The Clinical And Mechanism Research Of Angiogenesis In Adrenal Pheochromocytoma

OBJECTIVE: To explore the effects of vascular anatomy and variation of adrenal pheochromocytoma(PHEO)on surgical outcomes,establish a new surgical plan,reveal the mechanism of PHEO angiogenesis,and to discover new drug targets,which ultimately reduce intraoperative bleeding and facilitates surgical operation to benefit patients.METHODS:(1)A retrospective study of minimally invasive surgery of adrenal tumors in our department in 2017 was conducted to investigate the relationship between venous variants and preoperative items(tumor size,location,imaging features),intraoperative items(diameters of adrenal veins,surgery duration,bleeding volume),postoperative items(complication,hospitalization days,pathological diagnosis).(2)The clinical data of 15 patients with pheochromocytoma received surgical treatment from January 2018 to December 2018 who were supported by 3D visualization technology in our department were retrospectively analyzed.There were 7 males and 8 females.The patients were aged from 14 to 73 years old,with an average age of 42.5 years.The maximum diameter of the tumor was 3-17 cm,with an average 7.8 cm.Cases in the database were matched according to tumor size,disease type and surgical method,and were compared with 15 patients included in this study.Information such as operation time,intraoperative blood loss,postoperative hospitalization days and perioperative complications were recorded and statistically analyzed.(3)78 adrenal tumors specimens,including 20 adrenocortical adenoma(ACA)cases,22 adrenocortical carcinoma(ACC)cases,18 benign pheochromocytoma(BP)cases and 18 metastatic pheochromocytoma(MP)cases,were examined by immunohistochemistry for the expressions of HIF-2?,HIF-1?,VEGFR-2 and microvascular density(MVD).(4)Twelve surgical specimens of pheochromocytoma were grouped into high and low MVD group.They were also divided into rich blood supply and non-enriched blood supply group,according to CT manifestation.Comparative proteomic analysis based on LC–MS/MS and bioinformatics analysis were conducted for the discovery of protein involved in angiogenesis.Among the identified proteins,COX4I2 and PLAT were further verified using quantitive real-time PCR with 40 samples and immunohistochemistry with 27 samples.RESULTS:(1)We found variant venous anatomy in 62 of 303 adrenalectomies(20.5%),in which 58 had one central adrenal vein with an additional small vein;12 had a variant(usually the additional vein)joining the hepatic vein,the inferior vena cava,the lumbar vein or the inferior phrenic vein.Tumour with variant anatomy were associated with larger tumour size(5[3-7] cm vs 2[1.2-3] cm,P<0.001),larger diameters of adrenal veins(4[4-5] mm vs 4[3.5-4] mm,P=0.021),more adrenal medullary tumours(39% vs 8%,P<0.001),more estimated intraoperative blood loss(100[50-200] mL vs 50[20-100] mL,P<0.001),longer operation time(117[89-166] min vs 85[70-110] min,P<0.001)and longer length of hospitalization(4[4-5] days vs 4[3-5] days,P=0.004).(2)All 15 patients underwent surgery successfully.The median duration was 90 min(60-180 mins),the median intraoperative blood loss was 90 mL(20-200 mL),and the average postoperative hospital stay was(5.8±2.3)days.The 15 patients who were matched in the database also successfully completed the operation.The median operation time was 110 min(60-220 min),the median intraoperative blood loss was 150 mL(20-400 mL),and the average postoperative hospital stay was(5.1±3.1)days.Compared with the database matched cases,the operation time and intraoperative blood loss were significantly lower in this group(P<0.05).There was no significant difference in postoperative hospital stay and perioperative complications(P>0.05).(3)VEGFR-2 up-regulated in the ACC group(P=0.008)and MVD overexpressed in the ACC(45.06±23.23 vs 28.99±7.19,P=0.005)and MP(73.59±31.41 vs 44.21±28.45,P=0.006)group respectively,with significant difference.The expression of HIF-1? were positively related to HIF-2? in pheochromocytoma(?=0.719,P<0.01).In addition,HIF-1?,VEGFR-2 and MVD were independent risk factors for adrenal cortical malignancies,and MVD was independent risk factors for adrenal medullary tumor metastasis.(4)According to quantitative proteomics,206 proteins were discovered to be differentially regulated in the high MVD group compared to low MVD group.61 proteins were discovered to be significantly changed according to the rich blood supply group and the non-enriched ones detected with CT.25 proteins were filtered out by intersecting the result from MVD and CT grouping.COX4I2 was verified to be increased gradually with angiogenesis,while PLAT was shown to be decreased with angiogenesis in pheochromocytoma,which may contribute to pheochromocytoma angiogenesis by quantitive real-time PCR and immunohistochemistry.CONCLUSION: Adrenal venous variants are more likely to occur in pheochromocytoma,associated with poor surgical outcomes.Close attention should be paid to vascular structure during surgery.Hence more comprehensive pre-operative plans is required for better surgical outcomes.3D visualization is consistent with intraoperative evaluation,directly reflecting the vascular supply and peripheral structure of the tumor,which can reduces intraoperative bleeding and corresponding complications.The microvascular density of pheochromocytoma is closely related to the malignant tendency.The expression of COX4I2 and PLAT are associated with MVD,which may participated in pheochromocytoma angiogenesis.These can potentially serve as new targets to inhibit the angiogenesis of pheochromocytoma.