Chromosomal aberrations can be studied using many different techniques, such as Comparative Genomic Hybridization (CGH), Fluorescence in Situ Hybridization (FISH), and Representational Difference Analysis (RDA). Although chromosome CGH quickly became a standard method for cytogenetic studies, technical limitations restrict its usefulness as a comprehensive screening tool. Recently, the resolution of Comparative Genomic Hybridizations has been greatly improved using microarray technology. Substitution of the chromosome targets by a matrix consisting of an ordered set of defined nucleic acid target sequences greatly enhances the resolution and simplifies the analysis procedure, both of which are prerequisites for a broad application of CGH as a research and diagnostic tool. Array CGH has provided significant contributions to our understanding of chromosomal changes associated with tumor development and progression, making it possible to detect 40–80 kb regions of chromosomal gain and loss covering the entire human genome in a single experiment .
The genetic alterations involved in the pathogenesis of HL are still largely unknown. HRS cells in most if not all cases show numerical chromosome aberrations, as well as amplifications and deletions of chromosomal sub-regions [29, 30]. Several recurrent chromosomal alterations have been previously described but for most of these, identification of the relevant genes is still pending [29, 31]. Recently, Kluiver et al. performed serial analysis of gene expression (SAGE) and array-based comparative genomic hybridization (aCGH) to identify genes involved in the pathogenesis of classical Hodgkin lymphoma (cHL) . The comparison of SAGE libraries of cHL cell lines L428 and L1236 with those of germinal centre B cells revealed consistent overexpression of only 14 genes. In contrast, 141 genes were downregulated in both cHL cell lines, including many B cell and HLA genes and aCGH revealed gain of 2p, 7p, 9p, 11q and Xq and loss of 4q and 11q .
We studied tumor cell lines derived from HL and ALCL rather than primary lymphoma samples for the following reasons. Primary lymphomas are heterogeneous, with varying components such as infiltrating lymphocytes, invading blood vessels, and other stromal components which contribute to the extracted DNA and RNA and thus mask the signature of the neoplastic cells. The use of cell lines avoids the problem of having heterogeneous populations of cells complicating the analysis of the hybridization signals. Moreover, most of these cell lines preserve the phenotypic and differentiation related characteristics of lymphoma. Array-based-CGH was used to screen HL-derived cell lines (KMH2 and L428) and ALCL cell lines (DEL and SR-786) to identify chromosomal region gains and losses and gene copy number alterations that may reveal genes involved in the pathogenesis of HL and ALCL. Gene copy number gains and losses were observed on at least 12 chromosomes in all four cell lines investigated in this study. Assessment of copy number alterations with 26,819 DNA segments identified an average of 20 genetic alterations. These alterations defined 9 (45%) novel abnormal regions not previously reported in the literature. These novel regions may require further investigation in normal and tumor samples to eliminate the possibility of copy number variations within the normal human population. Of the recurrent MARs identified, 11 (55%) were identical to those determined in previously published studies of genetic alterations using lower resolution CGH methods than the sub-megabase resolution array-based CGH used in the current study.
The alterations in the main pathways responsible for controlling the cell cycle in HL have rarely been studied and poorly understood, mainly because the techniques used for molecular studies in this disease have been limited by the scarcity of malignant cells of interest . The analysis of cell-cycle regulation in different types of lymphoid and epithelial neoplasms reveals a relationship whereby increased clinical aggressiveness is associated with the accumulation of genetic and epigenetic alterations . In the current study, 3.6% of the genes reported to be differentially expressed in HL and ALCL cell lines were involved in cell cycle pathways. These include CDK6, PCNA, and ATM.
The mitogen-activated protein kinases (MAPKs) (also called extracellular signal-regulated kinase [ERK]) are a group of protein serine/threonine kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus [35, 36]. Zheng et al.  reported that the active phosphorylated form of MAPK/ERK is aberrantly expressed in cultured and primary HD cells. In the current study, 3.6% of the genes were found to represent the MAPK signaling pathway. These are DDIT3, HSPB1 and MYC. Recently it was reported that Myxoid/round cell liposarcoma (MLS/RCLS) may develop from cell types other than preadipocytes because the fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype when expressed in a primitive sarcoma cell line . The MLS/RCLS oncogene FUS-DDIT3 is the result of a translocation derived gene fusion between the splicing factor FUS and DDIT3. DDIT3 and FUS-DDIT3 show opposing transcriptional regulation of IL8 and suggest that FUS-DDIT3 may affect the synergistic activation of promoters regulated by the CCAAT/enhancer binding protein (C/EBP) beta and NFkappaB .
Certain (2.4%) of the genes were found to be involved in encoding tight junction proteins. These include CLDN4, PARD6G. The claudin (CLDN) genes encode a family of proteins important in tight junction formation and function . Recently, it was reported that CLDN, and CLDN4 are elevated in several epithelial malignancies such as those originating from the pancreas, bladder, thyroid, fallopian tubes, ovary, colon, breast, uterus, and prostate . Surprisingly, our data showed overexpression of CLDN4 in HL and ALCL. The finding of expression of these claudins in other tumors warrant further investigation for CLD4 as well as other claudins in HL and ALCL pathogenesis.
Another 3.6% of the genes were determined to be involved in the Jak/STAT signaling pathway. These include STAT1, JAK2 and MYC. The STAT proteins (Signal Transducers and Activators of Transcription), were identified in the last decade as transcription factors which are critical in mediating virtually all cytokine driven signaling . Several members of the STAT family of transcription factors, namely STAT3, STAT5 and STAT6, are frequently activated in HRS cells [42, 43]. STATs can be activated by cytokine receptors via the JAK kinases, by receptor tyrosine kinases (RTKs) and by 7 transmembrane receptors . In normal cells and in animals, ligand dependent activation of the STATs is a transient process, lasting for several minutes to several hours. In contrast, in many cancerous cell lines and tumors, where growth factor dysregulation is frequently at the heart of cellular transformation, the STAT proteins (in particular Stats 1, 3 and 5) are persistently tyrosine phosphorylated or activated . STATs can be divided into two groups according to their specific functions . One is made up of STAT2, STAT4, and STAT6, which are activated by a small number of cytokines and play a distinct role in the development of T-cells and in IFN signaling . The other group includes STAT1, STAT3, and STAT5, activated in different tissues by means of a series of ligands and involved in IFN signaling, development of the mammary gland, response to GH, and embryogenesis . This latter group of STATS plays an important role in controlling cell-cycle progression and apoptosis and thus contributes to oncogenesis. Although increased expression of STAT1 has been observed in many human neoplasia, this molecule can be considered a potential tumor suppressor, since it plays an important role in growth arrest and in promoting apoptosis . On the other hand, STAT3 and 5 are considered to be oncogenes, since they bring about the activation of cyclin D1, c-Myc, and bcl-xl expression, and are involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis. Recently, Cochet et al. reported that aberrant STAT activation in Hodgkin cells may promote cell survival and, as a consequence, facilitate oncogenic transformation .
Another interesting gene reported in this study is ING3 (inhibitor of growth family, member 3). The overexpression of ING3 was found to correlate with the gene copy number alterations at 7q11.1-q36.3 in HL cell lines (KMH2 and L428) and ALCL cell line (DEL) as shown in Table 1 and 2. The novel ING tumor-suppressor family proteins (ING1-5) have been recognized as the regulators of transcription, cell cycle checkpoints, DNA repair, apoptosis, cellular senescence, angiogenesis, and nuclear phosphoinositide signaling . Using stable clones of melanoma cells overexpressing ING3, Wang et al. showed that overexpression of ING3 significantly promoted UV-induced apoptosis . As there is no information reported in the literature about ING3 in HL and ALCL, further investigation is required to study the role of ING3 in HL and ALCL pathogenesis.
The use of the gene expression profiling of the same four cell lines used for array-based CGH proved to be useful in identifying differentially expressed genes in those chromosomal regions showing gene copy number alterations. A search for the involved genes located in these chromosomal regions can potentially shed light on the molecular pathogenesis of HL and ALCL. Our study found that different genes in the same altered region correspond very differently to gain and loss of genetic material, probably because of transcriptional regulation of each gene. A recent study compared gene expression levels with copy number alternations reported that gain of 2p and 9p did not result in overexpression of the proposed target genes c-REL and JAK2, but gain of two newly identified smallest region of overlap (SROs) on 7p and Xq did correlate with overexpression of FSCN1 and IRAK1 .
As can be seen from above (table 2), some of the genes in this study were previously reported to be involved in pathways of HL and ALCL pathogenesis. On the other hand, we propose that further analysis is required to investigate the role of overexpression of the other deregulated genes we have identified, that were not previously reported to be dysregulated in HL and ALCL.