| Objective: To detect the types of BRAF-V600 E and MAP2K1 gene mutations in Langerhans Cell Histiocytosis(LCH),combined with the analysis of the characteristics of clinical data in adults and children,to further improve the cells of Langerhans cells Understanding of the pathogenesis,clinical manifestations and targeted drug treatment of hyperplasia.Methods:(1)65 cases of LCH patients diagnosed and treated in the First Affiliated Hospital of Guangxi Medical University from January 1,2011 to January 31,2019 were taken as the research object,and paraffin-embedded tissue sections of 55 patients were collected in the pathology department.At the same time,10 patients with peripheral blood were collected from the patients,and the data of patients’ treatment,treatment and prognosis were collected;(2)DNA from paraffin-embedded tissues and peripheral blood of LCH patients was extracted,and exon 11 and exon 15 of BRAF gene and MAP2K1 were designed the primers of exon 2 and exon 3 of the gene were amplified by Polymerase Chain Reaction(PCR),and then the accuracy of the PCR results was detected by agarose gel electrophoresis,and the sequencing results were compared with the gene library Alignment of human BRAF and MAP2K1 gene sequences;(3)Detection of BRAF-V600 E gene locus and mutation type and mutation rate of MAP2K1 gene in 65 patients with LCH,analysis of chromosomal abnormalities of the target gene;(4)Combined with patient clinical data Understand the characteristics of LCH patients’ age,gender,clinical manifestations and affected organs,and analyze the relationship between BRAF-V600 E gene locus and MAP2K1 gene mutations and the patient’s age,gender,clinical manifestations,affected organs,locations and treatment.Results:(1)Of the 65 LCH patients treated at the First Affiliated Hospital of Guangxi Medical University,42 were male(64.6%)and 23 were female(35.4%).The sex ratio was male:female=1.83:1.The median age of male patients was 20.83 years old,and the median age of female patients was 18.46 years old.The proportion of males with LCH disease was relatively high,P=0.032.Among them,21(32.3%)were diagnosed at the age of 0-4.9,15(23.1%)at the age of 5-9.9,9(13.8%)at the age of 10-14.9,7(10.8%)at the age of 15-19.9,20-3 people diagnosed at the age of 24.9(4.7%),1 person diagnosed at the age of 25-29.9(1.5%),5 people diagnosed at the age of30-34.9(7.7%),2 people diagnosed at the age of 35-39.9(3.1%),40-44.9 One person(1.5%)was diagnosed at the age of 1 and one person(1.5%)was diagnosed at the age of over 45.There are many children with LCH in the age range of 0-5 years,accounting for 32.3%,which is the peak period of childhood onset,and the peak period of adult incidence is 30-35 years.(2)Of the 65 patients,45 cases(61.5%)involved single system(SS-LCH)and 20 cases(38.5%)involved multiple system(MS-LCH).Among the young people under 17 years old,there were 36 cases of SS-LCH and 18 cases of MS-LCH;among the patients over 17 years old,there were 9 cases of SS-LCH and 2 cases of MS-LCH;In 10 cases,among infants and young children under 4years old,70% were accompanied by damage to dangerous organs.The age of onset in the multi-system group was significantly smaller than that in the single-system group,and the prognosis of children with multi-system involvement was relatively poor.(3)The main symptoms of admission are painless tumors(21 cases),pain(13 cases),rash(12 cases),lame gait(10 cases),fever(5 cases),blurred vision(1 case),Diarrhea(1 case),yellow skin(1 case),polyuria(1 case),the first symptoms are painless masses,and more often involve bones.(4)65 patients involved 33 cases of bone(52.3%),10 cases of skin(15.4%),4 cases of submandibular gland(6.2%),3 cases of lung(4.6%),3 cases of parotid gland(4.6%),head and neck(Eyes,ears,oral cavity)3 cases(4.6%),lymph nodes 3 cases(4.6%),liver 2 cases(3%),spleen 2 cases(3%)and central nervous system 2 cases(3%),the skeleton is LCH is the most frequently involved organ,and its invasion into bone tissue has a better prognosis than that without invasion.(5)Of the 65 patients enrolled,15(23%)in the adult group had 3BRAF-V600 E gene site mutations(3/15)and 2 MAP2K1 gene mutations(2/15).20% and 13.3%;50 in the minor group(77%),20 cases of BRAF-V600 E gene mutation(20/50),8 cases of MAP2K1 gene mutation(8/50),the incidence rate was 40% and 16% respectively;The median age of BRAF-V600 E gene mutation is 11.3 years old,and the median age of MAP2K1 gene mutation is 7.5years old,suggesting that BRAF-V600 E gene locus and MAP2K1 gene mutation occur in younger patients,the difference is statistically significant(P=0.014).(6)In this study,paraffin-embedded tissues and peripheral blood were used to extract DNA from patients and sequenced genes.In paraffin-embedded tissues,18 cases of BRAF-V600 E gene mutations were detected in the juvenile group,8 cases of MAP2K1 gene mutations,3 cases of BRAF-V600 E gene site mutations were detected in the adult group,and 2 cases of MAP2K1 gene mutation were detected in peripheral blood.In the minor group,2 cases of BRAF-V600 E gene mutation were detected.The positive rate of gene mutation detected in paraffin-embedded tissue was higher than that in peripheral blood.(7)Gene sequencing results showed that 23 of 65 patients had BRAF-V600 E gene mutation(23/65),the mutation rate was 35.4%,and the mutation site was exon 15 c.1799T>A,resulting in Valine was changed to glutamic acid,20 cases of BRAF-V600 E gene mutations were detected in the juvenile group with a mutation rate of 87%,and 3 cases in the adult group with a mutation rate of 13%;mutations in the BRAF-V600 E gene site were negative Of the 10 patients with MAP2K1 gene point mutation(10/65),the mutation rate was 15.4%,and the mutation type was the second exon missense mutation p.C121S(chr15＿g.66729153T>A),p.G128V(chr15＿g.66729154G>T)and exon 3 deletion mutation p.E102＿l103del(the deletion sites are c.307＿312del and p.I103＿K104del).8 cases in the minor group with a mutation rate of 80%,2cases in the adult group with a mutation rate of 20%,including 5 cases with p.C121 S mutation,3 cases with p.G128 V mutation,1 case with p.C121 S and p.G128 V mutation,p.1 case of E102＿l103del mutation.(8)14 patients(60.9%)with bone involvement,4 patients with skin(17.5%),2 patients with lung(8.7%),1 patient with spleen(4.3%),and 1 lymph node with LCH patients with BRAF-V600 E gene mutation Cases(4.3%)and 1 case(4.3%)of extracranial intradural position.There were 14 patients with SS-LCH and 9 patients with MS-LCH.The BRAF-V600 E mutation status was not associated with organ involvement(P = 0.165),and was not associated with single-system or multi-system involvement(P = 0.216).Ten cases of MAP2K1 gene mutation.There were 4 cases of bone(40%),2 cases of skin(20%),2cases of lymph nodes(20%),1 case of lung(10%),1 case of submandibular gland(10%),4 cases of SS-LCH,MS-6 patients with LCH.MAP2K1 gene mutation was not associated with single-system or multi-system involvement(P= 0.548),but not with organ involvement(P = 0.638).There was no statistically significant difference between the treatment group and the non-mutation group using LCH-Ⅲ regimen(P ==0.394).Conclusion:1.The positive detection rate of gene mutation in paraffin-embedded tissue in this experiment is higher than that in peripheral blood.2.In this study,the BRAF-V600 E gene mutation rate was 35.4%,and the MAP2K1 gene mutation rate was 15.4%.The two gene mutations did not appear in the same patient at the same time.3.BRAF-V600 E and MAP2K1 gene mutations are not associated with organ involvement,and there is no statistical difference in the effect of treatment with LCH-Ⅲ regimen.4.The discovery of BRAF-V600 E and MAP2K1 gene mutations can provide evidence and direction for targeted drug therapy for LCH. |
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