Gerard R. Schaap

Conditionally Replicative Adenovirus with Tropism Expanded towards Integrins Inhibits Osteosarcoma Tumor Growth in Vitro and in Vivo

Purpose: The clinical course of osteosarcoma (OS) demands the development of new therapeutic options. Conditionally replicative adenoviruses (CRAds) represent promising agents for the treatment of solid tumors, because CRAds have an intrinsic replication capacity that allows in situ amplification and extensive tumor infection through lateral spread. The CRAd AdΔ24 has been developed to replicate selectively in cells with a defective retinoblastoma (Rb) pathway. Because genetic alterations in the Rb pathway are frequently observed in OS, AdΔ24 might be useful in the treatment of this cancer. Experimental Design: Because the lack of Coxsackie adenovirus receptor on OS cells limits the efficacy of CRAd treatment, we explored alternative target molecules on OS. Insertion of an Arg-Gly-Asp (RGD-4C) integrin-targeting motif into the adenovirus fiber knob expanded tropism toward the ανβ3 and ανβ5 integrins. The oncolytic capacity of the CRAd Ad5-Δ24RGD was tested on primary OS cells in vitro and in vivo. Results: The ανβ3and ανβ5 integrins are abundantly expressed on OS cells. RGD-mediated infection augmented adenovirus infection of primary OS cells by two orders of magnitude. Ad5-Δ24RGD was shown to be highly active in killing human OS cell lines, as well as primary cell cultures. Furthermore, intratumoral injections with Ad5-Δ24RGD into established human OS xenografts that were derived directly from a patient with OS refractory for chemotherapeutic treatment caused a significant tumor-growth delay. Furthermore, adenoviral particles could be detected in tumor cells 25 days posttumor injection. Conclusions: Targeting adenovirus toward integrins rendered OS cells more sensitive to killing by Ad5-Δ24RGD. These findings suggest that Ad5-Δ24RGD has potential for use in OS treatment.

Different susceptibility of osteosarcoma cell lines and primary cells to treatment with oncolytic adenovirus and doxorubicin or cisplatin

Despite improvements in treatment regimens for osteosarcoma (OS) patients, survival rate has not increased over the last two decades. New treatment modalities are therefore warranted. Preclinical results with conditionally replicative adenoviruses (CRAds) to treat OS are promising. One type of CRAd that was effective against OS cells is Ad5-Δ24RGD. In other types of cancer, CRAds have been shown to interact synergistically with chemotherapeutic agents. Chemotherapy for OS often includes doxorubicin and cisplatin. Therefore, we explored combination treatment of OS cell lines and primary OS cell cultures with Ad5-Δ24RGD and doxorubicin or cisplatin. On OS cell lines, combination treatment was additive to synergistic. Surprisingly, however, on seven of eight primary OS samples no such combination effects were observed. In contrast, in many cases chemotherapy even inhibited CRAd-mediated cell killing. The inhibitory effect of doxorubicin on Ad5-Δ24RGD in primary OS cells appeared to correlate with slow cell growth rate; reduced viral replication and absence of chemotherapy-induced G2 cell cycle arrest. Our results point to the possibility that, at least for OS, virotherapy and chemotherapy should best not be performed simultaneously. In general, our work underscores the importance of testing new genetic anticancer agents and treatment regimens on primary cancer specimens.

Functional ability and physical activity in children and young adults after limb-salvage or ablative surgery for lower extremity bone tumors

Aim of our study was to compare functional ability and physical activity in children and young adults who underwent surgery for a malignant bone tumor that was located around the knee.

Quality of life in young patients after bone tumor surgery around the knee joint and comparison with healthy controls.

This study aimed to compare the health related quality of life (HRQoL) of children and adolescents after malignant bone tumor surgery of the leg with healthy controls.

Identification of Novel Candidate Oncogenes in Chromosome Region 17p11.2-p12 in Human Osteosarcoma

Osteosarcoma is the most common primary malignancy of bone. The tumours are characterized by high genomic instability, including the occurrence of multiple regions of amplifications and deletions. Chromosome region 17p11.2–p12 is amplified in about 25% of cases. In previous studies, COPS3 and PMP22 have been identified as candidate oncogenes in this region. Considering the complexity and variation of the amplification profiles for this segment, the involvement of additional causative oncogenes is to be expected. The aim of the present investigation is to identify novel candidate oncogenes in 17p11.2–p12. We selected 26 of in total 85 osteosarcoma samples (31%) with amplification events in 17p11.2–p12, using quantitative PCR for 8 marker genes. These were subjected to high-resolution SNP array analysis and subsequent GISTIC analysis to identify the most significantly amplified regions. Two major amplification peaks were found in the 17p11.2–p12 region. Overexpression as a consequence of gene amplification is a major mechanism for oncogene activation in tumours. Therefore, to identify the causative oncogenes, we next determined expression levels of all genes within the two segments using expression array data that could be generated for 20 of the selected samples. We identified 11 genes that were overexpressed through amplification in at least 50% of cases. Nine of these, c17orf39, RICH2, c17orf45, TOP3A, COPS3, SHMT1, PRPSAP2, PMP22, and RASD1, demonstrated a significant association between copy number and expression level. We conclude that these genes, including COPS3 and PMP22, are candidate oncogenes in 17p11.2–p12 of importance in osteosarcoma tumourigenesis.

Colour Doppler ultrasound predicts chemotherapy response, but not survival in paediatric osteosarcoma

Background: Histological response to chemotherapy is an important prognostic factor in osteosarcoma, influencing therapeutic considerations. It would be advantageous to be able to assess chemotherapy response, and predict survival, prior to tumour resection. Colour Doppler US (CDUS) is non-invasive, non-demanding for the patient, and easy to plan. This makes the method especially suitable for children, who comprise the majority of patients. Objective: To establish the value of CDUS for pre-operative prediction of chemotherapy response and survival, using widely available US equipment. Materials and methods: CDUS was performed in 21 consecutive patients before and after chemotherapy. Peak systolic velocity (PSV) in the soft-tissue component of the tumour and quotient of resistive index (QRI) of the feeding artery and contralateral control were calculated. A pathologist, unaware of CDUS results, assessed the response to chemotherapy after resection. Results: QRI change after chemotherapy was significantly higher in histological responders. No correlation of QRI change with survival was found. There was no significant difference in PSV change comparing any subgroup. CDUS appeared useful in predicting chemotherapy response (sensitivity 83%, specificity 86%), especially for negative response (predictive value 92%). Survival could not be predicted accurately. Conclusions: CDUS can predict chemotherapy response, but not survival. The method could be useful in planning treatment prior to definitive surgery.

Focal chromosomal copy number aberrations identify CMTM8 and GPR177 as new candidate driver genes in osteosarcoma.

Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb). For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss). These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.

A patient with scapular Ewing sarcoma; 5-year follow-up after extracorporeal irradiation and re-implantation of the scapula, a case report

In 2007 a 9½-year-old boy was treated with resection, extracorporeal irradiation and re-implantation of the right scapula. He also received chemotherapy. During five year follow-up shoulder function remained largely intact. Subtotal resorption of the scapula occurred, leaving only the glenohumeral joint intact. Sensibility and strength are intact. To date there is no sign of local or metastatic recurrence.

Tenosynovial Giant Cell Tumors in Children: A Similar Entity Compared With Adults

Background Tenosynovial giant cell tumor (TGCT) is a rare, benign, monoarticular entity. Many case-series in adults are described, whereas TGCT is only incidentally reported in children. Therefore, its incidence rate and natural history in children are unknown. Questions/purposes (1) How many cases have been reported of this condition, and what were their characteristics? (2) What is the standardized pediatric incidence rate for TGCT? (3) Is there a clinical difference in TGCT between children and adults? (4) What is the risk of recurrence after open resection in children compared with adults? Methods Data were derived from three sources: (1) a systematic review on TGCT in children, seeking sources published between 1990 and 2016, included 17 heterogeneous, small case-series; (2) the nationwide TGCT incidence study: the Dutch pediatric incidence rate was extracted from this nationwide study by including patients younger than 18 years of age. This registry-based study, in which eligible patients with TGCT were clinically verified, calculated Dutch incidence rates for localized and diffuse-type TGCT in a 5-year timeframe. Standardized pediatric incidence rates were obtained by using the direct method; (3) from our nationwide bone and soft tissue tumor data registry, a clinical data set was derived. Fifty-seven children with histologically proven TGCT of large joints, diagnosed and treated between 1995 and 2015, in all four tertiary sarcoma centers in The Netherlands, were included. These clinically collected data were compared with a retrospective database of 423 adults with TGCT. Chi-square test and independent t-test were used to compare children and adults for TGCT type, sex, localization, symptoms before diagnosis, first treatment, recurrent disease, followup status, duration of symptoms, and time to followup. The Kaplan-Meier method was used to evaluate recurrence-free survival at 2.5 years. Results TGCT is seldom reported because only 76 pediatric patients (39 female), 29 localized, 38 diffuse, and nine unknown type, were identified from our systematic review. The standardized pediatric TGCT incidence rate of large joints was 2.42 and 1.09 per million person-years in localized and diffuse types, respectively. From our clinical data set, symptoms both in children and adults were swelling, pain, and limited ROM with a median time before diagnosis of 12 months (range, 1-72 months). With the numbers available, we did not observe differences in presentation between children and adults in terms of sex, symptoms before diagnosis, first treatment, recurrent disease, followup status, or median time to followup. The 2.5-year recurrence-free TGCT survival rate after open resection was not different with the numbers available between children and adults: 85% (95% confidence interval [CI], 67%-100%) versus 89% (95% CI, 83%-96%) in localized, respectively (p = 0.527) and 53% (95% CI, 35%-79%) versus 56% (95% CI, 49%-64%) in diffuse type, respectively (p = 0.691). Conclusions Although the incidence of pediatric TGCT is low, it should be considered in the differential diagnosis in children with chronic monoarticular joint effusions. Recurrent disease after surgical treatment of this orphan disease seems comparable between children and adults. With targeted therapies being developed, future research should define the most effective treatment strategies for this heterogeneous disease. Level of Evidence Level III, therapeutic study.