Role of SDHAF2 and SDHD in von Hippel–Lindau Associated Pheochromocytomas

Background

Pheochromocytomas (PCCs) develop from the adrenal medulla and are often part of a hereditary syndrome such as von Hippel–Lindau (VHL) syndrome. In VHL, only about 30 % of patients with a VHL missense mutation develop PCCs. Thus, additional genetic events leading to formation of such tumors in patients with VHL syndrome are sought. SDHAF2 (previously termed SDH5) and SDHD are both located on chromosome 11q and are required for the function of mitochondrial complex II. While SDHAF2 has been shown to be mutated in patients with paragangliomas (PGLs), SDHD mutations have been found both in patients with PCCs and in patients with PGLs.

Materials and methods

Because loss of 11q is a common event in VHL-associated PCCs, we aimed to investigate whether SDHAF2 and SDHD are targets. In the present study, 41 VHL-associated PCCs were screened for mutations and loss of heterozygosity (LOH) in SDHAF2 or SDHD. Promoter methylation, as well as mRNA expression of SDHAF2 and SDHD, was studied. In addition, immunohistochemistry (IHC) of SDHB, known to be a universal marker for loss of any part the SDH complex, was conducted.

Results and conclusions

LOH was found in more than 50 % of the VHL-associated PCCs, and was correlated with a significant decrease (p < 0.05) in both SDHAF2 and SDHD mRNA expression, which may be suggestive of a pathogenic role. However, while SDHB protein expression as determined by IHC in a small cohort of tumors was lower in PCCs than in the surrounding adrenal cortex, there was no obvious correlation with LOH or the level of SDHAF2/SDHD mRNA expression. In addition, the lack of mutations and promoter methylation in the investigated samples indicates that other events on chromosome 11 might be involved in the development of PCCs in association with VHL syndrome.     

Human Papillomavirus–Associated Neoplasms of the Head and Neck

Human papillomavirus (HPV) is an essential causal factor in a subset of head and neck neoplasms, most notably oropharyngeal squamous cell carcinoma, for which HPV infection has important diagnostic, prognostic, and therapeutic implications. This article summarizes the current understanding of HPV-associated neoplasms of the head and neck, including the recently described carcinoma with adenoid cystic-like features. Salient clinical, gross, and microscopic features are discussed, and the utility of specific ancillary studies is highlighted.     

Cost–Benefit Analysis of the Implementation of an Enhanced Recovery Program in Liver Surgery

Background

Enhanced recovery after surgery (ERAS) programs have been shown to ease the postoperative recovery and improve clinical outcomes for various surgery types. ERAS cost-effectiveness was demonstrated for colorectal surgery but not for liver surgery. The present study aim was to analyze the implementation costs and benefits of a specific ERAS program in liver surgery.

Methods

A dedicated ERAS protocol for liver surgery was implemented in our department in July 2013. The subsequent year all consecutive patients undergoing liver surgery were treated according to this protocol (ERAS group). They were compared in terms of real in-hospital costs with a patient series before ERAS implementation (pre-ERAS group). Mean costs per patient were compared with a bootstrap T test. A cost-minimization analysis was performed.

Results

Seventy-four ERAS patients were compared with 100 pre-ERAS patients. There were no significant pre- and intraoperative differences between the two groups, except for the laparoscopy number (n = 18 ERAS, n = 9 pre-ERAS, p = 0.010). Overall postoperative complications were observed in 36 (49 %) and 64 patients (64 %) in the ERAS and pre-ERAS groups, respectively (p = 0.046). The median length of stay was significantly shorter for the ERAS group (8 vs. 10 days, p = 0.006). The total mean costs per patient were €38,726 and €42,356 for ERAS and pre-ERAS (p = 0.467). The cost-minimization analysis showed a total mean cost reduction of €3080 per patient after ERAS implementation.

Conclusions

ERAS implementation for liver surgery induced a non-significant decrease in cost compared to standard care. Significant decreased complication rate and hospital stay were observed in the ERAS group.

     

Characterization of the Effects of Cyclooxygenase-2 Inhibition in the Regulation of Apoptosis in Human Small and Non–Small Cell Lung Cancer Cell Lines

Background Cyclooxygenase-2 enzyme (COX-2) is overexpressed in human non–small cell lung cancer (NSCLC) but is not expressed in small cell lung cancer. Selective COX-2 inhibitors have been shown to induce apoptosis in NSCLC cells, an effect which is associated with the regulation of intracellular MAP kinase (MAPK) signal pathways. Our aims were to characterize the effects of COX-2 inhibition by rofecoxib on apoptosis in human NSCLC and small cell lung cancer cell lines. Methods The human NSCLC cell line NCI-H2126 and small cell lung cancer cell line DMS-79 were used. Constitutive COX-2 protein levels were first determined by Western blot test. Levels of apoptosis were evaluated by using propidium iodide staining on FACScan analysis after incubation of NCI-H2126 and DMS-79 with p38 MAPK inhibitor SB202190 (25 μM), NF-κB inhibitor SN50 (75 μg/mL), and rofecoxib at 100 and 250 μM. All statistical analysis was performed by analysis of variance. Results Western blot test confirmed the presence of COX-2 enzyme in NCI-H2126 and absence in DMS-79. Interestingly, rofecoxib treatment demonstrated a dose-dependent increase in apoptosis in both cell lines. Given this finding, the effect of rofecoxib on NF-κB and p38 MAPK pathways was also examined. Apoptosis in both cell lines was unaltered by SN50, either alone or in combination with rofecoxib. A similar phenomenon was observed in NCI-H2126 cells treated with SB202190, either alone or in combination with rofecoxib. In contrast, p38 MAPK inhibition greatly upregulated DMS-79 apoptosis in a manner that was unaltered by the addition of rofecoxib. Conclusions Rofecoxib led to a dose-dependent increase in apoptosis in both tumor cell lines. This effect occurred independently of COX-2, NF-κB, and p38 MAPK pathways in DMS-79 cells. As such, rofecoxib must act on alternative pathways to regulate apoptosis in human small cell lung cancer cells. Presented at the Society of Surgical Oncology (SSO) Annual Meeting, New York, 18th-21st March 2004.     

Is Neutrophil Gelatinase–Associated Lipocalin an Optimal Marker of Renal Function and Injury in Liver Transplant Recipients?

Background

Recently, research has focused on the association of neutrophil gelatinase–associated lipocalin (NGAL) with acute and/or active kidney injury. However, it should be remembered that NGAL is involved in iron metabolism and antimicrobial defense mechanisms.

Methods

One hundred seven consecutive liver transplant recipients were included in this study. Plasma and urine NGAL levels were measured with the use of enzyme-linked immunosorbent assay. NGAL levels were studied as plasma concentrations (pNGAL), urine concentrations (uNGAL), urinary NGAL to creatinine ratio (uNGAL/Cr), and fractional NGAL secretion (fNGAL).

Results

pNGAL was found to be inversely correlated with estimated glomerular filtration rate (eGFR) and plasma cystatine C (pCysC) (r = −0.26 and P = .007, r = −0.38 and P = .00006, respectively). uNGAL was positively correlated with urinary cystatine C to creatinine ratio (uCysC/Cr) and fractional cystatine C excretion (fCysC) (r = 0.43 and P = .000004; r = 0.4 and P = .1; respectively). uNGAL/Cr was inversely correlated with hematocrit (Htc) and hemoglobin (Hb) (r = −0.35 and P = .0002; r = −0.39 and P = .00004; respectively), and positively correlated with uCysC/Cr (r = 0.5 and P < .0000001). fNGAL was directly correlated with uCysC/Cr and fCysC (r = 0.53 and P < .0000001; r = 0.39 and P = .00005; respectively) and inversely correlated with red blood cell count (RBC; r = −0.31 and P = .001). We observed significant differences of pNGAL, uNGAL/Cr, and fNGAL between sexes, with highest values of uNGAL, uNGAL/Cr, and fNGAL in women and pNGAL in men. In multivariate regression analysis, pNGAL was positively correlated with time elapsed from liver transplantation, neutrophil count, pCysC, and sex (β = 0.36 and P = .00001; β = 0.32 and P = .0001; β = 0.58 and P < .0000001; β = 0.17 and P = .025; respectively) and inversely correlated with patient's age (β = −0.18 and P = .02) with R = 0.67 and R² = 0.45, independently from blood glucose, eGFR, RBC, white blood cell count, Hb, uCysC, uCysC/Cr, and fCysC.

Conclusions

Plasma and urine NGAL levels are strongly correlated not only with kidney function parameters, but also with red and white blood cell parameters and patient's age and sex.     

Value of Multidetector Row CT in the Assessment of Longitudinal Extension of Cholangiocarcinoma—Correlation Between MDCT and Microscopic Findings

Background  A few authors have reported the value of multidetector row CT (MDCT) for evaluating the longitudinal extent of cholangiocarcinoma. They have not focused on CT attenuation of a tumor and actual tumor extent along the bile ducts. We designed the present study to analyze attenuation. Methods  Between January 2003 and July 2005, 113 consecutive patients with cholangiocarcinoma underwent a surgical resection following MDCT. Of these MDCT studies, 73 (perihilar cholangiocarcinoma, n = 62; middle and distal cholangiocarcinoma, n = 11) were suitable for analysis, and the patients were enrolled in the study. Patients were divided according to tumor hypoattenuation and hyperattenuation on MDCT. Histologic differentiation, desmoplastic reaction, and vascular density were microscopically compared with the tumor attenuation to differentiate the characteristics of the attenuation. The extent of cancer along the bile duct diagnosed by MDCT was compared with the actual extent determined by the microscopic findings. Results  Hyperattenuated tumor was observed in 40 patients. There was no difference in histologic differentiation, desmoplastic reaction, or vascular density between the hyperattenuated and hypoattenuated cholangiocarcinomas. Neither the proximal nor the distal borders between the normal and thickened bile duct wall could be determined in the 33 patients with hypoattenuated tumor; in contrast, an accurate assessment of extent of tumor was obtained in 76% of the proximal borders and 82% of the distal borders in the 40 patients with hyperattenuated tumor. Conclusions  Although the cause of the difference between the hyperattenuated and hypoattenuated cholangiocarcinoma still is unclear, MDCT can be an alternative to direct cholangiography in selected patients with hyperattenuated cholangiocarcinoma.     

Distinct Subsets of Noncoding RNAs Are Strongly Associated With BMD and Fracture,Studied in Weight-Bearing and Non–Weight-Bearing Human Bone

We investigated mechanisms resulting in low bone mineral density (BMD) and susceptibility to fracture by comparing noncoding RNAs (ncRNAs) in biopsies of non–weight-bearing (NWB) iliac (n = 84) and weight bearing (WB) femoral (n = 18) postmenopausal bone across BMDs varying from normal (T-score > −1.0) to osteoporotic (T-score ≤ −2.5). Global bone ncRNA concentrations were determined by PCR and microchip analyses. Association with BMD or fracture, adjusted by age and body mass index, were calculated using linear and logistic regression and least absolute shrinkage and selection operator (Lasso) analysis. At 10% false discovery rate (FDR), 75 iliac bone ncRNAs and 94 femoral bone ncRNAs were associated with total hip BMD. Eight of the ncRNAs were common for the two sites, but five of them (miR-484, miR-328-3p, miR-27a-5p, miR-28-3p, and miR-409-3p) correlated positively to BMD in femoral bone, but negatively in iliac bone. Of predicted pathways recognized in bone metabolism, ECM-receptor interaction and proteoglycans in cancer emerged at both sites, whereas fatty acid metabolism and focal adhesion were only identified in iliac bone. Lasso analysis and cross-validations identified sets of nine bone ncRNAs correlating strongly with adjusted total hip BMD in both femoral and iliac bone. Twenty-eight iliac ncRNAs were associated with risk of fracture (FDR < 0.1). The small nucleolar RNAs, RNU44 and RNU48, have a function in stabilization of ribosomal RNAs (rRNAs), and their association with fracture and BMD suggest that aberrant processing of rRNAs may be involved in development of osteoporosis. Cis-eQTL (expressed quantitative trait loci) analysis of the iliac bone biopsies identified two loci associated with microRNAs (miRNAs), one previously identified in a heel-BMD genomewide association study (GWAS). In this comprehensive investigation of the skeletal genetic background in postmenopausal women, we identified functional bone ncRNAs associated to fracture and BMD, representing distinct subsets in WB and NWB skeletal sites. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.     

Abrogation of Cbl–PI3K Interaction Increases Bone Formation and Osteoblast Proliferation

Cbl is an adaptor protein and E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and phosphorylated by Src family kinases. Phosphorylated CblY737 creates a binding site for the p85 regulatory subunit of phosphatidylinositol 3 kinase (PI3K) that also plays an important role in the regulation of bone homeostasis. To investigate the role of Cbl–PI3K interaction in bone homeostasis, we examined knock-in mice in which the PI3K binding site on Cbl was ablated due to the substitution of tyrosine 737 to phenylalanine (Cbl^YF/YF, YF mice). We previously reported that bone volume in these mice is increased due to decreased osteoclast function (Adapala et al., J Biol Chem 285:36745–36758, 19). Here, we report that YF mice also have increased bone formation and osteoblast numbers. In ex vivo cultures bone marrow-derived YF osteoblasts showed increased Col1A expression and their proliferation was also significantly augmented. Moreover, proliferation of MC3T3-E1 cells was increased after treatment with conditioned medium generated by culturing YF bone marrow stromal cells. Expression of stromal derived factor-1 (SDF-1) was increased in YF bone marrow stromal cells compared to wild type. Increased immunostaining of SDF-1 and CXCR4 was observed in YF bone marrow stromal cells compared to wild type. Treatment of YF condition medium with neutralizing anti-SDF-1 and anti-CXCR4 antibodies attenuated MC3T3-E1 cell proliferation. Cumulatively, these results show that abrogation of Cbl–PI3K interaction perturbs bone homeostasis, affecting both osteoclast function and osteoblast proliferation.     

Myeloperoxidase Peptide–Based Nasal Tolerance in Experimental ANCA–Associated GN

Less toxic treatment options for patients with myeloperoxidase (MPO)-ANCA–associated GN are needed. Using an established murine model of focal necrotizing GN mediated by autoimmunity to MPO (autoimmune anti–MPO GN), we assessed the capacity for nasal tolerance induced by nasal insufflation of the immunodominant nephritogenic MPO peptide (MPO_409–428) to attenuate this disease. Compared with mice that received an irrelevant immunodominant ovalbumin (OVA) peptide, OVA_323–339, mice that received MPO_409–428 were protected from the development of humoral and cell-mediated autoimmunity to full-length MPO and the development of GN. In mice with established anti–MPO autoimmunity, nasal insufflation of MPO_409–428 as a therapeutic attenuated anti–MPO GN. To investigate the nature of this induced tolerance, we isolated CD4⁺ T cells from the upper airway draining lymph nodes of both OVA_323–339- and MPO_409–428-tolerized mice. Adoptive transfer of CD4⁺ T cells from MPO_409–428- but not OVA_323–339-tolerized mice to animals with established anti–MPO autoimmunity attenuated the subsequent development of GN, confirming that the immunosuppression induced by these T cells is antigen specific. Ex vivo studies showed that nasal tolerance to MPO is mediated by both conventional and induced T regulatory cells. The strong homology between the pathogenic human MPO B cell epitope recognized by ANCA in patients with acute vasculitis and the nephritogenic murine T cell MPO epitope emphasizes the clinical relevance of this study.     

Role of Type 1 Diabetes–Associated SNPs on Risk of Autoantibody Positivity in the TEDDY Study

The Environmental Determinants of Diabetes in the Young (TEDDY) study prospectively follows 8,677 children enrolled from birth who carry HLA-susceptibility genotypes for development of islet autoantibodies (IA) and type 1 diabetes (T1D). During the median follow-up time of 57 months, 350 children developed at least one persistent IA (GAD antibody, IA-2A, or micro insulin autoantibodies) and 84 of them progressed to T1D. We genotyped 5,164 Caucasian children for 41 non-HLA single nucleotide polymorphisms (SNPs) that achieved genome-wide significance for association with T1D in the genome-wide association scan meta-analysis conducted by the Type 1 Diabetes Genetics Consortium. In TEDDY participants carrying high-risk HLA genotypes, eight SNPs achieved significant association to development of IA using time-to-event analysis (P < 0.05), whereof four were significant after adjustment for multiple testing (P < 0.0012): rs2476601 in PTPN22 (hazard ratio [HR] 1.54 [95% CI 1.27–1.88]), rs2292239 in ERBB3 (HR 1.33 [95% CI 1.14–1.55]), rs3184504 in SH2B3 (HR 1.38 [95% CI 1.19–1.61]), and rs1004446 in INS (HR 0.77 [0.66–0.90]). These SNPs were also significantly associated with T1D in particular: rs2476601 (HR 2.42 [95% CI 1.70–3.44]). Although genes in the HLA region remain the most important genetic risk factors for T1D, other non-HLA genetic factors contribute to IA, a first step in the pathogenesis of T1D, and the progression of the disease.     

Differential Association of BRCA1 and BRCA2 Genes with Some Breast Cancer–Associated Genes in Early and Late Onset Breast Tumors

Background: Accumulating evidence indicating more aggressive features of breast carcinoma (BC) in young women than their older counterparts have raised the question of whether these differences are present at the genetic level.Methods: For this purpose, we performed a comparative analysis of the frequency of deletions of BRCA1, BRCA2, BRCAX, TP53, ATM, and RB1 and amplification of Cyclin D1 and also studied the interrelation and prognostic significance of these genetic alterations in 30 early onset (40 years) and 33 late onset (>40 years) cases of BC. These gene alterations were also studied in 11 other types of breast lesions.Results: A differential pattern of alterations (deletion/amplification) was observed in the two age groups, with the sequence in younger women being BRCA1 (72%), TP53 (71%), ATM (64%), BRCA2 (62%), RB1 (60%), Cyclin D1 (43%), and BRCAX (24%) and that in the older group being TP53 (66%), RB1 (63%), BRCA1 (56%), ATM (53%), BRCA2 (45%), Cyclin D1 (24%), and BRCAX (23%). Similar, differential correlations were also seen with several clinicopathological parameters, prognosis, and combinations of alterations among these genes in the two age groups.Conclusions: Differential frequencies and interrelationships of genetic alterations and prognoses in these two age groups indicate that the molecular pathways for the development of tumors in both age groups may not be similar, though the ultimate effect is deregulation of cell cycle checkpoints and defects in the DNA repair pathway.