Early Eculizumab Withdrawal in Patients With Atypical Hemolytic Uremic Syndrome in Native Kidneys Is Safe and Cost-Effective: Results of the CUREiHUS Study.

Introduction: The introduction of eculizumab has improved the outcome in patients with atypical hemolytic uremic syndrome (aHUS). The optimal treatment strategy is debated. Here, we report the results of the CUREiHUS study, a 4-year prospective, observational study monitoring unbiased eculizumab discontinuation in Dutch patients with aHUS after 3 months of therapy. Methods: All pediatric and adult patients with aHUS in native kidneys and a first-time eculizumab treatment were evaluated. In addition, an extensive cost-consequence analysis was conducted. Results: A total of 21 patients were included in the study from January 2016 to October 2020. In 17 patients (81%), a complement genetic variant or antibodies against factor H were identified. All patients showed full recovery of hematological thrombotic microangiopathy (TMA) parameters after the start of eculizumab. A renal response was noted in 18 patients. After a median treatment duration of 13.6 weeks (range 2.1-43.9), eculizumab was withdrawn in all patients. During follow-up (80.7 weeks [0.0-236.9]), relapses occurred in 4 patients. Median time to first relapse was 19.5 (14.3-53.6) weeks. Eculizumab was reinitiated within 24 hours in all relapsing patients. At last follow-up, there were no chronic sequelae, i.e., no clinically relevant increase in serum creatinine (sCr), proteinuria, and/or hypertension in relapsing patients. The low sample size and event rate did not allow to determine predictors of relapse. However, relapses only occurred in patients with a likely pathogenic variant. The cost-effectiveness analysis revealed that the total medical expenses of our population were only 30% of the fictive expenses that would have been made when patients received eculizumab every fortnight. Conclusion: It is safe and cost-effective to discontinue eculizumab after 3 months of therapy in patients with aHUS in native kidneys. Larger data registries are needed to determine factors associated with suboptimal kidney function recovery during eculizumab treatment, factors to predict relapses, and long-term outcomes of eculizumab discontinuation.

The influence of onset of disease on exit from paid employment among workers in The Netherlands: A longitudinal register-based study with 9 years follow-up.

OBJECTIVES: This study investigates the influence of onset of disease on exit from paid employment and whether this differs across diseases and sociodemographic groups. METHODS: Register data from Statistics Netherlands on medication prescription was linked to information on employment status and demographics. Persons who were employed in 2009 and 2010 and who did not use medication for the selected disease in 2009 (N=5 889 036) were followed-up over nine years. Six diseases were identified based on medication prescription in 2010 and 2011: cardiovascular diseases, inflammatory diseases, diabetes mellitus, respiratory diseases, common mental disorders, and psychotic disorders. Four pathways out of paid employment were defined: disability benefits, unemployment, no income, and early retirement. Early exit from paid employment was defined as exiting paid employment before retirement age. Cause-specific Cox proportional hazards regression analyses were performed, with interaction terms for age, sex, and migration background. RESULTS: Onset of disease increased the likelihood of exit from paid employment, with the strongest associations for psychotic disorders [hazard ratio (HR) 2.88, 95% confidence interval (CI) 2.78-2.98] and common mental disorders (HR 2.00, 95% CI 1.97-2.03). Onset of disease was most strongly associated with disability benefits, followed by unemployment. The influence of common mental and psychotic disorders on disability increased until around middle-age, after which it decreased. The influence of mental health problems on exit from paid employment was stronger for persons with a non-native Dutch background and males. CONCLUSION: Onset of diseases, especially mental health disorders, is a risk for exiting paid employment before the retirement age. Effective interventions are needed to enhance an inclusive workforce and prevent involuntary loss of paid employment.

Chronic kidney disease ten years after pediatric allogeneic hematopoietic stem cell transplantation.

Chronic kidney disease (CKD) is an important sequela of hematopoietic stem cell transplantation (HSCT), but data regarding CKD after pediatric HSCT are limited. In this single center cohort study, we evaluated the estimated glomerular filtration rate (eGFR) dynamics, proteinuria and hypertension in the first decade after HSCT and assessed risk factors for CKD in 216 pediatric HSCT survivors, transplanted 2002-2012. The eGFR decreased from a median of 148 to 116 ml/min/1.73 m2 between pre-HSCT to ten years post-HSCT. CKD (KDIGO stages G2 or A2 or more; eGFR under 90 ml/min/1.73m2 and/or albuminuria) occurred in 17% of patients. In multivariate analysis, severe prolonged stage 2 or more acute kidney injury (AKI), with an eGFR under 60ml/min/1.73m2 and duration of 28 days or more, was the main risk factor for CKD (hazard ratio 9.5, 95% confidence interval 3.4-27). Stage 2 or more AKI with an eGFR of 60ml/min/1.73m2 or more and KDIGO stage 2 or more AKI with eGFR under 60ml/min/1.73m2 but recovery within 28 days were not associated with CKD. Furthermore, hematological malignancy as HSCT indication was an independent risk factor for CKD. One third of patients had both CKD criteria, one third had isolated eGFR reduction and one third only had albuminuria. Hypertension occurred in 27% of patients with CKD compared to 4.4% of patients without. Tubular proteinuria was present in 7% of a subgroup of 71 patients with available ß2-microglobulinuria. Thus, a significant proportion of pediatric HSCT recipients developed CKD within ten years. Our data stress the importance of structural long-term monitoring of eGFR, urine and blood pressure after HSCT to identify patients with incipient CKD who can benefit from nephroprotective interventions.

Optical limiting applications of resonating plasmonic Au nanoparticles in a dielectric glass medium.

Plasmonic nanostructures exhibiting high optical nonlinearities are widely used in the rapidly growing modern nanotechnology of nonlinear optics including biomedical applications due to their tunable plasmonic behavior. In this work, we investigate the nonlinear optical properties of uniformly distributed Au nanoparticles (NPs) embedded in pre-synthesized sodium-zinc borate glass by the well-known ion-exchange technique for optical limiting (OL) applications. Various techniques such as optical absorption spectroscopy, x-ray photoelectron spectroscopy, Transmission Electron Microscope (TEM), Photoluminescence, Time of Flight secondary mass spectroscopy and the Z scan technique were used for the characterization of these NPs. TEM confirmed spherically shaped Au NPs with varying sizes of up to 16 nm, in agreement with optical absorption spectroscopy. Nonlinear optical (NLO) properties of these Au NPs were investigated by using an open as well as close aperture Z scan technique which exhibited enhanced optical nonlinearities. The two-photon absorption (2PA) coefficients demonstrated an increasing trend while the OL threshold values demonstrated a decreasing trend as a function of heat treatment. The improved 2PA coefficients and decreased OL threshold values endorsed the Au NPs containing glasses as contending materials for the fabrication of promising optical limiters for the protection of eyes and other sensitive instruments from laser induced damages.

Cancer-ID: Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood.

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 µm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1 µm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic.

Enhanced adsorption of As(V) and Mn(VII) from industrial wastewater using multi-walled carbon nanotubes and carboxylated multi-walled carbon nanotubes.

The presence of As(V) and Mn(VII) in water beyond the permissible concentration allowed by World Health Organization (WHO) standard affects human beings, animals and the environment adversely. Hence, there is need for an efficient material to remove these potentially toxic elements from wastewater prior to discharge into water bodies. This research focused on the application of response surface method (RSM) assisted optimization of Fe-Ni/Activated carbon (AC) catalyst for the synthesis of MWCNTs. Also, the MWCNTs was carboxylated and the adsorption behaviors of both nano-adsorbents in the removal of As(V) and Mn(VII) from industrial wastewater was investigated through experimental and computational techniques. The prepared Fe-Ni/AC, MWCNTs and MWCNTs-OCH2CO2H were characterized using BET, TGA, FTIR, HRSEM, HRTEM, XRD and XPS. The result showed the BET surface area of Fe-Ni/AC, MWCNTs and MWCNTs-OCH2CO2H were obtained as 1100, 1250 and 1172 m2/g, respectively. Due to the enhanced impact of carboxylation, the adsorption capacity of As(V) and Mn(VII) removal increased from 200 to 192 mg/g for MWCNTs to 250 and 298 mg/g for MWCNTs-OCH2CO2H. The isotherm and kinetic models were best fitted by Langmuir and pseudo-second order kinetics, while the thermodynamic investigation found that the adsorption process was endothermic, spontaneous and chemisorptions controlled. The regeneration potential of MWCNTs and MWCNTs-OCH2CO2H after six repeated applications revealed good stability of adsorption efficiency. The study demonstrated optimization importance of Fe-Ni/AC catalyst design for MWCNTs adsorbents and the potentials of utilizing both MWCNTs and MWCNTs-OCH2CO2H in the removal of selected heavy metals from water and soil.

Use of amlodipine oral solution for the treatment of hypertension in children.

Background Amlodipine is a widely used antihypertensive agent for the treatment of paediatric hypertension, but the commercially available tablets are not suitable to treat young patients, who need lower, flexible dosages and a liquid formulation. Objective To determine the pharmacokinetic properties of amlodipine and the acceptability of a standardised, extemporaneous oral solution. Method A newly developed liquid formulation of amlodipine was administered to hypertensive children between the age of 6 months and 11 years. Using a limited sampling strategy, population PK analysis was performed using nonlinear mixed effects modelling. Results Nine children, with a median age of 2.9 years (IQR 1.8-8.4), receiving stable amlodipine therapy in a median dose of 0.15 mg kg-1 day-1 (IQR 0.11-0.18), were switched to study medication. The population pharmacokinetic model was able to accurately predict the clearance of amlodipine in the study population. Based on the final model, clearance was reduced by 31.2% (RSE: 10%) in females. Patient reported outcomes on taste from a five-point hedonic scale were available for five patients, who scored the taste from positive to slightly negative. Conclusion The results from the PK study and the acceptability assessment show that the amlodipine oral solution presented in this study offers an appropriate treatment option for young children.

Thermally induced structural metamorphosis of ZnO:Rb nanostructures for antibacterial impacts.

In this work, we report on the synthesis of pure and Rb doped ZnO (ZnO:Rb) nanoparticles by a simple combustion technique followed by thermal treatment in an open-air atmosphere. The prepared samples were characterized using UV-vis spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), photoluminescence, Raman spectroscopy and scanning electron microscopy. The wurtzite hexagonal phase structure of ZnO and a secondary phase of Rb2ZnO2 was observed after doping ZnO with Rb. FTIR and DSC confirmed the functional groups and the thermal stability of the ZnO samples. Field emission scanning electron microscope showed an irregular shaped agglomerated morphology for the ZnO:Rb samples. The chemical states of the undoped and Rb doped samples were identified using X-ray photoelectron spectroscopy for both pure and ZnO:Rb samples. In addition, ZnO:Rb samples exhibit good antimicrobial activities against Bacillus subtilis with a change in antibacterial behaviour as compared to pure ZnO structures indicating their multifunctional applications.

Maturation of adenovirus primes the protein nano-shell for successful endosomal escape.

The ability of adenoviruses to infect a broad range of species has spurred a growing interest in nanomedicine to use adenovirus as a cargo delivery vehicle. While successful maturation of adenovirus and controlled disassembly are critical for efficient infection, the underlying mechanisms regulating these processes are not well understood. Here, we present Atomic Force Microscopy nanoindentation and fatigue studies of adenovirus capsids at different maturation stages to scrutinize their dynamic uncoating properties. Surprisingly, we find that the early intermediate immature (lacking DNA) capsid is mechanically indistinguishable in both break force and spring constant from the mature (containing DNA) capsid. However, mature and immature capsids do display distinct disassembly pathways, as revealed by our mechanically-induced fatigue analysis. The mature capsid first loses the pentons, followed by either long-term capsid stability or abrupt and complete disassembly. However, the immature capsid has a stable penton region and undergoes a stochastic disassembly mechanism, thought to be due to the absence of genomic pressure. Strikingly, the addition of the genome alone is not sufficient to achieve penton destabilization as indicated by the penton stability of the maturation-intermediate mutant, G33A. Full penton destabilization was achieved only when the genome was present in addition to the successful maturation-linked proteolytic cleavage of preprotein VI. Therefore these findings strongly indicate that maturation of adenovirus in concert with genomic pressure induces penton destabilization and thus, primes the capsid for controlled disassembly. This latter aspect is critical for efficient infection and successful cargo delivery.

Photocatalytic degradation of local dyeing wastewater by iodine-phosphorus co-doped tungsten trioxide nanocomposites under natural sunlight irradiation.

In the present work, one-step green synthesis of WO3 based on the interaction of ammonium paratungstate and Spondias mombin leaves extract is reported. Different concentrations of iodine and phosphorus in the range of (2%, 5% and 10%) were firstly incorporated into the prepared WO3 nanoparticles to obtain Iodine doped and Phosphorus doped WO3 nanoparticles respectively. Subsequently, iodine and phosphorus co-doped WO3 nanocomposites was prepared using a wet impregnation method followed by calcination at high temperature. The nanomaterials were characterized by HRSEM, HRTEM, BET, UV-Visible, EDS, XRD and XPS. The photo-oxidation of dyeing wastewater by the synthesized WO3 nanomaterials were tested and assessed using Total organic carbon (TOC) and Chemical oxygen demand (COD) as indicator parameters. XRD and HRSEM analysis demonstrated the formation of only monoclinic phase of WO3 irrespective of the dopants. The UV-Visible diffuse reflectance spectroscopy showed the band gap energy of 2.61 eV for undoped WO3 and 2.02 eV for I-P co-doped WO3 nanocomposites. The surface area of I-P co-doped WO3 (416.18 m2/g) was higher than the undoped WO3 (352.49 m2/g). The XPS demonstrated interstitial and substitution of oxygen (O2-) vacancies in WO3 by I- and P3+ and formed I-P-WO(3-x). The I-P co-doped WO3 exhibited higher catalytic activities (93.4% TOC, 95.1% COD) than the undoped (54.9% TOC, 79.2% COD) due to the synergistic effects between the two dopants. The experimental data better fitted to pseudo-second order than first order and pseudo-first order model. This study demonstrated the enhanced photocatalytic performance of I-P co-doped WO3 nanocomposites under sunlight.

Recent Advances in Biological Single-Molecule Applications of Optical Tweezers and Fluorescence Microscopy.

Over the past two decades, single-molecule techniques have evolved into robust tools to study many fundamental biological processes. The combination of optical tweezers with fluorescence microscopy and microfluidics provides a powerful single-molecule manipulation and visualization technique that has found widespread application in biology. In this combined approach, the spatial (~nm) and temporal (~ms) resolution, as well as the force scale (~pN) accessible to optical tweezers is complemented with the power of fluorescence microscopy. Thereby, it provides information on the local presence, identity, spatial dynamics, and conformational dynamics of single biomolecules. Together, these techniques allow comprehensive studies of, among others, molecular motors, protein-protein and protein-DNA interactions, biomolecular conformational changes, and mechanotransduction pathways. In this chapter, recent applications of fluorescence microscopy in combination with optical trapping are discussed. After an introductory section, we provide a description of instrumentation together with the current capabilities and limitations of the approaches. Next we summarize recent studies that applied this combination of techniques in biological systems and highlight some representative biological assays to mark the exquisite opportunities that optical tweezers combined with fluorescence microscopy provide.

The Distress Thermometer for screening for severe fatigue in newly diagnosed breast and colorectal cancer patients.

OBJECTIVE: Internationally, the Distress Thermometer and associated Problem List are increasingly used in oncology as screening tools for psychological distress. Cancer-related fatigue is common but often overlooked in clinical practice. We examined if severe fatigue in cancer patients can be identified with the fatigue item of the Problem List. METHODS: Newly diagnosed breast (N = 334) and colorectal (N = 179) cancer patients were screened for severe fatigue, which was defined as having a positive score on the fatigue item of the Problem List. The Fatigue Severity subscale of the Checklist Individual Strength was used as gold standard measure for severe fatigue. RESULTS: In total, 78% of breast cancer patients and 81% of colorectal cancer patients were correctly identified with the fatigue item. The sensitivity was 89% in breast cancer patients and 91% in colorectal cancer patients. The specificity was 75% in breast cancer patients and 77% in colorectal cancer patients. The positive predictive value was 53% in breast cancer patients and 64% in colorectal cancer patients, whereas the negative predictive value was 95% in both tumor types. CONCLUSIONS: The fatigue item of the Problem List performs satisfactorily as a quick screening tool for severe fatigue. However, a positive screen should be followed up with a more thorough assessment of fatigue, ie, a questionnaire with a validated cutoff point. Given time pressure of clinicians, this already implemented and brief screening tool may prevent severe fatigue from going undetected in clinical practice.

Severe fatigue after treatment of ductal carcinoma in situ: A comparison with age-matched breast cancer survivors and healthy controls.

PURPOSE: Severe fatigue after treatment of ductal carcinoma in situ (DCIS) has not been studied before. The current study examined (i) the prevalence of severe fatigue in DCIS patients versus breast cancer survivors (BCS) and healthy controls (HC), (ii) quality of life and functioning of severely versus non-severely fatigued DCIS patients and BCS, and (iii) the association of fatigue with psychosocial and behavioral factors in DCIS patients. METHODS: 89 patients treated for DCIS were matched on age and gender to 67 BCS and 178 HC (ratio 1:1:2). Fatigue was measured with the Fatigue Severity subscale of the Checklist Individual Strength. RESULTS: 23% of DCIS patients, 25% of BCS, and 6% of HC were severely fatigued (DCIS versus HC: p < 0.001). Severely fatigued DCIS patients had a lower quality of life and were more impaired in all domains of functioning than non-severely fatigued DCIS patients. Sleep problems, dysfunctional cognitions regarding fatigue, avoidance of activities, all-or-nothing behavior, perceived lack of social support, DCIS-related coping problems, and fear of future cancer occurrence were related to fatigue. CONCLUSIONS: The prevalence of severe fatigue in DCIS patients was similar to BCS, but higher than in HC. Severely fatigued DCIS patients had a lower quality of life and more functional impairments. The psychosocial and behavioral fatigue-related factors in DCIS patients are known to perpetuate fatigue in BCS. These factors can be targeted in interventions for cancer-related fatigue. Our findings suggest that the same treatment elements might be applicable to severely fatigued DCIS patients.

Atomic force microscopy observation and characterization of single virions and virus-like particles by nano-indentation.

Structure and function of viruses are intimately related, and one of the goals in virology is to elucidate the mechanisms behind this relation. A variety of research endeavours is focused on studying these mechanisms and a relatively new technique in this field is Atomic Force Microscopy (AFM). Using AFM virions and virus-like particles can be imaged and manipulated at the single particle level. Here we review recent AFM nano-indentations studies unveiling for instance the mechanics of capsid-genome interactions, morphological changes that drive viral maturation, capsid stabilizing factors and viral uncoating. We show that in an increasing amount of literature a clear link between mechanics and infectivity is observed, which not only provides us with new fundamental insights into virology, but also provides ways to improve virus-like particles for applications in nanomedicine and nanotechnology.

Protein Nanocontainers from Nonviral Origin: Testing the Mechanics of Artificial and Natural Protein Cages by AFM.

Self-assembling protein nanocontainers are promising candidates for an increasingly wide scope of purposes. Their applications range from drug delivery vehicles and imaging agents to nanocompartments for controlled enzymatic activity. In order to exploit their full potential in these different fields, characterization of their properties is vital. For example, their mechanical properties give insight into the stability of a particle as a function of their internal content. The mechanics can be probed by atomic force microscopy nanoindentation, and while this single particle method is increasingly used to probe material properties of viral nanocages, it has hardly been used to characterize nonviral nanocages. Here we report nanoindentation studies on two types of nonviral nanocontainers: (i) lumazine synthase from Aquifex aeolicus (AaLS), which naturally self-assembles into icosahedral cages, and (ii) the artificial protein cage O3-33 originating from a computational design approach. In addition, we tested particles that had been engineered toward improved cargo loading capacity and compared these nanocages in empty and loaded states. We found that the thermostable AaLS cages are stiffer and resist higher forces before breaking than the O3-33 particles, but that mutations affecting the size of AaLS particles have a dramatic effect on their structural stability. Furthermore, we show that cargo packaging can occur while maintaining the cage's mechanical properties.

Management of the axilla after neoadjuvant chemotherapy for clinically node positive breast cancer: A nationwide survey study in The Netherlands.

BACKGROUND: Axillary pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is achieved in a substantial part of clinically node positive breast cancer patients. Treatment of the axilla after NAC varies widely, and new techniques to spare patients from an axillary lymph node dissection (ALND) are being introduced. METHODS: This Dutch nationwide survey regarding treatment of the initially clinically node positive axilla in patients receiving NAC was conducted amongst 148 surgical oncologists during November 2014-June 2015, to survey the diagnostic work-up, axillary mapping and willingness to omit ALND. RESULTS: Axillary ultrasound was considered a standard procedure in the diagnostic work-up by 99% of participants. The majority of 70% of participants stated that ALND could possibly be omitted in node positive patients with a favourable response to NAC. A positive correlation was observed between the total amount of patients treated, versus patients receiving NAC (P < 0.01). A total of 93 respondents performed axillary response evaluation after NAC, using imaging (72%), excision of localized lymph nodes (56%) or sentinel node biopsy (SNB; 45%). Decision-making in omitting ALND was influenced by the presence of N2-3 disease, patient age and type of breast surgery. Multivariable analysis showed that clinicians who administered NAC more often, were more likely to omit ALND (P < 0.01). DISCUSSION: The majority of surgeons are inclined to omit ALND in case of an axillary pCR. A large variety of techniques is being used to identify a pCR. The lack of consensus on this topic indicates the need for guidelines based on the best available evidence.

Building an artificial actin cortex on microscopic pillar arrays.

Eukaryotic cells obtain their morphology and mechanical strength from the cytoskeleton and in particular from the cross-linked actin network that branches throughout the whole cell. This actin cortex lies like a quasi-two-dimensional (2D) biopolymer network just below the cell membrane, to which it is attached. In the quest for building an artificial cell, one needs to make a biomimetic model of the actin cortex and combine this in a bottom-up approach with other "synthetic" components. Here, we describe a reconstitution method for such an artificial actin cortex, which is freely suspended on top of a regular array of pillars. By this immobilization method, the actin network is only attached to a surface at discrete points and can fluctuate freely in between. By discussing the method to make the micropillars and the way to reconstitute a quasi-2D actin network on top, we show how one can study an isolated, reconstituted part of a cell. This allows the study of fundamental interaction mechanisms of actin networks, providing handles to design a functional actin cortex in an artificial cell.

A prospective validation study of sentinel lymph node biopsy in multicentric breast cancer: SMMaC trial.

BACKGROUND: Multicentric breast cancer is often considered a contra-indication for sentinel lymph node (SLN) biopsy due to concerns with sensitivity and false negative rate. To assess SLN feasibility and accuracy in multicentric breast cancer, the multi-institutional SMMaC trial was conducted. METHODS: In this study 30 patients with multicentric breast cancer and a clinically negative axilla were prospectively included. Periareolar injection of radioisotope and blue dye was administered. In all patients SLN biopsy was validated by back-up completion axillary lymph node dissection. RESULTS: the SLN was successfully identified in 30 of 30 patients (identification rate 100%). The incidence of axillary metastases was 66.7% (20/30). The false negative rate was 0% (0/20) and the sensitivity was 100% (20/20). The negative predictive value was 100% (10/10). CONCLUSION: SLN biopsy in multicentric breast cancer seems feasible and accurate and should therefore be considered in patients with multicentric breast cancer and clinically negative axilla.

Discordance between number of scintigraphic and perioperatively identified sentinel lymph nodes and axillary tumour recurrence.

BACKGROUND: In breast cancer, sentinel node biopsy is considered the standard method to assess the lymph node status of the axilla. Preoperative identification of sentinel lymph nodes (SLN) is performed by injecting a radioactive tracer, followed by lymphoscintigraphy. In some patients there is a discrepancy between the number of lymphoscintigraphically identified sentinel nodes and the number of nodes found during surgery. We hypothesized that the inability to find peroperatively all the lymphoscintigraphically identified sentinel nodes, might lead to an increase in axillary recurrence because of positive SLNs not being removed. METHODS: Patients who underwent sentinel node biopsy between January 2000 and July 2010 were identified from a prospectively collected database. The number of lymphoscintigraphically and peroperatively identified sentinel nodes were reviewed and compared. Axillary recurrences were scored. RESULTS: 1368 patients underwent a SLN biopsy. Median follow up was 58.5 months (range 12-157). Patient and tumour characteristics showed no significant differences. In 139 patients (10.2%) the number of radioactive nodes found during surgery was less than preoperative scanning (group 1) and in 89.8% (N = 1229) there were equal or more peroperative nodes identified than seen lymphoscintigraphically (group 2). In group 1, 0/139 patients (0%) developed an axillary recurrence and in the second group this was 25/1229 (2.0%) respectively. No significant difference between groups regarding axillary recurrence, sentinel node status and distant metastasis was found. CONCLUSION: Axillary recurrence rate is not influenced by the inability to remove all sentinel nodes during surgery that have been identified preoperatively by scintigraphy.

Malignant melanoma cells acquire resistance to DNA interstrand cross-linking chemotherapeutics by p53-triggered upregulation of DDB2/XPC-mediated DNA repair.

Malignant melanoma is a cancer characterized by high chemoresistance although p53 is rarely mutated. Here, we show that p53 wild-type melanoma cells acquire resistance to cell death induced by fotemustine (FM), which is a representative of alkylating DNA interstrand cross-linking agents used in melanoma therapy. We show that drug-induced resistance is a result of p53-dependent upregulation of the nucleotide excision repair (NER) genes xeroderma pigmentosum complementation group C (XPC) and damaged DNA-binding protein 2 (DDB2), which stimulate the repair of DNA interstrand cross-links (ICLs) arising from O(6)-chloroethylguanine. Consequently, TP53 mutated cells are unable to repair ICLs, leading to prolonged ATM, ATR and checkpoint kinase 1 (CHK1) activation, and finally apoptosis. The roles of p53 and NER in ICL-triggered cell death were confirmed by knockdown of p53 and XPC. Upregulation of XPC and DDB2 in p53wt cells following a single drug treatment is a robust and sustained response that lasts for up to 1 week. Pretreatment with an inducing dose followed by a high and toxic dose of FM provoked an adaptive response as the killing outcome of the challenge dose was reduced. Upregulation of XPC and DDB2 was also observed in a melanoma mouse xenograft model following systemic administration of FM. Additionally, XPC and DDB2 induction occurred upon treatment with other cross-linking anticancer drugs, such as cisplatin and mafosfamide, indicating it is a general response of cancer cells to this group of chemotherapeutics. Collectively, the data indicate that p53-dependent upregulation of XPC and DDB2 is a key mechanism upon genotoxic stress, whereby melanoma cells acquire resistance towards DNA cross-linking agents. To our knowledge, this is the first demonstration of upregulation of NER following a single dose of a DNA interstrand cross-linker, which is a robust and long-lasting effect that impacts the killing response of cancer cells to subsequent treatments.