Purpose:

Treatment options for recurrent or refractory Ewing's sarcoma (ES) are limited. Vigil is a novel autologous tumor cell therapy expressing bi-shRNA furin/GMCSF plasmid, which previously demonstrated monotherapy activity in advanced ES. Herein we report safety and evidence of benefit to Vigil for ES as potential treatment.

Patients and Methods:

In this pilot trial, eligible patients with recurrent or refractory ES who failed initial standard-of-care therapy received treatment with temozolomide (TEM) 100 mg/m2/day oral and irinotecan (IRI) 50 mg/m2/day oral, Days 1 to 5, in combination with Vigil (1 × 106–107 cells/mL/day intradermal, Day 15), every 21 days (Vigil/TEM/IRI). Objective response rate (ORR) by RECIST v1.1, progression-free survival (PFS), and overall survival (OS) were assessed. Circulating tumor (ct) DNA analysis was done by patient-specific droplet digital PCR on baseline and serially collected on-treatment samples.

Results:

Eight of 10 enrolled patients were evaluable for safety and efficacy (mean age 24.6; 12.6–46.1 years old); 2 did not receive Vigil. Seven of 8 patients previously received TEM/IRI. No Vigil-related adverse events were reported. Common ≥Grade 3 chemotherapy-related toxicity included neutropenia (50%) and thrombocytopenia (38%). We observed two partial response patients by RECIST; both showed histologic complete response without additional cancer therapy. Median PFS was 8.2 months (95% confidence interval, 4.3–NA). Five patients showed stable disease or better for ≥6 months. Patient-specific EWS/FLI1 ctDNA was detectable in all 8 evaluable patients at baseline. Changes in ctDNA levels corresponded to changes in disease burden.

Conclusions:

Results demonstrated safety of combination Vigil/TEM/IRI.

Translational Relevance

Previously we have shown efficacy of single-agent Vigil in a phase I clinical trial, with a median overall survival (OS) of 24 months and 1-year OS rate of 73% compared with historical control of 23%. The efficacy of Vigil in combination with temozolomide/irinotecan (TEM/IRI) in recurrent Ewing's patients who had failed prior TEM/IRI treatment is demonstrated. There were no safety concerns with the combination and 2 patients demonstrated partial response and 3 patients achieved prolonged stable disease. We also investigated the use of ctDNA levels to monitor therapeutic response and found correlation with majority of patients, but in 2 responding patients results were variable. Analysis of ctDNA correlation to response in a larger cohort of patients is indicated. This work also further demonstrated safety of Vigil in patients with Ewing's sarcoma and justifies further study of Vigil with ctDNA assessment in Ewing's sarcoma.

Ewing's sarcoma (ES) is a malignant tumor of the bone and surrounding soft tissues occurring predominantly in young adults. It is classified as a peripheral neuroectodermal tumor (PNET), and is defined by specific chromosomal translocations resulting in gene fusions of EWSR1 with FLI1 (>80%), ERG (∼15%), and other transcription factors (<5%; ref. 1). The 5-year overall survival (OS) for patients with metastatic, recurrent ES is only about 15%. Several chemotherapy agents and combinations are widely used in attempt to prolong progression-free survival (PFS), however success is limited. The combination of irinotecan (IRI) and temozolomide (TEM), in relapsed patients with ES has demonstrated an objective response rate (ORR) of 55% and median PFS (mPFS) 5.5 months and median OS (mOS) up to 12 months has been observed (2). The IRI/TEM 1-year overall and event-free survival rates were 54.2% and 44.4%, respectively. Adding vincristine to IRI/TEM increased the ORR to 68.1%, but also increased toxicity without an additional survival advantage (3). Recent data from the rEECur trial indicate an even lower response in relapsed/refractory Ewing's patients receiving IRI/TEM of just 20%, median PFS of 4.7 months [95% confidence interval (CI), 3.4–5.7], and median OS of 13.9 months (95% CI, 10.6–18.1; ref. 4). This trial was designed to evaluate topotecan and cyclophosphamide, IRI/TEM, gemcitabine, and docetaxel or high-dose ifosfamide and the least two effective arms were dropped. At the first interim analysis, gemcitabine and docetaxel was dropped, and at the second interim analysis, IRI/TEM was determined to be less effective than the remaining two arms.

We have previously described use of a novel autologous tumor cell vaccine Vigil in patients with relapsed ES, which was constructed from harvested tumor tissue during palliatiave surgical intervention (5, 6). The patient-specific cancer vaccine incorporates a multigenic plasmid encoding the human immune-stimulatory GMCSF gene and a bifunctional short-hairpin RNA construct, which downregulates the proprotein convertase furin and its downstream targets TGFβ1 and TGFβ2 (7–9). Vigil is designed to enhance patient-specific cancer neoantigen expression via upregulation of MHC-II and dendritic cell interaction with effective, systemic antitumor immune responses.

In a phase I clinical trial, Vigil demonstrated a 73% 1-year OS rate compared with 23% in the matched control arm, with a median OS of 24 months (5). Given the limitations of treatment options available to patients with relapsed ES, we enrolled 10 patients and 8 evaluable patients received Vigil in a safety run-in cohort as part of a phase II clinical trial to explore safety and clinical benefit of Vigil combined with IRI/TEM.

We also explored relevance of detecting circulating tumor DNA (ctDNA) in blood samples collected from patients prior to therapy and on-therapy as a potential method for monitoring therapeutic response (10, 11).

Study design

This was a pilot study of Vigil in combination with TEM/IRI in patients with recurrent or refractory ES (NCT02511132) at three sites (Cleveland Clinic, Memorial Sloan Kettering Cancer Center, Texas Oncology). All patients included in the study signed informed consent for tissue procurement and treatment on the study according to the requirements of each individual institution and in accordance with the Declaration of Helsinki. Accrual period was 9.5 months.

Patient characteristics

Patients with the following characteristics were included: (i) histologically confirmed Ewing's sarcoma family of tumors (ESFT) and (ii) refractory or intolerant to at least first-line systemic chemotherapy. Patient characteristics including age, gender, Karnofsky performance status (KPS), prior therapies, toxicity, date of progression, response to therapy, date of last follow-up, or death were obtained from the patient clinical chart and collected in a study-specific case report form. Median follow-up time was 18.5 months (range, 5.7–29.5 months) from the time of treatment start.

Dose regimen and schedule

The plasmid construction, cGMP manufacturing, and tissue processing including Vigil construction were carried out as described previously (8, 9). On-study dosing was as follows: TEM 100 mg/m2/day oral and IRI 50 mg/m2/day oral, days 1 to 5, and Vigil 1 × 106 to 107 cells/mL/day intradermal, on Day 15, every 21 days. Gastrointestinal prophylaxis was permitted per protocol. Treatment was discontinued at progression or unacceptable toxicity, or once all constructed Vigil doses (4–12 doses/patient) were exhausted.

Safety and efficacy assessment

All patients who received at least one full cycle of Vigil/TEM/IRI were included in the evaluable patient population analysis for efficacy. Patients who received a single dose of all three agents were evaluable for safety. Safety was assessed and graded according to the Common Toxicity Criteria for Adverse Events (CTCAE) version 4.03.

Response (ORR) was assessed using the RECIST v1.1 (12). Radiologic assessment was performed locally (investigator-assessed) and confirmed by third party review (WorldCareClinical). Patients were assessed for response every 12 weeks and within 45 days of the last injection or disease recurrence or progression. Objective response was defined by complete response (CR) and partial response (PR). ORR was calculated as the proportion of patients achieving either CR or PR. Disease-free status was defined by confirmed complete remission by histology. PFS and OS were estimated according to the Kaplan and Meier method including 95% CIs and calculated from the first dose of study drug to tumor progression or death or last contact with the patient.

Plasma collection and processing for ctDNA analysis

Plasma samples were collected for every patient prior to Day 1 study dose administration at baseline and prior to Cycle 2. Plasma samples were also collected for some patients at up to seven additional timepoints, including prior to and during subsequent treatment cycles, end of treatment, and follow up, to evaluate the use of ctDNA fusion sequences for therapeutic monitoring and response to treatment.

Cell-free DNA was extracted from up to 2 mL of plasma (range, 0.5–2 mL) using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Tumor DNA was extracted from FFPE preserved samples (2–4 unstained slides) using the QIAamp DNA FFPE Tissue Kit (Qiagen). DNA quantification was performed using either Quant-iT PicoGreen dsDNA Assay Kit or Qubit dsDNA HS Assay Kit (both Thermo Fisher Scientific).

Tumor and cell-free DNA sequencing

To identify the patient-specific EWS/FLI translocation breakpoint, next-generation sequencing was performed on the first two timepoints of plasma for 8 patients (baseline and pre-cycle 2), and tumor samples for 7 patients. Library preparation was performed using KAPA Hyper Prep Kit (KAPA Biosystems) using barcoded adapters. Libraries were assessed by Bioanalyzer and quantified using the MiSeq Nano (Illumina). Hybrid capture sequencing with the TranSS-seq panel was performed and analyzed as described previously (10), with an average mean target coverage of 853× (range: 124–1,485×). All sequencing was performed using a HiSeq 2500 (Illumina).

ctDNA quantification by ddPCR

To quantify ctDNA content in plasma samples for pretreatment and all on-treatment samples collected on this study, PCR primers were designed with Primer3Plus (https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) to amplify the patient-specific translocations (Supplementary Table S1). Sample-specific primers were validated with tumor DNA or synthetic DNA (Integrated DNA Technologies). DNA extracted from the EW8 Ewing sarcoma cell line was used as a negative control for patient-specific primers. Bio-Rad designed primers for the RPP30 were used. Droplet digital PCR (ddPCR) was performed with 5 ng of DNA or pre-capture sequencing libraries using QX200 EvaGreen SuperMix and the QX200 Droplet Digital PCR system (Bio-Rad). Analysis was performed using the QuantaSoft program to determine the concentration of primer amplified DNA (Bio-Rad). Reactions were performed in duplicate. To determine the ctDNA content of each sample, we divided the number of translocation positive droplets by half the number of RPP30-positive droplets for each replicate. Replicates were averaged.

Data availability statement

Gradalis, Inc. will share data that support the findings of this study. Data sharing requests from qualified scientific and medical researchers can be sent to Gradalis, Inc. Requests will be reviewed by Gradalis, Inc. based on the medical and scientific merit of the project, purpose, and data availability. The clinical trial protocol and synopsis are available upon request.

Patient population

Ten patients were identified for the pilot study, however two were unevaluable for response to Vigil. One dropped out after registration and did not receive on-study (Vigil/TEM/IRI) treatment; another was enrolled and received first dose of on-study chemotherapy (TEM/IRI), however was taken off study by investigator due to rapid disease progression before receiving Vigil. All enrolled patients had successful Vigil manufacture. Eight patients were evaluable for safety and response to Vigil/TEM/IRI. The mean age was 24.6 years (range: 12.6–46.1 years). Five patients were ≥18 years, and 3 were <18 years of age. Four patients were identified as female and 4 identified as male. All 8 patients had a baseline Karnofsky score of ≥80%. The clinical characteristics are displayed in Table 1. All patients failed prior systemic treatment with one or more chemotherapy regimens: 1 patient received one prior line of systemic therapy, 7 patients received ≥2 systemic lines of treatment, furthermore 7 had previously been treated and failed TEM/IRI for relapsed disease.

Table 1.

Clinical characteristics of 8 evaluable patients with recurrent or refractory ES treated with Vigil and TEM/IRI.

Gender 
 Male 
 Female 
Age 
 Mean (years) 24.6 
 Median (years) 23.8 
 <18 years 
 ≥18 years 
Race 
 White 
Ethnicity  
 Not Hispanic/Latino 
 Hispanic/Latino 
Prior lines of systemic therapies 
 Mean 4.6 
 Median 4.5 
 Range 1a to 12 
Previous TEM/IRI  
 Yes 
 No 
KPS/Lansky status 
 ≥90 
 <90 
Vigil dose level 
 1 × 106 cells/mL 
 1 × 107 cells/mL 
Gender 
 Male 
 Female 
Age 
 Mean (years) 24.6 
 Median (years) 23.8 
 <18 years 
 ≥18 years 
Race 
 White 
Ethnicity  
 Not Hispanic/Latino 
 Hispanic/Latino 
Prior lines of systemic therapies 
 Mean 4.6 
 Median 4.5 
 Range 1a to 12 
Previous TEM/IRI  
 Yes 
 No 
KPS/Lansky status 
 ≥90 
 <90 
Vigil dose level 
 1 × 106 cells/mL 
 1 × 107 cells/mL 

Note: Two of 10 patients were not evaluable/did not complete or receive ≥1 cycle of Vigil/TEM/IRI due to rapid disease progression after enrollment and prior to treatment start.

aOne patient received VAC/IE plus local Rx to resistant disease.

Safety

Patients received a total of 69 (median = 10; mean = 8.6; range, 3–12 cycles) Vigil administrations. No Vigil treatment-related AEs were reported (Table 2). The median number of TEM/IRI cycles was 10.5 (total = 72; mean = 9; range, 4–12 cycles). One patient (167–3003) had delayed treatment due to thrombocytopenia, empyema, pneumonia, and respiratory failure; another (002–3005) had delayed treatment due to cerebral edema related to disease progression.

Table 2.

Adverse events of 8 evaluable patients with recurrent or refractory ES treated with Vigil and TEM/IRI.

Grade 1Grade 2Grade 3Grade 4Grade 5
AE terma#%#%#%#%#%
Blood and lymphatic system disorders 
 Anemia 13% 38% 13% 0% 0% 
 Febrile neutropenia 0% 0% 13% 0% 0% 
 Neutropenia 13% 0% 25% 25% 0% 
 Thrombocytopenia 13% 0% 13% 25% 0% 
Cardiac disorders 
 Pericardial effusion 13% 0% 0% 0% 0% 
 Tachycardia 63% 0% 0% 0% 0% 
Ear and labyrinth disorders 
 Hearing loss 13% 0% 0% 0% 0% 
Eye disorders 
 Blurred vision 25% 0% 0% 0% 0% 
Gastrointestinal disorders 
 Abdominal pain 25% 13% 0% 0% 0% 
 Blood in stool 13% 0% 0% 0% 0% 
 Constipation 13% 0% 0% 0% 0% 
 Diarrhea 50% 13% 0% 0% 0% 
 Nausea 63% 25% 0% 0% 0% 
 Toothache 13% 0% 0% 0% 0% 
 Vomiting 50% 25% 0% 0% 0% 
General disorders and administration site conditions 
 Chest pain 25% 0% 0% 0% 0% 
 Edema extremities 13% 0% 0% 0% 0% 
 Fatigue 63% 13% 0% 0% 0% 
 Fever 38% 13% 0% 0% 0% 
 Gait disturbance 0% 13% 0% 0% 0% 
 Pain 13% 0% 0% 0% 0% 
Infections and infestations 
 Empyema 0% 0% 0% 13% 0% 
 Pneumonia 0% 0% 0% 13% 0% 
 Rhinovirus infection 13% 0% 0% 0% 0% 
Shingles 0% 13% 0% 0% 0% 
Injury, poisoning, and procedural complications 
 Dermatitis radiation 0% 25% 0% 0% 0% 
Investigations 
 Alkaline phosphatase increased 25% 0% 0% 0% 0% 
 Weight loss 0% 13% 0% 0% 0% 
Metabolism and nutrition disorders 
 Anorexia 25% 25% 0% 0% 0% 
 Hyperglycemia 13% 0% 0% 0% 0% 
 Hyperkalemia 13% 0% 0% 0% 0% 
 Hypernatremia 13% 0% 0% 0% 0% 
 Hypoglycemia 13% 0% 0% 0% 0% 
 Hypokalemia 25% 0% 13% 0% 0% 
Musculoskeletal and connective tissue disorders 
 Arthralgia 13% 25% 0% 0% 0% 
 Back pain 13% 0% 0% 0% 0% 
 Bone pain 13% 0% 0% 0% 0% 
 Chest wall pain 13% 0% 13% 0% 0% 
 Flank pain 0% 13% 0% 0% 0% 
 Hip discomfort 13% 0% 0% 0% 0% 
Jaw pain 25% 0% 0% 0% 0% 
 Muscle weakness 0% 13% 0% 0% 0% 
 Musculoskeletal pain 13% 0% 0% 0% 0% 
 Pain in extremity 13% 13% 0% 0% 0% 
 Rib pain 0% 0% 13% 0% 0% 
 Swollen ankles 13% 0% 0% 0% 0% 
Neoplasms benign, malignant, and unspecified (incl. cysts and polyps) 
 Tumor pain 13% 0% 0% 0% 0% 
Nervous system disorders 
 Dizziness 13% 0% 0% 0% 0% 
 Edema cerebral 13% 0% 0% 0% 0% 
 Headache 13% 13% 0% 0% 0% 
 Peripheral neuropathy 0% 13% 0% 0% 0% 
 Taste alteration 13% 0% 0% 0% 0% 
Respiratory, thoracic and mediastinal disorders 
 Cough 25% 0% 0% 0% 0% 
 Dyspnea 13% 0% 0% 0% 0% 
 Nasal congestion 13% 0% 0% 0% 0% 
 Pneumonitis 13% 0% 0% 0% 0% 
 Respiratory failure 0% 0% 0% 13% 0% 
Skin and subcutaneous tissue disorders 
 Night sweats 13% 0% 0% 0% 0% 
 Pain of skin 13% 0% 0% 0% 0% 
Vascular disorders 
 Hypertension 13% 0% 0% 0% 0% 
 Hypotension 0% 13% 0% 0% 0% 
Grade 1Grade 2Grade 3Grade 4Grade 5
AE terma#%#%#%#%#%
Blood and lymphatic system disorders 
 Anemia 13% 38% 13% 0% 0% 
 Febrile neutropenia 0% 0% 13% 0% 0% 
 Neutropenia 13% 0% 25% 25% 0% 
 Thrombocytopenia 13% 0% 13% 25% 0% 
Cardiac disorders 
 Pericardial effusion 13% 0% 0% 0% 0% 
 Tachycardia 63% 0% 0% 0% 0% 
Ear and labyrinth disorders 
 Hearing loss 13% 0% 0% 0% 0% 
Eye disorders 
 Blurred vision 25% 0% 0% 0% 0% 
Gastrointestinal disorders 
 Abdominal pain 25% 13% 0% 0% 0% 
 Blood in stool 13% 0% 0% 0% 0% 
 Constipation 13% 0% 0% 0% 0% 
 Diarrhea 50% 13% 0% 0% 0% 
 Nausea 63% 25% 0% 0% 0% 
 Toothache 13% 0% 0% 0% 0% 
 Vomiting 50% 25% 0% 0% 0% 
General disorders and administration site conditions 
 Chest pain 25% 0% 0% 0% 0% 
 Edema extremities 13% 0% 0% 0% 0% 
 Fatigue 63% 13% 0% 0% 0% 
 Fever 38% 13% 0% 0% 0% 
 Gait disturbance 0% 13% 0% 0% 0% 
 Pain 13% 0% 0% 0% 0% 
Infections and infestations 
 Empyema 0% 0% 0% 13% 0% 
 Pneumonia 0% 0% 0% 13% 0% 
 Rhinovirus infection 13% 0% 0% 0% 0% 
Shingles 0% 13% 0% 0% 0% 
Injury, poisoning, and procedural complications 
 Dermatitis radiation 0% 25% 0% 0% 0% 
Investigations 
 Alkaline phosphatase increased 25% 0% 0% 0% 0% 
 Weight loss 0% 13% 0% 0% 0% 
Metabolism and nutrition disorders 
 Anorexia 25% 25% 0% 0% 0% 
 Hyperglycemia 13% 0% 0% 0% 0% 
 Hyperkalemia 13% 0% 0% 0% 0% 
 Hypernatremia 13% 0% 0% 0% 0% 
 Hypoglycemia 13% 0% 0% 0% 0% 
 Hypokalemia 25% 0% 13% 0% 0% 
Musculoskeletal and connective tissue disorders 
 Arthralgia 13% 25% 0% 0% 0% 
 Back pain 13% 0% 0% 0% 0% 
 Bone pain 13% 0% 0% 0% 0% 
 Chest wall pain 13% 0% 13% 0% 0% 
 Flank pain 0% 13% 0% 0% 0% 
 Hip discomfort 13% 0% 0% 0% 0% 
Jaw pain 25% 0% 0% 0% 0% 
 Muscle weakness 0% 13% 0% 0% 0% 
 Musculoskeletal pain 13% 0% 0% 0% 0% 
 Pain in extremity 13% 13% 0% 0% 0% 
 Rib pain 0% 0% 13% 0% 0% 
 Swollen ankles 13% 0% 0% 0% 0% 
Neoplasms benign, malignant, and unspecified (incl. cysts and polyps) 
 Tumor pain 13% 0% 0% 0% 0% 
Nervous system disorders 
 Dizziness 13% 0% 0% 0% 0% 
 Edema cerebral 13% 0% 0% 0% 0% 
 Headache 13% 13% 0% 0% 0% 
 Peripheral neuropathy 0% 13% 0% 0% 0% 
 Taste alteration 13% 0% 0% 0% 0% 
Respiratory, thoracic and mediastinal disorders 
 Cough 25% 0% 0% 0% 0% 
 Dyspnea 13% 0% 0% 0% 0% 
 Nasal congestion 13% 0% 0% 0% 0% 
 Pneumonitis 13% 0% 0% 0% 0% 
 Respiratory failure 0% 0% 0% 13% 0% 
Skin and subcutaneous tissue disorders 
 Night sweats 13% 0% 0% 0% 0% 
 Pain of skin 13% 0% 0% 0% 0% 
Vascular disorders 
 Hypertension 13% 0% 0% 0% 0% 
 Hypotension 0% 13% 0% 0% 0% 

Note: Treatment-emergent events are defined as those occurring after the first dose of Vigil. Subjects are counted only once per preferred term if multiple occurrences of the same AE.

Abbreviations: AE, adverse event; #, number of patients with AE; %, number of patients with AE/total number of patients.

aAll adverse events were unrelated to Vigil.

Response and survival

Despite prior disease progression on TEM/IRI, 2 of these patients (167–3003, 167–3006) had a PR to Vigil/IRI/TEM and 5 had prolonged duration of partial or stable response of >6 months (167–3003, 167–3006, 167–3010, 167–3012, 167–3014; Fig. 1). Both PR patients demonstrated RECIST PR (75% and 52% decrease in the sum of diameters of target lesions from baseline) as early as cycle 3. The 2 patients with PR were later identified as disease-free with confirmed histologic CR (1 patient at month 10 and the second at month 5.6) after treatment with Vigil/TEM/IRI. These patients did not receive additional cancer treatment after Vigil prior to histologic assessment. Patient 167–3003 passed away of noncancer causes and underwent autopsy with repeat biopsy of prior cancer locations that showed no histologic evidence of malignancy. The late continuation of further regression in these 2 patients after discontinuation of Vigil is suggestive of ongoing immune response after Vigil discontinuation.

Figure 1.

Swimmers plot of all patients (n = 8). Yellow arrows indicate patients who are still alive.

Figure 1.

Swimmers plot of all patients (n = 8). Yellow arrows indicate patients who are still alive.

Close modal

The overall median PFS was 8.2 months (95% CI, 4.3–NA). The 1-year OS rate was 62.5% (95% CI, 36.5–100). Median OS was 18.5 months (95% CI, 8.2–NA).

Quantification of patient-specific ctDNA and correlation to clinical responses to Vigil/TEM/IRI

The patient-specific EWS/FLI translocation breakpoint was identified in all patients (Table 3) and patient-specific ddPCR assays were successfully developed for each case. ddPCR was used to quantify ctDNA in plasma samples obtained prior to therapy and serially throughout the course of treatment with Vigil/TEM/IRI (10). ctDNA levels changed in concordance with changes in disease burden during the course of therapy.

Table 3.

Patient-specific fusions identified.

Patient IDFusion breakpointPredicted EWS exonPredicted FLI exonPredicted fusion type
EW-002–3005 chr22:29684364::chr11:128653233 
EW-167–3003 chr22:29684325::chr11:128657194 
EW-167–3006 chr22:29684240::chr11:128655185 
EW-167–3010 chr22:29688284::chr11:128678086 10 Non-1/2 
EW-167–3011 chr22:29684553::chr11:128646127 
EW-167–3012 chr22:29685406::chr11:128656298 8* (7) 1* 
EW-167–3013 chr22:29685923::chr11:128646587 8* (7) 2* 
EW-167–3014 chr22:29683596::chr11:128666151 
Patient IDFusion breakpointPredicted EWS exonPredicted FLI exonPredicted fusion type
EW-002–3005 chr22:29684364::chr11:128653233 
EW-167–3003 chr22:29684325::chr11:128657194 
EW-167–3006 chr22:29684240::chr11:128655185 
EW-167–3010 chr22:29688284::chr11:128678086 10 Non-1/2 
EW-167–3011 chr22:29684553::chr11:128646127 
EW-167–3012 chr22:29685406::chr11:128656298 8* (7) 1* 
EW-167–3013 chr22:29685923::chr11:128646587 8* (7) 2* 
EW-167–3014 chr22:29683596::chr11:128666151 

Note: DNA-level breakpoints of individual patient-specific EWS-FLI fusions, mapped to human genome assembly GRCh37.

Patient EW-167–3006 experienced a PR and demonstrated reduction from a high ctDNA baseline of 27.2% to a level <2% within 2 months after initiation of Vigil/TEM/IRI treatment (Fig. 2 EW-167–3006). ctDNA levels remained low throughout the remainder of the treatment course with Vigil. Tumor volume reduction measured by imaging correlated with ctDNA level drop and prolonged duration of response. Radiographic imaging confirmed response by CT (175–84 mm) and PET-CT. In addition, a biopsy of a responding pelvic lesion site also showed complete histologic response and residual necrotic tissue with no residual malignancy (Fig. 3).

Figure 2.

Monitoring of ctDNA percent levels (black) and tumor burden volume (blue) during the treatment course for all patients. Correlation of tumor response was demonstrated with ctDNA reduction and image (PET/CT) guidance. Vigil was administered on day 1 of each cycle. EOT, end of treatment; FU, 3-month follow up; Rx, palliative radiotherapy.

Figure 2.

Monitoring of ctDNA percent levels (black) and tumor burden volume (blue) during the treatment course for all patients. Correlation of tumor response was demonstrated with ctDNA reduction and image (PET/CT) guidance. Vigil was administered on day 1 of each cycle. EOT, end of treatment; FU, 3-month follow up; Rx, palliative radiotherapy.

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Figure 3.

Monitoring of tumor burden (PET/CT) in EW-167–3006. Target lesions in PET/CT scans are marked with white arrows. Tumor volumes in the left upper lung (AD) and left diaphragm (EH) are shown at baseline (A, E), month 3 (B, F), month 6 (C, G), and end of treatment (EOT; D, H). The biopsy of the bony pelvic lesion (I) showed a histologic CR with necrotic tissue and without any evidence of live tumor cells (J). Tumor volumes of all target lesions are shown in K.

Figure 3.

Monitoring of tumor burden (PET/CT) in EW-167–3006. Target lesions in PET/CT scans are marked with white arrows. Tumor volumes in the left upper lung (AD) and left diaphragm (EH) are shown at baseline (A, E), month 3 (B, F), month 6 (C, G), and end of treatment (EOT; D, H). The biopsy of the bony pelvic lesion (I) showed a histologic CR with necrotic tissue and without any evidence of live tumor cells (J). Tumor volumes of all target lesions are shown in K.

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ctDNA levels of two other patients with PR or stable disease (SD; EW-167–3003, EW-167–3014) were detectable at baseline but dropped below the limit of detection for ddPCR during the patients’ clinical responses (Fig. 2 EW-167–3003, EW-167–3014). After completion of Vigil therapy, ctDNA levels again rose suggesting the possibility of disease progression. In one of these patients, EW-167–3003, no evidence of disease progression was found in the prior sites of known disease, even at the time of autopsy (Fig. 4).

Figure 4.

Monitoring of tumor burden (CT and PET/CT) in EW-167–3003. Right lower lung lesions (AC) are marked with yellow arrows in CT scans at baseline (A), month 3 (B), and month 6 (C) of treatment. Posterior chest wall/spleen lesions (DH) are also marked with yellow arrows in CT scans (D, F, H) or yellow arrows in PET/CT scans (E, G) at baseline (D, E), month 3 (F, G), and month 6 (H) of treatment. Tumor volumes of target lesions are shown in I. Autopsy report showed a histological CR without any evidence of live tumor cells.

Figure 4.

Monitoring of tumor burden (CT and PET/CT) in EW-167–3003. Right lower lung lesions (AC) are marked with yellow arrows in CT scans at baseline (A), month 3 (B), and month 6 (C) of treatment. Posterior chest wall/spleen lesions (DH) are also marked with yellow arrows in CT scans (D, F, H) or yellow arrows in PET/CT scans (E, G) at baseline (D, E), month 3 (F, G), and month 6 (H) of treatment. Tumor volumes of target lesions are shown in I. Autopsy report showed a histological CR without any evidence of live tumor cells.

Close modal

Interestingly, patients EW-167–3010 and EW-167–3012 received medically indicated palliative radiation for symptom control while receiving on-study treatment. Radiotherapy was then followed by a decrease in ctDNA levels consistent with the reduction of tumor burden observed by imaging (Fig. 2 EW-167–3010, EW-167–3012).

Patient EW-167–3013 had very low ctDNA levels (0.32%) yet relatively high tumor burden at study entry. Local scans showed increase in target lesion size but below the definition of progressive disease (World Care, third-party assessment defined 19% increase maximum diameter) and was managed with other anticancer therapy 3.2 months after treatment initiation. ctDNA levels remained low throughout therapy indicating that this patient's tumor shed minimal amounts of ctDNA (Fig. 2 EW-167–3013). The 2 other patients that did not achieve PR or SD > 6 months (Fig. 2 EW-002–3005, EW-167–3011) had disease progression within 3 to <6 months of treatment start. ctDNA levels were elevated at baseline and remained elevated throughout treatment.

Management of recurrent or refractory ES is limited particularly in patients who have already failed treatment with TEM/IRI. Observation of two PRs and three prolonged SD in patients who have previously failed prior TEM/IRI to the Vigil/TEM/IRI regimen support proof of principle that Vigil may contribute to antitumor activity with TEM/IRI in ES. Vigil/TEM/IRI also showed no safety concerns with the addition of Vigil to TEM/IRI thereby justifying further exploration of Vigil in combination with TEM/IRI. It was also provocative that both patients achieving PR demonstrated eventual histologic CR without additional anticancer therapy. This is consistent with immune assessment of Vigil in phase I and phase II work demonstrating durable activation of enhanced ELISPOT response months after Vigil discontinuation.

Numerous studies have shown that ctDNA can be detected and quantified by identifying the allelic fraction of cancer-specific mutations in patients with solid malignancies. These ctDNA levels can be followed longitudinally and used as sensitive and minimally invasive markers for early cancer detection, tumor growth, and response assessment in multiple solid tumors, including colorectal cancer, breast cancer, and ES (11, 13–18). Oikkonen and colleagues (19) demonstrated that ctDNA (ERBB2) in high-grade serious ovarian cancer can be used to identify poorly responding patients after initial cycles of chemotherapy, which may help with early detection and clinically actionable alterations in the therapeutic area. In ES, multiple studies demonstrate that high ctDNA at diagnosis and persistent detection of ctDNA during therapy is associated with poor prognosis and that rising levels of ctDNA can be detected before clinical evidence of disease progression (10, 14, 18, 20, 21).

In this study, ctDNA was detected at baseline from all patients. In 2 patients with partial responses, ctDNA levels dropped below the limit of detection for ddPCR after initiation of Vigil therapy, but became detectable again after discontinuation of therapy suggesting disease progression. However imaging and histologic studies suggested that there was no residual disease at the sites being followed for these patients. One possible explanation is the presence of residual disease at another anatomic location or new sites of disease progression emerging after completion of Vigil, but more studies will be needed to confirm that these increases in ctDNA levels are predictive of new progression (13). ctDNA levels in patients who went on to achieve RECIST PR were decreased. Further studies in larger cohorts will be needed to validate the prognostic and predictive value of ctDNA biomarker studies for use in the setting of early-phase clinical trials in ES (22–24).

B.D. Crompton reports other support from Gradalis during the conduct of the study. L. Stanbery reports employment with Gradalis, Inc. E. Bognar reports employment with and ownership of Gradalis, Inc. stock. J. Nemunaitis reports employment with and ownership of Gradalis, Inc. stock. No disclosures were reported by the other authors.

P. Anderson: Resources, investigation, writing–review and editing. M. Ghisoli: Investigation, writing–review and editing. B.D. Crompton: Resources, data curation, formal analysis, investigation, writing–review and editing. K.S. Klega: Resources, formal analysis, investigation, writing–review and editing. L.H. Wexler: Resources, supervision, writing–review and editing. E.K. Slotkin: Resources, supervision, writing–review and editing. L. Stanbery: Resources, formal analysis, validation, investigation, writing–original draft, writing–review and editing. L. Manning: Resources, formal analysis, supervision, validation, investigation, writing–original draft, writing–review and editing. G. Wallraven: Resources, data curation, formal analysis, supervision, project administration, writing–review and editing. M. Manley: Data curation, project administration, writing–review and editing. S. Horvath: Data curation, project administration, writing–review and editing. E. Bognar: Resources, supervision, writing–original draft, writing–review and editing. J. Nemunaitis: Conceptualization, resources, supervision, investigation, methodology, writing–original draft, project administration, writing–review and editing.

The authors would like to acknowledge Brenda Marr for her competent and knowledgeable assistance in the preparation of the manuscript. No financial support was utilized for this study.

The publication costs of this article were defrayed in part by the payment of publication fees. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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Supplementary data