Post Transplant Monitoring with Chimerism

The role of the H&I laboratory is to provide clinicians with accurate information of the engraftment status post-transplant by quantitatively determining the proportion of donor and recipient derived cells in the patient post-transplant. Most H&I laboratories use Short Tandem Repeat (STR) testing for this. STR’s are short sequences of DNA, distributed throughout the genome which are repeated in tandem a variable number of times. The number of repeats of different STR markers varies between individuals, from 4 to 50 repeats for some STRs, giving a highly polymorphic system that can be used to uniquely identify donor derived DNA from patient derived DNA. With the exception of monozygotic twins, careful selection of a number of STR markers will enable most patients derived DNA to be distinguished from donor derived DNA.

Post stem cell transplant chimerism results are typically reported as % donor chimerism. A 100% donor chimera implies complete engraftment. A 0% donor chimerism implies no donor engraftment with all other percentages reported as mixed chimerism showing the proportion of donor engraftment. A longitudinal study of donor engraftment is of more value than a single static result and the H&I laboratory would typically test at agreed intervals and report a history of the chimeric status of the patient since transplant rather than a single test report. The relative changes in magnitude of the donor chimerism provide key information which helps clinicians to intervene and to monitor the patients’ response to such intervention. Intervention options include changes in immunosuppression regimes and donor lymphocyte infusion.

Longitudinal STR chimerism analysis is particularly useful in reduced intensity conditioning (RIC) regimes where initial mixed chimerism post-transplant is relatively common. The frequency of testing is agreed between the H&I laboratory and the transplant centre. For myeloablative regimens, this is typically weekly in the first month post-transplant followed by monthly testing. For RIC regimes this is typically monthly.

While STR analysis can be performed on whole blood, many H&I laboratories will offer lineage specific STR analysis, separating T and B cells from myeloid cells. This approach increases the sensitivity of the technique and has proved useful as in some cases of mixed chimerism, the initial myeloid mixed chimerism may dominate and mask clinically significant changes in other cell subsets.

The use of STR for chimerism analysis has also proved useful in the case of double cord transplants where it is possible to see a mixed chimera consisting of patient and one or both cords early in the post-transplant period before one cord eventually expands to 100% present in the patient.

Stable mixed chimerism post-transplant does not necessarily indicate a need to treat particularly in diseases such as Aplastic Anaemia (AA) and other non-malignant conditions especially where RIC regimes have been used. STR results showing increasing donor proportion is good whilst STR results showing increasing recipient proportion may indicate relapse or graft rejection and may indicate a need to treat. A reduction in donor chimerism post-transplant is potentially a sign of a failing graft and needs to be monitored closely. Weekly, fortnightly or at the very least monthly cell lineage based chimerism testing may be indicated if the donor chimerism level continues to fall. If the donor chimerism has not fallen too far it may be possible to save the graft by reducing GvHD prophylaxis if no GvHD present and/or by reducing immunosuppression if no infection present.

For non-malignant disease it may be possible that function remains acceptable even if donor chimerism has fallen. For malignant disease an MRD test may be indicated. A return to patient chimerism doesn’t necessarily mean return of the malignancy.

A DLI is not possible with a cord blood unit so a second transplant may be indicated. For matched sibling and MUD donor transplants a DLI would be indicated if donor chimerism continues to fall with second transplant with the same or a new donor as the next step though this may have some funding implications.

GvHD Post-Transplant

Overview

GvHD is the most frequent post allogeneic stem cell transplant complication and is a major cause of morbidity and mortality. GvHD is a consequence of activation of donor T lymphocytes by recipient antigen presenting cells. It requires three conditions, transplantation of immunocompetent donor cells, histo-incompatibility between recipient and donor and immunocompromised recipient such that the recipient cannot mount an adequate immune response against the donor cells. A number of randomised controlled trials have compared the incidence of GvHD in bone marrow and peripheral blood stem cell transplants in the related and unrelated setting. In allogeneic sibling transplantation, the incidence of acute GvHD was the same in the majority of studies. There was however an increased incidence of chronic GvHD with PBSC compared to BM. In the unrelated setting, matched cohort comparison of BM and PBSC transplants report no difference in the incidence of acute and chronic GvHD. These were not however randomised controlled trials.

GvHD develops in a three-phase process. In Phase 1 the effects of the conditioning regimen leads to tissue damage and the generation of pro inflammatory cytokines including TNFα and IL-1. In Phase 2, donor T lymphocytes are activated by host antigen presenting cells. The activated T cells produce cytokines including IL-2, leading to T cell expansion. Phase 3 is a cytokine storm in which a positive feedback loop of cytokine production leads to activation of effector cells such as CTLs and NK cells, which produce more pro inflammatory cytokines such as TNFα and IL-1.

GvHD has traditionally been classified into acute and chronic based mainly on the time to onset. GvHD arising before day 100 post-transplant was classified as acute and onset after day 100 classified as chronic. The current definition sets no time limits but is rather based on the presence of specific symptoms.

The organs primarily affected by GvHD are the skin, the gut and the liver. Skin GvHD manifests as a rash often affecting the palms and soles first, before spreading to the entire body surface. Gut involvement manifests as nausea and a watery diarrhoea, which in advanced disease can be bloody. Liver involvement can manifest as jaundice and is usually measured by the level of Bilirubin production.

Acute GvHD is traditionally classified in grades I to IV depending on the level of organ involvement. A new international classification of grades A to D is also in use. The traditional I – IV classification however continues to be the one most widely used. In brief, it classifies GvHD with only mild skin involvement as Grade I. Mild to moderate skin involvement with mild gut and/or liver involvement is classified as Grade II. Moderate to severe skin involvement with mild to moderate gut and/or liver involvement is classified as Grade III and Moderate to very severe skin involvement with moderate to severe gut and/or liver involvement is classified as Grade IV.

The two main mechanisms used to prevent GvHD are pre transplant graft T cell depletion of the stem cells and post-transplant immunosuppression of the patients. T cell depletion may be undertaken with for instance the use of alemtuzumab or Anti-Thymocyte Globulin (ATG). Graft T cell depletion is not very popular in the UK where patient immunosuppression appears to be the preferred method of GvHD prophylaxis. A common approach is to use a calcineurin inhibitor such as Cyclosporin-A (CsA) or Tacrolimus, in combination with short course Methotrexate (MTX). Another option is Sirolimus in combination with Tacrolimus and short course MTX. In reduced intensity transplants (RIC) Mycophenolate mofetil (MMF) has been used in combination with CsA.

Initial GvHD therapy post-transplant consists of a dose of Methyl-prednisolone.  Patients refractory to this primary treatment have a poorer prognosis. Secondary treatment is with higher doses of Methylprednisolone or other immunosuppressive drugs not already used as induction therapy, including Tacrolimus, MMF or Sirolimus, ATG, monoclonal antibodies.

Role of The H&I Laboratory in GvHD Cases

The Laboratory should undertake chimerism studies to determine level of engraftment and help stem cell unit assess if immunosuppression can be safely altered to increase GvHD treatment

• If there is skin involvement in the GvHD, a biopsy sample from the affected and a non-affected area of the skin should be undertaken to determine if there are donor cell in the affected area
• A number of biomarkers have been shown to be associated with the development of GvHD and it may be possible to test for these early post-transplant before clinical symptoms of GvHD are manifest. These include:
  • Suppression of tumorigenicity 2 (ST2)
  • Elafin
  • Regenerating islet-derived 3α (REG3α) is
  • The cytokine CX3CL1, levels of which have been shown to be predictive of the development of GvHD in day 0-50 post-transplant