Patients
Patients with GC and GEJ cancer will be recruited from eight university medical centers in seven different countries in EU (Spain, Netherlands, and Portugal) and LATAM (Argentina, Mexico, Chile, and Paraguay) from 2019 to 2023. Advanced stages will include state III and IV according to the American Joint Committee on Cancer (AJCC) stage system, GEJ will include Siewert type I and II tumors [22]. The clinical sites were selected based on their interest, expertise and geographical location, as well as the recruitment potential for the purpose of this study. Patient recruitment for each study is performed by coordinating a multidisciplinary team in each center including gastroenterologists, pathologists, surgeons, and medical oncologists. All patients should go through the informed consent procedure approved by each Institutional Research Board to participate in each of the sub studies.
Institutions and partners
LEGACy is a multi-institutional research approach performed by a team of four LATAM and seven EU organizations. The study members and centers can be found in Fig. 1 and in Supplementary Table 1. To ensure homogeneity and reproducibility of data collection by all different project partners the recruitment of subjects and the collection and handling of patient material and data has been standardized via use of a laboratory handbook. This handbook provides standard operating procedures on all processes and procedures pertaining to subjects, samples and assays included in the LEGACy project. All researchers, doctors, nurses, and data managers receive training on correct questionnaire administration, clinical data collection and biopsy collection before they begin work in the project.
Study design
LEGACy part 1
LEGACy part 1 is a case control study focused on identifying geographic variations in known and potential risk factors including detailed food consumption habits, BMI, socioeconomic status, smoking and alcohol use and family history of cancer. Within the study, cases are defined as adults with histologically confirmation of stage I-IV gastric adenocarcinoma (including GEJ cancer) within 6 months prior to inclusion, while controls are patients without gastric cancer from the same geographic regions as the cases, undergoing gastroscopy to rule out malignant disease. After signing informed consent forms, participants will be called by a trained member of the local LEGACy team who will record responses to the 30 min questionnaire in the eCRF. Epidemiologic data will be collected through a questionnaire. This questionnaire was modified and adapted according to the populations epidemiological facts of these regions to a shorter version to make a feasible approach in a 30 min call, including the most representative variables to measure and compare risk factors associated in EU and LATAM populations. A list of the parameters is provided in supplement 2. The data on risk factors will be compared between the different geographic regions and correlated with corresponding clinical data including age, sex, histology, disease stage and outcomes. Each recruiting center will include 100 cases and 100 control patients for the questionnaire, resulting in a total recruitment of 800 cases and 800 controls. Patients of this study are allowed to participate in other LEGACy studies when meeting all inclusion criteria as shown in Fig. 2.
LEGACy part 2
LEGACy part 2 is a tumor tissue sampling study focusing on multi-omics characterization of advanced GC in EU and LATAM populations. Data analyses include immunohistochemistry (IPATIMUP and GENPAT), genomics (VHIO and PUC), the immunome (VUMC and INCAN) and microbiome (IPATIMUP and PUC) of which the details can be found in Fig. 1. The patient population targeted of this study are treatment-naive advanced (stage III-IV) GC or GEJ adenocarcinomas. Subjects with gastroscopy indicated for a benign disease and confirmed absent of GC will be invited to participate as study controls.
From each patient included as a case eight tumor biopsies will be collected in formalin during gastroscopy, and in case of surgical tumor removal the resection specimen can be used as substitute. In cases, additional biopsies will be collected from non-tumoral gastric mucosa areas within four cm (2 in formalin, 1 snap frozen) and more than four cm around the tumor (2 in formalin). For controls, biopsies will be collected from gastric mucosa tissue at the corpus (2), antrum (2), and incisura (1) in formalin, and one extra antrum biopsy will be snap frozen in liquid nitrogen. For microbiome analysis, one of the gastric mucosa biopsies from each patient will be snap frozen in liquid nitrogen. Blood samples will be collected in one serum and 2 EDTA tubes of 6-10 ml each at the time of gastroscopy to isolate plasma, serum and non-viable peripheral blood mononuclear cells from both cases as controls. The biopsies collected and fixed in formalin will be paraffin-embedded (FFPE). All sample collections will be done in a standardized matter following a uniform lab manual. All FFPE tissues will be centrally collected at one destinated center, subjected to pathological examination by an expert pathologist and then distributed for further analyses. QC on each sample before and after RNA and DNA extraction will be performed, according to the different protocol requirements by the expert centers who perform the multi-omic determinations as indicated in Fig. 1.
Our first aim is to identify potential molecular subgroups that can be attributed to existing clinically relevant classification models, such as the TCGA molecular subgroups. Patients will therefore be stratified into subgroups based on the multi-omic variables that have tumor-driving potential and are distinctive for each specific subgroup. These features will be correlated with their corresponding clinical, pathological, and epidemiological characteristics. As a multi-omics method is not economically or practically applicable in clinical setting, a final cost-effective set of the most clinically relevant biomarkers will be proposed for validation in clinical practice.
The first half of the patients will be considered as training cohort. After analyzing the results of this cohort, the most relevant molecular biomarkers will be converted into more cost-effective panels and validated in the second half of the patients: the validation cohort. Based on GC incidence and presentation of advanced GC at each participating center in this proposal for the duration of 3 to 4 years it was decided to include 50 cases and 50 controls of each center (total 400 cases and 400 controls).
LEGACy part 3
This study consists of an online module for recording the knowledge of the general population on GC risk factors and symptoms and to provide education, as certain populations are reported to have low knowledge on risk factors [23]. The strategy is organized as follows: a) The participants consent their participation through an online informed consent form. b) The participants complete an online questionnaire on their knowledge of gastric cancer risk factors and symptoms and this information is recorded for further analysis by the LEGACy group. c) After completing the questionnaire, the subjects receive an informational brochure and a short video containing essential information about GC. d) After that, the same online questionnaire is provided after a few months to record the short- and long-term impact of this educational approach. The questionnaire lasts 15 minutes and was prepared by the LEGACy consortium partners and validated by the European Cancer Patient Coalition organization. The questionnaire and video is available in English, Spanish, Portuguese and Dutch. Finally, throughout this study, knowledge and best practices will be shared though educational training programs to share members’ expertise and train all involved project members. The courses will cover different aspects of GC including epidemiology, pathology, diagnosis, current and future treatments. Moreover, open access links to the online training courses are disseminated through the LEGACy website and social media channels to reach anyone interested to learn more about gastric cancer, to make a continuous and durable impact in GC research and clinical management.
Sample size considerations
Since there are a large number of specific factors to be analyzed with multiple techniques, and this is mostly a descriptive study, formal sample size calculations for the study as a whole were not feasible. The number of patients to include in the studies was decided upon from the following considerations:
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(i)
LEGACy part 1, 2 and 3 study sample size was based on the participating clinical centers capacity of recruitment along the project.
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(ii)
To get a better idea what sample size would be needed to give relevant results, we made an example calculation based on a RNA experiment in the two main groups. For example, we are planning to identify differential gene expression between two groups. Prior data indicates that the minimum average read counts among the differential genes in the control group is 5, the maximum dispersion is 0.5, and the ratio of the geometric mean of normalization factors is 1. Suppose that the total number of genes for testing is 20,000 and the top 100 genes are differentially expressed. If the desired minimum fold change is 1.5, we will need to study 200 subjects in each group to be able to reject the null hypothesis that the population means of the two groups are equal with probability (power) 0.8 using exact test. The FDR associated with this test of this null hypothesis is 0.01.
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(iii)
Based on (i) and the calculation in (ii) it was decided to include 200 cases and 200 controls for the training cohort, and to add another 200 cases and 200 controls for the validation cohort in LEGACy part 2 to be sure the generated results would be usable.
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(iv)
LEGACy part 1 is not limited to GC stage of disease. Therefore, it was decided to include two times the amount of patients included in part 2.