Clinical trial NCT01234766
Conditions: Lymphoma, Follicular
Sponsor: Dartmouth-Hitchcock Medical Center
Start date: 2010-10-01; Completion date: 2021-04-01
Enrollment: 39 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
The purpose of the study is to learn about the safety and effectiveness of treating follicular lymphoma with bendamustine and rituximab followed by radioimmunotherapy (RIT) using 90-yttrium (Y) ibritumomab tiuxetan.

The researchers will also test blood and bone marrow for the BCL2 gene-Jh that is a commonly found in people with follicular lymphoma (FL) and look at how the BCL2 gene-Jh responds to the study treatment.

Bendamustine is approved by the United States Food and Drug Administration (FDA) for the treatment of chronic lymphocytic leukemia and indolent B-cell non-Hodgkin's lymphoma (NHL) that has progressed during or within six months of treatment with rituximab or a rituximab-containing treatment regimen. Bendamustine is not approved by the FDA to treat follicular lymphoma.

Rituximab is approved by the FDA for the treatment of relapsed or refractory, low-grade or follicular, CD20-positive B-cell non-Hodgkin's lymphoma.

90-yttrium (Y) ibritumomab tiuxetan is approved by the FDA for the treatment of relapsed or refractory, low-grade or follicular B-cell NHL, including rituximab refractory follicular NHL. It is also approved for the treatment of follicular NHL that is previously untreated with radioimmunotherapy and that achieved a partial or complete response to first-line chemotherapy.

Study participants will will receive bendamustine and rituximab for up to 16 weeks. If participants' cancer responds well to the treatment with bendamustine and rituximab, they will receive up to 12 weeks of radioimmunotherapy (RIT). After the RIT is complete, participants will be asked to return to the clinic every 3 months for a maximum of 10 years for follow-up visits.

Detailed description:
STUDY OBJECTIVES

Primary Objective

\- To determine the complete response (CR) rate and overall response (OR) rate \[CR + partial response (PR) rate\] to a regimen of bendamustine and rituximab (B-R), followed by radioimmunotherapy (RIT) with 90-yttrium(Y) ibritumomab tiuxetan in subjects with untreated follicular lymphoma.

Secondary Objectives

* To characterize the safety profile of bendamustine and rituximab followed by 90-yttrium(Y) ibritumomab tiuxetan in subjects with untreated follicular lymphoma
* To determine the CR and OR rate after B-R
* To determine the CR and OR rate after 90-yttrium(Y) ibritumomab tiuxetan specifically the conversions from PR to CR
* To determine the progression-free survival (PFS)
* To determine time to next treatment

Exploratory Objectives

* To determine the molecular response after B-R as determined by qualitative polymerase chain reaction (PCR) of BCL2 from blood and bone marrow examination (required after B-R)
* To determine the molecular response after 90-yttrium(Y) ibritumomab tiuxetan radioimmunotherapy from blood and bone marrow examination (required after RIT)

BACKGROUND

Follicular lymphoma

Non-Hodgkin's lymphomas (NHL) encompass a group of malignancies of lymphocytes that vary in their histologic appearance, aggressiveness and response to therapy.

According to the American Cancer Society, NHL is the 6th most common cancer, with more than 50,000 new cases per year. Follicular lymphoma (FL) is the 2nd most common type of NHL accounting for approximately 20% of newly diagnosed NHL. FL is considered an indolent, but, incurable lymphoma. The goals of therapy are to treat symptomatic advanced stage disease to induce a maximum response with minimal toxicity. The optimal treatment of advanced stage follicular lymphoma (FL) remains to be determined. Combination chemotherapy is the standard frontline treatment option for this disease and the alkylating agent cyclophosphamide has been a common backbone in these combinations. The most common treatments for FL in the United States are rituximab combinations with chemotherapy such as cyclophosphamide, vincristine and prednisone (R-CVP) and cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). The NCCN guidelines also include fludarabine-based regimens, and radioimmunotherapy.

With the addition of immunotherapy (rituximab) to chemotherapy, the overall and complete response rates have improved.1-6 Furthermore, there is suggestive evidence that overall survival may be improved.

Radioimmunotherapy (RIT) is also effective as salvage therapy for indolent lymphoma and transformed lymphoma.7-9 In the first-line setting, RIT following chemotherapy can increase the CR rate and PFS.10-12

Rationale of combining bendamustine and rituximab with consolidation 90-yttrium(Y) ibritumomab tiuxetan

As mentioned above, the combination of bendamustine plus rituximab (B-R) appears to be non-inferior to R-CHOP as first-line treatment of indolent lymphomas including follicular and mantle cell lymphomas, while showing a better tolerability profile such as less alopecia, and potentially less cardiotoxicity, making it a rational choice for first line treatment of FL.17 When given after chemotherapy radioimmunotherapy can convert partial responses to complete responses and can prolong the PFS. The Follicular Lymphoma Ibritumomab tiuxetan (FIT) trial of consolidation Yttrium-90-Ibritumomab tiuxetan versus no additional therapy after first remission in advanced follicular lymphoma showed a prolongation of PFS (36 versus 13 months) in the RIT arm.12 The PFS was prolonged regardless of PR or CR after first-line therapy. The primary treatment included CVP, CHOP, fludarabine-based, and chlorambucil, with the minority of patients receiving rituximab. The results also showed that RIT converted 77% patients from PR to CR/unconfirmed CR (CRu).

An abbreviated course of CHOP-R followed by RIT has shown promise in patients with follicular lymphoma in a phase II trial reported recently.11 Of the 60 patients entering this trial 55 patients completed all protocol therapy. The median follow up was 19.7 months (range, 0.26-35.9 months). For intent-to-treat analysis, the complete response (CR) rate after CHOP-R, as assessed by CT and PET imaging, was 40% and 46%, respectively. After RIT, the CR rate improved, as assessed by CT and PET imaging, to 82% and 89%, respectively.

In this current study, we propose a first-line regimen for untreated FL using bendamustine and rituximab (B-R) (bendamustine 90mg/m2 on days 1 and 2 and Rituximab 375mg/m2 on Day 1 of a 28-day \[+2 days\] cycle) x 4 cycles followed by RIT; Zevalin (formerly Biogen Idec/Cell Therapeutics, now Spectrum).

The advantage of this treatment is that B-R has a better side effect profile including significantly less alopecia and less infectious complications. Currently bendamustine is not FDA-approved for first-line therapy for follicular lymphoma. 90-yttrium(Y) ibritumomab tiuxetan (Zevalin) radioimmunotherapy is FDA approved for patients with previously untreated follicular non-Hodgkin's Lymphoma (NHL), who achieve a partial or complete response to first-line chemotherapy. Evidence suggests that consolidation with RIT leads to a longer PFS. Since this specific combination has not been utilized in the first-line treatment of FL, it warrants investigation in the current study.

This trial will begin to establish a standard of care for the first-line treatment of follicular lymphoma. We hypothesize that bendamustine plus rituximab followed by RIT will contribute to among the highest CR rates seen in follicular lymphoma with relatively low toxicity. Based on the results of this trial, we would aim to open a larger trial for follicular lymphoma in a cooperative group setting, i.e. CALGB.

Correlative Studies Background

The BCL2 gene-Jh rearrangement is the common abnormality in FL t(14;18). This can be assessed by various PCR techniques.18,19

Patients can be assessed for this molecular abnormality in their bone marrow at baseline and following therapy. For instance in a similar Southwest Oncology Group study of chemotherapy followed by radioimmunotherapy using tositumomab/iodine I-131 tositumomab (Bexxar) for follicular lymphoma, patients were asked to undergo serial bone marrow aspirations at study entry, 4 weeks after the sixth cycle of CHOP (just before tositumomab/iodine I-131 tositumomab), and after tositumomab/iodine I-131 tositumomab for PCR testing.20 The mononuclear cell fraction was isolated from marrow aspirates by Ficoll-Hypaque sedimentation and cryopreserved for subsequent batch analysis using a double nested PCR assay to detect the major breakpoint region and the minor cluster region of the BCL2 gene. Samples were initially analyzed by fragment size using ethidium bromide gel electrophoresis of the PCR product and then transferred to nitrocellulose membranes for confirmation of the identity of the BCL2 translocation by Southern blotting. The adequacy of samples was demonstrated using beta-globin as a positive control housekeeping gene. Patients were considered to have attained a molecular remission if their marrow sample at study entry contained a detectable t(14;18) translocation that became undetectable after protocol treatment.

Arms: arm 1: Subjects will receive bendamustine and rituximab, followed by 90-yttrium (Y) Ibritumomab Tiuxetan
Interventions: intervention 1: Bendamustine intervention 2: Rituximab intervention 3: Y-90 ibritumomab
Intervention descriptions: intervention 1: 90mg/m2, IV - Days 1 and 2 of every cycle intervention 2: 375mg/m2, IV - Cycle 1 only: Day -7 (+1 day) Day 1 of every cycle intervention 3: 0.4mCi/kg, IV - Within 4 hours of rituximab, give over 10 minutes

Protocol documents:
Prot_SAP_000.pdf: 
Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
CLINICAL STUDY PROTOCOL D1015 
 
A Multicenter, Open Label, Phase II Study of Bendamustine and Rituximab followed by 
90-yttrium (Y) Ibritumomab Tiuxetan for Untreated Follicular Lymphoma  
(Fol-BRITe study) 
 
Protocol Number: 
 
 
 
D1015 
 
Indication: 
 
 
 
 
 
Untreated Follicular Lymphoma 
 
Phase:  
 
 
 
 
 
 
II 
 
Coordinating Center: 
 
 
Dartmouth-Hitchcock Medical Center 
One Medical Center Drive 
Lebanon, NH  03756 
 
Principal Investigator: 
Frederick Lansigan, MD 
 
 
Hematology/Oncology 
Dartmouth-Hitchcock Medical Center 
One Medical Center Drive 
Lebanon, NH  03756 
Phone: 603-650-5529 
Fax: 603-650-5830 
 
e-mail: Frederick.Lansigan@hitchcock.org  
 
Co-Investigators: 
 
Bassem Zaki, MD 
 
Radiation Oncology 
 
Dartmouth-Hitchcock Medical Center 
 
One Medical Center Drive 
 
Lebanon, NH 03756 
 
Phone: (603)650-6600 
 
e-mail: Bassem.I.Zaki@hitchcock.org 
 
 
 
Dennis Cooper, MD and Francine Foss, MD 
Yale Cancer Center 
20 York Street 
New Haven, CT 06520 
Phone: 203-737-4257 
e-mail: Dennis.Cooper@yale.edu 
 
 
Francine.Foss@yale.edu  
 
Adam Olszewski, MD  
University Medicine Foundation, Inc.  
Comprehensive Cancer Center 
593 Eddy St 
Providence, RI 02903  
Phone: 401-444-5435   Fax: 401-444-5256  
e-mail: aolszewski@lifespan.org  
 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
Matthew McKinney, MD 
Medicine-Oncology 
Duke University Medical Center  
DUMC Box 3872 
Durham, NC 27710  
Phone : 919-684-8964  
e-mail : Matthew.McKinney@duke.edu 
 
Helen F. Ryan, MD 
Maine Center for Cancer Medicine 
100 Campus Drive, Suite 108 
Scarborough, ME 04074 
Phone : 207-396-7600 Fax : 207-396-7986 
e-mail : Ryanh@mccm.org 
 
Molecular Science Correlatives: 
 
Gregory J. Tsongalis, Ph.D 
Dartmouth-Hitchcock Medical Center 
One Medical Center Drive 
Lebanon, NH  03756 
Phone (603) 650-7171 
Gregory.J.Tsongalis@hitchcock.org  
 
Statistician: 
 
 
Todd A. MacKenzie  
Dartmouth-Hitchcock Medical Center 
One Medical Center Drive 
Lebanon, NH  03756 
Todd.A.MacKenzie@Dartmouth.edu  
 
 
 
 
 
Protocol History: 
Original 
07 January 2010 
Amendment 1 
06 April 2010 
Amendment 2 
04 May 2010 
Amendment 3 
03 June 2010 
Amendment 4 
08 November 2010 
Amendment 5 
13 December 2010 
Amendment 6 
19 January 2011 
Amendment 7 
26 May 2011 
Amendment 8 
29 February 2012 
Amendment 9 
06 March 2013 
Amendment 10 
19 October 2016 
Amendment 11 
06 July 2018


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 i 
SCHEMA 
Informed Consent and Pre-study Evaluations 
↓ 
Eligibility Determination  
↓ 
Registration 
↓ 
Bendamustine + Rituximab (B-R) x 4 cycles 
 
Rituximab 
375mg/m2 
IV 
Cycle 1 only: Day -7 
(+1 day)  
Day 1 of every cycle 
1 cycle = 28 days (+2 days) 
X 
4 cycles 
 
Bendamustine 
90mg/m2 
IV 
Days 1 and 2 of 
every cycle 
 
Restaging 4-6 weeks after last dose of B-R 
(Although a maximum of 6 weeks is allowed, every effort should 
be made to restage as close to 4 weeks as possible.) 
↓ 
Radioimmunotherapy with 90-yttrium (Y) Ibritumomab Tiuxetan  
(as per institutional procedures) 6-12 weeks after last dose of B-R 
(Although a maximum of 12 weeks is allowed, every effort should be made to begin RIT as close to 6 
weeks after the last cycle of B-R as possible.) 
If participant meets criteria to receive RIT and platelet count is > 150,000/mm3:  
Rituximab 
250mg/m2 
IV 
Day 1 
Rituximab 
250mg/ m2 
IV 
Day 8 (+1 day) 
Y-90 ibritumomab 
0.4mCi/kg 
IV 
Within 4 hours of rituximab, give over 10 minutes 
 
If participant meets criteria to receive RIT and platelet count is 100,000-149,000/mm3: 
Rituximab 
250mg/m2 
IV 
Day 1 
Rituximab 
250mg/ m2 
IV 
Day 8 (+1 day) 
Y-90 ibritumomab 
0.3mCi/kg 
IV 
Within 4 hours of rituximab, give over 10 minutes 
 
↓ 
 
(End of Treatment) Restaging 12-16 weeks after 90-yttrium (Y) Ibritumomab Tiuxetan 
(Although a maximum of 16 weeks is allowed, every effort should be made to restage as close to 12 weeks as possible.) 
↓ 
Long-term follow-up until relapse, progression, alternative therapy or death 
↓ 
Effective 06July2018 – Annual follow-up until death 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 ii 
TABLE OF CONTENTS 
 
Page 
 
SCHEMA ........................................................................................................................................ i 
 
1. OBJECTIVES ..........................................................................................................................1 
1.1 
Primary Objectives ......................................................................................................1 
1.2 
Secondary Objectives ..................................................................................................1 
 
2. BACKGROUND ......................................................................................................................1 
2.1 
Follicular Lymphoma ..................................................................................................1 
2.2 
Bendamustine, Rituximab and 90-yttrium(Y) ibritumomab tiuxetan ....................2 
2.3 
Rationale .......................................................................................................................4 
2.4 
Correlative Studies Background .................................................................................5 
 
3. ELIGIBILITY CRITERIA .....................................................................................................6 
3.1 
Inclusion Criteria  ........................................................................................................6 
3.3 
Exclusion Criteria ........................................................................................................7 
3.4 
Inclusion of Women and Minorities ...........................................................................7 
 
4. REGISTRATION PROCEDURES ........................................................................................8 
4.1  
General Guidelines.......................................................................................................8 
4.2  
Registration Process.....................................................................................................8 
 
5. TREATMENT PLAN ..............................................................................................................9 
5.0  
Study Design .................................................................................................................9 
5.1  
Agent Administration ................................................................................................10 
5.2  
General Concomitant Medication and Supportive Care Guidelines ....................10 
5.3 
Duration of Therapy ..................................................................................................11 
5.4 
Duration of Follow Up ...............................................................................................11 
 
6. DOSING DELAYS/DOSE MODIFICATIONS ..................................................................12 
 
6.1       Starting Dose and Dose Modifications for Bendamustine ......................................12 
 
6.2 
Inclusion Criteria Prior to RIT…………………………………………………....12 
 
6.3 
Starting Dose of 90-yttrium(Y) ibritumomab tiuxetan ..........................................12 
 
6.4 
Postponement of Bendamustine and Rituximab Treatment ..................................13 
 
6.5 
Ancillary Therapy ......................................................................................................13 
 
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ............................13 
7.1  
Adverse Events ...........................................................................................................13 
7.2  
Adverse Event Definitions and Characteristics ......................................................19 
7.3  
Expedited Adverse Event Reporting ........................................................................20 
7.4  
Safety and Data Monitoring ......................................................................................21 
7.5  
On-Site Monitoring…………………………………………………………………21 
7.6  
Drug Accountability………………………………………………………………...21 
7.7  
Investigator Compliance………………………………..………………………….21 
 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 iii 
8. PHARMACEUTICAL INFORMATION ............................................................................23 
8.1  
Bendamustine  ............................................................................................................23 
8.2  
90-yttrium (Y) ibritumomab tiuxetan ......................................................................25 
8.3  
Rituximab ...................................................................................................................26 
 
9. CORRELATIVE/SPECIAL STUDIES ...............................................................................27 
9.1 
Qualitative PCR for BCL2 translocation ................................................................27 
 
10. STUDY CALENDAR ............................................................................................................29 
 
11. MEASUREMENT OF EFFECT 
 
11.1 
Assessment of Response ............................................................................................30 
11.2 
Definitions ..................................................................................................................30 
11.3 
Methods for Evaluation of Measurable Disease .....................................................31 
 
12. STATISTICAL CONSIDERATIONS .................................................................................32 
 
APPENDIX A: Correlative Science Blood and Marrow Samples Form ................................34 
APPENDIX B: Follicular Lymphoma International Prognostic Index ..................................35 
APPENDIX C: Safety and Data Monitoring Committee…......………………………...……36 
APPENDIX D: Correlative Studies……………………………………………………………38 
APPENDIX E: Karnofsky and ECOG Performance Status Conversion Table……………44 
APPENDIX F: Cockcroft-Gault Equation……………………………………………………45 
APPENDIX G: Lymphoma Response Guidelines……………………………………………46 
 
 
REFERENCES .............................................................................................................................47 
 
 
 
 
 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
1
1. STUDY OBJECTIVES 
 
1.1. 
Primary Objective 
1.1.1. To determine the complete response (CR) rate and overall response (OR) rate [CR 
+ partial response (PR) rate] to a regimen of bendamustine and rituximab (B-R), 
followed by radioimmunotherapy (RIT) with 90-yttrium(Y) ibritumomab tiuxetan 
in subjects with untreated follicular lymphoma. 
 
1.2. 
Secondary Objectives 
1.2.1. To characterize the safety profile of bendamustine and rituximab followed by 90-
yttrium(Y) ibritumomab tiuxetan in subjects with untreated follicular lymphoma  
1.2.2. To determine the CR and OR rate after B-R 
1.2.3. To determine the CR and OR rate after 90-yttrium(Y) ibritumomab tiuxetan 
specifically the conversions from PR to CR 
1.2.4. To determine the progression-free survival (PFS) 
1.2.5. To determine time to next treatment (TTNT) 
 
1.3. 
Exploratory Objectives 
1.3.1. To determine the molecular response after B-R as determined by qualitative 
polymerase chain reaction (PCR) of BCL2 from blood and bone marrow 
examination (required after B-R)  
1.3.2. To determine the molecular response after 90-yttrium(Y) ibritumomab tiuxetan 
radioimmunotherapy from blood and bone marrow examination (required after 
RIT) 
 
2. BACKGROUND 
 
2.1  
Follicular lymphoma 
 
Non-Hodgkin’s lymphomas (NHL) encompass a group of malignancies of lymphocytes that 
vary in their histologic appearance, aggressiveness and response to therapy. 
According to the American Cancer Society, NHL is the 6th most common cancer, with more 
than 50,000 new cases per year. Follicular lymphoma (FL) is the 2nd most common type of 
NHL accounting for approximately 20% of newly diagnosed NHL. FL is considered an 
indolent, but incurable lymphoma. The goals of therapy are to treat symptomatic advanced 
stage disease to induce a maximum response with minimal toxicity. The optimal treatment of 
advanced stage follicular lymphoma (FL) remains to be determined. Combination 
chemotherapy is the standard frontline treatment option for this disease and the alkylating 
agent cyclophosphamide has been a common backbone in these combinations. The most 
common treatments for FL in the United States are rituximab combinations with 
chemotherapy such as cyclophosphamide, vincristine and prednisone (R-CVP) and 
cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). The NCCN 
guidelines also include fludarabine-based regimens, and radioimmunotherapy.  
 
With the addition of immunotherapy (rituximab) to chemotherapy, the overall and complete 
response rates have improved.[1-6] Furthermore, there is suggestive evidence that overall 
survival may be improved. 
 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
2
Radioimmunotherapy (RIT) is also effective as salvage therapy for indolent lymphoma and 
transformed lymphoma.[7-9] In the first-line setting, RIT following chemotherapy can 
increase the CR rate and PFS.[10-12] 
 
2.2  
Bendamustine, Rituximab and 90-yttrium(Y) ibritumomab tiuxetan 
 
2.2.1 Bendamustine and Rituximab 
 
The chemical structure of bendamustine includes a bifunctional nitrogen mustard group, a 
benzimidazole ring, and a butyric acid side chain. It exhibits increased potency compared 
to other alkylators and has been shown to be active against cell lines which are resistant 
to other alkylating agents in pre-clinical experiments in vitro. These properties may be 
explained by a mechanism or mechanisms of action other than alkylation of DNA, 
possibly contributed by the presence of the benzimidazole ring and/or butyrate side chain 
or as a result of enhanced in vivo stability of the alkylating functionality. 
 
Bendamustine has been used in Germany for over twenty years for the treatment of 
non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, Hodgkin’s disease, multiple 
myeloma, and breast cancer. However, the drug has been relatively unknown in the 
United States until recent publications.[13] 
 
Bendamustine as a single agent for patients who have rituximab-refractory non-
Hodgkin’s lymphoma has shown efficacy. Friedberg et al. showed an overall response 
rate of 77% (15% complete response, 19% unconfirmed complete response, and 43% 
partial response).[14] The median duration of response was 6.7 months, 9.0 months in 
patients with indolent disease, and 2.3 months in patients with transformed disease. The 
most frequent nonhematologic adverse events included nausea, vomiting, fatigue, 
constipation, anorexia, fever, cough, and diarrhea. Grade 3 or 4 reversible hematologic 
toxicities included neutropenia (54%), thrombocytopenia (25%), and anemia (12%). 
Overall, single-agent bendamustine produced durable objective responses with acceptable 
toxicity in heavily pretreated patients with rituximab-refractory indolent NHL. 
 
Several randomized trials have shown that the addition of rituximab to chemotherapy 
results in higher response rates and longer time to progression and event-free survival in 
first-line FL or first relapse.  For example, a randomized study of 8 cycles of 
cyclophosphamide, vincristine and prednisone (CVP) or R-CVP showed that the overall 
response rate (OR) and complete response rate (CR) were 81% and 41% in the R-CVP 
arm compared to 57% and 10% in the CVP only arm, respectively.[5, 6]   Median time to 
treatment failure was 27 months in patients receiving R-CVP and 7 months in the CVP 
arm.  This trial also showed a trend toward improvement in overall survival (OS) in the 
R-CVP arm.  Similarly, the German Low-Grade Lymphoma Study Group (GLGLSG) 
showed in a randomized trial that R-CHOP was superior to CHOP with improved 
response rates (97% vs 90%), and longer time to treatment failure (not reached vs. 31 
months).[4]  Thus, it is widely accepted that rituximab added to chemotherapy is better 
than chemotherapy alone.  The choice of a first-line regimen is highly variable, however.  
While fludarabine based-regimens are also highly effective in the first-line setting as well 
as in relapsing patients (Table 1), stem cell mobilization may be impaired for future 
autologous stem cell transplants.[1, 2, 15] 
 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
3
Due to clinical data showing activity of the combination of bendamustine plus rituximab 
in non-Hodgkin’s lymphoma, a phase II study evaluated the combination of 
bendamustine plus rituximab in indolent non-Hodgkin’s lymphoma including follicular 
and mantle cell histologies.[16] The study by Robinson et al. evaluated bendamustine 
plus rituximab in 67 adults with relapsed, indolent B-cell or mantle cell lymphoma 
without documented resistance to prior rituximab. Patients received rituximab 375 mg/ 
m2 intravenously on day 1 and bendamustine 90 mg/m2 intravenously on days 2 and 3 of 
each 28-day cycle for four to six cycles. The overall response rate was 92% (41% 
complete response, 14% unconfirmed complete response, and 38% partial response). 
Median duration of response was 21 months and median progression-free survival time 
was 23 months. Outcomes were similar for patients with indolent or mantle cell 
histologies. The primary toxicity was myelosuppression (grade 3 or 4 neutropenia, 36%; 
grade 3 or 4 thrombocytopenia, 9%).  
 
For the first-line treatment of indolent lymphomas, the combination of bendamustine plus 
rituximab (B-R) was compared to R-CHOP.  Rummel et al. showed that B-R is non-
inferior to R-CHOP as first-line treatment of indolent lymphomas including follicular and 
mantle cell lymphomas, while showing a better tolerability profile such as less alopecia, 
and potentially less cardiotoxicity.[17]   A total of 546 patients were randomized to B-R 
vs. CHOP-R x 6 cycles.  The primary endpoint, progression-free survival (PFS), was not 
reached for the B-R arm and was 39 months in the R-CHOP arm. This difference was not 
statistically significant.  The overall response rate was 94% vs 93% respectively, and the 
rate of CR was similar, 41% versus 33%. In this trial, 52% of patients had FL.   
 
 
2.2.2 90-yttrium(Y) ibritumomab tiuxetan 
 
Technical advances have made it possible to link radionuclides such as yttrium-90 (90Y) 
to monoclonal antibodies to specifically target radiation to lymphoma cells. Yttrium-90 is 
a beta-emitting radionuclide that delivers 90% of its radiation (2.3 MeV) over a mean 
path length of 5 mm and has a half-life of 64 hours. These characteristics are particularly 
advantageous for treating bulky, poorly vascularized tumors and those with 
heterogeneous antigen expression.  
Ibritumomab tiuxetan (Zevalin; Spectrum Pharmaceuticals) is a short-course 
radioimmunotherapy that uses immunobiologic and radiolytic mechanisms of action to 
destroy both dividing and nondividing tumor cells. Ibritumomab is the murine, parent 
anti-CD20 antibody that was engineered to develop rituximab. Tiuxetan is a 
linker/chelator covalently attached to ibritumomab. Ibritumomab tiuxetan can chelate 
indium-111 (111In) for imaging or 90Y for therapy. Thus, the antibody specifically targets 
radiation to CD20+ cells while sparing normal nonlymphoid cells.  
Because rituximab immunotherapy is widely used to treat patients with NHL, a critical 
issue is whether patients treated previously with rituximab can respond to ibritumomab 
tiuxetan. This study tested the hypothesis that tumor resistance to an unconjugated anti-
CD20 antibody alone can be overcome by subsequent CD20-directed 
radioimmunotherapy. 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
4
 
Witzig et al. showed that ibritumomab tiuxetan radioimmunotherapy is effective in 
rituximab-refractory patients.[9] Eligible patients were refractory to rituximab; this was 
defined as no objective response to rituximab (375 mg/m2 weekly for 4 weeks) or time to 
progression (TTP) of = <6 months. The ibritumomab tiuxetan treatment regimen 
consisted of pretreatment with rituximab (250 mg/m2) intravenously on days 1 and 8 to 
deplete peripheral blood B cells, then yttrium-90 ibritumomab tiuxetan (0.4 mCi/kg; 
maximum, 32 mCi) intravenously on day 8, administered on an outpatient basis. An 
imaging/dosimetry dose of indium-111 ibritumomab tiuxetan (5 mCi) was injected after 
rituximab (day 1) in 28 patients. (The indium-111 and dosimetry bioscan are no longer 
required as per the FDA and revised package insert 11/2011). Fifty-seven patients were 
treated. The median age was 54 years, 74% had tumors > 5 cm, and all were extensively 
pretreated (median, four prior therapies; range, one to nine). The estimated radiation-
absorbed doses to healthy organs were below the study-defined limit in all patients 
studied with dosimetry. The overall response rate for the 54 patients with follicular NHL 
was 74% (15% complete responses and 59% partial responses). The Kaplan-Meier-
estimated TTP was 6.8 months (range, 1.1 to > 25.9 months) for all patients and 8.7 
months for responders. Adverse events were primarily hematologic; the incidence of 
grade 4 neutropenia, thrombocytopenia, and anemia was 35%, 9%, and 4%, respectively. 
The only significant toxicity was hematologic. 
 
2.3  
Rationale of combining bendamustine and rituximab with consolidation 90-
yttrium(Y) ibritumomab tiuxetan 
 
As mentioned above, the combination of bendamustine plus rituximab (B-R) appears to be 
non-inferior to R-CHOP as first-line treatment of indolent lymphomas including follicular 
and mantle cell lymphomas, while showing a better tolerability profile such as less alopecia, 
and potentially less cardiotoxicity, making it a rational choice for first line treatment of 
FL.[17]   
 
When given after chemotherapy, radioimmunotherapy can convert partial responses to 
complete responses and can prolong the PFS. The Follicular Lymphoma Ibritumomab 
tiuxetan (FIT) trial of consolidation Yttrium-90-Ibritumomab tiuxetan versus no additional 
therapy after first remission in advanced follicular lymphoma showed a prolongation of PFS 
(36 versus 13 months) in the RIT arm.[12] The PFS was prolonged regardless of PR or CR 
after first-line therapy. The primary treatment included CVP, CHOP, fludarabine-based, and 
chlorambucil, with the minority of patients receiving rituximab. The results also showed that 
RIT converted 77% patients from PR to CR/unconfirmed CR (CRu).  
 
An abbreviated course of CHOP-R followed by RIT has shown promise in patients with 
follicular lymphoma in a phase II trial reported recently.[11] Of the 60 patients entering this 
trial, 55 patients completed all protocol therapy. The median follow up was 19.7 months 
(range, 0.26-35.9 months). For intent-to-treat analysis, the complete response (CR) rate after 
CHOP-R, as assessed by CT and PET imaging, was 40% and 46%, respectively. After RIT, 
the CR rate improved, as assessed by CT and PET imaging, to 82% and 89%, respectively. 
 
In this current study, we propose a first-line regimen for untreated FL using bendamustine 
and rituximab (B-R) (bendamustine 90 mg/m2 on days 1 and 2 and Rituximab 375mg/m2 on 
Day 1 of a 28-day [+2 days] cycle) x 4 cycles followed by RIT; Zevalin (formerly Biogen 


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Idec/Cell Therapeutics, now Spectrum). 
 
The advantage of this treatment is that B-R has a better side effect profile including 
significantly less alopecia and less infectious complications. Currently bendamustine is not 
FDA-approved for first-line therapy for follicular lymphoma. 90-yttrium(Y) ibritumomab 
tiuxetan (Zevalin) radioimmunotherapy is FDA approved for patients with previously 
untreated follicular non-Hodgkin’s Lymphoma (NHL), who achieve a partial or complete 
response to first-line chemotherapy. Evidence suggests that consolidation with RIT leads to a 
longer PFS.  Since this specific combination has not been utilized in the first-line treatment 
of FL, it warrants investigation in the current study.  
 
This trial will begin to establish a standard of care for the first-line treatment of follicular 
lymphoma. We hypothesize that bendamustine plus rituximab followed by RIT will 
contribute to among the highest CR rates seen in follicular lymphoma with relatively low 
toxicity. Based on the results of this trial, we would aim to open a larger trial for follicular 
lymphoma in a cooperative group setting, i.e. CALGB. 
 
2.4 
Correlative Studies Background 
 
 
The BCL2 gene-Jh rearrangement is the common abnormality in FL t(14;18). This can be 
assessed by various PCR techniques.[18, 19] 
 
Patients can be assessed for this molecular abnormality in their bone marrow at baseline and 
following therapy. For instance in a similar Southwest Oncology Group study of 
chemotherapy followed by radioimmunotherapy using tositumomab/iodine I-131 
tositumomab  (Bexxar) for follicular lymphoma,  patients were asked to undergo serial bone 
marrow aspirations at study entry, 4 weeks after the sixth cycle of CHOP (just before 
tositumomab/iodine I-131 tositumomab), and after tositumomab/iodine I-131 tositumomab 
for PCR testing.[20] The mononuclear cell fraction was isolated from marrow aspirates by 
Ficoll-Hypaque sedimentation and cryopreserved for subsequent batch analysis using a 
double nested PCR assay to detect the major breakpoint region and the minor cluster region 
of the BCL2 gene using the method of Gribben.[18, 19] Samples were initially analyzed by 
fragment size using ethidium bromide gel electrophoresis of the PCR product and then 
transferred to nitrocellulose membranes for confirmation of the identity of the BCL2 
translocation by Southern blotting. The adequacy of samples was demonstrated using beta-
globin as a positive control housekeeping gene. Patients were considered to have attained a 
molecular remission if their marrow sample at study entry contained a detectable t(14;18) 
translocation that became undetectable after protocol treatment. 
 
 


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3. ELIGIBILITY CRITERIA 
 
 
3.1  
Inclusion Criteria 
 
3.1.1    Previously untreated, histologically confirmed follicular lymphoma  
classification grade 1, 2 or 3a (>15 centroblasts per high power field with  
centrocytes present) 
 
3.1.2 Ann Arbor stages of II to IV with either symptomatic or bulky disease (>5 
cm); or disease progression 
 
3.1.3 Male or female subject 18 years of age or older  
 
3.1.4 ECOG performance status <2  
 
3.1.5 Patients must have normal organ and marrow function as defined below: 
 
 
 
 
 
Adequate hematologic function 
 
 
 
 
 
- 
Absolute neutrophil count (ANC) 
>1,000/mm3 
 
 
 
 
 
- 
Platelet count  
 
 
 
 
 
>100,000/mm3 
 
 
 
 
 
- 
Patients with an ANC less than 1,000/mm3 and/or platelets below 
100,000/mm3 are still eligible for study entry as long as there is >50% 
bone marrow involvement with lymphoma 
 
Adequate hepatic function  
 
 
 
 
 
- 
Total bilirubin  
 
 
 
within normal institutional limits 
 
 
 
 
 
- 
AST(SGOT)  
 
 
 
<2.5 X institutional upper limit of normal 
 
 
 
 
 
- 
ALT(SGPT)  
 
 
 
<2.5 X institutional upper limit of normal 
 
 
 
 
 
 
Adequate renal function 
 
 
 
 
 
- 
Calculated creatinine clearance >40 mL/min/1.73m2 
 
 
 
 
 
 
Creatinine clearance should be calculated using the Cockcroft-Gault 
 
 
 
 
 
 
 
Equation (Appendix F) or per institutional standard 
 
 
 
 
3.1.6 Measureable disease with at least one lesion measuring > 2 cm in its greatest 
transverse diameter  
 
3.1.7    Female subjects of childbearing potential must have a negative pregnancy 
test (urine or serum b-HCG) at screening. Both male and female subjects must 
employ effective contraceptive measures prior to the start of therapy until 12 
months after the last dose of study drug. Men must agree not to father a child 
and agree to use effective birth control during therapy and for 12 months after 
the last dose of study drug, even if they have had a successful vasectomy, if 
their partner is of childbearing potential. 
 
3.1.8 Voluntary written informed consent must be given before performance of any 
study-related procedure, with the understanding that consent may be 
withdrawn by the subject at any time without prejudice to future medical care. 
The subject must have the ability to understand and the willingness to sign a 
written informed consent document. 
 


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3.3  
Exclusion Criteria 
 
3.3.1 Patients who have had prior chemotherapy or immunotherapy  
 
3.3.2 Patients receiving any other investigational agents 
 
3.3.3 Prior chemotherapy or monoclonal antibody therapy; prior radiation will be 
allowed if <25% of active bone marrow was exposed  
 
3.3.4 Patients with primary CNS lymphoma 
 
3.3.5 Patients with known HIV 
 
3.3.6 Treatment with therapeutic doses of systemic steroids within 4 weeks of 
beginning study treatment (cycle 1, day -7); topical use of corticosteroids and 
systemic replacement of corticosteroids for adrenal insufficiency are allowed 
 
3.3.7 Patients who have had malignant pleural, pericardial or peritoneal effusions at 
any time 
 
3.3.8 Patients with a known history of myelodysplastic syndrome (MDS) or found 
to have MDS on review of the staging bone marrow aspirate and biopsy  
 
3.3.9 Uncontrolled intercurrent illness including, but not limited to, ongoing or 
active infection, symptomatic congestive heart failure, unstable angina 
pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would, 
in the judgment of the investigator, limit compliance with study requirements 
 
3.3.10 Pregnant or lactating female subjects 
 
3.3.11 Concurrent active malignancy other than lymphoma or history of invasive 
malignancy within the past 5 years.  The only exception is completely excised, 
non-melanoma skin cancer 
 
3.3.12  Known Hepatitis B and/or Hepatitis C Infection 
 
3.3.13  Any other condition, that in the judgment of the investigator places the patient 
at unacceptable risk if he/she were to participant in the study 
 
3.4  
Inclusion of Women and Minorities 
 
Both men and women and members of all races and ethnic groups are eligible for this trial.  
 
 
 


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4. REGISTRATION PROCEDURES 
 
4.1 
General Guidelines 
 
 
Each participant must sign and date an informed consent form before undergoing any study-
specific procedures.  Contact the Project Manager at the Norris Cotton Cancer Center 
Clinical Research Office once consent is signed. The Project Manager will assign a 
participant identification number. Once a participant has completed all pre-treatment 
evaluations and eligibility criteria have been met, participants will be registered to the study.  
 
The registration form, PI or designee signed eligibility checklist, primary source 
documentation including pathologic documentation of the lymphoma, bone marrow 
examination, and staging studies should be faxed or emailed to the Project Manager. The 
Project Manager will send a confirmatory email that these materials have been received. 
 
Eligibility criteria will be verified by the Dartmouth PI or designee at the time of registration. 
Once the participant is determined by the PI or designee to be eligible, the subject will be 
registered. A completed registration form will be sent confirming registration. Each 
participant will then be entered into Velos by the Dartmouth Project Manager.   
 
4.2 
Registration Process 
 
To register a patient, the following documents need to be completed and faxed (603-650-
7799) or e-mailed to the Project Manager: 
 Investigator signed eligibility checklist 
 Lymphoma pathology report, bone marrow examination (when available), and                  
staging studies 
 Copy of required laboratory reports, including creatinine clearance calculation 
 Patient Enrollment Form from Spectrum Pharmaceuticals 
 Completed registration form 
 
Within 24 hours the Project Manager will: 
 Assign a participant study ID number 
 Fax or e-mail the participant study ID number to the participating site 
 Verify eligibility with the Dartmouth PI or designee 
 Register the participant to the study 
 Contact the participating site and confirm registration 
 


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5. TREATMENT PLAN 
 
 
5.0       Study design 
 
This multicenter, open label, single arm clinical trial has been designed to investigate 
bendamustine plus rituximab followed by 90-yttrium(Y) ibritumomab tiuxetan in patients 
with untreated follicular lymphoma requiring treatment. Upon successful completion of all 
screening procedures and registration, participants will be treated following the schema 
outlined below: 
 
In cycle 1 only, participants will receive rituximab 375mg/m2 on day -7 (+1 day).  For every 
cycle, participants will receive bendamustine 90 mg/m2 and rituximab 375 mg/m2 on day 1 
and bendamustine 90 mg/m2 on day 2.  A cycle will be 28 days (+2 days) and participants 
will be treated for a total of 4 cycles.  
 
Participants, who achieve at least a partial response (PR), achieve a platelet count 
>100,000/mm3, an ANC > 1500/mm3, and bone marrow infiltration less than 25% at 
restaging will be eligible for consolidation radioimmunotherapy (RIT) with 90-yttrium(Y) 
ibritumomab tiuxetan. RIT treatment will begin 6-12 weeks after the last cycle of B-R.  
Although a maximum of 12 weeks is allowed, every effort should be made to begin RIT as 
close to 6 weeks after the last cycle of B-R as possible.  Participants who either have stable 
disease (SD) or progression of disease or do not complete 4 cycles of bendamustine and 
rituximab treatment will not be eligible to receive RIT.   
 
The ibritumomab tiuxetan therapeutic regimen is given in 2 steps: Step 1 includes one 
infusion of rituximab on day 1. (Step 1 no longer requires the use of indium-111 or the 
dosimetric bioscan as of 11/2011 as per the FDA and new package insert 11/2011.) 
(http://zevalin.com/v3/pdf/Zevalin_Package_Insert.pdf) Step 2 is given on day 8 (+1 day) 
and consists of a second infusion of rituximab preceding Y-90 ibritumomab tiuxetan. 
Rituximab is administered to deplete peripheral blood CD20+ B-cells and to optimize 
biodistribution.  
 
Following RIT, a weekly CBC w/diff including platelet count is required until count 
recovery.  Every 4 weeks (starting from day 1) a physical exam, CBC w/ diff, serum 
chemistry, vitals, ECOG, and adverse event evaluation is required. Count recovery following 
RIT is defined as ANC >1000/mm3 and platelet count >100,000/mm3. 
 
 


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5.1  
Agent Administration 
 
Treatment will be administered on an outpatient basis. Reported adverse events and potential 
risks for bendamustine, rituximab and 90-yttrium(Y) ibritumomab tiuxetan are described in 
Section 7.  Appropriate dose modifications of bendamustine, rituximab and 90-yttrium(Y) 
ibritumomab tiuxetan are described in Section 6.  No investigational or commercial agents or 
therapies other than those described below may be administered with the intent to treat the 
patient's malignancy. 
 
 
REGIMEN DESCRIPTION 
Bendamustine and Rituximab 
 
Agent 
Premedications; 
Precautions 
 
Dose 
 
Route, Rate 
 
Schedule 
Cycle 
Length  
Rituximab 
Acetaminophen 650mg PO, 
diphenhydramine 25-50 mg 
PO or IV, dexamethasone 8-
12mg PO or IV 
375mg/m2 
IV , Rate: 
Administer per 
institutional 
guidelines 
Cycle 1 only: 
Day -7 (+1 
day) 
Day 1 of 
every cycle 
 1 cycle 
= 28 
days (+2 
days) 
 
Bendamustine 
Serotonin-receptor antagonist 
(ondansetron, granisetron, or 
equivalent antiemetic) is 
allowed 
90mg/m2 
IV, Rate: 
Administer per 
institutional 
guidelines 
Days 1 and 2 
of every 
cycle 
Radioimmunotherapy with Y-90 ibritumomab tiuxetan: 6-12 weeks after last dose of B-R 
Rituximab and Y-90 
ibritumomab tiuxetan 
 
(refer to package insert 
for Y-90 ibritumomab 
tiuxetan for details 
regarding administration, 
or refer to institutional 
protocols.) 
 
 
Step 1: Day 1: Rituximab 250 mg/m2 IV is administered first (see Rituximab 
package insert).  
 
Step 2: Day 8 (+1 day): a second dose of Rituximab 250 mg/m2 IV is given.  
 
If platelet count > 150,000/mm3: Within 4 hours of completing the rituximab 
infusion, give Y-90 ibritumomab tiuxetan 0.4 mCi/kg (14.8 MBq/kg) IV over 10 
minutes. The dose administered should be within 10% of the actual prescribed 
dose of Y-90 ibritumomab tiuxetan. 
If platelet count 100,000—149,000/mm3: Within 4 hours of completing the 
rituximab infusion, give Y-90 ibritumomab tiuxetan 0.3 mCi/kg (11.1 MBq/kg) 
IV over 10 minutes. The dose administered should be within 10% of the actual 
prescribed dose of Y-90 ibritumomab tiuxetan. 
 
The total Y-90 dose should not exceed 32 mCi (1184 MBq) regardless of the 
patient’s body weight. 
 
 
For rituximab dosing and administration, rituximab will be rounded to the nearest 10 mg. 
 
5.2  
General Concomitant Medication and Supportive Care Guidelines 
 
No formal clinical assessments of pharmacokinetic drug-drug interactions between 
bendamustine and other drugs have been conducted. Inhibitors of CYP1A2 (e.g., 
fluvoxamine, ciprofloxacin) have potential to increase plasma concentrations of 
bendamustine and decrease plasma concentrations of active metabolites. Inducers of 
CYP1A2 (e.g., omeprazole, smoking) have potential to decrease plasma concentrations of 
bendamustine and increase plasma concentrations of its active metabolites. Caution should be 
used if concomitant treatment with CYP1A2 inhibitors or inducers is needed. 
 
Prophylactic use of G-CSF or GM-CSF is discouraged. They are allowed in participants who 


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demonstrate the need for bone marrow support based on occurrence of prolonged neutropenia 
(Grade 4 leukopenia lasting at least one week, failure of WBC count to recover to at least a 
grade 1 toxicity by the next scheduled dose, or the occurrence of febrile neutropenia in a 
prior cycle of treatment.) 
 
Use of G-CSF or GM-CSF should not be used as a substitute for dose reduction. 
 
5.3  
Duration of Therapy 
 
In the absence of disease progression or unacceptable toxicity as deemed by the treating 
physician, treatment will continue for the planned duration of protocol therapy. Any 
participant with documented disease progression will be taken off of study treatments and 
removed from the protocol.   
 
Other conditions for withdrawal:  
If at any time the constraints of this protocol are detrimental to the participant’s health and/or 
the participant no longer wishes to continue protocol therapy, protocol therapy shall be 
discontinued. In this event, the Dartmouth PI shall be notified, and the reason(s) for 
discontinuation of therapy should be documented in the Case Report Form.  
 
5.4 
Duration of Follow Up 
 
Participants who receive any amount of study treatment will be followed every 3 months (+/- 
2 weeks) with a physical exam, vital signs, weight, ECOG, CBC with diff, platelets, and 
serum chemistry for the first 2 years. CT scans will be performed every 6 months for the first 
2 years.  Years 3 through 10, participants will be followed annually. If the participant 
receives alternative treatment, data will be collected on the type of alternative treatment and 
date received. This will allow determination of time to next treatment. Follow up will occur 
for a maximum of 10 years from study entry or death – whichever occurs first.  For those 
who do not receive Y90 ibritumumab tiuxetan but then receive maintenance rituximab, this 
does not constitute a new regimen, and patients should be followed for a maximum of 10 
years from study entry or death – whichever occurs first.  Rituximab should be recorded in 
the concomitant medications. 


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6. DOSING DELAYS/DOSE MODIFICATIONS 
 
6.1  Starting Dose and Dose Modifications for Bendamustine 
 
The starting dose of bendamustine will be 90 mg/m2 IV infusion over 30-60 minutes. The 
dose should be calculated based on the BSA of the patient on day 1 of every cycle. 
 
Participants who experience Grade 4 hematologic or Grade 3-4 non-hematologic toxicity 
after a cycle of B-R therapy will have their dose of bendamustine decreased to 60 mg/m2 IV 
for the remaining cycles.  
 
Participants who continue to experience Grade 4 hematologic or Grade 3-4 non-hematologic 
toxicity at this reduced dosage level should discontinue B-R. Any participant that does not 
meet criteria to receive RIT after 12 weeks from the last dose of B-R, will removed from 
study treatment and will be considered off study. (see section 5.4). 
 
 
 
 
 
 
Prophylactic use of G-CSF or GM-CSF is discouraged. They are allowed in participants who 
demonstrate the need for bone marrow support based on occurrence of prolonged neutropenia 
(Grade 4 leukopenia lasting at least one week, failure of WBC count to recover to at least 
Grade 1 toxicity by the next scheduled dose, or the occurrence of febrile neutropenia in a 
prior cycle of treatment.) 
 
Use of G-CSF or GM-CSF should not be seen as a substitute for dose reduction. 
 
6.2  Inclusion Criteria Prior to RIT 
 
6.2.1   
Patients must have achieved at least a partial response (PR), an ANC >1500/mm3, 
a platelet count >100,000/mm3 and less than 25% bone marrow involvement with lymphoma 
prior to the start of 90-yttrium (Y) ibritumomab tiuxetan.  
 
6.2.2 
Female subjects of childbearing potential must have a negative pregnancy test 
(urine or serum b-HCG) within 1 week prior to the start of treatment with Y-90 ibritumomab 
Tiuxetan. 
 
6.2.3 
 Patients must complete 4 cycles of bendamustine and rituximab treatment per 
protocol. 
 
      6.3  Starting Dose of 90-yttrium(Y) ibritumomab tiuxetan 
 
90-yttrium(Y) ibritumomab tiuxetan shall be given 6-12 weeks after completion of B-R 
provided the participant has achieved a platelet count of >100,000/mm3, an ANC > 
1500/mm3, and bone marrow infiltration less than 25%. Although a maximum of 12 weeks is 
allowed, every effort should be made to begin RIT as close to 6 weeks after the last cycle of 
B-R as possible. 
Dose Level 
Bendamustine Dose 
Baseline 
90 mg/m2 
-1 
60 mg/m2 


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On the actual day of RIT, for platelet counts > 150,000/mm3, Y-90 ibritumomab tiuxetan 
dose will be 0.4 mCi/kg (14.8 MBq/kg). 
 
On the actual day of RIT, for platelets count 100,000-149,000/mm3, Y-90 ibritumomab 
tiuxetan dose will be 0.3 mCi/kg (11.1 MBq/kg). 
 
6.4   Postponement of Bendamustine and Rituximab Treatment 
 
The prescribed time interval between each cycle of rituximab plus bendamustine is 28 days 
(+2 days).  Non-hematologic toxicities must have recovered to < grade 1 or to baseline 
before receiving the next treatment course. At the start of cycles 2 through 4, the absolute 
neutrophil count must be ≥1,000/mm3 and the platelet count must be ≥ 75,000/mm3 to 
proceed with treatment (unless the subject has documented >50% marrow involvement with 
lymphoma). If recovery criteria are not met within 2 weeks following the prescribed start of a 
treatment cycle (i.e. after 2 week delay), the patient will be re-evaluated by the investigator, 
and a decision will be made regarding further treatment with B-R. 
 
6.5 
Ancillary Therapy  
 
No other anti-tumor treatment is allowed during the course of the study. The investigator 
may prescribe supportive treatment for adverse events following the evaluation of the causal 
relationship of the symptom(s) to the study drug. Onset and duration of supportive treatment, 
including pre-infusion medications, should be recorded on the concomitant medication log 
and in the CRF.  
 
7. ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS  
 
Adverse event (AE) recording and reporting is a routine part of every clinical trial.  All AEs 
and SAEs, whether reported by the participant or noted by authorized study personnel, will 
be recorded in the participant’s medical record, on the AE or SAE log, and the case report 
forms. AE recording will begin on the first day of treatment (Cycle 1 Day -7) and end at the 
EOT visit. An ongoing AE will be followed until it returns to baseline. Abnormal lab values, 
even if they are expected, must be recorded appropriately as adverse events. Each site must 
record the onset and duration of each hematologic AE.  
The following list of AEs (Section 7.1) and the characteristics of an observed AE (Section 
7.2) may assist with determination of SAE reporting. 
 
7.1 
Adverse Events  
 
7.1.1 Clinical Trials Experience in NHL with Bendamustine 
 
The most common nonhematologic adverse reactions (≥30%) were nausea (75%), fatigue 
(57%), vomiting (40%), diarrhea (37%) and pyrexia (34%). The most common non-
hematologic Grade 3 or 4 adverse reactions (≥5%) were fatigue (11%), febrile 
neutropenia (6%), and pneumonia, hypokalemia and dehydration, each reported in 5% of 
patients. (Please refer to the package insert of Treanda®). 
 
In addition the following adverse events have been reported. 


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Tumor Lysis Syndrome 
Tumor lysis syndrome associated with bendamustine treatment has been reported in 
patients in clinical trials and in spontaneous reports. The onset tends to be within the first 
treatment cycle of bendamustine and, without intervention, may lead to acute renal failure 
and death. Preventive measures include maintaining adequate volume status, and close 
monitoring of serum chemistry, particularly potassium and uric acid levels. Allopurinol 
has also been used prior to or at the beginning of bendamustine therapy. However, there 
may be an increased risk of severe skin toxicity when bendamustine and allopurinol are 
administered concomitantly. 
 
Skin Reactions 
Skin reactions have been reported in clinical trials and postmarketing spontaneous 
reports. These events have included rash, toxic skin reactions, and bullous exanthema. 
Some events occurred when bendamustine was given in combination with other 
anticancer agents, so the precise relationship of the skin reactions to bendamustine 
treatment is uncertain. In a study of bendamustine (90 mg/m2) in combination with 
rituximab (study SDX-105-02), 1 case of toxic epidermal necrolysis (TEN) occurred. 
TEN has been associated with treatment with rituximab. Spontaneous reports of SJS and 
TEN, some fatal, have been reported when bendamustine was administered 
concomitantly with allopurinol and other medications known to cause these syndromes. 
The relationship to bendamustine cannot be determined. 
 
When skin reactions occur, they may be progressive and increase in severity with further 
treatment. Therefore, patients with skin reactions should be monitored closely. If skin 
reactions are severe or progressive, bendamustine treatment should be withheld or 
discontinued. 
 
 
 
 
 
 
 
 
 


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Comprehensive Non-Hematologic Toxicities Occurring in at least 5% of NHL patients 
treated with bendamustine. 
 
 
 
 
 
 
 
 


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7.1.2 Adverse Event List for 90-yttrium(Y) ibritumomab tiuxetan 
 
The following adverse reactions are possible with 90-yttrium(Y) ibritumomab tiuxetan. 
(Please refer to the package insert of Zevalin®). 
 
• Serious Infusion Reactions  
• Prolonged and Severe Cytopenias  
• Severe Cutaneous and Mucocutaneous Reactions  
• Secondary Leukemia and Myelodysplastic Syndrome  
 
The most common adverse reactions of the 90-yttrium(Y) ibritumomab tiuxetan 
therapeutic regimen are: neutropenia, thrombocytopenia, anemia, gastrointestinal 
symptoms (nausea, vomiting, abdominal pain, and diarrhea), increased cough, dyspnea, 
dizziness, arthralgia, anorexia, anxiety, and ecchymosis. 
 
The most serious adverse reactions of the 90-yttrium(Y) ibritumomab tiuxetan 
therapeutic regimen are prolonged and severe cytopenias, infections (predominantly 
bacterial in origin), hemorrhage while thrombocytopenic, severe cutaneous and 
mucocutaneous reactions, infusion reactions (bronchospasm and angioedema), myeloid 
malignancies and myelodysplasias.  
 


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Comprehensive Non-Hematologic Toxicities Occurring in at least 5% of NHL patients 
treated with 90-yttrium(Y) ibritumomab tiuxetan. 
 


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7.2 
Adverse Event Definitions and Characteristics  
 Adverse event (AE) or experience: An adverse event is any unfavorable sign, 
symptom, or disease temporally associated with the use of study therapy, whether 
or not considered related to the study therapy. 
 
 Adverse drug reaction (ADR): Any unexpected or dangerous reaction to a drug 
or an unwanted effect caused by the administration of a drug. 
 
 Serious adverse event or experience (SAE):  any AE/ADR occurring at any 
dose that results in any of the following outcomes: 
 
(a) Death; 
(b) A life-threatening AE/ADR (i.e., the patient/subject was, in the view of the 
initial reporter/investigator, at immediate risk of death from the AE/ADR as it 
occurred. It does not refer to an AE/ADR that hypothetically might have 
caused death if more severe); 
(c) Inpatient hospitalization or prolongation of existing hospitalization (Any 
adverse event requiring in-patient hospitalization i.e, hospitalization was 
required to treat or diagnose the AE/ADR, regardless of duration. A visit to 
the emergency room or hospital clinic would not be considered inpatient 
hospitalization; however, it could be considered serious if medical 
intervention was necessary in order to prevent a serious outcome); 
(d) A persistent or significant disability or incapacity (disability here means that 
there is a substantial disruption of a person’s ability to conduct normal life 
functions); 
(e) A congenital anomaly/birth defect. 
(f) An important medical event (i.e., AEs/ADRs that might not be immediately 
life-threatening, or result in death or hospitalization might be considered 
serious when, based upon appropriate medical and scientific judgment, they 
might jeopardize the patient/subject or might require medical or surgical 
intervention to prevent one of the other serious outcomes listed above); 
(g) Any suspected transmission via a medicinal product of an infectious agent;  
 
 
CTCAE term (AE description) and grade:  The descriptions and grading scales 
found in the revised NCI Common Terminology Criteria for Adverse Events 
(CTCAE) version 4.0 will be utilized for AE reporting.  All appropriate treatment 
areas should have access to a copy of the CTCAE version 4.0.  A copy of the 
CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep.cancer.gov). 
 
 
‘Expectedness’: AEs can be ‘Unexpected’ or ‘Expected’ (see Section 7.1 above) 
for expedited reporting purposes only.  ‘Expected’ AEs (the ASAEL) are bold 
and italicized in the CAEPR (Section 7.1.1). 
 
 


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 
Attribution of the AE: 
- 
Definite – The AE is clearly related to the study treatment. 
- 
Probable – The AE is likely related to the study treatment. 
- 
Possible – The AE may be related to the study treatment. 
- 
Unlikely – The AE is doubtfully related to the study treatment. 
- 
Unrelated – The AE is clearly NOT related to the study treatment. 
 
7.3 
Expedited Adverse Event Reporting 
 
Any serious adverse events which occur once the first dose of treatment has been 
administered or within 30 days of receiving the last dose of study medication, whether or not 
related to the study drug, must be reported by the investigator. In addition, any SAEs which 
occur as a result of protocol specific diagnostic procedures or interventions must also be 
reported.  
 
All serious adverse events as defined in 21 CFR 314.80 (whether initial or follow-up) must 
be reported to the IRB at local institution and the Project Manager at Dartmouth-Hitchcock 
Medical Center. Please send the SAE report by facsimile to Dartmouth-Hitchcock Medical 
Center (603-650-7799) or email the project manager within 1 business day of becoming 
aware of the SAE. 
 
As the sponsor, Dartmouth-Hitchcock Medical Center will report to the Dartmouth College 
Institutional Review Board (Committee for the Protection of Human Subjects), Cephalon 
(610-738-6396) and Spectrum Pharmaceuticals (617-679-2979) using the SAE Transmittal 
Forms provided by Cephalon and Spectrum, respectively.   
 
For the initial report to Spectrum, the Project Manager at DHMC will complete the provided 
Biogen Idec Adverse Event reporting form DSRM-F-18 v1.0 and fax to 617-679-2979 with 
all relevant documentation (laboratory tests, hospital admission/discharge summary, etc.)    
Please assure that participant identifiers are redacted from all documentation, except for 
participant study ID#, initials and date of birth.   
  
The initial SAE report must be reported within one business day of becoming aware of the 
SAE and comprise a full written summary detailing relevant aspects of the adverse events in 
question. Where applicable, information from relevant hospital case records and autopsy 
reports should be included. Follow-up information should be forwarded to Dartmouth-
Hitchcock Medical Center as soon as available. DHMC will send follow-up reports to 
Cephalon and Spectrum Pharmaceuticals.  
 
Instances of death or congenital abnormality, if brought to the attention of the investigator, 
AT ANY TIME after cessation of study medication, and linked by the investigator to a 
previous clinical trial, should be reported immediately to appropriate regulatory agencies. 
 
The Principal Investigator shall use his/her judgment to determine the relationship 
between the Serious Adverse Drug Experience and the Study Drug.  
 
The institution shall notify the IRB and Cephalon and Spectrum within one (1) business 
day, by facsimile, upon learning of the occurrence during the Study of: 
(a)  All Serious AE/ADR as defined in Section 7.2, regardless of causality; 
(b)  All Expedited AE/ADR of Interest as described in 7.3.1 
(c)  Any exposure of a pregnant Study participant to the Study Drug within thirty 


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Clinical Study Protocol D1015 version 06 July 2018 
 
 
21 
(30) days of exposure; 
(d)  A female partner of a male study participant becoming pregnant within thirty 
(30) days of exposure;     
(e)  Any medical event which may reasonably be believed to impair the integrity, 
validity or ongoing viability of the Study. 
 
All such occurrences listed in this section shall be reported to Cephalon and Spectrum 
Pharmaceuticals using the SAE Transmittal Form provided by Cephalon and Spectrum 
Pharmaceuticals. In the event the IRB requests additional safety information from the 
Sponsor-Investigator, the Sponsor-Investigator shall notify Cephalon and Spectrum of 
such requests within one (1) business day of receiving the IRB request. 
 
7.4 
Safety and Data Monitoring 
 
The Norris Cotton Cancer Center's Safety and Data Monitoring Committee (SDMC) will 
monitor the study progress on a quarterly basis according to the Cancer Center's guidelines.  
(See Appendix C). 
 
 
 
7.5 
On-Site Monitoring 
 
Representatives of Dartmouth-Hitchcock Medical Center must be allowed to visit all 
study site locations periodically to assess the regulatory compliance, data quality and 
study integrity. On site they will review study records and directly compare them with 
source documents, discuss the conduct of the study with the investigator, and verify that 
the facilities remain acceptable. They may request access to all regulatory documents, 
source documents, CRFs, and other study documentation for on-site inspection. Direct 
access to these documents must be guaranteed by the investigator, who must provide 
support at all times for these activities. 
 
 
7.6 
Investigator and Site Responsibility for Drug Accountability 
 
Accountability for the study drug at the trial site is the responsibility of the principal 
investigator at each site. Drug accountability records indicating the drug’s delivery date 
to the site, inventory at the site, use by each subject, or disposal of the drug, if approved 
by the principal investigator, will be maintained by the clinical site. Dartmouth-
Hitchcock or its designee will review drug accountability at the site on an ongoing basis. 
All material containing study drug will be treated and disposed of as hazardous waste in 
accordance with governing regulations. 
 
 
7.7  
Investigator Compliance 
 
The study shall be conducted as described in this approved protocol. All revisions to the 
protocol must be discussed with, and be prepared by, Dartmouth-Hitchcock. The 
investigator must not implement any deviation or change to the protocol without prior 
review and documented approval/favorable opinion from the IRB of an amendment, 
except where necessary to eliminate an immediate hazard(s) to study participants. Any 
significant deviation must be reported to the coordinating site, reported to the IRB if it 
meets their guidelines for reporting, and documented in the CRF. 
 


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Clinical Study Protocol D1015 version 06 July 2018 
 
 
22 
When immediate deviation from the protocol is required to eliminate an immediate 
hazard(s) to subjects, the investigator will contact Dartmouth-Hitchcock, or a designee, if 
circumstances permit, to discuss the planned course of action. Any departures from the 
protocol must be documented. If the deviation meets guidelines for reporting, it must be 
reported to the institutional IRB, as well as the Committee for the Protection of Human 
Subjects at Dartmouth.  The deviation form used for each institution can be submitted to 
the Project Manager at Dartmouth-Hitchcock Medical Center.  
 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
23 
8. PHARMACEUTICAL INFORMATION 
 
A list of the adverse events and potential risks associated with the investigational or 
commercial agents administered in this study can be found in Section 7.1. 
 
8.1 
Bendamustine (please see Treanda® package insert for more details) 
 
Availability 
Bendamustine is an investigational agent for the first line treatment of follicular lymphoma, 
and it is supplied to investigators by Cephalon, Inc. 
 
FDA approved indications 
Bendamustine is indicated for the treatment of patients with chronic lymphocytic leukemia, 
and for the treatment of patients with indolent B-cell non-Hodgkin’s lymphoma that has 
progressed during or within six months of treatment with rituximab or a rituximab-containing 
regimen. 
 
Description 
Bendamustine contains bendamustine hydrochloride, an alkylating drug, as the active 
ingredient. The chemical name of bendamustine hydrochloride is 1H-benzimidazole-2-
butanoic acid, 5-[bis(2-chloroethyl)amino]-1 methyl-, monohydrochloride. Its empirical 
molecular formula is C16H21Cl2N3O2 · HCl, and the molecular weight is 394.7. 
Bendamustine hydrochloride contains a mechlorethamine group and a benzimidazole 
heterocyclic ring with a butyric acid substituent. 
 
Bendamustine is intended for intravenous infusion only after reconstitution with 20 mL of 
Sterile Water for Injection, USP, and after further dilution with either 0.9% Sodium Chloride 
Injection, USP, or 2.5% Dextrose/0.45% Sodium Chloride Injection, USP. It is supplied as a 
sterile non-pyrogenic white to off-white lyophilized powder in a single-use vial. Each vial 
contains 100 mg of bendamustine hydrochloride and 170 mg of mannitol, USP. The pH of 
the reconstituted solution is 2.5 - 3.5. 
 
Mechanism of Action 
Bendamustine is a bifunctional mechlorethamine derivative containing a purine-like 
benzimidazole ring. Mechlorethamine and its derivatives form electrophilic alkyl groups. 
These groups form covalent bonds with electron-rich nucleophilic moieties, resulting in 
interstrand DNA crosslinks. The bifunctional covalent linkage can lead to cell death via 
several pathways. Bendamustine is active against both quiescent and dividing cells. The 
exact mechanism of action of bendamustine remains unknown. 
 
Pharmacokinetics 
Absorption 
Following a single IV dose of bendamustine hydrochloride Cmax typically occurred at the 
end of infusion. The dose proportionality of bendamustine has not been studied. 
 
Distribution 
In vitro, the binding of bendamustine to human serum plasma proteins ranged from 94-96% 
and was concentration independent from 1-50 μg/mL. Data suggest that bendamustine is not 
likely to displace or to be displaced by highly protein-bound drugs. The blood to plasma 


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Clinical Study Protocol D1015 version 06 July 2018 
 
 
24 
concentration ratios in human blood ranged from 0.84 to 0.86 over a concentration range of 
10 to 100 μg/mL indicating that bendamustine distributes freely in human red blood cells. In 
humans, the mean steady state volume of distribution (Vss) was approximately 25 L. 
 
Metabolism 
In vitro data indicate that bendamustine is primarily metabolized via hydrolysis to 
metabolites with low cytotoxic activity. In vitro, studies indicate that two active minor 
metabolites, M3 and M4, are primarily formed via CYP1A2. However, concentrations of 
these metabolites in plasma are 1/10 and 1/100 that of the parent compound, respectively, 
suggesting that the cytotoxic activity is primarily due to bendamustine. In vitro studies using 
human liver microsomes indicate that bendamustine does not inhibit CYP1A2, 2C9/10, 2D6, 
2E1, or 3A4/5. Bendamustine did not induce metabolism of CYP1A2, CYP2A6, CYP2B6, 
CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 enzymes in primary cultures of 
human hepatocytes. 
 
Elimination 
No mass balance study has been undertaken in humans. Preclinical radiolabeled 
bendamustine studies showed that approximately 90% of drug administered was recovered in 
excreta primarily in the feces. Bendamustine clearance in humans is approximately 700 
mL/minute. After a single dose of 120 mg/m2 bendamustine IV over 1-hour the intermediate 
t½ of the parent compound is approximately 40 minutes. The mean apparent terminal 
elimination t½ of M3 and M4 are approximately 3 hours and 30 minutes respectively. Little 
or no accumulation in plasma is expected for bendamustine administered on Days 1 and 2 of 
a 28-day cycle. 
 
Renal Impairment 
In a population pharmacokinetic analysis of bendamustine in patients receiving 120 mg/m2 
there was no meaningful effect of renal impairment (CrCL 40 - 80 mL/min, N=31) on the  
pharmacokinetics of bendamustine. Bendamustine has not been studied in patients with CrCL 
< 40 mL/min. These results are however limited, and therefore bendamustine should be used 
with caution in patients with mild or moderate renal impairment. Bendamustine should not be 
used in patients with CrCL < 40 mL/min.  
 
Hepatic Impairment 
In a population pharmacokinetic analysis of bendamustine in patients receiving 120 mg/m2 
there was no meaningful effect of mild (total bilirubin ≤ ULN, AST ≥ ULN to 2.5 x ULN, 
and/or ALP ≥ ULN to 5.0 x ULN, N=26) hepatic impairment on the pharmacokinetics of 
bendamustine. Bendamustine has not been studied in patients with moderate or severe 
hepatic impairment. These results are however limited, and therefore bendamustine should be 
used with caution in patients with mild hepatic impairment. Bendamustine should not be used 
in patients with moderate (AST or ALT 2.5 - 10 x ULN and total bilirubin 1.5 - 3 x ULN) or 
severe (total bilirubin > 3 x ULN) hepatic impairment.  
 
Effect of Age 
Bendamustine exposure (as measured by AUC and Cmax) has been studied in patients ages 
31 through 84 years. The pharmacokinetics of bendamustine (AUC and Cmax) were not 
significantly different between patients less than or greater than/equal to 65 years of age.  
 
 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
25 
Effect of Gender 
The pharmacokinetics of bendamustine were similar in male and female patients.  
 
Effect of Race 
The effect of race on the safety, and/or efficacy of bendamustine has not been established. 
Based on a cross-study comparison, Japanese subjects (n = 6) had on average exposures that 
were 40% higher than non-Japanese subjects receiving the same dose. The significance of 
this difference on the safety and efficacy of bendamustine in Japanese subjects has not been 
established. 
 
Pharmacokinetics/Pharmacodynamics 
Based on the pharmacokinetics/pharmacodynamics analyses of data from NHL patients, a 
correlation was observed between nausea and bendamustine Cmax. 
 
8.2  
90-yttrium (Y) ibritumomab tiuxetan 
 
Availability 
90-yttrium (Y) ibritumomab tiuxetan is commercially available.  It is supplied to 
investigators through coverage by the patients’ insurance. 
 
FDA indications 
90-yttrium (Y) ibritumomab tiuxetan (Zevalin) is indicated for the treatment of patients 
with relapsed or refractory, low-grade or follicular B-cell non-Hodgkin's lymphoma (NHL), 
including patients with rituximab refractory follicular NHL. It is in the NCCN compendium 
for the first-line treatment of follicular lymphoma. On 9/5/2009 the FDA expanded the label 
to include patients with previously untreated follicular non-Hodgkin’s Lymphoma (NHL), 
who achieve a partial or complete response to first-line chemotherapy. 
 
Mechanism of Action 
Ibritumomab tiuxetan binds specifically to the CD20 antigen (human B-lymphocyte-
restricted differentiation antigen, Bp35). The apparent affinity (KD) of ibritumomab tiuxetan 
for the CD20 antigen ranges between approximately 14 to 18 nM. The CD20 antigen is 
expressed on pre-B and mature B lymphocytes and on > 90% of B-cell non-Hodgkin’s 
lymphomas (NHL). The CD20 antigen is not shed from the cell surface and does not 
internalize upon antibody binding. The chelate tiuxetan, which tightly binds In-111 or Y-90, 
is covalently linked to ibritumomab. The beta emission from Y-90 induces cellular damage 
by the formation of free radicals in the target and neighboring cells. Ibritumomab tiuxetan 
binding was observed in vitro on lymphoid cells of the bone marrow, lymph node, thymus, 
red and white pulp of the spleen, and lymphoid follicles of the tonsil, as well as lymphoid 
nodules of other organs, such as the large and small intestines. 
 
Pharmacodynamics 
In clinical studies, administration of the Zevalin therapeutic regimen resulted 
in sustained depletion of circulating B cells. At four weeks, the median number of circulating 
B cells was zero (range, 0-1084/mm3). B-cell recovery began at approximately 12 weeks 
following treatment, and the median level of B cells was within the normal range (32 to 
341/mm3) by 9 months after treatment. Median serum levels of IgG and IgA remained within 
the normal range throughout the period of B-cell depletion. Median IgM serum levels 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
26 
dropped below normal (median 49 mg/dL, range 13-3990 mg/dL) after treatment and 
recovered to normal values by 6-months post therapy. 
 
Pharmacokinetics 
Pharmacokinetic and biodistribution studies were performed using In-111 Zevalin (5 mCi 
[185 MBq] In-111, 1.6 mg ibritumomab tiuxetan). In an early study designed to assess the 
need for pre-administration of unlabeled antibody, only 18% of known sites of disease were 
imaged when In-111 Zevalin was administered without unlabeled ibritumomab. When 
preceded by unlabeled ibritumomab (1.0 mg/kg or 2.5 mg/kg), In-111 Zevalin detected 
56% and 92% of known disease sites, respectively. These studies were conducted with a 
Zevalin therapeutic regimen that included unlabeled ibritumomab. 
 
In pharmacokinetic studies of patients receiving the Zevalin therapeutic regimen, the mean 
effective half-life for Y-90 activity in blood was 30 hours, and the mean area under the 
fraction of injected activity (FIA) vs. time curve in blood was 39 hours. Over 7 days, a 
median of 7.2% of the injected activity was excreted in urine. 
 
 
8.3  
Rituximab (refer to RITUXAN package insert for more information) 
 
Rituximab (Rituxan®) is a genetically engineered chimeric murine/human monoclonal 
antibody directed against the CD20 antigen found on the surface of normal and malignant 
B lymphocytes. 
 
It is indicated for the treatment of patients with relapsed or refractory, low-grade or follicular, 
CD 20-positive B-cell non-Hodgkin’s lymphoma. 
 
Rituximab is commercially available. 
 
Rituximab should be administered per the guidelines found in the package insert.


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
27 
9.  CORRELATIVE/SPECIAL STUDIES 
 
9.1 
Qualitative PCR for BCL2 translocation 
 
A bone marrow aspirate and biopsy, as well as a peripheral blood sample will be taken:  
 At screening 
 4-6 weeks after the last cycle of bendamustine and rituximab  
 12-16 weeks after 90-yttrium (Y) ibritumomab tiuxetan  
 
 
In order to standardize results, these research samples will be analyzed by polymerase chain 
reaction (PCR) for the bcl-2/JH t(14;18) translocation using a qualitative PCR assay at the 
Dartmouth-Hitchcock Department of Pathology – Molecular Pathology (by Gregory 
Tsongalis, Ph.D.) 
 
Correlative science kits will be provided to participating institutions and will include: 
1. EDTA anticoagulated vacutainer (3-5 mL whole blood) 
2. EDTA anticoagulated vacutainer (1-3 mL bone marrow) 
3. Forms (Appendix A) to be filled out indicating patient registration number, date and time 
sample taken, time point taken indicating “baseline”, “post-B-R”, or “post-RIT”, 
participating institution, responsible MD, and responsible local study coordinator.  
 
The bone marrow aspirate (lavender top) and peripheral blood (pink top) in EDTA tubes 
should be shipped the same day (Monday through Thursday) at room temperature (ambient). 
The lab cannot accept samples over the weekend. 
 
The specimens will be shipped via overnight courier (Mon-Thurs) to: 
 
Molecular Pathology Laboratory 
Room 483S01 
Borwell 4 East 
Dartmouth Hitchcock Medical Center 
1 Medical Center Drive 
Lebanon, NH 03756 
 
Tel# 603-650-8257 
 
In the event that a bone marrow aspirate was already performed prior to screening, DHMC 
will accept a frozen bone marrow aspirate, or non-decalcified paraffin embedded biopsy if 
completed within 30 days of screening. The aspirate must be frozen at -20 degrees Celsius. 
The sample can then be shipped to DHMC on dry ice. The non-decalcified paraffin 
embedded biopsy can be shipped at room temperature.  
 
9.2 Genetic Polymorphisms 
Within the context of this clinical trial, we plan to study specific genetic polymorphisms that 
have been known to correlate with clinical and molecular response to rituximab therapy, 
including polymorphisms in FcgammaRIIIa and FcgammaRIIa.  We hope to gain insight into 
molecular predictors of rituximab sensitivity and resistance. We also plan to learn about 
factors related to bendamustine sensitivity and resistance. Additionally, we will study if there 


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
28 
are polymorphisms or mutations of the CD20 protein that correlate with radioimmunotherapy 
sensitivity or resistance. See Appendix D for a detailed protocol on this correlative study. 
We plan to use separate research funds to do this specific correlative research.  


Fol-BRITe study  
 
 
 
Clinical Study Protocol D1015 version 06 July 2018 
 
 
29 
10. 
STUDY CALENDAR 
*Pre-Study evaluations are to be conducted within 2 weeks prior to start of treatment.  CT scans can be done 
within 4 weeks prior to the start of treatment. 
^Each cycle of B-R is 28 days (+2 days) for 4 cycles 
a. Serum chemistry requirements are Sodium, Potassium, Chloride, Bicarbonate, BUN, Creatinine, Magnesium, Phosphorus, Calcium, Total Protein, Albumin, Total Bilirubin, AST, 
ALT, LDH, uric acid. All labs are to be checked prior to the initiation of each cycle. Lab monitoring in between cycles of B-R will be left to the discretion of the investigator.  
b. The pre-study CT scan must be conducted within 4 weeks of starting treatment.  CT scans to measure target lesions will occur pre-study, 4-6 weeks after completion of B-R, and 12-16 
weeks after 90-(Y) ibritumomab tiuxetan. After this point, CT scans must be performed every 6 months for the first 2 years, then annually.  If the patient does not complete treatment 
per protocol, an EOT evaluation should occur within 28 days that the decision was made to take the patient off treatment. . 
c. For females of childbearing potential, a urine or serum B-HCG should be done at screening and within 1 week of the first dose of 90-(Y) ibritumomab tiuxetan.  
d. The bone marrow aspirate and biopsy should occur 4-6 weeks after the last dose of B-R. The research sample for both bone marrow aspirate and peripheral blood should be taken at 
this time. If a participant is not eligible to receive 90-yttrium Ibritumomab Tiuxetan, then a bone marrow biopsy does not need to be repeated at EOT. 
e. Although a maximum of 6 and 16 weeks are allowed for the restaging visits, respectively, every effort should be made to restage as close to 4 and 12 weeks, respectively. 
f. Although a maximum of 12 weeks is allowed to begin RIT, every effort should be made to begin RIT as close to 6 weeks after the last cycle of B-R as possible 
g. All participants will be followed after EOT every 3 months (+ 2 weeks) for 2 years and then annually for a maximum of 10 years from study entry or death, whichever occurs first. 
h. Count recovery following RIT is defined as ANC >1000/mm3 and platelet count  >100,000/mm3 
i.  CBC w/diff, plts will be monitored WEEKLY following RIT until count recovery.  These weekly labs can be drawn at a local facility for participant convenience if necessary.  
j. A bone marrow aspirate, or non-decalcified paraffin embedded biopsy will be accepted if done within 30 days of screening.  
k. Prestudy evaluation and procedures done within 7 days of C1D-7 are acceptable to be used for C1D-7 and do not need to be repeated. 
l. Effective October 19, 2016 – magnesium, phosphorus, and uric acid are no longer required during follow-up.  
 
   
 
Pre- 
Study* 
BR^ 
Cycle 1 
Day -7 
(+1 
day)k 
 
B-R^ 
Day 1 
Cycles 
1-4 
B-R^ 
Day 2 
Cycles 
1-4 
 
4-6 
weekse 
after 
B-R 
RIT  
Day 1  
(6-12 
weeksf 
after B-
R) 
RIT 
Day 8 
(+1 
day) 
 
Every 4 weeks 
after RIT until 
count 
recoveryh 
End of 
Treatment 
12-16 weekse 
after RITg 
Follow-Upg 
 
 
Rituximab 375mg/m2 
 
 
X 
X 
 
 
 
 
 
 
 
Bendamustine 
 
 
X 
X 
 
 
 
 
 
 
Rituximab 250mg/m2 
 
 
 
 
 
X 
X 
 
 
 
 
90-(Y) ibritumomab tiuxetan 
 
 
 
 
 
 
 
X 
 
 
 
 
Informed consent 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history and FLIPI 1 and 2 score 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concurrent meds 
 
X 
X 
X 
 
 
X 
 
X 
 
 
X 
 
X 
 
 
 
Physical exam 
 
X 
X 
 
X 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
Vital signs (Heart rate, blood pressure, 
temperature) 
 
X 
 
X 
 
X 
X 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
Height 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
 
X 
 
X 
 
X 
 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
ECOG Performance status 
 
X 
 
X 
 
X 
 
 
X 
 
X 
 
 
X 
 
X 
 
X 
 
CBC w/diff, pltsa 
 
X 
 
X 
 
X 
 
 
X 
 
X 
X 
 
Xi
 
 
X 
 
X 
 
Serum chemistrya 
 
X 
 
X 
 
X 
 
 
X 
 
X 
 
 
X 
 
X 
 
Xl 
B2 Microglobulin 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse event evaluation 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
CT Scanb 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
 
X 
 
B-HCGc 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
Bone Marrow Aspirate and Biopsyj 
 
X 
 
 
 
 
 
Xd 
 
 
 
 
 
 
X 
 
BCL2 evaluation (BM aspirate and 
peripheral blood) 
 
X 
 
 
 
 
 
Xd 
 
 
 
 
 
 
X 
 
 


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Clinical Study Protocol D1015 version 06 July 2018 
 
 
30 
11. 
MEASUREMENT OF EFFECT 
 
11.1 
Assessment of Response 
 
The International Response Criteria for non-Hodgkin’s Lymphoma will be used to assess 
response. (Cheson, JCO Vol 17, Issue 4 (April), 1999: 1244). CT scans will be used to 
determine the size of the target lesions. PET scans may be used at the discretion of the 
investigator, but should not be used as a replacement for CT scan.  
 
For the purposes of this study, patients should be evaluated by CT scan at baseline, after the 
completion of the last cycle of B-R, and 12-16 weeks after  receiving 90-(Y) ibritumomab 
tiuxetan. Follow up CT scans will occur every 6 months for 2 years, then annually. Any 
additional radiographic studies may be performed at the discretion of the investigator. Bi-
dimensional nodal measurements for lesions must be recorded on the CT report. A maximum 
of 6 lesions should be selected that are measurable in at least 2 perpendicular dimensions, 
representative of all involved organs/areas, suitable for accurate repeat measurements, and 
include mediastinal and retroperitoneal areas of disease.  
To define response, select sites and measure the two longest diameters. Multiply each node’s 
two longest diameters together, then Sum the Products of the greatest transverse Diameters 
(SPD). Calculate the percent increase or decrease. For percent decrease, calculate (baseline-
current disease assessment)/baseline. For percent increase, calculate (current disease 
assessment-smallest sum)/smallest sum. This will allow for accurate calculations to define 
response (see Appendix G) 
For this study, a CR and CRu will be categorized as CR for statistical analyses. 
 
11.2 
Definitions 
 
Response Criteria for Non-Hodgkin's Lymphoma 
Response Category Physical Examination 
Lymph 
Nodes 
 
Lymph Node 
Masses 
 
Bone Marrow 
CR 
Normal 
Normal 
Normal 
Normal 
CRu 
Normal 
Normal 
Normal 
Indeterminate 
Normal 
Normal 
> 75% 
decrease 
Normal or 
indeterminate 
PR 
Normal 
Normal 
Normal 
Positive 
Normal 
50% 
decrease 
50% 
decrease 
Irrelevant 
Decrease in 
liver/spleen 
50% 
decrease 
50% 
decrease 
Irrelevant 
Relapse/progression Enlarging 
liver/spleen; new sites 
New or 
increased 
New or 
increased 
Reappearance 
 
 


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Clinical Study Protocol D1015 version 06 July 2018 
 
 
31 
Progression-Free Survival (PFS) 
PFS is defined as the duration of time from start of treatment to time of progression or 
death from any cause whichever comes first. 
 
Time to next treatment (TTNT) 
TTNT is defined as the duration of time from end of study treatment (Y-90 ibritumomab 
tiuxetan) to next treatment either due to relapse or progression of disease, or death due to 
any cause, whichever comes first.  
 
11.3 Methods for Evaluation of Measurable Disease 
 
All measurements should be taken and recorded in metric notation using a ruler or calipers.  
All screening evaluations should be performed within 2 weeks of beginning of treatment, 
excluding CT scans which can be performed within 4 weeks of beginning treatment. 
 
The same method of assessment and the same technique should be used to characterize each 
identified and reported lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination when both methods have been used to assess 
the antitumor effect of a treatment. 
 
Clinical lesions  Clinical lesions will only be considered measurable when they are 
superficial (e.g., skin nodules and palpable lymph nodes).  In the case of skin lesions, 
documentation by color photography, including a ruler to estimate the size of the lesion, is 
recommended.  
 
CT is the preferred radiographic modality. PET scans can be performed in addition to CT 
at the discretion of the investigator.  
 
Conventional CT. This technique should be performed per institutional guidelines. Cuts of 10 
mm or less in slice thickness contiguously are recommended but not required.  It is also 
recommended that spiral CT should be performed using a 5 mm contiguous reconstruction 
algorithm.  This applies to tumors of the chest, abdomen, and pelvis.  Head and neck tumors 
and those of extremities may be done as per institutional guidelines. 
 
 


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Clinical Study Protocol D1015 version 06 July 2018 
 
 
32 
12. STATISTICAL CONSIDERATIONS 
 
12.1 
Study Design/Endpoints 
 
The primary endpoint is complete response (CR) rate. Historical complete response (CR) rate 
has been 35%. This rate will be considered as the null hypothesis.  The alternative hypothesis 
is that bendamustine-rituximab (B-R) followed by radioimmunotherapy (RIT) will increase 
the CR rate to 55%.  An optimal Simon two-stage design will be incorporated to allow an 
early futility look during the Phase II component while minimizing the expected total sample 
size. It is calculated to be 14 in the first stage. If more than 5 subjects have a CR then an 
additional stage of 25 patients will be added for a total of 39. The probability of early 
termination is 64% under the null hypothesis and 12% under the alternative. The expected 
sample size (i.e., average if this study was repeated many times) is 24.8. These calculations 
assume a type I error rate of 5% and power of 80%. 
 
The CR rate will calculated by CR + CRu divided by the # of evaluable subjects x 100%. 
 
The ORR will be calculated by CR + CRu + PR divided by the # of evaluable subjects x 
100%. 
 
Statistical analysis for the primary endpoint will include two pre-planned analyses: The first 
analysis will include only participants who complete all protocol therapy (4 cycles of 
bendamustine and rituximab followed by Y-90 ibritumomab tiuxetan). If a participant does 
not complete all study therapy, he/she will be replaced with another participant in a 1 to 1 
fashion until there are a total of 39 evaluable participants. Since the statistics for the primary 
endpoint are based around additive sequential treatment, we will be performing this analysis. 
 
The second analysis will include all participants who receive any portion of study therapy 
(intention-to-treat analysis). 
 
Both statistical analyses will be reported.  
 
12.2 
Sample Size/Accrual Rate 
 
39 evaluable participants. At the accrual rate of about 2 participants per month this trial 
should accrue in 20 months, and be completed in 28 months. Reporting of response rate 
would be possible at 31 months.  At the accrual rate of 4 participants per month, this trial 
should accrue in 10 months, and be completed in 18 months. Reporting of response rate 
would be possible at 21 months. With an expected dropout rate of 5 to 10%, 41 to 44 
participants will need to be accrued to this study to reach 39 evaluable participants. 
 
All participants will be evaluable for toxicity.  
 
12.3 Analysis of Secondary Endpoints 
 
The secondary endpoints are safety of this combination. The CTCAE v4.0 will be used to 
determine toxicities.  
 
In addition we will calculate the CR and OR rate after B-R and Y-90 ibritumomab tiuxetan. 
The conversions from PR to CR will be reported. 
 


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The progression-free survival and time to next treatment of all participants treated will be 
determined with adequate follow-up.  
 
12.4 Analysis of Exploratory Endpoints 
Molecular Correlations: The association of molecular Bcl-2 detection by PCR with CR and 
OR will be analyzed using Cox’s model with time-dependent covariates, as Bcl-2 is a 
longitudinal variable measured at three time-points. The hazard of complete, or partial 
response, the hazard of mortality, the hazard of progression or death, or time to next 
treatment or death, will be modeled based on longitudinal Bcl-2, and Wald test reported. In 
addition we shall use a joint longitudinal-survival model to characterize these associations. 
 
 
 


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APPENDIX A 
 
Correlative Science Blood and Marrow Samples Form 
 
ENCLOSE THIS FORM WITH SPECIMENS 
 
 
PATIENT IDENTIFICATION #: __________________________INITIALS:_____________ 
 
DATE SAMPLE TAKEN:________________________________ 
 
DATE SAMPLE SHIPPED:_______________________________ 
Indicate: 
□ Peripheral Blood  
 
 
Time sample taken:__________________ 
 
 
 
□ Bone Marrow  
 
 
 
Time sample taken:__________________ 
 
 
 
□ Paraffin-embedded biopsy 
Time sample taken: _________________ 
 
Indicate:  
□ “Baseline”  
 
 
 
□ “Post B-R” Date of Cycle 1 (Day 1) B-R ________________ 
 
 
 
 
 
 
 
 
Date of last Cycle (Day 1) of B-R ____________ 
 
 
 
□ “Post RIT” Date of RIT _______________________ 
Institution: ______________________________________________ 
 
Responsible MD: _________________________________________ 
 
Responsible Study Coordinator: ____________________________FAX#:________________ 
 
Please fax this form prior to enclosing in shipment to Fax # 603-650-7799 
 
Ship samples via overnight courier (Mon-Thurs) to: 
 
Molecular Pathology Laboratory 
Room 483S01 
Borwell 4 East 
Dartmouth Hitchcock Medical Center 
1 Medical Center Drive 
Lebanon, NH 03756 
Tel# 603-650-8257 
 
 
INTERNAL USE ONLY 
 
Date Received:__________________ Received by (Printed Name):______________________ 
 
Confirmation of receipt sent to fax#:_______________________    Date:_________________


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APPENDIX B 
 
Follicular Lymphoma International Prognostic Index (FLIPI) 
The Follicular Lymphoma International Prognostic Index uses five adverse factors to identify 
three risk groups (low, intermediate, and high) to provide clear differentiation of long term 
prognosis. 
 
FLIPI-1 
Age greater than 60 years? 
Ann Arbor Stage III or IV? 
Serum LDH greater than ULN? 
Number of nodal sites greater than 4? 
Pre-transfusion hemoglobin less than 12 g/dl? 
 
To calculate a FLIPI-1 score, answer “yes” or “no” to each question. Add up the number of 
“yes” responses. The total number of “yes” responses will be the FLIPI-1 score. A 0-1 score is 
“low risk” and OS at 10 years is estimated to be 70%. If the score is 2, this is characterized as 
“intermediate risk” and OS at 10 years is estimated to be 50%. If the score is 3 or greater, that is 
considered “high risk” and OS at 10 years is estimated to be 35%.  
 
 
FLIPI-2 
Beta 2 microglobulin greater than ULN? 
Longest diameter of largest involved lymph node greater than 6 cm? 
Bone marrow involvement? 
Pre-transfusion hemoglobin less than 12 g/dl? 
Age greater than 60 years? 
 
To calculate a FLIPI-2 score, answer “yes” or “no” to each question. Add up the number of 
“yes” responses. The total number of “yes” responses will be the FLIPI-2 score. A 0 score is 
“low risk” and PFS at 3 years is estimated to be 91%. If the score is 1-2, this is characterized as 
“intermediate risk” and PFS at 3 years is estimated to be 69%. If the score is 3 or greater, that is 
considered “high risk” and PFS at 3 years is estimated to be 51%.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX C 
 
Safety and Data Monitoring Committee (SDMC) 
 
The Safety and Data Monitoring Committee (SDMC) is the Data and Safety Monitoring Board 
for the Norris Cotton Cancer Center at Dartmouth. It is a multidisciplinary committee charged 
with overseeing monitoring of safety of participants, conduct, progress, and validity and integrity 
of the data of all clinical trials at NCCC at Dartmouth. The committee is chaired by the Director 
of the Clinical Research Office, and meets quarterly to review the progress and safety of all 
active research protocol that are not reviewed by another safety and data monitoring committee. 
If a study is already being monitored by a safety and data monitoring committee formed by a 
national cooperative group, a pharmaceutical sponsor, or a study specific committee for a phase 
III trial, the NCCC SDMC does not actively monitor the study. The SDMC oversees the process 
of adverse event reporting to assure that the requirements are met. The SDMC has the authority 
to require amendments, suspend or terminate any research activities that fall within its 
jurisdiction. The SDMC reports to the Clinical Cancer Review Committee (CCRC). The SDMC 
can institute any other appropriate conditions needed for subject safety.  
 
With the exception of the Office of Clinical Research (OCR) Regulatory and Compliance Officer 
who is a standing member, all members serve a five-year term. Committee membership and 
voting status are assigned by the Director of the NCCC. The Committee includes representatives 
from the following disciplines: medical oncology, hematology, radiation oncology, 
pharmacology, bio-statistics, ethics, oncology nursing and data management. Presence of three 
or more of the SDMC voting members constitutes a quorum. Members may be appointed on an 
ad hoc basis by the Chairman of the SDMC, should additional expertise be required in the review 
of certain studies. 
 
The SDMC meets on a quarterly basis. Special meetings may be convened when necessary, for 
urgent concerns regarding patient safety or data integrity. Administration of the SDMC 
(paperwork, meeting coordination, minutes) is provided by the Office of Clinical Research. 
Minutes reflect the members present, substantive issues discussed, voting results, and members 
abstaining due to conflict of interest. 
 
The frequency of SDMC monitoring for a particular study depends on the level of risk (low or 
high risk) assigned to the study by the Chairman of the CCRC, as described above. Low risk 
trials are reviewed on an annual basis by the SDMC. High risk trials are reviewed on a quarterly 
basis by the SDMC. The size and complexity of a trial also determines the frequency and level of 
review. Data and safety monitoring activities for each study continue until all patients have 
completed their treatment and all participants are beyond the time point at which study-related 
adverse events would likely be encountered. Since this is a multicenter study, this protocol will 
be reviewed on a quarterly basis. 
 
For each SDMC protocol review, summary information regarding toxicity and accrual patterns is 
prepared by the OCR staff, and submitted to the SDMC. The study Principal Investigator is 
required to review and sign all SDMC reports. Specific information submitted for review 
includes: 
• The expected and actual numbers of participants entered to date, as well as for the most recent 
reporting period 
• Number of participants treated 


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• Description of any changes to the study design since the last SDMC review 
• Exceptions in eligibility or treatment 
• Dates of participant enrollments 
• (Dose tier for each participant, for Phase I studies) 
• Best response to treatment for each participant, for Phase II and III studies 
• (Treatment arm for each participant, for Phase III studies) 
• The type and grade of adverse events for each participant using CTC 2.0 grading 
• The duration and outcome of adverse events for each participant 
• Current disease, vital, and study (on- or off-study) status of each participant 
• Copies of all adverse event reports submitted to the IRB since the last SDMC review are also  
      sent to the SDMC, so they can be reviewed for accuracy and timeliness 
• Significant literature reporting developments that may affect the safety of 
      participants or the ethics of the study 
• Results of any interim analyses required by the protocol 
• Copies of abstracts or papers written using study data 
 
Members receive this information approximately one week prior to Committee meetings, to 
allow for preliminary study and review. Each study is assigned to a specific Committee member 
for presentation during the Committee meeting. In the event that a Committee member is either a 
principal investigator or co-principal investigator for a study under review, or has any other 
conflict of interest (including substantial financial interest in the study sponsor agency), that 
member may be present to answer questions regarding the study, but must abstain from voting 
and leave the room prior to final deliberations and voting on that study. In the event that the 
Chairman is the principal investigator for the study, another member of the Committee will 
oversee the Committee deliberations and voting. 
 
The Committee may vote to take one of the following actions for each protocol reviewed: 
 
• Full Approval: enrollment may continue; no outstanding questions regarding toxicity or 
accrual 
 
• Conditional Approval: enrollment may continue conditional upon satisfactory response by the 
principal investigator to SDMC concerns regarding toxicities and/or accrual. 
 
• Suspension: enrollment immediately suspended pending principal investigator response 
SDMC concerns regarding toxicity and/or accrual patterns. 
 
• Closure: study closed due to unacceptable toxicity and/or accrual patterns. 
 
All SDMC decisions are conveyed in writing to the study principal investigator. Principal 
investigators may appeal SDMC decisions in writing to the chairman of the SDMC. If the 
decision regarding the appeal is unsatisfactory to the investigator, a second appeal may be made 
to the CCRC. 
 


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APPENDIX D 
 
Title: Polymorphisms associated with clinical and molecular responses in follicular 
lymphoma patients treated with rituximab and the novel agent bendamustine  
 
PrincipaI Investigator:  
Frederick Lansigan, MD 
 
 
 
 
Hematology/Oncology 
 
 
 
 
frederick.lansigan@hitchcock.org  
 
 
 
 
603-650-2967 
 
Co-Investigator:  
 
Gregory Tsongalis, MD 
 
 
 
 
Molecular Pathology 
 
 
 
 
gregory.tsongalis@hitchcock.org  
 
 
 
 
603-650-7171 
 
Statistician:  
 
Todd McKenzie, MD 
 
 
 
 
Biostatistics 
 
 
 
 
 
todd.a.mckenzie@hitchcock.org  
 
 
 
 
603-653-3542 
 
 
Abstract   
Rituximab is a monoclonal antibody (mAb) directed against the CD20 antigen present on 
lymphoma cells. Rituximab has revolutionalized the treatment follicular lymphoma, showing 
significantly better response rates, progression-free survival and overall survival when added to 
chemotherapy. Despite these impressive outcomes, rituxmab’s mechanisms of action are 
unknown. Rituximab works through many mechanisms, including antibody-dependent cellular 
cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct apoptotic effects by 
signaling through CD20.  
Polymorphisms of two IgG Fcγ receptors have been shown to predict clinical responses 
to rituximab therapy, presumably because of their role in ADCC.  The correlation of 
polymorphisms and response in patients treated with rituximab combined with chemotherapy, or 
“chemoimmunotherapy” is unclear. The D1015 clinical trial is studying the clinical and 
molecular response rates of follicular lymphoma patients to rituximab and bendamustine. As a 
scientific correlative to this ongoing study, we will determine how polymorphisms in Fcγ 
receptors correlate with clinical and molecular responses. We hypothesize that the response rates 
will be higher for patients with FcγRIIIA-158-V/V and FcγRIIa-131-H/H polymorphisms. If this 
correlation is confirmed it adds further credence that rituximab primarily acts by way of ADCC. 
Until recently, R-CHOP has been the preferred first-line chemoimmunotherapy for the treatment 
of follicular lymphoma. The clinical trial D1015 uses a novel alkylator/purine analog hybrid, 
bendamustine, in combination with rituximab. This novel combination has shown superiority 
when compared to R-CHOP. This study will be the first study of FcγR polymorphisms in 
follicular lymphoma patients treated with rituximab and the novel agent, bendamustine. 
 
 


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Background   
Rituximab is a chimeric IgG1 monoclonal antibody (mAb) directed against the B-cell 
surface antigen CD20 expressed on B-lymphocytes. Rituximab induces killing of normal and 
malignant B-cells expressing CD20. Rituximab’s proposed mechanisms include: 1) elicitation of 
antibody-dependent cellular cytotoxicity (ADCC), 2) induction of lymphoma cell death through 
complement-dependent cytolysis (CDC) and/or complement-dependent cellular toxicity, and 3) 
direct induction of apoptosis following engagement of CD20 by rituximab.[21]  
Clinical response to rituximab is dependent on specific FcγR polymorphisms. Cartron, et 
al. demonstrated that follicular lymphoma patients homozygous for FcγRIIIa-158-V/V [valine] 
had a significantly better clinical response rate (100%) compared to patients expressing 
heterozygous (V/F) or homozygous phenylalanine (F/F) genotype (67%).[22] In addition, Weng 
and Levy confirmed the FcγRIIIa-158-V/V association with clinical response in another cohort 
of follicular lymphoma patients.[23] They also reported that the polymorphism FcγRIIa-131-H/H 
[histidine] is  independently associated with response rate compared to R [arginine] carriers.[23] 
Similarly, in a study of Waldenstrom’s macroglobulinemia, another form of indolent lymphoma, 
response rates to rituximab correlated with FcγRIIIa-158 polymorphisms.[24] The response trend 
was higher for patients with FcγRIIIA-158-V/V, compared to heterozygous (V/F) or 
homozygous (F/F).[24] Thus, rituximab’s major mechanism of action seems to be through 
ADCC.   
In this model, the rituximab’s Fc portion binds to Fcγ receptors on effector cells, 
including natural killer cells, and granulocytes. This binding leads to the destruction of 
rituximab-bound B-cells, either by phagocytosis or by the release of cytotoxic granules contained 
in the effector cells. Functional allelic polymorphisms of the three classes of FcγR (FcγRI, 
FcγRII, and FcγRIII) exist in humans and these generate different receptor properties affecting 
binding affinity for the different IgG subclasses including IgG1.[21] Genetic polymorphisms 
corresponding to phenotype expression of valine (V) or phenylalanine (F) at amino acid 158 on 
the FcγRIIIa greatly influences the affinity of IgG1 to the Fcγ receptor. Studies have 
demonstrated the stronger binding of IgG1 antibody to homozygous FcγRIIIa-158V/V NK cells 
than to homozygous FcγRIIIa-158F/F or heterozygous NK cells.[25, 26] Similarly, 
polymorphisms related to the expression of histidine (H) or arginine (R) at amino acid 131 in 
FcγRIIa affects the binding affinity of IgG1.[27] This evidence supports that rituximab, an IgG1 
mAb, functions by ADCC due to higher affinity binding. 
When rituximab is combined with chemotherapy, response rates improve significantly 
compared to chemotherapy or rituximab alone. The addition of rituximab to every 
chemotherapeutic combination studied (CVP, CHOP, FND, FCM, bendamustine)[1, 2, 4-6, 15, 
17] has resulted in significant increase in the overall response rate and complete response rate. In 
addition, R-CVP has demonstrated a significant increase in overall survival when compared to 
CVP alone.[5] The mechanism of the added benefit of rituximab to chemotherapy is more 
elusive. For instance, in one study, when patients are sequentially treated with chemotherapy 
(CHOP) followed by rituximab, the association between FcγRIIIa and FcγRIIa is apparently 
lost.[28] However, in diffuse large B-cell lymphoma patients treated with R-CHOP, Kim, et al. 
demonstrated that the FcγRIIIa-158-V/V polymorphism was associated with responses and faster 
time to response.[29] Thus, it is still unclear whether rituximab works by ADCC in lymphoma 
patients treated with chemoimmunotherapy. It is also unclear whether rituximab acts differently 
when combined with chemotherapeutic agents other than CHOP, such as bendamustine. This 
study will provide useful data as to the mechanism of rituximab’s effect in follicular lymphoma 
patients treated with chemoimmunotherapy that incorporates the novel chemotherapeutic agent, 
bendamustine. Bendamustine is a purine analog/alkylator hybrid cytotoxic chemotherapy with 
significant clinical activity in lymphoma. It is highly active in refractory indolent B-cell 


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lymphoma as a single agent with an overall response rate of 75%.[30] In front-line combination 
with rituximab compared to R-CHOP, bendamustine significantly improves overall response 
rate, complete response rate, and progression-free survival. Bendamustine and rituximab 
chemoimmunotherapy results in far fewer toxicities including less infectious complications, and 
practically no alopecia.[17]  
Within the context of this clinical trial, we will examine specific genetic polymorphisms 
that correlate with clinical and molecular response the chemoimmunotherapy combination of 
bendamustine and rituximab. These results will provide additional knowledge addressing 
molecular predictors of rituximab sensitivity and resistance and will have important implications 
in personalizing immunotherapy for the treatment of follicular lymphoma. 
 
Specific Aims 
 
To determine the association of FcγRIIIa-158-V/V and FcγRIIa-131-H/H 
polymorphisms with clinical and molecular responses in follicular lymphoma patients 
treated with rituximab and bendamustine.  
 
Methods 
 
 
 
 
 
 
Patient samples will be collected and stored at study entry from consenting follicular 
lymphoma patients enrolled in D1015. In this proposed study we will examine FcγR (FcγRIIIa 
and FcγRIIa) polymorphisms known to correlate with response. In order to detect the FcγRIIIA 
polymorphisms, DNA will be extracted from peripheral blood mononuclear cells collected prior 
to treatment using a kit (Qiagen). Genotyping of FCGR3A-158V/F polymorphism will be 
performed as described by Cartron,[22] et al using a nested PCR followed by allele-specific 
restriction enzyme digestion. Briefly, 2 FCγRIIIA-specific primers (5’-
ATATTTACAGAATGGCACAGG-3’, 5’-GACTTGGTACCCAGGTTGAA-3’) will be used to 
amplify a 1.2 kilobase fragment containing the polymorphic site. The initial PCR assay will be 
performed with 1.25µg genomic DNA, 200ng of each primer, 200µM of each 
deoxyribonucleoside triphosphate (dNTP) and 1U Taq DNApolymerase. This first PCR will 
consist of 10 minutes at 95°C, then 35 cycles (each consisting of steps at 95°C for 1 minute, 
57°C for 1.5 minutes, and 72°C for 1.5 minutes), and 8 minutes at 72°C to achieve complete 
extension. The second PCR will use primers (5’-
ATCAGATTCGATCCTACTTCTGCAGGGGGCAT-3’, 5’-
ACGTGCTGAGCTTGAGTGATGGTGATGTTCAC-3’) amplifying a 94base pair (bp) 
fragment and creating an NlaIII restriction site only in the FCγRIIIa-158V allele. This nested 
PCR will be performed with 1µL of the amplified DNA, 150ng of each primer, 200µM of each 
dNTP, and 1U of Taq DNA polymerase. The first cycle will consist of 5 minutes at 95°C, then 
35 cycles (each consisting of steps at 95°C for 1 minute, 64°C for 1 minute, and 72°C for 1 
minute), and 9.5 minutes at 72°C to complete extension. The amplified DNA (10µL) will be 
digested with 10U NlaIII at 37°C for 12 hours and separated by electrophoresis on 8% 
polyacrylamide gel. After staining with ethidium bromide, DNA bands will be visualized under 
UV light. For homozygous FCγRIIIa-158F/F patients, only one undigested band (94 bp) should 
be visible. Three bands (94 bp, 61 bp, and 33 bp) should be seen in heterozygous individuals, 
whereas for homozygous FCγRIIIa-158V/V patients only 2 digested bands (61 bp and 33 bp) 
should be obtained. Similarly, genotyping of FCγRIIa-131-H/R will consist of PCR followed by 
allele-specific restriction enzyme digestion, also described by Cartron, et al.[22] 
 
Statistical Analysis 
The primary endpoint of the clinical trial within this study is complete response. The 
secondary endpoint is overall response (complete or partial). Statistical analysis of the clinical 


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41 
and molecular responses of the patients in the different genotypic groups will be compared by a 
two-tailed Fisher exact test, as well as an exact trend test (i.e., permutation version of chi-square 
test for trend). The latter trend test examines the association of number of alleles (0, 1 or 2) with 
the endpoint.  A logistic regression analysis including age >60, stage III or IV, bulky 
lymphadenopathy greater the 6cm, bone marrow involvement, number of extranodal sites >2, 
B2M, will be used to identify independent prognostic variables influencing clinical and 
molecular response.  
A total of 39 patients will be studied. We shall have sufficient power (i.e., 80%) to detect 
associations of complete response with presence of an allele (e.g., 1 or 2 copies vs 0) whose rate 
ratio is 3.5. For instance, if in truth 75% of patients with 1 or 2 copies have complete response 
versus 20% with 0 copies we shall have 80% power to obtain a significant finding (i.e., 
p<=0.05). This calculation assumes a complete response rate of 35% and genotype frequency of 
approximately 2 alleles: 15%, 1 allele: 45% and 0 alleles: 45%. 
 
Expected Results 
We anticipate finding an independent correlation between the FcgammaRIIIa-
158/FcgammaRIIa-131 polymorphisms and response. These results will define the effects of 
rituximab immunotherapy on follicular lymphoma, and learn the effects of rituximab within a 
combination regimen that includes bendamustine, a novel highly active drug. If this pilot study 
shows a correlation of these FcγR polymorphisms with response, it will substantiate findings by 
others that rituximab primarily acts through ADCC. Our results will be the first reported for 
rituximab in combination with the novel agent bendamustine. If this pilot study does not show a 
correlation with response rate, it may suggest that an alternative endpoint be used to correlate 
with these polymorphisms such as progression-free survival. 
 
Alternative Studies 
Alternatively, if no correlation is found between these polymorphisms and response, it 
would suggest that rituximab acts by other mechanisms in combination with chemotherapy and 
these mechanisms would need to be explored further. For instance, FcγRI may play a role in 
rituximab immunotherapy. FcγRI binding and its contribution to ADCC may be enhanced by 
cytokines such as IL-3, by qualitative effects on binding (termed “inside-out” regulation).[31] 
Apart from ADCC, a CDC mechanism directed through C1qA may also play a role in long-term 
response and PFS in rituximab-treated follicular lymphoma patients.[32] Pharmacogenetics 
relating to bendamustine therapy such as CYP1A2[33] or DNA repair enzyme 
polymorphisms[34, 35] may play a role in the responses to bendamustine. When used in 
combination with chemotherapy, rituximab may prime malignant cells to the effects of cytotoxic 
chemotherapy, as well as overcome chemotherapy resistance.[36] Rituximab may modulate the 
chemosensitivity of follicular lymphoma tumor cells by downregulation of Bcl-2 and Bcl-
XL[36] and this phenomenon would need to be explored further. Polymorphisms or mutations of 
the CD20 protein may correlate with rituximab sensitivity or resistance,[37] and can be further 
studied. Study of these alternative factors will enhance our understanding of targeted 
immunotherapy for follicular lymphoma.  
 
Feasibility 
An accrual of 39 patients is planned for D1015 over 2-3 years. This is a multicenter trial 
with an expected accrual rate of 1-2 patients per month. In one year, we expect 12-15 patients 
assessable for clinical and molecular response. The preliminary correlations between 
polymorphisms and response obtained from this study will be used for future grants. 
 


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Relevance of results to cancer and translational merit 
The use of FcγRIII and FcγRII polymorphism testing to predict outcomes of follicular 
lymphoma patients treated with rituximab in combination with chemotherapy has the capacity to 
contribute to the individualized care of the patient. Predictive genomic testing can play an 
integral role in choosing treatments that are highly effective and omitting agents that are not 
likely to have any effect. Successful demonstration of an association between FcγRIII and FcγRII 
polymorphisms and response to this novel combination of rituximab and bendamustine will 
enable predictive genomic testing for follicular lymphoma patients treated with this regimen. 
 
Future Directions 
There are now a new generation of anti-CD20 mAbs including ofatumumab and GA101. 
Ofatumumab is a fully humanized anti-CD20 mAb which is approved for use in refractory 
chronic lymphocytic leukemia, and is an exploratory treatment for follicular lymphoma. 
Ofatumumab binds to a different epitope of CD20 compared to rituximab, and its mechanism of 
action is unknown, but likely functions through ADCC and/or CDC. A multicenter collaborative 
clinical trials working group has been established that includes Dartmouth, Brown, and Yale. 
One of the ongoing clinical trials from this working group is studying the clinical response of 
ofatumumab combined with bortezomib in patients with low-grade B-cell NHL. The differences 
in immune mechanisms of rituximab and ofatumumab activity have not been well studied. The 
results and methods established from the current pilot proposal can be applied to the next 
generation CD20 mAbs to understand these differences. 
This pilot data would serve as the framework for extramural funding for these additional 
studies. 
 
References 
1. 
Bello C, Sotomayor EM. Novel Immunotherapeutic Approaches for Lymphomas. American Society of Hematology, Education Book 
2007:233-242. 
2. 
Cartron G, Dacheux L, Salles G, et al. Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc 
receptor FcgammaRIIIa gene. Blood. 2002;99:754-758. 
3. 
Weng WK, Levy R. Two immunoglobulin G fragment C receptor polymorphisms independently predict response to rituximab in 
patients with follicular lymphoma. J Clin Oncol. 2003;21:3940-3947. 
4. 
Treon SP, Hansen M, Branagan AR, et al. Polymorphisms in FcgammaRIIIA (CD16) receptor expression are associated with clinical 
response to rituximab in Waldenstrom's macroglobulinemia. J Clin Oncol. 2005;23:474-481. 
5. 
Koene HR, Kleijer M, Algra J, Roos D, von dem Borne AE, de Haas M. Fc gammaRIIIa-158V/F polymorphism influences the binding of 
IgG by natural killer cell Fc gammaRIIIa, independently of the Fc gammaRIIIa-48L/R/H phenotype. Blood. 1997;90:1109-1114. 
6. 
Wu J, Edberg JC, Redecha PB, et al. A novel polymorphism of FcgammaRIIIa (CD16) alters receptor function and predisposes to 
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Binstadt BA, Geha RS, Bonilla FA. IgG Fc receptor polymorphisms in human disease: implications for intravenous immunoglobulin 
therapy. J Allergy Clin Immunol. 2003;111:697-703. 
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Hiddemann W, Kneba M, Dreyling M, et al. Frontline therapy with rituximab added to the combination of cyclophosphamide, 
doxorubicin, vincristine, and prednisone (CHOP) significantly improves the outcome for patients with advanced-stage follicular lymphoma 
compared with therapy with CHOP alone: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood. 
2005;106:3725-3732. 
9. 
Forstpointner R, Dreyling M, Repp R, et al. The addition of rituximab to a combination of fludarabine, cyclophosphamide, 
mitoxantrone (FCM) significantly increases the response rate and prolongs survival as compared with FCM alone in patients with relapsed and 
refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. 
Blood. 2004;104:3064-3071. 
10. 
Wierda W, O'Brien S, Wen S, et al. Chemoimmunotherapy with fludarabine, cyclophosphamide, and rituximab for relapsed and 
refractory chronic lymphocytic leukemia. J Clin Oncol. 2005;23:4070-4078. 
11. 
Rummel MJ, von Gruenhagen U, Niederle N, et al. Bendamustine Plus Rituximab Versus CHOP Plus Rituximab in the First-Line-
Treatment of Patients with Follicular, Indolent and Mantle Cell Lymphomas: Results of a Randomized Phase III Study of the Study Group 
Indolent Lymphomas (StiL). ASH Annual Meeting Abstracts. 2008;112:2596-. 
12. 
Marcus R, Imrie K, Solal-Celigny P, et al. Phase III study of R-CVP compared with cyclophosphamide, vincristine, and prednisone 
alone in patients with previously untreated advanced follicular lymphoma. J Clin Oncol. 2008;26:4579-4586. 
13. 
Marcus R, Imrie K, Belch A, et al. CVP chemotherapy plus rituximab compared with CVP as first-line treatment for advanced follicular 
lymphoma. Blood. 2005;105:1417-1423. 
14. 
Czuczman MS, Koryzna A, Mohr A, et al. Rituximab in combination with fludarabine chemotherapy in low-grade or follicular 
lymphoma. J Clin Oncol. 2005;23:694-704. 
15. 
Carlotti E, Palumbo GA, Oldani E, et al. FcgammaRIIIA and FcgammaRIIA polymorphisms do not predict clinical outcome of follicular 
non-Hodgkin's lymphoma patients treated with sequential CHOP and rituximab. Haematologica. 2007;92:1127-1130. 


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16. 
Kim DH, Jung HD, Kim JG, et al. FCGR3A gene polymorphisms may correlate with response to frontline R-CHOP therapy for diffuse 
large B-cell lymphoma. Blood. 2006;108:2720-2725. 
17. 
Kahl BS, Bartlett NL, Leonard JP, et al. Bendamustine is effective therapy in patients with rituximab-refractory, indolent B-cell non-
Hodgkin lymphoma: results from a Multicenter Study. Cancer;116:106-114. 
18. 
Cheson BD, Horning SJ, Coiffier B, et al. Report of an international workshop to standardize response criteria for non-Hodgkin's 
lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999;17:1244. 
19. 
van der Poel CE, Karssemeijer RA, Boross P, et al. Cytokine induced immune complex binding to the high affinity IgG receptor, 
Fc{gamma}RI, in the presence of monomeric IgG. Blood:blood-2010-2004-280214. 
20. 
Racila E, Link BK, Weng WK, et al. A polymorphism in the complement component C1qA correlates with prolonged response 
following rituximab therapy of follicular lymphoma. Clin Cancer Res. 2008;14:6697-6703. 
21. 
Teichert J, Baumann F, Chao Q, et al. Characterization of two phase I metabolites of bendamustine in human liver microsomes and in 
cancer patients treated with bendamustine hydrochloride. Cancer Chemotherapy and Pharmacology. 2007;59:759-770. 
22. 
Rossi D, Rasi S, Franceschetti S, et al. Analysis of the host pharmacogenetic background for prediction of outcome and toxicity in 
diffuse large B-cell lymphoma treated with R-CHOP21. Leukemia. 2009;23:1118-1126. 
23. 
Leoni LM, Bailey B, Reifert J, et al. Bendamustine (Treanda) displays a distinct pattern of cytotoxicity and unique mechanistic 
features compared with other alkylating agents. Clin Cancer Res. 2008;14:309-317. 
24. 
Bonavida B. What signals are generated by anti-CD20 antibody therapy? Curr Hematol Malig Rep. 2006;1:205-213. 
25. 
Terui YMY, Mishima Y, Yokoyama M, et al. Point mutation of C-terminal region of CD20 molecule predicts rituximab-induced 
complement-dependent cytotoxicity and clinical response to rituximab in non-Hodgkin’s lymphoma. J Clin Oncol 2006;24(18s):7563. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX E 
Karnofsky and ECOG Performance Status Conversion Table 
ECOG 
KPS 
Performance Status 
0 
90-100 
Fully active, able to carry on all pre-disease performance without 
restriction. 
1 
70-80 
Restricted in physically strenuous activity but ambulatory and able to 
carry out work of a light or sedentary nature. 
2 
50-60 
Ambulatory and capable of all self care but unable to carry out any work 
activities. Up and about more than 50% of waking hours. 
3 
30-40 
Capable of only limited self care, confined to bed or chair more than 50% 
of waking hours 
4 
10-20 
Completely disabled. Cannot carry on any self care. Totally confined to a 
bed or chair. 
5 
0 
Dead 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX F 
Cockcroft-Gault Equation for Creatinine Clearance: 
Creatinine clearance is calculated from plasma creatinine (Cr), age, weight, and gender 
correction factor (GF; 0.85 in women, 1.0 in men). 
 
Cockcroft-Gault GFR = (140-age) * (Wt in kg) * (0.85 if female) / (72 * Cr)  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX G 
In order to correctly assess lymphoma response, please follow the guidelines: 
1. Determine lymphoma measurable disease 
a. Select a maximum of 6 lesions with the following features: 
i. Measurable in at least 2 perpendicular dimensions 
ii. Representative of all involved organs/areas 
iii. Suitable for accurate repeat measurements 
iv. Include mediastinal and retroperitoneal areas of disease 
2. Determine lymphoma non-measurable disease 
a. Assessable Disease 
i. Masses that cannot be measured bi-dimensionally 
ii. Organ involvement (liver and spleen) 
iii. Recorded as “present” at baseline, then “no change”, “increased”, 
“decreased”, “absent” at follow up 
b. Bone marrow status 
i. Positive or negative for lymphoma cells 
3. Report bi-dimensional nodal measurements 
a. Select sites and measure two longest diameters 
b. Multiply each node’s two longest diameters together 
c. Sum the Products of the greatest transverse Diameters (SPD or tumor size) 
d. Double check calculations 
e. Use correct units (cm) 
4. Calculating change: 
a. For response:  % Decrease=(baseline-current disease assessment)/baseline 
b. For progression: % Increase=(current disease assessment-smallest sum)/smallest 
sum 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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