Diagnosis and Prediction of Neuroendocrine Liver Metastases: A Protocol of Six Systematic Reviews

Background Patients with hepatic metastases from neuroendocrine tumors (NETs) benefit from an early diagnosis, which is crucial for the optimal therapy and management. Diagnostic procedures include morphological and functional imaging, identification of biomarkers, and biopsy. Objective The aim of six systematic reviews discussed in this study is to assess the predictive value of Ki67 index and other biomarkers, to compare the diagnostic accuracy of morphological and functional imaging, and to define the role of biopsy in the diagnosis and prediction of neuroendocrine tumor liver metastases. Methods An objective group of librarians will provide an electronic search strategy to examine the following databases: MEDLINE, EMBASE and The Cochrane Library (Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials (CENTRAL), Database of Abstracts of Reviews of Effects). There will be no restriction concerning language and publication date. The qualitative and quantitative synthesis of the systematic review will be conducted with randomized controlled trials (RCT), prospective and retrospective comparative cohort studies, and case-control studies. Case series will be collected in a separate database and only used for descriptive purposes. Results This study is ongoing and presents a protocol of six systematic reviews to elucidate the role of histopathological and biochemical markers, biopsies of the primary tumor and the metastases as well as morphological and functional imaging modalities for the diagnosis and prediction of neuroendocrine liver metastases. Conclusions These systematic reviews will assess the value and accuracy of several diagnostic modalities in patients with NET liver metastases, and will provide a basis for the development of clinical practice guidelines. Trial Registration The systematic reviews have been prospectively registered with the International Prospective Register of Systematic Reviews (PROSPERO): CRD42012002644; http://www.metaxis.com/prospero/full_doc.asp?RecordID=2644 (Archived by WebCite at http://www.webcitation.org/6LzCLd5sF), CRD42012002647; http://www.metaxis.com/prospero/full_doc.asp?RecordID=2647 (Archived by WebCite at http://www.webcitation.org/6LzCRnZnO), CRD42012002648; http://www.metaxis.com/prospero/full_doc.asp?RecordID=2648 (Archived by WebCite at http://www.webcitation.org/6LzCVeuVR), CRD42012002649; http://www.metaxis.com/prospero/full_doc.asp?RecordID=2649 (Archived by WebCite at http://www.webcitation.org/6LzCZzZWU), CRD42012002650; http://www.metaxis.com/prospero/full_doc.asp?RecordID=2650 (Archived by WebCite at http://www.webcitation.org/6LzDPhGb8), CRD42012002651; http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42012002651#.UrMglPRDuVo (Archived by WebCite at http://www.webcitation.org/6LzClCNff).

INTRODUCTION 2a-i) Problem and the type of system/solution Hormones secreted from NETs can be used as specific markers for NETs. Moreover, NETs express, store, and secrete characteristic neuronal proteins such as acid glycoprotein chromogranin A (a component of the membrane neurosecretory granula), neuron-specific-enolase (NSE), and synaptophysin [3,9]. These proteins derived from neuronal structures could serve as markers and are even positive in non-functioning NETs [1,3]. Since more than one half of NETs are non-secreting, these proteins play a crucial role [4]. Assessment of the different biochemical markers depends on various parameters, such as threshold cut-off level, detecting method of urine, serum or plasma as well as location of the primary tumour or metastases and extension of the disease. In consideration of the great number of evaluation parameters the comparison of studies is difficult [10,11].
Histopathological prognostic markers Ki67 is a monoclonal antibody, which was introduced in 1984 by Gerdes et al. [11]. It detects a growth rate depending on the nuclear antigen Ki67 which is only expressed during active cell cycle phases (S, G2 and M-phase). Ki67 is completely absent during the resting phase G0. Therefore, cell proliferation is assessed by the immunohistologic presence of Ki67 positive cells per area in stained tissue blocks [11]. For various human neoplasms such as breast, lung, and solid cancers, Ki67 proliferation index has been successfully established as a predictive marker [12,13]. The higher the cell proliferation, the greater is the probability for metastases with consecutively decreased patient survival. The primary location of NETs metastases is the liver [14,15,16,17]. The occurrence of hepatic metastases is a prognostic factor which strongly influences the survival of patients suffering from NET [18,19,20].
Genetic signatures and the presence of circulating tumour cells To stratify outcomes in patients undergoing resection of primary NET, a simple scoring system using tumour size, histological grade, nodal metastases, and resection margin status has been introduced [21]. Nevertheless, current classification systems for NETs other than PET fail to predict the clinical course and the response to treatment [22]. The discrepancy might be explained either by an insufficient accuracy of these classification systems or an adaptive NET behaviour [23]. These limitations of the pathologic classifications have led to the investigation of other predictive parameters based on genetic signatures as well as the presence of circulating tumour cells [24,25]. These novel predictive parameters have to be included in the classification systems in order to take in count the biological behaviour, the likelihood for developing metastases as well as the choice of treatment [25].
Imaging methods Imaging methods are used to diagnose neuroendocrine tumours (NETs) and their metastases [26]. Beside conventional morphologic imaging methods, functional imaging modalities have been introduced in order to improve accuracy in detecting NETs and liver metastases [27]. Functional imaging methods have their limitations with a great impact on a possible therapeutic strategy, where differentiation between pancreatic foci and neighbouring lymph nodes as well as exact demarcation of a suspicious focus to a liver segment is crucial [28]. Advanced techniques such as contrast-enhanced ultrasound may assist in earlier detection of hepatic metastases, and could therefore offer a wider therapeutic range either surgically, with radiofrequency thermal ablation or with systemic chemotherapy [29].

Liver biopsy
The most common site of neuroendocrine tumour (NET) metastases is the liver [30]. The presence of hepatic metastases is a strong prognostic factor for the survival of patients with NETs, regardless of the primary tumour site [31]. Histologic examination is the most sensitive diagnostic method and forms the basis for treatment decisions [32]. However, the value of the biopsy for treatment decision making involving primary NETs and their liver metastases is not well defined [33,34].
The aim of these six systematic reviews is to assess the predictive value of Ki67 index and other biomarkers, to compare the diagnostic accuracy of morphological and functional imaging, and to define the role of biopsy in the diagnosis and prediction of neuroendocrine tumour liver metastases. 2a-ii) Scientific background, rationale: What is known about the (type of) system Neuroendocrine tumours Neuroendocrine tumours (NETs) arise from the diffuse neuroendocrine system and therefore appear widespread over the whole body, especially in the gastrointestinal tract and the bronchopulmonary system [1,2]. NETs secreting hormones lead to a symptomatic disease. Non-secreting NETs may occur initially asymptomatic or with delayed symptoms due to progressive increase in tumour mass [3,4]. Therefore, differences in functional behaviour are the basis of a classification system categorising functioning and non-functioning NETs [4]. Other reported classification systems are based on embryological origin or histopathological findings. The World Health Organization (WHO) presented in 2010 a new classification on the basis of tumour grading using histopathological criteria such as Ki 67 index, mitotic count, and presence or absence of necrosis [5]. NETs are a relative rare disease with an incidence 1-3/100'000 [6,7]. The large range of reported incidence might be due to the fact that NETs often present initially asymptomatic and are often found accidentally or in autopsies [4]. Preponderantly, NETs emerge sporadically (>90%) and are traditionally assigned to multiple endocrine neoplasia type 1 (MEN1), neurofibromatosis-type 1 (NF1), and Von-Hippel-Lindau syndrome [1,4]. The clinical picture of NETs span over the different effects of excessive hormone secretion such as hypergastrinemia in Zollinger-Ellison Syndrome (ZES) with hyperchlorhydria, hyperinsulinemia in insulinoma, flushing and diarrhoea in the serotoninergic carcinoid syndrome. In the case of non-secreting NETs, symptoms present due to the adverse effects of the growing primary tumour or metastases [8].
Biochemical markers Hormones secreted from NETs can be used as specific markers for NETs. Moreover, NETs express, store, and secrete characteristic neuronal proteins such as acid glycoprotein chromogranin A (a component of the membrane neurosecretory granula), neuron-specific-enolase (NSE), and synaptophysin [3,9]. These proteins derived from neuronal structures could serve as markers and are even positive in non-functioning NETs [1,3]. Since more than one half of NETs are non-secreting, these proteins play a crucial role [4]. Assessment of the different biochemical markers depends on various parameters, such as threshold cut-off level, detecting method of urine, serum or plasma as well as location of the primary tumour or metastases and extension of the disease. In consideration of the great number of evaluation parameters the comparison of studies is difficult [10,11].
Histopathological prognostic markers Ki67 is a monoclonal antibody, which was introduced in 1984 by Gerdes et al. [11]. It detects a growth rate depending on the nuclear antigen Ki67 which is only expressed during active cell cycle phases (S, G2 and M-phase). Ki67 is completely absent during the resting phase G0. Therefore, cell proliferation is assessed by the immunohistologic presence of Ki67 positive cells per area in stained tissue blocks [11]. For various human neoplasms such as breast, lung, and solid cancers, Ki67 proliferation index has been successfully established as a predictive marker [12,13]. The higher the cell proliferation, the greater is the probability for metastases with consecutively decreased patient survival. The primary location of NETs metastases is the liver [14,15,16,17]. The occurrence of hepatic metastases is a prognostic factor which strongly influences the survival of patients suffering from NET [18,19,20].
Genetic signatures and the presence of circulating tumour cells To stratify outcomes in patients undergoing resection of primary NET, a simple scoring system using tumour size, histological grade, nodal metastases, and resection margin status has been introduced [21]. Nevertheless, current classification systems for NETs other than PET fail to predict the clinical course and the response to treatment [22]. The discrepancy might be explained either by an insufficient accuracy of these classification systems or an adaptive NET behaviour [23]. These limitations of the pathologic classifications have led to the investigation of other predictive parameters based on genetic signatures as well as the presence of circulating tumour cells [24,25]. These novel predictive parameters have to be included in the classification systems in order to take in count the biological behaviour, the likelihood for developing metastases as well as the choice of treatment [25].
Imaging methods Imaging methods are used to diagnose neuroendocrine tumours (NETs) and their metastases [26]. Beside conventional morphologic imaging methods, functional imaging modalities have been introduced in order to improve accuracy in detecting NETs and liver metastases [27]. Functional imaging methods have their limitations with a great impact on a possible therapeutic strategy, where differentiation between pancreatic foci and neighbouring lymph nodes as well as exact demarcation of a suspicious focus to a liver segment is crucial [28]. Advanced techniques such as contrast-enhanced ultrasound may assist in earlier detection of hepatic metastases, and could therefore offer a wider therapeutic range either surgically, with radiofrequency thermal ablation or with systemic chemotherapy [29].

Liver biopsy
The most common site of neuroendocrine tumour (NET) metastases is the liver [30]. The presence of hepatic metastases is a strong prognostic factor for the survival of patients with NETs, regardless of the primary tumour site [31]. Histologic examination is the most sensitive diagnostic method and forms the basis for treatment decisions [32]. However, the value of the biopsy for treatment decision making involving primary NETs and their liver metastases is not well defined [33,34].
The aim of these six systematic reviews is to assess the predictive value of Ki67 index and other biomarkers, to compare the diagnostic accuracy of morphological and functional imaging, and to define the role of biopsy in the diagnosis and prediction of neuroendocrine tumour liver metastases. METHODS 3a) CONSORT: Description of trial design (such as parallel, factorial) including allocation ratio The manuscript submitted is a protocol, therefore not applicable. 3b) CONSORT: Important changes to methods after trial commencement (such as eligibility criteria), with reasons The manuscript submitted is a protocol, therefore not applicable. 3b-i) Bug fixes, Downtimes, Content Changes The manuscript submitted is a protocol, therefore not applicable. 4a) CONSORT: Eligibility criteria for participants The manuscript submitted is a protocol, therefore not applicable.There were no restrictions in the literature search regarding the publication language or by publication date. The following study types were included: Randomised Controlled Trials (RCTs), prospective and retrospective comparative cohort and case-control studies and case series 4a-i) Computer / Internet literacy The manuscript submitted is a protocol, therefore not applicable. 4a-ii) Open vs. closed, web-based vs. face-to-face assessments: The manuscript submitted is a protocol, therefore not applicable. 4a-iii) Information giving during recruitment The manuscript submitted is a protocol, therefore not applicable. 4b) CONSORT: Settings and locations where the data were collected The manuscript submitted is a protocol, therefore not applicable. 4b-i) Report if outcomes were (self-)assessed through online questionnaires The manuscript submitted is a protocol, therefore not applicable. 4b-ii) Report how institutional affiliations are displayed The manuscript submitted is a protocol, therefore not applicable. 5) CONSORT: Describe the interventions for each group with sufficient details to allow replication, including how and when they were actually administered 5-i) Mention names, credential, affiliations of the developers, sponsors, and owners The manuscript submitted is a protocol, therefore not applicable. 5-ii) Describe the history/development process The manuscript submitted is a protocol, therefore not applicable.

5-iii) Revisions and updating
The manuscript submitted is a protocol, therefore not applicable. 5-iv) Quality assurance methods The manuscript submitted is a protocol, therefore not applicable. 5-v) Ensure replicability by publishing the source code, and/or providing screenshots/screen-capture video, and/or providing flowcharts of the algorithms used The manuscript submitted is a protocol, therefore not applicable.

5-vi) Digital preservation
The manuscript submitted is a protocol, therefore not applicable.

5-vii) Access
The manuscript submitted is a protocol, therefore not applicable. 5-viii) Mode of delivery, features/functionalities/components of the intervention and comparator, and the theoretical framework The manuscript submitted is a protocol, therefore not applicable.

5-ix) Describe use parameters
The manuscript submitted is a protocol, therefore not applicable.

5-x) Clarify the level of human involvement
The manuscript submitted is a protocol, therefore not applicable. 5-xi) Report any prompts/reminders used The manuscript submitted is a protocol, therefore not applicable. 5-xii) Describe any co-interventions (incl. training/support) The manuscript submitted is a protocol, therefore not applicable. 6a) CONSORT: Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed The manuscript submitted is a protocol, therefore not applicable. 6a-i) Online questionnaires: describe if they were validated for online use and apply CHERRIES items to describe how the questionnaires were designed/deployed The manuscript submitted is a protocol, therefore not applicable. 6a-ii) Describe whether and how "use" (including intensity of use/dosage) was defined/measured/monitored The manuscript submitted is a protocol, therefore not applicable. 6a-iii) Describe whether, how, and when qualitative feedback from participants was obtained The manuscript submitted is a protocol, therefore not applicable. 6b) CONSORT: Any changes to trial outcomes after the trial commenced, with reasons The manuscript submitted is a protocol, therefore not applicable. 7a) CONSORT: How sample size was determined 7a-i) Describe whether and how expected attrition was taken into account when calculating the sample size The manuscript submitted is a protocol, therefore not applicable. 7b) CONSORT: When applicable, explanation of any interim analyses and stopping guidelines The manuscript submitted is a protocol, therefore not applicable. 8a) CONSORT: Method used to generate the random allocation sequence The manuscript submitted is a protocol, therefore not applicable. 8b) CONSORT: Type of randomisation; details of any restriction (such as blocking and block size) The manuscript submitted is a protocol, therefore not applicable. 9) CONSORT: Mechanism used to implement the random allocation sequence (such as sequentially numbered containers), describing any steps taken to conceal the sequence until interventions were assigned The manuscript submitted is a protocol, therefore not applicable. 10) CONSORT: Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions The manuscript submitted is a protocol, therefore not applicable. 11a) CONSORT: Blinding -If done, who was blinded after assignment to interventions (for example, participants, care providers, those assessing outcomes) and how 11a-i) Specify who was blinded, and who wasn't The manuscript submitted is a protocol, therefore not applicable. 11a-ii) Discuss e.g., whether participants knew which intervention was the "intervention of interest" and which one was the "comparator" The manuscript submitted is a protocol, therefore not applicable. 11b) CONSORT: If relevant, description of the similarity of interventions The manuscript submitted is a protocol, therefore not applicable. 12a) CONSORT: Statistical methods used to compare groups for primary and secondary outcomes The manuscript submitted is a protocol, therefore not applicable. 12a-i) Imputation techniques to deal with attrition / missing values The manuscript submitted is a protocol, therefore not applicable. 12b) CONSORT: Methods for additional analyses, such as subgroup analyses and adjusted analyses The manuscript submitted is a protocol, therefore not applicable. RESULTS 13a) CONSORT: For each group, the numbers of participants who were randomly assigned, received intended treatment, and were analysed for the primary outcome The manuscript submitted is a protocol, therefore not applicable. 13b) CONSORT: For each group, losses and exclusions after randomisation, together with reasons The manuscript submitted is a protocol, therefore not applicable. 13b-i) Attrition diagram The manuscript submitted is a protocol, therefore not applicable. 14a) CONSORT: Dates defining the periods of recruitment and follow-up The manuscript submitted is a protocol, therefore not applicable. 14a-i) Indicate if critical "secular events" fell into the study period The manuscript submitted is a protocol, therefore not applicable. 14b) CONSORT: Why the trial ended or was stopped (early) The manuscript submitted is a protocol, therefore not applicable. 15) CONSORT: A table showing baseline demographic and clinical characteristics for each group The manuscript submitted is a protocol, therefore not applicable. 15-i) Report demographics associated with digital divide issues The manuscript submitted is a protocol, therefore not applicable. 16a) CONSORT: For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups 16-i) Report multiple "denominators" and provide definitions The manuscript submitted is a protocol, therefore not applicable. 16-ii) Primary analysis should be intent-to-treat The manuscript submitted is a protocol, therefore not applicable. 17a) CONSORT: For each primary and secondary outcome, results for each group, and the estimated effect size and its precision (such as 95% confidence interval) The manuscript submitted is a protocol, therefore not applicable. 17a-i) Presentation of process outcomes such as metrics of use and intensity of use The manuscript submitted is a protocol, therefore not applicable. 17b) CONSORT: For binary outcomes, presentation of both absolute and relative effect sizes is recommended The manuscript submitted is a protocol, therefore not applicable. 18) CONSORT: Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing pre-specified from exploratory The manuscript submitted is a protocol, therefore not applicable.

18-i) Subgroup analysis of comparing only users
The manuscript submitted is a protocol, therefore not applicable. 19) CONSORT: All important harms or unintended effects in each group The manuscript submitted is a protocol, therefore not applicable. 19-i) Include privacy breaches, technical problems The manuscript submitted is a protocol, therefore not applicable. 19-ii) Include qualitative feedback from participants or observations from staff/researchers The manuscript submitted is a protocol, therefore not applicable. DISCUSSION 20) CONSORT: Trial limitations, addressing sources of potential bias, imprecision, multiplicity of analyses 20-i) Typical limitations in ehealth trials The manuscript submitted is a protocol, therefore not applicable.There are several modalities for the diagnosis and prediction of neuroendocrine liver metastases, but there is a lack of consensual data on the subject. These six systematic reviews described in this protocol will elucidate the role and compare histopathological prognostic and biochemical markers, biopsies of the primary neuroendocrine tumour and NET liver metastases, morphological and functional imaging modalities. They will help to define clinical guidelines. 21) CONSORT: Generalisability (external validity, applicability) of the trial findings 21-i) Generalizability to other populations The manuscript submitted is a protocol, therefore not applicable. 21-ii) Discuss if there were elements in the RCT that would be different in a routine application setting The manuscript submitted is a protocol, therefore not applicable. 22) CONSORT: Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence 22-i) Restate study questions and summarize the answers suggested by the data, starting with primary outcomes and process outcomes (use) The manuscript submitted is a protocol, therefore not applicable. 22-ii) Highlight unanswered new questions, suggest future research The manuscript submitted is a protocol, therefore not applicable. Other information 23) CONSORT: Registration number and name of trial registry International Prospective Register of Systematic Reviews (PROSPERO): CRD42012002644, CRD42012002647, CRD42012002651, CRD42012002649, CRD42012002650, CRD42012002651. 24) CONSORT: Where the full trial protocol can be accessed, if available International Prospective Register of Systematic Reviews (PROSPERO): CRD42012002644, CRD42012002647, CRD42012002651, CRD42012002649, CRD42012002650, CRD42012002651. 25) CONSORT: Sources of funding and other support (such as supply of drugs), role of funders NO funding. X26-i) Comment on ethics committee approval The manuscript submitted is a protocol, therefore not applicable. x26-ii) Outline informed consent procedures