Characterization of Chromosomal Aberrations in Neuroblastoma Formalin-Fixed Paraffin-Embedded Specimens with Standard Arraycgh Procedure-Preliminary Experience

Neuroblastoma (NB) is one of the most common pediatric cancer (6% to 10%) [1]. Approximately 90% of patients are diagnosed before the age of 5 years old [2,3]. The primary form of NB is an extracranial solid tumor arisen from neural crest progenitor cells [1-4]. NB tumors have a very high tendency to form distant metastases [2]. The comprehensive characterization of tumor genetic and biological features is obligatory for NB patients [4]. These features determine the risk stratification and therapeutic decisions in treatment. The most unfavorable genetic factor in NB is MYCN oncogene amplification [2-7]. Moreover, tumor aggressiveness and progression is strongly associated with Segmental Chromosomal Aberrations (SCAs) observed in malignant cells [2,5,8]. Aggressive metastatic tumors are characterized by a high number of SCAs and a low number of Numerical Changes (NCAs). On the whole, genome-wide studies have demonstrated that critical chromosomal damages occur more frequently in older patients and that SCAs accumulate in an age-dependent manner, as supported by Schleiermacher and Coco [8-12].


Introduction
Neuroblastoma (NB) is one of the most common pediatric cancer (6% to 10%) [1]. Approximately 90% of patients are diagnosed before the age of 5 years old [2,3]. The primary form of NB is an extracranial solid tumor arisen from neural crest progenitor cells [1][2][3][4]. NB tumors have a very high tendency to form distant metastases [2]. The comprehensive characterization of tumor genetic and biological features is obligatory for NB patients [4]. These features determine the risk stratification and therapeutic decisions in treatment. The most unfavorable genetic factor in NB is MYCN oncogene amplification [2][3][4][5][6][7]. Moreover, tumor aggressiveness and progression is strongly associated with Segmental Chromosomal Aberrations (SCAs) observed in malignant cells [2,5,8]. Aggressive metastatic tumors are characterized by a high number of SCAs and a low number of Numerical Changes (NCAs). On the whole, genome-wide studies have demonstrated that critical chromosomal damages occur more frequently in older patients and that SCAs accumulate in an age-dependent manner, as supported by Schleiermacher and Coco [8][9][10][11][12].
According to the international guidelines the current gold standard for detecting MYCN oncogene amplification in NB tumor cells is the fluorescence in situ hybridization (FISH) analysis of interphase nuclei [5]. Other, critical copy number changes like 11q23 deletion and 17q gain can also be evaluated by FISH analysis [5, [13][14][15][16][17]. However, pan-genomic techniques like highresolution array-based comparative genomic hybridization (aCGH) or multiplex Ligation-Dependent Probe Amplification (MLPA) provide alternative methods for whole-genome screening and characterizing SCAs and NCAs in NB tumor cells [18][19][20][21]. These techniques are equivalent to thousands of FISH The main purpose of this study is evaluation the practical potential of routine use aCGH platform for detection of numerical and structural chromosomal changes in FFPE-derived DNA samples of NB primary tumor tissues.

Results
The aCGH evaluation was performed for 8 FFPE specimens. It was achieved a very good quality of genomic DNA with the described isolation kit. Moreover, there were no obstacles to achieving the required DNA quantity.
Abnormalities in the form of numerous NCAs were detected in 5 out of 8 cases. These tumors were identified as hyperdiploid. The most common NCAs were multiplication of whole chromosomes: 6,7,8,9,17,20,22. In 2 cases we have found also loss of whole chromosomes ( Table 1). All patients with the NCAs-positive aCGH profile were younger than 18 months.
Both, SCAs and NCAs were found in 2 out of 8 cases (Figure 1). These patients were older than 18 months, what was an unfavorable factor. Among SCAs we have found critical changes like 1p36 loss and 17q gain. The size of above structural abnormalities ranged from 5.8 Mb to 49.5 Mb. It was not an incidence of MYCN amplification (Table 1). One patient from the research group had a normal diploid aCGH profile, and was over 18-months-old.

Discussion
There is a well-known association between patient outcome and tumor ploidy in NB [2][3][4][5]. NB tumors with diploid DNA content are more likely to present in older children and with metastatic disease. These diploid tumor's cells are more likely to harbor MYCN gene amplification or other SCAs. In contrast, neartriploid DNA content is most commonly found in infant tumors that remain localized and are associated with more favorable outcome [2][3][4][5][6][7][8][9]12].
The most common type of SCAs in NB cells beside MYCN amplification is gain of the distal part of long arm of chromosome 17 which occurs in at least 50% of primary NB tumors. This abnormality is associated with poor prognosis, and is frequently coexisted with other biomarkers of aggressive disease such as MYCN amplification, older age, and chromosome 1p36 deletion. Loss of 1p36 region occurs in approximately a quarter of NB tumors and also predicted adverse outcome [2,3,7,11,13]. In addition, a recent INRG report on non-MYCN amplified tumors determined that it is not single genetic markers, but the overall segmental genomic profile of tumors that adds information to patient prognosis [11].

Conclusion
Whole-genome DNA copy number analyses have demonstrated that the overall genomic pattern adds significant prognostic information to clinical algorithms. Integration of molecular profiles and genomic markers that are reflective of tumor biology into future classification systems will allow more precise prognostication and refined treatment assignment.
Microarray profile for NB tumor. a) Segmental and numerical changes; b) Zoom in deletion of chromosome 1p36. Figure 1 Archival samples like FFPE specimens are an excellent source of DNA for aCGH processing with the advantage of simple handling, distribution and storage of tumors tissue. Moreover, aCGH analysis is cost-effective technology gives an opportunity to comprehensive characterization of neoplastic genome.
In conclusion, we believe that molecular karyotyping with use of standard-resolution cytogenetic microarrays can be recommended as a valuable method for detection of chromosomal aberrations in archival fixed samples of NB and other solid tumors.