Giovanni Ciriello: Good morning.
First of all, I want to thank the organizers for giving me the opportunity to present this
story today.
This story started on the latest phases of a TCGA breast cancer project, and it aimed
at exploring and trying to explain the diversity of a certain subset of breast tumors, and
I'm talking about luminal tumors, and in particular, luminal A.
As most of you know, breast cancer is an incredibly heterogeneous disease.
It can actually be thought of as a collection of distinct diseases.
And these diseases are mostly characterized by the status of hormone receptors ER and
PR and growth factors receptor HER2.
As you can see from the cartoon here, the largest fraction of breast tumors are positive
for the estrogen and progesterone receptor and negative for HER2.
And this definition largely identify what we call luminal breast cancer.
Luminal tumors have been further stratified using mRNA signature into luminal A and luminal
B, with luminal A being less aggressive, generally associated with a better prognosis, and overall,
the most frequently occurring breast cancer subtype in the population.
However, the great diversity and abundance of genomic data that has been recently become
available allowed us to do more in-depth, specific analysis on this subtype and identify
a diversity -- a great diversity -- yet to be explained.
So what we did was integrating multiple datasets from different studies, accounting for roughly
1,500 luminal tumors, 1,000 of which are luminal A. And from this dataset we have different
data type, but for almost all of them, we have copy number alterations, somatic mutation,
and mRNA expression.
So the first thing what we do was, we went comparing the well-established mRNA-driven
breast cancer subtype against copy number driven cluster identified in different dataset,
and I'm showing here the metabolic dataset and the TCGA one.
And what was quite interesting is that while basal and HER2 enriched breast tumors there
is an almost perfect one-to-one correspondence between these clusters.
Both luminal As and luminal B are associated with multiple and distinct copy number cluster
suggesting an intrinsic heterogeneity in terms of copy number alteration.
And a similar picture emerged when we went looking at somatic mutation.
Indeed, again, HER2 and basal are characterized by the highest number of mutation per sample
on average, indicated here by the blue bars, while luminal B, and especially luminal A,
I would say, are characterized by the highest number of genes mutated more frequently than
expected by chance, and these are indicated here by the red bars.
So, this tell us that these subtypes have a greater heterogeneity at the molecular level.
But this heterogeneity is not only molecular; indeed, luminal A is quite as heterogeneous
also in clinics.
Indeed, while it has the highest median overall survival, it also has the most variable one,
and you can see here from the box plot.
And it has also been shown that the risk of late mortality persists in this group even
after 10 years of initial diagnosis, and it is actually greater in these subtypes than
in the others in the long-term.
So, given this overall preliminary observation, we decided to focus specifically on this subtype
of breast cancer, the luminal A breast cancer, as it looks like the most heterogeneous, both
molecularly and clinically.
And we wanted to address some fundamental question, like can we link the variability
of certain clinical outcomes with the underlying molecular diversity.
And we started to address this question by looking at copy number alteration.
We went back to the TCGA dataset and we cluster luminal A tumors according to our copy number
levels across the whole genome for these patients.
And as you can see just by eye-balling this heat map, the diversity is pretty striking.
We found five major sub-groups, some of which are characterized by low-level or no copy
number alteration, while other has -- like this one in red here -- are characterized
by an abnormally high level of copy number alteration, and especially focal deletion
and amplification.
So we took the centroid of this cluster and we wanted to see if we could validate these
results in additional dataset.
And first we validate them in the metabolic dataset where we found similar clusters in
similar proportions, and especially in this dataset we had extensive follow-ups, so we
could do more reliable survival analysis, and we saw that the copy number high -- or
high genomic instable cluster is associated with the worst prognosis.
And I'm showing here the Kaplan-Meier for this -- specifically for this cluster in this
metabolic dataset here, and we went validating this finding on a third dataset where we found
a similar proportion, around 10 percent, of luminal A that have these genomic features
and are associated, again, with the worst prognosis.
So we were pretty excited because we started linking this molecular heterogeneity with
the clinical variability.
However, we ask ourselves if we could pinpoint specific genomic features that are enriched
in this subtype.
So I went comparing the copy number high luminal A tumors against all the other luminal As,
and what we found is that these tumors are highly enriched for P53 mutation, MYC focal
amplification, 20q gain, 5q loss, and are depleted for PIK3CA mutation but otherwise
characterize most of luminal A tumors.
We then look at genes with a differentially expressed between these two subgroups, and
we found that copy number high luminal As overexpress several regulator sub-mitosis
including both aurora kinases A and B, PIK1, CDK1, and several cyclins.
And they down regulate genes involved in DNA repair pathway, like P53 binding protein one,
MLH3, and several polymerases.
So all these features clearly point to a high proliferation, high genomic instability, and
well explain a match with phenotype that we observed in terms of overall survival.
At this point we wanted to see if we could have a similar comprehensive picture for the
other luminal A subtype.
We went looking first at somatic mutation, and we saw that the most frequently occurring
mutation in luminal A tumors -- and I'm referring here to PIK3CA and GATA3 in particular -- are
enriched in tumors that have low level of copy number alteration.
And, in particular, GATA3 is highly recurrent in tumors that present with 1q gain, 16q loss
pattern but characterize roughly 30 percent of luminal A tumors.
And this arrangement is even stronger when we looked specifically at hotspot mutation
in PIK3CA and GATA3.
We also saw that there is a strong association between MAP3K1 mutation and [unintelligible]
on chromosome eight than chromosome 16, but characterized a cluster here in the middle
that we call the chromosome eight-associated clusters.
As you may have noticed, several of these genes participate in similar signaling cascade,
so the obvious question was, which pathways are most deregulated in luminal A tumors and
in the luminal A subtypes.
We did a partial analysis using MEMo.
MEMo is a model that look for micro-pathways characterized by genomic alteration that occurs
in a mutually exclusive way.
I indicated here the link; the model is available at our website for download and use.
The first set of results of MEMo nicely confirmed what we already observed in the bigger breast
cancer dataset, which charts multiple genomic alteration, targeting AKT, microkinase, and
ROS/ERK signaling.
What we didn't find then and we could find now by focusing specifically on a single subtype,
was a set of multiple rare but mutually exclusive alteration affecting components of co-repressor
complexes, NCor, and SMRT.
You can see here several mutation, this time to be inactivating mutation, and generally
correlate with down regulation of the co-respective genes.
But why are co-repressor complexes relevant or important in ER-positive luminal tumors?
Well, in these tumors, proliferation is mostly driven by the estrogen receptor, and ER activity
can be blocked therapeutically using an estrogen antagonist called Tamoxifen, which recruits
co-repressor complexes to block ER transcriptional program.
However, it has been shown in breast cancer cells that lots of co-repressor complexes
may actually turn Tamoxifen into a promoter of ER-driven proliferation by recruiting co-activated
complexes instead.
So in this scenario, what we have now is a set of genomic alteration, where there -- which
are directly linked to loss of co-repressor complexes and may therefore predict lack of
response to endocrine therapy.
So, in conclusion, we dissected the genomics of luminal A tumors in multiple data-set to
explain the molecular and clinical heterogeneity.
We identified five major subtypes, actually, of luminal A tumors, which are characterized
by distinct copy number alteration and somatic mutation.
And, in particular, we found an atypical luminal A subtype characterized by high genomic instability,
P53 mutation, aurora kinases up-regulation, and associated with a poor prognosis.
Furthermore, we show you that luminal A hallmark mutation, specifically PIK3CA and GATA3, are
prevalent in tumors characterized by low level of copy number alteration, and we also identified,
by partial analysis, multiple rare but mutually exclusive alteration associated with loss
of co-repressor complexes.
And these alteration, as I just told you, are important as they may predict lack of
response to Tamoxifen treatment.
And with this, I'd like to conclude, and thanks all the people that collaborate -- that contributed
and are still contributing to this project.
And, in particular, Chris, Niki, and Rileen at cBio; and Chuck and Katie at UNC; and,
of course, the whole TCGA Breast Cancer Analysis Working Group for all of the analysis done
during the breast cancer project.
Thank you.
[applause]
Charles Perou: Any questions?
I, of course, always have one.
I wonder if we could look at those co-repressor complexes in the Wash U data from their paper
where they had endocrine responsiveness.
Did we look in that, did we see any of those?
Giovanni Ciriello: I didn't look at the response, I looked at
the mutations.
And.
yeah, and the --
Male speaker: Yeah I know those were rare mutations, probably
a couple in there.
Giovanni Ciriello: -- in the finger paint I show the only mutation
occurring with the TCGA dataset, but we found a mutation in these components, for sure NCor1.
I think one mutation in NCor2 and GPS2, also in their dataset.
So it's something...
Male speaker: We should look.
Giovanni Ciriello: And one thing that I didn't say is that these
mutations occur almost exclusively in luminal A tumors, so are highly enriched in this subtype.
Female Speaker: This is Angela from Harvard.
Do you see any deletions in the ER receptor?
Giovanni Ciriello: Deletion?
Female Speaker: Yes.
Giovanni Ciriello: A recurrent deletion -- this subgroup are
characterized by wide events [spelled phonetically].
Several, like -- wide deletion typically occurring in 16q if I remember correctly, 8p, and...
Female Speaker: But you didn't see any focal deletions in
that region?
Charles Perou: You mean deletions of the ER itself?
Giovanni Ciriello: Oh, of the ER itself?
No, not that I --
Charles Perou: I don't think so.
Giovanni Ciriello: Not off the top of my head.
I may have to double check it.
Charles Perou: All right.
Thank you, Giovanni.
So our next speaker --