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Diagnosing Fibrolamellar Carcinoma

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Published: December 2016

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Fibrolamellar carcinoma is a rare subtype of primary liver cancer, and the diagnosis can sometimes be difficult. A new test, developed at Mayo Clinic, uses FISH technology to identify the fusion gene, DNAJB1-PRKACA, reported in fibrolamellar carcinomas.

Presenters:

Rondell P. Graham, MBBS

  • Assistant Professor of Laboratory Medicine and Pathology at Mayo Clinic

Michael S. Torbenson, MD

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Introduction

Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.

Our speakers for this program are Dr. Rondell Graham, an Assistant Professor of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota. The presentation was developed in cooperation with Dr. Michael Torbenson. Dr. Graham provides an overview of a PRKACA FISH test that assists in differentiating fibrolamellar carcinoma from other subtypes of primary liver cancer. Thank you Dr. Graham for presenting with us today.

Thank you for the introduction.

Disclosures

I have no disclosures.

Utilization Management

As you view this presentation you will see how PRKACA FISH can be appropriately used to support the diagnosis of fibrolamellar carcinoma particularly in difficult cases and in small biopsies.

Objectives

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 improved 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 MML, 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.

Case

Our index case is that of a 32-year-old woman with a 7.4-centimeter 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. Please note the absence of cirrhosis.

Histologic sections revealed a proliferation of large cells with eosinophilic granular cytoplasm separated by ribbons of fibrosis. On high magnification, distinctive macronucleoli are seen.

Diagnosis: Fibrolamellar Carcinoma

We performed ancillary testing, which confirmed our histologic impression. I will describe the development of this ancillary test in subsequent slides, but (initiate animation) the diagnosis of this case is fibrolamellar carcinoma or the fibrolamellar variant of hepatocellular carcinoma.

Fibrolamellar Carcinoma is Positive for CK7 and CD68

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 variant of hepatocellular carcinoma is not associated with cirrhosis and has a typical immunophenotype–cytokeratin 7and CD68 coexpression. However, in some cases immunohistochemistry can show patchy or weak staining, which can give the surgical pathologist pause particularly in small biopsies.

Fibrolamellar Carcinoma is Characterized by DNAJB1-PRKACA

Recently fibrolamellar carcinoma has been shown to be characterized by a recurrent translocation formed by a heterozygous 400-kb deletion on the short arm of chromosome 19. The novel chimera formed from this genomic event is believed to be the genetic driver of fibrolamellar carcinoma and the encoded protein retains the kinase domain of PRKACA. At its initial description, this novel fusion transcript was found in all fibrolamellar carcinoma cases tested. Our group has subsequently shown that the fusion transcript is specific for fibrolamellar carcinoma in the context of primary liver tumors.

Novel FISH Probe to Detect PRKACA Rearrangement

We designed a novel fluorescence in situ hybridization assay to detect PRKACA rearrangement. In a normal nonneoplastic cell, one would anticipate 2 intact yellow signals corresponding to intact PRKACA loci. In fibrolamellar carcinoma, the 400-kb deletion would lead to loss of a single red probe giving rise to a separate green and an intact yellow signal seen here on the ideogram and here in the interphase FISH.

Cases of FLC

As part of the validation of the new FISH test, we retrieved 114 cases diagnosed as fibrolamellar carcinoma from various institutions in the United States and other parts of the world. Approximately 40% of the cases were from Mayo Clinic in Rochester. We also obtained a tissue microarray with 88 conventional hepatocellular carcinoma from colleagues in Germany.

Here at Mayo Clinic, Dr. Michael Torbenson and I reviewed the slides from these cases. We classified the cases in to cases that were typical of fibrolamellar carcinoma, cases which were possibly fibrolamellar carcinoma, and cases that were unlikely to be fibrolamellar carcinoma. Immunohistochemistry for cytokeratin 7 and CD68 were performed as previously described. We performed fluorescence in situ hybridization for PRKACA rearrangement using our newly developed probe set.

Typica

This is a representative photomicrograph of a typical fibrolamellar carcinoma characterized by neoplastic cells with abundant eosinophilic granular cytoplasm and prominent macronucleoli arranged in trabecula separated by ribbons of fibrosis. The corresponding FISH image shows separate green and intact yellow signals consistent with PRKACA rearrangement.

In some cases of otherwise typical fibrolamellar carcinoma, there were foci of, or extensive areas of, sheet-like growth without intratumoral fibrosis, which has been termed the solid variant or solid areas of fibrolamellar carcinoma. In addition, as shown in this needle biopsy, there are cases of fibrolamellar carcinoma, rare cases, with the characteristic cytomorphology, but there is no intratumor fibrosis and the neoplastic cells are arranged in a macrotrabecular configuration; this is an unusual feature of fibrolamellar carcinoma.

Solid with Intratumoral Histoiocytes

This slide shows another somewhat unusual pattern for fibrolamellar carcinoma. The neoplastic cells, in this case without prior treatment, have abundant eosinophilic granular cytoplasm and prominent macronucleoli. This tumor also shows no intratumoral fibrosis, but is somewhat more distinct in that it bears intratumoral histiocytes and, in this example, including giant cells.

Positive for Rearrangement

In the 93 cases that we classified as typical for fibrolamellar carcinoma, all showed the expected immunophenotype, but patchy CK7 and/or focal or limited CD68 expression were noted in approximately 10% of cases. Importantly, in these typical fibrolamellar carcinoma cases, the new FISH test was positive in 99% of them, showing separate green and intact yellow signals.

Possible FLC/ Unlikely FLC

We also encountered cases that were possibly fibrolamellar carcinoma. In this example in the upper panels, there is intratumoral fibrosis, but the cytoplasm is somewhat amphophilic rather than eosinophilic, and the nucleoli are not prominent. In the lower panels is an example of a tumor that we considered to be unlikely to be fibrolamellar carcinoma; there is marked nuclear pleomorphism and significant heterogeneity.

PRKACA Intact

This is a representative FISH image of an intact or normal PRKACA FISH result. There are 2 intact yellow signals per nucleus.

Of the 15 cases that were possibly fibrolamellar carcinoma, 10 showed PRKACA rearrangement with separate green signals, and 9 of these showed the characteristic CK7 and CD68 coexpression. The 1 PRKACA-rearranged case without CK7 and CD68 coexpression, was positive for CK7 but negative for CD68 in that small biopsy.

The 6 cases that were classified as unlikely to be fibrolamellar carcinoma were all negative for PRKACA rearrangement. One of these cases showed CK7 and CD68 coexpression. This was a scirrhous hepatocellular carcinoma with marked nuclear pleomorphism.

PRKACA FISH is Useful for Diagnosis

In summary, our data demonstrate that PRKACA confirmed the diagnosis of fibrolamellar carcinoma in 99% of cases. The single negative fibrolamellar carcinoma arose in the setting of the Carney Complex and the biallelic loss of PRKAR1A in that case provides an alternate mechanism of activation of the driver pathway in fibrolamellar carcinoma.

In the 21 cases where the histology was not typical of fibrolamellar carcinoma, PRKACA FISH allowed for the accurate diagnosis of fibrolamellar carcinoma in all 10 cases.

PRKACA Rearrangement is Specific for FLC

No PRKACA rearrangement was identified in any of the conventional hepatocellular carcinoma in the tissue microarray or in the normal tissues. When these data are combined with those from a prior publication by our group, PRKACA rearrangement has been absent in 113 conventional hepatocellular carcinoma.

Conclusion

In conclusion, detection of PRKACA rearrangement by FISH is an excellent diagnostic tool with a sensitivity of 99% and a specificity of 100% in the context of primary hepatocellular tumors. This test has the advantage of being informative with as few as 50 tumor nuclei. It is currently available from Mayo Medical Laboratories. Our approach to the diagnosis of fibrolamellar carcinoma thus includes a compatible morphology with the appropriate immunophenotype or detection of PRKACA rearrangement by FISH, which is very specific.

Thank you for your attention.

 

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