Canine Lymphoma --- The Prognostic Significance of Cytogenetic Changes.

摘要

Canine lymphoma accounts for nearly one quarter of all cancers in dogs. Although it is treatable with chemotherapy, as is true for NHL in humans, additional predictive factors are needed to generate more refined schemes that can offer reliable prognostic information. In recent years, cytogenetic evaluation has been used increasingly in the diagnosis and assessment of human NHLs. To determine the prognostic value of chromosomal aberrations identified in canine lymphoma, in Chapter 2 we analyzed 160 archival lymphoma specimens derived from a clinical trial, using interphase fluorescence in situ hybridization (FISH) analysis with genome integrated bacterial artificial chromosome contigs as probes for targeting nine recurrent chromosomal copy number aberrations. By the splitting-sample approach, the data obtained from 121 cases using regression analysis indicate that copy number aberration in one of the nine loci evaluated is significantly associated with diseasefree intervals of dog patients with lymphoma. A validation cohort of 39 cases was used to confirm the findings.;To provide reliable data in our subsequent gene expression analysis, required identification and validation of suitable canine reference genes. In Chapter 3, we used array comparative genomic hybridization (aCGH) data to guide the selection of new candidate reference genes and applied these genes to different tumor types, including canine lymphoma, histiocytic sarcoma and osteosarcoma. The stability of candidate reference genes and four conventional reference genes was accessed by three different programs. LOC611555 was identified as the most stable reference gene across the three tumor types. Comparison of gene stability between newly identified candidate genes and previously used housekeeping genes in multiple tumors, demonstrated a greater level of stability of the proposed new reference genes. In addition, our analysis also provided the most suitable combination of reference genes for multiple genes normalization for each type of tumor.;Moreover, to identify potential target genes located in these recurring aberrations identified in canine lymphoma, 11 genes were selected for further study based on their known tumor-associated roles and history of deregulation in human lymphomas. We characterized the gene expression patterns of these 11 tumor-associated genes in canine lymphomas and found that MYC was upregulated in lymphoma samples, whereas MEOX-2 and KIT were downregulated. We observed that KIT was highly expressed in several T-cell lymphoma cases, which suggests that these cases may be potential targets for KIT-specific treatment using tyrosine kinase inhibitors. Furthermore, by combining data of gene expression with cytogenetic changes, three gene-dosage regulated candidates were identified, including CDKN2A, TSC2 and STAT1. Deregulation of these genes may interfere with cell cycle control and signaling pathways that are involved in the pathogenesis of canine lymphoma.;MEOX-2 is a transcription factor whose involvement in many aspects of vascular cellular processes has been recognized, in particular as a regulator controlling the angiogenic transition in vascular endothelial cells. Due to the impact of MEOX-2 in development and cellular behavior, we were interested in investigating its expression pattern in tumors. A preliminary study in Chapter 4 showed that MEOX-2 expression was downregulated in all studied lymphoma cases, with no corresponding genetic deletion detected. In Chapter 5, we present evidence supporting our previous findings and further demonstrate that the CpG island of MEOX-2 was hypermethylated in all studied lymphoma cases, but not in normal lymph nodes. These findings suggest that MEOX-2 expression might be silenced by DNA methylation. Further studies are necessary to elucidate the significance of loss of MEOX-2 expression in canine lymphoma. The tumor-specific methylation pattern of MEOX-2 may have potential use as an epigenetic marker for molecular diagnosis of canine lymphoma.