Diagnosing Fibrolamellar Carcinoma

Expires: April 6, 2023

Photo of Kyriakos Chatzopoulos, M.D.Presenter

Rondell Graham, M.B.B.S.
Associate Professor of Laboratory Medicine and Pathology
Division of Anatomic Pathology
Mayo Clinic, Rochester, Minnesota

Questions?

Contact us: mcleducation@mayo.edu.

Transcript and References

Introduction

Hi, I’m Bobbi Pritt, Director of the Clinical Parasitology Lab and Vice Chair of Education in the Department of Laboratory Medicine and Pathology at Mayo Clinic. In this month’s “Hot Topic,” my colleague, Dr. Rondell Graham, provides an overview of a FRKACA FISH test that assists in differentiating fibrolamella 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. 

Disclosure

So with regards to this presentation, I have no conflicts of interest to disclose. 

Utilization Management

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.

Objectives

So 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. 

Index Case

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.   

Diagnosis: Fibrolamellar Carcinoma

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.

Fibrolamellar Carcinoma is Positive for CK7 and CD68

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 3 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. 

AJCC Staging system for HCC

In addition, there are 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 DNAJB1-PRKACA

So 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. 

Novel FISH Probe to Detect PRKACA Rearrangement

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. 

Cases of FLC

So in a normal PRKACA gene, you would anticipate to see 2 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 2 intact yellow signals in each nucleus.  In a case of fibrolamellar carcinoma, there’ll be loss of 1 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. 

Independent histologic review

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 3 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 2 panels above and some of them to be unlikely to be fibrolamellar carcinoma as seen in the 2 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 . . . I think all cases that were unlikely to be fibrolamellar carcinoma and in several cases which were possibly fibrolamellar carcinoma.  Here again you can see 15 cases of possible fibrolamellar carcinoma; 10 showed rearrangement, 5 showed the pattern that you can see here, there was intact PRKACA loci, and also you can see that 9 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 6 cases that were considered to be unlikely to be fibrolamellar carcinoma, none of them harbored PRKACA rearrangement, but 1 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. 

PRKACA FISH is Useful for Diagnosis

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 1 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. 

PRKACA Rearrangement is Specific for FLC in the context of a Hepatocellular Neoplasm

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. 

Well, There’s a Twist

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 IOPN

So 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. 

PRKAR1A loss may underlie Fibrolamellar carcinoma instead of PRKACA rearrangement

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.

Inactive Protein Kinase A

 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 immunophenotype, 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.

FLC Displays Distinctive

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. 

Conclusion

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.

References

  1. Ross H, Daniel H, Vivekanandan P, et al: Fibrolamellar carcinomas are positive for CD68. Mod Pathol 2011 Mar;24(3):390-395.
  2. Malouf G, Falissard B, Azoulay D, et al: Is histological diagnosis of primary liver carcinomas with fibrous stroma reproducible among experts? J Clin Pathol 2009 Jun;62(6):519-524.
  3. Yamashita S, Vauthey J, Kasb A, et al: Prognosis of fibrolamellar carcinoma compared to non-cirrhotic conventional hepatocellular carcinoma. J Gastrointest Surg 2016 Oct;20(10):1725-1731.
  4. Honeyman J, Simon E, Robine N, et al: Detection of a recurrent DNAJB1-PRKACA chimeric transcript in fibrolamellar hepatocellular carcinoma. Science 2014 Feb 28;343(6174):1010-1014.
  5. Graham R, Jin L, Knutson D, et al: DNAJB1-PRKACA is specific for fibrolamellar carcinoma. Mod Pathol 2015 Jun;28(6):822-829.
  6. Terracciano L, Tomillo L, Avoledo P, et al: Fibrolamellar hepatocellular carcinoma occurring 5 years after hepatocellular adenoma is a 14-year-old girl: a case report with comparative genomic hybridization analysis. Arch Pathol Lab Med 2004 Feb;128(2):222-226.
  7. Vyas M, Hechtman J, Zhang Y, et al: DNAJB1-PRKACA fusions occur in oncocytic pancreatic and biliary neoplasms and are not specific for fibrolamellar hepatocellular carcinoma. Mod Pathol 2019 Nov 1 epub adhead of print.
  8. Singhi A, Wood L, Parks E, et al: Recurrent rearrangements in PRKACA and PRKACB in intraductal oncocytic papillary neoplasms of the pancreas in bile duct. Gastroenterology 2020 Feb;158(3):573-582.
  9. Graham R, Fibrolamellar Carcinoma: What Is New and Why It Matters. Surg Pathol Clin. 2018 Jun;11(2):377-387.
MCL Education

MCL Education

This post was developed by our Education and Technical Publications Team.