Angeliki Pantazi: Hi, everyone.
So, today I’m going to present you some of the results from the data and the analysis
that Harvard CCC has done for various tumor types.
[inaudible commentary[
Male Speaker: Yes.
Angeliki Pantazi: So I’ll start with an overview of the data
that we have generated and our pipeline.
So, so far we have sequenced 14 -- subsets of 14 TCGA tumor types.
We have performed paired and whole-genome sequencing on coverage -- on a full coverage
of six to eight.
And this was done not only for -- to this end, but primarily to detect structure variants.
So, for our analysis, we used two tools: BreakDancer, which detects discordant read pairs, and also
Meerkat, which keeps the advantage of detecting the split read that spans the [unintelligible]
breakpoint.
So, for this analysis, I included data on 12 tumor types listed here.
In total, there were 1,200 tumors and their matched normal samples, either blood or tissue,
that were used.
In the majority of these tumor types, we had at least hundred samples in each case, and
for three, around 50 sample pairs.
So, here’s an overview of the distribution of all the sequence samples.
Regarding the number of structure variants that were detected, in total, we detected
almost 37,000 structural rearrangements, out of which something like 12,000 events were
gene-gene, meaning they had both breakpoints within gene region.
So, the most rearranged tumors were the gastroesophageal, bladder, and melanoma.
And the more quiet, as expected, was the thyroid.
So, we tried to test the specificity of our pipeline.
And we used different approaches.
For example, we gathered the events that were called by both BreakDancer and Meerkat -- so
they -- we could have more confidence in those; or events that they could also be cross-confirmed
with RNA-Seq data.
But ultimately, we did also lots of bench work with PCR amplifying of the junction fragment
with patient-specific primers.
So, we were able to cross-confirm or validate something like almost 80 percent of the events
that we detected.
So, then, in -- for this analysis, I included data on rearrangements that have to do with
the RAS pathway.
And I chose to do so because of the significance of this pathway.
We know that there are so many tumor types that have RAS aberrations, some to a high
extent like pancreas cancer; but practically almost in all TCGA working groups, we hear
about RAS-RAF aberrations that activate the RAS-MAP kinase pathway in these solid tumors.
So, we wanted to examine cases that still have RAS activation, but other RAS DNA [unintelligible]
are not obvious.
And we focused on these 12 tumor types that I’ve shown you.
And we’ve seen that almost 200 samples of what -- the cases of what we have sequenced
had at least one somatic gene-gene RAS rearrangement.
So, then, the question was, what was the relationship of these RAS rearrangement that we see in
comparison to other RAS aberrations?
But before coming to that, just to how we define the RAS pathway.
So, we heard yesterday about the needs to compile all the given, established knowledge
on certain cancer pathways.
Till we -- this goal has been reached, we are using the RAS pathway that NCI’s RAS
Initiative has put together.
And in red, there are the genes that comprise structural rearrangement in our data analysis.
So, that includes not only RTKs that draw most of the attention, but also genes in the
SOS-RAS-RAF axis, and also genes upstream of RAS that either inhibit, like NF1, or activate
RAS.
So, we then compared these cases with RAS rearrangement to cases we’ve -- other RAS
DNA aberrations.
Sorry.
And before coming to that, I’ll just show you some examples of the events that we’re
seeing.
So here you can see some cases of genes that are well known, like BRAF or RAF1, with structural
rearrangement, but also other interesting cases upstream of RAS, including disruption
of tumor suppressant genes like NF1.
So, at this panel, you can see with arrows the breakpoints in each gene, and then, in
red and blue color, expression data [unintelligible] course.
So, now I’m coming to the -- to the -- to the -- to the question whether the RAS rearrangements
[unintelligible] with other RAS somatic aberrations.
So, this is -- this subject has already been touched by another speaker yesterday, saying
that it seems that aberrations within one single cancer pathway seems to be mutually
exclusive across different cases.
And that’s what we also observe here, if we compare the RAS structural rearrangement
with RAS-RAF mutation or amplification.
So, we see that the RAS rearrangements are mutually exclusive to most of these somatic
aberrations.
We also heard about how structural variations can affect in different ways transcription.
Also, here’s some examples with structural rearrangements that affect practically the
3‘UTR in some well-known oncogenes like FGFR3 and FGFR2.
So, here in this slide I’ve got an example of a [unintelligible] location in a low-grade
glioma patient, where there is a fusion of FGFR3 with ELAVL3 gene.
So, the interesting and -- the interesting part, and the beauty of the DNA-based analysis,
is that you can actually see the original change that occurred in the genome and that
resulted later in the fusion transcript that can be oncogenic.
So, on the RNA level, we see the fusion of Exon 17 to -- of FGFR3 to Exon 2 of ELAVL3
gene.
But the actual genomic breakpoint lies within the 3‘UTR of FGFR3.
And due to alternative splicing, the final fusion transcript is missing its 3‘UTR.
So, now, there is already experimental data for other gene fusions like FGFR3-TACC3 and
how these are oncogenic because of the loss of microRNA control over FGFR3.
So, that could be, assumingly, a similar way through which this rearrangement and fusion
are oncogenic.
In a similar way, we detected an inversion in a gastric case, where FGFR2 is linked to
WDR11 gene.
So, in this case, the genomic breakpoint is in the last exon of FGFR2.
And interestingly, there is a new sub-codon within Exon 17.
And finally, FGFR2 is losing its 3‘UTR and acquires a new 3‘UTR practically, part of
which is Intron 17 and part of which is a fragment of the 3‘UTR of WDR11.
So, assumingly, this FGFR2 is also lacking control of microRNAs on its original 3‘UTR.
However, that case is -- would require experimental validation to show that there are oncogenic.
And we generally felt this was very important for all of the events that we detected.
So, that’s why we initiated a collaboration to try to functionally validate some of the
RAS rearrangements that we detected.
And for that, we’re current collaborating with Henry Lu and Kenneth Scott from Baylor
Institute.
And that group provides a very interesting and robust experimental pipeline to -- that
can validate mostly rearrangements that result into a fusion transcript that is oncogenic
and that drives cell proliferation.
So, this system takes advantage of a large set of gateway ORF cDNA clones, from which
it is possibly to PCR amplify the part of the gene that is needed.
And then, through a multi-site [unintelligible] recombination, they can be -- the final fusion
gene can be cloned into the destination vector and finally the expression vector.
So, these expression vectors are being transfected to Ba/F3 cell line, a mouse cell line that
is normally dependent on Interleukin-3.
So, once the cells are deprived from Interleukin-3, then the transformed cells can be selected.
And the oncogenic protein -- fusion protein can drive cell proliferation, which can be
measured by a luminescent cell viability assay.
So, some of our RAS rearrangements are already part of this pipeline.
So, I’ll show you some results for the TRAK1-RAF1 rearrangement, which occurs through an inversion
in Chromosome 3 in a melanoma patient.
So, both in the whole-genome sequencing data and in the RNAseq, we can see both products
of this rearrangement, meaning TRAK1-RAF1 and its reciprocal partner, RAF1-TRAK1.
However, only the TRAK1-RAF1, which, after all, contains the kinase domain, scores higher
in the cell viability assay comparable to the BCR-ABL fusion, and not the RAF1-TRAK1.
So, the TRAK1-RAF1 can promote cell proliferation in our cell system.
And furthermore, focusing on the RAS pathway and its activation, it does activate a RAS
pathway.
And here I’m showing you the increase of level of phosphorylated ERK in the transformed
cells.
Henry and Kenny also performed an assessment about drug sensitivity of the transformed
cells, and they were able to show that TRAK1-RAF1 cells do so -- are sensitive to the MEK inhibitor
Trametinib.
So to summarize, RAS mutations are found generally at a high frequency in many different cancers.
And we just propose that structural rearrangements also are a mechanism that alternatively can
activate the pathway.
So, in order to have the full view of the RAS activation across different tumors, we
could take them into consideration.
We’ve shown that RAS rearrangement are largely mutually exclusive to other RAS aberrations.
And we’re already putting our data in functional studies to show which events are functional
and which events can be prioritized.
So, this is a need for all data that come out from studies like the TCGA.
This need will be growing in the future, the need to functionally show that our high-quality
data have impact on the patients.
So, you can join me also at poster number 59 with more questions.
Thank you.
[applause]
Male Speaker: I have just a comment to make.
I really would advise some caution towards the thinking that the primary function of
this fusion is simply to eliminate 3-primum regulatory regions.
And there are several reason for this, certainly the most important one being that the simple
over-expression of the RTK gene in the case of FGFR is of a much less value in terms of
oncogenic role than the expression of the fusion.
What we typically see is that, in fact, it’s the fusion gene which is extremely transforming
the fusion protein and not the expression of the individual partner.
On the other hand, the 3-primum gene is clearly very important in providing multiple oncogenic
activities, such as [unintelligible], as well as aberrant activation of downstream signaling.
I mean, the idea that there is a simple activation of the canonical RTK pathway here, most of
the times, I should say, is not correct.
So, these fusions clearly have [unintelligible] functions most of the times, and the elimination
of regulatory regions is probably just an incident of the genetic rearrangement.
Angeliki Pantazi: Thank you for this input.
Unfortunately, the functional system that I’ve shown is not appropriate to test this.
However, I think the -- we should test more about what’s the functional impact and on
which cancer phenotype they’re actually contributing.
Female Speaker: Thank you, Angeliki.
Our next speaker is Juliann Shih.
She’s going to talk about somatic copy number alteration in uveal melanoma.
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