Genetic Profiling of MYCN non amplified Neuroblastoma by MLPA Omshree Shetty1, Mukta Ramadwar1, Mamta Gurav1, Trupti Pai 1, Vishakha Kamble1, Girish Chinnaswamy2, Tushar Vora2, Sajid Qureshi3, Sandeep Dhanavade1, Sonali Tambe1, Sangeeta Desai1 1-Molecular Pathology Division, Department of Pathology,2-Department of Medical Oncology, 3- Department of Surgical Oncology, Tata Memorial Centre, Mumbai, India Introduction Methods Results Results The study comprised of 32 patients of NB and molecular analysis could be performed on 28 cases. The median age of the study group 1year (Range: 1month-2 years) Infants n=11 (below 18 months) Male to female ratio 2.5:1 Predominant site affected : Abdomen MKI index - Low in 59% of the cases Unfavorable histology - 46.4% of the cases (n=13) As per International Neuroblastoma Staging System (INSS), 50% of the (n=14) cases – Stage 4 Neuroblastoma (NB) is a primitive neuroectodermal tumor, most common extra cranial solid neoplasm of childhood, presenting with genetic instability and genetically heterogeneous cell population. Highly variable disease ranging from small-large localized tumor (stages 1-3) to metastatic disease (stage 4) with ~50-60% children stage 4 at diagnosis. Prognosis depends on age and stage of disease ,overall cure rate~50%. For the past 30 years MYCN gene amplification (MNA) was only used for predicting the outcome of the disease.1 The current risk classification of patients with NB is based on the age of onset, the extent of disease at the time of diagnosis, MYCN amplification (MNA) status.2 MNA is considered the strongest prognostic factor and is routinely assessed for therapy stratification. Detailed genetic analysis of the NB is possible with latest methodologies such as Multiplex Ligation Probe amplification Assay (MLPA).3 This has led to the identification of new prognostic markers like loss of heterozygosity, segmental chromosomal aberrations (SCA). Table 2: Overview of Segmental Chromosomal Aberrations observed by MLPA analysis Neuroblastoma cases (FFPE) MYCN FISH Amplified Non Amplified ● DNA extraction ● Quality Assessment ●MLPA (P251/P252/P253) ●Data Analysis on Coffalyser software Segmental Chromosomal Aberrations No. of Cases MYCN amplified 4/28 (14.28%) MYCN non amplified 24/28 (85%) 2p gains/amplification 12/27* (44.44%) 17q gains/ amplification 19/27* (70.37%) 11q loss 8/27* (26.63%) 3p loss 11/27* (40.74%) 4p loss 4/27*(14.81%) 4q loss 7q gains 5/27*(18.51%) 14q loss 7/20# (35%) *- Ambiguous signals in one case, only 27 analyzed, #-data was available for 20 cases Molecular Analysis Fluorescence In situ Hybridization (FISH) was performed on all the cases of NB to ascertain the MYCN status. Dual colour probe set (Abbott Molecular Inc., Des Plaines, IL, USA), a mixture of LSI MYCN (2p24) labelled with Spectrum GreenTM and CEP 2 (2p11.1-q11.1) labelled with Spectrum Orange was used for studying the MYCN amplification . MYCN copy number are scored as either amplified (Nuclei containing an MYCN gene/CEP2 gene ratio of 4 or more) or non amplified depending on the ratio of the signals of MYCN gene and CEP2. After FISH evaluation MLPA was performed on all the cases. Representative sections with > 30% tumor were selected for MLPA analysis. MLPA probe mixes P251, P252 and P253 (MRC-Holland, The Netherlands) for Neuroblastoma panel was used. 2A 2B MLPA data obtained was correlated with clinical and histological parameters using SPSS software and Pearson’s Chi square test was used for statistical evaluation. 11q loss was significantly associated with high mitotic index of the tumor (p= 0.014) 17q gains showed significant association with high risk disease (p=0.045), all were of unfavorable histology. Gains in ALK gene was found to be associated with progressive disease with bone marrow involvement. Bone marrow was involved in 8/28 cases of which 5 cases showed gains in ALK gene and was significantly associated (p=0.029). Loss of 4q was found to be significantly associated with favorable histology of NB (p=0.014), clinical significance of which is unknown. Fig 2A: MYCN non-amplified case with 2 green signals denoting MYCN gene copy/ Nucleus and 2 red signals denoting CEP- 2/ Nucleus (1000X) Fig 2B: MYCN amplified case with 6-8 green signals denoting MYCN gene copy/ Nucleus and 2 red signals denoting CEP- 2/ Nucleus (1000X) Figure 3: MLPA data analyzed on coffalyser for probe mix P252. Fig 1: Neuroblastoma 2004 risk stratification 3A However there are a group of cases which are non MNA (MYCN non amplified) with poor prognosis. Spontaneous regression is often seen in localized infantile NB without MNA, however there has been incidences of local progression and progression to stage 4.2 It is essential to study the molecular profile of these group of NB which will help in better management of the non-MNA cases. In this study MLPA was used for molecular profiling of non-MNA NB and was correlated with the clinical findings. MLPA P251 P252 P253 1p 1q 3p 3q 11p 11q 2p 2q 17p 17q 4p 4q 7p 7q 9p 9q Conclusions 3B Aims 17q gains was found to be associated with the aggressive behavior with poor clinical out come in MYCN nonamplified NB. ALK gene gains was also correlated with the progressive disease. Segmental Chromosomal Aberrations observed in MYCN non amplified NB emphasize the need for genetic profiling of all the NBs irrespective of their MYCN status. MLPA analysis is an important and effective tool for molecular characterization of NB for prognostic and therapeutic implications To characterize the genetic abnormalities in MYCN non amplified (non- MNA) NBs Correlation of the genetic abnormalities of non-MNA NB with clinical and histological parameters. MLPA products were run on ABI 3500 Genetic Analyzer. Data analysis was performed using Coffalyser Software. Segmental Chromosomal Aberrations were reported as per SIOPEN Guidelines (as indicated in Table no.1). Materials & Methods 3C Table 1: Relation Dosage Quotient and Copy number (based on normal status of 2 copies) Clinical and histological review The present study was performed on formalin fixed paraffin embedded tissues of diagnostically confirmed Neuroblastoma cases (n=32) at Tata Memorial Hospital for MYCN FISH and MLPA analysis .Reactive lymph nodes (n=6) were used as reference samples. Histological evaluation, staging of the disease was performed as per International Neuroblastoma Pathology Classification (INPC) and International Neuroblastoma Risk Group (INRG) guidelines. Clinical details like age, treatment, stage of the disease, progression free survival, response to chemotherapy was retrieved from the electronic medical records of the hospital. References Probe Ratio( Dosage Quotient DQ) Copy Number status DQ = 0 0 copies (Homozygous deletion) 0.40<DQ<0.65 2 –1 copy (heterozygous deletion) 0.80<DQ<1.20 Normal (Identical to reference sample) 1.30<DQ<1.65 2—3 Copies (heterozygous duplication) 1.75<DQ<2.15 2—4 copies (or 1– 2 copies) All other values Ambiguous results Combaret V, Bréjon S et al.,Determination of 17q gain in patients with neuroblastoma by analysis of circulating DNA. Pediatr Blood Cancer. 2011 May;56(5):757-61. Ambros PF, Ambros I et al., International consensus for neuroblastoma molecular diagnostics: report from the International Neuroblastoma Risk Group (INRG) Biology Committee. Br J Cancer. 2009 May 5;100(9):1471-82. Ambros IM, Brunner B., A multilocus technique for risk evaluation of patients with neuroblastoma. Clin Cancer Res. 2011 Feb 15;17(4):792-804. Legends: 3A: Electropherogram of the sample for P252 probe mix. 3B. Sample Ratio chart for P252. 3C. Probe target details in P252