Cross sectional imaging of truncal and quadriceps muscles relates to different functional outcomes in cancer.

INTRODUCTION: Following the consensus definition of cancer cachexia, more studies are using CT scan analysis of truncal muscles as a marker of muscle wasting. However, how CT-derived body composition relates to function, strength and power in patients with cancer is largely unknown. AIMS: We aimed to describe the relationship between CT truncal (L3) skeletal muscle index (SMI) and MRI quadriceps cross sectional area with lower limb strength, power and measures of complex function. METHODS: Patients undergoing assessment for potentially curative surgery for oesophagogastric or pancreatic cancer were recruited from the regional upper gastrointestinal (UGI) or hepatopancreaticobiliary (HPB) multi-disciplinary team meetings. Maximum Isometric Knee Extensor Strength (IKES) and Maximum Leg Extensor Power (Nottingham Power Rig) (LEP) were used as measures of lower limb performance. Both Sit to Stand (STS) and Timed Up and Go (TUG) were used as measures of global complex muscle function. Muscle SMI was measured from routine CT scans at the level of the third lumbar vertebrae (L3) and MRI scan was used for the assessment of quadriceps muscles. Linear regression analysis was performed for CT SMI or MRI quadriceps as a predictor of each measure of performance. RESULTS: Forty-four patients underwent assessment. Height and weight were significantly related to function in terms of quadriceps power, while only weight was associated with strength (P < 0.001). CT SMI was not related to measures of quadriceps strength or power but had significant association with more complex functional measures (P = 0.006, R2 = 0.234 and 0.0019, R2 = 0.175 for STS and TUG respectively). In comparison, both gross and fat-subtracted measures of quadriceps muscle mass from MRI were significantly correlated with quadriceps strength and power (P < 0.001), but did not show any significant association with complex functional measures. CONCLUSION: CT SMI and MRI quadriceps have been shown to reflect different aspects of functional ability with CT SMI being a marker of global muscle function and MRI quadriceps being specific to quadriceps power and strength. This should therefore be considered when choosing outcome measures for trials or definitions of muscle mass and function.

Prediction of 90 Day and Overall Survival after Chemoradiotherapy for Lung Cancer: Role of Performance Status and Body Composition.

AIMS: If appropriate patients are to be selected for lung cancer treatment, an understanding of who is most at risk of adverse outcomes after treatment is needed. The aim of the present study was to identify predictive factors for 30 and 90 day mortality after chemoradiotherapy (CRT), and factors that were prognostic for overall survival. MATERIALS AND METHODS: A retrospective cohort study of 194 patients with lung cancer who had undergone CRT in South East Scotland from 2008 to 2010 was undertaken. Gender, age, cancer characteristics, weight loss, body mass index (BMI), performance status (Eastern Cooperative Oncology Group; ECOG) and computed tomography-derived body composition variables were examined for prognostic significance using Cox's proportional hazards model and logistic regression. RESULTS: The median overall survival was 19 months (95% confidence interval 16.3, 21.7). Four of 194 patients died within 30 days of treatment completion, for which there were no independent predictive variables; 22/194 (11%) died within 90 days of treatment completion. BMI < 20 and ECOG performance status ≥2 were independent predictors of death within 90 days of treatment completion (P = 0.001 and P = 0.004, respectively). Patients with either BMI < 20 or ECOG performance status ≥ 2 had an odds ratio of death within 90 days of 5.97 (95% confidence interval 2.20, 16.19), rising to an odds ratio of 13.27 (1.70, 103.47) for patients with both BMI < 20 and ECOG performance status ≥ 2. Patients with low muscle attenuation had significantly reduced overall survival (P = 0.004); individuals with low muscle attenuation had a median survival of 15.2 months (95% confidence interval 12.7, 17.7) compared with 23.0 months (95% confidence interval 18.3, 27.8) for those with high muscle attenuation, equating to a hazard ratio of death of 1.62 (95% confidence interval 1.17, 2.23, P = 0.003). CONCLUSION: Poor performance status, low BMI and low muscle attenuation identify patients at increased risk of premature death after CRT. Risk factors for adverse outcomes should inform personalised discussions with patients about the potential harms as well as the intended benefits of treatment.

ROMANA 3: a phase 3 safety extension study of anamorelin in advanced non-small-cell lung cancer (NSCLC) patients with cachexia.

BACKGROUND: Cancer anorexia-cachexia is a debilitating condition frequently observed in NSCLC patients, characterized by decreased body weight, reduced food intake, and impaired quality of life. Anamorelin, a novel selective ghrelin receptor agonist, has anabolic and appetite-enhancing activities. PATIENTS AND METHODS: ROMANA 3 was a safety extension study of two phase 3, double-blind studies that assessed safety and efficacy of anamorelin in advanced NSCLC patients with cachexia. Patients with preserved Eastern Cooperative Oncology Group ≤2 after completing 12 weeks (w) on the ROMANA 1 or ROMANA 2 trials (0-12 weeks) could enroll in ROMANA 3 and continue to receive anamorelin 100 mg or placebo once daily for an additional 12w (12-24 weeks). The primary endpoint of ROMANA 3 was anamorelin safety/tolerability (12-24 weeks). Secondary endpoints included changes in body weight, handgrip strength (HGS), and symptom burden (0-24 weeks). RESULTS: Of the 703 patients who completed ROMANA 1 and ROMANA 2, 513 patients entered ROMANA 3 (anamorelin, N = 345, mean age 62.0 years; placebo, N = 168; mean age 62.2 years). During ROMANA 3, anamorelin and placebo groups had similar incidences of treatment-emergent adverse events (TEAEs; 52.2% versus 55.7%), grade ≥3 TEAEs (22.4% versus 21.6%), and serious TEAEs (12.8% versus 12.6%). There were 36 (10.5%) and 23 (13.8%) deaths in the anamorelin and placebo groups, respectively; none were drug-related. Improvements in body weight and anorexia-cachexia symptoms observed in the original trials were consistently maintained over 12-24 weeks. Anamorelin, versus placebo, significantly increased body weight from baseline of original trials at all time points (P < 0.0001) and improved anorexia-cachexia symptoms at weeks 3, 6, 9, 12, and 16 (P < 0.05). No significant improvement in HGS was seen in either group. CONCLUSION: During the 12-24 weeks ROMANA 3 trial, anamorelin continued to be well tolerated. Over the entire 0-24w treatment period, body weight and symptom burden were improved with anamorelin. CLINICAL TRIAL REGISTRATION NUMBERS: ROMANA 1 (NCT01387269), ROMANA 2 (NCT01387282), and ROMANA 3 (NCT01395914).

Health-Related Quality of Life, Cachexia and Overall Survival After Major Upper Abdominal Surgery: A Prospective Cohort Study.

BACKGROUND AND AIMS: Major upper abdominal surgery is often associated with reduced health-related quality of life and reduced survival. Patients with upper abdominal malignancies often suffer from cachexia, represented by preoperative weight loss and sarcopenia (low skeletal muscle mass) and this might affect both health-related quality of life and survival. We aimed to investigate how health-related quality of life is affected by cachexia and how health-related quality of life relates to long-term survival after major upper abdominal surgery. MATERIALS AND METHODS: From 2001 to 2006, 447 patients were included in a Norwegian multicenter randomized controlled trial in major upper abdominal surgery. In this study, six years later, these patients were analyzed as a single prospective cohort and survival data were retrieved from the National Population Registry. Cachexia was derived from patient-reported preoperative weight loss and sarcopenia as assessed from computed tomography images taken within three months preoperatively. In the original trial, self-reported health-related quality of life was assessed preoperatively at trial enrollment and eight weeks postoperatively with the health-related quality of life questionnaire Short Form 36. RESULTS: A majority of the patients experienced improved mental health-related quality of life and, to a lesser extent, deteriorated physical health-related quality of life following surgery. There was a significant association between preoperative weight loss and reduced physical health-related quality of life. No association between sarcopenia and health-related quality of life was observed. Overall survival was significantly associated with physical health-related quality of life both pre- and postoperatively, and with postoperative mental health-related quality of life. The association between health-related quality of life and survival was particularly strong for postoperative physical health-related quality of life. CONCLUSION: Postoperative physical health-related quality of life strongly correlates with overall survival after major upper abdominal surgery.

Randomized open-label phase II study comparing oxycodone-naloxone with oxycodone in early return of gastrointestinal function after laparoscopic colorectal surgery.

BACKGROUND: Combined oral modified-release oxycodone-naloxone may reduce opioid-induced postoperative gut dysfunction. This study examined the feasibility of a randomized trial of oxycodone-naloxone within the context of enhanced recovery for laparoscopic colorectal resection. METHODS: In a single-centre open-label phase II feasibility study, patients received analgesia based on either oxycodone-naloxone or oxycodone. Primary endpoints were recruitment, retention and protocol compliance. Secondary endpoints included a composite endpoint of gut function (tolerance of solid food, low nausea/vomiting score, passage of flatus or faeces). RESULTS: Eighty-two patients were screened and 62 randomized (76 per cent); the attrition rate was 19 per cent (12 of 62), leaving 50 patients who received the allocated intervention with 100 per cent follow-up and retention (modified intention-to-treat cohort). Protocol compliance was more than 90 per cent. Return of gut function by day 3 was similar in the two groups: 13 (48 per cent) of 27 in the oxycodone-naloxone group and 15 (65 per cent) of 23 in the control group (95 per cent c.i. for difference -10·0 to 40·7 per cent; P = 0·264). However, patients in the oxycodone-naloxone group had a shorter time to first bowel movement (mean(s.d.) 87(38) h versus 111(37) h in the control group; 95 per cent c.i. for difference 2·3 to 45·4 h, P = 0·031) and reduced total (oral plus parenteral) opioid consumption (mean(s.d.) 78(36) versus 94(56) mg respectively; 95 per cent c.i. for difference -10·2 to 42·8 mg, P = 0·222). CONCLUSION: High participation, retention and protocol compliance confirmed feasibility. Potential benefits of oxycodone-naloxone in reducing time to bowel movement and total opioid consumption could be tested in a randomized trial. Registration number: NCT02109640 (https://www.clinicaltrials.gov/).

Influence of body composition profile on outcomes following colorectal cancer surgery.

BACKGROUND: Muscle depletion is characterized by reduced muscle mass (myopenia), and increased infiltration by intermuscular and intramuscular fat (myosteatosis). This study examined the role of particular body composition profiles as prognostic markers for patients with colorectal cancer undergoing curative resection. METHODS: Patients with colorectal cancer undergoing elective surgical resection between 2006 and 2011 were included. Lumbar skeletal muscle index (LSMI), visceral adipose tissue (VAT) surface area and mean muscle attenuation (MA) were calculated by analysis of CT images. Reduced LSMI (myopenia), increased VAT (visceral obesity) and low MA (myosteatosis) were identified using predefined sex-specific skeletal muscle index values. Univariable and multivariable Cox regression models were used to determine the role of different body composition profiles on outcomes. RESULTS: Some 805 patients were identified, with a median follow-up of 47 (i.q.r. 24·9-65·6) months. Multivariable analysis identified myopenia as an independent prognostic factor for disease-free survival (hazard ratio (HR) 1·53, 95 per cent c.i. 1·06 to 2·39; P = 0·041) and overall survival (HR 1·70, 1·25 to 2·31; P < 0·001). The presence of myosteatosis was associated with prolonged primary hospital stay (P = 0·034), and myopenic obesity was related to higher 30-day morbidity (P = 0·019) and mortality (P < 0·001) rates. CONCLUSION: Myopenia may have an independent prognostic effect on cancer survival for patients with colorectal cancer. Muscle depletion may represent a modifiable risk factor in patients with colorectal cancer and needs to be targeted as a relevant endpoint of health recommendations.

Enhanced Recovery After Surgery (ERAS) for gastrointestinal surgery, part 2: consensus statement for anaesthesia practice.

BACKGROUND: The present interdisciplinary consensus review proposes clinical considerations and recommendations for anaesthetic practice in patients undergoing gastrointestinal surgery with an Enhanced Recovery after Surgery (ERAS) programme. METHODS: Studies were selected with particular attention being paid to meta-analyses, randomized controlled trials and large prospective cohort studies. For each item of the perioperative treatment pathway, available English-language literature was examined and reviewed. The group reached a consensus recommendation after critical appraisal of the literature. RESULTS: This consensus statement demonstrates that anaesthesiologists control several preoperative, intraoperative and postoperative ERAS elements. Further research is needed to verify the strength of these recommendations. CONCLUSIONS: Based on the evidence available for each element of perioperative care pathways, the Enhanced Recovery After Surgery (ERAS®) Society presents a comprehensive consensus review, clinical considerations and recommendations for anaesthesia care in patients undergoing gastrointestinal surgery within an ERAS programme. This unified protocol facilitates involvement of anaesthesiologists in the implementation of the ERAS programmes and allows for comparison between centres and it eventually might facilitate the design of multi-institutional prospective and adequately powered randomized trials.

Enhanced Recovery After Surgery (ERAS) for gastrointestinal surgery, part 1: pathophysiological considerations.

BACKGROUND: The present article has been written to convey concepts of anaesthetic care within the context of an Enhanced Recovery After Surgery (ERAS) programme, thus aligning the practice of anaesthesia with the care delivered by the surgical team before, during and after surgery. METHODS: The physiological principles supporting the implementation of the ERAS programmes in patients undergoing major abdominal procedures are reviewed using an updated literature search and discussed by a multidisciplinary group composed of anaesthesiologists and surgeons with the aim to improve perioperative care. RESULTS: The pathophysiology of some key perioperative elements disturbing the homoeostatic mechanisms such as insulin resistance, ileus and pain is here discussed. CONCLUSIONS: Evidence-based strategies aimed at controlling the disruption of homoeostasis need to be evaluated in the context of ERAS programmes. Anaesthesiologists could, therefore, play a crucial role in facilitating the recovery process.

Risk factors, complications and survival after upper abdominal surgery: a prospective cohort study.

BACKGROUND: Preoperative weight loss and abnormal serum-albumin have traditionally been associated with reduced survival. More recently, a correlation between postoperative complications and reduced long-term survival has been reported and the significance of the relative proportion of skeletal muscle, visceral and subcutaneous adipose tissue has been examined with conflicting results. We investigated how preoperative body composition and major non-fatal complications related to overall survival and compared this to established predictors in a large cohort undergoing upper abdominal surgery. METHODS: From 2001 to 2006, 447 patients were included in a Norwegian multicenter randomized controlled trial in major upper abdominal surgery. Patients were now, six years later, analyzed as a single prospective cohort and overall survival was retrieved from the National Population Registry. Body composition indices were calculated from CT images taken within three months preoperatively. RESULTS: Preoperative serum-albumin <35 g/l (HR = 1.52, p = 0 .014) and weight loss >5 % (HR = 1.38, p = 0.023) were independently associated with reduced survival. There was no association between any of the preoperative body composition indices and reduced survival. Major postoperative complications were independently associated with reduced survival but only as long as patients who died within 90 days were included in the analysis. CONCLUSIONS: Our study has confirmed the robust significance of the traditional indicators, preoperative serum-albumin and weight loss. The body composition indices did not prove beneficial as global indicators of poor prognosis in upper abdominal surgery. We found no association between non-fatal postoperative complications and long-term survival.

The role of body composition evaluation by computerized tomography in determining colorectal cancer treatment outcomes: a systematic review.

BACKGROUND: Strong evidence indicates that excessive adipose tissue distribution or reduced muscle influence short-, mid-, and long-term colorectal cancer outcomes. Computerized tomography-based body composition (CTBC) analysis quantifies this in a reproducible parameter. We reviewed the evidence linking computerized tomography (CT) based quantification of BC with short and long-term outcomes in colorectal cancer (CRC). METHODS: A systematic review was performed according to PRISMA guidelines. A literature search was performed by two independent reviewers on all studies that included CTBC analysis in patients undergoing treatment for CRC using Medline, EMBASE, Google Scholar, and Cochrane databases. Outcomes of interest included short-term recovery, oncological outcomes, and survival. RESULTS: Seventy-five studies were identified; sixteen met the inclusion criteria. None were randomized controlled trials and all were cohort studies of small sample size. Several types of CTBC image analysis software were identified, reporting subcutaneous, visceral and skeletal muscle tissues. Visceral obesity and reduced muscle mass were categorical parameters quantified by CTBC analysis. Due to marked study heterogeneity, quantitative data synthesis was not possible. High visceral adipose tissue and reduced skeletal muscle resulted in poorer short-term recovery (eleven studies), poorer oncological outcomes (six studies), and poorer survival (six studies). CONCLUSIONS: CTBC techniques may be linked to outcomes in colorectal cancer patients, however larger studies are required. CTBC based assessment may allow early identification of high-risk patients, allowing early optimisation of patients undergoing cancer treatments.

Functional body composition and related aspects in research on obesity and cachexia: report on the 12th Stock Conference held on 6 and 7 September 2013 in Hamburg, Germany.

The 12th Stock Conference addressed body composition and related functions in two extreme situations, obesity and cancer cachexia. The concept of 'functional body composition' integrates body components into regulatory systems relating the mass of organs and tissues to corresponding in vivo functions and metabolic processes. This concept adds to an understanding of organ/tissue mass and function in the context of metabolic adaptations to weight change and disease. During weight gain and loss, there are associated changes in individual body components while the relationships between organ and tissue mass are fixed. Thus an understanding of body weight regulation involves an examination of the relationships between organs and tissues rather than individual organ and tissue masses only. The between organ/tissue mass relationships are associated with and explained by crosstalks between organs and tissues mediated by cytokines, hormones and metabolites that are coupled with changes in body weight, composition and function as observed in obesity and cancer cachexia. In addition to established roles in intermediary metabolism, cell function and inflammation, organ-tissue crosstalk mediators are determinants of body composition and its change with weight gain and loss. The 12th Stock Conference supported Michael Stocks' concept of gaining new insights by integrating research ideas from obesity and cancer cachexia. The conference presentations provide an in-depth understanding of body composition and metabolism.

Phase I/II trial of formoterol fumarate combined with megestrol acetate in cachectic patients with advanced malignancy.

PURPOSE: The aim of this study was to test the safety, tolerability and efficacy of a novel combination of an anabolic ß2-agonist and an appetite stimulant in patients with cancer cachexia. METHODS: Thirteen patients (M/F 5:8) with advanced malignancy and involuntary weight loss received oral formoterol (80 µg/day) and megestrol acetate (480 mg/day) for up to 8 weeks. Quadriceps size (MRI), quadriceps and hand-grip strength, lower limb extensor power, physical activity and quality of life were measured at baseline and at 8 weeks. Response criteria were specified pre-trial, with a major response defined as an increase in muscle size ≥ 4 % or function ≥ 10 %. RESULTS: Six patients withdrew before 8 weeks, reflecting the frail, comorbid population. In contrast, six out of seven (86 %) patients completing the course achieved a major response for muscle size and/or function. In the six responders, mean quadriceps volume increased significantly (left 0.99 vs. 1.05 L, p=0.012; right 1.02 vs. 1.06 L, p=0.004). There was a trend towards an increase in quadriceps and handgrip strength (p>0.05). The lack of appetite symptom score declined markedly (76.2 vs. 23.8; p=0.005), indicating improvement. Adverse reactions were few, the commonest being tremor (eight reports), peripheral oedema (three), tachycardia (two) and dyspepsia (two). CONCLUSIONS: In this frail cohort with advanced cancer cachexia, an 8-week course of megestrol and formoterol in combination was safe and well tolerated. Muscle mass and/or function were improved to a clinically significant extent in most patients completing the course. This combination regimen warrants further investigation in larger, randomized trials.

Genetic basis of interindividual susceptibility to cancer cachexia: selection of potential candidate gene polymorphisms for association studies.

Cancer cachexia is a complex and multifactorial disease. Evolving definitions highlight the fact that a diverse range of biological processes contribute to cancer cachexia. Part of the variation in who will and who will not develop cancer cachexia may be genetically determined. As new definitions, classifications and biological targets continue to evolve, there is a need for reappraisal of the literature for future candidate association studies. This review summarizes genes identified or implicated as well as putative candidate genes contributing to cachexia, identified through diverse technology platforms and model systems to further guide association studies. A systematic search covering 1986-2012 was performed for potential candidate genes / genetic polymorphisms relating to cancer cachexia. All candidate genes were reviewed for functional polymorphisms or clinically significant polymorphisms associated with cachexia using the OMIM and GeneRIF databases. Pathway analysis software was used to reveal possible network associations between genes. Functionality of SNPs/genes was explored based on published literature, algorithms for detecting putative deleterious SNPs and interrogating the database for expression of quantitative trait loci (eQTLs). A total of 154 genes associated with cancer cachexia were identified and explored for functional polymorphisms. Of these 154 genes, 119 had a combined total of 281 polymorphisms with functional and/or clinical significance in terms of cachexia associated with them. Of these, 80 polymorphisms (in 51 genes) were replicated in more than one study with 24 polymorphisms found to influence two or more hallmarks of cachexia (i.e., inflammation, loss of fat mass and/or lean mass and reduced survival). Selection of candidate genes and polymorphisms is a key element of multigene study design. The present study provides a contemporary basis to select genes and/or polymorphisms for further association studies in cancer cachexia, and to develop their potential as susceptibility biomarkers of cachexia.

A novel oral tracer procedure for measurement of habitual myofibrillar protein synthesis.

RATIONALE: Conventionally, myofibrillar protein synthesis is measured over time periods of hours. In clinical studies, interventions occur over weeks. Functional measures over such periods may be more representative. We aimed to develop a novel method to determine myofibrillar protein fractional synthetic rate (FSR) to estimate habitual rates, while avoiding intravenous tracer infusions. METHODS: Four healthy males were given 100 g water enriched to 70 Atom % with (2)H2O as a single oral bolus. Vastus-lateralis needle biopsies were performed and plasma samples collected, 3-13 days post-dose. (2)H enrichment in body water was measured in plasma using continuous flow isotope ratio mass spectrometry (IRMS). Myofibrillar protein was isolated from muscle biopsies and acid hydrolysed. (2)H enrichment of protein-bound and plasma-free alanine was measured by gas chromatography (GC)/pyrolysis/IRMS. Myofibrillar protein FSR was calculated (% day(-1)). RESULTS: The tracer bolus raised the initial enrichment of body water to 1514 ppm (2)H excess. Water elimination followed a simple exponential. The average elimination half-time was 8.3 days. Plasma alanine, labelled during de novo synthesis, followed the same elimination kinetics as water. The weighted average myofibrillar protein FSR from the four subjects was 1.38 % day(-1) (range, 1.0-1.9 % day(-1) ). CONCLUSIONS: Myofibrillar protein FSR was measured in free-living healthy individuals over 3-13 days. Using a single oral (2)H2O bolus, endogenous labelling of alanine occurred in a predictable manner giving estimates of synthesis comparable with published values. Furthermore, the protocol does not compromise the ability to measure other important metabolic processes such as total energy expenditure.

Muscle wasting in cancer.

Skeletal muscle loss appears to be the most significant clinical event in cancer cachexia and is associated with a poor outcome. With regard to such muscle loss, despite extensive study in a range of models, there is ongoing debate as to whether a reduction in protein synthesis, an increase in degradation or a combination of both is the more relevant. Each model differs in terms of key mediators and the pathways activated in skeletal muscle. Certain models do suggest that decreased synthesis accompanied by enhanced protein degradation via the ubiquitin proteasome pathway (UPP) is important. Murine models tend to involve rapid development of cachexia and may represent more acute muscle atrophy rather than the chronic wasting observed in humans. There is a paucity of human data both at a basic descriptive level and at a molecular/mechanism level. Progress in treating the human form of cancer cachexia can only move forwards through carefully designed large randomised controlled clinical trials of specific therapies with validated biomarkers of relevance to underlying mechanisms. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.

Patient optimization for gastrointestinal cancer surgery.

BACKGROUND: Although surgical resection remains the central element in curative treatment of gastrointestinal cancer, increasing emphasis and resource has been focused on neoadjuvant or adjuvant therapy. Developments in these modalities have improved outcomes, but far less attention has been paid to improving oncological outcomes through optimization of perioperative care. METHODS: A narrative review is presented based on available and updated literature in English and the authors' experience with enhanced recovery research. RESULTS: A range of perioperative factors (such as lifestyle, co-morbidity, anaemia, sarcopenia, medications, regional analgesia and minimal access surgery) are modifiable, and can be optimized to reduce short- and long-term morbidity and mortality, improve functional capacity and quality of life, and possibly improve oncological outcome. The effect on cancer-free and overall survival may be of equal magnitude to that achieved by many adjuvant oncological regimens. Modulation of core factors, such as nutritional status, systemic inflammation, and surgical and disease-mediated stress, probably influences the host's immune surveillance and defence status both directly and through reduced postoperative morbidity. CONCLUSION: A wider view on long-term effects of expanded or targeted enhanced recovery protocols is warranted.