Shui Yen Soh

Novel Karyotypes and Cyclin D1 Immunoreactivity in Clear Cell Sarcoma of the Kidney

Pathological diagnosis of clear cell sarcoma of the kidney (CCSK) is challenging as it resembles blastemal Wilms tumor (WT) and other pediatric sarcomas, and does not have any distinctive immunophenotype. The YWHAE-FAM22 translocation t(10;17)(q22;p13) has been reported in a subset of CCSK. This translocation also occurs in high-grade endometrial sarcoma, in which it is associated with cyclin D1 overexpression. Hence we seek to determine YWHAE-FAM22 translocation status and cyclin D1 immunoreactivity in a series of local CCSK cases. Of 8 CCSK cases from 7 patients identified, no CCSK had the YWHAE-FAM22 fusion transcript by reverse transcriptase–polymerase chain reaction. Novel karyotypes were identified for 2 cases: 1 had t(2;13)(q13; q22) and the other t(3:17)(q29;p11.2). Excluding a case with poor tissue section antigenicity, 7 of 7 CCSKs (100%) showed diffuse and strong nuclear cyclin D1 staining. Cyclin D1 immunohistochemistry was also performed on tissue microarrays of other pediatric renal tumors: blastemal areas of 18 WT cases were negative; 6 rhabdoid tumors and 1 metanephric adenoma showed patchy and weak staining; 3 mesoblastic nephromas and 18 of 29 neuroblastomas had positive staining. Cyclin D1 immunohistochemistry helps distinguish CCSK from blastemal WT and metanephric adenoma and rhabdoid tumors, but not from neuroblastomas and mesoblastic nephromas. Cyclin D1 overexpression in CCSK is not contingent on YWHAE-FAM22 translocation, and cyclin D1 inhibition may potentially be explored as a targeted therapeutic strategy in CCSK.

Platform Comparison for Evaluation of ALK Protein Immunohistochemical Expression, Genomic Copy Number and Hotspot Mutation Status in Neuroblastomas

ALK is an established causative oncogenic driver in neuroblastoma, and is likely to emerge as a routine biomarker in neuroblastoma diagnostics. At present, the optimal strategy for clinical diagnostic evaluation of ALK protein, genomic and hotspot mutation status is not well-studied. We evaluated ALK immunohistochemical (IHC) protein expression using three different antibodies (ALK1, 5A4 and D5F3 clones), ALK genomic status using single-color chromogenic in situ hybridization (CISH), and ALK hotspot mutation status using conventional Sanger sequencing and a next-generation sequencing platform (Ion Torrent Personal Genome Machine (IT-PGM)), in archival formalin-fixed, paraffin-embedded neuroblastoma samples. We found a significant difference in IHC results using the three different antibodies, with the highest percentage of positive cases seen on D5F3 immunohistochemistry. Correlation with ALK genomic and hotspot mutational status revealed that the majority of D5F3 ALK-positive cases did not possess either ALK genomic amplification or hotspot mutations. Comparison of sequencing platforms showed a perfect correlation between conventional Sanger and IT-PGM sequencing. Our findings suggest that D5F3 immunohistochemistry, single-color CISH and IT-PGM sequencing are suitable assays for evaluation of ALK status in future neuroblastoma clinical trials.

XAF1 promotes neuroblastoma tumor suppression and is required for KIF1Bβ-mediated apoptosis.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bβ, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bβ deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bβ. XAF1 silencing protects from NGF withdrawal and from KIF1Bβ-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions.

Characterization of anaplastic lymphoma kinase-positive medulloblastomas

Medulloblastomas are the most common pediatric malignant primary brain tumor. To our knowledge, there are no known critical and druggable tyrosine kinases in medulloblastomas, precluding the use of established tyrosine kinase inhibitors that have shown efficacy in other tumor types. We studied the expression of anaplastic lymphoma kinase (ALK), a well-characterized tyrosine kinase and drug target, in a cohort of medulloblastomas by immunohistochemistry, and identified three ALK-positive cases. Mutational analyses did not reveal a definite underlying genetic mechanism for the ALK expression, although one of the cases showed increased ALK copy number. Our findings have clinical implications and warrant further pharmacological and functional studies, as well as evaluation in larger patient cohorts, to fully characterize the value of ALK as a prognostic and predictive therapeutic marker in medulloblastomas.

Clinical, Pathological and Loss of Heterozygosity Differences in Wilms Tumors between Asian and non-Asian Children: Inter-Ethnic Differences in Wilms Tumors

Wilms tumor demonstrates significant interethnic epidemiological, histological and outcome differences, and is rare and poorly studied among Asians. We compared the clinicopathological, and loss of heterozygosity (LOH) profile and survival outcomes of Asian and non‐Asian patients with Wilms tumor. Clinical charts and histological slides from patients with malignant renal tumors over a period of 20 years were retrospectively reviewed. We adapted a genotyping assay to determine 1p36 and 16q21‐22 LOH in formalin‐fixed paraffin‐embedded (FFPE) specimens, and compared these characteristics between Asian and non‐Asian patients. Fifty‐three (79.1%) Asian and 14 (20.9%) non‐Asian patients had Wilms tumors. Compared to non‐Asians, Asians were younger (mean 4.6 and 4.0 years, respectively), had more equal gender distribution (female: male = 1.8 and 1.0, respectively), fewer tumors with unfavorable histology (25.0% and 4.1%, respectively, p = 0.05), and less advanced disease at presentation, yet similar nodal metastases rates (16.7% and 18.4%, respectively). No Asian patients had bilateral tumors. Our adapted genotyping assay accurately determined LOH in FFPE specimens <10 years post‐fixation. Among 30 Asian patients, 1p and 16q LOH were each detected in 5 (16.7%) patients, respectively—similar to rates reported in other ethnicities. Yet after similar treatment with National Wilms Tumor Study regimens, 15‐year event‐free and overall survival for Asian patients was 95.7% and 96.3% respectively. In summary, despite similar nodal metastasis and LOH rates, Asian patients had fewer unfavorable histology tumors, lower‐stage disease, and better survival outcomes. The bases for these differences and implications on treatment strategy for these patients warrant further study.

DTI fusion with conventional MR imaging in intra-operative MRI suite for paediatric brainstem glioma biopsy

Dear Editor-in-Chief, We read with interest Puget et al.’s series of stereotactic biopsy of diffuse intrinsic pontine glioma (DIPG*) as an integrated workflow for diagnosis and better disease understanding [1]. Despite advances in molecular knowledge, DIPG remains as one of the most challenging paediatric brain tumours. Median survival for afflicted children is less than 12 months from diagnosis [2]. In present times, there is growing consensus on the validity of biopsies for presumed DIPG patients. We are now cognizant of the need for tissue interrogation for urgent genomic information. Given the current neurosurgeon’s armamentarium, brainstem biopsy is now considered to be a safe procedure [1]. The authors report the use of diffusion tensor imaging (DTI) fusion with conventional MRI in the intra-operative MRI (iMRI) suite as a guide for neuronavigation in stereotactic biopsy. A 4-year-old female presented with right-sided gait ataxia, occulo-palsies and difficulty swallowing. Magnetic resonance imaging (MRI) of the neuroaxis reported diffuse enlargement of the pons with effacement of the surrounding parenchyma and fourth ventricle, as well as encasement of the basilar artery is suggestive of DIPG. There was no drop metastasis in the spine. A stereotactic biopsy was arranged in the iMRI suite which housed a 1.5 Tesla MRI machine. Procedural safety was the main consideration, with aims to avoid further neurological deficit and iatrogenic haemorrhage. Under general anaesthesia, the patient was positioned prone with head-pin fixation. She underwent a diffusion tensor imaging (DTI) sequence to delineate white matter tracts followed by T1weighted post-gadolinium MRI brain. Both sequences were fused at the in-house iMRI suite workstation (BrainlabTM) to plan an appropriate trajectory for the biopsy (see Fig. 1). Subsequently, a frame-based transcerebellar biopsy was performed with good tissue yield. In this case, surgical guidelines were largely adapted from Puget et al. [1]. Next, a T2weighted MRI scan was performed to assess for surgical complications and confirmation of the biopsy needle tract (see Fig. 2). Post-operatively, the patient had a transient right abducens nerve palsy that recovered within 24 h. Otherwise, the overall peri-operative period was uneventful. For most neurosurgeons, the concept of a brainstem biopsy can be dubitable. This is especially so in the context of firstly, a difficult tumour such as DIPG whereby limited therapeutic options are available and secondly, facing the consequences of possible surgical complications in a young child. Based on previous studies, the presumed risk profile of stereotactic brainstem biopsy reports up to 28% of operative morbidity [3]. Putting all these together, it is thus not inconceivable for the neurosurgeon to hesitate to proceed. DTI is an advanced imaging technique that evaluates white matter tracts in the brain. Infiltration or displacement of these fibre tracts by tumour can be detected by changes in the diffusion of water along the tracts, which is measured by fractional anisotropy [4]. Specifically, for DIPG, Prabhu et al.’s study reports that pre-treatment tumours tend to infiltrate and displace fibre tracts without complete disruption [5]. Although there are * Sharon Y. Y. Low sharon.low.y.y@singhealth.com.sg

Clinical management of pheochromocytoma and paraganglioma in Singapore: missed opportunities for genetic testing

Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors of the adrenal glands and paraganglia, occurring sporadically or as a range of hereditary tumor syndromes. About 30% of PPGLs are attributed to germline mutations. Clinical presentation, including localization, malignant potential, and age of onset, varies depending on the genetic background. Genetic testing for PPGLs is not well studied in Southeast Asia. We reviewed clinical management of PPGLs in Singapore, highlighting current gaps in clinical practice.

Critically Ill Children With Hemophagocytic Lymphohistiocytosis: A Case Series of 14 Patients.

Children with hemophagocytic lymphohistiocytosis (HLH) are at an increased risk of critical illness. In this study, we described the clinical characteristics of critically ill children with HLH and identify factors associated with poor clinical outcomes. Children who were diagnosed with HLH with emergent admission to Children’s Intensive Care Unit (CICU) between January 1, 2000 and October 31, 2015 were included. The primary outcome was CICU mortality. Over the 15-year period, there were 14 critically ill patients with HLH with 23 CICU admissions. Median age at HLH diagnosis was 8.2 years (interquartile range [IQR], 2.9 to 11.3). Overall CICU mortality was 8 of 23 CICU admissions (34.8%). Factors that were associated with CICU mortality in critically ill children with HLH identified in this study include: a worse median pediatric index of mortality 2 score (4.7% in survivors [IQR, 2.9% to 11.6%] vs. 2.4% [IQR, 1.2% to 4.3%]; P=0.031); higher median peak serum lactate level (mmol/L) within 24 hours of admission (5.6 [IQR, 2.7 to 17.4] vs. 1.6 [IQR, 1.2 to 2.8]; P=0.032); the need for mechanical ventilation (100% vs. 46.7%; P=0.019); inotropic support (87.5% vs. 20.0%; P=0.006); renal replacement therapy (50% vs. 0%; P=0.008); and blood product transfusion episodes (24.5 [IQR, 14.3 to 46.8] vs. 3.0 [IQR, 1.0 to 9.0]; P=0.002). Further studies are required to validate the factors that are associated with poor outcomes in critically ill children with HLH.

Abstract A47: Unusual suspects identified in neuroblastoma: An unexpected tumor suppression pathway

Developmental apoptosis of neural crest precursors is crucial in determining the final number of terminally differentiated cells such as sympathetic neurons. During neural development, cells undergo apoptosis as NGF becomes limiting. Aberrant developmental apoptosis is implicated in pediatric sympathetic nervous system tumors. When NGF becomes limiting, a developmental apoptotic pathway is activated which requires KIF1Bbeta. KIF1Bbeta is necessary and sufficient for apoptosis during NGF withdrawal. KIF1Bbeta maps to 1p36.2, a frequently deleted region in neuroblastomas, and a neural crest-derived cancer. We identified that KIF1Bbeta-induced apoptosis requires RNA/DNA helicase DHX9. KIF1Bbeta interacts with DHX9 to enhance translocation and accumulation of cytoplasmic DHX9 in the nucleus, resulting in transcription of apoptotic XAF1. Transcription-incompetent DHX9 is unable to potentiate KIF1Bbeta-induced cell death. Knockdown of DHX9 also protects from KIF1Bbeta-induced cell death whereas KIF1Bbeta loss-of-function domains or patient-associated point mutants are unable to translocate and accumulate cytoplasmic DHX9 in the nucleus, impairing XAF1 expression. Furthermore, XAF1 silencing protects from KIF1Bbeta-induced and NGF withdrawal-mediated apoptosis in vitro as well as promotes tumor growth in vivo whereas XAF1 overexpression is necessary and sufficient to induce apoptosis in vitro and delays tumor growth in vivo. Conditional knockout of KIF1Bbeta in the superior cervical ganglia neurons of mouse pups also specifically ablates XAF1 expression in vivo and ex vivo, suggesting that KIF1Bbeta and XAF1 are tightly regulated and act along the same pathway. Clinically, tissue microarray analysis of pre-treatment or post-treatment neuroblastoma patients who are 1p-intact or 1p-deleted, revealed strong prognostic significance of XAF1 expression in disease stratification. Our findings provide a mechanistic understanding on the development of neuroblastoma through unexpected candidates of tumorigenesis. Citation Format: Zhang9e Choo, Rachel Yu Lin Koh, Karin Wallis, Timothy Jia Wei Koh, Chik Hong Kuick, Veronica Sobrado, Amos Hong Pheng Loh, Shui Yen Soh, Susanne Schlisio, Kenneth Tou En Chang, Zhi Xiong Chen. Unusual suspects identified in neuroblastoma: An unexpected tumor suppression pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A47.

Neuroblastoma in Children Under 12 Months in Singapore—15-Year Experience and Outcomes From KKH

Background: Expectant observation of small adrenal lesions has shown promising results in recent studies. We present our 15 years outcome of managing infant neuroblastoma. Methods: All patients with neuroblastoma below the age of 1 year treated at the largest pediatric hospital in Singapore between 1998 and 2012 were identified. Results: Twenty-two patients were included in our study. Six were antenatally diagnosed. Nineteen (86%) patients had surgical resection of the tumor. Eight (36%) patients received chemotherapy as part of their treatment. Six patients were observed three of which had large adrenal tumors. Median follow-up in our series was 2.6 years. The 5 year overall survival was 90%. There were no recurrences and there were 2 deaths in our series. Conclusion: Our series shows excellent outcomes of infant neuroblastoma at our center. Careful observation of large tumors may be an option to avoid the morbidity of surgery.