| Medulloblastoma(MB)is the most common malignant embryonic tumor of the central nervous system(CNS)in childhood,accounting for about 10%of all CNS tumors in children.Most cases of MB originate in the midline of the cerebellum or in the vermis of the posterior fossa.The tumor is highly malignant and invasive,and is easily spread by cerebrospinal fluid.The patients of MB have poor prognosis and high mortality.MB is a highly heterogeneous tumor,which can be divided into:Classic MB.Desmoplastic/nodular(D/N)MB,MB with extensive nodular and Large/anaplastic(LC/A)MB.The newly published fifth edition of the WHO Classification of CNS tumors in 2021 clearly divides MB into four molecular subtypes:WNT-activated type,SHHactivated with TP53 wild type,SHH-activated with TP53 mutant type,and non-WNT/non-SHH type.The non-WNT/non-SHH types can be further divided into Group 3 and Group 4.which contain 8 subtypes.These four molecular subtypes have significantly different characteristics in clinic,pathological morphology and molecular biology.The prognosis of patients with different subtypes is significantly different.The WNT-activated type is rare to spread by leptomeningeal.Patients with the WNT-activated type are highly sensitive to postoperative radiotherapy and chemotherapy.The patients are rare to develop tumor metastasis,and have well prognosis.The 10year survival rate of children with WNT-activated MB can reach 95%.Therefore,it is of great clinical significance to conduct molecular classification and stratification diagnosis for MB patients,especially to screen out WNT-activated cases,so as to avoid the side effects caused by overtreatment.The common detection methods for MB molecular typing include NGS(Next generation sequencing)sequencing,DNA methylation sequencing,etc.Although high-throughput genetic testing methods such as NGS are more accurate than Immunohistochemistry(IHC)methods,the use is limited due to the high price.Therefore,it is of important diagnostic value to seek immunohistochemical markers with high specificity and sensitivity to replace high-throughput molecular detection.These markers contribute to conduct auxiliary typing of MB patients quickly and effectively,so as to assess patient prognosis and formulate appropriate treatment regimen.It is important to reduce the overtreatment of WNT activated MB patients.In the pathological diagnosis of MB molecular typing,the immunohistochemical indicators include β-catenin,GAB1(Grb2associated binders)and YAP1(Yes-associated protein 1).However,these indicators have not been widely used due to the unsatisfactory sensitivity and specificity.For example,the positive nuclear expression of β-catenin is a strong indicator of WNT-activated MB,but in practice,the positive nuclear expression of β-catenin is rarely seen even in the WNT-activated MB with CTNNB1 gene mutation confirmed by molecular detection.Therefore,whether there are other immunohistochemical indicators which can replace β-catenin in identification of WNT activated MB has become the direction of our research.β-catenin specifically interacts with lymphoid enhant-factor 1(LEF-1)in the nucleus to form a transcription complex which regulates WNT signaling pathways.Given the specific interaction between β-catenin and Lef-1,we infer that Lef-1 may be used as a recognition marker for WNTactivated MB.At present,Lef-1 immunohistochemistry has not been reported in MB.Therefore,in this study,we examined Lef-1 protein expression in MB and compared with β-catenin expression to predict WNT-activated MB.Non-WNT/non-SHH MB associated with high MYC expression is referred to the "extremely high risk" subtype,which has the highest malignancy and the worst prognosis.The overall survival rate of this type is less than 50%.Currently,patients with Group 3 MB with MYC amplification undergo intensive therapy,including surgical excision and whole-cerebrospinal irradiation,followed by intensive chemotherapy.Although,many patients still die or survive with suffering severe treatment-related side effects.Therefore,in order to quickly and efficiently assess risk stratification and prognosis in patients with MB,we further explored the relationship between immunohistochemical markers and clinicopathologic features,recurrence and metastasis in patients with MB.Recent studies have shown that Oligo2(oligodendrocyte lineage transcription factor 2)is a biomarker and an effective therapeutic target for high-risk MB subgroups with MYC amplification,which suggests poor prognosis for MB subgroups with MYC amplification.Therefore,in this study,we sfurther evaluated the application value of Oligo2 in determining the relapse risk of MB with different molecular subtypes by immunohistochemical methods.Research methods:Fifty-nine paraffin specimens of MB patients from 2017 to 2022 were collected from Qilu Hospital.Of these,51 cases have complete follow-up data and clinicopathological data.The sections were reviewed by two senior pathologists,and the histological classification was carried out after the pathological diagnosis was confirmed.(1)Fifty-one MB patients were grouped according to clinicopathologic data(age/sex/histological type/molecular type);(2)The molecular typing(including 62 MB-related genes and chromosomes)was performed on 51 MB paraffin tissues based on NGS;(3)The WNT-activated cases in NGS were further verified by CTNNB1 3Exon Sanger sequencing:(4)The expression of β-catenin,Lef-1,YAP1,GAB1,Oligo2 and Ki67 in 51 MB cases were detected by immunohistochemistry.(5)The recurrence and metastasis of the patients were obtained by telephone follow-up;(6)To analyze the relationship between the protein expression and molecular typing;to analyze the relationship between the protein expression and tumor recurrence.Results:(1)Among the 51 patients with MB,the median age was 10 years old.There were 28(54.9%)males and 23(45.1%)females,with a ratio of 1.2:1.There were 29 cases(56.8%)of patients with classic,15 cases(29.4%)of patients with pro-fibroplastic,7 cases(13.7%)of patients with anaplastic/macrocellular,and 0 cases of patients with extensive nodular.(2)37 cases were successfully sequenced by NGS sequencing,of which 7 cases were classified as WNT activated type,11 cases as SHH with TP53 wild type,1 cases as SHH with TP53 mutant type,and 18 cases as non-WNT/non-SHH type.(3)Four cases were successfully sequenced by Sanger,and the mutation sites of CTNNB1 exon 3 were as follows:001904.4:c.101 G>A,001904.4:c.94G>T.001904.4:c.98G>C,and 001904.4:c.109T>C.(4)The relationship between the expression of immunohistochemical markers and molecular typing.① β-catenin was expressed in tumor cell membrane or cytoplasm in all MB cases,and focal scattered expression was observed in nuclear positive cases(less than 1%of tumor cells).In the diffuse cell membrane/plasma background,it is difficult to find scattered positive nuclear expression of β-catenin.We regarded nuclear staining of β-catenin as positive expression.The statistical analysis showed that positive β-catenin nuclear staining was closely related to WNT activation type(P<0.0001).If 1%or more was the positive Cut-off value in the reference literature,the positive rate of β-catenin nuclear staining was 0,and there was no statistical correlation.② All the positive Lef-1 staining was nuclear positive.The positive rate of Lef-1 was 100%(7/7)in WNT type,5/12 in SHH type,and no expression in non-WNT/non-SHH type.Of the 21 MB patients with Lef-1 positive expression,7 were diffuse positive(positive cells greater than 50%)and 14 were focal positive(positive cells less than 50%).The histological morphology of the 7 cases with diffuse positive expression of Lef-1 was typical type and WNT type which was confirmed by NGS sequencing.There was a statistically significant correlation between Lef-1 positive expression and the molecular typing of MB(P<0.0001).Diffuse positive Lef-1 nuclear staining highly suggested the WNT-activated MB,which could be used as an alternative immunohistochemical marker for WNT-activated MB.③ YAP1 protein was localized in the nucleus.Among the 7 WNT-activated types,6 cases(85.7%)were positive for YAP1.Among the 12 cases of SHH activated type.6 cases(50%)were positive for YAP1 and 18 cases of non-WNT/SHH type were negative for YAP1.The expression of YAP1 was significantly different between WNT and non-WNT/non-SHH types(P<0.001).There was statistical difference between SHH type and non-WNT/non-SHH type(P<0.005).but there was no statistical difference between WNT type and SHH type(P>0.05).④GAB1 protein was localized in cytoplasmic plasma or cell membrane.In 7 cases of WNT activated type,4 cases(57.1%)were GAB1 positive,in 12 cases of SHH activated type,10 cases(83.3%)were GAB1 positive and in 18 cases of non-WNT/non-Shh type,6 cases(33.3%)were GAB1 positive.There was statistical difference in GAB1 expression between SHH and nonWNT/SHH types,but no statistical difference was found between WNT and SHH types,and between WNT and non-WNT/SHH types(P<0.05).(5)The relationship between the expression of immunohistochemical markers and the recurrence of patients.① The expression of Oligo2 in MB and its correlation with recurrence.No diffuse or partial expression pattern(greater than 10%)of Oligo2 was observed in all cases.In contrast with diffuse positive expression in glioma,the expression of Oligo2 in MB help to improve differential diagnosis.Oligo2 expression was found scattered in tumor cells in 20 of 51 MB patients(about 1%-5%positive cells).We chose 1%as the Cut-off value of Oligo2-positive expression.Among the 20 patients with recurrence,12 were Oligo2-positive,accounting for 60%,and among the 31 patients without recurrence,8 were Oligo positive,accounting for 25.8%.There was statistical difference between Oligo2 expression and relapse in MB patients(P<0.05).At the same time,we compared the expression of Oligo2 in different histological types and molecular classifications,and no statistical difference was found(P>0.05).② The relationship between Ki67 expression and recurrence in MB cases.The Ki67 proliferation index of 51 MB cases was between 20%and 90%.The mean value was(58.50±2.92)in the recurrence group and(43.87±2.05)in the survival group without recurrence.The expression of Ki67 was significantly different between the relapsed group and the non-relapsed group(P<0.001).At the same time,we compared the mean values of Ki67 expression in different histological types and molecular types,and no statistical difference was found(P>0.05).Conclusions:1.The expression of Lef-1 by immunohistochemistry can be used as a more sensitive marker than β-catenin to predict WNT-activated MB cases,which makes up for the difficult identification of β-catenin staining.Diffuse positive expression of Lef-1 highly suggests WNT-activated type.2.Oligo2 is usually not expressed in MB.This study found that the expression of Oligo2 in cells(1%-5%tumor cells positive)is closely related to the recurrence of MB patients,suggesting the role of Oligo2 expression in MB recurrence.3.The high expression of Ki67 was closely related to the recurrence of MB but not the histological types or molecular types,suggesting that Ki67 could be used as an independent predictor of recurrence. |
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