Desmoid tumors are proliferations of relatively benign appearing fibroblasts. Despite their histologic bland appearance, a significant subset of these tumors recurs aggressively and requires often debilitating surgery. Currently there are no molecular markers that predict the behavior of desmoid tumors.

The aims:

AIM 1: To generate an extensive molecular dataset covering gene expression profiling and gene mutation analysis of desmoid tumors and scar samples.

AIM 2: To identify gene changes in expression level (for mRNA and non-coding RNAs) and gene mutations that correlate with risk for recurrence.

AIM 3: To use the same approach to identify genetic markers that can distinguish scar tissue from recurrent desmoid.

AIM 4: To explore the molecular heterogeneity of desmoid tumors based on the analysis of multiple regions sampled from a single tumor.

AIM 5: To search for low frequency CTNNB1/APC mutations in “wild-type” tumors.

The purpose of this study will be to perform a very broad molecular search for markers that can be used to address two clinically highly relevant questions. Using a carefully selected group of cases with known clinical outcome, we will use stranded RNA-Seq data (for mutation and expression level analysis) to generate a dataset that will characterize desmoid tumors in the broadest sense possible. First, this dataset will be searched for markers that can distinguish desmoid tumors that can be followed by “watchful waiting” at the time of their initial biopsy from tumors that require aggressive surgical therapy. When desmoid tumors recur, the distinction between recurrent tumor and scar tissue from prior surgery can be very difficult by histology, and immunohistochemistry for beta-catenin is insufficient. In the second approach that will use the dataset mentioned above, we will compare RNA-Seq findings on scar tissue with those seen in desmoid tumors to identify markers that distinguish scar from desmoid in an effort to find a novel immunohistochemistry marker to discern these two entities and that can be used in surgical pathology practice.

We also plan to perform deep targeted sequencing of CTNNB1 and APC genes in samples with no detectable mutations in Sanger sequencing/RNA-Seq data. According to the recent findings of Crago et al. (Genes Chromosomes Cancer. 2015 Oct;54(10):606-15), many of desmoid tumors initially considered as wild-type appear to carry low frequency mutations in CTNNB1/APC genes that can be detected using next generation sequencing. Our set of samples comprises relatively high portion of wild-type cases (27%) therefore we aim to further investigate this issue in order to better characterize our samples.

This work will build on an already existing large dataset in the van de Rijn laboratory and will benefit from the experience that we have generated in our laboratory for the use of archival formalin-fixed, paraffin-embedded tissue in high throughput studies using next generation sequencing. The project greatly benefits from the collaboration with Dr. Kristen Ganjoo at Stanford University Hospital and from the collaboration with Dr. Raffi Avedian, orthopedic surgeon at Stanford. We have also started a new collaboration with Dr. Justin Cates and Dr. Thomas Stricker from Vanderbilt University, and with Dr. Chiara Colombo from Milan, Italy. We have previously performed quantitative measurement of the expression levels for all know human protein-encoding genes in 9 desmoid tumor samples, 4 scars and 42 non-desmoid fibroblastic lesions. The number of scar samples that we have analyzed was too low but the preliminary results were encouraging. Our current dataset includes 29 primary, 20 recurrent desmoid tumors and 15 scars from 26 patients.

LAY VERSION OF ABSTRACT- Next generation sequencing approach to desmoid tumors

Desmoid tumors are proliferations of relatively benign appearing fibroblasts. Despite their histologic bland appearance, a significant subset of these tumors recurs aggressively and requires often debilitating surgery. Currently there are no molecular markers that predict the behavior of desmoid tumors.

The aims:

AIM 1: To generate an extensive molecular dataset covering gene expression profiling and gene mutation analysis of desmoid tumors and scar samples.

AIM 2: To identify gene changes in expression level (for mRNA and non-coding RNAs) and gene mutations that correlate with risk for recurrence.

AIM 3: To use the same approach to identify genetic markers that can distinguish scar tissue from recurrent desmoid.

AIM 4: To explore the molecular heterogeneity of desmoid tumors based on the analysis of multiple regions sampled from a single tumor.

AIM 5: To search for low frequency CTNNB1/APC mutations in “wild-type” tumors.

The purpose of this study will be to perform a very broad molecular search for markers that can be used to address two clinically highly relevant questions. Using a carefully selected group of cases with known clinical outcome, we will use stranded RNA-Seq data (for mutation and expression level analysis) to generate a dataset that will characterize desmoid tumors in the broadest sense possible. First, this dataset will be searched for markers that can distinguish desmoid tumors that can be followed by “watchful waiting” at the time of their initial biopsy from tumors that require aggressive surgical therapy. When desmoid tumors recur, the distinction between recurrent tumor and scar tissue from prior surgery can be very difficult by histology, and immunohistochemistry for beta-catenin is insufficient. In the second approach that will use the dataset mentioned above, we will compare RNA-Seq findings on scar tissue with those seen in desmoid tumors to identify markers that distinguish scar from desmoid in an effort to find a novel immunohistochemistry marker to discern these two entities and that can be used in surgical pathology practice.

We also plan to perform deep targeted sequencing of CTNNB1 and APC genes in samples with no detectable mutations in Sanger sequencing/RNA-Seq data. According to the recent findings of Crago et al. (Genes Chromosomes Cancer. 2015 Oct;54(10):606-15), many of desmoid tumors initially considered as wild-type appear to carry low frequency mutations in CTNNB1/APC genes that can be detected using next generation sequencing. Our set of samples comprises a relatively high portion of wild-type cases (27%) therefore we aim to further investigate this issue in order to better characterize our samples.

This work will build on an already existing large dataset in the van de Rijn laboratory and will benefit from the experience that we have generated in our laboratory for the use of archival formalin-fixed, paraffin-embedded tissue in high throughput studies using next generation sequencing. The project greatly benefits from the collaboration with Dr. Kristen Ganjoo at Stanford University Hospital and from the collaboration with Dr. Raffi Avedian, orthopedic surgeon at Stanford. We have also started a new collaboration with Dr. Justin Cates and Dr. Thomas Stricker from Vanderbilt University, and with Dr. Chiara Colombo from Milan, Italy. We have previously performed quantitative measurement of the expression levels for all known human protein-encoding genes in 9 desmoid tumor samples, 4 scars and 42 non-desmoid fibroblastic lesions. The number of scar samples that we have analyzed was too low but the preliminary results were encouraging. Our current dataset includes 29 primary, 20 recurrent desmoid tumors and 15 scars from 26 patients.

LAY VERSION OF ABSTRACT- Next generation sequencing approach to desmoid tumors

This project is to develop the first validated PRO tool in desmoid tumors. The PRO tool will be developed and validated in partnership between MSKCC and Quintiles, a leading PRO company. Desmoid tumor (DT/DF) is a sarcoma of fibroblastic origin that afflicts children, adolescents and adults and has a median age of onset of 30 years. Desmoid tumors can occur in any anatomic location and threaten vital organs and limbs which often results in significant morbidity from organ failure, mutilating surgeries (and amputations), loss of limb function, pain and death. Surgery is associated with high rates of recurrences (~40%). In advanced disease, systemic therapies range from anti-estrogens to cytotoxic chemotherapies with variable response rates however no systemic therapies have been approved since pivotal trials evaluating survival or quality of life has never been conducted. In the last few years, a deeper biological understanding of this disease has led to promising drugs such as sorafenib, mTOR and Notch inhibitors that are currently in clinical trials. These and other drugs cause both tumor reduction and improvement in quality of life. Tumor reduction is measured by standard images and by established criteria (RECIST or WHO). However, the improvement in quality of life described by patients is lost as there are no validated tools to measure this drug effect. The classical regulatory (FDA) pathway to approving new drugs in cancer is demonstration of improvement in overall survival. Surrogates of overall survival include progression free survival and durable tumor responses. An emerging endpoint for regulatory approval is patient reported outcomes (PRO) tools. More recently, the FDA approved JakafiTM, a drug to treat myelodysplastic syndrome based on a PRO tool developed in MDS. We hypothesize that development of a PRO tool in desmoid tumors, will facilitate a new regulatory endpoint that can be used in all future clinical trials and will be a great investment of resources to advance the field for researchers worldwide.”

LAY VERSION OF ABSTRACT- Development of a patient reported outcome (PRO) tool in desmoid tumors

Desmoid tumors (DT) are locally invasive lesions that are difficult to treat using conventional therapies. While there is some success with chemotherapy in people who have recurrence of their tumors after surgery and/or radiation, much of the time the results from chemotherapy are temporary, and regrowth of tumor is seen. One approach to treatment is to develop a combination of medications that target different aspects of tumor growth, but have few side effects. Since desmoids are locally invasive but do not metastasize to other locations in the body, it is not necessary to eliminate every tumor cell, but instead to stabilize or shrink the tumor. We assembled a consortium of researchers from different backgrounds, and with complementary skills, whose ultimate goal is to develop a treatment to therapeutically target tumor cells in desmoid patients without causing serious side effects.

To achieve this goal, over the past two years, we screened drugs in the laboratory on cell lines from desmoid tumor patients to see which agents specifically target tumor cell viability and tested a first cohort of drugs in mice that develop desmoid tumors, identifying several agents already in use or close to being in use for patient care that could be repurposed for use in desmoid tumor treatment.

Over the remaining course of the proposal we plan to:

1. Complete the testing of drugs identified in the screen in a mouse model that is engineered to develop desmoids.
2. The agents identified will then be rescreening for drug combinations with synergistic effects.
3. A pilot multi-drug regimen will be tested in mice.

In the past two years we screened eight cell lines using the Maybridge HitFinderTM Collection; DIVERSetTM Collection (Chembridge); LOPAC (Sigma); BIOMOL collection; Prestwick collection (Prestwick Chemical); Seminatural compounds (AnalytiCon Discovery); and the NIH Clinical Collection (BioFocusDPI). This screening identified 45 compounds that inhibited cell proliferation in desmoid tumors by greater than 50% while not affecting normal fibroblast proliferation, and that also are either in use in patients or that have the potential to be rapidly developed for patient care. We then tested these agents using a collection of desmoid tumor cell cultures of different genetic etiologies, and based on this, as well as how available the selected agents are for clinical use today, prioritized 18 for testing in mice that develop desmoid tumors. We completed testing of nine agents, and found that five: Dasatinib (an oral Bcr-Abl tyrosine kinase inhibitor); A multi-target PI3K inhibitor; an FAK inhibitor; Letrozole; and a gamma secretase inhibitor. An additional nine drugs are currently being tested in the mice.

In the next year, we will undertake a rescreening of the five drugs that were effective in mice, by undertaking a sensitization test, in which we first treat the cells with the positively identified agents, and then rescreening them using the libraries. Agents that have a synergistic or additive effect will show lower cell viability than either agent alone. This approach will rapidly identify a multi-drug combination. We will then test the drug combinations in mice. Depending on the number of agents identified, we will either test all combinations, or if the number if too large, we will prioritize which to test based on a pharmacokinetic and then undertake analysis prioritizing agents that can be used in patients at the present time.

This data will identify new treatment regiments, which can rapidly be brought to patient care. In addition, it will identify new pathways activated by beta-catenin and new agents not yet in use for patient care that target desmoid tumors, both of which can be developed in future work which might ultimately identify even more effective therapeutic approaches.

LAY VERSION OF ABSTRACT- Collaboration for a Cure: Identifying new therapeutic targets for desmoid tumors