| PART 1:Clinical characteristics in patients with sellar Langerhans cellhistiocytosis and sellar germinomaBackground and objectiveThe differential diagnosis of central diabetes insipidus(CDI)is one of the difficult points in endocrinology,especially the patients with thicken pituitary stalk.The main reasons for thicken pituitary stalk include sellar Langerhans cell histiocytosis(LCH),sellar germinoma and lymphocytic hypophysitis.LCH and germinoma are difficult to distinguish for the lack of specific markers and biopsy is currently the only method of differential diagnosis.Biopsy is an invasive procedure which may probably cause irreversible damage of pituitary functions,therefore,early identification and treatment are significant for prognosis.This study aims at summarizing and comparing the clinical features of sellar LCH and sellar germinoma,and the results may help the clinical diagnosis.MethodsIn this retrospective study,181 patients confirmed sellar LCH(89)or sellar germinoma(92)seen at Peking Union Medical College Hospital from July 1993 to July 2017 were included.Clinical data were analyzed.T test was used to analyze normally distributed continuous variable data and Mann-Whitney U test was used for those in skewed distribution.Chi-square test was used to analyze the classified variable data.The bilateral p value less than 0.05 was considered statistically significant.Results89 LCH patients included 52 males and 37 females(1.4:1),and 92 germinoma patients included 38 males and 54 females(0.7:1).The median age of LCH patients was 23.0(1.0-53.0),while it was 12.5(4.0-35.0)in germinoma,and ages were statistically different.CDI was the most common primary symptom for both diseases.The median intervals between the initial symptoms and diagnosis were 23(1-131)months and 25(1-297)months,respectively.All of the LCH patients were diagnosed by biopsy,including 14 patients in sellar areas;83 germinoma patients were diagnosed by biopsy and 9 by clinical diagnostis.In pituitary functions,the most involved axis in LCH was hypothalamus-pituitary-gonad(HPG)axis dysfunction(48.3%),then it came hypothalamus-pituitary-thyroid(HPT)axis(39.3%),growth hormone-insulin-like growth factor 1(GH-IGF-1)axis(39.3%)and hypothalamus-pituitary-adrenal(HPA)axis(28.1%);while in germinoma they were HPT(67.4%),HPA(65.2%),HPG(51.1%)and GH-IGF-1(47.8%).In terms of hematological indicators,?-hCG(p=0.002)of LCH(median 0.1 mIU/ml(0-6.0 mIU/ml))was lower than that of germinoma(median 1.3 mIU/ml(0-40.4 mIU/ml)).In terms of cerebrospinal fluid(CSF)test,total cells(median7.O×106/L(0-7200.0×106/L),p=0.001)?white blood cells(median 1.5×106/L(0-8.0×106/L),p=0.002),monocytes(median 0.5×106/L(0-5.0×106/L),p<0.001)and ?-hCG(median 3.5 mIU/ml(0-6.6 mIU/ml),p=0.032)of LCH were lower than those of germinoma(total cells median 28.0×106/L(0-7815.0×106/L),white blood cells median 4.0×106/L(0-64.O×106/L),monocytes median 6.0×x106/L(0-52.0×106/L),?-hCG median 5.4 mIU/ml(0.2-36.3 mIU/ml)).In craniocerebral MRI examination,the most involved area was pituitary stalk,with 28 cases(47.5%)and 40cases(43.5%),respectively.The numbers of cases with pituitary and pituitary stalk involved were one(1.7%)and 16(17.4%)respectively;the numbers of cases with pituitary stalk and hypothalamus involved were 6(10.2%)and 9(9.8%)respectively;the numbers of cases with pituitary,pituitary stalk and hypothalamus involved were 6(10.2%)and 17(18.5%)respectively.ConclusionThis study showed the ratio of male to female and median age of LCH were significantly higher than those of germinoma.The total cells,white blood cells,monocytes and ?-HCG in CSF as well as serum ?-HCG of LCH were significantly lower than those of germinoma.The most involved axis in LCH was HPG,and HPT,GH-IGF-1,and HPA followed;while the sequence in germinoma was HPT,HPA,HPG and GH-IGF-1.There are no specific markers for differential diagnosis.PART 2:Cellular specific gene expression study in cerebrospinal fluid of sellar Langerhans cell histiocytosis and sellar germinomaBackgroundSellar LCH and sellar germinoma are differentiated by immunohistochemistry.The markers for LCH are protein CD1a,S100 and CD207,while those for germinoma are protein PLAP,OCT3/4 and SALL4.However,sellar biopsy is risky and may cause financial and psychological burden.And sometimes it is difficult due to the limited tissues.Previous studies have shown that it is possible to find tumor cells in CSF of LCH or germinoma patients,but the detection rate is very low and the application is limited.ObjectiveThis study tried to analyze gene expression of CSF cells in sellar LCH and sellar germinoma patients by Droplet Digital PCR(ddPCR)and explore a new alternative method for diagnosis.MethodsCSF and clinical data were collected from the patients suspected sellar LCH or sellar germinoma in Peking Union Medical College Hospital from February 2018 to May 2018.CSF from one patient with hydrocephalus was also collected as negative control.CSF was centrifuged and cell participation was taken for RNA extraction.Some CSF was detected for mRNA of CD1a,S100,CD207,PLAP,OCT3/4 and SALL4 by two-step ddPCR,which included reverse transcription and PCR.The remained specimen was detected by one-step ddPCR,in which reverse transcription and PCR were conducted together.Data were analyzed by QuantaSoft.ResultsFirstly,we explored the methods of analyzing gene expression in CSF cells by two-step and one-step ddPCR.By two-step ddPCR,the internal gene expression of Patients P#1-6 was high enough(5.96×105-108/ml CSF),suggesting the feasibility of two-step method in analyzing internal genes.Then some of the target genes of Patients P#1-6 were positive,suggesting the feasibility of two-step.method in analyzing target genes.Next we determined Patient P#9 with RNA quantity decreased in three repetitive experiments.The copy numbers were higher than those of the two-step method and similar in the three experiments,though those of S-100,CD207 and OCT3/4 in the third experiment were slightly lower.By analyzing the pictures,the RNA quantity of the second one was the best.CSF of negative control was repeated for twice and both results were negative.The results indicated that one-step method was feasible in analyzing gene expression and had good repeatability.The results of the 9 patients and one negative control were as follows.CSF from Patient P#1-6 was detected by two-step method-S100 and CD207 were positive in Patient P#1,with 23.2/m1 CSF and 128/m1 CSF respectively.S100 and CD207 were positive in Patient P#2,with 644/ml CSF and 140/ml CSF respectively.Patient P#1-2 had no confirmed diagnosis clinically.S100 and CD207 were positive in Patient P#3,with 57.4/m1 CSF and 84/m1 CSF respectively and she was suspected LCH clinically.CD1a,S100 and OCT3/4 were negative in Patient P#4-6,and Patient P#4 was suspected nodal glioma,and Patient P#5-6 had no confirmed diagnosis clinically.CSF from Patient P#7-9 was detected by one-step method.There was no positive events in Patient P#7 and she was diagnosed as granulosa cell tumor.CD 1a and CD207 were positive in Patient P#8,with 1080/ml CSF and 468/ml CSF respectively,and she was diagnosed as LCH by biopsy,as the immunohistochemistry showed S-100(+)and SALL4(-).CSF of Patient P#9 was repeated for three times,and CD1a,S100,CD207,PLAP and OCT3/4 were positive in each experiment.There was no positive events in CSF from the negative control.ConclusionBoth two-step and one-step methods were feasible for analyze specific gene expression in CSF cells and the one-step method was more efficient than two-step method.The methods had good specificity,sensitivity and repeatability.Patient P#1-3 and Patient P#8 showed LCH in experiment,and Patient P#3 was suspected LCH as well as Patient P#8 was diagnosed as LCH by biopsy.The markers for germinoma were positive in Patient P#9 and she was diagnosed as germinoma by biopsy.There were no positive results in Patient P#4-7 and the biopsy results were ganglioglioma in Patient P#4 and granulosa cell tumor in Patient P#7.The data were in line with clinical results. |
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