Chilean Gastric Cancer Task Force (FORCE 1)
Condition: Biomarkers
MeSH terms: Stomach Neoplasms
Study type: Observational [Patient Registry]

Summary: 
      Background. Gastric cancer (GC) is the world's second leading cause of neoplastic mortality.
      Genetic alterations, response to treatments and mortality rates are highly heterogeneous
      across different regions. In Chile, GC is the leading cause of cancer death, affecting 20 per
      100,000 people and >3,000 deaths/year. Clinical outcomes and response to "one size fits all"
      therapies are highly heterogeneous and thus a better stratification of patients may aid
      cancer treatment and response.

      Study design/methods. The Gastric Cancer Task Force (GCTF) is a Chilean collaborative,
      non-interventional retrospective study that seeks to stratify gastric adenocarcinomas (GACs)
      using retrospect clinical outcomes and genomic, epigenomic and protein alterations in a
      cohort of 200 patients. Tumor samples from the pathology department and the Cancer Center at
      UC Christus healthcare network at Pontificia Universidad Católica de Chile will be analyzed
      using a panel of 143 known cancer genes (Oncomine Comprehensive Assay) at the Center of
      Excellence of Precision Medicine (CEMP) in Santiago, Chile. Additionally, gene promoter
      methylation will be performed and selected clinically relevant proteins (e.g. PD-L1, Erb-2,
      VEGFR2 among others) will be assessed by Tissue Microarray, Epstein-Barr virus (EBV) status
      will also be assessed. Observations will be correlated to 120 clinical parameters, including
      general patient information, cancer history, laboratory studies, comorbidity index,
      chemotherapy, targeted therapies, efficacy and follow-up.

      Discussion. The development of a clinically meaningful classification that encompasses
      comprehensive clinical and molecular parameters may improve patient treatment, predict
      clinical outcomes, aid patient selection for clinical trials and offer insights into future
      preventive and/or therapeutic strategies.
    

Detailed description: 
      Participating entities: The Chilean Gastric Cancer Task Force (GCTF) is a collective effort
      between two principal identities: 1- The Center of Excellence of Precision Medicine (CEMP),
      which was established through a joint funding by the government agency for economic
      development (Corporacion de Fomento de la Produccion, CORFO) and Pfizer Chile and 2- The
      Center UC for Investigation in Oncology (CITO) based at the Pontificia Universidad Católica
      de Chile. Both entities are non-profit research organizations aimed at enhancing public
      education and implementing strategies to improve clinical outcomes in oncology treatment and
      cancer prevention.

      Primary objective: To stratify Chilean GC patients into prognostic subgroups, and to
      correlate therapy response according to clinical, protein, epigenetic and genetic alterations
      in a cohort of 200 GAC patients.

      Secondary objectives

        -  To determine the mutation profile in Chilean GC patients.

        -  To assess the percentage of Chilean GC patients that could benefit from currently
           available druggable targets (actionable genes).

        -  To correlate EBV presence to clinical parameters.

        -  To assess the expression levels of proteins associated with molecular stratifications
           and currently targeted therapies (eg. PD-L1 and antiangiogenics)

        -  To determine the profile of SNPs in the DPYD and TYMS genes in Chilean GC patients and
           their correlation with adverse events.

      Study design

      Ethics approval The GCTF is a non-interventional, collaborative, prospective non-concurrent
      study that seeks to stratify GAC patients based on their prognosis and therapy response. The
      study will strictly adhere to all legal requirements, regulations and general principles
      established by international agencies governing the ethical conduct in biomedical research on
      human subjects, following the good clinical practices and the declaration of Helsinki. The
      GCTF study protocol has been approved by the Ethics Committee of the University hospital
      (Pontificia Universidad Catolica de Chile , CEC MED UC approval number 16-046, resolution
      dated April 21st, 2016).

      Patient recruitment & characteristics Diagnosed GC patients will be recruited from the Red UC
      Christus network in Santiago, Chile. Patient recruitment and signing of informed consent
      forms, maintenance and monitoring of patient medical records, biological material and sample
      extractions will be managed by CITO.

      Patient and treatment history reveals that besides surgery and chemotherapy, approximately
      10% of patients received Trastuzumab (ERBB2 targeted therapy, also called Herceptin), another
      10% received immunotherapy including pembrolizumab and Ipilimumab (checkpoint inhibitors).
      Finally approximately 5% of patients received antiangiogenic therapy (consisting of VEGFR2
      targeted therapy with Ramucirumab). Additionally, histological analysis showed that
      approximately 50% of patients were classified as intestinal type, 30% as diffuse, and 20%
      were either mixed or undetermined.

      Inclusion criteria

        -  Adult male or female, aged >18 years

        -  Diagnosed with gastric cancer (histological or cytological)

        -  Attending health centers of the Red UC Christus network for at least 3 months with
           clinical follow-up

        -  Capable to read and speak Spanish

        -  Willing and able to provide written informed consent to the study that should be dated
           and signed at the time of enrollment.

      Exclusion criteria

      Patients:

        -  With small biopsy samples insufficient for analysis.

        -  Whose medical records cannot be collected or are unavailable.

        -  Without signed informed consent.

      Clinical data Clinical data from patients will be obtained by healthcare providers and
      entered into an online electronic platform. Samples will be coded and patient identity known
      only to the attending physician. Clinical variables are divided into sections: General
      Patient Information, Cancer History, Laboratory Studies, Comorbidity (Charlson) Index,
      Chemotherapy, Efficacy & Follow Up and Toxicity. Patient chemotherapy will be classified by:
      regime, number of cycles & time of treatment and chemotherapy dose-intensity during the first
      6 months. Chemotherapy regimens representing the first line chemotherapy prescribed to the
      patients. Full chemotherapy dose intensity during the first 6 months will be obtained through
      patient interviews and entered directly into the online platform. Finally, efficacy &
      follow-up and toxicity data obtained from patients.

      Main clinical outcomes Main outcomes will be inferred from obtained clinical data, these
      include overall survival, progression-free, and recurrence-free survival rates.

      Biological samples & Oncomine Comprehensive Assay Biological materials obtained at the Red UC
      Christus will be transported to CEMP in Santiago de Chile under standardized protocols. A
      total of 200 patient tumor samples will be obtained from archived Formalin Fixed Paraffin
      Embedded (FFPE) samples. Nucleic acids will be extracted using the RecoverAll kit (Thermo
      Fisher Cat #AM1975) and analyzed using the commercially available Oncomine Comprehensive
      Assay kit. This assay simultaneously analyzes DNA and RNA from samples allowing the
      assessment of 73 gene hotspots (based on DNA), 49 focal copy number variations (CNVs, DNA
      based), 26 full coding sequences (for mutations and CNV loss), and 22 gene fusion drivers
      (RNA). Notably, 72 of these genes are drug targets. Genomic raw data obtained (.vcf and .pdf
      files) will be stored and backed up in a local Data Center for subsequent genomic analysis.
      Upon publication of the findings of this study, the Oncomine results along with clinical
      classification of individual tumors will be made publicly available.

      Tissue Micro Array (TMA) Analysis The following genes will be further analyzed by a TMA using
      specific antibodies against: PD-L1 (Dako, Cat # SK00521), PD-L2 (Thermo Cat# B7-DC/CD273),
      Phosphorylated mTOR (Abcam Cat#AB118815), p53 (Cat # 5278074001), VEGFR2 (Abcam Cat
      #AB39256), Phosphorylated Akt (Thermo Cat #473), HER2 (Roche, Cat # 05278368001), p16 (Roche,
      Cat # 06695221001), Met (Abcam Cat #AB51067), HA-4 (Abcam Cat #AB24480) and four
      microsatellite markers (all from Roche): MLH1 (Cat # 06472966001), MSH2 (Cat # 05269270001),
      MSH6 (Cat # 5929911001), PMS2 (Cat # 06419216001). Manual TMA will be prepared as described
      previously. Briefly, paraffin blocks will be obtained and cut and stained by Hematoxylin &
      Eosin (H&E) in order to select the best histological area. Subsequently selected tissue area
      will be placed into the TMA by circling the identified area in the corresponding block.
      Cylindrical core biopsies will be extracted from each paraffin block using a 1 mm stylet and
      placed into a new recipient block. Selected adequate cases had tumors that occupied at least
      10% of the core area. Each case will be processed in triplicate to prevent tissue loss during
      cutting. Sections from each tissue array block will be cut, de-paraffinized and dehydrated
      for H&E and immunohistochemical procedures.

      Gene methylation Promoter gene methylation on six selected genes that have previously shown
      promoter regulation by methylation associated with GC will be assessed. Methylation analysis
      will include the reprimo gene and other mRNAs associated with GC progression. Analysis will
      be performed by bisulfite sequencing as described previously using the EZ DNA methylation
      Gold kit (Zymo Research) with minor modifications. Briefly, bisulfite treated DNA is
      amplified using specific PCR primers, with PCR products subsequently cloned and sequenced.

      EBV identification EBV subtypes in patient samples will be assessed using the chromogenic in
      situ hybridization (CISH) method with minor modifications.

      Single Nucleotide Polymorphism (SNP) analysis A significant proportion of GC patients can
      develop serious toxicity from 5-FU treatment including bone marrow suppression, neuropathy,
      low white blood cell numbers, fever, infections, nausea, vomiting, severe diarrhea, mouth and
      digestive tract inflammation, all of which are recorded in the patient history of other
      cohorts. Subtle personal and population changes in DNA, called SNPs can account for increases
      in the risk of 5-FU toxicity; 5-FU metabolism is predominantly hepatic, where the enzyme DPYD
      is responsible for metabolizing >80% of the drug, producing the inactive metabolite 5,6
      dihydroxy-5-FU. It is widely documented that a decreased DPYP activity is associated with
      severe toxicity. Non-metabolised fraction of 5-FU (20%) is transformed by a series of enzymes
      (e.g TP, TK), producing the active metabolites that will cause TYMS inhibition, thereby
      promoting DNA/RNA damage. Variations in TYMS and MTHFR genes (related to reduced folate
      synthesis, increased 5-FU effect), have been associated with toxicity by treatment with 5-FU.
      The approach that was used to select the genetic variants consisted of a search in the
      database, PharmGKB. A total of six non-synonymous SNPs will be analyzed: four of them
      comprise the DPYD gene, one for TYMS and one for MTHFR. Analysis will be performed using
      TaqMan™ SNP Genotyping Assay technology (Applied Biosystems™). SNPs will be assessed in DNA
      isolated from paraffin embedded patient samples.

      Sample size and statistical analysis

      The minimum sample number will be calculated in order to ensure the goals of the project are
      fully accomplished. Considering that approximately 90% of GC cases are indeed GAC, at 5%
      error rate and at 95% confidence interval the investigators originally projected a sample
      size of 200 patients. However the investigators have also considered a 15% rate of sample
      loss (defective samples or patient drop-out), which gave a total of 230 patients to be
      recruited.

      Standard descriptive statistics will be utilized to analyze qualitative and quantitative
      variables, such as relative and absolute frequencies, frequency tables, average, median,
      standard deviation, range and quartiles. A 95% confidence will be considered appropriate for
      analysis. Descriptive statistics will also be used to characterize the most relevant clinical
      parameters measured. The association of categorized variables will be performed by chi-square
      or Fisher's exact tests. One arm Analysis of Variance will compare continuous variables among
      groups. Survival outcome studies will be accomplished using the Kaplan-Meier method.
      Prognostic factors will be evaluated according to the Cox proportional hazards regression
      model.

      Principal component analysis (PCA) of the genes variants will be conducted and the
      association of the first principal components with a small pre-defined set of genomic
      alteration signatures will be assessed. To define molecular subgroups, the investigators will
      utilize unsupervised clustering. The correlation of the molecular subtypes with clinical data
      (e.g. age, gender, Lauren class) and clinical outcomes (e.g. overall survival, response rate)
      will be assessed. Moreover, supervised classification will be performed based on clinical
      outcomes and the resulting groups of both approaches will be compared with other reported
      molecular subtypes.

      Patient protection/written informed consent forms

      All parties guarantee the protection of the patients' personal records. Patient names are not
      included in any form in sheet-reports, publications, or in any type of publishable document
      derived from the study with the exception of documents required by law. Informed consent
      forms are elaborated strictly following legal and local regulations. The written informed
      consent forms, including all changes made throughout the study must be prospectively approved
      by the Internal Review Board/independent Ethics Committee, and CEMP prior to be incorporated
      into the study.

      The investigators, representatives or healthcare providers will obtain written informed
      consent forms from every patient or a legal representative before any specific activity of
      the study is performed.
    

Observational model: Cohort
Time perspective: Prospective
Sampling method: Probability Sample
Study population: 
        Chilean population diagned with gastric cancer
      
Biospecimen description: 
      RNA and DNA
    
Keywords: Gastric Cancer; gastric adenocarcinoma; cancer subtypes; prognosis; survival; molecular classification; chemotherapy; immunotherapy