Aanchal Kakkar

Integrating Molecular Subclassification of Medulloblastomas into Routine Clinical Practice: A Simplified Approach

Medulloblastoma (MB) is composed of four molecular subgroups viz. WNT, SHH, groups 3 and 4, identified using various high‐throughput methods. Translation of this molecular data into pathologist‐friendly techniques that would be applicable in laboratories all over the world is a major challenge. Ninety‐two MBs were analyzed using a panel of 10 IHC markers, real‐time PCR for mRNA and miRNA expression, and FISH for MYC amplification. β‐catenin, GAB1 and YAP1 were the only IHC markers of utility in classification of MBs into three subgroups viz. WNT (9.8%), SHH (45.6%) and non‐WNT/SHH (44.6%). mRNA expression could further classify some non‐WNT/SHH tumors into groups 3 and 4. This, however, was dependent on integrity of RNA extracted from FFPE tissue. MYC amplification was seen in 20% of non‐WNT/SHH cases and was associated with the worst prognosis. For routine diagnostic practice, we recommend classification of MBs into three subgroups: WNT, SHH and non‐WNT/SHH, with supplementation by prognostic markers like MYC for non‐WNT/SHH tumors. Using this panel, we propose a new three‐tier risk stratification system for MBs. Molecular subgrouping with this limited panel is rapid, economical, works well on FFPE tissue and is reliable as it correlates significantly with clinicopathological parameters and patient survival.

Does bony hyperostosis in intracranial meningioma signify tumor invasion? A radio-pathologic study

BACKGROUND While operating intracranial meningiomas, neurosurgeons commonly drill the hyperostotic bone and put the bone flap back. OBJECTIVE To determine whether bony changes seen in meningioma are due to tumor invasion or reactionary changes. MATERIALS AND METHODS This prospective study, conducted over 10 months (October 2010- July 2011) included consecutive patients with intracranial meningiomas. Preoperatively, computed tomography (CT) was done in all patients and reviewed by two neurosurgeons for associated bony hyperostosis. During surgery, a piece of bone showing hyperostosis was taken for histopathological evaluation for tumor invasion. In absence of hyperostosis, the bone sample was taken from the bone in contact with the dural attachment of the tumor. RESULTS This study included 40 consecutive patients who underwent resection for intracranial meningiomas. Radiological evidence of hyperostosis was present in 30 (75%) patients. On histopathological examination, tumor invasion of the bone was seen in eight (20%) patients. These included seven patients who demonstrated hyperostosis and one patient without hyperostosis. Convexity meningiomas (n=12) showed the highest rate of bony invasion (33.3%). CONCLUSIONS A significant number of patients with radiological hyperostosis have tumor invasion of the bone. The authors recommend that one should remove the bone (flap) whenever possible in order to achieve total excision of the tumor and use synthetic material to cover the defect.

Loss of heterozygosity on chromosome 10q in glioblastomas, and its association with other genetic alterations and survival in Indian patients

BACKGROUND Glioblastoma multiforme (GBM) is the most common malignant central nervous system neoplasm. Loss of heterozygosity (LOH) on chromosome 10q in these tumors has been found to show variable association with prognosis. AIM To evaluate LOH 10q status in cases of GBM, and to correlate these results with patient characteristics, other genetic alterations, and survival. MATERIAL AND METHODS Fresh tumor tissue and blood samples were obtained for 25 cases of GBM diagnosed over a 2-year period. LOH 10q assay was performed on blood and tumor DNA by a PCR-based method using four microsatellite markers. TP53 mutation analysis and fluorescence in situ hybridization for epidermal growth factor receptor (EGFR) were performed. Histopathology was reviewed and clinical data were analyzed. RESULTS LOH 10q was identified in 17 of 25 cases (68%). Losses were frequent with markers D10S1765 (12/20 informative cases; 60%) and D10S587 (12/17 informative cases; 70.5%) in the regions of 10q23.3 and 10q26.1, respectively. D10S540 for 10q25.1 showed LOH in 4/12 informative cases (33.3%) and D10S1770 for 10q26-ter in none of the 25 cases. LOH with D10S1765 at the PTEN gene locus was found to correlate with overall LOH 10q status (P = 0.001). LOH 10q was more common in patients older than 40 years (16/19, 84.2%) than in those below (1/6, 16.7%) (P = 0.006). One of three pediatric patients included demonstrated LOH 10q. Survival rates for patients with LOH were lower than for patients with retained heterozygosity. CONCLUSION LOH 10q is a frequent genetic abnormality in GBM in Indian patients, is seen more frequently in older adults, and its presence is associated with shorter survival. The single best marker to determine LOH 10q status is D10S1765 at the PTEN region.

Atypical teratoid/rhabdoid tumors: challenges and search for solutions

Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant embryonal central nervous system tumor commonly affecting children <3 years of age. It roughly constitutes 1%–2% of all pediatric central nervous system tumors. Recent data show that it is the most common malignant central nervous system tumor in children <6 months of age. Management of this aggressive tumor is associated with a myriad of diagnostic and therapeutic challenges. On the basis of radiology and histopathology alone, distinction of AT/RT from medulloblastoma or primitive neuroectodermal tumor is difficult, and hence this tumor has been commonly misdiagnosed as primitive neuroectodermal tumor for decades. Presence of a bulky heterogeneous solid-cystic mass with readily visible calcification and intratumor hemorrhage, occurring off-midline in children <3 years of age, should alert the radiologist toward the possibility of AT/RT. Presence of rhabdoid cells on histopathology and polyphenotypic immunopositivity for epithelial, mesenchymal, and neuroectodermal markers along with loss of expression of SMARCB1/INI1 or SMARCA4/BRG1 help in establishing a diagnosis of AT/RT. The optimal management comprises maximal safe resection followed by radiation therapy and multiagent intensive systemic chemotherapy. Gross total excision is difficult to achieve in view of the large tumor size and location and young age at presentation. Leptomeningeal spread is noted in 15%–30% of patients, and hence craniospinal irradiation followed by boost to tumor bed is considered standard in children older than 3 years. However, in younger children, craniospinal irradiation may lead to long-term neurocognitive and neuroendocrine sequel, and hence focal radiation therapy may be a pragmatic approach. In this age group, high-dose chemotherapy with autologous stem cell rescue may also be considered to defer radiation therapy, but this approach is also associated with significant treatment-related morbidity and mortality. Novel small molecule inhibitors hold promise in preclinical studies and should be considered in patients with relapsed or refractory tumor.

Expression of Cell Cycle-associated Proteins p53, pRb, p16, p27, and Correlation With Survival: A Comparative Study on Oral Squamous Cell Carcinoma and Verrucous Carcinoma.

Verrucous carcinoma (VC) is a well-differentiated form of squamous cell carcinoma (SCC) with better prognosis. Differences in molecular pathogenesis between the 2 have not been well-characterized. We conducted this study to evaluate immunohistochemical expression of cell-cycle regulatory proteins p53, pRb, p16, and p27 in SCC and VC, compare the expression in these 2 neoplasms, and assess if these markers have any diagnostic or prognostic value. Sixty cases of SCC with and without lymph node metastasis and 31 cases of VC were studied. Immunohistochemical analysis for p53, pRb, p16, and p27 was performed and the results were analyzed. SCC was most frequent in tongue (52%), whereas VC in buccal mucosa (81%). Mean age of SCC patients was significantly lower than in VC. Majority of SCCs were in stage III and IV (63%), whereas VCs were in stage I and II (84%). p53 immunopositivity was more frequent in SCC (65%) than in VC (23%) (P⩽0.001). VC had lower p53 as compared with well-differentiated SCC and SCC without lymph node metastasis. No significant difference was seen in pRb, p16, and p27 expression. Disease-free survival (DFS) at 1 year for SCC was 57% whereas it was 80% for VC (P=0.02). DFS and overall survival of SCC correlated with nodal status and stage; cell-cycle–associated protein expression had no association with DFS. To conclude, p53 immunoexpression differs in SCC and VC, suggesting different pathogenesis, and it may have some utility as an adjunct to morphology to differentiate between the 2. Expression of cell-cycle–associated proteins does not influence survival in SCC.

The expression of Cyclin D1, VEGF, EZH2, and H3K27me3 in Atypical Teratoid/Rhabdoid Tumors of the CNS: A Possible Role in Targeted Therapy.

Atypical teratoid/rhabdoid tumor (AT/RT) is an uncommon malignancy with a dismal outcome, which responds poorly to multimodality therapies. Animal studies have revealed Cyclin D1 as a possible therapeutic target. The addition of vascular endothelial growth factor (VEGF) inhibitors to chemotherapeutic regimens has shown promising results in pediatric central nervous system tumors. Enhancer of Zeste homolog 2 (EZH2) overexpression has been implicated in various cancers, including medulloblastomas. H3K27me3 is a new marker for pediatric high-grade gliomas. However, their role in AT/RT has not been evaluated sufficiently. We retrieved cases of AT/RT, and reviewed their clinical data and histopathologic features. Immunohistochemistry for Cyclin D1, VEGF, EZH2, and H3K27me3 was performed. Follow-up was noted when available. Fourteen cases of AT/RT were identified (mean age, 3.4 y; range, 10 mo to 8 y). Cyclin D1 immunopositivity was noted in all cases [labeling index (LI): 5% to 98%; mean, 41.3%]. VEGF positivity was seen in 83.3% of the cases. All cases showed EZH2 overexpression (mean LI, 74.3%; range, 32% to 96%). Reduction of H3K27me3 expression was noted in 63% of the cases, with no correlation with EZH2 LI. Two patients died of postoperative complications. Of the rest, follow-up was available for 7 (range, 7 to 120 wk): 1 achieved clinical remission, whereas 6 developed progressive disease, including 3 deaths. Varying degrees of immunoreactivity to Cyclin D1, VEGF, and EZH2 were noted in the majority of the AT/RTs, and detection of these markers may be of value in the development of novel therapeutic agents and in determining which patients can benefit from them. AT/RTs show reduction in H3K27me3 expression, independent of EZH2 expression, indicating that their interaction requires further evaluation.

Evaluation of 1p and 14q status, MIB-1 labeling index and progesterone receptor immunoexpression in meningiomas: Adjuncts to histopathological grading and predictors of aggressive behavior.

BACKGROUND Meningiomas are benign central nervous system tumors; however, significant fraction recurs, irrespective of histological grade. MATERIALS AND METHODS We performed fluorescence in-situ hybridization for 1p36 and 14q32, and immunohistochemistry for progesterone receptor (PR), p53 and MIB-1 on 84 meningiomas. RESULTS AND CONCLUSION Sixty-four were convexity tumors (30 grade I, 21 grade II, 13 grade III) and 20 petroclival (grade I; 10 with gross total resection (GTR), 10 with subtotal resection (STR)). Isolated 1p36 deletion was seen in 20% grade I, 28.6% grade II and 30.8% grade III convexity meningiomas, and isolated 14q deletion in one grade III convexity tumor. 1p/14q co-deletion was seen in none of grade I, 28.5% grade II and 30% grade III convexity meningiomas. PR immunoreactivity was less frequent in grade III tumors. Petroclival tumors did not show isolated deletion. However, 1p/14q co-deletion was seen in 20% of petroclival tumors with STR and in none with GTR. Frequency of chromosomal alterations and MIB-1 labeling index both increase with tumor grade. 1p/14q co-deletion is characteristic of grade II/III meningiomas, while PR immunoreactivity inversely correlates with grade, suggesting their use as surrogate markers for grading. Identification of 1p/14q co-deletion in grade I petroclival tumors with STR suggests that unresectable petroclival meningiomas are biologically more aggressive than their grade I convexity counterparts.

Mature Teratoma in Association with Neural Tube Defect (Occipital Encephalocele): Series of Four Cases and Review of the Literature

Both occipital encephalocele and teratomas are midline congenital malformations. Encephalocele is a form of neural tube defect in which there is a congenital defect of the cranium through which occurs a protrusion of brain matter or meninges, while teratoma is a tumor derived from all three germ layers. The association between occipital encephalocele and teratoma has not been reported to date. In the present study, the authors present a series of four such cases.

Alterations in BRAF gene, and enhanced mTOR and MAPK signaling in dysembryoplastic neuroepithelial tumors (DNTs).

OBJECTIVE Recently, BRAF V600E mutation, and activation of mTOR and MAPK pathways have been identified in various glial/glioneuronal tumors. Dysembryoplastic neuroepithelial tumors (DNTs) are epilepsy-associated glioneuronal neoplasms which have not been analyzed extensively in this respect. METHODS Sequencing for BRAF V600E mutation, analysis of BRAF copy number by qRT-PCR, and immunohistochemistry for mTOR (p-S6, p-4EBP1) and MAPK (p-MAPK) pathways were performed. RESULTS Sixty-four DNTs were identified, accounting for 15.1% of patients with drug-refractory epilepsy (mean age: 15.5 years). Duration of seizures ranged from 1 to 22 years. BRAF V600E mutation was identified in 3.7% of DNTs, while BRAF copy number gain was observed in 33.3%. mTOR-pathway activation indicated by p-S6 or p-4EBP1 immunopositivity was seen in 89.7% cases. Interestingly, p-S6 positivity was also seen in adjacent dysplastic cortex. p-MAPK immunopositivity was seen in 50% cases. MAPK and mTOR pathway activation was independent of BRAF alterations. All patients that underwent incomplete resection had Engel grade II-III outcomes (p<0.001). CONCLUSION BRAF alterations are frequent in DNTs, particularly BRAF copy number gain which is being reported for the first time in these tumors. Evidence of activation of mTOR and MAPK pathways suggests a role for altered signalling in DNT pathogenesis, and will pave the way for development of targeted therapies, particularly relevant for patients having persistent seizures after incomplete resection.

Study of clinicopathological features, hormone immunoexpression, and loss of ATRX and DAXX expression in pancreatic neuroendocrine tumors

ABSTRACT Objectives: Neuroendocrine tumors of the pancreas (PanNETs) are rare neoplasms, and not much is known about their pathogenesis. We aimed to evaluate ATRX/DAXX immunoexpression in PanNETs a cohort of well-characterized PanNETs. Methods: PanNETs diagnosed over a 10-year period were retrieved and clinicopathogical features reviewed. Immuohistochemistry for pancreatic hormones, and for ATRX and DAXX was performed. Results: Sixty-eight PanNETs were included (30 males and 38 females) with median age of 39 years. Histologically, there were 37 Grade 1 (54.4%), 27 Grade 2 (39.7%), and 4 Grade 3 (5.9%) cases. On immunostaining for hormones, insulin expression was most frequent (22 cases; 38.6%), followed by gastrin (7 cases; 12.3%); 25 cases (43.9%) were negative for all hormones. Loss of ATRX/DAXX immunoexpression was noted in 18 cases (39.1%), and was significantly more frequent in tumors larger than 5 cm. Lymphovascular invasion, infiltrative borders, and infiltration of adjacent organs were also more frequent in tumors with loss of ATRX/DAXX immunoreactivity. A little over half the tumors with ATRX/DAXX loss showed negative immunostaining for all hormones (55.6%). Conclusion: Loss of ATRX/DAXX expression is frequent in PanNETs, indicating a role in their pathogenesis. As ATRX/DAXX loss is more frequent in larger tumors, and in those with lymphovascular invasion, adjacent organ infiltration and infiltrative borders, this suggests that loss of ATRX/DAXX expression is a late event in pathogenesis and is associated with an aggressive phenotype. Immunohistochemical detection of ATRX/DAXX loss is a simple method for ATRX/DAXX evaluation and can easily be incorporated into routine pathological evaluation of PanNETs.