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Supplementary Information

Supplemental data for classification, subtype discovery and prediction of outcome in pediatric lymphoblastic leukemia by gene expression profiling

Section II: Patient Dataset

A total of 389 Pediatric acute leukemia samples were analyzed in this study, from which high quality gene expression data were obtained on 360 (93%). The successfully analyzed samples included: 332 diagnostic bone marrows (BM), 3 diagnostic peripheral bloods (PB), and 25 relapse ALL samples from BM or PB. 264 (79%) of the diagnostic ALL BM samples and all relapse samples were from patients treated on St. Jude Children's Research Hospital Total Therapy Studies XIIIA or XIIIB and correspond to 64% of the patients treated on these protocols. The details of these protocols have been previously published. 1,2 XIIIA ran from 12-20-91 to 8-23-94 and enrolled 165 patients, whereas XIIIB ran from 8-24-94 to 7-27-98 and enrolled 247 patients. No patients were lost to follow-up during treatment. When the databases were frozen for analysis, 100% and 93% of event-free survivors in studies XIIIA and XIIIB, respectively, had been seen within 12 months. The median (minimum, maximum) follow-up of the event-free survivors was 8.09 (6.59, 9.94) and 4.52 (2.37, 7.06) years for XIIIA and XIIIB, respectively. All other samples were obtained from patients treated on St. Jude Total Therapy Studies XI, XII, XIV, XV, or by best clinical management.

For the identification of gene expression profiles that predict specific genetic subtypes of ALL, 327 diagnostic BM samples were used. Inclusion in this dataset required availability of a cryopreserved diagnostic BM sample containing >75% blasts, and complete data from each of the following diagnostic studies: morphology, immunophenotype, cytogenetics, DNA ploidy, Southern blot for MLL gene rearrangements, and RT-PCR analysis for MLL-AF4, MLL-AF9, E2A-PBX1, TEL-AML1, and BCR-ABL. This final dataset includes diagnostic BM samples from XV (38), XIV (4), XIIIA (100), XIIIB (161), or from patients treated on one of our older protocols or by best clinical management (24).

The data sets used to identify expression profiles predicative of hematologic relapse and the development of therapy-induced AML are described below.

Table 10. Patient database [Diagnostic samples used for subtype classification (n=327)]

BCR-ABL subgroup (n=15)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  BCR-ABL-C1 T13B CCR   BCR-ABL-#4 T11 NA
  BCR-ABL-R1 T13A Heme Relapse   BCR-ABL-#5 T12 NA
  BCR-ABL-R2 T13A Heme Relapse   BCR-ABL-#6 T12 NA
  BCR-ABL-R3 T13B Heme Relapse   BCR-ABL-#7 T12 NA
  BCR-ABL-Hyperdip-R5 T13B Heme Relapse   BCR-ABL-#8 T14 NA
  BCR-ABL-#1 T13A Censored   BCR-ABL-#9 T15 NA
  BCR-ABL-#2 T13B Censored   BCR-ABL-Hyperdip-#10 T12 NA
  BCR-ABL-#3 T13B Censored        
E2A-PBX1 subgroup (n=27)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  E2A-PBX1-C1 T13A CCR   E2A-PBX1-#1 Others NA
  E2A-PBX1-C2 T13A CCR   E2A-PBX1-#2 Others NA
  E2A-PBX1-C3 T13A CCR   E2A-PBX1-#3 Others NA
  E2A-PBX1-C4 T13A CCR   E2A-PBX1-#4 Others NA
  E2A-PBX1-C5 T13A CCR   E2A-PBX1-#5 Others NA
  E2A-PBX1-C6 T13B CCR   E2A-PBX1-#6 Others NA
  E2A-PBX1-C7 T13B CCR   E2A-PBX1-#7 T11 NA
  E2A-PBX1-C8 T13B CCR   E2A-PBX1-#8 T11 NA
  E2A-PBX1-C9 T13B CCR   E2A-PBX1-#9 T12 NA
  E2A-PBX1-C10 T13B CCR   E2A-PBX1-#10 T12 NA
  E2A-PBX1-C11 T13B CCR   E2A-PBX1-#11 T14 NA
  E2A-PBX1-C12 T13B CCR   E2A-PBX1-#12 T15 NA
  E2A-PBX1-R1 T13B Heme Relapse   E2A-PBX1-#13 T15 NA
  E2A-PBX1-2M#1 T13B 2nd AML        
Hyperdip>50 subgroup (n=64)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  Hyperdip>50-C1 T13A CCR   Hyperdip>50-C33 T13B CCR
  Hyperdip>50-C2 T13A CCR   Hyperdip>50-C34 T13B CCR
  Hyperdip>50-C3 T13A CCR   Hyperdip>50-C35 T13B CCR
  Hyperdip>50-C4 T13A CCR   Hyperdip>50-C36 T13B CCR
  Hyperdip>50-C5 T13A CCR   Hyperdip>50-C37 T13B CCR
  Hyperdip>50-C6 T13A CCR   Hyperdip>50-C38 T13B CCR
  Hyperdip>50-C7 T13A CCR   Hyperdip>50-C39 T13B CCR
  Hyperdip>50-C8 T13A CCR   Hyperdip>50-C40 T13B CCR
  Hyperdip>50-C9 T13A CCR   Hyperdip>50-C41 T13B CCR
  Hyperdip>50-C10 T13A CCR   Hyperdip>50-C42 T13B CCR
  Hyperdip>50-C11 T13A CCR   Hyperdip>50-C43 T13B CCR
  Hyperdip>50-C12 T13A CCR   Hyperdip>50-R1 T13A Heme Relapse
  Hyperdip>50-C13 T13A CCR   Hyperdip>50-R2 T13A Heme Relapse
  Hyperdip>50-C14 T13A CCR   Hyperdip>50-R3 T13A Heme Relapse
  Hyperdip>50-C15 T13B CCR   Hyperdip>50-R4 T13B Heme Relapse
  Hyperdip>50-C16 T13B CCR   Hyperdip>50-R5 T13B Heme Relapse
  Hyperdip>50-C17 T13B CCR   Hyperdip>50-2M#1 T13A 2nd AML
  Hyperdip>50-C18 T13B CCR   Hyperdip>50-2M#2 T13B 2nd AML
  Hyperdip>50-C19 T13B CCR   Hyperdip>50-#1 T13A Censored
  Hyperdip>50-C20 T13B CCR   Hyperdip>50-#2 T13B Censored
  Hyperdip>50-C21 T13B CCR   Hyperdip>50-#3 Others NA
  Hyperdip>50-C22 T13B CCR   Hyperdip>50-#4 Others NA
  Hyperdip>50-C23 T13B CCR   Hyperdip>50-#5 T12 NA
  Hyperdip>50-C24 T13B CCR   Hyperdip>50-#6 T15 NA
  Hyperdip>50-C25 T13B CCR   Hyperdip>50-#7 T15 NA
  Hyperdip>50-C26 T13B CCR   Hyperdip>50-#8 T15 NA
  Hyperdip>50-C27-N T13B CCR   Hyperdip>50-#9 T15 NA
  Hyperdip>50-C28 T13B CCR   Hyperdip>50-#10 T15 NA
  Hyperdip>50-C29 T13B CCR   Hyperdip>50-#11 T15 NA
  Hyperdip>50-C30 T13B CCR   Hyperdip>50-#12 T15 NA
  Hyperdip>50-C31 T13B CCR   Hyperdip>50-#13 T15 NA
  Hyperdip>50-C32 T13B CCR   Hyperdip>50-#14 T15 NA
Hyperdip47-50 subgroup (n=23)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  Hyperdip47-50-C1 T13A CCR   Hyperdip47-50-C13 T13B CCR
  Hyperdip47-50-C2 T13A CCR   Hyperdip47-50-C14-N T13B CCR
  Hyperdip47-50-C3-N T13A CCR   Hyperdip47-50-C15 T13B CCR
  Hyperdip47-50-C4 T13A CCR   Hyperdip47-50-C16 T13B CCR
  Hyperdip47-50-C5 T13A CCR   Hyperdip47-50-C17 T13B CCR
  Hyperdip47-50-C6 T13B CCR   Hyperdip47-50-C18 T13B CCR
  Hyperdip47-50-C7 T13B CCR   Hyperdip47-50-C19 T13B CCR
  Hyperdip47-50-C8 T13B CCR   Hyperdip47-50-2M#1 T13A 2nd AML
  Hyperdip47-50-C9 T13B CCR   Hyperdip47-50-#1 T15 NA
  Hyperdip47-50-C10 T13B CCR   Hyperdip47-50-#2 T15 NA
  Hyperdip47-50-C11 T13B CCR   Hyperdip47-50-#3 T15 NA
  Hyperdip47-50-C12 T13B CCR        
Hypodip subgroup (n=9)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  Hypodip-C1 T13A CCR   Hypodip-C6 T13B CCR
  Hypodip-C2 T13A CCR   Hypodip-2M#1 T13A 2nd AML
  Hypodip-C3 T13B CCR   Hypodip-#1 T15 NA
  Hypodip-C4 T13B CCR   Hypodip-#2 T15 NA
  Hypodip-C5 T13B CCR        
MLL subgroup (n=20)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  MLL-C1 T13A CCR   MLL-2M#1 T13A 2nd AML
  MLL-C2 T13B CCR   MLL-2M#2 T13A 2nd AML
  MLL-C3 T13B CCR   MLL-#1 T13B Censored
  MLL-C4 T13B CCR   MLL-#2 T13B Censored
  MLL-C5 T13B CCR   MLL-#3 Others NA
  MLL-C6 T13B CCR   MLL-#4 Others NA
  MLL-R1 T13A Heme Relapse   MLL-#5 Others NA
  MLL-R2 T13A Heme Relapse   MLL-#6 T12 NA
  MLL-R3 T13B Heme Relapse   MLL-#7 T14 NA
  MLL-R4 T13B Heme Relapse   MLL-#8 T14 NA
Normal subgroup (n=18)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  Normal-C1-N T13A CCR   Normal-C10 T13B CCR
  Normal-C2-N T13A CCR   Normal-C11-N T13B CCR
  Normal-C3-N T13A CCR   Normal-C12 T13B CCR
  Normal-C4-N T13B CCR   Normal-R1 T13A Heme Relapse
  Normal-C5 T13B CCR   Normal-R2-N T13B Heme Relapse
  Normal-C6 T13B CCR   Normal-R3 T13B Heme Relapse
  Normal-C7-N T13B CCR   Normal-#1 T13A Censored
  Normal-C8 T13B CCR   Normal-#2 T13B Censored
  Normal-C9 T13B CCR   Normal-#3 T13B Censored
Pseudodip subgroup (n=29)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  Pseudodip-C1 T13A CCR   Pseudodip-C16-N T13B CCR
  Pseudodip-C2-N T13A CCR   Pseudodip-C17 T13B CCR
  Pseudodip-C3 T13A CCR   Pseudodip-C18 T13B CCR
  Pseudodip-C4 T13A CCR   Pseudodip-C19 T13B CCR
  Pseudodip-C5 T13A CCR   Pseudodip-R1-N T13A Heme Relapse
  Pseudodip-C6 T13A CCR   Pseudodip-#1 T13B Other Relapse
  Pseudodip-C7 T13A CCR   Pseudodip-#2 T13B Censored
  Pseudodip-C8 T13A CCR   Pseudodip-#3 Others NA
  Pseudodip-C9 T13A CCR   Pseudodip-#4 Others NA
  Pseudodip-C10 T13B CCR   Pseudodip-#5 T15 NA
  Pseudodip-C11 T13B CCR   Pseudodip-#6 T15 NA
  Pseudodip-C12 T13B CCR   Pseudodip-#7 T15 NA
  Pseudodip-C13 T13B CCR   Pseudodip-#8-N T15 NA
  Pseudodip-C14 T13B CCR   Pseudodip-#9 T15 NA
  Pseudodip-C15 T13B CCR        
T-ALL subgroup (n=43)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  T-ALL-C1 T13A CCR   T-ALL-C23 T13B CCR
  T-ALL-C2 T13A CCR   T-ALL-C24 T13B CCR
  T-ALL-C3 T13A CCR   T-ALL-C25 T13B CCR
  T-ALL-C4 T13A CCR   T-ALL-C26 T13B CCR
  T-ALL-C5 T13A CCR   T-ALL-R1 T13A Heme Relapse
  T-ALL-C6 T13A CCR   T-ALL-R2 T13B Heme Relapse
  T-ALL-C7 T13A CCR   T-ALL-R3 T13B Heme Relapse
  T-ALL-C8 T13A CCR   T-ALL-R4 T13B Heme Relapse
  T-ALL-C9 T13B CCR   T-ALL-R5 T13B Heme Relapse
  T-ALL-C10 T13B CCR   T-ALL-R6 T13B Heme Relapse
  T-ALL-C11 T13B CCR   T-ALL-2M#1 T13B 2nd AML
  T-ALL-C12 T13B CCR   T-ALL-#1 T13B Other Relapse
  T-ALL-C13 T13B CCR   T-ALL-#2 T13B Other Relapse
  T-ALL-C14 T13B CCR   T-ALL-#3 T13B Censored
  T-ALL-C15 T13B CCR   T-ALL-#4 T13B Censored
  T-ALL-C16 T13B CCR   T-ALL-#5 T15 NA
  T-ALL-C17 T13B CCR   T-ALL-#6 T15 NA
  T-ALL-C18 T13B CCR   T-ALL-#7 T15 NA
  T-ALL-C19 T13B CCR   T-ALL-#8 T15 NA
  T-ALL-C20 T13B CCR   T-ALL-#9 T15 NA
  T-ALL-C21 T13B CCR   T-ALL-#10 T15 NA
  T-ALL-C22 T13B CCR        
TEL-AML1 subgroup (n=79)
  Label@ Protocol# Outcome%   Label@ Protocol# Outcome%
  TEL-AML1-C1 T13A CCR   TEL-AML1-C41 T13B CCR
  TEL-AML1-C2 T13A CCR   TEL-AML1-C42 T13B CCR
  TEL-AML1-C3 T13A CCR   TEL-AML1-C43 T13B CCR
  TEL-AML1-C4 T13A CCR   TEL-AML1-C44 T13B CCR
  TEL-AML1-C5 T13A CCR   TEL-AML1-C45 T13B CCR
  TEL-AML1-C6 T13A CCR   TEL-AML1-C46 T13B CCR
  TEL-AML1-C7 T13A CCR   TEL-AML1-C47 T13B CCR
  TEL-AML1-C8 T13A CCR   TEL-AML1-C48 T13B CCR
  TEL-AML1-C9 T13A CCR   TEL-AML1-C49 T13B CCR
  TEL-AML1-C10 T13A CCR   TEL-AML1-C50 T13B CCR
  TEL-AML1-C11 T13A CCR   TEL-AML1-C51 T13B CCR
  TEL-AML1-C12 T13A CCR   TEL-AML1-C52 T13B CCR
  TEL-AML1-C13 T13A CCR   TEL-AML1-C53 T13B CCR
  TEL-AML1-C14 T13A CCR   TEL-AML1-C54 T13B CCR
  TEL-AML1-C15 T13A CCR   TEL-AML1-C55 T13B CCR
  TEL-AML1-C16 T13A CCR   TEL-AML1-C56 T13B CCR
  TEL-AML1-C17 T13A CCR   TEL-AML1-C57 T13B CCR
  TEL-AML1-C18 T13A CCR   TEL-AML1-R1 T13A Heme Relapse
  TEL-AML1-C19 T13A CCR   TEL-AML1-R2 T13A Heme Relapse
  TEL-AML1-C20 T13A CCR   TEL-AML1-R3 T13B Heme Relapse
  TEL-AML1-C21 T13A CCR   TEL-AML1-2M#1 T13A 2nd AML
  TEL-AML1-C22 T13A CCR   TEL-AML1-2M#2 T13A 2nd AML
  TEL-AML1-C23 T13A CCR   TEL-AML1-2M#3 T13A 2nd AML
  TEL-AML1-C24 T13A CCR   TEL-AML1-2M#4 T13B 2nd AML
  TEL-AML1-C25 T13A CCR   TEL-AML1-2M#5 T13B 2nd AML
  TEL-AML1-C26 T13A CCR   TEL-AML1-#1 T13B Other Relapse
  TEL-AML1-C27 T13A CCR   TEL-AML1-#2 T13A Censored
  TEL-AML1-C28 T13A CCR   TEL-AML1-#3 T13A Censored
  TEL-AML1-C29 T13B CCR   TEL-AML1-#4 T13B Censored
  TEL-AML1-C30 T13B CCR   TEL-AML1-#5 T15 NA
  TEL-AML1-C31 T13B CCR   TEL-AML1-#6 T15 NA
  TEL-AML1-C32 T13B CCR   TEL-AML1-#7 T15 NA
  TEL-AML1-C33 T13B CCR   TEL-AML1-#8 T15 NA
  TEL-AML1-C34 T13B CCR   TEL-AML1-#9 T15 NA
  TEL-AML1-C35 T13B CCR   TEL-AML1-#10 T15 NA
  TEL-AML1-C36 T13B CCR   TEL-AML1-#11 T15 NA
  TEL-AML1-C37 T13B CCR   TEL-AML1-#12 T15 NA
  TEL-AML1-C38 T13B CCR   TEL-AML1-#13 T15 NA
  TEL-AML1-C39 T13B CCR   TEL-AML1-#14 T15 NA
  TEL-AML1-C40 T13B CCR        

Label@ key:
Subtype Name-C# = Dx Sample of patient in CCR
Subtype Name-R# = Dx Sample of patient who developed a hematologic relapse
Subtype Name-# = Dx Sample used for subgroup classification only
Subtype Name-2M# = Dx Sample of patient who later developed 2nd AML
Subtype Name-N = Dx Sample in novel group

Protocol# key:
Protocol that patient was treated on

Outcome% key:
CCR = Continuous complete remission
Heme Relapse = Hematologic relapse
Other Relapse = Extramedullary relapse
2nd AML = Diagnostic samples of patients who later developed 2nd AML
Censored = Censored due to BM transplant, treated off protocol, or died in CR
NA = Not applicable, primarily because the patient was not treated on Total 13, and thus is excluded from the analysis used to identify gene expression profiles predictive of outcome

Diagnostic samples used for prediction of prognosis

In addition to the 201 CCR and 27 Heme Relapse cases listed in Table 10, five additional relapse cases were also included in the prognostic analysis. This gives a total of 233 cases for this analysis. These additional cases were not included in the subgroup prediction dataset because they did not meet the established criteria for the reasons listed below.

  Label Protocol Comment  
  BCR-ABL-R4 T13B Did not meet QC criteria because contained 70% blasts  
  MLL-R5 T13A Peripheral Blood Sample (90% blasts)  
  Normal-R4 T13B Molecular studies not performed  
  T-ALL-R7 T13A Peripheral Blood Sample (90% blasts)  
  T-ALL-R8 T13B Peripheral Blood Sample (90% blasts)  

Diagnostic Samples used for prediction of 2nd AML

In addition to the 201 CCR and 13 secondary AML cases listed in Table 10, three additional diagnostic marrow samples from patients who developed secondary AML were also included in the prognostic analysis. This gives a total of 217 cases used for this analysis. These additional cases were not included in the diagnostic data set because they did not meet the established criteria for the reasons listed below.

  Label Protocol Comment  
  Hyperdip>50-2M#3 T12 Non Total 13 diagnostic sample  
  Hypodip-2M#2 T13B No molecular studies performed  
  Hypodip-2M#3 T12 Non Total 13 diagnostic sample  

Relapsed Samples (n=25)

Twenty-five relapse samples were analyzed, 17 samples which were paired to the diagnostic samples listed in Table 10 and 8 additional non-paired relapse samples.