INTRODUCTION Gastric cancer is one of the most common cancers worldwide. Its incidence varies
      according to its geographic region, being more common in East Asia, Eastern Europe and South
      America. About 70% of gastric neoplasms occur in developing countries. Globally, it is
      estimated that in 2018 over 1 million cases were diagnosed with about 780,000 deaths, making
      gastric cancer the 5th in frequency and 3rd in mortality. In Brazil, 13,540 new cases were
      diagnosed in 2018, being the third leading cause of cancer death in Brazil in males. Most
      patients already have inoperable, advanced or metastatic disease at diagnosis, thus requiring
      palliative treatment. Over 90% of stomach tumors are adenocarcinomas and, in terms of
      histology, Lauren's classification subdivided gastric cancer into diffuse (undifferentiated)
      or intestinal cancer and both types have distinct clinical and pathological features. The
      diffuse type occurs in all age groups with equal gender distribution, involves the body or
      the entire stomach and has a greater tendency to invade the gastric wall (resulting in
      plastic linitis) and to metastasize, as well as a faster disease progression and worse
      prognosis. On the other hand, the intestinal type predominates in men and the elderly,
      occurring predominantly in the gastric antrum and notch. Although both subtypes are related
      to H. pylori infection, in the intestinal type the evolution of sequential preneoplastic
      alterations is observed, namely: atrophic gastritis / intestinal metaplasia; dysplasia and
      adenocarcinoma. Such a sequence may not occur in diffuse type. There is also a rare
      genetically related type called hereditary diffuse gastric cancer (HDGC) that is not related
      to H. pylori infection. Diffuse-type adenocarcinoma is the major histological subtype of
      HDGC. In addition to diffuse hereditary gastric cancer syndrome, gastric cancer is present in
      the description of several other hereditary syndromes, such as Lynch syndrome, juvenile
      polyposis syndrome, Peutz-Jeghers syndrome, Familial Adenomatous Polyposis,
      ataxia-telangiectasia syndrome, Li-Fraumeni and Xeroderma Pigmentosa. Regardless of
      association with genetic syndrome or even histological type, it is necessary to investigate
      the amplification of the human epidermal growth factor 2 (HER2) gene in the case of recurrent
      or metastatic gastric cancer. Today of well-defined importance in gastric cancer, HER2 was
      initially recognized 30 years ago as an amplified oncogene in over 20% of breast cancers and,
      after the genomic revolution, has recently been seen to amplify in many other cancers. HER2
      is a tyrosine kinase-like receptor that crosses the cell membrane of cells, with the
      extracellular half not coupling to ligands, but designed to recognize ligand-activated forms
      of family members as the factor of human epidermal growth (EGFR), HER3 or HER4, leading to
      dimerization with these receptors. The cytoplasmic part of HER2 and its partners contains a
      kinase domain that triggers HER2 kinase activity. The end result is phosphorylation at
      various tyrosine residues and recruitment of second messenger proteins for these
      phosphotyrosines. When the gene is amplified in cancer cells, the result is massive
      overexpression of HER2, which causes continuous activation of the various biological pathways
      that promote cell proliferation. For gastric cancer, positivity for HER2 expression, as
      assessed by immunohistochemistry (IHC) or in situ hybridization immunofluorescence (FISH),
      may vary between 8 and 36% between studies in the literature; However, those studies that
      considered only intestinal histology found positive expression of HER2 between 20 and 30% in
      patients' biopsies. A study conducted at AC Camargo identified 12% HER2 expression and 8%
      amplification in gastric tumors, with greater association with intestinal subtype and poor
      survival. Because it has a worse prognosis and has a target drug, treatment of tumors with
      HER2 amplification requires HER2-targeted agents, often in combination with chemotherapy or
      hormone therapies. Trastuzumab-associated chemotherapy (fluoro-uracil and platinum) is the
      first-line standard treatment for HER2-positive metastatic gastric cancer based on the phase
      III ToGA study (Trastuzumab for Gastric Cancer). This study recruited 594 patients and showed
      a median overall survival gain of 13.8 months for the combination versus 11.1 months in the
      chemotherapy arm alone (hazard ratio [HR], 0.74; 95% CI, 0.60 to 0.91; P = 0.0046). Survival
      gain was even more significant in the HER2 overexpressed subgroup (IHC 3+ or IHC 2+ with
      positive FISH), which achieved a median overall survival of 16 months. Regarding the second
      line, however, phase III studies failed to demonstrate the benefit of using anti-HER2
      therapy. In the TyTan study, the addition of lapatinib in the second line was not superior to
      chemotherapy alone. In the GATSBY study, the use of trastuzumab emtansine was also not
      superior to taxane chemotherapy. In this study, 77% of patients received first-line anti-HER2
      agents. Several clinical reasons may explain these disappointing results; One of these is
      resistance acquired after use of anti-HER2-based therapy. Another reason is the loss of HER2
      expression, as reported in a study by Pietroantonio et al that demonstrated loss of HER2
      positivity and overexpression in 32% of the tissue samples analyzed after anti-HER2 therapy.
      It is already known that in selective breast cancer treatment pressure can eradicate
      HER2-expressing clones and lead to proliferation of those HER2-negative clones, a phenomenon
      that can occur in gastric cancer because of the marked heterogeneity that exists in stomach
      tumors. In addition, it is known that HER2 expression discrepancy may exist between primary
      and metastatic or recurrent breast tumors (probably due to sample variability). In discordant
      cases, HER2-positive metastases from negative primary tumors are more frequent than the
      opposite. Therefore, it is recommended that metastatic disease on first relapse be biopsied
      as part of the investigation and HER2 status should be reevaluated. In cases of gastric
      cancer, this issue has not been widely explored.

      In this sense, the GASTHER 1 study investigated the role of HER2 expression reassessment in
      stomach cancer at primary, metastatic or recurrent sites in patients whose primary tumor was
      initially negative for HER2 expression. The results showed a positive rescue of 8.7%,
      confirming the relevant heterogeneity of HER2 status. This heterogeneity, however, may also
      be associated with the HER2 status assessment method. A prospective study in patients with
      localized gastric adenocarcinoma and treated with perioperative chemotherapy at our
      institution found 69.4% agreement for HER2 expression in primary tumor tissue biopsy and in
      paired circulating tumor cells (CTCs). HER2 in CTCs showed higher positivity compared to
      tumor tissue (43% x 11%). The positivity in HER2 CTCs was 60% for HER2-negative localized
      gastric cancer patients treated with perioperative chemotherapy whose disease recurred. Also,
      HER2 expression in CTCs correlated with progression-free survival, but in the tumor tissue
      the same relationship was not found.

      BACKGROUND Analysis of CTCs from the blood of patients with gastric adenocarcinoma may be
      useful to better understand the behavior of the disease, as well as patients more likely to
      respond to treatment, and may offer a less invasive way of investigating tumor dynamics. In
      addition, prospective evaluation of HER2 expression in CTCs has not been evaluated in
      metastatic gastric cancer, and the frequency with which this expression changes after
      first-line treatment with standard regimen with trastuzumab at the time of disease
      progression. Thus, this study is necessary to evaluate the frequency with which these
      phenomena occur and thus expand the knowledge of the dynamics of gastric cancer tumor
      biology.

      Hypothesis:

      Primary:

        1. There is disagreement in HER2 expression positivity between diagnostic tissue biopsy
           (preferably metastasis) and circulating tumor cells in metastatic intestinal cancer.

        2. Positivity for HER2 expression in circulating tumor cells in relapsed or metastatic
           gastric cancer may predict response to standard treatment with trastuzumab combination
           chemotherapy.

      Secondary:

        1. HER2 expression may have prognostic effect when positive in CTCs.

        2. HER2 expression in CTCs may be modified following treatment with anti-HER2 therapy.

      Objectives:

      Primary:

        1. Evaluate HER2 expression in circulating tumor cells of patients with relapsed or
           metastatic gastric cancer with negative HER2 expression on tissue biopsy.

        2. To evaluate the response to standard treatment with combination chemotherapy with
           trastuzumab in relapsed or metastatic gastric cancer with positive expression of HER2 on
           CTC and negative on tissue biopsy.

      Secondary:

        1. To verify the prognostic impact of HER2 positivity in circulating tumor cells in
           advanced gastric tumors;

        2. Evaluate HER2 expression in CTCs at the time of progression to standard first-line
           treatment with anti-HER2 therapy in patients who previously had HER2 positivity.