Chimaerism monitoring and therapeutic intervention

Transcription

1 Chimaerism monitoring and therapeutic intervention Dr Helena Lee, Transplantation Laboratory, Manchester Royal Infirmary

2 What is chimaerism testing? A method of monitoring the haematopoietic reconstitution of a recipients immune system post stem cell transplant. Can be carried out using a number of techniques. The method relies on genetic differences between recipient and donor. Used by transplant consultants to identify or confirm post transplant complications such as failed engraftment, graft rejection and disease relapse.

3 JACIE Accreditation: 2015 Chimaerism testing shall be performed in laboratories accredited for the techniques used Clinical decisions based on chimaerism result: may have potentially life-threatening consequences GVHD relapse risk graft failure Chimaerism testing - variety of methods Interlaboratory variability may be significant

4 Chimaerism techniques (1) Analysis Technique Advantages Problems Sensitivity (%) RFLP (Restriction Fragment Length Polymorphism) Highly Informative Time Consuming Labour Intensive DNA Hungry 5-10 Cytogenetics Time Consuming (Requires Cells in Metaphase) 5 Red Cell Polymorphism Simple and Accurate May be a Long Latency Period Looks at a Single Cell Lineage 1-5

5 Analysis Technique Advantages Chimaerism techniques Problems (2) Sensitivity (%) Analysis Technique Advantages Problems Sensitivity (%) X/Y FISH (Fluorescent in situ Hybridisation) Highly Sensitive Low Numbers of False Positive Results Only Applicable in Gender Mismatched Transplants Fluorescence-Based PCR-STR (Short Tandem Repeat) Real-Time PCR (Polymerase Chain Reaction) Robust, Fast High Quantitative Accuracy Highly Sensitive, Particularly using Lineage-Specific (LS) Approach Very High Sensitivity Rapid Results Labour Intensive Expensive False positive results in Single Nucleotide Polymorphism (SNP) Based Assays 1 5 Or (LS) Next Generation Sequencing Very high sensitivity At present the turnaround time of the assay is a couple of days? Droplet Digital PCR Very high sensitivity

6 Definition of Short Tandem Repeat CATA CATA CATA CATA CATA CATA CATA Short DNA sequence distributed in all the genome; in human, about 10 6 loci known Each marker is highly polymorphic, based on the number of repeats (Di, tri and tetra nucleotide repeat) Unknown function Initially used for forensic medicine.

7 STR-PCR Amplify pre and donor prior to transplant using multiplex approach Fluorescently labelled PCR products (NED, VIC, FAM, ROX) Panel of 8-24 markers From the pre-test select most informative markers at least 3 (NB double cord transplants and 2 nd transplant from 2 nd donor) Post-transplant sample can be peripheral blood, bone marrow or single cell lineage analysis. Amplify selected markers or multiplex (GenePrint 24)

8 Sizing of products Fragments separated according to size Laser excites the fluorescent labels on the PCR products CCD camera detects wavelength of the emission spectra Size standard included with every sample Unknown fragments are sized according to the generated sizing curve Fragment sizes related to known alleles for the patient and the donor

9 Engraftment Monitoring Donor Recipient ABI 3500 capillary electrophoresis

10 EFI Standards Version 6 Introduced October 2013 standard I6 Sensitivity and specificity of PCR DNA quantity Amplification bias from multiplex PCR Define criteria to accept results purity of the sorted population: must be documented and taken into account if this is not possible it must be clearly stated in the report

11 Why have guidelines?

12 Key recommendations 1 sample type Peripheral blood Bone marrow Single cell lineages Buccal

13 Key recommendations 2 lineage specific cell separation: cell lineage pertinent to disorder e.g. CD3, CD15, CD19, CD56 Purity assessment should be performed (also in EFI standard) by either flow cytometry or molecular methodology Sample should be processed within 24 hours of venepuncture Either positive or negative selection using specialised magnetic beads

14 Key recommendations 3 Minimum of 3 markers Skewing of STR markers: stutters, preferential amplification Coefficient of variation: below 5 where possible Reporting issues

15 Marker Selection Choose at least 3 Gold standard heterozygous for pre and donor - no stutter involvement i.e. at least 2 repeat units apart Non-informative both pre and donor share alleles Unbalanced amplification where one sample is homozygous and the other is heterozygous but no allele sharing Heterozygous but share an allele Share allele, one homozygous and one heterozygous

16 Analysis of results Recipient and donor homozygous, alleles are different D_ D and R Recipient and donor both heterozygous, alleles completely differ: D1 and D2 D1, D2, R1, R2 Recipient and donor both heterozygous, share one allele. Ignore the shared allele: D D and R

17 EQA results

18 EQA results

19 Limitations of chimaerism Identification of Informative Markers Method requires clear identification of recipient and donor derived immune repertoires. Extraction of Sufficient DNA Test often carried out on patients shortly after SCT when cell counts are low with atypical distributions depending on conditioning regimen Particularly important in lineage-specific analysis Application of Chimerism Result to Patient Care Sensitivity of test At what level of chimerism should the SCT consultant intervene? Which cell lineages are most informative?

20 Complete donor chimaerism Only donor DNA is found in a post transplant sample in all lineages. Often seen in the early post transplant period in full intensity conditioning. Monitoring should take place regularly in the early post transplant period to confirm stable engraftment.

21 Mixed chimaerism Both recipient and donor DNA detectable in posttransplant sample in all or majority of lineages. Mixed chimerism must be monitored closely as this is often the most dynamic chimeric state. Sensitivity of method often important in the detection of mixed chimerism above 95% (some centres say that this is full donor chimaerism)

22 Mixed chimaerism Mixed chimerism can be divided into 3 categories: 1.Decreasing recipient MC - Recipient DNA detected early post transplant that decreases over time. Seen in reduced intensity Txs. Requires close monitoring. 2.Stable MC Recipient and donor DNA remain in stasis over a lengthy period of time. Often a sign of true transplant tolerence. Rare. 3.Increasing recipient MC Clear increase of recipient DNA in sample. Worrying finding that may lead to alteration in immunosuppression or donor lymphocyte infusions in malignancies.

23 Split lineage chimaerism A form of mixed chimerism where only a single cell lineage is mixed. Importance of split chimerism depends on whether the recipient cells are malignant. Is the lineage displaying split chimerism the source of the malignant cells?

24 Single cell lineage analysis Test affected lineage if adequate then no need to intervene Correlation of donor T lymphocytes and GvHD In declining graft function if T cells are donor then reduce and stop immune suppression If T cells are recipient then intensify immune suppression Donor immunity recipient immunity

25 Autologous reconstituion Only recipient DNA found in post-transplant sample. Usually clinically indicated and urgent testing is requested. Often suggests failed engraftment. Patient will require reconditioning and a second transplant (HLA antibody screen). Second transplant may be from a different donor.

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28 Relapse Relapse Four factors for determining prognosis for relapsed malignancies: 1. Time elapsed from transplant to relapse 2. Disease type 3. Disease burden and site of relapse 4. Type of first transplant

29 Relapse Relapse 1. Leukaemia can recur in donor cells as a de novo event which can masquerade as a relapse 2. Patient s performance status is reduced by tissue damage induced by conditioning, post-transplant infection and GvHD 3. Treatment of relapse is managed on an individual basis 4. Multivariate analysis showed that day +30 whole blood and T-cell chimaerism levels were significantly associated with relapse and overall survival (Reshef et al., 2014 Philadelphia)

30 Donor lymphocyte infusion The theoretical principle of treatment of acute leukaemia by adoptive immunotherapy is to permit allogeneic, immumnologically competent cells to act against the host s leukaemia cells (Mathe et al., 1965) Recognition of GvL effect of allogeneic HPCT has prompted attempts at remission re-induction by adoptive immunotherapy with DLI in patients with relapsed disease

31 Treatment of mixed chimaerism Donor Lymphocyte Infusion (DLI) Common scenario in RIC transplants mixed chimaerism displayed in CD3 lineage Patients relapsing after SCT with few treatment options DLI is re-infusion of donor white cells post transplant not an option in cord blood transplants. Balance Graft versus Leukaemia with risk of GvHD In some conditions can re-induce remission status

32 Treatment of mixed chimaerism Reduction of Immunosuppression (IS) IS necessary for avoiding graft rejection Helps dampen the immune response to engraftment and post transplant complications such as GvHD. Too much IS may hinder engraftment Taper or withdraw IS, introduce chemotherapy appropriate for disase type and follow with DLI (acute leukaemia/mds) Then re-assess chimaeric status

33 Treatment of mixed chimaerism Second Transplant Option for those patients where DLIs ineffective Urgency in cases where primary non-engraftment failure is suspected Primary option for some non-malignant disorders (with RIC)

34 Other causes of mixed chimaerism CD8 autologous reconstitution T cells are largely recipient in nature Can be due to CD8 autologous reconstitution CMV-specific T cells are exclusively of recipient origin after R+D- T-cell depleted transplants CMV promotes recipient T-cell immunity following reduced-intensity T-cell depleted HSCT, significantly modulating chimerism status Sellar et al., Blood, 2015:

35 Manipulation of chimaerism Non-malignant disorders Mixed (stable) chimerism is tolerable e.g. in thalassaemia as long as red cell production is adequate, or in MPS as long as enzyme is adequate Second (usually reduced intensity) transplant where graft function is inadequate Rejection in SAA either very early post-transplant or later on following withdrawal of immunosuppression

36 Evidence for the utility of chimaerism testing

37 Pattern of engraftment in non-malignant disorders correlates with chimaerism level At three months, the pattern of engraftment (full donor vs mixed chimaerism) demonstrated a correlation with graft outcome in 91 patients (p<0.0001) Provides evidence for regular monitoring in early period post-transplant

38 The Manchester Algorithm The Manchester Algorithm

39 Troubleshooting results Loss of heterozygosity (LOH) Defect in DNA mismatch repair enzyme Malignant cells suffer alteration in enzyme and can t repair error Leads to LOH Pereira et al 2011 Human Immunology 72 (2011)

40 Case study - XIAP 14 year old M with XIAP Deletion in the SH2D1A gene - lack of modulation of IFN-γ, causing uncontrolled cell proliferation Presented 2012 with fever, rash, lymphadenopathy Colitis, cytopaenia, raised ferritin, coagulopathy, haemophagocytosis and falling counts, HLH Treatment is HPCT. Previous studies showed 86% mortality using MAC compared with 45% mortality using RIC. All patients with HLH at time of transplant died. Aim of transplant: achieve sufficient donor cell engraftment to prevent HLH occurring

41 Case study - XIAP Received MUD transplant July RIC conditioning: Fludarabine Treosulfan Alemtuzamab Post-transplant: Severe consumptive pancytopaenia. Recipient derived, donor directed anti-granulocyte antibodies. No response to steroids, immunoglobulin, plasma exchange and Rituximab Splenectomy ITU Recipient T cell driven HLH despite high % whole blood donor chimaerism (T cells 4.8% donor, whole blood 94.3%) NEVER GIVE DLI to paediatrics with non-malignant disorders GAVE DLI!!!! D+156 Follow-up: Recovery from HLH. Immediate neutrophil recovery and increasing CD3 donor chimaerism. Colitis resolved. Varghese et al., Journal of paediatric haematology and oncology 2015; 37:

42 Case Study XIAP - results Splenectomy DLI CD3 Whole Blood

43 Case study UK HAPLO TRIAL 62 yr old M with MDS RAEB2 Presented Jan 2013 with a stroke and cytopaenias Entered UK trial for AML 17. Normal cytogenetics Aim of transplant: use high dose cyclophosphamide to ensure engraftment following haploidentical transplant. 6 centres registered in UK, aim to recruit 78 patients. Alloreactive T cells (? Responsible for GvHD) are sensitive to toxic effects of cyclophosphamide Acute GvHD not increased in PBSC allograft = thought to be due to a skewing of the T-cell repertoire caused by exogenous G-CSF from a Th1 immunophenotype which is known to play a direct role in acute GVHD to an anti-inflammatory Th2 immunophenotype which can suppress acute GVHD.

44 Case study UK HAPLO TRIAL Transplanted Dec 2013 with 5/10 haploidentical sister as donor On d+28 his chimaerism started to drop His FBC dropped and he had low level CMV reactivation Required RC transfusions and GCSF maybe once a week BM revealed reduced cellularity, mild dyserythropoiesis, no significant dysplasia, no blasts. HLA antibody screen: NEGATIVE. Secondary engraftment failure (not previously reported). Outcome: having a double cord transplant in approx 3-4 weeks Update: double cord transplant April 14, patient passed away Nov 15 from CMV colitis

45 Case study UK HAPLO TRIAL

46 Case study: adult double cord 38 yr old M with PH positive ALL. WCC 155 at diagnosis. Transplanted 30/12/2015 with double cord. Treated off trial on UK ALL14. Mucositis and CMV reactivation. Neutrophils 0.12x10 9 /L one month post-hpct

47 Case study: adult double cord On d+28 his chimaerism showed 100% donor on PBL, CD3, CD15 and bone marrow. HLA antibody negative Engraftment syndrome

48 Acknowledgements Transplantation Laboratory team Prof Rob Wynn and clinical team RMCH Dr Eleni Tholouli and clinical team MRI