NEW YORK (GenomeWeb News) – In a paper scheduled to appear online this week in the Proceedings of the National Academy of Sciences, an international group of researchers led by investigators at the National Cancer Institute combined genome-wide copy number analysis with gene expression profiling to characterize hundreds of diffuse large B-cell lymphoma samples.
The researchers identified hundreds of chromosomal rearrangements affecting gene expression. Dozens of these rearrangements were particularly common to specific diffuse large B-cell lymphoma (DLBCL) subtypes, consistent with the notion that each of the three subtypes identified so far arises via distinct pathways and represents unique diseases. The work also holds promise for improving lymphoma prognosis, in some cases, since certain copy number changes were linked to different survival times.
“Together, these data provide genetic evidence that the DLBCL subtypes are distinct diseases that use different oncogenic pathways,” senior author Louis Staudt, a senior investigator in the metabolism branch at the National Cancer Institute, and his colleagues wrote. “[W]e identified chromosomal aberrations that were significantly more frequent in a particular DLBCL subtype than in others, and some of these aberrations were associated with clinical outcomes.”
In the past, gene expression profiles have been used to define three molecular subtypes of DLBCL, a cancer affecting the B lymphocytes — immune cells that produce antibodies. Some 40 percent to 50 percent of DLBCL, the most common form of non-Hodgkin’s lymphoma, can be cured with anthracycline-based chemotherapy, though post-chemotherapy survival rates vary by subtype.
Based on previous studies, the researchers suspected that they could uncover new cancer-related genes and pathways in the DLBCL subtypes by simultaneously determining the genome-wide alterations in copy number and gene expression associated with each. They used array-based comparative genomic hybridization to look for copy number changes in 203 DLBCL biopsy samples.
They also gauged gene expression in the same samples using Affymetrix U133 plus 2.0 arrays and developed a statistical algorithm to integrate the copy number and gene expression data.
The team detected 719 chromosomal changes that affected gene expression — 272 of these were recurrent, turning up in eight or more samples. When they focused on specific DLBCL subtypes to decrease the odds of encountering common copy number polymorphisms, the researchers identified 30 copy number changes that corresponded more closely to one of the three subtypes.
For instance, a quarter of the activated B-cell-like or ABC subtype samples contained a chromosome 19 amplification. This rearrangement — which was verified by quantitative PCR — was present in only about three percent of samples from the other two DLBCL subtypes. The ABC DLBCL subtype was also associated with other chromosomal abnormalities (deletions in the INK4A/ARF tumor suppressor locus, trisomy 3, and so on) that were less common in or absent from the other subtypes.
Using this new information, the team started unraveling the molecular pathways that were associated with each of the three DLBCL subtypes, highlighting suspicious rearrangements and expression changes. For example, they identified several instances in which copy number and gene expression changes affected genes coding for tumor suppressors, potential oncogenes, and transcription factors.
“Key to our analysis was the ability to correlate gene expression with copy number changes globally, allowing us to identify putative oncogenes and tumor suppressors affected by a change in copy number,” Staudt and his co-authors wrote.
And by recognizing these new pathways, the team was able to begin testing hypotheses in the cancer subtypes using RNA interference. For example, by knocking down a transcription factor called SPIB that was differentially expressed in the ABC DLBCL subtype, the researchers could kill three different ABC cell lines. In contrast, they saw little or no effect when they knocked down SPIB in cells from the other two DLBCL subtypes or in multiple myeloma cells.
Staudt and his team also identified eight copy number alterations that were significantly associated with the survival of those with DLBCL. In the ABC DLBCL subtype, trisomy 3 and INK4A/ARF deletions were both associated with overall survival time. Whereas the five-year survival rate was nearly 50 percent for ABC DLBCL individuals without the two rearrangements, it dropped to just nine percent for individuals with one or both.
“The broad gene expression signatures that distinguish these subtypes indicate that they probably arise from B cells at different stages of differentiation,” the authors wrote. And, they added, their research moves the field closer to understanding the pathways, pathogenesis, and prognoses for each. “As we integrate various forms of genomic analyses, the complexity of human cancer is finally coming into molecular clarity.”
At press time, the paper was not yet available on the PNAS website.