Molecular targets and novel therapeutic avenues in soft-tissue sarcoma

Soft-tissue sarcoma ( sts ) represents a heterogeneous group of rare tumours, and a significant number of affected patients will develop metastatic disease. Outcomes in the population with metastatic disease are generally poor, especially after progression on standard chemotherapy. The advent of personalized medicine has permitted oncologists to offer targeted treatment, thus addressing the limited treatment options and poor prognosis after progression on first-line chemotherapy. In this review, we delineate the existing data and therapeutic successes with respect to existing and emerging molecular targets in sts and options for immunotherapy in sts . Our review also summarizes emerging clinical trials that are currently recruiting patients.


INTRODUCTION
Soft-tissue sarcoma (sts) represents a heterogeneous group of rare tumours 1 . Of patients with early-stage disease treated with locoregional modalities such as surgery and radiation, approximately 40% will develop metastatic disease; and of patients presenting for the first time with sts, 10% will be diagnosed with metastatic disease 2 . Goals of treatment in the metastatic setting are to palliate symptoms and improve quality of life and survival. Doxorubicin with or without ifosfamide is the firstline standard of care for the treatment of metastatic sts. Angiosarcomas are also sensitive to anthracycline-based chemotherapy 3 , but taxane-based chemotherapy also has significant activity [4][5][6][7][8][9][10] .
For patients with metastatic disease, outcomes are generally poor, with overall survival ranging between 12 and 19 months, and little improvement in outcomes has been observed since the start of the 2000s 2 . There is therefore an unmet clinical need to develop and implement novel treatment modalities that will improve outcomes for patients with metastatic sts.
The advent of personalized medicine has permitted oncologists to offer targeted treatment, addressing the limited treatment options and poor prognosis after progression on first-line chemotherapy. In this review, we delineate the existing data and therapeutic successes with respect to existing and emerging molecular targets in sts. The stss with targetable molecular abnormalities in the adult population reviewed in detail here include desmoid tumours, tenosynovial giant-cell tumours, SMARCB1deficient sarcomas, and TRK fusion sarcomas. Nextgeneration sequencing (ngs) has allowed for the identification of driver mutations and potentially targetable molecular abnormalities in patients with limited treatment options, and recent data using ngs in the sts setting are also discussed in the review.

REVIEW Desmoid Tumours
Desmoid tumour, also known as aggressive fibromatosis, is a rare disease of fibroblastic proliferation, characterized by mutations of the CTNNB1 (beta-catenin) or APC (familial adenomatous polyposis) gene. Although this neoplasm lacks metastatic potential, it is often locally aggressive and is characterized by infiltrative growth of adjacent tissues into the mesentery, neurovascular structures, and visceral organs 1 . Patients diagnosed before the age of 40 are more likely to be women, and the most common site of onset in these patients is the abdominal wall. After the age of 40, these tumours develop in men and women equally, and no predilection for a particular tumour site is observed in older adults. Desmoid tumours are treated initially with active surveillance 11,12 , because about 20% of patients can experience spontaneous regression 13  symptoms that cannot be controlled with pain control measures, active therapy is offered. If the patient is a candidate for surgery and the desmoid tumour is located in the abdominal wall, surgery is generally the next step. It is important to note that rates of local recurrence are high 1 . If the desmoid tumour arises in other sites, such as the head-and-neck area, the extremities, or the intra-abdominal or pelvic areas, systemic therapy is usually offered. Typically given for 6-12 months, common cytotoxic chemotherapy regimens include low-dose vinblastine (or vinorelbine) and methotrexate for patients who have low burden-of-disease symptoms 14 , and liposomal doxorubicin for patients having more severe symptoms or experiencing progression after a prior regimen 15 .

Targeted Therapy in Desmoid Tumours
Imatinib: Imatinib has a limited role in patients with desmoid tumours. A phase ii study in which 38 patients with unresectable desmoid tumour received imatinib reported an overall response rate of 19% 16 , but no control group was used. A retrospective case series reviewed the response rate to imatinib combined with radiation therapy in 4 patients with unresectable or recurrent desmoid tumours; all patients either experienced a partial response or had stable disease 17 . Given the paucity of prospective data, the use of imatinib in patients with desmoid tumours cannot be recommended at this time.
As already mentioned, desmoid tumours are more likely to be diagnosed in women before the age of 40, often during pregnancy or within 1 year postpartum 18 . In support of those epidemiologic findings, immunohistochemical studies have demonstrated the presence of estrogen receptor beta in 90% of desmoid tumours 19 . The selective estrogen receptor modulator tamoxifen has been used in the treatment of desmoid tumours 20,21 , but a more recent publication showed no relationship between symptomatic benefit and the magnetic resonance imaging response with the use of tamoxifen 22 .
Sorafenib: Sorafenib, an oral multi-targeted receptor tyrosine kinase inhibitor, has shown activity in desmoid tumours. A retrospective analysis of 26 patients evaluated the efficacy of sorafenib in desmoid tumours that were unresectable or resectable only with amputation 23 . The most common primary site in the cohort was abdomen or pelvis. A significant proportion of patients (42%) received sorafenib in the first-line setting. Sorafenib was started at 400 mg daily and was decreased in cases of dose toxicity. As evaluated by the Response Evaluation Criteria in Solid Tumors (version 1.1), sorafenib was associated with a partial response rate of 25%, with 70% of patients having stable disease. Additionally, approximately 70% of patients reported a subjective decrease in pain and use of analgesics with sorafenib, with most of those clinical improvements being noted within 2 weeks of sorafenib initiation. The study also noted that the Response Evaluation Criteria in Solid Tumors might underestimate the treatment response and suggested that the signal from T2-weighted magnetic resonance imaging might be more clinically relevant and accurate.
That retrospective trial prompted Gounder et al. 24 to conduct a prospective double-blind randomized phase iii trial to evaluate the safety and efficacy of sorafenib in the treatment of desmoid tumours. The study randomized 87 adult patients with either symptomatic, recurrent, or primary desmoid tumour that was deemed unresectable to receive sorafenib at a dose of 400 mg daily or placebo. Patients who had previously received sorafenib were excluded from the trial. Crossover after progression was allowed for patients receiving placebo. The 2-year progression-free survival rate was 81% in the sorafenib group compared with 36% in the placebo group [hazard ratio for progression or death: 0.13; 95% confidence interval (ci): 0.05 to 0.31; p < 0.001]. Before crossover, the objective response rate was 33% in the sorafenib group (95% ci: 20% to 48%) and 20% in the placebo group (95% ci: 8% to 38%). The most frequently reported adverse effects in the sorafenib group were grade 1 or 2 rash, fatigue, hypertension, and diarrhea. Of the patients in the sorafenib group, 29% experienced a grade 3 adverse event, and the incidences of grade 4 thrombocytopenia and anemia were each 2%.
The trial demonstrated a role for sorafenib in delaying disease progression in patients with desmoid tumours, but also supported a role for observation in patients without symptoms, because 20% of the patients in the placebo group experienced spontaneous regression.

Gamma-Secretase Inhibitor PF-03084014:
A phase ii trial evaluated gamma-secretase inhibitor PF-03084014 (nirogacestat) in 17 patients with refractory desmoid tumours. Results were encouraging, with 5 patients (29%) experiencing a confirmed partial response, and another 5 patients having prolonged stable disease 25 . Those findings have led to a randomized placebo-controlled phase iii study of nirogacestat, which is currently ongoing (see NCT03785964 at https://ClinicalTrials.gov/).

Tenosynovial Giant-Cell Tumours
Tenosynovial giant-cell tumour (tgct), previously known as pigmented villonodular synovitis, is a rare and locally aggressive neoplasm of the joint or tendon sheath 1 . This neoplasm is characterized by the proliferation of synovial cells that initiate recruitment of inflammatory cells such as macrophages and histiocytes 1 . The tumours can be either localized or diffuse, the latter characterized by diffuse proliferation of the entire synovium, most commonly affecting the knee joint 26 . Although most of these neoplasms occur within the joints, a diffuse form can occur, and extraarticular forms have also been described 27 . Rarely, tgct can metastasize to the adjacent lymph nodes and to the lungs 28 . Localized tgct is curable with arthroscopic or open surgery, but the diffuse form of the disease is associated with a high rate of local recurrence 29 .

Targeted Therapy in TGCT
Pexidartinib: The neoplastic cells in giant-cell tumours can overexpress colony-stimulating factor 1 (csf1), with signalling between csf1 and the csf1 receptor (csf1r) 30,31 . Colony-stimulating factor 1 is often found on the membranes of immune cells. Given the recruitment of inflammatory cells by the neoplastic synovial cells implicated in the pathophysiology of tgct, it was hypothesized that inhibition of signalling between csf1 and csf1r could target the underlying cause of the disease.
A phase i study of 43 patients receiving pexidartinib, a dual inhibitor of csf1r and the c-Kit receptor tyrosine kinase, led to a multicentre single-cohort extension study of pexidartinib administrated at a dose of 1000 mg daily in 23 patients with tgct 32 . Patients with such tumours were included in the study if they had shown disease progression or if the tumour was recurrent or inoperable, or had required extensive surgery for complete resection. Most patients had disease within the knee. Of the 23 patients, 12 experienced a partial response, and 7 had stable disease, representing an overall response rate of 52% (95% ci: 32% to 73%) and a rate of disease control (complete response, partial response, or stable disease) of 83% (95% ci: 67% to 98%). Treatment-related adverse events occurred in 25% of the study population, and the most common adverse events included hair colour change, fatigue, nausea, dysgeusia, and periorbital edema. The most common grade 3 adverse event was elevated serum aspartate aminotransferase or alanine aminotransferase, which occurred in 3 patients.
A phase iii randomized controlled trial of pexidartinib in 174 patients with tgct was recently published in The Lancet 33 . The trial was conducted in two parts, the first being a randomized controlled trial comparing pexidartinib with placebo in patients with inoperable disease. Patients who completed the first part of the trial were eligible to enter the second part, in which they were offered open-label pexidartinib in an intention-to-treat analysis. Compared with 0% in the placebo group, 39% of the patients in the pexidartinib group achieved a response (95% ci: 27% to 52%; p < 0.0001). In a secondary endpoint analysis, patients receiving pexidartinib, compared with those in the placebo group, reported improved range of motion and physical functioning. Of the patients in the pexidartinib group, 13% experienced serious adverse events (a toxicity profile similar to that described in the phase i study outlined earlier), with elevations in liver enzymes being a frequent and important side effect.
Taken together, the data demonstrate that pexidartinib should be considered in the treatment of individuals with tgct and functional limitation where surgery is not possible.
Emactuzumab: A phase i study in 29 patients evaluated the use of emactuzumab (an immunoglobulin G1 subclass humanized recombinant monoclonal antibody directed against csf1r expressed on the surfaces of macrophages) in patients with unresectable tgct 34 . Expansion cohorts for the study are currently ongoing (see NCT01494688 at https://ClinicalTrials.gov/). In the safety expansion cohort, independent central review of magnetic resonance imaging was conducted for 28 of 29 patients, and independent central review of positron-emission tomography imaging, for 26 of 29 patients. Of the 28 reviewed patients, 24 (86%) achieved an objective response, and 19 (68%) achieved a partial response. Of the 26 reviewed patients, 24 (92%) achieved a metabolic response. Common adverse events after emactuzumab treatment were facial edema, fatigue, asthenia, and pruritus. Drug-induced lupus erythematous was a rare, but important, side effect.

Nilotinib:
Nilotinib is a multi-targeted tyrosine kinase inhibitor that has activity against csf1r 35 . Its efficacy was evaluated in a phase ii study involving 51 patients with advanced or unresectable tgct 36 . Although a high proportion of those patients were progression-free at 12 weeks, the overall response rate was lower than had been seen with pexidartinib and emactuzumab. Nearly all patients experienced at least 1 adverse event, and 11% experienced at least 1 grade 3 adverse event (headache, hepatic enzyme abnormalities, diarrhea).
Imatinib: Imatinib has weak activity against csf1r 35 . In a retrospective series of 29 patients with locally advanced or metastatic tgct, the objective response rate was 19%, and 74% of the patients reported symptomatic or functional improvement 37 . Aside from that retrospective series, case reports have described success with imatinib, but the data are currently insufficient to recommend this agent in the treatment of tgct.

Tazemetostat In Ini1-Deficient Sarcomas
Epithelioid sarcoma is a rare sts, and loss of Ini1 occurs in more than 90% of these tumours. Because Ini1 is a negative regulator of the oncogenic driver ezh2, inhibition of ezh2 was hypothesized to potentially be able to lead to tumour regression 38 . Tazemetostat, an inhibitor of ezh2, was evaluated in a phase ii study enrolling 62 patients with advanced Ini1-deficient epithelioid sarcoma. The overall response rate was 13%. Additionally, 35 patients had a best response of stable disease. Those results came from an interim analysis and remain unpublished.

Larotrectinib in TRK Fusion-Positive Sarcoma
Rarely, adults with sts have gene fusions involving NTRK. Larotrectinib, a selective NTRK inhibitor, was shown to be effective in a combined analysis of 55 patients with various NTRK fusion-positive solid tumours-an important subset of which consisted of patients with sts 39 . Of 11 patients with sts showing NTRK fusions, 10 experienced an objective response, and 1 achieved a complete response. No patient in the study discontinued larotrectinib because of treatment-related toxicity. The most common side effects included elevations in liver enzymes, fatigue, nausea, vomiting, dizziness, diarrhea, constipation, and cough.

MET/ALK Inhibition
Thus far, combined met and alk inhibition using crizotinib has been evaluated in a phase ii study of 20 patients with chemotherapy-refractory alveolar rhabdomyosarcoma 40 . More common in children and adolescents, alveolar rhabdomyosarcoma is a rare subtype of sts in adults, accounting for only 3% of cases. Unfortunately, the crizotinib study did not reveal any meaningful activity in the adult patient population. It is important to note that most patients in the study did not harbour an ALK mutation. A second study evaluated the role of met and alk inhibition in 53 patients with alveolar soft-part sarcoma, another orphan disease in adults 41 . That study showed important clinical activity in patients with MET-positive disease, most of whom had stable disease with treatment.

Pembrolizumab
Pembrolizumab, a PD-1 inhibitor, was tested in a phase ii trial involving 86 patients with refractory sts 42 . The primary endpoint of overall response was not met in the study, but pembrolizumab was associated with promising response rates in patients with undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. Results of expanded cohorts for those sts subtypes are awaited. A second phase ii study, this time in multiple cohorts of patients with sts, demonstrated limited activity for pembrolizumab in combination with cyclophosphamide 43 . The combination of nivolumab (a PD-1 inhibitor) and ipilimumab (a ctla-4 inhibitor) showed promising results in a phase ii trial involving patients with metastatic sarcoma (not limited to sts) 44 .
Between 1% and 5% percent of patients with sts have high microsatellite instability, which usually predicts response to immune checkpoint inhibitors targeting PD-1 or PD-L1 45 . Although not tested specifically in patients with sts, immune checkpoint inhibitors might reasonably be considered for patients with high microsatellite instability who progress on standard treatment.

New York Esophageal Squamous Cell Carcinoma 1
New York esophageal squamous cell carcinoma 1 (ny-eso-1) is a marker on both T cells and B cells that can be targeted by CMB305, an active immunotherapy regimen that uses dendritic cell-targeting lentiviral vectors. A phase ib study that enrolled 25 patients with treatment-refractory sts demonstrated interesting response rates, with favourable progression-free and overall survival outcomes 46 .

Adoptive T Cell Transfer
Adoptive transfer of T cells engineered to express ny-eso-1 was studied as part of an ongoing phase ib trial in patients with synovial sts, demonstrating, in 50% of patients (6 of 12), antitumour responses that were characterized by tumour shrinkage over several months. In the study, correlative science demonstrated that self-regenerating pools of CD8+ ny-eso-1 T cells produce a continuing supply of effector cells for several months 47 . Two other studies examining similar treatment are currently underway (see NCT01477021 and NCT01477021 at https://ClinicalTrials. gov/). Other targets for adoptive cell therapy such as mage-A3, mage-A4, and mage-A10 are also emerging and will be studied in clinical trials in the near future 48 .

Chimeric Antigen Receptor T Cell Therapy
Chimeric antigen receptors (cars) provide another way of invigorating the immune system to kill cancer cells. In this technique, T cells are modified and equipped with cars that redirect the antigen specificity of immune effector cells. Treatments can have important clinical activity, but sometimes cause severe adverse events by way of a cytokine storm. Use of this technique is currently being studied for many solid tumours, including a GD-2-targeting c a r in ganglioside-positive sarcomas (NCT02107963, NCT01953900) 48 .

Vaccine-Based Therapies
Vaccine-based therapies have been used as another method of stimulating the anticancer immune response. As mentioned earlier, the potential for using ny-eso-1 as a vaccinebased therapy is a promising emerging option. Another vaccine trial (of SYT-SSX breakpoint peptide vaccine) that enrolled 21 patients with synovial sts demonstrated encouraging results 49 .

Targeted Therapies
Approximately one third of patients with sts have specific genetic alterations, and it is therefore important to consider targeted therapy as a potential therapeutic avenue. A large study of 584 patients that sought to characterize genomic alterations in adult patients with sts was recently published. The authors found a median of 4 genomic alterations per patient. At least 1 targetable genomic alteration was found in 239 patients (41%) 50 . Another large study of 5635 patients examined the role of ngs in sts, finding meaningful clinical impacts. For example, nearly 10% of patients were found to have mutations suspicious for germline defects (including BRCA). In 16% of patients, treatment-linked alterations were found that are known to respond to a drug approved by the U.S. Food and Drug Administration, and in 7%, alterations were found that could make the patient eligible for a clinical drug trial. Of the patients in the study, 30% were enrolled to a clinical study, and partial and complete responses were seen in patients with alterations in NTRK, IDH1, BRAF, pi3k/mtor, and mdm2, among others. Furthermore, use of ngs altered the initial pathology diagnosis in 5% of patients enrolled to the study. In another study involving 20 patients with liposarcoma for whom ngs was used, 11 subsequently received mdm2 or cdk4 inhibition as part of a phase i clinical trial 51 . Meaningful clinical activity was observed in 7 patients who experienced stable disease. Although the possibilities are exciting, the incidence of actionable mutations leading to outcome-altering treatment is unknown, and given the cost of the technique, widespread use of ngs cannot be recommended at this time.

SUMMARY AND FUTURE DIRECTIONS
A significant proportion of patients with sts present with, or develop, metastatic disease. Given the poor outcomes in sts, novel therapeutic avenues have to be developed. Many clinical trials are actively recruiting patients with advanced sts (Table i). Promising options include the use of sorafenib in desmoid tumours, pexidartinib or emactuzumab in tgct, tazemetostat in Ini1-deficient sarcomas, and larotrectinib in TRK fusion-positive sarcomas. Immunotherapy is also proving to be an emerging and promising avenue for patients with advanced sts. Next-generation sequencing might provide insights for personalized therapy. Altogether, novel therapies in sts are proving to be an exciting and active area of cancer research, and more data are eagerly awaited.

CONFLICT OF INTEREST DISCLOSURES
We have read and understood Current Oncology's policy on disclosing conflicts of interest, and we declare the following interests: TA