Aggressive embryonic central nerve tumors, particularly medulloblastomas, are the most prevalent malignant brain tumor in children and continue to be a leading source of morbidity and death. About 20% of all primary CNS tumors are of this kind. ^[1] As per the WHO's 2016 categorization, they are classified as grade IV due to their aggressive behavior, strong proliferative potential, and poor results. ^[2] Medulloblastoma varieties have long been recognized and are primarily categorized according to radiological and histological criteria concerning age of start, metastatic dissemination, and residual tumor following surgery. It will be very useful to distinguish between positive and negative outcomes because vigorous multinodal treatment protocols have a high morbidity rate. ^[3]

Recent WHO 2016 Integrated diagnosis, which combines genotyping and histological type, has allowed us to divide medulloblastoma into molecular subgroups such as wingless (WNT), sonic hedgehog (SHH), and two more subgroups, group 3 and group 4. This has been accomplished after much work. This subclassification will be utilized in the near future to choose focused treatments and enhance comprehension of the disease's behavior. ^[4] However, SHH tumors, group 3 tumors, and group 4 cancers exhibit varied results, with the exception of the WNT group, which is consistently linked to improved survival. ^[5] The last three groups' overall survival (OS) did not differ significantly, according to a recent meta-analysis. Recently, our team was able to show that patients with SHH/TP53 mutant medulloblastomas have a distinct correlation with particular genetic catastrophic events (chromothripsis) believed that this particular link might help to explain the varied outcomes of patients with SHH medulloblastomas as well as the debate around the prognostic significance of TP53 mutations in medulloblastoma. ^[6]

In this work, we show that TP53 mutation status may be used to distinguish between groups of people with SHH medulloblastoma that have a favorable survival rate and those that have an exceedingly bad one. Patients with SHH/TP53 mutant medulloblastomas, in particular, fare significantly worse than those with SHH/TP53 wild-type tumors.The prognosis is better for infants and adults than for youngsters. ^[7]

FISH, artificial microarray, flow cytometry, and immunohistochemistry have all been employed to classify these groups, despite the fact that subgroup classification is crucial when p53 mutation is shown to be a prognostic marker. We have classified the SHH subgroup using the manual immunohistochemistry GAB-1 (IHC gab1). ^[8] To separate the SHH subtype, GAB-1 immunostaining was performed on all histologically and radiologically identified medulloblastomas every two months via phone calls with the patients and treating physicians.To distinguish between the SHH mutant type and the wild type, P53 immunohistochemistry was performed in each SHH (Gab-1positive) patient. The instances were being followed up on. ^[9].

AIMS & OBJECTIVES

1. To study the histological features of adult and paediatric medulloblastoma and classify them into the histological subtypes as defined by WHO -2016.                                                    
2. To  study  the  expression  profile  of   the immunohistochemical  marker  GAB-1 in medulloblastomas  and further segregate all GAB-1 positive SHH subgroup into SHH/TP53 mutant type and SHH/TP53 wild type by immunohistochemistry.

The S.C.B. MCH Cuttack Institutional Review Board gave its approval to every procedure used in this retrospective study. From October 2016 to November 2018, 48 medulloblastoma cases were obtained from the Department of General Pathology's database at S.C.B. MCH Cuttack.

Histopathological analysis was performed on all 48 cases of clinically and radiologically suspicious medulloblastoma biopsy tissues that were fixed in formalin.All of the GAB-1 positive SHHcases were further separated into SHH/TP53mutant or SHH/TP53wild types using the same manual immunohistochemistry. Histopathologically diagnosed cases of medulloblastomas were subjected to an immunohistochemical study of GAB-1 using a similar protocol of peroxidase and antiperoxidase technique in our setup. Only 46 of the 48 cases had enough paraffin-block tissues for further immunohistochemical analysis. The clinical workstation provided the clinical information. As indicated in the proforma on the overleaf, a thorough examination of the immunohistochemical and H&E-stained slides was conducted.

Statistical Analysis: Statistical analysis was done with the univariate and multivariate analysis. A p value of less than 0.05 was considered significant. The patient demographic details and the morphological features of each tumor subtype were analyzed. A pie chart for correlation between morphological subtypes and immunohistochemical markers was performed. The specificity and sensitivity GAB-1 among the desmoplastic and non-desmoplastic histological subtypes were calculated.

There were a total of 48 cases of medulloblastoma from October 2016 to November 2018. Of these 46 cases were included in the present study based on the defined inclusion and exclusion criteria that are outlined in the materials and method section.The study population included 12 adults (26%) and34 children (74%)(Figure 1). 

Figure 1: Age distribution among children and adults of medulloblastoma

The M:F ratio was approximately 2:1 with 69.5% (32 cases) males and 30.5% (14 cases) females (Figure 2). In children there was clear male preponderance with nearly two third of the cases being males 25/34(Figure 3), + whilst in adults there was a near equal gender distribution male 7cases and female 5 case (Figure4).

Figure 2: Gender distribution in medulloblastomas

Figure 3: Gender distribution among children

Figure 4: Gender distribution among adults.

Histological subtypes:

We found all five histological subtypes of medulloblastoma in our study population. The predominant subtype corresponded to the Classic variant at 58.7% (27cases).Desmoplastic medulloblastomas formed the next major group with 23.9 % (11 cases),followed by the Large Cell variant at 10.9% (5 cases). There were 4.3%(2 cases) and2.2%(1 case)of the Anaplastic and Medulloblastoma  with extensive nodularity (MBEN) subtypes respectively. (Figure 5)

Figure 5: Distribution of cases by histological subtype in the study population.

In adults although Classic variant was the predominant subtype, the Desmoplasticvariant came a close second constituting 50% and 33% of cases respectively (Figure 6 ).On the other hand in children, the Classic variant constituted nearly two thirds of the cases (62%) as opposed to only 20 % being Desmoplastic. (Figure 7)

Figure 6: Distribution of cases by histological subtype in adults

Figure 7: Distribution of cases by histological subtype in children.

Histological Features:

The Classic subtype was characterized by closely packed round to oval cells with hyperchromatic(carrot shaped) nuclei surrounded by scant cytoplasm and high mitotic activity.(Figures 18(a-e))

The Desmoplastic variant was characterized nodular pattern and reticulin free zones (pale islands) which were surrounded by densely packed cells with dense intercellular reticulin.(Figure 19.) Those cases with only dense collagenous and reticulin fibres without any nodular pattern were not classified as Desmoplastic or nodular variant.(Figure19).

The MBEN variant was characterized by expanded lobular architecture with enlarged reticulin free zones rich in neuropil like tissue.The tumour cells had uniform nuclei and clear cytoplasm resembling neurocytes.(Figure 20(a)-20(b)).

The Large Cell variant was characterized by monomorphous population of cells with large, round, vesicular nuclei, prominent nucleoli and variable amount of cytoplasm with abundant mitotic and apoptotic figures.(Figure 21(a)-21(b))

The Anaplastic variant was characterized by the presence of increased nuclear pleomorphism, nuclear moulding, cell-cell wrapping and high mitotic activity. Apoptosis was prominent in this variant. Presence of the above mentioned features in focal areas was not considered sufficient to diagnose an anaplastic variant.(Figure 22).

GAB-1 immunohistochemistry:

GAB-1 immunoexpression was seen in 26.1% (12) of SHH cases(Figures 24(b)-24(c)).

Figure 8: Distribution  of  Molecular Subtype SHH/NON SHH TYPE

GAB-1 immunoexpression was seen in 33 % children versus 67% of adults, this difference was statistically significant. (Figure 9)

Figure 9 : GAB-1 immunoexpression among children and adults.

GAB-1 immunoexpression and histological subtype (Figure 10)

Nearly three fifth of the cases were of the Desmoplastic variant (7cases), 4cases of classic variants and 1 case of large cell variant show   GAB-1 immunopositivity .

Figure 10: Prevalence of GAB-1 immunoexpression in the histological subtypes

P53   IMMUNOHISTOCHEMISTRY:

Out  of  46 cases  8  cases(18%)  were  P53 mutant type  showing  over expression , 24  cases(53%)   were  P53 wild type  showing normal expression,12 cases (26%)  were showing  complete  absence  and 2 cases(3%) showing  abnormal  cytoplasmic staining (Figure 11)

Figure 11: Prevalence  of  P53 immunoexpression  in  Medulloblastoma

Out  of  8 cases  of P53 mutant type 5 cases(62.5%) were SHH subgroup and 3 cases(37.5%) were NON SHH subgroup. (Figure 12)

Figure 12 : Prevalence  of  SHH subgroup  in P53 mutant type

Out  of  12 cases of  SHH subgroup medulloblastomas  5 cases(42%) were  P53 mutant type (Figure 13)

Figure 13: Prevalence of P53 mutant type in SHH subgroup medulloblastoma

Figure 14: CT Scan of medulloblastoma in right cerebellar hemisphere both noncontrast(A) and contrast(B)

Figure 15:CT Scan of medulloblastoma in midline of cerebellar hemisphere

Figure16(a):Coronal T1 weighted MRI(contrast) revealing an enhanced posterior midline cerebellar mass

Figure16(b):Sagittal view T1 weighted MRI(contrast) revealing an enhanced midline cerbellar mass compressing brain stem

Figure 17 : Gab-1 showing cytoplasmic positivity in Invasive ductal carcinoma,Breast as positive control(H&E,x100)

Figure 18(a): Classic medulloblastoma with closely packed cells(H&E,x100)

Figure 18(b):  Classic medulloblastoma with medium sized hyperchromatic nuclei and scanty cytoplasm(H&E,x400)

Figure 18(c):   classic medulloblastoma showing carrot shaped nuclei(H&E,x400)

Figure 18(d): Classic medulloblastoma with mitotic figures(H&E,x400)

Figure 18(e):Streaming in a Classic medulloblastoma with absent nodularity(H&E,x100)

Figure 19 : Desmoplastic/Nodular medulloblastoma highlighting pale nodule (H&E,x100)

Figure 20(a): Medulloblastoma with extensive nodularity(H&E,x40)

Figure 20(b): Medulloblastoma with extensive nodularity showing neurocytic differentiation(H&E,x200)

Figure 21(a): Large cell medulloblastoma with vesicular nuclei and prominent nucleoli(H&E,x400)

Figure 21(b): Large cell medulloblastoma with apoptotic bodies(H&E,x400)

Figure 22: Anaplastic medulloblastoma showing nuclear pleomorphism and brisk mitotic activity(H&E,x400)

Figure23: Classic medulloblastoma with Homer wright rosettes(HE,x400)

Figure24(a): Gab-1 immunonegativity in classic medulloblastoma(x400)

Figure 24(b): Gab-1 immunopositivity in internodular area(x100)

Figure24(c): Gab-1 cytoplasmic membrane immunopositivity(x400)

Figure 25(a): P53 immunonegative(x100)

Figure 25(b): P53 nuclear immunopositive(x100)

Figure 26: Gab-1 Immunomarker(Merck Millipore)

Figure 27: P53 Immunomarker(Ready to use by BioGenex)

Medulloblastomas are high grade embryonal tumours of the central nervous system accounting for nearly 20% of childhood brain tumors and less than 1% of adult CNS tumors.[10] ( Although high grade, current management strategies have resulted in better long-term survival. The 2007 WHO classification defines five histological subtypes of medulloblastoma, namely the Classic, Desmoplastic/nodular, Medulloblastoma with extensive nodularity, Anaplastic and Large Cell variants.[1,6] The developing cerebellum undergoes differentiation aided by signaling pathways such as SHH, Notch and WNTpathways.[11] A key mechanism involved, in tumorigenesis is believed to be dysregulation of some of these  signaling pathways.[12] The wingless (WNT) and sonic hedgehog (SHH) signaling pathways are prime amongst those incriminated and several studies have identified the involvement of these two pathways in different subsets of medulloblastomas reliably and consistently.

The present study was carried out with the aim of prognostic implication of TP53 mutation in SHH subgroup specific  Medulloblastoma as per the WHO 2016 classification using immunohistochemistry GAB-1(IHC GAB-1) marker and further segregated as SHH/TP53mutant and SHH/TP53wild type by immunohistochemistry P53(IHC P53).

Medulloblastomas are 10 times more common in children when compared to adults.[13] In the present study more than three fourths of the medulloblastomas were seen in children. The majority of the paediatric medulloblastomas in this study were seen between the ages of 5 and 16 years. The reported incidence of adult medulloblastoma is upto 30%. In our study 26% of medulloblastomas were seen in adults..[14] In the present study, a male preponderance was noted with twice as many males as females in the overall cohort. Amongst children, nearly two third of medulloblastomas were seen in males which is in keeping with the published literature.[15] In contrast there was an almost equal gender distribution in adults.

Histological subtypes:

Among the five histological subtypes the (WHO2016) Classic variant was found to be the predominant subtype constituting about 58.7%(27 cases) followed by the Desmoplastic variant 23.9% (11 cases) and the Large Cell variant at 10.9% (05 cases) The prevalence of the different subtypes is similar to that reported by others.[16]

Study reveals TP53 mutation remain a poor prognostic marker accounting treatment failure in SHH subgroup medulloblastoma receiving radiation therapy and SMO inhibitor(vismodegib,sonidegib).Further we found that failure cases response better to arsenic trioxide(GLI2 inhibitor) and Lithium (GSK3b inhibitor) which is exciting.                                                                       

Validation of  the study :

• Medulloblastoma more prevalent in children74%(34/46) than adult 26%(12/46),Smoll et al.[17] 
• Medulloblastoma were more common in male i.e 2/3rd of the female in children as compared to adult where gender distribution is near equal,Leary SES et al.[18-19]
• Prevalence of Desmoplastic variant of medulloblastoma more common in adult compared to children,Min et al.[20]
• GAB-1 positive SHH subgroup medulloblastoma were foun in 26.1% as compared to 30% in literature,Zhukova et al.[21]
• GAB-1 were more prevalent in Desmoplastic variant of medulloblastoma as reported in the literature.
• Somatic TP53 mutation  in 17.4%(8/46) of  cases  as  previously reported  by Zhukova et al .[21] i.e 16.9%.

Out  of  8 cases  of TP53  mutant type , 5cases 62.5%(5/8) were  identified as SHH subgroup type as reported  by Zhukova et al. [21-22]  i.e 67.9%. 

Medulloblastomas are primarily tumors of childhood and highly   aggressive. The recent 2016 integreted diagnosis classified medulloblastoma into molecular type i.e WNT/SHH/Group3/Group4 type. The recent advance in management protocol has improved the duration of survival and quality of life. WNT subgroup consistently associated with better prognosis but SHH subgroup shows heterogenous outcome and management of SHH type is challenging.GAB-1 antibody is useful molecular marker for the diagnosis of SHH subgroup medulloblastoma. Further segregation of SHH/TP53 mutant and SHH/TP53 wild type is required for favourable prognosis. The study demonstrated how combination of genomic and genetic analysis of brain tumors can predict differential response to therapies and provide insight into ways that favourable signalling pathways may be mimicked by pharmacological intervention.

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