Funded model development efforts

Timeframe: 2023 - 2025

Goals: Model development and understanding the requirements for FLC tumor formation

Principal Investigators: Benedetta Artegiani, PhD; and Delilah Hendriks, PhD

Study overview: In comparison with more common cancers, research models of FLC have been extremely limited. Drs. Artegiani and Hendriks have addressed this problem by creating “synthetic” models of FLC, generating cultures of normal cells as organoids (3D mini-organs). Specifically, they developed both liver cell organoids and techniques to genetically modify these organoids using CRISPR technology by which specific genetic changes can be introduced into normal cells.

In previous work, these investigators “knocked-in” the DNAJB1-PRKACA fusion gene into organoids. They found that this altered the normal hepatic cells to resemble FLC cancer cells in some respects, especially by changing patterns of gene expression in ways consistent with actual tumor cells. However, these gene-modified organoids did not fully transform into malignant cancer cells. While the DNAJB1-PRKACA fusion is clearly known to be the driver of FLC, and essential for the growth and survival of FLC tumor cells, there have been other suggestions that this fusion gene alone if not enough for the complete transformation to a robust FLC-like cancer. Similar observations were reported previously in studies of knock-in of the fusion oncogene into livers of young mice; in this case, at least one additional genetic change was required to yield aggressive tumor growth.

In this current study, the applicants propose to extend their initial findings in several ways. A major goal is to determine whether the introduction of additional mutations in cells containing the DNAJB1-PRKACA fusion can generate biological features more closely resembling fully transformed FLC cancer cells. The team will also determine whether introduction of the fusion gene and other genetic changes into normal ductal organoids gives more complete FLC-like transformation than in liver organoids. Finally, they propose to study influences of the tumor microenvironment on cells with the characteristic mutations of FLC by establishing co-cultures of the genetically modified cells with “mini-liver” cultures containing multiple cell types.

Successful accomplishment of the goals of this study should:

• reveal drivers of FLC that act in coordination with the fusion gene, which could lead to the discovery and design of new therapeutic approaches.
• build an understanding of the cause of observed heterogeneity among FLC patients
• create new disease models useful for the testing and development of FLC therapeutics.

Timeframe: 2023 - 2024

Goal: Immunocompetent mouse model development

Principal Investigator: Julien Sage, PhD

Study overview: Dr. Julien Sage has a very longstanding interest in FLC and is recognized as a world leader in the type of genetic modeling in mice proposed in this study. This study proposes to extend Dr. Sage's team’s previous work by taking four tumor lines generated from mouse tumors in immune-deficient mice, characterizing those tumors, propagating them in mice with intact immune systems to allow investigation of the interactions between FLC tumor cells and immune cells, which may be important for the development of future immunotherapies. In addition the study will use these models to test the response of FLC cells to a therapy targeting the cell cycle machinery, to stop the proliferation of FLC cells.

Currently, the absence of “pure” immunocompetent mouse models of FLC remains a conspicuous gap in FLC model systems. These new pre-clinical mouse models of FLC will complement human models and will hopefully help identify new therapeutic options in patients with FLC.

Timeframe: 2022 - 2023

Goal: Development of a novel human-derived liver progenitor cell line model of fibrolamellar carcinoma

Principal Investigator: Khashayar Vakili, MD

Study overview: Currently, very few cell-line models of FLC exist, so the development of additional cellular models is critical for the identification and testing of new therapies. Therefore, one of the aims of this proposal is to engineer normal liver cells in culture to express the DNAJB1-PRKACA gene fusion. This will be a novel model, which can provide insight into the mechanism of cancer development in addition to providing a platform for screening new therapeutic compounds. The second aim of this proposal is to assess the role of the mitochondria in FLC. Mitochondria are structures within cells that provide energy to the cells in addition to other vital functions. Mitochondria have been shown to develop abnormal characteristics in various types of cancers - leading to the hypothesis that they play a critical role in cancer survival and progression. Currently, there is very limited data available about the characteristics and role of mitochondria in FLC.

Timeframe: 2021 - 2022

Goal: Model development

Principal Investigator: Benedetta Artegiani, PhD

Study Overview: Research into FLC is complicated because of the limited experimental tools that can be used to study this rare cancer. While tests in laboratory animals such as mice are useful tools, they do not reflect the human disease due to inherent differences between mice and humans. Consequently, experimental models that can provide insights into human disease development and progression are highly sought after.

The overall goal of this research project was to build new models using human cells to study FLC. The researchers made use of lab-grown three-dimensional human mini-livers, so-called “organoids”. These organoids were grown from the two most important cells in the liver: the ductal cells and the hepatocytes. The researchers then precisely modified the DNA of the organoids using CRISPRCas9 technology, also called molecular scissors, to mimic the changes in the DNA that have been found in FLC.

By constructing and using such FLC models, the researchers hope to increase our current understanding of the origin of FLC tumors, and eventually create useful model systems for performing drug screens to identify new therapies.

Key Findings: In this study, the investigators successfully “knocked-in” FLC's characteristic DNAJB1-PRKACA fusion gene into organoids. They found that this altered the normal hepatic cells to resemble FLC cancer cells in some respects, especially by changing patterns of gene expression in ways consistent with actual tumor cells. However, these gene-modified organoids did not fully transform into malignant cancer cells. While the DNAJB1-PRKACA fusion is clearly known to be the driver of FLC, and essential for the growth and survival of FLC tumor cells, in this study at least one additional genetic change was required to yield aggressive tumor growth.

Artegiani and Hendriks found that using CRISPR to inactivate two additional genes in liver cell organoids generated fully transformed cancer cells - the inactivation of both copies of BAP1, known as a tumor suppressor gene, and both copies of PRKAR2A. The latter codes for the regulatory subunit of protein kinase A (PKA). Its loss leads to hyperactivity of PKA, the same enzyme that is dysregulated by the DNAJB1-PRKACA mutation. PRKACA codes for the active catalytic subunit of PKA, which normally is turned off by the regulatory (R) subunit and activated when the “second messenger” cyclic-AMP (cAMP) binds to R. In the absence of R subunits, the catalytic (C) subunit is active even without cAMP. The rare genetic loss of PRKAR2A (the Carney complex) has been associated with the development of the exceptional cases of FLC (approximately 1%) that do not have the classic fusion gene. An important feature of the organoids altered by inactivating BAP1 and PRKAR2A is that they more closely resemble primary FLC tumor samples and have properties in common with primitive progenitor cells found in biliary ducts.

The researchers concluded that although mutations in the PKA genes are crucial to the formation of FLC, they may not completely explain the disease development and progression. Their findings were published In May 2023 in Nature Communications. Click here to read and download the published journal article.

After the conclusion of this study, FLC funded a follow-on grant to determine whether the introduction of additional mutations in cells containing the DNAJB1-PRKACA fusion can generate biological features more closely resembling fully transformed FLC cancer cells. 

Benedetta Artegiani's and Delilah Hendriks' lab activites were also described in a general press articles in The Scientist in March 2023.

Timeframe: 2021

Goal: Generate a mouse model of FLC

Principal Investigator: Sean Ronnekleiv-Kelly, MD

Study overview: The purpose of this study was to generate a robust pre-clinical murine model of FLC that can provide a basis for understanding factors contributing to FLC formation, and for therapeutic development. The study proposed to develop a mouse model of FLC using a gene editing approach in a susceptible population, using hydrodynamic delivery of gene editing material to the liver via retro-orbital injection at age 7-8 weeks in mice with varying susceptibility to liver tumor formation. This included C57BL/6 mice, FVB mice, C3H mice, Balb/c mice and DBA/2 mice, which have a range of 2-3 fold lower vulnerability to 3-7 fold higher susceptibility to liver cancer development. In this manner, they wanted to identify if different susceptibilities to liver tumor formation causes earlier onset / more aggressive cancer, which could then ultimately improve the understanding of FLC development. In a second subset of the same mouse strains, they performed retro-orbital injection of the mice at age 2 weeks, a timeframe selected because the fusion gene mutation is an early somatic event in humans (i.e. peak age of FLC diagnosis is 21 years). Most previous studies targeted the DNAJB1-PRKACA mutation to the liver at age 7-8 weeks. The hope was that targeting the gene editing material to generate the fusion gene at 3 - 4 weeks (when liver cells are still actively dividing) would create a different tumor phenotype compared to existing models.

Results: The mouse models created by the introduction of the CRISPR/CAS9 to generate the fusion gene expressed the DNAJ-PRKACA mRNA as well as the fusion protein. Two groups of mice were injected, a control group at 7-8 weeks, which is the typical age of such manipulations and an experimental group at 3-4 weeks, since this age is more representative of the developmental age at which human subjects develop FLC.

The control group was evaluated at age 6 months and investigators did not identify tumor onset macroscopically or microscopically at that time. Some lesions were seen at 6 months of age that were more prominent at 10 months and appeared to match what was reported in a previous publication. The experimental group is being evaluated currently for histopathological abnormalities and tumor development.

Implications: This mouse model will be the first of its kind to express the fusion protein at an earlier time point in development. The development of the fusion protein at an earlier age should ideally give rise to a stronger tumor phenotype.

Timeframe: 2021 – 2022

Goal: Model development

Principal Investigator: Mark Yarchoan, MD

Study overview: Efforts to identify novel therapies for FLC have been confounded by a lack of preclinical models that accurately reflect the genetics and biology of the disease. This study aimed to establish and validate the first orthotopic, syngeneic, preclinical mouse model of FLC that reproduces the key biological behavior and tumor microenvironment (TME) of human FLC.

Because the tumors in such models grow in the context of an intact immune system, they are therefore appropriate models to study agents that act on the host immune system to enhance tumor immunity such as checkpoint inhibitors and immunotherapy agents. Once successfully established,the resulting preclinical model of FLC will be openly shared with the larger research community. This model could offer unprecedented opportunities to investigate mechanisms underlying FLC pathogenesis, and become a critical tool for investigating novel therapeutic strategies in FLC.

Key findings: The study team has successfully established a new pre-clinical model of FLC. They created a FLC-like murine cell line by inducing hepatoblast cells (TIB-75) to express the DNAJB1-PRKACA fusion and implanted the tumor line orthotopically into the livers of syngeneic BALB/c mice. In mice, these FLC-like tumors have growth kinetics, high levels of infiltrating lymphocytes, and histological features, such as fibrosis, that are consistent with the clinical phenotypes of FLC observed in humans.

The study team is committed to further describing and validating this new model. Additional planned analyses include characterizing the metabolomics of the FLC-like and parenteral cell lines. They intend to publish their findings, and are open to widely distributing the model within the FLC community. The hope is that this model can serve as the basis for in-depth investigation of the DNAJB1-PRKACA chimeric transcript-dependent pathway in the FLC tumor microenvironment, as well as efforts to drug the fusion and/or its downstream factors. Already, the model has proved instrumental in performing pre-clinical assessments of the impact of glutamine antagonist treatment in combination with checkpoint inhibition - work critical to the development of the upcoming DRP-104 clinical trial at Johns Hopkins.

Timeframe: 2021 – 2023

Goal: Develop a blood-based screening method to detect relapse in FLC patients

Principal Investigator: Gary S. Stein, PhD

Study overview: This study is focused on developing a blood-based screening method to detect relapse in FLC patients that have been diagnosed and treated by surgery. This screening protocol is intended to be non-invasive, to overcome limitations of current imaging strategies, and to be accessible and affordable for patients and their families. Currently, treatment of patients with FLC is limited by the understanding of how this cancer responds to therapies. Cancer physicians currently have limited ability to test for, and monitor patients’ response to therapy, and to detect disease progression. It is therefore important to have effective monitoring strategies for physicians to determine if patients are successfully remaining in remission, or if they are progressing towards the earliest stages of relapse. Catching relapsed tumors early, prior to spreads throughout the body, is critically important to increase FLC patient survival.

The study is evaluating the use of DnaJ-PKAc transcripts in circulating nucleated cells as a proxy for detecting the presence of circulating tumor cells (CTCs). A major challenge to detecting and quantitating CTCs is their extremely low abundance – roughly 1 to >100 per 7.5 mL of whole blood, compared to the billions of other cells typically found in blood. The objective of this initiative is to test whether the DnaJ-PKAc transcript can be detected in blood samples and to establish the threshold of transcript detection. We will utilize an engineered model of FLC that possesses the DnaJ-PKAc fusion gene as a substitute for actual CTCs. These pseudo-CTCs will be spiked into blood samples from normal donors to simulate whole blood from a metastatic or progressive disease scenario.

Timeframe: 2020 – 2023

Goal: Create a comprehensive list of potential drug targets for FLC

Principal Investigator: Jesse Boehm, PhD

Study overview: This project is part of the Broad Institute’s Rare Cancer Dependency Map Initiative. The project has three main goals to identify potential FLC therapeutics:

1. Developing new cell models of FLC for the research community. Harnessing the Broad Institute’s Cancer Cell Line Factory laboratory and its combinatorial media screening technology will allow the team to systematically determine the conditions necessary to grow FLC samples as three-dimensional organoid models. They will also work in coordination with the FCF-sponsored Biobank to create a unified pipeline by which any patient can direct tissue to FLC researchers.
2. Utilizing the developed cell culture models of FLC to create a comprehensive list of potential drug targets and to identify existing drugs that may have therapeutic potential against FLC.
3. Empowering the entire FLC research community by sharing all developed genomically characterized and clinically annotated cell models and by making all the data and biologist-friendly analysis tools freely available online, pre-publication at DepMap.org.

If fully successful, this effort will nominate high priority targets for drug discovery as well as new drug repurposing hypotheses.

Timeframe: 2020 – 2022

Goal: Model development

Principal Investigator: Nabeel Bardeesy, PhD

Study overview: While multiple FLC models have recently been developed, additional model development remains a priority for the FLC community. This effort aimed to develop multiple different FLC models as resources for the FLC research fibrolamellar cancer research community, collaborating with the Fibrolamellar Cancer Biobank established at Massachusetts General Hospital. Specimens from the biobank were used to attempt to create a series of transplant human FLC tumors grown in immune-deficient mice (patient-derived xenograft [PDX] models), three-dimensional cell culture models (3D tumor organoids), and cell lines in partnership with the Broad Institute.

Together with collaborators at the Broad Institute and throughout the FCF research network, the study team hoped to harness these newly developed models to identify genetic dependencies of the disease, better understand the molecular mechanisms underlying FLC formation and growth, and ultimately will set the stage for the development of new therapeutics.

Key Findings: Through this effort, the investigators successfully developed a new cell line model (FLX1) derived from a previously-established PDX model. The investigators reported high level of fusion protein expression in that FLX1 cell line and were able to successfully propagate it in lab as a functional cell line.

During the effort, organoids and patient derived 3D cultures proved more difficult to establish. In all, 28 patient tissue samples were received by the study team from the FCF biobank and other sources to drive PDX, cell line and organoid model development attempts. While 3 PDX models engrafted to a size allowing re-implantation and expansion, all were eventually lost to contamination, murine lymphoma or lack of fusion detection. A second established cell line (FLX2) had a histopathology more like HCC instead of FLC. For 3-dimensional organoids, growth in many attempts was good initially, but senesce (age deterioration) occurred after a limited number of passages in all cases, preventing the establishment of a useful model.

The successfully established FLX1 model has already been used by the study team to make significant advances in understanding the networks mediated by fusion signaling. Studies by Dr. Bardeesy using FLX1 have revealed that that DNAJ-PKAc inactivates three related protein kinases, the Salt-Inducible Kinases (SIKs), which in turn leads to the mitochondrial abnormalities observed in FLC. Currently, Dr. Bardeesy is exploring specifically how that DNAJ-PKAc/SIK pathway controls mitochondrial function and how the mitochondrial abnormalities contribute to cancerous growth in FLC.

Efforts are also underway to make this new FLX1 cell line broadly accessible to the research community as a research tool.

Timeframe: 2019 – 2021

Goal: Development of a novel human-derived liver progenitor cell line model of fibrolamellar carcinoma

Principal Investigator: Khashayar Vakili, MD

Study overview: Prior to this effort, the study team's lab had previously engineered a kidney cell line (HEK-DP) which contains the DNAJB1-PRKACA fusion gene found in FLC tumors. This cell line demonstrated interesting similarities to FLC tumors and served as a proof of concept for the development of additional cell lines. This effort applied apply the same strategy used to engineer the HEK-DP model to engineer normal human liver progenitor cells to express the DNAJB1-PRKACA fusion gene. These liver progenitor cells were to be grown as three-dimensional “organoid” cultures to better replicate in vivo conditions.

If successful, the study aimed to create a novel model to facilitate understanding how the fusion protein reprograms normal liver progenitor cells to become cancerous. Such understanding of the precise mechanisms underlying the formation of FLC could then provide insights to future therapeutic strategies.

Results: Following the successful expression of the fusion protein in HEK cells, the investigators introduced the CRISPR/CAS9 strategy in human-derived cholangiocyte cell cultures to create organoids carrying the fusion protein. However, the efficiency of the expression of the fusion protein was low. The team tried several strategies to increase the expression.

Once the most efficient method is identified, the team plans to conduct a single cell analysis to compare the fusion protein carrying cells with the normal cells in the same organoid.

Implications: This study demonstrated the successful adaptation of a strategy used in non-liver cells to generate specific mutation carrying organoids to liver progenitor cells. While much work remained to develop a usable model, the work was continued. Once these organoids are well characterized, they will be shared with the FLC community.

Timeframe: 2016 – 2019

Goal: Use zebrafish as a model system for fibrolamellar carcinoma to study the immune system

Principal Investigator: Sofia de Oliveira, PhD

Study overview: Few FLC animal models currently exist limiting our ability to study FLC in the context of a complete organism. While cell-based models are extremely useful, animal models allow scientists to study biological processes involving multiple organs and cell types, such as tumor immunology and metastasis. Zebrafish are a valuable tool to study many diseases including cancer and have been used as a model system by the genetics community for decades. They display remarkable similarities and share many genetic signatures with humans and have been used to study liver development, hepatocellular carcinoma, and several other liver disorders. This effort planned on developing a zebrafish model of FLC and harnessing this model to study how the immune system interacts with FLC.

Key findings: Expression of the zebrafish specific DNAJB1-PRKACA fusion protein in zebrafish embryos led to increased liver size (a.k.a. hepatomegaly) and mass formation in a small subset of adult zebrafish livers. Zebrafish larvae carrying the fusion protein showed an increased presence of inflammation-responsive cells (macrophages and neutrophils) in the liver microenvironment. Subsequent treatment of the zebrafish larvae with well-characterized anti-inflammatory drugs led to a decrease in liver size as well as a reduction in the numbers of macrophages and neutrophils in the liver.

This study established zebrafish as a potentially valuable model system with non-invasive live-imaging capabilities and scalability that could be utilized to study FLC disease mechanisms and identify potential therapeutic targets. Overall, the team's findings support the idea that non-resolving inflammation might be fueling the liver microenvironment and contributing to FLC pathology.

Details of the study were published and reported in Disease Models & Mechanisms (DMM), an Open Access biomedical research journal, in April 2020. The publication can be read or downloaded here.

Timeframe: 2016 – 2019

Goal: Develop a pre-clinical mouse model for fibrolamellar carcinoma

Principal Investigator: Dr. Julien Sage, PhD

Study overview: There are few effective therapies for FLC patients and development of improved therapeutics are hampered by the rarity of the disease and the challenge of including pediatric patients in many clinical trials. One solution to this problem is the development of accurate models of FLC. This study proposed to generate the first mouse model for FLC. Such models could be used to investigate the basic mechanisms of FLC development, identify new therapeutic targets, and to test novel therapeutic strategies.

The study had two specific aims:

1. Determining the consequences of DNAJB1-PKA expression during liver development. The team had already generated a transgenic mouse in which the DNAJB1-PRKACA gene fusion is inserted into the mouse genome in an inducible manner. By switching on the expression of DnaJ-PKA at specific stages of liver development, they planned to examine exactly how and when this fusion contributes to FLC formation.
2. Generating the first pre-clinical mouse model of FLC. To induce the development of FLC tumors in Rosa26LSL-DNAJB1-PRKACA-GFP mice, they planned to cross these transgenic mice with mice in which the Cre recombinase can be activated in liver stem/progenitor cells during liver development. They planned to follow tumor development in aging cohorts of mice, and to determine the accuracy of the model in comparison to human tumors using histopathological methods and by RNA sequencing.

For additional details see the article published by Stanford on FCF’s grant and collaboration.

Key findings: Since the initiation of the effort, two different studies published at the end of 2017 showed that activation of the fusion early in life is not necessary and that FLC can be modeled in mice after activation of the fusion in hepatocytes in young mice. Based on those studies, and to avoid duplicating published work, the team modified its research plan to focus on the tumor development effort and the development of better models of FLC. Their idea was to generate a number of aging mice with induction of the fusion and to develop cell lines or allografts with those mice.

At the conclusion of the study, the team had generated and begun to analyze a mouse model of FLC that is different from previous models. Approximately 20-25% of the genetically engineered mice developed tumors, but the growth of these tumors was slower than what was observed in humans. While that slow growth rate and the low penetrance are major impediments to performing pre-clinical treatment studies using the models, the hope is that the model can still be used to generate cell lines and allografts. To achieve that new goal, the mice need to be aged at least 2 years to wait for very large tumors to grow, which was not not anticipated by the initial proposal.

Timeframe: 2013 - 2014

Goal: Support costs related to establishment of new FLC biospecimen storage facility

Principal Investigator: Sandy Simon, PhD

Study background: Access to tumor tissue is critical to help cancer researchers understand what drives a disease and how the cancer responds to treatments. Because fibrolamellar is so rare, there has long been a shortage of FLC tissue available for research.

This funding request was to support the establishment of a new tissue repository for fibrolamellar carcinoma at The Rockefeller University, in new lab space built specifically for this purpose. The lab needed a new secure freezer to store the FLC samples and a secure computer to house de-identified patient data, which could then be correlated with the tissue samples to benefit research on outcomes and recurrence.

Results: The equipment was purchased and the new tissue repository successfully developed. These samples and data will be made available to collaborators and labs that are researching fibrolamellar, so that progress can be made without sequestration of resources or overlapping efforts.

Timeframe: 2012 - 2013

Goal: Support series of projects exploring the role of antibody involvement in FLC tumors

Principal Investigator: Sandy Simon, PhD

Study overview: This effort continued the encouraging early work begun on mice to see if researchers could take advantage of the immune system to identify potential ways to detect and eventually treat fibrolamellar. In particular, the team focused on identifying whether accumulation of antibodies within malignant tissue could provide a way to identify and track tumor cells as they grow and spread.

Specific activities included:

• Analyzing cancer tumors in mice, using slices of tissue. The goal of this effort was to characterize the accumulation of antibodies within tumors using tissue slices resected from a small animal models of different types of cancer.
• Analyzing tumors in mice, using whole animal imaging. Since a long-term goal is to use the antibody technology to detect and treat tumors in humans, this effort was focused on developing approaches that allow antibody observations to occur in living animals.
• Developing various nanoprobes to improve the whole animal imaging. These nanoprobes are molecules used to detect and visualize specific biological processes in living organisms. They are usually labeled with a radioactive or fluorescent tag, which allows them to be detected by imaging techniques such as positron emission tomography (PET) or fluorescence microscopy.
• Applying the findings from the mouse tumor work to human cancers, in particular FLC.

Results: The study analyzed the accumulation of a mouse’s antibodies (specifically an antibody known as IgG, or immunoglobin G) in their tumors. The models analyzed included several mouse models for liver cancer (HCC), two models for prostate cancer, two models for breast cancer and a model of skin cancer. Both transgenic models (generated by altering the genome of the mouse) and xenograft models (using tumors transplanted from one animal to another) were used.

The study clearly demonstrated that there is a very significant concentration of a patient’s (or
animal’s) antibodies across tumor types. Similar to the results in mice tumors, the study showed increased levels of a human patient's IgG in fibrolamellar carcinoma tumors relative to the levels in the adjacent normal tissue.

Results of the antibody accumulation analysis were published in May 2014 in Nature's Scientific Reports. The full text of the published article - "Endogenous Antibodies for Tumor Detection" - can be accessed here.

In whole animal imaging, the team examined both PET and fluorescence imaging approaches. While each alternative had different strengths and weaknesses, much work remains to identify an imaging approach that gives the maximum sensitivity, the fewest false positives and the fewest false negatives. Additional analyses, equipment improvements and probe modification efforts were identified for future investigation.

Implications: The study demonstrated that the enrichment of antibodies within malignant tissue, including FLC tissue, provides a potential means of identifying and tracking cancer cells as they mutate and diversify. Exploiting these antibodies for diagnostic and therapeutic purposes is therefore possible by using agents that bind to those antibodies.

Timeframe: 2010 - 2011

Goal: Build understanding of the immunology and cell biology of FLC using mouse models of cancer

Principal Investigator: Sandy Simon, PhD

Study background: This early study was designed to determine if researchers could take advantage of the immune system to identify potential ways to detect and eventually treat fibrolamellar.

The immune system is constantly screening bodies for transformed cells. Usually it recognizes these cells as foreign and triggers a programmed pathway of cell death called apoptosis. However, even though the immune system still can recognize tumor cells, mutations in tumor cells could disable the cell death pathway, so the immune system loses the ability to kill the tumor cell.

The study team's hypothesis was that this mechanism could potentially be leveraged in two ways:

• To use the immune system, and in particular antibodies, to detect micrometastases in the body
• To use the antibodies to deliver toxins directly to the tumors, instead of using general systemic chemotherapy approaches.

Results: During the initial effort, the team completed testing on:

• A genetically induced mouse model for hepatocellular carcinoma
• A chemically induced mouse model for hepatocellular carcinoma
• A genetically induced mouse model for prostate cancer.
• Four human patients samples of fibrolamellar heptocellular carcinoma.

The work was continued with a follow-on grant.

Timeframe: 2010 - 2011

Goal: Develop useful disease models to support subsequent research efforts

Principal Investigator: Yuzhou Wang, PhD

Study overview: Availability of model system to replicate the disease is an essential tool in development of therapeutics. These model systems can be used by researchers for mechanistic investigations, rapid drug screening, and many other purposes. Model systems such as primary cancer cell lines originate from tissue resected during biopsies and surgeries, or ascites fluid from metastasized cells. Similarly, patient-derived xenografts are models of cancer where the tissue or cells from a patient's tumor are implanted into immunodeficient mice.

The goal of this study was to:

1. Establish a FLC cell line from patient tissue samples
2. Establish a patient derived xenograft for drug screening studies
3. Characterize the microRNA population in FLC once a model was established.

Results: During the study, investigators attempted to establish a cell line from ascites fluid from a FLC patient. They were able to culture the fibrolamellar cells for several growth passages and demonstrated that the cells could be frozen for long term storage. Upon thawing of the frozen cells, viable cell cultures were generated that could be used for various experiments.

Using proprietary procedures, the lab had previously created person-to-mouse xenografts from various tumor tissues (other than FLC) with a high success rate. They attempted to apply these same techniques to FLC tissue samples. Unfortunately, their attempts to develop a fibrolamellar PDX model were unsuccessful.

Implications: While the lab's efforts to create FLC models were unsuccessful, additional efforts have been sponsored to develop such important research tools. 

Note: This study was one of four "Freedom of Pursuit" research awards to be distributed under the joint FCF/ICARE program active in 2010/2011. The use of those funds was unrestricted. Initially, the hope was that the Wang lab would use some of the funds to pursue microRNA research on FLC. However, those efforts remained focused on prostate cancer. FCF/ICARE's support of microRNA research was acknowledged in a 2011 paper about prostate cancer.

Since the conclusion of the ICARE effort, no grants issued by FCF have been unrestricted in their use.

Timeframe: 2010

Goal: Identify biomarkers for FLC

Principal Investigator: Michael Torbenson, MD (Currently at Mayo Clinic)

Study overview: Early identification of tumors is essential for aggressive treatment. The majority of fibrolamellar carcinoma (FLC) patients have metastatic disease at the time of diagnosis. Therefore, identification of biomarkers for FLC is essential. Using a broad collection of FLC pathology specimens, the study planned to identify diagnostic biomarkers for FLC. In addition, they investigated whether these biomarkers are linked to any genetic mutations or microRNA expression profiles unique to FLC.

Results: As a potential aide in diagnosis, the study team investigated the staining properties of CD68, a transmembrane glycoprotein present on lysosomes and endosomes (organelles that are involved in the transport of materials within cells). Cells like macrophages which are rich in lysosomes/endosomes are CD68 positive. The study team performed an analysis of CD68 within FLC and HCC samples. In all, the team analyzed 23 primary FLC and nine metastatic FLC from 24 individuals. Sixty-six tumor samples from HCC patient were analyzed as well. CD68 positivity was strongly associated with fibrolamellar carcinomas as compared to HCC.

The details of the study were published in Modern Pathology in March 2011. The full text of the article can be read here.

Implications: This study identified a biomarker, CD68, that is present on FLC samples and can be used in routine diagnostic surgical pathology. In addition, it may be of use in research studies by helping properly classify cases of fibrolamellar carcinoma. This is of importance because it can help ensure a uniform biological entity is being investigated across research groups.

Note: This study was one of four "Freedom of Pursuit" unrestricted research awards to be distributed under the joint FCF/ICARE program active in 2010/2011.