OBJECTIVES: To investigate the medium- and long-term efficacy of percutaneous mechanical thrombectomy (PMT) combined with stent implantation for treatment of iliac vein stenosis with lower extremity deep venous thrombosis (LEDVT). METHODS: Clinical and follow-up data of 125 patients with iliac vein stenosis and LEDVT who underwent PMT and stent implantation at five hospitals in northern Zhejiang province from January 2017 to June 2021 were collected. The thrombus clearance rate, thrombus recurrence rate, patency rate of iliac vein stents and post-thrombotic syndrome (PTS) occurrence rate were documented, and safety indicators such as bleeding, death, pulmonary embolism, stent fracture and displacement were assessed. RESULTS: Among 125 patients, for clearance of limb thrombosis, there were 8 cases of grade I (6.4%), 10 cases of grade II (8.0%), and 107 cases of grade III (85.6%). Patients were followed up for a median period of 74 months. According to the Villalta score, the recurrence rates of limb thrombosis at 12, 24 and 36 months were 8.48%, 8.93% and 10.91%; the iliac vein patency rates were 91.52%, 91.07%, and 89.09%; and the incidences of PTS were 5.08%, 5.36% and 6.36%, respectively. There were no major adverse events such as death, massive pulmonary embolism or severe hepatic and renal insufficiency, and no readmission intervention events due to stent fracture or other incidence were found. CONCLUSIONS PMT combined with iliac vein stent implantation is effective for patients with iliac vein stenosis complicated by LEDVT with good medium- and long-term efficacy and safety, which is worthy of clinical application.
Humans ; Stents ; Iliac Vein/pathology* ; Venous Thrombosis/surgery* ; Thrombectomy/methods* ; Female ; Male ; Middle Aged ; Aged ; Adult ; Constriction, Pathologic/surgery* ; Treatment Outcome ; Follow-Up Studies

Currently,electroencephalogram(EEG),functional near-infrared spectroscopy(fNIRS),and functional magnetic resonance imaging have been widely studied and applied to neuropsychiatric disorders.In recent years,the devices which can realize the simultaneous acquisition of EEG and fNIRS has been developed and gradually applied in the studies on neuropsychiatric disorders.The review provides an introduction of the techniques of synchronized detection and data analysis for EEG-fNIRS,summarizes the analysis methods and new findings of the recent studies of stroke,epilepsy,and other neuropsychiatric disorders using EEG-fNIRS,and also discusses the future research directions.

Objective:To construct a double-layer bone-on-a-chip containing bone matrix, with which the process of osteoblast and osteoclast differentiation in vitro is stimulated, aiming to provide a new platform for the development of osteoporosis medications. Methods:Software WorkSoild was used to design the double-layer and double-channel bone-on-a-chip and the template was fabricated by photolithography. With polydimethylsiloxane (PDMS) as the raw material, the main body of the chip was prepared by mold fabrication. The inlets and outlets of the four channels of the culture room were separated with bovine cortex bones and sealed with liquid storage columns. In the chip verification experiment, chips were divided into osteogenic and osteoclastic induction groups and osteogenic and osteoclastic control groups. In the osteogenic and osteoclastic induction groups, precursor cells of mouse embryonic osteoblast, MC3T3-E1 and mouse macrophage RAW264.7 were inoculated on the chip separately. Osteogenic induction lasted 14 days and osteoclastic induction 7 days. MC3T3-E1 cells and RAW264.7 cells were not induced in the osteogenic and osteoclastic control groups. The following indicators were observed: (1) Appearance and sealing performance of the chip: After the chip was prepared, photos were taken to observe its appearance and sealing tests were conducted to observe its sealing performance. (2) Biocompatibility: At 3 days after MC3T3-E1 cells were inoculated onto the chip and cultured and at 1, 3 and 5 days after RAW264.7 cells were inoculated onto the chip and cultured, the cell survival was observed with calcein acetoxymethyl ester/propidium iodide (AM/PI) staining and Cell Counting Kit 8 (CCK-8). (3) Osteogenic differentiation: Alkaline phosphatase (ALP) staining and alizarin red staining were performed on the cells in the osteogenic induction group to observe the osteogenic induction. RNA was collected from the osteogenic induction group and the osteogenic control group, the expression of osteoblast marker Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and type I collagen (COL1A1) was detected by real-time florescent quantitative PCR (qPCR), and the differentiation degree and osteogenic ability of osteoblasts were observed. (4) Osteoclast differentiation: tartrate-resistant acid phosphatase (TRAP) staining was performed on cells in the osteoclastic induction group to observe osteoclast differentiation. RNA was extracted from the osteoclastic induction group and the osteoclastic control group for qPCR of osteoclast differentiation-related genes, and the expression levels of the osteoclast marker gene TRAP, cathepsin K (CTSK) and dendritic cell specific transmembrane protein (DC-STAMP) were detected.Results:The double-layer bone-on-a-chip containing bone matrix was 3 cm×3 cm in size and transparent as a whole. The structure of the system on the chip system was compact and had no seepage. It was shown by calcein AM/PI staining that at 3 days after MC3T3-E1 cells and RAW264.7 cells were cultured, very few red fluorescent dead cells were found. CCK-8 test showed that within 5 days after being cultured, the cell viability was all above 90%, indicating that the biocompatibility of the chip was good and the cells could survive and proliferate normally. The results of ALP and alizarin red staining showed that MC3T3-E1 cells successfully differentiated into osteoblasts and produced calcified nodules in the osteogenic induction group at 14 days after the induction. The qPCR results showed that the relative expression level of RUNX2 in MC3T3-E1 cells in the osteogenic induction group was 4.98±0.74, which was significantly higher than that of the control group (0.99±0.03) ( P<0.01). The relative expression level of OCN in MC3T3-E1 cells was 7.98±0.76, which was significantly higher than that of the control group (1.00±0.06) ( P<0.01). The relative expression level of COL1A1 in MC3T3-E1 cells was 7.07±0.56, which was significantly higher than that of the control group (0.97±0.03) ( P<0.01). The TRAP staining results showed that the RAW264.7 cells in the osteoclastic induction group differentiated to giant multinucleated osteoclasts, and TRAP protein was expressed in large quantity in the osteoclasts. The results of qPCR showed that the relative expression level of TRAP in RAW264.7 cells in the osteoclastic induction group was 3.35±0.37, which was significantly higher than that of the control group (1.01±0.06) ( P<0.01). The relative expression level of CTSK in RAW264.7 cells was 3.46±0.79, which was significantly higher than that of the control group (1.01±0.05) ( P<0.01). The relative expression level of DC-STAMP in RAW264.7 cells was 1.92±0.12, which was significantly higher than that of the control group (0.98±0.08) ( P<0.01). Conclusions:The double-layer bone-on-a-chip containing bone matrix is compact in structure, can be cultured in vitro for a long time, has good biocompatibility and can be used for inducing osteogenic and osteoclast differentiation. Therefore, it is expected to provide a new research platform for exploring the mechanism of osteoporosis and medication screening.

In recent years, advancements in microfabrication technology and tissue engineering have propelled the development of a novel platform known as organoid-on-a-chip for drug screening and disease modeling. This platform integrates organoids and organ-on-a-chip technologies, emerging as a promising approach for in vitro modeling of human organs. Organ-on-a-chip leverages microfluidic device to simulate the physiological environment of specific organs, offering a more dynamic and flexible setting that can mimic a more comprehensive human biological context. However, the lack of functional vasculature has remained a major challenge in this technology. Vascularization is crucial for the long-term cultivation and in vitro modeling of organoids, which is of great significance in drug development and personalized medical approaches. The authors reviewed the research progress in the construction of vascularized organoid-on-a-chip including the methods for constructing in vitro vascularized models, vascularization of organoids, etc, which may serve as a reference for the construction of fully functional vascularized organoid-on-a-chip.

Objective:To compare the antiosteoporosis effect of conventional treatment and conventional treatment combined with Denosumab after percutaneous kyphoplasty (PKP) for osteoporotic vertebral compression fractures (OVCF).Methods:A retrospective cohort study was conducted to analyze the clinical data of 211 patients with OVCF admitted to the Second Affiliated Hospital of Soochow University from September 2020 to September 2022. All the patients were female, aged 56-90 years [(71.4±8.1)years]. The bone mineral density T-score of the lumbar spine was (-2.6±1.0)SD before operation. Fracture segments included T 1-T 9 in 45 patients, T 10-L 2 in 146, and L 3-L 5 in 69. Of all, 174 patients were treated with single-segment surgery, 25 with two-segment surgery and 12 with surgery involving three or more segments. According to the wishes of the patients, 107 patients were treated with daily oral administration of calcium and active Vitamin D after PKP (conventional treatment group) and 104 patients with Denosumab combined with the conventional treatment after PKP (Denosumab therapy group). The bone mineral density T-scores of the lumbar spine of the two groups were compared before surgery and at the last follow-up. The visual analogue scale (VAS) and Oswestry disability index (ODI) before surgery, at 3 days, 6 months after surgery, and at the last follow-up were evaluated and the refracture rate after surgery was detected. Possible adverse effects after medication during anti-osteoporosis treatment were observed in two the groups. Results:All the patients were followed up for 12-24 months [(13.5±2.0)months]. Before surgery, the bone mineral density T-score of the lumbar spine was (-2.7±1.1)SD in the Denosumab therapy group and (-2.5±0.8)SD in the conventional treatment group ( P>0.05). At the last follow-up, the bone mineral density T-score of the lumbar spine was (-2.1±1.1)SD in the Denosumab therapy group, significantly higher than (-2.5±0.9)SD in the conventional treatment group ( P<0.05). In the Denosumab therapy group, the bone mineral density T-score of the lumbar spine at the last follow-up was significantly increased compared to that before surgery ( P<0.01), while there was no significant difference in the conventional treatment group ( P<0.05). Before surgery and at 3 days after surgery, the VAS scores and ODI values were (8.5±0.9)points, (2.8±0.8)points, 48.7±4.8 and 25.6±4.0 in the Denosumab therapy group, which was not statistically different from those in the conventional treatment group [(8.5±1.3)points and (2.8±0.9)points, 47.9±7.0 and 25.9±3.7] ( P>0.05). At 6 months after surgery and at the last follow-up, the VAS scores and ODI values were (2.2±0.8)points, (1.7±0.8)points, 24.2±3.6 and 23.2±4.1 in the Denosumab therapy group, significantly lower than those of the conventional treatment group [(2.8±0.9)points, (2.8±1.1)points, 26.4±3.2 and 27.3±4.0] ( P<0.01). The VAS scores at each time point after surgery in both groups decreased significantly compared with those before surgery ( P<0.05). The VAS scores continued to decrease after surgery in the Denosumab therapy group ( P<0.05), while no significant difference was found among those at different time points in the conventional treatment group ( P>0.05). The ODI values at each time point after surgery in both groups significantly decreased compared to those before surgery ( P<0.05). The ODI values continued to decrease after surgery in the Denosumab therapy group ( P<0.05), while in the conventional treatment group, no significant difference was found between those at 6 months after surgery and those at 3 days after surgery ( P>0.05) and they were improved at the last follow-up compared with those at 3 days after surgery ( P<0.05). The refracture rate after surgery was 6.7% (7/104) in the Denosumab therapy group, significantly lower than 16.8% (18/107) in the conventional treatment group ( P<0.05). No serious complications were observed during the antiosteoporosis period in either group. Conclusion:Compared with daily oral administration of Calcium and active Vitamin D after PKP, the conventional treatment combined with Denosumab after PKP can effectively increase the bone density, relieve pain continuously, improve functional restoration, and reduce the risk of refracture in OVCF patients.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia seen in clinical settings, which has been associated with substantial rates of mortality and morbidity. However, clinically available drugs have limited efficacy and adverse effects. We aimed to investigate the mechanisms of action of andrographolide (Andr) with respect to AF. We used network pharmacology approaches to investigate the possible therapeutic effect of Andr. To define the role of Andr in AF, HL-1 cells were pro-treated with Andr for 1 h before rapid electronic stimulation (RES) and rabbits were pro-treated for 1 d before rapid atrial pacing (RAP). Apoptosis, myofibril degradation, oxidative stress, and inflammation were determined. RNA sequencing (RNA-seq) was performed to investigate the relevant mechanism. Andr treatment attenuated RAP-induced atrial electrophysiological changes, inflammation, oxidative damage, and apoptosis both in vivo and in vitro. RNA-seq indicated that oxidative phosphorylation played an important role. Transmission electron microscopy and adenosine triphosphate (ATP) content assay respectively validated the morphological and functional changes in mitochondria. The translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the molecular docking suggested that Andr might exert a therapeutic effect by influencing the Keap1-Nrf2 complex. In conclusions, this study revealed that Andr is a potential preventive therapeutic drug toward AF via activating the translocation of Nrf2 to the nucleus and the upregulation of heme oxygenase-1 (HO-1) to promote mitochondrial bioenergetics.
Animals ; Rabbits ; Atrial Fibrillation/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Signal Transduction ; NF-E2-Related Factor 2/pharmacology* ; Molecular Docking Simulation ; Oxidative Stress ; Energy Metabolism ; Mitochondria/metabolism* ; Inflammation/metabolism* ; Heme Oxygenase-1

Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of cancer death worldwide. It is urgent to develop new drugs to improve the prognosis of ESCC patients. Here, we found benzydamine, a locally acting non-steroidal anti-inflammatory drug, had potent cytotoxic effect on ESCC cells. Benzydamine could suppress ESCC proliferation in vivo and in vitro. In terms of mechanism, CDK2 was identified as a target of benzydamine by molecular docking, pull-down assay and in vitro kinase assay. Specifically, benzydamine inhibited the growth of ESCC cells by inhibiting CDK2 activity and affecting downstream phosphorylation of MCM2, c-Myc and Rb, resulting in cell cycle arrest. Our study illustrates that benzydamine inhibits the growth of ESCC cells by downregulating the CDK2 pathway.
Humans ; Benzydamine ; Esophageal Neoplasms/drug therapy* ; Esophageal Squamous Cell Carcinoma/drug therapy* ; Molecular Docking Simulation ; Phosphorylation ; Cell Proliferation ; Cell Line, Tumor ; Apoptosis ; Cyclin-Dependent Kinase 2

Objective:To design and construct a bone nonunion organoid on chip and explore the mechanism of aseptic bone nonunion.Methods:First a semi-open microfluidic chip was designed, on which human bone marrow mesenchymal stromal cells (BMSC), human fetal lung fibroblast 1, (HFL1) and human umbilical vein endothelial cells (HUVEC) were co-cultured, and a three-dimensional organ on chip system was established. Different proportions of HFL1 and HUVEC were co-cultured with BMSC, which were divided into the control group (HFL1∶HUVEC=1∶1), the fibrosis group (HFL1∶HUVEC=3∶1) and the vascularization group (HFL1∶HUVEC=1∶3). The osteogenic differentiation of BMSC was observed by alkaline phosphatase (ALP) and Alizarin red staining. The transcription level of osteogenic marker genes SP7, RUNX2, ALPL, and BGLAP, and vascularization related genes KDR and VWF were analyzed by qPCR. The expression levels of RUNX2 and ALP were determined by Western Blot. Results:In the co-culture system of BMSCs, HFL1, and HUVECs, BMSCs exhibited normal growth and apparent biomineralization behavior. Endothelial cells were capable of forming structured vascular networks, confirming the successful establishment of the system. Compared to the baseline group, the fibrotic group showed no significant decrease in BMSC osteogenic differentiation. The relative expression levels of the mineralization marker genes ALPL and BGLAP were 0.55±0.19 ( P<0.001) and 0.42±0.27 ( P<0.001), respectively. Vascularization genes KDR and VWF were downregulated, with relative expression levels of 0.49±0.17 ( P<0.001) and 0.49±0.21 ( P<0.001). In contrast, in the vascularized group, BMSC osteogenic differentiation genes SP7, RUNX2, ALPL, and BGLAP were upregulated, with relative expression levels of 2.91±0.52 ( P<0.001), 3.83±1.87 ( P<0.001), 3.22±1.29 ( P<0.001), and 5.21±1.46 ( P<0.001), respectively. Vascularization genes KDR and VWF were also upregulated, with relative expressions of 8.24±2.84 ( P<0.001) and 5.32±1.67 ( P<0.001). Western blot results indicated increased expression of RUNX2 and ALP in the vascularized group and decreased expression in the fibrotic group. Conclusion:The bone nonunion organoid on chip could partially simulate the local microenvironment of bone nonunion. Fibrosis may lead to a significant decrease in bone formation ability and vascularization level, which might be an important reason for the occurrence of aseptic bone nonunion.

Hepatic ischemia/reperfusion injury (HIRI) is a serious complication that occurs following shock and/or liver surgery. Gut microbiota and their metabolites are key upstream modulators of development of liver injury. Herein, we investigated the potential contribution of gut microbes to HIRI. Ischemia/reperfusion surgery was performed to establish a murine model of HIRI. 16S rRNA gene sequencing and metabolomics were used for microbial analysis. Transcriptomics and proteomics analysis were employed to study the host cell responses. Our results establish HIRI was significantly increased when surgery occurred in the evening (ZT12, 20:00) when compared with the morning (ZT0, 08:00); however, antibiotic pretreatment reduced this diurnal variation. The abundance of a microbial metabolite 3,4-dihydroxyphenylpropionic acid was significantly higher in ZT0 when compared with ZT12 in the gut and this compound significantly protected mice against HIRI. Furthermore, 3,4-dihydroxyphenylpropionic acid suppressed the macrophage pro-inflammatory response in vivo and in vitro. This metabolite inhibits histone deacetylase activity by reducing its phosphorylation. Histone deacetylase inhibition suppressed macrophage pro-inflammatory activation and diminished the diurnal variation of HIRI. Our findings revealed a novel protective microbial metabolite against HIRI in mice. The potential underlying mechanism was at least in part, via 3,4-dihydroxyphenylpropionic acid-dependent immune regulation and histone deacetylase (HDAC) inhibition in macrophages.

Objective:To compare the differences in distribution and prognosis of cervical cancer patients in the 2009 and 2018 editions of International Federation of Gynecology and Obstetrics (FIGO) staging, and to analyze the prognostic factors of cervical cancer patients.Methods:The clinical data of 524 cervical cancer patients admitted to the First Affiliated Hospital of Soochow University from January 2010 to December 2018 were retrospectively analyzed. The cases were staged according to the 2009 and 2018 FIGO staging, and the Kendall τb coefficient was calculated to compare the consistency of the distribution of the two stages. Kaplan-Meier was used for survival analysis, and log-rank test was used to test the difference of prognosis in each stage. Cox-regression was used to analyze the prognostic factors of cervical cancer patients.Results:In the 2009 FIGO edition of staging, 1 case of stage ⅠB1 was reduced to stage ⅠA1 due to the microscopic infiltration depth <5 mm, 51 cases of stage ⅠB1 were raised to stage ⅠB2 due to 2 cm4 cm. A total of 119 cases raised to stage ⅢC1 due to pelvic lymph node metastasis, and 11 cases raised to stage ⅢC2 due to para-aortic lymph node metastasis. The distribution of cases in the two stages was consistent (τb=0.61, P<0.001). There were statistically significant differences in overall survival (OS) ( χ2=107.13, P<0.001; χ2=93.02, P<0.001; χ2=92.74, P<0.001) and progression-free survival (PFS) ( χ2=91.95, P<0.001; χ2=77.69, P<0.001; χ2=73.27, P<0.001) among patients with different stages of FIGO in 2018 (ⅠA, ⅠB, Ⅱ, ⅢA, ⅢB, ⅢC1, ⅢC2, Ⅳ) and 2009 (ⅠA, ⅠB, Ⅱ, ⅢA, ⅢB, Ⅳ) and patients with different T stages (T 1, T 2, T 3, T 4). There were statistically significant differences in OS ( χ2=20.71, P<0.001) and PFS ( χ2=24.25, P<0.001) in 2018 FIGOⅠB and ⅢC stages, and there was a statistically significant difference in OS between stage ⅢC1 and stage ⅠB2 ( χ2=6.18, P=0.013). Multivariate analysis showed that age ( HR=1.88, 95% CI: 1.08-3.28, P=0.026), pathological type ( HR=2.11, 95% CI: 1.04-4.27, P=0.038), lymph node metastasis ( HR=2.18, 95% CI: 1.34-3.56, P=0.002), parametrial spread ( HR=2.56, 95% CI: 1.52-4.29, P<0.001), maximum tumor diameter ( HR=1.98, 95% CI: 1.18-3.30, P=0.009), squamous cell carcinoma antigen (SCCA) positive after treatment ( HR=4.49, 95% CI: 2.09-9.68, P<0.001) and Hemoglobin (HB) level before treatment ( HR=0.58, 95% CI: 0.35-0.96, P=0.035) were independent risk factors for OS in patients with cervical cancer. According to the 2018 FIGO stage, the 5-year OS rates of patients with stage ⅠB1, ⅠB2, ⅠB3 were 100%, 97.1% and 87.9% respectively, with a statistically significant difference ( χ2=7.79, P=0.020), and there was a statistically significant difference between stage ⅠB1 and ⅠB3 ( χ2=5.55, P=0.019). According to the 2009 FIGO stage, the 5-year OS rates of patients with stage ⅠB1 and ⅠB2 were 95.7% and 84.3% respectively, with a statistically significant difference ( χ2=9.08, P=0.003). For patients with 2018 FIGO stage ⅠB, SCCA positive after treatment ( HR=1 172.50, 95% CI: 10.37-132 554.51, P=0.003) was an independent risk factor for OS, and differentiation degree ( HR=0.09, 95% CI: 0.01-0.81, P=0.032), treatment method ( HR=0.17, 95% CI: 0.04-0.71, P=0.015) and SCCA positive after treatment ( HR=190.68, 95% CI: 14.27-2 547.67, P<0.001) were independent risk factors for PFS. For patients with 2018 FIGO stage ⅠB, stage ( HR=9.56, 95% CI: 2.38-38.32, P=0.001), SCCA positive after treatment ( HR=126.32, 95% CI: 12.36-1 290.60, P<0.001) and lymph node metastasis ( HR=20.07, 95% CI: 3.63-111.11, P=0.001) were independent risk factors for OS, and differentiation degree ( HR=0.11, 95% CI: 0.02-0.63, P=0.013), treatment method ( HR=0.22, 95% CI: 0.06-0.75, P=0.015) and SCCA positive after treatment ( HR=43.83, 95% CI: 7.94-241.84, P<0.001) were independent risk factors for PFS. According to the 2018 FIGO stage, the 5-year OS rates of patients with stage ⅡA and ⅡB were 95.7% and 75.6% respectively, with a statistically significant difference ( χ2=13.96, P<0.001). The 5-year PFS rates of patients with stage ⅡA and ⅡB were 83.1% and 67.1% respectively, with a statistically significant difference ( χ2=7.61, P=0.006). According to the 2009 FIGO stage, the 5-year OS rates of patients with stage ⅡA and ⅡB were 90.9% and 75.0% respectively, with a statistically significant difference ( χ2=8.85, P=0.003). The 5-year PFS rates of patients with stage ⅡA and ⅡB were 75.7% and 66.7% respectively, with a statistically significant difference ( χ2=4.07, P=0.044). For patients with 2018 FIGO stage Ⅱ, pathological type ( HR=20.28, 95% CI: 2.93-140.32, P=0.002) and stage ( HR=4.35, 95% CI: 1.02-18.55, P=0.047) were independent risks factors for OS. For patients with 2009 FIGO stage Ⅱ, pathological type ( HR=5.82, 95% CI: 1.62-20.94, P=0.007) was an independent risk factor for OS, and pathological type ( HR=3.09, 95% CI: 1.22-7.85, P=0.017) and lymph node metastasis ( HR=2.07, 95% CI: 1.22-3.51, P=0.007) were independent risk factors for PFS. For patients with 2018 FIGO stage Ⅲ, maximum tumor diameter ( HR=3.31, 95% CI: 1.45-7.56, P=0.005) and SCCA positive after treatment ( HR=4.67, 95% CI: 1.22-17.86, P=0.024) were independent risk factors for OS, and pathological type ( HR=4.15, 95% CI: 1.47-11.77, P=0.007) and SCCA positive after treatment ( HR=3.96, 95% CI: 1.45-10.86, P=0.007) were independent risk factors for PFS. Conclusion:The 2018 and 2009 FIGO staging have a good distribution consistency in the cervical cancer patients, and the 2018 FIGO stage ⅠB has more advantages in judging the prognosis, but stage ⅢC cannot accurately judge the prognosis. Lymph node metastasis and maximum tumor diameter are more important prognostic factors.