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August 2002, Volume 16, Number 8, Pages 1413-1418
Table of contents    Previous  Article  Next   [PDF]
Debate Round-Table
Chimerism testing and detection of minimal residual disease after allogeneic hematopoietic transplantation using the bioView (DuetÔ) combined morphological and cytogenetical analysis
A Shimoni1, A Nagler1, C Kaplinsky2, M Reichart3, A Avigdor1, I Hardan1, M Yeshurun1, M Daniely3, Y Zilberstein3, N Amariglio2, F Brok-Simoni2, G Rechavi2 and L Trakhtenbrot2

1Department of Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel

2Department of Hematology, Chaim Sheba Medical Center, Tel-Hashomer, Israel

3BioView Ltd, Nes-Ziona, Israel

Correspondence to: A Shimoni, Department of Bone Marrow Transplantation, Chaim Sheba Medical Center, Tel-Hashomer, Israel; Fax: 972 3 530 5298

Abstract

Recurrent disease remains a major obstacle to cure after allogeneic transplantation. Various methods have been developed to detect minimal residual disease (MRD) after transplantation to identify patients at risk for relapse. Chimerism tests differentiate recipient and donor cells and are used to identify MRD when there are no other disease-specific markers. The detection of MRD does not always correlate with relapse risk. Chimerism testing may also identify normal hematopoietic cells or other cells not contributing to relapse. In this study we report our initial experience with a novel system that provides combined morphological and cytogenetical analysis on the same cells. This system allows rapid automatic scanning of a large number of cells, thus increasing the sensitivity of detection of small recipient population. The clinical significance of MRD detection is improved by identifying the morphology of recipient cells. Identification of recipient characteristics within blasts predicts overt relapse in leukemia patients and precedes it by a few weeks to months. Identification within mature hematopoietic cells may not be closely associated with relapse. The system also allows chimerism testing after sex-mismatched transplants, within cellular subsets, with no need for sorting of cells. The system merits further study in larger scale trials.

Leukemia (2002) 16, 1413-1418. doi:10.1038/sj.leu.2402581

Keywords

chimerism; minimal residual disease; FISH; morphology

Introduction

Allogeneic hematopoietic cell transplantation is an effective treatment for advanced and high-risk hematological malignancies.1 However, recurrence of the underlying malignancy remains a major obstacle to cure. Detection of minimal residual disease (MRD) after transplantation can identify, in certain settings, patients who are at high risk for relapse. The timely application of additional therapeutic interventions to these patients may allow reduction of this risk. Immune-therapeutic interventions such as early withdrawal of immunosuppressive therapy given post-transplant for prevention of graft-versus-host disease (GVHD), and donor lymphocyte infusions (DLI) can induce potent graft-versus-leukemia (GVL) and graft-versus-tumor (GVT) effects and restore remission.2,3 These interventions are not without risks, as they may be complicated by life-threatening GVHD or marrow aplasia, and therefore should be applied only to those destined to relapse.4 The application of these interventions at minimal disease state is more effective and also safer than at overt relapse. Various methods have been developed for the detection of chimerism and MRD.5 The optimal timing and use of these tests is under debate and their clinical implications still controversial.6,7 This study reports our initial experience with a novel system that provides combined morphological and cytogentical analysis on the same cells for chimerism testing and detection of MRD. We describe how this system was used in three representative patients to illustrate the potential advantages over currently available detection methods.

Methods

BioView System (DuetÔ) provides combined analysis of cells by morphology, fluorescence in situ hybridization (FISH), and/or immunocytochemistry. Up to three parameters can be tested on the same cell (eg morphology and two rounds of FISH with different probes). This system allows rapid automatic scanning of a large number of cells for identification of small cellular populations by their morphology or cytogenetic characteristics, with high sensitivity. In brief, slides for DuetÔ are prepared from peripheral blood or bone marrow samples by density gradient centrifugation and cytospin. The slides are stained with May-Grunwald-Giemsa (MGG) and scanned with a bright-field microscope. The system saves the coordinates and images of all cells found on the slides for future reference during the next phases of analysis. The MGG staining is removed and FISH is applied to the same slide. Hybridization can then be removed and a second FISH with another probe can be applied to the same cells. After the FISH procedure, the slides are searches either manually or automatically for target cells containing the fluorescence signals. When the underlying malignancy is associated with a specific cytogenetic abnormality a specific FISH probe is used to identify the MRD population and the system relocates the morphology of cells harboring this abnormality. For sex-mismatched transplants, recipient cells presenting XY signals or XX signals are searched for and targeted. The system then characterizes the morphology of these cells for determination of their potential as MRD. Cells can be targeted by any combination of morphology, FISH or immunohistochemistry and the system can then present in parallel morphological and FISH images of target cells (Figures 1-3). The system is further discussed in the 'Method in Focus' appendix.

Results

Patient 1

A 54-year-old man was diagnosed with Philadelphia chromosome-positive acute lymphoblastic leukemia. FISH analysis of a bone marrow aspirate at the time of diagnosis revealed that 80% of the cells harbored the BCR/ABL fusion and 20% had a double BCR/ABL fusion, with the minor BCR/ABL breakpoint. The patient was given induction chemotherapy according to the GMALL protocol and achieved complete hematological and cytogenetical remission. He was then given high-dose chemotherapy with allogeneic stem cell transplantation from an HLA-matched sister. The patient was conditioned with high-dose busulfan (16 mg/kg). Fludarabine and anti-thymocyte globulin (ATG) were added in substitution to the standard high-dose cyclophosphamide due to cardiac dysfunction related to a prior myocardial infarction. The procedure was uncomplicated with prompt engraftment. Histological analysis of bone marrow aspirate performed on day +28 post transplant was compatible with complete remission with no conspicuous lymphoblasts. FISH analysis of this specimen revealed that 0.6% of 300 cells examined were recipient-derived XY cells. Further examination of 800 cells revealed 0.2% and 0.6% cells with single and double BCR/ABL fusion, respectively. We have used the DuetÔ system to test the lineage and morphology of residual host (XY) cells. Manual scanning of the slide marked with X and Y probes identified 44 XY cells. The DuetÔ system retrieved the images of these cells on the MGG staining. It was found that 36% were blasts and the other XY cells were defined as mature hematopoietic cells from all lineages. The X and Y probes were removed and a second FISH with a probe for BCR/ABL was performed on the same slide. This analysis revealed that the XY cells presenting as blasts also contained single or double BCR/ABL fusion, whereas all the mature XY hematopoietic cells contained normal BCR/ABL status (Figure 1). Two weeks later the patient presented with overt hematological relapse. He was given STI571 and 2 weeks later achieved complete remission. FISH analysis found 0.4% of 400 cells examined to be host XY cells and none harbored BCR/ABL fusion. Thirty thousand cells were automatically scanned with the DuetÔ system for Y signals. 57 XY cells were found (0.2%) and all were morphologically mature hematopoietic cells. The patient was given donor lymphocyte infusion (DLI) complicated by acute graft-versus-host disease. Six weeks later repeat analysis by FISH revealed no XY cells. Automatic analysis of 13000 cells with the DuetÔ system found 2 XY positive cells, which were normal neutrophils. This was compatible with further elimination of host hematopoiesis and residual disease with the allogeneic process induced by DLI.

Patient 2

Morphological assessment of host residual cells late after transplant may also be helpful. We have used the DuetÔ system to analyze disease status in a 52-year-old man with post-myelodysplasia acute myelogenous leukemia who was given allogeneic transplantation with standard BuCy ablative conditioning from an HLA-matched sister, as upfront therapy. Standard FISH analysis of a bone marrow aspirate at 1 year post transplant identified no recipient XY cells within 500 cells examined. Automatic scanning with the DuetÔ system of 75000 cells identified 5 XY cells. Morphological assessment of this minute XY population revealed that these were recipient non-hematopoietic cells, probably osteoblasts, rather than residual disease, and that further intervention is not required (Figure 2). The patient continues to be in remission 6 months later.

Patient 3

A 56-year-old man was diagnosed 5 years earlier with aggressive non-Hodgkin's lymphoma in stage IV-B bulky disease. He was given a few lines of chemotherapy with only partial response. He then underwent high-dose chemotherapy and autologous stem cell transplantation and achieved complete remission. However, approximately 4 years later he had massive bulky relapse, with cervical, supraclavicular and abdominal nodes, bone, and marrow disease. Re-induction chemotherapy was complicated by severe pneumonia and decreased performance status, but only partial response. We therefore decided to treat him with allogeneic transplantation from an HLA-matched sister, using non-myeloablative conditioning, in an attempt to induce graft-versus-lymphoma effect. The patient was given fludarabine (total 180 mg/m2) and intravenous busulfan (total 3.2 mg/kg) and no ATG. The course was uncomplicated. The patient became neutropenic and engrafted on day +16. We have used the DuetÔ system to follow chimerism status in different cellular subsets. On day +24 FISH analysis revealed that, overall, 4% of the cells examined were host XY cells. We have used the images of the MGG stained slide to mark different cellular subsets (eg lymphocytes, neutrophils, monocytes) by morphology. We could then analyze the FISH results only in the desired marked subset. The analysis showed that 3.2% of lymphocytes, and 2.9% of neutrophils were recipient XY cells. Repeat analysis during the first 2 months showed similar results. At 7 weeks post transplant re-evaluation of disease status showed residual Gallium-avid abdominal mass. We therefore started tapering cyclosporine therapy trying to convert the T cell chimerism to complete donor, and to achieve an allogeneic GVT response. On day +80 the DuetÔ analysis revealed that complete lymphocyte chimerism was achieved. Interestingly, 10 days later, the patient showed signs of graft-versus-host disease of the skin and liver, and corticosteroid treatment was initiated. One month later, repeat disease evaluation showed near resolution of the abdominal mass.

Discussion

Chimerism testing is used for routine documentation of engraftment and post-transplant follow-up of allogeneic transplant recipients. Donor and recipient cells can be distinguished by testing genetic markers that are determined prior to the transplant.2 In sex-mismatched transplants when the recipient and donor are of different gender, the proportion of female and male cells is usually determined by analysis of sex chromosome markers, most often with FISH.8,9 Assessment of variable number of tandem repeats (VNTR) or short tandem repeats (STR) by PCR has become the most valuable method for determination of chimerism in sex-matched transplants.10 Various methods have been developed for the detection of MRD. Morphological analysis of the marrow can identify residual disease occupying more than 5% of marrow space and thus lacks the necessary sensitivity for early detection. Also, it is often difficult to differentiate a small leukemia blast population from normal donor-derived hematopoietic blasts repopulating the marrow without using additional markers. Specific tests can detect MRD when there is a specific tumor marker such as a specific cytogenetical abnormality, a specific immunophenotype that can be identified by FACS, specific DNA or RNA sequences that can be amplified by PCR or a specific growth pattern in clonogenic assays. In the absence of a specific tumor marker for MRD, chimerism testing can be used to detect recipient-derived cells. The detection of recipient-derived cells may not always correlate with the risk for relapse. Recipient cells contributing to mixed chimerism (MC) belong to the malignant clone or may be normal hematopoietic cells, or even stromal cells. During the first few weeks after standard allogeneic transplantation recipient cells can still be detected when sensitive tests are used.9,11 These are mostly T cells surviving the conditioning regimen. Host cells may be detected at low levels (<1%) even later in the post-transplant course. MC is detected more often after non-myeloablative conditioning.2 Thus, tumor-specific markers probes are probably superior and more accurate than sex-mismatch probes for detection of MRD.12 However, even the detection of MRD with tumor-specific tests may not always be correlated with the risk of relapse. Positive PCR tests for BCR/ABL have been reported in healthy individuals.13 The t(14;18) translocation was detected in lineages other than B lymphocytes in patients with non-Hodgkin's lymphoma.14 There may be a threshold for MRD to be clinically significant. In several diseases, such as AML with t(8;21),15 long-lasting remissions have been observed in the presence of MRD detected by PCR, while in others, such as in acute promyelocytic leukemia, PCR positivity predicts relapse.16 Again, this may be related to the sensitivity of the specific PCR test and the threshold to clinical significance. Residual malignant cells may be at a dormant status17 lacking the potential to contribute to relapse, because of lack or gain of a second genetic alteration. In some diseases partial differentiation to more mature cells can occur and these cells may lack the potential to proliferate. MRD may be under immune surveillance, or the MRD cells be in an apoptotic phase rendering them irrelevant for disease recurrence. The sensitive PCR tests do not preserve cellular morphology, and therefore it is unclear which cells correlate with MRD in these different setting.

The DuetÔ system for combined simultaneous morphological and cytogenetical multiparametric analysis offers a few advantages that may help in disclosing the relevance of MRD detection. This system automatically searches a large number of cells (approximately 150000 cells per slide) for small cellular subsets with unique cytogentical or immune phenotype. Sensitivity for detection of small populations is increased. In a series of dilution experiments we have shown that the sensitivity for detection of a male cell in a female cell population is higher than 1:50000, however, due to limited specificity in this titer, best detection occurs between 1:10000 and 1:50000 (see appendix). As seen in the analysis of bone marrow samples from patient 1 (last analysis) and patient 2, DuetÔ system could detect very small populations of XY recipient cells not detected by routine FISH. The quantitative accuracy of FISH is dependent on the observed frequency and the number of cells scored, thus improved by scoring more cells.18 Scoring such a large number of cells is not practical with manual FISH but the DuetÔ automatic system is able to scan 10000 cells/min in bright-field microscopy and 2000-10000 cells/h in fluorescent microscopy, thus supporting the need for rapid and efficient scanning and increased sensitivity.

Sensitive tests are often associated with reduced specificity and a relatively high false positive rate. With standard FISH false positive scoring may occur due to non-specific hybridization of the probe. False positive scoring for chromosomal translocations may occur due to random spatial association and optical fusion.18 The DuetÔ system can potentially improve specificity by identifying the morphology of the cells in the population searched. Detection of FISH positivity, in a small population but with a uniform DuetÔ morphological appearance make it more likely to be a true positive, than when positive cells are scattered within different, unrelated lineages. The specificity of detection of MRD with nonspecific chimerism testing may also be improved. As discussed above, recipient cells can belong to the malignant clone (as in patient 1), can be normal hematopoietic cells (patient 3, patient 1 after treatment with STI571) or non-hematopoietic/stromal cells (such as osteoblasts, patient 2). The DuetÔ system allows differentiation between these options by the morphological appearance. We have shown in the analysis of patient 1, and two additional patients (data not shown), that identification of recipient characteristics (such as opposite gender cytogenetics) within blasts or immature cells predicts overt relapse and precedes it by a few weeks to a few months. In patient 1 separate FISH analyses disclosed a small XY recipient population (0.6%), and a small BCR/ABL positive population (0.8%) that could both be considered within the false positive rate of the FISH analysis. However, the DuetÔ system has shown that a large proportion of the XY recipient cells were blasts (and also BCR/ABL positive by a second FISH test on the same cells) and could thus predict that the patient is destined to relapse. The identification of a small recipient population within mature hematopoietic cells may not predict relapse. In three additional patients (data not shown) we have documented continuous remission in the presence of a small recipient population with mature morphology. Larger scale studies with a longer follow-up are needed to confirm the association between morphology of residual host cells and relapse risk in patients with no other unique markers for the malignant clone.

The system is relatively simple to operate, is mostly automatic, its turn-out time and costs are comparable to other systems for chimerism and MRD detection such as standard FISH and PCR and it may be suitable for large-scale testing. Other than the system's hardware and kits only standard equipment readily available in large laboratories is needed (see appendix).

In recent years the study of chimerism within different cellular subsets has gained in interest and popularity.19,20 This is of utmost importance after non-myeloablative transplants.2 It has been shown by some groups that complete chimerism within the T lymphocytes is required for induction of GVL, and therefore MC in this cellular subset may be associated with increased risk for relapse, especially of aggressive, rapidly growing malignancies.2,19 Chimerism testing of cellular subsets needs cumbersome and time-consuming sorting of cells with the theoretical potential to lose some cells during that process. As discussed in the appendix the preparation of slides for DuetÔ analysis does not cause lost or reduction of any cellular population. The DuetÔ system uses the MGG stained slide to locate lymphocytes and other cellular subsets and can also use immunocytochemical stains (such as anti-CD3 monoclonal antibodies) to locate these cells. In a second phase, FISH can be applied for chimerism testing in sex-mismatched transplants. The case presentation of patient 3 shows how the system was used to follow chimerism in a patient after non-myeloablative transplant and how conversion to complete chimerism following cyclosporine withdrawal predicted the occurrence of GVHD and GVT responses.

This report focuses on the use of DuetÔ following bone marrow transplantation, however, there may be many other implications. The system may also help study the cells and lineages that harbor a unique cytogenetical or immunophenotypical marker, such as BCR/ABL fusion (as seen in patient 1) and correlate their morphology with relapse risk after standard chemotherapy. It may help disclose the nature of MRD in different malignancies and why MRD detection is not always predictive of relapse. In certain settings small donor cell populations or micro-chimerism may be sought. For example, systemic lympho-hematopoietic microchimerism detected after solid organ transplantation may predict tolerance to the transplanted organ and direct the immunosuppressive therapy.21 Small maternal population contaminating cord-blood allografts may also be detected in a similar way and may have implications on post-transplantation risk for GVHD.

In conclusion, by adding morphological analysis of small populations of cells with malignancy or recipient-associated markers the DuetÔ system may improve the accuracy of chimerism and MRD testing, and delineate their clinical significance. This system merits further study in larger scale trials.

References

1 Thomas ED, Storb R, Clift RA, Fefer A, Johnson L, Nieman PE, Lender KG, Glucksberg H, Buckner CD. Bone marrow transplantation. N Engl J Med 1975; 292: 832-843. MEDLINE

2 Shimoni A, Nagler A. Non-myeloablative stem-cell transplantation (NST): chimerism testing as guidance for immune-therapeutic manipulations. Leukemia 2001; 15: 1967-1975. MEDLINE

3 Bader P, Klingebiel T, Schaudt A, Theurer-Mainka U, Handgretinger R, Lang P, Niethammer D, Beck JF. Prevention of relapse in pediatric patients with acute leukemias and MDS after allogeneic SCT by early immunotherapy initiated on the basis of increasing mixed chimerism: a single center experience of 12 children. Leukemia 1999; 13: 2079-2086. MEDLINE

4 Collins RH, Shpilberg O, Drobyski WR, Porter DL, Giralt S, Champlin R, Goodman SA, Wolff SN, Hu W, Verfaillie C, List A, Dalton W, Ognoskie N, Chetrit A, Antin JH, Nemunaitis J. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. J Clin Oncol 1997; 15: 433-444. MEDLINE

5 Toren A, Rechavi G, Nagler A. Minimal residual disease post bone-marrow transplantation for hemato-oncological diseases. Stem Cells 1996; 14: 300-311. MEDLINE

6 Lion T, Muller-Berat N. Chimerism testing after allogeneic stem cell transplantation: importance of timing and optimal technique for chimerism testing in different clinical-biological situations. Leukemia 1999; 13: 2059.

7 Lion T. Chimerism testing after allogeneic stem cell transplantation: importance of timing and optimal technique for chimerism testing in different clinical-biological situations. Leukemia 2001; 15: 292. MEDLINE

8 Esa A, Trakhtenbrot L, Hausmann M, Rauch J, Brok-Simoni F, Rechavi G, Ben-Bassat I, Cremer C. Fast-FISH detection and semi-automatic image analysis of numerical chromosome aberration in hematological malignancies. Anal Cell Pathol 1998; 16: 211-222. MEDLINE

9 Durnam DM, Anders KR, Fisher L, O'Quigley J, Bryant EM, Thomas ED. Analysis of the origin of marrow cells in bone marrow transplant recipients using a Y-chromosome-specific in situ hybridization assay. Blood 1989; 74: 2220-2226. MEDLINE

10 Thiede C, Bornhauser M, Oelschlagel U, Brendel C, Leo R, Daxberger H, Mohr B, Florek M, Kroschinsky F, Geissler G, Naumann R, Ritter M, Prange-Krex G, Lion T, Neubauer A, Ehninger G. Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers. Leukemia 2001; 15: 293-302. MEDLINE

11 Dubovski J, Daxberger H, Prinz D, Peters C, Matthes S, Gadner H, Lion T. Kinetics of chimerism during the early post-transplant period in pediatric patients with malignant and non-malignant hematologic disorders: implications for timely detection of engraftment, graft failure and rejection. Leukemia 1999; 13: 2060-2069.

12 Nagler A, Slavin S, Yarkoni S, Fejgin M, Amiel A. Detection of minimal residual disease after sex-mismatch bone marrow transplantation in chronic myelogenous leukemia by fluorescence in situ hybridization. Cancer Genet Cytogenet 1994; 73: 130-133. MEDLINE

13 Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV. The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease. Blood 1998; 92: 3362-3367. MEDLINE

14 Yarkoni S, Lishner M, Tangi H, Nagler A, Lorberboum-Galski H. B-cell non-Hodgkin's lymphoma: evidence for the t(14;18) translocation in all hematopoietic cell lineages. J Natl Cancer Inst 1996; 88: 973-979. MEDLINE

15 Jurlander J, Caligiuri MA, Ruutu T, Baer MR, Strout MP, Oberkircher AR, Hoffmann L, Ball ED, Frei-Lahr DA, Christiansen NP, Block AM, Knuutila S, Herzig GP, Bloomfield CD. Persistence of the AML1/ETO fusion transcript in patients treated with allogeneic bone marrow transplantation for t(8;21) leukemia. Blood 1996; 88: 2183-2191. MEDLINE

16 Perego RA, Marenco P, Bianchi C, Cairoli R, Urbano M, Nosari AM, Muti G, Morra E, Del Monte U. PML/RAR alpha transcripts monitored by polymerase chain reaction in acute promyelocytic leukemia during complete remission, relapse and after bone marrow transplantation. Leukemia 1996; 10: 207-212. MEDLINE

17 Greaves M. Silence of the leukemic clone. N Engl J Med 1997; 336: 367-369. MEDLINE

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Figures

Figure 1 Screen shot of the DuetÔ system in the analysis of a bone marrow aspirate from patient number 1, a male patient with Philadelphia-positive acute lymphoblastic leukemia transplanted with female donor cells, on day +28 after transplantation. (a) The right hand image shows the FISH analysis. A recipient cell (XY genotype - red and green signals, marked 1) and two donor cells (XX genotype - two green signals, marked 2, 3) are presented. The left hand image shows the morphology of the cells. The recipient cell is a lymphoblast (within the red rectangle). Donor cell 2 is a polymorphonuclear, donor cell 3 is a lymphocyte. (b) The same cells are shown after a second FISH using the BCR/ABL-ES probe. The XY recipient blast shows double BCR/ABL fusion (two yellow, two red and one green signals) while the PMN and the lymphocyte are normal (two red and two green signals). All the target XY cells snapped in this slide are shown in small images on the bottom left side of the screen.

Figure 2 Analysis of a bone marrow aspirate from patient 2, a male transplanted with female donor cells, 1 year after allogeneic transplantation. The right hand image shows a cell with recipient genotype (XY - one red and one green point). The left hand image shows the morphology of the cell suggesting it is an osteoblast.

Figure 3 Chimerism analysis of peripheral blood sample from patient 3, a male transplanted with female donor cells, on day +24 after allogeneic transplantation. The left hand image shows two lymphocytes. The right hand image shows that the cell marked 1 is of recipient genotype (XY - one red and one green point, marked 1) and the cell marked 2 is of donor genotype (XX genotype - two green signals). The polymorphonuclear in between and above the lymphocytes is also of recipient origin. Further analysis has shown that 3.2% of lymphocytes were recipient-derived.

Received 6 November 2001; accepted 19 March 2002
August 2002, Volume 16, Number 8, Pages 1413-1418
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