Despite recent developments in bottom‐up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein ...amounts. The suspension trapping method combines the advantage of efficient SDS‐based protein extraction with rapid detergent removal, reactor‐type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack—this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow‐through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.
Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related ...genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.
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•Glycolytic index in melanoma negatively correlates with response to anti-PD1 therapy•Blocking lactate transport or knock out of glycolytic genes improves checkpoint therapy•Diclofenac blocks the lactate transporters MCT1 and MCT4 in a COX-independent manner•Inhibition of glycolysis by MCT blockade does not impede T cell function
Renner et al. demonstrate a negative correlation between glycolytic activity in tumors and response to checkpoint therapy. Genetic blockade of glycolysis or pharmacological inhibition of the main lactate transporters MCT1 and MCT4 preserves T cell function, reverses tumor acidification, and augments response to checkpoint therapy.
Immune checkpoint inhibitors, including those targeting programmed cell death protein 1 (PD-1), are reshaping cancer therapeutic strategies. Evidence suggests, however, that tumor response and ...patient survival are determined by tumor programmed death ligand 1 (PD-L1) expression. We hypothesized that preconditioning of the tumor immune microenvironment using targeted, virus-mediated interferon (IFN) stimulation would up-regulate tumor PD-L1 protein expression and increase cytotoxic T cell infiltration, improving the efficacy of subsequent checkpoint blockade. Oncolytic viruses (OVs) represent a promising form of cancer immunotherapy. For brain tumors, almost all studies to date have used direct intralesional injection of OV, because of the largely untested belief that intravenous administration will not deliver virus to this site. We show, in a window-of-opportunity clinical study, that intravenous infusion of oncolytic human
(referred to herein as reovirus) leads to infection of tumor cells subsequently resected as part of standard clinical care, both in high-grade glioma and in brain metastases, and increases cytotoxic T cell tumor infiltration relative to patients not treated with virus. We further show that reovirus up-regulates IFN-regulated gene expression, as well as the PD-1/PD-L1 axis in tumors, via an IFN-mediated mechanism. Finally, we show that addition of PD-1 blockade to reovirus enhances systemic therapy in a preclinical glioma model. These results support the development of combined systemic immunovirotherapy strategies for the treatment of both primary and secondary tumors in the brain.
Objectives
To evaluate our clinical experience with percutaneous image‐guided radiofrequency ablation (RFA) of 200 renal tumours in a large tertiary referral university institution.
Patients and ...Methods
Image‐guided RFA (ultrasonography or computed tomography CT) of 200 renal tumours in 165 patients from June 2004 to 2012 was prospectively evaluated. Institutional Review Board approval was granted.
The treatment response and technical success were defined by absence of contrast enhancement within the tumour on contrast enhanced CT or magnetic resonance imaging.
Both major and minor complications, glomerular filtration rate (GFR) before and after RFA, the management and outcomes of the complications, as well as oncological outcome were prospectively documented.
Multivariate analysis was used to determine variables associated with major complications and also the percentage GFR change after RFA.
The overall (OS), 5‐year cancer‐specific (CSS), local recurrence‐free (LRFS) and metastasis‐free survival (MFS) rates are presented using the Kaplan–Meier curves.
Results
In all, 200 tumours were RF ablated with a mean (range) tumour size of 2.9 (1–5.6) cm and the mean (range) patient age was 67.7 (21–88.6) years with a mean follow‐up period of 46.1 months. The primary technical and overall technical success rate was 95.5% and 98.5%, respectively. Two independent predictors of successful RFA in a single sitting were tumour size (<3 cm) and exophytic location in multivariate logistic regression analysis.
Major complications included ureteric injury (six patients), calyceal‐cutaneous fistula (one), acute tubular necrosis (one) and abscess (two). Two independent predictors of ureteric injury were central location and lower pole position.
Within this cohort of patients, only four patients developed significant renal function deterioration i.e. >25% decreased in GFR. In all, 161 (98%) patients of the 165 patients have preservation of renal function. Any change in renal function after RFA was not influenced by tumour factors or solitary kidney status.
In our clinical series, this yielded a 5‐year OS, CSS, LRFS and MFS rates of 75.8%, 97.9%, 93.5% and 87.7% respectively.
Conclusions
Image‐guided RFA is a safe, nephron sparing and effective treatment for small renal cell carcinoma (RCC) tumours with a low rate of recurrence and has good 5‐year CSS and MFS rates.
To examine the effects on process of care and patient well-being, of the regular collection and use of health-related quality-of-life (HRQL) data in oncology practice.
In a prospective study with ...repeated measures involving 28 oncologists, 286 cancer patients were randomly assigned to either the intervention group (regular completion of European Organization for Research and Treatment of Cancer-Core Quality of Life Questionnaire version 3.0, and Hospital Anxiety and Depression Scale on touch-screen computers in clinic and feedback of results to physicians); attention-control group (completion of questionnaires, but no feedback); or control group (no HRQL measurement in clinic before encounters). Primary outcomes were patient HRQL over time, measured by the Functional Assessment of Cancer Therapy-General questionnaire, physician-patient communication, and clinical management, measured by content analysis of tape-recorded encounters. Analysis employed mixed-effects modeling and multiple regression.
Patients in the intervention and attention-control groups had better HRQL than the control group (P =.006 and P =.01, respectively), but the intervention and attention-control groups were not significantly different (P =.80). A positive effect on emotional well-being was associated with feedback of data (P =.008), but not with instrument completion (P =.12). A larger proportion of intervention patients showed clinically meaningful improvement in HRQL. More frequent discussion of chronic nonspecific symptoms (P =.03) was found in the intervention group, without prolonging encounters. There was no detectable effect on patient management (P =.60). In the intervention patients, HRQL improvement was associated with explicit use of HRQL data (P =.016), discussion of pain, and role function (P =.046).
Routine assessment of cancer patients' HRQL had an impact on physician-patient communication and resulted in benefits for some patients, who had better HRQL and emotional functioning.
Significant advances in our understanding of the biology of renal cell carcinoma (RCC) have been achieved in recent years. These insights have led to the introduction of novel targeted therapies, ...revolutionising the management of patients with advanced disease. Nevertheless, there are still no biomarkers in routine clinical use in RCC. Tools used routinely to determine prognosis have not changed over the past decade; classification remains largely morphology based; and patients continue to be exposed to potentially toxic therapy with no indication of the likelihood of response. Thus the need for biomarkers in RCC is urgent. Here, we focus on recent advances in our understanding of the genetics and epigenetics of RCC, and the potential for such knowledge to provide novel markers and therapeutic targets. We highlight on-going research that is likely to deliver further candidate markers as well as generating large, well-annotated sample banks that will facilitate future studies. It is imperative that promising candidates are validated using these resources, and in subsequent prospective clinical trials, so that future biomarkers may be used in the clinic to personalize patient care.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome ...sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T>T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.
Regularly collecting patient-reported outcomes (PROs) of health-related quality of life with feedback to oncologists may assist in eliciting and monitoring patients' problems during cancer treatment. ...This study examined how PRO feedback had an impact on patient-physician communication over time to gain a better understanding of how it may influence patient care.
Exploratory analyses were performed on a data set from a previous study. Patients were randomly assigned to intervention (regular completion of European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 and Hospital Anxiety and Depression Scale with feedback to oncologists), attention-control (completion of same questionnaires without feedback), and control (standard care) arms. The content of consultation audio recordings between 28 oncologists and 198 patients over four consecutive visits (792 consultations) was analyzed. Mixed-effects models and multivariate regressions were used to examine the longitudinal impact of the intervention on patient-physician communication, dynamics of patient-physician interaction, and the association between PROs and the content of clinic discussion.
Patients in the intervention arm discussed more symptoms over time compared with patients in the attention-control (P = .008) and control (P = .04) arms. No study arm effect was observed for function discussions. Discussion topics were predominantly raised by patients/relatives, regardless of arm allocation. Clinic discussions were associated with severity of patient-reported symptoms but not with patient-reported functional concerns.
A positive longitudinal impact of the intervention on symptom discussion was observed, but not for function discussion, suggesting that potentially serious problems may remain unaddressed. Training oncologists in responding to patient-reported functional concerns may increase the impact of this intervention.
ObjectivesTo describe the frequency and nature of symptoms in patients presenting with suspected renal cell carcinoma (RCC) and examine their reliability in achieving early ...diagnosis.DesignMulticentre prospective observational cohort study.Setting and participantsEleven UK centres recruiting patients presenting with suspected newly diagnosed RCC. Symptoms reported by patients were recorded and reviewed. Comprehensive clinico-pathological and outcome data were also collected.OutcomesType and frequency of reported symptoms, incidental diagnosis rate, metastasis-free survival and cancer-specific survival.ResultsOf 706 patients recruited between 2011 and 2014, 608 patients with a confirmed RCC formed the primary study population. The majority (60%) of patients were diagnosed incidentally. 87% of patients with stage Ia and 36% with stage III or IV disease presented incidentally. Visible haematuria was reported in 23% of patients and was commonly associated with advanced disease (49% had stage III or IV disease). Symptomatic presentation was associated with poorer outcomes, likely reflecting the presence of higher stage disease. Symptom patterns among the 54 patients subsequently found to have a benign renal mass were similar to those with a confirmed RCC.ConclusionsRaising public awareness of RCC-related symptoms as a strategy to improve early detection rates is limited by the fact that related symptoms are relatively uncommon and often associated with advanced disease. Greater attention must be paid to the feasibility of screening strategies and the identification of circulating diagnostic biomarkers.
Abstract Objectives This study sought to evaluate if diastolic pulmonary gradient (DPG) can predict survival in patients with pulmonary hypertension due to left heart disease (PH-LHD). Background ...Patients with combined post- and pre-capillary PH-LHD have worse prognosis than those with passive pulmonary hypertension. The transpulmonary gradient (TPG) and pulmonary vascular resistance (PVR) have commonly been used to identify high-risk patients. However, these parameters have significant shortcomings and do not always correlate with pulmonary vasculature remodeling. Recently, it has been suggested that DPG may be better a marker, yet its prognostic ability in patients with cardiomyopathy has not been fully assessed. Methods A retrospective cohort of 1,236 patients evaluated for unexplained cardiomyopathy at Johns Hopkins Hospital was studied. All patients underwent right heart catheterization and were followed until death, cardiac transplantation, or the end of the study period (mean time 4.4 years). The relationships between DPG, TPG, or PVR and survival in subjects with PH-LHD (n = 469) were evaluated with Cox proportional hazards regression and Kaplan-Meier analyses. Results DPG was not significantly associated with mortality (hazard ratio HR: 1.02, p = 0.10) in PH-LHD whereas elevated TPG and PVR predicted death (HR: 1.02, p = 0.046; and HR: 1.11, p = 0.002, respectively). Similarly, DPG did not differentiate survivors from non-survivors at any selected cut points including a DPG of 7 mm Hg. Conclusions In this retrospective study of patients with cardiomyopathy and PH-LHD, an elevated DPG was not associated with worse survival.
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