Background:
Autologous chondrocyte implantation, which uses passaged chondrocytes, commonly leads to the formation of fibrocartilage. When chondrocytes are passaged to increase cell numbers, they ...lose their phenotype and ability to form hyaline cartilage. The use of transforming growth factor β (TGFβ) to redifferentiate passaged chondrocytes has been validated in vitro; however, it is unknown if redifferentiated chondrocytes will enhance defect repair when implanted in vivo. Furthermore, fibrin gel is used in orthopaedic surgery as a fixative and scaffold and could be an appropriate carrier to enhance retention of cells in the repair site.
Purpose:
To investigate if passaged redifferentiated chondrocytes in fibrin gel have the ability to form cartilage tissue and if these redifferentiated cells will enhance the formation of hyaline cartilage in vivo when implanted into critical-size osteochondral defects.
Study Design:
Controlled laboratory study.
Methods:
Rabbit and human chondrocytes were serially passaged twice in monolayer culture. Twice-passaged cells were used directly (dedifferentiated) or redifferentiated in high-density culture with TGFβ3. Dedifferentiated or redifferentiated cells were mixed with fibrin gel to form fibrin clots, which were cultured in vitro to assess the use of fibrin gel as a scaffold or implanted in vivo in a critical-size osteochondral defect in New Zealand White rabbit knee joints. Rabbits were sacrificed 6 weeks after implantation, and tissues were assessed histologically and by immunohistochemistry.
Results:
Redifferentiation of passaged chondrocytes by means of 3-dimensional culture in the presence of TGFβ3 improved the formation of cartilaginous tissues in vitro, and culture in fibrin gel did not affect the cell phenotype. Implantation of dedifferentiated cells in vivo resulted in fibrocartilaginous repair tissues. Redifferentiated chondrocyte implants resulted in granulation tissues containing the hyaline cartilage marker collagen type 2.
Conclusion:
Redifferentiated chondrocytes will maintain their chondrogenic differentiation in fibrin clots. Implanted redifferentiated chondrocytes show a different reparative response than dedifferentiated chondrocytes and do not appear to enhance repair at an early time point. Another study of longer duration is required to assess tissue maturation over time.
Clinical Relevance:
Redifferentiation of passaged chondrocytes with TGFβ3 before implantation does not improve defect repair in the first 6 weeks.
Background:
Bioengineered cartilage is a developing therapeutic to repair cartilage defects. The matrix must be rich in collagen type II and aggrecan and mechanically competent, withstanding ...compressive and shearing loads. Biomechanical properties in native articular cartilage depend on the zonal architecture consisting of 3 zones: superficial, middle, and deep. The superficial zone chondrocytes produce lubricating proteoglycan-4, whereas the deep zone chondrocytes produce collagen type X, which allows for integration into the subchondral bone. Zonal and chondrogenic expression is lost after cell number expansion. Current cell-based therapies have limited capacity to regenerate the zonal structure of native cartilage.
Hypothesis:
Both passaged superficial and deep zone chondrocytes at high density can form bioengineered cartilage that is rich in collagen type II and aggrecan; however, only passaged superficial zone–derived chondrocytes will express superficial zone–specific proteoglycan-4, and only passaged deep zone–derived chondrocytes will express deep zone–specific collagen type X.
Study Design:
Controlled laboratory study.
Methods:
Superficial and deep zone chondrocytes were isolated from bovine joints, and zonal subpopulations were separately expanded in 2-dimensional culture. At passage 2, superficial and deep zone chondrocytes were seeded, separately, in scaffold-free 3-dimensional culture within agarose wells and cultured in redifferentiation media.
Results:
Monolayer expansion resulted in loss of expression for proteoglycan-4 and collagen type X in passaged superficial and deep zone chondrocytes, respectively. By passage 2, superficial and deep zone chondrocytes had similar expression for dedifferentiated molecules collagen type I and tenascin C. Redifferentiation of both superficial and deep zone chondrocytes led to the expression of collagen type II and aggrecan in both passaged chondrocyte populations. However, only redifferentiated deep zone chondrocytes expressed collagen type X, and only redifferentiated superficial zone chondrocytes expressed and secreted proteoglycan-4. Additionally, redifferentiated deep zone chondrocytes produced a thicker and more robust tissue compared with superficial zone chondrocytes.
Conclusion:
The recapitulation of the primary phenotype from passaged zonal chondrocytes introduces a novel method of functional bioengineering of cartilage that resembles the zone-specific biological properties of native cartilage.
Clinical Relevance:
The recapitulation of the primary phenotype in zonal chondrocytes could be a possible method to tailor bioengineered cartilage to have zone-specific expression.
Abstract With the success of immunotherapy in cancer, understanding the tumor immune microenvironment (TIME) has become increasingly important; however in pediatric brain tumors this remains poorly ...characterized. Accordingly, we developed a clinical immune-oncology gene expression assay and used it to profile a diverse range of 1382 samples with detailed clinical and molecular annotation. In low-grade gliomas we identify distinct patterns of immune activation with prognostic significance in BRAF V600E-mutant tumors. In high-grade gliomas, we observe immune activation and T-cell infiltrates in tumors that have historically been considered immune cold, as well as genomic correlates of inflammation levels. In mismatch repair deficient high-grade gliomas, we find that high tumor inflammation signature is a significant predictor of response to immune checkpoint inhibition, and demonstrate the potential for multimodal biomarkers to improve treatment stratification. Importantly, while overall patterns of immune activation are observed for histologically and genetically defined tumor types, there is significant variability within each entity, indicating that the TIME must be evaluated as an independent feature from diagnosis. In sum, in addition to the histology and molecular profile, this work underscores the importance of reporting on the TIME as an essential axis of cancer diagnosis in the era of personalized medicine.
When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, ...thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGFβ) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGFβ supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture.
Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGFβ. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically.
Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGFβ3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers.
This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.
Germline biallelic mutations in one of the mismatch repair genes, mutS homolog 2, mutS homolog 6, mutL homolog 1, or postmeiotic segregation increased 2, result in one of the most aggressive cancer ...syndromes in humans termed as constitutional mismatch repair deficiency (CMMRD). Individuals with CMMRD are affected with multiple tumors arising from multiple organs during childhood, and these individuals rarely reach adulthood without specific interventions. The most common tumors observed are central nervous system, hematological, and gastrointestinal malignancies. The incidence of CMMRD is expected to be high in low‐resource settings due to a high rate of consanguinity in these regions, and it is thought to be underrecognized and consequently underdiagnosed. This position paper is therefore important to provide a summary of the current situation, and to highlight the necessity of increasing awareness, diagnostic criteria, and surveillance to improve survival for patients and family members.
Replication repair deficiency (RRD) leading to hypermutation is an important driving mechanism of high-grade glioma (HGG) occurring predominantly in the context of germline mutations in ...RRD-associated genes. Although HGG presents specific patterns of DNA methylation corresponding to oncogenic mutations, this has not been well studied in replication repair-deficient tumors. We analyzed 51 HGG arising in the background of gene mutations in RRD utilizing either 450 k or 850 k methylation arrays. These were compared with HGG not known to be from patients with RRD. RRD HGG harboring secondary mutations in glioma genes such as
IDH1
and
H3F3A
displayed a methylation pattern corresponding to these methylation subgroups. Strikingly, RRD HGG lacking these known secondary mutations clustered together with an incompletely described group of HGG previously labeled “Wild type-C” or “Paediatric RTK 1”. Independent analysis of two comparator HGG cohorts showed that other RRD/hypermutant tumors clustered within these subgroups, suggesting that undiagnosed RRD may be driving some HGG clustering in this location. RRD HGG displayed a unique CpG Island Demethylator Phenotype in contrast to the CpG Island Methylator Phenotype described in other cancers. Hypomethylation was enriched at gene promoters with prominent demethylation in genes and pathways critical to cellular survival including cell cycle, gene expression, cellular metabolism, and organization. These data suggest that methylation arrays may provide diagnostic information for the detection of RRD HGG. Furthermore, our findings highlight the unique natural selection pressures in these highly dysregulated, hypermutant cancers and provide the novel impact of hypermutation and RRD on the cancer epigenome.
BackgroundResponse to immune checkpoint inhibition (ICI) is encouraging for patients with progressive, DNA replication-repair deficient, high-grade glioma (RRD-HGG).1 However, the clinical outcomes ...and biological mechanisms for subsequent immune-directed salvage approaches after progression on anti-PD1 monotherapy remain unknown.MethodsThe International RRD Consortium performed a registry study of patients managed using central molecular, genomic, radiological review and treatment recommendations between 2015–2021. Treatment after progression on anti-PD1 monotherapy included re-irradiation where feasible, and continuation of anti-PD1 with either anti-CTLA4 (ipilimumab), or a MEK-inhibitor (trametinib). Outcomes included radiological response (iRANO), toxicity, second progression-free (PFS2) and overall survival (OS2). Companion biomarkers were analyzed centrally.ResultsAmong 75 patients with RRD-HGG receiving PD-1 blockade, 20 remain progression-free at a median follow-up of 44.6-months. For 55 patients with 2nd-relapse/progressive tumors, continuation of ICI (n=38) resulted in median OS2 of 11.6-months (51% alive) versus 1.2-months when ICI was discontinued (n=17; no survivors, p<0.001). The combination of ipilimumab/nivolumab (n=24) resulted in response/stable disease in 75%, with median OS2 of 12.1-months. The addition of MEK-inhibitor led to response in 3/5 patients with prolonged survival. Re-irradiation improved OS2, especially for RRD-HGG with lower mutation burden (p=0.002), and those receiving ipilimumab (median OS2=33-months).Several important insights were gained from the biomarker-analyses. Survival was impacted by extreme mutation burden, but not genomic microsatellite instability. Delayed, sustained responses were observed in ultra-hypermutant RRD-HGG, associated with changes in mutational spectra and immune microenvironment. RRD-HGG showed elevated CTLA4 expression over time, explaining the responses to ipilimumab. The remarkable sensitivity to re-irradiation was explained by an absence of deleterious post-radiation indel signatures (ID8; COSMIC),2 suggesting selective immune-editing. Early radiological immune ‘flare’ was observed in 33% of patients on combined immunotherapy and radiation who did not demonstrate flare on monotherapy, suggesting immune-synergism. Enrichment of RAS-MAPK mutations in genomically unstable RRD-HGG explained responses to MEK-inhibitors. Additionally, reinvigoration of peripheral immune response was observed. In all cohorts, immune adverse events were a major cause of treatment interruption, with higher prevalence in patients with bi-allelic mismatch-repair deficiency vis-à-vis Lynch syndrome.ConclusionsWe provide mechanistic rationale for the sustained benefit in RRD-HGG from immune-directed/synergistic salvage. Our data suggest that the continuous mutagenesis renders hypermutant RRD-HGG susceptible to ICI beyond initial progression. The combination with re-irradiation and additional immune/targeted agents can maximize survival in these children and young adults. Future research should focus on biology-driven rational immunotherapy combinations that also result in lower toxicity to maximize patient benefit.AcknowledgementsAD would like to acknowledge the supports of the St Baldrick Foundation, Stand up to Cancer and Hold’em for Life Foundations for his fellowship and research.ReferencesDas A, Sudhaman S, Morgenstern D, et al. Genomic predictors of response to PD-1 inhibition in children with germline DNA replication repair deficiency. Nat Med. 2022;28:125–135.Kocakavuk E, Anderson KJ, Varn FS, et al. Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer. Nat Genet. 2021;53:1088–1096.Ethics ApprovalThe study was approved by the SickKids Research Ethics Board (REB number: 1000048813)ConsentConsent was obtained from study participants and/or their parents, as applicable.
Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair ...genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies.
•C-type natriuretic peptide (CNP) is an endogenous adipogenesis regulator.•CNP plasma levels and mRNA were evaluated, for the first time, in whole blood.•Normal and obese adolescents were enrolled.•A ...different trend was observed for CNP plasma levels and its expression.•Interesting area for new therapies.
C-type natriuretic peptide (CNP) is an endogenous adipogenesis regulator whose plasma levels in childhood are known, while no data are available on its expression. Our aim was to evaluate both CNP plasma levels and CNP system expression in whole blood obtained from normal-weight (N, n = 24) and obese (O, n = 16) adolescents (age:13.5 ± 0.4 years). Endothelial function was assessed measuring reactive hyperemia index (RHI). CNP plasma levels, evaluated with specific RIA, resulted significantly lower in O than in N (6.1 ± 0.8 vs.15.2 ± 1.3 pg/mL; p < 0.0001), while CNP/NPR-B/NPR-C mRNA, measured by Real-Time PCR, resulted similar in N (4.1 ± 1.7; 5.0 ± 1.6; 2.2 ± 0.9) and in O (4.3 ± 1.6; 3.5 ± 1.1; 2.3 ± 0.8). RHI was significantly lower in O than in N (1.4 ± 0.08 vs.2.1 ± 0.04, p < 0.0001). Dividing all subjects according to the RHI median value, irrespective of the presence or absence of obesity (Group 1 > 1.9, n = 23, Group 2 < 1.9, n = 17), CNP plasma concentrations resulted significantly (p = 0.014) higher in Group 1 (14.6 ± 1.6) than in Group 2 (7.5 ± 1.0), showing a significant correlation with RHI (p = 0.0026), while CNP mRNA expression was, surprisingly, higher in Group 2 (7.0 ± 2.3) than in Group 1 (1.8 ± 0.4; p = 0.02). NPR-B mRNA resulted similar in both Groups (4.3 ± 1.6; 4.7 ± 1.3) and NPR-C significantly higher in Group 2 (p = 0.02). Our data suggest different trends between CNP plasma levels and expression, assessed for the first time in whole blood, that could reflect changes occurring both at CNP transcriptional level in activated leukocytes due to inflammation, and at circulating levels, due to CNP paracrine/autocrine activities. This could represent an interesting area for new therapies able to modulate endothelial dysfunction.
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