Background and Aims

Achieving complete tumor resections without major complications remains challenging. Fluorescence guided surgery (FGS) with tumor-specific probes can visually assist the surgeon in discriminating tumorous from healthy tissue. However, tumor-specific probes are currently screened one-by-one and per tumor type. Predictive platforms to develop FGS probes in a tumor-specific and even patient-specific way are lacking. Here, we combine patient-derived organoids with 3D imaging technology to present a screening platform for FGS probes.

Methods

Both an adult and pediatric tumor organoid biobank were cultured, respectively breast cancer and neuroblastoma. Surface markers that are upregulated in neuroblastoma and/or breast cancer were identified based on RNA profiling, the human protein atlas and literature review. Probes targeting these surface markers were conjugated to six different fluorophores ranging between 488nm and 647nm. Together with the general marker efluor, they were used for seven-color 3D multispectral live imaging using confocal microscopy on living organoids. Using segmentation analysis by parallelization of 3D datasets (STAPL-3D) organoids were segmented and their fluorescent signals of the respective probes quantified. This high-throughput in vitro screening was validated by testing the three most promising FGS probes for neuroblastoma in vivo using a mouse xenograft model with tumors originating from neuroblastoma organoids.

Results

Our platform was able to simultaneously screen six different probes on two full organoid biobanks. Segmentation and quantification using STAPL-3D showed heterogeneous expression of markers on the organoid lines. The three most promising probes for neuroblastoma (GD2, L1cam and NCAM-1) were tested in vivo and all resulted in tumor-to-background ratios above 2.0, indicating good intraoperative visibility. For heterogenous tumors that are not covered with one probe, this platform offers the chance to screen most potential probe combinations.

Conclusions

We present a novel multiplex organoid-based 3D live imaging platform to screen FGS probes in a patient-specific manner with the potential to develop personalized FGS probes or probe combinations.

Background and Aims

Treatment strategies involving nodal basins for children and adolescents with melanoma are extrapolated from adult trials. There is increasing evidence that important clinical and biological differences exist between pediatric and adult melanoma, therefore these strategies should be studied. The purpose of this study was to determine the impact on disease outcomes with ultrasound surveillance (US) of involved nodal basins versus completion lymph node dissection (CLND) in children and adolescents with sentinel lymph node (SLN) positive melanoma.

Methods

Patients from 12 participating Pediatric Surgical Oncology Research Collaborative hospitals were included. Requirements included age <18 years and cutaneous melanoma diagnosed between 2010-2020. Data extracted included demographics, histopathology, surgical strategies including SLN and CLND, surveillance intervals, and survival information.

Results

249 patients were included, 52.2% (n=130) female, 90% (n=223) non-Hispanic White. 90.8% (n=226) underwent SLN biopsy, 50% (n=113) had at least 1 positive node. Median number of positive nodes was 1 (IQR: 1-2) and did not differ between US and CLND groups (p=0.20). Median Breslow depth was 2.5mm (IQR: 1.4-4.1) and did not differ between US and CLND groups (p=0.26). 59.3% (n=67) with positive SLN underwent CLND while 40.7% (n=46) underwent US surveillance of the nodal basin. Younger patients were more likely to undergo US surveillance (median age 96 months) than CLND (median age 142 months) (p=0.002). Of those who underwent CLND, 21% (n=14) had additional positive nodes removed. Overall, 11.3% (n=25) experienced disease recurrence: 7 primary, 7 nodal, 10 distant, 1 unknown. There was no difference in disease recurrence (11.4% vs 25%, p=0.08) or death from disease (2.3% vs 9.7%, p=0.29) for those who underwent US vs CLND, respectively.

Conclusions

Children and adolescents with cutaneous melanoma frequently have nodal metastases identified by SLN. Equivalent disease outcomes were observed with US surveillance and CLND following identification of a positive SLN.

Background and Aims

The treatment of neuroblastoma (NBL) presenting image defined risk factors (IDRF) pose significant surgical difficulties. Having known that a complete microscopic resection is unlikely in case of IDRF+, gross total resection (GTR) offers a chance for better prognosis. A reliable assessment of the residue after GTR is of utmost importance. Theoretically, ECT before the healing cascade developed prevents misinterpreting of a regenerative and healing process as a tumour remnant and vice versa. The aim of the study was to evaluate early computed tomography of tumour bed (ECT) as a tool to objectify the quality of GTR.

Methods

ECT was performed by the postoperative day 7 on stabilised patients, before any major regenerative reactions and possibility of an early relapse. Only children in whom GTR was subjectively assessed by the surgeon to be > 90% complete, were included. The study involved 61 children (31 local, 30 referred from other centres) who underwent GTR surgery for IDRF+ neuroblastoma (2018 – 2022). Images of ECT were segmented non-automatically, the boundaries of each tumour were marked manually, taking a layer thickness of 2.5 mm as the optimum. The total volumes of the primary masses and the post-operative residues were calculated and compared in rates and ml of remnants.

Results

The post-surgical, objectively measured remnants of tumours (ECT) ranged from 0 ml to 3,6 ml, completeness of GTR from 76,3% to 100% with median 91,8.

Conclusions

The subjective assessment of completeness of GTR by surgeons seems to offer an over optimistic view which may not find confirmation at objective assessment by ECT. Calculating the rate of resection and measuring volume of the post surgical residue describe the postoperative situation in different ways, thus should be used and analysed together. Authors emphasize on early assessment as that which shows direct post operative situation.

Background and Aims

To assess the outcomes of pediatric patients with Undifferentiated Embryonal Sarcoma of the Liver (UESL) treated according to successive European malignant mesenchymal tumors trials focusing on the effects of surgical margins, preoperative chemotherapy, use of radiotherapy (RT) and chemotherapy regimen.

Methods

This study included patients aged up to 25 years with a pathological diagnosis of UESL prospectively enrolled from 1995 to 2016 in the SIOP-MMT95, AIEOPX and NRSTS05 trials. The recommended treatment included conservative surgery at diagnosis or a biopsy followed by chemotherapy and delayed surgery. The decision to use RT was left to the treating center.

Results

Sixty-five patients met the study criteria with a median age at diagnosis of 8.7 years (0.6-20.8). Fifteen patients had T2 tumors, and one had lymph node spread. Stage distribution included IRS I (14), II (9), III (38), and IV (4). Upfront surgery (28) resulted in 5 operative spillages and 11 infiltrated surgical margins, whereas delayed surgery (37) resulted in no spillages (P= 0.0119) and 3 infiltrated margins(P=0.0114). All patients received post-operative chemotherapy, including anthracyclines in 47. Radiotherapy was administered in 15 patients. With a median follow-up of 78.6 months, 5 year OS and EFS were 90.1% (95%CI 79.2-95.5) and 89.1% (95%CI 78.4-94.6), respectively. Despite 3 out of 4 local relapses (3 others were metastatic) being associated with infiltrated surgical margins, we did not find evidence of an association between infiltrated margins and EFS (P=0.1607) . Similarly, T2 stage (P=0.3870), use of RT (P= 0.8731), and anthracycline-based chemotherapy (P= 0.1181), did not have an association with EFS.

Conclusions

Neoadjuvant chemotherapy with an alkylating agent regimen for pediatric patients with UESL increases the probability of complete surgical resection and decreases tumor spillage. The role of anthracyclines and radiotherapy for localized disease remains unclear and could be reserved for patients with infiltrated margins,tumor rupture, locoregional disease or distant extension.

Background and Aims

Extracellular vesicles (EVs) are spherical, lipid bilayer membrane vesicles, which are released by various cell types. They are found in distinct biofluids such as blood and urine. Although there are many known isolation techniques, there is currently no consensus for the optimal EV isolation method with respect to yield, quality, and purity. Furthermore, no data from pediatric solid tumor patients are available. In this study, we evaluated different EV isolation techniques and studied the application of EV as diagnostic tool in children suffering from rhabdomyosarcoma (RMS) and neuroblastoma (NB).

Methods

Four different isolation techniques (precipitation, size-exclusion, membrane affinity, and ultracentrifugation), were compared for EV isolation from plasma of healthy individuals (n=10). The most efficient technique was then used to analyze plasma samples from RMS (n=40) and NB (n=41) tumor patients. EV characterization included morphological analysis via electron-microscopy, determination of particle-size and concentration via nanoparticle tracking analysis. EV preparation quality, purity, and biomarker expression (GD2, CD146, CD171) was assessed using flow cytometry and Western Blotting.

Results

All EV isolation techniques were successfully tested. They showed approximately the same particle-size (115-147 nm) with a similar time requirement. The precipitation and membrane affinity methods showed the highest protein purity. The size-exclusion method had the highest values for the exosome markers CD9 (99%), CD63 (82%), and CD81 (97%) and showed higher purity on electron-microscopy. The tested biomarkers (CD146 and CD171) allowed to distinguish between NB patients and healthy individuals, which was not the case in RMS patients.

Conclusions

EVs seem to be a promising tool for non-invasive diagnosis and treatment monitoring in children suffering from NB. Further studies on thhis and other tumor etiologies including determination of expression patterns are needed in order to clarify the possible role of applying EVs in the clinical setting.

Background and Aims

Rhabdomyosarcoma of the perianal/perineal region (PRMS) is rare, with poor survival and limited understanding of the functional consequences of treatment.

Methods

SIOP MMT 95, Italian RMS 96, and EpSSG RMS 2005 studies were interrogated to identify factors that impact survival; in RMS 2005, functional outcomes were analyzed.

Results

Fifty patients (non-metastatic) were identified, median age 6.4 years (range 0.1-19.6): 29 male, 21 female. Tumors were >5cm in 33 patients. Histopathological subtype was alveolar in 35. Lymph nodes were involved in 23 patients. In RMS 2005, 16/21(76%) tested alveolar tumors had positive FOXO1-fusion status.

Diagnostic biopsy was performed in 37. Primary resection(13) was complete (R0) in 1. Delayed primary excision(16) was complete in 3.

Radiotherapy (RT) in 34/50 patients included external beam(28), brachytherapy(3), and both(3). Nodal RT was given in 16/23 N1 patients(70%).

Median follow up of alive patients (29) was 84.1 months (range 3.6-221.1).

Relapse or progression occurred in 24 patients (48%), 87% were fatal and most events (63%) were locoregional.

5-yr EFS was 47.8 (95%CI 32.8-61.3), and 5-yr OS was 52.6 (95%CI 36.7-66.2), with

age ≥ 10 years and tumor size > 5cm impacting 5-year EFS and OS (p<0.05).

Functional outcome data showed bowel, genito-urinary and psychological issues; fecal incontinence in 4/21 survivors, and urinary symptoms in 2/21.

Conclusions

About 60% of patients with non-metastatic PRMS survive; older patients and those with large tumors have the worst outcomes. Biopsy should be the initial procedure and definitive local therapy individualized. Quality-of-life and functional studies are needed to better understand the consequences of treatment.

Background and Aims

Nephron-sparing Surgery (NSS) is the surgical treatment of choice in children with stage V renal tumors. With increasing numbers of this approach, there is also an increase of tumor relapses in affected children. Aim of this study was to evaluate Redo-NSS in children with stage V disease, especially for centrally located tumor relapses.

Methods

We retrospectively analysed patients undergoing Redo-NSS for relapsed kidney tumors between 2009 and 2021 at our institution, which represents a national reference center within the SIOP-2001 and UMBRELLA study. The indication for Redo-NSS was established by a national and local multi-disciplinary tumorboard (MDT).

Results

During the observation period, 45 patients with bilateral disease underwent primary NSS (69 renal units). In the same period, 9 stage V patients receiver Redo-NSS: 5 girls, 4 boys, mean age at surgery: 58 months (12-137). Mean time between primary NSS and Redo-NSS was 25 months (4-104). All patients had only local recurrences.

Mean operative time for Redo-NSS was 195 minutes (137-260). R0 resection status was achieved in all children (intraoperative frozen sections). Histology after Redo-NSS showed diffuse anaplasia in 2 patients, which initially had low or intermediate risk tumors. These two patients died in the further course from combined second relapses. Two other patients had second relapses, one of them resected via NSS, the other child underwent tumor nephrectomy plus atypical liver resection. Thus 7/9 patients are alive without evidence of disease, an impaired renal function was observed in one child. Mean follow-up was 16 months (0-32).

Conclusions

With sufficient expertise of the interdisciplinary treating team, Redo-NSS for stage V renal tumors can be performed with satisfactory oncological and functional results. The occurrence of unfavourable tumor histology possibly represents a negative prognostic factor. Among other factors, this needs further evaluation in multicenter analyses.

Background and Aims

The most common primary extremity bone sarcomas include Ewing sarcoma (ES) and osteosarcoma (OS). Whereas ES being radio sensitive additional therapeutic measures were available for local control, historically surgery in the form of amputation was the mainstay for OS.

Herein, we describe progressive protocols used to obtain local control over the years for OS.

Methods

From 1962-1980 of the 139 cases enrolled only one underwent limb salvage surgery (LSS) with a local control rate of 95%. Based on the initial success by Rosen and Marcove using neoadjuvant chemotherapy followed by LSS and local control of over 95%, we instituted in 1980 a LSS program albeit using strict criteria. These included: a) age>13years or 75% of anticipated growth b) no distant metastasis c) intramedullary component <50% d) small extramedullary extension e) patient compliance.

Results

Between July 1981 and December 2020 of 398 patients enrolled, 368 patients underwent LSS (95%). Primary site was femur in 171 (46%). Median age was 10.8 years (range, 3-25). Range extent of resection was 7-35 cm. Five (1.3%) patients had a pathological fracture. Intraoperative complications included: a) hemorrhage defined as >10% blood volumen loss (25%), vascular injuries requiring repair (2%), peroneal or radial postoperative neuropathy (6.7%). All recovered within 4 months. Postoperative complications included: a) superficial wound infections (12%) b) implant removal due to refractory infection (2.4%) c) postoperative bleeding (2%) d) stem fracture (3.5%) e) implant loosening (4.3%) f) non-union allograft (0.8%) g) local recurrence (1.3%).

Conclusions

LSS is feasible in >95% of patients. Complications are mild and minimal. Patient satisfaction paramount importance. Early ambulation/rehabilitation are important facets. Major disadvantage cited are repeated surgeries for growth or complications (fracture, loosening, etc).