Clinical And Pathological Study Of Kasabach-Merritt Syndrome And Its Relationship With Related Growth Factors

Objectives Kasabach-Merritt syndrome(KMS)is clinically characterized as an enlarging vascular tumor complicated by profound thrombocytopenia and consumptive coagulopathy anemia.The etiology and pathogenesis of this disease are still unclear.The disease is different from infant hemangioma,whose symptoms are acute and progressively aggravate in short-term,the mortality rate is as high as 10%to 37%.There are painful lessons of the death due to misdiagnosis or mistreatment in the clinic.The case reports of KMS are scattered,and domestic or foreign large-scale case reports are rare.And the research of KMS is only limited to the discussion of treatment methods,the basic research progress is slow and rare,and there is no systematic and comprehensive study about the clinic and pathology of KMS.Recent years,many domestic and foreign studies have shown that angiogenic factors are the kind of substances that can induce or promote the formation of new capillary during the pathological evolution of hemangiomas.The most studied angiogenic factors are basic fibroblast growth factor(bFGF)and vascular endothelial growth factor(VEGF)in hemangiomas studies,their role is pretty positive in the pathogenesis of hemangioma.They are strongly stimulating factors on hemangioma endothelial cells,and play an essential role in the pathogenesis of hemangioma.With the development of study on bFGF and VEGF,they are playing a significant role in many physiological and pathological vascular proliferation processes.In vivo or in vitro the cultured vascular endothelial cells can form new small blood vessels under the induction of bFGF and VEGF.It is believed that bFGF and VEGF play an important role in the occurrence and development of hemangiomas.Many domestic and foreign scholars applied immunohistochemistry,in situ hybridization,RT-PCR and other methods to study the protein and mRNA expression of bFGF and VEGF at the different stages of hemangiomas,and they found that the protein and mRNA expressions of bFGF and VEGF in proliferative hemangioma tissues were significantly higher than those in degenerative phase,which suggested that bFGF and VEGF were involved in the proliferation of hemangiomas,and associated with the proliferation and degeneration of hemangiomas.Enzyme linked immunosorbent assay(ELISA)was used to detect the concentrations of bFGF and VEGF in serum and urine of infants with different stages hemangiomas and vascular malformations.The studies found that the concentrations of bFGF and VEGF in the serum and urine of proliferative hemangiomas,degenerative hemangiomas and vascular malformations were significantly different,which can be used as markers for the differential diagnosis of hemangiomas stages and the differential diagnosis of hemangioma and vascular malformation.However,the domestic or foreign studies of the relationship between the growth factors(such as bFGF,VEGF)and the KMS are rare,the studies including the bFGF,VEGF expression in KMS lesions,the bFGF and VEGF concentrations of the serum and urine in with infant KMS,the influence of changing concentrations of bFGF and VEGF in before and after the different therapies for KMS.First,we collected the clinical and pathological data of the KMS infants who are treated with surgical resection.Using HE staining and immunohistochemical methods to analyze the pathological results,discuss the histopathologic classification of KMS and the expression characteristics of the pathological tissue antigen markers in KMS for assisting the diagnosis of KMS,and explore the clinical and pathological features in KMS infants and preliminarily analyze of the predisposing factors leading to KMP.Secondly,we applied immunohistochemical methods to detect the expression of bFGF and VEGF in the KMS tissues,and the proliferative hemangiomas,explore the relationship between bFGF,VEGF and KMS,basically explore the role of bFGF and VEGF in the pathogenesis of KMS,and compared with the expression in hemangiomas and KMS,find more similarities and differences between hemangiomas and KMS.At last,we used enzyme linked immunosorbent assay(ELISA)to detect the changes of bFGF and VEGF in serum and urine of the KMS infants before and after surgical resection.Discuss the effects of surgery on the bFGF and VEGF concentrations in blood and urine of in the KMS infants,further explore the pathogenesis of KMS,provide theoretical basis and support for the promotion of surgical resection in treating KMS.Methods1.Collected the clinical data of 86 KMS infants who were treated with surgical resection.The tissue specimens were examined by HE staining and pathological examination for clinical and pathological diagnosis.Summarized the clinical features,risk factors and treatment results of various therapy methods.Applied immunohistochemical method to detect the antigenic markers(CD31,CD34,Factor VIII,FLI1,SMA,D2-40,GLUT-1,Ki-67,HHV8)in the KMS tissues,and used PBS instead of primary antibody as a negative control.The positive staining samples which were provided by the company was used as positive control.Analyzed the expression of the antigen markers in KMS tissue,and explored the histopathological characteristics of KMS.2.The total samples were 36 cases,including KMS(14cases)and proliferative hemangioma(22cases),All cases had no history of therapy before operation.The protein expression of bFGF and VEGF was detected with immunohistochemistiy methods in 22 specimens of proliferating hemangiomas,14 of KMS,and 8 of normal skin(control group).Compared the similarities and differences between KMS and proliferative hemangioma.3.Took serum and urine in the KMS infants who underwent surgical resection as experimental subjects.Used enzyme linked immunosorbent assay(ELISA)to identify bFGF,VEGF concentrations in serum and urine of the KMS infant at one day before surgery and 1 week,4 weeks after treatment.Compared the changes of bFGF and VEGF concentrations in serum and urine of the KMS cases after surgery.Results1.Clinic and pathology of KMS1.1 Clinical pathology classification:86 surgical specimens of KMS were pathologically examined.There were 69 kaposiform hemangioendothelioma(KHE)and 17 tufted angioma(TA).And there was no one case simultaneously met with the criterion of KHE and TA.No infant hemangioma was found in these cases.1.2 Pathological features:The diseased tissue involved multiple layers and/or multiple anatomical sites.The depth of infiltration ranged from superficial skin,subcutaneous tissues,to deep muscle or bone.Gross examination showed that the tumor typically looked dark red with fluent blood supply.Sporadic black or brown thrombosis,edema,fibrosis or proliferating lymphatic-like features were observed.There was indistinctive boundary with surrounding tissues.The tissue texture was tough or hard.Under the microscope,the KHE lesions were characterized by widely scattered small lobules or nodules composed of spindle endothelial cells in the dermis and subcutaneous tissue.The tumors were dominated by infiltrating isolated and fusional nodules.These spindle cells were associated with small endothelial lined vascular spaces to form slit-like and sieve-like spaces which looked like the Bauman cyst or like glomeruloid appearance.There was mild nuclear variation,but no significant nuclear atypia,or necrosis,with rare mitoses.It was often infiltrating into surrounding tissues,surrounded by lymphangioma-like hyperplasia.The TA lesions were characterized by widely scattered small tufts or lobules composed of plumps or spindle vascular endothelial cells and pericytes in the dermis and subcutaneous tissue.These tufts were apparently differentiable from fiber tissues,and appeared as "cannonballs",the round-shaped cells lined concentrically at low-power view.The spindle cells pushed against adjacent vessels and formed slit-like or crescent-shaped vascular space.Atypia neoplastic cells were not observed.The small abnormal veins or lymphatic vessels were identified at the edge of the lobules.No distant metastasis was found in KMS patient.1.3 Immunohistochemical phenotype:KMS were diffusely positive for vascular endothelial cell markers CD31,CD34,Factor Ⅷ,FLI1.Smooth muscle actin(SMA)was focally positive,which suggested the existed perivascular cells.D2-40(Podoplanin monoclonal antibody)was a kind of lymph endothelial cell marker.D2-40 was markedly immunoreactive in the peripheral area of proliferative capillaries,and mostly unreactive in the surrounding dilated vessels.Infant hemangioma(or placental tissue)specific marker GLUT-1(Glucose transporter 1)was negatively expressed.Immunohistochemical staining demonstrated that Ki67 labeling index ranged 3%-40%,with an average of 12%.KMS was negative expression for human herpes virus 8(HHV-8)by immunohistochemical staining.1.4 Treatment response or results:There were 46 males and 40 females(the ratio of male to female 1.15:1)in 86 KMS infants.Among them,82 cases were treated with glucocorticoids(drugs including prednisone,methylprednisolone,dexamethasone,etc.,regimens including oral,intravenous or local injection),and 31 cases were sensitive to glucocorticoids(about 37.8%,31/82),other drug regimens and non-surgical treatments are not very effective.Surgical methods were applied including 39 cases with completely resection and 47 cases with almost resection.84 cases(97.7%)of all were cured and 2 cases died from multiple organ failure after operations.The results showed that the sooner the surgery was done after the adjuvant and sufficient medical therapy,the easier the tumor was removed.And early surgical patients had better effect,fewer adverse reactions and complications than late surgical patients.2.Immunohistochemistry results of bFGF,VEGF:The protein expressions of bFGF,VEGF were positive in KMS and proliferation phase hemangiomas,and the expression in normal skin was negative.The expression levels of bFGF and VEGF in KMS and proliferative hemangiomas were higher than those in normal control group.(P<0.0083).The positive expression rates of bFGF,VEGF in KMS and proliferative phase both were 100%.From the view of the number and intensity in positive expression cell,the result of bFGF expression in KMS tissues was 4.42±0.437 were higher than that of proliferative hemangiomas 3.61±0.519;and the result of VEGF expression was 5.14±0.329 was higher than that of proliferative hemangioma 3.25±0.406,and the difference was statistically significant(P<0.0083).3.Concentrations of bFGF,VEGF in serum and urine of KMS infants before and after surgical resection3.1 Serum bFGF concentration test results:Results showed the bFGF concentrations in serum of KMS infants at one day before surgery,one week after surgery and four weeks after operation were(604.54±87.24)pg/ml,(390.74±46.59)pg/ml and(375.29±36.83)pg/ml respectively.There was significant difference between one day before operation and one week,four weeks after surgery(P<0.01).There is no significant difference between one week after surgery and four weeks after operation(P>0.05).3.2 Serum VEGF concentration test results:Results showed the VEGF concentrations in serum of KMS infants at one day before surgery,one week after surgery and four weeks after operation were(957.82±98.76)pg/ml,(402.3 6±67.58)pg/ml and(367.54±34.27)pg/ml respectively.There was significant difference between one day before operation and one week,four weeks after surgery(P<0.01).There is no significant difference between one week after surgery and four weeks after operation(P>0.05).3.3 Urine bFGF concentration test results:Results showed the bFGF concentrations in urine of KMS infants at one day before surgery,one week after surgery and four weeks after operation were(637.45±128.57)pg/ml,(413.62±57.91)pg/ml,(368.47±29.36)pg/ml respectively.There was significant difference between one day before operation and one week,four weeks after surgery(P<0.01).There is no significant difference between one week after surgery and four weeks after operation(P>0.05).3.4 Urine VEGF concentration test results:Results showed the VEGF concentrations in urine of KMS infants at one day before surgery,one week after surgery and four weeks after operation were(440.92±113.45)pg/ml,(307.92±56.73)pg/ml,and(256.74±38.97)pg/ml respectively.There was significant difference between one day before operation and one week,four weeks after surgery(P<0.01).There is no significant difference between one week after surgery and four weeks after operation(P>0.05).Conclusions1.There are two main clinical pathology types in 86 cases with Kasabach-Merritt syndrome,including 69 cases of kaposiform hemangioendothelioma(KHE)and 17 cases of tufted hemangioma(TA).There is no one case simultaneously that meets with the criterion of KHE and TA.No infant hemangioma is found in these cases.2.Immunohistochemical markers GLUT-1 and D2-40 contribute to the differentiation of KHE,TA and proliferative hemangioma.3.The data suggest that KMS lesions infiltrating into multiple layers and/or extending into multiple anatomic regions,and huge tumors play a key role on increasing risks of KMP.4.1t is concluded that early surgical treatment of KMS in pediatric patients is a reliable and priority option on the basis of adequate preoperative care.The patients will have fewer complications,lower morbidity,and faster recovery than the late stage and long-term patients with KMS.5.The high levels of bVGF,VEGE expression in KMS tissues indirectly indicate that bVGF,VEGE play an important role in occurrence and development of the infancy KMS.6.The expression of bFGF,VEGF in KMS tissues is higher than that in proliferative hemangioma,which can be used as a cytological marker for the identification of KMS and proliferative hemangioma.7.It is a simple and efficient method by using ELISA to detect bFGF,VEGF concentration in serum and urine of the KMS patients.It can be used as the method to dynamically monitor and evaluate the therapeutic effect,and to guide clinical treatment of the KMS.8.Surgical resection is an effective treatment for KMS infants.It can exert a great influence to the bFGF,VEGF concentration in serum and urine of the KMS patients.The results indirectly suggest that bFGF,VEGF play an important role in the pathogenesis of KMS.