicity against leukemic cells. Altogether, these findings suggest α-CD99-A192 enhances T cell activation and cytotoxic potential consistent with dual mechanisms of action for α-CD99-A192.

FLT3/CD99 Bispecific Antibody-Based Nanoparticles for Acute Myeloid Leukemia.

Cluster of differentiation 99 (CD99) is a receptor that is significantly upregulated in acute myeloid leukemia (AML). FMS-like tyrosine kinase 3 internal tandem duplication mutation in AML (FLT3-ITD AML) exhibits even higher levels of CD99 expression. Our group previously employed a novel peptide platform technology called elastin-like polypeptides and fused it with single-chain antibodies capable of binding to FLT3 (FLT3-A192) or CD99 (CD99-A192). Targeting either FLT3 or CD99 using FLT3-A192 or CD99-A192 led to AML cell death and reduced leukemia burden in AML mouse models. Here, we report on the development of a novel Co-Assembled construct that is capable of binding to both CD99 and FLT3 and the antileukemia activity of the bispecific construct in FLT3-ITD AML preclinical models. This dual-targeting Co-Assembled formulation exhibits cytotoxic effects on AML cells (AML cell lines and primary blasts) and reduced leukemia burden and prolonged survival in FLT3-ITD AML mouse models. Altogether, this study demonstrates the potential of an innovative therapeutic strategy that targets both FLT3 and CD99 in FLT3-ITD AML. SIGNIFICANCE: This study investigates a dual-targeting strategy in acute myeloid leukemia (AML), focusing on FLT3 and CD99. The approach demonstrates enhanced therapeutic potential, presenting a novel option for AML treatment.

Application of short tandem repeat (STR) genotyping in partial hydatidiform mole.

OBJECTIVE: The short tandem repeat (STR) technique, which is currently the most extensively applied genetic marker, works mainly due to the differences in DNA repeats, resulting in a rich population polymorphism and high genetic stability. This paper primarily investigated the application of STR genotyping in partial hydatidiform mole (PHM). METHODS: The clinical data of 31 PHM patients and 23 hydropic abortion patients diagnosed in the Pathology Department of Beijing Tsinghua Chang Gung Hospital from 2017 to 2022 were collected and retrospectively analyzed. The histomorphological features of H&E sections were observed. Immunohistochemical staining was performed to determine p57 protein levels. STR polymorphisms (STRPs), including 15 polymorphic loci and 1 sex recognition gene locus, were detected in tissue specimens, and the role of STR in the differential diagnosis of PHM was analyzed. RESULTS: In PHM cases, each STR locus of the PHM contained one maternal allele and two paternal alleles. Decidual tissue showed alleles of biparental origin. According to the Kappa's consistency test, the diagnosis made by STR showed good consistency (κ = 0.925, P < 0.001). CONCLUSIONS: STR genotyping is of great value in the diagnosis of PHM.

Effects of Vinyl Functionalized Silica Particles on Thermal and Mechanical Properties of Liquid Silicone Rubber Nanocomposites.

It is very important to develop a new method of preparing high-performance liquid silicone rubber-reinforcing filler. Herein, the hydrophilic surface of silica (SiO2) particles was modified by a vinyl silazane coupling agent to prepare a new type of hydrophobic reinforcing filler. The structures and properties of modified SiO2 particles were confirmed using Fourier-transform infrared spectroscopy (FT-IR), X-ray photoelectron spectrometer (XPS), specific surface area and particle size distribution and thermogravimetric analysis (TGA), the results of which demonstrated that the aggregation of hydrophobic particles is greatly reduced. Additionally, the effects of the vinyl-modified SiO2 particle (f-SiO2) content on the dispersibility, rheology, and thermal and mechanical properties of liquid silicone rubber (SR) composites were studied for application toward high-performance SR matrix. The results showed that the f-SiO2/SR composites possessed low viscosity and higher thermal stability, conductivity, and mechanical strength than of SiO2/SR composites. We believe that this study will provide ideas for the preparation of high-performance liquid silicone rubber with low viscosity.

A study evaluating liquid-based endometrial cytology test and transvaginal ultrasonography as a screening tool for endometrial cancer in 570 postmenopausal women.

OBJECTIVE: To evaluate the combination of transvaginal ultrasonography (TVS) and endometrial cytology test (ECT) as a potential diagnostic strategy for endometrial cancer and endometrial precancerous lesions in postmenopausal patients. METHODS: 570 postmenopausal patients admitted in our hospital due to abnormal bleeding or other symptoms and/or with endometrium thickness over 5 mm on ultrasound. The endometrial thickness was evaluated by TVS. Following obtainment with written consent, all patients underwent ECT, hysteroscopy and then dilatation and curettage (D&C). Cytological sampling was conducted by scratching the uterus cavity using SAP-1 and the samples were prepared as liquid-based smear using SurePath technology. The samples were stained using Papanicolaou method. The correlation between cytological diagnosis and TVS results with the D&C histological diagnosis was analyzed. The WHO classification was used for diagnosis. RESULTS: Sensitivity of ECT, TVS, ECT or TVS positive, ECT and TVS positive to diagnose atypical hyperplasia or worse were estimated at 80.7%, 86.8%, 97.4%, 70.2%, specificity at 94.7%, 20.4%, 17.5%, 88.4%, positive predictive value at 58.2%, 21.1%, 22.8%, 60.2%, negative predictive value at 94.4%, 86.1%, 96.4%, 92.2%, and accuracy at 84.6%, 33.7%, 33.5%, 84.7%, respectively. CONCLUSIONS: Transvaginal ultrasonography and Endometrial cytology test may be regarded as a effective first-line method in endometrial pathology detection in postmenopausal women.

[Correlations of p-AKT with PTEN, P53, HER-2 expressions in endometrial carcinoma and their relationship with patient survival].

OBJECTIVE: To investigate the correlation of phosphorylated protein kinase B(p-AKT) with phosphatase and tensin homolog(PTEN), P53,human epidermal growth factor receptor 2(HER-2) expressions in endometrial carcinoma and their significance. METHODS: The expressions of p-AKT with PTEN, HER-2, P53 and Ki67 were assessed in 95 endometrial carcinomas using immunohistochemistry. The biomarker expressions correlated with clinicopathologic variables and with patient survival. RESULTS: (1) p-AKT was positive in 53.7% (51/95) of tumors and was found to express almost similarly in endometrioid adenocarcinoma(EC) and non-enometrioid adenocarcinoma (NEC). There was no significant difference of patient survival between p-AKT positive and negative subgroups (P=0.757). (2) PTEN loss was found in 56.8%(54/95) of tumors, and occurred more often in EC(60.7%, 51/84) than in NEC (27.3%, 3/11), which was of statistical significance (P=0.035). The patients with PTEN loss had a longer survival than those without (P=0.015). (3) Although there was no significant correlation between p-AKT and PTEN expression, the extended analysis showed that the predictive value of PTEN loss in p-AKT positive subgroup (P=0.148) was lower than that in p-AKT negative expression subgroup (P=0.055). Meanwhile p-AKT positive and PTEN loss might have synergic effect on tumor proliferation, Ki67 positive rate was highest in PTEN+/p-AKT+ subgroup (40.0%) and lowest in PTEN-/p-AKT- subgroup (8.7%), which was of significant difference (P=0.015). (4) No correlation was found between p-AKT expression and P53 or HER-2 status (P>0.05). On the other hand, HER-2 and P53 positive correlated significantly (r=0.209, P=0.041) and occurred more frequently in NEC (45.5%, 100.0%) than in EC(6.0%, 42.9%)(P <0.05), which were found to predict poor survival (P<0.05). CONCLUSION: p-AKT was activated equally in EC and NEC. p-AKT positive alone might have limited effect on patient survival, however, p-AKT expression might lower the predictive value of PTEN loss in endometrial carcinoma. Moreover, p-AKT positive and PTEN loss might have synergic effect on tumor proliferation. On the other hand, as p-AKT expression did not have any correlations with PTEN, P53 and HER-2 status in this cohort, there might be other important factors involved in p-AKT activation in endometrial carcinoma. In sum, further investigation should be conducted in the targeted therapy based on p-AKT and associated molecular mechanism in advanced endometrial carcinoma.

The role of upper and lower genital tract microbiota alterations in term chorionamnionitis: A prospective study.

Objective: This study aimed to compare the dynamics of lower and upper genital tract microbiota in normal term pregnancy, histological chorioamnionitis (HCA), and clinical chorioamnionitis (CCA) patients to provide a reference for the diagnosis and treatment of chorioamnionitis (CAM) patients. Methods: We prospectively collected vaginal and cervical secretions, as well as placenta tissues, fetal membranes, and amniotic fluid from normal-term pregnant women, HCA and CCA patients. Then, we performed genomic DNA extraction and PCR amplification for all samples. The eligible samples were analyzed by 16S ribosomal RNA (16S rRNA) sequencing. Additionally, all placenta tissues were histopathologically examined, and neonatal pharyngeal swabs and placenta tissues from the HCA and CCA groups were subjected to microbial culture. Results: A total of 85 term pregnant women were enrolled in this study, including 34 in the normal group (N), 37 in the HCA group, and 14 in the CCA group. A total of 171 qualified samples were analyzed by 16S rRNA sequencing. The results suggested that the cervical microbiota was highly similar to the vaginal microbiota in normal term parturients, with Lactobacillus as the dominant bacterium. Moreover, there was no difference in the alpha and beta diversity of vaginal microbiota between the N, HCA, and CCA groups at the genus level. Besides, no significant differences were detected in cervical microbiome among the three groups. Regarding intrauterine microorganisms, the N and HCA groups had similar microbial composition but were different from the CCA group. No microbe was detected in the placental tissue of normal term parturients, while some microorganisms were found in the intrauterine amniotic fluid and fetal membrane samples. Regardless of cultivation or 16S rRNA sequencing, an extremely low microbial positive rate was detected in HCA and CCA intrauterine samples. Compared to the normal group, Lactobacillus was significantly reduced in the CCA group intrauterine, and Ureaplasma and Enterococcus increased with no statistically significant. Conclusion: The N, HCA and CCA groups had similar composition of vaginal and cervical microflora. Some normal-term pregnant women can harbor non-pathogenic microbiota in the uterine cavity. Sterile inflammation is more frequent than microbial-associated inflammation in term HCA and CCA parturients.

[Significance of phosphoinositide 3 kinase/AKT pathway alterations in endometrial carcinoma].

OBJECTIVE: To investigate the clinicopathologic and prognostic implications of phosphoinositide 3 kinase (PI3K)/AKT pathway alterations in endometrial cancers of Chinese women. METHODS: The expression of PTEN, p-AKT, and ER/PR was assessed in 71 cases of endometrial carcinoma by immunohistochemistry (EnVision method). The PIK3CA mutation at exon 9 and exon 20 was analyzed by PCR and direct sequencing in 34 tumors. RESULTS: (1) Of the 71 cases of endometrial carcinoma, 65 cases were endometrioid adenocarcinoma (EEC) and 6 cases were nonendometrioid adenocarcinoma (NEEC). PTEN loss of expression was found in 63.4% (45/71) of tumors, and more commonly occurred in EEC (66.2%, 43/65) than that in NEEC (2/6, P = 0.18). Patients with PTEN loss in their tumors (45 cases) had a better survival than those without (26 cases, P = 0.07). In ER negative subgroup, the patients with PTEN loss of expression (12 cases) had longer survival than those with normal PTEN expression (7 cases; P = 0.04). (2) The frequency of PIK3CA mutation was 41.2% (14/34) with a hot mutation spot at T544 in exon 9. PIK3CA mutations more commonly occurred in EEC (44.8%, 13/29) than in NEEC (1/5, P > 0.05). The mutations at exon 9 more commonly occurred in EEC, well- and moderately-differentiated EEC, and tumors at early stage (P > 0.05). On the contrary, in tumors at early stages, the frequency of mutations in exon 20 (14.3%, 4/28) was significantly lower than that at late stages (4/6, P = 0.01). (3) p-AKT was positive in 59.2% (42/71) of tumors that were more frequently found in EEC (60.0%, 39/65) than that in NEEC (3/6, P = 0.68). However, the significant difference of p-AKT expression was found between well- and moderately-differentiated EEC (75.0%, 21/28; 53.6%, 15/28) and poorly-differentiated EEC (3/9, P = 0.02). Moreover, p-AKT expression was significantly correlated with positive ER (r = 0.339, P = 0.00). CONCLUSIONS: Endometrial carcinoma patients with loss of PTEN and p-AKT positivity have a favorable prognosis. PIK3CA mutations at exon 9 or 20 may have different impact on the prognosis. The function of PTEN loss and p-AKT expression may vary according to different hormone status.

A risk score system for predicting complicated appendicitis and aid decision-making for antibiotic therapy in acute appendicitis.

BACKGROUND: Exclusive antibiotic therapy is a feasible treatment option for uncomplicated appendicitis, but the pre-treatment diagnosis of uncomplicated appendicitis is challenging. This study aimed to develop a risk score system to predict complicated appendicitis and aiding decision-making regarding antibiotic therapy for acute appendicitis. METHODS: The risk score system for predicting complicated appendicitis was constructed and validated by a surgical therapy cohort (n=543). Furthermore, we applied an independent antibiotic treatment cohort (n=169) to verify whether the risk score system could guide antibiotic treatment decision-making in patients with acute appendicitis (AA). RESULTS: A total of 543 patients were included in the surgical therapy cohort and was split into the primary (n=375) and validation (n=168) cohorts with repeated random sampling. In the primary cohort, multivariate analysis confirmed that periappendiceal fat stranding (PFS, P<0.001, OR =67.80), the C-reactive protein level (CRP ≥38 mg/L, P<0.001, OR =5.77) and the neutrophil-to-lymphocyte ratio (NLR ≥7, P<0.001, OR =3.51) were independent risk factors for complicated appendicitis. The PFS, CRP and NLR scores were 10.0, 4.0 and 3.0 points, respectively. Fourteen patients (3.7%, 14/375) and seven patients (4.2%, 7/168) with pathologically confirmed complicated appendicitis were classified as having uncomplicated appendicitis in the primary and validation cohorts based on the risk score system, respectively. In the independent antibiotic treatment cohort (n=169), the failure rate of antibiotic treatment was 49.2% and 5.3% for the risk score system predicted complicated AA and uncomplicated AA. Furthermore, the predictive accuracy of the risk score system for antibiotic treatment failure as measured by the area under the curve (AUC) was 0.823 (95% CI: 0.757-0.878). CONCLUSIONS: We found that the proposed risk score system based on biological and CT features not only enables the accurate identification of complicated appendicitis patients before pre-treatment but also serves to guide antibiotic treatment decisions.

Predictors and risk factors for intestinal necrosis in patients with mesenteric ischemia.

BACKGROUND: Mesenteric ischemia results in blood flow that is insufficient to meet metabolic demands and subsequent dysfunction of visceral organs, including arterial obstruction and venous thrombosis. Sustained mesenteric ischemia exhausts the ability of capillaries to provide oxygen and initiate an inflammatory reaction, and eventually leads to intestinal mucosal necrosis, a serious and potentially life-threatening condition. Therefore, it is essential that the predictors and risk factors for intestinal necrosis in patients with mesenteric thrombus are explored. METHODS: This study retrospectively enrolled 41 patients with mesenteric ischemia (including mesenteric vein embolism, mesenteric artery thrombosis, and portal vein thrombosis) who were admitted to the Department of Gastrointestinal Surgery, Beijing Tsinghua Changgung Hospital between May 2016 and October 2019; of the patients, 18 were further diagnosed with intestinal necrosis. Comparisons of symptoms, computed tomography angiography (CTA) features, and laboratory examination results were performed between mesenteric ischemia patients with and without intestinal necrosis. RESULTS: White blood cell count showed an excellent predictive ability for intestinal necrosis in patients with mesenteric ischemia, with an area under the receiver operating characteristic (ROC) curve of 0.772 (P=0.009). The four CTA features [pneumatosis (P=0.016), intestinal swelling (P=0.006), ascitic fluid (P<0.001), and decreased intestinal wall enhancement (P=0.004)] differed significantly between patients with and without intestinal necrosis. Peritonitis showed a strong association with intestinal necrosis (P=0.006) in the univariate analysis, and multivariate analysis further showed their association [odds ratio (OR): 8.53; 95%CI: 1.46-49.81; P=0.017]. CONCLUSIONS: White blood cell count is a potential predictor of intestinal necrosis. Peritonitis is a possible risk factor for intestinal necrosis in patients with mesenteric ischemia. A multi-center prospective study with a larger sample size needs to be performed to further investigate these findings.