Molecular Targeted NIR-II Probe for Image-Guided Brain Tumor Surgery.

Optical imaging strategies for improving delineation of glioblastoma (GBM) is highly desired for guiding surgeons to distinguish cancerous tissue from healthy and precious brain tissue. Fluorescence imaging (FLI) in the second near-infrared window (NIR-II) outperforms traditional NIR-I imaging with better tissue penetration, higher spatial and temporal resolution, and less auto fluorescence and scattering. Because of high expression in GBM and many other tumors, urokinase Plasminogen Activator Receptor (uPAR) is an attractive and well proven target for FLI. Herein we aim to combine the benefit of a NIR-II fluorophore with a high affinity uPAR targeting small peptide. A targeted NIR-II fluorescent probe was developed by conjugating an in-house synthesized NIR-II fluorophore, CH1055, and a uPAR targeting peptide, AE105. To characterize the in vivo distribution and targeting properties, a dynamic imaging was performed in orthotopic GBM bearing nude mice ( n = 8). Additionally, fluorescence guided surgery of orthotopic GBM was performed in living animals. CH1055-4Glu-AE105 was easily synthesized with >75% yield and >98% HPLC evaluated purity. The retention time of the probe on analytical HPLC was 15.9 min and the product was verified by mass spectrometry. Dynamic imaging demonstrated that the uPAR targeting probe visualized orthotopic GBM through the intact skull with a tumor-to-background ratio (TBR) of 2.7 peaking at 96 h. Further, the orthotopic GBM was successfully resected in small animals guided by the NIR-II FLI. By using a small uPAR targeting NIR-II probe, FLI allows us to specifically image and detect GBM. A real-time imaging setup further renders FLI guided tumor resection, and the probe developed in this work is a promising candidate for clinical translation.

Effect, Feasibility, and Clinical Relevance of Cell Enrichment in Large Volume Fat Grafting: A Systematic Review.

Large volume fat grafting is limited by unpredictable volume loss; therefore, methods of improving graft retention have been developed. Fat graft enrichment with either stromal vascular fraction (SVF) cells or adipose tissue-derived stem/stromal cells (ASCs) has been investigated in several animal and human studies, and significantly improved graft retention has been reported. Improvement of graft retention and the feasibility of these techniques are equally important in evaluating the clinical relevance of cell enrichment. We conducted a systematic search of PubMed to identify studies on fat graft enrichment that used either SVF cells or ASCs, and only studies reporting volume assessment were included. A total of 38 articles (15 human and 23 animal) were included to investigate the effects of cell enrichment on graft retention as well as the feasibility and clinical relevance of cell-enriched fat grafting. Improvements in graft retention, the SVF to fat (SVF:fat) ratio, and the ASC concentration used for enrichment were emphasized. We proposed an increased retention rate greater than 1.5-fold relative to nonenriched grafts and a maximum SVF:fat ratio of 1:1 as the thresholds for clinical relevance and feasibility, respectively. Nine studies fulfilled these criteria, whereof 6 used ASCs for enrichment. We found no convincing evidence of a clinically relevant effect of SVF enrichment in humans. ASC enrichment has shown promising results in enhancing graft retention, but additional clinical trials are needed to substantiate this claim and also determine the optimal concentration of SVF cells/ASCs for enrichment. LEVEL OF EVIDENCE: 4.

Incidence of In Situ and Invasive Melanoma in Denmark From 1985 Through 2012: A National Database Study of 24,059 Melanoma Cases.

IMPORTANCE: In Denmark, the incidence of malignant melanoma (MM) has doubled during the past 25 years, with an incidence of 29.5 and 31.7 per 100,000 person-years in 2012 for men and women, respectively. Understanding the nature of this increase in incidence is important to optimize prevention, early diagnosis, and treatment of in situ and invasive melanoma in Denmark. OBJECTIVE: To describe changes over time in the incidence and clinical and pathologic characteristics of in situ and invasive melanoma in Denmark from 1985 through 2012. DESIGN, SETTING, AND PARTICIPANTS: We used the official national Danish Melanoma Group database to describe all eligible, prospectively registered cases of in situ and invasive melanoma in Denmark from January 1, 1985, through December 31, 2012. Data analyses were performed from April 1, 2012, through January 31, 2013. MAIN OUTCOMES AND MEASURES: Estimated annual percentage changes (EAPCs) for men and women in European age-standardized incidence, age at diagnosis, and tumor region for in situ melanoma and MM. For MM only, melanoma type, Breslow thickness, ulceration, and mortality. RESULTS: We included 3299 cases of in situ melanoma and 20,760 cases of MM. The incidence (95% CI) of MM increased by 4.5% (3.6%-5.3%) for men and 4.3% (3.5%-5.2%) for women, which was especially pronounced in patients older than 60 years (EAPCs, 5.8% [4.7%-6.8%] and 4.8% [3.8%-5.9%], respectively), in thin (Breslow thickness, <0.75 mm) melanoma (EAPCs, 6.6% [5.0%-8.2%] and 6.1% [6.0%-7.1%], respectively), and in superficially spreading MM (EAPCs, 5.2% [4.3%-6.2%] and 4.7% [3.9%-5.7%], respectively). We found no significant EAPC in the incidence of melanomas with Breslow thickness greater than 2.00 mm in women, and relative ulceration rates (95% CI) declined in both sexes (EAPCs, -3.3% [-4.0% to -2.6%] in men and -3.4% [-4.0% to -2.8%] in women). More proximal tumor location occurred over time (P < .001). Incidence of in situ melanoma (95% CI) greatly increased (EAPCs, 14.0% [12.2%-15.8%] in men and 11.6% [10.2%-13.2%] in women) with changes over time in age and region (defined by codes in the International Statistical Classification of Diseases, Tenth Revision) similar to those for MM. Mortality related to MM increased in men (EAPC, 0.6% [0.1% to 1.2%]), whereas mortality in women (EAPC, -0.4% [-1.0% to 0.3%]) remained stable. CONCLUSIONS AND RELEVANCE: This study confirms a worldwide increase in melanoma incidence. Results may indicate the importance of secondary melanoma prevention in Denmark. Future efforts could intensify primary prevention aimed at young adults, adolescents, and children and maintain and target secondary prevention at the population older than 60 years.

Recurrence and survival after neck dissections in cutaneous head and neck melanoma.

INTRODUCTION: An important prognostic factor in head and neck melanoma is the status of the regional lymph nodes since the presence of metastatic disease in the nodes greatly aggravates the prognosis. There is no consensus on the surgical treatment algorithm for this group. Our aim was to study if there is a difference in nodal recurrence and survival after radical, modified or selective neck dissection. METHODS: A total of 57 patients treated for regional meta-stases of head and neck melanoma were analysed retrospectively with respect to type of neck dissection, use of sentinel node biopsy, nodal recurrence and survival. RESULTS: After a median 127-month (range: 22-290) follow-up period, we showed that there was no significant difference in nodal recurrence between three different dissection groups (11% for radical node dissection, 24% for modified radical node dissection and 23% for selective node dissection, p > 0.05). No significant difference in five-year survival was observed between the dissection types (56% for radical node dissection, 61% for modified radical node dissection and 48% for selective node dissection, p = 0.613). Multivariate and univariate analysis revealed that patients with metastatic deposits in sentinel nodes had a better survival than patients with clinically palpable nodes (five-year survival rate: 70% versus 36%, p = 0.008). CONCLUSION: The extent of neck dissection does not significantly influence the rate of recurrence or survival. This study indicates that there is a survival benefit for patients who undergo completion lymph node dissection following a positive sentinel node biopsy. FUNDING: not relevant. TRIAL REGISTRATION: not relevant.

Enrichment of autologous fat grafts with ex-vivo expanded adipose tissue-derived stem cells for graft survival: a randomised placebo-controlled trial.

BACKGROUND: Autologous fat grafting is increasingly used in reconstructive surgery. However, resorption rates ranging from 25% to 80% have been reported. Therefore, methods to increase graft viability are needed. Here, we report the results of a triple-blind, placebo-controlled trial to compare the survival of fat grafts enriched with autologous adipose-derived stem cells (ASCs) versus non-enriched fat grafts. METHODS: Healthy participants underwent two liposuctions taken 14 days apart: one for ASC isolation and ex-vivo expansion, and another for the preparation of fat grafts. Two purified fat grafts (30 mL each) taken from the second liposuction were prepared for each participant. One graft was enriched with ASCs (20 × 10(6) cells per mL fat), and another graft without ASC enrichment served as a control. The fat grafts were injected subcutaneously as a bolus to the posterior part of the right and left upper arm according to the randomisation sequence. The volumes of injected fat grafts were measured by MRI immediately after injection and after 121 days before surgical removal. The primary goal was to compare the residual graft volumes of ASC-enriched grafts with those of control grafts. This study is registered at www.clinicaltrialsregister.eu, number 2010-023006-12. FINDINGS: 13 participants were enrolled, three of whom were excluded. Compared with the control grafts, the ASC-enriched fat grafts had significantly higher residual volumes: 23·00 (95% CI 20·57-25·43) cm(3) versus 4·66 (3·16-6·16) cm(3) for the controls, corresponding to 80·9% (76·6-85·2) versus 16·3% (11·1-21·4) of the initial volumes, respectively (p<0·0001). The difference between the groups was 18·34 (95% CI 15·70-20·98) cm(3), equivalent to 64·6% (57·1-72·1; p<0·0001). No serious adverse events were noted. INTERPRETATION: The procedure of ASC-enriched fat grafting had excellent feasibility and safety. These promising results add significantly to the prospect of stem cell use in clinical settings, and indicate that ASC graft enrichment could render lipofilling a reliable alternative to major tissue augmentation, such as breast surgery, with allogeneic material or major flap surgery. FUNDING: Danish Cancer Society, Centre of Head and Orthopaedics Rigshospitalet, and Moalem Weitemeyer Bendtsen.

Pooled human platelet lysate versus fetal bovine serum-investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use.

BACKGROUND AIMS: Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS)). METHODS: ASCs from four healthy donors were cultured in either DMEM(pHPL) or DMEM(FBS), and the population doubling time (PDT) was calculated. ASCs from two of the donors were expanded in DMEM(pHPL) or DMEM(FBS) and cultured for the final week before harvesting with or without the addition of vascular endothelial growth factor. We assessed the chromosomal stability (through the use of array comparative genomic hybridization), the expression of ASC and endothelial surface markers and the differentiation and angiogenic potential of these cells. RESULTS: The ASCs that were cultured in pHPL exhibited a significantly shorter PDT of 29.6 h (95% confidence interval, 22.3-41.9 h) compared with those cultured in FBS, for which the PDT was 123.9 h (95% confidence interval, 95.6-176.2 h). Comparative genomic hybridization analyses revealed no chromosomal aberrations. Cell differentiation, capillary structure formation and cell-surface marker expression were generally unaffected by the type of medium supplement that was used or by the addition of vascular endothelial growth factor. CONCLUSIONS: We observed that the use of pHPL as a growth supplement for ASCs facilitated a significantly higher proliferation rate compared with FBS without compromising genomic stability or differentiation capacity.

Importance of mesenchymal stem cells in autologous fat grafting: a systematic review of existing studies.

Autologous fat grafting (lipofilling) enables repair and augmentation of soft tissues and is increasingly used both in aesthetic and reconstructive surgery. Autologous fat has several advantages, including biocompatibility, versatility, natural appearance, and low donor site morbidity. The main limitation is unpredictable graft resorption, which ranges from 25%-80%, probably as a result of ischaemia and lack of neoangiogenesis. To obviate these disadvantages, several studies have searched for new ways of increasing the viability of the transplanted tissue. One promising approach has been to enrich the fat graft with adipose tissue-derived mesenchymal stem cells (ASC) before transplantation. We have reviewed original studies published on fat transplantation enriched with ASC. We found four murine and three human studies that investigated the subject after a sensitive search of publications. In the human studies, so-called cell assisted lipotransfer (CAL) increased the ASC concentration 2-5 times compared with non-manipulated fat grafts, which caused a questionable improvement in survival of fat grafts, compared with that of traditional lipofilling. In contrast, in two of the murine studies ASC-concentrations were increased 1250 and 6250 times, respectively, by ASC ex vivo expansion, which resulted in considerably improved fat transplant survival as well as quality. This effect of high-level enrichment with ASC is thought to have been caused by paracrine signalling, cellular differentiation, or both. The surgical and tissue handling techniques used in lipofilling are well proved, but the added effect of high-level enrichment with ex vivo expanded ASC still needs to be investigated properly in human lipofilling studies, combined with a thorough follow up and matched control groups. In conclusion, ASC-enriched lipofilling theoretically has the potential for transforming lipofilling from a relatively unpredictable intervention into one in which the resorption rate, quality of tissue, and safety can be predicted, and possibly superior to prosthetic implantation.

[The genes disposing to malignant melanoma--a systematic review]. / Genetiske fund giver nye muligheder for udredning af arveligt malignt melanom.

Denmark is a high-risk country for malignant melanoma (MM).The incidence of MM has increased faster than the ones of any other cancer during the last ten years. 5-10% of the patients with MM report a family history of MM, and this is most likely caused by a combination of genetic and environmental factors. The genes disposing to MM are reviewed in this article and the challenges and ethical considerations concerning genetic counselling of families at high risk of MM are discussed.

Trends in the incidence of malignant melanoma in Denmark 1978-2007. Incidence on the island of Bornholm compared with the whole country incidence in Denmark.

INTRODUCTION: In Denmark, malignant melanoma is among the most rapidly increasing cancer types. Malignant melanoma accounts for the majority of skin cancer-related deaths. Sunshine is the main cause of the increase seen in melanoma incidence. Within Denmark, Bornholm is the area that receives most sunshine. It is therefore relevant to compare incidence data between Denmark and Bornholm. MATERIAL AND METHODS: Incidence data from the period 1978-2007 were extracted from The National Cancer Database. Incidence rates were analysed to determine any difference between Bornholm and Denmark in total. A prognosis was made based on extrapolation of incidence data from 1978-2007. RESULTS: Incidence rates increased exponentially in Denmark as well as on Bornholm. A significant annual increase in incidence rates over the 1978-2007 period was found. The difference in incidence was estimated to be 3.4%. The annual increase was significantly higher on Bornholm than in the rest of Denmark, a 20% increase was assessed. The increase in incidence rates in malignant melanoma is supported by the literature. The difference between Bornholm and Denmark is possibly due to differences in behaviour that translate into differences in UV exposure. CONCLUSION: If the current development continues, the incidence rate will increase more than 100% from 2007 to 2030. The effect of campaigns addressing behavioural sun exposure has yet to be recorded. UV exposure is expected to comprise the main reason for the difference in incidence rate between Bornholm and Denmark in total. More evidence is needed on the relation between malignant melanoma and UV radiation and socio-demographic conditions.

Sentinel node biopsy for melanoma: a study of 241 patients.

The aim of this study was to evaluate the sentinel node biopsy (SNB) technique for melanoma using both radiocolloid and blue dye in 241 clinically N0 patients with melanomas >1.0 mm, or thinner lesions exhibiting regression/ulceration. We showed that an increase in injected radioactivity increased both the number of visualized nodes at lymphoscintigraphy and the number of SNs removed surgically. At least one SN was removed in 98% (236) of patients, and all nodes were identified with the probe. Seventy-four per cent of the 194 patients injected with blue dye had stained SNs. In 46% (144) of the lymph node basins, there was a discrepancy between the nodes visualized at lymphoscintigraphy and the nodes removed at surgery. There were 38 unusually located nodes. Only eight of these were removed surgically; none contained metastases. SN metastases were detected in 22% (53) of patients. There were nine haematoxylin and eosin (HE)-negatives, all of which were found by immunohistochemistry. The false negative rate for the SNB procedure was 4% (2/55). The complication rate was 6% after SNB and 29% after complete node dissection. In conclusion, SN status is a strong prognostic factor in melanoma patients, and SNB has made the approach to radical lymphadenectomy more rational.