Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather ...than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors.
Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed.
TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse.
We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.
Purpose
Meta
-
18
Ffluorobenzylguanidine (
18
FmFBG) is a positron emission tomography (PET) radiotracer that allows for fast and high-resolution imaging of tumours expressing the norepinephrine ...transporter. This pilot study investigates the feasibility of
18
FmFBG PET-CT for imaging in neuroblastoma.
Methods
In a prospective, single-centre study, we recruited children with neuroblastoma, referred for
meta
-
123
Iiodobenzylguanidine (
123
ImIBG) scanning, consisting of total body planar scintigraphy in combination with single-photon emission computed tomography-CT (SPECT-CT). Within two weeks of
123
ImIBG scanning, total body PET-CTs were performed at 1 h and 2 h after injection of
18
FmFBG (2 MBq/kg). Detected tumour localisations on scan pairs were compared. Soft tissue disease was quantified by number of lesions and skeletal disease by SIOPEN score.
Results
Twenty paired
123
ImIBG and
18
FmFBG scans were performed in 14 patients (median age 4.9 years,
n
= 13 stage 4 disease and
n
= 1 stage 4S).
18
FmFBG injection was well tolerated and no related adverse events occurred in any of the patients. Mean scan time for
18
FmFBG PET-CT (9.0 min, SD 1.9) was significantly shorter than for
123
ImIBG scanning (84.5 min, SD 10.5),
p
< 0.01. Most tumour localisations were detected on the 1 h versus 2 h post-injection
18
FmFBG PET-CT. Compared to
123
ImIBG scanning,
18
FmFBG PET-CT detected a higher, equal, and lower number of soft tissue lesions in 40%, 55%, and 5% of scan pairs, respectively, and a higher, equal, and lower SIOPEN score in 55%, 30%, and 15% of scan pairs, respectively. On average, two more soft tissue lesions and a 6-point higher SIOPEN score were detected per patient on
18
FmFBG PET-CT compared to
123
ImIBG scanning.
Conclusion
Results of this study demonstrate feasibility of
18
FmFBG PET-CT for neuroblastoma imaging. More neuroblastoma localisations were detected on
18
FmFBG PET-CT compared to
123
ImIBG scanning.
18
FmFBG PET-CT shows promise for future staging and response assessment in neuroblastoma.
Trial registration
Dutch Trial Register NL8152.
Neuroblastoma is one of the most common pediatric cancers and a major cause of cancer-related death in infancy. Conventional therapies including high-dose chemotherapy, stem cell transplantation, and ...immunotherapy approach a limit in the treatment of high-risk neuroblastoma and prevention of relapse. In the last two decades, research unraveled a potential use of mesenchymal stromal cells in tumor therapy, as tumor-selective delivery vehicles for therapeutic compounds and oncolytic viruses and by means of supporting hematopoietic stem cell transplantation. Based on pre-clinical and clinical advances in neuroblastoma and other malignancies, we assess both the strong potential and the associated risks of using mesenchymal stromal cells in the therapy for neuroblastoma. Furthermore, we examine feasibility and safety aspects and discuss future directions for harnessing the advantageous properties of mesenchymal stromal cells for the advancement of therapy success.
BackgroundCurrent immunotherapy for patients with high-risk neuroblastoma involves the therapeutic antibody dinutuximab that targets GD2, a ganglioside expressed on the majority of neuroblastoma ...tumors. Opsonized tumor cells are killed through antibody-dependent cellular cytotoxicity (ADCC), a process mediated by various immune cells, including neutrophils. The capacity of neutrophils to kill dinutuximab-opsonized tumor cells can be further enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which has been shown in the past to improve responses to anti-GD2 immunotherapy. However, access to GM-CSF (sargramostim) is limited outside of Northern America, creating a high clinical need for an alternative method to stimulate dinutuximab responsiveness in the treatment of neuroblastoma. In this in vitro study, we have investigated whether clinically well-established granulocyte colony-stimulating factor (G-CSF) can be a potentially suitable alternative for GM-CSF in the dinutuximab immunotherapy regimen of patients with neuroblastoma.MethodsWe compared the capacity of neutrophils stimulated either in vitro or in vivo with GM-CSF or G-CSF to kill dinutuximab-opsonized GD2-positive neuroblastoma cell lines and primary patient tumor material. Blocking experiments with antibodies inhibiting either respective Fc gamma receptors (FcγR) or neutrophil integrin CD11b/CD18 demonstrated the involvement of these receptors in the process of ADCC. Flow cytometry and live cell microscopy were used to quantify and visualize neutrophil-neuroblastoma interactions.ResultsWe found that G-CSF was as potent as GM-CSF in enhancing the killing capacity of neutrophils towards neuroblastoma cells. This was observed with in vitro stimulated neutrophils, and with in vivo stimulated neutrophils from both patients with neuroblastoma and healthy donors. Enhanced killing due to GM-CSF or G-CSF stimulation was consistent regardless of dinutuximab concentration, tumor-to-neutrophil ratio and concentration of the stimulating cytokine. Both GM-CSF and G-CSF stimulated neutrophils required FcγRIIa and CD11b/CD18 integrin to perform ADCC, and this was accompanied by trogocytosis of tumor material by neutrophils and tumor cell death in both stimulation conditions.ConclusionsOur preclinical data support the use of G-CSF as an alternative stimulating cytokine to GM-CSF in the treatment of high-risk neuroblastoma with dinutuximab, warranting further testing of G-CSF in a clinical setting.
Cell-free DNA profiling using patient blood is emerging as a non-invasive complementary technique for cancer genomic characterization. Since these liquid biopsies will soon be integrated into ...clinical trial protocols for pediatric cancer treatment, clinicians should be informed about potential applications and advantages but also weaknesses and potential pitfalls. Small retrospective studies comparing genetic alterations detected in liquid biopsies with tumor biopsies for pediatric solid tumor types are encouraging. Molecular detection of tumor markers in cell-free DNA could be used for earlier therapy response monitoring and residual disease detection as well as enabling detection of pathognomonic and therapeutically relevant genomic alterations.
Conclusion
: Existing analyses of liquid biopsies from children with solid tumors increasingly suggest a potential relevance for molecular diagnostics, prognostic assessment, and therapeutic decision-making. Gaps remain in the types of tumors studied and value of detection methods applied. Here we review the current stand of liquid biopsy studies for pediatric solid tumors with a dedicated focus on cell-free DNA analysis. There is legitimate hope that integrating fully validated liquid biopsy–based innovations into the standard of care will advance patient monitoring and personalized treatment of children battling solid cancers.
What is Known:
•
Liquid biopsies are finding their way into routine oncological screening, diagnosis, and disease monitoring in adult cancer types fast.
•
The most widely adopted source for liquid biopsies is blood although other easily accessible body fluids, such as saliva, pleural effusions, urine, or cerebrospinal fluid (CSF) can also serve as sources for liquid biopsies
What is New:
•
Retrospective proof-of-concept studies in small cohorts illustrate that liquid biopsies in pediatric solid tumors yield tremendous potential to be used in diagnostics, for therapy response monitoring and in residual disease detection.
•
Liquid biopsy diagnostics could tackle some long-standing issues in the pediatric oncology field; they can enable accurate genetic diagnostics in previously unbiopsied tumor types like renal tumors or brain stem tumors leading to better treatment strategies
Background
The therapeutic use of
131
Imeta-iodobenzylguanidine (
131
IMIBG) is often accompanied by hematological toxicity, mainly consisting of persistent and severe thrombocytopenia. While MIBG ...accumulates in neuroblastoma cells via selective uptake by the norepinephrine transporter (NET), the serotonin transporter (SERT) is responsible for cellular uptake of MIBG in platelets. In this study, we have investigated whether pharmacological intervention with selective serotonin reuptake inhibitors (SSRIs) may prevent radiotoxic MIBG uptake in platelets without affecting neuroblastoma tumor uptake.
Methods
To determine the transport kinetics of SERT for
125
IMIBG, HEK293 cells were transfected with SERT and uptake assays were conducted. Next, a panel of seven SSRIs was tested in vitro for their inhibitory potency on the uptake of
125
IMIBG in isolated human platelets and in cultured neuroblastoma cells. We investigated in vivo the efficacy of the four best performing SSRIs on the accumulation of
125
IMIBG in nude mice bearing subcutaneous neuroblastoma xenografts. In ex vivo experiments, the diluted plasma of mice treated with SSRIs was added to isolated human platelets to assess the effect on
125
IMIBG uptake.
Results
SERT performed as a low-affinity transporter of
125
IMIBG in comparison with NET (
K
m
= 9.7 μM and 0.49 μM, respectively). Paroxetine was the most potent uptake inhibitor of both serotonin (IC
50
= 0.6 nM) and MIBG (IC
50
= 0.2 nM) in platelets. Citalopram was the most selective SERT inhibitor of
125
IMIBG uptake, with high SERT affinity in platelets (IC
50
= 7.8 nM) and low NET affinity in neuroblastoma cells (IC
50
= 11.940 nM). The in vivo tested SSRIs (citalopram, fluvoxamine, sertraline, and paroxetine) had no effect on
125
IMIBG uptake levels in neuroblastoma xenografts. In contrast, treatment with desipramine, a NET selective inhibitor, resulted in profoundly decreased xenograft
125
IMIBG levels (
p
< 0.0001). In ex vivo
125
IMIBG uptake experiments, 100- and 34-fold diluted murine plasma of mice treated with citalopram added to isolated human platelets led to a decrease in MIBG uptake of 54–76%, respectively.
Conclusion
Our study demonstrates for the first time that SSRIs selectively inhibit MIBG uptake in platelets without affecting MIBG accumulation in an in vivo neuroblastoma model. The concomitant application of citalopram during
131
IMIBG therapy seems a promising strategy to prevent thrombocytopenia in neuroblastoma patients.
PCR-based detection of minimal residual disease (MRD) in neuroblastoma (NB) is presently based on NB-specific transcripts. However, the expression of these targets varies between patients and upon ...treatment, and only PHOX2B is truly specific. RASSF1a is methylated (RASSF1a(M)) in NB, and we investigated whether it can serve as a specific and stable DNA MRD marker.
The RASSF1a(M)-specific quantitative real-time PCR was tested on control bone marrow (BM; n = 50), on 71 NB tumors, and on 159 clinical BM samples at diagnosis and at follow-up of 77 patients. Results were compared with a panel of RNA markers and correlated with prognosis.
RASSF1a(M) was present in all stage 4 and 4s tumors (n = 50) and in 86% stages 1 to 3 tumors (n = 21). The level of methylation in stage 4 NB was correlated with overall survival (P = 0.02). RASSF1a(M)-PCR was highly specific (only 1 amplification in 50 control samples tested in triplicate) and had a similar sensitivity as the RNA-based PCRs, as shown on clinical samples. Moreover, RASSF1a(M) enabled accurate quantification without need for the original tumor.
RASSF1a(M) is a novel, highly specific DNA marker for MRD detection in NB, equal to PHOX2B in specificity and sensitivity, and better suitable for MRD quantification. We propose to include RASSF1a(M) in further prospective MRD studies in NB alongside RNA MRD markers. In addition, this assay might also be applicable for detection of circulating tumor cells in patients with other cancers withRASSF1a(M) such as breast or lung cancer.
Background: Anti-GD2 based immunotherapy has improved overall (OS) and event free survival (EFS) for high-risk neuroblastoma (HR-NBL) patients. Here, we evaluate the long-term efficacy of anti-GD2 ...immunotherapy in combination with isotretinoin, GM-CSF, and IL-2. Methods: Dutch HR-NBL patients treated with immunotherapy according to the COG-ANBL0032 protocol (n = 47) were included and compared to historical controls (n = 37) treated with single-agent isotretinoin maintenance therapy. Survival time was calculated from start of the maintenance therapy. Results: The study and control group were similar concerning baseline characteristics. In the complete cohort, 5 year OS was 64 ± 7% and 49 ± 8% for the immunotherapy group and the control group, respectively (p = 0.16). Five year EFS was 57 ± 7% and 41 ± 8%, respectively (p = 0.16). In the subgroup of patients ≥ 18 months, 5-yr OS was 63 ± 8% and 39 ± 9, respectively (p = 0.04) and EFS 54 ± 8% and 29 ± 8%, respectively (p = 0.05). Landmark analysis for EFS with landmark point at 6 months after start of maintenance suggests a larger effect on the prevention of late than early events. Conclusions: This study is the first to confirm the results of the COG-ANBL0032 study in a cohort treated with a different induction regimen. Anti-GD2 immunotherapy prevents late events, most significantly in patients older than 18 months of age at diagnosis.
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