Minutes of the Hematopoietic Cell Transplantation Component (HCTC) Working Group (WG)
Saturday, October 10, 1998
Renaissance Hotel, Vancouver, BC, Canada


 Effie Petersdorf, Chair

Members Present: Domenico Adorno, Ekkehard Albert, Lee Ann Baxter-Lowe, Chaim Brautbar, Frank Christiansen, Vera Hauptfeld-Dolejsek, Shraga Goldman, Janet Hegland, Takeo Juji, A. Kuhrober, Ann Margaret Little, Martin Maiers, Steve Marsh, Carlheinz Muller, Machteld Oudshoorn, Noemi Farah Pereira, Colette Raffoux, E.C. Ren, David Sayer, Malcolm Simons, Marilyn Spyropoulen-Movrie, Jon van Rood, Judy Wade, Alois Wolpl

Minutes

1. Introduction to 13th IHWC.

Effie Petersdorf summarized the core and scientific components of the 13th IHWC. Pertinent to the HCTC are: Technology – SSOP (Dr. Carolyn Hurley); SBT (Dr. Marcel Tilanus); Database (Dr. John Hansen)

2. Background.

Effie Petersdorf summarized the background and significance of the HCTC. Recent data from the Seattle transplant program on allele matching for class I and II genes was presented.

Overall Goals of the HCTC were discussed and agreed upon:

  • Define genomic variation in unrelated stem cell transplants
  • Identify polymorphisms that function as clinical determinants
  • Establish an international repository of DNA and cells on retrospective and prospective transplants
  • Establish Informatics/database to support HLA and clinical outcome studies

Specific Aims (on consensus from the HCTC WG)

  • Define class I and class II allele mismatches which are well-tolerated
  • Define the relative importance of allele matching at HLA-A, B, C, DRB, DQB1, DPB1
  • Define the genes that encode minor determinants
  • Define the relative importance of HLA allele mismatching in haploidentical related versus unrelated transplants
  • Define the role of the non-shared parental haplotype in haploidentical related transplants inc
Conceptual Issues

In addition to well-defined scientific goals for the 2001 workshop, the HCTC component will begin to structure a long -term, ongoing strategy for future analysis of genomics in stem cell transplantation. We must begin to build the repository and databases necessary to sustain scientific work, realizing that our project may extend 10-15 years in the future, and not all scientific questions have been formulated at this time.

  • There should be no duplication of effort (in typing or in collection of clinical outcome data).
  • There should be flexibility in the addition of newly mapped genes for analysis.
  • There should be flexibility in the use of newly designed DNA methods for genomic testing.

3. Genes to be studied.

  • HLA-A, B, C, DRB, DQB1, DPB1
  • HA-1 and other minors
  • KIR
  • SNPs
  • microsatellites
  • Tap, LMP, CD1

Two new proposals under consideration:

 1. Role of IL-10 and TNF in clinical outcome: (Dr. Ben Bradley, Bristol)

Dr. Bradley's proposal to evaluate IL-10 and TNF in unrelated stem cell transplants was presented and copies give to WG.
  TO DO: Participants are asked to review and address specific questions directly to Ben Bradley and provide Effie Petersdorf with feedback.
  2. Capillary Leak Syndrome: (Dr. Andreas Kuhrober, Dusseldorf)

Dr. Kuhrober on behalf of Drs. Peter Wernet and Joachim Deeg presented the role of HLA and post-transplant capillary leak syndrome.

  TO DO: WG requested that Drs. Kuhrober, Wernet and Deeg provide specific information on a) supporting clinical and HLA data, b) sample size needed, c) techniques to be used, d) requirements for DNA/cells, e) raw data to be collected. The WG will subsequently evaluate proposal for inclusion into the HCTC studies.

  Single Nucleotide Polymorphisms (SNPs)

Dr. Dan Geraghty presented current research on SNPs identification and potential molecular methods that can be developed to type SNPs. The incorporation of SNPs and microsatellite studies in the Disease Component (Dr. Erik Thorsby) was discussed. The WG is interested in defining SNPs/microsatellites in a subset of the HCTC cases.

  TO DO: a) microsatellite protocol to be provided by Disease Component (Effie Petersdorf to request from Erik Thorsby and Benedicte Lie), b) as technology is developed, protocols for SNPs to be made available to HCTC (Geraghty to provide to Petersdorf).

  4. Methodologies.

Every donor-recipient pair will be typed at the allele level for HLA-A, B, C, DRB, DQB1, and DPB1. Consensus was reached on the following points:

  • Standardized 13th IHWC protocols will be used for high resolution typing. SSOP and SBT protocols will be made available in the next few months by the Technology Component. For SSOP, reagents will be available at reduced cost for workshop participants. For SBT, protocols will be available by December 1 for all class I and II genes at http://www.ihwc.org.


  • Provision for acceptance of other DNA methods for high resolution typing. Validation of high resolution typing is mandatory in order to preserve the integrity of the HLA database and assure integrity of the clinical outcome analysis. Evaluation of methods other than above will be made by a newly formed HCTC Methods Committee which include Dr. Lee Ann Baxter-Lowe, Dr. Frank Christiansen, Dr. Ekkehard Albert, Dr. Effie Petersdorf, Dr. Carolyn Hurley (Technology-SSOP) and Dr. Marcel Tilanus (Technology-SBT). The task of this committee will be to evaluate high-resolution methods and protocols that participating labs propose to use to type workshop samples.
  TO DO: Methods Committee will provide the HCTC WG with their recommendations as to a) the criteria to be used for evaluating protocols, and b) the acceptability of non-IHWC protocols submitted for consideration.
 
  • Raw data is to be collected on all retrospective and prospective samples. For SSOP typed samples, this would include the string of probe hits and misses. For SBT, the DNA sequence should be captured.


  • Other HLA data to be collected:
    • year sample typed
    • alleles assigned by contributing labs
    • CTLp - not discussed
    • HTLp - not discussed

  • SSP Issues
    • The WG recognized the invaluable role of SSP in group-specific amplification to type single alleles and to resolve ambiguous heterozygous combinations
  TO DO: Other issues. Should the Methods Committee and Core Technology Component consider the use of a workshop DNA panel for blinded QC of non-workshop protocols under consideration?

 
  • SSP used to arrive at allele typing for HCTC samples will need to have 1) specific sequence of SSP, 2) positive or negative hybridization results to each primer pair, 3) provision in database for fields for SSP data.

5. DNA and cells.

The WG recognizes the need to systematically collect and preserve DNA (and if possible, cells for LCL transfection) on all prospective transplant pairs. This philosophy must be conveyed to members of HLA laboratories and transplant centers.

The goal of the HCTC is to plan for long-range genomic analysis of unrelated stem cell transplants. These scientific-aims can be achieved only if labs and transplant centers make a personal commitment to save as much DNA/cells as feasible on both patient and donor. Pairs having insufficient DNA/cells for complete HLA analysis are essentially lost for outcome analysis as well as for genomic analysis.

The minimal criteria for DNA and cells was not discussed.

If we assure that in the worst case scenario a sample requires high-resolution typing of all six class I and II genes (HLA-A, B, C, DRB1, DQB1, DPB1), and factoring in need for repeat typing for failed amplifications, etc., 1.5-2 mg of genomic DNA will be needed! For cells, a minimum of two ampoules of 5 x 106 PBL are required in order to EBV-transform one and save one for future need.

Labs are also requested to freeze blood. Experience has indicated potential difficulty with subsequent amplification (especially for class I) depending on the method of DNA extraction used. Since a good portion of the samples will be tested in different labs, we need to maximize ability to perform amplification on the first try.

6. Sample size.

Sample size will depend on allele frequencies in a given study population and match/mismatches will be influenced by strength of linkage disequilibrium within HLA. Effie Petersdorf presented size calculations based on a worst case hypothetical scenario:

Define any two groups for comparison. For example,

Group 1: A*0201 versus A*0205 mismatches
Group 2: A*0201 versus A*0207 mismatches

OR

Group 1: HLA-A, B, C, DRB1, DQB1, DPB1 matched pairs
Group 2: HLA-A, B, DRB1, DQB1-matched, HLA-C mismatched pairs

OR

Group 1: Macintosh apples
Group 2: Granny Smith apples

If the number of samples in Group 1:Group 2 has a 1:4 ratio and if the "outcome" of Group 1 is 50% and the outcome of Group 2 is 40%, then 243 pairs are needed in Group 1 and 972 pairs are needed in Group 2 for 80% power (total 1215 pairs, alpha = .05). For 90% power, 324 pairs are needed in Group 1and 1296 pairs are needed in Group 2 (1620 pairs total; alpha = .05). Bottom line: a) depending on the allele frequencies in our populations, we may need several thousand study samples b) we must not miss collecting a single prospective transplant pair. We must start now.

7. Informatics/Database.

The NIH will support the 13th IHWC Informatics/Database. By December 1, 1998 all components must provide a complete list and description of raw data. The HCTC Component Working Group must address the following:
    What kinds of raw data should be collected?
    How do we want the data to be formatted for analysis?
    What questions will we ask of the database?
    What is the timeline to evaluate our data?

Here is a preliminary list of raw data that the WG agreed upon:

  • Identification number of sample
  • Serological assignments (if available)
  • Allele assignments made by contributing lab

SSOP typed samples
  • string of probe hits and misses (eg. 1811881181811880, where 1==negative, 8==positive,0==not tested
  • amplification primer sequence
  • protocol used

SBT typed samples
  • raw nucleotide sequence (not chromatograms)
  • amplification primer sequence
  • sequencing primer sequence
  • protocol used

SSP (In protocols where SSP is incorporated, capture):
  • sequence of primers
  • negative/positive amplification reactions

Queries required of database:
  • collapse allele name to broad group
  • sort based on allele name
  • sort based on probe hits or misses
  • sort based on nucleotides
  • link patient ( donor genetic data to sort on match status
  • sort based on clinical characteristics
Clinical data to be collected: (this list to be reviewed and discussed at ASH meeting)

  • demographic information in the patient and donor (race, age)
  • disease and stage
  • time for URD search (from start of formal search to identification of donor/transplant date)
  • pre-transplant therapy (drugs and dose)
  • transplant year
  • transplant regimens
  • engraftment
  • GVHD
  • relapse
  • survival
8. Clinical Issues.

The working group addressed issues of disease diagnosis as it pertains to asking different questions related to rejection, GVHD, relapse, survival. A working group meeting to discuss further clinical issues including the clinical database will be convened at the December 1998 ASH meeting in Miami.

The working group recognized the need to focus on specific HLA questions for the 2001 workshop and to define the questions of interest for long-range studies.

Potential Analyses for 2001:

In CML transplants, the definition of "tolerable" allele mismatches.

In acute leukemia, the role of the GVL effect according to HLA allele mismatch.

In non-neoplastic diseases, the influence of HLA on engraftment (SSA, thalasemia), chimerism.


The working group recognized that heterogeneity in HLA typing methods, patient/donor demographics, and clinical transplant regimens will impact our ability to discern genetic effects on clinical outcome. Therefore, there is rationale to study transplants performed from 1990 in order to decrease the amount of repeat high-resolution typing, and provide homogeneity for the recent changes in patient supportive care practices and transplant immunosuppressive regimens.

Other specific points discussed:

  • Separate analysis for T-cell replete from T-cell deplete transplants.
  • Survival may be the only evaluable clinical endpoint in our analysis due to the issues of multi-center interpretation of other clinical endpoints. This needs to be addressed to clinical transplanters in the working group.


9. Other Scientific Issues.

For related mismatched transplants, the working group will require:
1. Parental HLA data including haplotypes
2. Verification of sibling identity.

For the clinical transplanters to consider:
1. How reasonable/realistic is it to study endpoints other than survival?
2. Big question: how do we merge clinical databases?


Back to Overview
Back to Components

HOME
IHWG Contact Information