Project SATURN- a real-world evidence data collaboration with existing European datasets in Osteogenesis Imperfecta to support future therapies.

Regulatory marketing authorisation is not enough to ensure patient access to new medicinal products. Health Technology Assessment bodies may require data on effectiveness, relative effectiveness, and cost-effectiveness. Healthcare systems may require data on clinical utility, savings, and budget impact. Furthermore, the exact requirements of these bodies vary country by country and sometimes even region to region, resulting in a patchwork of different data requirements to achieve effective, reimbursed patient access to new therapies. In addition, clinicians require data to make informed clinical management decisions. This requirement is of key importance in rare diseases where there is often limited data and clinical experience at the time of regulatory approval.This paper describes an innovative initiative that is called Project SATURN: Systematic Accumulation of Treatment practices and Utilization, Real world evidence, and Natural history data for the rare disease Osteogenesis Imperfecta. The objective of this project is to generate a common core dataset by utilising existing data sources to meet the needs of the various stakeholders and avoiding fragmentation through multiple approaches (e.g., a series of individual national requests/approaches, and unconnected with the regulators' potential requirements). It is expected that such an approach will reduce the time for patient access to life-changing medications. Whilst Project SATURN applies to Osteogenesis Imperfecta, it is anticipated that the principles could also be applied to other rare diseases and reduce the time for patient access to new medications.

Defining priorities in the transition from paediatric to adult healthcare for rare bone disease patients: a dialogic approach.

The Italian patient association for Multiple Osteochondromas, Ollier Disease, and Maffucci Syndrome, Associazione Conto Alla Rovescia-ACAR Aps, conducted a mixed-methods study at its 2023 annual conference. The study included the Open Dialogue Approach and a feedback survey to identify the main priorities in the transitioning process from paediatric to adult healthcare for patients with Multiple Osteochondromas, Ollier Disease, and Maffucci Syndrome. The common needs identified by patients, families, caregivers, and healthcare professionals were coordination and continuity of care, patient empowerment and communication, social and practical support, and transition planning and support. This experience fostered a sense of collaboration and cooperation among stakeholders, helping to build trust and create a shared vision for improving the quality of care for these patients. Furthermore, it could be considered a starting point for other patient associations interested in using different approaches to identify the needs of their members and actively involve all stakeholders.

Providing high-quality care remotely to patients with rare bone diseases during COVID-19 pandemic.

During the COVID-19 outbreak, the European Reference Network on Rare Bone Diseases (ERN BOND) coordination team and Italian rare bone diseases healthcare professionals created the "COVID-19 Helpline for Rare Bone Diseases" in an attempt to provide high-quality information and expertise on rare bone diseases remotely to patients and healthcare professionals. The present position statement describes the key characteristics of the Helpline initiative, along with the main aspects and topics that recurrently emerged as central for rare bone diseases patients and professionals. The main topics highlighted are general recommendations, pulmonary complications, drug treatment, trauma, pregnancy, children and elderly people, and patient associations role. The successful experience of the "COVID-19 Helpline for Rare Bone Diseases" launched in Italy could serve as a primer of gold-standard remote care for rare bone diseases for the other European countries and globally. Furthermore, similar COVID-19 helplines could be considered and applied for other rare diseases in order to implement remote patients' care.

Insight into the Pathology of a COL1A1 Signal Peptide Heterozygous Mutation Leading to Severe Osteogenesis Imperfecta.

Osteogenesis imperfecta or "brittle bone disease" is a congenital disorder of connective tissue causing the bone to break easily. Around 85-90% of cases are due to autosomal dominant mutations in the genes encoding type I collagen, the major organic component of bone. Genotype-phenotype correlations have shown that quantitative defects of collagen type I lead to mild OI, whereas structural defects show a wide clinical range from mild to perinatal lethal. This may partially be explained by the type of amino acid substitution and the relative location in the domain structure. To fully understand the variability of the clinical manifestation and the underlying pathomechanisms, further investigations are required. Here we provide the first biochemical characterization of a mutation at the signal peptide cleavage site of COL1A1, a domain not yet characterized. By steady-state analysis, we observed reduced production of collagen type I. Furthermore, by pulse-chase analysis we detected delayed secretion and partial intracellular retention of collagen I. In the cellular fraction, the electrophoretic migration was abnormal; however, secreted type I collagen showed a normal migration pattern. The intracellular retention of collagen I was confirmed by immunofluorescent staining. Moreover, transmission electron microscopy of cultured fibroblasts revealed enlargement of ER cisternae. These results further support the hypothesis that mechanisms interfering with ER integrity play an important role in the pathology of severe OI.

In vitro gene and chromosome characterization of expanded bone marrow mesenchymal stem cells for musculo-skeletal applications.

OBJECTIVE: A number of studies have shown the role of expanded Bone Marrow-derived Mesenchymal Stem Cells in the repair and regeneration of musculo-skeletal tissues. The current European regulations define in vitro expanded cells for clinical purposes as substantially manipulated and include them in the class of Advanced-Therapy Medicinal Products to be manufactured in compliance with current Good Manufacturing Practice. Among the characteristics that such cells should display, genomic stability has recently become a major safety concern. The aim of this study is to perform a chromosomal and genetic characterization of Bone Marrow-derived Mesenchymal Stem Cells expanded in compliance with Good Manufacturing Practice for a potential clinical use in orthopaedics. MATERIALS AND METHODS: Mesenchymal Stem Cells, isolated from bone marrow, were expanded for six weeks in compliance with current Good Manufacturing Practice. DNA profiling analyses were applied to test cross-contamination absence. Genomic stability was evaluated by means of karyotyping, sequencing of TP53, p21/CDKN1A and MDM2 genes and the expression analysis of c-MYC and H-RAS oncogenes, p21/CDKN1A, TP53, p16/CDKN2A, RB1 and p27/CDKN1B tumor suppressor genes and hTERT gene. RESULTS: The DNA profiling analysis showed a unique genetic profile for each Mesenchymal Stem Cell culture, indicating the absence of cross-contamination. Karyotyping evidentiated some chromosomal abnormalities within the 10% limit set by the Cell Products Working Party review, except for one patient. In all cases, the molecular biology analyses did not revealed DNA point mutations, acquisition or changes in gene expression. hTERT levels were undetectable. CONCLUSIONS: Cultured Mesenchymal Stem Cells do not seem to be prone to malignant transformation. In fact, although some chromosomal aberrations were found, molecular biology analyses demonstrated that the expansion phase did not induce the acquisition of de novo genetic changes.

MicroRNA profiling of multiple osteochondromas: identification of disease-specific and normal cartilage signatures.

Multiple osteochondroma (MO) is a rare skeletal disease characterized by the formation of multiple benign cartilage-capped bone tumors; in 1-5% of patients, a malignant transformation into peripheral chondrosarcoma may occur. This disorder is characterized by a large spectrum of germline mutations scattered along EXT1/EXT2 genes, the presence of a significant percentage of patients without alterations in EXT genes, and a large phenotypic variability. The molecular basis of MO genetic and clinical heterogeneity, including the causes underlying malignant transformation, is currently unknown. This leads to the lack of appropriate diagnostic/prognostic markers as well as of therapeutic options. Recently, specific microRNAs (miRNAs) were reported to be involved in chondrogenesis and inflammatory cartilage diseases. We therefore hypothesized a role for microRNAs in cartilaginous tumors and investigated microRNA expression in osteochondroma and normal cartilage tissues to evaluate whether they could affect osteochondromas onset and/or clinical manifestations. Our results indicate that miRNAs differentially expressed in MO samples may hamper the molecular signaling responsible for normal differentiation of chondrocytes, contributing to pathogenesis and clinical outcome. Although further studies are needed to validate our observations and to identify targets of miRNAs, this is the first study reporting on miRNA expression in growth plate and its comparison with pathological conditions.

Genetic models of osteochondroma onset and neoplastic progression: evidence for mechanisms alternative to EXT genes inactivation.

Osteochondroma, the most common benign bone tumor, may occur as a sporadic lesion or as multiple neoplasms in the context of multiple osteochondromas syndrome. The most severe complication is malignant transformation into peripheral secondary chondrosarcoma. Although both benign conditions have been linked to defects in EXT1 or EXT2 genes, contradictory reports are present in the literature regarding the requirement of their biallelic inactivation for osteochondroma development. A major limitation of these studies is represented by the small number of samples available for the screening. Taking advantage of a large series of tissues, our aim was to contribute to the definition of a genetic model for osteochondromas onset and transformation. EXT genes point mutations and big deletions were analyzed in 64 tissue samples. A double hit was found in 5 out of 35 hereditary cases, 6 out of 16 chondrosarcomas and 2 recurrences; none of the 11 sporadic osteochondromas showed two somatic mutations. Our results clearly indicate that, in most cases, biallelic inactivation of EXT genes does not account for osteochondromas formation; this mechanism should be regarded as a common feature for hereditary osteochondromas transformation and as an event that occurs later in tumor progression of solitary cases. These findings suggest that mechanisms alternative to EXT genetic alteration likely have a role in osteochondromas pathogenesis.

Osteosarcoma of the pelvis.

AIMS: To describe the outcomes of a large number of patients with pelvic osteosarcoma, and to define the guidelines for appropriate treatment. METHODS: We reviewed 60 consecutive patients with primary pelvic high-grade osteosarcoma. The tumour involved the whole hemipelvis in 15 cases, while the most common location was the iliac wing in 29 cases (48.3%): 25 of these adjacent to or passing the sacroiliac joint. RESULTS: Thirty patients underwent surgery; there were 16 hindquarter amputations and 14 internal hemipelvectomies. All the patients who presented with metastasis died of their disease. In 18 cases wide margins were achieved, however, eight patients experienced local recurrence. Of the series, only eight patients are still alive. CONCLUSION: The use of intense chemotherapy and surgical wide margin, hardly seems to achieve local control, however, tumour necrosis was correlated with positive prognosis. When internal hemipelvectomy it is not safe enough, amputation must be considered, particularly for cases with sacrifice of the sciatic nerve roots or for older patients where a shorter surgical procedure can be less risky.

Involvement of INK4A gene products in the pathogenesis and development of human osteosarcoma.

BACKGROUND: The INK4A tumor suppressor gene plays a crucial role in the regulation of the G1 cell cycle phase. It encodes two transcripts, p16 and p14 alternate reading frame (ARF), involved in retinoblastoma protein (pRb)- and p53- cell growth control pathways, respectively. METHODS: To define the role of gene status and molecule expression involved in the INK4A regulatory system, immunohistochemistry, immunoblotting, and polymerase chain reaction (PCR) analysis were performed on 35 primary high grade osteosarcomas (OS). RESULTS: Although p16 and p14ARF proteins were found negative or weakly detectable in 60% and 57% of the cases respectively, INK4A gene analysis of exons 1alpha, 1beta and 2 did not reveal any deletion or mutation. However, methylation status of the 5'CpG promoter region, assessed by methylation-specific PCR, was found in 12 out of 21 OSs with negative or weak p16 expression. A statistical analysis based on pRb/p16 and p53/p14ARF staining status showed that pRb and p16 co-expression was inversely correlated to tumor relapse and was a marker for a more favorable prognosis. A statistically significant inverse correlation was found between wt-p53 and p14ARF expression. In the group of wt-p53 tumors, the loss of p14ARF was associated with a decreased expression of p21 protein, suggesting a down-regulation of the transcriptional activity of p53. CONCLUSIONS: The current results suggest that, in OS, the altered expression of INK4A products plays a primary role in the deregulation of both pRb and p53 cell growth control pathways, contributing to tumor pathogenesis and development.

Changes in p14(ARF) do not play a primary role in human chondrosarcoma tissues.

The locus encoding the tumor suppressor p16 has been found to code for a second, different protein. This protein, p14(ARF), has been shown to protect p53 from degradation. Like p16, its gene is often altered in different cancers. In this study, the first unique exon, exon 1 beta, of p14(ARF), has been studied in 22 chondrosarcoma tissues using polymerase chain reaction, DNA sequencing and methylation-specific polymerase chain reaction. One chondrosarcoma was found to have exon 1 beta homozygously deleted, but neither mutations nor methylations were found in any of the chondrosarcomas. This indicates that genetic changes of p14(ARF) are a rare event in chondrosarcoma.

Presence of telomerase activity in different musculoskeletal tumor histotypes and correlation with aggressiveness.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. Telomerase activity was observed and correlated with aggressiveness in different neoplasms such as breast, prostate, blood and brain cancers, among others. To investigate whether telomerase activity is an index of aggressiveness in bone and soft tissue lesions of the extremities, 66 biopsy samples from our tissue bank were studied. These samples included 43 high-grade sarcomas, 9 aggressive benign tumors and 14 totally benign lesions. The samples were collected from patients homogeneously treated at the Rizzoli Orthopaedic Institute with a follow-up ranging from 4 to 11 years (median, 7 years). A non-radioactive polymerase chain reaction-based enzyme-linked immunosorbent assay was used for the study. All tumors investigated were positive for telomerase activity. Among benign lesions, only 2 aneurysmal bone cysts showed higher telomerase activity than the cut-off point, whereas all the other benign lesions had lower activity. Our results indicate that high levels of telomerase activity in bone and soft tissue lesions correlate with more aggressive clinical behavior in patients treated with surgery alone. An interesting inverse correlation between telomerase activity and occurrence of pulmonary metastasis was detected in osteosarcoma patients treated with chemotherapy. A parallel increase of telomerase activity and malignancy was observed in the adipose and cartilagineous tissue lesions. Our data suggest that telomerase activity could be considered a marker of tumor aggressiveness for bone and soft tissue lesions. The results obtained in osteosarcoma samples suggest that low levels of telomerase activity may be predictive of the prognosis and should influence the therapeutic protocol.

Changes of the p16 gene but not the p53 gene in human chondrosarcoma tissues.

The role of two important tumour suppressor genes, p16 and p53, was evaluated in cartilaginous tumour tissues. Genomic DNA from 22 chondrosarcomas, 5 benign chondroid tumours, 1 sample of reactive proliferative cartilage and 2 samples of normal cartilage were analysed using polymerase chain reaction, single strand conformational polymorphism, DNA sequencing and methylation-specific polymerase chain reaction. The p16 gene was found to be partly methylated in 5 high-grade chondrosarcomas and homozygously deleted in 1 chondrosarcoma. Moreover, a polymorphism was detected in 3 malignant tumours, but not in benign tumours or normal cartilage. Analysis of the p53 gene revealed an unchanged structure in all samples. These findings show a role for p16, but not p53, in chondrosarcoma.

Angiogenesis in lipoma: An experimental study in the chick embryo chorioallantoic membrane.

Lipoma is one of the most common benign mesenchymal tumors. Its ability to trigger an angiogenic response is a critical step for its growth. Because adipose tissue serves as an important conduit for the vasculature, it is conceivable that the angiogenic properties of this tissue may modulate the growth of the vasculature in a paracrine manner. We investigated in vivo the angiogenic potential of bioptic fragments of human lipoma by using the chick embryo chorioallantoic membrane (CAM), a useful model for such an investigation. The angiogenic response in pathological and control implants was assessed on histologic sections by a morphometric method, 96 h after grafting. Results showed that pathological samples were surrounded by numerous allantoic vessels with a radially arranged pattern around the implant. The vascular counts in the CAMs treated with lipoma implants were comparable to that of FGF-2. The role played in vasoproliferative response by angiogenic cytokines (FGF-2, VEGF) released by adipocytes, by endogenous cytokines, such as FGF-2, stored in the CAM extracellular matrix and by angiogenic growth factors released by perivascular mononuclear cells around the newly-formed blood vessels, were supported by this study.

Secondary tumors in bone sarcomas after treatment with chemotherapy.

New oncologic treatments have improved survival in osteosarcoma and Ewing's sarcoma. However, these treatments may cause secondary malignancies after radiotherapy. This study evaluated the incidence of secondary malignancies after neoadjuvant chemotherapy. Between April 1972 and December 1990, 518 osteosarcoma and 299 Ewing's sarcoma patients entered neoadjuvant chemotherapy protocols. Follow-up records of all patients were analyzed and malignant tumors were reported. Nine patients developed another malignancy, including 5 leukemias, 1 astrocytoma, 1 liposarcoma, 1 parotid, and 1 breast carcinoma. Four leukemias were found in patients treated for osteosarcoma with chemotherapy, but not radiotherapy. Only one leukemia developed after Ewing's sarcoma treated with chemotherapy and radiotherapy. The incidence of leukemias is high, while the other tumors can be explained as unrelated cases. Incidence densities for leukemia were calculated for both groups of patients. Treated osteosarcoma patients seem to have a predisposition to develop leukemias, but whether this is chemotherapy induced needs to be investigated.

Efficacy of combined treatments in NIDDM patients with secondary failure to sulphonylureas. Is it predictable?

The treatment of NIDDM patients with secondary failure to sulphonylurea is a common problem. We performed a crossover study in 50 NIDDM patients with secondary failure to glibenclamide by comparing the addition to sulphonylurea of either a low-dose bedtime NPH insulin or a t.i.d. oral metformin and by analyzing treatment efficacy in relation to patient and disease characteristics. Both combined therapies clearly improved glycaemic control. HbA1 c were similarly reduced by the addition of either bedtime NPH insulin (7.6+/-0.34 vs 8.7+/-0.35, p<0.01) or metformin (7.6+/-0.22 vs 8.6+/-0.31, p<0.01). Also fasting plasma glucose (FPG) and post-prandial plasma glucose (PPPG) significantly decreased (p<0.01) with both treatments. Bed-time NPH insulin was more effective on FPG reduction than metformin (-36+/-2% vs -25+/-2%, p<0.01); in contrast, metformin addition was more effective on PPPG reduction than bedtime NPH insulin addition (-30+/-2% vs 20+/-3%, p<0.01). Serum cholesterol was marginally but significantly decreased after metformin (5.49+/-0.19 vs 5.91 +/-0.18 mM, p<0.05) but not after NPH insulin. Body weight increase was significantly greater after insulin addition than after metformin (1.47+/-0.25 Kg vs 0.64+/-0.17 p=0.02). All patients preferred the addition of metformin rather than NPH insulin. None of the measured clinical and metabolic variables (before treatment FPG and PPPG, HbA1 c, post-glucagon C-peptide levels, insulin sensitivity, patient age, BMI and diabetes duration) significantly correlated to the efficacy of the two combined treatments studied. In conclusion, in NIDDM patients with secondary failure to sulphonylureas the addition of either low-dose bedtime NPH insulin or t.i.d. metformin is similarly effective in improving glycaemic control. Metformin is better accepted by patients and provides a modest advantage in terms of body weight and cholesterol levels. The most common clinical and metabolic variables are not useful for predicting the efficacy of these two combined treatments.

Risk factors for local recurrences after limb-salvage surgery for high-grade osteosarcoma of the extremities.

BACKGROUND: Improvements in preoperative staging as well as in chemotherapeutic regimens have made limb-salvage surgery a reliable modality of treatment for high-grade osteosarcomas of the extremities, with local recurrences in most series of less than 10% after this type of surgery. The quality of surgical margins and local response to preoperative chemotherapy are known to be the most significant factors in recurrence [1, 8-10, 12], and complications related to the biopsy procedure may also be a significant factor. The study reported here comprised a histopathological analysis of our recurrent cases as part of an effort to identify the impact of each of the factors cited above. MATERIALS AND METHODS: Five hundred fourteen cases of high-grade, non-multicentric osteosarcoma of the extremities were treated at the Istituto Ortopedico Rizzoli between March 1983 and August 1991. In this study we analyzed 23 cases of local recurrence in patients with classic osteosarcoma who underwent limb-salvage procedures. RESULTS: In 15 cases we found correlation between the site of local recurrence and the site where the margins were less than wide. In five cases the recurrence was secondary to complications of the biopsy procedure (hematoma, delayed healing). In one case we suspect a previously undetected skip lesion. In the remaining two cases no clear explanation was found for the recurrence. There was also a statistically significant difference in the time of appearance of recurrences related to the tumor response to chemotherapy. CONCLUSIONS: For only two cases of recurrence was there no clear explanation. In one we suspect an undetected skip metastasis, and in the other there were certain factors which may have increased its risk of recurrence (non diagnostic trochar biopsy followed by an incisional biopsy, fair tumor necrosis, recurrence in a 'problem' anatomical site, i.e., the popliteal space). In the remaining cases the following factors were found to be directly related to the development of a local recurrence: a) the quality of the surgical margins, b) site of the biopsy as well as complications related to the biopsy procedure, c) local response to preoperative chemotherapy.

Altered G1 phase regulation in osteosarcoma.

Alterations in the normal cell cycle lead to abnormal cell proliferation and to tumor development. To explore the role of the cyclin D/Cdk4 complex and the retinoblastoma protein (pRb) in the growth and spread of osteoblastic osteosarcoma (OS), 40 tumor samples were selected. In 17 of these cases, lung metastases occurred during follow-up. Expression of pRb, cyclin D1 and its catalytic subunit, Cdk4, was studied by immunohistochemistry and immunoblotting. As controls, non-neoplastic tissues surrounding the tumor were used. The expression level and pattern were compared to clinical outcome. Cdk4 was over-expressed in 80% of OS, independently of clinical outcome, and showed an intense and uniform distribution in tumor cells compared to normal cells. However, co-immunoprecipitation of Cdk4 with cyclin D1 revealed low levels of cyclin D/Cdk4 complex; 20 of 40 OS examined had a negative or minimal immunostaining for active pRb. The probability of relapse was significantly higher in pRb-negative than in the -positive patients (p < 0.05). The ratio of unphosphorylated/hyperphosphorylated pRb was lower in relapsed patients than in patients with no evident disease, though the difference was not statistically significant. High levels of pRb/cyclin D1 were found in all samples exhibiting functional pRb expression. Our results show that G1 phase deregulation is involved in formation and development of OS. The expression levels of both pRb and cyclin D1 had a clear correlation with clinical outcome, suggesting that these parameters could be used as prognostic markers.

Chemotherapy-induced tumor necrosis as a prognostic factor in localized Ewing's sarcoma of the extremities.

PURPOSE: This study was performed to assess the prognostic value of the proposed histopathologic method to evaluate the response of the primary tumor to preoperative chemotherapy in Ewing's sarcoma. PATIENTS AND METHODS: The response to chemotherapy was evaluated from the specimens of 118 Ewing's sarcoma patients, who were preoperatively treated by chemotherapy alone. Responses were graded I to III (macroscopic viable tumor, microscopic viable tumor, and no viable tumor cells, respectively). Follow-up data were available for all patients, with a mean follow-up duration of 86 months (range, 30 to 158). RESULTS: A statistically highly significant difference was observed in outcome among the three groups of patients. For patients with total necrosis (grade III response), the estimated 5-year disease-free survival rate was 95%, in contrast to 68% for grade II responders and 34% for grade III responders (P < .0001). This difference was also confirmed when any single group was compared with the other groups. Among the parameters tested, patient age and the size of tumor had some prognostic value. CONCLUSION: The proposed histopathologic grading, to evaluate the effect of chemotherapy on the primary tumor, had the strongest correlation to clinical outcome. This method could therefore be used to identify patients with a high risk of recurrent disease. These patients could be randomized to receive alternative postoperative treatments to investigate whether more aggressive therapies will improve outcome.

Alveolar soft part sarcoma.

We present a rare case of alveolar soft-tissue sarcoma, with extensive bone invasion. The differential diagnosis with bone metastasis and primary bone tumors is discussed.