Hi, I’m Bobbi Pritt.
In this month’s “Hot Topic,” my colleague, Dr. Rondell Graham, provides an overview of
a PRKACA FISH test that assists in differentiating fibrolamellar hepatocellular carcinoma from
other subtypes of primary liver cancer.
I hope you enjoy this month’s Hot Topic, and I want to personally thank you for allowing
Mayo Clinic the opportunity to be a partner in your patient’s health care.
Thank you so much for that very kind introduction.
I’m delighted to be here today to share with you all.
So with regards to this presentation, I have no conflicts of interest to disclose.
As part of this presentation, you’ll see how PRKACA FISH can be used appropriately
to support a diagnosis of fibrolamellar carcinoma, and this is particularly useful in difficult
cases and in small biopsies, and I think you’ll be aware of the importance of the diagnosis
of fibrolamellar carcinoma in the clinical management of patients with this diagnosis.
Primary liver cancers are the only common cancers that have not experienced a reduction
in incidence or an improved prognosis in oncologic care.
The road to improve clinical outcomes will be paved by accurate diagnosis and use of
novel techniques to understand the biology of liver tumors and new means to treat them.
Today, I will be presenting a new fluorescence in situ hybridization assay available from
Mayo Clinic Laboratories, which is useful in the diagnosis of fibrolamellar carcinoma.
Specifically, I will use a case example to demonstrate our approach to the diagnosis
of fibrolamellar carcinoma.
Our index case is that of a 32-year-old woman with a 7.4-cm mass in the right lobe of the
liver.
The mass is multinodular in appearance, and macroscopic portal vein invasion can be seen
in this gross image on the right of the screen.
Please note the absence of cirrhosis, and these are illustrated by the yellow ovals,
the multinodular mass, the portal vein thrombus, and the normal background liver.
Histologically, the lesion is characterized by neoplastic cells with abundant eosinophilic
oncocytic cytoplasm with centrally placed nuclei characterized by open chromatid with
prominent macronucleoli.
The tumor cells are divided into trabecula, which are separated by ribbons or bands of
fibrosis.
On a higher magnification, one can appreciate the presence of prominent macronucleoli, abundant
eosinophilic cytoplasm as illustrated in this blue oval, and also in this field of view,
you can see pale bodies within the cytoplasm, particularly just above the blue oval.
So in this particular case, I’ll give you some time to think of the diagnosis, and this
certainly is a diagnosis of fibrolamellar carcinoma.
We performed ancillary testing, which confirmed our histologic impression, and I will describe
the development of these ancillary tests in the subsequent slides and their usefulness
in the diagnosis of fibrolamellar carcinoma.
So, fibrolamellar carcinoma was described in the mid-1950s, and it classically shows
the morphologic features seen in our index case and, in this inset, large eosinophilic
neoplastic cells with prominent macronucleoli separated by bands of intratumoral fibrosis.
This distinctive tumor is not associated with cirrhosis and has a typical immunophenotype
as characterized by coexpression of keratin 7 and CD68.
However, in some cases, immunohistochemistry can show patchy or weak staining which can
give the surgical pathologist pause, particularly in small biopsies.
Now, I wanted to highlight this literature from a group of excellent liver pathologists
published in 2009 where they examined 25 cases of primary liver carcinomas and looked for
the reproducibility of the diagnosis among these well-known experts.
You can see here from the kappa scores, 0.23 to 0.52, that the reproducibility of diagnosis
was modest at best, and poor at worst.
And interestingly, in 69% of cases, the experts believe that they had required no immunohistochemistry,
but their agreement in these cases where they thought they needed no ancillary studies was
actually even worse.
Now, in this data set, the diagnosis of fibrolamellar carcinoma was proffered 17 times, even though
the creators of the data set had only included three cases of fibrolamellar carcinoma.
So, I think the data from this paper are really important in that they illustrate that while
we know the classic features of fibrolamellar carcinoma really well, in practice, making
a diagnosis is quite challenging, the reproducibility of a diagnosis is quite challenging, and when
to use ancillary studies is quite challenging.
And so I think this Hot Topic and the tests that I will present in subsequent slides are
really timely and really useful.
In addition, there is a wealth of known data, particularly this study published in 2016,
showing that the AJCC staging for classification of hepatocellular carcinoma stratifies hepatocellular
carcinoma quite well.
You can see in the left-hand Kaplan-Meier curve a good stratification of stages I through
IV for hepatocellular carcinoma.
However, for fibrolamellar carcinoma, the system does not work very well, and there
is no statistically significant stratification of patients, indicating that the diagnosis
of fibrolamellar carcinoma has important implications for the expectation regarding the patient’s
prognosis.
And so when we sum up all of what I’ve said previously, we know that this is a diagnosis
that is important clinically, we know that reproducibly making this diagnosis is challenging,
and we know that the staging system for AJCC does not work really well.
Fibrolamellar carcinoma is characterized by a fusion gene most often encoding DNAJB1-PRKACA,
and, as you can see here in this illustration, this is due to approximately 400-Kb deletion
that brings together exon 1 of DNAJB1 with the coding portions of PRKACA, leaving the
protein kinase domain intact.
Now, this fusion transcript was initially identified in all fibrolamellar carcinoma
cases tested in this paper published in Science, and subsequently, we were able to report that
it is specific for a diagnosis of fibrolamellar carcinoma among primary liver tumors.
Now, this novel FISH probe that was used to detect fibrolamellar carcinoma, I’ll show
you in this next sequence of images the basis of this test and how it appears on the darkfield
microscopy.
So, in a normal PRKACA gene, you would anticipate to see two intact yellow signals as shown
on the ideogram away to the left, and on the right, you can see in darkfield microscopy,
you will see two intact yellow signals in each nucleus.
In a case of fibrolamellar carcinoma, there’ll be loss of one red signal, giving rise to
separate green and intact yellow as seen on the ideogram, and away on the right on the
darkfield microscopy, you’ll see separate green and intact yellow in each nucleus corresponding
to the rearrangement of PRKACA, and supporting a diagnosis of fibrolamellar carcinoma.
So, we set about the international study with collaborators from Europe, the Middle East,
South America, and also from centers across the United States—our Rochester location
is shown in the yellow color—and we collected cases of fibrolamellar carcinoma to really
examine molecular testing for the diagnosis of fibrolamellar carcinoma.
What we found in this study, including also a TMA from colleagues in Germany—which included
conventional hepatocellular carcinoma, fatty liver, and normal liver—we independently
classified these cases as typical fibrolamellar carcinoma, possible fibrolamellar carcinoma,
unlikely to be fibrolamellar carcinoma.
We performed immunohistochemistry, and we also performed FISH.
This is an example of a case that’s a typical fibrolamellar carcinoma.
You can see the tumor cells displaying the characteristic cytomorphology, and then the
bands of fibrosis dividing the tumor cells.
You can see in the panel on the right, separate green signals and intact yellow signals corresponding
to rearrangement of the PRKACA locus.
Here’s an image illustrating one of the interesting things we found in that study
where there were cases showing the characteristic cytomorphology, but you can see in this entire
10X field that there are no bands of fibrosis, and this is the so-called solid variant or
solid area of fibrolamellar carcinoma.
So, not all examples have those distinctive bands of fibrosis.
You can also see here on this image another case showing the characteristic cytomorphology,
but this needle biopsy is made even more tricky because you can see a macrotrabecular pattern,
so not only no intervening bands of fibrosis, but a macrotrabecular pattern that is much
more commonly associated with conventional hepatocellular carcinoma than with fibrolamellar
carcinoma.
Another example to illustrate solid sheets, no intervening intratumoral fibrosis in this
image, but a presence of multinucleated histiocytes scattered throughout this tumor—which is
a tumor without any prior treatment—to illustrate that there are some cases of fibrolamellar
carcinoma, as you’ve seen in the last three images, this slide and the prior slides, without
the characteristic intratumoral fibrosis—but all of them do show the characteristic cytology.
All of these cases show the typical FISH pattern for fibrolamellar carcinoma with separate
green signals and intact yellow signals in each nucleus, and in 93 cases which were classified
as typical based on the histology, 99% of them showed PRKACA rearrangement, confirming
the utility of this FISH test in making the diagnosis.
We also identified cases that previously, though called fibrolamellar carcinoma, we
considered some of them to possibly be fibrolamellar carcinoma as shown in the two panels above,
and some of them to be unlikely to be fibrolamellar carcinoma as seen in the two panels beneath.
This is an illustrative example of what the PRKACA FISH results look like in these cases,
and you can see that in cases that were unlikely to be fibrolamellar carcinoma, they showed
intact yellow signals in the nuclei corresponding to intact PRKACA loci, and this was the pattern
that was seen in most cases that were unlikely to be fibrolamellar carcinoma, and in several
cases which were possibly fibrolamellar.
Here, again, you can see 15 cases of possible fibrolamellar carcinoma; 10 showed rearrangement,
five showed the pattern that you can see here—there was intact PRKACA loci—and also, you can
see that nine of them showed the appropriate immunophenotype for a diagnosis of fibrolamellar
carcinoma—showing that the FISH test had a greater sensitivity to immunohistochemistry,
but also illustrating that some of those cases that were called possibly fibrolamellar carcinoma
were in fact not fibrolamellar carcinoma.
In the six cases that were considered to be unlikely to be fibrolamellar carcinoma, none
of them harbored PRKACA rearrangement, but one of them showed the immunophenotype you
would expect in a case of fibrolamellar carcinoma, showing a limitation of immunohistochemistry,
but also highlighting the specificity of the PRKACA rearrangement assay.
So PRKACA FISH is useful for diagnosis.
In 93 cases where the histology was typical, PRKACA FISH confirmed the diagnosis in 99%
of them.
There was one case of fibrolamellar carcinoma negative for rearrangement by FISH, and this
happened in the setting of the Carney complex, and I’ll elaborate on this some more in
some subsequent slides.
But in that particular case, we speculated initially, but subsequently were able to prove
that there was biallelic loss of PRKAR1A, which is the regulatory subunit of protein
kinase A that works in concert with PRKACA.
In the 21 cases where the histology was not typical for fibrolamellar carcinoma, in 10
cases we identified PRKACA rearrangement which, with some other orthogonal methods, we believe
were able to allow us to confirm a diagnosis of fibrolamellar carcinoma.
So, PRKACA rearrangement is specific for fibrolamellar carcinoma in the context of a hepatocellular
neoplasm.
No PRKACA rearrangement was identified in any of the control cases which we tested.
While there is new information available, a bit of a twist in the last year, which provided
new information and helped to refine our understanding of the utility of the PRKACA FISH assay.
PRKACA rearrangements are also found in intraductal oncocytic papillary neoplasms of the bile
duct and pancreas, IOPN, as published by the group at Memorial Sloan Kettering and a group
at University of Pittsburgh Medical Center.
So these neoplasms also harbor PRKACA rearrangements, and associated invasive carcinomas do as well.
Importantly, these tumors, while they harbor PRKACA rearrangements, do not show hepatocellular
differentiation, they do not produce bile, and expression of hepatocellular markers is
not well established in these tumors.
In fact, intraductal oncocytic papillary neoplasms of the pancreas, when associated with invasive
carcinomas, they are mucinous carcinomas and so do not closely mimic fibrolamellar carcinoma.
An additional twist that we have appreciated in the literature published from our group
here at Mayo Clinic is that PRKAR1A loss may underlie fibrolamellar carcinoma instead of
PRKACA rearrangement, and I’ll go into that in subsequent slides.
So if we look at this diagram, you can see that in the physiologic state, protein kinase
is composed of subunits: regulatory subunits in blue and catalytic subunits in red.
When there is an increase in cyclic AMP within the cells, this binds to the regulatory subunits,
which liberates the catalytic subunits to phosphorylate downstream targets.
If we apply this understanding of biologies in fibrolamellar carcinoma, it becomes intuitive
to appreciate that the PRKACA fusion results in activation of the catalytic subunits.
And so it’s possible that if you have normal catalytic subunits where there is no fusion
gene present in those cells, but you have loss of PRKAR1A, that you can potentially
have the same phenotype and the same biology at work.
Now, mutations in PRKAR1A underlie Carney complex, which was published by one of our
emeritus faculty, Aidan Carney, several years ago.
And so we undertook a study to review liver tumors in patients with the Carney complex
and used sporadic fibrolamellar carcinoma controls.
In that particular study, we were able to show that cases with the classic morphology
of fibrolamellar carcinoma, and also with the classic immunophenotype harbored loss
of PRKAR1A protein as shown in these panels.
Away on the right, you can see two examples, top and bottom both, without PRKAR1A expression
in the neoplastic cells, but retained expression within the Kupffer cells, and we published
this in the Journal of Hepatology in 2018.
You can also see a control fibrolamellar carcinoma which appears in the panel beneath, showing
the classic morphology, classic immunophenotyped.
It harbored the rearrangement by FISH, but intact expression of PRKAR1A.
So, this refines now our understanding of fibrolamellar carcinoma, it displays the distinctive
morphology, distinctive clinical features arising in young patients without cirrhosis,
and there is a characteristic immunophenotype.
But I think we can refine it by looking at this Venn diagram to show that all fibrolamellar
carcinomas are characterized by protein kinase A activation most commonly due to DNAJB1-PRKACA
fusion gene formation, which can be detected by fluorescence in situ hybridization and/or
alternatively by PRKAR1A loss which can be detected by immunohistochemistry.
So, in conclusion, detection of PRKACA rearrangement by FISH, available from Mayo Clinic Laboratories,
is an excellent diagnostic tool.
PRKAR1A immunohistochemistry is also available from Mayo Clinic Laboratories and can be useful
in diagnosis.
Our approach to the diagnosis of fibrolamellar carcinoma includes a compatible morphology
and detection of PRKACA rearrangement by FISH, which is useful and informative in cases with
as few as 50 cells, and it is highly likely to be positive.
We have confirmed that its sensitivity is in excess of 97%, and in those rare cases
where the FISH test is not positive, it’s because of a different underlying mechanism,
and the PRKAR1A immunostain is very informative in those cases in making a diagnosis of fibrolamellar
carcinoma.
With that, I’d like to thank you for your time.