Correspondence *Stem Cell Transplantation Unit, University of Würzburg, Josef-Schneider-Strae 2, 97080 Würzburg, Germany

Email stuhler_g@medizin.uni-wuerzburg.de

The case

A 21-year-old man was admitted to hospital with leukocytosis, thrombocytopenia, anemia and a deteriorating performance status. He reported no relevant medical history except the development of fever and night sweats a few days before presentation. On admission, he had elevated peripheral blood leukocytes (221,000/l; normal range 5,000–10,000/l) consisting of 94% lymphoblastic cells. Acute lymphoblastic leukemia (ALL) was suspected from microscopic examination of peripheral blood and bone-marrow smears and this diagnosis was confirmed by immunophenotyping. Cytogenetic analyses revealed Philadelphia chromosome positivity (t[9;22]) and the bcr–abl rearrangement was detected by polymerase-chain-reaction-based methods. Induction chemotherapy according to the German multicenter ALL (GMALL) protocol resulted in a complete hematological remission. Imatinib, a tyrosine-kinase inhibitor that specifically blocks the constitutively active bcr–abl fusion protein, was given at a starting dose of 600 mg per day in conjunction with induction chemotherapy.

After myeloablative conditioning, the patient was transplanted with a single unrelated cord blood (UCB) unit, (Table 1) because no suitable human leukocyte antigen (HLA)-identical related or unrelated donor could be identified. Despite the infusion of a sufficient number of nucleated cells, primary graft failure and minimal residual disease were documented by bone-marrow biopsy 44 days after the transplant (Figure 1A). His clinical situation was complicated by therapy-resistant invasive fungal infection of the lung, septicemia by Staphylococcus epidermidis and reactivation of cytomegalovirus (CMV; Figure 1B,C).

Figure 1 Hematological parameters and course of infectious complications during treatment.

(A) The course of the leukemic disease monitored by PCR is presented as the normalized bcr–abl:abl ratio. (B) Viral load in blood and cerebrospinal fluid as quantified by real-time PCR using CMV-specific primers. Arrows show time of cord-blood, stem-cell and T-cell transplantation and bars indicate duration of single agent or combined antiviral therapies. The threshold of real-time PCR is 600 copies/ml. (C) CT scans showing the course of the multidrug-resistant pulmonary fungal infection (arrows). (D) Mixed chimerism and relative values as assessed by short-tandem repeat PCR analysis. (E) The additive contribution of cord blood 2 and 3 and of the CD34-selected stem cells to the CD3⁺ T-cell, CD56⁺ NK-cell and granulocyte compartment in absolute numbers over time. Values are calculated using flow-cytometry techniques and HLA-specific antibodies to discriminate donors. Abbreviations: CMV, cytomegalovirus; CSF, cerebrospinal fluid; Gr, granulocytes; PCR, polymerase chain reaction.

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Table 1 HLA genotype of the recipient and respective donors.

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On day 44, a second conditioning course was initiated with the patient receiving the CD3-directed monoclonal OKT3 antibody and fludarabine for 5 and 3 days, respectively, followed by transplantation of two additional UCB units on day 49 (Table 1). The OKT3 antibody was chosen owing to its favorable pharmacokinetics—this antibody has an in vivo half life of only a few hours and so would not interfere with the newly established natural killer (NK) and T-cell compartment. To minimize the duration of neutropenia as a result of fungal pneumonia refractory to extensive antimycotic treatment, the patient was additionally transplanted with CD34-positive (CD34⁺)-selected stem cells from an unrelated third-party donor on day 50. The selected CD34⁺ stem cells matched 2 out of 10 of the recipient's original HLA molecules (Table 1). White blood cells exceeded 1,000/l on day 67 (18 and 17 days after double cord blood and third-party stem-cell rescue, respectively) with granulocyte-colony stimulating factor support. Mixed chimerism analysis and subsequent subpopulation analyses using flow cytometry¹ revealed that the granulocytes present were predominantly derived from third-party CD34⁺ stem cells and that the NK cells originated from cord blood 2 (Figure 1D,E). Cord blood 3 was shown to be the source of the initially very faint T-cell compartment, which in succession dominated hematopoiesis (Figure 1D,E).

Regressive pulmonary lesions were documented on CT scan after neutrophil uptake (Figure 1C) and CMV reactivation was controlled using foscarnet (Figure 1B). Despite prophylaxis with ciclosporin and mycophenolate mofetil for graft-versus-host disease (GVHD), grade I–II skin GVHD occurred on days 135 and 173, and was treated with short courses of systemic and topical steroids, respectively. A 2-log decrease in the bcr–abl:abl ratio was noticed 3 weeks after rescue transplantation and bcr–abl transcripts were no longer detectable 101 days after first and 52 days after rescue UCB transplantation (Figure 1A).

After transient complete recovery, the patient progressively developed cognitive deficits, clouding of consciousness and a severe slowing of baseline rhythm as detected by electroencephalography. Cerebral magnetic resonance tomography on day 104 revealed periventricular lesions corresponding to a high copy number of CMV and human herpesvirus 6 DNA and evidence for polyoma-virus infection in the cerebrospinal fluid (CSF; Figure 1B). Despite clearance of polyoma virus and human herpesvirus 6, and reduction of CMV burden in the CSF after cessation of immunosuppressive therapy and extensive treatment with cidofovir, foscarnet, ganciclovir, and a combination of the latter drugs (Figure 1B), neurological symptoms worsened dramatically. Consequently, a fifth transplantation was prepared by isolating T lymphocytes reactive to CMV pp65 antigen² from a third-party donor, matching 2 out of 10 of the recipient's original HLA-molecules (one HLA-A and one HLA-Cw molecule present on the infected tissues). The cells were infused on day 137, and within a few days a dramatic clinical improvement was observed. Recurrence of alpha rhythm was also noted; however, recovery was incomplete with persisting disturbance of short-term memory function.

The patient was discharged on day 178 after the first cord-blood transplantation, but a few weeks later readmission was necessary because of weight loss (approximately 12 kg over a 2–3 month period) and infectious complications, both of which were successfully treated. He again presented with wasting, fever, and severe neurological deficits 261 days after transplantation. CMV DNA was detected at a low copy number in peripheral blood but not in the CSF. Again, invasive fungal infection was diagnosed and turned out to be progressive despite extensive therapy. The patient died 282 days after the first transplantation, without evidence of recurrence of leukemic disease.

Discussion of diagnosis

ALL affects 1.4–1.8 per 100,000 people per year. Peak incidence is found in patients younger than 20 years or older than 75 years of age. Bcr–abl fusion proteins are detected in 25–30% of the patients and are associated with a devastating clinical course resulting in 3-year survival rates of less than 10%.³ Since the introduction of tyrosine-kinase-inhibitor treatment for bcr–abl-positive ALL, the number of patients responding to therapy has risen, but these increased response rates have not translated into improved survival rates.⁴

Treatment and management

The single curative option for bcr–abl-positive ALL is allogeneic stem-cell transplantation.³ UCB serves as an alternative source of stem cells for patients lacking an appropriate family or unrelated donor. UCB effectively restores hematopoiesis and provides permanent protection from relapse in many patients suffering from hematological malignancies.^{5, 6} Immaturity of UCB lymphocytes permits HLA disparities between donor and recipient without overt risk of severe GVHD. Importantly, the less rigorous HLA restrictions associated with UCB transplantation guarantees the instant availability of suitable cryopreserved grafts. Compared with peripheral blood stem-cell or bone-marrow transplantation, UCB transplantation is associated with prolonged neutropenia and a high risk of primary graft failure, attributed to the low number of nucleated cells within the graft.^{7, 8}

Rescue strategies for primary graft failure comprise a second transplantation with bone marrow, mobilized peripheral blood stem cells, UCB⁹ or stem cells from haploidentical donors.¹⁰ On the basis of reports of double UCB¹¹ and single UCB/third-party stem-cell grafts¹² in the primary transplant setting, a combination of double UCB and third-party stem-cell grafts were used in this patient to improve both the likelihood of UCB engraftment and the kinetics of neutrophil recovery. By use of this strategy, we confirmed previous findings indicating that CD34⁺ stem cells do successfully engraft irrespective of HLA concordance.¹²

The graft-versus-leukemia effect of UCB transplantation is traditionally attributed to T-cell function. In this case, we found a significant reduction of the bcr–abl:abl ratio 3 weeks after rescue transplantation in the absence of a detectable T-cell compartment but in the presence of NK cells. Potent NK-cell activity is the hallmark of T-lymphocyte-depleted, haploidentical stem-cell transplantation and we anticipated that the third-party stem cells would confer innate immunity. Surprisingly, we found that cord blood 2 contributed roughly 70% of the initial NK-cell compartment without contributing to T-cell or myeloid progeny.

NK-cell activity is governed by the balance of inhibitory and activating signals. Killer immunoglobulin-like receptors (KIR) are key negative regulators and ligation of KIR to their binding partners—the classical HLA-class I and, most importantly, HLA-C group 1 and 2 molecules—impedes target-cell destruction. Consequently, loss of HLA molecules sensitizes tumor or infected cells to lysis by NK cells. In the setting of haploidentical transplantation, the absence of a cognate KIR ligand on recipient cells elicits NK alloreactivity, which is significantly associated with tumor control.¹³ In this case, the recipient expressed all KIR ligand groups and NK alloreactivity was, therefore, not predicted. Target-cell destruction is the consequence of a complex molecular interaction at the interface between tumor and NK cells, and antileukemic activity of NK cells might be explained by downregulated KIR ligand expression on leukemic blasts. The observation that cord-blood-derived NK rather than T cells constitute the initial phase of antitumor activity is of great importance. On the basis of these findings, genetic information on potential KIR alloreactivity should be considered during the selection of cord-blood donors.

Viral infections such as human CMV reactivation are a common threat in cord-blood transplantation because of the immature T-cell compartment. CMV disease involving the central nervous system (CNS) is associated with a very poor prognosis. Currently available data indicate little benefit of either ganciclovir or foscarnet therapy or treatment with a combination of these drugs once CNS disease is established.¹⁴ Cidofovir can be used as second-line treatment, but does not significantly improve survival.

This patient developed CMV encephalitis despite ongoing antiviral therapy for CMV viremia (Figure 1B). On the basis of disappointing results following drug-based therapies, CMV-specific T cells were adoptively transferred to the patient. Since no T cells were available from the third-party or cord-blood donors, T cells reactive to CMV pp65 antigen were isolated from another third-party donor. The donor was chosen because of instant availability, of sufficient numbers of CMV-reactive T cells in peripheral blood and the matching of at least one HLA-class I restriction antigen.

Clinical improvement occurred rapidly after infusion of the specific T cells without evidence of GVHD. Low numbers of NK and T cells were detected in the CSF, but isolation of sufficient amounts of DNA to attribute the effect of the cells to the third-party donor was not possible. In the peripheral blood, the population was too small to be detected by chimerism analyses.

Although clinically suggestive, we cannot provide formal proof of activity of the third-party stem-cell-derived granulocytes, cord-blood-derived NK cells or CMV-specific third-party T lymphocytes. The chronology of cellular appearances, is, however, interesting, with an initial phase of NK-cell dominance, followed by the ascendancy of granulocytes during a second phase and a third phase of T-lymphocyte dominance. Importantly, NK-cell and specific third-party T-lymphocyte responses were transient and tightly controlled by the cord-blood graft contributing the T-cell compartment.

These findings indicate the possibility of new cellular therapies against viral and tumor-associated target structures. It might be possible to temporarily install high-avidity T cells from third-party donors that are reactive with tumor-associated antigens or lineage-specific markers such as CD19 or B-cell receptor-derived epitopes in the case of B-cell malignancies.^{15, 16} In addition, allorestricted¹⁷ or redirected T-cell receptor-transduced T cells could be considered for adoptive transfer. The temporary nature of the specific T-cell response might allow the undisturbed evolution of the donor's immune system, without the threat of GVHD, once the malignant cells are eradicated and tumor-specific T cells rejected.

Conclusions

The data presented here illustrate the therapeutic option of double cord-blood/third-party stem-cell rescue after primary graft failure and concerted and transient collaboration of multiple transplants for one patient beyond HLA restrictions. Mechanisms mediated by cord-blood-derived NK cells might significantly contribute to the control of high-risk ALL. Moreover, we suggest the possible clinical activity of virus-specific, partially HLA-class I-matched, third-party T lymphocytes against CMV-associated encephalitis.

Acknowledgments

The treatment protocol was set up by GS, HE and RH. Single-cell chimerism by flow cytometry and isolation of CMV-reactive T cells was performed by MS, TF and RH. HLA typing was performed by EK. The manuscript was written by BS and GS and critically reviewed by all authors. We are obliged to D Wernet at the University of Tübingen for selecting the third-party donor for the transfer of virus-specific T cells. We thank G Kögler from the cord-blood bank Düsseldorf/José Carreras Foundation for selecting the cord-blood units and for critically reviewing the manuscript. We would also like to thank A Schnack at the University of Würzburg for providing the CT scans, and V Kunzmann at the University of Würzburg for performing the CD34⁺ cell selection. OG Ottmann performed the MRD monitoring and contributed his experience and information from unpublished data concerning the tyrosine-kinase inhibitor imatinib in bcr–abl-positive ALL. We also thank V Rocha, Paris, for helpful discussions and key treatment suggestions.

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Subject areas under which this article appears: Hematology