Obinutuzumab treatment of a DLBCL patient subgroup
11597772 · 2023-03-07
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
C07K2317/41
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
G01N2800/52
PHYSICS
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
G01N2333/70596
PHYSICS
C12Q2600/106
CHEMISTRY; METALLURGY
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2039/545
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
The present invention relates to obinutuzumab (or its functional equivalents) for use in the treatment of a particular biomarker-defined DLBCL patient and a novel DLBCL patient subgroup, respectively. The present invention further relates to a method for treating DLBCL with obinutuzumab (or its functional equivalents) in a patient in need thereof, wherein said patient is a particular biomarker-defined DLBCL patient or belongs to a novel biomarker-defined DLBCL patient subgroup. The present invention further relates to the use of obinutuzumab (or its functional equivalents) for the preparation of a pharmaceutical composition for the treatment of DLBCL in the particular biomarker-defined DLBCL patient/novel DLBCL patient subgroup. The present invention further relates to a method for identifying a particular DLBCL patient/novel DLBCL patient subgroup and a method for diagnosing a novel form of DLBCL and a particular DLBCL patient/novel DLBCL patient subgroup, respectively.
Claims
1. A method of treating a patient having a diffuse large B-cell lymphoma (DLBCL), the method comprising administering to the patient a therapeutically effective amount of a humanized Type II anti-CD20 antibody, wherein a tumor sample from the patient has a germinal center B-cell-like (GCB) cell-of-origin (COO) status; wherein the humanized Type II anti-CD20 antibody comprises a heavy chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 1 and a light chain variable region comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 2; and wherein the GCB COO status is a strong GCB COO status identified by a linear predictor score (LPS) for the tumor sample that is less than 1141, and wherein the LPS is a weighted sum of the expression levels of the genes TNFRSF13B, LIMD1, IRF4, CREB3L2, PIM2, CYB5R2, RAB7L1, CCDC50, MME, SERPINA9, ASB13, MAML3, ITPKB, MYBL1, S1PR2, R3HDM1, WDR55, ISY1, UBXN4, and TRIM56 and is calculated according the formula:
LPS(X)=Σ.sub.ja.sub.jX.sub.j, wherein X.sub.j is the gene expression for gene j and a.sub.j is the coefficient for gene j.
2. The method of claim 1, wherein the humanized Type II anti-CD20 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2.
3. The method of claim 1, wherein the tumor sample from the patient further has (a) one or more mutations in one or more of CREBBP, EP300, MEF2B, MYC, EZH2, and TNFRSF14 and (b) one or both of (i) one or more genetic mutations in CD58 and (ii) an expression level of CD58 that is less than 5.3 log.sub.2(nRPKM).
4. The method of claim 1, wherein the tumor sample from the patient further has one or both of (i) a translocation of BCL2 and (ii) a high expression level of BCL2 as compared to a control.
5. The method of claim 1, wherein the patient reaches an improved clinical outcome as compared to a patient treated with rituximab.
6. The method of claim 5, wherein the clinical outcome is progression-free survival (PFS), overall survival (OS), or event-free survival (EFS).
7. The method of claim 1, wherein the humanized Type II anti-CD20 antibody comprises a glycoengineered Fc region.
8. The method of claim 7, wherein the humanized Type II anti-CD20 antibody has an increase in the fraction of non-fucosylated oligosaccharides attached to the glycoengineered Fc region relative to an anti-CD20 antibody that comprises a non-glycoengineered Fc region.
9. The method of claim 8, wherein the non-fucosylated oligosaccharides are bisected non-fucosylated oligosaccharides.
10. The method of claim 1, wherein the humanized Type II anti-CD20 antibody has an increased affinity for human FcγRIII receptors relative to an anti-CD20 antibody that comprises a non-glycoengineered Fc region or rituximab.
11. The method of claim 1, wherein the humanized Type II anti-CD20 antibody is capable of causing increased antibody-dependent cellular cytotoxicity (ADCC) relative to a non-glycoengineered antibody.
12. The method of claim 1, wherein the humanized Type II anti-CD20 antibody is capable of causing increased ADCC relative to rituximab.
13. The method of claim 1, wherein the antibody is obinutuzumab.
14. The method of claim 1, further comprising administering to the patient one or more additional cytotoxic agents or chemotherapeutic agents.
15. The method of claim 14, further comprising administering to the patient ionizing radiation enhancing the effects of the one or more cytotoxic agents or the one or more chemotherapeutic agents.
16. The method of claim 14, comprising administering to the patient the chemotherapeutic agents (i) cyclophosphamide; (ii) doxorubicin; (iii) vincristine; and (iv) prednisone or prednisolone.
17. The method of claim 1, wherein the humanized Type II anti-CD20 antibody is administered to the patient in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
18. The method of claim 1, wherein the humanized Type II anti-CD20 antibody is administered intravenously at a dose of 1000 mg every three weeks.
19. The method of claim 18, wherein the humanized Type II anti-CD20 antibody is administered on days 1, 8, and 15 of a first 21-day treatment cycle and on day 1 of one or more additional 21-day treatment cycles.
20. The method of claim 19, wherein 8 cycles of treatment are administered.
21. The method of claim 1, wherein the tumor sample from the patient further has an expression level of CD58 that is less than or equal to 5.2 log.sub.2(nRPKM).
22. The method of claim 1, wherein the expression levels of the genes are mRNA expression levels.
23. The method of claim 1, wherein a strong GCB COO status of a tumor sample from the patient is identified by a LPS for the tumor sample that is less than or equal to 1100.
24. The method of claim 23, wherein a strong GCB COO status of a tumor sample from the patient is identified by a LPS for the tumor sample that is less than or equal to 749.
25. The method of claim 24, wherein a strong GCB COO status of a tumor sample from the patient is identified by a LPS for the tumor sample that is less than or equal to 725.
26. The method of claim 1, wherein the tumor sample from the patient further has one or more of: (a) molecular follicular lymphoma (FL)-like features; (b) one or more mutations in one or more of CREBBP, EP300, MEF2B, MYC, EZH2, and TNFRSF14; (c) one or more mutations in CD58; (d) an expression level of CD58 that is less than 5.3 log.sub.2(nRPKM); (e) a translocation of BCL2; and (f) expression of BCL2 in at least 50% of tumor cells.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.
(2)
(3) Disposition of patients in GOYA.
(4) *Discontinued refers to patients who discontinued study (antibody) treatment.
(5) .sup.†Median observation time was 29 months in each group; completed treatment refers to patients who completed study (antibody) treatment.
(6) Patients were stratified at randomization by IPI score (low/low-intermediate, high-intermediate, and high-risk), planned number of CHOP cycles (8 vs. 6), and geographic region (Western Europe, Eastern Europe, South and Central America, North America, Asia, and others).
(7) G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone.
(8) Kaplan-Meier estimates of PFS and OS in GOYA.
(9)
(10) ABC, activated B-cell-like; CI, confidence interval; GCB, germinal-center B-cell-like; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; HR, hazard ratio; OS, overall survival; PFS, progression-free survival; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.
(11) Unstratified hazard ratios for investigator-assessed PFS in GOYA patient subgroups.
(12)
(13) ABC, activated B-cell-like; CI, confidence interval; DLBCL, diffuse large B-cell lymphoma;
(14) ECOG PS, Eastern Cooperative Oncology Group performance status; GCB, germinal-center B-cell-like; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; IPI, International Prognostic Index; KM, Kaplan-Meier; PFS, progression-free survival; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone.
(15) *Cases where ‘yes’ was ticked in the eCRF for extranodal involvement; 14 patients with extranodal sites 0 were ticked in error.
(16)
(17) Gazyva benefit in BCL2 translocated patients in GOYA
(18)
(19) Gazyva benefit in BCL2 protein expression positive patients in GOYA
(20)
(21) Gazyva benefit in BCL2 translocated patients that are BCL2 protein expression positive in GOYA
(22) Evaluation of treatment effect in quadrants defined by BCL2 IHC (pos/neg) and BCL2 FISH (pos/neg) in GOYA. Gazyva superiority shown to be specific to BCL2 IHC.sub.+/FISH.sub.+ pts.
(23) Gazyva benefit in subgroups of GCB defined by various cutoffs of the Linear Predictor Score in GOYA
(24)
(25)
(26)
(27)
(28) Gazyva benefit in GCB patients that are BCL2 translocated and BCL2 protein expression positive in GOYA.
(29)
(30) Gazyva benefit in CD58 mutated patients and/or patients with low CD58 gene expression in GOYA.
(31)
(32) Multivariate simulation optimization of the LPS cutoff for G-CHOP benefit over R-CHOP on progression-free survival in biomarker-evaluable pts with COO analysis (N=xx).
(33) LPS cutoff optimization for strong-GCB treatment effect in GOYA original data.
(34) *Multivariate HR adjusted for treatment, International Prognostic Index, number of chemotherapy cycles (6 or 8), and geographical region. Blue line, point estimate of the HR, yellow line, 95% CI; CI, confidence interval; COO, cell of origin; HR, hazard ratio; LPS, Linear Predictor Score.
(35) Molecular characterization of strong-GCB patients.sup.# in GOYA.
(36) Strong-GCB patients have significantly higher prevalence of FL somatic mutation hallmarks.
(37)
(38)
(39) .sup.# Other biomarkers evaluated to characterize strong-from weak-GCB, where no significant difference in prevalence rate was identified, were: by gene expression, stromal-1/2 gene signatures, immune-response 1/2 gene signatures, CD20, and PTEN; by protein expression, BCL2, MYC, and BCL2/MYC double-expressors; and by gene translocations, MYC translocations and BCL2/MYC double-hit. ABC, activated B-cell; DLBCL, diffuse large B-cell lymphoma; FDR, false discovery rate; FISH, fluorescence in situ hybridization; FL, follicular lymphoma; GCB, germinal center B-cell; NGS, next-generation sequencing.
(40)
(41) Distribution of optimal LPS cutoff across bootstrap samples Bootstrap multivariate simulations to further test robustness and generalizability of an optimal LPS identified using the “min.HR rule” Extreme peak across bootstrap samples is at LPS=725 LPS distribution with its unique peak, supports robustness of treatment effect signal Optimal LPS cutoff suggested for new potential confirmatory study is LPS≤725 Historically all GOYA biomarker analyses has defined strong-GCB as <749 (25% of pts in GOYA), including biomarker analyses presented in this OBRF n=4 pts with 725<LPS<749, all G-CHOP (1 event)
(42)
(43) Kaplan-Meier Estimates of Time to Next Anti-Lymphoma Treatment (Secondary Endpoint) in the Intent-To-Treat Population.
(44) CI, confidence interval; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; HR, hazard ratio; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone.
(45) Kaplan-Meier Estimates of Investigator-Assessed PFS by Treatment Arm in Patients With COO Data, Subgrouped by COO Subtype.
(46)
(47) ABC, activated B-cell-like; CI, confidence interval; COO, cell of origin; GCB, germinal-center B-cell-like; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; HR, hazard ratio; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone.
(48) The present invention also refers to the following tables.
(49) TABLE-US-00002 TABLE 1 Baseline Patient and Disease Characteristics (Intent-to-Treat Population) G-CHOP R-CHOP Characteristic (N = 706)* (N = 712)* Median age, years (range) 62.0 (18-86) 62.0 (18-83) Male - no. (%) 369 (52.3) 383 (53.8) Geographic region - no. (%) Asia 260 (36.8) 258 (36.2) Western Europe 211 (29.9) 215 (30.2) North America 109 (15.4) 107 (15.0) Eastern Europe 97 (13.7) 99 (13.9) Other 29 (4.1) 33 (4.6) ECOG PS - no. (%) n = 705 n = 712 0-1 618 (87.7) 613 (86.1) 2-3 87 (12.3) 99 (13.9) Ann Arbor stage - no. (%) n = 706 n = 711 I and II 170 (24.1) 171 (24.0) III and IV 536 (75.9) 540 (75.8) IPI risk group - no. (%) Low/low intermediate 376 (53.3) 409 (57.4) High-intermediate 221 (31.3) 192 (27.0) High 109 (15.4) 111 (15.6) Planned chemotherapy cycles - no. (%) 6 523 (74.1) 526 (73.9) 8 183 (25.9) 186 (26.1) LDH elevated - no. (%) n = 705 n = 708 415 (58.9) 401 (56.6) Extranodal involvement - no. (%).sup.† 484 (68.6) 468 (65.7) Bulky disease (7.5 cm) - no. (%) 261/703 (37.1) 262/710 (36.9) Cell of origin n = 471.sup.‡ n = 462.sup.‡ GCB 271 (57.5) 269 (58.2) ABC 125 (26.5) 118 (25.5) Unclassified 75 (15.9) 75 (16.2) ABC, activated B cell-like (subgroup); ECOG PS, Eastern Cooperative Oncology Group performance status; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; GCB, germinal-center B cell-like (subgroup); IPI, International Prognostic Index; LDH, lactate dehydrogenase; PET, positron emission tomography; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone. *n = 706 for G-CHOP and n = 712 for R-CHOP for all parameters unless otherwise specified. .sup.†Cases where ‘yes’ was ticked in the eCRF for extranodal involvement; 14 patients with extranodal sites 0 were ticked in error. .sup.‡COO subtype classification was missing for 485 patients (G-CHOP, 235; R-CHOP, 250); includes samples from China which could not be analyzed due to lack of an export license - analysis of these samples is planned in the near future.
(50) TABLE-US-00003 TABLE 2 Summary of Efficacy Endpoints (Intent-to-Treat Population) Investigator Assessment G-CHOP R-CHOP Endpoint N = 706) N = 712) Median observation time, months (range) 29.0 (0.1-56.6) 28.9 (0.1-56.2) Investigator-assessed PFS (primary endpoint) N = 706 N = 712 Patients with event, no. (%) 201 (28.5) 215 (30.2) 3-year PFS, % 69.6 66.9 Stratified HR (95% CI) - P value (log-rank)* 0.92 (0.76-1.11), P = .3868 IRC-assessed PFS N = 706 N = 712 Patients with event, no. (%) 171 (24.2) 186 (26.1) 3-year PFS, % 72.5 70.6 Stratified HR (95% CI), P value (log-rank)* 0.89 (0.72 to 1.10), P = .2736 OS N = 706 N = 712 Patients with event, no. (%) 126 (17.8) 126 (17.7) 3-year OS, % (95% CI) 81.2 (77.9 to 84.1) 81.4 (78.1 to 84.3) Stratified HR (95% CI)* 1.00 (0.78 to 1.28) DFS in patients with investigator-assessed CR n = 397 n = 369 Patients with event, no. (%) 77 (19.4) 64 (17.3) Stratified HR (95% CI)* 1.27 (0.91 to 1.77) Investigator-assessed EFS N = 706 N = 712 Events, no. (%) 236 (33.4) 250 (35.1) Stratified HR (95% CI)* 0.92 (0.77 to 1.11) Time to start of new anti-lymphoma treatment N = 706 N = 712 Patients with event, no. (%) 213 (30.2) 230 (32.3) Proportion event-free at 3 years, % (95% CI) 69.9 (66.2 to 73.2) 66.5 (62.7 to 70.1) Stratified HR (95% CI)* 0.92 (0.76 to 1.11) Investigator-assessed response (with PET) n = 669 n = 665 at end of treatment.sup.† ORR Proportion, no. (%) 518 (77.4) 518 (77.9) Percentage difference (95% CI) −0.47 (−5.01 to 4.08) CR Proportion, no. (%) 379 (56.7) 396 (59.5) Difference (95% CI) −2.90 (−8.27 to 2.48) Investigator-assessed response (without PET) N = 706 N = 712 at end of treatment.sup.† ORR Proportion, no. (%) 577 (81.7) 572 (80.3) Percentage difference (95% CI) 1.39 (−2.76 to 5.54) CR Proportion, no. (%) 248 (35.1) 241 (33.8) Difference (95% CI) 1.28 (−3.74 to 6.30) CI, confidence interval; CR, complete response; DFS, disease-free survival; EFS, event-free survival; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; HR, hazard ratio; IRC, Independent Review Committee; ORR, overall response rate; OS, overall survival; PET, positron emission tomography; PFS, progression-free survival; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone. *Stratification factors were International Prognostic Index score and planned number of CHOP cycles (6 or 8). .sup.†According to revised response criteria..sup.13
(51) TABLE-US-00004 TABLE 3 Summary of AEs Including Grade 3-5 and Serious AEs Reported by 5% of Patients in Either Group (At Preferred Term Level; Safety Population) G-CHOP R-CHOP (N = 704), No. (%) (N = 703), No. (%) Number of deaths for any reason 126 (17.9) 122 (17.4) Number of patients withdrawn from study 4 (0.6) 3 (0.4) due to an AE Patients with at least one AE 683 (97.0%) 657 (93.5) Grade 3-5 AE 519 (73.7) 455 (64.7) AE with fatal outcome* 41 (5.8) 30 (4.3) Serious AE 300 (42.6) 264 (37.6) Treatment-related AEs 639 (90.8) 596 (84.8) AEs leading to withdrawal of any treatment 84 (11.9) 60 (8.5) AEs leading to dose reduction for any 145 (20.6) 138 (19.6) treatment Grade 3-5 Serious Grade 3-5 Serious AEs AEs AEs AEs Blood and lymphatic system disorders Total number of patients with at least one 415 (58.9) 135 (19.2) 348 (49.5) 113 (16.1) AE Neutropenia 325 (46.2) 52 (7.4) 268 (38.1) 40 (5.7) Febrile neutropenia 123 (17.5) 81 (11.5) 107 (15.2) 72 (10.2) Leukopenia 96 (13.6) 10 (1.4) 71 (10.1) 5 (0.7) Anemia 51 (7.2) 9 (1.3) 53 (7.5) 6 (0.9) Infections and infestations Total number of patients with at least one 135 (19.2) 121 (17.2) 109 (15.5) 94 (13.4) AE Pneumonia 40 (5.7) 40 (5.7) 35 (5.0) 32 (4.6) AE, adverse event; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone. *Fatal AEs that were reported in more than one patient in either group, listed as preferred terms, were: septic shock (6 [0.9%] patients), pneumonia (5 [0.7%]), death (cause unknown; 3 [0.4%]), pulmonary embolism (2 [0.3%]) and cerebrovascular accident (2 [0.3%]) in the G-CHOP group and pneumonia (6 [0.9%]), sepsis (3 [0.4%]), cerebrovascular accident (2 [0.3%]) and death (cause unknown; 2 [0.3%]) in the R-CHOP group.
(52) TABLE-US-00005 TABLE 4 Effect of G-CHOP and R-CHOP on clinical outcomes in strong-GCB and all other patients Multivariate Analysis Strong-GCB Pts All Other Pts HR (G vs R)* [95% CI]; (n = 233) (n = 700) p-value R-CHOP: n = 121 R-CHOP: n = 341 3-yr rate (%) G-CHOP: n = 112 G-CHOP: n = 359 PFS 0.33 [0.18-0.63]; 0.99 [0.76-1.28]; p = 0.0007 p = 0.9117 R: 66%; G: 88% R: 66%; G: 66% EFS 0.47 [0.28-0.78]; 1.01 [0.79-1.29]; p = 0.00344 p = 0.9513 R: 59%; G: 80% R: 63%; G: 62% OS 0.41 [0.20-0.87]; 1.10 [0.79-1.53]; p = 0.019 p = 0.582 R: 79%; G: 92% R: 81%; G: 78% *Adjusted for treatment arm, International Prognostic Index, number of chemotherapy cycles (6 or 8), and geographic region CI, confidence interval; EFS, event-free survival; GCB, germinal center B-cell; HR, hazard ratio; OS, overall survival; PFS, progression-free survival (investigator-assessed); yr, year
(53) TABLE-US-00006 TABLE 5 Study Drug Exposure G-CHOP R-CHOP (N = 704), (N = 703), No. (%) No. (%) Number of obinutuzumab or rituximab doses 10 (1-10) 8 (1-8) received, median (range) Patients with modifications to any obinutuzumab 222 (31.5) 210 (29.9%) or rituximab dose* Patients with modifications to obinutuzumab or 192/702 (27.4) 155/703 (22.0) rituximab doses in cycle 1* Day 1 39/651 (6.0) 0 Day 8 41/624 (6.6) 0 Day 15 Patients with delays to obinutuzumab or 92 (13.1) 64 (9.1) rituximab doses of > 7 days Patients with 90% planned dose intensity of 671 (95.3) 697 (99.1) obinutuzumab or rituximab Patients with 90% planned dose intensity of Cyclophosphamide 642 (91.3) 647 (92.0) Doxorubicin 631 (89.8) 639 (90.9) Prednisone 662 (94.0) 643 (91.5) Vincristine 642 (91.3) 625 (88.9) Duration of exposure to obinutuzumab or 25.3 (1-32) 25.3 (0-32) rituximab, weeks, median (range) Cumulative dose of obinutuzumab or rituximab 10,000 5,133.5 in mg, median (range) (998-10,065) (515-8,084) G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. *Including interruptions to infusions and slowing of infusion rates.
(54) TABLE-US-00007 TABLE 6 Number (and %) of Patients Reporting AEs of Any Grade With an Incidence Rate of At Least 10% (At Preferred Term Level) in Either Treatment Arm, Listed by System Organ Class and Preferred Term (Safety Population) G-CHOP R-CHOP (N = 704), (N = 703), No. (%) No. (%) Blood and lymphatic system disorders Total number of patients with at least one AE 451 (64.1) 389 (55.3) Neutropenia 340 (48.3) 286 (40.7) Febrile neutropenia 127 (18.0) 108 (15.4) Leukopenia 115 (16.3) 87 (12.4) Anemia 95 (13.5) 99 (14.1) Gastrointestinal disorders Total number of patients with at least one AE 428 (60.8) 410 (58.3) Nausea 207 (29.4) 199 (28.3) Constipation 165 (23.4) 172 (24.5) Diarrhea 112 (15.9) 92 (13.1) Vomiting 103 (14.6) 74 (10.5) General disorders and administration site conditions Total number of patients with at least one AE 420 (59.7) 323 (45.9) Fatigue 137 (19.5) 123 (17.5) Pyrexia 142 (20.2) 83 (11.8) Chills 133 (18.9) 37 (5.3) Asthenia 71 (10.1) 76 (10.8) Injury, poisoning and procedural complications Total number of patients with at least one AE 281 (39.9) 204 (29.0) Infusion-related reaction 254 (36.1) 165 (23.5) Metabolism and nutrition disorders Total number of patients with at least one AE 202 (28.7) 170 (24.2)) Decreased appetite 97 (13.8) 71 (10.1) Nervous system disorders Total number of patients with at least one AE 336 (47.7) 299 (42.5) Peripheral neuropathy 88 (12.5) 89 (12.7) Headache 75 (10.7) 57 (8.1) Psychiatric disorders Total number of patients with at least one AE 107 (15.2) 83 (11.8) Insomnia 76 (10.8) 58 (8.3) Respiratory, thoracic and mediastinal disorders Total number of patients with at least one AE 232 (33.0) 197 (28.0) Cough 83 (11.8) 60 (8.5) Skin and subcutaneous tissue disorders Total number of patients with at least one AE 226 (32.1) 226 (32.1) Alopecia 145 (20.6) 142 (20.2) AE, adverse event; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; R-CHOP, rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone.
(55) TABLE-US-00008 TABLE 7 Summary of AEs of Particular Interest as Preferred Terms or Predefined Categories (Safety Population) All grades Grades 3-5 G-CHOP R-CHOP G-CHOP R-CHOP (N =704), (N =703), (N =704), (N =703), Category No. (%) No. (%) No. (%) No. (%) Infections* 379 (53.8) 310 (44.1) 135 (19.2) 109 (15.5) Opportunistic infections.sup.† 13 (1.8) 15 (2.1) 8 (1.1) 9 (1.3) Neutropenia.sup.‡ 398 (56.5) 338 (48.1) 384 (54.5) 324 (46.1) Infusion-related reactions.sup.§ 319 (45.3) 223 (31.7) 69 (9.8) 24 (3.4) Infusion-related reactions 273 (38.8) 174 (24.8) 53 (7.5) 16 (2.3) (antibody related).sup.§ Tumor lysis syndrome 4 (0.6) 4 (0.6) 4 (0.6) 4 (0.6) Cardiac events.sup.¶ 75 (10.7) 53 (7.5) 33 (4.7) 20 (2.8) Thrombocytopenia.sup.∥ 55 (7.8) 18 (2.6) 31 (4.4) 10 (1.4) Second malignancies** 15 (2.1) 15 (2.1) 12 (1.7) 13 (1.8) Hepatitis B reactivation.sup.†† 16 (2.3) 6 (0.9) 2 (0.3) 2 (0.3) Progressive multifocal 1 (0.1) 0 1 (0.1) 0 leukoencephalopathy Gastrointestinal 14 (2.0) 8 (1.1) 12 (1.7) 8 (1.1) perforation.sup.‡‡ Perforation events 7 (1.0) 7 (1.0) 6 (0.9) 7 (1.0) Abscesses/other 8 (1.1) 2 (0.3) 8 (1.1) 2 (0.3) Hemorrhagic events.sup.§§ 65 (9.2) 39 (5.5) 23 (3.3) 10 (1.4) AE, adverse event; G-CHOP, obinutuzumab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone; HBV, hepatitis B infection; MedDRA, Medical Dictionary for Regulatory Activities; R-CHOP rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone. *Any preferred term in the System Organ Class Infections and infestations. .sup.†Standardized MedDRA query. .sup.‡Neutropenia and associated complications reported as AEs, not including abnormal laboratory values. .sup.§Related to any infused treatment and occurring during/within 24 hours of infusion. .sup.¶Any preferred term in the System Organ Class Cardiac disorders. **Any preferred term in the System Organ Class Neoplasms benign, malignant and unspecified (including cysts and polyps) that started 6 months after the first study drug intake. .sup.††At least one of an increase in HBV DNA level of ≥100 IU/ml or an AE of hepatitis B reactivation. .sup.‡‡Standardized MedDRA query, comprising perforation events (preferred terms in the System Organ Class Gastrointestinal disorders) and abscesses and other events (preferred terms in other System Organ Classes). .sup.§§Standardized MedDRA query, comprising hemorrhagic cerebrovascular conditions, and hemorrhage (laboratory and non-laboratory terms).
EXAMPLE 1
General Materials and Methods
(56) Description of the NanoString LST Assay
(57) If not indicated to the contrary, the NanoString LST Assay was performed in the context of the invention in accordance with the manual of the distributor (NanoString Technologies, Inc., Seattle, Wash., USA). Further guidance as to the assay is provided in Scott (2014 and 2015 loc cit.).
(58) Overview of COO Assay
(59) NanoString's LST gene expression assay was developed to enable the identification of the COO subtypes on the nCounter® Analysis System. The nCounter® Gene Expression Assay together with the NanoString technology provide an ultra-sensitive and highly multiplexed method for detecting mRNAs with molecular barcodes called nCounter Reporter Probes without the use of reverse transcription or amplification (Geissloc. cit.). The detection of mRNA is based on digital detection and direct molecular barcoding of target molecules through the use of a color coded probe pair. The probe pair consists of a Reporter Probe, which carries the signal on its 5′ end, and a Capture Probe. The color codes carry six positions and each position can be one of four colors, thus allowing for a large diversity of tags that can be mixed together in a single well for direct hybridization to the target and yet still be individually resolved and identified during data collection. The NanoString Reporter and Capture Probe Technology employs the reporter probe that carries the color-coded barcode and the capture probe that allows the complex to be immobilized for data collection.
(60) Customized LST probe pairs are mixed with specimen RNA in massive excess to target mRNA to ensure that each target finds a probe pair. After hybridization, excess unbound probes are washed away, and the Target/Probe complexes are immobilized in the cartridge for data collection. Data Collection is carried out in the nCounter® Digital Analyzer. Digital images are processed and barcode counts are tabulated in a comma separated value (CSV) format ready for sample normalization and data analysis.
(61) Prior to data analyses, each sample is normalized to the reference counts generated from the synthetic RNA reference control transcript and the house keeping genes.
(62) Determination of COO Status in Patient Samples
(63) The LST assay generates gene expression data for each patient for 20 genes (see Table 8). Five of these genes are housekeeping reference genes, while the 15 other genes distinguish GCB from ABC, specifically eight genes are known to be overexpressed in ABC-like DLBCL, and seven genes are known to be overexpressed in GCB-like DLBCL (based on Scott 2014 loc.cit.
(64) TABLE-US-00009 TABLE 8 Twenty gene list in the NanoString LST Assay Type Genes Overexpressed in ABC- TNFRSF13B; LIMD1; IRF4; CREB3L2; like DLBCL PIM2; CYB5R2; RAB7L1; CCDC50 Housekeeping genes R3HDM1; WDR55; ISY1; UBXN4; Overexpressed in GCB- TRIM56MME; SERPINA9; ASB13; like DLBCL MAML3; ITPKB; MYBL1; S1PR2
(65) A Linear Predictor Score (LPS) is calculated for each patient based on the gene expression data. The LPS is a weighted sum of the gene expression of the 20 genes in the LST assay:
LPS(X)=Σ.sub.ja.sub.jX.sub.j,
(66) where X.sub.j the gene expression for gene j and a.sub.j is the coefficient for gene j.
(67) The LPS for each patient is then compared against predefined thresholds to determine the DLBCL COO subtype for a patient. For an individual LPS score, a probability is determined as to whether the tumor is likely to be part of the ABC subtype or the GCB subtype. Tumors with a probability of being ABC greater than 90% are considered ABC, whereas those with a probability of being GCB of greater than 90% are considered GCB. Tumors with a probability of being ABC or of being GCB of less than 90%, are considered Unclassified (Scott 2014 and 2015 loc. cit.)
(68) G-CHOP and R-CHOP Regimens
(69) Patients will receive treatment with one of two immunochemotherapy regimens: G-CHOP (investigational arm): CHOP chemotherapy combined with obinutuzumab R-CHOP (control arm): CHOP chemotherapy combined with rituximab
(70) Obinutuzumab and rituximab are considered the investigational medicinal products for the purposes of this protocol.
(71) In the investigational arm, obinutuzumab will be administered by IV infusion at an absolute (flat) dose of 1000 mg on Day 1 of each 21-day cycle for 8 cycles. During Cycle 1, obinutuzumab will also be infused on Days 8 and 15. Administration of obinutuzumab on days when both obinutuzumab and CHOP are to be given should be completed for at least 30 minutes before chemotherapy administration is started.
(72) CHOP chemotherapy may be given on the next day after obinutuzumab administration if the duration of the obinutuzumab infusion necessitates administration of the CHOP infusion the next day. CHOP chemotherapy will be given for a maximum of 6 or 8 cycles, as described in Section 4.5.3.a and Table 5. If only 6 cycles of CHOP chemotherapy are to be administered (see Table 5), Cycles 7 and 8 of obinutuzumab will be given as monotherapy on an every-21-day schedule.
(73) In the control arm of the study, rituximab at a dose of 375 mg/m.sup.2 will be administered by IV infusion on Day 1 of each 21-day cycle for 8 cycles. Rituximab will be administered prior to CHOP, and the infusion should be completed and patients observed for at least 30 minutes prior to starting CHOP. CHOP may be given on Day 2 if the duration of rituximab infusion necessitates administration of the CHOP infusion the next day. If only 6 cycles of CHOP chemotherapy are to be administered (see Table 5), Cycles 7 and 8 of rituximab will be given as monotherapy on an every-21-day schedule.
(74) The dose of the rituximab and chemotherapy should be calculated on the basis of a patient's body weight at the screening assessment (Day-14 to Day-1). For changes>10% in body weight from screening for all subsequent doses, the doses of rituximab and chemotherapy should be modified accordingly. The weight that triggered a dose adjustment will be taken as the new reference weight for future dose adjustments. As noted in Section 3.6.1.c, the use of G-CSF is recommended according to ASCO, EORTC, and ESMO guidelines for patients who are 60 years old and those with co-morbidities. It is strongly recommended in Cycle 1 for all patients treated with G-CHOP.
(75) Obinutuzumab (technical data)
(76) a Formulation
(77) Obinutuzumab is provided as a single-dose, sterile liquid formulation in a 50-mL pharmaceutical grade glass vial containing a nominal 1000 mg of obinutuzumab (G3 material). The formulated drug product consists of 25 mg/mL drug substance (G3) formulated in histidine, trehalose, and poloxamer 188. The vial contains 41 mL (with 2.5% overfill).
(78) For further details, see the Obinutuzumab Investigator's Brochure.
(79) b Handling and Storage
(80) The recommended storage conditions for the obinutuzumab drug product are between 2° C. and 8° C. protected from light. Chemical and physical in-use stability for obinutuzumab dilutions in 0.9% sodium chloride (NaCl) have been demonstrated for 24 hours at 2° C.-8° C. and at ambient temperature and ambient room lighting. The prepared diluted product should be used immediately. If not used immediately, in-use storage times and conditions prior to use are the responsibility of the user and would normally not be longer than 24 hours at 2° C.-8° C. Obinutuzumab should not be frozen or shaken. Mix gently. All transfer procedures require strict adherence to aseptic techniques. Do not use an additional in-line filter because of potential adsorption. For further details, see the Obinutuzumab Investigator's Brochure.
(81) c Obinutuzumab Dose and Schedule
(82) Obinutuzumab will be administered by IV infusion as an absolute (flat) dose of 1000 mg on Day 1 of each 21-day cycle for 8 cycles. Obinutuzumab will be administered prior to CHOP, and patients should be observed 30 minutes prior to starting CHOP. If CHOP is not completed on Day 1 because of the long duration of obinutuzumab therapy, CHOP chemotherapy may be administered on Day 2. During Cycle 1, obinutuzumab will also be infused on Days 8 and 15. If CHOP chemotherapy is not given at Cycles 7 and 8, obinutuzumab will be administered as monotherapy.
(83) d Obinutuzumab Preparation
(84) Obinutuzumab drug product intended for IV infusion is prepared by dilution of the drug product into an infusion bag containing 0.9% NaCl to the final drug concentration of 4 mg/mL. Using a 250-mL infusion bag containing 0.9% NaCl, withdraw and discard 40 mL of the sodium chloride. Withdraw 40 mL of obinutuzumab from a single glass vial and inject into the infusion bag (discard any unused portion of obinutuzumab left in the vial). Gently invert the infusion bag to mix the solution; do not shake.
(85) Administration sets with polyvinyl chloride (PVC), polyurethane (PUR), or polyethylene as product contact surface and IV bags with polyolefine, polypropylene (PP), PVC, or polyethylene, as product contact surface are compatible and may be used.
(86) Do not use obinutuzumab beyond the expiration date stamped on the carton.
(87) e Obinutuzumab Administration
(88) Obinutuzumab should be administered to patients in a clinical setting (inpatient or outpatient), where full emergency resuscitation facilities are immediately available and patients should be under close supervision of the investigator at all times. Do not administer as an IV push or bolus. After the end of the first infusion, the IV line or central venous catheter should remain in place for 2 hours in order to be able to administer IV drugs if necessary. If no adverse events occur after 2 hours, the IV line may be removed or the central venous catheter may be de-accessed. For subsequent infusions, access (either through an IV line or central venous catheter) should remain in place for at least 1 hour from the end of infusion, and if no adverse events occur after 1 hour, the IV access may be removed.
(89) Please refer to Section 4.3.5, General Precautions (for guidance on the use of pre-medication and prophylaxis of tumor lysis syndrome), prior to administration of obinutuzumab. Instructions for the first and subsequent infusions of obinutuzumab are presented in Table 1.
(90) TABLE-US-00010 TABLE 9 Administration of First and Subsequent Infusions of Obinutuzumab First Infusion (Day 1) Subsequent Infusions Begin infusion at and initial rate of 50 mg/hr. If a patient experienced an infusion reaction during If no infusion reaction occurs, increase the the prior infusion, start at the same rate as the first infusion rate in 50-mg/hr increments every infusion (50 mg/hr) and follow directions as noted. 30 minutes, to a maximum of 400 mg/hr. If the patient tolerated the prior infusion well If an infusion reaction develops, stop or (defined as an absence of Grade 2 reactions slow the infusion. Administer infusion-reaction during a final infusion rate of 100 mg/hr), begin medications and supportive care in accordance the infusion at a rate of 100 mg/hr. with institutional protocol. Resume the infusion If no infusion reaction occurs, increase the infusion at a 50% reduction in rate (the rate being used rate in 100-mg/hr increments every 30 minutes, at the time that the hypersensitivity or to a maximum of 400 mg/hr. infusion-related reaction occurred) if the If an infusion reaction develops, stop or slow the reaction has resolved. infusion. Administer infusion-reaction medications and supportive care in accordance with institutional protocol. Resume the infusion at a 50% reduction in rate (the rate being used at the time that the hypersensitivity or infusion-related reaction occurred) if the reaction has resolved.
(91) Obinutuzumab should be given as a slow IV infusion through a dedicated line. IV infusion pumps should be used to control the infusion rate of obinutuzumab. Do not administer as an IV push or bolus.
(92) On days when both obinutuzumab and CHOP are given, obinutuzumab will be administered prior to CHOP and patients should be observed 30 minutes prior to starting CHOP. CHOP chemotherapy may be administered the next day if it cannot be given on the same day as obinutuzumab administration. Prior to each obinutuzumab infusion that is given in combination with CHOP (Day 1 of Cycles 1-6 or Cycles 1-8), patients should take the Day 1 dose of oral prednisone (100 mg) specified for each cycle of the CHOP regimen. The prophylactic use of corticosteroids (e.g., 100 mg of IV prednisolone or equivalent) may also be considered for patients thought to be at high risk for IRRs, if deemed appropriate by the investigator, and should be also administered prior to the obinutuzumab infusion.
(93) For management of IRRs and anaphylaxis, see Section 4.3.6.a and Table 3.
(94) CHOP Chemotherapy
(95) CHOP is considered standard therapy for treatment of DLBCL. By center choice, sites will elect prior to study start whether they will be administering 8 cycles or 6 cycles of CHOP chemotherapy.
(96) a Dosage and Administration
(97) CHOP chemotherapy consists of IV cyclophosphamide, IV doxorubicin, vincristine administered by IV push, and oral prednisone or prednisolone. Standard CHOP will be administered for six to eight 21-day cycles. CHOP chemotherapy at Cycles 7 and 8 is to be administered according to a center's prospective choice for 6 versus 8 planned cycles. Cyclophosphamide 750 mg/m.sup.2 administered intravenously on Day 1 Doxorubicin 50 mg/m.sup.2 administered intravenously on Day 1 Vincristine 1.4 mg/m.sup.2 administered by IV push on Day 1 with a recommended cap of 2.0 mg Prednisone 100 mg/day orally (PO) on Days 1-5 Note: The dose of prednisone follows the National Comprehensive Cancer Network's recommendations (2010), which were based on Cruzman et al. 1999; Hiddemann et al. 2005. Prednisone may be replaced with prednisolone (1:1-conversion, 100 mg) in countries where prednisone is not available or is not the therapy of choice or it may be replaced with 80 mg methylprednisolone in countries or sites that do not have access to prednisone/prednisolone.
(98) CHOP will be administered according to the standard preparation and infusion procedures at each investigational site and after the rituximab or obinutuzumab infusion. Refer to the specific package inserts for preparation, administration, and storage guidelines. At the discretion of the investigator, the dose of vincristine may be capped at 2 mg. For patients who are ≥70 years old, the dose of vincristine may be capped at 1.5 mg. BSA may be capped at 2 m.sup.2 per institutional standards.
(99) When obinutuzumab or rituximab and CHOP are scheduled to be administered on the same day, it is recommended that prednisone (100 mg) be given prior to the obinutuzumab or rituximab infusion. Obinutuzumab or rituximab administration should be completed at least 30 minutes prior to administration of the CHOP (cyclophosphamide, vincristine, and adriamycin) infusions. If it is the strong preference of the investigator or of the site (e.g., for logistical reasons), the administration of rituximab or obinutuzumab is allowed one day prior to administration of CHOP with pre-medication (defined in Section 4.3.5). It is also allowed to split the antibody (rituximab or obinutuzumab) infusion over 2 days if the patient is at increased risk for an IRR (high tumor burden, high peripheral lymphocyte count). Also, in patients who experience an adverse event during obinutuzumab or rituximab infusion, administration of obinutuzumab may be continued on the following day if needed. If Cycle 1 Day 1 dose of obinutuzumab is split, both infusions must happen with appropriate pre-medication and at the First Infusion Rate (Please see Table 2). If CHOP is started later than the Day 1 of the cycle, then planned Day 1 of the next cycle should be calculated from the day when CHOP was actually initiated, in order to maintain the regular chemotherapy interval of 21 days.
EXAMPLE 2
Obinutuzumab or Rituximab (plus CHOP) in Previously Untreated DLBCL (Description of the Roche GOYA (B021005) Clinical Phase 3 Study)
(100) Patients
(101) Eligible patients were aged 18 years with: previously-untreated, histologically documented, CD20-positive DLBCL (as assessed by local pathology laboratory); 1 bi-dimensionally measurable lesion (>1.5 cm largest dimension on computed tomography [CT] scan); Eastern Cooperative Oncology Group performance status of 0-2; adequate hematologic, liver, and kidney function; left ventricular ejection fraction ≥50%; and an
(102) International Prognostic Index (IPI) score of 2 (and patients with an IPI score of 1 aged ≤60 years, with or without bulky disease, and those with an IPI score of 0 and bulky disease, ie, one lesion 7.5 cm). Full inclusion/exclusion criteria are detailed below. All patients provided written informed consent.
(103) Full Inclusion and Exclusion Criteria
(104) Patients had to meet the following criteria for study entry: 1. Written informed consent. 2. Previously untreated CD20-positive diffuse large B-cell lymphoma (DLBCL) histologically documented using the following: a. The pathology report must be available for review and a tissue block sent for retrospective central confirmation. b. Formalin-fixed paraffin-embedded tissue blocks are preferred; however, in countries using a different fixative, any tissue block available will be accepted and notation of the type of fixative included. c. If a tissue block is not available, 15 unstained slides (3-5 μm in thickness) will be accepted. d. The optional Roche clinical repository sample and required exploratory biomarker samples will be obtained from the same tissue block. If central confirmation is unable to be performed on submitted material, stained slides used for diagnosis may also be requested. 3. Patients in an International Prognostic Index (IPI) disease risk group that is one of the following: high, high-intermediate, or low-intermediate. a. Patients in the low-risk group are eligible but must have an IPI score of 1, irrespective of bulky disease, or IPI score of 0 with bulky disease, defined as one lesion ≥7.5 cm. b. Note: patients with IPI 1 due to age alone (ie, patients >60 years old with no other risk factors) with no bulky disease are not eligible for this trial. 4. At least one bi-dimensionally measurable lesion defined as >1.5 cm in its largest dimension on computed tomography scan. 5. Ability and willingness to comply with the study protocol procedures. 6. Age 18 years. 7. Eastern Cooperative Oncology Group performance status of 0, 1, or 2. 8. Left ventricular ejection fraction 50% on cardiac multiple-gated acquisition scan or cardiac echocardiogram. 9. Adequate hematologic function (unless due to underlying disease, as established by extensive bone marrow involvement or due to hypersplenism secondary to the involvement of the spleen by DLBCL per the investigator) defined as follows: a. Hemoglobin≥9 g/dl; b. Absolute neutrophil count≥1.5×10.sup.9/l; c. Platelet count≥75×10.sup.9/l. 10. For men who are not surgically sterile: agreement to use a barrier method of contraception during the treatment period and until ≥3 months after the last dose of obinutuzumab or rituximab, or according to institutional guidelines for CHOP chemotherapy, whichever is longer, and agreement to request that their partners use an additional method of contraception, such as oral contraceptives, intrauterine device, barrier method, or spermicidal jelly. 11. For women of reproductive potential who are not surgically sterile: agreement to use two adequate methods of contraception, such as oral contraceptives, intrauterine device, or barrier method of contraception in conjunction with spermicidal jelly during the treatment period and until ≥12 months after the last dose of obinutuzumab or rituximab, or according to institutional guidelines for CHOP chemotherapy, whichever is longer. Patients who met any of the following criteria were excluded from study entry: 1. History of severe allergic or anaphylactic reactions to humanized or murine monoclonal antibodies, or known sensitivity or allergy to murine products or to any component of CHOP or obinutuzumab. 2. Contraindication to any of the individual components of CHOP, including prior receipt of anthracyclines. 3. Patients with transformed lymphoma and patients with stage 3b follicular lymphoma. 4. Prior therapy for DLBCL, with the exception of nodal biopsy or local irradiation. 5. Prior treatment with cytotoxic drugs or rituximab for another condition (eg, rheumatoid arthritis) or prior use of an anti-CD20 antibody. 6. Prior use of any monoclonal antibody within 3 months of the start of cycle 1. 7. Corticosteroid use>30 mg/day of prednisone or equivalent, for purposes other than lymphoma symptom control. a. Patients receiving corticosteroid treatment with ≤30 mg/day of prednisone or equivalent must be documented to be on a stable dose of at least 4 weeks' duration prior to randomization (cycle 1, day 1). b. If glucocorticoid treatment is urgently required for lymphoma symptom control prior to the start of study treatment, prednisone 100 mg or equivalent could be given for a maximum of 5 days, but all tumor assessments must be completed prior to start of glucocorticoid treatment. 8. Primary central nervous system (CNS) lymphoma and secondary CNS involvement by lymphoma, mantle cell lymphoma, or histologic evidence of transformation to a Burkitt lymphoma, primary mediastinal DLBCL, primary effusion lymphoma, plasmablastic lymphoma, and primary cutaneous DLBCL. 9. Vaccination with live vaccines within 28 days prior to randomization. 10. Any investigational therapy within 28 days prior to the start of cycle 1. 11. History of other malignancy that could affect compliance with the protocol or interpretation of results. a. Patients with a history of curatively treated basal or squamous cell carcinoma, or melanoma of the skin or in-situ carcinoma of the cervix at any time prior to the study are eligible. b. Patients with any other malignancy that has been treated with surgery alone with curative intent and the malignancy has been in remission without treatment for 5 years prior to enrollment are eligible. 12. Evidence of significant, uncontrolled concomitant diseases that could affect compliance with the protocol or interpretation of results, including significant cardiovascular disease (such as New York Heart Association Class III or IV cardiac disease, myocardial infarction within the last 6 months, unstable arrhythmias, or unstable angina) or pulmonary disease (including obstructive pulmonary disease and history of bronchospasm). 13. Recent major surgery (within 4 weeks prior to the start of cycle 1), other than for diagnosis. 14. Any of the following abnormal laboratory values (unless any of these abnormalities are due to underlying lymphoma): a. Creatinine>1.5 times the upper limit of normal (ULN; unless creatinine clearance normal), or calculated creatinine clearance<40 ml/min (using the Cockcroft-Gault formula). b. Aspartate amino transferase or alanine amino transferase>2.5×the ULN. c. Total bilirubin≥1.5×the ULN: Patients with documented Gilbert disease may be enrolled if total bilirubin is ≤3.0×the ULN. d. International normalized ratio>1.5×the ULN in the absence of therapeutic anticoagulation. e. Partial thromboplastin time or activated partial thromboplastin time>1.5×the ULN in the absence of a lupus anticoagulant. 15. Known active bacterial, viral, fungal, mycobacterial, parasitic, or other infection (excluding fungal infections of nail beds) or any major episode of infection requiring treatment with intravenous antibiotics or hospitalization (relating to the completion of the course of antibiotics except if for tumor fever) within 4 weeks prior to the start of cycle 1. 16. Patients with suspected active or latent tuberculosis. a. Latent tuberculosis needs to be confirmed by positive interferon-gamma release assay. 17. Positive test results for chronic hepatitis B infection (defined as positive HBsAg serology). a. Patients with occult or prior hepatitis B infection (defined as positive total hepatitis B core antibody and negative HBsAg) may be included if HBV DNA is undetectable. These patients must be willing to undergo monthly DNA testing. 18. Positive test results for hepatitis C (HCV antibody serology testing). a. Patients positive for HCV antibody are eligible only if polymerase chain reaction is negative for HCV RNA. 19. Known history of HIV seropositive status. a. For patients with unknown HIV status, HIV testing will be performed at screening if required by local regulations. 20. Positive results for the human T-lymphotrophic 1 virus (HTLV). a. HTLV testing is required for patients at sites in endemic countries (Japan and Melanesia, and countries in the Caribbean basin, South America, Central America, and sub-Saharan Africa). 21. Patients with a history of confirmed progressive multifocal leukoencephalopathy. 22. Pregnancy or lactation. 23. Life expectancy of <12 months.
(105) Study Design and Treatments
(106) GOYA is a multicenter, open-label, randomized, phase 3 study. Patients were randomized (1:1 ratio) to receive eight 21-day cycles of either G (1000 mg intravenously [IV] on days 1, 8, and 15 of cycle 1, and on day 1 of cycles 2-8), or R (375 mg/m.sup.2 IV on day 1, cycles 1-8), plus 6 or 8 cycles of CHOP at the following doses: cyclophosphamide 750 mg/m.sup.2 IV (day 1); doxorubicin 50 mg/m.sup.2 IV (day 1); vincristine 1.4 mg/m.sup.2 IV (day 1, maximum 2.0 mg); and prednisone 100 mg/day orally (days 1-5). The number of CHOP cycles for both arms was agreed in advance with each study site; if only 6 CHOP cycles were administered, antibody was administered as monotherapy during cycles 7-8. Pre-planned radiotherapy at initial sites of bulky or extranodal disease was permitted within 8 weeks of day 1 of the last antibody cycle and after completion of end-of-treatment assessments. Details of pre-medications, permitted concomitant therapies, and permitted reasons for dose delays/reductions are given in the Supplementary Appendix. Randomization was via an interactive voice-response system with stratification according to the number of planned chemotherapy cycles (6/8 cycles of CHOP), IPI score, and geographic region (Western Europe, Eastern Europe, South and Central America, North America, and Asia and others).
(107) GOYA was conducted in accordance with the European Clinical Trial Directive (for European centers) and International Conference on Harmonization guidelines for Good Clinical Practice. The protocol was approved by the ethics committees of participating centers and was registered at ClinicalTrials.gov (NCT01287741).
(108) Pre-Medications, Dose Alterations, and Permitted Therapies
(109) All patients received pre-medication with oral acetaminophen and an antihistamine before administration of obinutuzumab (G) or rituximab (R). It was recommended that cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) administration be started at least 30 minutes after administration of G or R. In each cycle when G or R was given with CHOP, the day 1 dose of oral prednisone was administered before the antibody infusion. Additional glucocorticoid therapy could also be administered to patients considered at high risk of infusion-related reactions. Tumor lysis prophylaxis (adequate hydration and allopurinol) was recommended for patients with high tumor burden and who were considered to be at risk of tumor lysis. Treatment doses were delayed for up to 2 weeks in the event of grade 3 or 4 hematologic toxicity or grade 2-4 non-hematologic toxicity; if toxicity did not resolve, the patient was withdrawn from study treatment. Dose reductions were allowed for chemotherapy, but not for G or R.
(110) Prophylaxis with G-CSF was recommended for patients aged 60 years and/or with co-morbidities and during cycle 1 for all patients in the G-CHOP arm. G-CSF was also permitted for the treatment of neutropenia. Use of chemotherapy (other than CHOP), immunotherapy, hormone therapy (other than contraceptives, hormone-replacement therapy, or megestrol acetate), and any therapies intended for the treatment of lymphoma was prohibited (for radiotherapy, see main text).
(111) Study Endpoints and Assessments
(112) The primary study endpoint was investigator-assessed progression-free survival (PFS), defined as the time from the date of randomization until the first occurrence of disease progression, relapse, or death from any cause. To rule out bias and support the primary analysis, PFS was also assessed by an Independent Review Committee (IRC). Secondary endpoints included overall survival (OS), event-free survival (EFS), CR rate, overall response rate (ORR, including CR and partial response), disease-free survival (DFS), duration of response, time to next anti-lymphoma treatment (TTNT), and safety. PFS was also analyzed in DLBCL cell-of-origin (COO) subgroups (germinal-center B-cell-like [GCB], activated B-cell-like [ABC], and unclassified; exploratory analysis). COO classification was based on gene-expression profiling using the NanoString Research Use Only Lymphoma Subtyping Test (NanoString Technologies, Inc., Seattle, Wash., USA). Tumor response and progression were assessed by the investigator using regular clinical and laboratory examinations and CT scans, according to the Revised Response Criteria for Malignant Lymphoma (Cheson, J Clin Oncol 25, 2007, 579-586). For those patients with .sup.18F-fludeoxyglucose positron emission tomography (FDG-PET) scans (mandatory at sites with a PET scanner), a separate response assessment was performed incorporating the FDG-PET results. Primary endpoint analysis was based on the assessment of all patients using conventional CT scan. Response was evaluated 4-8 weeks (CT) or 6-8 weeks (FDG-PET) after last study treatment, or sooner in case of early discontinuation.
(113) Safety was assessed by monitoring and recording all adverse events (AEs) and serious AEs (SAEs), including abnormalities identified from laboratory evaluations, vital sign measurement, and physical examination. AEs were graded using National Cancer Institute Common Terminology Criteria for Adverse Events v4.0. Laboratory safety assessments included routine hematology and blood chemistry, and tests of immunologic parameters. An independent data monitoring committee (IDMC) performed periodic safety reviews.
(114) Statistical Analysis
(115) Sample size was calculated to allow detection of a 25% reduction in the risk of disease progression, relapse, or death with G-CHOP versus R-CHOP (ie, a PFS hazard ratio [HR] for G-CHOP over R-CHOP of 0.75), with a two-sided alpha level of 0.05 and 80% power. To achieve this, and allowing for an annual dropout rate of 5%, 405 PFS events were needed for the primary analysis, requiring enrollment of 1,400 patients over 3 years.
(116) Efficacy assessments were performed on the intent-to-treat (ITT) population, comprising all randomized patients. The safety analysis population included all patients who received any study drug (antibody or CHOP). Treatment comparison of PFS was performed using a two-sided level 0.05 stratified log-rank test. The Kaplan-Meier method was used to estimate PFS distribution for each treatment arm. Estimates of treatment effect were expressed as HRs using a stratified Cox proportional-hazards analysis, including 95% confidence intervals (Cis).
(117) The IDMC evaluated efficacy and safety at three formal interim analyses; two for futility and one for efficacy. Pre-planned subgroup analyses assessed the effect of selected baseline patient characteristics, including COO subtype, on PFS.
(118) Results
(119) Overview
(120) After 29 months' median observation, the number of investigator-assessed PFS events was similar with G (201, 28.5%) and R (215, 30.2%); stratified hazard ratio was 0.92 (95% confidence interval, 0.76 to 1.11; P=0.39); 3-year PFS rates were 70% and 67%, respectively. Secondary endpoints of independently-reviewed PFS, other time-to-event endpoints, and tumor response rates were similar between arms. In exploratory subgroup analyses, patients with germinal-center B-cell-like subtype had a better PFS than activated B-cell-like, irrespective of treatment. Frequencies of grade 3-5 adverse events (AEs; 73.7% vs. 64.7%) and serious AEs (42.6% vs. 37.6%) were higher with G-CHOP. Fatal AE frequencies were 5.8% for G-CHOP and 4.3% for R-CHOP. The most common AEs were neutropenia (G-CHOP, 48.3%; R-CHOP, 40.7%), infusion-related reactions (36.1%; 23.5%), nausea (29.4%; 28.3%), and constipation (23.4%; 24.5%).
(121) Patient Characteristics and Treatment
(122) Patients were enrolled at 207 centers in 29 countries. A total of 1,418 patients were randomized between July 2011 and June 2014 to receive either G-CHOP (n=706) or R-CHOP (n=712), and 1,188 patients (G-CHOP, 587; R-CHOP, 601) completed planned treatment (
(123) Demographic and baseline disease characteristics were well balanced between the two arms (Table 1). COO subgroup information was available for 933 patients; distribution by subtype was well-balanced and there were no clinically relevant differences between arms within COO subtypes. The reasons for missing COO information were: a restricted Chinese sample export license precluding biomarker assessments (n=252), CD20-positive DLBCL not confirmed by central laboratory (n=102; note that these patients were balanced between treatment arms: G-CHOP, n=53; R-CHOP, n=49), and missing/inadequate tissue (n=131). Median duration of exposure was 25.3 (range, 1-32) weeks for G and 25.3 (0-32) weeks for R. The dose intensity of G and R exceeded 90% for 95.3% and 99.1% of patients, respectively. Most patients in both arms (>88%) received more than 90% of the planned dose of each CHOP component. Antibody dose delays were more common in the G-CHOP arm: at least one delay of 5, 7 days (G-CHOP, 34.9%; R-CHOP, 30.0%) and of >7 days (G-CHOP, 13.1%; R-CHOP, 9.1%) (Table 5). New (unplanned) anti-lymphoma treatment was received by 103 patients (G-CHOP, 49; R-CHOP, 54) before disease progression, including radiotherapy for 23 patients with signs of residual disease after study treatment completion (G-CHOP, 9; R-CHOP, 14), and 227 patients (G-CHOP, 102; R-CHOP, 125) after disease progression.
(124) Efficacy
(125) As of Apr. 30, 2016, and after 29 months' median observation, the number of investigator-assessed PFS events in the ITT population was similar for G-CHOP (201, 28.5%) and R-CHOP (215, 30.2%), with stratified HR, 0.92 (95% CI, 0.76 to 1.11; P=0.3868). Estimated 3-year PFS rates were 69.6% and 66.9%, respectively (
(126) Secondary endpoints were consistent with the primary endpoint, with no clinically meaningful differences between treatment arms for IRC-assessed PFS or any other time-to-event endpoint (OS, EFS, DFS, and TTNT) (
(127) Subgroup and Exploratory Analyses
(128) The efficacy of G-CHOP versus R-CHOP (unstratified HR for investigator-assessed PFS) was generally similar across selected patient subgroups, including patients who received 6 versus 8 cycles of CHOP (
(129) Kaplan-Meier analysis of PFS in patients with different COO subtypes (irrespective of study treatment) suggested that the GCB subtype is associated with a better outcome than ABC or unclassified subtypes. The HRs for PFS were 1.71 (95% CI, 1.31 to 2.23) for the ABC-GCB comparison, 1.57 (95% CI, 1.14 to 2.15) for the unclassified-GCB comparison and 1.08 (95% CI 0.77 to 1.52) for the ABC-unclassified comparison (
(130) Safety
(131) In the safety population, the proportion of patients experiencing at least one AE of any grade was similar in the G-CHOP and R-CHOP arms (97.0% [683/704] and 93.5% [657/703], respectively) (Table 3). The most common AEs in both arms were neutropenia (G-CHOP, 48.3%; R-CHOP, 40.7%), infusion-related reactions (IRRs; 36.1%; 23.5%), nausea (29.4%; 28.3%), and constipation (23.4%; 24.5%) (Table 6). Grade 3-5 AEs were more common in the G-CHOP arm (73.7% [519/704] vs. 64.7% [455/703]), as were SAEs (42.6% [300/704] vs. 37.6% [264/703]). The most common grade 3-5 AEs in both arms were neutropenia (G-CHOP, 46.2%; R-CHOP, 38.1%), infections (19.2%; 15.5%), febrile neutropenia (17.5%; 15.2%), and leukopenia (13.6%; 10.1%) (Table 3).
(132) Analysis of AEs of particular interest showed that infections, neutropenia, IRRs, cardiac events, thrombocytopenia, and hemorrhagic events of any grade (as well as grade 3-5 AEs and SAEs) were more common with G-CHOP than R-CHOP (Table 7). Of note, rates of hepatitis B reactivation were higher with G-CHOP (2.3%) than R-CHOP (0.9%), the majority of events were grade 1 or 2, and grade 3 or 4 events were well balanced between the two arms (G-CHOP 0.3% vs R-CHOP 0.3%). All other AE groups of particular interest, namely opportunistic infections, tumor lysis syndrome, secondary malignancies, and gastrointestinal perforation (excluding abscesses) occurred at similar frequencies in the two arms (Table 7).
(133) A similar proportion of patients in each arm received at least one dose of G-CSF during the study (G-CHOP, 611 [86.5%]; R-CHOP, 586 [82.3%]).
(134) A higher proportion of patients in the G-CHOP arm than in the R-CHOP arm discontinued 1 component of the study treatment due to an AE (84 [11.9%] vs. 60 [8.5%]). Fatal AEs were experienced by 71 patients (G-CHOP, 5.8% [41/704]; R-CHOP, 4.3% [30/703]) and are detailed in Table 3.
(135) Discussion
(136) In the current study of patients with previously untreated DLBCL, G-CHOP and R-CHOP demonstrated similar efficacy for all time-to-event endpoints, and the primary study endpoint of investigator-assessed PFS was not met. The lack of superiority of G-CHOP over R-CHOP in a population with aggressive NHL contrasts with the results of studies evaluating G in CLL and FL. In the GALLIUM study, G-based induction and maintenance therapy significantly improved investigator-assessed PFS relative to R-based therapy in 1,202 previously untreated FL patients (Marcus loc. cit.). G also prolonged PFS relative to R in untreated CLL patients (n=663) when both were combined with chlorambucil in the phase 3 CLL11 study (Goede loc. cit.).
(137) Given the advantages of G-based therapy in FL and CLL patients, the lack of benefit of G-CHOP in DLBCL patients in GOYA was unexpected.lt might simply have resulted from the differences in biologic and clinical profiles between indolent lymphoproliferative diseases, such as FL and CLL, and aggressive ones, such as DLBCL (Lenz, N. Engl. J. Med. 362, 2010, 1417-1429; Lim loc. cit.). Indeed, obinutuzumab may be more beneficial in lymphomas that are less aggressive or, like FL, are derived from the germinal center. The trend towards a benefit of G-CHOP over R-CHOP in GOYA for the GCB subtype, which is derived from the germinal center and is known to be more like FL compared to other DLBCL subtypes (Morin, Nature 476 2011, 298-303; Shaffer, Nat. Rev. Immunol. 2, 2002, 920-932), with a more favorable prognosis and different immune microenvironment than the ABC and unclassified subtypes, appears to support this finding. The different mode of action of obinutuzumab and rituximab may also play a role in the differential benefit of these agents in FL and DLBCL, however, no data are yet available to support this statement. Ongoing analyses of GOYA biomarker data will provide further insight into these differences. Notably, dose interruptions and skipped doses in cycle 1 were more frequent with G-CHOP, reflecting a higher rate of AEs (IRRs and cytopenias); this might have contributed to the lack of efficacy benefit compared with R-CHOP.
(138) Since the dramatic improvement in outcomes after rituximab was first added to CHOP (Coiffier loc. cit.), no major advances have been made in the management of DLBCL patients. Randomized trials failed to show a benefit of shortening intervals between cycles (Cunningham, Lancet 381, 2013, 1817-1826), or from consolidation with high-dose chemotherapy and autologous stem cell transplantation (Stiff, N. Engl. J. Med. 369, 2013, 1681-1690; Schmitz, Lancet Oncol 13, 2012, 1250-1259); the addition of bortezomib to R-CHOP also failed to improve outcomes in a randomized trial of patients with non-GCB DLBCL (Leonard, Blood 126, 2015, 811), and maintenance with lenalidomide did not improve OS (Thieblemont, Blood 128, 2016, 471). Given the aggressive behavior of DLBCL, the substitution of rituximab by a new anti-CD20 antibody with a different mode of action may not be sufficient to overcome refractoriness to chemotherapy. Combinations of drug-conjugated antibody or anti-BCL2 agents with R-CHOP could hold more promise, as shown by preliminary results of recent phase 1/2 studies (Zelenetz, Blood 128, 2016, 3032; Tilly, Blood 128, 2016, 1853).
(139) Determination of COO status using gene-expression profiling has identified biologically distinct subtypes of DLBCL, including GCB and ABC origin subtypes (Lenz, Proc Natl Acad Sci USA 105, 2008, 13520-13525; Scott, J. Clin. Oncol. 33, 2015, 2848-2856). These molecular subtypes have important implications for oncogenesis and treatment outcome, as reflected by their inclusion in the current World Health Organization classification for DLBCL (Swerdlow, Blood 127, 2016, 2375-2390). Patients with the GCB subtype typically have more favorable outcomes, whereas the ABC subtype has been associated with inferior outcomes following chemotherapy or immunochemotherapy (including R-CHOP), and may represent a poor-risk subset of patients with unmet medical need (as shown in retrospective studies) (Lenz, Proc. Natl. Acad. Sci. USA loc. cit.; Scott 2015 loc. cit.). GOYA is the largest prospective study to assess the impact of COO on clinical outcomes. Comparison of PFS by COO subtype was consistent with a better outcome in GCB DLBCL, with the HR indicating a 70% increase in risk of disease progression in patients with ABC relative to GCB subtype. Outcome for the unclassified subgroup was similar to that of the ABC subgroup, which is in contrast to what has been reported in some prior studies (Scott 2015 loc. cit.). Interestingly, COO classification was not correlated with ORR and/or preliminary assessment of PFS in other prospectively defined studies, such as
(140) REMoDL-B (Davies, Blood 126, 2015, 812) or PYRAMID (Leonard loc. cit.), although these studies used different COO assays. Specific treatments aimed at COO subtypes of DLBCL may offer an alternative strategy for improving outcomes. Selectively targeting the B-cell receptor or NF-κB pathways, for example, may prove beneficial in DLBCL subtypes (ABC or non-GCB), as suggested by results of phase 2 studies that evaluated lenalidomide or ibrutinib with R-CHOP (Nowakowski, J Clin Oncol 33, 2015, 251-257; Vitolo, Lancet Oncol. 152014, 730-737; Younes, Lancet Oncol 15, 2014, 1019-1026). These strategies are currently being evaluated in randomized phase 3 studies.
(141) The profile and nature of the AEs reported among G-CHOP-treated patients was as expected, with no new safety signals. The incidence of grade 3-5 AEs, SAEs, and treatment discontinuations due to AEs was slightly higher in the G-CHOP group than in the R-CHOP group, in keeping with what has been reported in other studies. These discrepancies may be due to different structural and biologic properties of G and R.
(142) In conclusion, the current study demonstrated that G-CHOP did not improve PFS in a large population of patients with previously untreated DLBCL compared with R-CHOP, which remains the standard treatment for these patients. No new safety signals were identified.
EXAMPLE 3
Superiority of Obinutuzumab Over Rituximab in New Subgroups of DLBCL (Predictive Biomarker-Defined; Description and Results of Exploratory Analyses of the GOYA Clinical Phase 3 Study)
EXAMPLE 3.1
Gazyva™-CHOP is Superior to Ritixumab-CHOP in a Biomarker Defined Subset of DLBCL—Results from the Roche GOYA (B021005) ph3 Clinical Trial
(143) Summary
(144) Rituximab (R) plus CHOP chemotherapy is standard-of-care in previously untreated diffuse large B-cell lymphoma (DLBCL). Obinutuzumab (G) is a glycoengineered, type II anti-CD20 monoclonal antibody. GOYA was a randomized phase 3 study comparing G-CHOP and R-CHOP in previously untreated advanced-stage DLBCL.
(145) The GOYA trial in 1L DLBCL did not meet its primary endpoint: stratified HR, PFS: 0.92 (95% CI 0.76-1.12), but the GALLIUM trial demonstrated superiority of Gazyva over Rituximab in 1L FL (HR 0.66, 95% CI 0.51-0.85; currently in filing).
(146) In exploratory analyses of GOYA, superiority of Gazyva™ over Rituximab is seen in a subset of GCB DLBCL patients and/or also in patients with mutations in CD58 and/or low expression of CD58. This is the first time a Gazyva benefit has been identified in a biomarker defined subgroup of DLBCL.
(147) The results suggest that (a) subset(s) of GCB DLBCL patients that respond to Gazyva can be identified in several ways, e.g. by determining BCL2 translocation and BCL2 protein overexpression, and also by measuring gene expression profiling, e.g. Nanostring Cell of Origin (COO) assay using novel cutoffs for the Linear Predictor Score, LPS.
(148) Methods
(149) Cell-of-origin (COO) classification into subgroups germinal-center B-cell (GCB), activated B-cell (ABC), and Unclassified was based on gene-expression profiling using the NanoString Research Use Only Lymphoma Subtyping Test (NanoString Technologies, Inc., Seattle, Wash., USA).
(150) The Linear Predictor Score (LPS) is a continuous variable (weighted average for gene expression of the genes in the Nanostring Lymphoma Subtyping assay) with a range in GOYA from −1138 to 4504. Normally the LPS used to classify patients into COO subgroups GCB, ABC, Unclassified. Default COO algorithm uses a bayesian approach with GCB/ABC classification based on ≥90% cut-off on likelihood of being GCB or ABC (unclassified works as a buffer).
(151) The LPS has been analysed directly for clinical outcome for the first time. LPS was treated as a continuous variable for assessment of treatment effect (efficacy of G-CHOP vs. R-CHP) in exploratory un-specified analyses in the GOYA trial.
(152) BCL-2 translocations were assessed using the Bcl-2 Dual Color Break Apart (Vysis, Abbott Molecular) and also with the Foundation Medicine next-generation sequencing assay, FoundationOne Heme. BCL-2 protein expression was assessed using a Ventana investigational-use IHC assay (BCL2 antibody clone, 124). Whole-transcriptome gene expression in the GOYA trial was evaluated using the TruSeq® RNA Access Library Prep Kit. CD58 mutations were identified using the FoundationOne heme gene panel.
(153) Results
(154) Biomarker defined subgroups of DLBCL were identified that benefit from G (G-CHOP) over R (R-CHOP): BCL2 translocated patients (see
EXAMPLE 3.2
Superiority of Obinutuzumab Over Rituximab in a New Molecular FL-Like Subgroup of DLBCL—Results from the Phase 3 GOYA Trial
(155) Methods
(156) GOYA was an open-label, randomized phase 3 study comparing 1L G-CHOP with R-CHOP in 1418 DLBCL patients (pts). Biomarker testing was performed on formalin fixed, paraffin-embedded tumor tissue collected prior to treatment and tested retrospectively in central laboratories. COO was assessed using the NanoString Research Use Only LST (NanoString Technologies Inc., Seattle, Wash., USA) in 933 pts. Additional biomarker analyses used for molecualar characterization included DNA-targeted sequencing of 467 genes using the FoundationOne® Herne (FOH) panel (n=499 pts) and whole-transcriptome gene expression was evaluated using TruSeq® RNA sequencing in 552 pts. Vysis LSI Dual Color Break Apart ASH Probes were used to identify BCL2 translocations (n=644 pts; ASH cut-off, 50%), and the Ventana investigational-use IHC assay was used to assess BCL2 expression (BCL2 antibody clone, 124); BCL2.sub.+ IHC was defined as moderate/strong staining in ≥50% of tumor cells. Multivariate Cox regression and elastic net penalized regression (alpha=0.5) was used to evaluate biomarker treatment effects. In addition, bootstrap simulations were conducted to identify the optimal LPS (NanoString LST) to reflect the robustness of the observed treatment effectin GOYA and the generalizability of treatment effect to independent study populations. Multiple testing adjustment was done by estimating FDRs using the Benjamini-Hochberg procedure (significance, <5% FDR). Pathway enrichment analysis was performed using a hypergeometric test; by gene-set enrichment using gene sets defined by MSigDB Hallmarks and a curated FL somatic mutation hallmark gene set based on a recent published review. All pts consented to the biomarker analyses.
(157) Results
(158) Assessment of LPS as a continuous variable identified a subgroup of GCB pts that benefitted from G-CHOP. In particular, strong expression of a germinal-center gene expression profile (by LPS) was linked with a benefit in outcome from treatment with G-CHOP vs R-CHOP among pts in GOYA. Bootstrap simulations identified an optimal LPS cut-off (≤725) for predicting G-CHOP benefit as the 25% (233/933) of GOYA pts with the lowest LPS scores. These pts are referred to as particularly ‘strong-GCB’ pts, and comprised 43% (233/540) of evaluable GCB pts in GOYA. Strong-GCB pts treated with G-CHOP achieved significantly better clinical outcomes in terms of investigator-assessed progression-free (HR=0.33, p=0.0007), event-free (HR=0.47, p=0.003), and overall survival (HR=0.41, p=0.019) than those treated with R-CHOP (cf.
CONCLUSIONS
(159) By analyzing the data from the GOYA trial, identified was a new clinically and molecularly distinct subgroup of GCB DLBCL that comprises at least around 25% of all DLBCL pts, referred to as ‘strong-GCB’. This distinct subgroup is identifiable by gene-expression profiling (using, for example, an LPS cutoff of ≤725 on the Nanostring LST assay) and characterized by mutations that are also commonly identified in FL pts (cf. Morin loc. cit.). Treatment with G-CHOP confers substantial clinical benefit over R-CHOP in this new subset of IL DLBCL patients.
(160) The present invention refers the following nucleotide and amino acid sequences:
(161) TABLE-US-00011 SEQ ID NO: 1: Amino acid sequence of the heavy chain variable region of obinutuzumab. Mouse-human chimeric polypeptide. Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Tyr Ser Trp Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Phe Pro Gly Asp Gly Asp Thr Asp Tyr Asn Gly Lys Phe Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Val Phe Asp Gly Tyr Trp Leu Val Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 2: Amino acid sequence of the KV1 light chain variable region of obinutuzumab Mouse-human chimeric polypeptide Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Val Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn Leu Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val SEQ ID NO: 3: Nucleic acid sequence encoding the heavy chain variable region of obinutuzumab Mouse-human chimeric DNA caggtgcaat tggtgcagtc tggcgctgaa gttaagaagc ctgggagttc agtgaaggtc 60 tcctgcaagg cttccggata cgccttcagc tattcttgga tcaattgggt gcggcaggcg 120 cctggacaag ggctcgagtg gatgggacgg atctttcccg gcgatgggga tactgactac 180 aatgggaaat tcaagggcag agtcacaatt accgccgaca aatccactag cacagcctat 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc aagaaatgtc 300 tttgatggtt actggcttgt ttactggggc cagggaaccc tggtcaccgt ctcctca 357 SEQ ID NO: 4: Nucleic acid sequence encoding the KV1 light chain variable region of obinutuzumab Mouse-human chimeric DNA. gatatcgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gcccgccagc 60 attagctgca gagcctcttg ggtctagcaa cacagcaatg gcatcactta tttgtattgg 120 tacctgcaaa agccagggca gtctccacag ctcctgattt atcaaatgtc caaccttgtc 180 tctggcgtcc ctgaccggtt ctccggatcc gggtcaggca ctgatttcac actgaaaatc 240 agcagggtgg aggctgagga tgttggagtt tattactgcg ctcagaatct agaacttcct 300 tacaccttcg gcggagggac caaggtggag atcaaacgta cggtg 345 SEQ ID NO: 5: Amino acid sequence of the heavy chain of obinutuzumab. The variable region comprises amino acid positions 1 to 119. QVQLVQSGAE VKKPGSSVKV SCKASGYAFS YSWIHWVRQA PGQGLEWMGR 50 IFPGDGDTGY NGKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCARNV 100 FDGYWLVYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD 150 YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY 200 ICNVNHKPSN TKVDKKVEPK SCDKTHTCPP CPAPELLGGP SVFLFPPKPK 250 DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS 300 TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV 350 YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 400 DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 449 SEQ ID NO: 6: Amino acid sequence of the KV1 light chain of obinutuzumab. The variable region comprises amino acid positions 1 to 115. DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ 50' LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCAQNLELP 100’ YTFGGGTKVE IKRTVAAFSV FIFPPSDEQL KSGTASVVCL LHNFYPREAK 150’ VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE 200* VTHQGLSSPV TKSFNRGEC 219’ SEQ ID NO: 7: Nucleic acid sequence encoding the heavy chain variable region of obinutuzumab (B-HH6). CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAAGAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACGCCTTCAGCTATTCTTGGATCAATTGGGTGCGGCAGGCGCCTGGACAAGGGCTCGAGTG GATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATT ACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCA SEQ ID NO: 8: Nucleic acid sequence encoding the KV1 light chain variable region of obinutuzumab. GATATCGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCCGCCAGCATFAGCTGCA GGTCTAGCAAGAGCCTCTTGCACAGCAATGGCATCACTTATTTGTATTGGTACCTGCAAAAGCCAGGGCA GTCTCCACAGCTCCTGATTTATCAAATGTCCAACCTTGTCTCTGGCGTCCCTGACCGGTTCTCCGGATCC GGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCG CTCAGAATCTAGAACTTCCTTACACCTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTG SEQ ID NO: 9: Amino acid requence of the heavy chain of rituximab. Rituximab heavy chain chimeric. QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSY NQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10: Amino acid requence of the light chain of rituximab. Rituximab light chain chimeric. QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVR FSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVOLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11: Example of a nucleotide sequence encoding Homo sapiens (human) CD58 (variant 1). 1 gggccgccgg ctgccagccc agggcggggc ggagccctac ttctggccga ccgcgtaggc 61 ggtgcttgaa cttagggctg cttgtggctg ggcactcgcg cagaggccgg cccgacgagc 121 catggttgct gggagcgacg cggggcgggc cctgggggtc ctcagcgtgg tctgcctgct 181 gcactgcttt ggtttcatca gctgtttttc ccaacaaata tatggtgttg tgtatgggaa 241 tgtaactttc catgtaccaa gcaatgtgcc tttaaaagag gtcctatgga aaaaacaaaa 301 ggataaagtt gcagaactgg aaaattctga attcagagct ttctcatctt ttaaaaatag 361 ggtttattta gacactgtgt caggtagcct cactatctac aacttaacat catcagatga 421 agatgagtat gaaatggaat cgccaaatat tactgatacc atgaagttct ttctttatgt 481 gcttgagtct cttccatctc ccacactaac ttgtgcattg actaatggaa gcattgaagt 541 ccaatgcatg ataccagagc attacaacag ccatcgagga cttataatgt actcatggga 601 ttgtcctatg gagcaatgta aacgtaactc aaccagtata tattttaaga tggaaaatga 661 tcttccacaa aaaatacagt gtactcttag caatccatta tttaatacaa catcatcaat 721 cattttgaca acctgtatcc caagcagcgg tcattcaaga cacagatatg cacttatacc 781 cataccatta gcagtaatta caacatgtat tgtgctgtat atgaatggta ttctgaaatg 841 tgacagaaaa ccagacagaa ccaactccaa ttgattggta acagaagatg aagacaacag 901 cataactaaa ttattttaaa aactaaaaag ccatctgatt tctcatttga gtattacaat 961 ttttgaacaa ctgttggaaa tgtaacttga agcagctgct ttaagaagaa atacccacta 1021 acaaagaaca agcattagtt ttggctgtca tcaacttatt atatgactag gtgcttgctt 1081 tttttgtcag taaattgttt ttactgatga tgtagatact tttgtaaata aatgtaaata 1141 tgtacacaag tga SEQ ID NO: 12: Example of an amino acid sequence of Homo sapiens (human) CD58 (isoform 1). 1 mvagsdagra lgvlsvvcll hcfgfiscfs qqiygvvygn vtfhvpsnvp lkevlwkkqk 61 dkvaelense frafssfknr vyldtvsgsl tiynitssde deyemespni tdtmkfflyv 121 leslpsptlt caltngsiev qcmipehyns hrglimyswd cpmeqckrns tsiyfkmend 181 lpqkiqctls nplfnttssi ilttcipssg hsrhryalip iplavittci vlymngilkc 241 drkpdrtnsn SEQ ID NO: 13: Example of a nucleotide sequence encoding Homo sapiens (human) BCL2 (variant alpha). 1 tttctgtgaa gcagaagtct gggaatcgat ctggaaatcc tcctaatttt tactccctct 61 ccccgcgact cctgattcat tgggaagttt caaatcagct ataactggag agtgctgaag 121 attgatggga tcgttgcctt atgcatttgt tttggtttta caaaaaggaa acttgacaga 181 ggatcatgct gtacttaaaa aatacaacat cacagaggaa gtagactgat attaacaata 241 cttactaata ataacgtgcc tcatgaaata aagatccgaa aggaattgga ataaaaattt 301 cctgcatctc atgccaaggg ggaaacacca gaatcaagtg ttccgcgtga ttgaagacac 361 cccctcgtcc aagaatgcaa agcacatcca ataaaatagc tggattataa ctcctcttct 421 ttctctgggg gccgtggggt gggagctggg gcgagaggtg ccgttggccc ccgttgcttt 481 tcctctggga aggatggcgc acgctgggag aacagggtac gataaccggg agatagtgat 541 gaagtacatc cattataagc tgtcgcagag gggctacgag tgggatgcgg gagatgtggg 601 cgccgcgccc ccgggggccg cccccgcacc gggcatcttc tcctcccagc ccgggcacac 661 gccccatcca gccgcatccc gggacccggt cgccaggacc tcgccgctgc agaccccggc 721 tgcccccggc gccgccgcgg ggcctgcgct cagcccggtg ccacctgtgg tccacctgac 781 cctccgccag gccggcgacg acttctcccg ccgctaccgc cgcgacttcg ccgagatgtc 841 cagccagctg cacctgacgc ccttcaccgc gcggggacgc tttgccacgg tggtggagga 901 gctcttcagg gacggggtga actgggggag gattgtggcc ttctttgagt tcggtggggt 961 catgtgtgtg gagagcgtca accgggagat gtcgcccctg gtggacaaca tcgccctgtg 1021 gatgactgag tacctgaacc ggcacctgca cacctggatc caggataacg gaggctggga 1081 tgcctttgtg gaactgtacg gccccagcat gcggcctctg tttgatttct cctggctgtc 1141 tctgaagact ctgctcagtt tggccctggt gggagcttgc atcaccctgg gtgcctatct 1201 gggccacaag tgaagtcaac atgcctgccc caaacaaata tgcaaaaggt tcactaaagc 1261 agtagaaata atatgcattg tcagtgatgt accatgaaac aaagctgcag gctgtttaag 1321 aaaaaataac acacatataa acatcacaca cacagacaga cacacacaca cacaacaatt 1381 aacagtcttc aggcaaaacg tcgaatcagc tatttactgc caaagggaaa tatcatttat 1441 tttttacatt attaagaaaa aaagatttat ttatttaaga cagtcccatc aaaactcctg 1501 tctttggaaa tccgaccact aattgccaag caccgcttcg tgtggctcca cctggatgtt 1561 ctgtgcctgt aaacatagat tcgctttcca tgttgttggc cggatcacca tctgaagagc 1621 agacggatgg aaaaaggacc tgatcattgg ggaagctggc tttctggctg ctggaggctg 1681 gggagaaggt gttcattcac ttgcatttct ttgccctggg ggctgtgata ttaacagagg 1741 gagggttcct gtggggggaa gtccatgcct ccctggcctg aagaagagac tctttgcata 1801 tgactcacat gatgcatacc tggtgggagg aaaagagttg ggaacttcag atggacctag 1861 tacccactga gatttccacg ccgaaggaca gcgatgggaa aaatgccctt aaatcatagg 1921 aaagtatttt tttaagctac caattgtgcc gagaaaagca ttttagcaat ttatacaata 1981 tcatccagta ccttaagccc tgattgtgta tattcatata ttttggatac gcacccccca 2041 actcccaata ctggctctgt ctgagtaaga aacagaatcc tctggaactt gaggaagtga 2101 acatttcggt gacttccgca tcaggaaggc tagagttacc cagagcatca ggccgccaca 2161 agtgcctgct tttaggagac cgaagtccgc agaacctgcc tgtgtcccag cttggaggcc 2221 tggtcctgga actgagccgg ggccctcact ggcctcctcc agggatgatc aacagggcag 2281 tgtggtctcc gaatgtctgg aagctgatgg agctcagaat tccactgtca agaaagagca 2341 gtagaggggt gtggctgggc ctgtcaccct ggggccctcc aggtaggccc gttttcacgt 2401 ggagcatggg agccacgacc cttcttaaga catgtatcac tgtagaggga aggaacagag 2461 gccctgggcc cttcctatca gaaggacatg gtgaaggctg ggaacgtgag gagaggcaat 2521 ggccacggcc cattttggct gtagcacatg gcacgttggc tgtgtggcct tggcccacct 2581 gtgagtttaa agcaaggctt taaatgactt tggagagggt cacaaatcct aaaagaagca 2641 ttgaagtgag gtgtcatgga ttaattgacc cctgtctatg gaattacatg taaaacatta 2701 tcttgtcact gtagtttggt tttatttgaa aacctgacaa aaaaaaagtt ccaggtgtgg 2761 aatatggggg ttatctgtac atcctggggc attaaaaaaa aaatcaatgg tggggaacta 2821 taaagaagta acaaaagaag tgacatcttc agcaaataaa ctaggaaatt tttttttctt 2881 ccagtttaga atcagccttg aaacattgat ggaataactc tgtggcatta ttgcattata 2941 taccatttat ctgtattaac tttggaatgt actctgttca atgtttaatg ctgtggttga 3001 tatttcgaaa gctgctttaa aaaaatacat gcatctcagc gtttttttgt ttttaattgt 3061 atttagttat ggcctataca ctatttgtga gcaaaggtga tcgttttctg tttgagattt 3121 ttatctcttg attcttcaaa agcattctga gaaggtgaga taagccctga gtctcagcta 3181 cctaagaaaa acctggatgt cactggccac tgaggagctt tgtttcaacc aagtcatgtg 3241 catttccacg tcaacagaat tgtttattgt gacagttata tctgttgtcc ctttgacctt 3301 gtttcttgaa ggtttcctcg tccctgggca attccgcatt taattcatgg tattcaggat 3361 tacatgcatg tttggttaaa cccatgagat tcattcagtt aaaaatccag atggcaaatg 3421 accagcagat tcaaatctat ggtggtttga cctttagaga gttgctttac gtggcctgtt 3481 tcaacacaga cccacccaga gccctcctgc cctccttccg cgggggcttt ctcatggctg 3541 tccttcaggg tcttcctgaa atgcagtggt gcttacgctc caccaagaaa gcaggaaacc 3601 tgtggtatga agccagacct ccccggcggg cctcagggaa cagaatgatc agacctttga 3661 atgattctaa tttttaagca aaatattatt ttatgaaagg tttacattgt caaagtgatg 3721 aatatggaat atccaatcct gtgctgctat cctgccaaaa tcattttaat ggagtcagtt 3781 tgcagtatgc tccacgtggt aagatcctcc aagctgcttt agaagtaaca atgaagaacg 3841 tggacgtttt taatataaag cctgttttgt cttttgttgt tgttcaaacg ggattcacag 3901 agtatttgaa aaatgtatat atattaagag gtcacggggg ctaattgctg gctggctgcc 3961 ttttgctgtg gggttttgtt acctggtttt aataacagta aatgtgccca gcctcttggc 4021 cccagaactg tacagtattg tggctgcact tgctctaaga gtagttgatg ttgcattttc 4081 cttattgtta aaaacatgtt agaagcaatg aatgtatata aaagcctcaa ctagtcattt 4141 ttttctcctc ttcttttttt tcattatatc taattatttt gcagttgggc aacagagaac 4201 catccctatt ttgtattgaa gagggattca catctgcatc ttaactgctc tttatgaatg 4261 aaaaaacagt cctctgtatg tactcctctt tacactggcc agggtcagag ttaaatagag 4321 tatatgcact ttccaaattg gggacaaggg ctctaaaaaa agccccaaaa ggagaagaac 4381 atctgagaac ctcctcggcc ctcccagtcc ctcgctgcac aaatactccg caagagaggc 4441 cagaatgaca gctgacaggg tctatggcca tcgggtcgtc tccgaagatt tggcaggggc 4501 agaaaactct ggcaggctta agatttggaa taaagtcaca gaattaagga agcacctcaa 4561 tttagttcaa acaagacgcc aacattctct ccacagctca cttacctctc tgtgttcaga 4621 tgtggccttc catttatatg tgatctttgt tttattagta aatgcttatc atctaaagat 4681 gtagctctgg cccagtggga aaaattagga agtgattata aatcgagagg agttataata 4741 atcaagatta aatgtaaata atcagggcaa tcccaacaca tgtctagctt tcacctccag 4801 gatctattga gtgaacagaa ttgcaaatag tctctatttg taattgaact tatcctaaaa 4861 caaatagttt ataaatgtga acttaaactc taattaattc caactgtact tttaaggcag 4921 tggctgtttt tagactttct tatcacttat agttagtaat gtacacctac tctatcagag 4981 aaaaacagga aaggctcgaa atacaagcca ttctaaggaa attagggagt cagttgaaat 5041 tctattctga tcttattctg tggtgtcttt tgcagcccag acaaatgtgg ttacacactt 5101 tttaagaaat acaattctac attgtcaagc ttatgaaggt tccaatcaga tctttattgt 5161 tattcaattt ggatctttca gggatttttt ttttaaatta ttatgggaca aaggacattt 5221 gttggagggg tgggagggag gaagaatttt taaatgtaaa acattcccaa gtttggatca 5281 gggagttgga agttttcaga ataaccagaa ctaagggtat gaaggacctg tattggggtc 5341 gatgtgatgc ctctgcgaag aaccttgtgt gacaaatgag aaacattttg aagtttgtgg 5401 tacgaccttt agattccaga gacatcagca tggctcaaag tgcagctccg tttggcagtg 5461 caatggtata aatttcaagc tggatatgtc taatgggtat ttaaacaata aatgtgcagt 5521 tttaactaac aggatattta atgacaacct tctggttggt agggacatct gtttctaaat 5581 gtttattatg tacaatacag aaaaaaattt tataaaatta agcaatgtga aactgaattg 5641 gagagtgata atacaagtcc tttagtctta cccagtgaat cattctgttc catgtctttg 5701 gacaaccatg accttggaca atcatgaaat atgcatctca ctggatgcaa agaaaatcag 5761 atggagcatg aatggtactg taccggttca tctggactgc cccagaaaaa taacttcaag 5821 caaacatcct atcaacaaca aggttgttct gcataccaag ctgagcacag aagatgggaa 5881 cactggtgga ggatggaaag gctcgctcaa tcaagaaaat tctgagacta ttaataaata 5941 agactgtagt gtagatactg agtaaatcca tgcacctaaa ccttttggaa aatctgccgt 6001 gggccctcca gatagctcat ttcattaagt ttttccctcc aaggtagaat ttgcaagagt 6061 gacagtggat tgcatttctt ttggggaagc tttcttttgg tggttttgtt tattatacct 6121 tcttaagttt tcaaccaagg tttgcttttg ttttgagtta ctggggttat ttttgtttta 6181 aataaaaata agtgtacaat aagtgttttt gtattgaaag cttttgttat caagattttc 6241 atacttttac cttccatggc tctttttaag attgatactt ttaagaggtg gctgatattc 6301 tgcaacactg tacacataaa aaatacggta aggatacttt acatggttaa ggtaaagtaa 6361 gtctccagtt ggccaccatt agctataatg gcactttgtt tgtgttgttg gaaaaagtca 6421 cattgccatt aaactttcct tgtctgtcta gttaatattg tgaagaaaaa taaagtacag 6481 tgtgagatac tg SEQ ID NO 14:: Example of an amino acid sequence of Homo sapiens (human) BCL2 (isoform alpha). 1 mahagrtgyd nreivmkyih yklsqrgyew dagdvgaapp gaapapgifs sqpghtphpa 61 asrdpvarts plqtpaapga aagpalspvp pvvhltIrqa gddfsrryrr dfaemssqlh 121 ltpftargrf atvveelfrd gvnwgrivaf fefggymcve synremsply dnialwmtey 181 InrhIhtwiq dnggwdafve lygpsmrplf dfswIsIktlIslalvgaci tlgaylghk