GASTROGUIDE study

Early detection of peritoneal metastases in gastric cancer

GASTROGUIDE is a prospective multicentre study into the diagnostic value of tumour-guided cell-free DNA analysis (cftDNA) in peritoneal fluid in patients with gastric cancer.

Background

Of patients with gastric cancer, 30% have peritoneal metastases at diagnosis. This is associated with a poor prognosis, with a median survival of 9–10 months. Early detection of peritoneal metastases is therefore essential for an individual treatment plan, but is hampered by the limited sensitivity of current imaging. Conventional cytological analysis of peritoneal fluid in patients with gastric cancer has a low sensitivity (12–67%), resulting in frequent false-negative results. There is therefore a need for an accurate method to detect peritoneal spread at an early stage.

An alternative approach for detecting peritoneal metastases is analysis of cftDNA in abdominal (lavage) fluid obtained during diagnostic laparoscopy. Previous studies showed that cftDNA is detectable in peritoneal fluid in various types of cancer, and was more sensitive than conventional cytology for the detection of peritoneal metastases.

To investigate this further, a proof-of-principle study was carried out at the NKI-AvL into the feasibility of tumour-guided cfDNA detection in peritoneal fluid of patients with gastric cancer. This showed that tumour-guided cftDNA detection in abdominal (lavage) fluid of patients with gastric cancer is feasible, with a higher sensitivity than conventional cytology for detecting peritoneal disease (91% versus 64%). Notably, two patients in whom no peritoneal disease was seen at the time of diagnostic laparoscopy and in whom the conventional cytology of the abdominal (lavage) fluid was tumour-negative, but in whom cftDNA was detected in the lavage fluid, nevertheless developed peritoneal metastases within six months. This finding suggests that the detection of cftDNA in abdominal (lavage) fluid can predict the development of peritoneal metastasis.

Roles and responsibilities

Surgeon / OR team

  • Identify, inform and include the patient before laparoscopy
  • Laparoscopy + collection of peritoneal fluid, correctly labelled
  • Request the FFPE block from another centre (if applicable)
  • Send ascites, lavage fluid and biopsies separately per location
Keyhole operation (laparoscopy)

Local pathology laboratory

  • Receipt, registration & routine diagnostics
  • Separation of pellet and supernatant
  • Storage of study material + FFPE block
  • Shipment in batches to the central lab
Laboratory sample processing

Central lab (NKI-AvL)

  • Receipt, registration & archiving
  • Central molecular analyses
  • Feedback on logistical or quality problems
Laboratory processing

Patients

Inclusion criteria

Patients with a histologically proven adenocarcinoma of the stomach or gastro-oesophageal junction who undergo a diagnostic staging laparoscopy, or a primary resection with curative intent.

Gastric cancer illustration

Exclusion criteria

Patients with distant metastases and absence of the primary tumour.

Metastases in the liver

Patient pathway: from diagnosis to follow-up

Flowchart of the patient pathway: diagnosis, DLS, M1/M0 disease, follow-up

Surgery

Collection of peritoneal fluid (OR protocol)

Required materials

  • Sterile pathology container, 100 ml
  • Suction catheter, Ch. 14
  • 50 ml Luer-lock syringe
  • 500 ml NaCl 0.9%

Procedure

  • Inspection of the abdomen (patient in neutral position)
  • Collect ascites
  • Lavage of the left upper abdomen (reverse Trendelenburg) and the pelvis (Trendelenburg)
  • Rinse with 100 cc 0.9% NaCl, leave for 20 seconds
  • Take biopsies if indicated
  • Document the PCI score

Volume

  • Aim for 50 cc of fluid per collection site (left upper abdomen and pelvis)
  • At least 20 cc of fluid per collection site

Operation

Use the standard template for the operation report of the staging laparoscopy: DLS operation report template (Word).

Peritoneal Cancer Index (PCI) score form

Pathology

Collection and processing

1. Preparation

  • Keep cooled (ice or 4°C)
  • Centrifuge at 4°C
  • Record: collection and receipt time, volumes, centrifugation time, processor

2. Centrifugation

  • Transfer to a 15/50 ml Falcon tube
  • Centrifuge for 5 min at 300 × g
  • Use the same setting as for routine cytology

3. Separation

  • Pellet: routine cytology (also for GASTROGUIDE if sufficient)
  • Supernatant: for GASTROGUIDE
  • Pipette off carefully without disturbing the pellet
  • Label: study ID, centre, material, location, date, aliquot number

4. Storage

  • Freeze as soon as possible after cooling
  • Preferably −80°C (at least −20°C)
  • Aliquot preferably in 15 ml tubes
  • Record: volume, number of aliquots, temperature, freezer location

Volume

VolumeAction
<10 mLUse entirely for routine cytology; keep the supernatant after processing for GASTROGUIDE if possible
10–19 mLIn principle entirely for routine cytology; keep the supernatant after processing for GASTROGUIDE
20–29 mLDivide in advance: 10 mL for routine cytology, 10 mL for GASTROGUIDE (pellet and supernatant)
≥30 mLDivide in advance: 10 mL for routine cytology, the remaining mL for GASTROGUIDE (pellet and supernatant)

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