Analysis of PRAME immunocytochemistry in 109 acral malignant melanoma in situ.

AIMS: Preferentially expressed antigen in melanoma (PRAME) recently is a reliable immunohistochemistry (IHC) marker for distinguishing melanoma from other lesions. However, there are few articles focused on PRAME use in acral malignant melanoma, the most common type in Asians. This study investigated PRAME IHC expression in a large series of acral malignant melanoma in situ to add to the body of clinical knowledge. METHODS: PRAME IHC was performed in unequivocal cases of primary acral lentiginous melanoma in situ (ALMIS), subungual melanoma in situ (SMIS) and acral recurrent nevi as the control. PRAME tumour cell percentage positivity and intensity were expressed as categorised in a cumulative score by adding the quartile of positive tumour cells to intensity labelling. The final IHC expression was interpreted as negative (0-1), weak (2-3), moderate (4-5) or strong (6-7). RESULTS: In 91 ALMIS patients, 32 cases (35.16%) were strong, 37 (40.66%) were moderate and 22 (24.18%) were weak. In 18 SMIS patients, strong positivity of PRAME was observed in 4 (22.22%) cases, moderate in 10 (55.56%) and weak in the remaining 4 (22.22%). No melanoma sample was negative for PRAME. By comparison, only 2 of the 40 acral recurrent nevi cases were positive. CONCLUSIONS: Our study supports the ancillary value of PRAME for diagnosing ALMIS and SMIS with high sensitivity and specificity.

Construction and Identification of an NLR-Associated Prognostic Signature Revealing the Heterogeneous Immune Response in Skin Cutaneous Melanoma.

Background: Skin cutaneous melanoma (SKCM) is the deadliest dermatology tumor. Ongoing researches have confirmed that the NOD-like receptors (NLRs) family are crucial in driving carcinogenesis. However, the function of NLRs signaling pathway-related genes in SKCM remains unclear. Objective: To establish and identify an NLRs-related prognostic signature and to explore its predictive power for heterogeneous immune response in SKCM patients. Methods: Establishment of the predictive signature using the NLRs-related genes by least absolute shrinkage and selection operator-Cox regression analysis (LASSO-COX algorithm). Through univariate and multivariate COX analyses, NLRs signature's independent predictive effectiveness was proven. CIBERSORT examined the comparative infiltration ratios of 22 distinct types of immune cells. RT-qPCR and immunohistochemistry implemented expression validation for critical NLRs-related prognostic genes in clinical samples. Results: The prognostic signature, including 7 genes, was obtained by the LASSO-Cox algorithm. In TCGA and validation cohorts, SKCM patients with higher risk scores had remarkably poorer overall survival. The independent predictive role of this signature was confirmed by multivariate Cox analysis. Additionally, a graphic nomogram demonstrated that the risk score of the NLRs signature has high predictive accuracy. SKCM patients in the low-risk group revealed a distinct immune microenvironment characterized by the significantly activated inflammatory response, interferon-α/γ response, and complement pathways. Indeed, several anti-tumor immune cell types were significantly accumulated in the low-risk group, including M1 macrophage, CD8 T cell, and activated NK cell. It is worth noting that our NLRs prognostic signature could serve as one of the promising biomarkers for predicting response rates to immune checkpoint blockade (ICB) therapy. Furthermore, the results of expression validation (RT-qPCR and IHC) were consistent with the previous analysis. Conclusion: A promising NLRs signature with excellent predictive efficacy for SKCM was developed.

3D printed strontium-doped calcium phosphate ceramic scaffold enhances early angiogenesis and promotes bone repair through the regulation of macrophage polarization.

The vascularization of bone repair materials is one of the key issues that urgently need to be addressed in the process of bone repair. The changes in macrophage phenotype and function play an important role in the process of vascularization, and endowing bone repair materials with immune regulatory characteristics to enhance angiogenesis is undoubtedly a new strategy to improve the effectiveness of bone repair. In order to improve the effect of tricalcium phosphate (TCP) on vascularization and bone repair, we doped strontium ions (Sr) into TCP (SrTCP) and prepared porous 3D printed SrTCP scaffolds using 3D printing technology, and studied the scaffold mediated macrophage polarization and subsequent vascularization and bone regeneration. The results of the interaction between the scaffold and macrophages showed that the SrTCP scaffold can promote the polarization of macrophages from M1 to M2 and secrete high concentrations of VEGF and PDGF-bb cytokines, which shows excellent angiogenic potential. When human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophage-conditioned medium of SrTCP scaffold, HUVECs exhibited excellent early angiogenesis-promoting effects in terms of scratch healing, angiogenic gene expression, and in vitro tube formation performance. The results of in vivo bone repair experiments showed that the SrTCP scaffold formed a vascular network with high density and quantity in the bone defect area, which could increase the rate of new bone formation and advance the period of bone formation, and finally achieved a better bone repair effect. We observed a cascade effect in which Sr-doped SrTCP scaffold regulate macrophage polarization to enhance angiogenesis and promote bone repair, which may provide a new strategy for the repair of clinical bone defects.

A case of pagetoid reticulosis treated with topical photodynamic therapy.

A 70-year-old male presented with reddish brown, keratotic plaque on his left leg which had grown slowly for ten years. The histopathological examination revealed acanthosis and bands of lymphocytes with significant epidermotropism. The intraepidermal lymphocytes showed large, pleomorphic and hyperchromatic nuclei with distinct nucleoli and perinuclear halos, positive for CD3, CD4 and CD30, negative for CD8, CD20 and high in Ki67 index. Accordingly, the diagnosis of Pagetoid reticulosis was made. The patient was then treated with topical photodynamic therapy once a week sequential to partial in-situ resection and preliminary skin dermabrasion and finally achieved complete remission without obvious scars or recurrence after four sessions.

PLA2G6 Silencing Suppresses Melanoma Progression and Affects Ferroptosis Revealed by Quantitative Proteomics.

Although phospholipase A2 group VI (PLA2G6) is involved in oncogenesis in several human tumors, its expression and role in cutaneous malignant melanoma (CMM) pathogenesis remains unclear. Here, by using the Oncomine and CCLE online database, immunohistochemistry, RT-qPCR, and western blotting analysis, we revealed that PLA2G6 was markedly up-regulated in CMM tissues compared to nevus tissues, as well as remarkably increased in vitro in SK-MEL-28 and M14 melanoma cell lines compared to human melanocytes. In vivo, PLA2G6 was also elevated in nine melanoma tissues compared to adjacent tissues. To investigate the malignant behaviors of PLA2G6 in CMM, SK-MEL-28 and M14 cell lines with PLA2G6 stable knockdown by RNAi strategy were constructed. Through CCK8 and colony formation assays in vitro and xenograft tumor experiment in vivo, we found that knockdown of PLA2G6 dramatically inhibited cell proliferation. The results of scratch-wound and transwell assays suggested that the migration and invasion of melanoma cells were prominently suppressed after silencing PLA2G6. In addition, flow cytometry showed that the knockdown of PLA2G6 promoted the apoptosis rate of melanoma cells. To further explore the potential molecular mechanism, we used liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) proteomic and bioinformatics analysis. The GO and KEGG analysis suggested that the underlying mechanism of PLA2G6 in CMM might be associated with the ferroptosis pathway, and ferroptosis-related proteins were validated to be differentially expressed in PLA2G6 knockdown SK-MEL-28 and M14 cells. Together, these results suggested that PLA2G6 knockdown significantly inhibited cell proliferation, metastasis, and promoted apoptosis in melanoma. Our findings on the biological function of PLA2G6 and the underlying association between PLA2G6 and ferroptosis in melanoma may contribute to developing a potential therapeutic strategy for melanoma.

Preliminary application of 3D-printed individualised guiding templates for total hip arthroplasty in Crowe type IV developmental dysplasia of the hip.

OBJECTIVE: To evaluate the feasibility and accuracy of three-dimensional (3D)-printed individualised guiding templates in total hip arthroplasty (THA) for the treatment of developmental dysplasia of the hip (DDH). METHODS: 12 hips in 12 patients with Crowe type IV DDH were treated with THA. A 3D digital model of the pelvis and lower limbs was reconstructed using the computed tomography data of the patients. Preoperative surgical simulations were performed to determine the most suitable surgical planning, including femoral osteotomy and prosthesis placement. Based on the ideal surgical planning, individualised guiding templates were designed by software, manufactured using a 3D printer, and used in acetabulum reconstruction and femoral osteotomy during surgery. RESULTS: 12 patients were followed up for an average of 72.42 months (range 38-135 months). During surgery, the guiding template for each case was matched to the bony markers of the acetabulum and proximal femur. Preoperative and follow-up Harris Hip Scores were 34.2 ± 3.7 and 85.2 ± 4.2; leg-length discrepancy, 51.5 ± 6.5 mm and 10.2 ± 1.5 mm; and visual analogue scale scores, 6.2 ± 0.8 and 1.3 ± 0.3, respectively, with statistical difference. Shortened deformity and claudication of the affected limb were obviously improved after surgery. However, 1 patient had artificial hip dislocation 2 weeks after surgery, and another patient had sciatic nerve traction injury, both of whom recovered after physical treatment. CONCLUSIONS: Preoperative surgical simulation and 3D-printed individualised guiding templates can fulfil surgeon-specific requirements for the treatment of Crowe type IV DDH. Accurate THA can be achieved using 3D-printed individualised templates, which provide a new personalised surgical plan for the precise positioning and orientation of acetabular reconstruction and femoral osteotomy.

N6-methyladenosine reader YTHDF3 regulates melanoma metastasis via its 'executor'LOXL3.

BACKGROUND: A number of studies have demonstrated that N6-methyladenosine (m6A) plays a vital role in the pathological process of various tumours. Recently, it was found that m6A writers or erasers affect the tumourigenesis of melanoma. However, the relationship between m6A readers such as YTH domain family (YTHDF) proteins and melanoma was still elusive. METHODS: RT-qPCR, Western blot and immunohistochemistry were conducted to measure the expression level of YTH N6-methyladenosine RNA binding protein 3 (YTHDF3) and lysyl oxidase-like 3 (LOXL3) in melanoma tissues and cells. The effects of YTHDF3 and LOXL3 on melanoma were verified in vitro and in vivo. Multi-omics analysis including RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses was performed to identify the target. The interaction between YTHDF3 and LOXL3 was verified by RT-PCR, Western blot, MeRIP-qPCR, RIP-qPCR and CRISPR-Cas13b-based epitranscriptome engineering. RESULTS: In this study, we found that m6A reader YTHDF3 could affect the metastasis of melanoma both in vitro and in vivo. The downstream targets of YTHDF3, such as LOXL3, phosphodiesterase 3A (PDE3A) and chromodomain helicase DNA-binding protein 7 (CHD7) were identified by means of RNA-seq, MeRIP-seq, RIP-seq and mass spectrometry analyses. Besides, RT-qPCR, Western blot, RIP-qPCR and MeRIP-qPCR were performed for subsequent validation. Among various targets of YTHDF3, LOXL3 was found to be the optimal target of YTHDF3. With the application of CRISPR-Cas13b-based epitranscriptome engineering, we further confirmed that the transcript of LOXL3 was captured and regulated by YTHDF3 via m6A binding sites. YTHDF3 augmented the protein expression of LOXL3 without affecting its mRNA level via the enrichment of eukaryotic translation initiation factor 3 subunit A (eIF3A) on the transcript of LOXL3. LOXL3 downregulation inhibited the metastatic ability of melanoma cells, and overexpression of LOXL3 ameliorated the inhibition of melanoma metastasis caused by YTHDF3 downregulation. CONCLUSIONS: The YTHDF3-LOXL3 axis could serve as a promising target to be interfered with to inhibit the metastasis of melanoma.

Multi-stage controllable degradation of strontium-doped calcium sulfate hemihydrate-tricalcium phosphate microsphere composite as a substitute for osteoporotic bone defect repairing: degradation behavior and bone response.

Various requirements for the repair of complex bone defects have motivated to development of scaffolds with adjustable degradation rates and biological functions. Tricalcium phosphate (TCP) and calcium sulfate are the most commonly used bone repair materials in the clinic, how to better combine TCP and calcium sulfate and play their greatest advantages in the repair of osteoporotic bone defect is the focus of our research. In this study, a series of scaffolds with multistage-controlled degradation properties composed of strontium-doped calcium sulfate (SrCSH) and strontium-doped tricalcium phosphate (Sr-TCP) microspheres scaffolds were prepared, and their osteogenic activity,in vivodegradation and bone regeneration ability in tibia of osteoporotic rats were evaluated.In vitrostudies revealed that different components of SrCSH/Sr-TCP scaffolds significantly promoted the proliferation and differentiation of MC3T3-E1 cells, which showed a good osteogenic induction activity.In vivodegradation results showed that the degradation time of composite scaffolds could be controlled in a large range (6-12 months) by controlling the porosity and phase composition of Sr-TCP microspheres. The results of osteoporotic femoral defect repair showed that when the degradation rate of scaffold matched with the growth rate of new bone, the parameters such as bone mineral density, bone volume/total volume ratio, trabecular thickness, angiogenesis marker platelet endothelial cell adhesion molecule-1 and new bone formation marker osteocalcin expression were higher, which promoted the rapid repair of osteoporotic bone defects. On the contrary, the slow degradation rate of scaffolds hindered the growth of new bone to a certain extent. This study elucidates the importance of the degradation rate of scaffolds for the repair of osteoporotic bone defects, and the design considerations can be extended to other bone repair materials, which is expected to provide new ideas for the development of tissue engineering materials in the future.

Three-Dimensional Printing Technology for Surgical Correction of Congenital Scoliosis Caused by Hemivertebrae.

OBJECTIVE: This study aimed to explore the clinical application of three-dimensional (3D) printing technology in the surgical treatment of congenital scoliosis caused by hemivertebrae. METHODS: Twenty-four patients (11 in the 3D-printing group and 13 in the conventional group) with scoliosis secondary to a single hemivertebra were retrospectively reviewed. All patients underwent hemivertebrectomy and short-segment fixation. Virtual preoperative planning, operation simulation, and intraoperative application of 3D-printed patient-specific templates were performed in the 3D-printing group. Hemorrhage volume, operation time, transfusion, and complications were noted. Radiographic parameters were evaluated preoperatively, postoperatively, and at final follow-up. RESULTS: All patients had different degrees of successfully corrected scoliosis. There was a similar correction of the Cobb angle postoperatively between the 2 groups. The operation time, blood loss, transfusion, time for the insertion of each screw, accuracy of screw placement, and complication rate in the 3D-printing group were significantly superior to those in the control group. No patient experienced major complications. No significant correction loss or instrument dysfunction was observed during follow-up. CONCLUSIONS: As a viable and effective auxiliary technology, 3D printing makes it possible for surgery to meet both surgeon-specific and patient-specific requirements. 3D-printed individualized templates allow surgery for the correction of congenital scoliosis to enter a new stage of personalized precision surgery.

Controlled degradation of chitosan-coated strontium-doped calcium sulfate hemihydrate composite cement promotes bone defect repair in osteoporosis rats.

Strontium (Sr)-doped calcium sulfate hemihydrate (SrCSH) bioactive materials have been demonstrated to promote osteoporotic bone repair, being associated with the stimulation of bone formation and a reduction in bone resorption. However, the rapid degradation and absorption of SrCSH affects its clinical value. In order to delay the degradation time of SrCSH and improve the utilization of Sr2+, chitosan (CS)-coated SrCSH microspheres (CS-SrCSH) are prepared by electrostatic interaction between CS and SrCSH. X-ray diffraction analysis verifies that SrCSH coated by CS does not alter the phase composition of the SrCSH. It was observed that CS-SrCSH microspheres have uniform particle size. More importantly, the in vivo and in vitro degradation time of CS-SrCSH microspheres is significantly longer than that of SrCSH, and the release rate of Sr2+ is stable, achieving a sustained release effect. Furthermore, CS-SrCSH-based cement is used to repair critical-sized OVX rat tibial defects. The in vivo results reveal that CS-SrCSH exhibits a long-term capability for osteogenesis, angiogenesis and bone metabolism inhibition. In conclusion, the controllable degradation of CS-SrCSH-based cements described here could be beneficial for the repair of bone defects, especially in the osteoporotic bone.

Three-Dimensional-Printed Individualized Guiding Templates for Surgical Correction of Severe Kyphoscoliosis Secondary to Ankylosing Spondylitis: Outcomes of 9 Cases.

OBJECTIVE: To report the outcomes of severe kyphoscoliosis secondary to ankylosing spondylitis (AS) corrected with 3D-printed individualized guiding templates. METHODS: Computed tomography (CT) data of patients with severe kyphoscoliosis secondary to AS were used to reconstruct 3D models of the spine and to develop a surgical plan. An asymmetric wedge pedicle subtraction osteotomy (PSO) was simulated using medical computer design software. Before the actual surgery, continual surgical simulations were performed until the most suitable one was obtained, and personalized guiding templates were manufactured for the anticipated PSO. During operation, the osteotomy plane and trajectories for the pedicle screws were positioned by the designed patient-specific 3D-printed guiding templates. RESULTS: In this study, we reviewed 9 patients who underwent correction of kyphoscoliosis using a 3D-printed individualized guiding template and were followed for a median of 21.4 months (range, 9-36 months). The average correction at the site of osteotomy was 65.9°. No patient experienced severe complications, such as misplaced pedicle screws or neurologic complications. At the last follow-up, no patient exhibited implant dysfunction on radiography. CONCLUSIONS: Preoperative surgical simulation using 3D-printed templates is a viable technique that enables surgery to meet both patient- and surgeon-specific requirements for correction of severe thoracolumbar kyphoscoliosis. These 3D-printed templates can guide the performance of planned PSO to provide functional restoration of severe kyphoscoliosis secondary to AS.

[Precise orthormorphia of tibial angulation deformity and shortening deformity by using digital technology combined with circular external fixator].

Objective: To evaluate the effectiveness of precise orthormorphia of tibial angulation deformity and shortening deformity by using digital technology combined with external fixator. Methods: Twenty-six cases of tibial angulation deformity combined with shortening deformity were treated between June 2012 and August 2016, including 12 males and 14 females aged from 1 to 19 years with an average age of 16.5 years. There were 6 cases of congenital patella pseudoarthrosis, 1 case of fibrous dysplasia of femur and tibia, 3 cases of limb shortening deformity caused by infantile paralysis, 16 cases of fracture malunion. Limb shortening was 1.5-9.5 cm (mean, 6.2 cm) before operation. The deformity from three-dimensional perspective was analysed by digital technology, the surgical procedures of lengthening and osteotomy was simulated, the navigation templates were completed with computer aided design (CAD) and three-dimensional printing, and the external fixator was used to assist the lengthening of the tibia. X-ray films were regularly reviewed after operation to observe the new bone remolding, limb lengthening, load bearing line of lower limb, and recurrences of angulation. Results: All the patients were followed up 14-48 months (mean, 18.8 months). There was only 1 case of superficial pin site infection which was cured with oral antibiotics and pin site care with mild disinfectants, and no complication such as bone nonunion, equines deformity, or vascular nerve injury occurred. The deformity of tibia and load bearing line of lower limb had been completely recovered according to postoperative X-ray films at 1 week. All the cases achieved perfect limb length as with preoperative design. The bone mineralization time was 12-20 weeks (mean, 11.6 weeks), the external fixator removal time was 18-26 weeks (mean, 14.9 weeks), and the healing index was 21-78 d/cm (mean, 63.4 d/cm). The postoperative flexion range of the injured limb was 15° less than the unaffected extremity in 1 case, and the situation was improved significantly after some physical manipulation and exercise, who completed the limb lengthening and achieved the expected effectiveness finally. Conclusion: Precise orthormorphia of tibial deformity by using digital technology, and limb lengthening with the aid of external fixator can achieve good effectiveness with good reliability, invasiveness, and precision.

[Preliminary application of three dimensional printing personalized navigation template in assisting total elbow replacement for patients with elbow tumor].

Objective: To explore the clinical methods of resection of elbow tumor and total elbow replacement with custom personalized prosthesis based on three dimensional (3-D) printing navigation template. Methods: In August 2016, a 63-year-old male patient with left elbow joint tumor was treated, with the discovery of the left distal humerus huge mass over 3 months, with elbow pain, activity limitation of admission. Computer-assisted reduction technique combined with 3-D printing was used to simulate preoperative tumor resection, a customized personal prosthesis was developed; tumor was accurately excised during operation, and the clinical result was evaluated after operation. Results: The time was 46 minutes for tumor resection, and was 95 minutes for personalized implant and allograft bone without fluoroscopy. X-ray and CT examination at 1 week after operation showed good position of artificial elbow joint; the anteversion of ulna prosthesis was 30° and the elbow carrying angle was 15°, which were consistent with the simulated results before surgery. The finger flexion was normal at 1 month after operation; the range of motion was 0-130° for elbow flexion and extension, 80° for forearm pronation, and 80° for forearm supination. The elbow function was able to meet the needs of daily life at 7 months after operation, and no recurrence and metastasis of tumor were observed. Conclusion: For limb salvage of elbow joint, computer aided design can make preoperative surgical simulation; the navigation template can improve surgical precision; and the function of elbow joint can be reconstructed with customized and personlized prosthesis for total elbow replacement.