CHIC-Study CHemoImmunotherapy with early CNS … · CHIC protocol, , Protocol Code NLG-LBC05, EUDRACT No 2010-023125-38, Version 5 27.9.2012 ClinicalTrials.gov Identifier: NCT01325194

CHIC protocol, , Protocol Code NLG-LBC05, EUDRACT No 2010-023125-38, Version 5 27.9.2012 ClinicalTrials.gov Identifier: NCT01325194

Clinical phase II study protocol

Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 years with high risk (aaIPI≥ 2) Diffuse Large B-Cell

Lymphoma (NLG-LBC05)

CHIC-Study CHemoImmunotherapy with early CNS

prophylaxis

The Nordic Lymphoma Group Protocol Secretariat, Unit for Clinical Research Support at Oslo University Hospital, Radiumhospitalet, Pb 4953 Nydalen, 0424 Oslo, Norway. Telephone number: + 47 22 93 57 57 (09-15 Norwegian time).

CHIC protocol, Protocol Code NLG-LBC05, EUDRACT No 2010-023125-38, Version 5 27.9.2012 ClinicalTrials.gov Identifier: NCT01325194

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Writing committee Dr. Sirpa Leppä, (principal investigator) Dept Oncology Helsinki University Central Hospital, FIN-00029 Helsinki, Finland Phone: (358) 9 4711 Fax: (358) 9 471 73181 e-mail: [email protected]

Dr. Harald Holte (principal investigator) Dept. Oncology, Oslo University Hospital, Radiumhospitalet, N 0310 Oslo, Norway Phone: (47) 22 93 40 00 Fax: (47) 22 93 58 08 e-mail: [email protected]

Dr. Mats Jerkeman Dept. Oncology Lund University Hospital, SE 221 85 Lund, Sweden Phone: (46) 46 17 10 00 Fax (46) 46 17 60 80 e-mail: [email protected]

Dr. Sirkku Jyrkkiö Dept. Oncology Turku University Central Hospital, FIN-20520 Turku, Finland Phone: (358) 2-313 00 00 Fax: (358) 2 313 2809 e-mail: [email protected]

Dr. Judit Jørgensen Dept. Haematology,Århus University HospitalDK-8000 Århus, Denmark Phone: +4589497550 or +4589497551 Fax: +4589497597 e-mail: [email protected] Dr. Lars Møller Pedersen Dept Haematology Odense University Hospital, DK 5000 Odense, Denmark Phone: +45 4732 4801 Fax: +45 4635 3439 e-mail: [email protected]


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Dr. Magnus Björkholm Department of Medicine Karolinska hospital, S 171 76 Stockholm, Sweden Phone: (46) 8 5177 3851 Fax (46) 8 31 82 64 e-mail: [email protected] Dr. Øystein Fluge, Dept. Oncology Haukeland University Hospital, N-5021 Bergen, Norway Phone: (47) 55 97 2010 Fax: (47) 55 97 2046 e-mail: [email protected]

Members of the Molecular Study Group Sirpa Leppä (chair) Department of Oncology Helsinki University Central Hospital Finland e-mail: [email protected] Marja-Liisa Karjalainen-Lindsberg Department of Pathology Helsinki University Central Hospital Finland e-mail: [email protected] Mats Jerkeman Department of Oncology Lund University Hospital Sweden e-mail: [email protected] Jan Delabie Department of Pathology Oslo University Hospital, Radiumhospitalet Norway e-mail: [email protected] Dr. Øystein Fluge, Dept. Oncology Haukeland University Hospital, N-5021 Bergen, Norway e-mail: [email protected] Members of the Pathology Group Denmark Elisabeth Ralfkiær Dept. of Pathology 5444 Rigshospitalet DK-2100 København Ø, Denmark Fax: (+45) 35 45 63 80


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e-mail: [email protected] Finland Marja-Liisa Karjalainen-Lindsberg Department of Pathology Helsinki University Central Hospital Finland e-mail: [email protected] Norway Jan Delabie Department of Pathology Oslo University Hospital, Radiumhospitalet Montebello 0310 Oslo Norway e-mail: [email protected] Sweden Christer Sundström (chairman) Dept. of Pathology Uppsala University Hospital SE-75185 Uppsala, Sweden Fax: (+46) 18 662 665 e-mail: christer.sundströ[email protected] Statistician Knut Liestøl Dept of Informatics University of Oslo and Center for cancer biomedicine Oslo University Hospital Montebello 0310 Oslo email: [email protected]


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Contents 1. Synopsis of the study 6 2. Flow chart 9 3. Background and rationale 10 3.1. Clinical background and rationale 10 3.2. Risk versus benefit in the protocol 11 3.3. Molecular and biological background 11 4. Objectives of the study 12 5. Study duration and early study termination 13 6. Inclusion criteria 13 7. Exclusion criteria 14 8. Patient inclusion 15 9. Therapeutic regimens, toxicity and treatment modifications 15 9.1 Therapeutic regimen 15 9.2 Toxicity and treatment modifications during therapy 18 10. Disease assessment at trial entry 20 11. Disease assessment during and after therapy 21 12. Evaluation criteria and endpoints 22 13. Reporting adverse events 23 14. Statistical considerations 25 15. Ethical considerations 26 16. Publication policy 26 17. References 28 18. Appendix 1 - 10 31


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1. Study synopsis

Title Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less

than 65 years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma

Short title CHIC (CHemoImmunotherapy with early CNS prophylaxis)

Version 10.11.2010

Principal investigators Harald Holte and Sirpa Leppä

Clinical phase II

Planned sample size 170

Study design Multicentre trial

Number of centres Open for Nordic centres

Primary objectives Time to treatment failure from date of registration (3 year)

CNS relapse rate (1,5 year)

Secondary objectives Toxicity

Clinical response rate

Incidence of CNS relapse in CSF cytology neg/flow cytometry positive cases

Incidence of CNS relapse in a subgroup of patients with more than one extranodal

site and elevated LDH

Progression free survival (3 years)

Overall survival (3 years)

Molecular predictors

Inclusion criteria

Age ≥ 18 - < 65 years.

Histologically confirmed CD20+ diffuse large B-cell lymphoma based on WHO

2008 Lymphoma Classification

• Follicular lymphomas grade 3b is allowed

Patients in at least stage II with age adjusted IPI score of 2 or 3:

• Stage III /IV and elevated LDH

• Stage III/IV and WHO performance status 2 - 3

• Stage II and elevated LDH and WHO performance status 2 – 3

And/or patients with

• More than one extranodal site

• Testicular lymphoma, stage IIE and higher

• Paranasal sinus and orbital lymphoma with destruction of bone

• Large cell infiltration of the bone marrow

Previously untreated, except steroids allowed

WHO performance status 0-3

Written informed consent

• Patients with at least one of the following criteriae


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Inclusion criteria for

Swedish centres differ

from the other Nordic

countries

o More than one extranodal site and elevated LDH

o Testicular lymphoma, stage IIE and higher

o Paranasal sinus and orbital lymphoma

o Large cell infiltration of the bone marrow

o Epidural lymphoma

Exclusion criteria Severe cardiac disease: cardiac function grade 3-4

Impaired liver, renal or other organ function not caused by lymphoma, which will

interfere with the treatment schedule

Pregnancy/lactation

Men and women of reproductive potential not agreeing to use an acceptable method

of birth control during treatment and for six months after completion of treatment

Patients with other severe medical problems and with an expected short survival for

non-lymphoma reasons

Known HIV positivity

Uncontrolled infectious disease, including meningeal infection

Active cancer except basal cell carcinoma and cervical carcinoma in situ during the

last five years

Earlier treatment containing anthracyclins

Psychiatric or mental disorder which make the patient unable to give an informed

consent and/or adhere to the protocol

CNS disease as diagnosed by MRI or CSF cytology. Positive CSF flow cytometry

below diagnostic threshold level by cytology is allowed

Pleural or peritoneal fluid that cannot be drained safely

Conditions that may be compatible with impaired cerebrospinal fluid flow Hypersensitity to the active substance or any of the other ingredients

Patients participating in other clinical studies, unless followed for survival

Treatment Prephase

• Dexamethasone 10 mg x 2 for 3-6 days (max days -5- -0). Dexamethasone can

be substituted with betamethasone 8 mg x 2

• Vincristine 1.0 mg x1 single injection day -4

• Rituximab 375 mg / m2 day -4

Cycle 1

• HD-MTX 3.0 g/m2 iv (3h infusion) day -2 (For patients > 60 years 1.5 g/m2)

• R-CHOP day 1-5 (if s-MTX <1 µM/l at 42h)

• Pegfilgastrim sc day 3


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Cycle 2

• HD-MTX iv (3 h infusion) day 15 + R-CHOP day 15-19. R-CHOP15 is allowed

to start day 16 or day 17.

• Pegfilgastrim sc day 17*

Cycles 3 -6

• R-CHOEP-14 days 29-33, 43-47, 57-61, 71-75*

• Pegfilgastrim sc days 32, 46, 60, 74*

Cycle 7

• HD-Ara-C 3g/m2 x 2 for 2 days (in total four times) days 85-86* (For patients >

60 years 2 g/m2 x2 for 2 days).

• rituximab 375 mg / m2 day 85 or day 86*

• Pegfilgrastim sc day 88*

• *cycles 3-7 are postponed accordingly if R-CHOP-14 is given 1-2 days after sec.

HD-Mtx.

Radiotherapy is given after chemotherapy at the discretion of the individual centres.

Common indications are:

• Bulky disease (≥ 10 cm) at diagnosis

• PET positive residual disease, not eligible for biopsy at a localized site,

potentially curable by radiotherapy

Response evaluation According to Cheson 2007 criteria

CT covering neck, chest, abdomen and pelvis after 3 cycles and at the end of therapy

• PET should be performed at the end of therapy at least for the patients not in CR

according to CT

• Biopsy should be taken from PET positive sites if eligible

Expected number of

patients

170 evaluable

Statistics The primary objectives are to estimate the proportion of patients that are failure- and

CNS relapse-free at 3 and 1.5 years, respectively. The study results will also be

compared with historical CRY-04 data. Note though, that besides random errors,

there are also systematic differences that can lead to bias, when using historical

controls. Some of these will be accounted for by adjusting for known prognostic

factors, e.g. using stratification or logistic regression analysis.


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3. Background and rationale 3.1 Clinical background and treatment Diffuse large B-cell lymphoma (DLBCL) is a curable disease with combination chemotherapy. The outcome in DLBCL is variable, but can to some extent be predicted from clinical risk factors included in the IPI score (1). For several decades, a mainstay of therapy has been CHOP chemotherapy regimen, which is as effective as and less toxic than more intensive regimens (2-4). With a CHOP-like therapy, approximately 50% of all patients are cured. Corresponding percentage for low and high risk patients are 80% and 30%. Recently, combination of rituximab, a monoclonal antibody targeting CD20, with CHOP has led to a marked improvement of survival. The benefit of the addition of rituximab to eight cycles of CHOP given every three weeks (CHOP-21) was initially shown in a randomized study of the French GELA group for the elderly DLBCL patients (age>60 years) (5, 6). Three additional randomized trials of the German NHL Study group (RICOVER-60), joint effort of HOVON and NLG groups and US Intergroup) have confirmed this benefit (7-9). In the largest RICOVER-60 study (8), the patients received six or eight cycles of CHOP given every two weeks (CHOP-14) with or without eight rituximab infusions. Six cycles of CHOP in combination with eight applications of rituximab yielded the best results. In young patients with low risk (IPI 0-1) DLBCL, optimal treatment results have also been achieved in a European intergroup trial, the MInT study, using six cycles of CHOP21-like chemotherapy in combination with rituximab (10). Whether dose densification of R-CHOP from 21 to 14 days can improve the results in elderly patients is currently investigated by the GELA group and the British BNLI group. For young high risk DLBCL patients, five-year overall survival is approximately 60%, and benefit of rituximab or interval reduction from 21 to 14 days has not been specifically demonstrated. Neither has the randomized trials comparing conventional doses of chemotherapy with the high dose treatment (HDT) with stem cell transplantation convincingly shown an advantage for the HDT (11, 12). However, results

of a population-based study from British Columbia demonstrated the value of the addition of rituximab to CHOP-21 in all age and risk groups (13). German NHL Study Group in turn showed that CHOEP was better than CHOP for younger patients regarding progression free survival (14). The finding was confirmed in a subgroup analysis in the MInT Study, but after addition of rituximab, further benefit was not seen (10). Densification of CHOP or CHOEP to 14 days cycles neither improved the outcome (14). It is however important to note that only younger patients with low IPI score were included in these two German studies. It is argued that patients with risk factors have a disease with high proliferation rate, and that these patients may benefit from dose densification. Thus, it is possible that R-CHOEP-14 is a treatment with three favourable modifications from CHOP for younger patients with risk factors. In addition to high risk of systemic relapse, the patients with DLBCL are at risk for progression or relapse of their lymphoma in the CNS, the most frequent location being the leptomeninges. Several studies have shown that the risk of CNS progression or relapse is in the range of 4-6% (15-17). No study has demonstrated in a randomized fashion that CNS prophylaxis with i.t. or i.v. methotrexate prevents CNS-disease, but the lowest recorded frequency is demonstrated in a French study (18) with an isolated CNS recurrence rate of only 1.2%. In this study, high dose methotrexate was given at the end of the chemotherapy. Additionally, German NHL Studies have recently shown that the risk of CNS failure is reduced after addition of etoposide or rituximab to CHOP regimen (19, 20). According to several studies, patients with high tumour load including high IPI score, and extranodal involvement in more than one site are at the highest risk for developing CNS disease (16, 18-20).


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The toxicity and efficacy of R-CHOEP-14 regimen consolidated with late systemic CNS prophylaxis in young high risk DLBCL and follicular lymphoma grade III patients were recently investigated in a Nordic phase II study (CRY-04) (21). The patients less than 65 years old with age adjusted IPI 2 or 3 received six courses of R-CHOEP-14 followed by high dose cytarabine and high dose methotrexate. 158 eligible patients were included, and the final analysis will be performed early 2010. To date, however, early results indicate a low rate of toxic deaths (n=3) despite intensive therapy, and highly satisfactory response rates with 43%, 30% and 21% of the patients in CR, Cru, and PR, respectively. In addition, the majority of the PR patients were considered to have residual masses and not viable tumour tissue, and have been followed without further treatment. Seven patients have experienced a CNS relapse, all within 6 months after end of therapy, indicating that the CNS prophylaxis should be given earlier during therapy. The early appearance of CNS events in the CRY-04 study suggests that the patients had a subclinical disease at diagnosis. This study is being conducted to analyze the efficacy and toxicity of early CNS prophylaxis followed by R-CHOEP-14 regimen. The results will be compared to a recent CRY-04 study. Shifting of CNS prophylaxis to the beginning of the therapy offers a potential to overcome the subclinical disease and thus reduce the risk of early clinical CNS recurrence. As flow cytometry (FCM) can improve the sensitivity for detecting occult leptomeningeal disease over cytology (22, 23), FCM from cerebrospinal fluid will be incorporated into the staging procedures. One of the two primary objectives of the study is to further reduce the CNS relapse rate substantially from 5%. For further background information and procedures for handling the csf samples and FCM analysis, see Appendix 6. 3.2 Risk versus benefit in the protocol. A relapse or progression in the central nervous system in the majority of cases is lethal after a short period of time, and should be avoided if possible. CNS prophylaxis has proved beneficial in acute lymphoblastic leukemia and Burkitt lymphoma / leukemia, but the evidence from lymphoma protocols is more circumstantial (18). High dose methotrexcate and cytarabine have been shown to be administered safely in a previous Nordic protocol (21).. 3.3 Molecular background DLBCL is an aggressive disease, in which a correct diagnosis is essential for the outcome prediction and treatment planning. Despite recent advances in diagnostics (24), response evaluation (25), as well as in therapeutic options (5-8, 10, 14, 26), the response to treatment and outcome are still unpredictable. In DLBCL, clinically based IPI has been to date considered the only tool in classifying chemotherapy- and immunochemotherapy treated patients into low and high risk groups. However, the outcome within individual IPI risk groups can vary considerably, and there is a need for additional prognostic factors that could be used together with IPI to improve the outcome prediction of DLBCL patients. In recent years, major progress has been achieved in the knowledge of the molecular basis of DLBCL. Based on gene expression profiles, DLBCL is currently classified into distinct molecular subtypes. At least four major DLBCL entities, showing germinal center B-cell, activated B-cell-like, stromal-1 and 2 signatures, have been identified. The subgroups have significantly different outcome in response to chemo and immunochemotherapy (27-30). The aim of the molecular analyses in this study is to gain further insights into the clinical heterogeneity and prognosis of young high-risk DLBCL patients. The prognostic impact of both previously identified and novel signatures will be assessed. This will be performed by investigating the expression profiles of RNA and proteins relevant for DLBCL pathogenesis and prognosis. Furthermore, genetic, epigenetic and microRNA analyses will be used to complement the expression studies by identifying putative point mutations (both inherited and sporadic),


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deletions, amplifications, insertions, translocations, and silenced genes that may contribute to the clinical course of the disease.

The study material consists of

1) Fresh lymphoma tissue, 2) Paraffin embedded lymphoma tissue, 3) Cerebrospinal fluid 4) Serum 5) Plasma, and 6) Peripheral blood

All samples will be collected from patients prior to treatment.

Serum and plasma (samples 3-4) will also be collected after the 3rd course of chemotherapy; after the last course of chemotherapy.

In addition, lymphoma tissue, serum and plasma will be collected at the time of disease progression, whenever feasible.

A written patient informed consent for sampling will be required as part of the patient information before study entry. Preparation of samples, see Appendix 5, Molecular studies. 4. Objectives of the study Primary objectives To investigate Three-year Time to Treatment Failure (TTF) from the registration date. A definition of TTF is given

in chapter 12. Incidence and sites (parenchyma, leptomeninges, combined) of CNS relapse at 1,5 years Secondary objectives To investigate Toxicity Incidence of CNS relapse in CSF cytology negative flow cytometry positive cases Incidence of CNS relapse in a subgroup of patients with more than one extranodal site and elevated

LDH Clinical response rate Time to progression Overall survival Molecular factors important for clinical outcome


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5. Study duration and early study termination With participation from the four Nordic countries, and from the experience from the recent Nordic CRY-04 study, it is anticipated that patient accrual (170 eligible and evaluable patients) will occur within approximately 30 months.

The treatment period for each patient will be 14 weeks of chemotherapy, and – for patients given radiotherapy - additional four weeks.

Patients achieving a CR (or PR according to CT but PET negative) will be followed for event free survival every three months for the first two years, then every half year onward. A primary analysis on remission rates and toxicity will be performed when all patients have been followed for at least one year. The first complete analysis will be performed when all patients have reached the three-year time point and data from all of the study sites are received and the data integrity has been assured. Serious adverse events will be reported until 30 days after the last course of chemotherapy.

After 40 patients have completed therapy, the writing committee will perform the first interim analysis (safety analysis). Criteria for considering stopping the treatment: Efficacy: A CR rate of less than 60%. (Definite stopping criteriae based on conventional remission rates is difficult as most PR patients do not relapse as the residual masses may not contain viable tumor tissue). Toxicity: Unacceptable toxicity, including CNS toxicity, must be considered in view of the efficacy of the treatment, and also in this respect firm stopping rules are difficult to set. Specific toxicity related to the use of vincristine (peripheral neuropathy) is included in the CRF’s for treatment evaluation. 6. Inclusion criteria (for Swedish centres, see below) • Age ≥ 18 - < 65 years • Histologically confirmed CD20+ diffuse large B-cell lymphoma based on WHO 2008 Lymphoma

Classification. The following subgroups and variants may be included: ALK-positive large B-cell lymphoma, Mediastinal large B-cell lymphoma, Intravascular large B-cell lymphoma, T-cell/histiocyte rich large B-cell lymphoma. Follicular lymphomas grade 3b is allowed. Posttransplantation lymphoma, discordant or transformed lymphoma and lymphomas intermediate between DLBCL and Burkitt’s lymphoma are not allowed.

• Patients in at least stage II with age adjusted IPI score of 2 or 3:

o Stage III /IV and elevated LDH o Stage III/IV and WHO performance status 2 - 3 o Stage II and elevated LDH and WHO performance status 2 – 3

• And / or patients with o More than one extranodal site o Testicular lymphoma, stage IIE and higher o Paranasal sinus and orbital lymphoma o Large cell infiltration of the bone marrow

• Previously untreated, except steroids allowed • WHO performance status 0-3. • Written informed consent


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Inclusion criteria for Swedish centres • Age ≥ 18 - < 65 years

• Histologically confirmed CD20+ diffuse large B-cell lymphoma based on WHO 2008 Lymphoma Classification. The following subgroups and variants may be included: ALK-positive large B-cell lymphoma, Mediastinal large B-cell lymphoma, Intravascular large B-cell lymphoma, T-cell/histiocyte rich large B-cell lymphoma. Follicular lymphomas grade 3b is allowed. Posttransplantation lymphoma, discordant or transformed lymphoma and lymphomas intermediate between DLBCL and Burkitt’s lymphoma are not allowed.

• Patients in at least stage II with at least one of the following criteriae o More than one extranodal site and elevated LDH

o Testicular lymphoma, stage IIE and higher

o Paranasal sinus and orbital lymphoma

o Large cell infiltration of the bone marrow

o Epidural lymphoma

• Previously untreated, except steroids allowed

• WHO performance status 0-3.

• Written informed consent Number of centres 25-30 hospitals in the Nordic countries (Appendix 1)

7. Exclusion criteria

• Severe cardiac disease: cardiac function grade 3-4

• Impaired liver, renal or other organ function not caused by lymphoma, which will interfere with the treatment schedule.

• Pregnancy/lactation

• Men and women of reproductive potential not agreeing to use an acceptable method of birth

control during treatment and for six months after completion of treatment

• Patients with other severe medical problems and with an expected short survival for non-

lymphoma reasons

• Known HIV positivity

• Uncontrolled infectious disease, including meningeal infection

• Active cancer except basal cell carcinoma and cervical carcinoma in situ during the last five years


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• Earlier treatment containing anthracyclins

• Psychiatric or mental disorder which make the patient unable to give an informed consent and/or

adhere to the protocol

• CNS disease as diagnosed by MRI or CSF cytology. Positive CSF flow cytometry below

diagnostic threshold level by cytology is allowed

• Pleural or peritoneal fluid that cannot be drained safely • Conditions that may be compatible with impaired cerebrospinal fluid flow • Hypersensitity to the active substance or any of the other ingredients • Patients participating in other clinical studies, unless followed for survival

Pathology Patients may be included on the bases of the histological diagnosis of the local pathologist. The specimen will be reviewed by a central pathologist in each country (see page 17 and CRF form 12 and 13). The pathology review will include a panel of antibodies for the further subtyping of DLBCL according to known DLBCL subtypes and prognostic variants within the DLBCL NOS type. With respect to the latter, the reactivity for several antibodies will be scored positive or negative, and the appropriate cut-off values for scoring will be indicated. The subdivision of prognostic variants within DLBCL NOS will be made upon analysis of the study data, using various algorithms available. The best performing algorithm will then be chosen. 8. Patient inclusion Eligible patients will be registered at the NLG-LBC-05 Protocol Secretariat, Unit for Clinical Research Support at Oslo University Hospital, Radiumhospitalet, Pb 4953 Nydalen, 0424 Oslo, Norway by telephone contact Monday through Friday between 9-15 (10-16 Finnish time), whereupon The Inclusion form will be filled out. Please have the inclusion and exclusion criteria ready. Each patient will be assigned a specific patient number and a 3-letter initial based on the patient’s name on written confirmation from the Clinical research Support Unit. Patients with a positive CT or MR of the brain, but who otherwise are eligible, should be registered with initials and date of birth. Telephone number: + 47 22 93 57 57 Telefax number: + 47 22 50 91 99 9. Therapeutic regimens, toxicity and treatment modifications Prephase

• Dexamethasone 10 mg x 2 for 3-6 days (max days -5- -0). Dexamethasone can be substituted with

betamethasone 8 mg x 2.

• Vincristine 1.0 mg x1 single injection day -4

• Rituximab 375 mg / m2 day -4


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Cycle 1

• HD-MTX 3.0 g/m2 iv (3h infusion) day -2 (For patients > 60 years 1.5 g/m2)

• R-CHOP day 1-5 (if s-MTX <1 µM/l at 42h)

• Pegfilgastrim sc day 3

Cycle 2

• HD-MTX iv (3 h infusion) day 15 + R-CHOP day 15-19. R-CHOP may be delayed to day 16 or 17

• Pegfilgastrim sc day 17*

Cycles 3 -6

• R-CHOEP-14 days 29-33, 43-47, 57-61, 71-75*

• Pegfilgastrim sc days 32, 46, 60, 74*

Cycle 7

• HD-Ara-C 3g/m2 x 2 for 2 days (days 85-86*, in total four times) (For patients ≥ 60 years 2 g/m2 x2

for 2 days). Rituximab 375 mg / m2 day 85 or 86*

• Pegfilgrastim sc day 87*

*If R-CHOP at second course is postponed for one to two days, the subsequent cycles are postponed

accordingly

9.1. Therapeutic regimens Patients are given a prephase medication consisting of dexamethasone, rituximab and vincristine, followed by two cycles of high dose methotrexate with CHOP/CHOD and rituximab, and four cycles of CHOEP/CHOED with rituximab. In addition, one course of high dose cytarabine and rituximab is given. The courses should be given with a two-week interval if possible. High dose methotrexate Methotrexate 3 g/m2 i.v. as a 3 hour infusion.

Folic acid rescue (leucovorin) is given after 24 hours, see Appendix 4. NB: During administration of methotrexate, as few drugs as possible should be given due to possible interactions causing delayed mtx-excretion and increased toxicity (i.e. sulfa-trimethoprim, proton pump inhibitors)

CHOP + rituximab: Cyclophosphamide 750 mg/m2 i.v. day 1

Doxorubicin 50 mg/m2 i.v. day 1 Vincristine 1.4 mg/m2 (max. 2.0 mg) i.v. day 1 Prednisone 100 mg po days 1-5 Rituximab 375 mg/m2 i.v. day 1


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CHOEP + rituximab: Cyclophosphamide 750 mg/m2 i.v. day 1 Doxorubicin 50 mg/m2 i.v. day 1 Vincristine 1.4 mg/m2 (max. 2.0 mg) i.v. day 1 Etoposide 100 mg/m2 i.v. day 1-3 Prednisone 100 mg po p.o days 1-5

Rituximab 375 mg/m2 i.v. day 1

G-CSF Long lasting G-CSF (pegfilgrastim, Neulasta®) 48 hours after chemotherapy course 1 and 2, 24 hours after the 3-day CHOEP courses and the 2-day High dose AraC courses.

High dose cytarabine + rituximab Cytarabine 3 g/m2 i.v. x 2 for 2 days (in total four times).

Cytarabine is given as a 1-hour infusion every 12-hour in 500 ml glucose 5%. Corticosteroid eye droplets are given on days 1 – 3. Rituximab 375 mg/m2 i.v. may be given either of the two days cytarabine is given

For patients 60 – 64 years the doses are reduced as follows: High dose methotrexate Methotrexate 1,5 g/m2 i.v. as a 3 hour infusion. Folic acid

rescue (leucovorin) is given after 24 hours, see Appendix 4.

High dose cytarabine + rituximab: Cytarabine 2 g/m2 i.v. x 2 for 2 days (in total four times). Cytarabine is given as a 1-hour infusion every 12-hour in 500 ml glucose 5%. Corticosteroid eye droplets are given on days 1 – 3. Rituximab 375 mg/m2 i.v. may be given either of the two days cytarabine is given

Rituximab administration Rituximab is given as an i.v. infusion according to local routine regarding premedication, dose escalation and monitoring. Suggested guidelines: Rituximab is given as an i.v. infusion, diluted to 2 mg/ml in NaCl 0,9%. Vital signs (blood pressure, pulse, respiration and temperature) should be monitored every 15 min. during the first hour or until stable and then hourly until the infusion is discontinued and vital signs are stable. The initial dose should be 50 mg/hr for the first hour. If no adverse event is seen (see below), the dose may be escalated at 30 min. intervals with increment steps of 50 mg/hr, to a maximum of 400 mg/hr. Pneumocystis jirovecii prophylaxis Prophylaxis with trimethoprim-sulfa will be given 2 b.i.d. every Saturday and Sunday after the completion of cycle 2 and the excretion of methotrexate


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Radiotherapy Indications Radiotherapy will be given at the discretion of the individual centres. Common indications for giving radiotherapy after the completion of chemotherapy are: Bulky disease ( ≥ 10 cm) at diagnosis Localized PET positive lesions Residual disease, not eligible for biopsy at a localised site, potentially curable by radiotherapy Radiation dose

The radiation dose may be given according to the practice at the individual centres (36-45 Gy). 9.2. Toxicity and treatment modifications during therapy Anticipated rituximab toxicities and management The toxicities of rituximab are well known, and are to be handled according to local routine or according to guidelines given below. Rituximab is associated with cytokine release reactions, which may respond to adjustments in the infusion rate. An infusion related symptom complex consisting of fever and chills/rigors occurred in the majority of patients during the first infusion. Other occurring infusion-related symptoms include nausea, urticaria, fatigue, headache, pruritus, bronchospasm, dyspnoea, sensation of tongue or throat swelling (angioedema), rhinitis, vomiting, hypotension, flushing, and pain at disease sites. These reactions have generally occurred within 30 minutes to 2 hours of beginning the first infusion, and resolved with slowing or interruption of the rituximab infusion and with supportive care (IV saline, an antihistamine H1-receptor antagonist and paracetamol).

In the event that patients experience a fever spike (> 38°C), rigors, mucosal congestion or oedema, or a decline in blood pressure (> 30 mm systolic drop) during infusion with rituximab, the infusion should be temporarily discontinued and the patient observed. When the patient has recovered, the infusion should be restarted at 1/2 the previous rate. Treatment of infusion-related symptoms: an antihistamine H1-receptor antagonist and paracetamol is recommended. Additional treatment with bronchodilators or i.v. saline may be indicated. Medications for treatment of cytokine release reactions, such as epinephrine, antihistamines and corticosteroids should be available for immediate use in the event of a reaction during administration.

Infusions should be discontinued in the event of serious or life-threatening cardiac arrhythmias. Patients who develop clinically significant arrhythmias should undergo cardiac monitoring during and after subsequent infusions of rituximab. One should bear in mind that patients with pre-existing cardiac conditions including arrhythmias and angina have had recurrences of these events during rituximab therapy.


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Rituximab – dose adjustment and reporting serious adverse events There will be no reductions or escalations of the rituximab dose. If an infusion related reaction to rituximab is seen, the infusion rate of drug administration is altered as described above. The total dose administered remains the same in such cases. In case of a serious or life threatening reaction, the infusion should be terminated and such adverse event reported as described in chapter 13. If a serious or life threatening event is regarded as related to rituximab, the patient will be assigned further treatment without rituximab. Cytostatic agents Haematological and non-haematological toxicity will be graded according to the WHO Common Toxicity Criteria, (Appendix 3). In the case of vincristine associated neuropathy grade 2, the dose is reduced to 50%, and if grade 3, vincristine is stopped in all further cycles. In the case of etoposide-related grade 3-4 toxicities, etoposide may be withheld during subsequent cycles. Chemotherapy will be withheld in patients experiencing other grade 2 or 3 non-haematological toxicity besides alopecia, until the patient has recovered from the toxicity, i.e. criteria ≤ 1. In the case of nausea/vomiting, drug therapy may be continued with concomitant administration of appropriate antiemetics. If at the start of a subsequent chemotherapy course (day 15 from start of last treatment) cell numbers are too low (neutrophil counts < 1.0 x 109/l and/or platelet counts < 75 x 109/l), the next chemotherapy course should be postponed until acceptable values have been reached (neutrophils ≥ 1.0 x 109/l and/or platelets ≥ 75 x 109/l). Blood counts should be repeated no later than after three days. If acceptable values are achieved within one week, the subsequent course will be given with full dosage. If neutrophils and / or platelets counts still are low, the following dose modifications are applied, and the same reduced doses will then also be given if in following courses the values at day 15 are too low, as defined above. Reintroduction of G-CSF before next chemotherapy cycle is not recommended routinely, but should be recorded if given. Neutrophils (109/l)

Platelets (109/l)

Cyclophosphamide (%)

Doxorubicin (%)

Etoposide (%)

Vincristine (%)

Prednisone (%)

≥ 1.0 and > 75 100 100 100 100 100 *0.5-1.0 or *50 - 75 50 50 50 100 100 *< 0.5 or *< 50 0 0 0 100 100 *One week after schedule, see text above Table. The cytarabine dose is reduced to 50% if two of the three following criteria are reached (: 1) Serum creatinine level > 130 µmol/l 2) Alkaline phosphatase (ALP) > 3 times upper limit and 3) age > 40 years (27-28). When dose reductions otherwise are considered, one of the members of the writing committee should be contacted.


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Methotrexate. If serum creatinine is increased, analysis of glomerular filtration rate (GFR) is recommended. If GFR is lower than 60 ml/min, methotrexate administration is withheld. If GFR is ≥ 60, next course is given as planned, otherwise withheld. Criteria for discontinuation of study treatments In the absence of unacceptable toxicity or other cause for discontinuation (see below), patients will receive study treatment as outlined above. The following events are deemed sufficient cause to terminate study treatment. Severe (grade 4) toxicity The patients own wish to terminate study treatment If the responsible physician thinks a change of therapy would be in the best interest of the

patient

Investigations before, during and after treatment The disease status will be assessed prior to treatment start, after 3 cycles and after completion of the treatment schedule (see below). Positron Emission Tomography (PET) using F18 deoxyglucose is performed after fulfilment of treatment at least for the patients not in CR. Persistent, suspected lymphoma tissue should whenever possible be confirmed with a biopsy, otherwise the patient will be regarded as PR and second line therapy will be considered unless the lesion can be included in a limited radiation field (see schematic outline). 10. Disease assessment at trial entry (pre-therapeutic) NB: The following are minimum assessment requirements. Participating centres are free at any time to include additional investigations in accordance to local guidelines or at the discretion of the treating physician. Clinical history and full physical examination, including a recording of the WHO Performance Status. Laboratory tests: ESR, haemoglobin, leukocytes with a differential count, platelets, sodium, potassium, calcium, uric acid, glucose, creatinine, albumin, serum protein electrophoresis, IgG, IgA, IgM, LDH, ALAT, ALP and bilirubin (less than one week old). Routine urine analysis. HIV, EBV, CMV and hepatitis (A, B and C) serology (less than 4 weeks old). 5 ml serum and plasma and 2 x 5 ml whole blood (see Appendix 5) to be kept frozen (minus 70oC) for further analysis if the patient accepts to give tissue to be stored in a bio bank (see 3.2). ECG and evaluation of the left ventricular ejection fraction by echocardiography (ECHO) or radionuclide ventriculography (MUGA scintigraphy). ECHO or MUGA are not mandatory but recommended for all patients with identified or suspected cardiovascular disease. Radiological / imaging examinations: Chest X-ray (PA/lateral); CT scan of the whole body (brain, neck, thorax, abdomen and pelvis; other radiological examination as found indicated by the treating physician). MR of the brain instead of CT is allowed. PET/CT is recommended but not mandatory. All diagnostic images should be less than 4 weeks old at registration.


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Patients with a positive CT or MR of the brain who otherwise could have been included are to be registered with initials and date of birth. Histopathology and cytology examinations: - Lymph node or extra nodal tissue biopsy for morphology, immunhistochemistry and additional

molecular analyses. Part of the biopsy should be snap frozen and kept at –700C for molecular studies, (see 3.2 and Appendix 5).

- Bone marrow aspiration and biopsy for morphological and immunhistochemical analysis (less than 2 weeks old).

- Cerebrospinal fluid examination including cell count, cytology and flow cytometric immunophenotyping (less than 2 weeks old; see Appendix 6). However, lymphoma involvement is based on a positive cytology. Specimen for flow cytometry should be suspended in preservative (see Appendix 6; “Transfix (Cytomark, http://www.cytomark.co.uk; Immunostop SL, Salamanca, Spain)“ and shipped to Dep of Pathology, Lab for flowcytometry Radiumhospitalet Pb 4953 Nydalen 0424 Oslo, Norway (see separate instructions).

- Further tissue or fluid analysis according to clinical indication such as examination of pleural effusion, ascites, liver – and endoscopic biopsies.

11. Disease assessment during and after treatment Laboratory tests

- As before treatment, but without virus serological tests, serum protein elfo. and urine analysis after the third course and within one month after last chemotherapy course and after radiotherapy (if given).

- Serum and plasma for freezing after three courses and after end of treatment and at relapse / progression if feasible (see 3.2 and Appendix 5).

Clinical and radiological assessment including CT or FDG PET/CT of sites initially involved, and bone marrow biopsy if initially involved - After the 3rd course - After the last course (within one month) of chemotherapy. PET/CT is recommended for all patients

and it should be performed at least for the patients not in CR according to CT. If possible, PET positive lesions should be further analyzed with biopsy.

- After radiotherapy (for patient given radiotherapy as part of the primary treatment) Cerebrospinal fluid immunophenotyping if initially positive by FCM - Before DepoCyte administration at cycles 3 and 5 Clinical follow up - 4x per year during the first and second year of follow-up - 2x per year during the third, fourth and fifth year of follow-up. Lab. tests as above. Radiological investigations at follow up: CT after 6, 12 and 24 months of sites initially involved. CT abdomen in all cases after 12 and 24 months. X-ray of the thorax (if CT thorax is not performed) after 6, 12 and 24 months.


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Impaired cardiac function during therapy If a cardiac insufficiency is suspected and verified, e.g. as a significant drop in LVEF, so that further treatment according to protocol is not regarded to be in the patient’s interest, the patient is treated further according to the discretion of the physician. Treatment failure If not at least a PR is achieved after 3 cycles, the patient is off study, and may go on to second line treatment and possibly high dose chemotherapy. If progressive disease is encountered at any time during therapy, the patient also is off study and treated according to local tradition. 12. Evaluation criteria and endpoints A) Evaluation criteria follow those given by Cheson et al. (25). Complete remission (CR) Disappearance of all disease-related symptoms and measurable lesions, including normalisation of other abnormal initial parameters (if any), such as biochemical abnormalities definitely assignable to NHL (e.g. s-LDH), X-rays and bone marrow. A post-treatment residual mass of any size is permitted as long as it is PET negative. Alternatively, all lymph nodes must have regressed to ≤ 1.5 cm in their largest transverse diameter and to ≤ 1.0 cm for those nodes, which were 1.1 to 1.5 cm before treatment. Partial remission (PR) Decrease of at least 50% in the sum of the product of the two largest PET positive perpendicular diameters in all measurable and evaluable lesions and disappearance of disease-related symptoms with no lesion increasing 25% or more in size and without any new lesions appearing. No change/stable disease (NC) The patient does not qualify for complete or partial remission or progressive disease. Progressive disease (PD) Increase in size of 25% or more of the product of the two largest perpendicular diameters of one or more measurable and evaluable lesions during treatment or the occurrence of new lesions. Relapse During follow-up the occurrence of relapse in patients previously registered as being in CR will be registered. The following data will be registered: 1. Relapse yes/no

if yes: 2. Date of relapse 3. Site of relapse 4. Type of relapse: - in previously involved nodal area/-s - in previously uninvolved nodal area/-s - in previously involved extra nodal area/-s

- in previously uninvolved extra nodal area/-s


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- CNS relapse and site of relapse (leptomeningeal, parenchymal, combined) - Combined systemic and CNS relapse

NB: At relapse, every centre is free to initiate further treatment according to local guidelines. At this

point the patient will exit the study, although centres are encouraged to keep sending in follow-up forms to allow establishing the overall survival time.

B) Endpoints Time to treatment failure Interval between the registration date and the date of documented progression or lack of response, first relapse, death for any reason or discontinuation/change of therapy because of toxicity, whichever occurs first. Otherwise, patients will be censored at the last date they were known to be alive. For patients not responding at any time point on study treatment, TTF is defined as 1 day. Overall survival (all causes of death) The time from the registration date to the date of death. Patients still alive or lost to follow-up are censored at the last date they were known to be alive. Disease free survival The time from the first assessment within 2 months of completion of therapy that documents response to the date of disease progression. Otherwise, the patients are censored at the last date of follow up. Patients still alive in a CR or lost to follow-up are censored at the last date they were known to be alive. Patients who die due to causes other than NHL are censored at the date of death. Cause of death During the study and after its completion the cause of death will be registered according to the following cause-of-death criteria:

1. Lymphoma 2. Treatment toxicity 3. Secondary malignancy 4. Other disease not related to 1, 2 or 3 5. Other cause

Monitoring. Monitoring of the study will be performed according to the IHC-GCP guidelines (www.vadscorner.com/internet29.html). 13. Reporting adverse events All adverse events (see “Common Terminology Criteria for Adverse Events v3.0 (CTCA)” with excerpts Appendix 3 or http://ctep.cancer.gov ) occurring during the treatment period and until the end of the last treatment administration will be reported in the treatment evaluation form. Reporting serious adverse events Adverse events which are considered as serious (SAE) are those, which result in


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Death (including those due to progressive disease) A life threatening event Unplanned hospitalisation or prolongation of hospitalisation. (Hospitalization due to fever during

neutropenia or due to red blood cell transfusions is an expected event and is not necessarily an SAE unless considered to be life threatening).

Severe/permanent disability A congenital anomaly Unexpected SAE are those of which the nature or severity is not consistent with information in the relevant source documents. During the protocol treatment all deaths and all SAE’s that are life-threatening or unexpected must be reported on CRF 10 and faxed to Protocol Secretariat, Unit for Clinical Research Support at Oslo University Hospital, Radiumhospitalet, Pb 4953 Nydalen, 0424 Oslo, Norway within 24 hours after being aware of the event (telefax number: + 47 22 50 91 99). The original form is kept in the patient’s record. Sponsor is responsible to report all SAE’s that are both serious and unexpected (SUSARS; Suspected Unexpected Serious Adverse Reactions) to the National Medical Agency within 7 days for deaths or life threatening SUSARS, within 15 days for other SUSARS; form: http://www.cioms.ch/form/cioms.pdf. In circumstances where it is not possible to submit a complete report, an initial report may be made giving only the mandatory information. Initial reports must be followed-up by a complete report within a further 7 calendar days and sent to the Protocol Secretariat, Unit for Clinical Research Support at Oslo University Hospital, Radiumhospitalet, Pb 4953 Nydalen, 0424. All SAE Reports must be dated and signed by the responsible investigator or one of his/her authorised staff members. At any time after the completion of protocol treatment, unexpected Serious Adverse Events that are considered to be possibly related to protocol treatment and ANY death (regardless the cause) must also be reported using the same procedure, within 7 days after the SAE or death was known to the investigator. The investigator will decide whether the serious adverse event is related to the treatment (i.e. unrelated, unlikely, possible, probable, definitely and not assessable) and the deception will be recorded on the serious adverse event form. The investigator, using the following definitions, makes the assessment of causality:

RELATIONSHIP DESCRIPTION UNRELATED There is no evidence of any causal relationship UNLIKELY There is little evidence to suggest a causal relationship (e.g. the event did

not occur within a reasonable time after administration of the trial medication). There is another reasonable explanation for the event (e.g. the patients clinical condition, other concomitant treatments).

POSSIBLE There is some evidence to suggest a causal relationship (e.g. the event occurred within a reasonable time after administration of the trial medication). However, the influence of other factors may have contributed to the event (e.g. the patient’s clinical condition, other concomitant treatments).

PROBABLE There is evidence to suggest a causal relationship and the influence of other factors is unlikely.

DEFINITELY There is clear evidence to suggest a causal relationship and other possible


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contributing factors can be ruled out. NOT ASSESSABLE There is insufficient or incomplete evidence to make a clinical judgement

of the causal relationship. The Office for Clinical Research will forward all SAE reports within 24 hours of receipt to the study co-ordinator and the study central data manager. 14. Statistical considerations Statistics Endpoints and interim analysis The primary endpoints are three – year - time to treatment failure (TTF), and CNS relapse rate at 1.5 years. An interim analysis is planned after 40 patients have completed treatment and follow-up for response evaluation. If less than 60% of the patient respond, the study may be stopped. This might bias the final estimate, but since the level 60% is much lower than expected, the bias will probably be quite small. Analysis The main analysis will include those registered patients that have started the protocol treatment. 1. TTF as well as the secondary endpoints Overall Survival and Time to Progression, will be presented using Kaplan-Meier curves and the effect of clinical and biological prognostic factors will be analysed by means of Cox regression. Toxicity and incidence of CNS-relapse will be shown in descriptive tables. The study results will be compared to the recent CRY 04 study. The aims of the study are to at least achieve the 3 year TTF of the CRY04 study and with a substantial reduction of the CNS relapse rate with the use of early systemic and intrathecal CNS prophylaxis. A reduced CNS relapse rate in a phase II study with a historical control will not prove the importance of prophylaxis and should ideally be confirmed in an adequately sized phase III study. Such a study cannot be performed within the Nordic community within a reasonable time. Internationally it has not been possible to agree upon a treatment plan for a multicentre prospective randomized study due lack of conformity concerning treatment recommendations for this treatment group. In a recent study performed by the Nordic lymphoma group for the same patient population, including 156 patients, 3-year TTF was 67% (95% CI +/- 0.0.074). The sample size of 170 patients is chosen for this study so that a TTF will be estimated with a 95% CI of approximately 0.075. For a 50% reduction in CNS relapse to be statistically significant, more than 400 patients need to be included. This is not feasible within the Nordic area within a reasonable time frame. However, a comparison to a large US phase II study with similar inclusion criteria (see below), using i.t. CNS chemoprevention with DepoCyte, but without systemic CNS prophylaxis will be of interest. Further, the clinical significance of the ability to detect minimal involvement of the csf by FCM analysis will be evaluated.


26

Thus, the following sub analysis will be performed: -The relative frequencies of CNS relapse for patients with flow cytometry positive versus negative csf at initial staging. -CNS relapse frequencies of patients with the following risk factors as being the inclusion criteriae of a parallel US phase II study (being the same inclusion criteriae as for Swedish centres):

a. Elevated LDH and b. Involvement of more than one extranodal site

OR Disease at one of the following specific anatomic sites portending high risk of CNS relapse: testicular, orbital or sinus with destruction of bone, epidural, large cell infiltration of the bone marrow

Note though, that besides random errors, there are also systematic differences that can lead to bias, when using historical controls. Some of these will be accounted for by adjusting for known prognostic factors, e.g. using stratification or logistic regression analysis. Separate analyses will also be performed for those patients who have got treatment according to the protocol. 15. Ethical considerations Patient protection The responsible investigator will ensure that this study is conducted in agreement with either the declaration of Helsinki (Tokyo, Venice and Hong Kong amendments), or the laws and the regulations of the countries, whichever provide the greatest protection of the patient. The protocol has been written, and the study will be conducted according to the guidelines for Good Clinical Practice issued by the European Union. As a pre-requirement for implementation, the protocol will have to be approved by the local, regional or national Ethical Review Boards according to the existing national and local regulatory requirements. Informed consent All patients will be informed of the aims of the study, including the possible adverse events, the procedures involved and the possible hazards to which he/she will be exposed. They will be informed as to the strict confidentiality of their data, but that their medical records will be reviewed for trial purposes by authorised individuals other than their treating physician. It will be emphasised that the participation is voluntary and that the patient is allowed to refuse further participation in the protocol whenever he/she wants. This will not prejudice the patient’s subsequent care. Documented informed consent must be obtained for all patients included in the study before they are registered. In accordance with the guidelines of Good Clinical Practice the “consent must be documented either by the subject’s dated signature or by the signature of an independent witness who records the subject’s consent”.

16. Publication policy The writing committee, whose members are the same as those of the NLG-appointed Working Group on diffuse large B-cell lymphomas, is expected to contribute with the planning and writing of the protocol, and with the analysis and interpretation of the data. The chairman of the writing committee will be responsible for writing the first draft of the manuscript reporting treatment results and for editing the


27

manuscript according to the critical review by the writing committee. Co-authors will be the members of the NLG Working Group on diffuse large B-cell lymphomas and other investigators significantly contributing to the study. All manuscripts with any relation to the present protocol must be circulated for general information and comments to the members of the NLG Working Group on diffuse large B-cell lymphomas prior to submission for publication. All manuscripts will be distributed to the participating centres for the possibility of comments before submission. All study contributors (i.e. local investigators and other physicians including patients regardless of the number of patients included), will be listed in a special section of the article/-s. Reports focusing mainly, or entirely, on the results of special studies (e.g. molecular analyses) will be written by the person in charge of the specific analyses (first or last author). Co-authors will be the members of the NLG DLBCL working group and other investigators significantly contributing to the generation of data. Manuscripts based on this protocol will be written according to the fifth edition of the “Vancouver System: Uniform Requirements for Manuscripts Submitted to Medical Journals” (1997).


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17. References 1. IPI-Project. A predictive model for aggressive non-Hodgkin's lymphoma. The International Non-

Hodgkin's Lymphoma Prognostic Factors Project. N Engl J Med. 1993 Sep 30;329(14):987-94. 2. Cooper IA, Wolf MM, Robertson TI, Fox RM, Matthews JP, Stone JM, et al. Randomized

comparison of MACOP-B with CHOP in patients with intermediate-grade non-Hodgkin's lymphoma. The Australian and New Zealand Lymphoma Group. J Clin Oncol. 1994 Apr;12(4):769-78.

3. Fisher RI, Gaynor ER, Dahlberg S, Oken MM, Grogan TM, Mize EM, et al. Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin's lymphoma. N Engl J Med. 1993 Apr 8;328(14):1002-6.

4. Jerkeman M, Anderson H, Cavallin-Stahl E, Dictor M, Hagberg H, Johnson A, et al. CHOP versus MACOP-B in aggressive lymphoma--a Nordic Lymphoma Group randomised trial. Ann Oncol. 1999 Sep;10(9):1079-86.

5. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med. 2002 Jan 24;346(4):235-42.

6. Feugier P, Van Hoof A, Sebban C, Solal-Celigny P, Bouabdallah R, Ferme C, et al. Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d'Etude des Lymphomes de l'Adulte. J Clin Oncol. 2005 Jun 20;23(18):4117-26.

7. Habermann TM, Weller EA, Morrison VA, Gascoyne RD, Cassileth PA, Cohn JB, et al. Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J Clin Oncol. 2006 Jul 1;24(19):3121-7.

8. Pfreundschuh M, Schubert J, Ziepert M, Schmits R, Mohren M, Lengfelder E, et al. Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60). Lancet Oncol. 2008 Feb;9(2):105-16.

9. Sonneveld P, van Putten W, Biesma DH, Holte H, van Marwik Kooij M, Kramer MH, et al. Phase III trial of 2-weekly CHOP with rituximab for aggressive B-cell non-Hodgkin's lymphoma in elderly patients. Blood (ASH Meeting Abstracts). 2006;108:210.

10. Pfreundschuh M, Trumper L, Osterborg A, Pettengell R, Trneny M, Imrie K, et al. CHOP-like chemotherapy plus rituximab versus CHOP-like chemotherapy alone in young patients with good-prognosis diffuse large-B-cell lymphoma: a randomised controlled trial by the MabThera International Trial (MInT) Group. Lancet Oncol. 2006 May;7(5):379-91.

11. Gisselbrecht C, Lepage E, Molina T, Quesnel B, Fillet G, Lederlin P, et al. Shortened first-line high-dose chemotherapy for patients with poor-prognosis aggressive lymphoma. J Clin Oncol. 2002 May 15;20(10):2472-9.

12. Kaiser U, Uebelacker I, Abel U, Birkmann J, Trumper L, Schmalenberg H, et al. Randomized study to evaluate the use of high-dose therapy as part of primary treatment for "aggressive" lymphoma. J Clin Oncol. 2002 Nov 15;20(22):4413-9.

13. Sehn LH, Donaldson J, Chhanabhai M, Fitzgerald C, Gill K, Klasa R, et al. Introduction of Combined CHOP Plus Rituximab Therapy Dramatically Improved Outcome of Diffuse Large B-Cell Lymphoma in British Columbia. J Clin Oncol. 2005 Aug 1;23(22):5027-33.

14. Pfreundschuh M, Trumper L, Kloess M, Schmits R, Feller AC, Rudolph C, et al. Two-weekly or 3-weekly CHOP chemotherapy with or without etoposide for the treatment of young patients with


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good-prognosis (normal LDH) aggressive lymphomas: results of the NHL-B1 trial of the DSHNHL. Blood. 2004 Aug 1;104(3):626-33.

15. Bos GM, van Putten WL, van der Holt B, van den Bent M, Verdonck LF, Hagenbeek A. For which patients with aggressive non-Hodgkin's lymphoma is prophylaxis for central nervous system disease mandatory? Dutch HOVON Group. Ann Oncol. 1998 Feb;9(2):191-4.

16. Hollender A, Kvaloy S, Nome O, Skovlund E, Lote K, Holte H. Central nervous system involvement following diagnosis of non-Hodgkin's lymphoma: a risk model. Ann Oncol. 2002 Jul;13(7):1099-107.

17. van Besien K, Ha CS, Murphy S, McLaughlin P, Rodriguez A, Amin K, et al. Risk factors, treatment, and outcome of central nervous system recurrence in adults with intermediate-grade and immunoblastic lymphoma. Blood. 1998 Feb 15;91(4):1178-84.

18. Haioun C, Besson C, Lepage E, Thieblemont C, Simon D, Rose C, et al. Incidence and risk factors of central nervous system relapse in histologically aggressive non-Hodgkin's lymphoma uniformly treated and receiving intrathecal central nervous system prophylaxis: a GELA study on 974 patients. Groupe d'Etudes des Lymphomes de l'Adulte. Ann Oncol. 2000 Jun;11(6):685-90.

19. Boehme V, Schmitz N, Zeynalova S, Loeffler M, Pfreundschuh M. CNS events in elderly patients with aggressive lymphoma treated with modern chemotherapy (CHOP-14) with or without rituximab: an analysis of patients treated in the RICOVER-60 trial of the German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL). Blood. 2009 Apr 23;113(17):3896-902.

20. Boehme V, Zeynalova S, Kloess M, Loeffler M, Kaiser U, Pfreundschuh M, et al. Incidence and risk factors of central nervous system recurrence in aggressive lymphoma--a survey of 1693 patients treated in protocols of the German High-Grade Non-Hodgkin's Lymphoma Study Group (DSHNHL). Ann Oncol. 2007 Jan;18(1):149-57.

21. Holte H, Leppä S, Björkholm M, Flüge E, Jyrkkiö S, Delabie J, et al. R-CHOEP-14 X 6 Followed by Systemic CNS Prophylaxis for Diffuse Large B-Cell Lym-phoma (DLBCL)/Follicular Lymphoma (FL) Grade 3 with Age Adjusted IPI Score 2–3: Preliminary Results of a Nordic Lymphoma Group (NLG) Phase 2 Study Including 160 Patients Aged 18- 64 Years. Blood. 2008;112:Abstract 3604.

22. Hegde U, Filie A, Little RF, Janik JE, Grant N, Steinberg SM, et al. High incidence of occult leptomeningeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lymphomas at risk for central nervous system involvement: the role of flow cytometry versus cytology. Blood. 2005 Jan 15;105(2):496-502.

23. Quijano S, Lopez A, Manuel Sancho J, Panizo C, Deben G, Castilla C, et al. Identification of leptomeningeal disease in aggressive B-cell non-Hodgkin's lymphoma: improved sensitivity of flow cytometry. J Clin Oncol. 2009 Mar 20;27(9):1462-9.

24. Swerdlow SH, Campo E, Harris NL, Jaffe E, Pileri A, Stein H, et al. WHO Classification of tumours of hematopoietic and lymphoid tissues. Lyon: IARCH Press. 2008(4th Edition).

25. Cheson BD, Pfistner B, Juweid ME, Gascoyne RD, Specht L, Horning SJ, et al. Revised response criteria for malignant lymphoma. J Clin Oncol. 2007 Feb 10;25(5):579-86.

26. Pfreundschuh M, Trumper L, Kloess M, Schmits R, Feller AC, Rube C, et al. Two-weekly or 3-weekly CHOP chemotherapy with or without etoposide for the treatment of elderly patients with aggressive lymphomas: results of the NHL-B2 trial of the DSHNHL. Blood. 2004 Aug 1;104(3):634-41.

27. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000 Feb 3;403(6769):503-11.

28. Jais JP, Haioun C, Molina TJ, Rickman DS, de Reynies A, Berger F, et al. The expression of 16 genes related to the cell of origin and immune response predicts survival in elderly patients with


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diffuse large B-cell lymphoma treated with CHOP and rituximab. Leukemia. 2008 Oct;22(10):1917-24.

29. Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med. 2008 Nov 27;359(22):2313-23.

30. Rosenwald A, Wright G, Chan WC, Connors JM, Campo E, Fisher RI, et al. The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma. N Engl J Med. 2002 Jun 20;346(25):1937-47.


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CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05)

The Nordic Lymphoma Group Appendix 1 Nordic Lymphoma Group Investigators INVESTIGATORS – SWEDEN Dr Herman Nilsson-Ehle Dr Hans Hagberg Dept of Medicine Dept of Oncology Sahlgrenska University Hospital Uppsala University Hospital SE 416 85 Gothenburg, SE 751 85 Uppsala Sweden Sweden Phone +46-703468787 Phone +46 18 611 00 00 [email protected] [email protected] Dr Marie Nordström Dr Mats Jerkeman Center of Haematology Dept of Oncology Karolinska University Hospital University Hospital SE 171 76 Stockholm SE 221 85 Lund Sweden Sweden Phone +46 8 517 700 00 Phone + 46 46 17 10 00 [email protected] [email protected] Dr Franz Rommel Dr Eva Mrazek Dept of Hematology Department of Oncology University Hospital Karlstad Central hospital SE 581 85 Linköping SE 651 85 Karlstad SWEDEN SWEDEN Phone +46 13 22 20 00 Phone +46 54 61 50 00 [email protected] [email protected] Dr Martin Erlanson Dr. Lena Brandefors Dept of Medicine Dept of Medicine Norrlands University Hospital Sunderby Hospital SE 901 85 Umeå SE 971 80 Luleå Sweden Sweden Phone +46 90 785 00 00 Phone +46 920 28 20 00 [email protected] [email protected]


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CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group INVESTIGATORS - NORWAY Dr. Øystein Fluge Dept of Oncology Helse Bergen HF Haukeland sykehus N 5021 Bergen Norway Phone +47 55 97 20 10 E-mail: [email protected] Dr Harald Holte Dr Unn Merete Fagerli Dept of Oncology, Cancer Clinic Dep of Oncology Oslo University Hospital St. Olavs hospital HF Montebello 0310 Oslo N 7006 Trondheim Norway Norway Phone +47 22 93 40 00 Phone +47 73 86 78 36 E-mail: [email protected] E-mail: [email protected] Dr Peter Meyer Dr Martin Maisenhölder Dept of Haematology and Dept of Oncology Oncology Universitetssykehuset i Nord-Norge HF Helse Stavanger HF sykehuset N 9038 Tromsö 4011 Stavanger Norway Norway Phone +47 73 86 78 36 Phone +47 77 62 60 00 E-mail: [email protected] E-mail: [email protected]


33

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group INVESTIGATORS – FINLAND Dr. Sirpa Leppä, Dr. Tuula Lehtinen Dept Oncology Dept Oncology Helsinki University Central Hospital, P.O.Box 180 Tampere University Hospital. P.O.Box 2000 FIN-00029 HUCH FIN-33521 Tampere Finland Finland Phone: (358) 9 4711 Phone: (358) 3 247 3322 Fax: (358) 9 471 73181 Fax: (358) 3 247 3003 E-mail: [email protected] E-mail: [email protected] Dr. Sirkku Jyrkkiö Dr. Outi Kuittinen Dept Oncology and Radiotherapy Dept of Oncology Turku University Central Hospital, P.O. Box 52 Oulu University Hospital, P.O.Box 22 FIN-20521 Turku FIN-90220 Oulu Finland Finland Phone: (358) 2 313 00 00 Phone: (358) 8-315 2011 Fax: (358) 2 313 2809 Fax: (358) 8-31 53 127 E-mail: [email protected] E-mail: [email protected] Dr. Esa Jantunen Dr. Kaija Vasala Dept Medicine Dept of Oncology Kuopio University Hospital, P.O.Box 1777 Jyväskylä Central Hospital FIN-70211 Kuopio FIN-40620 Jyväskylä Finland Finland Phone: (358) 17 173 311 Phone : Fax: (358) 17 172 218 Fax: E-mail: [email protected] E-mail: [email protected]


34

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group INVESTIGATORS – DENMARK Dr. Bo Amdi Dept Haematology Odense University Hospital, Sdr. Boulevard 29, DK 5000 Odense, Denmark Phone: Fax: e-mail: [email protected] Dr. Judit Jørgensen Dept. Haematology, Aarhus University Hospital, Tage Hansens gade 2, DK-8000 Aarhus, Denmark Phone: +4589497511 Fax: e-mail: [email protected] Dr. Peter Brown Dept. Haematology L-4042 Rigshospitalet Blegdamsvej 9, 2100 København Ø Phone: +4535453545 e-mail: [email protected] Dr. Lars Munksgaard Dept. Of Haematology Roskilde Hospital 4000 Roskilde Phone: +45 4732 4811 e-mail: [email protected]


35

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group Appendix 2

WHO Performance Status Scale

Grade Performance status 0 Able to carry out all normal activities without restriction 1 Restricted in physically strenuous activity but ambulatory and able to carry out light work 2 Ambulatory and capable of all self-care but unable to carry out any work; up and about more than 50% of waking hours 3 Capable of only limited self-care; confined to bed or chair more than 50% of

waking hours

4 Completely disabled; cannot carry on any self-care; totally confined to bed or chair


36

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group Appendix 3 Common Terminology Criteria for Adverse Events v.3.0 (CTCAE) Grade Grade Grade Grade Grade 0 1 2 3 4 Hematological (adults) Hemoglobin LLN-10.0g/dL <10.0-8.0g/dL <8.0-6.5g/dL <6.5 g/dL Leukocytes (1000 mmol/mm³) 3.0-3.9 2.0-2.9 1.0-1.9 <1.0 Granulocytes (1000 mmol/mm³) 1.5-1.9 1.0-1.4 0.5-0.9 <0.5 Platelets (1000 mmol/mm³) 75-99 50-74 25-49 <25 Haemorrhage none petechiae mild blood loss gross blood loss debilitating blood loss Gastrointestinal Bilirubin > 1-1.5 x Na 1.5-3.0 x Nª 3.0-10 x Nª > 10 x Nª Aminotransferases (ASAT, ALAT >1-2.5 x Nª 2.6-5 x Nª 5.1-20 x Nª > 20 x Nª Alkaline phosphatase >1-2.5 x Nª >2.5-5 x Nª >5-20 x Nª > 20 x Nª Oral no change soreness/ erythema, ulcers; ulcers; requires alimentation erythema can eat solids liquid diet only not possible Nausea/vomiting none nausea transient vomiting requiring intractable extra therapy vomiting Nausea/vomiting with none nausea transient vomiting requiring intractable Prophylactic antiemetics vomiting extra therapy vomiting Diarrhoe none transient tolerable intolerable haemorrhagic < 2 days but >2 days requiring therapy dehydration Renal Blood creatinine >1-1.5 x Na >1.5-3.0 x Na >3.0-6 x Na >6 x Na 1+ 2-3+ 4+ Proteinuria no change < 0.3 g% 0.3-1.0 g% >1.0 g% nephrotic < 3 < 3-10 g/l > 10 g/l syndrome Haematuria no change microscopic gross gross + clots obstructive uropathy Pulmonary no change mild symptoms excertional dyspnoea at rest complete bed dyspnoe rest required Fever with drug none fever < 38 °C fever 38°C-40°C fever > 40°C fever with hypotension Allergic no change oedema bronchospasm; bronchospasm anaphylaxis no parenteral parenteral the- therapy rapy required


37

Appendix XI continued WHO RECOMMENDATIONS FOR GRADING OF ACUTE AND SUBACUTE TOXIC EFFECTS Grade Grade Grade Grade Grade 0 1 2 3 4 Cutaneous no change erythema dry desquamation moist desquama-exfoliative vesiculation, tion, ulceration dermatitis; pruritus necrosis requiring

surgical intervention Hair no change minimal hair moderate, patchy complete alopecia non-rev loss alopecia but reversible alopecia Infection (specify site) none minor infection moderate infection major infection major infection

with hypotension

Cardiac Rhythm no change sinus tachycardia,unifocal PVC, multifocal PVC ventricular >110 at rest arterial arrhythmia tachycardia Function no change asymptomatic, transient symptomatic symptomatic but abnormal symptomatic dysfunction dysfunction cardiac sign dysfunction; no responsive to non-responsive therapy required therapy to therapy Pericarditis no change asymptomatic symptomatic; tamponade tamponade; effusion no tap required tap required surgery required Neurotoxicity State of consciousness alert transient somnolence somnolence >50% coma lethargy >50% of waking of waking hours hours Peripheral none paraesthesias severe paraes- intolerable paralysis and/or decreased thesias and/or paraesthesias tendon reflexes mild weakness and/or marked motor loss Constipationb none mild moderate abdominal distension and distension vomiting Arachnoiditis Symptomatic, not Symptomatic Symptomatic, Life-threatening; interfering with (eg.photophobia, interfering with disabling (e.g., function; med. nausea). Interfer- ADL paraplegia) intervention ing with function, indicated not with ADL Painc none mild moderate severe intractable With prophylactic none mild moderate severe intractable analgetics a: Upper limit of normal value of population under study. b: This does not include constipation resulting from narcotics c: Only treatment-related pain is considered, not disease-related pain. The use of narcotics in grading pain, depending upon the tolerance level of the patient.


38

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group Appendix 4

High dose methotrexate 3 g/m2 (1.5 g /m2 patients 60 – 64 years) with leucovorin rescue. 1. The patient should be in a good fluid balance throughout the infusion and for the following days until

serum-methotrexate is below 0.1 µmol/l. 2. The urin-pH before start and until s-methotrexate > 0.1 µmol/l should be > 7. If not, give sodium

bicarbonate, preferably as i.v. infusion. 3. Time for start of leucovorin rescue is 24 h after start of methotrexate infusion. 4. Totally at least 8 doses of leucovorin is given or until the serum level is below 0.1 µmol/l.

At least the two first doses should be given intravenously. Thereafter, leucovorin can be given orally. 5. During administration of methotrexate as few other drugs as possible should be given due to possible

interactions causing delayed mtx-excretion and increased toxicity (i.e. trimethoprim-sulfa, proton pump inhibitors)

Measurements of serum methotrexate concentrations Analyses of methotrexate concentration in serum should be performed at least after 24, 48 and 72 hours after start of methotrexate infusion and of serum-creatinine before infusion and after 24 and 48 hours and if indicated • If the serum methotrexate excretion is delayed: Give extra dose of leucovorin, increase the

diuresis and make sure that urin-pH is ≥ 7 (if not – give NaHCO3 i.v.). • If severe nephrotoxicity occurs (>50% increase of s-creatinine compared to the value before

methotrexate infusion) hydration, alkalinisation and s-methotrexate measurements must be followed with special alertness.

Leucovorin after methotrexate 1.5 and 3 g/m2 – suggested leucovorin dosage and follow up form

Date

Time

Leucovorin dose

Time after start

of Mtx (hours)

Concentration of

Mtx (µmol/l)

Aim of Mtx

conc.

(µmol/l)

Serum-

creatinine

(µmol/l)

30 mg iv 24 <5

30 mg iv 36 XXXXX XXXXX

15 mg po 42 XXXXX XXXXX

15 mg po 48 <0.5




15 mg po 72 < 0.1


39

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group Appendix 5. Molecular studies Handling of biological material Tissue samples Part of the lymph node or extranodal tissue should be fixed in formaline, embedded in paraffin and used for histopathological diagnosis. Afterwards, material will be utilized for genetic (both germ line and genetic mutations; sequencing) and expression analyses, as well as construction of tissue microarrays using a broad panel of monoclonal antibodies and FISH. Part of the tissue material that will be used for sequencing and expression analyses should be snap frozen and stored at -70°C. Cerebrospinal fluid samples 2-4 ml of cerebrospinal fluid should be collected in a sterile test tube, centrifuged at 2,000g for 10 minutes at +4oC to remove cell debris and supernatant collected and stored at -70oC. (If a –700C-freezer is not available, use a –200C-freezer). Serum samples 5 ml of peripheral venous blood should be collected in a sterile test tube containing no anticoagulant. The tube will be stored at RT for 30-60 min after sampling to let blood clotting to take place. The sample is centrifuged at 2,000g for 10 minutes at +4°C, following which the top layer containing serum will be collected and stored at -70°C. (If a –700C-freezer is not available, use a–200C-freezer). Plasma samples 5.0 ml of peripheral venous blood will be collected in a sterile tube containing EDTA as an anticoagulant. The blood sample may be stored at +4°C for 0-120 min, followed by centrifugation at 2,000 g for 10 minutes at +4°C to separate the blood particles from plasma. The top layer containing plasma will be collected and stored at -70°C. (If a –700C-freezer is not available, use a –200C-freezer). Peripheral blood 2 x 5.0 ml of peripheral venous blood will be collected in a sterile tube containing EDTA as an anticoagulant. The sample will be frozen down at -70°C for later preparation of RNA and / or DNA. Study logistics All material should be first stored at the individual study centres and shipped on request as a frozen patch (fresh tissue, plasma, whole blood and serum), or normal mail (paraffin blocks) to the Department of Oncology, Helsinki University Central Hospital (Sirpa Leppä), or another address, which will be informed separately.


40

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group

Appendix 6. Flow Cytometric Analysis of cerebrospinal fluid (CSF) (To be shipped to Norway on “Transfix” according to instructions given elsewhere) Background information High occurrence of leptomeningeal involvement has been demonstrated in patients newly diagnosed with diffuse large B-cell lymphoma (DLBCL). Clinical features associated with CSF involvement include lymphoma in 2 or more extranodal sites, bone marrow involvement as well as the presence of circulating lymphoma cells. Currently, conventional cytology (CC) is the method of choice for the identification of leptomeningeal lymphoma. Despite its specificity, it is associated with a limited sensitivity with up to 20% to 60% false-negative results in paucicellular samples. Recent studies have shown that multiparameter flow cytometry markedly improves the sensitivity of the detection of neoplastic cells (2-5). Since the viability of cells in the CSF is rapidly decreasing after lumbar puncture, the immunophenotypic analysis of CSF requires standardized procedures for sample storage and transport, as well as labeling of the cells. Sample Collect 2-4mL CSF. It is imperative that the date as well as time of lumbar puncture is noted on the clinical request. The CSF liquid for flow cytometry analysis should be dripped directly into a tube with a conservative provided to all centers: Transfix (Cytomark, http://www.cytomark.co.uk; Immunostop SL, Salamanca, Spain): use 1/5 ratio of medium to CSF, the samples can be stored up to 48-72 hours at 4 C Selection of the antibody combination It is recommended to use one or more standardized antibody combinations tailored to the diagnosis of B lymphoma in the CSF. Four- to 8 color panels have been proposed in a consensus paper published in Current Protocols in Cytometry (4). Examples of such antibody combinations are given in table 1. The antibody combination (s) must be tested following previously described procedures (1,6). All reagents should be titrated to ensure an optimal signal to noise ratio (6). In addition, samples containing all reagents except one (fluorescence minus one (FMO) controls), samples containing the single reagents individually as well as a sample with all the reagents must be prepared to evaluate the presence of reagent interaction (1). Once the antibody combination is tested, it is recommended to use antibody cocktails in order to minimize errors during preparation.


41

Table 1. 8-, 6-, 5- and 4- color antibody panels Laser Blue Red Violet FITC PE PCX PECY7 APC APC7 PB PB 8-color panel 1. Unknown 8+λχ 56+κχ 4 19 3 + 14c 38 20 45 2. B λ CDx/Igx 19 10 κ CDx 20 45 6-color panel 1. Unknown 8+λχ 56+κχ 4+19 3 38 45 2. B λ CDx/Igx 19 10 κ 45 4-color panel 1. Unknown 8+λχ 56+κχ 4+19 3 2. B λ CDx/Igx 19/20 κ 3. B 20 10 45 19

A. Abbreviations: APC: allophycocyanin; APC7: APCCy7, B: B-cell lymphoma; FITC: fluoresceine isothyocyanate; PB: pacific blue; PE: phycoerytrine; PCX: PerCP Cy5.5: peridinin chlorophyll protein or PEC5: phycoerytrine cyanine 5 or PECy5.5: phycoerytrine cyanine 5.5, PECY7: phyoerytrine cyanine 7; PO: pacific orange B. The numbers indicate cluster of designation of the antibody or CD antigens C. A combination of two antibodies conjugated to the same fluorochrome is used; each antibody will be analyzed separately. D. Antibody combinations are taken from reference 4. Instrument Setup and Color Compensation Appropriate setup of the flow cytometer is performed according to the manufacturer´s recommendations. Software compensation, either on the instrument or off-line, must be used for multicolor compensation. The immunophenotypic analysis of white blood cells (WBC) in CSF In order to prevent cell loss in these paucicellular specimens, sample manipulation must be minimized. Therefore, specific methods have been developed for the staining of CSF cells with monoclonal antibodies to surface as well as intracytoplasmic antigens (4,5). An example of a staining procedure for labeling surface antigens is described below (4). Reagents Phosphate –buffer saline (PBS), pH 7.4, preferably filtered

Lysing buffer (commercial lysing buffer e.g. BD Bioscience or Beckman Coulter) Flow Buffer (commercial flow cytometer buffer e.g. BD Bioscience or Beckman Coulter) Rinse buffer (commercial flow cytometer rinse buffer e.g BD Bioscience or Beckman Coulter Antibody cocktail

Labeling of cell surface antigens with Moab A two –step approach is recommended for the flow cytometric analysis of WBC in CSF. First, a screenings tube as shown in table 1 can be run on 1/3 of the concentrated cell suspension. If no neoplastic B-cells are identified, the analysis is repeated on the remaining cell suspension to increase the sensitivity. If neoplastic B-cells are shown, a second analysis is performed in order to attempt a more complete characterization.

1. Add 2ml filtered PBS to the entire CSF sample and immediately centrifuge 8 minutes at 500 g, room temperature (RT). This step is sufficient to remove cytophilic antibodies which might present in some CSF samples

2. Aspirate the supernatant (do not decant in order to prevent excessive cell loss) and resuspend the cell pellet in 300 µl PBS

3. Transfer 100 µl of the concentrated cell suspension to a 12 x 75- mm tube


42

4. Add the exact volume of each of the antibody according to the appropriate Ab panel (or the exact volume of the antibody cocktail) and mix gently.

5. Incubate for 15 minutes in the dark 6. Add 2ml of lysing solution, mix gently and incubate for 5 minutes 7. Centrifuge 5 min at 500 g, room temperature 8. Aspirate the supernatant, and resuspend the cell pellet in Flow Buffer

Data acquisition

1. Clean the flow cytometer with rinse solution for 5 min, and wash with flow buffer for 5 minutes 2. Acquire a minimum of 100 000 cells or until the tube is empty. The time parameter can be included to gate out events

generated by air bubbles. Data analysis Data analysis can be performed with any of the commercial available analysis programs. A Boolean gating strategy is used to identify the malignant B-cells. Normal lymphocytes, monocytes and neutrophils present in the sample are used as positive and negative controls.

1. Define a gate of viable events using cd45 and forward light scatter 2. Identify the B-cells using a double gating strategy based on the expression of a B-cell lineage marker and side scatter

(ssc), as well as cd45 and ssc. 3. Analyze the B-cell data for phenotypic aberrancies. These may include changes in the intensity of cd19, cd20 and /or

CD22 expression, the lymphoma–specific ectopic expression of CD10 or cross-lineage expression of CD5 as well as changes in light scatter patterns. The restriction of light chain expression is analyzed in the total B-cell population as well as in the abnormal population if identified. Populations are identified as monoclonal when the kappa/ lambda ratio is above 4.0 or below .25, provided that at least 100 surface immunoglobulin positive B-cells have been measured. Please note that absence of light chain expression is observed in some cases of DLBCL.

4. The minimum cluster of abnormal B-cell events that is classified as CSF involvement varies depending on the number of parameters that is analyzed. Using a 6-color panel, a cluster of > 10 events is classified as positive. Using a 4-color analysis, a cluster of > 25 events is needed for a positive CSF. When > 10 and < 25 events are identified, the result should be reported as ‘suspicious for CSF involvement’

5. Repeat the analysis on the remaining 200µl cell suspension if no neoplastic B-cells are detected in the CSF. When the analysis is positive, a second Ab tube is run to confirm the results and for further characterization of the neoplastic B-cells

Reporting of the results A. Give a summary of the immunophenotypic results as follows:

1. Number of CD45+ events

2. Distribution / percentages of B-, T- and NK-cell + events within cd45+ events (if T- and NK-cell lineage markers are analyzed) Or Percentage of B-cell events within cd45+ events (if only B-cell markers are analyzed)

3. Percentage of neoplastic B-cells as well as of total B-cells within the cd45+ events. Report the immunophenotype of neoplastic B-cells OR No neoplastic B-cells are shown

B. Give a conclusion CSF involvement by DLBCL OR No CSF involvement by DLBCL


43

Saving of list mode files The list mode files must be saved in 2 separate copies in order to enable data reanalysis. References

1. N. Baumgarth and M. Roederer. A practical approach to multicolor flow cytometry for immunophenotyping. J of Immunological Methods 2000, 243: 77-97.

2. R Di Noto, G Scalia, G Abate et al. Critical role of multidimensional flow cytometry in detecting occult leptomeningeal disease in newly diagnosed aggressive B-cell lymphoma. Leukemia Research 2008, 32: 1196-1199.

3. U Hegde, A Filie, RF Little et al. High Incidence of occult leptomeningeal disease detected by flow cytometry in newly diagnosed aggressive B-cell lymphomas at risk for central nervous system involvement: role of flow cytometry versus cytology. Blood 2005, 105: 496-502.

4. J Kraan, Jw Gratama, C Haioun et al. Flow Cytometric Immunophenotyping of Cerebrospinal Fluid. Current

Protocols in Cytometry 2008, 6.25.1-6.25.16.

5. S Quijano, A Lopez, JM Sancho et al. Identification of Leptomeningeal Disease in Aggressive B-Cell Non-Hodgkin´s Lymphoma: Improved Sensitivity of Flow Cytometry. J Clin Oncol 2009, 27, 1-11.

6. Subira D, Castanon S, Aceituno E, et al. Flow Cytometric Analysis of Cerebrospinal Fluid Samples and its useulness

in Routine Clinical Practice. Am J Clin Pathol 2002, 117:952-958. Titering antibodies. Current Protocols in Cytometry 1997, 4.1.1-4.1.13.

CHIC protocol, , Protocol Code NLG-LBC05, EUDRACT No 2010-023125-38, Version 5 27.9.2012 ClinicalTrials.gov Identifier: NCT01325194

Appendix 7. Flow chart

Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 years with high risk (aaIPI≥2)

Diffuse Large B-Cell Lymphoma (NLG-LBC05) CHIC-STUDY

Baseline Before

each course

Before DepoCyte

cycle 3

Evaluations after 3 cycles

Before DepoCyte

cycle 5

After the last course (within

one month)

After Radiotherapy

10)

Follow up month 3

Follow up month 6

Follow up month 9

Follow up month 12

Date Clinical history x Physical examination (Height, weight, temp, bloodpressure, pulse)

x x x x x x x x x

WHO performance status x x x x x x x x x ECG x Echocardiography (ECHO) OR MUGA 1) x Chest X-ray (PA/lateral) x CT scan of brain, neck, thorax, abdomen and pelvis 2) x x 7) x 11) x 11) x 11) x 11) PET/CT (recommended but not mandatory) x x 9) Lymp node or extra nodal tissue biopsy 3) x Bone marrow aspiration and biopsy 4) x x 8) Cerebrospinal fluid examinations 5) x x 6) x 6) Routine urine analyses x Serology HIV, EBV, CMV, hepatitis (A,B and C) x Serum protein electrophoresis x ESR, haemoglobin, leukocytes with differential count, platelets, sodium, potassium, calcium, uric acid, glucose, creatinine, albumin, IgG, IgA, IgM, LDH, ALAT, ALP and bilirubin

x x x x x x x x x

CSF, Serum, plasma, whole blood (see Appendix 5 and 3.2) frozen

x

5erum, plasma (see Appendix 5 and 3.2) frozen x x 1) ECHO or MUGA are not mandatory but recommended for all patients with identified or suspected cardiovascular disease 2) Other radiological examination as found indicated by the treating phycisian 3) Morphology, immunhistochemistry and additional molecular analyses. Part of the biopsy should be snapt frozen and kept at -700C for molecular studies, (see 3.2 and Appendix 5) 4) Morphological and immunhistochemical analysis 5) Including cell count, cytology and flow cytometric immunophenotyping (see Appendix 6). However, lymphoma involvment is based on a positive cytology. 6) If initially positive 7) CT of sites initially involved 8) If initially involved 9) Is recommended for all patients. Performed at least for the patients not in CR according to CT. If possible, PET positive lesions should be further analyzed with biopsy. 10) For patients given radiotherapy as part of the primary treatment 11) CT thorax, abdomen and pelvis. Neck if involed at baseline. Remember to; collect lymphoma tissue, serum and plasma at the time of disease progression, whenever feasible (see 3.2. and Appendix 5)


45

Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 years with high risk (aaIPI≥2)

Diffuse Large B-Cell Lymphoma (NLG-LBC05)

CHIC-STUDY

Year 2 3 4 5 Month 15 18 21 24 30 36 42 48 54 60 Date: Physical examination (Height, weight, temp, bloodpressure, pulse)

x x x x x x x x x x

WHO performance status x x x x x x x x x x CT scan 2) X 11) ESR, haemoglobin, leukocytes with differential count, platelets, sodium, potassium, calcium, uric acid, glucose, creatinine, albumin, IgG, IgA, IgM, LDH, ALAT, ALP and bilirubin

x x x x x x x x x x


46

CHIC Study: Dose densified chemoimmunotherapy with early CNS prophylaxis in patients less than 65 (66?) years with high risk (aaIPI≥ 2) Diffuse Large B-Cell Lymphoma (NLG-LBC05) The Nordic Lymphoma Group

Appendix 8. PET uptake intensity score

Negative 1 no uptake 2 uptake ≤ mediastinum

Positive 3 uptake > mediastinum but ≤ liver 4 moderately increased uptake compared to liver at any

site 5 markedly increased uptake compared to liver at any site


47

APPENDIX 9

Pharmacovigilance Agreement with Mundipharma

1. Sponsor will be responsible for reporting all required safety information to the concerned regulatory authorities and concerned ethics committees in accordance with Directive 2001/20/EC of the European Parliament and of the Council of 4 April 2001 (EU Clinical Trials Directive) and ICH E6, Guideline for Good Clinical Practice and local implementing texts.

2. The clinical study report, publication in the scientific literature or other results of a final review of the safety data, respectively should be provided to Mundipharma not later than 3 months after completion of the study.


48

Appendix 10 Protocol changes Amendment I (28.11.2011) Revision: New inclusion criterion for Danish and Swedish centres: Epidural lymphoma Rationale: Epidural location of lymphoma is considered as a risk factor for CNS recurrence New text: - included in the synopsis under inclusion criteria for Danish and Swedish centres on page 7 - included in the chapter 6 on page 14. Revision: New exclusion criterion: Conditions that may be compatible with impaired cerebrospinal fluid flow Rationale: Impaired flow may increase DepoCyte toxicity and reduce efficacy New text: -included in the synopsis under exclusion criteria on page 7 -included in the chapter 7 on page 15 Revision: Addition of two rituximab courses (one extra during prephase on day -4 and one during 7th course with high-dose cytarabine) Rationale: It is by now widely accepted that high risk patients with DLBCL or FL grade 3B should receive at least eight courses of rituximab. New text: rituximab 375 mg / m2 is added -in the synopsis under the heading treatment – prephase and treatment - cycle 7 on pages 7-8 -included on the flow chart similarly on page 9, -included in the chapter 9 prephase and cycle 7 on pages 15-16, and the chapter 9.1 therapeutic regimens on pages 16-17. -Flow chart on page 9 revised accordingly. Revision: Increased dose of dexamethasone during CHOD or CHOED courses. Dexamethasone can be substituted with equipotent amount of betamethasone Rationale: dexamethasone 4 mg x 2 is recommended dose before and during DepoCyte administration, but dose not corresponding to the corticosteroid dose in the CHOP course; prednisolone 500 mg x 1. Thus, dexamethasone dose is accordingly increased to 10 mg x 2 for 5 days. Corresponding betamethasone dose is 8 mg x2 for 5 days. New text, chapter 9.1 -CHOD and CHOED courses: Dexamethasone 10 mg x 2 daily p.o. day 1-5 or betamethasone 8 mg x 2 replaces old text on page 17: dexamethasone 4 mg x 2 daily p.o. days 1-5. -DepoCyte administration: Dexamethasone 10 mg x 2 replaces dexamethasone 4 mg x 2 on page 17. Revision: Option to delay second R-CHOP course with 1-2 days. Rationale: A few patients have experienced serious mucosal toxicity, delayed methotrexate excretion and temporarily reduced renal function. This may be due to simultaneous administration of methotrexate and CHOP chemotherapy, but may also be due to administration of other drugs at the time methotrexate was given, i.e. proton ion pump inhibitors. The writing committee has decided to give the investigators the option to delay CHOP course 1-2 days. At the same time the protocol text pinpoints that as few drugs as possible should be given during methotrexate administration. New text:


49

-Synopsis, new text included under cycle 2 on page 8: R-CHOP15 is allowed to start day 16 or day 17. -Synopsis, new text on page 8: *cycles 3-7 are postponed accordingly if R-CHOP-14 is given 1-2 days after sec. H-Mtx. -Chapter 9, cycle 2, new text on page 17: R-CHOP may be delayed to day 16 or 17. -Flow chart on page 9 revised accordingly.

Revision: Revised Common Terminology Criteria for Adverse Events v.3.0 (CTCAE) (Appendix 3) Rationale: Previous minimun and/or maximum values for hemoglobin, bilirubin, aminotransferases, alkaline phosphatase and creatinine used for grading AEs have been revised in the table of Appendix 3 on page 37 New text: A revised table has been included Revision: Revised methotrexate excretion table (Appendix 4) Rationale: Previous predicted methotrexate excretion values have been revised in the table of Appendix 4 on page 39. New text: A revised table has been included Revision: Sampling of cerebrospinal fluid for freezing and subsequent study analysis (Appendix 5). Rationale: Protein analysis of cerebrospinal fluid in patients with malignant lymphoma with and without CSF involvement is a new research area and should be available for further studies. New text: Sampling of cerebrospinal fluid included in the chapter 3.3 on page 12 and Appendix 5 on page 40.

Revision: Revised instructions to handle biological material (Appendix 5) Rationale: Clarification and specification of molecular substudies according to chapter molecular background New text: -Appendix 5, page 40, chapter tissue samples: material will be utilized for genetic (both germ line and sporadic mutations; sequencing) and expression analyses, as well as construction of tissue microarrays using a broad panel of monoclonal antibodies and FISH. Part of the tissue material that will be used for sequencing and expression analyses should be snap frozen and stored at -70°C. -Appendix 5, page 40, chapter serum samples: 5 ml of peripheral venous blood should be collected in a sterile test tube containing no anticoagulant. The tube will be stored at RT for 30-60 min after sampling to let blood clotting to take place. Revision: Inclusion of signature page of sponsor and national coordinators. Rationale: signature page should be included in the protocol according to GCP regulations New text: signature page included in the protocol on page 1.


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Amendment II (27.9.2012) Revision: Administration of DepoCyte omitted from the protocol. Rationale: Deficiencies have been identified at the finished product manufacturer site of DepoCyte, in particular, a lack of assurance of the sterility in the manufacturing process giving rise to a possible sterility risk of failure. Since DepoCyte has no indication in the prophylactic setting of CNS lymphoma, and is investigational drug in the protocol, it is not ethically acceptable to continue administering the drug. When pure product will be available, DepoCyte will be reimplemented to the protocol. New text: A revised synopsis, flowchart and text do not contain any information on DepoCyte. Revision: CHOED courses changed to CHOEP (Dxm changed to Prednisone in courses 1, 3 and 5). Rational: CHOEP is the standard chemotherapy regimen, in which P corresponds to prednisone, whereas Dxm (D) was previously used in courses 3 and 5 as a pre- and postmedication for Depocyte according to instructions of the manufacturer. New text: -Revised synopsis on pages 7-8. -Revised text in chapter 9.1 on pages 16-17. Revision: Number of study population increased from 150 to 170. Rationale: It is estimated that 20 patients will be included to the study before DepoCyte can be reimplemented to the protocol and due to statistical considerations the total number of patients treated with DepoCyte should be 150. New text: - revised text in the synopsis under inclusion criteria on page 6 and 8. - revised text in the chapters 5 and 14 under Study duration and early study termination and Statistical considerations on pages 13 and 25, respectively. Revision: Inclusion criteria for Danish centres have been revised to be the same as in Finland and Norway Rationale: To unify inclusion criteria in different countries and to improve recruitment. New text: - revised text in the synopsis under inclusion criteria on page 7 - revised text in the chapter 6 on page 14. Revision: List of local PIs in Denmark has been updated (Appendix 1 on page 34).

Documents

CHIC-Study CHemoImmunotherapy with early CNS … · CHIC protocol, , Protocol Code NLG-LBC05, EUDRACT No 2010-023125-38, Version 5 27.9.2012 ClinicalTrials.gov Identifier: NCT01325194