Long-term outcomes to neoadjuvant pembrolizumab based on pathological response for patients with resectable stage III/IV cutaneous melanoma.

BACKGROUND: While neoadjuvant immunotherapy for melanoma has shown promising results, the data have been limited by a relatively short follow-up time, with most studies reporting 2-year outcomes. The goal of this study was to determine long-term outcomes for stage III/IV melanoma patients treated with neoadjuvant and adjuvant programmed cell death receptor 1 (PD-1) inhibition. PATIENTS AND METHODS: This is a follow-up study of a previously published phase Ib clinical trial of 30 patients with resectable stage III/IV cutaneous melanoma who received one dose of 200 mg IV neoadjuvant pembrolizumab 3 weeks before surgical resection, followed by 1 year of adjuvant pembrolizumab. The primary outcomes were 5-year overall survival (OS), 5-year recurrence-free survival (RFS), and recurrence patterns. RESULTS: We report updated results at 5 years of follow-up with a median follow-up of 61.9 months. No deaths occurred in patients with a major pathological response (MPR, <10% viable tumor) or complete pathological response (pCR, no viable tumor) (n = 8), compared to a 5-year OS of 72.8% for the remainder of the cohort (P = 0.12). Two of eight patients with a pCR or MPR had a recurrence. Of the patients with >10% viable tumor remaining, 8 of 22 patients (36%) had a recurrence. Additionally, the median time to recurrence was 3.9 years for patients with ≤10% viable tumor and 0.6 years for patients with >10% viable tumor (P = 0.044). CONCLUSIONS: The 5-year results from this trial represent the longest follow-up of a single-agent neoadjuvant PD-1 trial to date. Response to neoadjuvant therapy continues to be an important prognosticator with regard to OS and RFS. Additionally, recurrences in patients with pCR occur later and are salvageable, with a 5-year OS of 100%. These results demonstrate the long-term efficacy of single-agent neoadjuvant/adjuvant PD-1 blockade in patients with a pCR and the importance of long-term follow-up for these patients. TRIAL REGISTRATION: Clinicaltrials.gov, NCT02434354.

Overexpression of protein kinase B/AKT induces phosphorylation of p70S6K and 4E-BP1 in goat fetal fibroblasts.

Protein kinases regulate many processes, including cell growth, metabolism, molecular interactions, and cell proliferation. Protein kinase B (PKB)/AKT (v-AKT mouse thymoma viral oncogene homolog) is an upstream component of mammalian target of rapamycin (mTOR) signaling and mediates pathophysiological processes in several signaling pathways. This study aimed to construct and overexpress a eukaryotic goat AKT expression vector in goat fetal fibroblasts and examine the effects of AKT on the phosphorylation of p70S6K and 4E-BP1. AKT was subcloned into the expression vector pIRES2-DsRed2 to generate pIRES2-DsRed2-AKT, which was transfected into goat fetal fibroblasts with LipofectamineTM 2000. AKT was measured by reverse transcription-polymerase chain reaction in the transgenic cells, and the expression of AKT and phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) were analyzed by Western blot. Cell clones that stably emitted red fluorescence were obtained after transfection for 48 h, and the exogenous gene was verified. Exogenous AKT was transcribed, and AKT was overexpressed, inducing the phosphorylation of p70S6K (Thr389) and 4E-BP1 (Thr37/46) in goat fetal fibroblasts. Thus, the overexpression of AKT activates mTOR signaling in goat cells.

Proximal femoral nails antirotation, Gamma nails, and dynamic hip screws for fixation of intertrochanteric fractures of femur: A meta-analysis.

BACKGROUND: Which surgical strategy is the best one for intertrochanteric fractures remains a controversial issue. Dynamic hip screw (DHS) and Gamma nail were commonly used but often associated with some complications, such as fixation failure and implant-related fractures. Meanwhile, proximal femoral nail anti-rotation (PFNA) fixation has recently been developed for minimally invasive surgery to reduce the complications rate. To facilitate the clinical decision-making, we conducted an updated meta-analysis to discuss the optimal treatment of intertrochanteric fractures aiming to determine which implant gives the lower rates of blood loss, complications (peri-implant fracture, fixation failure, infection, thromboembolic), reoperation, and mortality, as well as the minimal duration related to surgery (fluoroscopic exposure, surgery and hospital stay). PATIENTS AND METHODS: Seven electronic databases were searched for randomized controlled trials (including OVID, Springer, Google Scholar, PubMed, Cochrane library, Embase, and Web of Science). Fourteen studies with 1983 patients were included. The modified Jadad Scale was used to assess the methodological quality of these studies. Risk of bias in the included studies was assessed using the Cochrane Risk of Bias tool. Comparison among the three groups was based on twelve indicators, including operative time, fluoroscopy time, operative blood loss, length of hospital stays, wound infection or hematoma, pneumonia, thromboembolic complications, fixation failure, operative fracture of femur, later fracture of femur, reoperation, and mortality. RESULTS: (1) PFNA group versus DHS group: PFNA was associated with less blood loss (mean difference (MD) -253.86, 95% CI -270.25 to 237.47; P<0.00001) and lower rate of fixation failure (MD 0.20, 95% CI 0.07 to 0.59; P=0.004), but led to more fluoroscopy time (MD 2.11, 95% CI 1.78 to 2.43; P<0.00001). (2) PFNA group versus Gamma nail group: PFNA led to less blood loss (MD -55.30, 95% CI -60.07 to -50.53; P<0.00001), shorter fluoroscopy time (MD -0.50, 95% CI -0.55 to -0.45; P<0.00001) and length of hospital stay (MD -0.20, 95% CI -0.27 to -0.13; P<0.00001). (3) DHS group versus Gamma nail group: DHS was associated with lower rate of operative fracture of femur (MD 0.31, 95% CI 0.11 to 0.89; P=0.03), later fracture of femur (MD 0.16, 95% CI 0.06 to 0.43; P=0.0004), and reoperation (MD 0.49, 95% CI 0.27 to 0.88; P=0.02), but caused more blood loss (MD 29.49, 95% CI 8.27 to 50.70; P=0.006). In contrast, there was no difference regarding operative time, infection hematoma, pneumonia, thromboembolic events, and mortality. DISCUSSION: PFNA should be a priority choice for treatment of intertrochanteric fractures with minimal rate of fixation failure, less blood loss and shorter length of hospital stay. DHS has distinct advantages over Gamma nail with lower rate of plant-related complications and should be preferred device for intertrochanteric fractures. However, owing to the low quality evidence currently available, more high-quality RCTs are needed to confirm these findings. LEVEL OF EVIDENCE: Level II.

Effects of L-arginine on the proliferation of human renal mesangial cells and production of extracellular matrix.

AIM: To investigate the effect of L-arginine (L-arg) on the proliferation of human mesangial cells and production of collagen. METHODS: The influence of L-arg on the cell proliferation was determined by MTT assay, immunocytochemical detection of expression of proliferative cell nuclear antigen (PCNA), and flow cytometrical analysis of cell cycle. Procollagen III and total collagen level in the supernatant and expression of collagen IV mRNA in human mesangial cells were determined by radioimmunoassay, hydroxyproline colorimetric assay, and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: L-Arg induced inhibition of human mesangial cell lines (HMCL) in a concentration- and time-dependent manner. Immunocytochemical study for PCNA showed the number of cells was decreased, though the percentage of PCNA positive cells was increased in L-arg-treated group. Flow cytometrical analysis showed that cells in S and G2/-M phases were markedly increased in L-arg-treated group compared with those in control group. Furthermore, L-arg significantly inhibited the production of procollagen III and total collagen in the supernatants determined by radioimmunoassay and hydroxyproline colorimetric assay (P < 0.05 and 0.01, respectively) and inhibited the expression of collagen IV mRNA determined by RT-PCR (P < 0.01). CONCLUSION: L-arg could exert an inhibitory effect on the proliferation of human mesangial cells and production of extracellular components, which strongly suggested its potential therapeutic role in the chronic renal scarring.

Selective removal of glycosylation sites from sex hormone-binding globulin by site-directed mutagenesis.

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with high affinity for sex steroid hormones. It contains two N-linked carbohydrate chains and one O-linked oligosaccharide per subunit, but their functional significance is not known. Site-directed mutagenesis of a human SHBG cDNA has enabled us to selectively disrupt the known glycosylation sites individually and in various combinations. The mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells, and it was found that the presence of carbohydrates is not an absolute requirement for the secretion of SHBG from these cells, but the absence of both N-linked oligosaccharides reduced the amount of SHBG in the culture medium. In addition, the affinity and specificity of SHBG for steroid ligands was unaffected by the lack of one or more carbohydrate chains. Proportionally greater amounts (26-31%) of the mutants lacking a single N-linked carbohydrate chain failed to interact with Concanavalin-A. (Con-A) compared to normal SHBG produced by CHO cells (15%). Western analysis demonstrated that both consensus sites for N-glycosylation are used and that the typical heavy [mol wt (M(r)), approximately 51,000] and light (M(r), approximately 47,000) subunit size-heterogeneity was maintained regardless of the absence of an O-linked carbohydrate at residue 7. Furthermore, the SHBG mutants containing only one N-linked oligosaccharide comprise only a single subunit with a M(r) of approximately 47,000. This suggests that the heavy subunit contains two N-linked oligosaccharides, while only one of these sites is used on the light subunit. The M(r) of the various SHBG mutants were also examined by gel filtration, and this indicated that they are all produced as homodimers and that carbohydrates are not involved in subunit association.