Discusión de los Resultados

Lymphoma-containing material at diagnosis and material from follow-up biopsies should be collected for molecular biology studies.

The polymerase chain reaction (PCR) assay for the rearrangement of the immunoglobulin heavy chain genes will be performed to provide a molecular marker to evaluate the follow-up biopsies, especially in primary gastric localisations, as already applied in the previous LY03/IELSG 3 trial (Ref. 13).

The presence of the t(11;18)(q21;q21) translocation may have a prognostic significance (Ref. 2) and will be evaluated by reverse-transcription polymerase chain reaction (RT-PCR) assay on RNA derived from the diagnostic biopsies. Aliquots of DNA for molecular biology studies will be stocked. Whenever it is possible, three unstained slides will be also collected.

METHODS OF TISSUE SAMPLE COLLECTION

• Using liquid nitrogen

The specimen is put in a sterile Nunc vial and submerged in liquid nitrogen; store at –80°C. • Using RNAlater TM (Ambion) Tissue collection RNA stabilization solution

The dissected fresh tissue is submerged in approximately 5 volumes of RNAlater (p.e, 0.5 g of tissue in 2.5 ml of solution). With RNAlater, RNA will be stable for one day at 37°C, one week at 25°C, one month at 4°C, or indefinitely at –20°C.

• Using cell culture medium

Operation specimens should be immersed in culture medium or in sterile balanced salt solution, such as isotonic sodium chloride. Tissue retains viability for 2-3 days. Samples have to be shipped immediately. • Paraffin-embedded material

Two 1.5 ml Eppendorf tubes are prepared: one for DNA extraction and one for RNA extraction. They are prepared with five 10µm sections of tumour-containing paraffin-embedded sections. Please note for RNA extraction: use RNAse-free microcentrifuge tube and clean the microtome blades with acetone.

For both DNA and RNA, extra-care has to be taken to avoid cross-contamination among the different samples. Separate parts of the blade should be used for cutting different blocks; the same part of the blade must not be used to cut more than one block.

BLOOD SAMPLE COLLECTION

Heparin is the anticoagulant of choice. EDTA may also be used. Five ml peripheral blood in EDTA and bone marrow aspirate sample in EDTA at study entry have to be collected. Follow up molecular studies are only appropriate for those patients with a marker at diagnosis.

Cells pellet can be extracted from blood and bone marrow samples and stored at -80°C.

Protocol to extract pellet from blood for DNA analysis

1. Transfer the anticoagulated blood in a 50 ml sterile Falcon vial

2. Add 10 ml of Lysis Buffer (0.32 M sucrose, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1% Triton X-100)

3. Vortex

4. Centrifuge 3000 rpm for 10-15’ 5. Discard the supernatant 6. Add 10 ml of Lysis Buffer 7. Vortex

8. Centrifuge 3000 rpm for 10-15’ 9. Discard the supernatant 10. Add 10 ml of PBS 1x; 11. Vortex

12. Centrifuge 3000 rpm for 10-15’ 13. Discard the supernatant 14. Put the pellet at -80°C.

SAMPLE SHIPMENT

Before the sample shipment each investigator should contact the IELSG Coordinating Office ([email protected]) in order to arrange the shipment details.

ADDENDUM 1

MARCH 31, 2003 - Version 3.1

This addendum refers to the interpretation of the Inclusion Criteria 4.1.3 and clarifies the eligibility criteria for gastric MALT lymphoma.

The following patients with gastric MALT lymphoma can be entered:

1. H. pylori-negative cases, either de novo (non pre-treated) or at relapse following local therapy (i.e., surgery, radiotherapy or antibiotics).

2. H. pylori-positive cases at diagnosis, who failed antibiotic therapy, including patients with:

• clinical (endoscopic) and histological evidence of disease progression at any time post H. pylori eradication

• stable disease with persistent lymphoma at ≥ 1 year post H. pylori eradication • relapse (without H. pylori re-infection), after a remission

• patients who failed either first line antibiotics or further local treatment (surgery or radiotherapy)

IELSG 19 PROTOCOL – version n. 3.4.1 – 13.01.2009 page 42

ADDENDUM 2

MAY 15, 2006 - Version 3.2

Addition of a third arm (Rituximab alone) in the study IELSG 19

Based on the results of the interim analysis, which showed no difference in response rate and toxicity between the two study arms of Chlorambucil alone vs. Chlorambucil plus Rituximab and on the now published data on the activity of Rituximab alone in extranodal marginal non-lymphoma of MALT type, this amendment to the IELSG 19 study is

implemented:

- A third treatment arm (namely, arm C with the administration of Rituximab alone), is introduced.

- Randomization will proceed with a 1:1:6 allocation until a total additional number of approximately new 150 patients will be recruited in each of the three arms.

- The protocol version nr. 3.1, May 15, 2006, is provided, which takes into account these changes.

- A new version of the informed consent form, will have to be prepared by the local investigators according to the local EC and authorities requirements. - Patients randomized in arm C will receive Rituximab 375mg/m2 i.v. on day

1,8,15, 22, 56, 84,112 and 140 as described on page 11 of the study protocol (version 3.1). Rituximab will be supplied by Roche International Ltd. in labeled vials and will be distributed by the local/national Roche representatives.

ADDENDUM 3

APRIL 9, 2008 - Version 3.3

Update of the stability and storage instructions of rituximab

According to the Roche Investigator’s Brochure 12th version, Feb. 2007, R045-2294, page 23, we have modified the APPENDIX G (page 37) as follows:

Prepared infusion solutions of rituximab are stable for 24 hours in the refrigerator (2-8°C) and

at room temperature for additional 12 hours.

AUGUST 12, 2008 – Version 3.4

This addendum refers to a minor amendment due to a typographical error

Appendix G (page 38) the double reference If WBC ≤ 50 X 109/l on the table has been cancelled.

JANUARY 13, 2009 – Version 3.4.1

This addendum refers to a non substantial amendment regarding the modalities of dilution of the drug

According to Roche instructions as reported on the “Summary of product characteristics” (page 61) we have updated the dilution modalities.

Appendix G (page 37) has been modified as follows:

The prescribed dose of Rituximab should be diluted in the amount of ml NaCl 0.9% needed to achieve a final concentration of 1 to 4 mg/ml rituximab.