Richard J.E. Skipworth

Using transcriptomics to identify and validate novel biomarkers of human skeletal muscle cancer cachexia

BackgroundCancer cachexia is a multi-organ tissue wasting syndrome that contributes to morbidity and mortality in many cancer patients. Skeletal muscle loss represents an established key feature yet there is no molecular understanding of the disease process. In fact, the postulated molecular regulators of cancer cachexia originate largely from pre-clinical models and it is unclear how these translate to the clinical environment.MethodsRectus abdominis muscle biopsies were obtained from 65 upper gastrointestinal (UGI) cancer patients during open surgery and RNA profiling was performed on a subset of this cohort (n = 21) using the Affymetrix U133+2 platform. Quantitative analysis revealed a gene signature, which underwent technical validation and independent confirmation in a separate clinical cohort.ResultsQuantitative significance analysis of microarrays produced an 83-gene signature that was able to identify patients with greater than 5% weight loss, while this molecular profile was unrelated to markers of systemic inflammation. Selected genes correlating with weight loss were validated using quantitative real-time PCR and independently studied as general cachexia biomarkers in diaphragm and vastus lateralis from a second cohort (n = 13; UGI cancer patients). CaMKIIβ correlated positively with weight loss in all muscle groups and CaMKII protein levels were elevated in rectus abdominis. TIE1 was also positively associated with weight loss in both rectus abdominis and vastus lateralis muscle groups while other biomarkers demonstrated tissue-specific expression patterns. Candidates selected from the pre-clinical literature, including FOXO protein and ubiquitin E3 ligases, were not related to weight loss in this human clinical study. Furthermore, promoter analysis identified that the 83 weight loss-associated genes had fewer FOXO binding sites than expected by chance.ConclusionWe were able to discover and validate new molecular biomarkers of human cancer cachexia. The exercise activated genes CaMKIIβ and TIE1 related positively to weight-loss across muscle groups, indicating that this cachexia signature is not simply due to patient inactivity. Indeed, excessive CaMKIIβ activation is a potential mechanism for reduced muscle protein synthesis. Our genomics analysis also supports the view that the available preclinical models do not accurately reflect the molecular characteristics of human muscle from cancer cachexia patients.

Physical Activity Monitoring: A Responsive and Meaningful Patient-Centered Outcome for Surgery, Chemotherapy, or Radiotherapy?

CONTEXT In surgical and clinical oncology, there is a growing need for patient-centered outcomes that are responsive, meaningful, and fit for purpose. OBJECTIVES The aim of this study was to validate physical activity (PA) monitoring as a responsive outcome measure at different stages of disease and treatment, by verifying correlations between PA, performance score, and quality of life (QoL). METHODS Daily life PA of 162 cancer patients, monitored by a device that records time sitting/lying, time standing, time walking, number of steps taken, and walking cadence, was compared with 20 healthy volunteers. In a subgroup of patients, functional status and QoL were assessed using the World Health Organization/Eastern Cooperative Oncology Group and the Karnofsky Performance Status scores and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-C30 (EORTC QLQ-C30) questionnaire. RESULTS The PA of patients with resectable gastrointestinal cancer did not differ significantly from controls. In contrast, patients with advanced cancer took 45% fewer steps and spent an extra 2.8 hours/day lying/sitting (P=0.001). Patients undergoing neoadjuvant chemotherapy and surgery (5-6 weeks after operation) experienced a similar reduction in PA. There were significant correlations between PA and the physical and role domains as well as fatigue subscale of the EORTC QLQ-C30 scale. CONCLUSION Objective PA scores correlate significantly with disease stage, functional status, and QoL of patients with cancer. Therefore, activity monitoring can make meaningful objective estimates of patient function in response to cancer and its treatment and may provide surrogate outcomes of QoL.

Cachexia, survival and the acute phase response

Purpose of reviewTo review current knowledge of the relationship between cytokines, the acute phase response (APR) and the development of cachexia. Recent findingsCytokines important in the initiation of the APR are also important in the genesis of cachexia. The presence of an APR or high circulating levels of pro-inflammatory cytokines are known to be related to adverse outcome in cancer patients. Studies of host cytokine genotype have demonstrated that specific host cytokine polymorphisms are associated with both the development of cachexia and reduced patient survival. The desire to be able to predict accurately survival in cancer patients has led to the description of various APR-based prognostic scoring systems. SummaryCachexia is an important clinical problem affecting up to two thirds of cancer patients. It results from anorexia and metabolic change and leads to loss of both adipose tissue and lean body mass, particularly in the skeletal muscle compartment. An APR is seen in half of cancer patients at presentation, and is most often determined clinically by raised plasma C-reactive protein concentrations. Adverse outcome and shortened survival have been linked to the presence of an APR. This article explores the cause and consequences of the APR in cancer cachexia.

Is There a Human Homologue to the Murine Proteolysis-Inducing Factor?

Purpose: A tumor-derived proteolysis-inducing factor (PIF) is suggested to be a potent catabolic factor in skeletal muscle of mice and humans. We aimed to establish the clinical significance of PIF in cancer patients and to elucidate its structural features. Experimental Design: PIF was detected in human urine using a monoclonal antibody (mAb) and related to clinical outcomes. PIF immunoaffinity-purified using the mAb was purified/separated using reverse-phase high-performance liquid chromatography and two-dimensional electrophoresis. Ten human cancer cell lines were tested for expression of mRNA encoding PIF core peptide. Results: PIF immunoreactivity was present in 160 of 262 patients with advanced cancers of the lung, esophagus/stomach, and other organs. In a Kaplan-Meier survival analysis of 181 lung cancer patients, PIF was unrelated to survival; PIF status was also unrelated to skeletal muscle loss confirmed by computed tomography imaging. PIF was seen in 16 of 24 patients with chronic heart failure and thus is not exclusive to malignant disease. In-gel digestion and mass spectrometric analysis of immunoaffinity purified PIF from cancer patients consistently identified human albumin and immunoglobulins. We showed nonspecific binding of purified albumin and immunoglobulins to the anti-PIF mAb, which is thus not a useful tool for PIF detection or purification in humans. Finally, the human PIF core peptide was detected in human cancer cell lines using reverse transcription-PCR and nucleotide sequencing; however, none of the amplified products had a site for the glycosylation critical to the proteolysis-inducing activity of murine PIF. Conclusions: A putative human homologue of murine PIF and its role in human cancer cachexia cannot be verified.

Suppression of Skeletal Muscle Turnover in Cancer Cachexia: Evidence from the Transcriptome in Sequential Human Muscle Biopsies

Purpose: The mechanisms underlying muscle wasting in patients with cancer remain poorly understood, and consequently there remains an unmet clinical need for new biomarkers and treatment strategies. Experimental Design: Microarrays were used to examine the transcriptome in single biopsies from healthy controls (n = 6) and in paired biopsies [pre-resection baseline (weight-loss 7%) and 8 month post-resection follow-up (disease-free/weight-stable for previous 2 months)] from quadriceps muscle of patients with upper gastrointestinal cancer (UGIC; n = 12). Results: Before surgery, 1,868 genes were regulated compared with follow-up (false discovery rate, 6%). Ontology analysis showed that regulated genes belonged to both anabolic and catabolic biologic processes with overwhelming downregulation in baseline samples. No literature-derived genes from preclinical cancer cachexia models showed higher expression in baseline muscle. Comparison with healthy control muscle (n = 6) revealed that despite differences in the transcriptome at baseline (941 genes regulated), the muscle of patients at follow-up was similar to control muscle (2 genes regulated). Physical activity (step count per day) did not differ between the baseline and follow-up periods (P = 0.9), indicating that gene expression differences reflected the removal of the cancer rather than altered physical activity levels. Comparative gene expression analysis using exercise training signatures supported this interpretation. Conclusions: Metabolic and protein turnover–related pathways are suppressed in weight-losing patients with UGIC whereas removal of the cancer appears to facilitate a return to a healthy state, independent of changes in the level of physical activity. Clin Cancer Res; 18(10); 2817–27. ©2012 AACR.

Sexual dimorphism modulates the impact of cancer cachexia on lower limb muscle mass and function

BACKGROUND & AIMS There is a sparsity of data on the impact of cachexia on human muscle function. This study examined the relationship between cachexia, quality of life and the mass/function/mechanical quality of lower limb skeletal muscle in gastrointestinal cancer patients. METHODS Quadriceps strength and lower limb power were measured in 54 patients with gastrointestinal cancer (n = 24 ≥ 10% weight-loss) and 18 healthy controls. Quadriceps cross-sectional area was measured in 33/54 patients and in all controls using MRI. Muscle mechanical quality was defined as quadriceps strength/unit quadriceps cross-sectional area. Quality of life was assessed using the EORTC QLQ-C30. Patients with weight-loss ≥ 10% were classified as cachectic. RESULTS In male cachectic patients, quadriceps strength (p = 0.003), lower limb power (p = 0.026), quadriceps cross-sectional area (p = 0.019) and muscle quality (p = 0.008) were reduced compared with controls. In female cachectic patients, quadriceps strength (p = 0.001) and muscle quality (p = 0.001) were reduced compared with controls. Physical function (p = 0.013) and fatigue (p = 0.004) quality of life scores were reduced in male cachectic compared with non-cachectic patients, but not in females. CONCLUSIONS Muscle quality is reduced in cancer patients. The degree of impairment of lower limb muscle mass, quality and function and the impact on quality of life varies with weight-loss and sex.

Increased expression of phosphorylated forms of RNA-dependent protein kinase and eukaryotic initiation factor 2α may signal skeletal muscle atrophy in weight-losing cancer patients

Previous studies suggest that the activation (autophosphorylation) of dsRNA-dependent protein kinase (PKR) can stimulate protein degradation, and depress protein synthesis in skeletal muscle through phosphorylation of the translation initiation factor 2 (eIF2) on the α-subunit. To understand whether these mediators are important in muscle wasting in cancer patients, levels of the phospho forms of PKR and eIF2α have been determined in rectus abdominus muscle of weight losing patients with oesophago-gastric cancer, in comparison with healthy controls. Levels of both phospho PKR and phospho eIF2α were significantly enhanced in muscle of cancer patients with weight loss irrespective of the amount and there was a linear relationship between phosphorylation of PKR and phosphorylation of eIF2α (correlation coefficient 0.76, P=0.005). This suggests that phosphorylation of PKR led to phosphorylation of eIF2α. Myosin levels decreased as the weight loss increased, and there was a linear relationship between myosin expression and the extent of phosphorylation of eIF2α (correlation coefficient 0.77, P=0.004). These results suggest that phosphorylation of PKR may be an important initiator of muscle wasting in cancer patients.

Interaction of gonadal status with systemic inflammation and opioid use in determining nutritional status and prognosis in advanced pancreatic cancer

PurposeHypogonadism has been linked with systemic inflammation and opioid use in males with advanced cancer. We aimed to investigate the interaction of gonadal status with systemic inflammation and opioids in determining nutritional status and prognosis in advanced pancreatic cancer.MethodsOne hundred and seventy-five patients (92 males, 83 postmenopausal females) with unresectable pancreatic cancer were studied. Serum sex steroids (total testosterone [TT], calculated free testosterone [cFT], oestradiol, sex hormone binding globulin), gonadotropins (follicle-stimulating hormone and luteinising hormone) and pro-inflammatory mediators (C-reactive protein [CRP], interleukin-6 [IL-6], soluble tumour necrosis factor receptor 75 [sTNFR75]) were measured, and nutritional assessment and opioid use recorded.ResultsSeventy-three percent of males were hypogonadal (by cFT definition). cFT correlated positively with BMI (r2 = 0.349; p < 0.001) and grip strength (r2 = 0.229; p = 0.034) and inversely with weight loss (r2 = −0.287; p = 0.007), CRP (r2 = −0.426; p < 0.001) and IL-6 (r2 = −0.303; p = 0.004). CRP (p = 0.007), opioid dosage (p = 0.009) and BMI (p = 0.005) were independent determinants of cFT on ANOVA. Hypogonadal males demonstrated worsened survival compared with eugonadal patients (TT: OR of death = 2.87; p < 0.001; cFT: OR = 2.26; p = 0.011). Furthermore, male opioid use was associated with decreased TT (p < 0.001) and cFT (p < 0.001) and worsened survival (OR = 1.96; p = 0.012). In contrast, 18% of postmenopausal females exhibited premenopausal (“hyperoestrogenic”) oestradiol levels. Oestradiol correlated positively with sTNFR75 (r2 = 0.299; p = 0.008). CRP (p < 0.001) was an independent determinant of oestradiol. Hyperoestrogenic females demonstrated worsened survival compared with eugonadal patients (OR = 2.43; p = 0.013).ConclusionsIn males with pancreatic cancer, systemic inflammation and opioid use are associated with hypogonadism. Male hypogonadism and female hyperoestrogenism are associated with shortened survival in advanced pancreatic cancer.

Plasma MIC-1 correlates with systemic inflammation but is not an independent determinant of nutritional status or survival in oesophago-gastric cancer

Background:Macrophage inhibitory cytokine-1(MIC-1) is a potential modulator of systemic inflammation and nutritional depletion, both of which are adverse prognostic factors in oesophago-gastric cancer (OGC).Methods:Plasma MIC-1, systemic inflammation (defined as plasma C-reactive protein (CRP) of ⩾10 mg l–1 or modified Glasgow prognostic score (mGPS) of ⩾1), and nutritional status were assessed in newly diagnosed OGC patients (n=293). Healthy volunteers (n=35) served as controls.Results:MIC-1 was elevated in patients (median=1371 pg ml–1; range 141–39 053) when compared with controls (median=377 pg ml–1; range 141–3786; P<0.001). Patients with gastric tumours (median=1592 pg ml–1; range 141–12 643) showed higher MIC-1 concentrations than patients with junctional (median=1337 pg ml–1; range 383–39 053) and oesophageal tumours (median=1180 pg ml–1; range 258–31 184; P=0.015). Patients showed a median weight loss of 6.4% (range 0.0–33.4%), and 42% of patients had an mGPS of ⩾1 or plasma CRP of ⩾10 mg l–1 (median=9 mg l–1; range 1–200). MIC-1 correlated positively with disease stage (r2=0.217; P<0.001), age (r2=0.332; P<0.001), CRP (r2=0.314; P<0.001), and mGPS (r2=0.336; P<0.001), and negatively with Karnofsky Performance Score (r2=−0.269; P<0.001). However, although MIC-1 correlated weakly with dietary intake (r2=0.157; P=0.031), it did not correlate with weight loss, BMI, or anthropometry. Patients with MIC-1 levels in the upper quartile showed reduced survival (median=204 days; 95% CI 157–251) when compared with patients with MIC-1 levels in the lower three quartiles (median=316 days; 95% CI 259–373; P=0.036), but MIC-1 was not an independent prognostic indicator.Conclusions:There is no independent link between plasma MIC-1 levels and depleted nutritional status or survival in OGC.

Evaluating potential biomarkers of cachexia and survival in skeletal muscle of upper gastrointestinal cancer patients

In order to grow the potential therapeutic armamentarium in the cachexia domain of supportive oncology, there is a pressing need to develop suitable biomarkers and potential drug targets. This pilot study evaluated several potential candidate biomarkers in skeletal muscle biopsies from a cohort of upper gastrointestinal cancer (UGIC) patients.