Abstract 5897: Single cell-derived analysis of desmoid tumors for studying tumor-stroma interactions

<div><p>The interaction between neoplastic and stromal cells within a tumor mass plays an important role in cancer biology. However, it is challenging to distinguish between tumor and stromal cells in mesenchymal tumors because lineage-specific cell surface markers typically used in other cancers do not distinguish between the different cell subpopulations. Desmoid tumors consist of mesenchymal fibroblast-like cells driven by mutations stabilizing beta-catenin. Here we aimed to identify surface markers that can distinguish mutant cells from stromal cells to study tumor–stroma interactions. We analyzed colonies derived from single cells from human desmoid tumors using a high-throughput surface antigen screen, to characterize the mutant and nonmutant cells. We found that CD142 is highly expressed by the mutant cell populations and correlates with beta-catenin activity. CD142-based cell sorting isolated the mutant population from heterogeneous samples, including one where no mutation was previously detected by traditional Sanger sequencing. We then studied the secretome of mutant and nonmutant fibroblastic cells. PTX3 is one stroma-derived secreted factor that increases mutant cell proliferation via STAT6 activation. These data demonstrate a sensitive method to quantify and distinguish neoplastic from stromal cells in mesenchymal tumors. It identifies proteins secreted by nonmutant cells that regulate mutant cell proliferation that could be therapeutically.</p>Significance:<p>Distinguishing between neoplastic (tumor) and non-neoplastic (stromal) cells within mesenchymal tumors is particularly challenging, because lineage-specific cell surface markers typically used in other cancers do not differentiate between the different cell subpopulations. Here, we developed a strategy combining clonal expansion with surface proteome profiling to identify markers for quantifying and isolating mutant and nonmutant cell subpopulations in desmoid tumors, and to study their interactions via soluble factors.</p></div>

Desmoid Tumors in a Dutch Cohort of Patients With Familial Adenomatous Polyposis

10561 Background: The biologic heterogeneity of soft tissue sarcomas (STS) complicates treatment. Metastatic propensity may be determined by gene expression patterns that do not correlate well with morphology. In earlier studies, gene expression patterns were identified that distinguish 2 subsets of clear cell renal carcinoma (RCC), serous ovarian carcinoma (OVCA), and aggressive fibromatosis (AF). We reported the use of a gene set derived from these three studies to separate 73 high grade STS into groups with different probabilities of developing metastatic disease (PrMet). We wished to confirm our findings using an independent data set. Methods: We utilized these gene sets, hierarchical clustering (HC), Kaplan-Meier, and log-rank analyses to examine the Affymetrix HU_133 expression profiles of 309 STS. Results: HC using a pooled gene set derived from the AF-, RCC-, and OVCA-gene sets identified subsets of the STS samples. Kaplan-Meier analysis revealed differences in PrMet between the clusters defined by the first branch point of the clustering dendrogram (p=0.048), and also among the 4 different clusters defined by the second branch points (p<0.0001). Analysis also revealed differences in PrMet between the leiomyosarcomas (LMS), dedifferentiated liposarcomas (LipoD), and undifferentiated pleomorphic sarcomas (UDS) (p=0.0004). HC of the LipoD and UDS samples with the pooled probe set divided the samples into 2 groups with different PrMet (p=0.013, and 0.0002, respectively). HC of the UDS samples also showed 4 groups with different PrMet (p=0.0007). In contrast, HC found no subgroups of the LMS samples. Each individual gene set (AF-, RCC-, and OVCA-) separated the UDS samples into subsets of different metastatic outcome, but only the AF- gene set separated the LipoD samples, and no gene set identified LMS subsets. Conclusions: These data confirm our earlier studies and suggest that this approach may allow the identification of more than 2 subsets of high grade STS, each with distinct clinical behavior, and may be useful to stratify STS in clinical trials and in patient management.

Desmoid tumors (DTs), derived from the abdomen, are a type of rare and aggressive borderline tumor exhibiting high recurrence and malignant potential. The aim of the present study was to investigate the clinicopathological and molecular characteristics of abdominal DT in a Chinese population and to provide clues for selecting the optimal treatment strategy for different types of abdominal DT. The clinicopathological data of 15 consecutive patients with DT was collected. Matched fresh-frozen tumor tissues and peripheral blood samples were used to detect mutations of adenomatous polyposis coli gene (APC), β-catenin (CTNNB1) and MutY DNA glycosylase (MUTYH) using Sanger sequencing. Pearson's test was conducted to analyze the differences between sporadic DT and familial adenomatous polyposis (FAP) associated with DT. Time to progress (TTP) and overall survival curves were estimated using the Kaplan-Meier method and compared using the log-rank test. A review of the patient clinicopathological characteristics revealed that FAP-associated DT exhibited a higher rate of abdominal surgery history (P=0.011), with no significant differences in any other characteristics. Sequencing revealed that mutations in the APC, CTNNB1 and MUTYH genes were common in DT, and only one patient harbored no mutations in these genes. Survival analyses revealed that patients with FAP exhibited shorter TTP (P=0.030). Log-rank test demonstrated a tendency towards shorter TTP in the cases where an R2 resection was performed (P=0.072) and a tendency towards poor prognosis in the cases of DT associated with FAP (P=0.087). In conclusion, Abdominal DTs were prone to occur in patients with FAP with a history of abdominal surgery. Mutations in the APC, CTNNB1 and MUTYH genes were detected in patients with DTs. To the best of our knowledge, this is the first study of abdominal DT occurrence in patients with MUTYH-associated FAP. The prognosis of DT associated with FAP may be worse compared with that of sporadic DT.

Isolated loss of external rotation of the shoulder with the arm at the side is a rare clinical finding in patients presenting with a shoulder problem. Most patients with shoulder stiffness are first seen with a loss of mobility involving at least two planes of motion that usually results from primary or secondary adhesive capsulitis1,2. Soft-tissue tumors of the shoulder girdle are rare3. Among soft-tissue tumors, desmoid tumors are very rare and they most frequently occur in the shoulder girdle and the thigh of adolescents and young adults4. Desmoid tumors are slow-growing benign fibrous neoplasms arising from soft tissue and muscle. They have a notoriously high recurrence rate5,6. Adequate surgical resection can be challenging, and early diagnosis is intuitively beneficial in facilitating surgical management before the tumor has invaded and distorted the regional anatomy. We present the cases of two patients with a desmoid tumor originating in the subscapularis muscle who were first seen with vague progressive shoulder pain and a clinical examination pertinent for only marked limitation of active and passive external rotation of the shoulder with the arm at the side. The patients were informed that data concerning their cases would be submitted for publication. CASE 1. A fifty-three-year-old woman who was right-hand dominant and worked as an administrator presented with progressive pain in the left shoulder, including mild night pain, of insidious onset occurring over a two-year period. The pain was described as a dull ache of variable intensity deep within the shoulder. She reported that she had not had any fevers or other systemic symptoms. The only prior treatment was a subacromial injection of lidocaine and corticosteroid, which had been given by her primary care physician, and it had provided no relief. Plain radiographs …

Distinguishing between neoplastic (tumor) and non-neoplastic (stromal) cells within mesenchymal tumors is particularly challenging, because lineage-specific cell surface markers typically used in other cancers do not differentiate between the different cell subpopulations. Here, we developed a strategy combining clonal expansion with surface proteome profiling to identify markers for quantifying and isolating mutant and nonmutant cell subpopulations in desmoid tumors, and to study their interactions via soluble factors.

Desmoid tumors (aggressive fibromatosis) are locally invasive soft tissue tumors that lack the ability to metastasize. There are no directed therapies or standard treatment plan, and chemotherapeutics, radiation, and surgery often have temporary effects. The majority of desmoid tumors are related to T41A and S45F mutations of the beta-catenin encoding gene (CTNNB1). Using broad spectrum metabolomics, differences were investigated between paired normal fibroblast and desmoid tumor cells from affected patients. There were differences identified, also, in the metabolomics profiles associated with the two beta-catenin mutations, T41A and S45F. Ongoing drug screening has identified currently available compounds which inhibited desmoid tumor cellular growth by more than 50% but did not affect normal fibroblast proliferation. Two drugs were investigated in this study, and Dasatinib and FAK Inhibitor 14 treatments resulted in unique metabolomics profiles for the normal fibroblast and desmoid tumor cells, in addition to the T41A and S45F. The biochemical pathways that differentiated the cell lines were aminoacyl-tRNA biosynthesis in mitochondria and cytoplasm and signal transduction amino acid-dependent mTORC1 activation. This study provides preliminary understanding of the metabolic differences of paired normal and desmoid tumors cells, their response to desmoid tumor therapeutics, and new pathways to target for therapy.

Desmoid tumors are rare benign monoclonal fibroblastic tumors. Their aggressiveness is local with no potential for metastasis or dedifferentiation. Here we report on a 61-year-old patient who presented a locally advanced breast desmoid tumor diagnosed 20 years after post-operative radiotherapy for breast carcinoma. After 2 years of medical treatment, a high-grade undifferentiated pleomorphic soft tissue sarcoma arose within the desmoid tumor. Despite extensive surgery removing both tumors, the patient showed locoregional relapse by the sarcoma, followed by multimetastatic progression, then death 25 months after the surgery. The arising of a soft tissue sarcoma in a desmoid tumor is an exceptional event since our case is the fourth one reported so far in literature. It reinforces the need for timely and accurate diagnosis when a new mass develops in the region of a preexisting desmoid tumor, and more generally when a desmoid tumor modifies its clinical or radiological aspect.

<div>Abstract<p>Desmoid tumors are nonmalignant neoplasms of mesenchymal origin that mainly contain fibroblast lineage cells. These tumors often occur in patients with familial adenomatous polyposis (FAP) coli who have germ line mutations in the <i>APC</i> gene. Given emerging data that has implicated multipotent mesencyhmal stromal cells (MSC) in the origin of mesenchymal tumors, we hypothesized that desmoid tumors may arise in patients with FAP after MSCs acquire somatic mutations during the proliferative phase of wound healing. To test this idea, we examined 16 desmoid tumors from FAP-associated and sporadic cases, finding that all 16 of 16 tumors expressed stem cell markers, whereas matching normal stromal tissues were uniformly negative. Desmoid tumors also contained a subclass of fibrocytes linked to wound healing, angiogenesis, and fibrosis. Using an MSC cell line derived from an FAP-associated desmoid tumor, we confirmed an expected loss in the expression of adenomatous polyposis coli (APC) and the transcriptional repressor BMI-1 while documenting the coexpression of markers for chondrocytes, adipocytes, and osteocytes. Together, our findings argue that desmoid tumors result from the growth of MSCs in a wound healing setting that is associated with deregulated Wnt signaling due to APC loss. The differentiation potential of these MSCs combined with expression of BMI-1, a transcriptional repressor downstream of Hedgehog and Notch signaling, suggests that desmoid tumors may respond to therapies targeting these pathways. <i>Cancer Res; 72(1); 346–55. ©2011 AACR</i>.</p></div>

<div>Abstract<p>Desmoid tumors are nonmalignant neoplasms of mesenchymal origin that mainly contain fibroblast lineage cells. These tumors often occur in patients with familial adenomatous polyposis (FAP) coli who have germ line mutations in the <i>APC</i> gene. Given emerging data that has implicated multipotent mesencyhmal stromal cells (MSC) in the origin of mesenchymal tumors, we hypothesized that desmoid tumors may arise in patients with FAP after MSCs acquire somatic mutations during the proliferative phase of wound healing. To test this idea, we examined 16 desmoid tumors from FAP-associated and sporadic cases, finding that all 16 of 16 tumors expressed stem cell markers, whereas matching normal stromal tissues were uniformly negative. Desmoid tumors also contained a subclass of fibrocytes linked to wound healing, angiogenesis, and fibrosis. Using an MSC cell line derived from an FAP-associated desmoid tumor, we confirmed an expected loss in the expression of adenomatous polyposis coli (APC) and the transcriptional repressor BMI-1 while documenting the coexpression of markers for chondrocytes, adipocytes, and osteocytes. Together, our findings argue that desmoid tumors result from the growth of MSCs in a wound healing setting that is associated with deregulated Wnt signaling due to APC loss. The differentiation potential of these MSCs combined with expression of BMI-1, a transcriptional repressor downstream of Hedgehog and Notch signaling, suggests that desmoid tumors may respond to therapies targeting these pathways. <i>Cancer Res; 72(1); 346–55. ©2011 AACR</i>.</p></div>

Desmoid tumors (DT) are rare, benign, fibroblastic neoplasm with challenging histological diagnosis. DTs can occur sporadically or associated with the familial adenomatous polyposis coli (FAP). Most sporadic DTs are associated with β-catenin gene (CTNNB1) mutations, while mutated APC gene causes FAP disease. microRNAs (miRNAs) are involved in many human carcinogenesis.The miRNA profile was analyzed by microarray in formalin-fixed, paraffin-embedded (FFPE) specimens of 12 patients (8 sporadic, 4 FAP-associated) and 4 healthy controls. One hundred and one mRNAs resulted dysregulated, of which 98 in sporadic DTs and 8 in FAP-associated DTs, 5 were shared by both tumors. Twenty-six miRNAs were then validated by RT-qPCR in 23 sporadic and 7 FAP-associated DT samples matched with healthy controls. The qPCR method was also used to evaluate the CTNNB1 mutational status in sporadic DTs. The correlation between sporadic DTs and miRNA expression showed that miR-21-3p increased in mutated versus wild-type DTs, while miR-197-3p was decreased. The mRNA expression of Tetraspanin3 and Serpin family A member 3, as miR-21-3p targets, and L1 Cell Adhesion Molecule, as miR-197-3p target, was also evaluate. CTNNB1 mutations associated to miRNA dysregulation could affect the genesis and the progression of this disease and help histological diagnosis of sporadic DTs.

Introduction: Desmoid tumor is a rare aggressive tumor where there is no satisfying systemic treatment. Previous studies showed increased expression of transforming growth factor-β (TGF-β) in the desmoid tumor. Here, we analyzed genomic and transcriptomic data of tumor samples from patients with desmoid tumor to evaluate therapeutic applicability of inhibition of TGF-β response. Methods: We collected pre-treatment desmoid tumor samples from 31 patients in Yonsei cancer center, (YCC)-desmoid cohort and performed targeted next-generation sequencing and whole RNA sequencing. To compare the data with other cancer types, we used whole exome sequencing and whole RNA sequencing data from sarcoma patients from Yonsei Cancer Center (YCC-sarcoma cohort, n = 17 and n =53, respectively) and The Cancer Genome Atlas (TCGA, n = 9,235). Transcriptomic data was normalized throughout the three cohorts to reduce batch effect. To evaluate the enrichment of TGF-β responsive signature (TBRS) in tumor and microenvironment, we calculated the mean expression values of fibroblast-TBRS (F-TBRS) from previous study. Results: In the two sarcoma cohorts (YCC-desmoid and YCC-sarcoma cohorts), desmoid tumor samples had the highest F-TBRS score among the various types of sarcoma. When compared with other cancer types in TCGA, desmoid tumor samples showed the higher F-TBRS than all other cancer types except for pancreatic adenocarcinoma. In the desmoid tumor samples, CTNNB1, GNAQ, and APC mutations were the top 3 frequent mutations (23, 19, and 2 samples, respectively). In the merged two sarcoma cohorts, CTNNB1/APC mutation and GNAQ mutation were both associated with higher enrichment in F-TBRS (p < 0.001, respectively). Conclusion: Desmoid tumors are enriched with expression of genes associated with TGF-β response of fibroblast compared with other cancer types including other sarcomas. In addition, CTNNB1, APC and GNAQ mutations were associated with higher enrichment in TBRS. Therapeutic intervention to decrease the TGF-β response of fibroblast by using TGF-β receptor inhibitor may show clinical benefit in patients with desmoid tumors. Citation Format: Changhee Park, Kum-Hee Yun, Sanghoon Song, Jin Kyung Lee, Jiyeon Ryu, Bitna Oh, Sunjin Hwang, Ki Baik Hahm, Seung-Jin Kim, Seung Hyun Kim, Chan-Young Ock, Hyo-Song Kim. Genomic and transcriptomic analyses of desmoid tumor reveals enrichment of transforming growth factor beta responsive signature [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2250.

BackgroundInternational experts recommended pazopanib to treat a 9 year old boy with a recurrent desmoid tumour. The tumour had progressed, leading to blockage of the airway and dysphagia. The child had previously received three lines of intravenous chemotherapy as well as surgery and local radiotherapy. Treatment intent was to give an effective oral therapy with minimal side effects aiming to maintain a reasonable quality of life and prevent further life threatening respiratory compromise. As the child had an unsafe swallow, the drug would need to be given via a gastrostomy tube.Pharmacy InputA literature review was completed to establish the evidence to support the use and dose of pazopanib to treat desmoid type tumours in children.1 2 Attempts made to find a commercial supply of pazopanib liquid yielded no results. In the absence of any data or experience from other principal children’s cancer centres in the UK, first principles were used to review the physico-chemical properties of the molecule. Given the highly insoluble nature of pazopanib, an extemporaneous formulation for a pazopanib suspension was developed using Orasweet®, by adapting a formula suggested by the University of Oklahoma.3 A risk assessment for the preparation process was completed and a mini isolator was re-commissioned for this purpose following appropriate testing by Quality Control. The Department of Pharmacology at Newcastle University were contacted to establish if therapeutic drug level analysis might be possible. Local approval for pazopanib use was obtained from the Drug and Therapeutic Group and an IFR was submitted to NHS England.ChallengesIt was challenging to establish a dose regimen since the recommended paediatric dose from Phase I studies of pazopanib were dependent on the formulation used.1 4 Pharmacokinetic sampling was not possible as no assay had been developed in the UK. The lack of availability of any commercial or compassionate use liquid preparation meant that the only way of giving the drug to this child was to prepare an extemporaneous preparation, despite paucity of evidence to support the stability of the preparation. Funding was not approved by NHS England; local funding was agreed.Outcome and DiscussionThe child commenced treatment with the suspension in October 2018, administered via the gastrostomy tube. By July 2019 the patient had completed 10 cycles of therapy. Treatment was well tolerated. Minor side effects included abdominal and leg pain, vomiting, and a change in hair colour. The patient has had a good clinical response and a recent scan has shown substantial improvements in morphological appearance and size of the tumour.These results indicate that a locally prepared extemporaneous oral chemotherapy suspension can be successfully used to deliver treatment for a rare type of children’s cancer. Pharmacy colleagues from across the department collaborated to facilitate this novel treatment option.ReferencesGlade Bender J, Lee A, Reid J, et al. Phase I pharmacokinetic and pharmacodynamic study of pazopanib in children with soft tissue sarcoma and other refractory solid tumors: a children’s oncology group phase I consortium report. JCO 2011;31:3034–3043.Agresta L, Lee H, Turpin B, et al. Pazopanib therapy for desmoid tumors in adolescent and young adult patients. Pediatr Blood Cancer 2018;65:e26968.Allen L Jr. Pazopanib 50 mg/ml oral suspension. US Pharm 2015;40:61–62.Heath E, Forman K, Malburg L, et al. A phase I pharmacokinetic and safety evaluation of oral pazopanib dosing administered as crushed tablet or oral suspension in patients with advanced solid tumors. Invest New Drugs 2012;30:1566–1574.

The development of desmoid fibromatosis after tumor resection may mimic local recurrence. To our knowledge, this phenomenon has not been reported after extremity sarcoma resection. We report four cases of desmoid-type fibromatosis ("desmoid tumors") mimicking local recurrence after extremity sarcoma resection. We retrospectively reviewed the records of patients treated for extremity sarcoma by our orthopedic oncology service from 2014 to 2019 and identified four patients with biopsy-proven desmoid tumors. We extracted clinical, pathologic, radiographic, and operative data for the primary neoplasms and desmoid tumors. Four patients with postresection surveillance magnetic resonance imaging suspicious for local recurrence underwent further analysis showing desmoid tumors. Patients underwent image-guided needle biopsy, with specimens demonstrating fibromatosis-type histologic characteristics. Two cases were β-catenin positive. Desmoid tumors were managed with observation. No patient had experienced local or distant recurrence of the primary tumor at a mean follow-up of 30 months after resection (range, 23-34 months); none underwent surgery for symptoms of desmoid tumors. Desmoid tumors should be considered part of the differential diagnosis when assessing patients with radiographic concern for postresection local recurrence of extremity bone and soft-tissue sarcoma. An image-guided needle biopsy can inform diagnosis and management.

Imaging and intervention for soft tissue tumours in the era of locoregional therapies and immunotherapy.